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

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ASPECTS OF THE SEED BIOLOGY OF ORCHARD-PRODUCED SITKA SPRUCE SEEDS  by  K o w i t Chaisurisri B . S c . (Forestry), Kasetsart University, 1 9 8 0 IVl.Sc. ( F o r e s t r y ) , K a s e t s a r t U n i v e r s 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 THE R E Q U I R E M E N T S FOR T H E DEGREE O F DOCTOR OF PHILOSOPHY in THE FACULTY OF G R A D U A T E STUDIES (Faculty of Forestry)  W e accept this thesis as c o n f o r m i n g to the required standard  T H E UNIVERSITY O F BRITISH C O L U M B I A July 1992 ® Kowit Chaisurisri, 1 9 9 2  In  presenting  degree freely  at  this  the  available  copying  of  department publication  in  partial  fulfilment  of  the  University  of  British  Columbia,  I  agree  for  this or of  thesis  reference  thesis by  this  for  his thesis  and  study.  scholarly  or for  her  financial  of  fbres'l'  geîein*-eS  The University of British Columbia Vancouver, Canada  Date  DE-6  (2/88)  AMjîn>  4.  gain  shall  that  agree  purposes may  representatives.  permission.  Department  I further  requirements  be  It not  that  the  be  an  advanced  Library shall  permission for  granted  is  for  by  understood allowed  the  make  extensive  head  that  without  it  of  copying my  my or  written  Abstract  B i o l o g i c a 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 t u d i e d for o n e s e e d o r c h a r d located 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 Island, B . C . T h e s e a s p e c t s i n c l u d e : t h e d e t e r m i n a t i o n t h e l e v e l o f g e n e t i c d i v e r s i t y in t h e  orchard  p o p u l a t i o n a n d its c o m p a r i s o n t o t h e s p e c i e s ' r a n g e e s t i m a t e s , t h e e s t i m a t i o n o f t h e d e g r e e of i n b r e e d i n g in t h e o r c h a r d , s e e d a n d c o n e c r o p p a r e n t a l b a l a n c e o v e r t w o  crop y e a r s , the  g e n e t i c control of g e r m i n a t i o n p a r a m e t e r s a n d s e e d s i z e , the i m p a c t of s i m u l a t e d  long-term  s t o r a g e on the genetic c o n s t i t u t i o n of bulk s e e d c r o p s , a n d the relationships b e t w e e n s e e d size a n d g e n e t i c o r i g i n o n t h e i r s e e d l i n g a t t r i b u t e s . It w a s f o u n d t h a t : (1 ) M o s t o f t h e p o l y m o r p h i c a l l o z y m e markers investigated 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 ratio, a n d no l i n k a g e b e t w e e n a n y pair of t h e s e l o c i w a s o b s e r v e d ; (2) T h e l e v e l o f g e n e t i c d i v e r s i t y p r e s e n t in t h e s e e d o r c h a r d p o p u l a t i o n i n d i c a t e d t h a t p h e n o t y p i c s e l e c t i o n o f p a r e n t t r e e s d i d  not  r e d u c e t h e l e v e l o f d i v e r s i t y ; (3) S i g n i f i c a n t r a t e s o f 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 h e s e e d orchard population  (t„ = 0.875).  M o s t o f t h i s i n b r e e d i n g o c c u r r e d in l o w e r b r a n c h e s ;  (4)  Parental imbalance w a s f o u n d for the t w o c r o p s investigated, h o w e v e r , i m p r o v e m e n t over time w a s o b s e r v e d , i n d i c a t i n g a n a g e e f f e c t ; (5) G e r m i n a t i o n p a r a m e t e r s ( G C , P V a n d G V ) a n d s e e d s i z e w e r e u n d e r g e n e t i c c o n t r o 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 h e r i t a b i l i t i e s o f 0 . 7 4 - 0 . 7 8 a n d 0 . 3 6 , r e s p e c t i v e l y ; (6) A r t i f i c i a l a g i n g o f s e e d (i.e., a c c e l e r a t e d a g i n g ) i n d i c a t e d t h a t r e d u c t i o n in r a t e s o f g e r m i n a t i o n w e r e c l o n e - s p e c i f i c ; a n d (7) S e e d s i z e s h o w e d n o e f f e c t o n s e e d l i n g attributes, but a significant effect on germination speed 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 2 . 2 Materials 2.3 Results  9 and Methods  10  and Discussion  16  2 . 3 . 1 Monomorphic 2 . 3 . 2 Segregation  Loci of Polymorphic  2 . 3 . 3 Summary 2 . 3 . 4 Linkage  16 Loci  16 23  Analyses  24  2 . 4 Conclusion  27  2 . 5 References  29  Chapter 3. Genetic Diversity  33  3.1 Introduction  33  2.2 Materials 3 . 2 . 1 Isozyme 2.2.2  Natural  and Methods Assay Populations  34 35 35  3 . 2 . 3 Data Analysis  35  3.3 Results  35  and Discussion  3 . 4 References  47  Chapter 4. Mating System  50  4.1 Introduction 4.2 Materials 4.3 Results  50 and Methods  51  and Discussion  53  4 . 3 . 1 y4//e//c Frequencies  53  4 . 3 . 2 Outcrossing  53  Rate  4 . 4 References  64  Chapter 5 . Parental Balance  69  5.1 Introduction 5.2 Materials  69 and Methods  70  b.3 Results  and Discussion  71  5.3.1  1988  Orchard  Crop  71  5 . 3 . 2 1990  Orchard  Crop  74  5 . 4 References  85  Chapter 6. Accelerated A g i n g  87  6.1 Introduction  87  6 . 2 Materials  and Methods  88  6 . 2 . 1 Accelerated  Aging  89  6 . 2 . 2 Germination  Test  89  Analysis  90  6.2.3  Statistical  6.3 Results  and Discussion  6 . 4 References Chapter 7 . Germination  91 99 101  7.1 Introduction 7 . 2 Materials 7 . 2 . 1 Seed  101 and Methods  Weight  7 . 2 . 2 Germination 7.2.3  Statistical  7.3 Results  102 Test  103  Analysis  103  and Discussion  7.4 References  8.1 Introduction  8 . 3 Results  104 118  Chapter 8. Seed Size and Seedling Attributes  8.2 Materials  102  121 121  and Methods  121  and Discussion  123  8 . 4 References Chapter 9. Conclusions  127 129  L i s t of T a b l e s  Table 2. /.  Page L i s 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 , a n d G e l B u f f e r s y s t e m s used for Sitka spruce electrophoresis  12  E l e c t r o p h o r e t i c P r o c e d u r e : A . b u f f e r s y s t e m ( E l - K a s s a b y et al. 1 9 8 2 a ) a n d B. stain recipes (Yeh and O ' M a l l e y , 1980)  13  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 , allele 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 , a n d r e f e r e n c e s t o i n h e r i t a n c e s t u d i e s in o t h e r c o n i f e r s  17  L o g - l i k e l i h o o d 6 - t e s t s o n s e g r e g a t i o n r a t i o s of 1 3 p o l y m o r p h i c l o c i in S i t k a spruce seeds  20  G o o d n e s s - o f - f i t a n a l y s e s f o r p a i r - w i s e c o m b i n a t i o n s of l o c i in S i t k a s p r u c e . L o g - l i k e l i h o o d t e s t s j o i n t i n d e p e n d e n c e a s s o r t m e n t (G,). P a r t i t i o n e d tests t e s t s e g r e g a t i o n at l o c u s A (xi), B (xl) a n d j o i n t s e g r e g a t i o n Off)  25  2.6.  T e s t of i n d e p e n d e n c e f o r t h e PGM-/.•PGM-2 c o m b i n a t i o n  26  2.7.  T h r e e - w a y log-likelihood combination  2.2.  2.3.  2.4.  2.5.  3.1.  test  for  independence  for  the  PGM-1:6PG-2 28  L o c a t i o n of 1 0 l U F R O n a t u r a l p o p u l a t i o n s of S i t k a s p r u c e t o w h i c h r e s u l t s from the seed orchard were compared  36  A l l e l i c f r e q u e n c i e s at 1 3 l o c i in 1 0 l U F R O n a t u r a l p o p u l a t i o n s ( Y e h a n d E l K a s s a b y , 1 9 8 0 ) a n d s e e d o r c h a r d ( S . O . ) of S i t k a s p r u c e  37  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 n a t u r a l p o p u l a t i o n s a n d s e e d o r c h a r d  39  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 l e l e s p e r l o c u s , a n d p e r c e n t a g e o f l o c i p o l y m o r p h i c in 1 0 l U F R O n a t u r a l p o p u l a t i o n s a n d s e e d orchard  41  G e n e - d i v e r s i t y statistics estimates for 1 3 p o l y m o r p h i c loci a v e r a g e d o v e r 1 0 n a t u r a l p o p u l a t i o n s a n d o v e r 11 p o p u l a t i o n s ( 1 0 n a t u r a l a n d o n e s e e d orchard)  43  Genetic distances b e t w e e n Sitka spruce populations, 10 natural populations and a s e e d orchard (SO)  44  Allelic frequencies for outcrossing pollen and maternal gene pools for s a m p l e s obtained f r o m the upper and lower c r o w n portions a n d c o m b i n e d for a Sitka spruce seed orchard  54  3.2.  3.5.  3.6.  4.1.  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 o f o u t c r o s s i n g f o r s a m p l e s obtained f r o m the upper a n d l o w e r levels of the c r o w n of clonal S i t k a s p r u c e t r e e s in a m a n a g e d s e e d o r c h a r d  55  E s t i m a t e s o f p o l l e n - a l l e l i c f r e q u e n c i e s f o r 3 ( w i t h PGM-2) a n d 2 l o c i ( w i t h o u t PGM-2) c o r r e s p o n d i n g t o m a t e r n a l g e n o t y p e s ( n u m b e r o f p a r e n t t r e e s in parentheses)  57  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 maternal g e n o t y p e s , a n d o u t c r o s s i n g rate ( t „ )  58  4.5.  E f f e c t o f c r o w n l e v e l 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  60  4.6.  Comparison populations  4.3.  4.4.  5.1.  5.2.  5.3.  5.4.  6.1.  7. / .  of  mating-system  estimates  in  seed  orchard  and  natural 62  C o n e a n d filled s e e d p r o d u c t i o n p o p u l a t i o n n u m b e r (N,,)  in t h e  1988  crop,  and  their  effective 73  A N O V A : e s t i m a t e s of variance 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 production  75  1 9 9 0 c o n e , s e e d , filled s e e d , v i a b l e s e e d p r o d u c t i o n , p o p u l a t i o n n u m b e r (N,,)  79  and their  effective  A N O V A : e s t i m a t e s of variance 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 production  81  V a r i a t i o n in g e r m i n a t i o n of 6 S i t k a 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 a g i n g ( A A ) a n d s e e d p r e t r e a t m e n t ( v a l u e s are p e r c e n t a g e s o f t h e t o t a l m e a n squares)  92  Estimation  of  variance  components,  significance  level, and  broad-sense  h e r i t a b i l i t i e s (h^) f o r i n d i v i d u a l s e e d w e i g h t o f 1 8 S i t k a s p r u c e c l o n e s 7.2.  7.3.  7.4.  7.5.  8.1.  E s t i m a t i o n of variance 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 level for  106  germination  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  109  E s t i m a t i o n of v a r i a n c e c o m p o n e n t s , s i g n i f i c a n c e l e v e l , a n d b r o a d - s e n s e h e r i t a b i l i t i e s (h^) f o r g e r m i n a t i o n p a r a m e t e r s u s i n g u n s o r t e d s e e d s o f 1 8 S i t k a spruce clones  110  E s t i m a t i o n of variance 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 level for p a r a m e t e r s u s i n g s o r t e d 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  germination 114  E s t i m a t i o n of variance 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 level for  germination  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  116  V a r i a t i o n in d i a m e t e r (Dia), h e i g h t (Ht), s h o o t d r y w e i g h t ( S D W ) , r o o t d r y w e i g h t ( R D W ) , total dry w e i g h t ( T D W ) , and shoot/root dry w e i g h t ratio (S/R) of S i t k a s p r u c e s e e d l i n g s f r o m t w o s e e d s i z e s of 18 c l o n e s  124  8.2.  S t u d e n t - N e w m a n - K e u l s m u l t i p l e - r a n g e t e s t s f o r d i a m e t e r at r o o t c o l l a r , h e i g h t , s h o o t dry w e i g h t , root dry w e i g h t , total dry w e i g h t , a n d r o o t - s h o o t ratio of eight-month-old seedlings from 18 Sitka spruce clones  125  List of Figures Figure 1.1.  2.1.  3.1.  5.1.  5.2.  5.3.  5.4.  5.5.  5.6.  6.1.  6.2.  6.3.  6.4.  Page D i s t r i b u t i o n o f Sitica s p r u c e a n d l o c a t i o n s Powells, 1965)  of o r c h a r d  parent  trees  (After 4  B a n d i n g p a t t e r n s a n d their allelic d e s i g n a t i o n s f o r 1 4 a l l o z y m e l o c 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 t o t h e r e l a t i v e m i g r a t i o n d i s t a n c e . T h e s h a d e d line r e p r e s e n t s a h e t e r o d i m e r . T h e d a s h e d line r e p r e s e n t s a null allele (n)  15  P h e n o g r a m of S i t k a s p r u c e differentiation b a s e d o n N e i ' s ( 1 9 7 8 ) u n b i a s e d g e n e 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 u s i n g t h e unweighted-pair-groupmethods algorithm ( U P G M ) with N e i ' s (1978) unbiased genetic distances  45  1 9 8 8 c o n e c r o p p a r e n t a l b a l a n c e c u r v e b a s e d o n all o r c h a r d c l o n e s (N = 1 3 9 ) , indicating cotributions of the s a m p l e d c l o n e s  72  Rank order for 2 2 Sitka-spruce 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 yields  76  C u m u l a t i v e c o n e a n d filled 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 k a s p r u c e c l o n e s , 1 9 8 8 c r o p . S t r a i g h t line r e p r e s e n t s e q u a l c o n t r i b u t i o n  77  1 9 9 0 c o n e c r o p p a r e n t a l b a l a n c e c u r v e b a s e d o n all o r c h a r d c l o n e s (N = 1 3 9 ) , indicating c o n t r i b u t i o n s of the s a m p l e d c l o n e s  78  Rank order for 18 Sitka spruce c l o n e s , 1 9 9 0 crop, b a s e d on c o n e and seed yields  82  C u m u l a t i v e c o n e , filled s e e d s , t o t a l s e e d a n d v i a b l e s e e d p r o d u c t i o n c u r v e s f o r 18 Sitka spruce clones, 1 9 9 0 crop. Straight line r e p r e s e n t s equal contribution  83  A v e r a g e g e r m i n a t i o n (21 d a y s ) o f u n s t r a t i f i e d 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 after a c c e l e r a t e d aging  93  A v e r a g e g e r m i n a t i o n (21 d a y s ) o f s t r a t i f i e d 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 after a c c e l e r a t e d aging  94  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 o f 6 S i t k a s p r u c e c l o n e s a f t e r a c c e l e r a t e d aging  95  C u r v e s s h o w i n g g e r m i n a t i o n of u n s t r a t i f i e d , s t r a t i f i e d , a n d m o i s t u r e  content  of a c c e l e r a t e d - a g e d s e e d s ; a v e r a g e f o r 6 c l o n e s  96  7. / .  S e e d w e i g h t distribution c u r v e s of 18 S i t k a s p r u c e c l o n e s  105  7.2.  D i f f e r e n c e s of s e e d w e i g h t in s m a l l a n d 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 154  108  7.3.  G e r m i n a t i o n c u r v e s for clones  unsorted-unstratified  seeds  of  18  Sitl<a  spruce Ill  7.4.  G e r m i n a t i o n c u r v e s f o r u n s o r t e d - s t r a t i f i e d s e e d s o f 1 8 Sitl<a s p r u c e c l o n e s . . .  112  7.5.  D i f f e r e n c e s in g e r m i n a t i o n rate o f u n s t r a t i f i e d a n d s t r a t i f i e d s e e d s o f c l o n e n o s . 4 1 1 and 5 1 6 ( l o w a n d higfi d o r m a n t s e e d s , r e s p e c t i v e l y )  113  Acknowledgements  It is m y g r e a t p l e a s u r e t o e x p r e s s m y a p p r e c i a t i o n t o t h e o r g a n i z a t i o n s a n d p e o p l e w h o h a v e in v a r i o u s w a y s c o n t r i b u t e d  to this thesis. This study w a s made possible by A S E A N -  C a n a d a Forest Tree Seed Centre (CIDA). Canadian Pacific Forest Products Ltd. and Forestry C a n a d a , Pacific Forestry C e n t r e , provided research facilities. W e s t e r n Tree S e e d L t d . , Blind B a y , B r i t i s h 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 a n d D r . 0 . S z i k l a i h a v e provided  support  and encouragement  throughout the study.  M y supervisor.  Dr. Y . A . El-  K a s s a b y , has p r o v i d e d a stimulating s o u r c e of ideas a n d inspiration t h r o u g h o u t the s t u d y . O t h e r committee m e m b e r s , Drs. J . E . Carlson, D . G . W . E d w a r d s , D.P. Lavender, and D.T. Lester, also p r o v i d e d a d v i c e a n d a s s i s t a n c e w h e n r e q u i r e d . M s . S . A . B a r n e s 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 t h e m a n u s c r i p t . M s . K . P . B r a d y a s s i s t e d in s e e d c o l l e c t i o n a n d s e e d l i n g h a r v e s t . T h e p e o p l e at S a a n i c h F o r e s t r y C e n t r e h a v e m a d e m y s t u d y at t h e c e n t r e a p l e a s a n t o n e . Finally, I a m indebted to m y family and friends; w i t h o u t their s u p p o r t s , this thesis would  not  have been completed.  K o m k h a m , is i n v a l u a b l e .  The patience  and understanding  of m y  friend,  M s . S.  Chapter 1 General Introduction  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 p o p u l a t i o n s c o n s i s t i n g o f g e n e t i c a l l y s u p e r i o r t r e e s w h i c h  are i s o l a t e d t o r e d u c e p o l l i n a t i o n f r o m o u t s i d e s o u r c e s a n d are m a n a g e d t o p r o v i d e a b u n d a n t , e a s i l y h a r v e s t e d s e e d c r o p s {Zobel et al.,  frequent,  1 9 5 8 ) . B y d e f i n i t i o n , s e e d o r c h a r d s are  d e s i g n e d a n d m a n a g e d t o p r o d u c e s e e d s t h a t are s u p e r i o r in v a l u e t o t h o s e o b t a i n e d f r o m s e e d p r o d u c t i o n a r e a s o r r a n d o m u n i m p r o v e d c o l l e c t i o n s ( A s k e w , 1 9 8 8 ) . In c o n i f e r s , i n b r e e d i n g c a u s e s r e d u c t i o n in g r o w t h a n d s e e d l i n g s u r v i v a l ( F r a n k l i n , 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 have  been d e s i g n e d to  prevent  inbreeding,  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.,  inbreeding  h a s still b e e n r e p o r t e d  for  many  1 9 7 5 ; S h a w and A l l a r d , 1 9 8 2 ; Griffin, 1 9 8 4 ; Ritland  a n d E l - K a s s a b y , 1 9 8 5 ; O m i a n d A d a m s , 1 9 8 6 ) . A l s o , it h a s b e e n o b s e r v e d t h a t o n l y c e r t a i n clones may contribute Mûller-Stark,  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 9 0 ; B o e s etal., a restricted  g e n e s t o o r c h a r d s e e d c r o p s ( J o n s s o n et al.,  1 9 7 6 ; Griffin,  1982;  1 9 8 9 ; El-Kassaby and Reynolds,  1 9 9 1 ) . In c o m p a r i s o n t o n a t u r a l p o p u l a t i o n s , s e e d o r c h a r d s u s u a l l y c o n t a i n  n u m b e r of parents, therefore,  the risk of l o s s of genetic variability for  future  p l a n t a t i o n s is of c o n c e r n ( B o u v a r e l , 1 9 7 0 ; A d a m s , 1 9 8 1 ) . B e c a u s e t r e e s a r e o f t e n i n t r o d u c e d into highly h e t e r o g e n e o u s natural e n v i r o n m e n t s  (Hamrick,  1991), genetic diversity  c u l t i v a t e d f o r e s t is e s s e n t i a l t o e n a b l e t h e m t o c o u n t e r b a l a n c e t h e e f f e c t s o f  in t h e  environmental  c h a n g e s ( K l e i n s c h m i t , 1 9 7 9 ; G r e g o r i u s , 1 9 8 9 ) . S u p e r i o r i t y of t h e o r c h a r d - p r o d u c e d s e e d s c a n b e j u d g e d b y q u a l i t y a n d q u a n t i t y , w h i c h is a f u n c t i o n o f t h e p e r c e n t a g e o f f i l l e d s e e d , a n d t h e g e n e t i c c o m p o s i t i o n o f t h e s e e d c r o p ( A s k e w , 1 9 8 8 ) . In t h i s r e s p e c t , t h e q u a l i t y o f t h e o r c h a r d s e e d c r o p s c a n b e u s e d t o d e t e r m i n e if t h e p r i m a r y o b j e c t i v e of t h e s e e d o r c h a r d h a s b e e n m e t a n d to e x a m i n e the breeding b e h a v i o u r of the o r c h a r d p o p u l a t i o n . Selective breeding programs for northern  coniferous s p e c i e s have e m p h a s i z e d the  a d d i t i v e g e n e t i c e f f e c t s in r a n d o m m a t i n g p o p u l a t i o n s w i t h i n 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 o f t h e v a r i a t i o n in t h e s p e c i e s r a n g e t o m a x i n n i z e g e n e t i c r e t u r n s ( Y e h a n d R a s m u s s e n , 1 9 8 5 ) . Breeding p r o c e s s e s h a v e been reported to n a r r o w the g e n e t i c b a s e of the species while improving commercial values (Francis, 1981 ; N a m k o o n g and 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 g e n e t i c d i v e r s i t y in t h e b r e e d i n g p o p u l a t i o n of a n y c o m m e r c i a l l y - v a l u a b l e species should  be a priority of  any breeding  program  (Hamrick,  1991)  to  maintain  the  o p p o r t u n i t y f o r f u t u r e s e l e c t i o n ( A d a m s , 1 9 8 1 ). T h e r e f o r e , it is c r u c i a l t o d e t e r m i n e t h i s e f f e c t at t h e e a r l y s t a g e s o f a b r e e d i n g p r o g r a m t o m i n i m i z e  losses since forest  tree  breeding  p r o g r a m s are b a s e d o n t h e g e n e t i c v a r i a b i l i t y of n a t u r a l o r i n t r o d u c e d p o p u l a t i o n s . T o d a t e , t h e i m p a c t of d o m e s t i c a t i o n o n forest trees 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  a g r i c u l t u r a l c r o p p l a n t s h a s r e s u l t e d in l o s s of g e n e t i c v a r i a b i l i t y ( E l l s t r a n d a n d M a r s h a l l , 1 9 8 5 ) . E l e c t r o p h o r e s i s of i s o z y m e s m a k e s the s t u d y of genetic variation a n d the  mating  s y s t e m in p l a n t p o p u l a t i o n s p o s s i b l e at t h e s e e d c r o p s t a g e ( C l e g g , 1 9 8 0 ) . In f o r e s t r y ,  this  t e c h n i q u e h a s b e e n u s e d in m a n y a s p e c t s o f f o r e s t tree p o p u l a t i o n g e n e t i c s ( H a m r i c k et  al.,  1 9 7 9 ) i n c l u d i n g e x a m i n i n g g e n e t i c v a r i a t i o n ( H a m r i c k et al., 1 9 7 9 ; Y e h a n d E l - K a s s a b y , 1 9 8 0 ; Y e h a n d O ' M a l l e y , 1 9 8 0 ; Y e h et al., 1 9 8 6 ) a n d m a t i n g s y s t e m s ( S h a w at al., 1 9 8 1 ; S h a w a n d Allard,  1982;  Ritland  and  El-Kassaby,  1985).  Germination  tests  can  provide  an  early  a s s e s s m e n t of p o t e n t i a l f i e l d p e r f o r m a n c e of t h e s e e d s w h i l e t h e p o t e n t i a l o f s e e d s t o r a b i l i t y c a n be obtained f r o m vigour t e s t s ( A s s o c i a t i o n of Official 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 h a s b e e n u s e d t o t e s t f o r p o t e n t i a l s e e d s t o r a b i l i t y in a g r i c u l t u r a l c r o p s ( D e l o u c h e a n d B a s k i n , 1 9 7 3 ) . This m e t h o d has been applied to s o m e forest tree s e e d s as w e l l (Pitel, 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 , t h e r e s p o n s e of s e e d s t o a c c e l e r a t e d a g i n g 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 O f f i c i a l S e e d A n a l y s t s , 1 9 8 3 ; B l a n c h e et al., S i t k a s p r u c e (Picea  sitchensis  North America (Powells, 1965).  1988, 1990).  ( B o n g . ) C a r r . ) is t h e l a r g e s t o f t h e s p r u c e s g r o w i n g in  Its r a n g e , c h a r a c t e r i z e d b y a l o n g f r o s t - f r e e  period,  precipitation, high humidity and moderate temperatures, o c c u p i e s a n a r r o w strip about m i l e s l o n g a l o n g t h e P a c i f i c c o a s t f r o m t h e K o d i a k I s l a n d s in s o u t h e r n A l a s k a t o  high 1,800  northwestern  C a l i f o r n i a (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 b e l t , v a r y i n g in w i d t h f r o m  a f e w m i l e s in C a l i f o r n i a t o a b o u t 1 3 0 m i l e s in A l a s k a . S i t k a s p r u c e is r a r e l y f o u n d a b o v e 1 , 0 0 0 f e e t ( P o w e l l s , 1 9 6 5 ; R o c h e a n d F o w l e r , 1 9 7 5 ) . It is t h e o n l y N o r t h A m e r i c a n s p r u c e t h a t h a s g r o w n w e l l f o r c e n t u r i e s a s a n e x o 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 , a n d 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 t o b e c o m e a l a n d r a c e . In B r i t a i n , I r e l a n d , a n d s o m e p a r t s o f N o r t h A m e r i c a , it is a m a j o r s p e c i e s f o r t i m b e r a n d p u l p p r o d u c t i o n ( R o c h e a n d F o w l e r , 1975). In B r i t i s h C o l u m b i a , S i t k a 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 (British 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 ) ; h o w e v e r , its b r e e d i n g p r o g r a m is in a n e a r l y s t a g e o f d e v e l o p m e n t (Yeh and R a s m u s s e n ,  1985).  Since 1 9 7 1 , four  Sitka spruce seed orchards  have  been  e s t a b l i s h e d ( H a n s o n , 1 9 8 5 ; B r i t i s h 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 ) , a n d o n l y o n e s e e d o r c h a r d is in t h e 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 ) . D u e t o S i t k a s p r u c e w e e v i l (Pissodes  strobi  ( P e c k . ) ) a t t a c k ( M a c S i u r t a i n , 1 9 8 1 ; A l f a r o , 1 9 8 2 ; 1 9 8 9 ) , r e f o r e s t a t i o n o f t h e s p e c i e s is l i m i t e d t o l o w h a z a r d a r e a s ( H e p p e r a n d W o o d , 1 9 8 4 ) . H o w e v e r , a s t u d y o f w e e v i l r e s i s t a n c e in S i t k a s p r u c e i n d i c a t e s t h a t t h e r e is a h i g h p o t e n t i a l t o o v e r c o m e t h i s p r o b l e m t h r o u g h  genetic  i m p r o v e m e n t (Ying, 1 9 9 1 ) . A t this developmental stage, information c o n c e r n i n g the orchard p o p u l a t i o n s a n d t h e i r i m p a c t o n s e e d p r o d u c t i o n is e s s e n t i a l f o r o r c h a r d m a n a g e m e n t . 1.2  Objectives T h e o b j e c t i v e s of this s t u d y are: 1 . 2 . 1 . t o d e t e r m i n e t h e e x t e n t of g e n e t i c v a r i a t i o n in t h e s e e d o r c h a r d p o p u l a t i o n a n d c o m p a r e it t o t h e p u b l i s h e d r e p o r t s o f v a r i a t i o n l e v e l in n a t u r e , 1 . 2 . 2 . t o e s t i m a t e t h e level of i n b r e e d i n g in t h e 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 orchard parental balance b a s e d o n c o n e v s . s e e d c r o p , 1 . 2 . 4 . t o s t u d y t h e e f f e c t o f l o n g t e r m s t o r a g e of s e e d s o n t h e g e n e t i c m a k e u p of seed crops.  Figure  1.1.  Distribution of Sitka spruce and locations of orchard parent trees (After Foweils, 1965).  1 . 2 . 5 . t o d e t e r m i n e t i i e g e n e t i c c o n t r o l of g e r m i n a t i o n p a r a m e t e r s a n d 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 seed sizing o n g e r m i n a t i o n and s u b s e q u e n t s e e d l i n g attributes.  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 a n d g e n e 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 c o n i f e r s . In Evolution today, Proc. Intern. Congress. System. Evol. Biol., 2 n d . , p p . 4 0 1 - 4 1 5 . A l f a r o , R.I. 1 9 8 2 . F i f t y - y e a r - o l d S i t k a s p r u c e p l a n t a t i o n s w i t h a h i s t o r y o f i n t e n s i v e a t t a c k . J. Entomol. Soc. B r i t i s h C o l u m b i a 7 9 : 6 2 - 6 5 .  weevil  A l f a r o , R.I. 1 9 8 9 . 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D e v e l o p m e n t o f p l a n t g e n o t y p e s f o r m u l t i p l e c r o p p i n g s y s t e m s . In Plant Breeding II, e d . K . J . F r e y , p p . 1 7 9 - 2 3 1 . A m e s : T h e I o w a S t a t e U n i v e r s i t y P r e s s . F r a n k l i n , E . C . 1 9 7 0 . S u r v e y of m u t a n t f o r m s a n d i n b r e e d i n g d e p r e s s i o n in s p e c i e s o f t h e f a m i l y Pinaceae.  S o u t h e a s t e r n F o r . 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 i b u t i o n of p h e n o t y p i c v a r i a t i o n t o g e n e t i c o r e n v i r o n m e n t a l v a r i a t i o n 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 a n d D. R u d i n , 1 - 1 6 . H e i d e l b e r g : S p r i n g e r Verlage. G r i f f i n , A . R . 1 9 8 2 . C l o n a l v a r i a t i o n in r a d i a t a pine 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 a n d s e e d p r o d u c t i o n t r a i t s . Aust. J. For. Res. 1 2 : 2 9 5 - 3 0 2 . G r i f f i n , A . R . 1 9 8 4 . C l o n a l v a r i a t i o n in r a d i a t a p i n e s e e d o r c h a r d s . II. F l o w e r i n g Aust. J. For. Res. / 4 : 2 7 1 - 2 8 1 .  phenology.  H a m r i c k , J . L . 1 9 9 1 . A l l o z y m e diversity of natural s t a n d s v e r s u s s e e d o r c h a r d loblolly pine. Paper presented at the 23rd biennial meeting of Canadian Tree Improvement Association, August 19-23, 1991. Ottawa. H a m r i c k , J . L . , Y . B . L i n h a r t a n d 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 i s t o r y c h a r a c t e r i s t i c s a n d e l e c t r o p h o r e t i c a l l y d e t e c t a b l e g e n e t i c v a r i a t i o n in p l a n t s . 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 Printer for British  Columbia. H e p p e r , D . G . a n d P . M . W o o d . 1 9 8 4 . Vancouver Forest Region Sitka spruce results (1982-19831 with recommandations for planting Sitka spruce. V a n c o u v e r F o r e s t R e g i o n . Internal R e p . P M - V - 5 .  weevil survey B . C . M i n . For.,  J o n s s o n , A . , I. E k b e r g a n d 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 o f Pinus L. Stud.  For.  Sue.  sylvestris  135:1-38.  K l e i n s c h m i t , J . 1 9 7 9 . L i m i t a t i o n s f o r r e s t r i c t i o n of t h e g e n e t i c v a r i a t i o n . Sivae 67.  Genet.  28:61-  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 , U n i v e r s i t y of British C o l u m b i a , F a c u l t y of Forestry, V a n c o u v e r , B . C .  M ù l l e r - S t a 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 c o n i f e r s e e d o r c f i a r d s . I. IVIating p r o b a b i l i t y in a s e e d o r c h a r d of Pinus sylvetris L. Silvae Genet. 3 1 : 1 8 8 - 1 9 7 . N a m k o o n g , G . , a n d J . H . R o b e r d s . 1 9 8 2 . S h o r t - t e r m l o s s o f n e u t r a l a l l e l e s in s m a l l - p o p u l a t i o n b r e e d i n g . Silvae  Genet.  31:1-6.  O m i , S . K . a n d W . T . A d a m s . 1 9 8 6 . V a r i a t i o n in s e e d s e t a n d p r o p o r t i o n s o f 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 , a n d t o p p r u n i n g in a D o u g l a s - 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 t e l , J . A . 1 9 8 0 . A c c e l e r a t e d a g i n g s t u d i e s of s e e d s o f j a c k p i n e (Pinus banksiana Lamb.) and red o a k (Quercus rubra L.). In Proc. International Symposium on Forest tree seed storage, l U F R O W o r k i n g party 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 . Pitel, pp. 4 0 - 5 4 . P e t a w a w a National Forestry Institute, C h a l k River, Ontario. R i t l a n d , K. a n d Y . A . E l - K a s s a b y . 1 9 8 5 . T h e n a t u r e o f i n b r e e d i n g in a s e e d o r c h a r d o f D o u g l a s fir a s s h o w n b y a n e f f i c i e n t m u l t i l o c u s m o d e l . Theor. Appl. Genet. 71:375-384. 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 26:1-15.  For.  Serv.  Res. Pap.  WO-  S c h m i d t l i n g , R . C . 1 9 8 3 . G e n e t i c v a r i a t i o n in f r u i t f u l n e s s in a l o b l o l l y p i n e s e e d o r c h a r d . Genet. 3 2 : 7 6 - 8 0 .  Silvae  Shaw,  using  D.V. and R.W. Allard. 1 9 8 2 . i s o z y m e m a r k e r s . Theor. Appl.  E s t i m a t i o n o f o u t c r o s s i n g r a t e s in D o u g l a s - f i r Genet. 6 2 : 1 1 3 - 1 2 0 .  S h a w , D . V . , A . L . K a h l e r a n d R . W . A l l a r d . 