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Isozyme patterns of a selected Pseudotsuga menziesii (mirb.) franco population El-Kassaby, Yousry Aly 1980

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c.l ISOZYME PATTERNS OF A SELECTED PSEUDOTSUGA MENZIESII (MIRB.) FRANCO POPULATION by YOUSRY ALY , EL-^KASSABY B.Sc, Genetics (Hons), Alexandria U n i v e r s i t y , 1970 M.Sc, Genetics, Tanta Un i v e r s i t y , 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of FORESTRY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July, 1980 (c) Yousry A. .El-Kassaby In presenting t h i s thesis i n p a r t i a l f u l f i l l m e n t of the require-ments for an advanced degree at the University of B r i t i s h Columbia, I agree that the Li b r a r y s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s thesis for s c h o l a r l y purposes may be granted to the head of my Faculty or h i s representative. I t i s understood that copying or p u b l i c a t i o n of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Faculty of Forestry The U n i v e r s i t y of B r i t i s h Columbia VANCOUVER, B.C. CANADA V6T 1W5 ABSTRACT Open p o l l i n a t e d seed samples were c o l l e c t e d from 42 Douglas-fir trees i n September 1978. The i n d i v i d u a l i t y of cone l o t s and subsequent seed l o t s had been retained i n these trees. The trees are located i n the University of B r i t i s h Columbia Research Forest, Haney, B.C. and were studied previously f o r phenology, growth, and flower and cone production by G r i f f i t h (1968). Isozyme v a r i a t i o n s were studied (using the g e l electrophoresis technique) f or both the haploid megagametophyte and the d i p l o i d embryo tissues at 27 l o c i , coding f o r 18 d i f f e r e n t enzymes for each tree separately. The objectives of t h i s study were: 1. to study the mode of inheritance f o r these l o c i ; 2. to determine the linkage r e l a t i o n s h i p s among these l o c i ; 3. to estimate the outcrossing rate (t) f o r t h i s population; 4. to determine the most e f f e c t i v e sample s i z e f o r estimating a l l e l i c frequencies; 5. to study the amount and organization of isozyme v a r i a t i o n i n t h i s population and compare the r e s u l t s with the v a r i a t i o n f o r some quan t i t a t i v e t r a i t s , and 6. to study the a s s o c i a t i o n between isozyme genotypes and quantitative t r a i t s . The r e s u l t s from the inheritance analyses f o r the 14 heterozygous enzyme systems showed that these electrophoretic variants segregated i n a co-dominant fashion with d i s t i n c t simple Mendelian expression. The i i i linkage study yielded two t i g h t l y linked pairs [(AAT-2:PGI-2) and(AAT-3: SOD)] with recombination frequencies of 1.5 and 22.4 percent r e s p e c t i v e l y . In addition, seven loosely l i n k e d p a i r s were detected with recombination frequencies varying between 32.7 and 41.9 percent. I t was not possible to study three-point linkage due to the lack of appropriate combinations. Conditional p r o b a b i l i t i e s were used to estimate the outcrossing rate (t) i n the population using four enzyme systems. The estimated outcrossing rate was 0.9 with a standard deviation of 0.11, giving an inbreeding rate of 10 percent. By the minimum sampling variance c r i t e r i o n i t was e s t i -mated that sample sizes between 42 and 60 trees from the base population are optimal to obtain r e l i a b l e estimates for the a l l e l i c frequencies. S t r i k i n g agreement on the appointment of genetic v a r i a t i o n was found between r e s u l t s obtained from the gene d i v e r s i t y analysis for the electrophoretic data and the analysis of variance for seven d i f f e r e n t q uantitative t r a i t s . These two independent sets of information confirmed that the majority of the v a r i a t i o n existed within and not between populations. F i n a l l y , the a s s o c i a t i o n between the mother trees' genotypes and the performance of t h e i r h a l f - s i b f a m i l i e s showed that the mother trees appeared to exert minimal influence on those c h a r a c t e r i s t i c s analysed. The p o l l e n parent contribution to the genetic c o n s t i t u t i o n of the progenies should be investigated and i t i s recommended that isozyme studies should be extended to f u l l - s i b progenies. i v TABLE OF CONTENTS Page ABSTRACT . ....... i i TABLE OF CONTENTS i v LIST OF TABLES v i LIST OF FIGURES v i i i LIST OF PLATES i x ACKNOWLEDGEMENTS x 1. INTRODUCTION 1 2. MATERIALS AND METHODS 6 2.1 Seed source 6 2.2 Biochemical methods 6 2.3 Progeny test 14 3. RESULTS AND DISCUSSION 16 3.1 Inheritance 16 3.1.1 Monomorphic enzymes 19 3.1.2 Polymorphic enzymes 19 3.1.2.1 Acid phosphatase (APH) 19 3.1.2.2 Aconitase (ACO) 21 3.1.2.3 Aspartate aminotransferase (AAT) ..... 22 3.1.2.4 Diaphorase (DIA) 23 3.1.2.5 Esterase (EST) 24 3.1.2.6 Glucose-6-phosphate dehydrogenase (G6P) 25 3.1.2.7 Hexoseaminidase (HA) 26 3.1.2.8 I s o c i t r a t e dehydrogenase (IDH) 27 V Page 3.1.2.9 Malate dehydrogenase (MDH) 28 3.1.2.10 Malic enzyme (ME) 30 3.1.2.11 Phosphoglucose isomerase (PGI) 31 3.1.2.12 Phosphoglucomutase (PGM) 32 3.1.2.13 6-Phosphogluconic dehydrogenase (6PG) 33 3.1.2.14 Superoxide dismutase (SOD) 34 3.1.3 Summary and conclusion 35 3.2 Linkage 48 3.3 Estimation of the outcrossing rate (t) 59 3.4 E f f e c t of sample s i z e on the p r e c i s i o n of the estimate of a l l e l e frequency 64 3.5 Gene d i v e r s i t y within the " G r i f f i t h " population 78 3.6 Association between isozyme genotypes and quanti-t a t i v e characters 92 4. SUMMARY AND CONCLUSIONS 102 5. LITERATURE CITED 104 APPENDICES 1. Chi-square r e s u l t s f o r linkage analyses 112 2. A l l e l e frequencies, t h e i r standard error estimates, and t-values f o r d i f f e r e n t sample sizes f o r PGI-2, MDH-3, MDH-4 l o c i f o r the three random runs (A, B, and C) . 124 3. A l l e l i c v a r i a t i o n of 27 l o c i i n the four e l e v a t i o n a l classes 134 v i LIST OF TABLES Table - Page 1 The gel and electrode buffers and t h e i r e l e c t r i c power 12 conditions'. 2. The eighteen enzyme stained f o r , abbreviations, buffer 13 systems, and s t a i n recipes. 3 Genotypes of heterozygous l o c i f o r 27 trees included 17 i n the inheritance and linkage studies. 4 Subunit structures of homomeric and heteromeric 18 isozymes i n heterozygotes, where one a l l e l e deter-mines polypeptide subunit a i and the other a l l e l e polypeptide subunit a 2 . 5 Segregation of a l l e l i c variants at the APH-2 locus. 20 6 Segregation of a l l e l i c variants at the ACO locus. 21 7 Segregation of a l l e l i c variants at the heterozygous 23 AAT l o c i . 8 Segregation of a l l e l i c variants at the heterozygous 24 DIA l o c i . 9 Segregation of a l l e l i c variants at the EST-1 locus. 25 10 Segregation of a l l e l i c variants at the G6P locus. 26 11 Segregation of a l l e l i c variants at the HA locus. 27 12 Segregation of a l l e l i c variants at the IDH locus. 28 13 Segregation of a l l e l i c variants at the heterozygous 29 MDH l o c i . 14 Segregation of a l l e l i c variants at the ME locus. 31 15 Segregation of a l l e l i c v ariants at the PGI-2 locus. 31 16 Segregation of a l l e l i c variants at the PGM locus. 33 17 Segregation of a l l e l i c variants at the heterozygous 34 6PG l o c i . 18 Segregation of a l l e l i c variants at the SOD l o c i . 35 V l l Table Page 19 Test of linkage between polymorphic l o c i . 52 20 Test of linkage between polymorphic l o c i from 53 combined data. 21 Test of linkage between polymorphic l o c i from 54 combined data. 22 Estimated outcrossing rate. 63 23 Review of d i f f e r e n t sample sizes used for 66 d i f f e r e n t species. 24 Analysis of variance for the analysis of between 84 and within elevation v a r i a t i o n . 25 Analysis of gene d i v e r s i t y and degree of d i f f e r e n - 87 t i a t i o n at 27 l o c i between d i f f e r e n t elevations. 26 Estimates of the variance components and t h e i r 90 l e v e l of s i g n i f i c a n c e for the d i f f e r e n t seven characters f o r both analyses (balanced and unbalanced). 