Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Aspects of the seed Biology of orchard-produced sitka spruce seeds Chaisurisri, Kowit 1992

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_1992_fall_chaisurisri_kowit.pdf [ 4.47MB ]
Metadata
JSON: 831-1.0075160.json
JSON-LD: 831-1.0075160-ld.json
RDF/XML (Pretty): 831-1.0075160-rdf.xml
RDF/JSON: 831-1.0075160-rdf.json
Turtle: 831-1.0075160-turtle.txt
N-Triples: 831-1.0075160-rdf-ntriples.txt
Original Record: 831-1.0075160-source.json
Full Text
831-1.0075160-fulltext.txt
Citation
831-1.0075160.ris

Full Text

A S P E C T S O F T H E S E E D B I O L O G Y O F O R C H A R D - P R O D U C E D S I T K A S P R U C E S E E D S by K o w i t Cha i su r i s r i B . S c . (Fores t ry ) , K a s e t s a r t Un i ve rs i t y , 1 9 8 0 IVl.Sc. (Fores t ry ) , K a s e t s a r t Un i ve rs i t y , 1 9 8 3 A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y in T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Facu l ty of Fores t ry ) W e a c c e p t th is t hes i s as c o n f o r m i n g to the requ i red s t anda rd T H E U N I V E R S I T Y O F BRIT ISH C O L U M B I A J u l y 1 9 9 2 ® K o w i t C h a i s u r i s r i , 1 9 9 2 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of fbres' l ' geîein*-eS The University of British Columbia Vancouver, Canada Date AMjîn> 4. DE-6 (2/88) Abstract Bio log ica l a s p e c t s o f o r c h a r d - p r o d u c e d S i t k a s p r u c e (Picea sitchensis (Bong.) Carr . ) s e e d s w e r e s tud ied for one s e e d o r cha rd l oca ted o n the S a a n i c h P e n i n s u l a of V a n c o u v e r Is land, B . C . T h e s e a s p e c t s i nc lude : the de te rm ina t i on the level of gene t i c d i ve rs i t y in the o r cha rd popu la t i on and its c o m p a r i s o n to the s p e c i e s ' range e s t i m a t e s , the es t ima t i on of the degree of i nb reed ing in t he o r c h a r d , s e e d and c o n e c r o p parenta l ba l ance ove r t w o c r o p y e a r s , the gene t i c con t ro l of ge rm ina t i on pa rame te rs and s e e d s i z e , the i m p a c t o f s imu la ted long - te rm s to rage on the gene t i c cons t i t u t i on of bu lk s e e d c r o p s , and the re la t i onsh ips b e t w e e n s e e d s ize and gene t i c or ig in on thei r seed l i ng a t t r ibu tes . It w a s f o u n d that : (1 ) M o s t of t he p o l y m o r p h i c a l l o z y m e marke rs i nves t i ga ted s e g r e g a t e d a c c o r d i n g to the e x p e c t e d M e n d e l i a n rat io, and no l inkage b e t w e e n any pair of t h e s e loc i w a s o b s e r v e d ; (2) T h e level o f gene t i c d i ve rs i t y p resen t in the s e e d o r cha rd popu la t i on i nd i ca ted tha t p h e n o t y p i c se l ec t i on of parent t rees d id not r e d u c e the leve l of d i ve rs i t y ; (3) S ign i f i can t ra tes of o u t c r o s s i n g w e r e d e t e c t e d in t he s e e d o r c h a r d popu la t i on ( t „ = 0 . 8 7 5 ) . M o s t of th is i nb reed ing o c c u r r e d in l o w e r b r a n c h e s ; (4) Parenta l imba lance w a s f o u n d fo r the t w o c r o p s i nves t i ga ted , h o w e v e r , i m p r o v e m e n t ove r t ime w a s o b s e r v e d , i nd ica t ing an age e f fec t ; (5) G e r m i n a t i o n pa rame te rs ( G C , P V a n d G V ) and s e e d s i ze w e r e under gene t i c con t ro l w i t h e s t i m a t e s of b r o a d - s e n s e her i tab i l i t ies of 0 . 7 4 - 0 . 7 8 and 0 . 3 6 , r espec t i ve l y ; (6) A r t i f i c ia l ag ing of s e e d ( i .e. , a c c e l e r a t e d ag ing) i nd i ca ted that reduc t ion in ra tes of ge rm ina t i on w e r e c l o n e - s p e c i f i c ; and (7) S e e d s i ze s h o w e d no e f fec t o n seed l i ng a t t r i bu tes , bu t a s ign i f i can t e f f ec t o n ge rm ina t i on s p e e d w a s o b s e r v e d . Table of Contents Page Abstract ii Table of Contents iii List of Tables vi List of Figures ix Acknowledgements xl Chapter 1. General Introduction 1 1.1 Introduction 1 ^ .2 Objectives 3 1.3 References 6 Chapter 2 . Genetics of Allozyme Variants 9 2.1 Introduction 9 2 . 2 Materials and Methods 10 2.3 Results and Discussion 1 6 2 .3 .1 Monomorphic Loci 1 6 2 . 3 . 2 Segregation of Polymorphic Loci 1 6 2 . 3 . 3 Summary 2 3 2 . 3 . 4 Linkage Analyses 2 4 2 . 4 Conclusion 2 7 2 . 5 References 2 9 Chapter 3. Genetic Diversity 3 3 3.1 Introduction 3 3 2.2 Materials and Methods 3 4 3 .2 .1 Isozyme Assay 3 5 2.2.2 Natural Populations 3 5 3 . 2 . 3 Data Analysis 3 5 3.3 Results and Discussion 3 5 3 . 4 References 4 7 Chapter 4. Mating System 5 0 4.1 Introduction 5 0 4.2 Materials and Methods 51 4.3 Results and Discussion 5 3 4 .3 .1 y4//e//c Frequencies 5 3 4 . 3 . 2 Outcrossing Rate 5 3 4 . 4 References 6 4 Chapter 5 . Parental Balance 6 9 5.1 Introduction 6 9 5.2 Materials and Methods 7 0 b.3 Results and Discussion 71 5 .3 .1 1988 Orchard Crop 71 5 . 3 . 2 1990 Orchard Crop 7 4 5 .4 References 8 5 Chapter 6. Accelerated Aging 8 7 6.1 Introduction 8 7 6 . 2 Materials and Methods 8 8 6 .2 .1 Accelerated Aging 8 9 6 . 2 . 2 Germination Test 8 9 6.2.3 Statistical Analysis 9 0 6.3 Results and Discussion 91 6 . 4 References 9 9 Chapter 7 . Germination 101 7.1 Introduction 101 7 . 2 Materials and Methods 1 0 2 7.2 .1 Seed Weight 1 0 2 7 . 2 . 2 Germination Test 1 0 3 7.2.3 Statistical Analysis 1 0 3 7.3 Results and Discussion 1 0 4 7.4 References 1 1 8 Chapter 8. Seed Size and Seedling Attributes 121 8.1 Introduction 121 8.2 Materials and Methods 121 8 . 3 Results and Discussion 1 2 3 8 .4 References 1 2 7 Chapter 9. Conclusions 1 2 9 Lis t of T a b l e s Table P a g e 2 . / . L is t of e n z y m e s , E n z y m e C o m m i s s i o n R e f e r e n c e n u m b e r s , and G e l Buf fe r s y s t e m s u s e d fo r S i t ka s p r u c e e l ec t r opho res i s 12 2.2. E lec t ropho re t i c P r o c e d u r e : A . buf fer s y s t e m ( E l - K a s s a b y et al. 1 9 8 2 a ) and B. s ta in rec ipes (Yeh and O ' M a l l e y , 1 9 8 0 ) 1 3 2.3. D e s c r i p t i o n o f g a m e t o p h y t i c p h e n o t y p e , al lele d e s i g n a t i o n s o f S i t k a s p r u c e e n z y m e s , and re fe rences to inher i tance s tud ies in o the r c o n i f e r s 17 2.4. Log - l i ke l i hood 6 - t e s t s o n seg rega t i on ra t ios of 13 p o l y m o r p h i c loc i in S i t ka s p r u c e s e e d s 2 0 2.5. G o o d n e s s - o f - f i t a n a l y s e s fo r pa i r -w i se c o m b i n a t i o n s of loc i in S i t ka s p r u c e . Log - l i ke l i hood t e s t s jo int i n d e p e n d e n c e a s s o r t m e n t (G,). Par t i t i oned t e s t s tes t s e g r e g a t i o n at l o c u s A (xi), B (xl) and jo in t s e g r e g a t i o n Off) 2 5 2.6. T e s t of i n d e p e n d e n c e for the PGM-/.•PGM-2 c o m b i n a t i o n 2 6 2 . 7 . T h r e e - w a y log- l i ke l ihood tes t for i n d e p e n d e n c e fo r t he PGM-1:6PG-2 c o m b i n a t i o n 2 8 3.1. Loca t i on of 1 0 l U F R O natura l popu la t i ons of S i t ka s p r u c e to w h i c h resu l t s f r o m the s e e d o r cha rd w e r e c o m p a r e d 3 6 3.2. A l l e l i c f r e q u e n c i e s at 1 3 loc i in 1 0 l U F R O natura l p o p u l a t i o n s (Yeh and E l -K a s s a b y , 1 9 8 0 ) and s e e d o r cha rd (S .O . ) of S i t k a s p r u c e 3 7 3.3. R a n g e of a l l o z y m e f r equenc ies in natura l popu la t i ons a n d s e e d o r cha rd 3 9 3.4. M e a n h e t e r o z y g o s i t y per l o c u s , m e a n n u m b e r of a l le les per l o c u s , a n d p e r c e n t a g e o f loc i p o l y m o r p h i c in 10 l U F R O natura l p o p u l a t i o n s and s e e d o r cha rd 41 3.5. G e n e - d i v e r s i t y s ta t i s t i cs e s t i m a t e s fo r 1 3 p o l y m o r p h i c loc i a v e r a g e d o v e r 1 0 natura l popu la t i ons and ove r 11 popu la t i ons (10 na tura l a n d one s e e d orchard) 4 3 3.6. G e n e t i c d i s t a n c e s b e t w e e n S i t ka s p r u c e p o p u l a t i o n s , 1 0 na tura l p o p u l a t i o n s and a s e e d o r c h a r d (SO) 4 4 4.1. A l l e l i c f r e q u e n c i e s fo r o u t c r o s s i n g po l len and mate rna l gene p o o l s fo r s a m p l e s ob ta ined f r o m the upper and l o w e r c r o w n po r t i ons a n d c o m b i n e d fo r a S i t k a s p r u c e s e e d o r c h a r d 5 4 4.2. S i n g l e - l o c u s (t^) a n d m u l t i l o c u s (t^) e s t i m a t e s of o u t c r o s s i n g fo r s a m p l e s ob ta ined f r o m the upper and l o w e r leve ls o f the c r o w n of c lona l S i t ka s p r u c e t rees in a m a n a g e d s e e d o r cha rd 5 5 4.3. E s t i m a t e s of po l len-a l le l ic f r e q u e n c i e s for 3 (w i th PGM-2) and 2 loc i (w i thou t PGM-2) c o r r e s p o n d i n g to mate rna l g e n o t y p e s (number of parent t rees in pa ren theses ) 5 7 4.4. N u m b e r of e m b r y o g e n o t y p e s s c o r e d c o r r e s p o n d i n g to ma te rna l g e n o t y p e s , and o u t c r o s s i n g rate ( t „ ) 5 8 4.5. E f fec t of c r o w n level o n o u t c r o s s i n g e s t i m a t e s in s e e d o r c h a r d s 6 0 4.6. C o m p a r i s o n of m a t i n g - s y s t e m e s t i m a t e s in s e e d o r c h a r d and natura l popu la t i ons 6 2 5.1. C o n e and f i l led s e e d p r o d u c t i o n in the 1 9 8 8 c r o p , and their e f fec t i ve popu la t i on n u m b e r (N,,) 7 3 5.2. A N O V A : e s t i m a t e s of va r i ance c o m p o n e n t s of 1 9 8 8 S i t k a s p r u c e s e e d o r c h a r d p r o d u c t i o n 7 5 5.3. 1 9 9 0 c o n e , s e e d , f i l led s e e d , v iab le s e e d p r o d u c t i o n , and thei r e f fec t i ve popu la t i on n u m b e r (N,,) 7 9 5.4. A N O V A : e s t i m a t e s of va r i ance c o m p o n e n t s of 1 9 9 0 S i t k a - s p r u c e s e e d o r c h a r d p r o d u c t i o n 81 6.1. Va r i a t i on in ge rm ina t i on of 6 S i t ka s p r u c e c l o n e s f o l l o w i n g a c c e l e r a t e d ag ing ( A A ) a n d s e e d p re t rea tmen t (va lues are p e r c e n t a g e s of the to ta l m e a n squa res ) 9 2 7. / . Es t ima t i on of va r i ance c o m p o n e n t s , s i g n i f i c a n c e leve l , a n d b r o a d - s e n s e her i tab i l i t ies (h^) fo r i nd iv idua l s e e d w e i g h t of 1 8 S i t k a s p r u c e c l o n e s 1 0 6 7 .2 . Es t ima t i on of va r i ance c o m p o n e n t s , and s i g n i f i c a n c e level fo r ge rm ina t i on p a r a m e t e r s of 1 8 S i t k a s p r u c e c l o n e s 1 0 9 7.3. Es t ima t i on of va r i ance c o m p o n e n t s , s i g n i f i c a n c e leve l , and b r o a d - s e n s e her i tabi l i t ies (h^) for ge rm ina t i on pa rame te rs us ing u n s o r t e d s e e d s of 18 S i t k a s p r u c e c l o n e s 1 1 0 7.4. Es t ima t i on of va r i ance c o m p o n e n t s , and s i g n i f i c a n c e leve l fo r ge rm ina t i on pa rame te rs u s i n g so r t ed s e e d s of 1 8 S i t k a s p r u c e c l o n e s 1 1 4 7.5. Es t ima t i on of va r i ance c o m p o n e n t s a n d s i g n i f i c a n c e leve l fo r ge rm ina t i on pa rame te rs of 1 8 S i t k a s p r u c e c l o n e s 1 1 6 8.1. Va r i a t i on in d iamete r (Dia), he igh t (Ht), s h o o t d ry w e i g h t ( S D W ) , roo t d ry w e i g h t ( R D W ) , to ta l d ry w e i g h t ( T D W ) , and s h o o t / r o o t d ry w e i g h t rat io (S/R) of S i t k a s p r u c e seed l i ngs f r o m t w o s e e d s i z e s of 18 c l o n e s 1 2 4 8.2. S t u d e n t - N e w m a n - K e u l s mu l t ip le - range t e s t s fo r d iamete r at roo t co l la r , he igh t , s h o o t d ry w e i g h t , root d ry w e i g h t , to ta l d ry w e i g h t , and r oo t - shoo t rat io of e i gh t -mon th -o l d s e e d l i n g s f r o m 1 8 S i t ka s p r u c e c l o n e s 1 2 5 List of Figures Figure Page 1.1. D is t r ibu t ion of Sitica s p r u c e and l oca t i ons of o r cha rd paren t t rees (A f te r P o w e l l s , 1 9 6 5 ) 4 2.1. Band ing pa t te rns and their a l le l ic d e s i g n a t i o n s fo r 1 4 a l l o z y m e loc i in S i t k a s p r u c e . T h e n u m b e r s a b o v e b a n d s refer to the re lat ive m ig ra t ion d i s t a n c e . T h e s h a d e d l ine rep resen ts a he te rod imer . T h e d a s h e d l ine rep resen ts a nul l al lele (n) 1 5 3.1. P h e n o g r a m of S i t ka s p r u c e d i f fe rent ia t ion b a s e d o n N e i ' s ( 1 9 7 8 ) u n b i a s e d gene t i c d i s t a n c e . C l u s t e r s are p r o d u c e d us ing the u n w e i g h t e d - p a i r - g r o u p -m e t h o d s a lgo r i thm ( U P G M ) w i t h N e i ' s ( 1 9 7 8 ) u n b i a s e d gene t i c d i s t a n c e s 4 5 5.1. 1 9 8 8 c o n e c rop parenta l ba l ance c u r v e b a s e d o n al l o r cha rd c l o n e s (N = 1 3 9 ) , ind ica t ing co t r i bu t i ons of the s a m p l e d c l o n e s 7 2 5.2. R a n k order for 2 2 S i t k a - s p r u c e c l o n e s , 1 9 8 8 c r o p , b a s e d on c o n e a n d s e e d y ie lds 7 6 5.3. C u m u l a t i v e c o n e and f i l led s e e d p r o d u c t i o n c u r v e s of 2 2 S i t ka s p r u c e c l o n e s , 1 9 8 8 c r o p . S t ra igh t l ine rep resen ts equa l con t r i bu t i on 7 7 5.4. 1 9 9 0 c o n e c r o p parenta l ba l ance c u r v e b a s e d o n all o r cha rd c l o n e s (N = 1 3 9 ) , ind ica t ing con t r i bu t i ons of the s a m p l e d c l o n e s 7 8 5.5. R a n k order fo r 1 8 S i t k a s p r u c e c l o n e s , 1 9 9 0 c r o p , b a s e d o n c o n e a n d s e e d y ie lds 8 2 5.6. C u m u l a t i v e c o n e , f i l led s e e d s , to ta l s e e d and v iab le s e e d p r o d u c t i o n c u r v e s fo r 18 S i t ka s p r u c e c l o n e s , 1 9 9 0 c r o p . S t ra igh t l ine rep resen ts equa l con t r i bu t i on 8 3 6.1. A v e r a g e ge rm ina t i on (21 days ) of uns t ra t i f i ed s e e d s of 6 S i t k a s p r u c e c l o n e s af ter acce le ra ted ag ing 9 3 6.2. A v e r a g e ge rm ina t i on (21 days ) of s t ra t i f ied s e e d s o f 6 S i t k a s p r u c e c l o n e s af ter acce le ra ted ag ing 9 4 6.3. M o i s t u r e - c o n t e n t c u r v e s of s e e d s of 6 S i t ka s p r u c e c l o n e s af ter a c c e l e r a t e d ag ing 9 5 6.4. C u r v e s s h o w i n g ge rm ina t i on of uns t ra t i f i ed , s t ra t i f i ed , and m o i s t u r e c o n t e n t of a c c e l e r a t e d - a g e d s e e d s ; ave rage for 6 c l o n e s 9 6 7. / . S e e d w e i g h t d is t r ibu t ion c u r v e s of 18 S i t ka s p r u c e c l o n e s 1 0 5 7.2. D i f f e rences of s e e d w e i g h t in sma l l and large s o r t e d s e e d s f r o m c l o n e n o s . 5 and 1 5 4 1 0 8 7.3. G e r m i n a t i o n c u r v e s for unso r ted -uns t ra t i f i ed s e e d s of 18 Sitl<a s p r u c e c l o n e s I l l 7.4. G e r m i n a t i o n c u r v e s fo r unsor ted-s t ra t i f i ed s e e d s of 18 Sitl<a s p r u c e c l o n e s . . . 1 1 2 7 .5 . D i f f e rences in ge rm ina t i on rate of uns t ra t i f ied and s t ra t i f ied s e e d s of c l o n e n o s . 4 1 1 and 5 1 6 ( l ow and higf i d o r m a n t s e e d s , respec t i ve l y ) 1 1 3 A c k n o w l e d g e m e n t s It is m y great p leasure to e x p r e s s m y app rec ia t i on to the o rgan i za t i ons a n d peop le w h o have in va r i ous w a y s con t r i bu ted to th is t h e s i s . T h i s s t u d y w a s m a d e p o s s i b l e by A S E A N -C a n a d a Fores t T ree S e e d Cen t re (C IDA) . C a n a d i a n Pac i f i c Fo res t P r o d u c t s L t d . a n d Fo res t r y C a n a d a , Pac i f i c Fo res t r y C e n t r e , p rov ided resea rch fac i l i t i es . W e s t e r n T ree S e e d L t d . , B l ind B a y , Br i t i sh C o l u m b i a , a s s i s t e d in s e e d e x t r a c t i o n . M r . P. W a s u w a n i c h and Dr . 0 . Sz i k la i h a v e p r o v i d e d s u p p o r t and e n c o u r a g e m e n t t h r o u g h o u t the s t u d y . M y s u p e r v i s o r . Dr . Y . A . E l -K a s s a b y , has p r o v i d e d a s t imu la t i ng s o u r c e of i deas and insp i ra t ion t h r o u g h o u t t he s t u d y . O the r c o m m i t t e e m e m b e r s , D rs . J . E . C a r l s o n , D . G . W . E d w a r d s , D . P . L a v e n d e r , and D.T . Les te r , a l so p r o v i d e d a d v i c e and a s s i s t a n c e w h e n requ i red . M s . S . A . Ba rnes a s s i s t e d in i s o e n z y m e a s s a y a n d r e v i e w e d the m a n u s c r i p t . M s . K . P . B rady a s s i s t e d in s e e d c o l l e c t i o n and seed l i ng harves t . T h e peop le at S a a n i c h Fo res t r y Cen t re have m a d e m y s t u d y at the cen t re a p leasan t o n e . F ina l ly , I a m indeb ted to m y fami l y and f r i ends ; w i t h o u t thei r s u p p o r t s , th is t hes i s w o u l d not have been c o m p l e t e d . T h e pa t ience and u n d e r s t a n d i n g of m y f r i e n d , M s . S . K o m k h a m , is i nva luab le . C h a p t e r 1 G e n e r a l In t roduc t ion 1.1 Introduction S e e d o r c h a r d s are p r o d u c t i o n popu la t i ons c o n s i s t i n g of gene t i ca l l y supe r i o r t rees w h i c h are i so la ted to reduce po l l ina t ion f r o m o u t s i d e s o u r c e s and are m a n a g e d to p rov ide f requen t , a b u n d a n t , eas i l y h a r v e s t e d s e e d c r o p s {Zobel et al., 1 9 5 8 ) . By de f i n i t i on , s e e d o r c h a r d s are d e s i g n e d and m a n a g e d to p r o d u c e s e e d s tha t are super io r in va lue to t h o s e ob ta i ned f r o m s e e d p r o d u c t i o n a reas or r a n d o m u n i m p r o v e d co l l ec t i ons ( A s k e w , 1 9 8 8 ) . In c o n i f e r s , inbreed ing c a u s e s reduc t i on in g r o w t h and seed l i ng su rv i va l (Frank l in , 1 9 7 0 ) . E v e n t h o u g h s e e d o r c h a r d s h a v e been d e s i g n e d to p reven t i nb reed ing , inb reed ing has st i l l been repor ted for m a n y c o n i f e r o u s s e e d o r c h a r d s ( B r o w n et al., 1 9 7 5 ; S h a w and A l l a r d , 1 9 8 2 ; Gr i f f i n , 1 9 8 4 ; R i t land and E l - K a s s a b y , 1 9 8 5 ; O m i and A d a m s , 1 9 8 6 ) . A l s o , it has been o b s e r v e d tha t on l y cer ta in c l o n e s m a y con t r i bu te g e n e s to o r cha rd s e e d c r o p s ( J o n s s o n et al., 1 9 7 6 ; Gr i f f i n , 1 9 8 2 ; Mû l l e r -S ta rk , 1 9 8 2 ; S c h m i d t l i n g , 1 9 8 3 ; E l - K a s s a b y et al., 1 9 8 9 ; E l - K a s s a b y and R e y n o l d s , 1 9 9 0 ; B o e s etal., 1 9 9 1 ) . In c o m p a r i s o n to natura l p o p u l a t i o n s , s e e d o r c h a r d s usua l l y con ta i n a res t r i c ted n u m b e r of pa ren ts , there fo re , the r isk of l oss of gene t i c var iab i l i ty for fu ture p lan ta t i ons is of c o n c e r n (Bouvare l , 1 9 7 0 ; A d a m s , 1 9 8 1 ) . B e c a u s e t rees are o f ten i n t r oduced in to h igh ly h e t e r o g e n e o u s natura l e n v i r o n m e n t s (Hamr ick , 1 9 9 1 ) , g e n e t i c d i ve rs i t y in the cu l t i va ted fo res t is essen t i a l to enab le t h e m to c o u n t e r b a l a n c e the e f f ec t s of env i r onmen ta l c h a n g e s (K le inschmi t , 1 9 7 9 ; G r e g o r i u s , 1 9 8 9 ) . Super io r i t y of the o r c h a r d - p r o d u c e d s e e d s c a n be j u d g e d by qua l i t y and quan t i t y , w h i c h is a f u n c t i o n of the p e r c e n t a g e of f i l led s e e d , and the gene t i c c o m p o s i t i o n of the s e e d c r o p ( A s k e w , 1 9 8 8 ) . In th is r espec t , the qua l i t y of the o rcha rd s e e d c r o p s c a n be u s e d to de te rm ine if the p r imary ob jec t i ve of the s e e d o r c h a r d has been me t a n d to e x a m i n e the b reed ing behav iou r of the o r c h a r d p o p u l a t i o n . S e l e c t i v e b reed ing p r o g r a m s fo r no r the rn c o n i f e r o u s s p e c i e s h a v e e m p h a s i z e d the add i t i ve gene t i c e f f ec t s in r a n d o m ma t ing popu la t i ons w i th in s e e d o r c h a r d s . T h e s e require a t h o r o u g h u n d e r s t a n d i n g of the var ia t ion in the s p e c i e s range to maxinnize gene t i c re tu rns (Yeh and R a s m u s s e n , 1 9 8 5 ) . B reed ing p r o c e s s e s have been repor ted to n a r r o w the gene t i c base of t he s p e c i e s wh i l e i m p r o v i n g c o m m e r c i a l va lues (F ranc is , 1 9 8 1 ; N a m k o o n g a n d R o b e r d s , 1 9 8 2 ) . T h e m a i n t e n a n c e of gene t i c d ive rs i t y in the b reed ing popu la t i on of a n y c o m m e r c i a l l y - v a l u a b l e s p e c i e s s h o u l d be a pr ior i ty of any b reed ing p r o g r a m (Hamr ick , 1 9 9 1 ) to ma in ta in the o p p o r t u n i t y fo r fu tu re se lec t i on ( A d a m s , 1981 ). T h e r e f o r e , it is c ruc ia l t o de te rm ine th is e f fec t at the ear ly s t a g e s of a b reed ing p r o g r a m to m in im ize l o s s e s s i n c e fo res t t ree b reed ing p r o g r a m s are b a s e d o n the gene t i c var iabi l i ty of natura l or i n t r oduced p o p u l a t i o n s . T o da te , the i m p a c t of d o m e s t i c a t i o n o n fo res t t rees has not been e x a m i n e d . H o w e v e r , d o m e s t i c a t i o n of agr icu l tu ra l c rop p lan ts has resu l ted in l o s s of gene t i c var iab i l i ty (E l ls t rand and M a r s h a l l , 1 9 8 5 ) . E lec t ropho res i s of i s o z y m e s m a k e s the s t u d y of gene t i c va r ia t ion and the ma t i ng s y s t e m in p lant popu la t i ons poss ib l e at the s e e d c r o p s tage ( C l e g g , 1 9 8 0 ) . In f o res t r y , th is t e c h n i q u e has been u s e d in m a n y a s p e c t s of f o res t t ree popu la t i on g e n e t i c s (Hamr i ck et al., 1 9 7 9 ) i nc lud ing e x a m i n i n g gene t i c var ia t ion (Hamr ick et al., 1 9 7 9 ; Y e h and E l - K a s s a b y , 1 9 8 0 ; Y e h and O ' M a l l e y , 1 9 8 0 ; Y e h et al., 1 9 8 6 ) and ma t i ng s y s t e m s ( S h a w at al., 1 9 8 1 ; S h a w and A l l a r d , 1 9 8 2 ; R i t land and E l - K a s s a b y , 1 9 8 5 ) . G e r m i n a t i o n t e s t s c a n p rov i de an ear ly a s s e s s m e n t of po tent ia l f ie ld p e r f o r m a n c e of the s e e d s wh i l e the po ten t ia l of s e e d s to rab i l i t y c a n be ob ta ined f r o m v igour t e s t s ( A s s o c i a t i o n of Of f i c ia l S e e d A n a l y s t s , 1 9 8 3 ) . A c c e l e r a t e d a g i n g has been u s e d to t es t for po ten t ia l s e e d s torab i l i t y in agr i cu l tu ra l c r o p s ( D e l o u c h e and B a s k i n , 1 9 7 3 ) . T h i s m e t h o d has been app l ied to s o m e fo res t t ree s e e d s as w e l l (P i te l , 1 9 8 0 ; B l a n c h e et al., 1 9 8 8 , 1 9 9 0 ) . H o w e v e r , the r e s p o n s e of s e e d s to a c c e l e r a t e d ag ing is s p e c i e s -s p e c i f i c ( A s s o c i a t i o n of Of f i c ia l S e e d A n a l y s t s , 1 9 8 3 ; B l anche et al., 1 9 8 8 , 1 9 9 0 ) . S i t ka s p r u c e (Picea sitchensis (Bong.) Carr . ) is the la rgest of the s p r u c e s g r o w i n g in N o r t h A m e r i c a (Powe l l s , 1 9 6 5 ) . Its range , cha rac te r i zed by a long f ros t - f ree p e r i o d , h igh p rec ip i t a t i on , h igh humid i t y and mode ra te t e m p e r a t u r e s , o c c u p i e s a n a r r o w s t r ip a b o u t 1 , 8 0 0 m i les l ong a long the Pac i f i c c o a s t f r o m the K o d i a k Is lands in s o u t h e r n A l a s k a t o n o r t h w e s t e r n Ca l i f o rn ia (Figure 1.1). It is c l o s e l y a s s o c i a t e d w i t h a c o a s t a l f o g bel t , va r y i ng in w i d t h f r o m a f e w mi les in Ca l i f o rn ia t o abou t 1 3 0 mi les in A l a s k a . S i t ka s p r u c e is rarely f o u n d a b o v e 1 , 0 0 0 fee t (Powe l l s , 1 9 6 5 ; R o c h e and F o w l e r , 1 9 7 5 ) . It is the on ly N o r t h A m e r i c a n s p r u c e tha t h a s g r o w n w e l l fo r cen tu r i es as an exo t i c s p e c i e s in E u r o p e , S c a n d i n a v i a , and N e w Z e a l a n d . In s o m e p l a c e s , S i t k a s p r u c e h a s a d a p t e d to b e c o m e a land race . In Br i ta in , I re land, and s o m e par ts of No r th A m e r i c a , it is a major s p e c i e s fo r t imber and pu lp p r o d u c t i o n (Roche and F o w l e r , 1 9 7 5 ) . In Br i t ish C o l u m b i a , S i t ka s p r u c e is a c o m m e r c i a l l y - i m p o r t a n t s p e c i e s (Br i t ish C o l u m b i a M in i s t r y of Fo res t s , 1 9 9 1 ) ; h o w e v e r , its b reed ing p r o g r a m is in an ear ly s t a g e of d e v e l o p m e n t (Yeh and R a s m u s s e n , 1 9 8 5 ) . S i n c e 1 9 7 1 , f ou r S i t ka s p r u c e s e e d o r c h a r d s have been es tab l i shed ( H a n s o n , 1 9 8 5 ; Br i t ish C o l u m b i a M in i s t r y of F o r e s t s , 1 9 9 0 ) , and on ly one s e e d o r c h a r d is in the p r o d u c t i o n p h a s e ( H a n s o n , 1 9 8 5 ) . Due to S i t ka s p r u c e w e e v i l (Pissodes strobi (Peck.) ) a t tack ( M a c S i u r t a i n , 1981 ; A l f a r o , 1 9 8 2 ; 1 9 8 9 ) , re fo res ta t i on of the s p e c i e s is l imi ted to l o w haza rd a reas (Hepper and W o o d , 1 9 8 4 ) . H o w e v e r , a s t u d y of w e e v i l r es i s t ance in S i t ka s p r u c e ind ica tes tha t there is a h igh po ten t ia l to o v e r c o m e th is p r o b l e m t h r o u g h gene t i c i m p r o v e m e n t (Y ing , 1 9 9 1 ) . A t th is d e v e l o p m e n t a l s t a g e , i n fo rma t i on c o n c e r n i n g the o rcha rd popu la t i ons and thei r i m p a c t o n s e e d p r o d u c t i o n is essen t i a l fo r o r c h a r d m a n a g e m e n t . 1.2 Objectives T h e ob jec t i ves of th is s t u d y are : 1 . 2 . 1 . t o de te rm ine the ex ten t of gene t i c var ia t ion in the s e e d o r c h a r d popu la t i on and c o m p a r e it to the pub l i shed repor ts of va r ia t ion level in na tu re , 1 .2 .2 . to es t ima te the level of i nb reed ing in the s e e d o r c h a r d , 1 .2 .3 . to a s s e s s the s e e d o r cha rd parenta l ba lance b a s e d o n c o n e v s . s e e d c r o p , 1 .2 .4 . to s t u d y the e f fec t of long te rm s to rage of s e e d s o n the gene t i c m a k e u p of s e e d c r o p s . Figure 1.1. Distribution of Sitka spruce and locations of orchard parent trees (After Foweils, 1965). 1 .2 .5 . to de te rm ine t i ie gene t i c con t ro l of ge rm ina t i on pa rame te rs and s e e d s i z e , and 1 .2 .6 . to s t u d y the i m p a c t of s e e d s i z ing o n ge rm ina t i on and s u b s e q u e n t seed l i ng a t t r ibu tes . 1.3 References A d a m s , W . T . 1 9 8 1 . P o p u l a t i o n g e n e t i c s and gene c o n s e r v a t i o n in P a c i f i c N o r t f i w e s t con i f e r s . In Evolution today, Proc. Intern. Congress. System. Evol. Biol., 2 n d . , pp . 4 0 1 - 4 1 5 . A l f a r o , R.I. 1 9 8 2 . F i f t y -year -o ld S i t k a s p r u c e p lan ta t ions w i t h a h i s to ry of i n tens i ve w e e v i l a t tack . J. Entomol. Soc. Br i t ish C o l u m b i a 7 9 : 6 2 - 6 5 . A l f a r o , R.I. 1 9 8 9 . S t e m d e f e c t s in S i t ka s p r u c e i n d u c e d by S i t k a s p r u c e w e e v i l , Pissodes strobi (Peck) . In Proc. lUFRO Working Group on insects affecting reforestation, e d . R.I. A l f a r o and S . G . G l o v e r , pp . 1 7 7 - 1 8 5 , V a n c o u v e r , B . C . A s k e w , G . R . 1 9 8 8 . Es t ima t i on of g a m e t e poo l c o m p o s i t i o n s in c lona l s e e d o r c h a r d s . Silvae Genet. 3 7 : 2 2 7 - 2 3 2 . A s s o c i a t i o n of Of f i c ia l S e e d A n a l y s t s 1 9 8 3 . Seed vigour testing handbook. Con t r i bu t i on N o . 3 2 . A s s o c i a t i o n of Of f i c ia l S e e d A n a l y s t s . Br i t i sh C o l u m b i a M i n i s t r y of F o r e s t s . 1 9 9 1 . Annual report 1989-90. V i c t o r i a , B . C . Br i t i sh C o l u m b i a M i n i s t r y of F o r e s t s . 1 9 9 0 . Coastal seed transfer guidelines. B . C . For . S e r v . S i l v i cu l tu re . B o e s , T . K . , J . R . B rand ie , and W . R . Love t t . 1 9 9 1 . Cha rac te r i za t i on of f l o w e r i n g p h e n o l o g y and s e e d y ie ld in a Pinus sylvestris c l ona l s e e d o r cha rd in N e b r a s k a . Can. For. Res. 2 1 : 1 7 2 1 - 1 7 2 9 . B r o w n , A . H . D . , A . C . M a t h e s o n , and C . G . A l d r i d g e . 1 9 7 5 . Es t ima t i on of t he ma t i ng s y s t e m of Eucalyptus obliqua L 'Hér i t . by us ing a l l o z y m e p o l y m o r p h i s m s . Aust. J. Bot. 2 3 : 9 3 1 -9 4 9 . B l a n c h e , C . A . , W . W . E l a m , J . D . H o d g e s , F.T. Bonne r , and A . C . M a r q u e z . 1 9 8 8 . A c c e l e r a t e d ag ing of s e l e c t e d t ree s e e d s . In Proc. 10th north American forest biology workshop, ed . J . W o r r a l l , J . L o o - D i n k i n s , and D . P . Les te r , pp . 3 2 7 - 3 3 4 . V a n c o u v e r , Br i t ish C o l u m b i a . B l a n c h e , C . A . , W . W . E l a m , and J . D . H o d g e s . 1 9 9 0 . A c c e l e r a t e d ag ing of Quercus nigra s e e d : b i o c h e m i c a l c h a n g e s and app l i cab i l i t y as a v igour tes t . Can. J. For. Res. 2 0 : 1 6 1 1 -1 6 1 5 . B o u v a r e l , P. 1 9 7 0 . T h e c o n s e r v a t i o n of gene r e s o u r c e s in f o res t t r ees . In Genetic resources in plants: Their exploration and conservation, e d . O . H . F ranke l and E. Benne t t , pp . 5 2 3 -5 3 0 . O x f o r d : B l a c k w e l l . C l e g g , M . T . 1 9 8 0 . M e a s u r i n g p lant ma t i ng s y s t e m s . Bioscience 3 0 : 8 1 4 - 8 1 8 . D e l o u c h e , J . C . and C . C . B a s k i n . 1 9 7 3 . A c c e l e r a t e d ag ing t e c h n i q u e s fo r p red ic t ing the re lat ive s to rab i l i t y of s e e d lo ts . Seed Sci & Technol. 1 : 4 2 7 - 4 5 2 . E l - K a s s a b y , Y . A . , A . M . K . Fash le r , and M . C r o w n . 1 9 8 9 . Va r i a t i on in f ru i t f u lness in a Doug las - f i r s e e d o r cha rd and its e f fec t o n c r o p m a n a g e m e n t d e c i s i o n s . Silvae Genet. 3 8 : 1 1 3 - 1 2 1 . E l - K a s s a b y , Y . A . , a n d S . R e y n o l d s . 1 9 9 0 . R e p r o d u c t i v e p h e n o l o g y , pa ren ta l b a l a n c e , and s u p p l e m e n t a l m a s s po l l ina t ion in a S i t k a - s p r u c e s e e d - o r c h a r d . For. Ecol. Manage. 3 1 : 4 5 - 5 4 . E l l s t rand , N . C . a n d D.L . M a r s h a l l . 1 9 8 5 . T h e i m p a c t s of d o m e s t i c a t i o n o n d is t r ibu t ion of a l l o z y m e var ia t ion w i t h i n and a m o n g cu l t i va rs of r ad i sh , Raphanus sativus L. Theor. Appl. Genet. 6 9 : 3 9 3 - 3 9 8 . F o w e i l s , H . A . 1 9 6 5 . Silvias of forest trees of the United States. U S D A For . S e r v . Ag r i cu l t u re H a n d b o o k N o . 2 7 1 . F r a n c i s , C . A . 1 9 8 1 . D e v e l o p m e n t of p lant g e n o t y p e s fo r mul t ip le c r o p p i n g s y s t e m s . In Plant Breeding II, e d . K . J . F rey , pp . 1 7 9 - 2 3 1 . A m e s : T h e Iowa S t a t e U n i v e r s i t y P r e s s . F rank l i n , E . C . 1 9 7 0 . S u r v e y of mu tan t f o r m s and inb reed ing d e p r e s s i o n in s p e c i e s of the fami l y Pinaceae. S o u t h e a s t e r n For . E x p . S t a . , USDA Forest Service Res. Paper S E - 6 1 : 1 - 2 1 . G r e g o r i u s , H-R. 1 9 8 9 . T h e a t t r ibu t ion of p h e n o t y p i c var ia t ion to gene t i c or e n v i r o n m e n t a l var ia t ion in e c o l o g i c a l s t u d i e s . In Genetic effects of air pollutants in forest tree populations, e d . F. S c h o l z , H . -R . G r e g o r i u s and D. R u d i n , 1 -16 . H e i d e l b e r g : Sp r i nge r V e r l a g e . Gr i f f i n , A . R . 1 9 8 2 . C l o n a l var ia t ion in rad iata p ine s e e d o r c h a r d s : l . S o m e f l o w e r i n g , c o n e and s e e d p r o d u c t i o n t ra i ts . Aust. J. For. Res. 1 2 : 2 9 5 - 3 0 2 . Gr i f f i n , A . R . 1 9 8 4 . C l o n a l var ia t ion in rad iata p ine s e e d o r c h a r d s . II. F l o w e r i n g p h e n o l o g y . Aust. J. For. Res. / 4 : 2 7 1 - 2 8 1 . H a m r i c k , J . L . 1 9 9 1 . A l l o z y m e d ive rs i t y of natura l s t a n d s v e r s u s s e e d o r c h a r d lob lo l l y p ine . Paper presented at the 23rd biennial meeting of Canadian Tree Improvement Association, A u g u s t 1 9 - 2 3 , 1 9 9 1 . O t t a w a . H a m r i c k , J . L . , Y . B . L inhar t and J . B . M i t t o n . 1 9 7 9 . R e l a t i o n s h i p s b e t w e e n l i fe h i s to ry cha rac te r i s t i c s and e lec t rophore t i ca l l y de tec tab le gene t i c va r ia t i on in p lan ts . Ann. Rev. Ecol. Syst. 1 0 : 1 7 3 - 2 0 0 . H a n s o n , P. 1 9 8 5 . Seed orchards of British Columbia. V i c t o r i a , B . C . : Q u e e n ' s Pr in ter fo r Br i t ish C o l u m b i a . H e p p e r , D . G . and P . M . W o o d . 1 9 8 4 . Vancouver Forest Region Sitka spruce weevil survey results (1982-19831 with recommandations for planting Sitka spruce. B . C . M i n . For . , V a n c o u v e r Fo res t R e g i o n . Internal R e p . P M - V - 5 . J o n s s o n , A . , I. Ekbe rg and G . E r i k s s o n . 1 9 7 6 . F l o w e r i n g in a s e e d o r c h a r d of Pinus sylvestris L. Stud. For. Sue. 1 3 5 : 1 - 3 8 . K l e i n s c h m i t , J . 1 9 7 9 . L im i ta t i ons for res t r i c t ion of the gene t i c va r ia t i on . Sivae Genet. 2 8 : 6 1 -6 7 . M a c S i u r t a i n , M . P . 1 9 8 1 . Distribution, management, variability and economics of Sitka spruce (Picea sitchensis (Bong.) Carr.) in coastal British Columbia. M . S c . T h e s i s , Un i ve r s i t y of Br i t ish C o l u m b i a , Facu l t y of Fo res t r y , V a n c o u v e r , B . C . Mùl le r -S ta rk , G . 1 9 8 2 . R e p r o d u c t i v e s y s t e m s in con i fe r s e e d o rc f i a rds . I. IVIating probab i l i t y in a s e e d o r cha rd of Pinus sylvetris L. Silvae Genet. 31 :1 88-1 9 7 . N a m k o o n g , G . , and J . H . R o b e r d s . 1 9 8 2 . Sho r t - t e rm loss of neut ra l a l le les in sma l l - popu la t i on b reed ing . Silvae Genet. 3 1 : 1 - 6 . O m i , S . K . and W . T . A d a m s . 1 9 8 6 . Va r ia t i on in s e e d se t and p ropo r t i ons of o u t c r o s s e d p r o g e n y w i t h c l o n e s , c r o w n p o s i t i o n , and t op p run ing in a Doug las - f i r s e e d o r c h a r d . Can. J. For. Res. 1 6 : 5 0 2 - 5 0 7 . P i te l , J . A . 1 9 8 0 . A c c e l e r a t e d ag ing s tud ies of s e e d s of j ack p ine (Pinus banksiana Lamb. ) and red oak (Quercus rubra L.). In Proc. International Symposium on Forest tree seed storage, l U F R O W o r k i n g par ty on S e e d P r o b l e m s , e d . B . S . P . W a n g and J . A . P i te l , pp . 4 0 - 5 4 . P e t a w a w a Na t iona l Fo res t r y Inst i tute, C h a l k R ive r , O n t a r i o . R i t l a n d , K. and Y . A . E l - K a s s a b y . 1 9 8 5 . T h e na ture of i nb reed ing in a s e e d o r c h a r d of D o u g l a s -fir as s h o w n by an e f f i c ien t mu l t i l ocus m o d e l . Theor. Appl. Genet. 7 1 : 3 7 5 - 3 8 4 . R o c h e , L. a n d D . P . F o w l e r . 1 9 7 5 . G e n e t i c s of S i t k a s p r u c e . USDA For. Serv. Res. Pap. W O -2 6 : 1 - 1 5 . S c h m i d t l i n g , R . C . 1 9 8 3 . G e n e t i c var ia t ion in f ru i t f u lness in a lob lo l ly p ine s e e d o r c h a r d . Silvae Genet. 3 2 : 7 6 - 8 0 . S h a w , D . V . and R . W . A l l a r d . 1 9 8 2 . Es t ima t i on of o u t c r o s s i n g ra tes in Doug las - f i r us ing i s o z y m e marke rs . Theor. Appl. Genet. 6 2 : 1 1 3 - 1 2 0 . S h a w , D . V . , A . L . Kah le r and R . W . A l l a r d . 