1 9 8 1 . A m u l t i l o c u s e s t i m a t o r of m a t i n g p a r a m e t e r s in p l a n t 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 .  system  Y e h , F . C . a n d Y . A . E l - K a s s a b y . 1 9 8 0 . E n z y m e v a r i a t i o n in n a t u r a l p o p u l a t i o n s of S i t k a s p r u c e (Picea sitchensis). 1. G e n e t i c v a r i a t i o n p a t t e r n s a m o n g t r e e s 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 v a r i a t i o n in n a t u r a l p o p u l a t i o n s o f D o u g l a s - f i r , Pseudotsuga menziesii ( M i r b . ) F r a n c o , f r o m B r i t i s h C o l u m b i a . 1. G e n e t i c v a r i a t i o n p a t t e r n s 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 . K h a l i l , Y . A . E l - K a s s a b y a n d D . C . T r u s t . 1 9 8 6 . A l l o z y m e v a r i a t i o n in Picea mariana f r o m N e w f o u n d l a n d : genetic diversity, population s t r u c t u r e , a n d analysis of d i f f e r e n t i a t i o n . Can. J. For. Res. 1 6 : 7 1 3 - 7 2 0 . Y e h , F . C . a n d S . R a s m u s s e n . 1 9 8 5 . H e r i t a b i l i t y o f h e i g h t g r o w t h in 1 0 - y e a 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 . 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 t o t h e w h i t e p i n e w e e v i l in S i t k a s p r u c e . B.C. Res. Notes N o . 1 0 6 : 1 - 1 7 .  Min.  For.  Z o b e l , B . J . , J . Barber, C . L . B r o w n and T . O . Perry. 1 9 5 8 . S e e d orchards - their 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 l e c t r o p h o r e s i s is a p r o c e s s w h e r e p r o t e i n s are f o r c e d t o m i g r a t e t h r o u g h a n i n t r o d u c e d  m o l e c u l a r s i e v i n g m e d i u m under the influence of an electric current (Markert a n d M o l l e r , 1 9 5 9 ) . S i n c e its i n t r o d u c t i o n t o p o p u l a t i o n g e n e t i c s b y 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 h a s b e e n u s e d e x t e n s i v e l y t o s t u d y t h e g e n e t i c v a r i a t i o n in a w i d e a r r a y of living o r g a n i s m s . Unlike m o r p h o l o g i c a l traits, a l l o z y m e s exhibit c o d o m i n a n t e x p r e s s i o n a n d their m o d e of inheritance usually f o l l o w s s i m p l e 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 v a r i a t i o n h a s b e e n s t u d i e d e x t e n s i v e l y in b a c t e r i a ( M i l k m a n , 1 9 7 3 ) , h u m a n s ( H a r r i s , 1 9 6 6 ) , a n n u a l s ( C l e g g a n d A l l a r d , 1 9 7 2 ) , a n d f o r e s t t r e e s ( H a m r i c k et al.,  1979).  F o r e s t g e n e t i c i s t s h a v e u s e d i s o z y m e s in p r o v e n a n c e r e s e a r c h ( F a l k e n h a g e n , 1 9 8 5 ) , t o s t u d y h y b r i d z o n e s ( C o p e s a n d B e c k w i t h , 1 9 7 7 ) , m a t i n g s y s t e m s ( S h a w et al.,  1981 ; Ritland and El-  K a s s a b y , 1 9 8 5 ) , pollen migration (Smith and A d a m s , 1 9 8 3 ; El-Kassaby and Ritland, 1 9 8 6 ) , pollination biology ( W e b b e r a n d Y e h , 1 9 8 7 ) , a n d t o d e t e r m i n e the validity 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.,  1988).  In c o n i f e r s , s e e d s , m a t u r e t i s s u e , a n d d o r m a n t b u d s c a n b e u s e d f o 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 i n h e r i t a n c e s t u d i e s , 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 t h e s e e d h a s b e e n u s e d (Lundkvist, 1 9 7 7 ; El-Kassaby  a/., 1 9 8 2 a ; C h e l i a k a n d Pitel, 1 9 8 4 ) . M e g a g a m e t o p h y t i c tissue  is s u i t a b l e m a t e r i a l f o r i n h e r i t a n c e s t u d i e s s i n c e it is h a p l o i d , t h u s a l l o w i n g d i r e c t o b s e r v a t i o n o f M e n d e l i a n s e g r e g a t i o n at h e t e r o z y g o u s i s o z y m e l o c i ( G u r i e s a n d L e d i g , 1 9 7 8 ) .  Simple  extraction p r o c e d u r e s c a n be applied 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 not c o n t a i n large a m o u n t s o f s e c o n d a r y m e t a b o l i t e s . 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 , related s p e c i e s o f t e n r e s e m b l e one a n o t h e r in g e n e r a l i s o z y m e c h a r a c t e 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 f e r in 1 ) t h e n u m b e r s o f l o c i f o r t h e s a m e e n z y m e ( A d a m s a n d J o l y , 1 9 8 0 a ; E l - K a s s a b y , 1 9 8 1 ), 2) t h e b a n d p h e n o t y p e s  f o r s i m i l a r l o c i ( A d a m s a n d 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 h e i n t e r a c t i o n  of  p r o d u c t s at t w o l o c i ( E l - K a s s a b y , 1 9 8 1 ) , a n d 4) t h e e x p r e s s i o n of m o d i f i e r l o c i ( H a r r y , 1 9 8 3 ) . T o avoid misinterpretation,  it is i m p o r t a n t t o d o c u m e n t  band inheritance before  beginning  e l e c t r o p h o r e t i c a n a l y s e s in a s p e c i e s ( R u d i n , 1 9 7 6 ; M i l l a r , 1 9 8 5 ) . Population-genetics analyses s u c h as the estimation ( S h a w et al., information  of m a t i n g s y s t e m  parameters  1 9 8 1 ; Ritland and El-Kassaby, 1985) and genetic distance (Nei, 1975)  require  f r o m a n u m b e r of i n d e p e n d e n t l o c i . T h e r e f o r e , l i n k e d l o c i s h o u l d n o t 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 r e l i m i n a r y quantitatively  s t e p in t h e  use of i s o z y m e s as markers  of  c o n t r o l l e d 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 a c t i o n s b e t w e e n l o c i are i m p o r t a n t in  establishing s u c h m a p s (Vallejos and T a n k s l e y , 1 9 8 3 ) . This study  reports  an a n a l y s i s of  inheritance  and linkage  relationships  based 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 l o c 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 s u e o f S i t k a s p r u c e . T h e o b j e c t i v e s o f t h i s c h a p t e r a r e ; 1) t o p r e s e n t t h e m o d e o f i n h e r i t a n c e  of  a l l o z y m e m a r k e r s in S i t k a s p r u c e , a n d 2) t o d e t e r m i n e t h e l i n k a g e r e l a t i o n s h i p s a m o n g t h e s e m a r k e r s . T h i s i n f o r m a t i o n is a p r e r e q u i s i t e f o r s t u d i e s r e p o r t e d in t h e f o l l o w i n g t w o  chapters  on gene diversity and the mating s y s t e m . 2 . 2 Materials  and  Methods  C a n a d i a n P a c i f i c F o r e s t P r o d u c t s L t d . p r o v i d e d t h e s e e d s f o r t h i s s t u d y f r o m its S i t k a s p r u c e s e e d o r c h a r d l o c a t e d in S a a n i c h t o n , B r i t i s h C o l u m b i a ( l a t i t u d e 4 8 ° 3 5 ' N ,  longitude  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 (averaging 9 . 3 r a m e t s per clone) s e l e c t e d f r o m elevations 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 Island, W a s h i n g t o n and Oregon (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  u n e q u a l b l o c k s . In S e p t e m b e r 1 9 9 0 , w i n d - p o l l i n a t e d  in a r a n d o m s i n g l e - t r e e m i x o v e r  three  seeds were collected from 134 seed  o r c h a r d c l o n e s . F i v e 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 i d e n t i t y of w h i c h w a s c a r e f u l l y 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 d r i e d at r o o m t e m p e r a t u r e a n d e x t r a c t e d b y h a n d . S e e d s a m p l e s w e r e l a b e l l e d a n d k e p t at 2 ° C u n t i l u s e d .  S e e d s f r o m i n d i v i d u a l c l o n e s w e r e s o a k e d in w a t e r f o r 2 4 h r s . at r o o m  temperature,  d r a i n e d , a n d a l l o w e d t o g e r m i n a t e . F r o m e a c h o f t h e 1 3 4 c l o n e s , at l e a s t e i g h t g e r m i n a n t s w i t h a  radicle  approximately  equal  to  the  length  of  the  seed  were  dissected  to  separate  m e g a g a m e t o p h y t e s f r o m t h e e m b r y o s . E l e c t r o p h o r e 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 a n d O ' M a l l e y (1980), and El-Kassaby  a/. ( 1 9 8 2 a ) , u s i n g h o r i z o n t a l 1 1 . 5 % ( w / v ) s t a r c h g e l s . T w o  different gel buffer s y s t e m s (C-Tris/citrate, p H . 7 . 0 ; and D-Tris/citrate:Lithium borate, p H . 8.5) w e r e u s e d (Table  2.2)  a n d e l e v e n e n z y m e s y s t e m s w e r e i n v e s t i g a t e d ( 7 a 6 / e 2.1).  Staining  recipes and procedures f o l l o w e d those reported by Y e h and O ' M a l l e y (1980), and El-Kassaby at al. ( 1 9 8 2 a ) (Table  2.2).  F o r s o m e l o c i w h e r e t h e initial s c r e e n i n g i n d i c a t e d l a r g e a m o u n t s  of variation, an additional 1 6 m e g a g a m e t o p h y t e / e m b r y o pairs w e r e a s s a y e d per c l o n e . The following  n o m e n c l a t u r e w a s u s e d : a n e n z y m e a n d its b a n d p h e n o t y p e s  i d e n t i f i e d b y t h e e n z y m e ' s a b b r e v i a t i o n in italic c a p i t a l l e t t e r s (Table e n z y m e w a s defined by the e n z y m e ' s band migration multiple  2.1);  were  e a c h l o c u s of an  and noted abbreviation and,  where  loci 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 loci w e r e n u m b e r e d , w i t h  the most-anodally-migrating  locus designated as " 1 . " Within e a c h l o c u s , the  most-frequent  allele w a s a s s i g n e d t h e v a l u e o f 1 0 0 . O t h e r a l l e l e s o f t h e l o c u s w e r e d e s i g n a t e d b y  mobility  v a l u e e x p r e s s e d r e l a t i v e t o t h e m o s t - f r e q u e n t a l l e l e . A n allele l a c k i n g s t a i n a c t i v i t y (null), a n d o b s e r v e d at o n l y o n e 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 t e s t t h e null h y p o t h e s i s o f a 1:1 s e g r e g a t i o n ratio a m o n g t h e 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 , s e g r e g a t i o n d a t a 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 t h a t h a d t h e s a m e allele c o m b i n a t i o n a n d t e s t e d u s i n g t h e l o g - l i k e l i h o o d G - t e s t ( S o k a l a n d Rohlf, 1 9 8 1 ) . A G value w a s calculated to test the heterogeneity  in t h e o b s e r v e d ratio in  gametic arrays f r o m different c l o n e s . T o a v o i d b i a s in C h i - s q u a r e c a l c u l a t i o n s w h e r e s a m p l e s i z e s a r e t o o s m a l l , n o m o r e t h a n 2 0 % of t h e e x p e c t e d cell f r e q u e n c i e s s h o u l d b e l e s s t h a n 5 . 0 ( C o c h r a n , 1 9 5 4 ) . T h e r e f o r e ,  Table 2.1.  Gel Buffer  L i s 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 , a n d G e l B u f f e r s y s t e m s used for Sitka s p r u c e electrophoresis  Enzyme  Abbreviation  EC Number  AAT GDH LAP  G6P IDH MDH  2.6.1.1 1.4.1.3 3.4.1.1 4.2.1.3 3.1.1.2 1.1.1.49 1.1.1.42 1.1.1.37  PGI PGM 6PG  5.3.1.9 2.7.5.1 1.1.1.44  System" C  D  Aspartate amino-transferase Glutamate dehydrogenase Leucine aminopeptidase Aconitase Esterase Glucose 6-phosphate dehydrogenase Isocitrate d e h y d r o g e n a s e Malate dehydrogenase Phosphoglucose isomerase Phosphoglucomutase 6-Phosphogluconic dehydrogenase  ° G e l b u f f e 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.  (1982a).  AGO EST  Table  2.2.  E l e c t r o p h o r e t i c P r o c e d u r e : A . b u f f e r s y s t e m ( E l - K a s s a b y et al. 1 9 8 2 a ) a n d B. s t a i n r e c i p e s ( Y e h a n d O ' M a l l e y , 1 9 8 0 )  A . BUFFER S Y S T E M Power  Buffer System  Gel  Electrode  C  Tris/citrate p H 7 . 0 (TC)  0 . 1 3 M Tris and 0 . 0 4 3 M citric acid (anhydrous)  1:10 dilution of electrode buffer  6 0 m A ( « 1 6 0 V) until tracking d y e has migated 5 c m .  D  Tris/citrate: Li/borate p H 8.1 ( R W )  0 . 0 6 M Li/hydroxide and 0.3 M Boric acid (pH 8 . 1 )  0.3 M Tris, 0 . 0 0 5 M Citric acid (anhydrous) and 1 % electrode buffer (pH 8 . 5 )  250 V ( « 6 0 mA) until t r a c k i n g d y e has migated 5 c m .  B. S T A I N RECIPES Enzyme AAT  ACO  Stain C o m p o n e n t  Isocitric d e h y d r o g e n a s e NADP 1 % M a g n e s i u m chloride NBT EST  200 100 200 30 200  (w/v)  PMS 0 . 2 M P h o s p h a t e buffer, p H 6.4 a-Naphthyl acetate" /9-Naphthyl acetate" {' a n d  GDH  30 1  Tris-HCI, pH 8.0 Pyridoxal 5-Phosphate L-Aspartic acid o-Ketoglutaric acid Fast Blue B B salt Tris-HCI, pH 8.0 cis A c o n i t i c acid  10 5 30 50 50 100 30 400  NAD  30 30 5 30 200 1 10  PMS Tris-HCL Glocose-6-phosphate 1 % M a g n e s i u m chloride NADP Ml 1 PMS  mg unit mg ml mg mg ml mg mg  in 5 m l a c e t o n e )  Fast Blue RR salt Tris-HCI, pH 8.0 Monosodium Glutamate NBT  G6P  40 10 1  ml mg mg mg mg ml  (w/v)  10 5  mg ml mg mg mg mg ml mg ml mg mg mg  Table 2.2.  (Continued)  Enzyme IDH  LAP  MDH  Stain C o m p o n e n t Tris-HCI, pH 8.0 DL-lsocitric acid NADP 1 % iVlagnesium chloride (w/v) Ml 1 PMS A m i n o p e p t i d a s e buffer 0 . 4 % L-Leucine B-Naphthyl amide solution Black K salt Tris-HCI, pH 8.0 Malate solution T r i s - H C I (pH 8 . 0 ) NAD NBT  PGI  PGM  PMS Tris-HCI, pH 8.0 D-Fructose 6-Phosphate Glucose-6-phosphate dehydrogenase NADP 1 % M a g n e s i u m chloride (w/v) Ml 1 PMS Tris-HCI, pH 8.0 a-D-Glucose 1-phosphate a-D-Glucos 1,6-diphosphate Glucose-6-phosphate dehydrogenase NADP 1 % M a g n e s i u m chloride (w/v) MTT PMS  6PG  Tris-HCI, pH 8.0 6-phosphogluconic acid NADP 1 % M a g n e s i u m chloride MTT PMS  (w/v)  30 100 10 1 10 5 30 5 20 30 45 45 10 10 5 30 25 10 10 1 10 5 30 100 0.5 10 10 1 10  ml mg mg ml mg mg ml ml mg ml ml ml mg mg mg ml mg unit mg ml mg mg ml mg mg unit mg ml mg  5 10 10 10 1 10  mg ml  5  mg  mg mg ml mg  AAT-1  AGO  AAT-2  EST  GDH  116  "îôcT  221.  100  100  87  96  75  100  70 PGM-1  PGI-2  MDH-1  IDH  G6P-1  100 114  100  inn  100 100  100  „  6PG-2  6PG- -1  LAP-1  PGM-2  J2i.  n  113 100  100  86 100  Figure  2.1.  117 ••' "  90  B a n d i n g p a t t e r n s a n d their allelic d e s i g n a t i o n s f o r 1 4 a l l o z y m e l o c 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 t o t h e r e l a t i v e m i g r a t i o n d i s t a n c e . T h e s h a d e d line r e p r e s e n t s a h e t e r o d i m e r . T h e d a s h e d line r e p r e s e n t s a null allele (n).  ,0  ...  linkage relationships w e r e p o s t u l a t e d f r o m the s e g r e g a t i o n data of pairs of a l l o z y m e loci only w h e r e the s a m p l e size w a s greater than 2 0 s e e d s per c l o n e . T h e statistical p r o c e d u r e s u s e d to test for linkage w e r e : the o b s e r v e d gene segregation for  each locus w a s  u s e d to  calculate the  expected  number  for  e a c h of the  two-locus  segregation g r o u p s (Bailey, 1 9 6 1 ) , Chi-square a n a l y s e s to test for linkage (Rudin a n d Ekberg, 1 9 7 8 ) , and three-way log-likelihood test for independence (Sokal and Rohlf, 1 9 8 1 ) . 2 . 3 Resu/ts  and  Discussion  In g e n e r a l , i s o z y m e a c t i v i t y in S i t k a s p r u c e r e s e m b l e d t h a t f o u n d in r e l a t e d s p e c i e s , b o t h in t h e n u m b e r of l o c i f o r g i v e n e n z y m e s a n d in b a n d p h e n o t y p e s ( n u m b e r s o f b a n d s a n d patterns  of  stain  intensity)  (see  Tab/e  2.3  for  review).  Additional  zones that  appeared  i n c o n s i s t e n 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 a n d 1 4 p o l y m o r p h i c l o c i r e s o l v e d c o n s i s t e n t l y in t h e 1 3 4 c l o n e s s t u d i e d (Table 2.4).  T h e n u m b e r of alleles o b s e r v e d a n d  v e r i f i e d at p o l y m o r p h i c l o c i v a r i e d f r o m t w o t o f o u r , a n d b a n d p h e n o t y p e s at a l o c u s v a r i e d f r o m null t o d o u b l e - b a n d e d (Figure  2.1).  F o u r e n z y m e s (ACO,  EST, GDH, a n d /D/V) h a d a s i n g l e  z o n e o f a c t i v i t y , w h i l e t h e rest h a d m u l t i p l e 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, MDH-2,  a n d MDH-3  PGI-1,  loci w e r e unclear a n d too i n c o n s i s t e n t for reliable 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,  a n d LAP-2)  w e r e i n v a r i a n 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 t h e r c o n i f e r s , w h e r e M e n d e l i a n s e g r e g a t i o n h a s b e e n s h o w n (Tab/e  2.3). 2 . 3 . 2 Segregation Aspartate  of Po/ymorphic  Aminotransferase  Loci  (AAT)  T h r e e z o n e s o f a c 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 v a r i a n t s ( 1 0 0 a n d 9 2 ) in t h e 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 only one clone w a s o b s e r v e d w i t h this variant. T w o variants ( 1 0 0 a n d 20)  were  o b s e r v e d at AAT-2  AAT-2  (Figure  2.1).  A n a d d i t i o n a l b a n d w a s l o c a t e d m i d w a y b e t w e e n the  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 , allele d e s i g n a t i o n s of S i t k a s p r u c e e n z y m e s , a n d r e f e r e n c e s t o i n h e r i t a n c e s t u d i e s in o t h e r c o n i f e r s  Locus  AAT-1  Gametophytic phenotype  AAT-2  Single Single  ACQ  Single  Alleles  Gametophytic enzyme structure  100, 92 100, 20  Dimer Dimer  100,104,  Monomer  96  References reporting inheritance in o t h e r c o n i f e r s p e c i e s G u r i e s a n d 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 a n d D a n c i k , 1 9 8 3 ; B o y l e a n d M o r g e n s t e r n , 1 9 8 5 ; C h e l i a k a n d P i t e l , 1 9 8 5 ; E l - K a s s a b y et al., 1 9 8 7 ; P i t e l et al., 1 9 8 7 ; E r n s t et al., 1 9 8 7 ; P e r r y a n d K n o w l e s , 1 9 8 9 ; X i e etal., 1 9 9 1 Guries and Ledig, 1 9 7 8 ; Y e h and Layton,1979; A d a m s and Joly, 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 a n d Dancik, 1 9 8 3 ; Cheliak a n d Pitel, 1 9 8 5 ; Millar, 1 9 8 5 ; Harry, 1 9 8 6 ; S t r a u s s a n d C o n k l e , 1 9 8 6 ; E r n s t 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 e f al., 1 9 9 1 R u d i n a n d R a s m u s o n , 1 9 7 3 ; L u n d k v i s t , 1 9 7 7 ; E l - K a s s a b y et al., 1 9 8 1 ; Strauss and Conkle, 1 9 8 6 ; Lewandowski and Mejnartowicz, 1990  EST  Single  100,116, 87, 75  GDH  Single  100,137, 71  A d a m s a n d J o l y , 1 9 8 0 a ; N e a l e a n d 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 ; King a n d Dancik, 1 9 8 3 ; Boyle a n d Morgenstern, 1 9 8 5 ; S t r a u s s a n d 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 ; E r n s t et al., 1 9 8 7 ; Pitel etal., 1 9 8 7 ; P e r r y a n d 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., 1 9 9 1  G6P-1  Single  100,100'  O'Malley a / . , 1 9 7 9 ; E l - K a s s a b y ef a/., 1 9 8 1 ; Neale a n d 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 a n d D a n c i k , 1 9 8 3 ; C h e l i a k e f al., 1 9 8 4 ; B o y l e a n d M o r g e n s t e r n , 1 9 8 5 ; C h e l i a k a n d P i t e 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 a n d Mejnartowicz, 1 9 9 0  IDH  Single  100,108  Dimer  G u r i e s a n d 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 a n d L a y t o n , 1 9 7 9 ; A d a m s a n d J o l y , 1 9 8 0 a ; E c k e r t et al., 1 9 8 1 ; E l - K a s s a b y et al., 1 9 8 1 ; N e a l e a n d 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 a n d D a n c i k , 1 9 8 3 ; C h e l i a k a n d P i t e l , 1 9 8 4 ; C h e l i a k et al., 1 9 8 4 ; N e a l e etal., 1 9 8 4 ; B o y l e a n d M o r g e n s t e r n , 1 9 8 5 ; M i l l a r , 1 9 8 5 ;  Table 2.3. Locus  (Continued) Gametopiiytic  Alleles  phenotype  LAP-1  Gametophytic enzyme structure  References reporting inheritance in o t h e r c o n i f e r s p e c i 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 ; E l - K a s s a b y ef a/., 1 9 8 7 ; E r n s t et al., 1 9 8 7 ; Pitel et al., 1 9 8 7 ; P e r r y a n d 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 a n d M e j n a r t o w i c z , 1 9 9 0 ; X i e et al., 1 9 9 1 Tigerstedt, 1 9 7 3 ; Lundkvist, 1 9 7 4 ; Simonsen and Wellendorf, 1 9 7 5 ;  Single Null  100, n  MDH-1  Single  100,114, 9 2 , 81  Monomer  Simonsen and Wellendorf, 1 9 7 5 ; Guries and Ledig, 1 9 7 8 ; O'Malley et al., 1 9 7 9 ; Y e h a n d L a y t o n , 1 9 7 9 ; A d a m s a n d J o l y , 1 9 8 0 a ; E l K a s s a b y et al., 1 9 8 1 ; N e a l e a n d 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 a n d D a n c i k , 1 9 8 3 ; C h e l i a k etal., 1 9 8 4 ; H a r r y , 1 9 8 3 ; N e a l e et al., 1 9 8 4 ; B o y l e a n d M o r g e n s t e r n , 1 9 8 5 ; C h e l i a k a n d P i t e l , 1 9 8 5 ; Millar, 1 9 8 5 ; Harry, 1 9 8 6 ; S t r a u s s and C o n k l e , 1 9 8 6 ; ElK a s s a b y et al., 1 9 8 7 ; Pitel et al., 1 9 8 7 ; P e r r y a n d 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 a n d M e j n a r t o w i c z , 1 9 9 0  PGI-2  Single  100,117  Dimer  S i m o n s e n a n d W e l l e n d o r f , 1 9 7 5 ; O ' M a l l e y et al.,  Monomer  G u r i e s a n d 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 a n d J o l y , 1 9 8 0 a ; E c k e r t et al., 1 9 8 1 ; N e a l e a n d A d a m s , 1 9 8 1 ; K i n g a n d Dancik, 1 9 8 3 ; Cheliak and Pitel, 1 9 8 5 ; Millar, 1 9 8 5 ; Harry, 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 a n d Mejnartowicz, 1990  93  1 9 7 9 ; Y e h and  L a y t o n , 1 9 7 9 ; A d a m s a n d 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 a n d D a n c i k , 1 9 8 3 ; N e a l e et al., 1 9 8 4 ; Boyle and M o r g e n s t e r n , 1 9 8 5 ; Cheliak and Pitel, 1 9 8 5 ; Millar, 1 9 8 5 ; Harry, 1 9 8 6 ; Strauss and Conkle, 1 9 8 6 ; El-Kassaby a/., 1987; E r n s t et al., 1 9 8 7 ; P e r r y a n d 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., 1 9 9 1  PGM-1  Single  100, 90, 75  Monomer  G u r i e s a n d 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 a n d L a y t o n , 1 9 7 9 ; A d a m s a n d J o l y , 1 9 8 0 a ; E c k e r t et al., 1 9 8 1 ; N e a l e a n d  PGM-2  Single  100,  Monomer  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 a n d D a n c i k , 1 9 8 3 ; N e a l e et al., 1 9 8 4 ; B o y l e a n d M o r g e n s t e r n , 1 9 8 5 ; C h e l i a k a n d P i t e l , 1 9 8 5 ; Millar, 1 9 8 5 ; Harry, 1 9 8 6 ; S t r a u s s and C o k l e , 1 9 8 6 ; ElK a s s a b y et al., 1 9 8 7 ; Pitel et al., 1 9 8 7 ; P e r r y a n d K n o w l e s , 1 9 8 9 ;  86  Table 2.3. Locus  (Continued) Gametophytic phenotype  6PG-1  Single  6PG-2  Single, Double  Alleles  Gametophytic enzyme structure  100,113, 86 100,117, 90  Dimer Dimer  References reporting inheritance in o t h e r c o n i f e r s p e c i e s A d a m s etal., 1 9 9 0 ; X i e etal., 1 9 9 1 Simonsen and Wellendorf, 1 9 7 5 ; Guries and Ledig, 1 9 7 8 ; O'Malley et al., 1 9 7 9 ; Y e h a n d L a y t o n , 1 9 7 9 ; A d a m s a n d 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 i n g a n d D a n c i k , 1 9 8 3 ; C h e l i a k a n d P i t e l , 1 9 8 4 ; C h e l i a k et al., 1 9 8 4 ; N e a l e et al., 1 9 8 4 ; Boyle a n d M o r g e n s t e r n , 1 9 8 5 ; Cheliak a n d Pitel, 1 9 8 5 ; Millar, 1 9 8 5 ; Harry, 1 9 8 6 ; El-Kassaby ef a/., 1 9 8 7 ; Ernst ef a/., 1 9 8 7 ; P e r r y a n d K n o w l e s , 1 9 8 9 ; X i e etal., 1 9 9 1  Table 2.4.  L o g - l i k e l i h o o d G - t e s t s o n s e g r e g a t i o n r a t i o s o f 1 3 p o l y m o r p h i c l o c i in S i t k a spruce seeds  Locus  Genotype  Observed Ratio  P o o l e d G° (df = 1)  AAT-1 AAT-2 ACQ  100:92 100:20 100:104  4:4  0.000 1.168 0.728 5.437'  EST  G6P-1 GDH IDH LAP-1 MDH-1  PGI-2 PGM-1 PGM-2 6PG-1 6PG-2  100:96 100:116 100:87 100:75 100:100' 100:137 100:71 100:108 100:n 100:114 100:92 100:81 100:117 100:93 100:90 100:75 100:86 100:113 100:86 100:117 100:90  77:91 184:168 32:16 67:69 60:44 23:25 669:611 54:74 20:20 11:13 79:81 27:13 101:131 9:15 102:90 83:61 235:229 25:23 446:410 380:228 12:12 598:562 12:4  H e t e r o g e n e i t y G^  df  _ 17.319 64.939* 7.791 15.354 7.922 7.712 113.671** 8.239 1.680  0.029 2.471 0.083 10.894" 3.138 0.000 0.167  c  20 43 5 1 12 5 75 10 1 —  28.287  0.025 5.005* 3.890' 1.516 0.750 3.374  6.889** 26.899** —  15.965 7.178 57.493** 6.932**  0.078 0.083 1.514  74.430* 128.409** 0.756 122.639**  38.406" 0.000 1.117  -  4.186*  19 1 11 —  12 7 25 1 53 39 1 66  -  * S i g n i f i c a n t at 5 % . " " S i g n i f i c a n t at 1 % . ' P o o l e d G v a l u e s i n d i c a t e t h e o v e r a l l d e v i a t i o n f r o m 1:1  ratio.  '° H e t e r o g e n e i t y G v a l u e s i n d i c a t e t h e a m o u n t of h e t e r o g e n e i t y in t h e s e g r e g a t i o n r a t i o a m o n g mother trees. " A n a l y s i s is b a s e d o n s i n g l e t r e e d a t a .  a n d AAJ-3 Joly,  loci, and this zone h a s been interpreted a s an interlocus heterodimer ( A d a m s a n d  1 9 8 0 a ; Cheliak and Pitel, 1 9 8 4 ; E l - K a s s a b y , 1 9 8 1 ) . This interlocus band w a s only  o b s e r v e d f o r t h e y4/4r-2-20M/4 7 - 3 - 1 0 0 a n d n o t b e t w e e n AAT-2-\Ç)()IAAJ-3AQQ. f o r t h i s a n o m a l y is u n k n o w n at p r e s e n 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 d i f f e r e n t f r o m t h e e x p e c t e d 1:1 ratio b y p o o l e d G a n d h e t e r o g e n e i t y  T h e reason is n o t s i g n i f i c a n t l y  G, i n d i c a t i n g t h a t e a c h  z o n e is c o n t r o l l e d b y a s i n g l e l o c u s . Aconitase  (ACO)  O n e z o n e of a c t i v i t y w a s o b s e r v e d in ACO w i t h t h r e e s i n g l e - b a n d e d p h e n o t y p e s ( 9 6 , 100,  and 104). The pooled  G value for the genotype  100:96  w a s significant,  but the  h e t e r o g e n e i t y G v a l u e w a s n o t s i g n i f i c a n t , i n d i c a t i n g t h a t t h e d e v i a t i o n f r o m 1:1 ratio is c a u s e d b y a s m a l l e x c e s s o f g a m e t o p h y t e s c a r r y i n g allele " 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 significant and significant results for the pooled a n d heterogeneity tests, respectively 2.4),  i n d i c a t i n g t h a t t h e s e g r e g a t i o n at t h i s l o c u s d o e s n o t f o l l o w t h e e x p e c t e d  1:1  (Table ratio  because clones s h o w e d a heterogeneous segregation. Esterase  (EST)  O n e zone of activity w i t h four single-banded variants ( 7 5 , 8 7 , 1 0 0 , a n d 1 1 6 ) w a s observed  (Figure  2.1).  Neither  the pooled  nor heterogeneity  G of the three  observed  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 i g n i f i c a n t , t h u s c o n f i r m i n g t h e 1:1 ratio s e g r e g a t i o n (Table Glutamate  dehydrogenase  O n e zone of activity  2.4).  (GDH) w i t h three  single-banded variants  (71, 1 0 0 and 137) were  o b s e r v e d at t h i s l o c u s . N e i t h e r t h e p o o l e d n o r h e t e r o g e n e i t y G v a l u e o f t h e 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 i g n i f i c a n t (Table  2.4),  indicating that the genetic m o d e of this i s o z y m e w a s  1:1 ratio of s e g r e g a t i o n , a s e x p e c t e d . Glucose-6-phosphate  dehydrogenase  (G6P)  T h e r e w e r e t w o z o n e s of e n z y m e a c t i v i t y o b s e r v e d f o r g e l s s t a i n e d f o r G6P. H o w e v e r , the most-cathodally-migrating  z o n e (G6P-2)  w a s unclear and inconsistent for scoring. Both  p o o l e d a n d h e t e r o g e n e i t y G v a l u e s o f t h e h é t é r o z y g o t e ( 1 0 0 : 1 0 0 ' ) at t h e G6P-1 s i g n i f i c a n t (Table  2.4),  Isocitrate  i n d i c a t i n g a d e p a r t u r e f r o m t h e e x p e c t e d 1:1  dehydrogenase  locus were  ratio.  (IDH)  O n l y o n e z o n e of activity  w a s o b s e r v e d in t h i s e n z y m e s y s t e m (Figure  2.1).  One  h e t e r o z y g o u s g e n o t y p e o f t w o s i n g l e - b a n d e d v a r i a n t s ( 1 0 0 a n d 1 0 8 ) w a s o b s e r v e d in a s i n g l e c l o n e (Table  2.4).  Segregation analysis w a s not significant, indicating that the mode  of  i n h e r i t a n c e at t h i s 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 o f a c t i v i t y w e r e o b s e r v e d (LAP-1 LAP-1  h a d t w o a l l e l e s ( 1 0 0 a n d null (n)) (Figure  o f t h e h é t é r o z y g o t e (LAP-1)  2.1).  a n d LAP-2),  LAP-2  was monomorphic.  Both pooled and heterogeneity G values  w e r e n o n s i g n i f i c a n t (Table  2.4),  t h u s c o n f i r m i n g 1:1 s e g r e g a t i o n  ratio. Malate  dehydrogenase  Three  z o n e s of  (MDH)  activity  c a t h o d a l l y - m i g r a t i n g z o n e s (MDH-2  were  observed for  a n d MDH-3)  this  enzyme  system. However,  were u n c l e a r a n d i n c o n s i s t e n t , a n d t h e y w e r e  e x c l u d e d f r o m t h e s t u d y . F o u r , s i n g l e - b a n d e d v a r i a n t s w e r e o b s e r v e d at t h e MDH-1 9 2 , 1 0 0 , a n d 1 1 4 ) (Figure 2.4).  2.1).  two  locus (81,  One clone w a s heterozygous for the " 1 0 0 : 8 1 " genotype  (Table  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 t o t h e 1:1 e x p e c t e d r a t i o . B o t h o f t h e " 1 0 0 : 1 1 4 " a n d  " 1 0 0 : 9 2 " genotypes gave significant pooled and heterogeneity G tests indicating the presence o f a d e p a r t u r e f r o m t h e 1:1 e x p e c t e d ratio (Table Phosphoglucose  isomerase  2.4).  (PGI)  T w o z o n e s of a c t i v i t y w e r e o b s e r v e d f o r t h e e n z y m e s y s t e m (PGI-1  a n d PGI-2).  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 (PGI-2) w a s c l e a r a n d c o n s i s t e n t f o 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 f o r t h i s l o c u s (Figure  2.1).  Neither the pooled nor heterogeneity G  v a l u e w a s s i g n i f i c a n t , c o n f i r m i n g t h e ratio 1:1 o f s e g r e g a t i o n (Table Phosphoglucomutase  (PGM)  2.4).  T w o z o n e s of a c t i v i t y w e r e o b s e r v e d in t h i s e n z y m e s y s t e m . T h r e e a n d t w o s i n g l e b a n d e d v a r i a n t s w e r e o b s e r v e d f o r PGM-1  a n d PGM-2,  r e s p e c t i v e l y (Figure  w a s n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h e e x p e c t e d 1:1 significant  heterogeneity  for  b o t h l o c i (Table  2.4),  2.1).  Pooled G test  ratio; h o w e v e r , trees s h o w e d highly and should thus  not  be a c c e p t e d as  Mendelian. 6-Phosphogloconic  dehydrogenase  T w o z o n e s of activity  (6PG)  w e r e observed on gels stained for this e n z y m e . The  a n o d a l l y - m i g r a t i n g b a n d of b o t h l o c i (6PG-1  a n d 6PG-2)  presented double-banded phenotypes,  w h i l e t h e c a t h o d a l l y - m i g r a t i n g b a n d p r e s e n t e d s i n g l e - b a n d e d p h e n o t y p e s (Figure v a r i a n t s ( 8 6 , 1 0 0 , a n d 1 1 3 ) w e r e o b s e r v e d at 6PG-1 only  observed for  o n e tree  and  it  most-  (Figure  segregated following  2.1).  2.1).  Three  The genotype 1 0 0 : 8 6 w a s  expectations  (Table  2.4).  The  " 1 0 0 : 1 1 3 " g e n o t y p e , on the other h a n d , deviated 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 significant p o o l e d a n d h e t e r o g e n e i t y G t e s t s f o r 6PG-1.  T h e g e n o t y p e " 1 0 0 : 1 1 7 " of 6PG-2  s i g n i f i c a n t p o o l e d G t e s t , b u t t r e e s 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  gave a non2.4)  and so  t h i s l o c u s c a n n o t b e c o n f i r m e d a s 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 f o r o n l y o n e t r e e a n d d i d n o t f o l l o w t h e e x p e c t e d 1:1 r a t i o (Table 2.3.3  2.4).  