27 A summary of the 63 d i f f e r e n t analyses made to 96 compare d i f f e r e n t genotypes within each isozyme system, and also between d i f f e r e n t l e v e l s of heterozygosity. v i i i LIST OF FIGURES Figure • Page 1 Map showing the l o c a t i o n of Douglas-fir trees 8 used i n t h i s study. 2, Assembly for h o r i z o n t a l starch gel electrophoresis. 10 3 Diagram of c h a r a c t e r i s t i c isozyme patterns expected 18 i n heterozygotes i n the case of enzymes which are monomers, dimers, trimers, and tetramers. 4 I l l u s t r a t i o n of the r e l a t i v e migration of allozymes 37 for the following l o c i : APH-2, ACO, AAT-2, AAT-3, DIA-1, DIA-2, EST-1, G6P, HA, and IDH. 5 I l l u s t r a t i o n of the r e l a t i v e migration of allozymes 39 for the following l o c i : MDH-1, MDH-3, MDH-4, ME, PGI-2, PGM, 6PG-1, 6PG-2, and SOD. 6 Pair-wise combinations between the f i v e l o c i included 56 i n the f i r s t n e t - l i k e group. 7 Pair-wise combinations between the four l o c i included 57 i n the second n e t - l i k e group. 8 Standard error estimates of a l l e l i c frequencies for 71 PGI-2 locus, for the d i f f e r e n t sample s i z e s . 9 Standard error estimates of a l l e l i c frequencies f o r 73 MDH-3 locus, for the d i f f e r e n t sample s i z e s . 10 Standard error estimates of a l l e l i c frequencies f o r 77 MDH-4 locus, for the d i f f e r e n t sample s i z e s . 11 A summary of the f i v e Duncan's multiple range t e s t s . 98 i x LIST-OF.PLATES Plate Page Photographs of gels stained f o r the following 41 enzyme systems: APH, ACO, and AAT. Photographs of gels stained f o r the following 43 enzyme systems: DIA, EST, and G6P. Photographs of gels stained f o r the following 45 enzyme systems: IDH and PGM, MDH, and ME. Photographs of gels stained f o r the following 47 enzyme systems: PGI, PGM, and 6PG. X ACKNOWLEDGEMENTS Without the e f f o r t , encouragement, and guidance of several people, t h i s thesis would not have been possible. I wish to extend my sincere thanks to my supervisor, Dr. 0. S z i k l a i , Faculty of Forestry, for sharing h i s time, wisdom, and experience i n his r o l e as an academic advisor. The moral support he gave me throughout the study, his guidance, understanding, patience and the a d d i t i o n a l e f f o r t he made to make my studies a warm and rewarding experience are h e a r t i l y acknowledged. I am indebted to Dr. F.C. Yeh, B r i t i s h Columbia Forest Service, Research D i v i s i o n , for providing lab f a c i l i t i e s , chemicals, photo lab assistance, and the valuable advice i n the course of data c o l l e c t i o n and a n a l y s i s . In addition, acknowledgement i s extended to Dr. A. Kozak, Faculty of Forestry, for recommending the s t a t i s t i c a l analyses used herein. Special thanks are extended to my Committee Members: Dr. K. Cole (Botany), Dr. A. Kozak (Forestry), Dr. G. Weetman (Forestry), and Dr. F.C. Yeh ( B r i t i s h Columbia Forest Service) for t h e i r invaluable suggestions and advice. Thanks are due to Ms. D. Chipman, Mrs. A. Fashler, Ms. H. Mouktar, and Mr. C. Layton for t h e i r lab assistance, Mr. L. S z i k l a i f o r c o l l e c t i n g the research materials, Ms. G. Ho and Mr. M. El-Sharkawi f o r t h e i r computing assistance, Mrs. A. Fashler again, Mr. G. Nelson, Mr. S. Omule, Mr. J . Parminter, and my fellow graduate students f o r x i t h e i r encouragement and support, Ms. P. Yuen for typing the manuscript. I also thank Dr. M.H. El-Lakany, Faculty of Ag r i c u l t u r e , U n i v e r s i t y of Alexandria, Egypt f o r introducing me to Dr. S z i k l a i and to t h i s f i e l d . F i n a n c i a l assistance was given i n the form of Faculty of Forestry teaching a s s i s t a n t s , U n i v e r s i t y of B r i t i s h Columbia Graduate Fellowship, the McPhee Fellowship fund, the McPhee Forestry Award, and the Youth Summer Employment Program of the Province of B r i t i s h Columbia. I am g r a t e f u l to a l l those that made these possible. F i n a l l y , I owe my deepest gratitude to my family and my wife L e i l a , who have undergone many s a c r i f i c e s to make i t a l l possible, L e i l a you are the queen of your species. THE MEMORY OF MY MOTHER 1 1. INTRODUCTION Basic research i n f o r e s t tree genetics i s an absolute necessity to a t t a i n two important objectives i n f o r e s t tree improvement (Snow and D u f f i e l d , 1940). These are the c o l l e c t i o n of information on the mode of inheritance of economically s i g n i f i c a n t c h a r a c t e r i s t i c s and the pro-duction of i n d i v i d u a l s with c e r t a i n desirable c h a r a c t e r i s t i c s . There-fore, the.study of genetic v a r i a t i o n and i t s mode of inheritance within a tree species of commercial value i s an important component i n an under-standing of the genetic structure of forest tree populations. T r a d i t i o n a l methods of assessing genetic v a r i a t i o n i n f o r e s t trees with biometrical tools have provided a c e r t a i n amount of information on quantitative c h a r a c t e r i s t i c s which are c o n t r o l l e d by a large but unknown number of genes. However, the introduction of the isozyme technique -a combination of electrophoresis and histochemical s t a i n i n g - makes i t possible to study t h i s v a r i a t i o n at the gene l e v e l as w e l l . The s i g n i f i c a n c e of the isozyme concept i n the study of natural populations was not f u l l y appreciated when i t was f i r s t introduced by Markert and Moller i n 1959. Recently, intensive use of t h i s technique began, independently and simultaneously, by several research groups. S p e c i f i c reviews re l a t e d to f o r e s t r y which concerned applications and general t h e o r e t i c a l background were reported by Lever and Burley (1974) and Feret and Bergmann (1976). A l l a r d et_ a l . (1975) pointed out the main advantages and the potentials of t h i s technique. They emphasized the following points: 1. I t makes a large number of s i n g l e gene characters a v a i l a b l e for s t u dies. 2 2. I t reveals i n d i v i d u a l a l l e l e s (allozymes) at each locus. 3. Most banding patterns are i n h e r i t e d codominantly. 4. I t i s applicable to a wide range of plant and animal species. Two opposing theories f o r explaining the maintenance of isozyme v a r i a t i o n i n natural populations emerged i n the l a t e 1960's and early 1970's. The n e u t r a l i t y theory, which was introduced by Kimura (1969), King and Jukes (1969), Kimura and Ohta (1971 and 1974), and others, stated that isozyme variants are s e l e c t i v e l y neutral or nearly n e u t r a l . This theory proposed that random genetic d r i f t i s responsible for main-ta i n i n g p r o t e i n polymorphisms i n natural populations. The s e l e c t i o n theory, on the other hand, was advocated by Dobzhansky (1970), Marshall and A l l a r d (1970), Dobzhansky and Ayala (1973), Lewontin (1973), Johnson (1973), Ayala (1974), Nevo (1978), and others. I t r e l i e d on the f a c t that isozyme - manifested l o c i as well as other l o c i are representative of the genome, and therefore to a great extent are subject to s e l e c t i o n . U n t i l 1970 no reports were a v a i l a b l e which elucidated genetic co n t r o l of isozymes i n forest tree species. Since then, however, numerous reports have been published on t h i s subject (Bartels, 1971; Conkle, 1971; Feret and S t a i r s , 1971; Lundkvist, 1975; Rudin, 1975; Simonsen and Wellendorf, 1975; Guries and Ledig, 1978; Rudin and Ekberg, 1978; Mitton ejt a l . , 1979; and O'Malley e_t al., 1979). Several studies of isozyme v a r i a t i o n between and within Douglas-fir populations were reported recently (Muhs, 1974; Copes, 1975; Yang et a l . , 1977; and Yeh and O'Malley, 1978 and 1980); yet, no work has been done on inheritance of the isozymes i n t h i s species. 