1 9 8 1 . A mu l t i l ocus e s t i m a t o r of ma t i ng s y s t e m pa rame te rs in p lant popu la t i ons . Proc. Nat. Acad. Sci. U S A 7 8 : 1 2 9 8 - 1 3 0 2 . Y e h , F . C . and Y . A . E l - K a s s a b y . 1 9 8 0 . E n z y m e var ia t ion in natura l p o p u l a t i o n s of S i t ka s p r u c e (Picea sitchensis). 1. G e n e t i c var ia t ion pa t te rns a m o n g t rees f r o m l U F R O p r o v e n a n c e s . Can. J. For. Res. 1 0 : 4 1 5 - 4 2 2 . Y e h , F . C . a n d D. O ' M a l l e y . 1 9 8 0 . E n z y m e var ia t ion in na tura l p o p u l a t i o n s of Doug las - f i r , Pseudotsuga menziesii (Mirb.) F r a n c o , f r o m Br i t i sh C o l u m b i a . 1. G e n e t i c var ia t ion pa t te rns in c o a s t a l p o p u l a t i o n s . Silvae Genet. 2 9 : 8 3 - 9 2 . Y e h , F . C , M . A . K . Kha l i l , Y . A . E l - K a s s a b y and D . C . T rus t . 1 9 8 6 . A l l o z y m e var ia t ion in Picea mariana f r o m N e w f o u n d l a n d : gene t i c d i ve rs i t y , popu la t i on s t r uc tu re , and ana l ys i s of d i f fe ren t ia t ion . Can. J. For. Res. 1 6 : 7 1 3 - 7 2 0 . Y e h , F . C . and S . R a s m u s s e n . 1 9 8 5 . Her i tab i l i ty of he igh t g r o w t h in 10 - yea r -o l d S i t ka s p r u c e . Can. J. Genet. Cytol. 2 7 : 7 2 9 - 7 3 4 . Y i n g , C . C . 1 9 9 1 . G e n e t i c R e s i s t a n c e to the w h i t e p ine w e e v i l in S i t ka s p r u c e . B.C. Min. For. Res. Notes N o . 1 0 6 : 1 - 1 7 . Z o b e l , B . J . , J . Barber , C . L . B r o w n and T . O . Per ry . 1 9 5 8 . S e e d o r c h a r d s - the i r c o n c e p t and m a n a g e m e n t . J. For. 5 6 : 8 1 5 - 8 2 5 . Chapter 2 Genetic Control of Allozyme Variants in Sitka Spruce 2.1 Introduction E lec t r opho res i s is a p r o c e s s w h e r e p ro te ins are f o r c e d to m ig ra te t h r o u g h an i n t r oduced m o l e c u l a r s i e v i n g m e d i u m unde r the in f l uence of an e lec t r i c cu r ren t (Marke r t and Mo l l e r , 1 9 5 9 ) . S i n c e i ts i n t roduc t i on to popu la t i on g e n e t i c s by L e w o n t i n a n d H u b b y ( 1 9 6 6 ) a n d H u b b y a n d L e w o n t i n ( 1 9 6 6 ) , it has been u s e d e x t e n s i v e l y to s t u d y the gene t i c var ia t ion in a w i d e ar ray of l i v ing o r g a n i s m s . Un l ike m o r p h o l o g i c a l t ra i ts , a l l o z y m e s exh ib i t c o d o m i n a n t e x p r e s s i o n and thei r m o d e of i nher i tance usua l l y f o l l o w s s imp le M e n d e l i a n g e n e t i c s . A l l o z y m e var ia t ion has been s tud ied e x t e n s i v e l y in bac te r ia ( M i l k m a n , 1 9 7 3 ) , h u m a n s (Harr is , 1 9 6 6 ) , annua l s (C legg and A l l a r d , 1 9 7 2 ) , and fo res t t rees (Hamr i ck et al., 1 9 7 9 ) . Fo res t gene t i c i s t s have u s e d i s o z y m e s in p r o v e n a n c e resea rch ( F a l k e n h a g e n , 1 9 8 5 ) , to s t u d y hybr id z o n e s ( C o p e s and B e c k w i t h , 1 9 7 7 ) , ma t i ng s y s t e m s ( S h a w et al., 1981 ; R i t l and and E l -K a s s a b y , 1 9 8 5 ) , po l len m ig ra t ion (Smi th and A d a m s , 1 9 8 3 ; E l - K a s s a b y and R i t l and , 1 9 8 6 ) , po l l i na t ion b io l ogy (Webbe r and Y e h , 1 9 8 7 ) , and t o de te rm ine the va l id i ty of c o n t r o l l e d c r o s s e s ( A d a m s et al., 1 9 8 8 ) . In c o n i f e r s , s e e d s , ma tu re t i s sue , and d o r m a n t b u d s c a n be u s e d fo r i s o z y m e a n a l y s i s . H o w e v e r , in m o s t inher i tance s tud ies , m e g a g a m e t o p h y t i c t i s s u e f r o m the s e e d has been used (Lundkv i s t , 1 9 7 7 ; E l - K a s s a b y a / . , 1 9 8 2 a ; C h e l i a k a n d P i te l , 1 9 8 4 ) . M e g a g a m e t o p h y t i c t i s s u e is su i tab le mater ia l for inher i tance s tud ies s i n c e it is hap lo id , t hus a l l o w i n g d i rec t o b s e r v a t i o n of M e n d e l i a n seg rega t i on at h e t e r o z y g o u s i s o z y m e loc i (Gur ies and L e d i g , 1 9 7 8 ) . S i m p l e ex t rac t i on p r o c e d u r e s c a n be app l ied b e c a u s e the m e g a g a m e t o p h y t i c t i s s u e d o e s no t con ta i n large a m o u n t s of s e c o n d a r y me tabo l i t es . D e s p i t e the a d v a n t a g e of e n z y m e e l e c t r o p h o r e s i s , re la ted s p e c i e s o f ten r e s e m b l e one ano the r in genera l i s o z y m e cha rac te r i s t i c s . H o w e v e r , s p e c i e s c a n d i f fer in 1 ) the n u m b e r s of loc i fo r the s a m e e n z y m e ( A d a m s and J o l y , 1 9 8 0 a ; E l - K a s s a b y , 1 9 8 1 ), 2) t he b a n d p h e n o t y p e s for s imi lar loc i ( A d a m s and J o l y , 1 9 8 0 a ; E l - K a s s a b y et al. 1 9 8 2 a ) , 3) t he in te rac t ion of p r o d u c t s at t w o loc i ( E l - K a s s a b y , 1 9 8 1 ) , and 4) the e x p r e s s i o n of mod i f i e r loc i (Harry, 1 9 8 3 ) . T o a v o i d m is in te rp re ta t i on , it is impor tan t to d o c u m e n t band inher i tance be fo re beg inn ing e lec t ropho re t i c a n a l y s e s in a s p e c i e s (Rud in , 1 9 7 6 ; M i l la r , 1 9 8 5 ) . Popu la t i on -gene t i c s a n a l y s e s s u c h as the es t ima t i on of ma t i ng s y s t e m paramete rs ( S h a w et al., 1 9 8 1 ; R i t land and E l - K a s s a b y , 1 9 8 5 ) and gene t i c d i s t a n c e (Ne i , 1 9 7 5 ) require i n fo rma t ion f r o m a n u m b e r of i ndependen t l oc i . T h e r e f o r e , l inked loc i s h o u l d not be u s e d . L i n k a g e m a p s of i s o z y m e s are a p re l im inary s tep in t he use of i s o z y m e s as markers of quan t i ta t i ve l y con t ro l l ed c h a r a c t e r s . R e c o m b i n a t i o n f r ac t i ons b e t w e e n loc i are impor tan t in es tab l i sh i ng s u c h m a p s (Val le jos and T a n k s l e y , 1 9 8 3 ) . T h i s s t u d y repor ts an a n a l y s i s of inher i tance a n d l inkage re la t i onsh ips b a s e d on s e g r e g a t i o n of 1 4 p o l y m o r p h i c loc i f r o m 11 e n z y m e s y s t e m s in m e g a g a m e t o p h y t i c t i s sue of S i t k a s p r u c e . T h e ob jec t i ves of th is c h a p t e r are ; 1) to p resen t the m o d e of inher i tance of a l l o z y m e marke rs in S i t ka s p r u c e , and 2) to de te rm ine the l inkage re la t i onsh ips a m o n g these m a r k e r s . T h i s i n fo rmat ion is a p re requ is i te for s tud ies repor ted in the f o l l o w i n g t w o chap te rs o n gene d ive rs i t y and the ma t i ng s y s t e m . 2 . 2 Materials and Methods C a n a d i a n Pac i f i c Fo res t P r o d u c t s L t d . p rov ided the s e e d s fo r th i s s t u d y f r o m its S i t ka s p r u c e s e e d o r cha rd l oca ted in S a a n i c h t o n , Br i t i sh C o l u m b i a ( lat i tude 4 8 ° 3 5 ' N , long i tude 1 2 3 ° 2 4 ' W ) . T h e o r cha rd c o n s i s t s of 1 3 9 c l o n e s (averag ing 9 . 3 r ame ts per c lone) se lec ted f r o m e leva t i ons b e t w e e n 0 and 4 1 5 m on w e s t e r n V a n c o u v e r Is land, W a s h i n g t o n and O r e g o n (Figure 1.1). T h e o r cha rd w a s e s t a b l i s h e d in 1971 in a r a n d o m s ing le - t ree m i x ove r three unequa l b l o c k s . In S e p t e m b e r 1 9 9 0 , w ind -po l l i na ted s e e d s w e r e c o l l e c t e d f r o m 1 3 4 s e e d o r c h a r d c l o n e s . F ive s e e d - c o n e s w e r e r a n d o m l y c o l l e c t e d f r o m e a c h c l o n e . T h e ident i ty of w h i c h w a s ca re fu l l y ma in ta i ned . C o n e s a m p l e s w e r e dr ied at r o o m t e m p e r a t u r e a n d ex t rac ted by h a n d . S e e d s a m p l e s w e r e labe l led and kept at 2 ° C unt i l u s e d . S e e d s f r o m ind iv idua l c l o n e s w e r e s o a k e d in w a t e r for 2 4 h rs . at r o o m tempera tu re , d ra i ned , and a l l o w e d to ge rm ina te . F r o m e a c h of the 1 3 4 c l o n e s , at least e ight ge rm inan t s w i t h a rad ic le a p p r o x i m a t e l y equa l to the leng th of the s e e d w e r e d i s s e c t e d to separa te m e g a g a m e t o p h y t e s f r o m the e m b r y o s . E lec t rophore t i c t e c h n i q u e s f o l l o w e d t h o s e of Y e h and O ' M a l l e y ( 1 9 8 0 ) , and E l - K a s s a b y a/. ( 1 9 8 2 a ) , us ing hor i zon ta l 1 1 . 5 % (w/v) s t a r c h ge l s . T w o d i f fe rent gel buf fer s y s t e m s (C-Tr i s / c i t ra te , p H . 7 . 0 ; and D-T r i s / c i t ra te :L i th ium bora te , p H . 8 .5) w e r e used (Table 2.2) and e leven e n z y m e s y s t e m s w e r e i nves t i ga ted (7a6/e 2.1). S ta in i ng rec ipes and p r o c e d u r e s f o l l o w e d t h o s e repor ted by Y e h and O ' M a l l e y ( 1 9 8 0 ) , a n d E l - K a s s a b y at al. ( 1 9 8 2 a ) (Table 2.2). For s o m e loc i w h e r e the init ial s c r e e n i n g i nd i ca ted large a m o u n t s of va r i a t i on , an add i t iona l 1 6 m e g a g a m e t o p h y t e / e m b r y o pa i rs w e r e a s s a y e d per c l o n e . T h e f o l l o w i n g n o m e n c l a t u r e w a s u s e d : an e n z y m e and its b a n d p h e n o t y p e s w e r e ident i f ied by the e n z y m e ' s abb rev ia t i on in i tal ic cap i ta l le t ters (Table 2.1); e a c h l o c u s of an e n z y m e w a s de f i ned by the e n z y m e ' s band mig ra t ion and n o t e d abb rev ia t i on a n d , w h e r e mul t ip le loc i o c c u r r e d for an e n z y m e , the e n z y m e p h e n o t y p e s and loc i w e r e n u m b e r e d , w i t h the mos t -anoda l l y -m ig ra t i ng l o c u s des igna ted as " 1 . " W i t h i n e a c h l o c u s , the mos t - f r equen t al lele w a s a s s i g n e d the va lue of 1 0 0 . O the r a l le les of the l o c u s w e r e d e s i g n a t e d by mob i l i t y va lue e x p r e s s e d re lat ive to the mos t - f r equen t a l le le. A n al lele l ack ing s ta in ac t i v i t y (null), and o b s e r v e d at on l y one l o c u s w a s d e s i g n a t e d w i t h l o w e r - c a s e " n " (Figure 2.1). T o tes t the nul l h y p o t h e s i s o f a 1:1 seg rega t i on rat io a m o n g the g a m e t o p h y t e s f r o m h e t e r o z y g o u s c l o n e s , seg rega t i on da ta of e a c h p o l y m o r p h i c l o c u s w e r e p o o l e d a c r o s s all c l o n e s tha t had the s a m e al lele c o m b i n a t i o n and tes ted u s i n g the log - l i ke l ihood G- tes t (Soka l and Roh l f , 1 9 8 1 ) . A G va lue w a s ca l cu la ted to tes t the he te rogene i t y in the o b s e r v e d rat io in g a m e t i c a r rays f r o m d i f ferent c l o n e s . T o a v o i d b ias in C h i - s q u a r e ca l cu la t i ons w h e r e s a m p l e s i z e s are t o o s m a l l , no m o r e t han 2 0 % of the e x p e c t e d cel l f r e q u e n c i e s shou ld be less t han 5 . 0 ( C o c h r a n , 1 9 5 4 ) . The re fo re , Table 2.1. L is t of e n z y m e s , E n z y m e C o m m i s s i o n R e f e r e n c e n u m b e r s , and G e l Buf fe r s y s t e m s u s e d for S i t ka s p r u c e e l ec t r opho res i s G e l Buf fe r S y s t e m " E n z y m e A b b r e v i a t i o n E C N u m b e r C A s p a r t a t e am ino - t r ans fe rase AAT 2 .6 .1 .1 G l u t a m a t e d e h y d r o g e n a s e GDH 1 . 4 . 1 . 3 Leuc ine a m i n o p e p t i d a s e LAP 3 . 4 . 1 . 1 D A c o n i t a s e AGO 4 . 2 . 1 . 3 Es te rase EST 3 . 1 . 1 . 2 G l u c o s e 6 - p h o s p h a t e d e h y d r o g e n a s e G6P 1 . 1 . 1 . 4 9 Isoc i t ra te d e h y d r o g e n a s e IDH 1 . 1 . 1 . 4 2 M a l a t e d e h y d r o g e n a s e MDH 1 . 1 . 1 . 3 7 P h o s p h o g l u c o s e i s o m e r a s e PGI 5 . 3 . 1 . 9 P h o s p h o g l u c o m u t a s e PGM 2 .7 .5 .1 6 - P h o s p h o g l u c o n i c d e h y d r o g e n a s e 6PG 1 . 1 . 1 . 4 4 ° G e l bu f fe r s y s t e m s f o l l o w e d E l - K a s s a b y et al. ( 1 9 8 2 a ) . Table 2.2. E lec t ropho re t i c P r o c e d u r e : A . buf fer s y s t e m ( E l - K a s s a b y et al. 1 9 8 2 a ) and B. s ta in rec ipes (Yeh and O ' M a l l e y , 1 9 8 0 ) A . BUFFER S Y S T E M Buf fer P o w e r S y s t e m E lec t rode G e l C T r i s /c i t ra te p H 7 . 0 (TC) D T r i s / c i t ra te : L i /bo ra te p H 8.1 (RW) 0 . 1 3 M Tr i s and 0 . 0 4 3 M c i t r ic ac id (anhydrous) 0 . 0 6 M L i / hyd rox i de and 0 . 3 M Bor ic ac id (pH 8.1) 1 :10 d i lu t ion of e l ec t rode bu f fe r 0 . 3 M T r i s , 0 . 0 0 5 M C i t r i c ac id (anhydrous) and 1 % e lec t rode buf fer (pH 8.5) 6 0 m A ( « 1 6 0 V) unt i l t r ack ing d y e has m iga ted 5 c m . 2 5 0 V ( « 6 0 m A ) unti l t r ack ing d y e has m iga ted 5 c m . B. STAIN RECIPES E n z y m e S ta in C o m p o n e n t AAT T r i s - H C I , p H 8 . 0 3 0 ml P y r i d o x a l 5 - P h o s p h a t e 1 m g L - A s p a r t i c ac id 2 0 0 m g o -Ke tog lu ta r i c ac id 1 0 0 m g Fas t B lue B B sa l t 2 0 0 m g ACO T r i s - H C I , p H 8 . 0 3 0 ml c i s A c o n i t i c ac id 2 0 0 m g Isoc i t r ic d e h y d r o g e n a s e 4 0 uni t N A D P 1 0 m g 1 % M a g n e s i u m ch lo r ide (w/v) 1 ml N B T 1 0 m g P M S 5 m g EST 0 . 2 M P h o s p h a t e buf fer , p H 6 . 4 3 0 ml a - N a p h t h y l ace ta te " 5 0 m g /9-Naphthy l ace ta te " 5 0 m g {' and in 5 ml ace tone) Fas t B lue RR sa l t 1 0 0 m g GDH T r i s - H C I , p H 8 . 0 3 0 ml M o n o s o d i u m G l u t a m a t e 4 0 0 m g N A D 3 0 m g N B T 3 0 m g P M S 5 m g G6P T r i s - H C L 3 0 ml G l o c o s e - 6 - p h o s p h a t e 2 0 0 m g 1 % M a g n e s i u m ch lo r ide (w/v) 1 ml N A D P 1 0 m g M l 1 1 0 m g P M S 5 m g Table 2.2. (Con t inued) E n z y m e S ta in C o m p o n e n t IDH T r i s - H C I , p H 8 . 0 3 0 m l D L - l s o c i t r i c ac id 1 0 0 m g N A D P 1 0 m g 1 % iV lagnes ium ch lo r ide (w/v) 1 ml M l 1 1 0 m g P M S 5 m g LAP A m i n o p e p t i d a s e buf fer 3 0 ml 0 . 4 % L -Leuc ine B -Naph thy l a m i d e so lu t i on 5 m l B l a c k K sa l t 2 0 m g MDH T r i s - H C I , p H 8 . 0 3 0 ml M a l a t e so lu t i on 4 5 m l T r i s -HCI (pH 8.0) 4 5 m l N A D 1 0 m g N B T 1 0 m g P M S 5 m g PGI T r i s - H C I , p H 8 . 0 3 0 m l D - F r u c t o s e 6 - P h o s p h a t e 2 5 m g G l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e 1 0 uni t N A D P 1 0 m g 1 % M a g n e s i u m ch lo r ide (w/v) 1 m l M l 1 1 0 m g P M S 5 m g PGM T r i s - H C I , p H 8 . 0 3 0 ml a - D - G l u c o s e 1 -phospha te 1 0 0 m g a - D - G l u c o s 1 ,6 -d iphospha te 0 . 5 m g G l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e 1 0 uni t N A D P 1 0 m g 1 % M a g n e s i u m ch lo r ide (w/v) 1 m l M T T 1 0 m g P M S 5 m g 6PG T r i s - H C I , p H 8 . 0 1 0 m l 6 - p h o s p h o g l u c o n i c ac id 1 0 m g N A D P 1 0 m g 1 % M a g n e s i u m ch lo r ide (w/v) 1 m l M T T 1 0 m g P M S 5 m g AAT-1 AAT-2 "îôcT 70 AGO 100 221. 96 EST 116 100 87 75 GDH 100 G6P-1 inn 100 IDH 100 MDH-1 114 PGI-2 100 J 2 i . „ PGM-1 100 , 0 . . . PGM-2 100 LAP-1 100 n 6PG-113 100 -1 86 6PG-2 117 100 ••' " 9 0 Figure 2.1. B a n d i n g pat terns a n d their a l le l ic d e s i g n a t i o n s fo r 1 4 a l l o z y m e loc i in S i t k a s p r u c e . T h e n u m b e r s a b o v e b a n d s refer to the re la t ive m ig ra t ion d i s t a n c e . T h e s h a d e d line rep resen ts a he te rod imer . T h e d a s h e d l ine rep resen ts a nul l al lele (n). l i nkage re la t i onsh ips w e r e pos tu l a ted f r o m the seg rega t i on da ta of pa i rs of a l l o z y m e loc i on ly w h e r e the s a m p l e s i ze w a s greater than 2 0 s e e d s per c l o n e . T h e s ta t i s t i ca l p r o c e d u r e s u s e d to tes t for l inkage w e r e : the o b s e r v e d gene seg rega t i on fo r e a c h l o c u s w a s u s e d to ca l cu la te the e x p e c t e d n u m b e r fo r e a c h of the t w o - l o c u s seg rega t i on g r o u p s (Bai ley, 1 9 6 1 ) , C h i - s q u a r e a n a l y s e s to tes t fo r l inkage (Rudin a n d E k b e r g , 1 9 7 8 ) , and t h r e e - w a y log- l i ke l ihood tes t fo r i n d e p e n d e n c e (Soka l a n d Roh l f , 1 9 8 1 ) . 2 . 3 Resu/ts and Discussion In gene ra l , i s o z y m e ac t i v i t y in S i t ka s p r u c e r e s e m b l e d tha t f o u n d in re la ted s p e c i e s , bo th in the n u m b e r of loc i for g i ven e n z y m e s and in band p h e n o t y p e s (numbers of b a n d s and pa t te rns of s ta in in tens i ty) (see Tab/e 2.3 fo r r ev iew) . A d d i t i o n a l z o n e s tha t appea red i ncons i s t en t l y in s o m e s y s t e m s w e r e e x c l u d e d . T w o m o n o m o r p h i c and 14 p o l y m o r p h i c loc i r e s o l v e d c o n s i s t e n t l y in the 1 3 4 c l o n e s s tud ied (Table 2.4). T h e n u m b e r of a l le les o b s e r v e d and ver i f ied at p o l y m o r p h i c loc i var ied f r o m t w o to fou r , and b a n d p h e n o t y p e s at a l o c u s var ied f r o m nul l to d o u b l e - b a n d e d (Figure 2.1). Four e n z y m e s (ACO, EST, GDH, and /D/V) had a s ing le z o n e of ac t i v i t y , w h i l e the rest had mul t ip le z o n e s . T h e b a n d p h e n o t y p e s at G6P-2, PGI-1, MDH-2, and MDH-3 loc i w e r e unc lear and too i ncons i s t en t fo r re l iable s c o r i n g . 2 .3 .1 /Monomorphic Loci T w o m o n o m o r p h i c z o n e s (AAT-3, and LAP-2) w e r e invar ian t . T h e b a n d p h e n o t y p e s in t h e s e z o n e s r e s e m b l e d t h o s e of o ther c o n i f e r s , w h e r e M e n d e l i a n seg rega t i on h a s b e e n s h o w n (Tab/e 2.3). 2 . 3 . 2 Segregation of Po/ymorphic Loci Aspartate Aminotransferase (AAT) Three z o n e s o f ac t i v i t y w e r e o b s e r v e d in AA T. T h e m o s t - a n o d a l z o n e (AA T-1) p r o d u c e d t w o var ian ts ( 1 0 0 a n d 92) in the m e g a g a m e t o p h y t e s . N o n - s i g n i f i c a n t s e g r e g a t i o n w a s f o u n d b e c a u s e on ly one c l o n e w a s o b s e r v e d w i t h th is var ian t . T w o va r ian ts ( 1 0 0 a n d 20) w e r e o b s e r v e d at AAT-2 (Figure 2.1). A n add i t iona l band w a s l oca ted m i d w a y b e t w e e n the AAT-2 Table 2.3. D e s c r i p t i o n of g a m e t o p h y t i c p h e n o t y p e , al lele des igna t i ons of S i t ka s p r u c e e n z y m e s , and re fe rences to inher i tance s tud ies in o ther con i f e rs L o c u s G a m e t o p h y t i c A l l e l es G a m e t o p h y t i c R e f e r e n c e s repor t ing inher i tance p h e n o t y p e e n z y m e s t ruc tu re in o ther con i fe r s p e c i e s AAT-1 AAT-2 ACQ EST GDH G6P-1 Sing le S ing le S ing le S ing le S ing le S ing le IDH Sing le 1 0 0 , 9 2 1 0 0 , 2 0 1 0 0 , 1 0 4 , 9 6 1 0 0 , 1 1 6 , 8 7 , 7 5 1 0 0 , 1 3 7 , 71 1 0 0 , 1 0 0 ' 1 0 0 , 1 0 8 D imer D imer M o n o m e r D imer G u r i e s and L e d i g , 1 9 7 8 ; O ' M a l l e y etal., 1 9 7 9 ; E l - K a s s a b y e f a / . , 1 9 8 1 ; E l - K a s s a b y et al., 1 9 8 2 a ; K i n g and Danc i k , 1 9 8 3 ; Boy le and M o r g e n s t e r n , 1 9 8 5 ; Che l iak and Pi te l , 1 9 8 5 ; E l - K a s s a b y et al., 1 9 8 7 ; Pi te l et al., 1 9 8 7 ; Erns t et al., 1 9 8 7 ; Per ry and K n o w l e s , 1 9 8 9 ; X i e etal., 1991 G u r i e s and L e d i g , 1 9 7 8 ; Y e h and L a y t o n , 1 9 7 9 ; A d a m s and J o l y , 1 9 8 0 a ; E l - K a s s a b y et al., 1 9 8 1 ; E l - K a s s a b y et al., 1 9 8 2 a ; K i n g and Danc i k , 1 9 8 3 ; Che l i ak and P i te l , 1 9 8 5 ; Mi l la r , 1 9 8 5 ; Har ry , 1 9 8 6 ; S t r a u s s and C o n k l e , 1 9 8 6 ; Ernst etal., 1 9 8 7 ; Pitel et al., 1 9 8 7 ; A d a m s et al., 1 9 9 0 ; X i e ef al., 1991 Rud in and R a s m u s o n , 1 9 7 3 ; Lundkv i s t , 1 9 7 7 ; E l - K a s s a b y et al., 1 9 8 1 ; S t r a u s s and C o n k l e , 1 9 8 6 ; L e w a n d o w s k i and M e j n a r t o w i c z , 1 9 9 0 A d a m s and J o l y , 1 9 8 0 a ; Nea le and A d a m s , 1 9 8 1 ; E l - K a s s a b y et al., 1 9 8 2 a ; K i n g and Danc i k , 1 9 8 3 ; Boy le and M o r g e n s t e r n , 1 9 8 5 ; S t r a u s s and C o n k l e , 1 9 8 6 ; E l - K a s s a b y et al., 1 9 8 7 ; Erns t et al., 1 9 8 7 ; Pi te l etal., 1 9 8 7 ; Per ry and K n o w l e s , 1 9 8 9 ; A d a m s et al., 1 9 9 0 ; X i e etal., 1991 O ' M a l l e y a / . , 1 9 7 9 ; E l - K a s s a b y ef a / . , 1 9 8 1 ; Nea le and A d a m s , 1 9 8 1 ; E l - K a s s a b y etal., 1 9 8 2 a ; K i n g and Danc i k , 1 9 8 3 ; Che l iak e f al., 1 9 8 4 ; Boy le and M o r g e n s t e r n , 1 9 8 5 ; Che l iak and P i te l , 1 9 8 5 ; E l -K a s s a b y et al., 1 9 8 7 ; A d a m s et al., 1 9 9 0 ; L e w a n d o w s k i and M e j n a r t o w i c z , 1 9 9 0 G u r i e s and L e d i g , 1 9 7 8 ; O ' M a l l e y et al., 1 9 7 9 ; Y e h and L a y t o n , 1 9 7 9 ; A d a m s and J o l y , 1 9 8 0 a ; Ecker t et al., 1 9 8 1 ; E l - K a s s a b y et al., 1 9 8 1 ; Nea le and A d a m s , 1 9 8 1 ; E l - K a s s a b y e r a / . , 1 9 8 2 a ; K i n g and Danc i k , 1 9 8 3 ; Che l iak and Pi te l , 1 9 8 4 ; Che l iak et al., 1 9 8 4 ; Nea le etal., 1 9 8 4 ; Boy le and M o r g e n s t e r n , 1 9 8 5 ; Mi l la r , 1 9 8 5 ; Table 2.3. (Con t inued) L o c u s G a m e t o p i i y t i c A l l e l es G a m e t o p h y t i c p h e n o t y p e e n z y m e s t ruc tu re R e f e r e n c e s repor t ing inher i tance in o ther con i fe r s p e c i e s LAP-1 Sing le Nu l l MDH-1 Sing le PGI-2 Sing le PGM-1 PGM-2 Sing le S ing le 1 0 0 , n 1 0 0 , 1 1 4 , 9 2 , 81 1 0 0 , 1 1 7 9 3 1 0 0 , 9 0 , 7 5 1 0 0 , 8 6 Har ry , 1 9 8 6 ; S t r a u s s and C o n k l e , 1 9 8 6 ; E l - K a s s a b y e f a / . , 1 9 8 7 ; Ernst et al., 1 9 8 7 ; Pitel et al., 1 9 8 7 ; Per ry and K n o w l e s , 1 9 8 9 ; A d a m s et al., 1 9 9 0 ; L e w a n d o w s k i and M e j n a r t o w i c z , 1 9 9 0 ; X i e et al., 1991 M o n o m e r T ige rs ted t , 1 9 7 3 ; L u n d k v i s t , 1 9 7 4 ; S i m o n s e n and We l l endo r f , 1 9 7 5 ; G u r i e s and L e d i g , 1 9 7 8 ; O ' M a l l e y et al., 1 9 7 9 ; A d a m and J o l y , 1 9 8 0 a ; Ecker t et al., 1 9 8 1 ; Nea le and A d a m s , 1 9 8 1 ; K i n g and Danc i k , 1 9 8 3 ; Che l iak and P i te l , 1 9 8 5 ; Mi l la r , 1 9 8 5 ; Har ry , 1 9 8 6 ; Pitel et al., 1 9 8 7 ; A d a m s et al., 1 9 9 0 ; L e w a n d o w s k i and M e j n a r t o w i c z , 1 9 9 0 M o n o m e r S i m o n s e n and We l l endo r f , 1 9 7 5 ; G u r i e s and Led ig , 1 9 7 8 ; O ' M a l l e y et al., 1 9 7 9 ; Y e h and L a y t o n , 1 9 7 9 ; A d a m s and J o l y , 1 9 8 0 a ; E l -K a s s a b y et al., 1 9 8 1 ; Nea le and A d a m s , 1 9 8 1 ; E l - K a s s a b y et al., 1 9 8 2 a ; K ing and Danc i k , 1 9 8 3 ; Che l iak etal., 1 9 8 4 ; Ha r r y , 1 9 8 3 ; Nea le et al., 1 9 8 4 ; Boy le and M o r g e n s t e r n , 1 9 8 5 ; Che l i ak and P i te l , 1 9 8 5 ; Mi l lar , 1 9 8 5 ; Har ry , 1 9 8 6 ; S t r a u s s and C o n k l e , 1 9 8 6 ; E l -K a s s a b y et al., 1 9 8 7 ; Pitel et al., 1 9 8 7 ; Per ry and K n o w l e s , 1 9 8 9 ; A d a m s et al., 1 9 9 0 ; L e w a n d o w s k i and M e j n a r t o w i c z , 1 9 9 0 D imer S i m o n s e n and We l l endo r f , 1 9 7 5 ; O ' M a l l e y et al., 1 9 7 9 ; Y e h and L a y t o n , 1 9 7 9 ; A d a m s and J o l y , 1 9 8 0 a ; E l - K a s s a b y et al., 1 9 8 1 ; E l -K a s s a b y et al., 1 9 8 2 a ; K i n g and Danc i k , 1 9 8 3 ; Nea le et al., 1 9 8 4 ; Boy le and M o r g e n s t e r n , 1 9 8 5 ; Che l iak and P i te l , 1 9 8 5 ; Mi l la r , 1 9 8 5 ; Ha r r y , 1 9 8 6 ; S t r a u s s and C o n k l e , 1 9 8 6 ; E l - K a s s a b y a / . , 1 9 8 7 ; E rns t et al., 1 9 8 7 ; Per ry and K n o w l e s , 1 9 8 9 ; A d a m s et al., 1 9 9 0 ; X i e et al., 1991 M o n o m e r G u r i e s and L e d i g , 1 9 7 8 ; O ' M a l l e y et al., 1 9 7 9 ; Y e h and L a y t o n , 1 9 7 9 ; A d a m s and J o l y , 1 9 8 0 a ; Ecker t et al., 1 9 8 1 ; Nea le and M o n o m e r A d a m s , 1 9 8 1 ; E l - K a s s a b y et al., 1 9 8 2 a ; K i n g and Danc i k , 1 9 8 3 ; Nea le et al., 1 9 8 4 ; Boy le and M o r g e n s t e r n , 1 9 8 5 ; Che l iak and P i te l , 1 9 8 5 ; Mi l la r , 1 9 8 5 ; Ha r r y , 1 9 8 6 ; S t r a u s s and C o k l e , 1 9 8 6 ; E l -K a s s a b y et al., 1 9 8 7 ; Pitel et al., 1 9 8 7 ; Per ry and K n o w l e s , 1 9 8 9 ; Table 2.3. (Cont inued) L o c u s G a m e t o p h y t i c p h e n o t y p e A l l e l e s G a m e t o p h y t i c e n z y m e s t ruc tu re Re fe rences repor t ing inher i tance in o ther con i fe r s p e c i e s 6PG-1 6PG-2 Sing le S ing le , Doub le 1 0 0 , 1 1 3 , 8 6 1 0 0 , 1 1 7 , 9 0 D imer D imer A d a m s etal., 1 9 9 0 ; X i e etal., 1991 S i m o n s e n and We l lendo r f , 1 9 7 5 ; G u r i e s and Led ig , 1 9 7 8 ; O ' M a l l e y et al., 1 9 7 9 ; Y e h and L a y t o n , 1 9 7 9 ; A d a m s and J o l y , 1 9 8 0 a ; E l -K a s s a b y etal., 1 9 8 1 ; E l - K a s s a b y e f a / . , 1 9 8 2 a ; K ing and Danc i k , 1 9 8 3 ; Che l i ak and Pi te l , 1 9 8 4 ; Che l i ak et al., 1 9 8 4 ; Nea le et al., 1 9 8 4 ; Boy le and M o r g e n s t e r n , 1 9 8 5 ; Che l i ak and Pi te l , 1 9 8 5 ; Mi l la r , 1 9 8 5 ; Har ry , 1 9 8 6 ; E l - K a s s a b y e f a / . , 1 9 8 7 ; Ernst ef a / . , 1 9 8 7 ; Per ry and K n o w l e s , 1 9 8 9 ; X i e etal., 1991 Table 2.4. Log - l i ke l i hood G- tes t s o n seg rega t i on ra t ios of 1 3 p o l y m o r p h i c loc i in S i t ka s p r u c e s e e d s L o c u s G e n o t y p e O b s e r v e d Rat io P o o l e d G° (df = 1) H e t e r o g e n e i t y G^ df AAT-1 1 0 0 : 9 2 4 : 4 0 . 0 0 0 _ c AAT-2 1 0 0 : 2 0 7 7 : 9 1 1 . 1 6 8 1 7 . 3 1 9 2 0 ACQ 1 0 0 : 1 0 4 1 8 4 : 1 6 8 0 . 7 2 8 6 4 . 9 3 9 * 4 3 1 0 0 : 9 6 3 2 : 1 6 5 . 4 3 7 ' 7 . 7 9 1 5 EST 1 0 0 : 1 1 6 6 7 : 6 9 0 . 0 2 9 1 5 . 3 5 4 1 1 0 0 : 8 7 6 0 : 4 4 2 . 4 7 1 7 . 9 2 2 12 1 0 0 : 7 5 2 3 : 2 5 0 . 0 8 3 7 . 7 1 2 5 G6P-1 1 0 0 : 1 0 0 ' 6 6 9 : 6 1 1 1 0 . 8 9 4 " 1 1 3 . 6 7 1 * * 7 5 GDH 1 0 0 : 1 3 7 5 4 : 7 4 3 . 1 3 8 8 . 2 3 9 1 0 1 0 0 : 7 1 2 0 : 2 0 0 . 0 0 0 1 . 6 8 0 1 IDH 1 0 0 : 1 0 8 1 1 : 1 3 0 . 1 6 7 — LAP-1 1 0 0 : n 7 9 : 8 1 0 . 0 2 5 2 8 . 2 8 7 19 MDH-1 1 0 0 : 1 1 4 2 7 : 1 3 5 . 0 0 5 * 6 . 8 8 9 * * 1 1 0 0 : 9 2 1 0 1 : 1 3 1 3 . 8 9 0 ' 2 6 . 8 9 9 * * 11 1 0 0 : 8 1 9 : 1 5 1 . 5 1 6 — — PGI-2 1 0 0 : 1 1 7 1 0 2 : 9 0 0 . 7 5 0 1 5 . 9 6 5 12 1 0 0 : 9 3 8 3 : 6 1 3 . 3 7 4 7 . 1 7 8 7 PGM-1 1 0 0 : 9 0 2 3 5 : 2 2 9 0 . 0 7 8 5 7 . 4 9 3 * * 2 5 1 0 0 : 7 5 2 5 : 2 3 0 . 0 8 3 6 . 9 3 2 * * 1 PGM-2 1 0 0 : 8 6 4 4 6 : 4 1 0 1 . 5 1 4 7 4 . 4 3 0 * 5 3 6PG-1 1 0 0 : 1 1 3 3 8 0 : 2 2 8 3 8 . 4 0 6 " 1 2 8 . 4 0 9 * * 3 9 1 0 0 : 8 6 1 2 : 1 2 0 . 0 0 0 0 . 7 5 6 1 6PG-2 1 0 0 : 1 1 7 5 9 8 : 5 6 2 1 . 1 1 7 1 2 2 . 6 3 9 * * 6 6 1 0 0 : 9 0 1 2 : 4 4 . 1 8 6 * - -* S ign i f i can t at 5 % . " "S ign i f i can t at 1 % . ' P o o l e d G va l ues ind ica te the overa l l dev ia t i on f r o m 1:1 rat io. '° He te rogene i t y G va l ues ind ica te the a m o u n t of he te rogene i t y in the seg rega t i on rat io a m o n g mo the r t rees . " A n a l y s i s is b a s e d o n s ing le t ree da ta . and AAJ-3 l oc i , and th is z o n e has been in terpre ted as an in te r locus he te rod imer ( A d a m s and J o l y , 1 9 8 0 a ; Che l i ak and P i te l , 1 9 8 4 ; E l - K a s s a b y , 1 9 8 1 ) . T h i s i n te r l ocus b a n d w a s on ly o b s e r v e d fo r the y4/4r-2-20M/4 7 - 3 - 1 0 0 and not b e t w e e n AAT-2-\Ç)()IAAJ-3AQQ. T h e reason fo r th i s a n o m a l y is u n k n o w n at p resen t . T h e o b s e r v e d s e g r e g a t i o n ax AAT-2 is no t s ign i f i can t l y d i f fe ren t f r o m the e x p e c t e d 1:1 rat io by poo led G and he te rogene i t y G, i nd i ca t i ng tha t e a c h z o n e is con t ro l l ed by a s ing le l o c u s . Aconitase (ACO) O n e z o n e of ac t i v i t y w a s o b s e r v e d in ACO w i t h th ree s i n g l e - b a n d e d p h e n o t y p e s (96 , 1 0 0 , a n d 1 0 4 ) . T h e poo led G va lue for the g e n o t y p e 1 0 0 : 9 6 w a s s ign i f i can t , but the he te rogene i t y G va lue w a s not s ign i f i can t , i nd ica t ing tha t the dev ia t i on f r o m 1:1 rat io is c a u s e d by a s m a l l e x c e s s of g a m e t o p h y t e s ca r r y ing al lele " 1 0 0 . " T h e g e n o t y p e " 1 0 0 : 1 0 4 " g a v e n o n -s ign i f i can t and s ign i f i can t resu l ts fo r the poo led and he te rogene i t y t e s t s , r e s p e c t i v e l y (Table 2.4), i nd ica t ing that the seg rega t i on at th is l o c u s d o e s not f o l l o w the e x p e c t e d 1:1 rat io b e c a u s e c l o n e s s h o w e d a h e t e r o g e n e o u s s e g r e g a t i o n . Esterase (EST) O n e z o n e of ac t i v i t y w i t h four s i ng l e -banded va r ian ts ( 7 5 , 8 7 , 1 0 0 , a n d 116 ) w a s o b s e r v e d (Figure 2.1). Ne i the r the p o o l e d nor he te rogene i t y G of t he th ree o b s e r v e d h e t e r o z y g o u s g e n o t y p e s w a s s ign i f i can t , t hus c o n f i r m i n g the 1:1 rat io s e g r e g a t i o n (Table 2.4). Glutamate dehydrogenase (GDH) O n e z o n e of ac t i v i t y w i t h th ree s i ng l e -banded va r ian ts ( 7 1 , 1 0 0 a n d 137) w e r e o b s e r v e d at th is l o c u s . Ne i ther the p o o l e d nor he te rogene i t y G va lue of the t w o h e t e r o z y g o u s g e n o t y p e s w a s s ign i f i can t (Table 2.4), i nd ica t ing tha t the gene t i c m o d e of th is i s o z y m e w a s 1:1 rat io of s e g r e g a t i o n , as e x p e c t e d . Glucose-6-phosphate dehydrogenase (G6P) There w e r e t w o z o n e s of e n z y m e ac t iv i t y o b s e r v e d fo r ge ls s t a i ned fo r G6P. H o w e v e r , the m o s t - c a t h o d a l l y - m i g r a t i n g z o n e (G6P-2) w a s unc lea r and i n c o n s i s t e n t fo r s c o r i n g . Bo th poo led and he te rogene i t y G va l ues of the h é t é r o z y g o t e ( 1 0 0 : 1 0 0 ' ) at the G6P-1 l o c u s w e r e s ign i f i can t (Table 2.4), i nd ica t ing a depar tu re f r o m the e x p e c t e d 1:1 rat io . Isocitrate dehydrogenase (IDH) O n l y one z o n e of ac t i v i t y w a s o b s e r v e d in th is e n z y m e s y s t e m (Figure 2.1). O n e h e t e r o z y g o u s g e n o t y p e of t w o s i ng l e -banded va r ian ts ( 1 0 0 and 108 ) w a s o b s e r v e d in a s ing le c l o n e (Table 2.4). S e g r e g a t i o n ana l ys i s w a s not s i gn i f i can t , i nd i ca t i ng tha t the m o d e of inher i tance at th is l o c u s f o l l o w s M e n d e l i a n l a w . Leucine aminopeptidase (LAP) T w o z o n e s of ac t i v i t y w e r e o b s e r v e d (LAP-1 and LAP-2), LAP-2 w a s m o n o m o r p h i c . LAP-1 had t w o a l le les ( 1 0 0 and nul l (n)) (Figure 2.1). Bo th poo led and he te rogene i t y G va lues of the hé té rozygo te (LAP-1) w e r e nons ign i f i can t (Table 2.4), t hus c o n f i r m i n g 1:1 seg rega t i on rat io. Malate dehydrogenase (MDH) Three z o n e s of ac t i v i t y w e r e o b s e r v e d fo r th is e n z y m e s y s t e m . H o w e v e r , t w o ca thoda l l y -m ig ra t i ng z o n e s (MDH-2 and MDH-3) were unc lea r and i n c o n s i s t e n t , and t hey w e r e e x c l u d e d f r o m the s t u d y . Four , s i n g l e - b a n d e d va r ian ts w e r e o b s e r v e d at the MDH-1 l o c u s ( 8 1 , 9 2 , 1 0 0 , and 114) (Figure 2.1). O n e c l o n e w a s h e t e r o z y g o u s fo r the " 1 0 0 : 8 1 " g e n o t y p e (Table 2.4). T h i s c l o n e s e g r e g a t e d a c c o r d i n g to the 1:1 e x p e c t e d rat io . B o t h o f the " 1 0 0 : 1 1 4 " and " 1 0 0 : 9 2 " g e n o t y p e s g a v e s ign i f i can t poo led and he te rogene i t y G t e s t s i nd i ca t i ng the p r e s e n c e of a depar tu re f r o m the 1:1 e x p e c t e d rat io (Table 2.4). Phosphoglucose isomerase (PGI) T w o z o n e s of ac t i v i t y w e r e o b s e r v e d fo r the e n z y m e s y s t e m (PGI-1 and PGI-2). T h e m o s t - c a t h o d a l l y - m i g r a t i n g zone (PGI-2) w a s c lear and c o n s i s t e n t fo r s c o r i n g . T w o h e t e r o z y g o u s g e n o t y p e s w e r e o b s e r v e d fo r th is l o c u s (Figure 2.1). Ne i the r the p o o l e d nor he te rogene i t y G va lue w a s s ign i f i can t , c o n f i r m i n g the rat io 1:1 of s e g r e g a t i o n (Table 2.4). Phosphoglucomutase (PGM) T w o z o n e s of ac t i v i t y w e r e o b s e r v e d in th is e n z y m e s y s t e m . Th ree a n d t w o s ing le -b a n d e d va r ian ts w e r e o b s e r v e d for PGM-1 and PGM-2, r espec t i ve l y (Figure 2.1). P o o l e d G t es t w a s no t s ign i f i can t l y d i f ferent f r o m the e x p e c t e d 1:1 rat io; h o w e v e r , t rees s h o w e d h igh ly s ign i f i can t he te rogene i t y for bo th loc i (Table 2.4), and s h o u l d t h u s no t be a c c e p t e d as M e n d e l i a n . 6-Phosphogloconic dehydrogenase (6PG) T w o z o n e s of ac t i v i t y w e r e o b s e r v e d o n ge ls s t a i ned fo r th is e n z y m e . T h e m o s t -anoda l l y -m ig ra t i ng band of bo th loc i (6PG-1 and 6PG-2) p resen ted d o u b l e - b a n d e d p h e n o t y p e s , w h i l e the ca thoda l l y -m ig ra t i ng band p resen ted s i n g l e - b a n d e d p h e n o t y p e s (Figure 2.1). Th ree va r ian ts (86 , 1 0 0 , and 113) w e r e o b s e r v e d at 6PG-1 (Figure 2.1). T h e g e n o t y p e 1 0 0 : 8 6 w a s on ly o b s e r v e d fo r one tree and it seg rega ted f o l l o w i n g e x p e c t a t i o n s (Table 2.4). T h e " 1 0 0 : 1 1 3 " g e n o t y p e , on the o ther h a n d , dev ia ted f r o m e x p e c t a t i o n s and p r o d u c e d s ign i f i can t p o o l e d and he te rogene i t y G t e s t s fo r 6PG-1. T h e g e n o t y p e " 1 0 0 : 1 1 7 " of 6PG-2 g a v e a n o n -s ign i f i can t p o o l e d G tes t , but t rees s e g r e g a t e d in a h e t e r o g e n e o u s f a s h i o n (Table 2.4) and s o th is l o c u s c a n n o t be c o n f i r m e d as 1:1 s e g r e g a t i o n . T h e " 1 0 0 : 9 0 " g e n o t y p e w a s o b s e r v e d fo r on l y one t ree and d id not f o l l o w the e x p e c t e d 1:1 rat io (Table 2.4). 2 . 3 . 3 Summary Eight of the 2 0 poo led G tes t s s h o w e d s ign i f i can t depar tu re f r o m the e x p e c t e d 1:1 rat io . T h i s is 4 0 pe rcen t h igher t han the e x p e c t e d seg rega t i on at 5 % . S ign i f i can t depa r tu res f r o m e x p e c t e d seg rega t i on ra t ios are re la t ive ly c o m m o n in s t ud ies of con i fe r i s o z y m e s (Harry, 1 9 8 6 ; S t r a u s s and C o n k l e , 1 9 8 6 ) . T h e s e dev ia t i ons m a y resul t f r o m seve ra l c a u s e s , i nc lud ing i s o z y m e s be ing under the con t ro l of mo re than one l o c u s , me io t i c d is to r t ion or in tera l le l ic i n te rac t ion , or o b s e r v e d var ia t ion not be ing under gene t i c con t ro l (Feret , 1 9 7 1 ; E l - K a s s a b y , 1 9 8 1 ) . 2 . 3 . 