Summary  Eight of the 2 0 p o o l e d G tests s h o w e d significant departure f r o m the e x p e c t e d  1:1  r a t i o . T h i s is 4 0 p e r c e n t h i g h e r t h a n t h e e x p e c t e d s e g r e g a t i o n at 5 % . S i g n i f i c a n t d e p a r t u r e s f r o m e x p e c t e d s e g r e g a t i o n r a t i o s are r e l a t i v e l y c o m m o n in s t u d i e s o f c o n i f e r i s o z y m e s ( H a 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 ) . T h e s e deviations m a y result f r o m several c a u s e s , including i s o z y m e s b e i n g u n d e r t h e c o n t r o l of m o r e t h a n o n e l o c u s , m e i o t i c d i s t o r t i o n o r i n t e r a l l e l i c interaction, or o b s e r v e d variation not being under g e n e t i c c o n t r o l (Feret, 1 9 7 1 ; E l - K a s s a b y , 1981).  2 . 3 . 4 Linkage  Analyses  Of the 1 0 possible pairwise 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 loci s c o r e d , c l o n e s h a d at l e a s t o n e 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 2.5).  20  (Table  O n e of the eight c o m b i n a t i o n s significantly deviated f r o m joint s e g r e g a t i o n ( 1 : 1 : 1 : 1 ) .  T h e s e w a s PGM-1:PGM-2 Results  were  (Table grouped  2.5). into  "combinations  without  significant  linkage",  and  "combinations with significant linkage." Combinations  without  significant  linkage:  this group contained 6 different combinations  of p a i r e d l o c i ; t h e n u m b e r o f c l o n e s w i t h i n c o m b i n a t i o n s v a r i e d b e t w e e n o n e a n d s e v e n . N o v a r i a t i o n f r o m t h e e x p e c t e d s e g r e g a t i o n ratio w a s o b s e r v e d . Chi-square analyses for the  detection  of linkage revealed three  combinations  for  h o m o g e n e o u s d a t a . D a t a f r o m s i n g l e c l o n e s o b s e r v e d at o n e l o c u s d i f f e r e d s i g n i f i c a n t l y f r o m t h e e x p e c t e d 1:1  r a t i o s (PGM-2:6PG-2  a n d PGM-1-.GDH).  S u c h d e v i a t i o n s are d u e e i t h e r t o  c h a n c e or to the differential viability of g a m e t e s carrying different alleles ( A d a m s and J o l y , 1980b). Combinations 2.5):  with significant  linkage:  this g r o u p c o n t a i n e d 2 t y p e s of o n e e a c h  " 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 i g n i f i c a n t o n G, b u t n o n - s i g n i f i c a n t on/l)  (Table and  " c o m b i n a t i o n s w i t h significant deviation from the e x p e c t e d segregation ratios" (significant on b o t h G | andxDChi-square tests for linkage on 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 data w e r e s i g n i f i c a n t f o r PGM-1 :PGM-2  ( 8 . 5 9 5 , P > 0 . 0 1 ) (Table  a n u n e q u a l c o n t r i b u t i o n of l o c u s PGM-1 2  combination.  A  three-way  2.5).  Partitioned A^-tests failed to detect  t o t h e d i s t o r t i o n of j o i n t i n d e p e n d e n c e o f PGM-1  log-likelihood  test  for  i n d e p e n d e n c e of  c o m b i n a t i o n i n d i c a t e d t h a t i n t e r a c t i o n a m o n g t r e e s at l o c u s PGM-1 o f t h i s c o m b i n a t i o n (Table  2.6).  the  PGM-1  :PGM:PGM-2  a c c o u n t e d for the distortion  T h e r e f o r e , l i n k a g e at t h i s l o c u s c a n n o t b e c o n f i r m e d e i t h e r .  Table 2.5. G o o d n e s s - o f - f i t a n a l y s e s f o r p a i r - w i s e c o m b i n a t i o n s of l o c i in S i t k a s p r u c e . L o g - l i k e l i h o o d t e s t s j o i n t i n d e p e n d e n c e a s s o r t m e n t (G|). P a r t i t i o n e d t e s t s t e s t s e g r e g a t i o n at l o c u s A (/|), B (xl) a n d j o i n t s e g r e g a t i o n (xl)  L o c u s Pair (A-B)  Segregation Class  N o . of Clones AB  PGI-2:PGM-2 PGI-2:6PG-2 PGM-1: PGM-2 PGM-1:6PG-2 PGM-2:6PG-2 PGI-2:PGM-1 PGM-1 :GDH PGM-2:GDH "=Not s i g n i f i c a n t . ' S i g n i f i c a n t at 5 % . " S i g n i f i c a n t at 1 %.  Ab  aB  ab  19 20 56 34  18 25 28 24  38 6  25 6 9 4  3 4 7  23  12  31 37  5 7 1 1 1  35  20 47 27  59 6 5 8  G, (df)  46 6 1 6  9 6  xl (df = 1)  xl  xl  (df = 1)  (df = 1)  7.320(7)"=  0.055"=  2.000"=  1.483"=  13.634(10)™ 61.142(21)"  0.375"=  0.375"= 1.928"= 2.700"= 4.023" 0 0.666"= 0.666"=  2.543"= 8.595" 0.056"= 0.251"= 0  31.178(13)"" 25.973(19)"=  -  0 0.133"= 10.500" 0 6.000' 0.666"=  1.510"= 0.018"=  Table 2.6.  T e s t of i n d e p e n d e n c e f o r t l i e PGM-1 :PGM-2  Hypothesis Test Clone Clone Locus Clone  X X A X  Locus A Locus B X Locus Locus A  df independence independence B independence x L o c u s B interaction  Clone X Locus A x Locus B independence ™ Not significant. "  combination  S i g n i f i c a n t at 1 % .  G  6  35.68" 11.85™ 8.77" 4.84™  19  61.14"  6 6 1  T h r e e - w a y log-likelihood tests applied 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 deviate  significantly  f r o m joint  i n d e p e n d e n c e a t PGM-1:6PG-2  independence  ( Table 2.7).  by /^-tests  indicated  deviation  from  to  joint  This combination did not s h o w significant distortion  f r o m i n d e p e n d e n c e d u e t o i n t e r a c t i o n a m o n g c l o n e a n d t h e t w o l o c i . T h e r e f o r e , PGM-1  should  n o t b e 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 o f t h e a l l o z y m e v a r i a t i o n in t h e 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 c o n t r o l l e d  b y 14 loci, 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 s e g r e g a t i o n ratio a n d the remaining 9 loci g a v e significant deviation f r o m the e x p e c t e d ratio. T h e r e w a s a lack of joint segregation f o r m o s t s t u d i e d l o c i , b u t G6P anû MDH  gave c o n s i s t e n t d i s t o r t e d s e g r e g a t i o n . T h e r e f o r e , t h e y  c o u l d n o t b e i n c l u d e d in t h e m a t i n g s y s t e m s t u d y (see C h e l i a k et al., f u r t h e r i n d i c a t e d t h e i n c o n s i s t e n c y o f l o c i PGM-1. a n a l y s i s of t h e S i t k a s p r u c e m a t i n g s y s t e m .  1984). Linkage analyses  T h e r e f o r e , t h e s e l o c i w e r e n o t u s e d in  Table 2.7.  T h r e e - w a y log-likelihood test for i n d e p e n d e n c e for the combination  Hypothesis Test Clone Clone Locus Clone  X X A X  Locus A Locus B X Locus Locus A  df independence independence B independence x L o c u s B interaction  Clone X Locus A x Locus B independence ™Not s i g n i f i c a n t . * S i g n i f i c a n t at 5 % . * * S i g n i f l e a n t at 1 % .  4 4  PGM-2:6PG-2  G  1 4  12.16* 13.50** 0.06™ 5.46™  13  31.18**  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 o f a l l o z y m e v a r i a n t s in l o b l o l l y p i n e . J. 71:33-40. A d a m s , W . T . and R . J . J o l y . 1 9 8 0 b . Linkage relationships a m o n g twelve l o b l o l l y p i n e . J. Heredity 71:199-202.  Heredity  a l l o z y m e l o c i in  A d a m s , W . T . , D . B . N e a l e a n d C . A . L o o p s t r a . 1 9 8 8 . 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I n h e r i t a n c e of a l l o z y m e v a r i a n t s in c o a s t a l D o u g l a s - f i r {Pseudotsuga menziesii var. menziesii). Can. J. Genet. Cytol. 2 4 : 3 2 5 - 3 3 5 .  El-Kassaby, Y . A . , F.C. Y e h and 0 . Sziklai. 1 9 8 2 b . Linkage relationships a m o n g 19 polymorphic a l l o z y m e l o c i in c o a s t a l D o u g l a s - 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 . a n d K. R i t l a n d . 1 9 8 6 . L o w l e v e l s o f p o l l e n c o n t a m i n a t i o n in a D o u g l a s - f i r s e e d o r c h a r d a s d e t e c t e d b y 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 . Parkinson a n d F.T. Portlock. 1 9 8 7 . A l l o z y m e inheritance, h e t e r o z y g o s i t y a n d 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 , British C o l u m b i a . Heredity 58:173-181. E r n s t , S . G . , D . E . K e a t h l e y a n d J . W . H a n o v e r . 1 9 8 7 . I n h e r i t a n c e o f i s o z y m e s in s e e d a n d b u d t i s s u e s of b l u e a n d E n g e l m a n n s p r u c e . Genome 29:239-246. F a l k e n h a g e n , E . R . 1 9 8 5 . I s o z y m e s t u d i e s in p r o v e n a n c e r e s e a r c h o f f o r e s t t r e e s . Theor. Genet. 6 9 : 3 3 5 - 3 4 7 . F e r e t , P . P . 1 9 7 1 . I s o z y m e v a r i a t i o n in Picea 20:46-50.  glauca  ( M o e n c h ) V o s s s e e d l i n g s . S/7vae  Appl.  Genet.  G u r i e s , R . P . a n d F . T . L e d i g . 1 9 7 8 . I n h e r i t a n c e o f 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 c h p i n e (Pinus rigida M i l l . ) . Heredity 40:27-32. H a m r i c k , J . L . , Y . B . L i n h a r t a n d 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 i s t o r y c h a r a c t e r i s t i c s a n d e l e c t r o p h o r e t i c a l l y d e t e c t a b l e g e n e t i c v a r i a t i o n in p l a n t s . Ann. Rev. Ecol. Syst. 1 0 : 1 7 3 - 2 0 0 . H a r 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 .  Harry, D . E . 1 9 8 3 . Identification of a l o c u s m o d i f y i n g the e l e c t r o p h o r e t i c mobility of malate 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 implications for p o p u l a t i o n s t u d i e s . Biochem. Genet. 2 1 : 4 1 7 - 4 3 4 . H a r r y , D . E . 1 9 8 6 . I n h e r i t a n c e a n d l i n k a g e o f i s o z y m e v a r i a n t s in i n c e n s e - c e d a r . J. 77:261-266. Hubby,  Heredity  J . L . and R . C . L e w o n t i n . 1 9 6 6 . A m o l e c u l a r a p p r o a c h to the s t u d y of genie h e t e r o z y g o s i t y in n a t u r a l p o p u l a t i o n s . I. T h e n u m b e r o f a l l e l e s at d i f f e r e n t l o c i in Drosophila pseudoobscura. Genetics 54:577-594.  K i n g , J . N . , a n d B . P . D a n c i k . 1 9 8 3 . I n h e r i t a n c e a n d l i n k a g e o f i s o z y m e s in w h i t e s p r u c e 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 . a n d L. M e j n a r t o w i c z . 1 9 9 0 . I n h e r i t a n c e o f a l l o z y m e s in Larix Silvae Genet. 3 9 : 1 8 4 - 1 8 8 .  decidua  (Picea  Mill.  L e w o n t i n , R . C . and J . L . H u b b y . 1 9 6 6 . A m o l e c u l a r a p p r o a c h to the s t u d y of genie h e t e r o z y g o s i t y in n a t u r a l p o p u l a t i o n s . II. A m o u n t o f v a r i a t i o n a n d d e g r e e o f h e t e r o z y g o s i t y in n a t u r a l p o p u l a t i o n s of Drosophila pseudoobscura. Genetics 54:595609.  L u n d k v i s t , K. 1 9 7 4 . I n l i e r i t a n c e of l e u c i n e 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 76:91-96.  K.  Hereditas  L u n d k v i s t , K. 1 9 7 7 . I n h e r i t a n c e of e s t e r a s e in n e e d l e s a n d e n d o s p e r m o f N o r w a y s p r u c e abies K . ) . Hereditas 87:27-32.  (Picea  M a r k e r t , C . L . a n d F. M o l l e r . 1 9 5 9 . M u 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 a n d s p e c i e s s p e c i f i c p a t t e r n s . 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 l e c t r o p h o r e t i c v a r i a t i o n in Escherichia 182:1024-1026.  coli from  natural s o u r c e s .  M i l l a r , C.I. 1 9 8 5 . I n h e r i t a n c e o f a l l o z y m e v a r i a n t s in B i s h o p p i n e (Pinus Bioch. Genet. 23:933-946.  muricuta  Science  D. D o n ) .  N e a l e D . B . a n d W . T . A d a m s . 1 9 8 1 . I n h e r i t a n c e o f i s o z y m e v a r i a n t s in s e e d t i s s u e s o f 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 , a n d W . T . A d a m s . 1 9 8 4 . I n h e r i t a n c e of n e e d l e t i s s u e i s o z y m e s in D o u g l a s - f i r . Can. J. Genet. Cytol. 2 6 : 4 5 9 - 4 6 8 . Nei,  M.  1 9 7 5 . Molecular Publishing C o .  population  genetics  and  evolution.  Amsterdam:  North-Holland  O ' M a l l e y , D . M . , F . W . A l l e n f o r f a n d G . M . B l a k e . 1 9 7 9 . I n h e r i t a n c e of i s o z y m e v a r i a t i o n a n d h e t e r o z y g o s i t y in Pinus ponderosa. Bioch. Genet. 17:233-250. P e r r y , D . J . a n d P. K n o w l e s . 1 9 8 9 . I n h e r i t a n c e a n d l i n k a g e r e l a t i o n s h i p s o f a l l o z y m e s o f e a s t e r n 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 O n t a r i o . Genome 32:245-250. P i t e l , J . A . , W . M . C h e l i a k a n d J . B a r r e t t . 1 9 8 7 . I n h e r i t a n c e o f a l l o z y m e s in a b l a c k s p r u c e diallel c r o s s . Silvae  Genet.  36:149-153.  R i t l a n d , K. a n d Y . A . E l - K a s s a b y . 1 9 8 5 . T h e n a t u r e o f i n b r e e d i n g in a s e e d o r c h a r d o f D o u g l a s fir a s s h o w n b y a n e f f i c i e n t m u l t i l o c u s m o d e l . Theor. Appl. Genet. 71:375-384. R u d i n , D. a n d B. R a s m u s o n . 1 9 7 3 . G e n e t i c v a r i a t i o n in e s t e r a s e f r o m n e e d l e s o f Pinus L. Hereditas 73:89-98.  silvestris  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 a n d s e l e c t i o n a p p l i c a t i o n o f i s o z y m e s in tree b r e e d i n g . 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 . Bordeaux: INRA. R u d i n , D. a n d I. E c k b e r g . 1 9 7 8 . L i n k a g e s t u d i e s in Pinus sylvestris a l l o z y m e s . Silvae Genet. 2 7 : 1 - 1 2 .  L. u s i n g m a c r o g a m e t o p h y t e  S h a w , D . V . , A . L . Kahler a n d R . W . A l l a r d . 1 9 8 1 . A m u l t i l o c u s e s t i m a t o r of m a t i n g p a r a m e t e r s in p l a n t p o p u l a t i o n s . Proc.  Nat.  Acad.  Sci.  system  USA 78:1298-1302.  S i m o n s e n , V . a n d H . W e l l e n d o 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 t h e s e e d e n d o s p e r m o f S i t k a s p r u c e (Picea sitchensis [ B o n g . ] C a r r . ) . Forest Tree Improv. ( D e n m a r k ) 9 : 1 - 2 1 .  S m i t h , D . B . a n d W . T . A d a m s . 1 9 8 3 . IVleasuring p o l l e n c o n t a m i n a t i o n in c l o n a l s e e d o r c h a r d s w i t h t h e a i d o f g e n e t i c m a r k e r s . In Proc. 17th South. For. Tree Improv. Conf., p p . 6 9 77. Athens: Univ. Georgia. Sokal,  R.R. a n d F . J . Rohlf. 1 9 8 1 . B i o m e t r y : T h e principles a n d practice of biological research. 2nd ed. N e w York: W . H . Freeman and C o .  statistics  in  S t r a u s s , S . H . a n d 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 n k a g e , a n d d i v e r s i t y o f a l l o z y m e s in k n o b c o n e p i n e . Theor. Appl. Genet. 7 2 : 4 8 3 - 4 9 3 . T i g e r s t e d t , P . M . A . 1 9 7 3 . S t u d i e s o n i s o z y m e v a r i a t i o n in m a r g i n a l a n d c e n t r a l p o p u l a t i o n s of Picea abies. Hereditas 75:47-60. V a l l e j o s , C E . a n d 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 o f i s o z y m e m a r k e r s a n d c o l d t o l e r a n c e in a n i n t r a s p e c i f i c c r o s s o f t o m a t o . Theor. Appl. Genet. 66:241-274. W e b b e r , J . E . a n d F . C . Y e h . 1 9 8 7 . T e s t o f t h e f i r s t - o n , f i r s t - i n p o l l i n a t i o n h y p o t h e s i s in c o a s t a l D o u g l a s - f i r . Can. J. For. Res. 1 7 : 6 3 - 6 8 . X i e , C . Y . , B . P . Dancik and F . C . Y e h . 1 9 9 1 . Inheritance orientalis. J. Heredity 82:329-334.  a n d l i n k a g e o f i s o z y m e s in  Thuja  Y e h , F . C . a n d C . L a y t o n . 1 9 7 9 . T h e o r g a n i z a t i o n of g e n e t i c v a r i a b i l i t y in c e n t r a l a n d m a r g i n a l p o p u l a t i o n s o f l o d g e p o l e p i n e , Pinus contorta s s p . latifolia. Can. J. Genet. Cytol. 24:487-503. Y e h , F . C . a n d D . M . O ' M a l l e y . 1 9 8 0 . E n z y m e v a r i a n t s in n a t u r a l p o p u l a t i o n s o f D o u g l a s - f i r , Pseudotsuga menziesii (Mirb) F r a n c o , f r o m B r i t i s h C o l u m b i a . 1. G e n e t i c v a r i a t i o n p a t t e r n s 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 .  Chapter 3 G e n e t i c D i v e r s i t y in S e e d O r c h a r d a n d N a t u r a l P o p u l a t i o n s o f S i t k a S p r u c e  3.1  Introduction F o r e s t tree  breeding programs  are b a s e d o n t h e  genetic  variability  of  natural  or  i n t r o d u c e d p o p u l a t i o n s . T h e r e f o r e , m a i n t a i n i n g a large g e n e t i c d i v e r s i t y at a n e a r l y s t a g e is e s s e n t i a l if t h e o p p o r t u n i t i e s f o r s e l e c t i o n in t h e f u t u r e are t o b e m a i n t a i n e d a n d i n b r e e d i n g minimized ( A d a m s , 1 9 8 1 ) . H o w e v e r , the breeding p r o c e s s has been f o u n d to n a r r o w  the  g e n e t i c b a s e w h i l e i m p r o v i n g c o m m e r c i a l v a l u e s ( F r a n c i s , 1 9 8 1 ) . D o m e s t i c a t i o n in a g r i c u l t u r a l c r o p p l a n t s h a s r e s u l t e d in a l o s s of g e n e t i c v a r i a b i l i t y ( E l l s t r a n d a n d M a r s h a l l , 1 9 8 5 ) . I s o z y m e a n a l y s i s o f i n b r e d c r o p p l a n t s s u c h a s b a r l e y s h o w l o s s in g e n e t i c v a r i a b i l i t y  during  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 wild populations to land races to cultivars ( B r o w n and C l e g g , 1 9 8 3 ) . T h e r e f o r e , t h e m a i n t e n a n c e o f g e n e t i c d i v e r s i t y in t h e b r e e d i n g p o p u l a t i o n o f  any  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 b e a p r i o r i t y of a n y b r e e d i n g p r o g r a m ( H a m r i c k ,  1991).  T r e e s are o f t e n i n t r o d u c e d i n t o h i g h l y h e t e r o g e n e o u s n a t u r a l e n v i r o n m e n t s ( H a m r i c k ,  1991),  s o g e n e t i c d i v e r s i t y in t h e c u l t i v a t e d f o r e s t is e s s e n t i a l f o r t h e i r a b i l i t y t o c o u n t e r b a l a n c e t h e effects of e n v i r o n m e n t a l c h a n g e s (Kleinschmit, 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 g e n e t i c v a r i a t i o n in a p a r t i c u l a r s p e c i e s is i m p o r t a n t , n o t o n l y f o r a breeding p r o g r a m , but also for effective  genetic c o n s e r v a t i o n (Millar a n d M a r s h a l l , 1 9 9 1 ) .  T h e r e f o r e , t h e m a i n t e n a n c e of h i g h l e v e l s of g e n e t i c d i v e r s i t y in s e e d o r c h a r d s a s c o m p a r e d t o n a t u r a l p o p u l a t i o n s is i m p o r t a n t . While e v i d e n c e e x i s t s indicating that a l l o z y m e loci m a y not be an entirely  unbiased  s a m p l e o f g e n e t i c v a r i a b i l i t y ( L e i g h B r o w n a n d 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 o f i s o z y m e s is a better  method  for providing a faster a s s e s s m e n t of g e n e t i c variation t h a n  quantitative  a p p r o a c h w h i c h requires a series of controlled c r o s s e s . H o w e v e r , patterns of a l l o z y m e variation in p l a n t s  seem to  be at  least roughly  morphological markers and quantitative  c o m p a r a b l e to t h o s e f o u n d  for  genes  controlling  traits (Clegg a n d A l l a r d , 1 9 7 2 ; H a m r i c k a n d A l l a r d ,  1 9 7 5 ; F o w l e r a n d M o r r i s , 1 9 7 7 ; E l - K a s s a b y a n d S z i k l a i , 1 9 8 2 ) . S t a r c h g e l - e l e c t r o p h o r e s i s of i s o z y m e s h a s b e e n u s e d in e l u c i d a t i n g {Pseudotsuga  menziesii,  patterns  1 9 8 3 ; Picea  variation  in m a n y  conifers  Y e h a n d O ' M a l l e y , 1 9 8 0 ; Li a n d A d a m s , 1 9 8 9 ; Picea sitchensis,  a n d E l - K a s s a b y , 1 9 8 0 ; Pinus nigra Arnold, et al.,  of population  mariana,  Y e h et al.,  N i k o l i é a n d T u c i é , 1 9 8 3 ; Pinus monticola,  Yeh  Steinhoff  1 9 8 6 ; Boyle and Morgenstern, 1 9 8 7 ;  Pinuspungens,  G i b s o n a n d H a m r i c k , 1 9 9 1 ) . P a t t e r n s of a l l o z y m e v a r i a t i o n c a n b e f o r m u l a t e d in t e r m s o f t h e d i s t r i b u t i o n o f g e n e t i c d i v e r s i t y 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 , t h e k n o w l e d g e o f w h i c h is essential for long t e r m g e n e 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 u d i e s of a l l o z y m e v a r i a t i o n o n S c o t s p i n e {Pinus sylvestris abies  L.) ( Y a z d a n i et al.,  1985), and N o r w a y spruce  (L.) K a r s t . ) ( G ô m ô r y , 1 9 9 2 ) s t a n d s h a v e s h o w n t h e e x i s t e n c e o f g e n e t i c  {Picea  differentiation  b e t w e e n natural and m a n - m a d e populations. H o w e v e r , genetic differentiation b e t w e e n natural a n d p r o d u c t i o n (seed orchards) p o p u l a t i o n s of c o n i f e r s has not b e e n d e t e r m i n e d . T h i s s t u d y r e p o r t s t h e a p p o r t i o n m e n t of g e n e t i c v a r i a t i o n in a s e e d o r c h a r d a n d in n a t u r a l p o p u l a t i o n s o f Sitka spruce. 3 . 2 Materials  and  This study  Methods u s e d starch-gel e l e c t r o p h o r e s i s of  i s o z y m e s to determine  the  genetic  d i v e r s i t y in a S i t k a s p r u c e s e e d o r c h a r d o w n e d b y C a n a d i a n P a c i f i c F o r e s t P r o d u c t s L t d . T h e seed  orchard  123°24'W)  is  located  in  a n d c o n s i s t s of  Saanichton,  British  Columbia  1 3 9 clones (averaging  9.3  (latitude  ramets  48°35'N,  longitude  per clone) s e l e c t e d  from  e l e v a t i o n s b e t w e e n 0 a n d 4 1 5 m o n w e s t e r n V a n c o u v e r I s l a n d , 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 c h a r d w a s e s t a b l i s h e d in 1 9 7 1  b l o c k s . In S e p t e m b e r  1 9 9 0 , wind-pollinated  in a r a n d o m s i n g l e - t r e e m i x o v e r t h r e e seeds were collected from 134  unequal  seed-orchard  c l o n e s that p r o d u c e d sufficient s e e d s for the s t u d y . Five 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 a n d t h e i r i d e n t i t i e s 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 d r i e d at r o o m t e m p e r a t u r e e x t r a c t e d b y h a n d . S e e d s a m p l e s b y c l o n e w e r e k e p t at 2 ° C u n t i l u s e d .  and  T h e a p p o r t i o n m e n t of g e n e t i c d i v e r s i t y w a s c o m p a r e d t o t h a t d e t e r m i n e d f o r 1 0 l U F R O (International U n i o n of Forest R e s e a r c h Organization) natural p o p u l a t i o n s b y Y e h a n d ElKassaby (1980). 3 . 2 . 1 Isozyme  Assay  E i g h t m e g a g a m e t o p h y t e s f r o m g e r m i n a n t s of e a c h c l o n e w e r e s u b j e c t e d t o i s o z y m e a s s a y f o r 1 3 l o c 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 a n d E l - K a s s a b y ( 1 9 8 0 ) . Details of  t h e 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 t o t a l o f 1 3 4 o f t h e 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 a n d t h e allelic 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 b y Y e h a n d E l Kassaby (1980) on 10 lUFRO Sitka spruce populations. 3 . 2 . 2 Natural  Populations  T e n l U F R O p o p u l a t i o n s s t u d i e d b y Y e h a n d E l - K a s s a b y (1 9 8 0 ) , r e p r e s e n t i n g t h e n a t u r a l r a n g e of S i t k a s p r u c e p o p u l a t i o n s (Table 3 . 2 . 3 Data  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 population.  Analysis  Allelic frequencies for  each population  were  calculated from the  inferred  diploid  g e n o t y p e s of s a m p l e t r e e s . O b s e r v e d a n d 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 i e r a r c h i c a l d i v e r s i t y statistics (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 group-method algorithm ( U P G M A ) cluster analysis (Sneath and Sokal, 1973) were calculated f r o m allelic f r e q u e n c i e s w i t h t h e 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 a n d S e l a n d e r , 1 9 8 1 ) and G E N E S T A T - P C computer program (Whitkus, 1988). 3 . 3 Results  and  Discussion  W h e n t h e allelic f r e q u e n c i e s of 1 0 n a t u r a l p o p u l a t i o n s ( Y e h a n d E l - K a s s a b y , 1 9 8 0 ) a n d t h e s e e d o r c h a r d w e r e c o m p a r e d , t h e s e e d o r c h a r d a n d n a t u r a l p o p u l a t i o n s a r e s i m i l a r in m o s t of t h e a l l e l e s p r e s e n t e d (Table 3.2 a n d Table 3.3). 6PG-2-63  O n l y t h r e e a l l e l e s , G6P-1-^^0,  IDH-88,  o b s e r v e d in n a t u r a l p o p u l a t i o n s w e r e n o t d e t e c t e d in t h e s e e d o r c h a r d (Table  T w o of t h e s e t h r e e a l l e l e s (/D/7-88 a n d 6PG-2-63)  and 3.3).  w e r e v e r y rare in t h e n a t u r a l p o p u l a t i o n s .  In f a c t t h e y w e r e p r e s e n t in o n l y 2 a n d 1 p o p u l a t i o n s o u t of t h e 1 0 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 o f 1 0 l U F R O n a t u r a l p o p u l a t i o n s of S i t k a s p r u c e t o w h i c h r e s u l t s f r o m t h e seed orchard were compared"  lUFRO No. 3024 3030 3040 3044 3049 3058 3062 3003 3008 3012  Location  Duck Cr., Alaska W a r d L., A l a s k a Usk Ferry, B . C . Inverness, B.C. Link R d . , B . C . Salmon Bay, B.C. B i g Q u a l i c u m R., B . C . Forks, W a s h . Hoquiam, Wash. Necanicum, Ore.  ° After Yeh and El-Kassaby (1980).  Elev. (m)  Latitude  Longitude  30 15 137  58°22'N 55°25'N 54°46'N 54°12'N 53°30'N 50°23'N 49°23'N 48°04'N 47°05'N 45°49'N  134°35'W 131°42'W 128°15'W 130°15'W 132°10'W 125°57'W 124°37'W 124°18'W 124°03'W 123°46'W  15 90 0 0 137 7 46  Table 3.2.  A l l e l i c f r e q u e n c i e s at 1 3 l o c i in 1 0 l U F R O n a t u r a l p o p u l a t i o n s ( Y e h a n d E l - K a s s a b y , 1 9 8 0 ) a n d s e e d o r c h a r d ( S . O . ) of S i t k a spruce  Population  Locus AAT-1  Allele  3024  3030  3040  3044  3049  100 92  1.000 0.000 0.986 0.014  1.000 0.000 1.000 0.000 0.616 0.384  1.000 0.000 1.000 0.000 0.850 0.000 0.150 0.641 0.154  1.000 0.000 0.905 0.095 0.821 0.179 0.000 0.800 0.100 0.100 0.000  1.000 0.000 0.881 0.119 1.000 0.000 0.000 0.554  AAT-2  100 71  ACO  100 104 96 100  EST  116 87 75 GDH  G6P-1  IDH  100 137 71 100 110 118 100 108 88  MDH-1  100 114 92 81  PGI-2  100 117 93  0.731 0.269 0.000 0.851 0.055 0.094 0.000 0.862 0.138 0.000 0.612 0.000 0.388 0.911 0.077  0.000 0.722 0.000 0.278 0.000 0.875 0.125 0.000 0.570 0.063 0.367  0.205 0.000 0.963 0.037 0.000 0.707 0.000  0.975 0.025 0.000 0.788 0.000 0.212  0.900  0.293 0.947  1.000  0.100 0.000 1.000  0.053 0.000 1.000  0.000 0.000  0.000 0.000  0.000 0.975 0.000 0.025  0.000 1.000 0.000 0.000  0.000 0.000 0.000  0.000 0.000  1.000 0.000 0.000  1.000 0.000 0.000  0.012  1.000 0.000 0.000 1.000 0.000  0.203 0.243 0.000 0.875 0.113 0.012 0.797  3058  3062  3003  3008  3012  S.O.  1.000  1.000 0.000 0.888 0.112  1.000 0.000 1.000 0.000 0.812  1.000 0.000 0.858 0.142 0.712  1.000 0.000 0.840 0.160 0.727  0.996 0.004  0.188 0.000 0.684 0.177  0.275 0.013 0.800 0.150 0.050 0.000  0.273 0.000 0.824  0.000 1.000 0.000 0.862 0.138 0.000 0.850 0.150 0.000 0.000 0.863 0.000 0.137 0.375  0.000 0.203  0.050 0.575  1.000 0.000  1.000 0.000 0.000  0.000 1.000 0.000 0.000 0.000 0.875 0.143 0.000  1.000 0.000 0.000 0.000 1.000 0.000 0.000  0.811 0.189 0.000 0.718 0.128 0.154 0.000 0.924  0.139  0.076  0.000 0.925 0.075  0.000 0.684  0.000 0.597  0.000  0.195  0.316 1.000  0.208 1.000  0.000 0.000 1.000 0.000  0.000 0.000 1.000 0.000 0.000  0.000 0.000 0.925 0.012 0.063  1.000 0.000 0.000 0.506 0.000 0.494 1.000 0.000 0.000  0.000 0.909  1.000 0.000 0.000 0.000 0.974  0.091 0.000  0.013 0.013  0.038 0.138 0.000 1.000 0.000 0.000 0.363 0.213 0.424 0.949 0.000 0.051 1.000 0.000 0.000 0.000 1.000 0.000 0.000  0.925 0.075 0.769 0.209 0.022 0.821 0.078 0.078 0.023 0.944 0.049 0.007 0.601 0.000 0.399 0.996 0.004 0.000 0.940 0.004 0.052 0.004 0.929 0.045 0.026  (Table  3.2.  Continued) Population  Locus  Allele  3024  3030  3040  3044  3049  3058  3062  3003  3008  3012  S.O.  PGM-1  100  0.846 0.154  0.923 0.077  0.628 0.372  0.923 0.077  0.888 0.112  0.838 0.162  0.888 0.087  0.937  0.863 0.137  0.877  0.000 0.987  0.000 1.000 0.000 0.936 0.064  0.785 0.215 0.000 1.000 0.000 0.760  0.000 1.000 0.000 0.947 0.053 0.000 0.724  0.000 1.000 0.000 0.913 0.087 0.000  0.000  0.000  0.000 0.645 0.000 0.341 0.014  0.000 1.000 0.000 0.900 0.100 0.000 0.625  0.025 1.000  0.240 0.000 0.760 0.000 0.240  0.000 1.000 0.000 0.875 0.125  PGM-2 6PG-1  6PG-2  90 75 100 86 100 113 86 100 117 90 63  0.013 0.815 0.185 0.000 0.461 0.000 0.539 0.000  0.000 1.000 0.000 0.000 0.000  0.263 0.013  0.625 0.350 0.025 0,000  0.300 0.075 0.000  0.000 0.757 0.243 0.000 0.590 0.219 0.191 0.000  0.063 0.000 1.000 0.000 0.886 0.114  0.000 1.000 0.000 0.974  0.116 0.007 0.769 0.231 0.832  0.026 0.000  0.153 0.015  0.000  0.878 0.108 0.014  0.660 0.336 0.004  0.000  0.000  0.000  0.000 0.708 0.292  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 n a t u r a l p o p u l a t i o n s ' a n d s e e d o r c h a r d  Natural Locus  AAT-1 AAT-2 ACO  EST  GDH  G6P-1  IDH  MDH-1  PGI-2  PGM-1  PGM-2 6PG-1  6PG-2  Orchard  Allele Max.  Min.  Ave.  Freq.  100 92 100 71 100 104  1.000 0.000 1.000 0.160 1.000 0.384  1.000 0.000 0.935 0.064 0.794  96 100 116 87  0.150 0.851 0.203 0.278 0.000 1.000 0.138 0.137 0.797  1.000 0.000 0.840 0.000 0.616 0.000 0.000 0.554  0.115 0.140 0.000 0.926 0.058 0.014  1.000 0,000 0.000 0.000  0.000 0.000 0.000 0.862 0.000 0.000 0.363 0.000 0.203 0.900 0.000 0.000 1.000 0.000 0.000 0.000  0.996 0.004 0.925 0.075 0.769 0.209 0.022 0.821 0.078 0.078 0.023 0.944 0.049 0.007 0.601 0.000 0.399 0.996 0.004  1.000 0.143  0.875 0.000  0.965 0.025  0.063 0.937 0.372  0.000 0.628 0.063 0.000 0.987  0.010 0.851 0.145 0.002  75 100 137 71 100 110 118 100 108 88 100 114 92 81 100 117 93 100 90 75 100 86 100 113 86  0.213 0.575 1.000 0.100 0.051  0.025 1.000 0.013 0.974  0.189 0.016 0.744  0.599 0.052 0.347 0.970 0.023 0.006  0.243 0.000  0.026 0.000  0.998 0.001 0.876 0.123 0.000  100 117  1.000 0.350  0.461 0.000  0.701 0.153  90  0.539 0.014  0.000 0.000  0.143 0.001  63  0.000 0.757  0.000 0.940 0.004  1.000 0.000 0.000 0.000  0.052 0.004 0.929 0.045  " D i f f e r e n c e s in allelic f r e q u e n c y w h e r e +  = gained and -  =  lost.  —  + 0.004  -  —  -  —  + 0.023 —  -  -0.052  0,006 —  + 0.004 + 0.052 + 0.004 —  -  0.026 0.877  —  0.116 0.007  —  0.769 0.231 0.832 0.153  —  0.015 0.660  ' Y e h and El-Kassaby (1980).  Gain/loss"  -  • 0.01 5 —  0.336 0.004  -  0.000  0.001  w i t h i n t h e s e t h r e e p o p u l a t i o n s , t h e i r f r e q u e n c i e s r a n g e d f r o m 0 . 0 1 2 t o 0 . 0 5 1 (Table 3.2). G6P-1-'\  1 0 , o n t h e o t h e r h a n d , w a s p r e s e n t in 3 p o p u l a t i o n s w i t h a f r e q u e n c y r a n g i n g f r o m  0 . 0 5 0 t o 0 . 2 1 3 (Table  3.2),  £ 5 7 - 7 5 , MDH-1-^^4,-92,  b u t w a s n o t p r e s e n t in t h e s e e d o r c h a r d . S i x a l l e l e s , a n d - 8 1 , a n d 6PG-1-86  AAT-1-92,  o b s e r v e d in t h e s e e d o r c h a r d w e r e  d e t e c t e d in t h e n a t u r a l p o p u l a t i o n s . H o w e v e r , s o m e o f t h e m w e r e rare a l l e l e s (Table Table  The  3.3).  