3 Since these isozymes were used extensively as gene markers, and due to the increasing number of isozyme l o c i , i t became f e a s i b l e to reveal the multilocus organization between these l o c i . Studies along t h i s l i n e w i l l provide useful information about the d i s t r i b u t i o n of these markers i n the genome, help to answer questions pertaining to non-random as s o c i a t i o n (linkage) between a v a i l a b l e l o c i , and reveal mechanisms which may be responsible for maintaining these blocks of i n t e r a c t i n g sets of l o c i (Clegg et_ aL., 1972). A few attempts to detect linkage among polymorphic l o c i have been reported f o r a small number of tree species with equivocal r e s u l t s (Simonsen and Wellendorf, 1975; Feret and Witter, 1977; Guries and Ledig, 1978; Rudin and Ekberg, 1978; O'Malley et a l . , 1979; and Adams and J o l y , 1980). Genetic a r c h i t e c t u r e of the next generations w i l l be determined, to a large extent, by the genetic structure of previous generations and by the type of mating system which ex i s t s i n the species i n question. Therefore, an important p r e r e q u i s i t e f o r understanding and manipulating the genetic structure of a species i s a knowledge of i t s mating system. Extensive estimations of the outcrossing and inbreeding rates have been reported i n annual plant species (Jain, 1979). However, quantitative estimation f o r outcrossing rates f o r forest tree i s rare. Isozyme techniques would, however, f a c i l i t a t e estimation of the outcrossing rate i n f o r e s t tree populations (Brown et a l . , 1975; MulTer, 1976; and Rudin et a l . , 1977). Allozyme frequencies have often been frequently used to study amount and organization of genetic v a r i a t i o n i n natural populations. The number of trees sampled from each population was v a r i a b l e (see 4 review i n Section 3.4). I n s u f f i c i e n t sample s i z e often led to inaccurate estimates of population a l l e l e frequencies. Therefore, questions about the optimal number of i n d i v i d u a l s which should be sampled from the base population to obtain r e l i a b l e a l l e l i c frequency estimates should be determined. The recent use of isozyme studies of genetic v a r i a b i l i t y i n natural populations of conifers (see review b y Feret and Bergmann, 1976 and Rudin, 1976) has permitted researchers to investigate many basic questions of evolutionary biology. Among these i s the study of the d i s t r i b u t i o n of genetic v a r i a t i o n within and between l o c a l populations (O'Malley et a l . , 1979; Yeh and Layton, 1979; and Yeh and O'Malley, 1980). These studies considered the e n t i r e range of species d i s t r i b u t i o n and were, therefore, r e s t r i c t e d to genetic d i f f e r e n t i a t i o n at the macro-geographical l e v e l (see Section 3.5). Studies on the micro-genetic l e v e l are also needed to t i e the information f o r the v a r i a t i o n on the gene l e v e l with the quantitative t r a i t s which are the end product of t h i s genetic v a r i a b i l i t y . Responding to the obvious lack of comprehensive reports concerning the isozyme v a r i a t i o n between i n d i v i d u a l trees i n a sing l e population, t h i s study was i n i t i a t e d to elucidate t h i s v a r i a t i o n and to answer some of these questions with the following objectives: 1. To study the mode of inheritance of 18 d i f f e r e n t isozyme systems. 2. To determine the linkage r e l a t i o n s h i p s among these l o c i . 3. To estimate the outcrossing rate (t) f o r t h i s population; 4. To determine the most e f f e c t i v e sample s i z e f o r estimating a l l e l i c frequencies. 5 5. To study the amount and organization of isozyme v a r i a t i o n i n t h i s population, and compare the r e s u l t s with v a r i a t i o n for some quantitative t r a i t s ; and 6. To study the asso c i a t i o n between isozyme genotypes and quan t i t a t i v e t r a i t s . 6 2. MATERIALS AND METHODS 2.1 Seed source Cone samples from 42 Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) trees were c o l l e c t e d at the University of B r i t i s h Columbia Research Forest, Haney, B.C. (Figure 1) during early September 1978. These trees are part of a Douglas-fir population that G r i f f i t h (1968) studied and described f or phenology, growth, and flower and cone production. Between 20 to 30 cones were c o l l e c t e d from each parent tree. The i n d i v i d u a l i t y of cone l o t s and subsequent seed l o t s had been retained i n these trees. Cones were a i r dried i n the laboratory at room tempera-ture. Seeds were extracted, dewinged and cleaned by hand 6-10 days l a t e r . F i l l e d seeds were separated from empty ones using the s o f t X-ray technique and stored at 0°C - 2°C u n t i l further use. 2.2 Biochemical methods Biochemical work was c a r r i e d out at the Population Genetics Laboratory, Research Branch of the B r i t i s h Columbia Mini s t r y of Forests, V i c t o r i a , B.C. Seeds from each tree were hydrated f o r 24 hours at room temperature, surface d r i e d , and r e f r i g e r a t e d f o r l a t e r use. Hydrated seeds are meta-b o l i c a l l y more a c t i v e than dry, dormant seeds (O'Malley et a l . , 1979). For estimating parental genotypes, eight megagametophytes (haploid tissue) per parent tree were assayed f o r each enzyme. Assuming a one-to-one segregation of allozymes i n a heterozygous parent tree, the 7 FIGURE 1. Map showing the l o c a t i o n of Douglas-fir trees used i n t h i s study. 8 122 33" 00" o f o u t c r o s s i n g r a t e s e c t i o n . _ I I 9 p r o b a b i l i t y of m i s c l a s s i f y i n g a heterozygote at a p a r t i c u l a r locus i s K—1 (1/2) where K i s the number of megagametophytes analyzed per parent tree (Tigerstedt, 1973). Megagametophytes were i n d i v i d u a l l y homogenized i n 0.5 ml auto-analyser cups (Elkay Products, Worcester, Massachusetts) containing one drop of extraction b u f f e r , pH 7.5. This buffer was prepared with: 10 ml T r i s / c i t r a t e Electrophoresis buffer; 80 ml Water; 5 ml of 10 mg/ml nicotinamide adenine dinucleatide phosphate (NADP); 5 ml of 10 mg/ml nicotinamide adenine dinucleotide (NAD); 0.018 g ascorbic acid; 0.034 g ethylenediamine t e t r a a c e t i c acid (EDTA) ; 0.100 g bovine serum albumin; and 5 drops 2-mercaptoethanol (Yeh and O'Malley, 1980). Samples were absorbed onto wicks and inserted into a s l o t cut two centimeters from one end of the starch g e l . Each gel accommodated 50 samples. Horizontal starch gels were prepared from e l e c t r o s t a r c h ( E l e c t r o s t a r c h Co., Madison, Wisconsin) at a 12.5% (W/V) concentration (75 g starch/600 ml gel b u f f e r ) . The starch was cooked, degassed and poured into gel molds (21 cm long, 9.5 cm wide and 1 cm t h i c k ) . These were allowed to cool to room temperature and wrapped i n a t h i n p l a s t i c sheet ("Saran Wrap"). Runs were standardized by monitoring the migration of a tracking dye ( d i l u t e red food colouring). Genotype standards were also added, when needed, to check for new va r i a n t s . Once samples were loaded, a l l but one centimeter at eit h e r end of the gels was covered with "Saran Wrap" to prevent dessic a t i o n during the run. Gels were then placed on electrode trays with the o r i g i n ( i . e . samples) at the cathode. Electrode wicks ("handi-wipes") were placed on the unwrapped ends of the gel and 10 appropriate current applied u n t i l the tracking dye had migrated 5-10 mm from the o r i g i n . The wicks were then removed and frozen i c e packs were applied onto the gel surface to prevent d i s t o r t i o n and denaturing of proteins from excessive heat. Electrophoresis was continued u n t i l the tracking dye had migrated the appropriate distance (Table 1). The assembly f o r h o r i z o n t a l starch gel electrophoresis i s presented i n Figure 2. FIGURE 2: Assembly f o r h o r i z o n t a l starch g e l electrophoresis. Key: A, starch g e l ; B, glass plate) C, wicks; D, p l a s t i c sheet; E, platinum electrode; F, electrode vessel; and G, bridge b u f f e r . Upon completion of the run, gels were removed, trimmed and s l i c e d f o r s t a i n i n g . S t r i p s of " p l e x i g l a s s " one millimeter thick were used to s l i c e the g e l . The s l i c i n g procedure involved placing a