4 Linkage Analyses Of the 1 0 poss ib l e pa i rw i se c o m p a r i s o n s a m o n g the 5 p o l y m o r p h i c loc i s c o r e d , 2 0 c l o n e s had at least one d o u b l y h e t e r o z y g o u s l o c u s , p r o d u c i n g 8 p o s s i b l e c o m b i n a t i o n s (Table 2.5). O n e of the e ight c o m b i n a t i o n s s ign i f i can t l y dev ia ted f r o m jo int seg rega t i on ( 1 : 1 : 1 : 1 ) . T h e s e w a s PGM-1:PGM-2 (Table 2.5). R e s u l t s w e r e g r o u p e d into " c o m b i n a t i o n s w i t h o u t s ign i f i can t l i n k a g e " , and " c o m b i n a t i o n s w i t h s ign i f i can t l i n kage . " Combinations without significant linkage: th is g roup c o n t a i n e d 6 d i f fe rent c o m b i n a t i o n s of pa i red l oc i ; the n u m b e r of c l o n e s w i t h i n c o m b i n a t i o n s var ied b e t w e e n one and s e v e n . N o var ia t ion f r o m the e x p e c t e d seg rega t i on rat io w a s o b s e r v e d . C h i - s q u a r e a n a l y s e s fo r the de tec t i on of l inkage revea led th ree c o m b i n a t i o n s for h o m o g e n e o u s d a t a . Da ta f r o m s ing le c l o n e s o b s e r v e d at one l o c u s d i f fe red s ign i f i can t l y f r o m the e x p e c t e d 1:1 ra t ios (PGM-2:6PG-2 and PGM-1-.GDH). S u c h dev i a t i ons are due ei ther to c h a n c e or to the d i f ferent ia l v iab i l i ty of g a m e t e s ca r r y ing d i f fe rent a l le les ( A d a m s and J o l y , 1 9 8 0 b ) . Combinations with significant linkage: th is g roup c o n t a i n e d 2 t y p e s of o n e e a c h (Table 2.5): " c o m b i n a t i o n s w i t h h e t e r o g e n e o u s d a t a " (s ign i f icant o n G, but non -s i gn i f i can t on/l) and " c o m b i n a t i o n s w i t h s ign i f i can t dev ia t i on f r o m the e x p e c t e d s e g r e g a t i o n ra t i os " (s ign i f icant o n bo th G| andxD-C h i - s q u a r e t e s t s for l inkage o n t w o - l o c u s c o m b i n a t i o n s w i t h h e t e r o g e n e o u s da ta w e r e s ign i f i can t fo r PGM-1 :PGM-2 ( 8 . 5 9 5 , P > 0 . 0 1 ) (Table 2.5). Par t i t i oned A^- tes ts fa i led to de tec t an unequa l con t r i bu t i on of l ocus PGM-1 to the d is to r t ion of jo in t i n d e p e n d e n c e of PGM-1 :PGM-2 c o m b i n a t i o n . A t h r e e - w a y log- l i ke l ihood tes t for i n d e p e n d e n c e of the PGM-1 :PGM-2 c o m b i n a t i o n ind ica ted tha t in te rac t ion a m o n g t rees at l o c u s PGM-1 a c c o u n t e d fo r the d is to r t ion of th is c o m b i n a t i o n (Table 2.6). T h e r e f o r e , l i nkage at th is l o c u s c a n n o t be c o n f i r m e d ei ther. Table 2.5. G o o d n e s s - o f - f i t a n a l y s e s for pa i r -w ise c o m b i n a t i o n s of loc i in S i t ka s p r u c e . Log- l i ke l ihood t e s t s jo in t i ndependence a s s o r t m e n t (G|). Par t i t i oned t e s t s tes t seg rega t i on at l o c u s A (/|), B (xl) and jo int seg rega t i on (xl) L o c u s Pair N o . of Seg rega t i on C l a s s G, xl xl xl (A-B) C l o n e s (df) (df = 1) (df = 1) (df = 1) A B A b a B ab PGI-2:PGM-2 3 2 3 12 19 18 7.320(7)"= 0.055"= 2.000"= 1.483"= PGI-2:6PG-2 4 31 2 0 2 0 2 5 1 3 . 6 3 4 ( 1 0 ) ™ 0.375"= 0.375"= 2.543"= PGM-1: PGM-2 7 3 7 4 7 5 6 2 8 6 1 . 1 4 2 ( 2 1 ) " 0 1.928"= 8 . 5 9 5 " PGM-1:6PG-2 5 3 5 2 7 3 4 2 4 3 1 . 1 7 8 ( 1 3 ) " " 0.133"= 2.700"= 0.056"= PGM-2:6PG-2 7 5 9 4 6 3 8 2 5 25 .973(19) "= 1 0 . 5 0 0 " 4 . 0 2 3 " 0.251"= PGI-2:PGM-1 1 6 6 6 6 - 0 0 0 PGM-1 :GDH 1 5 1 9 9 - 6 . 0 0 0 ' 0.666"= 1.510"= PGM-2:GDH 1 8 6 6 4 - 0.666"= 0.666"= 0.018"= "=Not s ign i f i can t . ' S i gn i f i can t at 5 % . " S i g n i f i c a n t at 1 % . Table 2.6. T e s t of i n d e p e n d e n c e fo r t l ie PGM-1 :PGM-2 c o m b i n a t i o n H y p o t h e s i s T e s t df G C l o n e X L o c u s A i n d e p e n d e n c e 6 3 5 . 6 8 " C l o n e X L o c u s B i n d e p e n d e n c e 6 11 .85™ L o c u s A X L o c u s B i n d e p e n d e n c e 1 8 . 7 7 " C l o n e X L o c u s A x L o c u s B in te rac t ion 6 4 .84™ C l o n e X L o c u s A x L o c u s B i n d e p e n d e n c e 19 6 1 . 1 4 " ™ N o t s ign i f i can t . " S ign i f i can t at 1 % . T h r e e - w a y log- l i ke l ihood t e s t s app l ied to the c o m b i n a t i o n w h i c h w a s not f o u n d to dev ia te s ign i f i can t l y f r o m jo int i n d e p e n d e n c e by / ^ - t e s t s i nd i ca ted dev ia t i on f r o m jo int i n d e p e n d e n c e at PGM-1:6PG-2 ( Table 2.7). T h i s c o m b i n a t i o n d id no t s h o w s ign i f i can t d i s to r t i on f r o m i n d e p e n d e n c e due to in te rac t ion a m o n g c lone and the t w o loc i . T h e r e f o r e , PGM-1 s h o u l d not be u s e d in m a t i n g - s y s t e m a n a l y s e s . 2 . 4 Conclusion M o s t of the a l l o z y m e var ia t ion in the 1 3 4 S i t k a s p r u c e c l o n e s s t u d i e d w a s con t ro l l ed by 14 l oc i , 5 of w h i c h f o l l o w e d the e x p e c t e d M e n d e l i a n seg rega t i on rat io a n d the rema in ing 9 loc i gave s ign i f i can t dev ia t i on f r o m the e x p e c t e d rat io . The re w a s a lack of jo in t seg rega t i on fo r m o s t s t ud ied l oc i , but G6P anû MDH gave c o n s i s t e n t d i s to r ted s e g r e g a t i o n . The re fo re , t hey c o u l d not be i nc l uded in the ma t i ng s y s t e m s t u d y (see Che l i ak et al., 1 9 8 4 ) . L inkage a n a l y s e s fu r ther i nd ica ted the i n c o n s i s t e n c y of loc i PGM-1. T h e r e f o r e , t h e s e loc i w e r e not u s e d in ana l ys i s of the S i t ka s p r u c e ma t i ng s y s t e m . Table 2.7. T h r e e - w a y log- l i ke l ihood tes t for i n d e p e n d e n c e fo r the PGM-2:6PG-2 c o m b i n a t i o n H y p o t h e s i s T e s t df G C l o n e X L o c u s A i n d e p e n d e n c e 4 1 2 . 1 6 * C l o n e X L o c u s B i n d e p e n d e n c e 4 1 3 . 5 0 * * L o c u s A X L o c u s B i n d e p e n d e n c e 1 0 .06™ C l o n e X L o c u s A x L o c u s B in te rac t ion 4 5.46™ C l o n e X L o c u s A x L o c u s B i n d e p e n d e n c e 1 3 3 1 . 1 8 * * ™Not s ign i f i can t . * S ign i f i can t at 5 % . * *S ign i f lean t at 1 %. 2 . 5 References A d a m s , W . T . a n d R . J . J o l y . 1 9 8 0 a . G e n e t i c s of a l l o z y m e var ian ts in lob lo l l y p ine . J. Heredity 7 1 : 3 3 - 4 0 . A d a m s , W . T . and R . J . J o l y . 1 9 8 0 b . L inkage re la t i onsh ips a m o n g t w e l v e a l l o zyme loc i in lob lo l ly p ine . J. Heredity 7 1 : 1 9 9 - 2 0 2 . A d a m s , W . T . , D .B . Nea le and C . A . L o o p s t r a . 1 9 8 8 . V e r i f y i n g con t ro l l ed c r o s s e s in con i fe r t ree-i m p r o v e m e n t p r o g r a m s . Silvae Genet. 3 7 : 1 4 7 - 1 5 2 . A d a m s , W . T . , D .B . Nea le , A . H . D o e r k s e n and D .B . S m i t h . 1 9 9 0 . Inher i tance and l inkage of i s o z y m e var ian ts f r o m s e e d and vege ta t i ve bud t i s s u e s in c o a s t a l Doug las - f i r (Pseudotsuga menziesii Mat. menziesii {M\rb.) F ranco ) . Silvae Genet. 3 9 : 1 5 3 - 1 6 7 . Ba i ley , N . T . J . 1 9 6 1 . Introduction to mathematical theory of genetic linkage. L o n d o n : O x f o r d Un i ve rs i t y P r e s s . B o y l e , T . J . B . and E .K . M o r g e n s t e r n . 1 9 8 5 . Inher i tance and l inkage re la t i onsh ips of s o m e i s o z y m e s of b lack s p r u c e in N e w B r u n s w i c k . Can. J. For. Res. 1 5 : 9 9 2 - 9 9 6 . Che l i ak , W . M . , K. M o r g a n , B . P . Danc i k , C . S t r o b e c k and F . C . H . Y e h . 1 9 8 4 . S e g r e g a t i o n of a l l o z y m e s in m e g a g a m e t o p h y t e s of v iab le s e e d f r o m a natura l popu la t i on of J a c k p ine , Pinus banksiana L a m b . Theor. Appl. Genet. 6 9 : 1 4 5 - 1 5 1 . Che l i ak , W . M . a n d J . A . P i te l . 1 9 8 4 . G e n e t i c con t ro l of a l l o z y m e va r ian ts in ma tu re t i s s u e s of w h i t e s p r u c e t rees . J. Heredity 7 5 : 3 4 - 4 0 . Che l i ak , W . M . and J . A . P i te l . 1 9 8 5 . Inher i tance and l inkage of a l l o z y m e s in Larix laricina. Silvae Genet. 3 4 : 1 4 2 - 1 4 8 . C l e g g , M . T . and R . W . A l l a r d . 1 9 7 2 . Pa t te rns of gene t i c d i f fe ren t ia t ion in the s l ende r w i l d oa t s p e c i e s Avena barbata. Proc. Natl. Acad. Sci. U S A 6 9 : 1 8 2 0 - 1 8 2 4 . C o c h r a n , W . G . 1 9 5 4 . S o m e m e t h o d s fo r s t r eng then ing the c o m m o n t e s t s . Biometrics 1 0 : 4 1 7 - 4 5 1 . C o p e s , D.L . and R . C . B e c k w i t h . 1 9 7 7 . I sozyme iden t i f i ca t ion of Picea glauca, P. sitchensis and P. / t / f z / / p o p u l a t i o n s . Bot. Gaz. 1 3 8 ( 4 ) : 5 1 2 - 5 2 1 . Ecke r t , R .T . , R . J . J o l y and D .B . Nea le . 1 9 8 1 . G e n e t i c s of i s o z y m e var ian ts and l inkage re la t i onsh ips a m o n g a l l o z y m e loc i in 3 5 eas te rn w h i t e p ine c l o n e s . Can. J. For. Res. 1 1 : 5 7 3 - 5 7 9 . E l - K a s s a b y , Y . A . 1 9 8 1 . G e n e t i c in terpre ta t ion of ma la te d e h y d r o g e n a s e i s o z y m e s in s o m e con i fe r s p e c i e s . J. Heredity 7 2 : 4 5 1 - 4 5 2 . E l - K a s s a b y , Y . A . , F . C . Y e h a n d 0 . Sz i k l a i . 1 9 8 1 . E s t i m a t i o n of the o u t c r o s s i n g rate o f D o u g l a s -fir [Pseudotsuga menziesii var. menziesii). Silvae Genet. 3 0 : 1 8 2 - 1 8 4 . E l - K a s s a b y , Y . A . , F . C . Y e h and 0 . Sz i k l a i . 1 9 8 2 a . Inher i tance of a l l o z y m e va r ian ts in coas ta l Doug las - f i r {Pseudotsuga menziesii var. menziesii). Can. J. Genet. Cytol. 2 4 : 3 2 5 - 3 3 5 . E l - K a s s a b y , Y . A . , F . C . Y e h and 0 . Sz i k l a i . 1 9 8 2 b . L inkage re la t i onsh ips a m o n g 19 p o l y m o r p h i c a l l o z y m e loc i in c o a s t a l Doug las - f i r (Pseudotsuga menziesii var. menziesii). Can. J. Genet. Cytol. 2 4 : 1 0 1 - 1 0 8 . E l - K a s s a b y , Y . A . and K. R i t l and . 1 9 8 6 . L o w leve ls of po l len c o n t a m i n a t i o n in a Doug las - f i r s e e d o r c h a r d as d e t e c t e d by a l l o z y m e m a r k e r s . Silvae Genet. 3 5 : 2 2 4 - 2 2 9 . E l - K a s s a b y , Y . A . , M . D . M e a g h e r , J . P a r k i n s o n a n d F.T. Po r t l ock . 1 9 8 7 . A l l o z y m e inher i tance , h e t e r o z y g o s i t y and o u t c r o s s i n g rate a m o n g Pinus monticola near L a d y s m i t h , Br i t ish C o l u m b i a . Heredity 5 8 : 1 7 3 - 1 8 1 . E rns t , S . G . , D .E . K e a t h l e y and J . W . H a n o v e r . 1 9 8 7 . Inher i tance of i s o z y m e s in s e e d and bud t i s s u e s of b lue and E n g e l m a n n s p r u c e . Genome 2 9 : 2 3 9 - 2 4 6 . F a l k e n h a g e n , E .R. 1 9 8 5 . I sozyme s tud ies in p r o v e n a n c e resea rch of f o res t t rees . Theor. Appl. Genet. 6 9 : 3 3 5 - 3 4 7 . Fere t , P . P . 1 9 7 1 . I s o z y m e var ia t ion in Picea glauca ( M o e n c h ) V o s s s e e d l i n g s . S/7vae Genet. 2 0 : 4 6 - 5 0 . G u r i e s , R . P . and F.T. L e d i g . 1 9 7 8 . Inher i tance of s o m e p o l y m o r p h i c i s o z y m e s in p i t ch p ine (Pinus rigida M i l l . ) . Heredity 4 0 : 2 7 - 3 2 . H a m r i c k , J . L . , Y . B . L inhar t and J . B . M i t t o n . 1 9 7 9 . R e l a t i o n s h i p s b e t w e e n life h is to ry cha rac te r i s t i c s and e lec t rophore t i ca l l y de tec tab le gene t i c var ia t ion in p lan ts . Ann. Rev. Ecol. Syst. 1 0 : 1 7 3 - 2 0 0 . Har r i s , H . 1 9 6 6 . E n z y m e p o l y m o r p h i s m s in m a n . Proc. Roy. Soc, Ser. B 1 6 4 : 2 9 8 - 3 1 0 . Ha r r y , D .E . 1 9 8 3 . Ident i f ica t ion of a l o c u s m o d i f y i n g the e lec t ropho re t i c mob i l i t y of ma la te d e h y d r o g e n a s e i s o z y m e s in i n c e n s e - c e d a r (Calocedrus decurrens) and imp l i ca t i ons fo r popu la t i on s t u d i e s . Biochem. Genet. 2 1 : 4 1 7 - 4 3 4 . Ha r r y , D .E . 1 9 8 6 . Inher i tance and l inkage of i s o z y m e var ian ts in i n c e n s e - c e d a r . J. Heredity 7 7 : 2 6 1 - 2 6 6 . H u b b y , J . L . and R . C . L e w o n t i n . 1 9 6 6 . A mo lecu la r a p p r o a c h to the s t u d y of gen ie h e t e r o z y g o s i t y in natura l popu la t i ons . I. T h e n u m b e r of a l le les at d i f fe ren t loc i in Drosophila pseudoobscura. Genetics 5 4 : 5 7 7 - 5 9 4 . K i n g , J . N . , a n d B . P . D a n c i k . 1 9 8 3 . Inher i tance a n d l inkage of i s o z y m e s in w h i t e s p r u c e (Picea glauca). Can. J. Genet. Cytol. 2 5 : 4 3 0 - 4 3 6 . L e w a n d o w s k i , A . and L. M e j n a r t o w i c z . 1 9 9 0 . Inher i tance of a l l o z y m e s in Larix decidua M i l l . Silvae Genet. 3 9 : 1 8 4 - 1 8 8 . L e w o n t i n , R . C . and J . L . H u b b y . 1 9 6 6 . A mo lecu la r a p p r o a c h to the s t u d y of gen ie h e t e r o z y g o s i t y in natura l popu la t i ons . II. A m o u n t of var ia t ion a n d d e g r e e of h e t e r o z y g o s i t y in natura l popu la t i ons of Drosophila pseudoobscura. Genetics 5 4 : 5 9 5 -6 0 9 . L u n d k v i s t , K. 1 9 7 4 . In l ier i tance of leuc ine a m i n o p e p t i d a s e i s o z y m e s in Picea abies K. Hereditas 7 6 : 9 1 - 9 6 . L u n d k v i s t , K. 1 9 7 7 . Inher i tance of es te rase in need les and e n d o s p e r m of N o r w a y s p r u c e (Picea abies K. ) . Hereditas 8 7 : 2 7 - 3 2 . M a r k e r t , C . L . and F. Mo l l e r . 1 9 5 9 . Mu l t i p l e f o r m s of e n z y m e s : t i s s u e , o n t o g e n e t i c and s p e c i e s s p e c i f i c pa t te rns . Proc. Natl. Acad. Sci. U S A 4 5 : 7 5 3 - 7 6 3 . M i l k m a n , R. 1 9 7 3 . E lec t rophore t i c var ia t ion in Escherichia coli from na tura l s o u r c e s . Science 1 8 2 : 1 0 2 4 - 1 0 2 6 . M i l l a r , C.I . 1 9 8 5 . Inher i tance of a l l o z y m e va r ian ts in B i s h o p p ine (Pinus muricuta D. Don ) . Bioch. Genet. 2 3 : 9 3 3 - 9 4 6 . Nea le D .B . and W . T . A d a m s . 1 9 8 1 . Inher i tance of i s o z y m e var ian ts in s e e d t i s s u e s of b a l s a m fir (Abies balsamea). Can. J. Bot. 5 9 : 1 2 8 5 - 1 2 9 1 . N e a l e , D . B . , J . C . W e b b e r , and W . T . A d a m s . 1 9 8 4 . Inher i tance of need le t i s s u e i s o z y m e s in Doug las - f i r . Can. J. Genet. Cytol. 2 6 : 4 5 9 - 4 6 8 . N e i , M . 1 9 7 5 . Molecular population genetics and evolution. A m s t e r d a m : No r th -Ho l l and Pub l i sh ing C o . O ' M a l l e y , D . M . , F . W . A l l en fo r f and G . M . B lake . 1 9 7 9 . Inher i tance of i s o z y m e var ia t ion and h e t e r o z y g o s i t y in Pinus ponderosa. Bioch. Genet. 1 7 : 2 3 3 - 2 5 0 . Per ry , D . J . and P. K n o w l e s . 1 9 8 9 . Inher i tance and l inkage re la t i onsh ips of a l l o z y m e s of eas te rn w h i t e c e d a r (Thuja occidentalis) in n o r t h w e s t e r n On ta r i o . Genome 3 2 : 2 4 5 - 2 5 0 . P i te l , J . A . , W . M . Che l i ak and J . Barret t . 1 9 8 7 . Inher i tance of a l l o z y m e s in a b lack s p r u c e dial lel c r o s s . Silvae Genet. 3 6 : 1 4 9 - 1 5 3 . R i t l and , K. a n d Y . A . E l - K a s s a b y . 1 9 8 5 . T h e na ture of i nb reed ing in a s e e d o r c h a r d of D o u g l a s -fir as s h o w n by an e f f i c ien t mu l t i l ocus m o d e l . Theor. Appl. Genet. 7 1 : 3 7 5 - 3 8 4 . R u d i n , D. and B. R a s m u s o n . 1 9 7 3 . G e n e t i c var ia t ion in e s t e r a s e f r o m need les o f Pinus silvestris L. Hereditas 7 3 : 8 9 - 9 8 . R u d i n , D. 1 9 7 6 . B i o c h e m i c a l g e n e t i c s and se lec t i on app l i ca t i on of i s o z y m e s in t ree b reed ing . In Proc. the lUFRO joint meeting of working parties on population and ecological genetics, breeding theory, biochemical genetics and progeny testing, pp . 1 4 5 - 1 6 4 . B o r d e a u x : I N R A . R u d i n , D. and I. E c k b e r g . 1 9 7 8 . L i nkage s tud ies in Pinus sylvestris L. u s i n g m a c r o g a m e t o p h y t e a l l o z y m e s . Silvae Genet. 2 7 : 1 - 1 2 . S h a w , D . V . , A . L . Kah le r a n d R . W . A l l a r d . 1 9 8 1 . A mu l t i l ocus e s t i m a t o r of ma t i ng s y s t e m pa rame te rs in p lant popu la t i ons . Proc. Nat. Acad. Sci. U S A 7 8 : 1 2 9 8 - 1 3 0 2 . S i m o n s e n , V . and H . We l l endo r f . 1 9 7 5 . S o m e p o l y m o r p h i c i s o e n z y m e s in the s e e d e n d o s p e r m of S i t ka s p r u c e (Picea sitchensis [Bong. ] Car r . ) . Forest Tree Improv. (Denmark ) 9 : 1 - 2 1 . S m i t h , D .B . and W . T . A d a m s . 1 9 8 3 . IVleasuring po l len c o n t a m i n a t i o n in c l ona l s e e d o r c h a r d s w i t h the a id of gene t i c m a r k e r s . In Proc. 17th South. For. Tree Improv. Conf., pp . 6 9 -7 7 . A t h e n s : Un i v . G e o r g i a . S o k a l , R .R . and F . J . Roh l f . 1 9 8 1 . B iome t r y : T h e p r inc ip les and p rac t i ce of s ta t i s t i cs in b io log ica l r e s e a r c h . 2 n d e d . N e w Y o r k : W . H . F r e e m a n and C o . S t r a u s s , S . H . and W . T . C o n k l e . 1 9 8 6 . S e g r e g a t i o n , l i nkage, and d i ve rs i t y of a l l o z y m e s in k n o b c o n e p ine . Theor. Appl. Genet. 7 2 : 4 8 3 - 4 9 3 . T i ge r s ted t , P . M . A . 1 9 7 3 . S t u d i e s o n i s o z y m e var ia t ion in marg ina l a n d cen t ra l popu la t i ons of Picea abies. Hereditas 7 5 : 4 7 - 6 0 . Va l l e j os , C E . and S . D . T a n k s l e y . 1 9 8 3 . S e g r e g a t i o n of i s o z y m e m a r k e r s and c o l d to le rance in an in t raspec i f i c c r o s s of t o m a t o . Theor. Appl. Genet. 6 6 : 2 4 1 - 2 7 4 . W e b b e r , J . E . and F . C . Y e h . 1 9 8 7 . T e s t of the f i r s t -on , f i rs t - in po l l i na t ion h y p o t h e s i s in coas ta l Doug las - f i r . Can. J. For. Res. 1 7 : 6 3 - 6 8 . X i e , C . Y . , B . P . Danc i k and F . C . Y e h . 1 9 9 1 . Inher i tance and l inkage of i s o z y m e s in Thuja orientalis. J. Heredity 8 2 : 3 2 9 - 3 3 4 . Y e h , F . C . and C . L a y t o n . 1 9 7 9 . T h e o rgan iza t i on of gene t i c var iab i l i ty in cen t ra l and marg ina l popu la t i ons of l odgepo le p ine , Pinus contorta s s p . latifolia. Can. J. Genet. Cytol. 2 4 : 4 8 7 - 5 0 3 . Y e h , F . C . and D . M . O ' M a l l e y . 1 9 8 0 . E n z y m e var ian ts in natura l p o p u l a t i o n s of Doug las - f i r , Pseudotsuga menziesii (Mirb) F r a n c o , f r o m Br i t ish C o l u m b i a . 1. G e n e t i c var ia t ion pa t te rns in c o a s t a l popu la t i ons . Silvae Genet. 2 9 : 8 3 - 9 2 . C h a p t e r 3 G e n e t i c D i ve rs i t y in S e e d O r c h a r d and Natu ra l P o p u l a t i o n s of S i t k a S p r u c e 3.1 Introduction Fores t t ree b reed ing p r o g r a m s are b a s e d o n the gene t i c var iab i l i ty o f natura l or i n t r oduced p o p u l a t i o n s . The re fo re , ma in ta in ing a large gene t i c d i ve rs i t y at an ear ly s tage is essen t i a l if the oppo r tun i t i es fo r se lec t i on in the fu tu re are to be ma in ta i ned and inb reed ing m i n i m i z e d ( A d a m s , 1 9 8 1 ) . H o w e v e r , the b reed ing p r o c e s s has been f o u n d to n a r r o w the gene t i c base wh i l e i m p r o v i n g c o m m e r c i a l va lues (F ranc is , 1 9 8 1 ) . D o m e s t i c a t i o n in agr icu l tu ra l c r o p p lan ts has resu l ted in a l o s s of gene t i c var iabi l i ty (E l ls t rand and M a r s h a l l , 1 9 8 5 ) . I sozyme a n a l y s i s of inbred c r o p p lan ts s u c h as bar ley s h o w l oss in gene t i c var iab i l i ty dur ing the d o m e s t i c a t i o n s e q u e n c e f r o m w i l d popu la t i ons to land r a c e s to cu l t i va rs ( B r o w n and C l e g g , 1 9 8 3 ) . The re fo re , the m a i n t e n a n c e of gene t i c d i ve rs i t y in t he b reed ing p o p u l a t i o n of any c o m m e r c i a l l y va luab le s p e c i e s s h o u l d be a pr ior i ty of any b reed ing p r o g r a m (Hamr i ck , 1 9 9 1 ) . T r e e s are o f ten i n t r oduced into h igh ly h e t e r o g e n e o u s natura l e n v i r o n m e n t s (Hamr i ck , 1 9 9 1 ) , s o gene t i c d i ve rs i t y in the cu l t i va ted fo res t is essen t i a l fo r the i r abi l i ty to c o u n t e r b a l a n c e the e f f ec t s of e n v i r o n m e n t a l c h a n g e s (K le inschmi t , 1 9 7 9 ; G r e g o r i u s , 1 9 8 9 ) . K n o w l e d g e of gene t i c var ia t ion in a par t icu lar s p e c i e s is impor tan t , no t on l y for a b reed ing p r o g r a m , but a lso for e f fec t i ve gene t i c c o n s e r v a t i o n (Mi l lar a n d M a r s h a l l , 1 9 9 1 ) . T h e r e f o r e , the m a i n t e n a n c e of h igh leve ls of gene t i c d i ve rs i t y in s e e d o r c h a r d s as c o m p a r e d to natura l popu la t i ons is impor tan t . Wh i l e e v i d e n c e ex i s t s i nd ica t ing tha t a l l o z y m e loc i m a y no t be an ent i re ly u n b i a s e d s a m p l e of gene t i c var iab i l i ty (Leigh B r o w n and L a n g l e y , 1 9 7 9 ) , e l e c t r o p h o r e s i s of i s o z y m e s is a bet ter m e t h o d fo r p rov id ing a fas te r a s s e s s m e n t of g e n e t i c va r ia t ion t h a n quan t i ta t i ve a p p r o a c h w h i c h requ i res a se r ies o f con t ro l l ed c r o s s e s . H o w e v e r , pa t te rns of a l l o z y m e var ia t ion in p lan ts s e e m to be at least rough ly c o m p a r a b l e to t h o s e f o u n d fo r g e n e s con t ro l l i ng m o r p h o l o g i c a l ma rke rs and quan t i ta t i ve t rai ts (C legg and A l l a r d , 1 9 7 2 ; H a m r i c k and A l l a r d , 1 9 7 5 ; F o w l e r and M o r r i s , 1 9 7 7 ; E l - K a s s a b y and S z i k l a i , 1 9 8 2 ) . S t a r c h ge l -e l ec t ropho res i s of i s o z y m e s has been u s e d in e luc ida t ing pa t te rns of popu la t i on var ia t ion in m a n y con i f e r s {Pseudotsuga menziesii, Y e h and O ' M a l l e y , 1 9 8 0 ; Li and A d a m s , 1 9 8 9 ; Picea sitchensis, Y e h and E l - K a s s a b y , 1 9 8 0 ; Pinus nigra Arnold, N iko l ié a n d T u c i é , 1 9 8 3 ; Pinus monticola, S te inho f f et al., 1 9 8 3 ; Picea mariana, Y e h et al., 1 9 8 6 ; Boy le and M o r g e n s t e r n , 1 9 8 7 ; Pinuspungens, G i b s o n and H a m r i c k , 1 9 9 1 ) . Pa t te rns of a l l o z y m e var ia t ion c a n be f o r m u l a t e d in t e r m s of the d is t r ibu t ion of gene t i c d i ve rs i t y w i t h i n and a m o n g p o p u l a t i o n s , the k n o w l e d g e of w h i c h is essen t i a l for long t e rm gene c o n s e r v a t i o n ( A d a m s , 1 9 8 1 ) . C o m p a r a t i v e s t ud ies of a l l o z y m e var ia t ion on S c o t s p ine {Pinus sylvestris L.) (Yazdan i et al., 1 9 8 5 ) , and N o r w a y s p r u c e {Picea abies (L.) Kars t . ) ( G ô m ô r y , 1 9 9 2 ) s t a n d s have s h o w n the e x i s t e n c e of gene t i c d i f fe rent ia t ion b e t w e e n natura l and m a n - m a d e popu la t i ons . H o w e v e r , gene t i c d i f fe rent ia t ion b e t w e e n natura l a n d p r o d u c t i o n (seed o rcha rds ) popu la t i ons of c o n i f e r s has no t been d e t e r m i n e d . T h i s s t u d y repor ts the a p p o r t i o n m e n t of gene t i c var ia t ion in a s e e d o r cha rd and in natura l popu la t i ons of S i t ka s p r u c e . 3 . 2 Materials and Methods T h i s s t u d y u s e d s ta rch -ge l e l ec t r opho res i s of i s o z y m e s to de te rm ine the gene t i c d i ve rs i t y in a S i t k a s p r u c e s e e d o r cha rd o w n e d by C a n a d i a n P a c i f i c Fo res t P r o d u c t s L t d . T h e s e e d o r cha rd is l oca ted in S a a n i c h t o n , Br i t i sh C o l u m b i a ( lat i tude 4 8 ° 3 5 ' N , long i tude 1 2 3 ° 2 4 ' W ) and c o n s i s t s of 1 3 9 c l o n e s (averag ing 9 . 3 r ame ts per c lone) s e l e c t e d f r o m e leva t i ons b e t w e e n 0 and 4 1 5 m on w e s t e r n V a n c o u v e r Is land, W a s h i n g t o n and O r e g o n {Figure 1.1). T h e o r cha rd w a s es tab l i shed in 1971 in a r a n d o m s ing le - t ree m i x ove r th ree unequa l b l o c k s . In S e p t e m b e r 1 9 9 0 , w ind -po l l i na ted s e e d s w e r e c o l l e c t e d f r o m 1 3 4 s e e d - o r c h a r d c l o n e s tha t p r o d u c e d su f f i c ien t s e e d s fo r the s t u d y . F ive s e e d - c o n e s per c l o n e w e r e r a n d o m l y c o l l e c t e d and thei r ident i t ies ma in ta i ned . C o n e s a m p l e s w e r e dr ied at r o o m tempera tu re and e x t r a c t e d by h a n d . S e e d s a m p l e s by c l o n e w e r e kept at 2 ° C unt i l u s e d . T h e a p p o r t i o n m e n t of gene t i c d ive rs i t y w a s c o m p a r e d to tha t d e t e r m i n e d fo r 1 0 l U F R O ( Internat ional U n i o n of Fo res t R e s e a r c h Organ iza t ion ) natura l p o p u l a t i o n s by Y e h and E l -K a s s a b y ( 1 9 8 0 ) . 3 .2 .1 Isozyme Assay Eight m e g a g a m e t o p h y t e s f r o m ge rm inan t s of e a c h c lone w e r e s u b j e c t e d to i s o z y m e a s s a y for 1 3 loc i (Table 3.2), f o l l o w i n g the m e t h o d s of Y e h and E l - K a s s a b y ( 1 9 8 0 ) . Deta i ls of the m e t h o d s are p r o v i d e d in C h a p t e r 2. A to ta l of 1 3 4 of the o r c h a r d ' s 1 3 9 c l o n e s w e r e g e n o t y p e d and the al le l ic f r e q u e n c i e s w e r e c o m p a r e d w i t h t h o s e p u b l i s h e d by Y e h and E l -K a s s a b y ( 1 9 8 0 ) o n 10 l U F R O S i t ka s p r u c e popu la t i ons . 3 . 2 . 2 Natural Populations T e n l U F R O popu la t i ons s tud ied by Y e h and E l - K a s s a b y (1 9 8 0 ) , r ep resen t i ng the natura l range of S i t ka s p r u c e popu la t i ons (Table 3.1), w e r e c o m p a r e d w i t h the o r c h a r d popu la t i on . 3 . 2 . 3 Data Analysis Al le l i c f r e q u e n c i e s for e a c h popu la t i on w e r e ca l cu la ted f r o m the in fer red d ip lo id g e n o t y p e s of s a m p l e t rees . O b s e r v e d and e x p e c t e d h e t e r o z y g o s i t i e s , h ie ra rch ica l d i ve rs i t y s ta t i s t i cs (Nei , 1 9 7 3 ) , u n b i a s e d - g e n e t i c - d i s t a n c e m e a s u r e s (Nei , 1 9 7 8 ) , a n d u n w e i g h e d - p a i r -g r o u p - m e t h o d a lgo r i thm ( U P G M A ) c lus te r ana l ys i s (Snea th and S o k a l , 1 9 7 3 ) w e r e ca l cu la ted f r o m al le l ic f r e q u e n c i e s w i t h the B I O S Y S - 1 c o m p u t e r p r o g r a m ( S w o f f o r d and S e l a n d e r , 1 9 8 1 ) a n d G E N E S T A T - P C c o m p u t e r p r o g r a m ( W h i t k u s , 1 9 8 8 ) . 3 . 3 Results and Discussion W h e n the al le l ic f r equenc ies of 1 0 natura l popu la t i ons (Yeh and E l - K a s s a b y , 1 9 8 0 ) and the s e e d o r cha rd w e r e c o m p a r e d , the s e e d o r cha rd a n d natura l p o p u l a t i o n s are s imi la r in m o s t of the al le les p resen ted (Table 3.2 and Table 3.3). O n l y th ree a l le les , G6P-1-^^0, IDH-88, and 6PG-2-63 o b s e r v e d in natura l popu la t i ons w e r e not d e t e c t e d in the s e e d o r c h a r d (Table 3.3). T w o of t h e s e th ree a l le les ( /D/7-88 and 6PG-2-63) w e r e ve ry rare in t he na tu ra l p o p u l a t i o n s . In fac t t hey w e r e p resen t in on ly 2 and 1 popu la t i ons ou t of the 10 s t u d i e d , r e s p e c t i v e l y . E v e n Table 3.1. L o c a t i o n of 10 l U F R O natura l popu la t i ons of S i t k a s p r u c e to w h i c h resu l t s f r o m the s e e d o r cha rd w e r e c o m p a r e d " l U F R O L o c a t i o n E lev . La t i tude L o n g i t u d e N o . (m) 3 0 2 4 D u c k C r . , A l a s k a 3 0 5 8 ° 2 2 ' N 1 3 4 ° 3 5 ' W 3 0 3 0 W a r d L., A l a s k a 1 5 5 5 ° 2 5 ' N 1 3 1 ° 4 2 ' W 3 0 4 0 U s k Fer ry , B . C . 1 3 7 5 4 ° 4 6 ' N 1 2 8 ° 1 5 ' W 3 0 4 4 Inve rness , B . C . 15 5 4 ° 1 2 ' N 1 3 0 ° 1 5 ' W 3 0 4 9 L ink R d . , B . C . 9 0 5 3 ° 3 0 ' N 1 3 2 ° 1 0 ' W 3 0 5 8 S a l m o n B a y , B . C . 0 5 0 ° 2 3 ' N 1 2 5 ° 5 7 ' W 3 0 6 2 Big Q u a l i c u m R., B . C . 0 4 9 ° 2 3 ' N 1 2 4 ° 3 7 ' W 3 0 0 3 F o r k s , W a s h . 1 3 7 4 8 ° 0 4 ' N 1 2 4 ° 1 8 ' W 3 0 0 8 H o q u i a m , W a s h . 7 4 7 ° 0 5 ' N 1 2 4 ° 0 3 ' W 3 0 1 2 N e c a n i c u m , O r e . 4 6 4 5 ° 4 9 ' N 1 2 3 ° 4 6 ' W ° A f t e r Y e h and E l - K a s s a b y ( 1 9 8 0 ) . Table 3.2. A l l e l i c f r equenc ies at 1 3 loc i in 1 0 l U F R O natura l popu la t i ons (Yeh and E l - K a s s a b y , 1 9 8 0 ) and s e e d o rcha rd (S .O. ) of S i t ka s p r u c e Popu la t i on L o c u s A l l e l e 3 0 2 4 3 0 3 0 3 0 4 0 3 0 4 4 3 0 4 9 3 0 5 8 3 0 6 2 3 0 0 3 3 0 0 8 3 0 1 2 S.O. AAT-1 100 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 0 . 9 9 6 92 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 4 AAT-2 100 0 . 9 8 6 1 . 0 0 0 1 . 0 0 0 0 . 9 0 5 0 . 8 8 1 1 . 0 0 0 0 . 8 8 8 1 . 0 0 0 0 . 8 5 8 0 . 8 4 0 0 . 9 2 5 71 0 . 0 1 4 0 . 0 0 0 0 . 0 0 0 0 . 0 9 5 0 . 1 1 9 0 . 0 0 0 0 . 1 1 2 0 . 0 0 0 0 . 1 4 2 0 . 1 6 0 0 . 0 7 5 ACO 100 0 . 7 3 1 0 . 6 1 6 0 . 8 5 0 0 . 8 2 1 1 . 0 0 0 0 . 8 6 2 0 . 8 1 1 0 . 8 1 2 0 . 7 1 2 0 . 7 2 7 0 . 7 6 9 1 0 4 0 . 2 6 9 0 . 3 8 4 0 . 0 0 0 0 . 1 7 9 0 . 0 0 0 0 . 1 3 8 0 . 1 8 9 0 . 1 8 8 0 . 2 7 5 0 . 2 7 3 0 . 2 0 9 96 0 . 0 0 0 0 . 0 0 0 0 . 1 5 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 1 3 0 . 0 0 0 0 . 0 2 2 EST 100 0 .851 0 . 7 2 2 0 . 6 4 1 0 . 8 0 0 0 . 5 5 4 0 . 8 5 0 0 . 7 1 8 0 . 6 8 4 0 . 8 0 0 0 . 8 2 4 0 .821 116 0 . 0 5 5 0 . 0 0 0 0 . 1 5 4 0 . 1 0 0 0 . 2 0 3 0 . 1 5 0 0 . 1 2 8 0 . 1 7 7 0 . 1 5 0 0 . 0 3 8 0 . 0 7 8 87 0 . 0 9 4 0 . 2 7 8 0 . 2 0 5 0 . 1 0 0 0 . 2 4 3 0 . 0 0 0 0 . 1 5 4 0 . 1 3 9 0 . 0 5 0 0 . 1 3 8 0 . 0 7 8 75 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 2 3 GDH 100 0 . 8 6 2 0 . 8 7 5 0 . 9 6 3 0 . 9 7 5 0 . 8 7 5 0 . 8 6 3 0 . 9 2 4 0 . 9 2 5 1 . 0 0 0 1 . 0 0 0 0 . 9 4 4 1 3 7 0 . 1 3 8 0 . 1 2 5 0 . 0 3 7 0 . 0 2 5 0 . 1 1 3 0 . 0 0 0 0 . 0 7 6 0 . 0 7 5 0 . 0 0 0 0 . 0 0 0 0 . 0 4 9 71 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 1 2 0 . 1 3 7 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 7 G6P-1 100 0 . 6 1 2 0 . 5 7 0 0 . 7 0 7 0 . 7 8 8 0 . 7 9 7 0 . 3 7 5 0 . 6 8 4 0 . 5 9 7 0 . 5 0 6 0 . 3 6 3 0 .601 110 0 . 0 0 0 0 . 0 6 3 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 5 0 0 . 0 0 0 0 . 1 9 5 0 . 0 0 0 0 . 2 1 3 0 . 0 0 0 118 0 . 3 8 8 0 . 3 6 7 0 . 2 9 3 0 . 2 1 2 0 . 2 0 3 0 . 5 7 5 0 . 3 1 6 0 . 2 0 8 0 . 4 9 4 0 . 4 2 4 0 . 3 9 9 IDH 100 0 . 9 1 1 0 . 9 0 0 0 . 9 4 7 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 0 . 9 4 9 0 . 9 9 6 1 0 8 0 . 0 7 7 0 . 1 0 0 0 . 0 5 3 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 4 88 0 . 0 1 2 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 5 1 0 . 0 0 0 MDH-1 100 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 0 . 9 4 0 1 1 4 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 4 92 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 5 2 81 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 4 PGI-2 1 0 0 0 . 9 7 5 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 0 . 8 7 5 1 . 0 0 0 0 . 9 2 5 0 . 9 0 9 0 . 9 7 4 1 . 0 0 0 0 . 9 2 9 1 1 7 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 1 4 3 0 . 0 0 0 0 . 0 1 2 0 . 0 9 1 0 . 0 1 3 0 . 0 0 0 0 . 0 4 5 9 3 0 . 0 2 5 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 6 3 0 . 0 0 0 0 . 0 1 3 0 . 0 0 0 0 . 0 2 6 (Table 3.2. Con t i nued ) Population Locus Al le le 3024 3030 3040 3044 3049 3058 3062 3003 3008 3012 S . O . PGM-1 100 0 . 8 4 6 0 . 9 2 3 0 . 7 8 5 0 . 6 2 8 0 . 9 2 3 0 . 8 8 8 0 . 8 3 8 0 . 8 8 8 0 . 9 3 7 0 . 8 6 3 0 . 8 7 7 90 0 . 1 5 4 0 . 0 7 7 0 . 2 1 5 0 . 3 7 2 0 . 0 7 7 0 . 1 1 2 0 . 1 6 2 0 . 0 8 7 0 . 0 6 3 0 . 1 3 7 0 . 1 1 6 75 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 2 5 0 . 0 0 0 0 . 0 0 0 0 . 0 0 7 PGM-2 100 0 . 9 8 7 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 0 . 7 6 9 86 0 . 0 1 3 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 2 3 1 6PG-1 100 0 . 8 1 5 0 . 9 3 6 0 . 7 6 0 0 . 9 4 7 0 . 8 7 5 0 . 9 1 3 0 . 9 0 0 0 . 7 5 7 0 . 8 8 6 0 . 9 7 4 0 . 8 3 2 113 0 . 1 8 5 0 . 0 6 4 0 . 2 4 0 0 . 0 5 3 0 . 1 2 5 0 . 0 8 7 0 . 1 0 0 0 . 2 4 3 0 . 1 1 4 0 . 0 2 6 0 . 1 5 3 86 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 1 5 6PG-2 100 0 . 4 6 1 1 . 0 0 0 0 . 7 6 0 0 . 7 2 4 0 . 6 4 5 0 . 6 2 5 0 . 6 2 5 0 . 5 9 0 0 . 7 0 8 0 . 8 7 8 0 . 6 6 0 117 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 2 6 3 0 . 0 0 0 0 . 3 5 0 0 . 3 0 0 0 . 2 1 9 0 . 2 9 2 0 . 1 0 8 0 . 3 3 6 90 0 . 5 3 9 0 . 0 0 0 0 . 2 4 0 0 . 0 1 3 0 . 3 4 1 0 . 0 2 5 0 . 0 7 5 0 . 1 9 1 0 . 0 0 0 0 . 0 1 4 0 . 0 0 4 63 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 1 4 0 , 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 CO 00 Table 3.3. R a n g e of a l l o z y m e f r e q u e n c i e s in natura l p o p u l a t i o n s ' and s e e d o r c h a r d Na tu ra l O r c h a r d L o c u s A l l e le M a x . M i n . A v e . F r e q . G a i n / l o s s " AAT-1 1 0 0 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 0 . 9 9 6 — 9 2 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 4 + 0 . 0 0 4 AAT-2 1 0 0 1 . 0 0 0 0 . 8 4 0 0 . 9 3 5 0 . 9 2 5 -71 0 . 1 6 0 0 . 0 0 0 0 . 0 6 4 0 . 0 7 5 -ACO 1 0 0 1 . 0 0 0 0 . 6 1 6 0 . 7 9 4 0 . 7 6 9 -1 0 4 0 . 3 8 4 0 . 0 0 0 0 . 1 8 9 0 . 2 0 9 -9 6 0 . 1 5 0 0 . 0 0 0 0 . 0 1 6 0 . 0 2 2 -EST 1 0 0 0 . 8 5 1 0 . 5 5 4 0 . 7 4 4 0 . 8 2 1 — 1 1 6 0 . 2 0 3 0 . 0 0 0 0 . 1 1 5 0 . 0 7 8 -8 7 0 . 2 7 8 0 . 0 0 0 0 . 1 4 0 0 . 0 7 8 — 7 5 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 2 3 + 0 . 0 2 3 GDH 1 0 0 1 . 0 0 0 0 . 8 6 2 0 . 9 2 6 0 . 9 4 4 — 1 3 7 0 . 1 3 8 0 . 0 0 0 0 . 0 5 8 0 . 0 4 9 -71 0 . 1 3 7 0 . 0 0 0 0 . 0 1 4 0 . 0 0 7 -G6P-1 1 0 0 0 . 7 9 7 0 . 3 6 3 0 . 5 9 9 0 . 6 0 1 -1 1 0 0 . 2 1 3 0 . 0 0 0 0 . 0 5 2 0 . 0 0 0 - 0 . 0 5 2 1 1 8 0 . 5 7 5 0 . 2 0 3 0 . 3 4 7 0 . 3 9 9 -IDH 1 0 0 1 . 0 0 0 0 . 9 0 0 0 . 9 7 0 0 . 9 9 6 1 0 8 0 . 1 0 0 0 . 0 0 0 0 . 0 2 3 0 . 0 0 4 8 8 0 . 0 5 1 0 . 0 0 0 0 . 0 0 6 0 . 0 0 0 0 , 0 0 6 MDH-1 1 0 0 1 . 0 0 0 1 . 0 0 0 1 . 0 0 0 0 . 9 4 0 — 1 1 4 0 , 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 4 + 0 . 0 0 4 9 2 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 5 2 + 0 . 0 5 2 81 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 4 + 0 . 0 0 4 PGI-2 1 0 0 1 . 0 0 0 0 . 8 7 5 0 . 9 6 5 0 . 9 2 9 — 1 1 7 0 . 1 4 3 0 . 0 0 0 0 . 0 2 5 0 . 0 4 5 -9 3 0 . 0 6 3 0 . 0 0 0 0 . 0 1 0 0 . 0 2 6 — PGM-1 1 0 0 0 . 9 3 7 0 . 6 2 8 0 . 8 5 1 0 . 8 7 7 -9 0 0 . 3 7 2 0 . 0 6 3 0 . 1 4 5 0 . 1 1 6 — 7 5 0 . 0 2 5 0 . 0 0 0 0 . 0 0 2 0 . 0 0 7 -PGM-2 1 0 0 1 . 0 0 0 0 . 9 8 7 0 . 9 9 8 0 . 7 6 9 — 8 6 0 . 0 1 3 0 . 0 0 0 0 . 0 0 1 0 . 2 3 1 -6PG-1 1 0 0 0 . 9 7 4 0 . 7 5 7 0 . 8 7 6 0 . 8 3 2 -1 1 3 0 . 2 4 3 0 . 0 2 6 0 . 1 2 3 0 . 1 5 3 8 6 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 1 5 • 0 .01 5 6PG-2 1 0 0 1 . 0 0 0 0 . 4 6 1 0 . 7 0 1 0 . 6 6 0 — 1 1 7 0 . 3 5 0 0 . 0 0 0 0 . 1 5 3 0 . 3 3 6 -9 0 0 . 5 3 9 0 . 0 0 0 0 . 1 4 3 0 . 0 0 4 6 3 0 . 0 1 4 0 . 0 0 0 0 . 0 0 1 0 . 0 0 0 0 . 0 0 1 ' Y e h and E l - K a s s a b y ( 1 9 8 0 ) . " D i f f e rences in al le l ic f r e q u e n c y w h e r e + = ga ined and - = los t . w i t h i n t hese th ree p o p u l a t i o n s , thei r f r e q u e n c i e s ranged f r o m 0 . 0 1 2 to 0 . 0 5 1 (Table 3.2). T h e G6P-1-'\ 1 0 , o n the o ther h a n d , w a s p resen t in 3 popu la t i ons w i t h a f r e q u e n c y rang ing f r o m 0 . 0 5 0 to 0 . 2 1 3 (Table 3.2), but w a s not p resen t in the s e e d o r c h a r d . S i x a l le les , AAT-1-92, £ 5 7 - 7 5 , MDH-1-^^4,-92, and - 8 1 , and 6PG-1-86 o b s e r v e d in the s e e d o r c h a r d w e r e not d e t e c t e d in the natura l popu la t i ons . H o w e v e r , s o m e of t h e m w e r e rare a l le les (Table 3.2 and Table 3.3). T h e s e resu l ts p robab l y are due to the d i f f e rences in s o u r c e range b e t w e e n the o r c h a r d and the 1 0 natura l p o p u l a t i o n s . O r c h a r d c l o n e s w e r e s e l e c t e d ma in ly o n V a n c o u v e r Is land, wh i l e the s a m p l e s of natura l popu la t i ons w e r e c o l l e c t e d r a n g e - w i d e f r o m s o u t h e r n A l a s k a to no r the rn O r e g o n , i nc lud ing on ly t w o popu la t i ons o n V a n c o u v e r Is land. The re fo re , s a m p l i n g b read th m a y be respons ib l e fo r t hese d i f f e rences . S u c h d i f f e rences w e r e o b s e r v e d a l s o in N o r w a y s p r u c e ( G o m o r y , 1 9 9 2 ) and Po r t -Or fo rd c e d a r , Chamaecyparis lawsoniana (Mi l lar and M a r s h a l l , 1 9 9 1 ) . M e a n h e t e r o z y g o s i t y of natura l popu la t i ons ranged f r o m 0 . 