3.2  T h e s e r e s u l t s p r o b a b l y are d u e t o t h e d i f f e r e n c e s in s o u r c e r a n g e b e t w e e n  not and the  orchard and the 1 0 natural populations. O r c h a r d c l o n e s w e r e selected mainly on V a n c o u v e r Island, w h i l e the s a m p l e s of natural p o p u l a t i o n s 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 northern O r e g o n , including only t w o populations o n V a n c o u v e r Island. Therefore, s a m p l i n g breadth m a y be r e s p o n s i b l e for these differences. S u c h differences 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 ) a n d P o r t - O r f o r d c e d a r , Chamaecyparis  lawsoniana  (Millar and M a r s h a l l , 1 9 9 1 ) . Mean  heterozygosity  0.216 + 0.062  (average  of  natural  0.184 ±0.020)  0 . 2 3 0 + 0 . 0 4 4 p e r l o c u s (Table 3.4).  populations per  locus  ranged  while  the  from  0.160 ±0.050  seed  orchard  to  presented  T h e m e a n h e t e r o z y g o s i t y p e r l o c u s w a s h i g h e r in t h e s e e d  o r c h a r d t h a n in t h e n a t u r a l s t a n d s , b u t t h i s d i f f e r e n c e w a s n o t s i g n i f i c a n t . W h e n t h e n u m b e r o f a l l e l e s per l o c u s w a s c o m p a r e d , n a t u r a l p o p u l a t i o n s a n d t h e s e e d o r c h a r d s h o w e d s i g n i f i c a n t d i f f e r e n c e s in a v e r a g e allele n u m b e r p e r l o c u s . N a t u r a l p o p u l a t i o n s p r e s e n t e d a r a n g e b e t w e e n 1.62 ± 0 . 1 8 a n d 2 . 0 0 + 0 . 1 6 (average 1.82 ± 0 . 1 2 ) alleles per l o c u s w h i l e the s e e d o r c h a r d s h o w e d 2 . 7 7 ± 0 . 2 0 a l l e l e s per l o c u s (Table  3.4).  T h e p e r c e n t a g e o f p o l y m o r p h i c l o c i in t h e  natural populations 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 % ) , while the p o l y m o r p h i s m o f l o c i in t h e s e e d o r c h a r d w a s 8 4 . 6 2 % (this c o m p a r i s o n is r e s t r i c t e d t o o n l y 13  polymorphic  loci). T h e s e figures  indicate that the  genetic  diversity  found  in  natural  p o p u l a t i o n s w a s r e t a i n e d o r i n c r e a s e d in t h e s e e d o r c h a r d . T h e g e n e t i c d i v e r s i t y o f S i t k a s p r u c e ( s e e d o r c h a r d i n c l u d e d ) e q u a l s t h e a v e r a g e f o r g y m n o s p e r m s a s a w h o l e (see H a m r i c k et 1979).  al.,  Table 3.4.  Population  3024 3030 3040 3044 3049 3058 3062 3003 3008 3012 Ave. Orchard  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 l e l e s p e r l o c u s , a n d p e r c e n t a g e of l o c i p o l y m o r p h i c in 1 0 l U F R O n a t u r a l p o p u l a t i o n s a n d seed orchard  Mean Heterozygosity per l o c u s  M e a n N o . of Alleles per locus  0.209 ±0.050 0.160 + 0.054 0.189±0.054  2.00 + 0.16 1.62±0.18 1.69±0.17 1.77 + 0 . 2 0 1.85 + 0 . 2 2 1.69 + 0.21 1.92 + 0.21 1.92 + 0 . 2 4 1.85±0.22 1.85 + 0 . 2 2 1.82 + 0 . 1 2 2.77±0.20  0.167±0.050 0.187 + 0.054 0.162±0.052 0 . 2 0 5 + 0.051 0.216±0.062 0.177±0.053 0.171 ± 0 . 0 5 5 0.184±0.020 0.230±0.044  P e r c e n t a g e of Loci Polymorphic 84.62 53.85 61.54 61.54 61.54 53.85 69.23 61.54 61.54 61.54 63.08±8.73 84.62  Gene 0.201 ± 0 . 0 5 1  diversity [Table  (H,) 3.5),  of  Sitl<a  with  the  spruce  estimated  relative  amount  from of  natural  genetic  populations  differentiation  was  among  p o p u l a t i o n s (G,,) o f 0 . 0 8 2 ± 0 . 0 1 6 . H o w e v e r , w h e n t h e s e e d o r c h a r d w a s i n c l u d e d , t h e g e n e d i v e r s i t y (H,) w a s i n c r e a s e d t o 0 . 2 0 6 ± 0 . 0 5 1 i.e. 2 . 5 % h i g h e r . T h e r e l a t i v e a m o u n t o f g e n e t i c d i f f e r e n t i a t i o n a m o n g p o p u l a t i o n s after t h e i n c l u s i o n o f t h e 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 % increase. The improvement  in d i v e r s i t y w h e n t h e o r c h a r d p o p u l a t i o n w a s  added  indicated that the seed orchard p o s s e s s e d genie diversity higher than the m e a n of the rangew i d e p o p u l a t i o n . H o w e v e r , t h e s e differences w e r e not significant. T h e proportion of the total gene diversity w i t h i n populations of S i t k a 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 total g e n e diversity, the s a m e as Douglas-fir ( A d a m s , 1 9 8 1 ) , w h e r e o n l y 8 . 6 % of total diversity  was  among populations. W h e n t w o p o p u l a t i o n s p o s s e s s i d e n t i c a l d i v e r s i t y , it d o e s n o t n e c e s s a r i l y m e a n t h a t t h e y h a v e t h e s a m e g e n e in c o m m o n ( G r e g o r i u s , 1 9 7 8 ) . T h e a m o u n t o f t h e d i f f e r e n c e c a n be o b t a i n e d f r o m g e n e t i c - d i s t a n c e a n a l y s i s {Table  3.6).  Genetic-distance values indicate that  population 3 0 6 2 and 3 0 0 8 presented the minimal genetic distance ( 0 . 0 0 5 ) while the s e e d orchard and population 3 0 2 4 provided the maximal genetic distance (0.044). H o w e v e r , the average genetic distance a m o n g the 10 l U F R O natural populations w a s about 0 . 0 2 0 ± 0 . 0 0 9 , while the average genetic distance a m o n g these 10 populations and the seed orchard w a s 0.029±0.009.  T h e d i f f e r e n c e is a b o u t 4 % . S u r p r i s i n g l y , c l u s t e r a n a l y s i s i n d i c a t e d t h a t t h e  s e e d o r c h a r d w a s d i s t i n c t f r o m all n a t u r a l p o p u l a t i o n s s t u d i e d {Figure 3.1).  P e r h a p s the greatest  g e n e t i c d i f f e r e n c e s of t h e s e e d o r c h a r d w e r e d u e p a r t i a l l y t o t h e f a c t t h a t t h e s e e d o r c h a r d harboured more variation from m a n y geographic populations than any other natural population a l o n e . H o w e v e r , t h i s is e x p e c t e d d u e t o t h e c l u s t e r i n g of s i m i l a r g e n o t y p e s w i t h i n p o p u l a t i o n s ( B r u n e i a n d R o d o l p h e , 1 9 8 5 ; E l - K a s s a b y et al.,  1987).  Unlike agricultural crop plants (Brown and C l e g g , 1 9 8 3 ; Ellstrand and M a r s h a l l , 1 9 8 5 ) , t h e r e d u c t i o n o f g e n e t i c d i v e r s i t y d u e t o p h e n o t y p i c s e l e c t i o n is m i n o r f o 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 statistics e s t i m a t e s for 1 3 poiymorpfiic loci a v e r a g e d over 1 0 natural p o p u l a t i o n s a n d o v e r 11 p o p u l a t i o n s {10 n a t u r a l a n d o n e s e e d o r c h a r d )  Dl,  h;  Gt  Locus 10P"  IIP"  10P  IIP  10P  IIP  10P  IIP  AAT-1 AAT-2  0.000 0.121 0.336 0.415 0.140 0.521 0.057  0.000 0.113 0.310 0.396 0.132  0.000 0.063 0.068 0.043 0.048 0.112 0.062  0.035 0.215 0.401  0.000 0.008 0.026 0.019 0.008 0.043 0.003 0.000 0.006 0.016 0.000 0.010 0.076  0.000 0.008 0.023 0.018 0.007  0.478 0.054  0.001 0.115 0.315 0.389 0.130 0.478 0.050 0.010 0.072 0.237  0.000 0.064  ACO EST GDH G6P-1 IDH MDH-1 PGI-2 PGM-1  0.001 0.123 0.338 0.407 0.137  0.188 0.045  0.017 0.006  PGM-2 6PG-1 6PG-2  0.255 0.003 0.218 0.472  0.538 0.053 0.011 0.076 0.251 0.044 0.224 0.474  Ave. S.E.=  0.201 0.051  0.206 0.051  0.000 0.071  0.000 0.065 0.239 0.003 0.208 0.396 0.184 0.046  0.060 0.003 0.001 0.004 0.014 0.009 0.009 0.073 0.018 0.006  ^ Total gene diversity. ^ Gene diversity within populations. ^ Gene diversity a m o n g populations. •* R e l a t i v e a m o u n t o f g e n e t i c d i f f e r e n t i a t i o n a m o n g p o p u l a t i o n s . ' 10 natural populations. " 1 1 p o p u l a t i o n s (natural and s e e d orchard). " Chakraborty (1974).  0.076 0.046 0.056 0.083 0.058 0.000 0.079 0.061 0.000 0.046 0.160 0.082 0.016  0.099 0.058 0.057 0.201 0.039 0.153 0.086 0.016  Table 3.6.  Population 3024 3030 3040 3044 3049 3058 3062 3003 3008 3012 SO  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 p o p u l a t i o n s , 1 0 n a t u r a l p o p u l a t i o n s a n d a s e e d o r c h a r d ( S O )  3024  3030  3040  3044  3049  3058  3062  3003  3008  -  0.032 0.018 0.031 0.023 0.029 0.020 0.016 0.027 0.033 0.044  0.023  -  0.026 0.036 0.029 0.019  0.016 0.010 0.027  0.023 0.017  0.011 0.023 0.026 0.034  0.011 0.035  0.013  0.026 0.023 0.006 0.018 0.017 0.021 0.024  -  0.038 0.015 0.014 0.032 0.038 0.039  -  -  0.012 0.017  0.006  0.006 0.015 0.032  0.005 0.015 0.020  -  0.014 0.020 0.014  0.007 0.026  3012  SO  3024 3040 3049 3003 3030 3012 3044 3058 3062 3008 ORCHARD  0.04  0.03  0.02  0.01  0 . 0 0  DISTANCE Figure  3.1. P h e n o g r a m o f S i t k a s p r u c e d i f f e r e n t i a t i o n b a s e d o n N e i ' s ( 1 9 7 8 ) u n b i a s e d genetic distance. Clusters are produced using the unweighted-pair-groupmethod alogorithm ( U P G M ) with Nei's (1978) unbiased genetic distances.  H o w e v e r , t h e s e e d o r c h a r d s t u d i e d h e r e is still in t h e f i r s t s t a g e o f d o m e s t i c a t i o n ,  therefore,  r e d u c t i o n m i g h t be o b s e r v e d after a c o m p l e t e c y c l e of recurrent s e l e c t i o n . T h i s h a s been o b s e r v e d in o t h e r c o n i f e r s ( D o u g l a s - f i r , A d a m s , 1 9 8 1 ; S c o t s p i n e , S z m i d t a n d M u o n a , 1 9 8 5 ; radiata pine, M o r a n a n d Bell, 1 9 8 7 ; loblolly pine, H a m r i c k ,  1991).  V a r i o u s s t u d i e s o f p o l l i n a t i o n p a t t e r n s in f o r e s t t r e e s e e d o r c h a r d s i n d i c a t e t h a t o f t e n o n l y a l i m i t e d n u m b e r of m a l e c l o n e s c o n t r i b u t e a c t i v e l y t o t h e n e x t g e n e r a t i o n ( J o n s s o n 1 9 7 6 ; G r 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.,  etal.,  1 9 8 2 ; R o b e r d s et al., 1 9 9 1 ) . A l s o ,  a disproportion of female clones contributing to seed production w a s o b s e r v e d a n d this varied f r o m y e a r - t o - y e a r ( E l - K a s s a b y et al., 1 9 8 9 ; C h a p t e r 5). T h e o r e t i c a l l y , g e n e t i c d i v e r s i t y s h o u l d b e r e d u c e d in s e e d o r c h a r d s . B u t , it is p o s s i b l e t h a t t h e a m o u n t o f g e n e t i c d i v e r s i t y  retained  a m o n g o r c h a r d c l o n e s is still large ( A d a m s , 1 9 8 1 ) . D a t a f r o m o t h e r c o n i f e r s s u g g e s t t h a t h o m o z y g o t e e x c e s s , d u e t o s e l f i n g o r c o n s a n g u i n e o u s m a t i n g in s e e d o r c h a r d s , p r e s e n t a t t h e e m b r y o s t a g e h a d b e e n r e m o v e d after a f e w y e a r s o f f i e l d g r o w t h ( N e a l e , 1 9 8 5 ; S z m i d t a n d 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 a v a i l a b l e t o d a t e i n d i c a t e s t h a t c o n i f e r s in g e n e r a l p o s s e s s a g r e a t d e a l of g e n e t i c d i v e r s i t y ( H a m r i c k et al.,  1979).  M o r a n et al. ( 1 9 8 0 ) c a u t i o n e d a b o u t t h e l o s s of g e n e t i c v a r i a b i l i t y t h a t o c c u r s in s e e d orchards comprising a l o w number of clones. This study indicated that a production population o f 1 3 9 c l o n e s in S i t k a s p r u c e is s u f f i c i e n t t o p r e v e n t t h e l o s s of g e n e t i c v a r i a b i l i t y . M o r e o v e r , supplemental m a s s pollination c a n be used to broaden the genetic base by introducing desirable g e n o t y p e s i n t o s e e d o r c h a r d s ( W o e s s n e r a n d F r a n k l i n , 1 9 7 3 ) . C o n s e q u e n t l y , a n i n c r e a s e in t h e gene diversity of a seed o r c h a r d ' s c r o p s c a n be obtained directly (El-Kassaby, 1 9 9 1 ) .  3.4  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 a n d g e n e 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 c o n 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 a n d J . L . R e v e a l , p p . 4 0 1 - 4 1 5 , P i t t s b u r g h : H u n t Inst. B i o l . D o c , C a r n e g i e - M e l l o n U n i v . 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P a t t e r n s of g e n e t i c d i f f e r e n t i a t i o n in t h e s e l e n d e r w i l d o a 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 . 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 a n d g e n e t i c d i v e r s i t y - s h o u l d w e b e c o n c e r n e d ? Paper presented at the 23rd biennial meeting of Canadian Tree Improvement Association, August 19-23, 1991. Ottawa. E l - K a s s a b y , Y . A . a n d 0 . S z i k l a i . 1 9 8 2 . G e n e t i c v a r i a t i o n o f a l l o z y m e a n d q u a n t i t a t i v e t r a i t s in a s e l e c t e d D o u g l a s - f i r (Pseudotsuga menziesii var. menziesii {Mirb.) F r a n c o ) p o p u l a t i o n . For. Ecol. Manage. 4:115-126. 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. Portlock. 1 9 8 7 . 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B I O S Y S - 1 : a F O R T R A N program for the c o m p r e h e n s i v e a n a l y s i s of e l e c t r o p h o r e t i c d a t a in p o p u l a t i o n g e n e t i c s a n d s y s t e m a t i c s . J. Heredity 72:281-283. W h i t k u s , R. 1 9 8 8 . M o d i f i e d v e r s i o n o f G E N E S T A T : A p r o g r a m f o r c o m p u t i n g g e n e t i c s t a t i s t i c s f r o m allelic f r e q u e n c y d a t a . P/ant Genet. News/etter 4:10. W o e s s n e r , R . A . and E . G . Franklin. 1 9 7 3 . C o n t i n u e d reliance on wind-pollinated southern pine s e e d o r c h a r d s - is it r e a s o n a b l e ? 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. Exp. Stat, and Lousiana State Univ. Y a z d a n i , R. 0 . M u o n a , D. R u d i n , a n d A . E . S z m i d t . 1 9 8 5 . G e n e t i c s t r u c t u r e of a Pinus sy/vestris L. s e e d - t r e e s t a n d a n d n a t u r a l l y r e g e n e r a t e d 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 . a n d Y . A . E l - K a s s a b y . 1 9 8 0 . E n z y m e v a r i a t i o n in n a t u r a l p o p u l a t i o n s o f S i t k a s p r u c e {Picea sitchensis). 1. G e n e t i c v a r i a t i o n p a t t e r n s a m o n g t r e e s 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 . Yeh,  F . C . a n d D. O ' M a l l e y . 1 9 8 0 . E n z y m e v a r i a t i o n in n a t u r a l p o p u l a t i o n s o f D o u g l a s - f i r , Pseudotsuga menziesii (Mirb.) F r a n c o , f r o m B r i t i s h C o l u m b i a . 1. G e n e t i c v a r i a t i o n p a t t e r n s 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 . K h a l i l , Y . A . E l - K a s s a b y a n d D . C , T r u s t . 1 9 8 6 . A l l o z y m e v a r i a t i o n in Picea mariana f r o m N e w f o u n d l a n d : genetic diversity, population s t r u c t u r e , a n d analysis of d i f f e r e n t i a t i o n . Can. J. For. Res. 1 6 : 7 1 3 - 7 2 0 .  Chapter 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 v a r i a t i o n in a p o p u l a t i o n is a r e f l e c t i o n of t h e n a t u r e a n d e x t e n t o f g e n e t i c  transmission to s u c c e s s i v e generations by m e a n s of the mating s y s t e m (Clegg, 1 9 8 0 ) . Plant s p e c i e s w h i c h practice a high degree of cross-fertilization are more genetically variable than t h o s e t h a t p r a c t i c e s e l f - f e r t i l i z a t i o n ( H a m r i c k et al., 1 9 7 9 ) . C o n i f e r o u s t r e e s a r e c o n s i d e r e d t o be a m o n g t h e m o s t - h e t e r o z y g o u s plants k n o w n , at least at t h e level of e n z y m e  variation  ( H a m r i c k , 1 9 8 2 ) . H i g h o u t c r o s s i n g r a t e s , e x t e n s i v e g e n e f l o w , a n d 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 i g h e r l e v e l s o f g e n e t i c v a r i a t i o n ( H a m r i c k et al., 1 9 7 9 ) l e a d i n g t o a h i g h  proportion  o f h e t e r o z y g o u s i n d i v i d u a l s in t h a t p o p u l a t i o n . H e t e r o z y g o u s i n d i v i d u a l s a r e p o s t u l a t e d t o be more vigourous than h o m o z y g o u s individuals a n d m a y be more tolerant to a wider range of environmental  c o n d i t i o n s , perhaps d u e to their greater metabolic flexibility ( B r o w n , 1 9 7 9 ;  M i t t o n et al., 1 9 8 1 ) . F e a t u r e s o f i n b r e e d i n g d e p r e s s i o n in c o n i f e r s a r e w e l l k n o w n ( F r a n k l i n , 1 9 7 0 ) a n d r e s u l t in r e d u c t i o n s in v i a b l e s e e d y i e l d (Pinus sylvestris, menziesii, and  Pseudotsuga  S o r e n s e n , 1 9 7 1 ; O r r - E w i n g , 1 9 7 6 ; W o o d s a n d H e a m a n , 1 9 8 9 ; Pinus radiata.  Lindgren,  1985),  seedling vigour  (Douglas-fir,  ( D o u g l a s - f i r , S o r e n s e n a n d M i l e s , 1 9 7 4 ; Pinus 1 9 8 4 ; Picea Picea  Sarvas, 1962;  omorika,  pungens.  radiata,  Sorensen and Miles, W i l c o x , 1 9 8 3 ; Picea  Griffin  1974),  growth  pungens,  Cram,  Geburek, 1 9 8 6 ) , and survival (Douglas-fir, S o r e n s e n a n d M i l e s , 1 9 7 4 ;  C r a m , 1 9 8 4 ) . T h e r e f o r e , in s e e d o r c h a r d s , t h e e n h a n c e m e n t of o u t c r o s s i n g  should be accentuated (Denison a n d Franklin, 1 9 7 5 ) . M a n y f a c t o r s h a v e b e e n c o n s i d e r e d t o a f f e c t t h e rate of o u t c r o s s i n g in a p o p u l a t i o n ( C l e g g , 1 9 8 0 ) . T h e s e i n c l u d e : a g e ( C h e l i a k e f a / . , 1 9 8 5 a ; S n y d e r etal.,  1 9 8 5 ; Shea,  1987),  tree size ( S h e a , 1 9 8 7 ) , d e n s i t y (Levin a n d K e r s t e r , 1 9 7 4 ; Farris a n d M i t t o n , 1 9 8 4 ; S h e a , 1987),  maternal  heterozygosity  (Shea,  1987),  cone production  (Shea,  1987),  pollination  e n v i r o n m e n t ( E l - K a s s a b y a n d D a v i d s o n , 1 9 9 1 ), a n d t e m p o r a l d i s t r i b u t i o n ( C h e l i a k etal,  1985a;  S n y d e r e r a / . , 1 9 8 5 ; G i b s o n a n d H a m r i c k , 1 9 9 1 ; E l - K a s s a b y e r a / . , 1 9 9 2 ) . G e n e t i c v a r i a t i o n in  p o l l e n f e c u n d i t y a n d f l o r a l p h e n o l o g y c a n b e f a c t o r s a f f e c t i n g o u t c r o s s i n g rate a s w e l l ( E r i k s s o n et al.,  1 9 7 3 ; G r 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 c o n i f e r s e e d  o r c h a r d s , p o l l e n c o n t a m i n a t i o n w a s o b s e r v e d t o inflate o u t c r o s s i n g e s t i m a t e s ( F r i e d m a n a n d A d a m s , 1 9 8 5 a , b; E l - K a s s a b y a n d R i t l a n d , 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 management practices have s h o w n that pollen contamination  a s w e l l a s i n b r e e d i n g c a n be  reduced (El-Kassaby and Davidson, 1990). U n d e r s t a n d i n g the breeding structure of conifer 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 better o r c h a r d d e s i g n a n d m a n a g e m e n t that will p r o m o t e intermating a m o n g the s e l e c t e d 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 e d m a n a n d A d a m s , 1 9 8 5 a ) . In t h i s s t u d y ,  o u t c r o s s i n g a n d i n b r e e d i n g at u p p e r a n d l o w e r c r o w n l e v e l s w e r e e s t i m a t e d in a c l o n a l S i t k a spruce seed orchard. 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 P a c i f i c F o r e s 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 c h a r d is l o c a t e d in S a a n i c h t o n , B r i t i s h C o l u m b i a ( l a t i t u d e longitude 1 2 3 ° 2 4 ' W )  48°35'N,  a n d c o n s i s t s of 1 3 9 c l o n e s ( a v e r a g i n g 9 . 3 r a m e t s p e r c l o n e ) s e l e c t e d  from elevations b e t w e e n 0 and 4 1 5 m on western V a n c o u v e r Island, W a s h i n g t o n and Oregon (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 i n g l e - t r e e m i x o v e r t h r e e  u n e q u a l b l o c k s . T r e e s are s p a c e d 3 m a p a r t a n d k e p t at a p p r o x i m a t e l y 4 m in h e i g h t b y t o p p r u n i n g . T h e s e e d o r c h a r d is 1 0 k m a w a y f r o m t h e n e a r e s t S i t k a s p r u c e s t a n d , t h e r e f o r e , t h e S i t k a s p r u c e b a c k g r o u n d p o l l e n is n e g l i g i b l e in t h e o r c h a r d a r e a ( 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 i n d - p o l l i n a t e d s e e d s f r o m t r e e s p r o d u c i n g s u f f i c i e n t s e e d s f o r t h e s t u d y w e r e c o l l e c t e d f r o m o n e of t h e o r c h a r d ' s t h r e e 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 p e r c e n t o f t h e t o t a l o r c h a r d c l o n e s . F i v e s e e d c o n e s f r o m t h e u p p e r a n d l o w e r c r o w n of c o n e - b e a r i n g p o r t i o n s o f t h e t r e e s w e r e c o l l e c t e d r a n d o m l y a n d t h e i r i d e n t i t i e s 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 d r i e d at r o o m t e m p e r a t u r e a n d e x t r a c t e d b y h a n d . S e e d s a m p l e s w e r e l a b e l l e d a n d k e p t at 2 ° C u n t 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 ( I n ) a n d 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 t h e m e t h o d s o u t l i n e d in C h a p t e r 2 . T h r e e e n z y m e s y s t e m s (PGI2, PGM-2  a n d GDH) w e r e s e l e c t e d f o r e s t i m a t i o n 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 t h e i r  M e n d e l i a n s e g r e g a t i o n patterns a n d apparent lack of linkage (Chapter 2). M a t e r n a l g e n o t y p e s w e r e i n f e r r e d f r o m t h e s e g r e g a t i o n o f 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 p e r tree: 8 f r o m e a c h of the upper a n d l o w e r c r o w n s e g m e n t s . T h e probability of  incorrectly  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 o n e l o c u s is (72)'""^', w h e r e n = t h e n u m b e r o f s e e d s a n a l y z e d (16) ( T i g e r s t e d t ,  1 9 7 3 ) . A t a n d e m a s s a y of t h e h a p l o i d m e g a g a m e t o p h y t e a n d t h e  diploid  e m b r y o tissue revealed the pollen contribution. Single  and  outcrossed-pollen  multilocus allelic  population  frequencies  (p)  estimates were  of  outcrossing  estimated  using  the  rate  (t^  and  t^)  and  maximum-likelihood  p r o c e d u r e of R i t l a n d a n d 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 mating 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 statistically m o r e efficient 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 a n d 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 ( N e a l e a n d A d a m s , 1 9 8 5 ) , e s p e c i a l l y w h e n r e l a t i v e l y f e w l o c i are a s s a y e d . A m u l t i l o c u s e s t i m a t e o f o u t c r o s s i n g is c o n s i d e r e d m o r e a c c u r a t e a n d l e s s s e n s i t i v e t o v i o l a t i o n s o f t h e m o d e l a s s u m p t i o n s s o t h a t a g r e a t e r n u m b e r o f o u t c r o s s e s m a y be i d e n t i f i e d w i t h c e r t a i n t y ( S h a w et al.,  1981).  In t h i s s t u d y it w a s a s s u m e d t h a t : 1 ) f e r t i l i z a t i o n e v e n t s are a m i x t u r e of r a n d o m o u t c r o s s i n g a n d s e l f - f e r t i l i z a t i o n ; 2) t h e r e is n o s e l e c t i o n b e t w e e n f e r t i l i z a t i o n a n d c e n s u s ; 3) t h e rate of o u t c r o s s i n g is i n d e p e n d e n t a m o n g l o c i (for m u l t i l o c u s e s t i m a t e s ) a n d 4) allelic f r e q u e n c i e s in t h e o u t c r o s s i n g - p o l l e n p o o l are i d e n t i c a l o v e r t h e p o p u l a t i o n o f m o t h e r t r e e s ( F y f e a n d B a i l e y , 1 9 5 1 ; S h a w et al.,  1981).  A C h i - s q u a r e c o n t i n g e n c y test (Sokal and Rohlf, 1981 ) w a s u s e d to test the difference in t h e e s t i m a t e s of p o l l e n allelic f r e q u e n c i e s f r o m u p p e r a n d l o w e r c r o w n p o r t i o n s .  4 . 3 Results  and  Discussion  4 . 3 . 1 Allelic  Frequencies  P o l l e n allelic f r e q u e n c i e s w e r e s l i g h t l y d i f f e r e n t b e t w e e n t h e u p p e r a n d l o w e r c r o w n s (Table  4.1).  H o w e v e r , no significant differences b e t w e e n the upper and lower outcrossing-  p o l l e n p o o l s w a s o b s e r v e d . T h e allelic f r e q u e n c i e s o f s a m p l e d p o l l e n a n d t h e m a t e r n a l g e n e p o o l s w e r e c o m p a r e d t o d e t e r m i n e if t h e e f f e c t i v e p o l l e n p o o l w a s r e p r e s e n t a t i v e o f t h e a d u l t m a t e r n a l t r e e s o f t h a t b l o c k (Table 4.1).  A s i g n i f i c a n t d i f f e r e n c e f o r 1 (PGM-2)  out of 3 studied  loci b e t w e e n the t w o gene pools w a s o b s e r v e d , indicating that pollen f l o w f r o m the other t w o o r c h a r d b l o c k s c o n t r i b u t e d t o t h e s t u d i e d b l o c k . In c o n i f e r s , c o n t r i b u t i o n s of m a l e a n d f e m a l e p a r e n t s t o g a m e t i c p o o l s h a s b e e n o b s e r v e d t o be a s y m m e t r i c a l , i.e., s o m e t r e e s  contributed  m o r e o f t h e e l e c t r o p h o r e t i c a l l y h o m o l o g o u s allele t o t h e i r p r o g e n y w h e n u s e d a s a m a l e p a r e n t a n d l e s s w h e n u s e d a s a f e m a l e p a r e n t , ( M u l l e r - S t a r c k , 1 9 8 2 ; M o r a n et al., 1 9 8 3 ; C h e l i a k a n d P i t e l , 1 9 8 4 ) , i n d i c a t i n g t h e e x i s t e n c e of d i f f e r e n t s e l e c t i v e v a l u e s a s s o c i a t e d w i t h a r r a y s d e r i v e d f r o m t h e m a l e a n d f e m a l e ( C h e l i a k et al., The  gametic  1985b).  d i s c r e p a n c y in allelic f r e q u e n c i e s a m o n g d i f f e r e n t  gene pools  (maternal  and  o u t c r o s s i n g ) c o u l d b e c a u s e d b y s e v e r a l o t h e r f a c t o r s . T h e s e i n c l u d e : d i f f e r e n c e s in p o l l e n 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 p o l l e n p a r e n t s ( M o r a n et al., S t a r c k et al.,  1 9 8 3 , E l - K a s s a b y a n d R i t l a n d , 1 9 8 6 ; E l - K a s s a b y et al.,  1 9 8 0 ; Muller-  1 9 8 8 ; Erickson 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 s e l e c t i o n of m a l e r e p r o d u c t i v e s u c c e s s ( E l - K a s s a b y a n d R i t l a n d , 1 9 9 2 ) , differences a m o n g o u t c r o s s i n g pollen p o o l s , a n d / o r the p r e s e n c e of g a m e t i c o r z y g o t i c ( p o s t m a t i n g ) s e l e c t i o n ( B r o w n et al., 4 . 3 . 2 Outcrossing  1 9 7 5 ; Y e h et al.,  1983).  Rate  Single- a n d m u l t i l o c u s e s t i m a t e s of o u t c r o s s i n g r a n g e d f r o m 6 5 % to 9 4 % a n d f r o m 6 5 % t o 8 8 % ) , r e s p e c t i v e l y (Table 4.2), PGM-2  all e s t i m a t e s w e r e s i g n i f i c a n t at t h e P<  0 . 0 5 level. The  l o c u s gave the l o w e s t estimates for both upper and l o w e r c r o w n . The data f r o m  GDH  Table 4.1.  Allelic frequencies for outcrossing pollen and maternal gene pools for s a m p l e s obtained f r o m the upper and lower c r o w n portions and c o m b i n e d for a Sitka spruce seed orchard  Locus  Allele  Pollen  PGI-2  PGM-2  GDH  Maternal Upper  Lower  0.992 0.007  93  0.950 0.025 0.025  0.001  0.990 0.009 0.001  0.982 0.017 0.001  100 86  0.852 0.148  0.956 0.044  0.963 0.037  0.967  100 116 71  0.934  0.941 0.027 0.032  0.947 0.018 0.035  0.950 0.020 0.030  100 117  0.033 0.033  Combined  0.033  Table 4.2.  S i n g l e - l o c u s ({3) a n d m u l t i l o c u s [Xj e s t i m a t e s o f o u t c r o s s i n g for s a m p l e s o b t a i n e d f r o m the upper a n d l o w e r levels of the c r o w n o f c l o n a l S i t k a s p r u c e t r e e s in a m a n a g e d s e e d o r c h a r d  Locus  Upper  Lower  PGI-2  0.906»  0.839  0.968  PGM-2  0.769  0.649  0.773  GDH  0.940  -  0.999  \  0.872  0.744  0.913  0.866  0.648  0.875  61  61  61  427  427  915  # of t r e e s # of s e e d s  Combined  " 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^ a n d t^) are s i g n i f i c a n t at P< 0 . 0 5 (i.e., t ^ t l . O ) .  l o c u s o f t h e l o w e r c r o w n d i d n o t c o n v e r g e (Table 4.2), o f s e e d s per c l o n e u s e d ( B r o w n et al., Apparently,  the  single-locus  w h i c h c o u l d be due to the s m a l l s a m p l e  1985). outcrossing  estimate  is  affected  by  population  s u b s t r u c t u r i n g in n a t u r a l p o p u l a t i o n s . H o w e v e r , s u c h e f f e c t w a s n o t 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 a n d A l l a r d , 1 9 8 2 ) .  B i j i s m a et al.  (1986)  f o u n d a n e x c e s s of  h o m o g a m e t i c f e r t i l i z a t i o n 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 i m e w a s o n e o f t h e c a u s e s o f 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 m a i z e . T h e r e f o r e , t o d e t e r m i n e t h e p o s s i b l e c a u s e o f t h e 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 e s t i m a t e at l o c u s PGM-2, to determine  if m a t e r n a l  genotypes affected  a complementary analysis w a s conducted  this estimate.  The maternal  population  was  s u b d i v i d e d i n t o t h r e e c l a s s e s a c c o r d i n g t o t h e i r g e n o t y p e s (i.e., 1 0 0 : 1 0 0 , 1 0 0 : 8 6 , a n d 8 6 : 8 6 ) a n d t h e m u l t i l o c u s o u t c r o s s i n g rate f o r 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 ( w i t h t h e PGM-2  l o c u s i n c l u d e d a n d e x c l u d e d ) (Table 4.4).  t h a t o b s e r v e d allelic f r e q u e n c i e s at l o c u s PGM-2  T h e Chi-square test for pollen pool indicates are h i g h l y s i g n i f i c a n t l y d i f f e r e n t f r o m t h e  e x p e c t e d allelic f r e q u e n c i e s , w h i l e t h e e s t i m a t e s f o r o t h e r l o c i d o n o t d i f f e r s i g n i f i c a n t l y in b o t h a n a l y s e s (Table  4.3  a n d Table  allele ( 1 0 0 ) at l o c u s PGM-2  4.4.)  T h e result s u g g e s t s that the f r e q u e n c y of the  common  is in e x c e s s o f t h e f r e q u e n c i e s o f o t h e r a l l e l e s in all m a t e r n a l  g e n o t y p e s ( 1 0 0 : 1 0 0 , 1 0 0 : 8 6 , a n d 8 6 : 8 6 ) . T h i s is p r o b a b l y d u e t o n o n r a n d o m m a t i n g ( B i j i s m a et al.,  1 9 8 6 ) c a u s e d b y g e n o t y p i c s e l e c t i o n f o r f e r t i l i z a t i o n . A n t h e s i s o f t r e e s p o s s e s s i n g allele  1 0 0 m a y h a v e been s y n c h r o n i z e d w i t h the peak of f e m a l e receptivity or/and the  p r e s e n c e of g a m e t i c  in t h e s e e d o r c h a r d ,  s e l e c t i o n . A s s u m i n g t h a 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  m a t e r n a l g e n o t y p e s , t h e e s t i m a t e of p o p u l a t i o n 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 t o m a t e r n a l g e n o t y p e s at l o c u s PGM-2  w a s v e r i f i e d a c c o r d i n g t o F a l c o n e r ( 1 9 8 6 , p. 1 0 2 ) , M = a(p-q)  2 d p q , w h e r e M = p o p u l a t i o n m e a n , a ( g e n o t y p i c v a l u e of h o m o z y g o t e 1 0 0 : 1 0 0 ) ( g e n o t y p i c v a l u e of h é t é r o z y g o t e 1 0 0 : 8 6 )  +  = 0.092, d  = 0 . 0 4 6 , p ( g e n o t y p i c f r e q u e n c y o f allele 1 0 0 )  =  0 . 7 7 9 , a n d q ( g e n o t y p i c f r e q u e n c y of allele 8 6 ) = 0 . 2 2 1 . If b o t h a l l e l e s 1 0 0 a n d 8 6 s h o w e d i n c o m p l e t e 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 e s t i m a t e for g e n o t y p e 1 0 0 : 8 6 s h o u l d  Table 4.3.  