weight on top of the g e l , putting s l i c i n g s t r i p s on e i t h e r side of the g e l , holding f i s h i n g l i n e (1 kg test) against these s t r i p s and drawing i t through the g e l . Once a s l i c e had been made, another p a i r of s l i c i n g s t r i p s was added and the procedure repeated. I t was possible to get 11 between s i x and eight s l i c e s from a sin g l e gel including the top and bottom s l i c e s , which were discarded. The s l i c e s were placed i n the appropriate enzyme s t a i n i n g s o l u t i o n and incubated at 37°C u n t i l banding patterns were scorable. Each megagametophyte y i e l d e d a s u f f i c i e n t sample to assay f o r 18 enzyme systems. Within these 18 systems i t was possible to resolve 27 d i f f e r e n t l o c i using only four starch gel buffers and four electrophoretic bu f f e r s . These buffers and t h e i r running conditions are given i n Table 1. Table 2 l i s t s the enzymes scored and describes s t a i n i n g procedures. Two terms w i l l be commonly used and should be defined. These are isozyme (isoenzyme) and allozyme. Isozymes are defined as multiple molecular forms of a given enzyme while allozymes designate variant enzymes produced by d i f f e r e n t a l l e l e s of one and the same gene locus (Prakash et a l . , 1969). The isozyme nomenclature adopted f o r th i s study was modified from Allendorf and Utter (1978). An abbreviation designates the enzyme system and the locus ( l o c i ) f o r which i t i s coded. Enzyme systems s p e c i f i e d by more than one locus were given hyphenated numerals to i d e n t i f y the l o c i . The locus coding f o r the most anodally migrating proteins was designated one, the next as two and so on. A l l e l i c v ariants (or allozymes) were i d e n t i f i e d by a superscript according to t h e i r r e l a t i v e e l e c t r o p h o r e t i c m o b i l i t y . One a l l e l e , generally the most common, was a r b i t r a r i l y designated 100. Other a l l e l e s , and t h e i r corresponding allozymes, were assigned numerical values according to t h e i r migration r e l a t i v e to the 100 form. A l l e l e s coding f o r allozymes with no s t a i n i n g a c t i v i t y ( s i l e n t a l l e l e s ) were designated as n u l l TABLE 1: The gel and electrode buffers and their e l e c t r i c power conditions. Buffer system Described by Electrode buffer Gel buffer E l e c t r i c power , H l s t i d i n e / c i t r a t e P „ , 0 m d e s and Harris (1*6, 0 £ M c i t r i c - d ^ t r i s o d i u , .005 « M s t i d i n e . ^ d . u s t 6 0 * ^ ( H C ) 0.41 M c i t r i c acid (anhydrous) migrated 5 cm. B. Morphollne/citrate PH , 1 Clayton and T r e t i a . (1972, O ^ M c i t r i c ^ c i d ^ a n h y d r o u s ) . . . d i l u t i o n of electrode ^ £ 5 0 * ) ^ aminopropyl)-morpholine C. T r i s / c i t r a t e , i / b o r a t e PH S , Kidgway « a l . (1.0) O . O ^ l i t h i ^ y d r o . i d e and 0,S« T r i s , . 0 0 5 ^ ^ ^ H J - > t K W> 1% electrode buffer, pH 8.5 0. T r i s / c i t r a t e P „ , 0 S i c i l i a n o and Shaw 0.13±H Tr i s a . O . O . M . . d i l u t i o n of electrode J - C - i a O V ^ t i ^ t r a c , -13 TABLE 2: The eighteen enzymes stained for, abbreviations, buffer systems, and stain recipes. Abbreviation Buffer1 Stain2 NADP/^NBT/SHTT/5 systems buffer NAD3 MgCl2 PMS PMS Other components 1 Acid phosphates 2 Aconitase 3 Aspartate aalno-transferase 5 Esterase 6 Glutaaate dehydrogenase 7 Glucose 6-phosphate dehydrogenase 8 Clyceraldehyde-3-phosphate dehydrogenase 9 Hexoaearn!nidase6 10 leocitrate dehydrogenase 11 Malate dehydrogenase 12 Malic enzyme 13 Mannoae-6-phosphate isomeraae 14 Peptidase 15 Phosphoglucose isooerase 16 Phosphoglucomutase 17 6-Phosphoglucooic dehydrogenase 16 Superoxide dlaautase ACO AAT GDH G6P GAPDH IDH MDH C 1 D 1 C 1 A 5 (5 ml) D 1 / • A 1(25 ml) / / B 2(25 ml) • / C 1 (5 ml) / • 150 mg Na-a-Napthyl Acid Phosphate 5 al IX Mg CI 75 ng Past Garnet GBC Salt 5 ml 5* Cls-Aconlcic Acid (U/V) 40 u IDH 1 mg Pyrldoxal-5-phoaphate 200 mg L-Aspartlc Acid 100 ng a-Ketoglutaria Acid 200 mg PaBt Blue BB Salt 1 ng 2,6-Dlchlorophenol-Indophenol 25 mg KADH 10 mg KIT 50 mg a-napthyl acetate \ Dissolved In 1 50 mg B-napthyl acetate f Acetone 100 mg Fast Blue RR Salt 400 mg L-Clutaalc Acid 200 mg Clucose-6-phosphate 270 mg Pructose-l,6-dlphosphate 75 mg Arsenic acid 100 u Aldolase 10 I ; 4-Methylumbelllferyl-N-Acetyl-B-D-Glucosamlnlde 200 mg DL-Isocltrlc Acid 25 ml -SM DL-Halic Acid pH 7.0 25 ml .5K DL-Mallc Acid pH 7.0 25 ml Monnose-6-Phoaphate 100 n PCI 20 v G6PDH 80 mg L-Luecylglycyl-Glyclne 10 mg Crude Peroxidase 10 mg Snake Venom (Crotalus Atrox) 50 mg 0-Dlanisldlne D:HC1 5 ml IX Mg Cl 2 25 mg Fructoae-6-Phosphate 10 v C6PDH 300 mg Glucose-l-Phosphate 0.5 ml Glucose-1, 6-Phosphate (10 mg/100 ml H20) 50 u G6PDH 10 mg Phosphogluconlc Acid (Naj Salt) 2.5 ag PMS Scored on gels stained for GDH activity; SOD appeared as white bands against a blue back ground. 1 See Table 1. ^ Stain buffers: Five different stain buffers were used, unless otherwise specified 50 ml of each of the buffers was used: #1. 0.2 M Trls-Hcl pH 8.0 #2. MC Electrode buffer #3. 0.2 M Na-Acetate pH 5.0 #4. 0.2 H Phosphate pH 6.4 #5. 0.05 M Citrate - 0.05 M Phosphate pH 4.0 10 mg NAD unless otherwise indicated. 10 mg NADP unless otherwise Indicated. 10 mg NBT or MTT plus 5 mg PMS unless otherwise indicated. Fluorescent under long-wave U.V. 14 ( i . e . APH-2nU^''"' and MDH-3nU"''"'") . For example the a l l e l e at the most anodal i s o c i t r a t e dehydrogenase (IDH) locus coding f o r an allozyme migrating 122 22% further than the common form was designated IDH 2.3 Progeny test This work was c a r r i e d out at the G.S. A l l e n , Forest Genetics -Tree Seed Laboratory, Faculty of Forestry, University of B r i t i s h Columbia, B.C. Seeds from each tree were s t r a t i f i e d using the method recommended by Cleary et a l . (1978). S t r a t i f i e d seeds were used i n the germination test following A l l e n and Bientjes (1954). Four r e p l i c a t i o n s , each of 15 seeds from each parent tree were made and a random arrangement of the germination pads on the germination tables was applied. The test was c a r r i e d out for four weeks at an incubation temperature of 25°C. Light was applied for a period of 12 hours every day. Germinating seeds were counted d a i l y during the four week period. Germination percent was c a l -culated f o r each r e p l i c a t i o n separately and a mean for each tree was computed. Since f i l l e d s t r a t i f i e d seeds were used, germination per-formance was uniform and germination percent varied between 56.66 percent (tree #55) and 98.33 percent (tree #3). Following germination t e s t s , germinants were planted i n d i v i d u a l l y i n polyethylene foam blocks which contain 240 c a v i t i e s (2.5 cm diameter and 11 cm depth) f i l l e d with sand and clay i n 1:1 r a t i o . The blocks were placed i n a growth chamber which was set f o r 12 hours of continuous i l l u m i n a t i o n . Light i n t e n s i t y was 3500-foot candles. A temperature of 25°C was maintained during the i l l u m i n a t i o n period and 15°C was maintained during the dark period. Although r e l a t i v e humidity was not c o n t r o l l e d , i t was i n close c o r r e l a t i o n with temperature changes; 50-60 percent 15 r e l a t i v e humidity occurred during the i l l u m i n a t i o n period and 80-100 percent during the dark period. A randomized complete design was used with four r e p l i c a t i o n s . Each parent tree was represented by f i v e seedlings per r e p l i c a t i o n . These blocks were placed randomly on the shelf of the growth chamber at a distance of one meter from the i l l u m i n a t i o n source and were rearranged randomly every seven days to further reduce biases that might have been caused by v a r i a t i o n s i n the distance from observa-t i o n window or c i r c u l a t i o n fans. Seedlings were watered twice every day during the te s t period to prevent drought. Hoagland's s o l u t i o n was provided once every week instead of i r r i g a t i o n by water. The test t e r -minated a f t e r eight weeks. Seedlings were then