1 6 0 ± 0 . 0 5 0 to 0 . 2 1 6 + 0 . 0 6 2 (average 0 . 1 8 4 ± 0 . 0 2 0 ) per l o c u s w h i l e the s e e d o r cha rd p resen ted 0 . 2 3 0 + 0 . 0 4 4 per l o c u s (Table 3.4). T h e m e a n h e t e r o z y g o s i t y per l o c u s w a s h igher in the s e e d o r c h a r d t han in the natura l s t a n d s , but th is d i f fe rence w a s no t s i gn i f i can t . W h e n the n u m b e r of a l le les per l ocus w a s c o m p a r e d , natura l popu la t i ons and the s e e d o r c h a r d s h o w e d s ign i f i can t d i f f e rences in ave rage al lele n u m b e r per l o c u s . Na tu ra l p o p u l a t i o n s p r e s e n t e d a range b e t w e e n 1 .62 ± 0 . 1 8 and 2 . 0 0 + 0 . 1 6 (average 1 .82 ± 0 . 1 2 ) a l le les per l o c u s w h i l e the s e e d o r cha rd s h o w e d 2 . 7 7 ± 0 . 2 0 a l le les per l o c u s (Table 3.4). T h e p e r c e n t a g e of p o l y m o r p h i c loc i in the natura l popu la t i ons ranged b e t w e e n 5 3 . 8 5 % to 8 4 . 6 2 % (average 6 3 . 0 8 ± 8 . 7 3 % ) , wh i l e the p o l y m o r p h i s m of loc i in the s e e d o r cha rd w a s 8 4 . 6 2 % (this c o m p a r i s o n is res t r i c ted to on ly 1 3 p o l y m o r p h i c loc i ) . T h e s e f igu res ind ica te that the gene t i c d i ve rs i t y f o u n d in natura l popu la t i ons w a s re ta ined or i n c r e a s e d in the s e e d o r c h a r d . T h e gene t i c d i ve rs i t y of S i t k a s p r u c e (seed o rcha rd inc luded) equa l s the ave rage for g y m n o s p e r m s as a w h o l e (see H a m r i c k et al., 1 9 7 9 ) . Table 3.4. M e a n h e t e r o z y g o s i t y per l o c u s , m e a n n u m b e r of a l le les per l o c u s , and p e r c e n t a g e of loc i p o l y m o r p h i c in 1 0 l U F R O natura l popu la t i ons a n d s e e d o r c h a r d M e a n M e a n N o . of P e r c e n t a g e of Popu la t i on H e t e r o z y g o s i t y A l l e l e s L o c i per l o c u s per l o c u s P o l y m o r p h i c 3 0 2 4 0 . 2 0 9 ± 0 . 0 5 0 2 . 0 0 + 0 . 1 6 8 4 . 6 2 3 0 3 0 0 . 1 6 0 + 0 . 0 5 4 1 . 6 2 ± 0 . 1 8 5 3 . 8 5 3 0 4 0 0 . 1 8 9 ± 0 . 0 5 4 1 . 6 9 ± 0 . 1 7 6 1 . 5 4 3 0 4 4 0 . 1 6 7 ± 0 . 0 5 0 1 .77 + 0 . 2 0 6 1 . 5 4 3 0 4 9 0 . 1 8 7 + 0 . 0 5 4 1 .85 + 0 . 2 2 6 1 . 5 4 3 0 5 8 0 . 1 6 2 ± 0 . 0 5 2 1 .69 + 0 .21 5 3 . 8 5 3 0 6 2 0 . 2 0 5 + 0 . 0 5 1 1 .92 + 0 .21 6 9 . 2 3 3 0 0 3 0 . 2 1 6 ± 0 . 0 6 2 1 .92 + 0 . 2 4 6 1 . 5 4 3 0 0 8 0 . 1 7 7 ± 0 . 0 5 3 1 . 8 5 ± 0 . 2 2 6 1 . 5 4 3 0 1 2 0 . 1 7 1 ± 0 . 0 5 5 1 .85 + 0 . 2 2 6 1 . 5 4 A v e . 0 . 1 8 4 ± 0 . 0 2 0 1 .82 + 0 . 1 2 6 3 . 0 8 ± 8 . 7 3 O r c h a r d 0 . 2 3 0 ± 0 . 0 4 4 2 . 7 7 ± 0 . 2 0 8 4 . 6 2 G e n e d i ve rs i t y (H,) of Sitl<a s p r u c e e s t i m a t e d f r o m natura l p o p u l a t i o n s w a s 0 . 2 0 1 ± 0 . 0 5 1 [Table 3.5), w i t h the re la t ive a m o u n t of gene t i c d i f fe ren t ia t ion a m o n g popu la t i ons (G,,) of 0 . 0 8 2 ± 0 . 0 1 6 . H o w e v e r , w h e n the s e e d o r cha rd w a s i n c l u d e d , the gene d i ve rs i t y (H,) w a s i n c r e a s e d to 0 . 2 0 6 ± 0 . 0 5 1 i.e. 2 . 5 % h igher . T h e re la t ive a m o u n t o f gene t i c d i f fe ren t ia t ion a m o n g popu la t i ons af ter the i nc lus ion of the s e e d o r c h a r d w a s 0 . 0 8 6 + 0 . 0 1 6 , a 4 . 9 % i nc rease . T h e i m p r o v e m e n t in d i ve rs i t y w h e n the o r c h a r d p o p u l a t i o n w a s a d d e d i nd i ca ted tha t the s e e d o r cha rd p o s s e s s e d gen ie d i ve rs i t y h igher t han the m e a n o f the range-w i d e p o p u l a t i o n . H o w e v e r , t hese d i f f e rences w e r e not s ign i f i can t . T h e p ropo r t i on of the to ta l gene d ive rs i t y w i t h i n popu la t i ons of S i t ka s p r u c e w a s a p p r o x i m a t e l y 9 1 . 4 % of the to ta l gene d i ve rs i t y , the s a m e as Doug las - f i r ( A d a m s , 1 9 8 1 ) , w h e r e on l y 8 . 6 % of to ta l d i ve rs i t y w a s a m o n g popu la t i ons . W h e n t w o popu la t i ons p o s s e s s ident ica l d i ve rs i t y , it d o e s not n e c e s s a r i l y m e a n that t h e y have the s a m e gene in c o m m o n (Grego r ius , 1 9 7 8 ) . T h e a m o u n t of the d i f f e rence c a n be ob ta i ned f r o m gene t i c -d i s t ance a n a l y s i s {Table 3.6). G e n e t i c - d i s t a n c e v a l u e s ind ica te tha t popu la t i on 3 0 6 2 and 3 0 0 8 p resen ted the m in ima l gene t i c d i s t a n c e ( 0 . 0 0 5 ) w h i l e the s e e d o r c h a r d and popu la t i on 3 0 2 4 p rov ided the m a x i m a l gene t i c d i s t a n c e ( 0 . 0 4 4 ) . H o w e v e r , the ave rage gene t i c d i s t ance a m o n g the 10 l U F R O natura l popu la t i ons w a s abou t 0 . 0 2 0 ± 0 . 0 0 9 , w h i l e the ave rage gene t i c d i s t a n c e a m o n g these 1 0 p o p u l a t i o n s a n d the s e e d o r c h a r d w a s 0 . 0 2 9 ± 0 . 0 0 9 . T h e d i f fe rence is abou t 4 % . Su rp r i s i ng l y , c l us te r a n a l y s i s i nd i ca ted tha t the s e e d o r cha rd w a s d is t inc t f r o m all natura l popu la t i ons s t u d i e d {Figure 3.1). P e r h a p s the g rea tes t gene t i c d i f f e rences of the s e e d o r cha rd w e r e due part ia l ly to the f ac t tha t the s e e d o r cha rd ha rbou red more var ia t ion f r o m m a n y g e o g r a p h i c popu la t i ons t h a n any o the r na tu ra l popu la t i on a lone . H o w e v e r , th is is e x p e c t e d due to the c lus te r i ng of s imi la r g e n o t y p e s w i t h i n popu la t i ons (Brunei and R o d o l p h e , 1 9 8 5 ; E l - K a s s a b y et al., 1 9 8 7 ) . Un l ike agr icu l tu ra l c r o p p lan ts ( B r o w n and C l e g g , 1 9 8 3 ; E l l s t rand a n d M a r s h a l l , 1 9 8 5 ) , the reduc t i on of gene t i c d i ve rs i t y due to p h e n o t y p i c s e l e c t i o n is m ino r fo r S i t k a s p r u c e . Table 3.5. G e n e - d i v e r s i t y s ta t i s t i cs e s t i m a t e s for 1 3 p o i y m o r p f i i c loc i a v e r a g e d ove r 1 0 natura l popu la t i ons and ove r 11 popu la t i ons {10 natura l a n d one s e e d orchard) h; Dl, G t L o c u s 1 0 P " I I P " 1 0 P I I P 1 0 P I I P 1 0 P I I P AAT-1 0 . 0 0 0 0 . 0 0 1 0 . 0 0 0 0 . 0 0 1 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 0 . 0 0 0 AAT-2 0 . 1 2 1 0 . 1 2 3 0 . 1 1 3 0 . 1 1 5 0 . 0 0 8 0 . 0 0 8 0 . 0 6 4 0 . 0 6 3 ACO 0 . 3 3 6 0 . 3 3 8 0 . 3 1 0 0 . 3 1 5 0 . 0 2 6 0 . 0 2 3 0 . 0 7 6 0 . 0 6 8 EST 0 . 4 1 5 0 . 4 0 7 0 . 3 9 6 0 . 3 8 9 0 . 0 1 9 0 . 0 1 8 0 . 0 4 6 0 . 0 4 3 GDH 0 . 1 4 0 0 . 1 3 7 0 . 1 3 2 0 . 1 3 0 0 . 0 0 8 0 . 0 0 7 0 . 0 5 6 0 . 0 4 8 G6P-1 0 . 5 2 1 0 . 5 3 8 0 . 4 7 8 0 . 4 7 8 0 . 0 4 3 0 . 0 6 0 0 . 0 8 3 0 . 1 1 2 IDH 0 . 0 5 7 0 . 0 5 3 0 . 0 5 4 0 . 0 5 0 0 . 0 0 3 0 . 0 0 3 0 . 0 5 8 0 . 0 6 2 MDH-1 0 . 0 0 0 0 . 0 1 1 0 . 0 0 0 0 . 0 1 0 0 . 0 0 0 0 . 0 0 1 0 . 0 0 0 0 . 0 9 9 PGI-2 0 . 0 7 1 0 . 0 7 6 0 . 0 6 5 0 . 0 7 2 0 . 0 0 6 0 . 0 0 4 0 . 0 7 9 0 . 0 5 8 PGM-1 0 . 2 5 5 0 . 2 5 1 0 . 2 3 9 0 . 2 3 7 0 . 0 1 6 0 . 0 1 4 0 . 0 6 1 0 . 0 5 7 PGM-2 0 . 0 0 3 0 . 0 4 4 0 . 0 0 3 0 . 0 3 5 0 . 0 0 0 0 . 0 0 9 0 . 0 0 0 0 . 2 0 1 6PG-1 0 . 2 1 8 0 . 2 2 4 0 . 2 0 8 0 . 2 1 5 0 . 0 1 0 0 . 0 0 9 0 . 0 4 6 0 . 0 3 9 6PG-2 0 . 4 7 2 0 . 4 7 4 0 . 3 9 6 0 . 4 0 1 0 . 0 7 6 0 . 0 7 3 0 . 1 6 0 0 . 1 5 3 A v e . S.E.= 0 . 2 0 1 0 . 0 5 1 0 . 2 0 6 0 . 0 5 1 0 . 1 8 4 0 . 0 4 6 0 . 1 8 8 0 . 0 4 5 0 . 0 1 7 0 . 0 0 6 0 . 0 1 8 0 . 0 0 6 0 . 0 8 2 0 . 0 1 6 0 . 0 8 6 0 . 0 1 6 ^ To ta l g e n e d i ve rs i t y . ^ G e n e d i ve rs i t y w i t h i n p o p u l a t i o n s . ^ G e n e d i ve rs i t y a m o n g popu la t i ons . •* Re la t i ve a m o u n t of gene t i c d i f fe rent ia t ion a m o n g p o p u l a t i o n s . ' 1 0 natura l p o p u l a t i o n s . " 1 1 popu la t i ons (natural and s e e d o rcha rd ) . " C h a k r a b o r t y ( 1 9 7 4 ) . Table 3.6. G e n e t i c d i s t a n c e s b e t w e e n Sitl<a s p r u c e popu la t i ons , 10 natura l popu la t i ons and a s e e d o rcha rd (SO) Popu la t i on 3 0 2 4 3 0 3 0 3 0 4 0 3 0 4 4 3 0 4 9 3 0 5 8 3 0 6 2 3 0 0 3 3 0 0 8 3 0 1 2 S O 3 0 2 4 -3 0 3 0 0 . 0 3 2 3 0 4 0 0 . 0 1 8 0 . 0 2 3 -3 0 4 4 0 . 0 3 1 0 . 0 2 6 0 . 0 1 6 3 0 4 9 0 . 0 2 3 0 . 0 3 6 0 . 0 1 0 0 . 0 2 6 -3 0 5 8 0 . 0 2 9 0 . 0 2 9 0 . 0 2 7 0 . 0 2 3 0 . 0 3 8 -3 0 6 2 0 . 0 2 0 0 . 0 1 9 0 . 0 1 3 0 . 0 0 6 0 . 0 1 5 0 . 0 1 2 -3 0 0 3 0 . 0 1 6 0 . 0 2 3 0 . 0 1 1 0 . 0 1 8 0 . 0 1 4 0 . 0 1 7 0 . 0 0 6 -3 0 0 8 0 . 0 2 7 0 . 0 1 7 0 . 0 2 3 0 . 0 1 7 0 . 0 3 2 0 . 0 0 6 0 . 0 0 5 0 . 0 1 4 3 0 1 2 0 . 0 3 3 0 . 0 1 1 0 . 0 2 6 0 . 0 2 1 0 . 0 3 8 0 . 0 1 5 0 . 0 1 5 0 . 0 2 0 0 . 0 0 7 S O 0 . 0 4 4 0 . 0 3 5 0 . 0 3 4 0 . 0 2 4 0 . 0 3 9 0 . 0 3 2 0 . 0 2 0 0 . 0 1 4 0 . 0 2 6 3 0 2 4 3 0 4 0 3 0 4 9 3 0 0 3 3 0 3 0 3 0 1 2 3 0 4 4 3 0 5 8 3 0 6 2 3 0 0 8 O R C H A R D 0 . 0 4 0 . 0 3 0 . 0 2 0 . 0 1 0 . 0 0 D I S T A N C E Figure 3.1. P h e n o g r a m of S i t k a s p r u c e d i f fe rent ia t ion b a s e d o n N e i ' s ( 1 9 7 8 ) u n b i a s e d gene t i c d i s t a n c e . C l u s t e r s are p r o d u c e d us ing the u n w e i g h t e d - p a i r - g r o u p -m e t h o d a l ogo r i t hm ( U P G M ) w i t h N e i ' s ( 1 9 7 8 ) u n b i a s e d gene t i c d i s t a n c e s . H o w e v e r , t he s e e d o r cha rd s tud ied here is st i l l in the f i rs t s tage of d o m e s t i c a t i o n , the re fo re , r educ t i on m igh t be o b s e r v e d af ter a c o m p l e t e c y c l e of recur ren t s e l e c t i o n . T h i s has been o b s e r v e d in o ther c o n i f e r s (Doug las- f i r , A d a m s , 1 9 8 1 ; S c o t s p ine , S z m i d t a n d M u o n a , 1 9 8 5 ; rad ia ta p ine , M o r a n and Be l l , 1 9 8 7 ; lob lo l ly p ine , H a m r i c k , 1 9 9 1 ) . V a r i o u s s t u d i e s of po l l ina t ion pa t te rns in fo res t t ree s e e d o r c h a r d s ind ica te that o f ten on l y a l imi ted n u m b e r of ma le c l o n e s con t r i bu te ac t i ve l y to the nex t gene ra t i on ( J o n s s o n etal., 1 9 7 6 ; Gr i f f i n , 1 9 8 2 ; M u l l e r - S t a r c k , 1 9 8 2 ; O ' R e i l l y etal., 1 9 8 2 ; R o b e r d s et al., 1 9 9 1 ) . A l s o , a d i sp ropo r t i on of f ema le c l o n e s con t r i bu t i ng to s e e d p r o d u c t i o n w a s o b s e r v e d a n d th is var ied f r o m yea r - t o -yea r ( E l - K a s s a b y et al., 1 9 8 9 ; C h a p t e r 5). Theo re t i ca l l y , gene t i c d i ve rs i t y s h o u l d be r e d u c e d in s e e d o r c h a r d s . Bu t , it is poss ib l e tha t the a m o u n t of gene t i c d i ve rs i t y re ta ined a m o n g o r cha rd c l o n e s is sti l l large ( A d a m s , 1 9 8 1 ) . Da ta f r o m o ther c o n i f e r s s u g g e s t that h o m o z y g o t e e x c e s s , due to se l f ing or c o n s a n g u i n e o u s ma t i ng in s e e d o r c h a r d s , p resen t at the e m b r y o s tage had been r e m o v e d after a f e w yea rs of f ie ld g r o w t h (Nea le , 1 9 8 5 ; S z m i d t and M u o n a , 1 9 8 5 ; Y a z d a n i et al., 1 9 8 5 ; G ô m ô r y , 1 9 9 2 ) . A l l e v i d e n c e ava i lab le to da te ind ica tes tha t con i f e rs in genera l p o s s e s s a great dea l of gene t i c d i ve rs i t y (Hamr i ck et al., 1 9 7 9 ) . M o r a n et al. ( 1 9 8 0 ) c a u t i o n e d abou t the l oss of gene t i c var iab i l i ty tha t o c c u r s in s e e d o r c h a r d s c o m p r i s i n g a l o w n u m b e r of c l o n e s . T h i s s t u d y i nd i ca ted that a p r o d u c t i o n popu la t i on of 1 3 9 c l o n e s in S i t k a s p r u c e is su f f i c ien t to p reven t the l oss of gene t i c var iab i l i ty . M o r e o v e r , s u p p l e m e n t a l m a s s po l l ina t ion c a n be u s e d to b r o a d e n the gene t i c base by i n t r o d u c i n g des i rab le g e n o t y p e s in to s e e d o r c h a r d s ( W o e s s n e r and F rank l i n , 1 9 7 3 ) . C o n s e q u e n t l y , an i nc rease in the g e n e d ive rs i t y of a s e e d o r c h a r d ' s c r o p s c a n be ob ta i ned d i rec t l y ( E l - K a s s a b y , 1 9 9 1 ) . 3 . 4 References A d a m s , W . T . 1 9 8 1 . Popu la t i on g e n e t i c s and gene c o n s e r v a t i o n in Pac i f i c N o r t f i w e s t con i f e r s . In Evolution today, Proc. 2nd. Intern. Congress System. Evol. Biol., e d . G . G . E . S c u d d e r and J . L . R e v e a l , pp . 4 0 1 - 4 1 5 , P i t t sbu rgh : H u n t Inst. B io l . D o c , C a r n e g i e - M e l l o n Un iv . B o y l e , T . J . B . and E .K . M o r g e n s t e r n . 1 9 8 7 . S o m e a s p e c t s of the popu la t i on s t r uc tu re of b lack s p r u c e in cen t ra l N e w B r u n s w i c k . Silvae Genet. 3 6 : 5 3 - 6 0 . B r o w n , A . H . D . and M . T . C l e g g . 1 9 8 3 . I sozyme a s s e s s m e n t of p lant gene t i c r e s o u r c e s . In Isozymes: Current Topics in Biology and Medical Research, vol.11, p p . 2 8 5 - 2 9 5 . N e w Y o r k : A l a n R. L i s s . B rune i , D. and F. R o d o l p h e . 1 9 8 5 . G e n e t i c n e i g h b o u r h o o d s t ruc tu re in a p o p u l a t i o n of Picea abies L. Theor. Appl. Genet. 7 1 : 1 0 1 - 1 1 0 . C h a k r a b o r t y , R. 1 9 7 4 . A note o n N e i ' s m e a s u r e of gene d ive rs i t y in a s u b s t r u c t u r e d p o p u l a t i o n . Humangenetik 2 1 : 8 5 - 8 8 . C l e g g , M . T . and R . W . A l l a r d . 1 9 7 2 . Pa t te rns of gene t i c d i f fe ren t ia t ion in the s e l e n d e r w i l d oat s p e c i e s Avena barbata. Proc. Natl. Acad. Sci. U S A 6 9 : 1 8 2 0 - 1 8 2 4 . E l - K a s s a b y , Y . A . 1 9 9 1 . D o m e s t i c a t i o n and gene t i c d i ve rs i t y - s h o u l d w e be c o n c e r n e d ? Paper presented at the 23rd biennial meeting of Canadian Tree Improvement Association, A u g u s t 1 9 - 2 3 , 1 9 9 1 . O t t a w a . E l - K a s s a b y , Y . A . and 0 . Sz i k l a i . 1 9 8 2 . G e n e t i c var ia t ion of a l l o z y m e and quan t i t a t i ve t ra i ts in a s e l e c t e d Doug las - f i r (Pseudotsuga menziesii var. menziesii {Mirb.) F ranco) popu la t i on . For. Ecol. Manage. 4 : 1 1 5 - 1 2 6 . E l - K a s s a b y , Y . A . M . D . M e a g h e r , J . P a r k i n s o n , and F.T. Po r t l ock . 1 9 8 7 . A l l o z y m e inhe r i t ance , h e t e r o z y g o s i t y and o u t c r o s s i n g rate a m o n g Pinus monticola near L a d y s m i t h , Br i t ish C o l u m b i a . Heredity 5 8 : 1 7 3 - 1 8 1 . E l - K a s s a b y , Y . A . , A . M . K . F lash ier , and M . C r o w n . 1 9 8 9 . Va r ia t i on in f ru i t f u lness in a D o u g l a s -fir s e e d o r cha rd and its e f fec t on c r o p - m a n a g e m e n t d e c i s i o n s . Silvae Genet. 3 8 : 1 1 3 -1 2 1 . E l l s t rand , N . C . and D.L . M a r s h a l l . 1 9 8 5 . T h e i m p a c t s of d o m e s t i c a t i o n o n d i s t r i bu t i on of a l l o zyme var ia t ion w i t h i n and a m o n g cu l t i va rs of r ad i sh , Raphanus sativus L. Theor. Appl. Genet. 6 9 : 3 9 3 - 3 9 8 . F o w l e r , D . P . and R . W . M o r r i s . 1 9 7 7 . G e n e t i c d i ve rs i t y in red p ine : E v i d e n c e fo r l o w gen ie h e t e r o z y g o s i t y . Can. J. For. Res. 7 : 3 4 3 - 3 4 7 . F r a n c i s , C . A . 1 9 8 1 . D e v e l o p m e n t of p lant g e n o t y p e s fo r mul t ip le c r o p p i n g s y s t e m s . In Plant Breeding II, e d . K . J . F rey , pp . 1 7 9 - 2 3 1 . A m e s : T h e Iowa S ta te Un i ve r s i t y P r e s s . G i b s o n , J . P . and J . L . Hamr i ck . 1 9 9 1 . G e n e t i c D ive rs i t y and s t ruc tu re in Pinus pugens (table m o u n t a i n pine) popu la t i ons . Can. J. For. Res. 2 1 : 6 3 5 - 6 4 2 . G ô m ô r y , D. 1 9 9 2 . E f fec t of s t a n d or ig in on t l ie gene t i c d ive rs i t y of N o r w a y s p r u c e (Picea abies Kars t . ) popu la t i ons . For. Ecol. Manage, (in p ress ) . G r e g o r i u s , H . -R . 1 9 7 8 . T h e c o n c e p t of gene t i c d ive rs i t y and its f o rma l re la t ionsh ip to h e t e r z y g o s i t y and gene t i c d i s t a n c e . Math. BioSci. 4 1 : 2 5 3 - 2 7 1 . G r e g o r i u s , H-R. 1 9 8 9 . T h e a t t r ibu t ion of p h e n o t y p i c va r ia t ion to gene t i c or e n v i r o n m e n t a l var ia t ion in e c o l o g i c a l s t u d i e s . In Genetic effects of air pollutants in forest tree populations, e d . F. S c h o l z , H . -R . G r e g o r i u s and D. R u d i n , 1 -16 . H e i d e l b e r g : Sp r inge r V e r l a g e . Gr i f f i n , A . R . 1 9 8 2 . C l o n a l var ia t ion in rad iata p ine s e e d o r c h a r d s . I. S o m e f l o w e r i n g , c o n e and s e e d p r o d u c t i o n t ra i ts . Aust. For. Res. 1 2 : 2 9 5 - 3 0 2 . H a m r i c k , J . L . 1 9 9 1 . A l l o z y m e d ive rs i t y of natura l s t a n d s v e r s u s s e e d o r c h a r d lob lo l l y p ine . Paper presented at the 23rd biennial meeting of Canadian Tree Improvement Association, A u g u s t 1 9 - 2 3 , 1 9 9 1 . O t t a w a . H a m r i c k , J . L . and R . W , A l l a r d . 1 9 7 5 . Co re l a t i ons b e t w e e n quan t i ta t i ve c h a r a c t e r s a n d e n z y m e g e n o t y p e s in Avena barbata. Evolution 2 9 : 4 3 8 - 4 4 2 . H a m r i c k , J . L . , Y . B . L inhar t , and J . B . M i t t o n . 1 9 7 9 . R e l a t i o n s h i p s b e t w e e n life h is to ry cha rac te r i s t i c s and e lec t ropho re t i ca l l y -de tec tab le gene t i c var ia t ion in p lan ts . Ann. Rev. Ecol. Syst. 1 0 : 1 7 3 - 2 0 0 J o n s s o n , A . , I. Ekbe rg and G . E r i k s s o n . 1 9 7 6 . F l o w e r i n g in a s e e d o r cha rd of Pinus sylvestris L. Stud. For. Sue. 1 3 5 : 1 - 3 8 . K l e i n s c h m i t , J . 1 9 7 9 . L im i ta t i ons fo r res t r i c t ion o f t he gene t i c va r ia t i on . Sivae Genet. 2 8 : 6 1 -6 7 . Le igh B r o w n , A . J . and C H . Lang ley . 1 9 7 9 . Rééva lua t ion of level of gene t i c h e t e r z y g o s i t y in natura l popu la t i on of Drosophila melanogaster by t w o - d i m e n s i o n a l e l e c t r o p h o r e s i s . Proc. Natl. Acad. Sci. U S A 7 6 : 2 3 8 1 - 2 3 8 4 . L i , P. and W . T . A d a m s . 1 9 8 9 , R a n g e - w i d e pa t te rns of a l l o z y m e var ia t ion in Doug las - f i r (Pseudotsuga menziesii). Can. J. For. Res. 1 9 : 1 4 9 - 1 6 1 . M i l la r , C. I . a n d K . A . M a r s h a l l . 1 9 9 1 . A l l o z y m e var ia t ion of P o r t - O r f o r d - c e d a r ( C h a m a e c y p a r i s l a w s o n i a n a ) : Imp l ica t ions fo r gene t i c c o n s e r v a t i o n . For. Sci. 3 7 : 1 0 6 0 - 1 0 7 7 . M o r a n , G . F . and J . C . Be l l . 1 9 8 7 . T h e or ig in and gene t i c d i ve rs i t y of Pinus radiata in A u s t r a l i a . Theor. Appl. Genet. 7 3 : 6 1 6 - 6 2 2 . M o r a n , G . F . , J . C . Be l l , and A . C . M a t h e s o n . 1 9 8 0 . T h e gene t i c s t ruc tu re and leve ls of i nb reed ing in a Pinus radiata D. D o n s e e d o r c h a r d . Silvae Genet. 2 9 : 1 9 0 - 1 9 3 . Mu l l e r -S ta r k , G . 1 9 8 2 . R e p r o d u c t i v e s y s t e m s in con i f e r s e e d o r c h a r d s . I. M a t i n g probab i l i t y in a s e e d o r cha rd of Pinus sylvetris L. Silvae Genet. 3 1 : 1 8 8 - 1 9 7 . N e a l e , D .B . 1 9 8 5 . G e n e t i c imp l i ca t i ons of s h e l t e r w o o d regenera t ion of Doug las - f i r in S o u t h w e s t O r e g o n . For. Sci. 3 1 : 9 9 5 - 1 0 0 5 . N e i , M. 1 9 7 3 . A n a l y s i s of gene d ive rs i t y in s u b d i v i d e d p o p u l a t i o n s . Proc. Nat. Acad. Sci. U S A 7 0 : 3 3 2 1 - 3 3 2 3 . N e i , M . 1 9 7 8 . Es t ima t i on of ave rage f i e te rzygos i t y and gene t i c d i s t a n c e f r o m a sma l l number of i nd i v idua ls . Genetics 8 9 : 5 8 3 - 5 9 0 . N iko l ié , D. and N . T u c i é . 1 9 8 3 . I s o e n z y m e var ia t ion w i t h i n a n d a m o n g p o p u l a t i o n s of Eu ropean b lack p ine {Pinus nigra A r n o l d ) . Si/vae Genet. 3 2 : 8 0 - 8 9 . O ' R e i l l y , C O . , W . H . Parker and S . E . Barker . 1 9 8 2 . E f fec t of po l l i na t ion per iod and s t rob i lus n u m b e r on r a n d o m mat ing in a c l ona l s e e d o r cha rd of Picea marina. Si/vae Genet. 3 1 : 9 0 - 9 4 . R o b e r d s , J . H . , S . T . F r i e d m a n and Y . A . E l - K a s s a b y . 1 9 9 1 . E f fec t i ve n u m b e r of po l len pa ren ts in c lona l s e e d o r c h a r d s . T/ieor. App/. Genet. 8 2 : 3 1 3 - 3 2 0 . S te inho f f , R . J . , D . G . J o y c e and L. F ins . 1 9 8 3 . I sozyme var ia t ion in Pinus montico/a. Can. J. For. Res. 1 3 : 1 1 2 2 - 1 1 3 2 . S z m i d t , A . E . and 0 . M u o n a . 1 9 8 5 . G e n e t i c e f f ec t s of S c o t s p ine {Pinus sy/vestris L.) d o m e s t i c a t i o n . Lect. Notes Biomat/i. 6 0 : 2 4 1 - 2 5 2 . S n e a t h , P . H . A . and R.R. S o k a l . 1 9 7 3 . Numericai taxonomy. S a n F r a n c i s c o : W . H . F r e e m a n . S w o f f o r d , D.L . and R . B . Se lande r . 1 9 8 1 . B I O S Y S - 1 : a F O R T R A N p r o g r a m for the c o m p r e h e n s i v e ana l ys i s of e lec t rophore t i c da ta in popu la t i on g e n e t i c s and s y s t e m a t i c s . J. Heredity 7 2 : 2 8 1 - 2 8 3 . W h i t k u s , R. 1 9 8 8 . M o d i f i e d ve r s i on of G E N E S T A T : A p r o g r a m fo r c o m p u t i n g gene t i c s ta t i s t i cs f r o m al le l ic f r e q u e n c y d a t a . P/ant Genet. News/etter 4 : 1 0 . W o e s s n e r , R . A . and E . G . F rank l i n . 1 9 7 3 . C o n t i n u e d re l iance o n w i n d - p o l l i n a t e d s o u t h e r n p ine s e e d o r c h a r d s - is it r easonab le? In Proc. 12th South. For. Tree /mprov. Conf., p p . 6 4 -7 3 , L o u s i a n a : S o u t h . For . E x p . S ta t , and L o u s i a n a S ta te U n i v . Y a z d a n i , R. 0 . M u o n a , D. R u d i n , and A . E . S z m i d t . 1 9 8 5 . G e n e t i c s t ruc tu re of a Pinus sy/vestris L. seed- t ree s t a n d and natura l ly regenera ted u n d e r s t o r e y . For. Sci. 3 1 : 4 3 0 - 4 3 6 . Y e h , F . C . and Y . A . E l - K a s s a b y . 1 9 8 0 . E n z y m e var ia t ion in natura l p o p u l a t i o n s of S i t ka s p r u c e {Picea sitchensis). 1. G e n e t i c var ia t ion pa t te rns a m o n g t rees f r o m l U F R O p r o v e n a n c e s . Can. J. For. Res. 1 0 : 4 1 5 - 4 2 2 . Y e h , F . C . and D. O ' M a l l e y . 1 9 8 0 . E n z y m e var ia t ion in natura l p o p u l a t i o n s of Doug las - f i r , Pseudotsuga menziesii (Mirb.) F r a n c o , f r o m Br i t ish C o l u m b i a . 1. G e n e t i c var ia t ion pa t te rns in c o a s t a l p o p u l a t i o n s . Si/vae Genet. 2 9 : 8 3 - 9 2 . Y e h , F . C , M . A . K . Kha l i l , Y . A . E l - K a s s a b y and D . C , T rus t . 1 9 8 6 . A l l o z y m e var ia t ion in Picea mariana f r o m N e w f o u n d l a n d : gene t i c d ive rs i t y , popu la t i on s t r uc tu re , and ana l ys i s of d i f fe ren t ia t ion . Can. J. For. Res. 1 6 : 7 1 3 - 7 2 0 . C h a p t e r 4 M a t i n g S y s t e m in a C l o n a l S i t k a S p r u c e S e e d O r c h a r d 4.1 Introduction G e n e t i c var ia t ion in a popu la t i on is a re f lec t ion of the nature and ex ten t of gene t i c t r a n s m i s s i o n to s u c c e s s i v e gene ra t i ons by m e a n s o f the ma t i ng s y s t e m ( C l e g g , 1 9 8 0 ) . P lant s p e c i e s w h i c h p rac t i ce a h igh degree of c ross - fe r t i l i za t i on are m o r e gene t i ca l l y var iab le t han t h o s e tha t p rac t i ce se l f - fe r t i l i za t ion (Hamr ick et al., 1 9 7 9 ) . C o n i f e r o u s t rees are c o n s i d e r e d to be a m o n g the m o s t - h e t e r o z y g o u s p lan ts k n o w n , at leas t at the leve l of e n z y m e var ia t ion (Hamr i ck , 1 9 8 2 ) . H igh o u t c r o s s i n g ra tes , e x t e n s i v e g e n e f l o w , and large n e i g h b o u r h o o d s i z e s s u p p o r t h igher leve ls of gene t i c var ia t ion (Hamr i ck et al., 1 9 7 9 ) lead ing to a h igh p ropor t i on of h e t e r o z y g o u s ind iv idua ls in that popu la t i on . H e t e r o z y g o u s ind iv idua ls are p o s t u l a t e d to be m o r e v i g o u r o u s t han h o m o z y g o u s ind iv idua ls and m a y be more to le rant to a w i d e r range of e n v i r o n m e n t a l c o n d i t i o n s , pe rhaps due to thei r g rea ter me tabo l i c f lex ib i l i t y ( B r o w n , 1 9 7 9 ; M i t t o n et al., 1 9 8 1 ) . Fea tu res of inb reed ing d e p r e s s i o n in con i f e rs are we l l k n o w n (Frank l in , 1 9 7 0 ) and resul t in r educ t i ons in v iab le s e e d y ie ld (Pinus sylvestris, S a r v a s , 1 9 6 2 ; Pseudotsuga menziesii, S o r e n s e n , 1971 ; O r r - E w i n g , 1 9 7 6 ; W o o d s and H e a m a n , 1 9 8 9 ; Pinus radiata. Gr i f f in and L i n d g r e n , 1 9 8 5 ) , seed l i ng v i gou r (Doug las - f i r , S o r e n s e n and M i l e s , 1 9 7 4 ) , g r o w t h (Doug las - f i r , S o r e n s e n and M i l e s , 1 9 7 4 ; Pinus radiata, W i l c o x , 1 9 8 3 ; Picea pungens, C r a m , 1 9 8 4 ; Picea omorika, G e b u r e k , 1 9 8 6 ) , and s u r v i v a l (Doug las - f i r , S o r e n s e n a n d M i l e s , 1 9 7 4 ; Picea pungens. C r a m , 1 9 8 4 ) . The re fo re , in s e e d o r c h a r d s , t he e n h a n c e m e n t of o u t c r o s s i n g s h o u l d be a c c e n t u a t e d (Den ison and Frank l in , 1 9 7 5 ) . M a n y f a c t o r s have been c o n s i d e r e d to a f fec t the rate of o u t c r o s s i n g in a popu la t i on ( C l e g g , 1 9 8 0 ) . T h e s e i nc lude : age (Che l iak e f a / . , 1 9 8 5 a ; S n y d e r etal., 1 9 8 5 ; S h e a , 1 9 8 7 ) , t ree s i ze ( S h e a , 1 9 8 7 ) , d e n s i t y (Levin and K e r s t e r , 1 9 7 4 ; Farr is and M i t t o n , 1 9 8 4 ; S h e a , 1 9 8 7 ) , ma te rna l h e t e r o z y g o s i t y ( S h e a , 1 9 8 7 ) , c o n e p r o d u c t i o n ( S h e a , 1 9 8 7 ) , po l l ina t ion e n v i r o n m e n t ( E l - K a s s a b y and D a v i d s o n , 1991 ), and t e m p o r a l d i s t r ibu t ion (Che l iak etal, 1 9 8 5 a ; S n y d e r e r a / . , 1 9 8 5 ; G i b s o n and Hamr i ck , 1 9 9 1 ; E l - K a s s a b y er a / . , 1 9 9 2 ) . G e n e t i c var ia t ion in po l len f e c u n d i t y a n d f lora l p h e n o l o g y c a n be f a c t o r s a f fec t i ng o u t c r o s s i n g rate as w e l l (E r i ksson et al., 1 9 7 3 ; Gr i f f i n , 1 9 8 2 ; A d a m s , 1 9 8 3 ; E l - K a s s a b y et al., 1 9 8 4 , 1 9 8 8 ) . In con i f e r s e e d o r c h a r d s , po l len c o n t a m i n a t i o n w a s o b s e r v e d to inf late o u t c r o s s i n g e s t i m a t e s (F r iedman and A d a m s , 1 9 8 5 a , b; E l - K a s s a b y and R i t l and , 1 9 8 6 ; E l - K a s s a b y e f a / . , 1 9 8 9 ) . H o w e v e r , o r c h a r d -m a n a g e m e n t p rac t i ces have s h o w n tha t po l len c o n t a m i n a t i o n as w e l l as i nb reed ing c a n be r e d u c e d ( E l - K a s s a b y and D a v i d s o n , 1 9 9 0 ) . U n d e r s t a n d i n g the b reed ing s t ruc tu re of con i fe r s e e d o r c h a r d s c a n lead to d e v e l o p m e n t of bet ter o r c h a r d d e s i g n and m a n a g e m e n t tha t w i l l p romo te in te rmat ing a m o n g the se lec ted g e n o t y p e s ( S h e n , et al., 1 9 8 1 ; A d a m s , 1 9 8 3 ; F r i edman and A d a m s , 1 9 8 5 a ) . In th is s t u d y , o u t c r o s s i n g and inb reed ing at upper and l o w e r c r o w n leve ls w e r e e s t i m a t e d in a c l ona l S i t ka s p r u c e s e e d o r c h a r d . 4 . 2 Materials and Methods T h e s t u d y w a s c o n d u c t e d in C a n a d i a n Pac i f i c Fo res t P r o d u c t s L t d . S i t k a s p r u c e s e e d o r c h a r d . T h e s e e d o r cha rd is l oca ted in S a a n i c h t o n , Br i t ish C o l u m b i a ( lat i tude 4 8 ° 3 5 ' N , l ong i tude 1 2 3 ° 2 4 ' W ) and c o n s i s t s of 1 3 9 c l o n e s (averag ing 9 . 3 rame ts per c lone) s e l e c t e d f r o m e leva t i ons b e t w e e n 0 and 4 1 5 m o n w e s t e r n V a n c o u v e r Is land, W a s h i n g t o n a n d O r e g o n (Figure 1.1). T h e o r cha rd w a s es tab l i shed in 1971 in a r a n d o m s ing le - t ree m i x ove r three unequa l b l o c k s . T r e e s are s p a c e d 3 m apar t and kept at a p p r o x i m a t e l y 4 m in he igh t by t op -p run ing . T h e s e e d o r cha rd is 1 0 k m a w a y f r o m the neares t S i t k a s p r u c e s t a n d , the re fo re , the S i t k a s p r u c e b a c k g r o u n d po l len is neg l ig ib le in the o r cha rd a rea ( E l - K a s s a b y a n d R e y n o l d s , 1 9 9 0 ) . In S e p t e m b e r 1 9 9 0 , w ind -po l l i na ted s e e d s f r o m t rees p r o d u c i n g su f f i c i en t s e e d s fo r the s t u d y w e r e co l l ec ted f r o m one of the o r c h a r d ' s th ree b l o c k s . T h e s t u d i e d b l o c k c o n s i s t e d of 61 c l o n e s , w h i c h is a p p r o x i m a t e l y 4 6 percen t of the to ta l o r c h a r d c l o n e s . F ive s e e d c o n e s f r o m the uppe r and l o w e r c r o w n of c o n e - b e a r i n g po r t i ons of the t rees w e r e c o l l e c t e d r a n d o m l y and the i r ident i t ies w e r e m a i n t a i n e d . C o n e s a m p l e s w e r e dr ied at r o o m tempe ra tu re a n d ex t rac ted by h a n d . S e e d s a m p l e s w e r e label led and kept at 2 ° C unt i l u s e d . S t a r c h g e l - e l e c t r o p h o r e s i s o f m e g a g a m e t o p h y t i c ( In) and c o r r e s p o n d i n g e m b r y o n i c (2n) t i s s u e s w e r e a s s a y e d f o l l o w i n g the m e t h o d s ou t l i ned in C h a p t e r 2 . Th ree e n z y m e s y s t e m s (PGI-2, PGM-2 and GDH) w e r e s e l e c t e d fo r es t ima t i on of m a t i n g - s y s t e m p a r a m e t e r s , b a s e d o n thei r M e n d e l i a n seg rega t i on pa t te rns and apparen t lack of l inkage (Chap te r 2) . M a t e r n a l g e n o t y p e s w e r e in ferred f r o m the seg rega t i on of a l l o z y m e s in m e g a g a m e t o p h y t i c t i s s u e o f 1 6 s e e d s per t ree : 8 f r o m e a c h of the upper and l o w e r c r o w n s e g m e n t s . T h e p robab i l i t y of i ncor rec t l y c l a s s i f y i n g a h é t é r o z y g o t e at a n y one l o c u s is (72)'""^', w h e r e n = the n u m b e r of s e e d s ana l yzed (16) (T igers ted t , 1 9 7 3 ) . A t a n d e m a s s a y of the hap lo id m e g a g a m e t o p h y t e a n d the d ip lo id e m b r y o t i s sue revea led the po l len con t r i bu t i on . S ing le and mu l t i l o cus popu la t i on es t ima tes of o u t c r o s s i n g rate (t^ and t^) and o u t c r o s s e d - p o l l e n al lel ic f r e q u e n c i e s (p) w e r e e s t i m a t e d u s i n g the m a x i m u m - l i k e l i h o o d p r o c e d u r e of R i t l and and E l - K a s s a b y ( 1 9 8 5 ) . T h i s p r o c e d u r e is b a s e d o n a m u l t i l o c u s , m i x e d -ma t ing s y s t e m m o d e l w h i c h w a s s h o w n to be s ta t i s t i ca l l y m o r e e f f i c ien t t h a n the " o b s e r v e d o u t c r o s s " m o d e l ( S h a w and A l l a r d , 1 9 8 2 ) or " d e t e c t a b l e o u t c r o s s " m o d e l (Neale and A d a m s , 1 9 8 5 ) , espec ia l l y w h e n re la t ive ly f e w loc i are a s s a y e d . A m u l t i l o c u s es t ima te of o u t c r o s s i n g is c o n s i d e r e d more a c c u r a t e and less sens i t i ve to v io la t i ons of the m o d e l a s s u m p t i o n s s o tha t a greater n u m b e r of o u t c r o s s e s m a y be ident i f ied w i t h ce r ta in ty ( S h a w et al., 1 9 8 1 ) . In th is s t u d y it w a s a s s u m e d that : 1 ) fer t i l i za t ion even t s are a mix tu re of r a n d o m o u t c r o s s i n g and se l f - fe r t i l i za t ion ; 2) there is no se lec t i on b e t w e e n fer t i l i za t ion and c e n s u s ; 3) the rate of o u t c r o s s i n g is i ndependen t a m o n g loc i (for mu l t i l o cus es t ima tes ) and 4) al le l ic f r e q u e n c i e s in the o u t c r o s s i n g - p o l l e n poo l are iden t i ca l ove r t he popu la t i on of m o t h e r t rees (Fyfe a n d Ba i ley , 1 9 5 1 ; S h a w et al., 1 9 8 1 ) . A C h i - s q u a r e c o n t i n g e n c y tes t (Soka l and Roh l f , 1981 ) w a s u s e d to tes t the d i f fe rence in the es t ima tes of po l len al le l ic f r e q u e n c i e s f r o m upper and l o w e r c r o w n po r t i ons . 4 . 3 Results and Discussion 4 .3 .1 Allelic Frequencies Pol len al le l ic f r e q u e n c i e s w e r e s l igh t ly d i f fe rent b e t w e e n the upper and l o w e r c r o w n s (Table 4.1). H o w e v e r , no s ign i f i can t d i f f e rences b e t w e e n the uppe r and l o w e r o u t c r o s s i n g -po l len poo l s w a s o b s e r v e d . T h e al le l ic f r equenc ies of s a m p l e d po l len a n d the mate rna l gene p o o l s w e r e c o m p a r e d to de te rm ine if the e f fec t i ve po l len poo l w a s rep resen ta t i ve o f the adul t ma te rna l t rees of tha t b l o c k (Table 4.1). A s ign i f i can t d i f f e rence fo r 1 (PGM-2) ou t o f 3 s tud ied loc i b e t w e e n the t w o gene poo l s w a s o b s e r v e d , i nd ica t ing that po l len f l o w f r o m the o ther t w o o r c h a r d b l o c k s con t r i bu ted to the s tud ied b l ock . In c o n i f e r s , c o n t r i b u t i o n s of ma le and fema le pa ren ts to g a m e t i c poo l s has been o b s e r v e d to be a s y m m e t r i c a l , i .e., s o m e t rees con t r i bu ted m o r e of the e lec t rophore t i ca l l y h o m o l o g o u s al lele to thei r p r o g e n y w h e n u s e d as a ma le parent and l ess w h e n u s e d as a f e m a l e paren t , (Mu l l e r -S ta r ck , 1 9 8 2 ; M o r a n et al., 1 9 8 3 ; Che l i ak and P i te l , 1 9 8 4 ) , ind ica t ing the e x i s t e n c e of d i f ferent se lec t i ve va l ues a s s o c i a t e d w i t h game t i c a r rays de r i ved f r o m the ma le and fema le (Chel iak et al., 1 9 8 5 b ) . T h e d i s c r e p a n c y in al le l ic f r e q u e n c i e s a m o n g d i f ferent gene poo l s (materna l and o u t c r o s s i n g ) c o u l d be c a u s e d by severa l o ther f a c t o r s . T h e s e inc lude : d i f f e rences in po l len p r o d u c t i o n or p h e n o l o g i c a l d e v e l o p m e n t a m o n g po l len pa ren ts ( M o r a n et al., 1 9 8 0 ; Mu l le r -S t a r c k et al., 1 9 8 3 , E l - K a s s a b y and R i t l and , 1 9 8 6 ; E l - K a s s a b y et al., 1 9 8 8 ; E r i c k s o n and A d a m s , 1 9 9 0 ) , f r e q u e n c y - d e p e n d e n t se lec t i on of ma le rep roduc t i ve s u c c e s s ( E l - K a s s a b y and R i t l an d , 1 9 9 2 ) , d i f f e rences a m o n g o u t c r o s s i n g po l len p o o l s , and /o r the p r e s e n c e of game t i c or z y g o t i c (post mat ing) se lec t i on ( B r o w n et al., 1 9 7 5 ; Y e h et al., 1 9 8 3 ) . 4 . 3 . 2 Outcrossing Rate S ing le - and mu l t i l ocus e s t i m a t e s of o u t c r o s s i n g ranged f r o m 6 5 % to 9 4 % a n d f r o m 6 5 % to 88%), r espec t i ve l y (Table 4.2), all es t ima tes w e r e s ign i f i can t at the P< 0 . 0 5 leve l . T h e PGM-2 l o c u s g a v e the l o w e s t e s t i m a t e s fo r bo th upper and l o w e r c r o w n . T h e da ta f r o m GDH Table 4.1. A l l e l i c f r e q u e n c i e s fo r o u t c r o s s i n g po l len and mate rna l gene p o o l s fo r s a m p l e s ob ta ined f r o m the upper and l o w e r c r o w n po r t i ons and c o m b i n e d fo r a S i t k a s p r u c e s e e d o r cha rd Po l len L o c u s A l l e le M a t e r n a l U p p e r L o w e r C o m b i n e d PGI-2 1 0 0 0 . 