E s t i m a t e s o f p o i l e n - a l l e l i c f r e q u e n c i e s f o r 3 ( w i t h PGM-2)  a n d 2 l o c i ( w i t h o u t PGM-2)  c o r r e s p o n d i n g to maternal g e n o t y p e s  ( n u m b e r o f p a r e n t t r e e s in p a r e n t h e s e s )  Locus  Allele  Maternal Genotype W i t h PGM-2 100:100 (74)  100:86 (42)  86:86  0.924 0.038  GDH  100 137 71  0.946 0.020 0.034  0.960 0.019 0.021  PGI-2  100 117  0.991  0.992 0.007  PGM-2  93 100 86  ™ Not significant, " S i g n i f i c a n t at P <  0.008 0.001 0.999 0.001  0.001.  Without  0.001 0.899 0.101  100:100 (74)  100:86  0.544™  0,946 0.020 0.034  0.965 0.015 0.020  0.089™  0.991 0.008  0.989 0.010  0.928 0.036 0.036 0.999 0.001  0.001  0.001  0.000  (6)  0.038 0.999 0.001 0.000 0.787 0.213  PGM-2  20.308"  -  (42)  -  86:86 (6)  -  -  x'  0.666™  0,120™  -  Table  4.4.  Maternal Genotype  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 maternal g e n o t y p e s , a n d o u t c r o s s i n g rate (t^n)  No. Tree  Embryo Genotype 100:100  100:86  100:100  74  581  11  100:86  42  171  86:86  6  ^ With and without  -  PGM-2.  t^ 86:86  With  Without  -  0.908  0.949  140  25  0.862  0.901  34  14  0.724  0.730  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 this g e n o t y p e w a s 0 . 8 6 2 , yielding a population m e a n o f 0 . 8 8 3 , i n d i c a t i n g t h a t allele 1 0 0 is d o m i n a n t o v e r allele 8 6 f o r t h i s trait. T h e r e f o r e , if t h i s g e n e t i c m o d e l is v a l i d , g e n o t y p i c s e l e c t i o n c o u l d a c c o u n t f o r t h e 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 selection 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 a n d a particular  allele m i g h t a c c o u n t f o r t h i s d i f f e r e n c e . In m a i z e , h o m o g a m e t i c f e r t i l i z a t i o n w a s o b s e r v e d t o contribute  t o l o w o u t c r o s s i n g , a n d it w a s c o n c l u d e d t h a t t h e e f f e c t  genotypes  in f a v o u r  of early f l o w e r i n g  of o n e h o m o z y g o t e  w a s associated with  ( B i j i s m a et  al.,  1986).  Also,  h e t e r o g e n e i t y in t h e p o l l e n p o o l w a s a t t r i b u t e d t o t h e o b s e r v e d l o w o u t c r o s s i n g rate f o r a s i n g l e l o c u s in a T a b l e M o u n t a i n p i n e (Pinus  pungens)  population (Gibson and Hamrick,  1991).  In t h i s 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 t h e s a m e s e t o f e m b r y o s s o t h a t v a r i a t i o n in t h e a c t u a l p r o p o r t i o n s o f s e l f e d a n d o u t c r o s s e d o f f s p r i n g d i d n o t c o n t r i b u t e t o t h e 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 , t h e v a r i a b i l i t y m u s t be d u e t o e i t h e r r a n d o m v a r i a t i o n or violations of the a s s u m p t i o n s intrinsic to the estimation 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 i n g l e a n d m u l t i l o c u s o u t c r o s s i n g - r a t e e s t i m a t e s f o r t h e s t u d i e d b l o c k w e r e d i f f e r e n t f r o m 1.0 ( c o m p l e t e o u t c r o s s i n g ) , i n d i c a t i n g t h e p r e s e n c e o f s e l f i n g in t h e o r c h a r d b l o c k . D i f f e r e n c e s in e s t i m a t e s of o u t c r o s s i n g r e l a t e d t o c r o w n l e v e l s r e p o r t e d a u t h o r s are s u m m a r i z e d in Table 4.5.  by  other  T h e o b s e r v e d differences m a y be due to self-fertilization  b e i n g m o r e f r e q u e n t in l o w e r t h a n in u p p e r c r o w n s . H o w e v e r , W o o d s a n d H e a m a n  (1989)  c o n t e n d e d t h a t 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 , t h e t y p e o f r e l a t e d m a t i n g r e s u l t i n g in empty  seeds  is i m p o s s i b l e  to  determine.  Therefore,  selfing  estimates  are  likely  to  be  conservative. T h e p r e s e n c e o f i n b r e e d i n g d i f f e r e n c e s in u p p e r v s . l o w e r c r o w n s s u g g e s t s t h a t if t h e S i t k a s p r u c e o r c h a r d is h i g h l y p r o d u c t i v e , t h e n s e e d c o l l e c t i o n s h o u l d b e m a d e o n l y f r o m u p p e r c r o w n s . H o w e v e r , w h e n t h e d e m a n d f o r s e e d s is h i g h , i n c r e a s i n g f e r t i l i z a t i o n consequently  reduced  self-fertilization  in  the  lower  crown  might  be  success and,  accomplished  by  Table 4.5.  E f f e c t of c r o w n l e v e l 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  Species  Douglas-fir  Sitka spruce  S.O. composition  Crown level  Reference  t.  Clonal  Upper Lower  0.916 0.918  0.955 0.950  Seedling  Upper Lower  0.901 0.884  0.932 0.901  Clonal  Upper Lower  Clonal  Upper Lower  Clonal  Upper Lower  0.872 0.744  E l - K a s s a b y etal.  (1986)  0.920 0.890  S h a w and Allard (1982)  0.999 0.871  Omi and A d a m s (1986)  0.866 0.648  Present study  supplemental m a s s pollination (El-Kassaby and R e y n o l d s , 1 9 9 0 ; El-Kassaby and D a v i d s o n , 1990). T h e r e d u c t i o n of i n b r e d s e e d s f r o m s e e d o r c h a r d s is i m p o r t a n t . plantings, inbred s e e d l i n g s c a n survive (Ritland a n d E l - K a s s a b y ,  In  1985).  non-competitive However,  inbred  individuals m a y be virtually eliminated by natural s e l e c t i o n due to poor s e e d l i n g vigour during n u r s e r y a n d p l a n t a t i o n p r o c e s s e s , a s f o u n d in S c o t s p i n e (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 plantation e s t a b l i s h m e n t higher than the c o s t of e x c l u d i n g inbred seedlings from plantings. T h e i m p l i c a t i o n of p o l l e n 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 o f i n t e r e s t i n g . If s e e d o r c h a r d s f o r d i f f e r e n t s e e d z o n e s are l o c a t e d c l o s e t o e a c h o t h e r , c a r e s h o u l d b e t a k e n t o a v o i d p o l l e n c o n t a m i n a t i o n . S e p a r a t i o n by sufficient d i s t a n c e a n d / o r buffer as a barrier for pollen migration s h o u l d be c o n s i d e r e d . S i t k a s p r u c e in B r i t i s h C o l u m b i a is in t h e e a r l y s t a g e o f b r e e d i n g , a n d o p e n - p o l l i n a t i o n p r o g e n y t e s t i n g is a v i a b l e o p t i o n f o r t h e s p e c i e s ' b r e e d i n g p r o g r a m ( Y e h a n d R a s m u s s e n , 1 9 8 5 ) . H o w e v e r , a h i g h p r o p o r t i o n of i n b r e d p r o g e n y in t h e t e s t i n g p o p u l a t i o n c a n r e s u l t in a n o v e r e s t i m a t i o n of additive genetic variance a n d genetic gains ( 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 single s e e d crop for a p r o g e n y test m a y retain substantial i n b r e e d i n g . T h i s m a y substantially affect the e s t i m a t e d genetic g a i n . T h e r e f o r e , a mixture of s e v e r a l s e e d y e a r s f o r o p e n - p o l l i n a t i o n p r o g e n y t e s t s is m o r e a p p r o p r i a t e ( K i n g et al.,  1 9 8 4 ) or a d j u s t i n g  the  g e n e t i c p a r a m e t e r s t o a c c o u n t f o r t h e o b s e r v e d l e v e l of i n b r e e d i n g w i l l b e n e c e s s a r y ( A s k e w a n d E l - K a s s a b y , 1 9 9 2 ) . W h e r e h i g h i n b r e e d i n g in t h e 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 t h e c o l l e c t i o n f r o m this portion will also i m p r o v e reliability. 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 a n d t h e i r n a t u r a l - p o p u l a t i o n c o u n t e r p a r t s is e s s e n t i a l in u n d e r s t a n d i n g c h a n g e s of m a t i n g s t r u c t u r e in a r t i f i c i a l p o p u l a t i o n s . T h i s w i l l p r o v i d e substantial information for better orchard 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 reported 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 t o b e h i g h e r t h a n n a t u r a 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 o f 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 a n d n a t u r a l p o p u l a t i o n s  Species Douglas-fir Natural Seed orchard Black spruce Natural Seed orchard  Reference  0.910 0.758 0.940 0.934  0.900 0.887 0.910 0.925  Neale and A d a m s (1985) Yeh and Morgan (1987) S h a w and Allard (1982) E l - K a s s a b y etal. (1988)  0.932  0.924  0.940  0.840  Boyle and Morgenstern (1986) B a r r e t t et al. ( 1 9 8 7 )  T h e d i f f e r e n c e s in o u t c r o s s i n g e s t i m a t e s b e t w e e n  natural populations and s e e d orchards  i n d i c a t e s t h a t p o p u l a t i o n s t r u c t u r e (i.e., t h e p h y s i c a l a r r a n g e m e n t o f t h e r e l a t e d a n d u n r e l a t e d i n d i v i d u a l s w i t h i n a p o p u l a t i o n ) h a s a f f e c t e d t h e rate o f o u t c r o s s i n g ( E n n o s a n d C l e g g , 1 9 8 2 ; E l l s t r a n d a n d F o s t e r , 1 9 8 3 ; R u d i n et al., 1 9 8 6 ; E l - K a s s a b y a n d D a v i d s o n , 1 9 9 0 ) . In f a c t , s e e d orchards provide  lower  inbreeding levels than  natural  populations  ( R u d i n et  al.,  1986).  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Genet, (in p r e s s ) . E l - K a s s a b y , Y . A . , K. R i t l a n d , A . M . K . F a s h l e r , a n d W . J . B . D e v i t t . 1 9 8 8 . T h e e f f e c t o f r e p r o d u c t i v e p h e n o l o g y u p o n t h e m a t i n g s y s t e m o f a D o u g l a s - 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 , a n d R. Y a z d a n i . 1 9 8 9 . L e v e l s o f 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 in t w o Pinus sylvestris L. s e e d o r c h a r d s in n o r t h e r n S w e d e n . Scand. J. For. Res. 4 : 4 1 49. E l l s t r a n d , N . C . a n d K . W . F o s t e r . 1 9 8 3 . I m p a c t of p o p u l a t i o n s t r u c t u r e o n t h e apparent o u t c r o s s i n g rate of g r a i n 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 . a n d M . T . C l e g g . 1 9 8 2 . 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G e n e t i c c h a n g e b e t w e e n life s t a g e s in Pinus sylvestris: a l l o z y m e v a r i a t i o n in s e e d s a n d p l a n t e d s e e d l i n g s . Silvae Genet. 3 6 : 3 9 - 4 2 .  Mûller-Starck, G . 1 9 8 2 . S e x u a l l y a s y m m e t r i c fertility s e l e c t i o n a n d partial self-fertilization. 2 . C l o n a l g a m e t i c c o n t r i b u t i o n s t o t r e e o f f s p r i n g o f S c o t s p i n e s e e d o r c h a r d . Silva Fenn. 16:99-106. M u l l e r - S t a r c k , G . , M . Z i e h e a n d H . H . H a t t e m e r . 1 9 8 3 . R e p r o d u c t i v e s y s t e m s in c o n i f e 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 s e l e c t i o n m o n i t o r e d at a n M P g e n e 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 . I n b r e e d i n g e f f e c t s o n e s t i m a t i o n o f g e n e t i c a d d i t i v e v a r i a n c e . For. 12:8-13.  Sci.  N e a l e , D . B . a n d W . T . A d a m s . 1 9 8 5 . T h e m a t i n g s y s t e m in n a t u r a l a n d s h e l t e r w o o d s t a n d s of D o u g l a s - f i r . Theor. Appl. Genet. 7 1 : 2 0 1 - 2 0 7 . O m i , S . K . a n d W . T . A d a m s . V a r i a t i o n in s e e d s e t a n d p r o p o r t i o n s o f 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 , a n d t o p p r u n i n g in a D o u g l a s - f i r s e e d o r c h a r d . Can. J. For. Res. 16:502-507. O r r - E w i n g , A . L . 1 9 7 6 . I n b r e e d i n g D o u g l a s - f i r t o t h e S 3 g e n e r a t i o n . Silvae  Genet.  25:179-183.  R i t l a n d , K. a n d Y . A . E l - K a s s a b y . 1 9 8 5 . T h e n a t u r e o f i n b r e e d i n g in a s e e d o r c h a r d o f D o u g l a s fir a s s h o w n b y a n e f f i c i e n t m u l t i l o c u s m o d e l . Theor. Appl. Genet. 71:375-384. R u d i n , D. 0 . M u o n a , a n d 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 h e m a t i n g s y s t e m o f sylvestris in n a t u r a l s t a n d s a n d s e e d o r c h a r d s . Hereditas 104:15-19. S a r v a s , R. 1 9 6 2 . I n v e s t i g a t i o n s o n t h e f l o w e r i n g a n d s e e d c r o p s o f Pinus Inst. For. Fenn. 5 3 : 1 - 1 9 8 . Shaw,  Pinus  sylvestris.  Comm.  D . V . a n d R . W . A l l a r d . 1 9 8 2 . E s t i m a t i o n of o u t c r o s s i n g r a t e s in D o u g l a s - f i r i s o z y m e m a r k e r s . Theor. Appl. Genet. 6 2 : 1 1 3 - 1 2 0 .  S h a w , D . V . , A . L . K a h l e r a n d R . W . A l l a r d . 1 9 8 1 . A m u l t i l o c u s e s t i m a t o r of m a t i n g p a r a m e t e r s in p l a n t p o p u l a t i o n s . Proc.  Nat.  Acad.  Sci.  using  system  USA 78:1298-1302.  S h e a , K . L . 1 9 8 7 . E f f e c t s o f p o p u l a t i o n s t r u c t u r e a n d 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 r a t e s in E n g e l m a n n s p r u c e a n d s u b a l p i n e fir. Evolution 41:124-136. S h e n , H . - H . , D. R u d i n a n d D. L i n d g r e n . 1 9 8 1 . S t u d y o f t h e p o l l i n a t i o n p a t t e r n in a s c o t p i n e s e e d o r c h a r d b y m e a n s of i s o z y m e a n a l y s i s . Silvae  Genet.  30:7-15.  S n y d e r , T . P . , D . A . S t e w a r d a n d A . F . Strickler. 1 9 8 5 . T e m p o r a l a n a l y s i s of breeding structure in j a c k p i n e (Pinus Sokal,  banksiana  L a m b . ) . Can. J. For. Res.  15:1159-1166.  R . R . a n d F . J . R o h l f . 1 9 8 1 . Biometry: The principles and practice biological research. 2nd ed. N e w York: W.H.Freeman and C o .  S o r e n s e n , F . C . 1 9 7 1 . E s t i m a t e of self-fertility Silvae Genet. 2 0 : 1 1 5 - 1 2 0 .  of  statistics  in  in c o a s t a l D o u g l a s - f i r f r o m i n b r e e d i n g s t u d i e s .  S o r e n s e n , F . C . a n d R . S . M i l e s . 1 9 7 4 . Self-pollination effects on D o u g l a s - f i r a n d p o n d e r o s a pine s e e d s a n d s e e d l i n g s . Silvae Genet. 2 3 : 1 3 5 - 1 3 8 .  Squillace, A . E . 1 9 7 4 . A v e r a g e genetic correlations a m o n g offspring from open-pollinated forest t r e e s . Silvae Genet. 2 3 : 1 4 9 - 1 5 6 . T i g e r s t e d t , P . M . A . 1 9 7 3 . S t u d i e s o n i s o z y m e v a r i a t i o n in m a r g i n a l a n d c e n t r a l p o p u l a t i o n s o f Picea abies. Hereditas 75:47-60. W i l c o x , M . D . 1 9 8 3 . I n b r e e d i n g d e p r e s s i o n a n d g e n e t i c v a r i a n c e e s t i m a t e d f r o m self- a n d c r o s s p o l l i n a t e d f a m i l i e s 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 . Effect of different inbreeding levels on filled s e e d p r o d u c t i o n in D o u g l a s - f i r . Can. J. For. Res. 1 9 : 5 4 - 5 9 . Y e h , F . C , A . B r u n e , W . M . C h e l i a k a n d 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 citriodora in a s e e d - p r o d u c t i o n a r e a . Can. J. For. Res. 1 3 : 1 0 5 1 - 1 0 5 5 .  Eucalyptus  Y e h , F . C , a n d 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 m u l t i l o c u s a s s o c i a t i o n s in a n a t u r a l p o p u l a t i o n o f 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 a n d S . R a s m u s s e n . 1 9 8 5 . H e r i t a b i l i t y of h e i g h t g r o w t h in 1 0 - y e a 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 .  Chapter 5 P a r e n t a 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 l a n t a t i o n of g e n e t i c a l l y - s u p e r i o r i n d i v i d u a l s i n t e n s i v e l y m a n a g e d  for s e e d production and d e s i g n e d to p r o m o t e inter-clonal m a t i n g while preventing e x t r a n e o u s p o l l e n ( Z o b e l et al.,  1 9 5 8 ) . T h e q u a l i t y o f 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 t o b e h i g h a n d  their genetic diversity maintained 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 f o r p a n m i x i a are r e q u i r e d a s f o l l o w s : 1 ) e q u a l i t y o f m a l e a n d f e m a l e g a m e t e s a m o n g t h e o r c h a r d c l o n e s , 2) r e p r o d u c t i v e 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 m a t i n g (i.e., a n y p o l l e n o f a c l o n e h a s t h e s a m e p r o b a b i l i t y of r e a c h i n g a n o v u l e of a n y c l o n e ) , 4) n o i n c o m p a t i b i l i t y a n d s e l e c t i o n b e t w e e n f e r t i l i z a t i o n a n d g e r m i n a t i o n o f s e e d s , a n d 5) m i n i m a l o r n o p o l l e n c o n t a m i n a t i o n ( E r i k s s o n 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 t h a t s e e d o r c h a r d s  o f t e n d e v i a t e f r o m t h e " i d e a l " e x p e c t a t i o n s . D i f f e r e n c e s in r e p r o d u c t i v e o u t p u t (Pinus G r i f f i n , 1 9 8 2 ; Pinus taeda, R o b e r d s et al.,  S c h m i d t l i n g , 1 9 8 3 ; Pseudotsuga  1 9 9 1 ; Pinus  orchard's effective  sylvestris,  B o e s et al.,  menziesii,  1991)  radiata.  E l - K a s s a b y ef a/., 1 9 8 9 ;  have been reported. T h u s , the  p o p u l a t i o n n u m b e r is e x p e c t e d t o b e l o w e r t h a n t h e a c t u a l  population  n u m b e r . T h e c o n c e p t of e f f e c t i v e p o p u l a t i o n n u m b e r c a n b e u s e d t o d e m o n s t r a t e t h e d e v i a t i o n o f m a t i n g b e h a v i o u r f r o m a n i d e a l i z e d b r e e d i n g p o p u l a t i o n ( F a l c o n e r , 1 9 8 6 ) . It a l s o h a s b e e n r e p o r t e d t h a t t h e e f f e c t i v e n u m b e r of a n a t u r a l p o p u l a t i o n is a l w a y s l e s s t h a n t h e n u m b e r of adults 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 ) unequal n u m b e r s of m a l e s a n d f e m a l e s , 2) t e m p o r a l v a r i a t i o n in p o p u l a t i o n n u m b e r , a n d 3) g r e a t e r t h a n b i n o m i a l o r P o i s s o n v a r i a b i l i t y in t h e n u m b e r o f p r o g e n y p e r p l a n t ( C r o w a n d D e n n i s t o n , 1 9 8 8 ) . F a c t o r s i n f l u e n c i n g 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 n c l u d e g e n e t i c c o m p o n e n t s , c l o n e s i z e ( n u m b e r o f r a m e t p e r c l o n e ) , tree s i z e , p e r i o d i c i t y , d i f f e r e n c e s b e t w e e n  reproductive  e n e r g y (i.e., n u m b e r of s e e d c o n e s ) a n d r e p r o d u c t i v e s u c c e s s (i.e., n u m b e r o f f i l l e d s e e d s )  ( G r i f f i n , 1 9 8 2 , 1 9 8 4 ; O ' R e 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 a n d E l - K a s s a b y , 1 9 9 0 ; B o e s et al.,  1991).  T h e c u m u l a t i v e c o n e - y i e l d c u r v e is c o m m o n l y u s e d t o a s s e s s p a r e n t a l b a l a n c e in s e e d o r c h a r d s (Griffin, 1 9 8 2 ) . R e y n o l d s and E l - K a s s a b y (1990) used cumulative s e e d - c r o p data to a s s e s s p a r e n t a l b a l a n c e in a D o u g l a s - f i r s e e d o r c h a r d , a n d f o u n d t h e c u m u l a t i v e s e e d - y i e l d c u r v e is a b e t t e r p a r a m e t e r t h a n c o n e - y i e l d t o a s s e s s p a r e n t a l b a l a n c e in t e r m o f g e n e t i c diversity and family representation. In t h i s s t u d y , t h e p a r e n t a l b a l a n c e a n d f e m a l e e f f e c t i v e p o p u l a t i o n n u m b e r b a s e d o n c o n e , s e e d , a n d v i a b l e 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 orchard were assessed. 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 P a c i f i c F o r e s 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 c h a r d is l o c a t e d in S a a n i c h t o n , B r i t i s h C o l u m b i a ( l a t i t u d e  48°35'N,  l o n g i t u d e 1 2 3 ° 2 4 ' W ) a n d c o n s i s t s of 1 3 9 c l o n e s ( a v e r a g i n g 9 . 3 r a m e t s p e r c l o n e ) s e l e c t e d f r o m elevations 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 Island, 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 i n g l e - t r e e m i x o v e r t h r e e  u n e q u a l b l o c k s . T r e e s are s p a c e d 3 m a p a r t a n d k e p t at a p p r o x i m a t e l y 4 m in h e i g h t b y t o p p r u n i n g . T h e s e e d o r c h a r d is 1 0 k m a w a y f r o m t h e n e a r e s t S i t k a s p r u c e s t a n d , s o t h e S i t k a s p r u c e b a c k g r o u n d p o l l e n is n e g l i g i b l e in t h e o r c h a r d ' s a r e a ( E l - K a s s a b y a n d R e y n o l d s , 1 9 9 0 ) . D u r i n g 1 9 8 8 a n d 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 o f e v e r y r a m e t w a s r e c o r d e d . In t h i s s t u d y , s e e d s w e r e c o l l e c t e d f r o m 9 6 trees of 2 2 c l o n e s ( 1 9 8 8 crop) and 1 4 2 trees of  18  c l o n e s ( 1 9 9 0 c r o p ) . W h e r e p o s s i b l e , a s a m p l e of f i v e c o n e s w a s r a n d o m l y t a k e n f r o m e a c h t r e e f o r s e e d e x t r a c t i o n . C o n e s a m p l e s w e r e air d r i e d , a n d 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 a n d c l e a n e d b y h a n d . N u m b e r s o f filled a n d e m p t y s e e d ( i d e n t i f i e d b y X - r a y ) w e r e r e c o r d e d . T o t a l s e e d y i e l d s w e r e a s s e s s e d o n t h e 1 9 9 0 s e e d c r o p (i.e., c l o n a l s e e d c r o p s ) . T h e w e i g h t of e a c h individual 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  total s e e d c r o p w a s c a l c u l a t e d by dividing bulk s e e d w e i g h t of e a c h c l o n e b y the c o r r e s p o n d i n g a v e r a g e i n d i v i d u a l s e e d w e i g h t . In a d d i t i o n , g e r m i n a t i o n t e s t s w e r e c o n d u c t e d t o p r o v i d e v i a b l e s e e d p r o d u c t i o n d a t a (see C h a p t e r 7 f o r g e r m i n a t i o n t e s t d e s c r i p t i o n ) . O n e - w a y A N O V A w a s u s e d to a n a l y z e 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 . D u e t o a c l o s e r e l a t i o n s h i p a m o n g f i l l e d s e e d s (r = 1 . 0 0 , P < 0 . 0 1 ) , t o t a l s e e d s (r = 0 . 8 1 , P < 0 . 0 1 ), a n d v i a b l e s e e d d a t a ( e s t i m a t e d b y m u l t i p l y i n g t h e c l o n a l g e r m i n a t i o n c a p a c i t y b y t h e n u m b e r of f i l l e d s e e d s ) , f i l l e d - s e e d d a t a o n l y w e r e u s e d in t h e a n a l y s i s . T h e  relationship  between clonal cone and seed crops w a s assessed by Pearson's product-moment  correlation  (Sokal a n d Rohlf, 1 9 8 1 ) . B r o a d - s e n s e heritability w a s e s t i m a t e d a c c o r d i n g to F a l c o n e r ( 1 9 8 6 , p.113). Data on c o n e n u m b e r s , total s e e d s , filled s e e d s , a n d viable s e e d p r o d u c t i o n p r o v i d e d t h e e s t i m a t e s of f e m a l e e f f e c t i v e p o p u l a t i o n 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,,=  female effective population number,  Xj = p r o p o r t i o n of ith c l o n e c o n t r i b u t i o n t o t h e o r c h a r d c r o p 5 . 3 Results  and  5.3.1  1988  Discussion 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 for this s t u d y w e r e a representative s a m p l e of the p r o d u c t i o n r a n g e in t h e s e e d o r c h a r d (Figure  5.1),  indicating that this s u b s e t of c l o n e s will  provide an u n b i a s e d estimate for the w h o l e o r c h a r d . T h e relationship b e t w e e n c o n e a n d filleds e e d p r o d u c t i o n is e x p e c t e d t o be s i g n i f i c a n t a n d p o s i t i v e . T h e p r o d u c t - m o m e n t  correlation  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 p o s i t i v e a n d s i g n i f i c a n t (r = 0 . 8 6 , n = 2 2 , P < 0 . 0 1 ) . T h e c o e f f i c i e n t of d e t e r m i n a t i o n w a s h i g h (r^ = 0 . 7 5 ) , i n d i c a t i n g t h a t t h i s r e l a t i o n s h i p a c c o u n t s f o r a l a r g e p r o p o r t i o n o f v a r i a t i o n in t h e d a t a . H o w e v e r , t h e a v e r a g e 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 varied 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 a n d 4 6 , 2 0 0 s e e d s per c l o n e f o r c l o n e n u m b e r s 1 8 4 a n d 3 6 , r e s p e c t i v e l y (Table  5.1).  1,749 In f a c t .  ..^^  1nn  90  6 3 / ^  80  70 ^ 3 2 0 ^  /  ^60 O cc o ^ o o  ^  y  /  y  f  /  50  • • •  40 r  36,187 A ABO  •  30  20  / /  ^ •  /  1  /  I / J  10  CONE CROP  f  1 /  y  /  •  /  36 ...  1  0 0  SAMPLED CLONES  •  10  20  1  1  1  1  1  1  1  30  40  50  60  70  80  90  % CLONE  F/yure 5. / .  1988 cone crop parental balance curve based on ail orchard clones (N = 139), indicating contributions of the sampled clones.  100  Table  5.1.  C o n e a n d f i l l e d s e e d p r o d u c t i o n in t h e 1 9 8 8 c r o p , a n d t h e i r e f f e c t i v e p o p u l a t i o n n u m b e r (N^,)  C r o p (No.) No.  1 2 3 4 5 6 7 8 9 10 11 12 13 14  Clone No.  5 13 15  156 25 162  20 27 36 40 46 49 50 53 64 71 92  175 35 488 39 34 449 203 55 19 14  15 16 17  98 124  18 19 20 21 22  153 184 187  N., Ne,/N  Cones  143  196 413  145 151 78 32 34 12 428 30 93 10 0.45  Filled Seeds 16,688 3,208 10,501 10,167 4,286 46,200 3,292 3,100 25,304 35,384 6,872 3,126 2,562 12,052 21,806 8,843 3,102 5,183 1,749 22,764 3,749 10,361 11 0.50  3 7 a n d 3 8 p e r c e n t o f t h e v a r i a t i o n in t h e c o n e c r o p a n d t h e s e e d c r o p , r e s p e c t i v e l y , w e r e d u e t o c l o n a l d i f f e r e n c e (Table production  was 0.37  5.2).  and 0 . 3 8 ,  In o t h e r w o r d s , b r o a d - s e n s e h e r i t a b i l i t y f o r c o n e a n d s e e d respectively. The remaining variation  in c o n e a n d s e e d  p r o d u c t i o n is c a u s e d b y w i t h i n - c l o n e v a r i a t i o n (i.e., a m o n g r a m e t s w i t h i n a c l o n e ) . T h e c h a n g e in r a n k b e t w e e n c o n e a n d s e e d p r o d u c t i o n s p r o v i d e s i n f o r m a t i o n r e g a r d i n g r e p r o d u c t i v e e n e r g y a n d r e p r o d u c t i v e s u c c e s s (Figure  5.2).  For e x a m p l e , clone number 2 0 , ranked 5 as a c o n e  p r o d u c e r , w a s ranked 1 0 as a s e e d p r o d u c e r . O n l y six c l o n e s out of the 2 2 s t u d i e d (clone n o s . 36,  1 2 4 , 5 3 , 2 7 , 71  and 184)  maintained the s a m e rank as c o n e and seed producers.  H o w e v e r , rank c h a n g e s were generally small w h e n they o c c u r r e d . P a r e n t a l b a l a n c e e s t i m a t e s b a s e d o n e i t h e r c o n e or s e e d c r o p s (Figure 5.3),  w e r e similar  a n d g a v e r a t i o s of " 3 8 / 8 0 " (i.e, 3 8 % o f t r e e s p r o d u c e d 8 0 % o f t h e c r o p ) f o r c o n e s a n d " 4 4 / 8 0 " for s e e d s . T h i s indicated that the proportion of effective a n d actual f e m a l e n u m b e r s d e v i a t e d f r o m a n i d e a l s e e d o r c h a r d in t e r m s o f f e m a l e c o n t r i b u t i o n in t h e 1 9 8 8 s e e d c r o p , i.e., N , , / N w e r e 0 . 4 5 a n d 0 . 5 o n c o n e a n d f i l l e d - s e e d c r o p s , r e s p e c t i v e l y (Table  5.1).  In o t h e r  w o r d s , the actual population number w a s t w i c e the effective population number. T h u s , the c o n e c r o p c o u l d be u s e d t o p r e d i c t t h e p a r e n t a l b a l a n c e o f s e e d p r o d u c t i o n in t h i s 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 f o r t h i s s t u d y a l s o w e r e a r e p r e s e n t a t i v e s a m p l e of t h e p r o d u c t i o n r a n g e in t h e s e e d o r c h a r d (Figure  5.4),  indicating that this s u b s e t of c l o n e s will  provide an u n b i a s e d estimate 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 correlation 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 p o s i t i v e a n d s i g n i f i c a n t (r = 0 . 8 1 , n = 1 8 , / ' < 0 . 0 1 ). T h e c o e f f i c i e n t o f d e t e r m i n a t i o n w a s h i g h (r^ = 0 . 6 6 ) , i n d i c a t i n g t h a t t h e r e l a t i o n s h i p b e t w e e n c o n e a n d s e e d p r o d u c t i o n a c c o u n t s f o r t h e l a r g e p r o p o r t i o n of v a r i a t i o n in t h e d a t a s i m i l a r t o t h a t in t h e 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 varied b e t w e e n 3 8 (clone n o . 5 1 6 ) a n d 5 9 5 (clone n o . 4 2 1 ) c o n e s per c l o n e , w h 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 filled s e e d per c l o n e r a n g e d b e t w e e n 2 , 8 4 4 ( c l o n e n o . 1 5 4 ) a n d 7 7 , 8 2 8 ( c l o n e n o . 4 2 1 ) (Table  5.3).  In f a c t .  Table 5.2.  A N O V A : e s t i m a t e s of v a r i a n c e c o m p o n e n t s o f 1 9 8 8 Sitl^a s p r u c e s e e d o r c l i a r d production  S o u r c e of Variation  df  Expected' Mean Squares  A m o n g Clones  21  al  Within Clones  76  ol  " '  + 4.452a^  Variance Components  (%)  Cone Crop  Seed Crop  37.073"  38.445"  62.927  61.555  S i g n i f i c a n t at P < 0 . 0 1 . = variance due to differences a m o n g ramets w i t h i n the s a m e c l o n e ; to differences a m o n g clones.  = variance due  CLONE SEED NO. YIELD  CONE CLONE YIELD NO.  36  46,200  -  50  35,384  -  - - -  49  25,304  -  - - -187  22,764  20  . , -  98  21,806  162  15  - - -  5  16,688  156  5  -  92  12,052  151  98  ~ -  15  10,501  145  92  - - -413  10,361  93  413  78  124  55  53  39  40  35  448  36  -  449  49  -  428  187  203  50  175  - •-  -  ^ -  20  10,167  124  8,843  53  6,872  -153  5,183  27  27  4,286  34  153  , - -196  3,749  33  46  ^  -  40  3,292  32  143  -  -  13  3,208  30  196  '  -  64  3,126  25  13  ~ -143  3,102  19  64  -  -  46  3,100  14  71  - - -  71  2,562  12  184  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 a n d filled s e e d p r o d u c t i o n c u r v e s o f 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 . S t r a i g h t line r e p r e s e n t s equal contribution.  0 L 0  I  I  I  I  I  \  I  I  I  10  20  30  40  50  60  70  80  90  % CLONE Figure 5.4.  1 9 9 0 cone crop parental balance curve based on all orchard clones (N = 139), indicating contributions of the sampled clones.  100  Table 5.3.  1 9 9 0 c o n e , s e e d , filled s e e d , viable s e e d p r o d u c t i o n , and their effective p o p u l a t i o n n u m b e r (N,,)  No.  Clone No.  C r o p (Number) Cones  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 N., Ne,/N  5 15 20 24 44 61 68 92 105 113 154 405 411 416 421 514 516 521  254 172 206 442 220 219 176 163 195 243 49 282 112 55 595 53 38 211  Total Seeds 183,433 70,069 115,038 284,292 125,079 122,966 76,869 48,654 80,095 165,116 18,335 192,221 43,103 38,219 373,709 18,615 17,963 116,554  Filled Seeds 21,853 10,306 20,244 13,535 16,060 27,130 21,146 11,195 22,998 36,036 2,844 44,216 10,138 7,969 77,828 4,328 4,994  Viable Seeds 21,202 9,945 19,583 12,817 14,458 25,971 19,335 10,857 21,618 34,865 2,627 43,549 9,479 7,826 75,693 4,222  28,576  4,645 26,840  12.54  10.93  10.68  10.52  0.70  0.61  0.59  0.58  3 3 . 5 a n d 4 1 . 0 p e r c e n t o f t h e v a r i a t i o n in c o n e c r o p a n d s e e d c r o p , r e s p e c t i v e l y , w a s d u e t o c l o n a l d i f f e r e n c e s (Table  5.4).  T h u s , the e s t i m a t e d b r o a d - s e n s e heritability of c o n e p r o d u c t i o n  w a s 0 . 3 4 , w h i l e t h e b r o a d - s e n s e h e r i t a b i l i t y o f 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 t h a t t h e l o w e s t c o n e - a n d s e e d - p r o d u c e r s ( c l o n e n o s . 