examined for the follow-ing c h a r a c t e r i s t i c s : (a) number of cotyledons, (b) number of needles, (c) length of hypocotyl to 1.0 mm, (d) length of e p i c o t y l to 1.0 mm, (e) t o t a l height to 1.0 mm, (f) root dry weight to 0.001 g, and (g) shoot dry weight to 0.001 g. 16 3. RESULTS AND DISCUSSION 3.1 Inheritance Isozyme v a r i a t i o n was assayed i n megagametophytes and i n embryo tissues from 27 trees. Two c r i t e r i a were used to se l e c t those trees: the f i r s t was that each tree should be heterozygous for at l e a s t three allozyme l o c i , the second c r i t e r i o n was the number of seeds a v a i l a b l e from each tree. Tree #119 was included although i t was heterozygous for only two l o c i , i n order to resolve for the DIA-1 locus, which i s normally d i f f i c u l t to resolve. These trees and t h e i r genotypes are l i s t e d i n Table 3. Megagametophytes (N) from a heterozygous mother-tree can possess ei t h e r one of the two a l t e r n a t i v e electrophoretic variants which segregate as a l l e l e s at a s i n g l e locus. Embryos (2N), on the other hand, can ei t h e r be homozygous or heterozygous at a s i n g l e locus. In the case of monomeric enzymes, the isozyme pattern observed i n the heterozygote i s the sum of corresponding homozygotes. However, multimeric enzymes have one or more hybrid zones i n heterozygotes which are not present i n e i t h e r homozygote. The subunit structure of primary isozymes that may be generated i n such heterozygotes varies according.to the number of subunits which occur i n isozyme molecules, as shown i n Table 4 and Figure 3. In the case of dimers there are two homomeric and one hetero-meric forms; i n trimers there are two homomeric and two heteromeric forms; and i n tetramers there are two homomeric and three heteromeric forms (Brewer, 1970). Eighteen enzyme systems were analysed from a sample of 47 to 78 TABLE 3: Genotypes of heterozygous l o c i for 27 trees included i n the inheritance and linkage studies. Tree i AAT-2 AAT-3 ACO APH-2 DIA- DIA-2 EST-1 G6P HA IDH MDH-1 MDH-3 MDH-4 KE PGI-2 PGM 6PG-1 6PG-2 SOD 1 3 4 5 10 •14 18 20 23 29 33 37 53 55 66 68 92 11'. 117 118 119 121 123 124 127 128 142 100/112 100/130 77/100 72/100 72/100 87/100 • 87/100 77/100 87/100 87/100 72/87 87/100 100/120 100/112 77/100 72/87 null/72 77/100 87/100 77/100 72/87 100/130 72/87 82/100 72/100 100/112 100/130 77/100 nuil/72 null/100 100/120 100/112 100/112 100/130 77/100 82/100 87/100 72/100 72/37 79/100 90/100 85/110 92/110 92/100 90/100 92/100 80/100 92/100 92/100 92/100 90/100 92/110 92/110 100/110 90/100 92/110 92/110 90/100 92/110 90/100 92/100 90/100 100/110 90/100 100/110 90/100 92/110 90/100 100/110 92/100 92/100 100/105 100/140 90/100 100/140 65/100 100/140 100/140 65/100 100/140 100/140 100/140 100/122 90/100 90/100 100/122 100/140 140/170 100/122 100/122 100/140 100/122 90/100 100/170 90/100 100/140 100/170 100/170 100/170 84/100 100/130 84/100 100/129 84/100 75/100 94/105 94/100 84/100 85/100 8.5/100 85/100 100/125 100/105 94/100 94/100 94/100 100/122 33/100 85/100 100/129 84/100 75/100 100/129 null/100 84/100 75/100 85/100 75/100 85/100 75/100 85/100 100/125 94/100 100/107 100/129 94/100 85/100 100/125 100/105 100/129 85/100 100/129 85/100 94/100 null/100 100/122 18 TABLE 4: Subunit structures of homomeric and heteromeric isozymes i n heterozygotes, where one a l l e l e determine polypeptide subunit a 1 and the other a l l e l e polypeptide subunit ex. The isozymes expected i n the cases of monomers, dimers, trimers and tetramers are shown (Brewer, 1970). Monomer Dirtier Trimer Tetramer Homomer 1 1 Heteromers 1 2 l 1 ? 12 2 1 1 1 2 1 1 2 2 1 2 2 2 Homomer 2 2 2 2 2 2 2 2 2 FIGURE 3: Diagram of c h a r a c t e r i s t i c isozyme patterns expected i n heterozygotes i n the case of enzymes which are monomers, dimers, trimers, and tetramers. HQMQZYGOTE HETERQZYG07E ICMQZTC07E MONOPER DIMER TRIftR TEKWER 19 megagametophytes for each tree. Segregation of a l l e l e s at a p a r t i c u l a r locus was determined by chi-square tests against a predicted one-to-one segregation r a t i o . Each enzyme system studied w i l l be described with respect to i t s multilocus organization, subunit ( t e r t i a r y ) structure, and a l l e l i c v a r i a t i o n . 3.1.1 Monomorphic enzymes Four monomorphic enzymes were observed i n t h i s Douglas-fir stand. These included glutamate dehydrogenase (GDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with one zone of a c t i v i t y ; mannose-6-phosphate isomerase (MPI) showed two zones of a c t i v i t y ; and peptidase (PEP) exhibited three zones of a c t i v i t y . Each zone of a c t i v i t y for the above four monomorphic enzymes was represented by a sing l e i n v a r i a t e band. Information which could be obtained about the number of a l l e l e s or subunit structure of these enzymes i s l i m i t e d as a r e s u l t of t h e i r monomorphic nature. Mitton e_t a l . (1979), using mature needle t i s s u e as a protein source, reported that the GDH locus i n ponderosa pine (Pinus  ponderosa) was a dimeric enzyme. Harris and Hopkinson (1976), using human ti s s u e , reported that MPI was monomeric while PEP was monomeric or dimeric f o r d i f f e r e n t peptidase forms. 3.1.2 Polymorphic enzymes To study polymorphic enzymes i t was assumed that at l e a s t one locus was heteromorphic i n t h i s group of enzymes. 3.1.2.1 Acid phosphatase (APH) There were two zones of a c t i v i t y on gels stained f o r APH:APH-1 and APH-2. The APH-1 locus was represented by two bands which mimicked 20 the v a r i a t i o n i n APH~2; therefore the APH^l locus was not included i n the study. Rudin and Ekberg (1978), however, were able to detect v a r i a t i o n i n the APH^l locus i n Scots pine (Pirius s y l v e s t r i s ) using d i f f e r e n t running conditions. Four a l l e l e s were detected at APH-2 (Figure 4,A), including one that was n u l l . Plate 1,A i s a photograph of a gel showing two of four a l l e l e s detected at the APH-2 locus (72/100). Results of segregation analyses f o r d i f f e r e n t a l l e l e combinations are presented i n Table 5 f o r 19 d i f f e r e n t trees. TABLE 5: Segregation of a l l e l i c variants at the APH-2 locus. # of mega. A l l e l i c Observed 2 . t Tree if ^ . . . . X -value; anal. combination segregation 1 47 3 55 68 68 123 68 4 58 5 58 10 48 14 58 23 58 53 58 121 58 18 57 33 58 55 56 66 68 127 56 37 58 92 68 118 58 72/100 87/100 72/87 null/72 null/100 23:24 0.021 33:22 2.200 40:28 2.118 30:38 0.941 27:31 0.276 29:29 0.000 24:24 1.724 23:35 2.483 33:25 1.103 34:24 1.724 24:34 1.724 30:27 0.158 29:29 0.000 29:27 0.071 30:38 0.941 28:28 0.000 26:32 0.621 37:31 0.529 34:24 1.724 f 2 None of the above x ^values were s i g n i f i c a n t at P<=0.05. 21 It was not possible to resolve f o r APH i n d i p l o i d t i s s u e of Douglas-fir. However, Harris and Hopkinson (1976) working with APH isozymes i n human tis s u e s , found a homodimer for two l o c i and a hetero-dimer subunit structure for a t h i r d locus. Subunit structure of APH was a dimer i n Norway spruce (Picea abies) (Lundkvist, 1975). 3.1.2.2 Aconitase (ACO) The ACO enzyme showed two zones of a c t i v i t y describing two d i f f e r e n t isozymes; the anodal zone was ACO and the other one was IDH. The IDH enzyme also tended to show up on several gels stained f o r other enzymes (e.g. PGM). Information regarding t h i s condition i s further described i n the section on PGM (3.1.2.12). Two a l l e l e s were detected at ACO (Figure 4,B). Plate 1,B i s a photograph of a gel showing ACO and IDH a c t i v i t y when stained f o r ACO. Results of segregation analyses fo r the only possible a l l e l e combination are presented i n Table 6 for seven d i f f e r e n t trees. TABLE 6: Segregation of a l l e l i c variants at the ACO locus. Tree # # of mega, anal. A l l e l i c combination Observed segregation 2 . t X rvalue 1 47 77/100 21:26 0.532 10 57 31:26 0.439 33 58 30:28 0.069 53 58 33:25 1.110 55 57 32:25 0.860 92 68 36:32 0.236 128 58 23:35 2.483 None of the above x -values were s i g n i f i c a n t at P<0.05. 