9 5 0 0 . 9 9 2 0 . 9 9 0 0 . 9 8 2 1 1 7 0 . 0 2 5 0 . 0 0 7 0 . 0 0 9 0 . 0 1 7 9 3 0 . 0 2 5 0 . 0 0 1 0 . 0 0 1 0 . 0 0 1 PGM-2 1 0 0 0 . 8 5 2 0 . 9 5 6 0 . 9 6 3 0 . 9 6 7 8 6 0 . 1 4 8 0 . 0 4 4 0 . 0 3 7 0 . 0 3 3 GDH 1 0 0 0 . 9 3 4 0 . 9 4 1 0 . 9 4 7 0 . 9 5 0 1 1 6 0 . 0 3 3 0 . 0 2 7 0 . 0 1 8 0 . 0 2 0 71 0 . 0 3 3 0 . 0 3 2 0 . 0 3 5 0 . 0 3 0 Table 4.2. S i n g l e - l o c u s ( { 3 ) and mu l t i l ocus [Xj e s t i m a t e s of o u t c r o s s i n g for s a m p l e s ob ta i ned f r o m the upper and l o w e r leve ls of the c r o w n of c l ona l S i t ka s p r u c e t rees in a m a n a g e d s e e d o r c h a r d L o c u s U p p e r L o w e r C o m b i n e d PGI-2 0 . 9 0 6 » 0 . 8 3 9 0 . 9 6 8 PGM-2 0 . 7 6 9 0 . 6 4 9 0 . 7 7 3 GDH 0 . 9 4 0 - 0 . 9 9 9 \ 0 . 8 7 2 0 . 7 4 4 0 . 9 1 3 0 . 8 6 6 0 . 6 4 8 0 . 8 7 5 # of t rees 61 61 61 # of s e e d s 4 2 7 4 2 7 9 1 5 "A l l o u t c r o s s i n g rate e s t i m a t e s (t^ and t^) are s ign i f i can t at P< 0 . 0 5 ( i .e. , t ^ t l . O ) . l o c u s of the l o w e r c r o w n d id not c o n v e r g e (Table 4.2), w h i c h c o u l d be due to t he s m a l l s a m p l e of s e e d s per c l o n e u s e d ( B r o w n et al., 1 9 8 5 ) . A p p a r e n t l y , the s i ng l e - l ocus o u t c r o s s i n g es t ima te is a f f ec ted by popu la t i on s u b s t r u c t u r i n g in natura l popu la t i ons . H o w e v e r , s u c h e f fec t w a s not o b s e r v e d in s e e d o r c h a r d s of the s a m e s p e c i e s ( S h a w and A l l a r d , 1 9 8 2 ) . B i j i sma et al. ( 1 9 8 6 ) f o u n d an e x c e s s of h o m o g a m e t i c fer t i l i za t ion of g e n o t y p e s a s s o c i a t e d w i t h f l o w e r i n g t ime w a s one o f the c a u s e s of l o w s i n g l e - l o c u s o u t c r o s s i n g in ma i ze . T h e r e f o r e , to de te rm ine the p o s s i b l e c a u s e of the l o w s i n g l e - l o c u s o u t c r o s s i n g es t ima te at l ocus PGM-2, a c o m p l e m e n t a r y ana l ys i s w a s c o n d u c t e d to de te rm ine if ma te rna l g e n o t y p e s a f fec ted th is es t ima te . T h e mate rna l popu la t i on w a s s u b d i v i d e d into th ree c l a s s e s a c c o r d i n g to thei r g e n o t y p e s ( i .e., 1 0 0 : 1 0 0 , 1 0 0 : 8 6 , and 8 6 : 8 6 ) and the mu l t i l ocus o u t c r o s s i n g rate for e a c h g e n o t y p i c c l a s s w a s e s t i m a t e d t w i c e (wi th the PGM-2 l o c u s i nc l uded and exc luded) (Table 4.4). T h e C h i - s q u a r e tes t fo r po l len poo l i nd i ca tes tha t o b s e r v e d al le l ic f r equenc ies at l ocus PGM-2 are h igh ly s ign i f i can t l y d i f fe rent f r o m the e x p e c t e d al lel ic f r e q u e n c i e s , wh i l e the e s t i m a t e s fo r o ther loc i do not d i f fer s i gn i f i can t l y in bo th a n a l y s e s (Table 4.3 and Table 4.4.) T h e resul t s u g g e s t s tha t the f r e q u e n c y of the c o m m o n al lele (100) at l ocus PGM-2 is in e x c e s s of the f r e q u e n c i e s of o ther a l le les in all ma te rna l g e n o t y p e s ( 1 0 0 : 1 0 0 , 1 0 0 : 8 6 , and 8 6 : 8 6 ) . T h i s is p robab l y due to n o n r a n d o m m a t i n g (Bi j isma et al., 1 9 8 6 ) c a u s e d by g e n o t y p i c se lec t i on fo r fe r t i l i za t ion . A n t h e s i s of t rees p o s s e s s i n g al lele 1 0 0 m a y have been s y n c h r o n i z e d w i t h the peak of f ema le recep t i v i t y in the s e e d o r c h a r d , o r / a n d the p r e s e n c e of g a m e t i c s e l e c t i o n . A s s u m i n g tha t o u t c r o s s i n g is a s s o c i a t e d w i t h mate rna l g e n o t y p e s , the es t ima te of popu la t i on m e a n o u t c r o s s i n g c o r r e s p o n d i n g to mate rna l g e n o t y p e s at l ocus PGM-2 w a s ver i f ied a c c o r d i n g to Fa l cone r ( 1 9 8 6 , p. 1 0 2 ) , M = a(p-q) + 2 d p q , w h e r e M = popu la t i on m e a n , a (geno typ ic va lue of h o m o z y g o t e 1 0 0 : 1 0 0 ) = 0 . 0 9 2 , d (geno typ i c va lue of h é t é r o z y g o t e 1 0 0 : 8 6 ) = 0 . 0 4 6 , p (geno typ i c f r e q u e n c y of al lele 100 ) = 0 . 7 7 9 , and q (geno typ ic f r e q u e n c y of al lele 86) = 0 . 2 2 1 . If bo th a l le les 1 0 0 a n d 8 6 s h o w e d i ncomp le te d o m i n a n c e for o u t c r o s s i n g , the o u t c r o s s i n g es t ima te fo r g e n o t y p e 1 0 0 : 8 6 s h o u l d Table 4.3. Es t ima tes of po i len-a l le l ic f r e q u e n c i e s fo r 3 (w i th PGM-2) and 2 loc i (w i thout PGM-2) c o r r e s p o n d i n g to materna l g e n o t y p e s (number of parent t rees in pa ren theses ) L o c u s A l le le Ma te rna l G e n o t y p e W i t h PGM-2 W i t h o u t PGM-2 1 0 0 : 1 0 0 (74) 1 0 0 : 8 6 (42) 8 6 : 8 6 (6) 1 0 0 : 1 0 0 (74) 1 0 0 : 8 6 (42) 8 6 : 8 6 (6) x' GDH 1 0 0 0 . 9 4 6 0 . 9 6 0 0 . 9 2 4 0 .544™ 0 , 9 4 6 0 . 9 6 5 0 . 9 2 8 0 .666™ 1 3 7 0 . 0 2 0 0 . 0 1 9 0 . 0 3 8 0 . 0 2 0 0 . 0 1 5 0 . 0 3 6 71 0 . 0 3 4 0 . 0 2 1 0 . 0 3 8 0 . 0 3 4 0 . 0 2 0 0 . 0 3 6 PGI-2 1 0 0 0 . 9 9 1 0 . 9 9 2 0 . 9 9 9 0 .089™ 0 . 9 9 1 0 . 9 8 9 0 . 9 9 9 0 ,120™ 1 1 7 0 . 0 0 8 0 . 0 0 7 0 . 0 0 1 0 . 0 0 8 0 . 0 1 0 0 . 0 0 1 9 3 0 . 0 0 1 0 . 0 0 1 0 . 0 0 0 0 . 0 0 1 0 . 0 0 1 0 . 0 0 0 PGM-2 1 0 0 0 . 9 9 9 0 . 8 9 9 0 . 7 8 7 2 0 . 3 0 8 " - - - -8 6 0 . 0 0 1 0 . 1 0 1 0 . 2 1 3 - -™ N o t s ign i f i can t , " S ign i f i can t at P < 0 . 0 0 1 . Table 4.4. N u m b e r of e m b r y o g e n o t y p e s s c o r e d c o r r e s p o n d i n g to ma te rna l g e n o t y p e s , and o u t c r o s s i n g rate (t^ n) M a t e r n a l N o . E m b r y o G e n o t y p e t^ G e n o t y p e Tree 1 0 0 : 1 0 0 1 0 0 : 8 6 8 6 : 8 6 W i t h W i t h o u t 1 0 0 : 1 0 0 7 4 581 11 - 0 . 9 0 8 0 . 9 4 9 1 0 0 : 8 6 4 2 171 1 4 0 2 5 0 . 8 6 2 0 . 9 0 1 8 6 : 8 6 6 - 3 4 14 0 . 7 2 4 0 . 7 3 0 ^ W i t h and w i t h o u t PGM-2. be 0 . 8 1 6 . H o w e v e r , the o u t c r o s s i n g for th is g e n o t y p e w a s 0 . 8 6 2 , y ie ld ing a popu la t i on m e a n of 0 . 8 8 3 , i nd ica t ing tha t al lele 1 0 0 is d o m i n a n t ove r al lele 8 6 fo r th i s trai t . T h e r e f o r e , if th is g e n e t i c m o d e l is va l i d , g e n o t y p i c se lec t i on c o u l d a c c o u n t fo r the l o w s i n g l e - l o c u s o u t c r o s s i n g at l o c u s PGM-2. O t h e r w i s e , g a m e t i c se lec t i on or a s s o c i a t i o n b e t w e e n f l o w e r i n g and a par t icu lar al le le m igh t a c c o u n t fo r th is d i f f e rence . In ma i ze , h o m o g a m e t i c fe r t i l i za t ion w a s o b s e r v e d to con t r i bu te to l o w o u t c r o s s i n g , and it w a s c o n c l u d e d that the e f f ec t w a s a s s o c i a t e d w i t h g e n o t y p e s in f avou r of ear ly f l o w e r i n g of one h o m o z y g o t e (B i j isma et al., 1 9 8 6 ) . A l s o , he te rogene i t y in the po l len poo l w a s a t t r ibu ted to the o b s e r v e d l o w o u t c r o s s i n g rate for a s ing le l o c u s in a T a b l e M o u n t a i n p ine (Pinus pungens) popu la t i on ( G i b s o n a n d H a m r i c k , 1 9 9 1 ) . In th is s t u d y , all e s t i m a t e s w e r e m a d e f r o m the s a m e se t of e m b r y o s s o tha t var ia t ion in the ac tua l p ropo r t i ons of se l f ed and o u t c r o s s e d o f f sp r i ng d id not c o n t r i b u t e to the o b s e r v e d s i n g l e - l o c u s e s t i m a t e s of t. T h u s , the var iabi l i ty m u s t be due to e i ther r a n d o m var ia t ion or v io la t i ons of the a s s u m p t i o n s in t r ins ic to the es t ima t i on p r o c e d u r e ( S h a w a n d A l l a r d 1 9 8 2 ) . T h e s ing le and mu l t i l o cus o u t c r o s s i n g - r a t e es t ima tes fo r the s t u d i e d b l o c k w e r e d i f fe rent f r o m 1.0 ( comp le te o u t c r o s s i n g ) , i nd i ca t i ng the p r e s e n c e of se l f i ng in the o r c h a r d b l ock . D i f f e rences in e s t i m a t e s of o u t c r o s s i n g re la ted to c r o w n leve ls repo r ted by o ther au tho rs are s u m m a r i z e d in Table 4.5. T h e o b s e r v e d d i f f e rences m a y be due to se l f - fe r t i l i za t ion be ing more f requen t in l o w e r t han in upper c r o w n s . H o w e v e r , W o o d s and H e a m a n ( 1 9 8 9 ) c o n t e n d e d that w i t h o u t c o n t r o l l e d - c r o s s i n g s t u d i e s , the t y p e of re la ted ma t i ng resu l t ing in e m p t y s e e d s is i m p o s s i b l e to de te rm ine . The re fo re , se l f i ng e s t i m a t e s are l ikely to be c o n s e r v a t i v e . T h e p r e s e n c e of i nb reed ing d i f f e rences in upper v s . l o w e r c r o w n s s u g g e s t s tha t if the S i t ka s p r u c e o r cha rd is h igh ly p r o d u c t i v e , then s e e d co l l ec t i on s h o u l d be m a d e on l y f r o m upper c r o w n s . H o w e v e r , w h e n the d e m a n d for s e e d s is h i g h , i nc reas ing fe r t i l i za t ion s u c c e s s a n d , c o n s e q u e n t l y r e d u c e d se l f - fe r t i l i za t ion in the l o w e r c r o w n m igh t be a c c o m p l i s h e d by Table 4.5. E f fec t of c r o w n level o n o u t c r o s s i n g e s t i m a t e s in s e e d o r c h a r d s S p e c i e s S . O . c o m p o s i t i o n C r o w n leve l t. R e f e r e n c e Doug las - f i r C l o n a l U p p e r L o w e r 0 . 9 1 6 0 . 9 1 8 0 . 9 5 5 0 . 9 5 0 E l - K a s s a b y etal. ( 1986 ) S e e d l i n g Uppe r L o w e r 0 . 9 0 1 0 . 8 8 4 0 . 9 3 2 0 . 9 0 1 C l o n a l U p p e r L o w e r - 0 . 9 2 0 0 . 8 9 0 S h a w and A l l a r d (1982 ) C l o n a l U p p e r L o w e r - 0 . 9 9 9 0 . 8 7 1 O m i and A d a m s (1986 ) S i t k a s p r u c e C l o n a l Uppe r L o w e r 0 . 8 7 2 0 . 7 4 4 0 . 8 6 6 0 . 6 4 8 P resen t s t u d y s u p p l e m e n t a l m a s s po l l ina t ion ( E l - K a s s a b y and R e y n o l d s , 1 9 9 0 ; E l - K a s s a b y and D a v i d s o n , 1 9 9 0 ) . T h e reduc t i on of inbred s e e d s f r o m s e e d o r c h a r d s is impor tan t . In n o n - c o m p e t i t i v e p lan t i ngs , inbred seed l i ngs c a n su rv i ve (Ri t land and E l - K a s s a b y , 1 9 8 5 ) . H o w e v e r , inbred ind iv idua ls m a y be v i r tua l ly e l im ina ted by natura l se l ec t i on due to poo r seed l i ng v igour du r ing nu rse ry and p lan ta t ion p r o c e s s e s , as f o u n d in S c o t s p ine (Pinus sylvestris L.) ( M u o n a et al., 1 9 8 7 ) , m a k i n g the c o s t of p lan ta t ion e s t a b l i s h m e n t h igher t han the c o s t of e x c l u d i n g inbred s e e d l i n g s f r o m p lan t ings . T h e imp l i ca t i on of po l len f l o w b e t w e e n o r c h a r d b l o c k s is of i n te res t i ng . If s e e d o r c h a r d s fo r d i f fe rent s e e d z o n e s are l oca ted c l o s e to e a c h o ther , ca re s h o u l d be t aken to a v o i d po l len c o n t a m i n a t i o n . Sepa ra t i on by su f f i c ien t d i s t a n c e and /o r buf fer as a barr ier fo r po l len m ig ra t ion s h o u l d be c o n s i d e r e d . S i t ka s p r u c e in Br i t ish C o l u m b i a is in the ear ly s t a g e of b r e e d i n g , and open -po l l i na t i on p r o g e n y tes t i ng is a v iab le op t i on fo r the s p e c i e s ' b reed ing p r o g r a m (Yeh and R a s m u s s e n , 1 9 8 5 ) . H o w e v e r , a h igh p ropor t i on of inbred p r o g e n y in the tes t i ng popu la t i on c a n resu l t in an o v e r e s t i m a t i o n of add i t i ve gene t i c va r i ance a n d gene t i c ga ins ( N a m k o o n g , 1 9 6 6 ; S q u i l l a c e , 1 9 7 4 ) . S e e d s f r o m a s ing le s e e d c rop for a p r o g e n y tes t m a y reta in subs tan t i a l i n b r e e d i n g . T h i s m a y subs tan t i a l l y a f fec t the es t ima ted gene t i c ga in . T h e r e f o r e , a m ix tu re of seve ra l s e e d yea rs fo r open-po l l i na t ion p r o g e n y tes t s is more appropr ia te (K ing et al., 1 9 8 4 ) or ad jus t i ng the gene t i c pa rame te rs to a c c o u n t for the o b s e r v e d level of i nb reed ing wi l l be n e c e s s a r y ( A s k e w and E l - K a s s a b y , 1 9 9 2 ) . W h e r e h igh inb reed ing in the l o w e r c r o w n is k n o w n , e x c l u d i n g the c o l l e c t i o n f r o m th is por t ion w i l l a l so i m p r o v e rel iabi l i ty . C o m p a r i n g o u t c r o s s i n g in s e e d o r c h a r d s and thei r na tu ra l -popu la t i on c o u n t e r p a r t s is essen t i a l in unde rs tand ing c h a n g e s of ma t i ng s t ruc tu re in ar t i f ic ia l p o p u l a t i o n s . T h i s w i l l p rov ide subs tan t i a l i n fo rma t ion for bet ter o r cha rd d e s i g n a n d m a n a g e m e n t . F e w s t u d i e s h a v e repor ted o u t c r o s s i n g e s t i m a t e s f r o m s e e d o r c h a r d s to be h igher t han natura l p o p u l a t i o n s (Table 4.6). Table 4.6. C o m p a r i s o n of m a t i n g - s y s t e m e s t i m a t e s in s e e d o r c h a r d and natura l popu la t i ons S p e c i e s R e f e r e n c e Doug las - f i r Na tu ra l S e e d o r cha rd B lack s p r u c e Natu ra l S e e d o rcha rd 0 . 9 1 0 0 . 7 5 8 0 . 9 4 0 0 . 9 3 4 0 . 9 3 2 0 . 9 4 0 0 . 9 0 0 0 . 8 8 7 0 . 9 1 0 0 . 9 2 5 0 . 9 2 4 0 . 8 4 0 Nea le and A d a m s ( 1 9 8 5 ) Y e h a n d M o r g a n ( 1 9 8 7 ) S h a w a n d A l l a r d ( 1 9 8 2 ) E l - K a s s a b y etal. ( 1 9 8 8 ) Boy le and M o r g e n s t e r n ( 1 9 8 6 ) Barret t et al. ( 1 9 8 7 ) T h e d i f f e rences in o u t c r o s s i n g es t ima tes b e t w e e n natura l p o p u l a t i o n s and s e e d o r c h a r d s i nd i ca tes that popu la t i on s t ruc tu re (i .e., the p h y s i c a l a r rangemen t of the re la ted a n d unre la ted ind iv idua ls w i t h i n a popu la t ion) has a f f ec ted the rate of o u t c r o s s i n g (Ennos and C l e g g , 1 9 8 2 ; E l l s t rand and Fos te r , 1 9 8 3 ; Rud in et al., 1 9 8 6 ; E l - K a s s a b y and D a v i d s o n , 1 9 9 0 ) . In f ac t , s e e d o r c h a r d s p rov ide l o w e r i nb reed ing leve ls than natura l p o p u l a t i o n s (Rud in et al., 1 9 8 6 ) . T h e r e f o r e , k n o w l e d g e of ma t i ng s t ruc tu re in natura l popu la t i ons of S i t k a s p r u c e is requ i red fo r t es t i ng the va l id i ty of th is p h e n o m e n o n in the s p e c i e s , and fo r fu r ther o r c h a r d i m p r o v e m e n t . 4 . 4 References A d a m s , W . T . 1 9 8 3 . A p p l i c a t i o n of i s o z y m e s in t ree b reed ing . In Isozymes in plant genetics and breeding, Par t A , e d . S . D . T a n k s l e y and T . J . O r t o n , p p . 3 8 1 - 4 0 0 . A m s t e r d a m : E lsev ier S c i e n c e P u b l i s h e r s , B . V . A s k e w , G . R . and Y . A . E l - K a s s a b y . 1 9 9 2 . Es t ima t i on of add i t i ve va r i ance f r o m w ind -po l l i na ted s e e d o r c h a r d s e e d s o u r c e s . For. Sci. (in p ress ) . Barret t , J . W . , P. K n o w l e s , and W . M . Che l iak . 1 9 8 7 . T h e ma t i ng s y s t e m in a b lack s p r u c e c lona l s e e d o r c h a r d . Can. J. For. Res. 1 7 : 3 9 7 - 3 8 2 . B i j i sma , R., R . W . A l l a r d , and A . L . Kah le r . 1 9 8 6 . N o n r a n d o m mat ing in an open -po l l i na ted ma ize p o p u l a t i o n . Genetics 1 1 2 : 6 6 9 - 6 8 0 . B o y l e , T . J . B . and E .K . M o r g e n s t e r n . 1 9 8 6 . Es t ima tes of o u t c r o s s i n g ra tes in s i x popu la t i ons of b lack s p r u c e in cen t ra l N e w B r u n s w i c k . Silvae Genet. 3 5 : 1 0 2 - 1 0 6 . B r o w n , A . H . D . 1 9 7 9 . E n z y m e p o l y m o r p h i s m in p lant p o p u l a t i o n s . Theor. Pop. Biol. 1 5 : 1 - 4 2 . B r o w n , A . H . D . , A . C . M a t h e s o n , and C . G . A l d r i d g e . 1 9 7 5 . Es t ima t i on of the ma t i ng s y s t e m of Eucalyptus obliqua L 'Hér i t . by us ing a l l o z y m e p o l y m o r p h i s m s . Aust. J. Bot. 2 3 : 9 3 1 -9 4 9 . B r o w n , A . D . H . , S . C . H . Barret t and G . F . M o r a n . 1 9 8 5 . M a t i n g s y s t e m es t ima t i on in fo res t t rees : m o d e l s , m e t h o d s and m e a n i n g s . In Population Genetics in Forestry. Lecture Notes in Biometrics N o . 6 0 , e d . H . -R . G r e g o r i u s , pp . 3 2 - 4 9 . N e w Y o r k : S p r i n g e r - V e r l a g . Che l i ak , W . M . , and J . A . P i te l . 1 9 8 4 . G e n e t i c con t ro l of a l l o z y m e va r ian ts in ma tu re t i s s u e s of Picea glauca ( M o e n c h ) V o s s . J. Heredity 7 4 : 3 4 - 4 0 . Che l i ak , W . M . , B . P . Danc i k , K. M o r g a n , F . C . H . Y e h , and C . S t r o b e c k . 1 9 8 5 a . T e m p o r a l var ia t ion and the ma t i ng s y s t e m in a natura l popu la t i on of j ack p ine . Genetics 1 0 9 : 5 6 9 -5 8 4 . Che l i ak , W . M . , J . A . P i te l , and G . M u r r a y , 1 9 8 5 b . P o p u l a t i o n s t ruc tu re and the ma t i ng s y s t e m of w h i t e s p r u c e . Can. J. For. Res. 1 5 : 3 0 1 - 3 0 8 . C l e g g , M . T . 1 9 8 0 . M e a s u r i n g p lant ma t ing s y s t e m s . Bioscience 3 0 : 8 1 4 - 8 1 8 . C r a m , W . H . 1 9 8 4 . S o m e e f fec t s of sel f - , c r o s s - , a n d open -po l l i na t i on in Picea pugens. Can. J. Bot. 6 2 : 3 9 2 - 3 9 5 . D e n i s o n , N . P . and E . C . F rank l in . 1 9 7 5 . Po l len m a n a g e m e n t . In S e e d o r c h a r d s , e d . R. Fau lkner . G.B. For. Common. Bull. N o . 5 4 : 9 2 - 1 0 0 . E l - K a s s a b y , Y . A . 1 9 9 1 . A s s e s s m e n t of i nb reed ing and gene t i c var ia t ion in a w e s t e r n red ceda r s e e d o r c h a r d . Report submitted to B.C. Ministry of Forests. 18 p. (unpub l i shed) . E l - K a s s a b y , Y . A . , and R. D a v i d s o n . 1 9 9 0 . Impact of c r o p m a n a g e m e n t p rac t i ces o n the s e e d c rop gene t i c qua l i t y in a Doug las - f i r s e e d o r c h a r d . Silvae Genet. 3 9 : 2 3 0 - 2 3 7 . E l - K a s s a b y , Y . A . and D a v i d s o n . 1 9 9 1 . Impac t of po l l ina t ion e n v i r o n m e n t man ipu la t i on o n the apparen t o u t c r o s s i n g rate in a Doug las - f i r s e e d o r c h a r d . Heredity 6 6 : 5 5 - 5 9 . E l - K a s s a b y , Y . A . , A . M . K . Fash le r , and 0 . S z i k l a i . 1 9 8 4 . R e p r o d u c t i v e p h e n o l o g y and its i m p a c t on gene t i ca l l y i m p r o v e d s e e d p r o d u c t i o n in a Doug las - f i r s e e d o r c h a r d . Silvae Genet. 3 3 : 1 2 0 - 1 2 5 . E l - K a s s a b y , Y . A . , M . D . M e a g h e r and R. D a v i d s o n . 1 9 9 2 . T e m p o r a l va r ia t ion in the o u t c r o s s i n g rate in a natura l s t and of W e s t e r n w h i t e p ine . Silvae Genet, (in p ress ) . E l - K a s s a b y , Y . A . , J . P a r k i n s o n and J . B . Dev i t t . 1 9 8 6 . T h e e f fec t of c r o w n s e g m e n t o n the ma t i ng s y s t e m in a Doug las - f i r (Pseudotsuga menziesii (Mirb. ) F ranco) s e e d o r c h a r d . Silvae Genet. 3 5 : 1 4 9 - 1 5 5 . E l - K a s s a b y , Y . A . and S . R e y n o l d s . 1 9 9 0 . R e p r o d u c t i v e p h e n o l o g y , parenta l b a l a n c e , and s u p p l e m e n t a l m a s s po l l ina t ion in a S i t k a s p r u c e s e e d - o r c h a r d . For. Ecol. Manage. 3 1 : 4 5 - 5 4 . E l - K a s s a b y , Y . A . and K. R i t l and . 1 9 8 6 . T h e re la t ion of o u t c r o s s i n g a n d c o n t a m i n a t i o n to r ep roduc t i ve p h e n o l o g y and s u p p l e m e n t a l m a s s po l l ina t ion in a Doug las - f i r s e e d o r c h a r d . Silvae Genet. 3 5 : 2 4 0 - 2 4 4 . E l - K a s s a b y , Y . A . and K. R i t l and . 1 9 9 2 . F r e q u e n c y - d e p e n d e n t ma le rep roduc t i ve s u c c e s s in a p o l y c r o s s of Doug las - f i r . Theor. Appl. Genet, (in p ress ) . E l - K a s s a b y , Y . A . , K. R i t l and , A . M . K . Fash le r , and W . J . B . Dev i t t . 1 9 8 8 . T h e e f fec t of r ep roduc t i ve p h e n o l o g y u p o n the ma t i ng s y s t e m of a Doug las - f i r s e e d o r c h a r d . Silvae Genet. 3 7 : 7 6 - 8 2 . E l - K a s s a b y , Y . A . , D. R u d i n , and R. Y a z d a n i . 1 9 8 9 . Leve l s of o u t c r o s s i n g and c o n t a m i n a t i o n in t w o Pinus sylvestris L. s e e d o r c h a r d s in nor thern S w e d e n . Scand. J. For. Res. 4 : 4 1 -4 9 . E l l s t rand , N . C . and K . W . Fos te r . 1 9 8 3 . Impact of popu la t i on s t ruc tu re o n the apparen t o u t c r o s s i n g rate of gra in s o r g h u m (Sorghum bicolor). Theor. Appl. Genet. 6 6 : 3 2 3 - 3 2 7 . E n n o s , R . A . and M . T . C l e g g . 1 9 8 2 . E f fec t of popu la t i on s u b s t r u c t u r i n g o n e s t i m a t e s of o u t c r o s s i n g rate in p lant p o p u l a t i o n s . Heredity 4 8 : 2 8 3 - 2 9 2 . E r i c k s o n , V . J . and W . T . A d a m s . 1 9 9 0 . M a t i n g s y s t e m var ia t ion a m o n g ind iv idua l r ame ts in a Doug las - f i r s e e d o r c h a r d . Can. J. For. Res. 2 0 : 1 6 7 2 - 1 6 7 5 . E r i k s s o n , G . , A . J o n s s o n and D. L i ndg ren . 1 9 7 3 . F l o w e r i n g in a c l ona l tr ial of Picea abies Ka r s t . Stud. For. Suec. N o . 1 1 0 : 5 - 4 5 . F a l c o n e r , D . S . 1 9 8 6 . Introduction to quantitative genetics. 2 n d e d . N e w Y o r k : L o n g m a n Sc ien t i f i c & T e c h n i c a l . Far r i s , M . A . and J . B . M i t t o n . 1 9 8 4 . Popu la t i on dens i t y , o u t c r o s s i n g ra te, and h é t é r o z y g o t e super io r i t y in p o n d e r o s a p ine . Evolution 3 8 : 1 1 5 1 - 1 1 5 4 . Frank l i n , E . C . 1 9 7 0 . S u r v e y of mu tan t f o r m s and inb reed ing d e p r e s s i o n in s p e c i e s of the fami l y Pinaceae. S o u t h e a s t e r n For . E x p . S t a . , USDA For. Serv. Res. Pap. S E - 6 1 : 1 - 2 1 F r i e d m a n , S . T . and W . T . A d a m s . 1 9 8 5 a . Es t ima t i on of g e n e f l o w into t w o s e e d o r c h a r d s of lob lo l ly p ine (Pinus taeda L.). Ttieor. Appl. Genet. 6 9 : 6 0 9 - 6 1 5 . F r i e d m a n , S . T . and W . T . A d a m s . 1 9 8 5 b . Leve ls of o u t c r o s s i n g in t w o lob lo l l y p ine s e e d o r c h a r d s . Silvae Genet. 3 4 : 1 5 7 - 1 6 2 . Fy fe , J . L . and N .T . Ba i ley . 1 9 5 1 . P lant b reed ing s tud ies in l e g u m i n o u s f o rage c r o p s . I. natura l c r o s s - b r e e d i n g in w in te r b e a n s . J. Agr. Sci. 4 1 : 3 7 1 - 3 7 8 . G e b u r e k , T . 1 9 8 6 . S o m e resu l ts of i nb reed ing d e p r e s s i o n in Serb ian s p r u c e (Picea omorika (Pane.) Purk . ) . Silvae Genet. 3 5 : 1 6 9 - 1 7 2 . G i b s o n , J . P . and J . L . H a m r i c k . 1991 He te rogene i t y in po l len al lele f r e q u e n c i e s a m o n g c o n e s , w h o r l s , and t rees of tab le m o u n t a i n p ine (Pinus pugens). Amer. J. Bot. 7 8 ( 9 ) : 1 2 4 4 -1 2 5 1 . Gr i f f i n , A . R . 1 9 8 2 . C l o n a l var ia t ion in rad iata p ine s e e d o r c h a r d s . I. S o m e f l o w e r i n g , c o n e and s e e d p r o d u c t i o n t ra i ts . Aust. For. Res. 1 2 : 2 9 5 - 3 0 2 . Gr i f f i n , A . R . and D. L i ndg ren . 1 9 8 5 . E f fec t of i nb reed ing o n p r o d u c t i o n of f i l led s e e d in Pinus radiata- expe r imen ta l resu l ts and a m o d e l of gene a c t i o n . Theor. Appl. Genet. 7 1 : 3 3 4 -3 4 3 . H a m r i c k , J . L . 1 9 8 2 . P lant popu la t i on g e n e t i c s and e v o l u t i o n . Amer. J. Bot. 6 9 : 1 6 8 5 - 1 6 9 3 . H a m r i c k , J . L . , Y . B . L inhar t and J . B . M i t t o n . 1 9 7 9 . R e l a t i o n s h i p s b e t w e e n life h is to ry cha rac te r i s t i c s and e lec t rophore t i ca l l y de tec tab le gene t i c va r ia t ion in p l an t s . Ann. Rev. Ecol. Syst. 1 0 : 1 7 3 - 2 0 0 . K i n g , J . N . , B . P . D a n c i k , and N .K Dhi r . 1 9 8 4 . G e n e t i c s t ruc tu re and m a t i n g s y s t e m of w h i t e s p r u c e (Picea glauca) in a s e e d p r o d u c t i o n a rea . Can. J. For. Res. 1 4 : 6 3 9 - 6 4 3 . L e v i n , D . A . and H . W . Ke rs te r . 1 9 7 4 . G e n e f l o w in s e e d p lan ts . In Evolutionary biology, v o l . 7 . E d . T . D o b z h a n s k y , M . T . H e c h t and W . C . S tee re , pp . 1 3 9 - 2 2 0 . N e w Y o r k : P l e n u m P r e s s . M i t t o n , J . B . , P. K n o w l e s , K . B . S t u r g e o n , Y . B . L inhar t and A . D a v i s . 1 9 8 1 . A s s o c i a t i o n b e t w e e n h e t e r o z y g o s i t y and g r o w t h rate va r iab les in th ree w e s t e r n fo res t t rees . In Proc. Symposium on isozymes of North American forest trees and insects, e d . M . T . C o n k l e , pp . 2 7 - 3 4 . U S D A Fores t S e r v i c e . General Technical Report P S W - 4 8 . M o r a n , G . F . , J . C . Bell and A . C . M a t h e s o n . 1 9 8 0 . T h e gene t i c s t r uc tu re and leve ls of i nb reed ing in Pinus radiata D. D o n s e e d o r c h a r d . Silvae. Genet. 2 9 : 1 9 0 - 1 9 3 . M o r a n , G . F . , J . C . Be l l , and A . J . Hi l l iker . 1 9 8 3 . Grea te r me io t i c r e c o m b i n a t i o n in ma le v s . f ema le g a m e t e s in Pinus radiata. J. Heredity 7 4 : 6 2 . M u o n a , 0 . , R. Y a z d a n i and D. R u d i n . 1 9 8 7 . G e n e t i c c h a n g e b e t w e e n l i fe s t a g e s in Pinus sylvestris: a l l o z y m e var ia t ion in s e e d s and p lan ted s e e d l i n g s . Silvae Genet. 3 6 : 3 9 - 4 2 . Mûl le r -S ta rck , G . 1 9 8 2 . S e x u a l l y a s y m m e t r i c fert i l i ty se l ec t i on and par t ia l se l f - fe r t i l i za t ion . 2 . C l o n a l game t i c con t r i bu t i ons to t ree o f f sp r i ng of S c o t s p ine s e e d o r c h a r d . Silva Fenn. 1 6 : 9 9 - 1 0 6 . M u l l e r - S t a r c k , G . , M . Z i ehe a n d H . H . Ha t t emer . 1 9 8 3 . R e p r o d u c t i v e s y s t e m s in con i fe r s e e d o r c h a r d s . 2 . R e p r o d u c t i v e se lec t i on mon i t o red at an M P gene l o c u s in Pinus sylvestris L. Theor. Appl. Genet. 6 5 : 3 0 9 - 3 1 6 . N a m k o o n g , G . 1 9 6 6 . Inbreed ing e f fec ts o n es t ima t i on of gene t i c add i t i ve va r i ance . For. Sci. 1 2 : 8 - 1 3 . N e a l e , D . B . and W . T . A d a m s . 1 9 8 5 . T h e ma t i ng s y s t e m in na tura l and s h e l t e r w o o d s t a n d s of Doug las - f i r . Theor. Appl. Genet. 7 1 : 2 0 1 - 2 0 7 . O m i , S . K . and W . T . A d a m s . Va r i a t i on in s e e d se t and p ropo r t i ons of o u t c r o s s e d p r o g e n y w i t h c l o n e s , c r o w n p o s i t i o n , and top p run ing in a Doug las - f i r s e e d o r c h a r d . Can. J. For. Res. 1 6 : 5 0 2 - 5 0 7 . O r r - E w i n g , A . L . 1 9 7 6 . Inbreed ing Doug las - f i r t o the S 3 gene ra t i on . Silvae Genet. 2 5 : 1 7 9 - 1 8 3 . R i t l a n d , K. and Y . A . E l - K a s s a b y . 1 9 8 5 . T h e nature of i nb reed ing in a s e e d o r c h a r d of D o u g l a s -fir as s h o w n by an e f f i c ien t mu l t i l ocus m o d e l . Theor. Appl. Genet. 7 1 : 3 7 5 - 3 8 4 . R u d i n , D. 0 . M u o n a , and R. Y a z d a n i . 1 9 8 6 . C o m p a r i s o n o f t he ma t i ng s y s t e m of Pinus sylvestris in natura l s t a n d s and s e e d o r c h a r d s . Hereditas 1 0 4 : 1 5 - 1 9 . S a r v a s , R. 1 9 6 2 . Inves t iga t ions on the f l o w e r i n g and s e e d c r o p s of Pinus sylvestris. Comm. Inst. For. Fenn. 5 3 : 1 - 1 9 8 . S h a w , D . V . and R . W . A l l a r d . 1 9 8 2 . Es t ima t i on of o u t c r o s s i n g ra tes in Doug las - f i r us ing i s o z y m e marke rs . Theor. Appl. Genet. 6 2 : 1 1 3 - 1 2 0 . S h a w , D . V . , A . L . Kah le r a n d R . W . A l l a r d . 1 9 8 1 . A mu l t i l o cus e s t i m a t o r of ma t i ng s y s t e m pa rame te rs in p lant p o p u l a t i o n s . Proc. Nat. Acad. Sci. U S A 7 8 : 1 2 9 8 - 1 3 0 2 . S h e a , K . L . 1 9 8 7 . E f f ec t s of popu la t i on s t ruc tu re and c o n e p r o d u c t i o n o n o u t c r o s s i n g ra tes in E n g e l m a n n s p r u c e a n d suba lp ine fir. Evolution 4 1 : 1 2 4 - 1 3 6 . S h e n , H . - H . , D. Rud in and D. L i n d g r e n . 1 9 8 1 . S t u d y of the po l l ina t ion pa t te rn in a s c o t p ine s e e d o r cha rd by m e a n s of i s o z y m e a n a l y s i s . Silvae Genet. 3 0 : 7 - 1 5 . S n y d e r , T . P . , D . A . S t e w a r d and A . F . S t r i ck le r . 1 9 8 5 . T e m p o r a l a n a l y s i s of b reed ing s t ruc tu re in jack p ine (Pinus banksiana L a m b . ) . Can. J. For. Res. 1 5 : 1 1 5 9 - 1 1 6 6 . S o k a l , R .R. and F . J . Roh l f . 1 9 8 1 . Biometry: The principles and practice of statistics in biological research. 2 n d e d . N e w Y o r k : W . H . F r e e m a n and C o . S o r e n s e n , F . C . 1 9 7 1 . Es t ima te of sel f - fer t i l i ty in c o a s t a l Doug las - f i r f r o m inb reed ing s tud ies . Silvae Genet. 2 0 : 1 1 5 - 1 2 0 . S o r e n s e n , F . C . and R . S . M i l e s . 1 9 7 4 . Se l f -po l l i na t ion e f f ec t s on Doug las - f i r and p o n d e r o s a pine s e e d s and s e e d l i n g s . Silvae Genet. 2 3 : 1 3 5 - 1 3 8 . S q u i l l a c e , A . E . 1 9 7 4 . A v e r a g e gene t i c co r re la t ions a m o n g o f f sp r i ng f r o m open-po l l i na ted fo res t t rees . Silvae Genet. 2 3 : 1 4 9 - 1 5 6 . T ige rs ted t , P . M . A . 1 9 7 3 . S t u d i e s on i s o z y m e var ia t ion in marg ina l a n d cen t ra l popu la t i ons of Picea abies. Hereditas 7 5 : 4 7 - 6 0 . W i l c o x , M . D . 1 9 8 3 . Inbreed ing d e p r e s s i o n and gene t i c va r i ance e s t i m a t e d f r o m self- and c r o s s -po l l ina ted fam i l i es of Pinus radiata. Silvae Genet. 3 2 : 8 9 - 9 6 . W o o d s , J . H . and J . C . H e a m a n . 1 9 8 9 . E f fec t of d i f ferent i nb reed ing leve ls on f i l led s e e d p r o d u c t i o n in Doug las - f i r . Can. J. For. Res. 1 9 : 5 4 - 5 9 . Y e h , F . C , A . B rune , W . M . Che l i ak and D . C C h i p m a n . 1 9 8 3 . M a t i n g s y s t e m s of Eucalyptus citriodora in a s e e d - p r o d u c t i o n a rea . Can. J. For. Res. 1 3 : 1 0 5 1 - 1 0 5 5 . Y e h , F . C , and K. M o r g a n . 1 9 8 7 . M a t i n g s y s t e m a n d mu l t i l o cus a s s o c i a t i o n s in a natura l popu la t i on of Pseudotsuga menziesii (Mirb.) F r a n c o . Theor. Appl. Genet. 7 3 : 7 9 9 - 8 0 8 . Y e h , F . C and S . R a s m u s s e n . 1 9 8 5 . Her i tab i l i ty of he igh t g r o w t h in 10 -yea r -o l d S i t k a s p r u c e . Can. J. Genet. Cytol. 2 7 : 7 2 9 - 7 3 4 . C h a p t e r 5 Paren ta l B a l a n c e in a S i t k a S p r u c e S e e d O r c h a r d 5.1 Introduction A s e e d o r c h a r d is a p lan ta t ion of gene t i ca l l y - supe r io r i nd i v idua ls i n tens i ve l y m a n a g e d fo r s e e d p r o d u c t i o n and d e s i g n e d to p r o m o t e in te r -c lona l m a t i n g wh i l e p reven t i ng e x t r a n e o u s po l len (Zobel et al., 1 9 5 8 ) . T h e qua l i ty of o r c h a r d - p r o d u c e d s e e d s is e x p e c t e d to be h igh and thei r gene t i c d i ve rs i t y ma in ta ined t h r o u g h o u t s u c c e s s i v e g e n e r a t i o n s . T o p r o m o t e o u t c r o s s i n g , c o n d i t i o n s fo r p a n m i x i a are requ i red as f o l l o w s : 1 ) equa l i t y of ma le and f ema le g a m e t e s a m o n g the o r c h a r d c l o n e s , 2) rep roduc t i ve p h e n o l o g y s y n c h r o n y , 3) r a n d o m ma t ing (i.e., a n y po l len of a c l o n e h a s the s a m e probab i l i t y of reach ing an ovu le of any c lone ) , 4) no incompat ib i l i t y a n d se l ec t i on b e t w e e n fer t i l i za t ion and ge rm ina t i on of s e e d s , a n d 5) m in ima l or no po l len c o n t a m i n a t i o n (E r i ksson et al., 1 9 7 3 ) . H o w e v e r , it is c o m m o n l y o b s e r v e d tha t s e e d o r c h a r d s o f ten dev ia te f r o m the " i d e a l " e x p e c t a t i o n s . D i f f e rences in r ep roduc t i ve ou tpu t (Pinus radiata. Gr i f f i n , 1 9 8 2 ; Pinus taeda, S c h m i d t l i n g , 1 9 8 3 ; Pseudotsuga menziesii, E l - K a s s a b y e f a / . , 1 9 8 9 ; R o b e r d s et al., 1 9 9 1 ; Pinus sylvestris, B o e s et al., 1 9 9 1 ) have been repo r ted . T h u s , the o r c h a r d ' s e f fec t i ve popu la t i on n u m b e r is e x p e c t e d to be l o w e r t han the ac tua l popu la t i on n u m b e r . T h e c o n c e p t of e f fec t i ve popu la t i on n u m b e r c a n be u s e d t o d e m o n s t r a t e the dev ia t i on of ma t i ng b e h a v i o u r f r o m an idea l ized b reed ing popu la t i on (Fa lconer , 1 9 8 6 ) . It a l so has been repor ted that the e f fec t i ve n u m b e r of a natura l popu la t i on is a l w a y s less t han the n u m b e r of adu l t s of r e p r o d u c i n g age for one or m a n y r e a s o n s : 1 ) unequa l n u m b e r s of m a l e s and f e m a l e s , 2) t empo ra l var ia t ion in popu la t i on number , and 3) greater t han b inomia l or P o i s s o n var iabi l i ty in the n u m b e r of p r o g e n y per p lant ( C r o w and D e n n i s t o n , 1 9 8 8 ) . F a c t o r s i n f l uenc ing c o n e p r o d u c t i o n in s e e d o r c h a r d s i nc lude gene t i c c o m p o n e n t s , c l o n e s i ze (number of ramet per c l one ) , t ree s i z e , pe r iod ic i t y , d i f f e rences b e t w e e n rep roduc t i ve ene rgy (i.e., n u m b e r of s e e d cones ) and rep roduc t i ve s u c c e s s ( i .e., n u m b e r of f i l led seeds) (Gr i f f in , 1 9 8 2 , 1 9 8 4 ; O 'Re i l l y e f a / . , 1 9 8 2 ; S c h m i d t i n g , 1 9 8 3 ; E l - K a s s a b y ef a / . , 1 9 8 4 , 1 9 8 9 ; B y r a m et al., 1 9 8 6 ; R e y n o l d s and E l - K a s s a b y , 1 9 9 0 ; B o e s et al., 1 9 9 1 ) . T h e c u m u l a t i v e cone -y i e l d cu r ve is c o m m o n l y u s e d to a s s e s s parenta l ba l ance in s e e d o r c h a r d s (Gr i f f in , 1 9 8 2 ) . R e y n o l d s and E l - K a s s a b y ( 1 9 9 0 ) u s e d c u m u l a t i v e s e e d - c r o p da ta to a s s e s s parenta l ba lance in a Doug las - f i r s e e d o r c h a r d , and f o u n d the c u m u l a t i v e seed -y ie l d c u r v e is a bet ter pa ramete r t han c o n e - y i e l d to a s s e s s paren ta l ba l ance in t e rm of gene t i c d i ve rs i t y and fam i l y rep resen ta t i on . In th is s t u d y , the parenta l ba lance and f ema le e f fec t i ve popu la t i on n u m b e r b a s e d o n c o n e , s e e d , and v iab le s e e d s of t w o - s e p a r a t e s e e d - p r o d u c t i o n y e a r s in a S i t k a - s p r u c e s e e d o r c h a r d w e r e a s s e s s e d . 5 . 2 Materials and Methods T h e s t u d y w a s c o n d u c t e d in C a n a d i a n Pac i f i c Fo res t P r o d u c t s L t d . S i t k a s p r u c e s e e d o r c h a r d . T h e s e e d o r cha rd is l oca ted in S a a n i c h t o n , Br i t i sh C o l u m b i a ( lat i tude 4 8 ° 3 5 ' N , l ong i tude 1 2 3 ° 2 4 ' W ) and c o n s i s t s of 1 3 9 c l o n e s (averag ing 9 . 