5 1 4 a n d 1 5 4 , r e s p e c t i v e l y ) are n o t t h e s a m e c l o n e (Table  5.3);  t h e f o r m e r is m o r e e f f i c i e n t in p r o d u c i n g f i l l e d s e e d t h a n t h e  latter. Rank changes within and between cone and seed producer classes were also observed in t h e 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 (clone no. 24) a n d 6 (clone no. 44)  a s c o n e p r o d u c e r s r a n k e d 11 a n d 1 0 a s s e e d p r o d u c e r s , r e s p e c t i v e l y . H o w e v e r , m o s t o f t h e poor cone producers remained poor seed producers.  (Figure  Parental balance estimates b a s e d on either c o n e c r o p or viable s e e d s w e r e  similar  5.6)  seeds,  and gave  the  ratios  of  "58/80"  and  "56/80"  for  cone  and  viable  r e s p e c t i v e l y . E s t i m a t e s b a s e d o n filled a n d t o t a l s e e d c r o p s a l s o g a v e s i m i l a r r a t i o s o f " 5 1 / 8 0 " a n d " 4 9 / 8 0 , " r e s p e c t i v e l y . T h e proportions of female effective a n d actual n u m b e r s w e r e 0 . 7 0 , 0 . 6 1 , 0 . 5 9 a n d 0 . 5 8 w h e n the estimations w e r e b a s e d o n c o n e , total s e e d s , filled s e e d s , and v i a b l e s e e d p r o d u c t i o n (Table  5.3),  respectively.  T h e p a r e n t a l b a l a n c e r e c o r d s f o r t h i s s e e d o r c h a r d in 1 9 8 5 , 1 9 8 6 a n d 1 9 8 7  were  " 2 0 / 8 0 , " " 3 5 / 8 0 " a n d " 4 2 / 8 0 " ( E l - K a s s a b y a n d R e y n o l d s , 1 9 9 0 ) , r e s p e c t i v e l y . In t h i s s t u d y , t h e p a r e n t a l b a l a n c e s f o r 1 9 8 8 a n d 1 9 9 0 w e r e " 4 2 / 8 0 " a n d " 4 8 / 8 0 " (Figure 5.4),  respectively. This indicates an improvement  5.1  and  in p a r e n t a l b a l a n c e w i t h a g e . A  i m p r o v e m e n t h a s a l s o b e e n o b s e r v e d in l o b l o l l y p i n e ( B y r a m et al.,  Figure similar  1 9 8 6 ) , a n d in D o u g l a s - 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 h e r i t a b i l i t y f o r r e p r o d u c t i o n t r a i t s in t h i s s t u d y r a n g e s  from 0 . 3 4 to 0.41  i n d i c a t i n g m o d e r a t e g e n e t i c c o n t r o l s i m i l a r t o t h a t f o u n d in l o b l o l l y p i n e  ( B y r a m et al.,  1986).  In c o n c l u s i o n , m a t e r n a l c o n t r i b u t i o n  to seed production  in t h i s s e e d o r c h a r d  was  i m b a l a n c e d . H o w e v e r , a n i m p r o v e m e n t in p a r e n t a l b a l a n c e w i t h a g e w a s o b s e r v e d . F e m a l e  Table 5.4.  A N O V A : e s t i m a t e s of variance 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 orcliard production  S o u r c e of Variation  A m o n g Clones Within Clones  df  Expected^ Mean Squares  17  al  124  ol  +  7.869a^  Variance Components  (%)  Cone Crop  Seed Crop  33.548"  41.014"  66.452  58.986  " S i g n i f i c a n t at P < 0 . 0 1 . ^ al = v a r i a n c e d u e t o d i f f e r e n c e s a m o n g r a m e t s w i t h i n t h e s a m e c l o n e ; al = v a r i a n c e d u e t o d i f f e r e n c e s a m o n g c l o n e s .  CONECLONE YIELD N O . 595  421  T  442  24  282  405  254  5  243  CLONE NO. \  _  ^  _  "  113  ^  ^  44  219  61  211  521  206  ""^  -405  44,216  -113  36,036  -  521 2 8 , 5 7 6  -  61 2 7 , 1 3 0  "  ^  ^\  ^  " "  -105  22,998  -  5 21,853  -  68 21,146  -  -  20 20,244  "  -  20  —  " "  \  220  421 7 7 , 8 2 8  -  \ \  SEED YIELD  ^  - '  ^  '  ^ ^  N  '  - '  "  195  105  -  -  44  16,060  176  68  -  -  24  13,535  172  15  -  -  92 11,195  163  92  -  -  15  10,306  112  411  -  -411  10,138  55  416  -  -416  7,969  53  514  -  -516  4,994  49  154  -  -514  4,328  38  516  ^154  2,844  Figure  5.5.  -  -  -  -  Rank order for 18 Sitka-spruce c l o n e s , 1 9 9 0 c r o p , based on cone and seed yields.  % CLONE  Figure  5.6.  C u m u l a t i v e c o n e , filled s e e d s , t o t a l s e e d a n d p o t e n t i a l s e e d l i n g production c u r v e s for 1 8 Sitka spruce c l o n e s , 1 9 9 0 crop. S t r a i g h t line r e p r e s e n t s e q u a l c o n t r i b u t i o n .  effective  population  number  provided  tfie  estimate  of  deviation  from  the  ideal  equal  c o n t r i b u t i o n ; h o w e v e r , this parameter did not provide insight into the relationship b e t w e e n reproductive energy and reproductive success. This study and El-Kassaby and Reynolds (1990) have indicated that clonal contribution t o s e e d c r o p s in t h e s t u d i e d S i t k a s p r u c e s e e d o r c h a r d v a r i e s f r o m y e a r t o y e a r ; h o w e v e r , f e c u n d i t y is a g e n e t i c a l l y c o n t r o l l e d trait in c o n i f e r s ( J o n s s o n et al.,  1976; El-Kassaby, 1989).  T h e r e f o r e , certain 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 m a s s - p o l l i n a t i o n ( S M P ) has p r o v e n to be a vital tool for i m p r o v i n g r e p r o d u c t i v e s u c c e s s for Sitka s p r u c e due to the reproductive 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 male and female strobili (El-Kassaby and Reynolds, 1 9 9 0 ) . S M P could improve parental balance as well as reproductive s u c c e s s . T o i m p r o v e p a r e n t a l c o n t r i b u t i o n , t h e f o l l o w i n g s t r a t e g i e s c o u l d be a p p l i e d : a) t h e u s e of S M P p o l l e n 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 h u s t h e i r g a m e t e s are o v e r e m p h a s i z e d in t h e s e e d c r o p t h r o u g h t h e i r m a l e p a r t , a n d b) h a r v e s t i n g t h e c r o p i n t o s u b s e t s t h a t c o n s i s t s of h i g h , i n t e r m e d i a t e , a n d p o o r p r o d u c e r s , t h u s t h e p a r e n t a l b a l a n c e w i t h i n e a c h s u b s e t w i l l be better than the entire o r c h a r d c r o p .  5.4  References  B o e s , T . K . , J . R . Brandie, a n d W . R . Lovett. 1 9 9 1 . C h a r a c t e r i z a t i o n 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 i e l d in a P i n u s s y l v e s t r i s c l o n a l s e e d o r c h a r d in N e b r a s k a . Can. For. Res. 21:1721-1729. Byram,  T . D . , W . J . L o w e , and J . A . McGriff. 1 9 8 6 . Clonal and annual variation p r o d u c t i o n in l o b l o l l y p i n e 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 variance effective Evolution 42(31:482-495. C r o w , J . F . a n d M . K i m u r a . 1 9 7 0 . An introduction H a p p e r a n d R o w P u b l i s h e r s , 5 9 1 p.  to population  genetic  population  theory.  in  cone  numbers.  N e w York:  E l - K a s s a b y , Y . A . 1 9 8 9 . G e n e t i c s o f D o u g l a s - 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 r e a l i t i e s . In Proc. 20th Southern Forest Tree Improvement Conference, pp. 8 7 - 1 0 9 . Charleston, South Carolina. E l - K a s s a b y , Y . A . , A . M . K . F a s h l e r , a n d M . C r o w n . 1 9 8 9 . V a r i a t i o n in f r u i t f u l n e s s in a D o u g l a s - f i r s e e d o r c h a r d a n d its e f f e c 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. 38:113-121. El-Kassaby, Y . A . , and S . Reynolds. 1 9 9 0 . Reproductive phenology, parental balance, and s u p p l e m e n t a l m a s s p o l l i n a t i o n 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. 31:45-54, E r i k s s o n , G . , A . J o n s s o n , a n d 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 trial o f Picea K a r s t . Stud. For. Suec. 110:1-45. F a l c o n e r , D . S . 1 9 8 6 . Introduction Scientific & Technical.  to quantitative  genetics.  2nd ed. N e w York:  abies  Longman  G r i f f i n , A . R . 1 9 8 2 . C l o n a l v a r i a t i o n in r a d i a t a p i n e 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 a n d s e e d p r o d u c t i o n t r a i t s . Aust.  J. For.  Res.  12:295-302.  G r i f f i n , A . R . 1 9 8 4 . C l o n a l v a r i a t i o n in r a d i a t a pine s e e d o r c h a r d s . II. F l o w e r i n g Aust. J. For. Res. 1 4 : 2 7 1 - 2 8 1 .  phenology.  J o n s s o n , A . , I. E k b e r g , a n d 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 o f Pinus L. Stud. For. Suec. 135:1-38. O ' R e i l l y , C , W . H . Parker, a n d J . E . Barker. 1 9 8 2 . Effect of pollination period a n d n u m b e r o n r a n d o m m a t i n g in a c l o n a l s e e d o r c h a r d o f Picea mariana. Silvae 31:90-94.  sylvestris  strobili Genet.  R e y n o l d s , S , a n d Y . A . E l - K a s s a b y . 1 9 9 0 . P a r e n t a l b a l a n c e in D o u g l a s - f i r s e e d o r c h a r d - c o n e c r o p 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 e d m a n and Y . A . E l - K a s s a b y . 1 9 9 1 . Effective n u m b e r of pollen parents in c l o n a 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 v a r i a t i o n in f r u i t f u l n e s s Genet. 3 2 : 7 6 - 8 0 .  in a l o b l o l l y p i n e s e e d o r c h a r d .  Silvae  S c h o e n , D . J . , D. D e n t i , a n d S . C . S t e w a r t . 1 9 8 6 . S t r o b i l u s p r o d u c t i o n in a c l o n a 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 f o 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 . Sokal,  R . R . a n d F . J . R o h l f . 1 9 8 1 . Biometry: The principles and practice biological research. 2nd ed. N e w York: W.H.Freeman and C o .  of  statistics  in  Z o b e l , B . J . , J . Barber, C . L . B r o w n , and T . O . Perry. 1 9 5 8 . S e e d orchards - Their 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 6 E f f e c 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 o n 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 u s u a l l y c o n d u c t e d a s part o f s e e d - q u a l i t y t e s t i n g . W h e r e a s t h e  s t a n d a r d g e r m i n a t i o n t e s t is b a s e d o n a n e s t i m a t e o f t h e m a x i m a l p o t e n t i a l f o r s e e d v i a b i l i t y , o r t h e a b i l i t y o f a s e e d t o p r o d u c e a n o r m a l p l a n t u n d e r f a v o r a b l e c o n d i t i o n s , it is n o t a d e q u a t e f o r a s s e s s i n g f i e l d e m e r g e n c e ( M c D o n a l d , 1 9 8 0 ) . A v i g o u r t e s 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 more appropriate germinate  for testing  s e e d e m e r g e n c e s i n c e it i m p l i e s t h e  ability  of the  seed  to  under both favourable and unfavourable conditions (Kneebone, 1 9 7 6 ) . Possible  c a u s e s of v a r i a t i o n in t h e l e v e l o f s e e d v i g o u r i n c l u d e (1 ) g e n e 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 a n d n u t r i t i o n o f t h e m o t h e r p l a n t , (3) s t a g e o f m a t u r i t y at h a r v e s t , (4) s e e d s i z e , w e i g h t specific gravity,  (5)  m e c h a n i c a l integrity,  (6) d e t e r i o r a t i o n  a n d a g i n g , a n d (7)  or  pathogens  ( A s s o c i a t i o n of O f f i c i a l S e e d A n a l y s t s , 1 9 8 3 ) . V i a b i l i t y in s e e d s h a s b e e n f o u n d t o b e h i g h e s t at t h e t i m e o f p h y s i o l o g i c a l  maturity,  a n d to decline w i t h age. D e l o u c h e and Baskin ( 1 9 7 3 ) have d e s c r i b e d s e e d deterioration encompassing  initial  membrane  degradation  and ending  with  l o s s of  germinability.  as The  s y m p t o m s o f t h i s d e t e r i o r a t i o n m a y i n c l u d e a d e c r e a s e in m e t a b o l i c a c t i v i t y , s u s c e p t i b i l i t y t o s t r e s s , r e d u c e d rate o f g e r m i n a t i o n a n d s e e d l i n g g r o w t h , d e c r e a s e d s t o r a b i l i t y , i n f e r i o r  plant  d e v e l o p m e n t a n d y i e l d , r e d u c e d e m e r g e n c e p o t e n t i a l , a n d i n c r e a s e s in s e e d l i n g a b n o r m a l i t i e s . T h e p r o c e s s e s of s e e d d e t e r i o r a t i o n  in a p o p u l a t i o n are i n d e p e n d e n t a m o n g t h e  individual  s e e d s , and the time c o u r s e for deterioration ranges from d a y s to y e a r s . T h u s , the germination p e r c e n t a g e of a s e e d l o t d e c r e a s e s w i t h t i m e in p r o p o r t i o n t o t h e n u m b e r o f i n d i v i d u a l s e e d s t h a t are n o l o n g e r g e r m i n a b l e ( D e l o u c h e a n d B a s k i n , 1 9 7 3 ) . D i f f e r e n c e s in t h e d e g r e e o f s e e d d e t e r i o r a t i o n c a n be r e v e a l e d t h r o u g h a v i g o u r t e s t . T h i s i n d i c a t e s w h e t h e r t h e d i f f e r e n c e s 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 i a l S e e d A n a l y s t s 1 9 8 3 ) .  S e e d vigour lias been dennonstrated to be heritable ( 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 ) , a n d v a r i e s a c c o r d i n g t o f i e l d w e a t h e r i n g ( P a s c a l a n d E l l i s , 1 9 7 8 ; P o t t s et 1978;  N d i m a n d e etal.,  al.,  1981) and storage conditions (Wein and K u e n e m a n , 1 9 8 1 ; Minor and  P a s c a l , 1 9 8 2 ) . In s o m e p l a n t s , t h i s trait is i n h e r i t e d m a t e r n a l l y ( K u e n e m a n , 1 9 8 3 ) . W h e n s e e d s age, not only d o e s their physiological activity c h a n g e , but also their c h r o m o s o m a l  structure  c h a n g e s (Roberts, 1 9 7 2 ; Pitel, 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 n c l u d e d in s t r e s s t e s t s , h a s b e e n e f f e c t i v e l y  used to  e s t i m a t e s e e d v i g o u r a n d s t o r a b i l i t y in b o t h a n n u a l a n d p e r e n n i a l p l a n t s ( D e l o u c h e a n d B a s k i n , 1973;  Pitel, 1 9 8 0 ;  B l a n c h e et  al.,  1988,  1990).  While  relatively  little is k n o w n  about  a c c e l e r a t e d a g i n g in t r e e s e e d s , t h i s s t u d y e m p l o y e d t h e s t a n d a r d p r o c e d u r e d e v e l o p e d f o r a g r i c u l t u r a l s e e d s t o d e t e r m i n e if a c c e l e r a t e d a g i n g c o u l d b e u s e d t o e s t i m a t e t h e v i g o u r o f orchard-produced Sitka spruce seeds. 6 . 2 Materials  and  Methods  C a n a d i a n Pacific Forest P r o d u c t s Ltd. provided the s e e d s for this s t u d y f r o m the Sitka s p r u c e s e e d o r c h a r d l o c a t e d in S a a n i c h t o n , B r i t i s h C o l u m b i a ( l a t i t u d e 4 8 ° 3 5 ' N ,  longitude  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 ( a v e r a g i n g 9 . 3 r a m e t s p e r c l o n e s ) s e l e c t e d from elevations between 0 m and 4 1 5 m on western O r e g o n (Figure  1.1).  V a n c o u v e r Island, W a s h i n g t o n  and  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 i n g l e - t r e e m i x o v e r  three unequal blocks. In S e p t e m b e r 1 9 9 0 , w i n d - p o l l i n a t e d 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 f o r t h e s t u d y . T h e c l o n a l identity of the s e e d s w a s maintained during s e e d e x t r a c t i o n , after w h i c h the s e e d s w e r e k e p t at 2 ° C u n t i l u s e d . T h e y w e r e t h e n s u b j e c t e d t o a c c e l e r a t e d a g i n g a c c o r d i n g t o t h e A O S A ' s s e e d - v i g o u r - t e s t i n g p r o c e d u r e ( A s s o c i a t i o n of Official S e e d A n a l y s t s , 1 9 8 3 ) . In t h i s  study,  it is a s s u m e d t h a t  (1)  the  p r o c e s s e s of  seed deterioration  under  a c c e l e r a t e d a g i n g c o n d i t i o n s are s i m i l a r t o t h o s e u n d e r n a t u r a l c o n d i t i o n s - o n l y t h e rate of d e t e r i o r a t i o n is i n c r e a s e d ( D e l o u c h e a n d B a s k i n , 1 9 7 3 ) , a n d (2) t h e d e c l i n e in g e r m i n a t i o n after  a c c e l e r a t e d a g i n g is a s s o c i a t e d w i t h t h e initial d e g r e e of d e t e r i o r a t i o n of t h e s e e d l o t s , i.e., h i g h v i g o u r l o t s w i l l s h o w little d e c l i n e 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 l o t s w i l l s h o w a m a r k e d d e c l i n e in g e r m i n a t i o n ( B a s k i n , 1 9 7 7 ) . 6 . 2 . 1 Accelerated  Aging  A pilot s t u d y c o n d u c t e d o n a s i n g l e c l o n e u n d e r f o u r t e m p e r a t u r e s : 3 5 , 3 7 . 5 , 4 0 a n d 4 5 ° C (data n o t s h o w n ) i n d i c a t e d 3 7 . 5 ° C t o b e t h e a p p r o p r i a t e t e m p e r a t u r e f o r a c c e l e r a t e d a g i n g in S i t k a s p r u c e . T h i s t e m p e r a t u r e is l o w e r t h a n t h a t p r e s c r i b e d f o r a c c e l e r a t e d a g i n g ( A s s o c i a t i o n o f O f f i c i a l S e e d A n a l y s t s , 1 9 8 3 ) . B l a n c h e et al. ( 1 9 8 8 ) a l s o r e c o m m e n d e d t h e u s e of l o w e r t e m p e r a t u r e s w h e n a c c e l e r a t e d a g i n g is a p p l i e d t o t r e e s e e d s . In t h i s s t u d y , a c c e l e r a t e d a g i n g w a s a p p l i e d at 3 7 . 5 ° C f o r 8 a g i n g p e r i o d s f r o m 0 t o 21 d a y s at 3 - d a y i n t e r v a l s . E a c h t e s t c o n s i s t e d o f t e n s a m p l e s o f 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 s e p a r a t e l y 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 . o f w a t e r . S a m p l e s w e r e o b t a i n e d in s u c c e s s i o n s o t h a t t h e s a m p l e s f o r t h e l o n g e s t 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 (21 d a y s ) w e r e p l a c e d in t h e i n c u b a t o r f i r s t a n d t h e s h o r t e s t - a g e d s a m p l e s (3 d a y s ) w e r e t h e l a s t . A t t h e e n d of t h e t e s t all s a m p l e s w e r e r e m o v e d a n d t w o s a m p l e s from e a c h accelerated aging treatment w e r e u s e d for moisture-content  determination  ( I n t e r n a t i o n a l 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 r e m a i n i n g s a m p l e s (8) w e r e k e p t at r o o m t e m p e r a t u r e o v e r n i g h t t o r e d u c e t h e e f f e c t of r a p i d t e m p e r a t u r e c h a n g e o n s e e d g e r m i n a t i o n , a s r e c o m m e n d e d b y B o u r l a n d a n d Ibrahim ( 1 9 8 2 ) . A l l t h e s e e d s a m p l e s ( m o l d y a n d n o n - m o l d y s e e d s ) w e r e c l e a n e d u n d e r r u n n i n g , t e p i d , w a t e r f o r 2 m i n u t e s prior t o t h e g e r m i n a t i o n t e s t . 6 . 2 . 2 Germination  Test  A l l g e r m i n a t i o n t e s t s w e r e c o n d u c t e d under I S T A rules (International  Seed Testing  A s s o c i a t i o n , 1 9 8 5 ) . Four samples were germinated without stratification, while the remaining f o u r w e r e s o a k e d in w a t e r f o r 2 4 hr, t h e n s t r a t i f i e d at 2 ° C f o r 21 d a y s b e f o r e g e r m i n a t i o n . F o r 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 g h t l y - l i d d e d , c l e a r p l a s t i c g e r m i n a t i o n b o x lined w i t h m o i s t e n e d c e l l u l o s e w a d d i n g ( K i m p a k ) a n d filter p a p e r , t h e n p l a c e d in a g e r m i n a t o r s e t at  3 0 ° C f o r a n 8 hr d a y , a n d 2 0 ° C f o r a 1 6 hr n i g h t . L i g h 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 d u r i n g t h e d a y b y 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 every  day for  Association,  21  days and classified as normal  or a b n o r m a l  (International S e e d  Testing  1985).  The germination data w e r e calculated and e x p r e s s e d as germination c a p a c i t y (GC), the p e r c e n t a g e o f s e e d s t h a t h a d g e r m i n a t e d at t h e e n d o f t h e t e s t ; p e a k v a l u e ( P V ) , t h e m a x i m u m quotient derived b y dividing daily 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 t h e m e a n d a i l y g e r m i n a t i o n of t h e m o s t - v i g o r o u s c o m p o n e n t o f t h e s e e d l o 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 t h e t a n g e n t d r a w n t h r o u g h t h e o r i g i n o f t h e s i g m o i d c u r v e representing a typical c o u r s e of germination)  (Czabator, 1962); and germination  value  ( G V ) , t h e c o m b i n a t i o n of b o t h t h e s p e e d a n d c o m p l e t e n e s s o f g e r m i n a t i o n i n t o a s i n g l e v a l u e (Czabator,  1962).  6 . 2 . 3 Statistical  Analysis  T h e a n a l y s i s of v a r i a n c e u s e d w a s b a s e d o n t h e f o l l o w i n g n e s t e d - f a c t o r i a l ,  additive,  linear m o d e l ; Yij„ = // + Ci + P, + C P , + T / C , i „ + P T / C , „ „ + e,i,,„ w h e r e /y = o v e r a l l m e a n . Ci = t h e e f f e c t o f ith c l o n e , i =  1-6 ( r a n d o m  P| = t h e e f f e c t jth s e e d p r e t r e a t m e n t , j =  effect),  1-2 ( f i x e d  effect),  CPi, = t h e e f f e c t of i n t e r a c t i o n b e t w e e n c l o n e s a n d s e e d p r e t r e a t m e n t s , T/C,i||^ = t h e e f f e c t of k t h a g i n g t i m e w i t h i n c l o n e s , k =  1-8 ( f i x e d  effect),  PT/C,,,jk = t h e e f f e c t of i n t e r a c t i o n b e t w e e n s e e d p r e t r e a t m e n t s a n d a g i n g times within clone, and  = residual term, 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 using an ad-fioc p r o c e d u r e for finding appropriate transformation to normalize the calculated response variables and achieve homogeneity  of  variances. 6 . 3 Results  and  Discussion  T h e v a r i a t i o n in t h e e f f e c t o f a c c e l e r a t e d a g i n g d u e t o c l o n a l d i f f e r e n c e s w a s h i g h l y s i g n i f i c a n t (P < 0 . 0 1 ) (Table  6.1).  C l o n a l v a r i a t i o n in g e r m i n a t i o n p a r a m e t e r s r a n g e d b e t w e e n  3 0 . 9 6 % ( G O a n d 4 1 . 2 2 % (GV) of total variation. Variation due to s e e d pretreatment w a s not s i g n i f i c a n t f o r P V a n d G V . It w a s s i g n i f i c a n t (P < 1 8 . 2 6 % of t o t a l v a r i a t i o n (Table  6.1).  0.05)  for G C , a c c o u n t i n g for 2 . 4 6  to  Variation due to the length of the a c c e l e r a t e d - a g i n g  period w a s highly significant, a c c o u n t i n g for the largest proportion of the total variation, r a n g i n g f r o m 4 6 . 4 7 % ) in G C t o 5 0 . 2 3 % ) in G V . C l o n a l r e s p o n s e t o t h e v a r i o u s  treatments  (pretreatment, a c c e l e r a t e d aging a n d their interaction) w a s c o n s i s t e n t a s e x p r e s s e d by the l o w r e s i d u a l t e r m (i.e. r e p l i c a t i o n - t o - r e p l i c a t i o n v a r i a t i o n ) (Table  6.1).  It a p p e a r s t h a t 3 t o 6 d a y s o f a c c e l e r a t e d a g i n g e n h a n c e g e r m i n a t i o n o f u n s t r a t i f i e d a n d s t r a t i f i e d s e e d s in s o m e c l o n e s (Figure  6.1 a n d Figure  6.2.), b u t g e r m i n a t i o n g r a d u a l l y d e c l i n e s  w i t h longer treatment. G e r m i n a t i o n e n h a n c e m e n t due to short-time a c c e l e r a t e d aging might be of operational i m p o r t a n c e . S e e d m o i s t u r e 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 a g i n g (on a v e r a g e f r o m 6 . 7 % t o 3 3 % , b a s e d o n d r y w e i g h t ) a n d t h e n r e m a i n e d r e l a t i v e l y s t a b l e t o d a y 1 2 (Figure  6.3).  S e e d m o i s t u r e c o n t e n t i n c r e a s e d dramatically after 1 2 d a y s of treatment, p r o b a b l y b e c a u s e cell m e m b r a n e s b e c o m e s e v e r e l y d a m a g e d , a n d v i a b i l i t y is s u b s e q u e n t l y l o s t . T h i s d e c l i n e in g e r m i n a t i o n w a s a s s o c i a t e d w i t h t h e i n c r e a s e in m o i s t u r e c o n t e n t (Figure  6.4).  Correlations  b e t w e e n a v e r a g e G C a n d m o i s t u r e c o n t e n t are n e g a t i v e a n d h i g h l y s i g n i f i c a n t (P< stratified (r=  - 0 . 9 4 ) a n d u n s t r a t i f i e d (r = - 0 . 9 4 ) s e e d s (Figure  0 . 0 1 ) for  6.4).  The period w h e n accelerated aging e n h a n c e s s e e d germination has been termed the " c o n d i t i o n e d s t a g e " by Bird and R e y e s ( 1 9 6 7 ) , while the term "deteriorated s t a g e " w a s u s e d  Table  6.1.  V a r i a t i o n in g e r m i n a t i o n o f 6 Sitita 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 a g i n g ( A A ) a n d s e e d p r e t r e a t m e n t p e r c e n t a g e s of the total m e a n squares)  S o u r c e of Variation  Degrees of Freedom  Germination Parameters^  Expected M e a n Squares^ GC  C l o n e (C)  (C-1) =  5  al  +  P r e t r e a t m e n t (P)  (P-1) =  1  al  + Zlal,  C  (C-1)(P-1)  X  P  =  A A - T i m e w i t h i n C l o n e (T/C)  C(T-1)=  P  C(T-1)(P-1) =  X  T/C  Residual ™ Not significant. " S i g n i f i c a n t at P <  CPT(N-I)  5  of +  42 42  = 288  QAal  22ol,  al  +  8af,,  ol  +  4a^,,,  al  +  ( v a l u e s are  1920,  PV  GV  30.96**  34.70""  41.22""  18.26*  12.17™  2.46™  2.07""  3.14""  3.65"*  46.47""  48.09""  50.23**  1.70"  1.20""  1.54""  0.54  0.70  0.90  0.05.  •" S i g n i f i c a n 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 nested-factorial e x p e r i m e n t a 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 ; t h e p e r c e n t a g e of s e e d s t h a t h a d g e r m i n a t e d at t h e e n d o f t h e t e s t ( A r c s i n ) . P V = P e a k v a l u e ; a m a t h e m a t i c a l e x p r e s s i o n of t h e b r e a k of a s i g m o i d c u r v e r e p r e s e n t i n g a t y p i c a l c o u r s e of g e r m i n a t i o n (1 -1 / ( X -i-1 )). G V = Germination value, (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  Figure  6.2.  6 9 12 15 ACCELERATED AGING TIME (DAYS)  18  21  Average germination (21 days) of stratified seeds of 6 Sitka spruce clones after accelerated aging.  I  0  Figure  \  3  6.3.  \  \  \  I  6 9 12 15 ACCELERATED AGING TIME (DAYS)  \  18  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 k a s p r u c e c l o n e s after accelerated aging.  L  21  Figure  6.4. Curves shovy/ing germination of unstratified, stratified, and moisture content, of accelerated-aged seeds; average for 6 clones.  for the period w h e n germination d e c r e a s e s until the s e e d d i e s . T h e g e r m i n a t i o n t r e n d s  after  a c c e l e r a t e d a g i n g o b s e r v e d in t h i s s t u d y fit t h e 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 o f seed germination (Quercus  d u e t o a s h o r t p e r i o d o f a c c e l e r a t e d a g i n g w a s o b s e r v e d in w a t e r  nigra L.) ( B l a n c h e et ai.,  oak  1 9 9 0 ) . S u c h e n h a n c e m e n t is n o r m a l f o r a n n u a l s e e d p l a n t s  ( B o u r l a n d a n d I b r a h i m , 1 9 8 2 ) , a n d is d u e t o t h e i n c r e a s e in m o i s t u r e c o n t e n t t h a t b r i n g s t h e level of hydration c l o s e r to the minimal requirement 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 s o a c c o u n t s f o r t h i s e n h a n c e m e n t ( P i t e 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 , t h e e x t e n t of g e r m i n a t i o n e n h a n c e m e n t in r e s p o n s e t o a c c e l e r a t e d a g i n g is i n f l u e n c e d b y t h e initial s e e d q u a l i t y ( B l a n c h e etal.,  1990), including moisture content  (McDonald, 1977; Toa, 1979). T h e f i r s t e v e n t d u r i n g t h e " d e t e r i o r a t e d s t a g e " is b e l i e v e d t o b e c a u s e d b y a l o s s o f m e m b r a n e i n t e g r i t y ( B a s a v a r a j a p p a et al.,  1 9 9 1 ) ; c e l l p e r m e a b i l i t y is i n c r e a s e d , a l l o w i n g large  q u a n t i t i e s o f c e l l u l a r c o m p o n e n t s t o d i f f u s e o u t w h e n t h e 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 a n d P r e s l e y , 1 9 6 3 ) . T h e i n c r e a s e in m e m b r a n e p e r m e a b 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 d u e t o c h a n g e s in t h e m o l e c u l a r s t r u c t u r e of t h e 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 , 1974;  P i t e l , 1 9 8 0 ) . T h e i n c r e a s e in m e t a b o l i s m d u r i n g a g i n g d e p l e t e s f o o d r e s e r v e s a n d ,  s u b s e q u e n t l y , s e e d v i g o u r d e c l i n e s ( B l a n c h e et al., differential  1 9 9 0 ) . T h e l o s s of s e e d viability  and  s u r v i v a l of b i o t y p e s d u e t o a c c e l e r a t e d a g i n g w a s f o u n d t o b e a s s o c i a t e d w i t h  s e l e c t i o n a n d g e n e t i c s h 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 t o a c c e l e r a t e d a g i n g v a r i e s a c c o r d i n g t o s p e c i e s ( B e n n e t t - L a r t e y , 1 9 9 1 ; B l a n c h e et al.,  1988), population (Milby and J o h n s o n , 1989), family  (Bourland and  I b r a h i m , 1 9 8 2 ) a n d i n d i v i d u a l s e e d s ( D e l o u c h e a n d B a s k i n , 1 9 7 3 ) , a n d is s i m i l a r t o s e e d s t h a t h a v e b e e n s t o r e d f o r a l o n g t i m e . In S i t k a s p r u c e , t h e o b s e r v e d d i f f e r e n c e s in t h e e f f e c t s o f accelerated  aging on seed germination  suggest that the  vigour  of  orchard  s e e d s varies  a c c o r d i n g t o c l o n e . C o n s e q u e n t l y , a c c e l e r a t e d a g i n g is p r o b a b l y a p p l i c a b l e f o r i n d e x i n g s e e d v i g o u r in S i t k a s p r u c e .  In p r a c t i c e , w l i e n s e e d s c o l l e c t e d f r o m d i f f e r e n t o r c h a r d c l o n e s are b u l k e d i n t o a s i n g l e s e e d l o t , long-term s t o r a g e of this s e e d l o t might reduce genetic diversity. S i n c e the contribution of p a r e n t t r e e s t o s e e d p r o d u c t i o n in t h e o r c h a r d f r o m w h i c h t h e s e e d s t e s t e d in t h i s 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 a n d R e y n o l d s , 1 9 9 0 ) s o m e c l o n e s might be eliminated by long term-storage. For agricultural c r o p s , g e r m i n a t i o n f o l l o w i n g a c c e l e r a t e d aging a n d periods of storage 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 l o t s t h a t h a v e h i g h s u r v i v a l a f t e r a c c e l e r a t e d a g i n g s t o r e d w e l l , w h i l e l o t s t h a t w e r e s e v e r e l y r e d u c e d in their g e r m i n a t i o n b y a c c e l e r a t e d a g i n g d e c l i n e d r a p i d l y in s t o r a g e ( D e l o u c h e a n d B a s k i n , 1 9 7 3 ) . T o - d a t e , f e w if a n y s u c h d a t a are a v a i l a b l e f o r tree 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 i a l S e e d A n a l y s t s . 1 9 8 3 . Seed 3 2 . A s s o c i a t i o n of Official S e e d A n a l y s t s .  vigor  testing  handbooif..  Contribution No.  Basavarajappa, B . S . , H . S . Shetty, and H . S . Prakash. 1 9 9 1 . M e m b r a n e deterioration and other 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 a c c e l e r a t e d a g e i n g o f m a i z e s e e d s . Seed Sci. & Technol. 19:279-286. B a s k i n , C . C . 1 9 7 7 . V i g o r t e s t m e t h o d s - A c c e l e r a t e d aging. Assoc. 51:42-52.  Off  Seed Anal.  Newslett.  Bennett-Lartey, S . O . 1 9 9 1 . T h e longevity 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 c o n t r o l l e d t e m p e r a t u r e a n d m o i s t u r e 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 r d , L . S . a n d A . A . R e y e s . 1 9 6 7 . E f f e c t s of c o t t o n s e e d q u a l i t y o n s e e d a n d 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 . Cotton Council, Memphis, Tennessee.  