22 Aconitase has a monomeric structure i n humans (Slaughter e_t a l . , 1975 and Zouros, 1976); but i t was not possible to confirm i t s subunit structure i n Douglas-fir. 3.1.2.3 Aspartate aminotransferase (AAT) There were three zones of a c t i v i t y on gels stained for AAT:AAT-1, AAT-2, and AAT-3, which migrated anodally. In addition, there was a fourth zone of AAT a c t i v i t y cathodal to the o r i g i n that matched v a r i a -t i o n i n AAT-3 (Figure 4,C and D). A s i m i l a r r e s u l t was found i n p i t c h pine (Pinus r i g i d a ) by Guries and Ledig (1978) and i n ponderosa pine (O'Malley et a l . , 1979). Whether both AAT-3 and the cathodal AAT band were co n t r o l l e d by the same locus, or whether they represented two t i g h t l y l inked l o c i remains unknown at present. Plate 1,G i s a photograph of a gel showing two of the three a l l e l e s detected at AAT-2 and AAT-3. The AAT-1 locus was monomorphic i n t h i s population. Three and two a l l e l e s were detected at the AAT-2 and AAT-3 l o c i , r e s p e c t i v e l y . Results of segregation analyses are presented i n Table 7. AAT isozymes displayed three-banded patterns i n the heterozygous embryos, suggesting that AAT enzyme molecules are dimers. This agrees with the subunit structure of AAT described for other species: brown trout (Salmo trutta) by Allendorf et a l . (1977), p i t c h pine (Guries and Ledig, 1978), d i f f e r e n t human tissues (Harris and Hopkinson, 1976), ponderosa pine (O'Malley et a l . , 1979), and Scots pine (Rudin, 1975). 23 TABLE 7, Segregation of a l l e l i c variants at the heterozygous AAT l o c i . Tree // Locus //of mega, anal. A l l e l i c combination Observed segregation X^-value"f* 20 AAT-2 58 100/130 28:30 0.069 33 . 58 28:30 0.069 92 68 41:27 2.882 123 68 33:35 0.059 68 68 82/100 33:35 0.059 142 58 32:26 0.621 20 AAT-3 58 100/130 28:30 0.069 66 68 35:33 0.059 92 68 34:34 0.000 128 68 32:26 0.621 'None of the above 2 . X -values were s i g n i f i c a n t at P < 0.05. 3.1.2.4 Diaphorase (DIA) There were two zones of a c t i v i t y on gels stained f o r DIA: DIA-1 and DIA-2. The DIA-1 locus was very d i f f i c u l t to resolve although i t was resolved i n some trees, two of which were heterozygous f or t h i s locus. The number of heterozygous trees f o r these two l o c i was very l i m i t e d . Only two trees were heterozygous f o r DIA-1 and one tree was heterozygous f o r DIA^-2. Two a l l e l e s were detected at each locus (Figure 4, E and F ) . Plate 2,A i s a photograph of a gel showing the two st a i n i n g zones f or t h i s enzyme. Results of segregation analyses are presented i n Table 8. The DIA isozymes displayed a monomeric structure i n human tissues (Harris and Hopkinson, 1976), but i t was not possible to confirm t h i s subunit structure i n Douglas-fir. 24 TABLE 8: Segregation of a l l e l i c variants at the heterozygous DIA l o c i . Tree # Locus # of mega, anal. A l l e l i c combination Observed segregation X^-value 29 DIA-1 49 100/120 30:19 2.469 119 58 24:34 1.724 3 DIA-2 55 79/100 20:35 4.091* Sig n i f i c a n c e ; *, P<0.05. 3.1.2.5 Esterase (EST) There were two zones of a c t i v i t y on gels stained f o r EST: EST-1 and EST-2. EST-1 stained darkly and consistently, while EST-2 stained f a i n t l y or was absent on most gels. Therefore, the EST-2 locus was excluded from t h i s study. Four a l l e l e s were detected at EST-1 (Figure 4,G). Plate 2,B i s a photograph of a gel showing a l l four a l l e l e s . I t was possible to test for segregation i n only four out of s i x possible com-binations. Results of segregation analyses are presented i n Table 9. Heterozygous embryos for the EST-1 locus were i d e n t i f i e d by t h e i r two-banded patterns suggesting that i t i s a monomer. This agrees with the subunit structure of EST described f o r other species: Norway spruce (Bartels, 1971), some human tissues (Harris and Hopkinson, 1976), and wild oats (Avena barbata) by Marshall and A l l a r d (1969) . 25 TABLE 9: Segregation of a l l e l i c variants at the EST-1 locus. # of mega. A l l e l i c Observed 2 , t , , . . X -value anal. combination segregation 10 58 95/100 35:i 2 3 2.483 14 58 29:29 0.000 18 58 30:28 0.069 20 58 27:31 0.276 23 58 28:30 0.069 127 56 28:28 0.000 142 58 31:27 0.276 5 58 95/110 29:29 0.000 33 58 27:31 0.276 37 58 36:22 3.379 55 58 25:33 1.103 68 68 30:38 0.941 114 58 24:34 1.724 117 68 31:37 0.529 123 68 28:40 2.118 53 58 100/100 31:27 0.276 118 58 32:26 0.621 119 58 27:31 0.276 124 55 26:29 0.164 4 58 85/110 32:26 0.621 ^None of the above 2 X -values were s i g n i f i c a n t at P<0.05. 1.1.2. 6 Glucose-6-phosphate dehydrogenase (G6P) There was a s i n g l e dark zone of a c t i v i t y on gels stained f o r G6P. There were two or three other l i g h t l y s t a i n i n g zones that were not included i n the study because the r e s o l u t i o n of these zones was not consistent, thus making i n t e r p r e t a t i o n d i f f i c u l t . Three a l l e l e s were detected at the G6P "locus (Figure 4,H). Plate 2,C i s a photograph of a gel showing two of the three a l l e l e s detected i n t h i s population. 26 Results of segregation analyses are presented i n Table 10. TABLE 10: Segregation of a l l e l i c variants at the G6P locus. # of mega. A l l e l i c Observed 2 .. t -i , . ^ . X -value anal. combination segregation t. 1 57 90/100 34:23 2.123 10 65 34:31 0.138 23 68 30:38 0.941 53 68 33:35 0.059 68 78 42:36 0.462 114 68 33:35 0.059 117 78 35:43 0.821 118 68 30:38 0.941 121 78 47:31 3.282 123 78 39:39 0.000 14 68 80/100 31:37 0.529 None of the above 2 . X -values were s i g n i f i c a n t at P < 0.05. G6P displayed three-banded patterns i n the heterozygous embryos, suggesting that the G6P enzyme molecules are dimers. This agrees with the subunit structure of G6P described i n human tissues by Harris and Hopkinson (1976) . 3.1.2.7 Hexoseaminidase (HA) Gels stained f o r the HA enzyme showed only one zone of a c t i v i t y under UV l i g h t . Three a l l e l e s were detected at t h i s locus (Figure 4,1). Results of segregation analyses are presented f o r 15 d i f f e r e n t trees i n Table 11. I t was not possible to i n t e r p r e t the pattern of HA i n heterozygous embryos because bands were bl u r r e d . 27 TABLE 11: Segregation of a l l e l i c variants at the HA locus. Tree # # of mega, anal. A l l e l i c combination Observed segregation X^-value 3 55 •100/140 22:33 2.200 4 58 32:26 0.621 5 58 23:35 2.483 10 58 30:28 0.069 18 58 31:27 0.276 20 58 18:40 8.345** 23 58 31:27 0.276 53 58 34:24 1.724 114 58 31:27 0.276 123 68 35:33 0.059 117 68 100/170 36:32 0.235 124 55 29:26 0.164 128 58 34:24 1.724 55 58 140/170 31:27 0.276 Sig n i f i c a n c e ; **, P<0.01. 3.1.2.8 I s o c i t r a t e dehydrogenase (IDH) There was a s i n g l e zone of a c t i v i t y on gels stained f o r IDH. Faint IDH patterns could be detected on many gels stained for NADP-dependent enzymes. Four a l l e l e s were detected at the IDH locus (Figure 4,J). Plate 3,A i s a photograph of a gel stained f o r both IDH and PGM enzymes and showing only two of four IDH a l l e l e s detected i n th i s population. Results of segregation analyses are presented for eight d i f f e r e n t trees i n Table 12. 28 TABLE 12: Segregation of a l l e l i c variants at the IDH locus. Tree # # of mega, anal. A l l e l i c combination Observed segregation 2 i + X -value 23 68 100/122 28.:40 2.118 37 68 28:40 2.118 66 78 43:35 0.821 92 75 43:32 1.613 114 68 35:33 0.059 4 68 65/100 34:34 0.000 10 68 31:37 0.529 3 65 90/100 32:33 0.015 None of the above x -values were s i g n i f i c a n t at P<0.05. Heterozygous embryos showed a three-banded pattern which indicated the IDH isozyme subunit structure i s a dimer. This agrees with the sub-unit structure of IDH described f o r other species: mosquitoes (Culex  pipiens) by Cheng et a l . (1977), p i t c h pine (Guries and Ledig, 1978), human tissues (Harris and Hopkinson, 1976), and ponderosa pine (O'Malley et a l . , 1979). 