3 r ame ts per c lone) s e l e c t e d f r o m e leva t i ons b e t w e e n 0 and 4 1 5 m o n w e s t e r n V a n c o u v e r Is land, W a s h i n g t o n a n d O r e g o n (Figure 1.1). T h e o rcha rd w a s es tab l i shed in 1971 in a r a n d o m s ing le - t ree m i x o v e r th ree u n e q u a l b l o c k s . T rees are s p a c e d 3 m apar t and kept at a p p r o x i m a t e l y 4 m in he igh t by top -p r u n i n g . T h e s e e d o r cha rd is 1 0 k m a w a y f r o m the neares t S i t k a s p r u c e s t a n d , s o the S i t ka s p r u c e b a c k g r o u n d po l len is neg l ig ib le in the o r c h a r d ' s area ( E l - K a s s a b y and R e y n o l d s , 1 9 9 0 ) . Dur ing 1 9 8 8 and 1 9 9 0 h a r v e s t s , c o n e p r o d u c t i o n of e v e r y ramet w a s r e c o r d e d . In th is s t u d y , s e e d s w e r e co l l ec ted f r o m 9 6 t rees of 2 2 c l o n e s ( 1 9 8 8 crop) and 1 4 2 t rees of 18 c l o n e s ( 1 9 9 0 c rop) . W h e r e p o s s i b l e , a s a m p l e of f i ve c o n e s w a s r a n d o m l y t aken f r o m e a c h t ree for s e e d ex t r ac t i on . C o n e s a m p l e s w e r e air d r i ed , and s e e d s w e r e e x t r a c t e d , d e w i n g e d and c l e a n e d by h a n d . N u m b e r s of f i l led and e m p t y s e e d ( ident i f ied by X - ray ) w e r e r e c o r d e d . T o t a l s e e d y ie lds w e r e a s s e s s e d on the 1 9 9 0 s e e d c r o p ( i .e., c l ona l s e e d c r o p s ) . T h e w e i g h t of e a c h ind iv idua l s e e d , c o n s i s t i n g of 1 0 0 s e e d s per c l o n e , w a s d e t e r m i n e d , t h e n the to ta l s e e d c r o p w a s ca l cu la ted by d iv id ing bu lk s e e d w e i g h t of e a c h c l o n e by the c o r r e s p o n d i n g a v e r a g e ind iv idua l s e e d w e i g h t . In add i t i on , ge rm ina t i on tes t s w e r e c o n d u c t e d to p rov ide v iab le s e e d p r o d u c t i o n da ta (see C h a p t e r 7 fo r ge rm ina t i on tes t desc r i p t i on ) . O n e - w a y A N O V A w a s used to ana l yze the s e e d and c o n e c o u n t s of 1 9 8 8 a n d 1 9 9 0 . Due to a c l o s e re la t ionsh ip a m o n g f i l led s e e d s (r = 1 .00 , P < 0 . 0 1 ) , to ta l s e e d s (r = 0 . 8 1 , P < 0 .01 ), and v iab le s e e d da ta (es t imated by mu l t ip l y ing the c lona l ge rm ina t i on c a p a c i t y by the n u m b e r of f i l led s e e d s ) , f i l l ed -seed da ta on ly w e r e used in the a n a l y s i s . T h e re la t ionsh ip b e t w e e n c lona l c o n e and s e e d c r o p s w a s a s s e s s e d by P e a r s o n ' s p r o d u c t - m o m e n t co r re la t ion (Soka l and Roh l f , 1 9 8 1 ) . B r o a d - s e n s e her i tabi l i ty w a s es t ima ted a c c o r d i n g to F a l c o n e r ( 1 9 8 6 , p . 1 1 3 ) . Da ta on c o n e n u m b e r s , to ta l s e e d s , f i l led s e e d s , and v iab le s e e d p r o d u c t i o n p rov ided the e s t i m a t e s of f ema le e f fec t i ve popu la t i on n u m b e r (N^,) ( C r o w a n d K i m u r a , 1 9 7 0 , p. 3 2 4 ) : "-^^ w h e r e N,,= f ema le e f fec t i ve popu la t i on n u m b e r , Xj = p ropor t i on of ith c l o n e con t r i bu t i on to the o r c h a r d c r o p 5 . 3 Results and Discussion 5.3.1 1988 Orchard Crop T h e 2 2 r a n d o m l y s e l e c t e d c l o n e s fo r th is s t u d y w e r e a rep resen ta t i ve s a m p l e of the p r o d u c t i o n range in the s e e d o r c h a r d (Figure 5.1), i nd ica t ing tha t th i s s u b s e t of c l o n e s wi l l p rov ide an u n b i a s e d es t ima te for the w h o l e o r c h a r d . T h e re la t ionsh ip b e t w e e n c o n e and f i l led-s e e d p r o d u c t i o n is e x p e c t e d to be s ign i f i can t and pos i t i ve . T h e p r o d u c t - m o m e n t co r re la t ion b e t w e e n c o n e and s e e d c r o p s w a s pos i t i ve and s ign i f i can t (r = 0 . 8 6 , n = 2 2 , P < 0 . 0 1 ) . T h e coe f f i c i en t of de te rm ina t i on w a s h igh (r^ = 0 . 7 5 ) , ind ica t ing tha t th is re la t ionsh ip a c c o u n t s for a large p ropor t i on of var ia t ion in the d a t a . H o w e v e r , the ave rage n u m b e r o f c o n e s a n d s e e d s per ramet p r o d u c e d by the 2 2 c l o n e s var ied b e t w e e n 12 and 4 8 8 c o n e s , a n d b e t w e e n 1 , 7 4 9 and 4 6 , 2 0 0 s e e d s per c l one for c l o n e n u m b e r s 1 8 4 and 3 6 , r e s p e c t i v e l y (Table 5.1). In fac t . 1 n n 90 ..^^ 80 6 3 / ^ 70 ^ 3 ^ 2 0 ^ ^ 6 0 O cc o ^ 50 o o 40 / y / y f / • • • 30 36,187 A A B O r • 20 / ^ / • / y 1 / I / C O N E CROP 10 / f J / 1 • / • 36 SAMPLED CLONES 0 1 ... 1 1 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100 % CLONE F/yure 5. / . 1988 cone crop parental balance curve based on ail orchard clones (N = 139), indicating contributions of the sampled clones. Table 5.1. C o n e and f i l led s e e d p r o d u c t i o n in the 1 9 8 8 c r o p , a n d the i r e f fec t i ve popu la t i on n u m b e r (N^,) C r o p (No.) N o . C l o n e N o . C o n e s Fi l led S e e d s 1 5 1 5 6 1 6 , 6 8 8 2 1 3 2 5 3 , 2 0 8 3 15 1 6 2 1 0 , 5 0 1 4 2 0 1 7 5 1 0 , 1 6 7 5 2 7 3 5 4 , 2 8 6 6 3 6 4 8 8 4 6 , 2 0 0 7 4 0 3 9 3 , 2 9 2 8 4 6 3 4 3 , 1 0 0 9 4 9 4 4 9 2 5 , 3 0 4 1 0 5 0 2 0 3 3 5 , 3 8 4 11 5 3 5 5 6 , 8 7 2 12 6 4 19 3 , 1 2 6 1 3 71 1 4 2 , 5 6 2 14 9 2 1 4 5 1 2 , 0 5 2 15 9 8 151 2 1 , 8 0 6 16 1 2 4 7 8 8 , 8 4 3 17 1 4 3 3 2 3 , 1 0 2 1 8 1 5 3 3 4 5 , 1 8 3 19 1 8 4 12 1 , 7 4 9 2 0 1 8 7 4 2 8 2 2 , 7 6 4 21 1 9 6 3 0 3 , 7 4 9 2 2 4 1 3 9 3 1 0 , 3 6 1 N . , 1 0 11 Ne,/N 0 . 4 5 0 . 5 0 3 7 and 3 8 pe rcen t of the var ia t ion in the c o n e c r o p and the s e e d c r o p , r e s p e c t i v e l y , w e r e due to c l ona l d i f f e rence (Table 5.2). In o ther w o r d s , b r o a d - s e n s e her i tabi l i ty fo r c o n e and s e e d p r o d u c t i o n w a s 0 . 3 7 and 0 . 3 8 , r espec t i ve l y . T h e rema in ing var ia t ion in c o n e and s e e d p r o d u c t i o n is c a u s e d by w i t h i n - c l o n e var ia t ion ( i .e., a m o n g rame ts w i t h i n a c l one ) . T h e c h a n g e in rank b e t w e e n c o n e and s e e d p r o d u c t i o n s p rov i des in fo rmat ion regard ing rep roduc t i ve energy a n d rep roduc t i ve s u c c e s s (Figure 5.2). For e x a m p l e , c l one n u m b e r 2 0 , r anked 5 as a c o n e p r o d u c e r , w a s ranked 10 as a s e e d p r o d u c e r . O n l y s i x c l o n e s ou t of the 2 2 s tud ied (c lone n o s . 3 6 , 1 2 4 , 5 3 , 2 7 , 71 and 184) ma in ta ined the s a m e rank as c o n e and s e e d p r o d u c e r s . H o w e v e r , rank c h a n g e s w e r e genera l l y sma l l w h e n they o c c u r r e d . Paren ta l ba lance e s t i m a t e s b a s e d o n e i ther c o n e or s e e d c r o p s (Figure 5.3), w e r e s imi lar and g a v e ra t ios of " 3 8 / 8 0 " (i.e, 3 8 % of t rees p r o d u c e d 8 0 % of the c rop) fo r c o n e s and " 4 4 / 8 0 " fo r s e e d s . T h i s i nd i ca ted that the p ropo r t i on of e f fec t i ve a n d ac tua l f e m a l e n u m b e r s dev ia ted f r o m an ideal s e e d o r c h a r d in t e r m s of f ema le con t r i bu t i on in the 1 9 8 8 s e e d c r o p , i .e., N , , /N w e r e 0 . 4 5 and 0 . 5 o n c o n e and f i l l ed -seed c r o p s , r espec t i ve l y (Table 5.1). In o ther w o r d s , the ac tua l popu la t i on n u m b e r w a s t w i c e the e f fec t i ve popu la t i on n u m b e r . T h u s , the c o n e c r o p c o u l d be u s e d to p red ic t the parenta l ba l ance of s e e d p r o d u c t i o n in th is o r c h a r d . 5 . 3 . 2 1990 Orchard Crop T h e 1 8 r a n d o m l y s e l e c t e d c l o n e s fo r th is s t u d y a l so w e r e a rep resen ta t i ve s a m p l e of the p r o d u c t i o n range in the s e e d o r cha rd (Figure 5.4), i nd ica t ing tha t th is s u b s e t of c l o n e s wi l l p rov ide an u n b i a s e d es t ima te for the w h o l e o r c h a r d . T h e p r o d u c t - m o m e n t co r re la t i on b e t w e e n c o n e a n d s e e d c r o p s w a s pos i t i ve and s ign i f i can t (r = 0 . 8 1 , n = 1 8 , / ' < 0 .01 ). T h e coe f f i c ien t of de te rm ina t i on w a s h igh (r^ = 0 . 6 6 ) , ind ica t ing tha t the re la t ionsh ip b e t w e e n c o n e and s e e d p r o d u c t i o n a c c o u n t s for the large p ropo r t i on of var ia t ion in the da ta s im i la r to tha t in the 1 9 8 8 s e e d c r o p . T h e n u m b e r of c o n e s p r o d u c e d by the 18 c l o n e s var ied b e t w e e n 3 8 (c lone n o . 5 1 6 ) and 5 9 5 (c lone n o . 4 2 1 ) c o n e s per c l o n e , wh i l e the e s t i m a t e d a v e r a g e n u m b e r of f i l led s e e d per c l o n e ranged b e t w e e n 2 , 8 4 4 (c lone no . 154) and 7 7 , 8 2 8 (c lone n o . 4 2 1 ) (Table 5.3). In fac t . Table 5.2. A N O V A : e s t i m a t e s of va r i ance c o m p o n e n t s of 1 9 8 8 Sit l^a s p r u c e s e e d o rc l i a rd p r o d u c t i o n S o u r c e of Va r i a t i on df E x p e c t e d ' M e a n S q u a r e s V a r i a n c e C o m p o n e n t s (%) C o n e C r o p S e e d C r o p A m o n g C l o n e s 21 al + 4 . 4 5 2 a ^ 3 7 . 0 7 3 " 3 8 . 4 4 5 " W i t h i n C l o n e s 7 6 ol 6 2 . 9 2 7 6 1 . 5 5 5 " S ign i f i can t at P < 0 . 0 1 . ' = va r iance due to d i f f e rences a m o n g rame ts w i t h i n the s a m e c l o n e ; = va r i ance due to d i f f e rences a m o n g c l o n e s . C O N E CLONE YIELD NO. 448 36 -449 49 -428 187 -203 50 -175 162 156 151 145 20 15 5 98 92 93 413 78 124 55 53 39 40 35 27 34 153 33 46 32 143 30 196 25 13 19 64 14 71 12 184 CLONE SEED NO. YIELD - • - 36 46,200 - - 50 35,384 - - - 49 25,304 - - - 1 8 7 22,764 . , - 98 21,806 - - - 5 16,688 - - 92 12,052 ~ - 15 10,501 - - - 4 1 3 10,361 ^ - 20 10,167 124 8,843 - - - 53 6,872 - - 1 5 3 5,183 27 4,286 , - - 1 9 6 3,749 ^ - 40 3,292 - - 13 3,208 ' - 64 3,126 ~ - 1 4 3 3,102 - - 46 3,100 - - - 71 2,562 - 184 1,749 Figure 5.2. Rank order for 22 Sitka-spruce clones, 1988 crop, based on cone and seed yields. Figure 5.3. C u m u l a t i v e c o n e and f i l led s e e d p roduc t ion c u r v e s of 2 2 S i t k a sp ruce c l o n e s , 1 9 8 8 c rop . S t ra igh t l ine represen ts equa l con t r ibu t ion . 0 L I I I I I \ I I I 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 % C L O N E Figure 5.4. 1990 cone crop parental balance curve based on all orchard clones (N = 139), indicating contributions of the sampled clones. Table 5.3. 1 9 9 0 c o n e , s e e d , f i l led s e e d , v iab le s e e d p r o d u c t i o n , and thei r e f fec t i ve popu la t i on n u m b e r (N,,) N o . C l o n e C r o p (Number) N o . C o n e s To ta l Fi l led V iab le S e e d s S e e d s S e e d s 1 5 2 5 4 1 8 3 , 4 3 3 2 1 , 8 5 3 2 1 , 2 0 2 2 15 1 7 2 7 0 , 0 6 9 1 0 , 3 0 6 9 , 9 4 5 3 2 0 2 0 6 1 1 5 , 0 3 8 2 0 , 2 4 4 1 9 , 5 8 3 4 2 4 4 4 2 2 8 4 , 2 9 2 1 3 , 5 3 5 1 2 , 8 1 7 5 4 4 2 2 0 1 2 5 , 0 7 9 1 6 , 0 6 0 1 4 , 4 5 8 6 61 2 1 9 1 2 2 , 9 6 6 2 7 , 1 3 0 2 5 , 9 7 1 7 6 8 1 7 6 7 6 , 8 6 9 2 1 , 1 4 6 1 9 , 3 3 5 8 9 2 1 6 3 4 8 , 6 5 4 1 1 , 1 9 5 1 0 , 8 5 7 9 1 0 5 1 9 5 8 0 , 0 9 5 2 2 , 9 9 8 2 1 , 6 1 8 1 0 1 1 3 2 4 3 1 6 5 , 1 1 6 3 6 , 0 3 6 3 4 , 8 6 5 11 1 5 4 4 9 1 8 , 3 3 5 2 , 8 4 4 2 , 6 2 7 12 4 0 5 2 8 2 1 9 2 , 2 2 1 4 4 , 2 1 6 4 3 , 5 4 9 1 3 4 1 1 1 1 2 4 3 , 1 0 3 1 0 , 1 3 8 9 , 4 7 9 1 4 4 1 6 5 5 3 8 , 2 1 9 7 , 9 6 9 7 , 8 2 6 1 5 4 2 1 5 9 5 3 7 3 , 7 0 9 7 7 , 8 2 8 7 5 , 6 9 3 1 6 5 1 4 5 3 1 8 , 6 1 5 4 , 3 2 8 4 , 2 2 2 17 5 1 6 3 8 1 7 , 9 6 3 4 , 9 9 4 4 , 6 4 5 1 8 521 2 1 1 1 1 6 , 5 5 4 2 8 , 5 7 6 2 6 , 8 4 0 N . , 1 2 . 5 4 1 0 . 9 3 1 0 . 6 8 1 0 . 5 2 Ne,/N 0 . 7 0 0 .61 0 . 5 9 0 . 5 8 3 3 . 5 and 4 1 . 0 pe rcen t of the var ia t ion in c o n e c rop and s e e d c r o p , r espec t i ve l y , w a s due to c l ona l d i f f e rences (Table 5.4). T h u s , the es t ima ted b r o a d - s e n s e her i tab i l i ty of c o n e p r o d u c t i o n w a s 0 . 3 4 , wh i l e the b r o a d - s e n s e her i tabi l i ty of s e e d p r o d u c t i o n w a s 0 . 4 1 . It is n o t e w o r t h y to m e n t i o n that the l o w e s t c o n e - and s e e d - p r o d u c e r s (c lone n o s . 5 1 4 and 1 5 4 , respec t i ve ly ) are not the s a m e c l o n e (Table 5.3); the fo rmer is mo re e f f i c ien t in p r o d u c i n g f i l led s e e d than the lat ter. R a n k c h a n g e s w i t h i n and b e t w e e n c o n e and s e e d p r o d u c e r c l a s s e s w e r e a l so o b s e r v e d in the 1 9 9 0 c r o p (Figure 5.5). T h e c l o n e s ranked n u m b e r 2 (c lone no . 24 ) a n d 6 (c lone no . 44) as c o n e p r o d u c e r s ranked 11 and 10 as s e e d p r o d u c e r s , r espec t i ve l y . H o w e v e r , m o s t of the p o o r c o n e p r o d u c e r s rema ined poo r s e e d p r o d u c e r s . Parenta l ba lance es t ima tes b a s e d on e i ther c o n e c r o p or v iab le s e e d s w e r e s imi lar (Figure 5.6) and g a v e the rat ios of " 5 8 / 8 0 " and " 5 6 / 8 0 " fo r c o n e and v iab le s e e d s , r espec t i ve l y . Es t ima tes b a s e d o n f i l led and to ta l s e e d c r o p s a l so g a v e s im i la r ra t ios of " 5 1 / 8 0 " and " 4 9 / 8 0 , " r espec t i ve l y . T h e p ropo r t i ons of f ema le e f fec t i ve and ac tua l n u m b e r s w e r e 0 . 7 0 , 0 . 6 1 , 0 . 5 9 and 0 . 5 8 w h e n the es t ima t i ons w e r e b a s e d o n c o n e , to ta l s e e d s , f i l led s e e d s , and v iab le seed p r o d u c t i o n (Table 5.3), r espec t i ve l y . T h e parenta l ba lance reco rds for th is s e e d o r cha rd in 1 9 8 5 , 1 9 8 6 and 1 9 8 7 w e r e " 2 0 / 8 0 , " " 3 5 / 8 0 " and " 4 2 / 8 0 " ( E l - K a s s a b y and R e y n o l d s , 1 9 9 0 ) , r espec t i ve l y . In th is s t u d y , the parenta l b a l a n c e s fo r 1 9 8 8 and 1 9 9 0 w e r e " 4 2 / 8 0 " and " 4 8 / 8 0 " (Figure 5.1 and Figure 5.4), r espec t i ve l y . T h i s ind ica tes an i m p r o v e m e n t in parenta l ba l ance w i t h age . A s imi lar i m p r o v e m e n t has a l so been o b s e r v e d in lob lo l ly p ine (By ram et al., 1 9 8 6 ) , a n d in Doug las - f i r ( E l - K a s s a b y et al., 1 9 8 9 ) . B r o a d - s e n s e her i tabi l i ty for r ep roduc t i on t ra i ts in th is s t u d y ranges f r o m 0 . 3 4 to 0 .41 ind ica t ing modera te gene t i c con t ro l s imi la r to tha t f o u n d in lob lo l l y p ine (By ram et al., 1 9 8 6 ) . In c o n c l u s i o n , ma te rna l con t r i bu t i on to s e e d p r o d u c t i o n in th is s e e d o r c h a r d w a s i m b a l a n c e d . H o w e v e r , an i m p r o v e m e n t in parenta l ba lance w i t h age w a s o b s e r v e d . Fema le Table 5.4. A N O V A : e s t i m a t e s of va r i ance c o m p o n e n t s of 1 9 9 0 S i t k a - s p r u c e s e e d o rc l i a rd p r o d u c t i o n S o u r c e of Va r i a t i on df Expec ted^ M e a n S q u a r e s V a r i a n c e C o m p o n e n t s (%) C o n e C r o p S e e d C r o p A m o n g C l o n e s 17 al + 7 . 8 6 9 a ^ 3 3 . 5 4 8 " 4 1 . 0 1 4 " W i t h i n C l o n e s 1 2 4 ol 6 6 . 4 5 2 5 8 . 9 8 6 " S ign i f i can t at P < 0 . 0 1 . ^ al = va r i ance due to d i f f e rences a m o n g rame ts w i t h i n the s a m e c l o n e ; al = va r i ance due to d i f f e rences a m o n g c l o n e s . C O N E C L O N E Y I E L D N O . 5 9 5 4 2 1 T 4 4 2 2 4 2 8 2 4 0 5 2 5 4 5 2 4 3 1 1 3 2 2 0 4 4 2 1 9 61 2 1 1 5 2 1 -2 0 6 2 0 -1 9 5 1 0 5 -1 7 6 6 8 -1 7 2 15 -1 6 3 9 2 -1 1 2 4 1 1 -5 5 4 1 6 -5 3 5 1 4 -4 9 1 5 4 -3 8 5 1 6 C L O N E S E E D N O . Y I E L D - 4 2 1 7 7 , 8 2 8 \ _ ^ -- 4 0 5 4 4 , 2 1 6 \ _ — - 1 1 3 3 6 , 0 3 6 \ ^ " " - 521 2 8 , 5 7 6 ^ ""^ " " ^ \ ^ \ ^ " " - 61 2 7 , 1 3 0 - 1 0 5 2 2 , 9 9 8 - 5 2 1 , 8 5 3 " ^ ^ ' N - ' ^ ^ ' - ' " - 6 8 2 1 , 1 4 6 - 2 0 2 0 , 2 4 4 - 4 4 1 6 , 0 6 0 - 2 4 1 3 , 5 3 5 - 9 2 1 1 , 1 9 5 - 1 5 1 0 , 3 0 6 - 4 1 1 1 0 , 1 3 8 - 4 1 6 7 , 9 6 9 - - - - - 5 1 6 4 , 9 9 4 - 5 1 4 4 , 3 2 8 ^ 1 5 4 2 , 8 4 4 Figure 5.5. Rank order for 18 S i t ka -sp ruce c l o n e s , 1 9 9 0 c r o p , b a s e d on c o n e and s e e d y ie lds . % C L O N E Figure 5.6. C u m u l a t i v e c o n e , f i l led s e e d s , to ta l s e e d and potent ia l seed l i ng p roduc t i on c u r v e s for 1 8 S i t k a sp ruce c l o n e s , 1 9 9 0 c r o p . S t ra igh t l ine represents equa l con t r ibu t ion . e f fec t i ve popu la t i on n u m b e r p rov ided tf ie es t ima te of dev ia t i on f r o m the ideal equa l c o n t r i b u t i o n ; h o w e v e r , th is pa ramete r d id not p rov ide ins igh t in to the re la t ionsh ip b e t w e e n rep roduc t i ve ene rgy and rep roduc t i ve s u c c e s s . T h i s s t u d y and E l - K a s s a b y and R e y n o l d s ( 1 9 9 0 ) have i nd i ca ted tha t c lona l con t r i bu t i on t o s e e d c r o p s in the s tud ied S i t ka s p r u c e s e e d o r cha rd va r ies f r o m year to yea r ; h o w e v e r , f e c u n d i t y is a gene t i ca l l y con t ro l l ed trai t in con i f e r s ( J o n s s o n et al., 1 9 7 6 ; E l - K a s s a b y , 1 9 8 9 ) . T h e r e f o r e , ce r ta in c l o n e s m a y c o n s i s t e n t l y p r o d u c e large or s m a l l s e e d c r o p s . S u p p l e m e n t a l -mass -po l l i na t i on ( S M P ) has p r o v e n to be a v i ta l t oo l fo r i m p r o v i n g rep roduc t i ve s u c c e s s for S i t k a s p r u c e due to the rep roduc t i ve p h e n o l o g y d i s p l a c e m e n t b e t w e e n ma le and f ema le s t rob i l i ( E l - K a s s a b y and R e y n o l d s , 1 9 9 0 ) . S M P c o u l d i m p r o v e parenta l ba l ance as we l l as rep roduc t i ve s u c c e s s . T o i m p r o v e parenta l con t r i bu t i on , the f o l l o w i n g s t ra teg ies c o u l d be app l i ed : a) the use of S M P po l len m i x e s f r o m l o w s e e d - c o n e p r o d u c e r s , t hus thei r g a m e t e s are ove r e m p h a s i z e d in the s e e d c r o p t h r o u g h their male part , and b) ha rves t i ng the c r o p into s u b s e t s tha t c o n s i s t s of h i g h , i n te rmed ia te , and poo r p r o d u c e r s , t h u s the parenta l ba l ance w i t h i n e a c h s u b s e t w i l l be be t te r t han the ent i re o r cha rd c r o p . 5 . 4 References B o e s , T . K . , J . R . B rand ie , and W . R . Love t t . 1 9 9 1 . Cha rac te r i za t i on of f l o w e r i n g p h e n o l o g y and s e e d y ie ld in a P inus sy l ves t r i s c lona l s e e d o r cha rd in N e b r a s k a . Can. For. Res. 2 1 : 1 7 2 1 - 1 7 2 9 . B y r a m , T . D . , W . J . L o w e , and J . A . M c G r i f f . 1 9 8 6 . C l o n a l a n d annua l va r ia t ion in c o n e p r o d u c t i o n in lob lo l ly p ine s e e d o r c h a r d s . For. Sci. 3 2 : 1 0 6 7 - 1 0 7 3 . C r o w , J . F . and C . D e n n i s t o n . 1 9 8 8 . Inbreeding and va r i ance e f fec t i ve popu la t i on n u m b e r s . Evolution 4 2 ( 3 1 : 4 8 2 - 4 9 5 . C r o w , J . F . and M . K i m u r a . 1 9 7 0 . An introduction to population genetic theory. N e w Y o r k : Happe r and R o w Pub l i she rs , 591 p. E l - K a s s a b y , Y . A . 1 9 8 9 . G e n e t i c s of Doug las - f i r s e e d o r c h a r d s : e x p e c t a t i o n s a n d real i t ies . In Proc. 20th Southern Forest Tree Improvement Conference, pp . 8 7 - 1 0 9 . C h a r l e s t o n , S o u t h C a r o l i n a . E l - K a s s a b y , Y . A . , A . M . K . Fash le r , and M . C r o w n . 1 9 8 9 . Va r i a t i on in f ru i t f u lness in a Doug las - f i r s e e d o r cha rd and its e f fec t o n c r o p m a n a g e m e n t d e c i s i o n s . Silvae Genet. 3 8 : 1 1 3 - 1 2 1 . E l - K a s s a b y , Y . A . , and S . R e y n o l d s . 1 9 9 0 . R e p r o d u c t i v e p h e n o l o g y , parenta l b a l a n c e , and s u p p l e m e n t a l m a s s po l l ina t ion in a S i t k a - s p r u c e s e e d - o r c h a r d . For. Ecol. Manage. 3 1 : 4 5 - 5 4 , E r i k s s o n , G . , A . J o n s s o n , and D. L i n d g r e n . 1 9 7 3 . F l o w e r i n g in a c l o n e tr ial of Picea abies Kars t . Stud. For. Suec. 1 1 0 : 1 - 4 5 . F a l c o n e r , D . S . 1 9 8 6 . Introduction to quantitative genetics. 2 n d e d . N e w Y o r k : L o n g m a n Sc ien t i f i c & T e c h n i c a l . Gr i f f i n , A . R . 1 9 8 2 . C l o n a l var ia t ion in rad iata p ine s e e d o r c h a r d s : l . S o m e f l o w e r i n g , c o n e and s e e d p roduc t i on t ra i ts . Aust. J. For. Res. 1 2 : 2 9 5 - 3 0 2 . Gr i f f i n , A . R . 1 9 8 4 . C lona l var ia t ion in rad iata p ine s e e d o r c h a r d s . II. F l o w e r i n g p h e n o l o g y . Aust. J. For. Res. 1 4 : 2 7 1 - 2 8 1 . J o n s s o n , A . , I. E k b e r g , and G . E r i k s s o n . 1 9 7 6 . F l o w e r i n g in a s e e d o r c h a r d of Pinus sylvestris L. Stud. For. Suec. 1 3 5 : 1 - 3 8 . O ' R e i l l y , C , W . H . Parker , and J . E . Barker . 1 9 8 2 . E f fec t of po l l i na t ion per iod and st robi l i n u m b e r o n r a n d o m ma t i ng in a c lona l s e e d o r cha rd of Picea mariana. Silvae Genet. 3 1 : 9 0 - 9 4 . R e y n o l d s , S , and Y . A . E l - K a s s a b y . 1 9 9 0 . Paren ta l ba lance in Doug las - f i r s e e d o r c h a r d - cone c rop v s . s e e d c r o p . Silvae Genet. 3 9 : 4 0 - 4 2 . R o b e r d s , J . H . , S . T . F r i edman and Y . A . E l - K a s s a b y . 1 9 9 1 . E f fec t i ve n u m b e r of po l len paren ts in c lona l s e e d o r c h a r d s . Theor. Appl. Genet. 8 2 : 3 1 3 - 3 2 0 . S c h m i d t l i n g , R . C . 1 9 8 3 . G e n e t i c var ia t ion in f ru i t f u lness in a lob lo l ly p ine s e e d o r c h a r d . Silvae Genet. 3 2 : 7 6 - 8 0 . S c h o e n , D . J . , D. Den t i , and S . C . S t e w a r t . 1 9 8 6 . S t rob i l us p r o d u c t i o n in a c lona l w h i t e s p r u c e s e e d o r c h a r d : e v i d e n c e fo r u n b a l a n c e d m a t i n g . Silvae Genet. 3 5 : 2 0 1 - 2 0 5 . S o k a l , R .R . and F . J . Roh l f . 1 9 8 1 . Biometry: The principles and practice of statistics in biological research. 2 n d e d . N e w Y o r k : W . H . F r e e m a n and C o . Z o b e l , B . J . , J . Barber , C . L . B r o w n , and T . O . Per ry . 1 9 5 8 . S e e d o r c h a r d s - The i r c o n c e p t and m a n a g e m e n t . J. For. 5 6 : 8 1 5 - 8 2 5 . C h a p t e r 6 E f f ec t s of A c c e l e r a t e d A g i n g T r e a t m e n t s on S e e d s of S i x S i t k a S p r u c e O r c h a r d C l o n e s 6.1 Introduction G e r m i n a t i o n t e s t s are usua l l y c o n d u c t e d as part of seed -qua l i t y t es t i ng . W h e r e a s the s t a n d a r d ge rm ina t i on tes t is b a s e d on an es t ima te of the m a x i m a l po ten t ia l fo r s e e d v iab i l i ty , or the abi l i ty of a s e e d to p r o d u c e a no rma l p lant unde r f avo rab le c o n d i t i o n s , it is no t adequa te fo r a s s e s s i n g f ie ld e m e r g e n c e ( M c D o n a l d , 1 9 8 0 ) . A v i gou r tes t b a s e d o n s t r e s s c o n d i t i o n s is mo re appropr ia te fo r t es t i ng s e e d e m e r g e n c e s i n c e it imp l ies the abi l i ty of the s e e d to ge rm ina te under bo th f avou rab le and un favou rab le c o n d i t i o n s ( K n e e b o n e , 1 9 7 6 ) . P o s s i b l e c a u s e s of va r ia t ion in the level of s e e d v igour i nc lude (1 ) gene t i c c o n s t i t u t i o n , (2) e n v i r o n m e n t and nut r i t ion of the mo the r p lant , (3) s tage of matu r i t y at ha rves t , (4) s e e d s i z e , w e i g h t or s p e c i f i c g rav i ty , (5) m e c h a n i c a l in tegr i ty , (6) de ter io ra t ion and a g i n g , and (7) p a t h o g e n s ( A s s o c i a t i o n of O f f i c ia l S e e d A n a l y s t s , 1 9 8 3 ) . V iab i l i t y in s e e d s has been f o u n d to be h ighes t at the t ime of p h y s i o l o g i c a l matur i t y , a n d to dec l ine w i t h age . D e l o u c h e and Bask in ( 1 9 7 3 ) have d e s c r i b e d s e e d de te r io ra t ion as e n c o m p a s s i n g ini t ial m e m b r a n e deg rada t ion and end ing w i t h l o s s of ge rm inab i l i t y . T h e s y m p t o m s of th is de te r io ra t ion m a y inc lude a d e c r e a s e in me tabo l i c ac t i v i t y , suscep t i b i l i t y to s t r e s s , r e d u c e d rate of ge rm ina t i on and seed l i ng g r o w t h , d e c r e a s e d s to rab i l i t y , in fer ior p lant d e v e l o p m e n t and y i e l d , r e d u c e d e m e r g e n c e po ten t ia l , and i n c r e a s e s in seed l i ng abno rma l i t i es . T h e p r o c e s s e s of s e e d de ter io ra t ion in a popu la t i on are i n d e p e n d e n t a m o n g the ind iv idua l s e e d s , and the t ime c o u r s e for de ter io ra t ion ranges f r o m d a y s to y e a r s . T h u s , the ge rm ina t i on pe rcen tage of a seed lo t d e c r e a s e s w i t h t ime in p ropo r t i on to t he n u m b e r of i nd iv idua l s e e d s tha t are no longer ge rm inab le (De louche and B a s k i n , 1 9 7 3 ) . D i f f e rences in the deg ree of s e e d de ter io ra t ion c a n be revea led t h rough a v igour tes t . T h i s i nd i ca tes w h e t h e r the d i f f e rences s t e m f r o m s e e d p r o c e s s i n g or are g e n o t y p e - s p e c i f i c ( A s s o c i a t i o n of O f f i c ia l S e e d A n a l y s t s 1 9 8 3 ) . S e e d v igou r l i as been dennonst ra ted to be her i tab le ( D i c k s o n , 1 9 8 0 ; K n e e b o n e , 1 9 7 6 , M c D a n i e l , 1 9 7 3 ) , and va r ies a c c o r d i n g to f ie ld w e a t h e r i n g (Pasca l and El l is , 1 9 7 8 ; Po t t s et al., 1 9 7 8 ; N d i m a n d e etal., 1 9 8 1 ) and s to rage c o n d i t i o n s (Wein and K u e n e m a n , 1 9 8 1 ; M i n o r and P a s c a l , 1 9 8 2 ) . In s o m e p lan ts , th is trai t is inher i ted mate rna l l y ( K u e n e m a n , 1 9 8 3 ) . W h e n s e e d s a g e , no t on ly d o e s thei r phys i o l og i ca l ac t iv i t y c h a n g e , but a l so thei r c h r o m o s o m a l s t ruc tu re c h a n g e s (Rober t s , 1 9 7 2 ; P i te l , 1 9 8 0 ) . A c c e l e r a t e d a g i n g , a m e t h o d i nc luded in s t r e s s t e s t s , has been e f fec t i ve l y u s e d to es t ima te s e e d v igou r and s torab i l i t y in bo th annua l and perenn ia l p lan ts ( D e l o u c h e and B a s k i n , 1 9 7 3 ; P i te l , 1 9 8 0 ; B l anche et al., 1 9 8 8 , 1 9 9 0 ) . W h i l e re la t ive ly l itt le is k n o w n abou t a c c e l e r a t e d ag ing in t ree s e e d s , th is s t u d y e m p l o y e d the s t anda rd p r o c e d u r e d e v e l o p e d for agr icu l tu ra l s e e d s to de te rm ine if acce le ra ted ag ing c o u l d be u s e d to es t ima te the v igour of o r c h a r d - p r o d u c e d S i t k a s p r u c e s e e d s . 6 . 2 Materials and Methods C a n a d i a n Pac i f i c Fo res t P r o d u c t s L td . p r o v i d e d the s e e d s fo r th is s t u d y f r o m the S i t ka s p r u c e s e e d o r cha rd l oca ted in S a a n i c h t o n , Br i t i sh C o l u m b i a ( lat i tude 4 8 ° 3 5 ' N , long i tude 1 2 3 ° 2 4 ' W ) . T h e o r cha rd c o n s i s t s of 1 3 9 c l o n e s (averag ing 9 . 3 rame ts per c l ones ) s e l e c t e d f r o m e leva t i ons b e t w e e n 0 m and 4 1 5 m o n w e s t e r n V a n c o u v e r Is land, W a s h i n g t o n and O r e g o n (Figure 1.1). T h e o r c h a r d w a s e s t a b l i s h e d in 1971 in a r a n d o m s ing le - t ree m i x ove r th ree unequa l b l o c k s . In S e p t e m b e r 1 9 9 0 , w ind -po l l i na ted s e e d s f r o m 6 c l o n e s w e r e c o l l e c t e d fo r the s t u d y . T h e c l ona l ident i ty of the s e e d s w a s ma in ta ined du r ing s e e d e x t r a c t i o n , a f ter w h i c h the s e e d s w e r e kept at 2 ° C unt i l u s e d . T h e y w e r e t hen s u b j e c t e d to a c c e l e r a t e d ag ing a c c o r d i n g to the A O S A ' s seed -v i gou r - t es t i ng p rocedu re ( A s s o c i a t i o n of O f f i c ia l S e e d A n a l y s t s , 1 9 8 3 ) . In th is s t u d y , it is a s s u m e d that (1) the p r o c e s s e s of s e e d de te r io ra t ion under a c c e l e r a t e d ag ing c o n d i t i o n s are s imi lar to t h o s e under natura l c o n d i t i o n s - on l y the rate of de te r io ra t ion is i n c r e a s e d (De louche and B a s k i n , 1 9 7 3 ) , and (2) the dec l i ne in ge rm ina t i on af ter a c c e l e r a t e d ag ing is a s s o c i a t e d w i t h the init ial deg ree of de te r io ra t ion of t he s e e d l o t s , i .e. , h i gh -v igou r lots wi l l s h o w litt le dec l i ne in g e r m i n a t i o n , w h e r e a s l o w - v i g o u r lo ts w i l l s h o w a marked dec l i ne in ge rm ina t i on (Bask in , 1 9 7 7 ) . 6 .2 .1 Accelerated Aging A pi lot s t u d y c o n d u c t e d on a s ing le c l o n e under fou r t e m p e r a t u r e s : 3 5 , 3 7 . 5 , 4 0 and 4 5 ° C (data no t s h o w n ) ind ica ted 3 7 . 5 ° C to be the appropr ia te t empe ra tu re fo r acce le ra ted ag ing in S i t ka s p r u c e . T h i s t empera tu re is l o w e r t han tha t p resc r i bed fo r a c c e l e r a t e d ag ing ( A s s o c i a t i o n of Of f i c ia l S e e d A n a l y s t s , 1 9 8 3 ) . B l anche et al. ( 1 9 8 8 ) a lso r e c o m m e n d e d the use of l o w e r t empe ra tu res w h e n acce le ra ted ag ing is app l ied to t ree s e e d s . In th is s t u d y , acce le ra ted ag ing w a s app l ied at 3 7 . 5 ° C for 8 ag ing pe r i ods f r o m 0 to 21 d a y s at 3 - d a y in te rva ls . E a c h tes t c o n s i s t e d of ten s a m p l e s of 1 0 0 s e e d s . E a c h s a m p l e w a s p l a c e d separa te ly o n a w i r e - m e s h s c r e e n in a t i g h t l y - c l o s e d , c o v e r e d i n c u b a t i o n b o x c o n t a i n i n g 5 0 m l . of w a t e r . S a m p l e s w e r e ob ta ined in s u c c e s s i o n s o tha t the s a m p l e s fo r the l onges t a c c e l e r a t e d - a g i n g t rea tmen t (21 days ) w e r e p l aced in the i ncuba to r f i rs t and the s h o r t e s t - a g e d s a m p l e s (3 days ) w e r e the last . A t the end of the tes t all s a m p l e s w e r e r e m o v e d and t w o s a m p l e s f r o m e a c h acce le ra ted ag ing t rea tmen t w e r e u s e d for m o i s t u r e - c o n t e n t de te rm ina t i on ( Internat ional S e e d T e s t i n g A s s o c i a t i o n , 1 9 8 5 ) . T h e rema in ing s a m p l e s (8) w e r e kept at r o o m tempe ra tu re ove rn igh t to reduce the e f fec t of rap id t empera tu re c h a n g e o n s e e d g e r m i n a t i o n , as r e c o m m e n d e d by Bou r l and and Ibrahim ( 1 9 8 2 ) . A l l the s e e d s a m p l e s (mo ldy and n o n - m o l d y seeds ) w e r e c l e a n e d under runn ing , t ep id , w a t e r for 2 m inu tes pr ior to the g e r m i n a t i o n tes t . 6 . 2 . 2 Germination Test A l l ge rm ina t i on t e s t s w e r e c o n d u c t e d under I S T A ru les ( Internat ional S e e d T e s t i n g A s s o c i a t i o n , 1 9 8 5 ) . Four s a m p l e s w e r e ge rm ina ted w i t h o u t s t ra t i f i ca t ion , w h i l e the rema in ing f ou r w e r e s o a k e d in w a t e r for 2 4 hr, t hen s t ra t i f ied at 2 ° C fo r 21 d a y s be fo re g e r m i n a t i o n . For g e r m i n a t i o n , e a c h s a m p l e w a s s p r e a d in a t i gh t l y - l i dded , c lear p las t i c ge rm ina t i on b o x l ined w i t h m o i s t e n e d ce l l u lose w a d d i n g (K impak) and f i l ter paper , t hen p l a c e d in a ge rm ina to r se t at 3 0 ° C fo r an 8 hr d a y , and 2 0 ° C for a 1 6 hr n ight . L igh t , at a p p r o x i m a t e l y 1 , 0 0 0 lux, w a s p r o v i d e d dur ing the day by m e a n s of c o o l - w h i t e f l u o r e s c e n t t u b e s . G e r m i n a n t s w e r e c o u n t e d eve ry day for 21 d a y s and c l ass i f i ed as no rma l or a b n o r m a l ( Internat ional S e e d T e s t i n g A s s o c i a t i o n , 1 9 8 5 ) . T h e ge rm ina t i on da ta w e r e ca l cu la ted and e x p r e s s e d as ge rm ina t i on c a p a c i t y ( G C ) , the p e r c e n t a g e of s e e d s tha t had ge rm ina ted at the e n d of the tes t ; peak va lue ( P V ) , the m a x i m u m quo t ien t de r i ved by d iv id ing da i ly the a c c u m u l a t e d n u m b e r of g e r m i n a n t s b y the c o r r e s p o n d i n g n u m b e r of d a y s ( w h i c h is the m e a n dai ly ge rm ina t i on of the m o s t - v i g o r o u s c o m p o n e n t of the seed lo t , a m a t h e m a t i c a l e x p r e s s i o n of the tangen t d r a w n t h r o u g h the or ig in o f the s i g m o i d c u r v e rep resen t ing a t yp i ca l c o u r s e of germina t ion) (Czaba to r , 1 9 6 2 ) ; a n d ge rm ina t i on va lue ( G V ) , the c o m b i n a t i o n of bo th the s p e e d and c o m p l e t e n e s s of ge rm ina t i on in to a s ing le va lue (Czaba to r , 1 9 6 2 ) . 6 . 2 . 3 Statistical Analysis T h e ana l ys i s of va r i ance used w a s b a s e d on the f o l l o w i n g n e s t e d - f a c t o r i a l , add i t i ve , l inear m o d e l ; Yij„ = / / + Ci + P, + C P , + T / C , i „ + PT/C,„ „ + e,i,,„ w h e r e /y = overa l l m e a n . Ci = the e f fec t of ith c l o n e , i = 1-6 ( random e f fec t ) , P| = the e f fec t j th s e e d p re t rea tment , j = 1-2 ( f ixed e f fec t ) , CPi, = t he e f fec t of in te rac t ion b e t w e e n c l o n e s and s e e d p r e t r e a t m e n t s , T/C,i||^ = the e f fec t of k th ag ing t ime w i th i n c l o n e s , k = 1-8 ( f ixed e f fec t ) , PT/C,,,jk = the e f fec t of in te rac t ion b e t w e e n s e e d p r e t r e a t m e n t s and ag ing t imes w i t h i n c l o n e , and = res idua l t e r m , 1 = 1-4. Data t r a n s f o r m a t i o n s w e r e c o n d u c t e d us ing an ad- f ioc p r o c e d u r e fo r f i nd ing appropr ia te t r ans fo rma t i on to no rma l i ze the ca l cu la ted r e s p o n s e var iab les a n d a c h i e v e h o m o g e n e i t y of v a r i a n c e s . 6 . 3 Results and Discussion T h e var ia t ion in the e f fec t of acce le ra ted ag ing due to c l ona l d i f f e rences w a s h igh ly s ign i f i can t (P < 0 . 0 1 ) (Table 6.1). C l o n a l var ia t ion in ge rm ina t i on p a r a m e t e r s ranged b e t w e e n 3 0 . 9 6 % ( G O and 4 1 . 2 2 % (GV) of to ta l va r ia t ion . Va r ia t i on due to s e e d p re t rea tmen t w a s not s ign i f i can t for P V and G V . It w a s s ign i f i can t (P < 0 . 0 5 ) for G C , a c c o u n t i n g fo r 2 . 4 6 to 1 8 . 2 6 % of to ta l var ia t ion (Table 6.1). Va r ia t i on due to the length of the acce le ra ted -ag ing pe r iod w a s h igh ly s ign i f i can t , a c c o u n t i n g fo r the la rgest p ropo r t i on of t he to ta l va r ia t i on , rang ing f r o m 46 .47%) in G C to 50 .23%) in G V . C l o n a l r e s p o n s e to the va r i ous t r ea tmen ts (pre t reatment , acce le ra ted ag ing and their in teract ion) w a s c o n s i s t e n t as e x p r e s s e d by the l o w res idua l te rm (i.e. rep l i ca t ion- to - rep l i ca t ion var iat ion) (Table 6.1). It appea rs that 3 to 6 d a y s of acce le ra ted ag ing e n h a n c e ge rm ina t i on of uns t ra t i f i ed and s t ra t i f ied s e e d s in s o m e c l o n e s (Figure 6.1 and Figure 6.2.), but ge rm ina t i on g radua l l y dec l i nes w i t h longer t rea tmen t . G e r m i n a t i o n e n h a n c e m e n t due to sho r t - t ime a c c e l e r a t e d ag ing migh t be of opera t i ona l i m p o r t a n c e . S e e d mo is tu re c o n t e n t w a s i n c r e a s e d at 3 d a y s of a c c e l e r a t e d ag ing (on ave rage f r o m 6 . 7 % to 3 3 % , b a s e d o n d ry we igh t ) and then rema ined re la t ive ly s tab le to d a y 12 (Figure 6.3). S e e d mo i s tu re c o n t e n t i n c r e a s e d d ramat i ca l l y af ter 12 d a y s of t r ea tmen t , p r o b a b l y b e c a u s e cel l m e m b r a n e s b e c o m e seve re l y d a m a g e d , and v iab i l i ty is s u b s e q u e n t l y los t . T h i s dec l i ne in ge rm ina t i on w a s a s s o c i a t e d w i t h the inc rease in mo is tu re c o n t e n t (Figure 6.4). Co r re la t i ons b e t w e e n ave rage G C and mo i s tu re con ten t are nega t i ve and h igh ly s i gn i f i can t (P< 0 . 