under  seedling National  Blanche, C . A . , W . W . E l a m , J . D . H o d g e s , F.T. Bonner, 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 a g i n g o f s e l e c t e d t r e e s e e d s . In Proc. 10th north American forest biology workshop, ed. J . Worrall, J . L o o - D i n k i n s , and D . P . Lester, pp. 3 2 7 - 3 3 4 . V a n c o u v e r , British Columbia. B l a n c h e , C . A . , W . W . E l a m , a n d J . D . H o d g e s . 1 9 9 0 . A c c e l e r a t e d a g i n g o f Ouercus nigra seed: b i o c h e m i c a l c h a n g e s a n d a p p l i c a b i l i t y a s a v i g o r t e s 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 . a n d A . A . L . I b r a h i m . 1 9 8 2 . E f f e c 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 c o t t o n c u l t i v a r s . Crop Sci 22:637-640.  on six  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 value: A n index c o m b i n i n g s p e e d a n d c o m p l e t e n e s s of pine s e e d g e r m i n a t i o n . For. Sci 8:386-396. Delouche, 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 aging t e c h n i q u e s for predicting r e l a t i v e s t o r a b i l i t y of s e e d l o t s . Seed Sci. & Technol. 1:427-452. 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 q u a l i t y . HortSci.  the  15:771-774.  El-Kassaby, Y . A . , and S . Reynolds. 1 9 9 0 . Reproductive phenology, parental balance, and s u p p l e m e n t a l m a s s p o l l i n a t i o n 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. 31:45-54. I n t e r n a t i o n a l 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 . I n t e r n a t i o n a l r u l e s f o r s e e d t e s t i n g . Seed Technol. 13:299-513. K n e e b o n e , W . R . 1 9 7 6 . S o m e g e n e t i c a s p e c t s of s e e d v i g o r . J. Seed 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 r a s t r u c t u r e 1:417-425.  Technol.  d u r i n g s e n e s c e n c e . Seed  K u e n e m a n , E . A . 1 9 8 3 . G e n e t i c c o n t r o l of s e e d l o n g e v i t y in s o y b e a n s . Crop  Sci.  &  1:86-97. Sci  Sci  &  Technol.  23:5-8.  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 u e n c i n g s e e d v i g o r : B i o c h e m i s t r y of h e t e r o s i s . Sci. & Techno/. 1:25-50.  Seed  M c D o n a l d , M . B . , J r . 1 9 7 7 . T h e influence of s e e d m o i s t u r e o n the a c c e l e r a t e d aging s e e d vigor t e s t . J. Seed  Techno/.  2:18-28.  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 o f s e e d q u a l i t y . HortSci.  15:184-788.  M c W i l l i a m , J . R . a n d B. G r i f f i n g . 1 9 6 5 . T e m p e r a t u r e - d e p e n d e n t Bio/. Sci. 1 8 : 5 6 9 - 5 8 3 .  h e t e r o s i s in m a i z e . Aust.  J.  M i l b y , T . H . , a n d F . L . J o h n s o n . 1 9 8 9 . V a r i a t i o n in r e s p o n s e t o s t r a t i f i c a t i o n a n d a g e i n g in g a m a g r a s s s e e d s f r o m d i f f e r e n t g e o g r a p h i c l o c a t i o n s . Seed Sci. & Techno/. 17:413419. Minor, H.C., and E.C. Pascal. t r o p i c a l c o n d i t i o n s . Seed  1 9 8 2 . V a r i a t i o n in s t o r a b i l i t y Sci.  & Techno/.  of s o y b e a n s under  simulated  10:131-139.  N d i m a n d e , B . N . , H . C . W e i n , a n d 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 d e t e r i o r a t i o n in t h e t r o p i c s . T h e role o f p h y s i o l o g i c a l f a c t o r s a n d f u n g a l p a t h o g e n s . Fie/d Crops Res. 4 : 1 1 3 121. P a s c a l , E . H . II, a n d M . A . Ellis. 1 9 7 8 . V a r i a t i o n in s e e d q u a l i t y c h a r a c t e r i s t i c s o f g r o w n s o y b e a n s . Crop Sci. 1 8 : 8 3 7 - 8 4 0 .  tropically  P i t e l , J . A . 1 9 8 0 . A c c e l e r a t e d a g i n g s t u d i e s o f s e e d s o f j a c k p i n e (Pinus ban/^siana Lamb.) and red o a k (Quercus rubra L.). In Proc. internationa/ Symposium on Forest tree seed storage, l U F R O W o r k i n g party 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 . Pitel, pp. 4 0 - 5 4 . P e t a w a w a National Forestry Institute, C h a l k River, Ontario. 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 , a n d J . C . D e l o u c h e . 1 9 7 8 . S o m e i n f l u e n c e s 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 q u a 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 , genetical a n d m e t a b o 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 o s s of v i a b i l i t y . In Viabi/ity of seeds, e d . E . H . R o b e r t s , p p . 2 5 3 - 3 0 6 . L o n d o n : C h a p m a n a n d Hall. S c h n a t h o r s t , W . C , a n d J . T . Presley. 1 9 6 3 . P r o n e n e s s of deteriorated c o t t o n s e e d to d e c a y and s e e d l i n g d i s e a s e s r e l a t e d t o 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., p p . 5 7 - 5 8 . N a t i o n a 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 . Simola,  L . K . 1 9 7 4 . U l t r a s t r u c t u r e c h a n g e s in s e n e s c e n c e . Stud. For. Suec. 1 1 9 : 1 - 2 2 .  the  s e e d s of  Pinus  sy/vestris  L.  S t o y a n o v a , S . D . 1 9 9 1 . G e n e t i c shifts a n d variations of gliadins i n d u c e d b y s e e d a g e i n g . Sci. & Techno/. 19:363-371.  during  Seed  T o a , K . J . 1 9 7 9 . A n evaluation of alternative m e t h o d s of a c c e l e r a t e d a g i n g s e e d vigor test for s o y b e a n s . J. Seed Techno/. 3:30-40. W e i n , H . C , a n d 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 d e t e r i o r a t i o n in t h e t r o p i c s . V a r i e t a l d i f f e r e n c e s a n d t e c h n i q u e s f o r s c r e e n i n g . Fie/d  Crops  Res.  4:123-132.  Chapter 7 E f f e c t of S e e d S i z e o n G e r m i n a t i o n o f 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 r o u t i n e l y u s e d t o e s t i m a t e v i a b i l i t y a n d g e r m i n a t i v e e n e r g y o f s e e d s . T h e  pattern  of s e e d g e r m i n a t i o n v e r i f i e s q u a l i t y w i t h r e f e r e n c e t o 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 i a 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 t h e n a t u r a 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 b e s o w n is u n s t a b l e , e v e n in t h e g r e e n h o u s e , a s t a n d a r d g e r m i n a t i o n t e s t n o r m a l l y is c o n d u c t e d u s i n g u n i f o r m c o n d i t i o n s . T h u s , the germination test often o v e r - e s t i m a t e s field p e r f o r m a n c e of s e e d s . T h e p a t t e r n of s e e d g e r m i n a t i o n in a s p e c i e s v a r i e s a c c o r d i n g t o s e e d s o u r c e ( A l l e n , 1 9 6 1 ) , f a m i l y ( B r a m l e t t et al.,  1 9 8 3 ; E l - K a s s a b y et al.,  G e n t l e , 1 9 5 9 ; D e M a t o s M a l a v a s i et al.,  1992), pretreatments  (Kozlowski and  1 9 8 5 ; Pitel a n d W a n g , 1 9 8 5 ) , s e e d maturity (Allen,  1 9 5 8 a , b; E d w a r d s , 1 9 8 0 ; E d w a r d s a n d 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  preconditioning  during s e e d d e v e l o p m e n t (Koller, 1 9 6 2 ; S a w h n e y a n d N a y l o r , 1 9 7 9 ; S a w h n e y a n d N a y l o r , 1 9 8 0 ; Naylor, 1 9 8 3 ) , and s e e d size (Spurr, 1 9 4 4 ; S h o u l d e r s , 1 9 6 1 ; Burgar, 1 9 6 4 ;  Wulff,  1 9 7 2 ; Dunlap and Barnett, 1 9 8 3 ; Helium, 1 9 9 0 ) . S e e d s i z e h a s b e e n f o u n d t o be u n d e r s t r o n g g e n e t i c i n f l u e n c e ( H e l i u m , 1 9 7 6 ; S i l e n a n d 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 ; W u l f f , 1 9 8 6 ; B a g c h i et al.,  1 9 9 0 ) t h a t is m a i n l y m a t e r n a l  (Perry, 1 9 7 6 ; R o a c h and Wulff, 1 9 8 7 ; T y s o n , 1 9 8 9 ) . G e o g r a p h i c variation also influences seed s i z e . W i t h i n a s p e c i e s , s e e d s i z e is c o r r e l a t e d w i t h d r y n e s s o f t h e s i t e . A s d r y n e s s i n c r e a s e s , s e e d s i z e i n c r e a s e s ( B a k e r , 1 9 7 2 ) . In a d d i t i o n , y e a r - t o - y e a r v a r i a t i o n in s e e d s i z e h a s b e e n o b s e r v e d in n o b l e fir (Abies (Pseudotsuga  menziesii  procera  (M\rb.)  Rehd.) (Sorensen and Franklin, 1977) and Douglas-fir  Franco) (Silen and O s t e r h a u s , 1 9 7 9 ) . W i t h i n a plant,  variation  in s e e d s i z e m a y b e c a u s e d b y p o s i t i o n of s e e d s in t h e p l a n t i n f l o r e s c e n c e ( C a v e r s a n d H a r p e r , 1 9 6 6 ; D a t t a et al.,  1 9 7 0 ) , or f r u i t ( L i n c k , 1 9 6 1 ; S c h a a l , 1 9 8 0 ) . In c o n i f e r s , t h e b i g g e s t s e e d s  u s u a l l y o c c u r in t h e m i d d l e p o r t i o n of t h e c o n e . T h e r e l a t i o n s h i p b e t w e e n s e e d s i z e a n d g e r m i n a t i o n c a n be v a r i a b l e . S e e d s i z e h a d little e f f e c t o n t h e g e r m i n a t i o n p a t t e r n in D o u g l a s - f i r ( L a v e n d e r , 1 9 5 8 ) , l o b l o l l y p i n e (Pinus  taeda  L.)  ( D u n l a p a n d B a r n e t t , 1 9 8 3 ) , N o r w a y s p r u c e (Picea J a p a n e s e red p i n e (Pinus densiflora  L. K a r s t . ) ( A n d e r s s o n , 1 9 6 5 ) , a n d  Sieb. & Zucc.) (Choi and K i m , 1 9 6 9 ) , but significant effects  w e r e o b s e r v e d in s l a s h p i n e (Pinus elliottii glauca  abies  Engelm.) (Shoulders, 1961 ) and white spruce  (Picea  (Moench) Voss) (Ackerman and G o r m a n , 1969). In t h i s s t u d y , t h e e f f e c t s of c l o n e s , s e e d s i z e a n d s e e d p r e t r e a t m e n t o n g e r m i n a t i o n 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 are r e p o r t e d . 7 . 2 Materials  and  Methods  Canadian Pacific Forest Products Ltd. provided the s e e d s for this study from the Sitka s p r u c e s e e d o r c h a r d l o c a t e d in S a a n i c h t o n , B r i t i s h C o l u m b i a (latitude 4 8 ° 3 5 ' N ,  longitude  1 2 3 ° 2 4 ' W ) . T h e orchard c o n s i s t s of 1 3 9 c l o n e s (averaging 9 . 3 r a m e t s per c l o n e s ) s e l e c t e d from elevations b e t w e e n 0 m and 4 1 5 m on western V a n c o u v e r Island, 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 i n g l e - t r e e m i x o v e r  three unequal blocks. In S e p t e m b e r 1 9 9 0 , w i n d - p o l l i n a t e d 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 l o n a l identities of the s e e d s w e r e maintained during 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 p o r t i o n s . O n e portion w a s s o r t e d into t w o size c l a s s e s , large (> and small (<  1.41  1.41  mm.)  mm.) using a 1 4 - m e s h s c r e e n , while the other w a s kept u n s o r t e d . Both  u n s o r t e d a n d s o r t e d s e e d p o r t i o n s w e r e k e p t at 2 ° C u n t i l u s e d . S i z e a n d w e i g h t are t h e m a i n p a r a m e t e r s u s e d in s e e d s o r t i n g , a n d t h e s e p a r a m e t e r s are h i g h l y c o r r e l a t e d in D o u g l a s - f i r ( S i l e n a n d O s t e r h a u s , 1 9 7 9 ) . W h e r e a s s i z i n g is l e s s t i m e consuming  when  sorting  a seedlot,  weighing  is m o r e  accurate  for  individual seed-size  d e t e r m i n a t i o n s . In t h i s s t u d y , w e i g h i n g a n d s i z i n g w e r e u s e d . 7 . 2 . 1 Seed  Weight  I n d i v i d u a l 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 , a n d 5 0 l a r g e a n d s m a l l s o r t e d 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 nearest 0.01 m g to determine  patterns  of  weigfit  distribution  among  c l o n e s and of  sizes  within  c l o n e s . Filled s e e d s  (determined by X-ray analysis) only were used. 7 . 2 . 2 Germination  Test  E i g h t r a n d o m s a m p l e s o f 1 0 0 s e e d s e a c h w e r e t a k e n f r o m u n s o r t e d a n d s o r t e d (large a n d small) s e e d s of e a c h c l o n e a n d s u b j e c t e d to a s t a n d a r d g e r m i n a t i o n test. Four of the eight s a m p l e s w e r e s o a k e d in w a t e r f o r 2 4 h r s . , d r a i n e d , a n d s t r a t i f i e d ( p r e c h i l l e d ) f o r 21 d a y s at 2 ° C . Stratified and unstratified s a m p l e s w e r e germinated simultaneously. For germination, seed s a m p l e s w e r e s p r e a d in t i g h t l y - l i d d e d , c l e a r p l a s t i c g e r m i n a t i o n b o x e s l i n e d w i t h m o i s t e n e d c e l l u l o s e w a d d i n g ( K i m p a k ) a n d filter p a p e r , a n d p l a c e d in a g e r m i n a t o r s e t at a n a l t e r n a t i n g t e m p e r a t u r e o f 3 0 ° C f o r 8 h r s . f o l l o w e d b y 2 0 ° C f o r 1 6 h r s . L i g h t , at a p p r o x i m a t e l y  1,000  lux, w a s p r o v i d e d during the high-temperature period 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 . Germinants were  counted every day for  21  d a y s a n d classified as normal or  abnormal  a c c o r d i n g to the I S T A (International 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 ) rules. R e s u l t s w e r e e x p r e s s e d a s (i) g e r m i n a t i o n c a p a c i t y ( G C ) , t h e p e r c e n t a g e of s e e d s t h a t h a d g e r m i n a t e d at t h e e n d o f t h e t e s t ; (ii) p e a k v a l u e ( P V ) , t h e m a x i m u m q u o t i e n t d e r i v e d b y d i v i d i n g d a i l y t h e a c c u m u l a t e d n u m b e r o f g e r m i n a n t s b y t h e c o r r e s p o n d i n g n u m b e r o f d a y s , w h i c h is t h e m e a n d a i l y g e r m i n a t i o n o f t h e m o s t v i g o r o u s c o m p o n e n t s o f a s e e d l o t ( C z a b a t o r , 1 9 6 2 ) , a n d (iii) g e r m i n a t i o n v a l u e ( G V ) , t h e c o m b i n a t i o n of s p e e d a n d c o m p l e t e n e s s o f g e r m i n a t i o n i n t o a single index (Czabator, 1 9 6 2 ) . 7 . 2 . 3 Statistical  Analysis  Data transformations were c o n d u c t e d using an ad-hoc procedure for finding appropriate t r a n s f o r m a t i o n s to normalize the c a l c u l a t e d r e s p o n s e variables a n d a c h i e v e h o m o g e n e i t y of variances. The germination parameters (GC, P V , and G V ) were then analyzed using analysis of variance ( A N O V A ) . A n a l y s e s were separated into t w o parts:  (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 t o e s t i m a t e g e n e t i c c o m p o n e n t s o f s e e d w e i g h t (Table  7.1)  a n d g e r m i n a t i o n p a r a m e t e r s o f u n s o r t e d S i t k a s p r u c e s e e d s (Table  7.3).  Broad-  s e n s e h e r i t a b i l i t i e s of t h e s e p a r a m e t e r s w e r e d e t e r m i n e d u s i n g t h e 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 o f a n i m a l b r e e d e r s ( F a l c o n e r , 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 a s m u l t i p l e m e a s u r e m e n t s per c l o n e . (2) A f a c t o r i a l e x p e r i m e n t w i t h 3 l e v e l s , (i) s e e d s i z e ( S , 2 c l a s s e s p l u s u n s o r t e d s e e d s ) , (ii) c l o n e ( C , 1 8 c l o n e s ) , a n d (iii) p r e t r e a t m e n t s (P, 2 t r e a t m e n t s ) , w a s u s e d t o a s s e s s t h e e f f e c t o f s e e d s i z e o n g e r m i n a t i o n p a r a m e t e r s . T h e m o d e l f o r t h i s A N O V A is a s 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 = o v e r a l l m e a n s Si = e f f e c t o f s e e d s i z e ( f i x e d e f f e c t ) , i = Ci = c l o n e e f f e c t ( r a n d o m e f f e c t ) , j =  1-3,  1-18,  Pi^ = p r e t r e a t m e n t e f f e c t ( f i x e d e f f e c t ) , k =  1-2,  SCij = e f f e c t of i n t e r a c t i o n b e t w e e n s e e d s i z e a n d c l o n e , PCjn = e f f e c t of i n t e r a c t i o n b e t w e e n p r e t r e a t m e n t a n d c l o n e , SPii^ = e f f e c t of i n t e r a c t i o n b e t w e e n s e e d s i z e a n d p r e t r e a t m e n t ( f i x e d e f f e c t ) , SCPijk = t h e e f f e c t of i n t e r a c t i o n a m o n g s e e d s i z e , c l o n e a n d p r e t r e a t m e n t , a n d eiiji,,! = r e s i d u a l t e r m , 1 = 7 . 3 Results  and  1-4.  Discussion  T h e d i f f e r e n c e b e t w e e n t h e l a r g e s t a n d s m a l l e s t s e e d s is s m a l l (range 0 . 0 2 0 0 0 . 0 3 1 3 m g in s e e d w e i g h t ) (Figure  to  7.1). H o w e v e r , t h i s v a r i a t i o n is h i g h l y s i g n i f i c a n t (P < 0 . 0 1 )  a n d a c c o u n t e d f o r 3 6 . 1 8 % o f t o t a l v a r i a t i o n (Table  7.1).  T h e largest a m o u n t of total variation,  6 3 . 8 2 % , is d u e t o d i f f e r e n c e s 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 i n t o 2 size c l a s s e s , w e i g h t d i f f e r e n c e s b e t w e e n s m a l l a n d large varied 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 t h e s m a l l e s t d i f f e r e n c e 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 h a s t h e  Figure  7.1. S e e d w e i g h t d i s t r i b u t i o n 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.  E s t i m a t i o n of v a r i a n c e c o m p o n e n t s , s i g n i f i c a n c e l e v e l , a n d b r o a d - s e n s e h e r i t a b i l i t i e s (h^) f o r i n d i v i d u a 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  S o u r c e of Variation A m o n g Clones Within Clones  df  (C-1)=  Expected^ Mean Squares 17  of +  lOOa^  C(N-1) = 1 7 8 2  Variance Component of S e e d W e i g h t 36.18" 63.82 0.36  ^ al = v a r i a n c e a m o n g c l o n e s ; al " S i g n i f i c a n t at P < 0 . 0 1 .  = variance within clones.  l a r g e s t d i f f e r e n c e (Figure  7.2.). T h e e s t i m a t e d b r o a d - s e n s e h e r i t a b i l i t y f o 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^ =  0.36).  Variation pretreatment,  in g e r m i n a t i o n  of  unsorted seeds  c l o n e , a n d t h e i r i n t e r a c t i o n (Table  is h i g h l y  7.2).  significant  (P  <  0.01)  for  S e e d pretreatments a c c o u n t for the  l a r g e s t p r o p o r t i o n o f v a r i a t i o n (range 7 4 . 1 2 - 9 3 . 8 5 % ) , w h i l e c l o n a l v a r i a t i o n a c c o u n t s o n l y f o r a s m a l l proportion of total variation  (range 3 . 0 7 - 5 . 9 1 % ) .  The interaction  between  seed  pretreatment 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 total variation. To make the clonal effect more discernible, complementary analyses were calculated for  unstratified  germination  and  stratified  seeds  (Table  7.3).  p a r a m e t e r s are h i g h l y s i g n i f i c a n t (P <  For unstratified 0.01)  seeds,  differences  in  w i t h e s t i m a t e s of b r o a d - s e n s e  h e r i t a b i l i t y (h^) of all p a r a m e t e r s r a n g i n g f r o m 0 . 7 6 t o 0 . 7 9 (Table  7.3).  For stratified seeds,  t h e d i f f e r e n c e s r e m a i n h i g h l y s i g n i f i c a n t (P < 0 . 0 1 ) , w i t h b r o a d - s e n s e h e r i t a b i l i t y e s t i m a t e s r a n g i n g f r o m 0 . 7 4 t o 0 . 7 8 (Table  7.3).  T h e s e d i f f e r e n c e s in t h e h e r i t a b i l i t y e s t i m a t e s f o r  u n s t r a t i f i e d a n d s t r a t i f i e d s e e d s are c o n s i d e r e d m i n i m a l in t h i s r a n g e . For u n s t r a t i f i e d  s e e d s , germination 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  d u r a t i o n of t h e s e t e s t s (Figure  7.3).  the  C l o n a l d i f f e r e n c e s in g e r m i n a t i o n c a p a c i t y are s u b s t a n t i a l  (range 4 5 . 7 5 t o 9 8 . 0 0 % ) . W h e n s e e d s w e r e s t r a t i f i e d (Figure  7.4),  v a r i a t i o n in g e r m i n a t i o n is  g r e a t l y r e d u c e d ( r a n g e 9 0 . 0 0 t o 9 8 . 5 0 % ) , s i n c e s t r a t i f i c a t i o n e n h a n c e d g e r m i n a t i o n rate f o r all 1 8 c l o n e s . T r a d i t i o n a l l y , s t r a t i f i c a t i o n temperate  species  (Edwards,  1980);  has been used to o v e r c o m e seed d o r m a n c y for  the  r e s p o n s e of  seeds  to  stratification  indicates  " d o r m a n c y . " C l o n a l d i f f e r e n c e s in g e r m i n a t i o n p a t t e r n s b e t w e e n u n s t r a t i f i e d a n d s t r a t i f i e d s e e d s i n d i c a t e t h e p r e s e n c e o f s o m e d e g r e e of d o r m a n c y (Figure  7.3 a n d Figure  7.4).  When  g e r m i n a t i o n o f u n s t r a t i f i e d a n d s t r a t i f i e d s e e d s w e r e c o m p a r e d f o r i n d i v i d u a l c l o n e s , it w a s f o u n d t h a t r e s u l t s v a r i e d 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 f o r 0 . 0 9 - 1 . 3 9 % of t o t a l v a r i a t i o n in g e r m i n a t i o n , w h i l e c l o n a l d i f f e r e n c e s a c c o u n t f o r 2 . 9 4 - 5 . 1 3 % (Table  7.4).  H o w e v e r , t h i s s m a l l a m o u n t o f v a r i a t i o n in  Figure  7.2. D i f f e r e n c e s in s e e d w e i g h t o f s m a l l a n d large s o r t e d s e e d s f r o m clone nos. 5 and 154.  Table  7.2.  E s t i m a t i o n o f v a r i a n c e 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 l e v e l for g e r m i n a t i o n 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  S o u r c e of Variation  df  Expected^  Germination Parameters^  Mean Squares GC  P r e t r e a t m e n t (P)  (P-1) =  1  ol  +  4afp +  C l o n e (C)  (C-1) =  17  ol  +  Sal  (P-1)(C'1) = 17  ol  +  PC(N-1) = 108  ol  C  X  P  Residual  ^ 0p = v a r i a n c e a m o n g p r e t r e a t m e n t ; al  720,  PV  GV  74.12"  93.85"  92.17"  5.91"  3.07"  3.23"  14.06"  1.85"  3.05"  1.23  1.55  5.91  = variance a m o n g clones; o^, = variance due to interaction  between  p r e t r e a t m e n t a n d c l o n e ; al = v a r i a n c e w i t h i n p r e t r e a t m e n t 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 , t h e p e r c e n t a g e o f s e e d s t h a t h a d g e r m i n a t e d at t h e e n d o f t h e t e s t ( A r c s i n ) . PV = P e a k 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 o f t h e b r e a k o f a s i g m o i d c u r v e r e p r e s e n t i n g a t y p i c a l c o u r s e o f g e r m i n a t i o n ( s q u a r e r o o t (X + 0 . 5 ) ) . GV = G e r m i n a t i o n V a l u e ( C z a b a t o r 1 9 6 2 ) , (no t r a n s f o r m a t i o n ) . " S i g n i f i c a n t at P < 0 . 0 1 .  Table  7.3.  E s t i m a t i o n o f v a r i a n c e c o m p o n e n t s , s i g n i f i c a n c e l e v e l , a n d b r o a d - s e n s e h e r i t a b i l i t i e s (h^) f o r g e r m i n a t i o n  parameters  u s i n g u n s o r t e d 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 Variation  df  A m o n g Clones  (C-1) =  17  Within Clones  C(N-1)=  54  h^  Unstratified  Expected' Mean Squares  ol  Stratified PV  GV  ^GC  PV  GV  78.87"  76.13"  77.22"  74.14"  11.iv  74.34"  21.13  23.87  22.78  25.86  12.22  25.66  0.79  0.76  0.77  0.74  GC  0.78  ' al = v a r i a n c e a m o n g c l o n e s ; o f = v a r i a n c e 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 , t h e p e r c e n t a g e of s e e d s t h a t h a d g e r m i n a t e d at t h e e n d of t h e t e s t ( A r c s i n e ) . PV = P e a k 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 o f t h e b r e a k o f a s i g m o i d c u r v e r e p r e s e n t i n g a t y p i c a l c o u r s e of g e r m i n a t i o n (no t r a n s f o r m a t i o n ) . GV = G e r m i n a t i o n V a l u e ( C z a b a t o r 1 9 6 2 ) , (no t r a n s f o r m a t i o n ) . " S i g n i f i c a n t at P < 0 . 0 1 .  0.74  0  3  6  9  12  15  18  21  TIME (DAYS) Figure  7.3.  Germination c u r v e s for unsorted-unstratified 18 Sitka spruce clones.  s e e d s of  0  3  6  9  12  15  18  21  TIME (DAYS) Figure  7.4.  G e r m i n a t i o n c u r v e s f o r u n s o r t e d - s t r a t i f i e d s e e d s of Sitka spruce clones.  18  Figure  7.5. D i f f e r e n c e s in g e r m i n a t i o n rate o f u n s t r a t i f i e d a n d s t r a t i f i e d s e e d s of clone nos. 4 1 1 and 5 1 6 (low and high d o r m a n t s e e d s , respectively).  Table  7.4.  E s t i m a t i o n of v a r i a n c e 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 l e v e l f o r g e r m i n a t i o n p a r a m e t e r s u s i n g s o r t e d s e e d s o f 1 8 S i t k a spruce clones  S o u r c e of Variation  df  Germination Parameters^  Expected' Mean Squares GC  S e e d S i z e (S)  (S-1) =  2  of  +  C l o n e (C)  (C-1) =  17  of  +  P r e t r e a t m e n t (P)  (P-1) =  1  ol  + 12a^, +  8of, +  1440,  24CT^  2160,  PV  GV  0.09™  1.39'*  0.76**  5.13**  2.94**  3.88*'  72.45**  89.84"  83.71"  SxC  (S-1)(C-1)=  34  ol  +  0.36*  0.27"  1.03"  SxP  (S-1)(P-1)=  2  ol  + 720,,  0.80**  1.81**  0.83"  CxP  (C-1)(P-1)=  17  ol  + 12afp  9.83**  2.14*'  6.03"  SxCxP  (S-1)(C-1)(P-1)=34  ol  +  7.16'  0.37**  2.58"  4.17  1.22  1.17  Residual  SCP(N-1)=324  ol  ' 03 = v a r i a n c e a m o n g s e e d s i z e s ; ol = v a r i a n c e a m o n g c l o n e s ; 0, = v a r i a n c e b e t w e e n s e e d p r e t r e a t m e n t s ; al,. = v a r i a n c e o f i n t e r a c t i o n e f f e c t b e t w e e n s e e d s i z e a n d c l o n e ; 0,, = v a r i a n c e o f i n t e r a c t i o n e f f e c t b e t w e e n s e e d s i z e a n d s e e d p r e t r e a t m e n t ; (jf, = v a r i a n c e o f i n t e r a c t i o n e f f e c t b e t w e e n c l o n e a n d s e e d p r e t r e a t m e n t ; of^, = v a r i a n c e of i n t e r a c t i o n e f f e c t a m o n g s e e d s i z e , c l o n e a n d s e e d p r e t r e a t m e n t ; ol = v a r i a n c e 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 , t h e p e r c e n t a g e o f s e e d s t h a t h a d g e r m i n a t e d at t h e e n d o f t h e t e s t ( A r c s i n ) . PV = P e a k 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 o f t h e b r e a k of a s i g m o i d c u r v e r e p r e s e n t i n g a t y p i c a l c o u r s e o f g e r m i n a t i o n (no t r a n s f o r m a t i o n ) . GV = G e r m i n a t i o n V a l u e ( C z a b a t o r 1 9 6 2 ) , (no t r a n s f o r m a t i o n ) . ™ N o n s i g n i f i c a n t ; " S i g n i f i c a n t at P < 0 . 0 5 ; ** S i g n i f i c a n t at P < 0 . 0 1 .  all g e r m i n a t i o n  parameters  s i g n i f i c a n t ( P < 0 . 0 1 ) (Table  among 7.4).  seed sizes  (except  GC) and among  c l o n e s is  highly  O n c e again, pretreatment a c c o u n t e d for the largest amount  of v a r i a t i o n ( r a n g e : 7 2 . 4 5 - 8 9 . 8 4 % ) in g e r m i n a t i o n (Table  7.4).  T o make seed size and clonal  effects more discernible, a s e c o n d , c o m p l e m e n t a r y , analysis w a s c o n d u c t e d separately for u n s t r a t i f i e d a n d s t r a t i f i e d s e e d s (Table  7.5).  For unstratified s e e d s , size a c c o u n t s for a very  s m a l l , i n s i g n i f i c a n t , p r o p o r t i o n o f t o t a l v a r i a t i o n (range 0 . 8 9 - 1 . 1 6 % )  (Table  7.5).  p r o p o r t i o n , 7 4 % , is d u e t o a m o n g - c l o n e v a r i a t i o n a n d is h i g h l y s i g n i f i c a n t (P< 7.5).  T h e largest 0.01)  (Table  A l t h o u g h t h e e f f e c t of s e e d s i z e o n g e r m i n a t i o n is n o t s i g n i f i c a n t , t h e e f f e c t o f i n t e r a c t i o n  b e t w e e n c l o n e a n d s e e d s i z e is h i g h l y s i g n i f i c a n t , i n d i c a t i n g t h a t g e r m i n a t i o n p a r a m e t e r s of different s e e d sizes differ a m o n g the 1 8 c l o n e s . For stratified  s e e d s , the  effects  p a r a m e t e r s are h i g h l y s i g n i f i c a n t (Table  of both 7.5).  s e e d s i z e a n d c l o n e o n all  germination  T h i s i n d i c a t e s t h a t t h e e f f e c t o f s e e d s i z e is  significant for the rapidity of S i t k a s p r u c e s e e d g e r m i n a t i o n . D u n l a p a n d Barnett ( 1 9 8 3 ) f o u n d a s i m i l a r i n f l u e n c e of s e e d s i z e in l o b l o l l y p i n e . G e n e t i c v a r i a t i o n in g e r m i n a t i o n in c o n i f e r s h a s b e e n r e p o r t e d t o b e u n d e r maternal  genetic  control  (Bramlett  et  al..  1983;  El-Kassaby  et  al.,  1992;  Hoff,  strong 1987).  G e r m i n a t i o n is i n f l u e n c e d b y m a t e r n a l e f f e c t s in m o s t p l a n t s s i n c e a m a j o r p o r t i o n o f t h e s e e d c o m p o n e n t s ( s e e d c o a t (2n), e n d o s p e r m ( I n ) , a n d half o f t h e e m b r y o (In)) c o n t r i b u t e d (Perry, 1 9 7 6 ; Ellner, 1 9 8 6 ; E l - K a s s a b y  are m a t e r n a l l y  a / . , 1 9 9 2 ) . V a r i a t i o n in s e e d g e r m i n a t i o n  w a s c o n s i d e r e d t o be a n a d a p t a t i o n f o r s u r v i v a l u n d e r 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 ( J a i n , 1982). S e e d - d o r m a n c y m e c h a n i s m s v a r y a m o n g s p e c i e s , s o d i f f e r e n t s e e d p r e t r e a t m e n t s are r e q u i r e d . V a r i a t i o n in s e e d d o r m a n c y a m o n g f a m i l i e s w i t h i n a s p e c i e s h a s b e e n r e p o r t e d f o r w e s t e r n w h i t e p i n e (Pinus monticola  Dougl.) (Hoff, 1 9 8 7 ) , the g e r m i n a t i o n of w h i c h i m p r o v e d  w i t h d u r a t i o n of s t r a t i f i c a t i o n . In c o n t r a s t , H e i t ( 1 9 6 1 ) c o n c l u d e d t h a t n e i t h e r s t r a t i f i c a t i o n n o r c h e m i c a l p r e t r e a t m e n t is r e q u i r e d f o r g e r m i n a t i o n o f S i t k a s p r u c e s e e d s . H o w e v e r , t h e p r e s e n t  Table  7.5.  E s t i m a t i o n o f v a r i a n c e 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 level f o r g e r m i n a t i o n p a r a m e t e r s o f 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 Variation  df  Expected' Mean Squares  S e e d S i z e (S)  (S-1) =  2  ai  +  Aài,  C l o n e (C)  (C-1) 1 =  17  ài  +  Mal  (S-1)(C-1) =  34  ài  +  4(7f.  S C ( N - 1 11 = 2 1 5  ol  S  X  C  Residual  +  Unstratified  720,  Stratified  ^GC  PV  0.89"=  1.16"=  1.09"=  9.39"*  38.50"  11.89"  74.33"  73.75*"  74.30"  38.93"  41.89*'  56.23"  7.01**  5.89"  6.53""  5.74"  23.82"  0.18  19.20  GV  18.08  GC  0.00 53.69  PV  13.87  GV  8.06  ' 0s, = v a r i a n c e a m o n g s e e d s i z e s ; o f = v a r i a n c e a m o n g c l o n e s ; of^ = v a r i a n c e of i n t e r a c t i o n e f f e c t b e t w e e n s e e d s i z e a n d c l o n e ; of = variance within clones. ^ GC = G e r m i n a t i o n C a p a c i t y , t h e p e r c e n t a g e o f s e e d s t h a t h a d g e r m i n a t e d at t h e e n d o f t h e t e s t ( A r c s i n ) . PV = P e a k 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 o f t h e b r e a k o f a s i g m o i d c u r v e r e p r e s e n t i n g a t y p i c a l c o u r s e of g e r m i n a t i o n (no t r a n s f o r m a t i o n ) GV = G e r m i n a t i o n V a l u e ( C z a b a t o r 1 9 6 2 ) , (no t r a n s f o r m a t i o n ) "= N o n s i g n i f i c a n t " S i g n i f i c a n t at P < 0 . 0 1 .  study demonstrates that stratification p e r i o d of  stratification  of  at l e a s t  is e s s e n t i a l f o r u n i f o r m g e r m i n a t i o n in t h i s s p e c i e s . A 21  d a y s is r e c o m m e n d e d  d i f f e r e n c e s d u e , p r o b a b l y , t o d i f f e r e n t i a l d o r m a n c y (Figure  to  reduce the  7.3 a n d Figure  germination  7.4).  