3.1.2.9 Malate dehydrogenase (MDH) Two zones of a c t i v i t y were detected on gels stained f o r MDH. Var i a t i o n i n the most anodal zone appeared to be cont r o l l e d by three l o c i (MDH-1, MDH-2, and MDH-3) while v a r i a t i o n i n the slower zone was determined by a s i n g l e locus (MDH-4). In addition to these four l o c i the MDH-2 and the MDH-3 l o c i formed a heterodimer band i n the megagame-tophyte, r e s u l t i n g i n a three-banded pattern f o r haploid t i s s u e . 29 Interpretation f o r the enzyme was d i f f i c u l t and required a trained eye due to the overlapping of the d i f f e r e n t v a r i a n t s . The MDH-2 locus was monomorphic i n t h i s population. Three a l l e l e s were detected f o r each of the other three l o c i i ncluding a n u l l a l l e l e at MDH-3 (Figures 5,A, B, and C). Plate 3,B i s a photograph of a gel showing v a r i a t i o n i n MDH-3 and MDH-4. Results of segregation analyses are presented f o r 17 d i f f e r e n t trees i n Table 13. TABLE 13: Segregation of a l l e l i c variants at the heterozygous MDH l o c i . „ T # of mega. A l l e l i c Observed 2 . Tree # Locus - , ° . . . . Y -value anal. combination segregation * 14 29 114 117 1 3 4 18 55 114 92 142 4 55 114 117 118 3 53 66 121 124 127 MDH-1 MDH-3 MDH-4 58 58 58 68 47 47 55 58 58 58 58 68 57 58 50 58 68 58 55 58 68 58 54 56 83/100 100/105 70/100 hull/100 75/100 100/130 34:24 1.724 29:29 0.000 27:31 0.276 32:36 0.236 25:22 0.191 25:22 0.191 29:26 0.164 33:25 1.103 33:25 1.103 35:23 2.483 30:28 0.069 30:38 0.941 23:34 2.123 24:34 1.724 25:25 0.000 24:34 1.724 32:36 0.235 31:27 0.276 35:20 4.091* 27:31 0.276 32:36 0.235 30:28 0.069 27:27 0.000 29:27 0.071 Sign i f i c a n c e ; *, P<0.05. 30 Heterozygous embryos f o r the MDH-3 locus displayed three-banded patterns, suggesting that i t s subunit structure i s dimeric. The MDH-1 locus was a monomeric enzyme. Subunit structure for MDH-2 and MDH-4 was d i f f i c u l t to study because the MDH-2 locus was monomorphic and the sta i n i n g of the MDH-4 locus was very f a i n t i n the embryo. The number of MDH l o c i has been found to d i f f e r among various species. Two l o c i were observed i n brown trout (Allendorf et a l . , 1977), p i t c h pine (Guries and Ledig, 1978), human ti s s u e (Harris and Hopkinson, 1976), Scots pine (Rudin and Ekberg, 1978), and Sitk a spruce (Picea s i t c h e n s i s (Bong.) Carr.) (Simonsen and Wellendorf, 1975). Three l o c i were observed i n Sitka spruce (Yeh and El-Kassaby, 1980). Four l o c i were found i n ponderosa pine (0'Malley et a l . , 1979), Douglas-fir (Yeh and O'Malley, 1978 and 1980), and lodgepole pine (Pinus contorta var. l a t i f o l i a ) (Yeh and Layton, 1979) . 3.1.2.10 Malic enzyme (ME) There were two zones of a c t i v i t y on gels stained f o r ME. One was ME and the more, anodal zone was MDH-1. The method used to confirm t h i s observation i s described i n d e t a i l i n section (3.1.2.12). Three a l l e l e s were detected at t h i s locus (Figure 5,D). Plate 3,C i s a photograph of a gel showing two of three a l l e l e s detected i n the population and the MDH-1 locus. Results of segregation analyses f o r nine d i f f e r e n t trees are presented i n Table 14. The ME molecules displayed a tetrameric structure i n human tissues (Harris and Hopkinson, 1976), but i t was not possible to confirm t h i s structure i n Douglas-fir. 31 TABLE 14: Segregation of a l l e l i c variants at the ME locus. „ it of mega. A l l e l i c Observed 2 , f Tree it . & , . ' . . X -value anal. combination segregation 10 58 85/100 35:23 2.483 14 58 28:30 0.069 18 58 28:30 0.069 117 68 36:32 0.235 118 58 28:30 0.069 123 68 39:29 1.471 124 55 29:26 0.164 127 56 32:24 1.143 1 47 100/130 25:22 0.191 t 2 None of the above x -values were s i g n i f i c a n t at P <0.05. 3.1.2.11 Phosphoglucose isomerase (PGI) There were two zones of a c t i v i t y on gels stained f o r PGI: PGI-1 and PGI-2. PGI-1 was f a i n t and inconsistent i n s t a i n i n g i n t e n s i t y and was not included i n th i s study. PGI-2 exhibited occasional variants f o r two a l l e l e s (Figure 5,E). Plate 4,A i s a photograph of a gel showing these a l l e l e s . Results of segregation analyses f o r PGI-2 f o r three d i f f e r e n t trees are presented i n Table 15. TABLE 15: Segregation of a l l e l i c variants at the PGI-2 locus. „ ,, it of mega. A l l e l i c Observed 2 , . t Tree it .. 6 ... ^ • X -value anal. combination segregation A 18 58 100/125 26:32 0.621 118 58 24:34 1.724 123 68 34:34 0.000 t 2 None of the above x -values were s i g n i f i c a n t at P<0.05. 32 Heterozygous embryos for the PGI-2 locus displayed t r i p l e -banded patterns, suggesting that PGI-2 enzyme molecules are dimers. This was i n agreement with the subunit structure f o r PGI observed i n p i t c h pine (Guries and Ledig, 1978), and Sit k a spruce (Somonsen and Wellendorf, 1975) . 3.1.2.12 Phosphoglucomutase (PGM) Two zones of a c t i v i t y were detected on gels stained for PGM: PGM-1 and PGM-2. PGM-1 stained much more intensely than PGM-2. I t appeared that the PGM-2 mimicked v a r i a t i o n at IDH. Previously, i t was mentioned that IDH enzyme tended to show up on many gels stained f o r NADP-dependent enzymes. Since the st a i n i n g s o l u t i o n used f or PGM contained NADP, atte n t i o n was turned to i n t e r -p r e t a t i o n of gels with NADP-dependent enzymes. A check was made by doing the following: (a) Staining one s l i c e of gel for IDH enzyme. (b) Staining another s l i c e of the same gel for PGM enzyme. (c) Staining a t h i r d s l i c e of the same gel for IDH and PGM concurrently. By comparing migration distances for each band i n these three gels, i t was d e f i n i t e l y shown that the PGM-2 locus was i n fact IDH and not a second PGM locus as reported previously i n p i t c h pine (Guries and Leding, 1978), i n Ponderosa pine (Mitton et a l . , 1979), and i n S i t k a spruce (Simonsen and Wellendorf, 1975 and Yeh and El-Kassaby, 1980). Three a l l e l e s were detected at the PGM locus (Figure 5,F). Plate 4,B i s a photograph of gel showing only two of three a l l e l e s detected. Results of segregation analyses f o r ten d i f f e r e n t trees are presented i n Table 16. 33 TABLE 16: Segregation of a l l e l i c variants at the PGM locus. Tree # # of mega, anal. A l l e l i c combination Observed segregation 2 i + X -value 10 67 94/100 37:30 0.731 23 68 36:32 0.235 29 68 41:27 2.882 53 68 38:30 0.941 118 68 37:31 0.529 121 78 33:45 1.846 127 66 28:38 1.515 18 • 66 100/105 38:28 1.515 123 78 40:38 0.051 5 68 94/105 37:31 0.529 None of the above x -values were s i g n i f i c a n t at P<0.05. Heterozygous embryos displayed two-banded patterns suggesting that PGM enzyme molecules are monomeric. This was i n agreement with the reported structure of PGM i n human tissues (Harris and Hopkinson, 1976) and i n ponderosa pine (Mitton et a l . , 1979). 3.1.2.13 6-Phosphogluconic dehydrogenase (6PG) There were two zones of a c t i v i t y on gels stained for 6PG: 6PG-1 and 6PG-2. Three a l l e l e s were detected at 6PG-1 and two a l l e l e s at 6PG-2 (Figure 5,G and H). Plate 4,C i s a photograph of a gel showing heteromorphic 6PG-1 and monomorphic 6PG-2. Results of segregation analyses are presented i n Table 17. 34 TABLE 17: Segregation of a l l e l i c variants at the heterozygous 6PG l o c i . Tree •.#. . Locus # of mega, anal. A l l e l i c combination Observed segregation 2. . . t X -value 114 6PG-1 68 85/100 26:42 3.765 118 68 100/107 39:29 1.471 3 6PG-2 65 100/122 33:32 0.015 68 78 37:41 0.205 ^None of the above X^-values were s i g n i f i c a n t at P < 0.05. Heterozygous embryos displayed three-banded patterns for each locus, suggesting that the 6PG enzyme molecules are dimers. This was supported by the reported structure for 6PG i n human ti s s u e (Harris and Hopkinson, 1976). 3.1.2.14 Superoxide dismutase(SOD) Superoxide dismutase has also been termed tetrazolium oxidase ( L i p p i t t and F r i d o v i c h , 1973 and Lorimer, 1979). SOD appeared as a s i n g l e white band on gels stained with tetrazolium dye. Three a l l e l e s were detected at t h i s locus (Figure 5,1). Results of segregation analyses are presented for two d i f f e r e n t trees in.Table 18. Heterozygous embryos displayed three-banded patterns suggesting that SOD molecules are dimers. This was i n agreement with the reported subunit structure f o r SOD i n dogs (Canis latrans) (Baur and Schorr, 1969) and lodgepole pine (Yeh and Layton, 1979). 