0 1 ) for s t ra t i f ied ( r= - 0 . 9 4 ) and uns t ra t i f i ed (r = -0 .94 ) s e e d s (Figure 6.4). T h e per iod w h e n a c c e l e r a t e d ag ing e n h a n c e s s e e d ge rm ina t i on has been t e r m e d the " c o n d i t i o n e d s t a g e " by Bi rd and R e y e s ( 1 9 6 7 ) , w h i l e t he t e rm "de te r i o ra ted s t a g e " w a s u s e d Table 6.1. Va r i a t i on in ge rm ina t i on of 6 Si t i ta s p r u c e c l o n e s f o l l o w i n g acce le ra ted ag ing ( A A ) and s e e d p re t rea tment (va lues are p e r c e n t a g e s of the to ta l m e a n squares) S o u r c e of Va r i a t i on Deg rees of F r e e d o m E x p e c t e d M e a n Squares^ G e r m i n a t i o n Parameters^ G C P V G V C l o n e (C) (C-1) = 5 al + QAal 3 0 . 9 6 * * 3 4 . 7 0 " " 4 1 . 2 2 " " P re t rea tmen t (P) (P-1) = 1 al + Zlal, + 1 9 2 0 , 1 8 . 2 6 * 12 .17™ 2.46™ C X P (C-1)(P-1) = 5 o f + 22ol, 2 . 0 7 " " 3 . 1 4 " " 3 . 6 5 " * A A - T i m e w i th i n C l o n e (T/C) C ( T - 1 ) = 4 2 al + 8a f , , 4 6 . 4 7 " " 4 8 . 0 9 " " 5 0 . 2 3 * * P X T / C C(T-1 ) (P-1 ) = 4 2 ol + 4a^ , , , 1 . 7 0 " 1 .20" " 1 .54"" Res idua l C P T ( N - I ) = 2 8 8 al 0 . 5 4 0 . 7 0 0 . 9 0 ™ N o t s ign i f i can t . " S ign i f i can t at P < 0 . 0 5 . •" S ign i f i can t at P < 0 . 0 1 . ^ E x p e c t e d m e a n s q u a r e s c o m p u t e d f r o m the nes ted- fac to r ia l expe r imen ta l d e s i g n . ^ G C = G e r m i n a t i o n c a p a c i t y ; the pe rcen tage of s e e d s that had ge rm ina ted at the end of the tes t (A rcs in ) . P V = Peak va lue ; a m a t h e m a t i c a l e x p r e s s i o n of the break of a s i g m o i d cu r ve represen t ing a t yp i ca l c o u r s e of ge rm ina t ion (1 -1 / (X -i-1 )). G V = G e r m i n a t i o n va lue , ( 1 - 1 / ( X + 1 ) ) . CD Figure 6.1. Average germination (21 days) of unstratified seeds of 6 Sitka spruce clones after accelerated aging. 0 3 6 9 12 15 18 21 ACCELERATED AGING TIME (DAYS) Figure 6.2. Average germination (21 days) of stratified seeds of 6 Sitka spruce clones after accelerated aging. I \ \ \ \ I \ L 0 3 6 9 12 15 18 21 ACCELERATED AGING TIME (DAYS) Figure 6.3. Mo i s tu re -con ten t c u r v e s of s e e d s of 6 S i t k a s p r u c e c l o n e s af ter acce le ra ted ag ing . Figure 6.4. Curves shovy/ing germination of unstratified, stratified, and moisture content, of accelerated-aged seeds; average for 6 clones. f o r the per iod w h e n ge rm ina t i on d e c r e a s e s unt i l the s e e d d ies . T h e ge rm ina t i on t r ends af ter a c c e l e r a t e d ag ing o b s e r v e d in th is s t u d y f i t the t w o s t a g e s d e s c r i b e d a b o v e . E n h a n c e m e n t of s e e d ge rm ina t i on due to a sho r t pe r iod of a c c e l e r a t e d ag ing w a s o b s e r v e d in w a t e r oak (Quercus nigra L.) (B lanche et ai., 1 9 9 0 ) . S u c h e n h a n c e m e n t is no rma l f o r annua l s e e d p lan ts (Bour land and Ibrah im, 1 9 8 2 ) , and is due to the i nc rease in mo i s t u re c o n t e n t tha t b r ings the level of hyd ra t i on c l o s e r to the m in ima l requ i rement for s e e d g e r m i n a t i o n . T h e b r e a k d o w n of p o l y m e r i c s t o r a g e c o m p o u n d s a l so a c c o u n t s fo r th is e n h a n c e m e n t (Pi te l , 1 9 8 0 ; B l a n c h e et al., 1 9 8 8 , 1 9 9 0 ) . H o w e v e r , the ex ten t of ge rm ina t i on e n h a n c e m e n t in r e s p o n s e to acce le ra ted ag ing is i n f l uenced by the init ial s e e d qua l i ty (B lanche etal., 1 9 9 0 ) , i nc lud ing m o i s t u r e c o n t e n t ( M c D o n a l d , 1 9 7 7 ; T o a , 1 9 7 9 ) . T h e f i rst even t du r ing the "de te r io ra ted s t a g e " is be l ieved to be c a u s e d by a l oss of m e m b r a n e in tegr i ty (Basavara jappa et al., 1 9 9 1 ) ; ce l l pe rmeab i l i t y is i n c r e a s e d , a l l o w i n g large quan t i t i es of ce l lu lar c o m p o n e n t s to d i f fuse out w h e n the s e e d is p l a c e d in w a t e r ( S c h n a t h o r s t and P res l ey , 1 9 6 3 ) . T h e i nc rease in m e m b r a n e permeab i l i t y in a c c e l e r a t e d - a g e d s e e d s is p o s s i b l y due t o c h a n g e s in the mo lecu la r s t ruc tu re of the m e m b r a n e ( K o o s t r a , 1 9 7 3 ; S i m o l a , 1 9 7 4 ; P i te l , 1 9 8 0 ) . T h e inc rease in m e t a b o l i s m du r ing ag ing dep le tes f o o d rese rves a n d , s u b s e q u e n t l y , s e e d v igour dec l i nes (B lanche et al., 1 9 9 0 ) . T h e l oss of s e e d v iab i l i ty and d i f ferent ia l su rv i va l of b i o t ypes due to acce le ra ted ag ing w a s f o u n d to be a s s o c i a t e d w i t h se l ec t i on and gene t i c sh i f t s in g e r m p l a s m a c c e s s i o n s ( S t o y a n o v a , 1 9 9 1 ) . T h e r e s p o n s e of s e e d s to a c c e l e r a t e d ag ing var ies a c c o r d i n g to s p e c i e s (Benne t t -La r tey , 1 9 9 1 ; B lanche et al., 1 9 8 8 ) , popu la t i on (M i lby and J o h n s o n , 1 9 8 9 ) , f am i l y (Bour land and Ibrah im, 1 9 8 2 ) and ind iv idua l s e e d s (De louche and B a s k i n , 1 9 7 3 ) , and is s im i la r to s e e d s that h a v e been s to red for a long t ime . In S i t ka s p r u c e , the o b s e r v e d d i f f e rences in the e f f ec t s of acce le ra ted ag ing o n s e e d ge rm ina t i on s u g g e s t tha t the v i gou r of o r c h a r d s e e d s var ies a c c o r d i n g to c l o n e . C o n s e q u e n t l y , acce le ra ted ag ing is p robab l y app l i cab le fo r i ndex i ng s e e d v igour in S i t ka s p r u c e . In p rac t i ce , w l i e n s e e d s co l l ec ted f r o m d i f ferent o r cha rd c l o n e s are bu l ked into a s ing le seed lo t , l ong- te rm s to rage of th is seed lo t m igh t r educe gene t i c d i ve rs i t y . S i n c e the con t r i bu t i on of parent t rees to s e e d p r o d u c t i o n in the o r cha rd f r o m w h i c h the s e e d s t e s t e d in th is s t u d y w a s o b s e r v e d to be u n b a l a n c e d ( E l - K a s s a b y and R e y n o l d s , 1 9 9 0 ) s o m e c l o n e s m igh t be e l im ina ted by l ong t e r m - s t o r a g e . For agr icu l tu ra l c r o p s , ge rm ina t i on f o l l o w i n g acce le ra ted ag ing and pe r i ods of s to rage are c l o s e l y a s s o c i a t e d , i .e. , t h o s e lo ts tha t h a v e h igh su rv i va l af ter a c c e l e r a t e d ag ing s to red w e l l , wh i l e lo ts tha t w e r e seve re l y r e d u c e d in their ge rm ina t i on by a c c e l e r a t e d ag ing dec l i ned rap id ly in s to rage (De louche and B a s k i n , 1 9 7 3 ) . T o - d a t e , f e w if any s u c h da ta are ava i lab le fo r t ree s e e d s . 6 . 4 References A s s o c i a t i o n of O f f i c ia l S e e d A n a l y s t s . 1 9 8 3 . Seed vigor testing handbooif.. Con t r i bu t i on N o . 3 2 . A s s o c i a t i o n of Of f i c ia l S e e d A n a l y s t s . B a s a v a r a j a p p a , B . S . , H . S . S h e t t y , and H . S . P r a k a s h . 1 9 9 1 . M e m b r a n e de te r io ra t ion and o ther b i o c h e m i c a l c h a n g e s , a s s o c i a t e d w i t h acce le ra ted age ing of ma i ze s e e d s . Seed Sci. & Technol. 1 9 : 2 7 9 - 2 8 6 . B a s k i n , C . C . 1 9 7 7 . V i g o r tes t m e t h o d s - A c c e l e r a t e d aging. Assoc. Off Seed Anal. Newslett. 5 1 : 4 2 - 5 2 . Benne t t -La r tey , S . O . 1 9 9 1 . T h e longev i t y of p e a , s u n f l o w e r a n d g r o u n d n u t s e e d s under con t ro l l ed t empera tu re and mo is tu re c o n t e n t c o n d i t i o n s . Trop. Sci. 3 1 : 9 - 1 9 . B i rd , L . S . and A . A . R e y e s . 1 9 6 7 . E f fec ts of c o t t o n s e e d qua l i t y o n s e e d a n d seed l i ng c h a r a c t e r i s t i c s . In Proc. Beltw/ide Cotton Prod. Res. Conf., p p . 1 9 9 - 2 0 6 . Na t i ona l C o t t o n C o u n c i l , M e m p h i s , T e n n e s s e e . B l a n c h e , C . A . , W . W . E l a m , J . D . H o d g e s , F.T. B o n n e r , and A . C . M a r q u e z . 1 9 8 8 . A c c e l e r a t e d ag ing of s e l e c t e d t ree s e e d s . In Proc. 10th north American forest biology workshop, ed . J . W o r r a l l , J . L o o - D i n k i n s , and D . P . Les te r , pp . 3 2 7 - 3 3 4 . V a n c o u v e r , Br i t ish C o l u m b i a . B l a n c h e , C . A . , W . W . E l a m , and J . D . H o d g e s . 1 9 9 0 . A c c e l e r a t e d ag ing of Ouercus nigra seed: b i o c h e m i c a l c h a n g e s and app l icab i l i t y as a v igor tes t . Can. J. For. Res. 2 0 : 1 6 1 1 - 1 6 1 5 . B o u r l a n d , F . M . and A . A . L . Ibrah im. 1 9 8 2 . E f fec t s of a c c e l e r a t e d ag ing t r ea tmen ts o n s i x c o t t o n cu l t i va rs . Crop Sci 2 2 : 6 3 7 - 6 4 0 . C z a b a t o r , F . J . 1 9 6 2 . G e r m i n a t i o n va lue : A n index c o m b i n i n g s p e e d and c o m p l e t e n e s s of p ine s e e d g e r m i n a t i o n . For. Sci 8 : 3 8 6 - 3 9 6 . D e l o u c h e , J . C . and C . C . B a s k i n . 1 9 7 3 . A c c e l e r a t e d ag ing t e c h n i q u e s for p red ic t i ng the re lat ive s to rab i l i t y of s e e d lo ts . Seed Sci. & Technol. 1 : 4 2 7 - 4 5 2 . D i c k s o n , M . H . 1 9 8 0 . G e n e t i c a s p e c t s of s e e d qua l i t y . HortSci. 1 5 : 7 7 1 - 7 7 4 . E l - K a s s a b y , Y . A . , and S . R e y n o l d s . 1 9 9 0 . R e p r o d u c t i v e p h e n o l o g y , paren ta l b a l a n c e , and s u p p l e m e n t a l m a s s po l l ina t ion in a S i t k a - s p r u c e s e e d - o r c h a r d . For. Ecol. Manage. 3 1 : 4 5 - 5 4 . In ternat ional S e e d T e s t i n g A s s o c i a t i o n . 1 9 8 5 . Internat ional ru les fo r s e e d t e s t i n g . Seed Sci. & Technol. 1 3 : 2 9 9 - 5 1 3 . K n e e b o n e , W . R . 1 9 7 6 . S o m e gene t i c a s p e c t s of s e e d v igor . J. Seed Technol. 1 : 8 6 - 9 7 . K o o s t r a , P. 1 9 7 3 . C h a n g e s in s e e d u l t ras t ruc tu re dur ing s e n e s c e n c e . Seed Sci & Technol. 1 : 4 1 7 - 4 2 5 . K u e n e m a n , E . A . 1 9 8 3 . G e n e t i c con t ro l of s e e d longev i t y in s o y b e a n s . Crop Sci 2 3 : 5 - 8 . M c D a n i e l , R . G . 1 9 7 3 . G e n e t i c f a c t o r s i n f l uenc ing s e e d v igor : B i o c h e m i s t r y of he te ros i s . Seed Sci. & Techno/. 1 : 2 5 - 5 0 . M c D o n a l d , M . B . , J r . 1 9 7 7 . T h e in f luence of s e e d mo i s tu re o n the a c c e l e r a t e d ag ing s e e d v igor tes t . J. Seed Techno/. 2 : 1 8 - 2 8 . M c D o n a l d , M . B . , J r . 1 9 8 0 . A s s e s s m e n t of s e e d qua l i t y . HortSci. 1 5 : 1 8 4 - 7 8 8 . M c W i l l i a m , J . R . and B. Gr i f f i ng . 1 9 6 5 . T e m p e r a t u r e - d e p e n d e n t he te ros i s in m a i z e . Aust. J. Bio/. Sci. 1 8 : 5 6 9 - 5 8 3 . M i l b y , T . H . , and F.L. J o h n s o n . 1 9 8 9 . Va r ia t i on in r e s p o n s e to s t ra t i f i ca t ion and age ing in g a m a g r a s s s e e d s f r o m d i f ferent g e o g r a p h i c l o c a t i o n s . Seed Sci. & Techno/. 1 7 : 4 1 3 -4 1 9 . M i n o r , H . C . , and E . C . P a s c a l . 1 9 8 2 . Va r ia t i on in s to rab i l i t y of s o y b e a n s under s imu la ted t rop ica l c o n d i t i o n s . Seed Sci. & Techno/. 1 0 : 1 3 1 - 1 3 9 . N d i m a n d e , B . N . , H . C . W e i n , and E . A . K u e n e m a n . 1 9 8 1 . S o y b e a n s e e d de te r io ra t ion in the t r o p i c s . T h e role of phys i o l og i ca l f ac to r s and f unga l p a t h o g e n s . Fie/d Crops Res. 4 : 1 1 3 -1 2 1 . P a s c a l , E . H . II, and M . A . El l is . 1 9 7 8 . Va r ia t i on in s e e d qua l i t y c h a r a c t e r i s t i c s of t rop ica l l y g r o w n s o y b e a n s . Crop Sci. 1 8 : 8 3 7 - 8 4 0 . P i te l , J . A . 1 9 8 0 . A c c e l e r a t e d ag ing s tud ies of s e e d s of j ack p ine (Pinus ban/^siana Lamb . ) and red oak (Quercus rubra L.). In Proc. internationa/ Symposium on Forest tree seed storage, l U F R O W o r k i n g par ty o n S e e d P r o b l e m s , e d . B . S . P . W a n g and J . A . P i te l , pp . 4 0 - 5 4 . P e t a w a w a Na t iona l Fo res t r y Inst i tute, C h a l k R i ve r , On ta r i o . P o t t s , H . C , J . D u a n g p a t r a , W . G . H a i r s t o n , and J . C . D e l o u c h e . 1 9 7 8 . S o m e i n f l uences of h a r d s e e d e d n e s s o n s o y b e a n s e e d qua l i t y . Crop Sci. 1 8 : 2 2 1 - 2 2 4 . R o b e r t s , E . H . 1 9 7 2 . C y t o l o g i c a l , gene t i ca l and me tabo l i c c h a n g e s a s s o c i a t e d w i t h l oss of v iab i l i ty . In Viabi/ity of seeds, e d . E . H . R o b e r t s , pp . 2 5 3 - 3 0 6 . L o n d o n : C h a p m a n and Ha l l . S c h n a t h o r s t , W . C , and J . T . P res ley . 1 9 6 3 . P r o n e n e s s of de te r io ra ted c o t t o n s e e d to d e c a y and seed l i ng d i s e a s e s re la ted to p h y s i c o - b i o c h e m i c a l f a c t o r s . In Proc. Be/twide Cotton Prod. Res. Conf., pp . 5 7 - 5 8 . Na t iona l C o t t o n C o u n c i l , M e m p h i s , T e n n e s s e e . S i m o l a , L .K . 1 9 7 4 . U l t ras t ruc tu re c h a n g e s in the s e e d s of Pinus sy/vestris L. dur ing s e n e s c e n c e . Stud. For. Suec. 1 1 9 : 1 - 2 2 . S t o y a n o v a , S . D . 1 9 9 1 . G e n e t i c sh i f t s and va r ia t ions of g l iad ins i n d u c e d by s e e d a g e i n g . Seed Sci. & Techno/. 1 9 : 3 6 3 - 3 7 1 . T o a , K . J . 1 9 7 9 . A n eva lua t i on of a l te rnat ive m e t h o d s o f a c c e l e r a t e d ag ing s e e d v igor tes t for s o y b e a n s . J. Seed Techno/. 3 : 3 0 - 4 0 . W e i n , H . C , and E . A . K n u e n e m a n . 1 9 8 1 . S o y b e a n s e e d de te r io ra t ion in the t r o p i c s . Var ie ta l d i f f e rences and t e c h n i q u e s for s c r e e n i n g . Fie/d Crops Res. 4 : 1 2 3 - 1 3 2 . C h a p t e r 7 E f fec t of S e e d S i z e on G e r m i n a t i o n of Sitl<a S p r u c e S e e d s 7.1 Introduction G e r m i n a t i o n is rou t ine ly u s e d to es t ima te v iab i l i ty and ge rm ina t i ve e n e r g y of s e e d s . T h e pa t te rn of s e e d ge rm ina t i on ver i f ies qua l i ty w i t h re fe rence to s e e d s o u r c e ( A s s o c i a t i o n of O f f i c ia l S e e d A n a l y s t s , 1 9 7 0 ) . A l t h o u g h the natura l e n v i r o n m e n t in w h i c h s e e d s w i l l be s o w n is uns tab le , e v e n in the g r e e n h o u s e , a s t anda rd ge rm ina t i on tes t no rma l l y is c o n d u c t e d us ing u n i f o r m c o n d i t i o n s . T h u s , the ge rm ina t i on tes t o f ten o v e r - e s t i m a t e s f ie ld p e r f o r m a n c e of s e e d s . T h e pa t te rn of s e e d ge rm ina t i on in a s p e c i e s var ies a c c o r d i n g to s e e d s o u r c e (A l len , 1 9 6 1 ) , f am i l y (Bramlet t et al., 1 9 8 3 ; E l - K a s s a b y et al., 1 9 9 2 ) , p re t rea tmen ts ( K o z l o w s k i and G e n t l e , 1 9 5 9 ; De M a t o s M a l a v a s i et al., 1 9 8 5 ; Pi te l a n d W a n g , 1 9 8 5 ) , s e e d matu r i t y (A l l en , 1 9 5 8 a , b; E d w a r d s , 1 9 8 0 ; E d w a r d s and E l - K a s s a b y , 1 9 8 8 ) , e n v i r o n m e n t a l p recond i t i on ing dur ing s e e d d e v e l o p m e n t (Kol ler , 1 9 6 2 ; S a w h n e y and N a y l o r , 1 9 7 9 ; S a w h n e y and N a y l o r , 1 9 8 0 ; Nay lo r , 1 9 8 3 ) , and s e e d s i ze (Spurr , 1 9 4 4 ; S h o u l d e r s , 1 9 6 1 ; Bu rga r , 1 9 6 4 ; Wu l f f , 1 9 7 2 ; D u n l a p and Barne t t , 1 9 8 3 ; H e l i u m , 1 9 9 0 ) . S e e d s i ze has been f o u n d to be unde r s t r o n g gene t i c i n f l uence (He l i um, 1 9 7 6 ; S i l en and O s t e r h a u s , 1 9 7 9 ; L i n d g r e n , 1 9 8 2 ; Wu l f f , 1 9 8 6 ; B a g c h i et al., 1 9 9 0 ) tha t is ma in l y ma te rna l (Perry , 1 9 7 6 ; R o a c h and Wu l f f , 1 9 8 7 ; T y s o n , 1 9 8 9 ) . G e o g r a p h i c var ia t ion a l so i n f l u e n c e s s e e d s i ze . W i t h i n a s p e c i e s , s e e d s ize is co r re la ted w i t h d r y n e s s of the s i te . A s d r y n e s s i n c r e a s e s , s e e d s i ze i n c r e a s e s (Baker , 1 9 7 2 ) . In add i t i on , yea r - to -yea r var ia t ion in s e e d s i ze h a s been o b s e r v e d in nob le fir (Abies procera Rehd . ) ( S o r e n s e n and F rank l i n , 1 9 7 7 ) and Doug las - f i r (Pseudotsuga menziesii (M\rb.) F ranco) (Si len and O s t e r h a u s , 1 9 7 9 ) . W i t h i n a p lan t , var ia t ion in s e e d s ize m a y be c a u s e d by pos i t i on of s e e d s in the p lant i n f l o r e s c e n c e ( C a v e r s and Harper , 1 9 6 6 ; Da t ta et al., 1 9 7 0 ) , or f rui t (L inck, 1961 ; S c h a a l , 1 9 8 0 ) . In c o n i f e r s , the b i g g e s t s e e d s usua l l y o c c u r in the m idd le por t ion of the c o n e . T h e re la t ionsh ip b e t w e e n s e e d s i ze and ge rm ina t i on c a n be var iab le . S e e d s i ze had little e f fec t o n the ge rm ina t i on pat tern in Doug las - f i r (Lavender , 1 9 5 8 ) , lob lo l l y p ine (Pinus taeda L.) (Dun lap and Barne t t , 1 9 8 3 ) , N o r w a y s p r u c e (Picea abies L. Ka rs t . ) ( A n d e r s s o n , 1 9 6 5 ) , and J a p a n e s e red p ine (Pinus densiflora S i e b . & Z u c c . ) (Cho i and K i m , 1 9 6 9 ) , but s i gn i f i can t e f fec ts w e r e o b s e r v e d in s l a s h pine (Pinus elliottii Enge lm. ) (Shou lde r s , 1961 ) and w h i t e s p r u c e (Picea glauca ( M o e n c h ) V o s s ) ( A c k e r m a n and G o r m a n , 1 9 6 9 ) . In th is s t u d y , the e f fec ts of c l o n e s , s e e d s i ze and s e e d p re t rea tmen t o n ge rm ina t i on of o r c h a r d - p r o d u c e d S i t ka s p r u c e s e e d s are repo r ted . 7 . 2 Materials and Methods C a n a d i a n Pac i f i c Fo res t P r o d u c t s L t d . p r o v i d e d the s e e d s fo r th is s t u d y f r o m the S i t ka s p r u c e s e e d o r cha rd l oca ted in S a a n i c h t o n , Br i t ish C o l u m b i a ( lat i tude 4 8 ° 3 5 ' N , long i tude 1 2 3 ° 2 4 ' W ) . T h e o rcha rd c o n s i s t s of 1 3 9 c l o n e s (averag ing 9 . 3 r ame ts per c l ones ) s e l e c t e d f r o m e leva t i ons b e t w e e n 0 m and 4 1 5 m on w e s t e r n V a n c o u v e r Is land, W a s h i n g t o n and O r e g o n (Figure 1.1). T h e o r c h a r d w a s e s t a b l i s h e d in 1 9 7 1 in a r a n d o m s ing le - t ree m i x ove r th ree unequa l b l o c k s . In S e p t e m b e r 1 9 9 0 , w ind -po l l i na ted s e e d s w e r e c o l l e c t e d f r o m 1 8 c l o n e s . T h e c lona l ident i t ies of the s e e d s w e r e ma in ta ined dur ing s e e d e x t r a c t i o n . S e e d s f r o m e a c h c l o n e w e r e d i v i d e d into t w o po r t i ons . O n e por t ion w a s so r t ed into t w o s i ze c l a s s e s , large (> 1.41 mm.) and sma l l ( < 1.41 mm.) us ing a 1 4 - m e s h s c r e e n , wh i l e the o ther w a s kept u n s o r t e d . Bo th u n s o r t e d and so r t ed s e e d po r t i ons w e r e kept at 2 ° C unt i l u s e d . S i ze and w e i g h t are the ma in pa rame te rs u s e d in s e e d s o r t i n g , a n d t h e s e pa rame te rs are h igh ly co r re la ted in Doug las - f i r (Si len and O s t e r h a u s , 1 9 7 9 ) . W h e r e a s s i z i ng is l ess t ime c o n s u m i n g w h e n sor t ing a seed lo t , w e i g h i n g is mo re a c c u r a t e for i nd iv idua l s e e d - s i z e de te rm ina t i ons . In th is s t u d y , w e i g h i n g and s i z ing w e r e u s e d . 7 .2 .1 Seed Weight Indiv idual s e e d s f r o m r a n d o m s a m p l e s of 1 0 0 u n s o r t e d s e e d s , and 5 0 large and sma l l so r t ed s e e d s , f r o m e a c h of the 18 c l o n e s , w e r e w e i g h e d to the neares t 0 .01 m g to de te rm ine pa t te rns of we ig f i t d i s t r ibu t ion a m o n g c l o n e s and of s i zes w i t h i n c l o n e s . Fi l led s e e d s (de te rmined by X - r a y ana lys is ) on l y w e r e u s e d . 7 . 2 . 2 Germination Test Eight r a n d o m s a m p l e s of 1 0 0 s e e d s e a c h w e r e t aken f r o m u n s o r t e d and so r ted (large a n d smal l ) s e e d s of e a c h c l o n e and sub jec ted to a s t a n d a r d ge rm ina t i on tes t . Fou r of the e ight s a m p l e s w e r e s o a k e d in w a t e r for 2 4 h r s . , d ra i ned , and s t ra t i f ied (prechi l led) f o r 21 d a y s at 2 ° C . S t ra t i f i ed and uns t ra t i f ied s a m p l e s w e r e ge rm ina ted s i m u l t a n e o u s l y . For g e r m i n a t i o n , s e e d s a m p l e s w e r e sp read in t i gh t l y - l i dded , c lear p las t i c ge rm ina t i on b o x e s l ined w i t h m o i s t e n e d ce l l u l ose w a d d i n g (K impak) and f i l ter paper , and p laced in a ge rm ina to r se t at an a l ternat ing t empera tu re of 3 0 ° C for 8 h rs . f o l l o w e d by 2 0 ° C for 16 h rs . L igh t , at a p p r o x i m a t e l y 1 , 0 0 0 lux , w a s p rov ided dur ing the h igh - tempera tu re per iod by m e a n s of c o o l - w h i t e f l u o r e s c e n t t u b e s . G e r m i n a n t s w e r e c o u n t e d eve ry d a y fo r 21 d a y s and c l ass i f i ed as no rma l or abno rma l a c c o r d i n g to the I S T A ( Internat ional S e e d T e s t i n g A s s o c i a t i o n , 1 9 8 5 ) ru les . R e s u l t s w e r e e x p r e s s e d as (i) ge rm ina t i on c a p a c i t y ( G C ) , the pe rcen tage of s e e d s tha t had ge rm ina ted at the e n d of the tes t ; (ii) peak va lue ( P V ) , the m a x i m u m quo t ien t de r i ved by d iv id ing da i ly the a c c u m u l a t e d n u m b e r of g e r m i n a n t s by the c o r r e s p o n d i n g n u m b e r of d a y s , w h i c h is the m e a n da i ly ge rm ina t i on of the m o s t v i g o r o u s c o m p o n e n t s of a seed lo t ( C z a b a t o r , 1 9 6 2 ) , and (iii) ge rm ina t i on va lue ( G V ) , the c o m b i n a t i o n of s p e e d and c o m p l e t e n e s s of ge rm ina t i on into a s ing le i ndex (Czaba to r , 1 9 6 2 ) . 7 . 2 . 3 Statistical Analysis Data t r a n s f o r m a t i o n s w e r e c o n d u c t e d us ing an a d - h o c p r o c e d u r e fo r f i nd ing appropr ia te t r a n s f o r m a t i o n s to no rma l i ze the c a l c u l a t e d r e s p o n s e va r iab les a n d a c h i e v e h o m o g e n e i t y of v a r i a n c e s . T h e ge rm ina t i on pa rame te rs ( G C , P V , and G V ) w e r e t h e n a n a l y z e d us ing ana l ys i s o f va r i ance ( A N O V A ) . A n a l y s e s w e r e s e p a r a t e d in to t w o pa r t s : (1 ) S i m p l e o n e - w a y A N O V A w a s u s e d to es t ima te gene t i c c o m p o n e n t s of s e e d w e i g h t (Table 7.1) and ge rm ina t i on pa rame te rs of unso r t ed S i t ka s p r u c e s e e d s (Table 7.3). B r o a d -s e n s e her i tab i l i t ies of t h e s e pa rame te rs w e r e de te rm ined u s i n g the m u l t i p l e - m e a s u r e m e n t s c o n c e p t of an ima l b reeders (Fa lconer , 1 9 8 6 , p. 1 2 7 ) . R e p l i c a t i o n s w e r e u s e d as mul t ip le m e a s u r e m e n t s per c l o n e . (2) A fac to r ia l e x p e r i m e n t w i t h 3 l eve ls , (i) s e e d s i ze (S, 2 c l a s s e s p lus unso r t ed s e e d s ) , (ii) c l o n e (C, 1 8 c l o n e s ) , and (iii) p re t rea tmen ts (P, 2 t r ea tmen ts ) , w a s u s e d to a s s e s s the e f fec t of s e e d s ize on ge rm ina t i on pa rame te rs . T h e m o d e l for th is A N O V A is as f o l l o w s : Yij„ = // + S, + C, + SC„ + P , + C P j , + S P ; , + S C P , , + e„j,„ w h e r e /j = overa l l m e a n s Si = e f fec t of s e e d s ize ( f ixed e f fec t ) , i = 1-3, Ci = c l o n e e f fec t ( random e f fec t ) , j = 1 -18 , Pi^  = p re t rea tment e f fec t ( f ixed e f fec t ) , k = 1-2, SCij = e f fec t of in te rac t ion b e t w e e n s e e d s i ze and c l o n e , PCjn = e f fec t of in te rac t ion b e t w e e n p re t rea tmen t a n d c l o n e , SPii^ = e f fec t of in te rac t ion b e t w e e n s e e d s i ze a n d p re t rea tmen t ( f ixed e f fec t ) , SCPijk = the e f fec t of in te rac t ion a m o n g s e e d s i z e , c l o n e and p re t rea tment , and eiiji,,! = res idua l t e r m , 1 = 1-4. 7 . 3 Results and Discussion The d i f fe rence b e t w e e n the largest and sma l l es t s e e d s is s m a l l ( range 0 . 0 2 0 0 to 0 . 0 3 1 3 m g in s e e d we igh t ) (Figure 7.1). H o w e v e r , th is var ia t ion is h igh ly s ign i f i can t (P < 0 .01 ) a n d a c c o u n t e d for 3 6 . 1 8 % of to ta l var ia t ion (Table 7.1). T h e la rges t a m o u n t of to ta l va r ia t ion , 6 3 . 8 2 % , is due to d i f f e rences w i t h i n c l o n e s . W h e n s e e d s f r o m e a c h c l o n e w e r e s o r t e d into 2 s i ze c l a s s e s , w e i g h t d i f f e rences b e t w e e n sma l l and large var ied c o n s i d e r a b l y f r o m c l o n e to c l o n e . C l o n e n o . 5 h a s the sma l l es t d i f fe rence in s e e d w e i g h t , w h i l e c l o n e n o . 1 5 4 has the Figure 7.1. S e e d w e i g h t d is t r ibu t ion c u r v e s of 1 8 Sitl<a s p r u c e c l o n e s . o Table 7.1. Es t ima t i on of va r i ance c o m p o n e n t s , s i gn i f i cance leve l , and b r o a d - s e n s e her i tab i l i t ies (h^) fo r ind iv idua l s e e d w e i g h t of 1 8 S i t ka s p r u c e c l o n e s S o u r c e of Va r i a t i on df Expec ted^ M e a n S q u a r e s V a r i a n c e C o m p o n e n t of S e e d W e i g h t A m o n g C l o n e s ( C - 1 ) = 17 of + l O O a ^ 3 6 . 1 8 " W i t h i n C l o n e s C (N-1 ) = 1 7 8 2 6 3 . 8 2 0 . 3 6 ^ al = va r iance a m o n g c l o n e s ; al = va r i ance w i t h i n c l o n e s . " S ign i f i can t at P < 0 . 0 1 . la rgest d i f fe rence (Figure 7.2.). T h e es t ima ted b r o a d - s e n s e her i tab i l i ty fo r S i t k a s p r u c e s e e d w e i g h t is m o d e r a t e (h^ = 0 . 3 6 ) . Va r ia t i on in ge rm ina t i on of unso r ted s e e d s is h igh ly s ign i f i can t (P < 0 . 0 1 ) for p re t rea tmen t , c l o n e , and thei r in te rac t ion (Table 7.2). S e e d p re t rea tmen ts a c c o u n t for the la rgest p ropo r t i on of var ia t ion (range 7 4 . 1 2 - 9 3 . 8 5 % ) , w h i l e c l ona l va r ia t ion a c c o u n t s on ly fo r a s m a l l p ropo r t i on of to ta l var ia t ion (range 3 . 0 7 - 5 . 9 1 % ) . T h e in te rac t ion b e t w e e n s e e d p re t rea tment a n d c l o n e a c c o u n t s for 1 . 8 5 - 1 4 . 0 6 % . of to ta l va r ia t i on . T o m a k e the c lona l e f fec t mo re d i sce rn ib le , c o m p l e m e n t a r y a n a l y s e s w e r e c a l c u l a t e d fo r uns t ra t i f i ed and s t ra t i f ied s e e d s (Table 7.3). For uns t ra t i f i ed s e e d s , d i f f e rences in ge rm ina t i on pa rame te rs are h igh ly s ign i f i can t (P < 0 . 0 1 ) w i t h e s t i m a t e s of b r o a d - s e n s e her i tabi l i ty (h^) of all pa rame te rs rang ing f r o m 0 . 7 6 to 0 . 7 9 (Table 7.3). For s t ra t i f ied s e e d s , the d i f f e rences rema in h igh ly s ign i f i can t (P < 0 . 0 1 ) , w i t h b r o a d - s e n s e her i tab i l i ty e s t i m a t e s rang ing f r o m 0 . 7 4 to 0 . 7 8 (Table 7.3). T h e s e d i f f e rences in the her i tabi l i ty e s t i m a t e s for uns t ra t i f ied and s t ra t i f ied s e e d s are c o n s i d e r e d m in ima l in th i s range . For uns t ra t i f i ed s e e d s , ge rm ina t i on of u n s o r t e d s e e d s w a s i n c o m p l e t e w i t h i n the dura t ion of t h e s e tes t s (Figure 7.3). C l o n a l d i f f e rences in ge rm ina t i on c a p a c i t y are subs tan t i a l ( range 4 5 . 7 5 to 9 8 . 0 0 % ) . W h e n s e e d s w e r e s t ra t i f ied (Figure 7.4), var ia t ion in ge rm ina t i on is great ly r e d u c e d (range 9 0 . 0 0 to 9 8 . 5 0 % ) , s i n c e s t ra t i f i ca t ion e n h a n c e d ge rm ina t i on rate fo r all 1 8 c l o n e s . T rad i t i ona l l y , s t ra t i f i ca t ion h a s been u s e d t o o v e r c o m e s e e d d o r m a n c y fo r t empe ra te s p e c i e s ( E d w a r d s , 1 9 8 0 ) ; the r e s p o n s e of s e e d s to s t ra t i f i ca t ion ind ica tes " d o r m a n c y . " C l o n a l d i f f e rences in ge rm ina t ion pa t te rns b e t w e e n uns t ra t i f i ed and s t ra t i f ied s e e d s ind ica te the p r e s e n c e of s o m e degree of d o r m a n c y (Figure 7.3 and Figure 7.4). W h e n ge rm ina t i on of uns t ra t i f i ed and s t ra t i f ied s e e d s w e r e c o m p a r e d fo r ind iv idua l c l o n e s , it w a s f o u n d tha t resu l t s va r ied a m o n g c l o n e s (Figure 7.5). S e e d - s i z e a c c o u n t s for 0 . 0 9 - 1 . 3 9 % of to ta l va r ia t ion in g e r m i n a t i o n , w h i l e c lona l d i f f e rences a c c o u n t for 2 . 9 4 - 5 . 1 3 % (Table 7.4). H o w e v e r , th is s m a l l a m o u n t of var ia t ion in Figure 7.2. D i f f e rences in s e e d w e i g h t o f sma l l and large so r ted s e e d s f r om c lone nos . 5 and 1 5 4 . Table 7.2. Es t ima t i on of va r i ance c o m p o n e n t s , and s i gn i f i cance level for ge rm ina t ion pa ramete rs of 18 S i t ka s p r u c e c l o n e s S o u r c e of Va r i a t i on df Expec ted^ M e a n S q u a r e s G e r m i n a t i o n Paramete rs^ GC PV GV Pre t rea tmen t (P) (P-1) = 1 ol + 4afp + 7 2 0 , 7 4 . 1 2 " 9 3 . 8 5 " 9 2 . 1 7 " C l o n e (C) (C-1) = 17 ol + Sal 5 . 9 1 " 3 . 0 7 " 3 . 2 3 " C X P (P -1 ) (C '1 ) = 17 ol + 1 4 . 0 6 " 1 . 8 5 " 3 . 0 5 " R e s i d u a l P C ( N - 1 ) = 1 0 8 ol 5.91 1 .23 1 .55 ^ 0p = va r iance a m o n g p re t rea tmen t ; al = va r i ance a m o n g c l o n e s ; o^ , = va r iance due to in te rac t ion b e t w e e n p re t rea tmen t and c l o n e ; al = va r iance w i t h i n p re t rea tment w i t h i n c l o n e s . ^ GC = G e r m i n a t i o n C a p a c i t y , the pe rcen tage of s e e d s that had ge rm ina ted at the end of the tes t (A rcs in ) . PV = Peak V a l u e , a m a t h e m a t i c a l e x p r e s i o n of the break of a s i g m o i d cu r ve rep resen t ing a t yp i ca l c o u r s e o f ge rm ina t i on (square root (X + 0 .5) ) . GV = G e r m i n a t i o n V a l u e (Czaba to r 1 9 6 2 ) , (no t rans fo rma t i on ) . " S ign i f i can t at P < 0 . 0 1 . Table 7.3. Es t ima t i on of va r iance c o m p o n e n t s , s i gn i f i cance leve l , and b road -sense her i tabi l i t ies (h^) for ge rm ina t i on pa ramete rs us ing unso r t ed s e e d s of 1 8 S i t k a s p r u c e c l o n e s S o u r c e of Va r i a t i on df E x p e c t e d ' M e a n S q u a r e s Unst ra t i f ied St ra t i f ied ^GC PV GV GC PV GV A m o n g C l o n e s (C-1) = 17 7 8 . 8 7 " 7 6 . 1 3 " 7 7 . 2 2 " 7 4 . 1 4 " 11.iv 7 4 . 3 4 " W i t h i n C l o n e s C ( N - 1 ) = 5 4 ol 2 1 . 1 3 2 3 . 8 7 2 2 . 7 8 2 5 . 8 6 12.22 2 5 . 6 6 h^ 0 . 7 9 0 . 7 6 0 . 7 7 0 . 7 4 0 . 7 8 0 . 7 4 ' al = va r i ance a m o n g c l o n e s ; o f = va r iance w i t h i n c l o n e s . ^ GC = G e r m i n a t i o n C a p a c i t y , the p e r c e n t a g e of s e e d s that had ge rm ina ted at the end of the tes t (A rcs ine ) . PV = Peak V a l u e , a m a t h e m a t i c a l e x p r e s i o n of the break of a s i g m o i d cu r ve represen t ing a t yp ica l c o u r s e of ge rm ina t i on (no t rans fo rma t i on ) . GV = G e r m i n a t i o n V a l u e (Czaba to r 1 9 6 2 ) , (no t rans fo rma t ion ) . " S ign i f i can t at P < 0 . 0 1 . 0 3 6 9 12 1 5 1 8 21 T I M E ( D A Y S ) Figure 7.3. Ge rm ina t i on c u r v e s for unsor ted-uns t ra t i f ied s e e d s of 1 8 S i t k a sp ruce c l ones . 0 3 6 9 1 2 1 5 1 8 21 T I M E ( D A Y S ) Figure 7.4. Ge rm ina t i on c u r v e s fo r unsor ted-s t ra t i f ied s e e d s of 1 8 S i t k a sp ruce c l ones . Figure 7.5. D i f fe rences in germina t ion rate o f unst ra t i f ied and s t ra t i f ied s e e d s of c lone nos . 4 1 1 and 5 1 6 ( low and h igh do rman t s e e d s , respec t i ve ly ) . Table 7.4. Es t ima t ion of va r i ance c o m p o n e n t s , and s ign i f i cance level fo r germina t ion pa ramete rs us ing so r ted s e e d s of 18 S i t ka s p r u c e c l o n e s S o u r c e of Va r ia t i on df E x p e c t e d ' M e a n S q u a r e s G e r m i n a t i o n Parameters^ GC PV GV S e e d S i ze (S) (S-1) = 2 o f + 8 o f , + 1 4 4 0 , 0 .09™ 1 . 3 9 ' * 0 . 7 6 * * C l o n e (C) (C-1) = 17 o f + 2 4 C T ^ 5 . 1 3 * * 2 . 9 4 * * 3 . 8 8 * ' P re t rea tmen t (P) (P-1) = 1 ol + 1 2 a ^ , + 2 1 6 0 , 7 2 . 4 5 * * 8 9 . 8 4 " 8 3 . 7 1 " S x C ( S - 1 ) ( C - 1 ) = 3 4 ol + 0 . 3 6 * 0 . 2 7 " 1 . 0 3 " S x P (S -1 ) (P -1 )= 2 ol + 7 2 0 , , 0 . 8 0 * * 1 .81** 0 . 8 3 " C x P (C -1 ) (P -1 )= 17 ol + 12afp 9 . 8 3 * * 2 . 1 4 * ' 6 . 0 3 " S x C x P ( S - 1 ) ( C - 1 ) ( P - 1 ) = 3 4 ol + 7 . 1 6 ' 0 . 3 7 * * 2 . 5 8 " R e s i d u a l S C P ( N - 1 ) = 3 2 4 ol 4 . 1 7 1 .22 1 .17 ' 03 = va r iance a m o n g s e e d s i z e s ; ol = va r iance a m o n g c l o n e s ; 0, = va r iance b e t w e e n s e e d p re t rea tmen ts ; al,. = va r iance of in te rac t ion e f fec t b e t w e e n s e e d s ize and c l o n e ; 0,, = var iance of in terac t ion e f fec t b e t w e e n s e e d s ize and s e e d p re t rea tment ; (jf, = va r i ance of in te rac t ion e f fec t b e t w e e n c lone and s e e d p re t rea tment ; of^, = va r iance of in te rac t ion e f fec t a m o n g s e e d s i z e , c l o n e and s e e d p re t rea tment ; ol = va r i ance w i th in c l o n e s . ^ GC = G e r m i n a t i o n C a p a c i t y , the p e r c e n t a g e of s e e d s that had ge rm ina ted at the end of the tes t (Arcs in ) . PV = Peak V a l u e , a m a t h e m a t i c a l e x p r e s i o n of the break of a s i g m o i d cu r ve represen t ing a t yp i ca l c o u r s e of ge rm ina t ion (no t rans fo rma t ion ) . GV = G e r m i n a t i o n V a l u e (Czaba to r 1 9 6 2 ) , (no t rans fo rmat ion ) . ™ N o n s ign i f i can t ; " S ign i f i can t at P < 0 . 0 5 ; ** S ign i f i can t at P < 0 . 0 1 . all ge rm ina t i on pa rame te rs a m o n g s e e d s i z e s (excep t G C ) and a m o n g c l o n e s is h igh ly s ign i f i can t ( P < 0 . 0 1 ) (Table 7.4). O n c e aga in , p re t rea tmen t a c c o u n t e d fo r t he la rges t a m o u n t of var ia t ion ( range: 7 2 . 4 5 - 8 9 . 8 4 % ) in ge rm ina t i on (Table 7.4). T o m a k e s e e d s i ze and c lona l e f f ec t s mo re d i sce rn ib le , a s e c o n d , c o m p l e m e n t a r y , ana l ys i s w a s c o n d u c t e d sepa ra te l y for uns t ra t i f i ed and s t ra t i f ied s e e d s (Table 7.5). For uns t ra t i f ied s e e d s , s i ze a c c o u n t s fo r a very s m a l l , i ns ign i f i can t , p ropo r t i on of to ta l var ia t ion (range 0 . 8 9 - 1 . 1 6 % ) (Table 7.5). T h e largest p ropo r t i on , 7 4 % , is due to a m o n g - c l o n e var ia t ion and is h igh ly s ign i f i can t (P< 0 . 