C o r r e l a t i o n s b e t w e e n s e e d s i z e a n d g e r m i n a t i o n h a v e b e e n r e p o r t e d in s p e c i e s s u c h a s Hyptis  suaveolens  ( W u l f f , 1 9 7 2 ) , Pinus strobus  ( S p u r r , 1 9 4 4 ) a n d Acacia  holosericea  (Helium,  1 9 9 0 ) . In c o n t r a s t , n o s i g n i f i c a n t r e a l i z e d g a i n in f a v o u r o f large s e e d s o v e r s m a l l s e e d s w a s o b s e r v e d in t h e g e r m i n a t i o n o f m a n y o t h e r s p e c i e s ( B u r g a r , 1 9 6 4 ; L a r s o n , 1 9 6 3 ; A c k e r m a n a n d 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 u n s o r t e d s e e d s is r e c o m m e n d e d t o m a x i m i z e g e n e t i c diversity (Helium, 1 9 7 6 ; L i n d g r e n , 1 9 8 2 ; Silen a n d O s t e r h a u s , 1 9 7 9 ) . T h e effect of s e e d size in S i t k a s p r u c e is c o n s i d e r e d m i n i m a l in c o m p a r i s o n t o t h e a m o n g - a n d w i t h i n - c l o n e in g e r m i n a t i o n .  variation  7.4  References  A c k e r m a n , , R . F . a n d J . R . G o r m a n . 1 9 6 9 . E f f e c t of s e e d w e i g h t o n t h e s i z e o f l o d g e p o l e p i n e a n d w h i t e s p r u c e c o n t a i n e r p l a n t i n g 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 a n d s e e d s t u d i e s in N o r w a y s p r u c e . Stud.  For. Suec.  23:1-214.  A l l e n , G . S . 1 9 5 8 a . F a c t o r s affecting the viability a n d germination b e h a v i o u r of c o n i f e r o u s s e e d . P a r t I. C o n e a n d s e e d m a t u r i t y , Tsuga heterophylla ( R a f n . ) S a r g . For. Chron. 34:266-274. A l l e n , G . S . 1 9 5 8 b . F a c t o r s affecting the viability a n d g e r m i n a t i o n b e h a v i o u r of c o n i f e r o u s s e e d . P a r t II. C o n e a n d s e e d m a t u r i t y , Pseudotsuga menziesii ( M i r b . ) 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 D o u g l a s - f i r s e e d f o r p r o v e n a n c e . Proc. 26:388-403.  Int.  Seed  A s s o c i a t i o n o f O f f i c i a l S e e d A n a l y s t s . 1 9 7 0 . R u l e s f o r t e s t i n g s e e d s , Proc. Assoc. Anal. 6 0 : 1 - 1 1 6 .  Test.  Assoc.  Office.  Seed  spp.  Silvae  B a g c h i , S . K . , D . N . J o s h i , a n d D . S . R a w a t . 1 9 9 0 . V a r i a t i o n in s e e d s i z e of Acacia Genet. 3 9 : 1 0 7 - 1 1 0 .  B a k e r , H . G . 1 9 7 2 . S e e d w e i g h t in r e l a t i o n t o e n v i r o n m e n t a l c o n d i t i o n s in C a l i f o r n i a . 53:997-1010.  Ecology  Bramlett, D.L., T . R . Dell and W . D . Pepper. 1 9 8 3 . G e n e t i c and maternal i n f l u e n c e s o n Virginia p i n e s e e d g e r m i n a t i o n . Silvae Genet. 3 2 : 1 - 4 . B u r g a r , R . J . 1 9 6 4 . T h e e f f e c t of s e e d s i z e o n g e r m i n a t i o n , s u r v i v a l a n d initial 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 , , a n d J . L . H a r p e r . 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 obtusifolius. J. Ecol. 5 4 : 3 6 7 - 3 8 2 .  crispus  and  Rumex  C h o i , S . K . , a n d K . C . K i m . 1 9 6 9 . S t u d i e s o n t h e c h a r a c t e r i s t i c s o f s e l e c t e d p l u s - t r e e s . 11. T h e d i f f e r e n c e in s e e d g e r m i n a t i o n c a p a c i t y b e t w e e n p l u s - t r e e 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 v a l u e : A n i n d e x c o m b i n i n g s p e e d a n d c o m p l e t e n e s s of p i n e 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 a t t a , S . C , M . E v e n a r i , a n d Y . G u t t e r m a n . 1 9 7 0 . T h e h e t e r o p l a s t y o f Aegilopsis IsraelJ. Bot. 1 9 : 4 6 3 - 4 8 3 .  ovata  L.  De M a t o s M a l a v a s i , M . , S . G . Stafford and D.P. Lavender. 1 9 8 5 . Stratifying, partially redrying and storing Douglas-fir seeds: effects on growth and physiology during germination. Ann. Sci. For. 4 2 : 3 7 1 - 3 8 4 . Dunlap,  J . R . , and J . P . Barnett. 1 9 8 3 . Influence of s e e d size o n g e r m i n a t i o n a n d early d e v e l o p m e n t of l o b l o l l y p i n e [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 . a n d Y . A . E l - K a s s a b y . 1 9 8 8 . Effect of f l o w e r i n g p h e n o l o g y , date of c o n e collection, cone-storage treatment and seed pretreatment and seed pretreatment on y i e l d a n d g e r m i n a t i o n of s e e d s f r o m a D o u g l a s - f i r s e e d o r c h a r d . For. Ecol. Manage. 25:17-29. E d w a r d s , D . G . W . 1 9 8 0 . M a t u r i t y a n d quality of tree s e e d s - a state-of-the-art Sci. & Technol. 8:625-657.  review.  Seed  E l - K a s s a b y , Y . A . , D . G . W . E d w a r d s a n d D . W . Taylor. 1 9 9 2 . G e n e t i c c o n t r o l of germination p a r a m e t e r s in D o u g l a s - f i r a n d its i m p o r t a n c e f o r d o m e s t i c a t i o n . Silvae Genet, (in p r e s s ) .  E l l n e r , 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 a n d p a r e n t - o f f s p r i n g 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 . F a l c o n e r , D . S . 1 9 8 6 . Introduction S o n s , Inc.  to quantitative  genetics.  2nd ed. N e w York: J o h n Wiley &  Heit, C E . 1 9 6 1 . Laboratory germination and r e c o m m e n d e d testing m e t h o d s for 16 spruce Picea s p e c i e s . Proc. Assoc. Off. Seed Anal. 5 1 : 1 6 5 - 1 7 1 . Helium,  A . K . 1976. Grading 27(1):16,17,23.  seed  by  weight  in  white  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 p o p u l a t i o n o f Acacia 20:927-933.  spruce.  Tree  holoserica.  Planters'  Can.  J.  Notes  For.  Res.  H o f f , R . J . 1 9 8 7 . D o r m a n c y in Pinus monticola s e e d related to stratification time, s e e d coat, a n d g e n e t i c s . Can. J. For. Res. 1 7 : 2 9 4 - 2 9 8 . International 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 . International rules for s e e d testing 1 9 8 5 . Sci. & Technol. 13:299-513.  Seed  J a i n , S . K . 1 9 8 2 . V a r i a t i o n a n d a d a p t i v e role o f s e e d d o r m a n c y in s o m e a n n u a 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 . K o l l e r , D. 1 9 6 2 . P r e c o n d i t i o n i n g o f g e r m i n a t i o n in l e t t u c e at t i m e o f f r u i t r i p e n i n g . Amer. Bot. 4 9 : 8 4 1 - 8 4 4 .  J.  K o z l o w s k i , T . T . a n d A . C . G e n t l e . 1 9 5 9 . Influence 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 , a n d O x y g e n u p t a k e o f E a s t e r n w h i t e p i n e s e e d . For. Sci 5 : 3 8 9 - 3 9 5 . Larson,  M . M . 1 9 6 3 . Initial r o o t d e v e l o p m e n t o f g e r m i n a t i o n d a t e a n d s i z e o f s e e d . For. Sci  p o n d e r o s a pine s e e d l i n g s as related 9:456-460.  L a v e n d e r , D . P . 1 9 5 8 . V i a b i l i t y of D o u g l a s - f i r s e e d a f t e r s t o r a g e in t h e c o n e s . Oregon Lands Research Center Res. Notes N o . 3 1 : 1 - 9 . L i n c k , 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 o f t h e f r u i t o f Pisum Phytomorphology 11:79-84.  sativum  var.  to  Forest  Alaska.  L i n d g r e n , D. 1 9 8 2 . F r a c t i o n a t i o n o f s e e d o r c h a r d i m p l i c a t i o n s . Silva Fenn. 1 6 : 1 5 6 - 1 6 0 .  seeds by  weight  does have  genetic  N a y l o r , J . M . 1 9 8 3 . S t u d i e s o n t h e g e n e t i c c o n t r o l o f 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 . P e r r y , T . O . 1 9 7 6 . M a t e r n a l e f f e c t s o n t h e e a r l y p e r f o r m a n c e o f t r e e 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 a n d F. T . L a s t , p p . 4 7 3 - 4 8 1 . N e w York: A c a d e m i c Press. Pitel, J . P . and B . S . P . W a n g . 1 9 8 5 . Physical and c h e m i c a l treatments to improve laboratory g e r m i n a t i o n of w e s t e r n w h i t e p i n e s e e d s . Can. J. For. Res. 1 5 : 1 1 8 7 - 1 1 9 0 . Roach,  D.A. and R.D. Wulff. 18:209-235.  1987.  Maternal  effects  in p l a n t s . Ann.  Rev.  Ecol.  Syst.  S a w h n e y , R. a n d J . M . N a y l o r . 1 9 7 9 . D o r m a n c y s t u d i e s in s e e d o f Avena fatua. 9. D e m o n s t r a t i o n of g e n e t i c v a r i a b i l i t y a f f e c t i n g t h e r e s p o n s e t o t e m p e r a t u r e d u r i n g 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. a n d J . M . N a y l o r . 1 9 8 0 . D o r m a n c y s t u d i e s in s e e d oi Avena fatua. 1 2 . I n f l u e n c e of t e m p e r a t u r e o n g e r m i n a t i o n b e h a v i o u r o f n o n d o r m a n t f a m i l i e s . Can. J. Bot. 58:578-581. 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 a n d s e e d s i z e in Lupinus 67:703-709.  texensis.  Amer.  J.  Bot.  S h o u l d e r s , E. 1 9 6 1 . E f f e c t o f s e e d s i z e o n g e r m i n a t i o n , g r o w t h , a n d s u r v i v a l o f s l a s h p i n e . J. For. 5 9 : 3 6 3 - 3 6 5 . S i l e n , R., a n d C . O s t e r h a u s . 1 9 7 9 . R e d u c t i o n of g e n e t i c b a s e b y s i z i n g of b u l k e d D o u g l a s - f i r s e e d l o t s . Tree Planters'  Notes  30:24-30.  S o r e n s e n , F . C , a n d J . F . Franklin. 1 9 7 7 . Influence of y e a r of c o n e c o l l e c t i o n o n s e e d w e i g h t a n d 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 f e c t of s e e d w e i g h t a n d s e e d o r i g i n o n t h e e a r l y d e v e l o p m e n t o f e a s t e r n w h i t e p i n e . 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 c o n t r o l o f s e e d w e i g h t in f l a x (Linum i m p l i c a t i o n s . Theor. Appl. Genet. 7 7 : 2 6 0 - 2 7 0 . W u l f f , R. 1 9 7 2 . Ecology  Intrapopulational  usitatissimum)  v a r i a t i o n in t h e g e r m i n a t i o n o f s e e d s in Hyptis  and possible  suaveolens.  54:646-649.  W u l f f , R . D . 1 9 8 6 . S e e d s i z e v a r i a t i o n in Desmodium s i z e . J. Ecol. 7 4 : 8 7 - 9 7 .  paniculatum.  I. F a c t o r s a f f e c t i n g s e e d  Chapter 8 E f f e c t s of S e e d S i z e o n S e e d l i n g A t t r i b u t e s  8.1  Introduction The efficient  management.  u s e o f s e e d s in s e e d l i n g p r o d u c t i o n  S e e d s i z i n g is o n e m e t h o d  of a t t a i n i n g  s e e d l i n g s p e r u n i t w e i g h t o f s e e d s ( B e l c h e r et al.,  is a m a j o r o b j e c t i v e f o r  s o w i n g uniformity,  producing  more  1984) and reducing seedling competition.  S e e d s i z e is a m a j o r trait in a g r i c u l t u r a l - c r o p i m p r o v e m e n t  ( H a r p e r et al.,  u n d e r s t r o n g g e n e t i c i n f l u e n c e (Fehr a n d W e b e r , 1 9 6 8 ; V o i g t et al., and Wulff,  nursery  1 9 7 0 ) , s i n c e it is  1 9 6 6 ; Perry, 1 9 7 6 ; Roach  1 9 8 7 ; T y s o n , 1 9 8 9 ) . T h e reported effects of s e e d size o n seedling g r o w t h  in  c o n i f e r s h a v e v a r i e d . A p o s i t i v e c o r r e l a t i o n o f s e e d s i z e w i t h s e e d l i n g s i z e w a s o b s e r v e d in s e v e r a l p i n e s ( S p u r r , 1 9 4 4 ; R i g h t e r , 1 9 4 5 ) a n d w h i t e s p r u c e (Picea S e e d s of m e d i u m size f r o m  s l a s h p i n e (Pinus  elliottii)  glauca)  (Burgar,  1964).  (Shoulders, 1961)  produced  bigger  s e e d l i n g s t h a n d i d e i t h e r s m a l l or large s e e d s . S u c h a r e l a t i o n s h i p l a s t e d f r o m 5 m o n t h s in caribaea  ( T o o n et al.,  1 9 9 1 ) , t o 5 y e a r s in p o n d e r o s a p i n e (Pinus  ponderosa)  Pinus  (Ackerman and  G o r m a n , 1 9 6 9 ) . In c o n t r a s t , n o c o r r e l a t i o n h a s b e e n f o u n d in m a n y o t h e r s t u d i e s o n c o n i f e r s , e v e n at e a r l y s e e d l i n g 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 i n c l a 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 a n d W e n n y , 1 9 8 7 ) . T h e d i s t r i b u t i o n o f s e e d w e i g h t in a s e e d l o t h a s been o b s e r v e d to vary a m o n g s e e d trees a n d y e a r s of s e e d c o l l e c t i o n (Helium, 1 9 7 6 ; Silen a n d Osterhaus, 1979). In t h i s s t u d y , t h e e f f e c t of s e e d s i z e o n t h e a t t r i b u t e s  of 8 - m o n t h - o l d  Sitka spruce  s e e d l i n g s is r e p o r t e d . 8 . 2 Materials  and  Methods  C a n a d i a n Pacific Forest P r o d u c t s Ltd. provided the s e e d s for this s t u d y f r o m the Sitka s p r u c e s e e d o r c h a r d l o c a t e d in S a a n i c h t o n , B r i t i s h C o l u m b i a ( l a t i t u d e 4 8 ° 3 5 ' N ,  longitude  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 ( a v e r a g i n g 9 . 3 r a m e t s p e r c l o n e s ) s e l e c t e d from elevations 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 Island, 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 i n g l e - t r e e m i x o v e r  three unequal blocks. In S e p t e m b e r 1 9 9 0 , w i n d - p o l l i n a t e d 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 l o n a l identities of the s e e d s w e r e maintained during 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 size c l a s s e s , large (>  1.41  mm) and small (<  1.41  mm), using 14-mesh  s c r e e n . S o r t e d s e e d s w e r e k e p t at 2 ° C u n t i l u s e d . S e e d s f r o m b o t h s i z e c l a s s e s w e r e s o a k e d in w a t e r f o r 2 4 h r s . , a n d t h e n  stratified  (prechilled) f o r 21 d a y s p r i o r t o 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 n u r s e r y p r a c t i c e s , t h e stratified  s e e d s w e r e s o w n into 3 1 3 B s t y r o b l o c k s (65 c m ^  1 6 0 c a v i t i e s p e r b l o c k ) 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 u s i n g r o w - w i t h i n - s t y r o b l o c k s a s r e p l i c a t i o n s (8 s e e d l i n g s p e r r e p l i c a t i o n ) . S e e d l i n g s in t h e o u t e r r o w s o f e a c h s t y r o b l o c k w e r e u s e d a s t h e T h e seedlings w e r e g r o w n a c c o r d i n g to the operational g r o w i n g  buffer.  regime for Sitka  s p r u c e . O n e m o n t h after s o w i n g , a c o m b i n a t i o n o f f e r t i l i z e r s a n d w a t e r w a s a p p l i e d o n a w e e k l y b a s i s until the s e e d l i n g s 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 e v e r y w e e k s o t h a t a n y e f f e c t o f s y s t e m a t i c e r r o r d u e t o p o s i t i o n o n t h e n u r s e r y b e n c h w o u l d be reduced. E i g h t m o n t h s after  s o w i n g , seedlings were harvested. The growing  medium  was  w a s h e d f r o m t h e r o o t s , a n d t h e s e e d l i n g w a s c u t at t h e r o o t c o l l a r . D i a m e t e r at r o o t c o l l a r a n d s h o o t l e n g t h w e r e m e a s u r e d . R o o t s a n d s h o o t s w e r e d r i e d f o r 2 4 h r s . at 9 0 ° C a n d d r y w e i g h t per seedling w a s determined. S u b s e q u e n t l y , shoot-root ratios w e r e c a l c u l a t e d . T h e data set w a s s u b j e c t e d to a n a l y s i s of variance ( A N O V A ) f o l l o w i n g the additive linear m o d e l : Y,ii,„ = / / + C, + Sj + CS,  + R/S,„ + CR/Si„„ + e„„,  w h e r e fj = o v e r a l l m e a n s C, = c l o n e e f f e c t ( r a n d o m e f f e c t ) , i =  1-18,  Sj = e f f e c t of s e e d s i z e (fixed e f f e c t ) , j =  1-2,  CS|| = effect of interaction b e t w e e n s e e d size a n d c l o n e .  R/S,j,^ = e f f e c t of r e p l i c a t i o n w i t h i n s e e d s i z e , k =  1-6,  CR/Siijik = t h e e f f e c t o f i n t e r a c t i o n b e t w e e n c l o n e a n d r e p l i c a t i o n w i t h i n s e e d size, and  = residual term, 1 =  The S t u d e n t - N e w m a n - K e u l s range test  1-8. w a s used to compare the  m e a n s of each  growth  parameter. 8 . 3 Resu/ts  and  Discussion  N o s i g n i f i c a n t e f f e c t of s e e d s i z e o n s e e d l i n g g r o w t h w a s o b s e r v e d (Table c l o n e v a r i a t i o n f o r all p a r a m e t e r s w a s h i g h l y s i g n i f i c a n t (P<  8.1).  Among-  0 , 0 1 ) , and a c c o u n t e d for 5 . 1 3 %  o f t h e t o t a l s u m of s q u a r e s f o r d r y w e i g h t a n d 1 2 . 2 8 % f o r h e i g h t (Table  8.1).  This variation  is v e r y s m a l l in c o m p a r i s o n t o t h e a m o u n t o f v a r i a t i o n w i t h i n r e p l i c a t i o n s ( f r o m 5 8 . 7 5 %  to  7 8 . 5 5 % for height and s h o o t dry weight). 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 different replications as well as genetic variation w i t h open-pollination families c o u l d be c o n s i d e r e d as c a u s e s o f v a r i a t i o n in s e e d l i n g a t t r i b u t e s . 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 t o b e r e l a t e d t o e a c h o t h e r (r^=  0.98). Clone no. 411 produced the highest mean height, s h o o t dry weight,  a n d t o t a l d r y w e i g h t (Table  8.2).  C l o n e n o s . 1 5 4 a n d 4 1 6 p r o v i d e d t h e h i g h e s t m e a n s in r o o t  dry w e i g h t a n d s h o o t - r o o t ratio, r e s p e c t i v e l y . C l o n a l differences a m o n g the m e a n s of t h e s e a t t r i b u t e s are w i t h i n t h e s t o c k s p e c i f i c a t i o n s f o r 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 s e e d l i n g s (1991  British C o l u m b i a M i n i s t r y of F o r e s t s Culling S t a n d a r d s , Silviculture B r a n c h , V i c t o r i a ,  B . C . ) , therefore, indicating no operational significance to the n u r s e r y m a n . T h e l a c k of a n y s i g n i f i c a n t e f f e c t o f s e e d s i z e o n s e e d l i n g g r o w t h in S i t k a s p r u c e 8.1)  (Table  agrees w i t h the findings for other conifers (Langdon, 1 9 5 8 ; L a v e n d e r , 1 9 5 8 ; Sinclair,  1973;  M a n n , 1 9 7 9 ; Sluder, 1 9 7 9 ; Dumroese and W e n n y ,  1987). The positive  correlation  b e t w e e n s e e d s i z e a n d e a r l y s t a g e s o f s e e d l i n g 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 ( S h o u l d e r s , 1 9 6 1 ; S i n c l a i r , 1 9 7 3 ; T o o n et al., species.  F o r e x a m p l e , in Pinus  1991) declined w i t h age, w i t h s o m e variation according to caribaea  Morelet  v a r hondurensis  Barrett  a n d G o l f e r i , the  Table 8.1.  V a r i a t i o n in d i a m e t e r (Dia), h e i g h t (Ht), s h o o t d r y w e i g h t ( S D W ) , r o o t d r y w e i g h t ( R D W ) , t o t a l d r y w e i g h t ( T D W ) , a n d s h o o t / r o o t d r y - w e i g h t ratio ( S / R ) o f S i t k a s p r u c e s e e d l i n g s f r o m t w o s e e d s i z e s o f 1 8 c l o n e s  S o u r c e of Variation  df  Expected^ Mean Squares  S u m S q u a r e (%) Dia.  Ht.  6.60"  12.28"*  SDW  RDW  TDW  S/R  5.19"  7.48**  5.13""  10.76*"  17  ol  +  +  1  ol  +  + 1 4 4 a f , , + 48CT?, + 8 6 4 0 3  0.00  0.04™  0.14™  0.02™  0.11™  0.09™  C X S  17  ol  +  +  1.74™  2.66™  1.58™  1.91™  1.32™  3.56"  R e p / S i z e (R/S)  10  ol  -1- 8 a ^ . s  + 144af,3  1.59*  2.88"  1.69"  0.59™  1.30™  1.34™  C X R/S  170  ol  + 8of.3  13.72**  23.39**  12.85"  20.59""  13.74"  18.99""  Residual  1426  ol  76.35  58.75  78.55  69.05  78.40  65.26  C l o n e s (C) S e e d s i z e (S)  2Qol  48af,  not significant, s i g n i f i c a n t at P < 0 . 0 5 . s i g n i f i c a n t at P < 0 . 0 1 . C = 1 8 , S = 2 , R = 6. al = v a r i a n c e a m o n g c l o n e s ; 0 , = v a r i a n c e b e t w e e n s e e d s i z e s ; o f , = v a r i a n c e d u e t o i n t e r a c t i o n b e t w e e n c l o n e s a n d s e e d s i z e s ; of/5 = v a r i a n c e a m o n g r e p l i c a t i o n w i t h i n s e e d s i z e s ; of,/, = v a r i a n c e d u e t o i n t e r a c t i o n b e t w e e n c l o n e s a n d r e p l i c a t i o n w i t h i n seed sizes; of = residual term.  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  Clone M e a n s (mm) 405 421 514 411 92 521 5 61 105 154 15 44 416 20 24 516 113 68 '  3.04a^ 2.94 b 2.91 be 2 . 9 0 bo 2 . 8 8 bod 2.87 bed 2 . 8 6 bcde 2 . 8 4 bcdef 2 . 8 3 bcdef 2 . 8 3 bcdef 2 . 8 0 bcdef 2 . 7 9 edef 2 . 7 8 edef 2.76 def 2 . 7 5 def 2 . 7 5 def 2.73 ef 2.70 f  Clone 411 514 405 61 5 24 15 421 521 20 92 516 44 68 113 416 154 105  Means (cm) 31.37a 29.30 b 29.14 b 2 8 . 6 5 be 2 8 . 4 8 bed 2 8 . 2 3 bcde 2 7 . 9 7 ede 2 7 . 8 7 edef 2 7 . 8 7 edef 2 7 . 8 4 edef 2 7 . 5 9 edefg 2 7 . 5 0 cdefg 2 7 . 2 8 defg 27.02 efg 27.01 efg 26.67 fg 26.57 g 26.48 g  Shoot Dry Weight  Root Dry weight  Total Dry Weight  Shoot-Root Ratio  Clone  Means (g.)  Clone  Means (g.)  Clone  Means (g.)  Clone  411 514 405 5 421 154 516 44 92 521 68 105 24 15 416 20 61 113  1 .82a 1 .78ab 1 .77abc 1 .74abc 1 .72abcd 1 .69abcde 1 .67 bcdef 1 .67 bcdef 1 .67 bcdef 1 .65 bcdef 1 .65 bcdef 1 .64 bcdef 1 .63 bcdef 1 .62 edef 1 .61 edef 1 .58 def 1 .54 ef 1.53 f  154 44 516 20 5 405 521 92 105 514 411 421 24 68 61 15 113 416  0.62a 0.60ab 0.60ab 0.60ab 0.59abc 0.58abe 0.57abed 0.57abed 0.57abed 0.57abed 0 . 5 6 bcde 0 . 5 4 ede 0 . 5 4 ede 0 . 5 3 de 0 . 5 2 de 0.51 ef 0.51 ef 0.47 f  411 405 514 5 154 516 44 421 92 521 105 68 20 24 15 416 61 113  2.38a 2.36a 2.35a 2.34ab 2.31ab 2.27abc 2.27abe 2.26abed 2.24abed 2.22abede 2.21 abode 2.18abede 2.18abcde 2,17abede 2 . 1 3 bcde 2 . 0 9 ede 2.07 de 2.04 e  416 411 15 421 68 514 405 113 24 61 92 521 5 105 516 44 154 20  Means 3.56a 3.35 b 3.31 be 3 . 3 0 be 3.26 be 3 . 1 9 bed 3.11 ede 3.09 edef 3 . 0 8 edef 3 . 0 0 defg 2.98 defg 2.96 defgh 2.95 defgh 2.93 defgh 2.87 efgh 2.84 fgh 2.80 gh 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 m m ; target = 2.6 m m ; Height: min = 14 e m , target = 18 c m ; Root dry weight: min = 0 . 5 g, target = 0.7 g). ^ Clones sharing a c o m m o n letter are not significantly different at P < 0 . 0 5 .  p o s i t i v e , s t r o n g , c o r r e l a t i o n o b s e r v e d in 2 - m o n t h - o l d ( T o o n et al.,  seedlings disappeared after 7  months  1 9 9 1 ) . Similarly, this effect also d i s a p p e a r e d after 3 m o n t h s (Sinclair, 1 9 7 3 ) or  o n e y e a r ( L a v e n d e r , 1 9 5 8 ) in D o u g l a s - f i r , 3 y e a r s in s l a s h p i n e ( S h o u l d e r s , 1 9 6 1 ), a n d 5 y e a r s in p o n d e r o s a p i n e ( A c k e r m a n a n d G o r m a n , 1 9 6 9 ) . R i g h t e r ( 1 9 4 5 ) f u r t h e r i n d i c a t e d t h a t t h e r e w a s no relationship b e t w e e n seed weight and inherent seedling vigour. It is i n t e r e s t i n g t o n o t e t h a t 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 r e l a t e d t o S i t k a s p r u c e , a positive relationship b e t w e e n seed size and seedling size w a s o b s e r v e d by Burgar  (1964).  T h e l a c k of a n y e f f e c t of s e e d s i z e o n s e e d l i n g s i z e at 8 m o n t h s in S i t k a s p r u c e in t h i s s t u d y indicates that extrapolating b e t w e e n e v e n related 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 effect of c l o n e on seedling attributes c a n be greater than the effect of s e e d size.  8.4  References  A c k e r m a n , R . F . a n d J . R . G o r m a n . 1 9 6 9 . Effect of s e e d w e i g h t on the s i z e of lodgepole pine a n d w h i t e s p r u c e c o n t a i n e r - p l a n t i n g s t o c k . Pulp Pap. Mag. Can. 7 0 ( c ) : 1 6 7 - 1 6 9 . Belcher, E . W . , G . N . L e a c h , and H . H . G r e s h a m . 1 9 8 4 . Sizing slash pine s e e d s as a nursery p r o c e d u r e . Tree Planters' Notes 3 5 : 5 - 1 0 . B u r g a r , R . J . 1 9 6 4 . T h e e f f e c t of s e e d s i z e o n g e r m i n a t i o n , s u r v i v a l a n d initial g r o w t h in w h i t e s p r u c e . For. Chron. 41:93-97. D u m r o e s e , R . K . a n d 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 c o n t a i n e r i z e d n u r s e r y . West. J. Appl. For. 2 : 1 2 8 - 1 3 0 .  w h i t e pine in a  F e h r , W . R . , a n d C R . W e b e r . 1 9 6 8 . M a s s s e l e c t i o n b y s e e d s i z e a n d s p e c i f i c g r a v 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 . H a r p e r , J . L . , P . H . L o v e l l , a n d K . G . M o o r e . 1 9 7 0 . T h e s h a p e s a n d s i z e s o f s e e d s . Ann. Ecol. Syst. 1 1 : 3 2 7 - 3 5 6 . Helium,  A.K. 1976. 27:16,17,23.  Grading  seed  by  weight  in  white  spruce.  Tree  Planters'  Rev.  Notes  L a n g d o n , O . G . 1 9 5 8 . C o n e a n d s e e d s i z e of s o u t h F l o r i d a s l a s h pine a n d t h e i r e f f e c t s s e e d l i n g s i z e a n d s u r v i v a l . J. For. 5 6 : 1 2 2 - 1 2 7 .  on  L a v e n d e r , D . P . 1 9 5 8 . E f f e c t of s e e d s i z e o n D o u g l a s - f i r s e e d l i n g s . O r e g o n F o r e s t L a n d s R e s e a r c h C e n t e r , Research Note N o . 3 2 : 1 - 9 . M a n n , W . F . , J r . 1 9 7 9 . R e l a t i o n s h i p s o f s e e d s i z e , n u m b e r o f c o t y l e d o n s , a n d initial g r o w t h o f s o u t h e r n p i n e s . Tree Planters' Notes 30:22-26. P e r r y , T . O . 1 9 7 6 . M a t e r n a l e f f e c t s o n t h e e a r l y p e r f o r m a n c e of t r e e p r o g e n i e s . In Tree physiology and yield improvement, ed. M . G . R . Cannell and F.T. Last, pp. 4 7 3 - 4 8 1 . N e w York: A c a d e m i c Press. R i g h t e r , F.I. 1 9 4 5 . P i n u s : T h e r e l a t i o n s h i p of s e e d s i z e a n d s e e d l i n g s i z e t o i n h e r e n t v i g o u r . J. For. 4 3 : 1 3 1 - 1 3 7 . R o a c h , D . A . a n d R . D . W u l f f . 1 9 8 7 . M a t e r n a l e f f e c t s in p l a n t s . Ann. 235.  Rev.  Ecol.  Syst.  18:209-  S h o u l d e r s , E. 1 9 6 1 . E f f e c t of s e e d s i z e o n g e r m i n a t i o n , g r o w t h , a n d s u r v i v a l o f s l a s h p i n e . J. For. 5 9 : 3 6 3 - 3 6 5 . S i l e n , R., a n d C . O s t e r h a u s . 1 9 7 9 . R e d u c t i o n of g e n e t i c b a s e b y s i z i n g o f b u l k e d s e e d l o t s . Tree Planters' Notes 30:24-30. Sinclair, W . A .  1973.  19:105-108.  Development  of w e i g h t  variation  Douglas-fir  in D o u g l a s - f i r s e e d l i n g s . For.  Sci.  S l u d e r , E . R . 1 9 7 9 . T h e e f f e c t s of s e e d a n d s e e d l i n g s i z e o n s u r v i v a l a n d g r o w t h of l o b l o l l y p i n e . Tree Planters' Notes 3 0 : 2 5 - 2 8 . S p u r r , S . H . 1 9 4 4 . Effect of s e e d w e i g h t a n d s e e d origin on the early d e v e l o p m e n t of eastern w h i t e p i n e . J. Am. Arbor. 2 5 : 4 6 7 - 4 8 1 . Toon,  P . G . , R . J . Haines and M . J . Dieters. 1 9 9 1 . Relationship b e t w e e n seed weight, g e r m i n a t i o n t i m e a n d s e e d l i n g h e i g h t g r o w t h in Pinus caribaea M o r e l e t v a r . hondurensis B a r r e t t a n d G o l f a r i . Seed Sci. & Technol. 19:397-402.  T y s o n , H . 1 9 8 9 . G e n e t i c c o n t r o l o f s e e d w e i g h t in f l a x (Linum i m p l i c a t i o n s . Theor. Appl. Genet. II.IQO-TJO.  usitatissimum)  and possible  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 . I n h e r i t a n c e o f s e e d s i z e in S o r g h u m , vulgare P e r s . Crop Sci. 6 : 5 8 2 - 5 8 6 .  Sorghum  Chapter 9 Conclusions  S e v e r a l biological a s p e c t s of S i t k a 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  investigated  in a s e e d o r c h a r d l o c a t e d o n t h e S a a n i c h P e n i n s u l a o f V a n c o u v e r I s l a n d , B . C . It w a s f o u n d t h a t : 1 ) Of 1 6 e n z y m e loci that c o u l d be s c o r e d reliably, t w o variation while  1 4 a p p e a r e d t o e a c h p o s s e s s at l e a s t t w o  exhibited  no  detectable  a l l o z y m e v a r i a n t s . T h e m o d e of  inheritance for m o s t of the p o l y m o r p h i c loci 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 ,  although  11 o u t o f 2 4 allelic c o m b i n a t i o n s d i s p l a y e d m a r k e d s e g r e g a t i o n d i s t o r t i o n ; 2) N o l i n k a g e w a s o b s e r v e d f o 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 l a c k o f i n d e p e n d e n t s e g r e g a t i o n b e t w e e n o n e s e t of l o c i {PGM-1  •.PGM-2)  w a s attributed to their  apparent linkage; 3) G e n e d i v e r s 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 i g h in b o t h t h e studied seed orchard and 1 0 natural populations previously studied by Y e h and El-Kassaby ( 1 9 8 0 ) . T h e o r c h a r d population s h o w e d significantly higher n u m b e r of alleles per l o c u s a n d p e r c e n t a g e of p o l y m o r p h i c loci. S a m p l i n g breadth of the s e e d o r c h a r d ' s parent trees has been identified as the major c a u s e for the o b s e r v e d i n c r e a s e d level of g e n e t i c 4) S i g n i f i c a n t  variability;  levels of inbreeding, b a s e d o n single- a n d m u l t i - l o c u s  estimates  of  o u t c r o s s i n g ( R i t l a n d a n d E l - K a s s a b y , 1 9 8 5 ) e x i s t e d in t h e o r c h a r d ' s s e e d c r o p . T h i s s i g n i f i c a n t i n b r e e d i n g l e v e l w a s g r e a t l y a f f e c t e d b y t h e p r e s e n c e of a s i g n i f i c a n t l e v e l o f i n b r e e d i n g in t h e 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 o f PGM-2  indicated the p r e s e n c e of n o n - r a n d o m mating  c a u s e d b y g e n o t y p i c s e l e c t i o n f o r f e r t i l i z a t i o n (i.e., h o m o z y g o t i c f e r t i l i z a t i o n ) s i m i l a r t o t h a t o b s e r v e d o n c o r n ( B i j i s m a et a/., 5)  Parental imbalance  1986); was  observed for  the  two  crop  years  studied.  Maternal  c o n t r i b u t i o n b a s e d o n s e e d - c o n e yield w a s similar to that b a s e d o n filled s e e d yield, h o w e v e r , m o s t c l o n e s did not maintain their 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 yield w a s made. Effective female population number ( C r o w and K i m u r a , 1970) provided an estimate  o f t h e d e v i a t i o n f r o m t h e i d e a l e q u a l c o n t r i b u t i o n , h o w e v e r , it d i d n o t p r o v i d e i n s i g h t i n t o t h e relationship between reproductive energy and reproductive s u c c e s s ; 6) S e e d s i z e a n d g e r m i n a t i o n p a r a m e t e r s ( G C , P V a n d G V ) w e r e u n d e r g e n e t i c c o n t r o l . B r o a d - s e n s e heritability w a s moderate (0.36) for s e e d size and high ( 0 . 7 4 - 0 . 7 8 ) for germination traits.  Seed  pretreatment  (i.e.,  stratification)  was  s u c c e s s f u l in  achieving  germination  uniformity; 7) S e e d v i g o u r t e s t (i.e., a c c e l e r a t e d a g i n g ) p r o d u c e d s i g n i f i c a n t c l o n a l d i f f e r e n c e s in g e r m i n a t i o n p a r a m e t e r s i n d i c a t i n g t h a t t h e d e g r e e o f s e e d d e t e r i o r a t i o n is c l o n e - s p e c i f i c . T h e s h o r t t r e a t m e n t t e s t (i.e., 3 - 6 d a y s ) p r o d u c e d a n i n c r e a s e in g e r m i n a t i o n 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 decline. T h e i m p r o v e m e n t of g e r m i n a t i o n traits after short treatment time needs to be investigated for operational exploitation; 8) U n l i k e o t h e r s p r u c e s p e c i e s , s e e d s i z e s h o w e d n o s i g n i f i c a n t e f f e c t o n s e e d l i n g a t t r i b u t e s in S i t k a s p r u c e . C l o n a l d i f f e r e n c e s w e r e s i g n i f i c a n t , b u t t h e s e d i f f e r e n c e s w e r e w i t h i n the operational specifications for the species.  

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