0 1 ) (Table 7.5). A l t h o u g h the e f fec t of s e e d s ize on ge rm ina t i on is not s ign i f i can t , the e f fec t of in te rac t ion b e t w e e n c lone and s e e d s ize is h igh ly s ign i f i can t , i nd ica t ing tha t ge rm ina t i on pa rame te rs of d i f fe rent s e e d s i z e s d i f fer a m o n g the 1 8 c l o n e s . For s t ra t i f ied s e e d s , the e f f ec t s of bo th s e e d s i ze and c l o n e o n all ge rm ina t i on pa rame te rs are h igh ly s ign i f i can t (Table 7.5). T h i s i nd i ca tes tha t the e f fec t o f s e e d s i ze is s ign i f i can t fo r the rap id i ty of S i t ka s p r u c e s e e d ge rm ina t i on . D u n l a p and Barne t t ( 1 9 8 3 ) f o u n d a s imi la r i n f l uence of s e e d s i ze in lob lo l ly p ine . G e n e t i c var ia t ion in ge rm ina t i on in con i f e r s has been repo r ted t o be under s t rong ma te rna l gene t i c con t ro l (Bramlet t et al.. 1 9 8 3 ; E l - K a s s a b y et al., 1 9 9 2 ; Ho f f , 1 9 8 7 ) . G e r m i n a t i o n is i n f l uenced by mate rna l e f f ec t s in m o s t p lan ts s i n c e a major po r t ion of the s e e d c o m p o n e n t s (seed c o a t (2n), e n d o s p e r m ( In ) , and half of the e m b r y o ( In)) are materna l l y con t r i bu ted (Perry, 1 9 7 6 ; El lner, 1 9 8 6 ; E l - K a s s a b y a / . , 1 9 9 2 ) . Va r i a t i on in s e e d ge rm ina t i on w a s c o n s i d e r e d to be an adap ta t i on for su rv i va l under e x t r e m e e n v i r o n m e n t a l c o n d i t i o n s ( Ja in , 1 9 8 2 ) . S e e d - d o r m a n c y m e c h a n i s m s vary a m o n g s p e c i e s , s o d i f ferent s e e d p re t rea tmen ts are requ i red . Va r ia t i on in s e e d d o r m a n c y a m o n g fami l ies w i t h i n a s p e c i e s has been repor ted for w e s t e r n w h i t e p ine (Pinus monticola Doug l . ) (Hoff , 1 9 8 7 ) , the ge rm ina t i on of w h i c h i m p r o v e d w i t h dura t ion of s t ra t i f i ca t ion . In con t ras t , Hei t ( 1961 ) c o n c l u d e d tha t ne i ther s t ra t i f i ca t ion nor c h e m i c a l p re t rea tment is requ i red for ge rm ina t i on of S i t ka s p r u c e s e e d s . H o w e v e r , the p resen t Table 7.5. Es t ima t i on of va r i ance c o m p o n e n t s and s i gn i f i cance level for ge rm ina t ion pa ramete rs of 18 S i t ka s p r u c e c l o n e s S o u r c e of Va r ia t i on df E x p e c t e d ' M e a n S q u a r e s Unst ra t i f ied St ra t i f ied ^GC PV GV GC PV GV S e e d S i ze (S) (S-1) = 2 ai + Aài, + 7 2 0 , 0.89"= 1.16"= 1.09"= 9 . 3 9 " * 3 8 . 5 0 " 1 1 . 8 9 " C l o n e (C) (C-1) 1 = 17 ài + Mal 7 4 . 3 3 " 7 3 . 7 5 * " 7 4 . 3 0 " 3 8 . 9 3 " 4 1 . 8 9 * ' 5 6 . 2 3 " S X C (S-1) (C-1) = 3 4 ài + 4 ( 7 f . 7 . 0 1 * * 5 . 8 9 " 6 . 5 3 " " 0 . 0 0 5 . 7 4 " 2 3 . 8 2 " Res idua l SC(N-11 1 = 2 1 5 ol 0 . 1 8 1 9 . 2 0 1 8 . 0 8 5 3 . 6 9 1 3 . 8 7 8 . 0 6 ' 0s, = va r iance a m o n g s e e d s i z e s ; o f = va r iance a m o n g c l o n e s ; of^ = va r iance of in te rac t ion e f fec t b e t w e e n s e e d s i ze and c l one ; o f = va r iance w i t h i n c l o n e s . ^ GC = G e r m i n a t i o n C a p a c i t y , the pe rcen tage of s e e d s that had ge rm ina ted at the end of the tes t (A rcs in ) . PV = Peak V a l u e , a m a t h e m a t i c a l e x p r e s i o n of the break of a s i g m o i d cu r ve represen t ing a t yp i ca l c o u r s e of ge rm ina t ion (no t rans fo rmat ion ) GV = G e r m i n a t i o n V a l u e (Czaba to r 1 9 6 2 ) , (no t rans fo rmat ion ) "= N o n s ign i f i can t " S ign i f i can t at P < 0 . 0 1 . s t u d y d e m o n s t r a t e s tha t s t ra t i f i ca t ion is essen t ia l fo r un i f o rm ge rm ina t i on in th is s p e c i e s . A pe r iod of s t ra t i f i ca t ion of at leas t 21 d a y s is r e c o m m e n d e d t o r e d u c e the ge rm ina t i on d i f f e rences d u e , p robab l y , to d i f ferent ia l d o r m a n c y (Figure 7.3 a n d Figure 7.4). Cor re la t i ons b e t w e e n s e e d s ize and ge rm ina t i on have been repo r ted in s p e c i e s s u c h as Hyptis suaveolens (Wul f f , 1 9 7 2 ) , Pinus strobus (Spur r , 1 9 4 4 ) a n d Acacia holosericea (He l ium, 1 9 9 0 ) . In c o n t r a s t , no s ign i f i can t rea l ized ga in in f a v o u r of large s e e d s ove r s m a l l s e e d s w a s o b s e r v e d in the ge rm ina t i on of m a n y o ther s p e c i e s (Burgar , 1 9 6 4 ; L a r s o n , 1 9 6 3 ; A c k e r m a n and G o r m a n , 1 9 6 9 ) . T h e r e f o r e , s o w i n g unso r t ed s e e d s is r e c o m m e n d e d to m a x i m i z e gene t i c d i ve rs i t y (He l i um, 1 9 7 6 ; L i n d g r e n , 1 9 8 2 ; S i l en a n d O s t e r h a u s , 1 9 7 9 ) . T h e e f fec t of s e e d s ize in S i t k a s p r u c e is c o n s i d e r e d m in ima l in c o m p a r i s o n to the a m o n g - and w i t h i n - c l o n e var ia t ion in g e r m i n a t i o n . 7 . 4 References A c k e r m a n , , R . F . and J . R . G o r m a n . 1 9 6 9 . E f fec t of s e e d w e i g h t o n the s i ze of l odgepo le p ine and w h i t e s p r u c e con ta ine r p lan t ing s t o c k . Pulp Pap. Mag. Can. 7 0 : 1 6 7 - 1 6 9 . A n d e r s s o n , E. 1 9 6 5 . C o n e and s e e d s tud ies in N o r w a y s p r u c e . Stud. For. Suec. 2 3 : 1 - 2 1 4 . A l l e n , G . S . 1 9 5 8 a . F a c t o r s a f fec t i ng the v iabi l i ty and ge rm ina t i on b e h a v i o u r of c o n i f e r o u s s e e d . Par t I. C o n e and s e e d matu r i t y , Tsuga heterophylla (Rafn.) S a r g . For. Chron. 3 4 : 2 6 6 - 2 7 4 . A l l e n , G . S . 1 9 5 8 b . F a c t o r s a f fec t i ng the v iab i l i ty a n d ge rm ina t i on b e h a v i o u r of c o n i f e r o u s s e e d . Par t II. C o n e and s e e d matu r i t y , Pseudotsuga menziesii (Mirb. ) F r a n c o . For. Chron. 3 4 : 2 7 5 - 2 8 2 . A l l e n , G . S . 1 9 6 1 . T e s t i n g Doug las - f i r s e e d for p r o v e n a n c e . Proc. Int. Seed Test. Assoc. 2 6 : 3 8 8 - 4 0 3 . A s s o c i a t i o n of Of f i c ia l S e e d A n a l y s t s . 1 9 7 0 . Ru les fo r t es t i ng s e e d s , Proc. Assoc. Office. Seed Anal. 6 0 : 1 - 1 1 6 . B a g c h i , S . K . , D . N . J o s h i , and D . S . R a w a t . 1 9 9 0 . Va r i a t i on in s e e d s i ze of Acacia spp. Silvae Genet. 3 9 : 1 0 7 - 1 1 0 . Baker , H . G . 1 9 7 2 . S e e d w e i g h t in re lat ion to e n v i r o n m e n t a l c o n d i t i o n s in Ca l i f o rn i a . Ecology 5 3 : 9 9 7 - 1 0 1 0 . B ramle t t , D .L . , T . R . Del l and W . D . Peppe r . 1 9 8 3 . G e n e t i c and mate rna l i n f l u e n c e s o n V i rg in ia p ine s e e d ge rm ina t i on . Silvae Genet. 3 2 : 1 - 4 . Burgar , R . J . 1 9 6 4 . T h e e f fec t of s e e d s i ze on g e r m i n a t i o n , su rv i va l and init ial g r o w t h in w h i t e s p r u c e . For. Chron. 4 1 : 9 3 - 9 7 . C a v e r s , P . B , , and J . L . Harper . 1 9 6 6 . G e r m i n a t i o n p o l y m o r p h i s m in Rumex crispus and Rumex obtusifolius. J. Ecol. 5 4 : 3 6 7 - 3 8 2 . C h o i , S . K . , and K . C . K i m . 1 9 6 9 . S t u d i e s on the c h a r a c t e r i s t i c s of s e l e c t e d p l us - t r ees . 11. T h e d i f fe rence in s e e d ge rm ina t i on c a p a c i t y b e t w e e n p lus- t ree c l o n e s . Res. Rep. Inst. For. Genet. K o r e a 7 : 8 1 - 9 0 . C z a b a t o r , F . J . 1 9 6 2 . G e r m i n a t i o n va lue : A n index c o m b i n i n g s p e e d and c o m p l e t e n e s s of p ine s e e d ge rm ina t i on . For. Sci. 8 : 3 8 6 - 3 9 6 . D a t t a , S . C , M . Evena r i , and Y . G u t t e r m a n . 1 9 7 0 . T h e he te rop las t y of Aegilopsis ovata L. IsraelJ. Bot. 1 9 : 4 6 3 - 4 8 3 . De M a t o s M a l a v a s i , M . , S . G . S ta f fo rd and D . P . L a v e n d e r . 1 9 8 5 . S t ra t i f y i ng , par t ia l ly redry ing and s to r ing Doug las - f i r s e e d s : e f f ec t s on g r o w t h and p h y s i o l o g y du r ing g e r m i n a t i o n . Ann. Sci. For. 4 2 : 3 7 1 - 3 8 4 . Dun lap , J . R . , and J . P . Barnet t . 1 9 8 3 . In f luence of s e e d s i ze o n g e r m i n a t i o n and ear ly d e v e l o p m e n t of lob lo l ly p ine [Pinus taeda L.) g e r m i n a n t s . Can. J. For. Res. 1 3 : 4 0 - 4 4 . E d w a r d s , D . G . W . and Y . A . E l - K a s s a b y . 1 9 8 8 . E f fec t of f l o w e r i n g p h e n o l o g y , da te of c o n e c o l l e c t i o n , c o n e - s t o r a g e t r ea tmen t and s e e d p re t rea tmen t and s e e d p re t rea tment on y ie ld and ge rm ina t i on of s e e d s f r o m a Doug las - f i r s e e d o r c h a r d . For. Ecol. Manage. 2 5 : 1 7 - 2 9 . E d w a r d s , D . G . W . 1 9 8 0 . Ma tu r i t y and qua l i t y of t ree s e e d s - a s ta te -o f - the-ar t r e v i e w . Seed Sci. & Technol. 8 : 6 2 5 - 6 5 7 . E l - K a s s a b y , Y . A . , D . G . W . E d w a r d s and D . W . Tay lo r . 1 9 9 2 . G e n e t i c con t ro l of ge rm ina t i on pa rame te rs in Doug las - f i r a n d i ts i m p o r t a n c e for d o m e s t i c a t i o n . Silvae Genet, (in p ress ) . El lner , S . 1 9 8 6 . G e r m i n a t i o n d i m o r p h i s m s and pa ren t -o f f sp r ing c o n f l i c t in s e e d g e r m i n a t i o n . J. Theor. Biol. 1 2 3 : 1 7 3 - 1 8 5 . Fa l cone r , D . S . 1 9 8 6 . Introduction to quantitative genetics. 2 n d e d . N e w Y o r k : J o h n W i l e y & S o n s , Inc. He i t , C E . 1 9 6 1 . Labo ra to ry ge rm ina t i on and r e c o m m e n d e d tes t i ng m e t h o d s fo r 1 6 s p r u c e Picea s p e c i e s . Proc. Assoc. Off. Seed Anal. 5 1 : 1 6 5 - 1 7 1 . H e l i u m , A . K . 1 9 7 6 . G r a d i n g s e e d by w e i g h t in w h i t e s p r u c e . Tree Planters' Notes 2 7 ( 1 ) : 1 6 , 1 7 , 2 3 . H e l i u m , A . K . 1 9 9 0 . S e e d e c o l o g y in a popu la t i on of Acacia holoserica. Can. J. For. Res. 2 0 : 9 2 7 - 9 3 3 . Ho f f , R . J . 1 9 8 7 . D o r m a n c y in Pinus monticola s e e d re la ted to s t ra t i f i ca t ion t ime , s e e d coa t , and g e n e t i c s . Can. J. For. Res. 1 7 : 2 9 4 - 2 9 8 . In ternat ional S e e d T e s t i n g A s s o c i a t i o n . 1 9 8 5 . In ternat ional ru les for s e e d tes t i ng 1 9 8 5 . Seed Sci. & Technol. 1 3 : 2 9 9 - 5 1 3 . J a i n , S . K . 1 9 8 2 . Va r ia t i on and adap t i ve role of s e e d d o r m a n c y in s o m e annua l g r a s s l a n d s p e c i e s . Bot. Gaz. 1 4 3 : 1 0 1 - 1 0 6 . Ko l le r , D. 1 9 6 2 . P recond i t i on i ng of ge rm ina t i on in le t tuce at t ime of f ru i t r i pen ing . Amer. J. Bot. 4 9 : 8 4 1 - 8 4 4 . K o z l o w s k i , T . T . and A . C . G e n t l e . 1 9 5 9 . In f luence of the s e e d c o a t o n g e r m i n a t i o n , w a t e r a b s o r b t i o n , and O x y g e n up take of Eas te rn w h i t e p ine s e e d . For. Sci 5 : 3 8 9 - 3 9 5 . L a r s o n , M . M . 1 9 6 3 . Initial root d e v e l o p m e n t of p o n d e r o s a p ine s e e d l i n g s as re la ted to ge rm ina t i on date and s ize of s e e d . For. Sci 9 : 4 5 6 - 4 6 0 . L a v e n d e r , D . P . 1 9 5 8 . V iab i l i t y of Doug las - f i r s e e d af ter s t o rage in the c o n e s . Oregon Forest Lands Research Center Res. Notes N o . 3 1 : 1 - 9 . L i nck , A . J . 1 9 6 1 . T h e m o r p h o l o g i c a l d e v e l o p m e n t of the f ru i t of Pisum sativum var . Alaska. Phytomorphology 1 1 : 7 9 - 8 4 . L i n d g r e n , D. 1 9 8 2 . F rac t i ona t i on of s e e d o r cha rd s e e d s by w e i g h t d o e s h a v e gene t i c imp l i ca t i ons . Silva Fenn. 1 6 : 1 5 6 - 1 6 0 . N a y l o r , J . M . 1 9 8 3 . S t u d i e s o n the gene t i c con t ro l of s o m e p h y s i o l o g i c a l p r o c e s s e s in s e e d s . Can. J. Bot. 6 1 : 3 5 6 1 - 3 5 6 7 . Pe r r y , T . O . 1 9 7 6 . M a t e r n a l e f f ec t s o n the ear ly p e r f o r m a n c e of t ree p r o g e n i e s . In Tree physiology and yield improvement, e d . M . G . R. C a n n e l l and F. T. Las t , pp . 4 7 3 - 4 8 1 . N e w Y o r k : A c a d e m i c P r e s s . P i te l , J . P . and B . S . P . W a n g . 1 9 8 5 . P h y s i c a l and c h e m i c a l t r e a t m e n t s to i m p r o v e labora to ry ge rm ina t i on of w e s t e r n wh i t e p ine s e e d s . Can. J. For. Res. 1 5 : 1 1 8 7 - 1 1 9 0 . R o a c h , D . A . and R . D . Wu l f f . 1 9 8 7 . M a t e r n a l e f f ec t s in p l an t s . Ann. Rev. Ecol. Syst. 1 8 : 2 0 9 - 2 3 5 . S a w h n e y , R. and J . M . Nay lo r . 1 9 7 9 . D o r m a n c y s tud ies in s e e d of Avena fatua. 9 . D e m o n s t r a t i o n of gene t i c var iab i l i ty a f fec t ing the r e s p o n s e to t empera tu re dur ing s e e d d e v e l o p m e n t . Can. J. Bot. 5 7 : 5 9 - 6 3 . S a w h n e y , R. and J . M . Nay lo r . 1 9 8 0 . D o r m a n c y s tud ies in s e e d oi Avena fatua. 1 2 . In f luence of t empera tu re o n ge rm ina t ion behav iou r of n o n d o r m a n t fam i l i es . Can. J. Bot. 5 8 : 5 7 8 - 5 8 1 . S c h a a l , B . A . 1 9 8 0 . R e p r o d u c t i v e c a p a c i t y and s e e d s ize in Lupinus texensis. Amer. J. Bot. 6 7 : 7 0 3 - 7 0 9 . S h o u l d e r s , E. 1 9 6 1 . E f fec t of s e e d s i ze o n g e r m i n a t i o n , g r o w t h , and su rv i va l o f s l a s h p ine. J. For. 5 9 : 3 6 3 - 3 6 5 . S i l e n , R., and C . O s t e r h a u s . 1 9 7 9 . R e d u c t i o n of gene t i c base by s i z i ng of bu l ked Doug las - f i r s e e d lo ts . Tree Planters' Notes 3 0 : 2 4 - 3 0 . S o r e n s e n , F . C , and J . F . F rank l in . 1 9 7 7 . In f luence of yea r of c o n e co l l ec t i on o n s e e d w e i g h t and c o t y l e d o n n u m b e r in Abies procera. Silvae Genet. 2 6 : 4 1 - 4 3 . S p u r r , S . H . 1 9 4 4 . E f fec t of s e e d w e i g h t and s e e d or ig in o n the ear ly d e v e l o p m e n t of eas te rn w h i t e p ine . J. Am. Arbor. 2 5 : 4 6 7 - 4 8 1 . T y s o n , H . 1 9 8 9 . G e n e t i c con t ro l of s e e d w e i g h t in f lax (Linum usitatissimum) and poss ib l e imp l i ca t i ons . Theor. Appl. Genet. 7 7 : 2 6 0 - 2 7 0 . Wu l f f , R. 1 9 7 2 . In t rapopu la t iona l var ia t ion in the ge rm ina t i on of s e e d s in Hyptis suaveolens. Ecology 5 4 : 6 4 6 - 6 4 9 . Wu l f f , R . D . 1 9 8 6 . S e e d s ize var ia t ion in Desmodium paniculatum. I. F a c t o r s a f f ec t i ng s e e d s i ze . J. Ecol. 7 4 : 8 7 - 9 7 . C h a p t e r 8 E f f ec t s of S e e d S i z e on S e e d l i n g A t t r i b u t e s 8.1 Introduction T h e e f f i c ien t use of s e e d s in seed l i ng p r o d u c t i o n is a major ob jec t i ve fo r nu rse ry m a n a g e m e n t . S e e d s i z i ng is one m e t h o d of a t ta in ing s o w i n g un i f o rm i t y , p r o d u c i n g more s e e d l i n g s per uni t w e i g h t of s e e d s (Be lcher et al., 1 9 8 4 ) and reduc ing seed l i ng c o m p e t i t i o n . S e e d s i ze is a major trait in agr i cu l tu ra l -c rop i m p r o v e m e n t (Harper et al., 1 9 7 0 ) , s i n c e it is under s t rong gene t i c i n f l uence (Fehr and W e b e r , 1 9 6 8 ; V o i g t et al., 1 9 6 6 ; Pe r r y , 1 9 7 6 ; R o a c h and Wu l f f , 1 9 8 7 ; T y s o n , 1 9 8 9 ) . T h e repor ted e f f ec t s of s e e d s i ze o n seed l i ng g r o w t h in c o n i f e r s have va r ied . A pos i t i ve cor re la t ion of s e e d s i ze w i t h seed l i ng s ize w a s o b s e r v e d in seve ra l p ines (Spurr , 1 9 4 4 ; R igh te r , 1 9 4 5 ) and w h i t e s p r u c e (Picea glauca) (Burgar , 1 9 6 4 ) . S e e d s of m e d i u m s i ze f r o m s l a s h p ine (Pinus elliottii) ( S h o u l d e r s , 1 9 6 1 ) p r o d u c e d b igger seed l i ngs than d id e i ther sma l l or large s e e d s . S u c h a re la t ionsh ip las ted f r o m 5 m o n t h s in Pinus caribaea (Toon et al., 1 9 9 1 ) , to 5 yea rs in p o n d e r o s a p ine (Pinus ponderosa) ( A c k e r m a n and G o r m a n , 1 9 6 9 ) . In con t ras t , no cor re la t ion h a s been f o u n d in m a n y o the r s t u d i e s o n con i f e r s , e v e n at ear ly seed l i ng a g e s ( L a n g d o n , 1 9 5 8 ; L a v e n d e r , 1 9 5 8 ; S inc la i r , 1 9 7 3 ; M a n n , 1 9 7 9 ; S l u d e r , 1 9 7 9 ; D u m r o e s e and W e n n y , 1 9 8 7 ) . T h e d is t r i bu t ion of s e e d w e i g h t in a seed lo t has been o b s e r v e d to vary a m o n g s e e d t rees and yea rs of s e e d co l l ec t i on (He l i um, 1 9 7 6 ; S i l en and O s t e r h a u s , 1 9 7 9 ) . In th is s t u d y , the e f fec t of s e e d s i ze o n the a t t r ibu tes of 8 - m o n t h - o l d S i t k a s p r u c e seed l i ngs is repo r ted . 8 . 2 Materials and Methods C a n a d i a n Pac i f i c Fo res t P r o d u c t s L td . p rov ided the s e e d s fo r th is s t u d y f r o m the S i t ka s p r u c e s e e d o r c h a r d l oca ted in S a a n i c h t o n , Br i t ish C o l u m b i a ( lat i tude 4 8 ° 3 5 ' N , long i tude 1 2 3 ° 2 4 ' W ) . T h e o r c h a r d c o n s i s t s of 1 3 9 c l o n e s (averag ing 9 . 3 r ame ts per c l ones ) s e l e c t e d f r o m e leva t i ons b e t w e e n 0 m and 4 1 5 m on w e s t e r n V a n c o u v e r Is land, W a s h i n g t o n and O r e g o n (Figure 1.1). T h e o r cha rd w a s e s t a b l i s h e d in 1971 in a r a n d o m , s ing le - t ree mix ove r th ree unequa l b l o c k s . In S e p t e m b e r 1 9 9 0 , w ind -po l l i na ted s e e d s w e r e c o l l e c t e d f r o m 1 8 c l o n e s . T h e c lona l ident i t ies of the s e e d s w e r e ma in ta ined dur ing s e e d e x t r a c t i o n . S e e d s f r o m e a c h c l o n e w e r e s o r t e d into t w o s i ze c l a s s e s , large (> 1.41 mm) and sma l l ( < 1.41 m m ) , u s i n g 1 4 - m e s h s c r e e n . S o r t e d s e e d s w e r e kept at 2 ° C unt i l u s e d . S e e d s f r o m bo th s ize c l a s s e s w e r e s o a k e d in w a t e r fo r 2 4 h r s . , a n d t h e n s t ra t i f ied (prechi l led) for 21 d a y s pr ior to s o w i n g . F o l l o w i n g s t a n d a r d c o m m e r c i a l nu r se ry p r a c t i c e s , the s t ra t i f ied s e e d s w e r e s o w n in to 3 1 3 B s t y r o b l o c k s (65 c m ^ 1 6 0 cav i t i es per b lock) in a c o m p l e t e l y r a n d o m i z e d d e s i g n us ing r o w - w i t h i n - s t y r o b l o c k s as rep l i ca t ions (8 seed l i ngs per rep l ica t ion) . S e e d l i n g s in the ou te r r o w s of e a c h s t y r o b l o c k w e r e u s e d as the buf fer . T h e seed l i ngs w e r e g r o w n a c c o r d i n g to the opera t iona l g r o w i n g reg ime for S i t ka s p r u c e . O n e m o n t h af ter s o w i n g , a c o m b i n a t i o n of fer t i l izers and w a t e r w a s app l i ed o n a w e e k l y bas i s unt i l the seed l i ngs w e r e h a r v e s t e d . T h e s t y r o b l o c k s w e r e r e - r a n d o m i z e d eve ry w e e k s o that any e f fec t of s y s t e m a t i c error due to pos i t i on on the nu rse ry b e n c h w o u l d be r e d u c e d . E ight m o n t h s af ter s o w i n g , seed l i ngs w e r e h a r v e s t e d . T h e g r o w i n g m e d i u m w a s w a s h e d f r o m the roo ts , and the seed l i ng w a s cu t at the root co l la r . D iame te r at root co l la r and s h o o t length w e r e m e a s u r e d . R o o t s and s h o o t s w e r e dr ied for 2 4 h rs . at 9 0 ° C and dry w e i g h t per seed l i ng w a s d e t e r m i n e d . S u b s e q u e n t l y , shoo t - r oo t ra t ios w e r e c a l c u l a t e d . T h e da ta se t w a s s u b j e c t e d to ana l ys i s of va r i ance ( A N O V A ) f o l l o w i n g the add i t i ve l inear m o d e l : Y,ii,„ = / / + C, + Sj + CS, + R /S , „ + CR/S i „ „ + e „ „ , w h e r e fj = overa l l m e a n s C, = c l o n e e f fec t ( random ef fec t ) , i = 1 -18 , Sj = e f fec t of s e e d s ize ( f ixed e f fec t ) , j = 1-2, CS | | = e f fec t of in te rac t ion b e t w e e n s e e d s ize and c l o n e . R/S,j,^ = e f fec t of rep l i ca t ion w i t h i n s e e d s i z e , k = 1-6, CR/Siijik = the e f fec t of in te rac t ion b e t w e e n c l o n e and rep l i ca t ion w i t h i n s e e d s i z e , and = res idua l t e r m , 1 = 1-8. T h e S t u d e n t - N e w m a n - K e u l s range tes t w a s u s e d to c o m p a r e the m e a n s o f e a c h g r o w t h pa ramete r . 8 . 3 Resu/ts and Discussion N o s ign i f i can t e f fec t of s e e d s i ze on seed l i ng g r o w t h w a s o b s e r v e d (Table 8.1). A m o n g -c l o n e var ia t ion fo r all pa ramete rs w a s h igh ly s ign i f i can t (P< 0 , 0 1 ) , and a c c o u n t e d fo r 5 . 1 3 % of the to ta l s u m of s q u a r e s for d ry w e i g h t and 1 2 . 2 8 % for he igh t (Table 8.1). T h i s var ia t ion is ve ry sma l l in c o m p a r i s o n to the a m o u n t of var ia t ion w i t h i n rep l i ca t ions ( f rom 5 8 . 7 5 % to 7 8 . 5 5 % for he igh t and s h o o t d ry we igh t ) . T h e r e f o r e , c o m p e t i t i o n a m o n g s e e d l i n g s of d i f ferent rep l i ca t ions as we l l as gene t i c var ia t ion w i t h open-po l l i na t i on fami l i es c o u l d be c o n s i d e r e d as c a u s e s of var ia t ion in seed l i ng a t t r ibu tes . H o w e v e r , t h e s e p a r a m e t e r s a p p e a r e d to be re lated to e a c h o ther (r^= 0 . 9 8 ) . C l o n e no . 4 1 1 p r o d u c e d the h ighes t m e a n he igh t , s h o o t d ry w e i g h t , and to ta l d ry w e i g h t (Table 8.2). C l o n e n o s . 1 5 4 and 4 1 6 p r o v i d e d the h i ghes t m e a n s in root d ry w e i g h t and shoo t - r oo t rat io, r espec t i ve l y . C l o n a l d i f f e rences a m o n g the m e a n s of t hese a t t r ibu tes are w i t h i n the s t o c k s p e c i f i c a t i o n s for 3 1 3 B s t y r o b l o c k s , 1 + 0 S i t k a s p r u c e seed l i ngs (1991 Br i t ish C o l u m b i a M i n i s t r y of Fo res t s Cu l l i ng S t a n d a r d s , S i l v i cu l t u re B r a n c h , V i c t o r i a , B . C . ) , t he re fo re , ind ica t ing no opera t iona l s i gn i f i cance to the n u r s e r y m a n . T h e lack of any s ign i f i can t e f fec t of s e e d s i ze on seed l i ng g r o w t h in S i t k a s p r u c e (Table 8.1) ag rees w i t h the f i nd ings for o ther con i f e rs ( L a n g d o n , 1 9 5 8 ; L a v e n d e r , 1 9 5 8 ; S inc la i r , 1 9 7 3 ; M a n n , 1 9 7 9 ; S lude r , 1 9 7 9 ; D u m r o e s e and W e n n y , 1 9 8 7 ) . T h e p o s i t i v e co r re la t ion b e t w e e n s e e d s ize and ear ly s t a g e s of seed l i ng g r o w t h o b s e r v e d in s o m e c o n i f e r s (Shou lde rs , 1 9 6 1 ; S inc la i r , 1 9 7 3 ; T o o n et al., 1 9 9 1 ) dec l i ned w i t h age , w i t h s o m e va r ia t i on a c c o r d i n g to s p e c i e s . For e x a m p l e , in Pinus caribaea Mo re le t var hondurensis Barret t a n d G o l f e r i , the Table 8.1. Va r i a t i on in d iamete r (Dia), he igh t (Ht), s h o o t d ry w e i g h t ( S D W ) , root d ry w e i g h t ( R D W ) , to ta l d ry w e i g h t ( T D W ) , and shoo t / r oo t d r y - w e i g h t rat io (S/R) of S i t ka s p r u c e seed l i ngs f r o m t w o s e e d s i zes of 18 c l o n e s S o u r c e of d f E x p e c t e d ^ S u m Squa re (%) Va r ia t i on M e a n S q u a r e s D ia . Ht . S D W R D W T D W S / R C l o n e s (C) 17 ol + + 2Qol 6 . 6 0 " 1 2 . 2 8 " * 5 . 1 9 " 7 . 4 8 * * 5 . 1 3 " " 1 0 . 7 6 * " S e e d s ize (S) 1 ol + + 1 4 4 a f , , + 48CT?, + 8 6 4 0 3 0 . 0 0 0 .04™ 0.14™ 0.02™ 0.11™ 0.09™ C X S 17 ol + + 4 8 a f , 1.74™ 2.66™ 1.58™ 1.91™ 1.32™ 3 . 5 6 " R e p / S i z e (R/S) 1 0 ol -1- 8a ^ . s + 1 4 4 a f , 3 1.59* 2 . 8 8 " 1 .69" 0 .59™ 1.30™ 1.34™ C X R / S 1 7 0 ol + 8 o f . 3 1 3 . 7 2 * * 2 3 . 3 9 * * 1 2 . 8 5 " 2 0 . 5 9 " " 1 3 . 7 4 " 1 8 . 9 9 " " R e s i d u a l 1 4 2 6 ol 7 6 . 3 5 5 8 . 7 5 7 8 . 5 5 6 9 . 0 5 7 8 . 4 0 6 5 . 2 6 not s ign i f i can t , s ign i f i can t at P < 0 . 0 5 . s ign i f i can t at P < 0 . 0 1 . C = 1 8 , S = 2 , R = 6. al = va r i ance a m o n g c l o n e s ; 0 , = va r i ance b e t w e e n s e e d s i z e s ; o f , = va r iance due to in te rac t ion b e t w e e n c l o n e s and s e e d s i z e s ; of/5 = va r i ance a m o n g rep l ica t ion w i t h i n s e e d s i z e s ; of,/, = va r iance due to in te rac t ion b e t w e e n c l o n e s and rep l ica t ion w i t h i n s e e d s i z e s ; o f = res idua l t e r m . ro Table 8.2. Student-Newman-Keuls multiple-range tests for diameter at root collar, height, shoot dry weight, root dry weight, total dry weight, and root-shoot ratio of eight-month-old seedlings from 18 Sitka spruce clones' Diameter Height Shoot Dry Weight Root Dry weight Total Dry Weight Shoot-Root Ratio Clone Means (mm) Clone Means (cm) Clone Means (g.) Clone Means (g.) Clone Means (g.) Clone Means 4 0 5 3.04a^ 411 31 .37a 411 1 .82a 154 0.62a 411 2.38a 416 3.56a 421 2 .94 b 514 2 9 . 3 0 b 5 1 4 1 .78ab 44 0.60ab 4 0 5 2.36a 411 3.35 b 514 2.91 be 4 0 5 29 .14 b 4 0 5 1 .77abc 516 0.60ab 514 2 .35a 15 3.31 be 411 2 .90 bo 61 28 .65 be 5 1 .74abc 20 0.60ab 5 2.34ab 421 3.30 be 92 2 .88 bod 5 28 .48 bed 421 1 .72abcd 5 0.59abc 154 2.31ab 68 3.26 be 521 2.87 bed 24 2 8 . 2 3 bcde 154 1 .69abcde 4 0 5 0.58abe 516 2.27abc 5 1 4 3.19 bed 5 2 .86 bcde 15 27 .97 ede 516 1 .67 bcdef 521 0.57abed 4 4 2.27abe 4 0 5 3.11 ede 61 2 .84 bcdef 421 27 .87 edef 4 4 1 .67 bcdef 92 0.57abed 421 2.26abed 113 3.09 edef 105 2 .83 bcdef 521 27 .87 edef 92 1 .67 bcdef 105 0.57abed 92 2.24abed 24 3.08 edef 154 2 .83 bcdef 20 27 .84 edef 521 1 .65 bcdef 514 0.57abed 521 2.22abede 61 3.00 defg 15 2 .80 bcdef 92 27 .59 edefg 68 1 .65 bcdef 411 0.56 bcde 105 2.21 abode 92 2.98 defg 44 2 .79 edef 516 2 7 . 5 0 cdefg 105 1 .64 bcdef 421 0.54 ede 68 2.18abede 521 2.96 defgh 416 2 .78 edef 44 27 .28 defg 24 1 .63 bcdef 24 0.54 ede 20 2.18abcde 5 2 .95 defgh 20 2.76 def 68 27 .02 efg 15 1 .62 edef 68 0 .53 de 24 2,17abede 105 2 .93 defgh 24 2 .75 def 113 27.01 efg 4 1 6 1 .61 edef 61 0.52 de 15 2 .13 bcde 516 2.87 efgh 516 2 .75 def 416 26 .67 fg 20 1 .58 def 15 0.51 ef 4 1 6 2.09 ede 44 2.84 fgh 113 2 .73 ef 154 26 .57 g 61 1 .54 ef 113 0.51 ef 61 2.07 de 154 2 .80 gh 68 2 .70 f 105 26 .48 g 113 1 .53 f 416 0.47 f 113 2.04 e 20 2.72 h ' 1991 British Columbia Ministry of Forests Culling Standard for 3 1 3 B styrobloek Sitka spruce 1 +0 seedlings (Diameter: min = 2.2 mm; target = 2.6 mm; Height: min = 14 em, target = 18 cm ; Root dry weight: min = 0.5 g, target = 0.7 g). ^ Clones sharing a common letter are not significantly different at P < 0 .05 . pos i t i ve , s t r o n g , co r re la t ion o b s e r v e d in 2 - m o n t h - o l d s e e d l i n g s d i s a p p e a r e d af ter 7 m o n t h s (Toon et al., 1 9 9 1 ) . S im i la r l y , th is e f fec t a lso d i s a p p e a r e d af ter 3 m o n t h s (S inc la i r , 1 9 7 3 ) or one yea r (Lavender , 1 9 5 8 ) in Doug las - f i r , 3 yea rs in s l a s h p ine ( S h o u l d e r s , 1 9 6 1 ), and 5 yea rs in p o n d e r o s a p ine ( A c k e r m a n and G o r m a n , 1 9 6 9 ) . R igh te r ( 1 9 4 5 ) fu r ther i nd i ca ted that there w a s no re la t ionsh ip b e t w e e n s e e d w e i g h t and inherent seed l i ng v igou r . It is i n te res t ing to no te that in w h i t e s p r u c e , a s p e c i e s c l o s e l y re la ted to S i t k a s p r u c e , a pos i t i ve re la t ionsh ip b e t w e e n s e e d s ize and seed l i ng s i ze w a s o b s e r v e d by Burgar ( 1 9 6 4 ) . T h e lack of any e f fec t of s e e d s ize o n seed l i ng s i ze at 8 m o n t h s in S i t ka s p r u c e in th is s t u d y i nd i ca tes that ex t rapo la t i ng b e t w e e n e v e n re lated s p e c i e s c a n be p r o b l e m a t i c . T h i s s t u d y has s h o w n that the e f fec t of c l o n e on seed l i ng a t t r ibu tes c a n be greater t han the e f fec t of s e e d s i z e . 8 . 4 References A c k e r m a n , R . F . and J . R . G o r m a n . 1 9 6 9 . E f fec t of s e e d w e i g h t on the s i ze of l odgepo le p ine and w h i t e s p r u c e con ta ine r -p lan t i ng s t o c k . Pulp Pap. Mag. Can. 7 0 ( c ) : 1 6 7 - 1 6 9 . Be l che r , E . W . , G . N . L e a c h , and H . H . G r e s h a m . 1 9 8 4 . S i z i n g s l a s h p ine s e e d s as a nu rse ry p r o c e d u r e . Tree Planters' Notes 3 5 : 5 - 1 0 . Burgar , R . J . 1 9 6 4 . T h e e f fec t of s e e d s i ze o n g e r m i n a t i o n , su rv i va l and init ial g r o w t h in w h i t e s p r u c e . For. Chron. 4 1 : 9 3 - 9 7 . D u m r o e s e , R . K . and D. L. W e n n y . 1 9 8 7 . S o w i n g s i z e d s e e d of w e s t e r n w h i t e pine in a con ta i ne r i zed nu rse ry . West. J. Appl. For. 2 : 1 2 8 - 1 3 0 . Fehr , W . R . , and C R . W e b e r . 1 9 6 8 . M a s s se lec t i on by s e e d s i ze and s p e c i f i c g rav i t y in s o y b e a n p o p u l a t i o n s . Crop Sci. 8 : 5 5 1 - 5 5 4 . Ha rpe r , J . L . , P . H . Love l l , and K . G . M o o r e . 1 9 7 0 . T h e s h a p e s and s i z e s of s e e d s . Ann. Rev. Ecol. Syst. 1 1 : 3 2 7 - 3 5 6 . H e l i u m , A . K . 1 9 7 6 . G r a d i n g s e e d by w e i g h t in w h i t e s p r u c e . Tree Planters' Notes 2 7 : 1 6 , 1 7 , 2 3 . L a n g d o n , O . G . 1 9 5 8 . C o n e and s e e d s ize of s o u t h F lor ida s l a s h pine and thei r e f f ec t s on seed l i ng s ize and s u r v i v a l . J. For. 5 6 : 1 2 2 - 1 2 7 . L a v e n d e r , D . P . 1 9 5 8 . E f fec t of s e e d s ize o n Doug las - f i r seed l i ngs . O r e g o n Fo res t L a n d s R e s e a r c h Cen te r , Research Note N o . 3 2 : 1 - 9 . M a n n , W . F . , J r . 1 9 7 9 . Re la t i onsh ips of s e e d s i z e , n u m b e r of c o t y l e d o n s , and init ial g r o w t h of s o u t h e r n p ines . Tree Planters' Notes 3 0 : 2 2 - 2 6 . Per ry , T . O . 1 9 7 6 . M a t e r n a l e f fec ts o n the ear ly p e r f o r m a n c e of t ree p r o g e n i e s . In Tree physiology and yield improvement, e d . M . G . R . Canne l l and F .T . L a s t , pp . 4 7 3 - 4 8 1 . N e w Y o r k : A c a d e m i c P r e s s . R igh te r , F.I. 1 9 4 5 . P i n u s : T h e re la t ionsh ip of s e e d s i ze and seed l i ng s ize to inherent v igour . J. For. 4 3 : 1 3 1 - 1 3 7 . R o a c h , D . A . and R . D . Wu l f f . 1 9 8 7 . M a t e r n a l e f fec ts in p lan ts . Ann. Rev. Ecol. Syst. 1 8 : 2 0 9 -2 3 5 . S h o u l d e r s , E. 1 9 6 1 . E f fec t of s e e d s i ze o n g e r m i n a t i o n , g r o w t h , and su rv i va l of s l a s h p ine . J. For. 5 9 : 3 6 3 - 3 6 5 . S i l e n , R., and C . O s t e r h a u s . 1 9 7 9 . R e d u c t i o n of gene t i c base by s i z ing of bu l ked Doug las - f i r s e e d lo ts . Tree Planters' Notes 3 0 : 2 4 - 3 0 . S inc la i r , W . A . 1 9 7 3 . D e v e l o p m e n t of w e i g h t var ia t ion in Doug las - f i r s e e d l i n g s . For. Sci. 1 9 : 1 0 5 - 1 0 8 . S l u d e r , E .R. 1 9 7 9 . T h e e f f ec t s of s e e d and seed l i ng s i ze o n su rv i va l and g r o w t h of lob lo l ly p ine . Tree Planters' Notes 3 0 : 2 5 - 2 8 . S p u r r , S . H . 1 9 4 4 . E f fec t of s e e d w e i g h t and s e e d or ig in on the ear ly d e v e l o p m e n t of eas te rn w h i t e p ine . J. Am. Arbor. 2 5 : 4 6 7 - 4 8 1 . T o o n , P . G . , R . J . H a i n e s and M . J . D ie te rs . 1 9 9 1 . Re la t i onsh ip b e t w e e n s e e d w e i g h t , ge rm ina t i on t ime and seed l i ng he igh t g r o w t h in Pinus caribaea M o r e l e t var . hondurensis Barret t and Go l f a r i . Seed Sci. & Technol. 1 9 : 3 9 7 - 4 0 2 . T y s o n , H . 1 9 8 9 . G e n e t i c con t ro l of s e e d w e i g h t in f l ax (Linum usitatissimum) and poss ib l e imp l i ca t i ons . Theor. Appl. Genet. II.IQO-TJO. V o i g t , R .L . , C O . G a r d n e r , O . J . W e b s t e r . 1 9 6 6 . Inher i tance of s e e d s i ze in S o r g h u m , Sorghum vulgare P e r s . Crop Sci. 6 : 5 8 2 - 5 8 6 . C h a p t e r 9 C o n c l u s i o n s S e v e r a l b io log ica l a s p e c t s of S i t ka s p r u c e o r c h a r d - p r o d u c e d s e e d s w e r e i nves t i ga ted in a s e e d o r c h a r d l o c a t e d o n the S a a n i c h P e n i n s u l a of V a n c o u v e r Is land, B . C . It w a s f o u n d that : 1 ) O f 1 6 e n z y m e loc i that c o u l d be s c o r e d re l iab ly , t w o exh ib i t ed no de tec tab le var ia t ion wh i l e 14 a p p e a r e d to e a c h p o s s e s s at leas t t w o a l l o z y m e va r ian ts . T h e m o d e of i nher i tance for m o s t of the p o l y m o r p h i c loc i c o n f o r m e d to M e n d e l i a n e x p e c t a t i o n s , a l t hough 11 ou t of 2 4 al le l ic c o m b i n a t i o n s d i sp l ayed m a r k e d seg rega t i on d i s t o r t i on ; 2) N o l inkage w a s o b s e r v e d fo r 1 6 d o u b l y h e t e r o z y g o u s c o m b i n a t i o n s , h o w e v e r , a lack of i ndependen t seg rega t i on b e t w e e n one set of loc i {PGM-1 •.PGM-2) w a s a t t r ibu ted to their appa ren t l i nkage ; 3) G e n e d i ve rs i t y a n d h e t e r o z y g o s i t y e s t i m a t e s w e r e c o m p a r a t i v e l y h igh in bo th the s t u d i e d s e e d o r c h a r d and 1 0 natura l popu la t i ons p rev i ous l y s tud ied by Y e h and E l - K a s s a b y ( 1 9 8 0 ) . T h e o r c h a r d popu la t i on s h o w e d s ign i f i can t l y h igher n u m b e r of a l le les per l o c u s and p e r c e n t a g e o f p o l y m o r p h i c l oc i . S a m p l i n g b read th of the s e e d o r c h a r d ' s paren t t rees has been ident i f ied as the major c a u s e fo r the o b s e r v e d i n c r e a s e d level of gene t i c var iab i l i ty ; 4) S ign i f i can t leve ls of i nb reed ing , b a s e d o n s ing le - and mu l t i - l ocus e s t i m a t e s of o u t c r o s s i n g (Ri t land and E l - K a s s a b y , 1 9 8 5 ) ex i s t ed in the o r c h a r d ' s s e e d c r o p . T h i s s ign i f i can t i nb reed ing leve l w a s great ly a f f ec ted by the p r e s e n c e of a s ign i f i can t leve l of i nb reed ing in the l o w e r b r a n c h e s . S i n g l e - l o c u s e s t i m a t e s of PGM-2 i nd i ca ted the p r e s e n c e of n o n - r a n d o m mat ing c a u s e d by g e n o t y p i c se l ec t i on for fer t i l i za t ion ( i .e., h o m o z y g o t i c fer t i l izat ion) s im i la r to that o b s e r v e d on c o r n (Bi j isma et a/., 1 9 8 6 ) ; 5) Paren ta l i m b a l a n c e w a s o b s e r v e d fo r the t w o c r o p y e a r s s t u d i e d . M a t e r n a l con t r i bu t i on b a s e d o n s e e d - c o n e y ie ld w a s s imi la r to that b a s e d o n f i l led s e e d y i e l d , h o w e v e r , m o s t c l o n e s d id not ma in ta in thei r ranks w h e n the c o m p a r i s o n b e t w e e n c o n e v s . s e e d y ie ld w a s m a d e . E f fec t i ve f e m a l e popu la t i on n u m b e r ( C r o w and K i m u r a , 1 9 7 0 ) p r o v i d e d an es t ima te of the dev ia t i on f r o m the ideal equa l con t r i bu t i on , h o w e v e r , it d id not p rov ide ins igh t in to the re la t ionsh ip b e t w e e n rep roduc t i ve ene rgy and rep roduc t i ve s u c c e s s ; 6) S e e d s ize and ge rm ina t i on pa rame te rs ( G C , P V and G V ) w e r e under g e n e t i c c o n t r o l . B r o a d - s e n s e her i tab i l i ty w a s m o d e r a t e (0 .36) fo r s e e d s i ze and h igh ( 0 . 7 4 - 0 . 7 8 ) fo r ge rm ina t i on t ra i ts . S e e d p re t rea tment ( i .e., s t ra t i f i ca t ion) w a s s u c c e s s f u l in a c h i e v i n g ge rm ina t i on un i fo rm i t y ; 7) S e e d v igour tes t ( i .e., a c c e l e r a t e d aging) p r o d u c e d s ign i f i can t c l ona l d i f f e rences in ge rm ina t i on pa rame te rs ind ica t ing that the degree of s e e d de te r io ra t ion is c l o n e - s p e c i f i c . T h e sho r t t r ea tmen t t es t ( i .e., 3 -6 days ) p r o d u c e d an i nc rease in ge rm ina t i on p a r a m e t e r s w h i l e longer t r e a t m e n t s w e r e a s s o c i a t e d w i t h m a r k e d dec l i ne . T h e i m p r o v e m e n t of ge rm ina t i on t ra i ts a f ter sho r t t r ea tmen t t ime needs to be i nves t i ga ted fo r ope ra t i ona l exp lo i t a t i on ; 8) Un l i ke o ther s p r u c e s p e c i e s , s e e d s i ze s h o w e d no s ign i f i can t e f fec t o n seed l i ng a t t r ibu tes in S i t ka s p r u c e . C l o n a l d i f f e rences w e r e s ign i f i can t , but t h e s e d i f f e rences w e r e w i t h i n the opera t iona l s p e c i f i c a t i o n s for the s p e c i e s . 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0075160/manifest

Comment

Related Items