35 TABLE 18: Segregation of a l l e l i c variants at the SOD locus. .. # of mega. A l l e l i c Observed 2 , t ee f i , . . X -value anal. combination segregation 20 58 33/100 31:27 0.276 29 58 85/100 30:28 0.069 None of the above x -values were s i g n i f i c a n t at P<0.05. 3.1.3 Summary and conclusion 2 From a t o t a l of 146 x -te s t s analysed only three showed deviation from the expected one-to-one r a t i o . These three are: (a) HA locus for the 100/140 combination i n tree # 20. (b) MDH-4 locus f o r the 100/130 combination i n tree # 3. (c) DIA-2 locus f o r the 79/100 combination i n tree # 3. There were other trees at HA and MDH-4 l o c i having the same allozyme combination that showed no deviation from the expected segregation r a t i o . One possible reason f o r the s i g n i f i c a n t deviation from the expected r a t i o i s that a bias could e x i s t f o r one a l l e l e at the expense of the other one i n the sample of megagametophytes analysed (sampling e r r o r ) . More seeds are needed to test t h i s hypothesis. Another reason could be that there was d i f f e r e n t i a l v i a b i l i t y of gametes carrying d i f f e r e n t a l l e l e s . Only tree # 3 was heterozygous at the DIA-2 locus. I f hypotheses for deviant segregation at MDH-4 were acceptable for tree # 3, they could also explain the r e s u l t f o r the DIA-2 locus. A linkage r e l a t i o n s h i p between these two l o c i could account f o r deviant segregation of these 36 FIGURE 4(A-J). I l l u s t r a t i o n of the r e l a t i v e migration of allozymes for the following l o c i . A: APH-2; B: ACO; C: AAT-2; D: AAT-3; E: DIA-1; F: DIA-2; G: EST-1; H: G6P; I: HA; J : IDH. A I 1 0 0 — A A T - 2 2 1 , 2  3 1 0 0  2 S 2  ORIGIN B D ^ BO 9 0 J 1 0 0  38 FIGURE 5(A-I). I l l u s t r a t i o n of the r e l a t i v e migration of allozymes for the following l o c i . A: MDH-1; B: MDH-3; C: MDH-4; D: ME; E: PGI-2; F: PGM; G: 6PG-1; H: 6PG-2; I: SOD. 39 A 2100 MDH- 1 I 8 3 - — . 3 1 0 0  3»° 4100 4 oo B ORIGIN MDH-3 ,100 2»o 3 1 0 0  3 7° 4100 ORIGIN ORIGIN ORIGIN H ) 6 P G - 2 .100 o!22 'too " ~ ' 2 MDH-4 ME — — 130 2100 D 100 B5 3 oo ORIGIN 4100 4B 0  4 7 S _ ORIGIN 40 PLATE 1 (A-C). Photographs of gels stained f o r the following enzyme systems: A: APH-2 allozymes; showing segregation f o r eight i n d i v i d u a l megagametophytes from tree # 123. Sample # 9 to the l e f t i s a standard (100). B: ACO gel showing monomorphic condition f o r trees # 119 and # 123; f i r s t eight samples represent megagametophytes from one mother tree. Sample # 9 to the l e f t side i s a standard (100). C: AAT-2 and AAT-3 allozymes; showing 20 megagametophytes from tree # 92. 4 1 42 PLATE 2 (A-C). Photographs of gels stained for the following enzyme systems: A: DIA-2 allozymes; showing 20 megagametophytes from tree # 3. DIA-1 was monomorphic. B: EST-1 allozymes. The f i r s t group shows eight megagametophytes from tree # 5, heterozygous f o r the (85/100) combination followed by a standard (100). The second group (tree # 9) represents i d e n t i c a l (85/100) genotype. The t h i r d group i s from tree # 10, that heterozygous for the (95/100) combination. Notice the f a i n t zone at the bottom of the gel (EST-2). C: G6P allozyme; showing two groups of 10 megagametophytes from tree # 53. Notice two f a i n t l y stained zones i n the g e l . 43 OlA .fl B EST-1 6 < P 44 PLATE 3 (A-C). Photographs of gels stained f o r the following enzyme systems: A: IDH and PGM isozymes; f i v e groups of 8 megagametophytes from f i v e d i f f e r e n t trees. Sample # 9 to the l e f t of each group i s a standard (100). B: MDH isozymes; two groups of 10 megagametophytes from tree # 4. Notice: a - v a r i a t i o n i n MDH-3 and MDH-4. b - the heterodimer band between these two l o c i . C: ME allozymes; two groups of 10 megagametophytes from tree # 127. The monomorphic MDH-1 locus i s represented by the f a i n t s t a i n i n g zone. MOM 1M 46 PLATE 4 (A-C). Photographs of gels stained f o r the following enzyme systems: A: PGI isozyme; two groups of 8 megagame-tophytes from tree # 10 and tree # 22, sample # 9 i n each group i s a standard (100). Notice the f i r s t s t a i n i n g zone which i s PGI-1. B: PGM isozyme; two groups of 10 megagametophytes from tree # 127. Notice the very f a i n t s t a i n i n g zone i n the bottom of the g e l . C: 6PG isozyme; two groups of 10 megagametophytes from tree # 118 showing heterozygous and homozygous 6PG-1 and homozygous 6PG-2 locus. 47 48 d i f f e r e n t l o c i i n Tree # 3. Summarizing the r e s u l t s of chi-square analyses, with an under-standing of the b i o l o g i c a l nature of haploid megagametophyte t i s s u e , i t seems l o g i c a l to assume that these allozymes exhibited d i s t i n c t simple Mendelian expression i n t h e i r mode of inheritance i n a co-dominant fashion. This simple system of inheritance w i l l f a c i l i t a t e the use of allozymes as gene markers i n p r a c t i c a l tree improvement programs. 3.2 Linkage Multilocus organization i n forest trees has gained a t t e n t i o n as an increasing number of l o c i become a v a i l a b l e . Obtaining more informa-t i o n about linkage r e l a t i o n s h i p s increases the value of isozymes as a new research t o o l i n forest genetics studies. For example, a knowledge of linkage r e l a t i o n s h i p s w i l l enable the assessment of the d i s t r i b u t i o n of a v a i l a b l e l o c i i n a genome, help to determine whether non-random associations of allozymes e x i s t , and f i n d whether e p i s t a t i c s e l e c t i o n i s responsible for maintaining these blocks of i n t e r a c t i n g sets of l o c i (Clegg et a l . , 1972). Such knowledge w i l l also aid i n answering questions pertaining to the d i s t r i b u t i o n and maintenance of genie v a r i a t i o n i n natural populations (Langley et a l . , 1974 and Zouros e_t a l . , 1974) . Linkage studies and gene mapping have been rare i n l o n g - l i v e d organisms such as forest trees as a r e s u l t of a lack of a v a i l a b l e gene markers. Recently, however, the use of electrophoretic technique has f a c i l i t a t e d studies of linkage r e l a t i o n s h i p s among numerous l o c i i n forest tree species. 49 Attempts to detect linkage among polymorphic l o c i have been reported for a few tree species with equivocal r e s u l t s . Negative r e s u l t s ( i . e . no linkage) have been reported f o r the following species: Norway spruce (Bergmann, 1974 and Lundkvist, 1974), p i t c h pine (Guries and Ledig, 1978), S i t k a spruce (Simonsen and Wellendorf, 1975), and V i r g i n i a pine (Pinus virginiana) (Feret and Witter, 1977). P o s i t i v e r e s u l t s ( i . e . linkage) have been reported f o r l o b l o l l y pine (Pinus taeda) (Adams and J o l y , 1980), p i t c h pine (Guries ^ t ^ a l . , 1978), ponderosa pine (O'Malley et a l . , 1979), and Scots pine (Rudin and Ekberg, 1978)-. Materials and Methods Data were c o l l e c t e d from each i n d i v i d u a l megagametophyte at a l l heterozygous l o c i f o r each enzyme system. The observed number of two-locus a l l e l i c segregants was recorded for each of the four expected classes. This process was repeated f o r each pair of heterozygous l o c i f o r each tree separately. A heterozygous tree for n l o c i w i l l give n(n-l)/2 possible pair-combinations. The s t a t i s t i c a l procedures used to test f or linkage are described as follows: The observed gene segregation for each locus was used to c a l c u l a t e the expected numbers for each of the two-locus segregants (Bailey, 1961) and was followed by chi-square analyses to test f or linkage. Assuming a A B genotype of a parent tree 1 1 f o r two l i n k e d l o c i , then the possible A 2 ^2 isozyme types of gametes are: A i / B x A i / B 2 A 2/B! A 2/B 2 parental type crossing over types parental type Expected N PA PB N PA QB N QA PB N QA QB Observed a b c d where: , , a + b , PA n~ ' QA = 1 " PA a + c n  PB = ~n— ' QB = 1 " PB The equation for the chi-square analysis i s : X L = K A + D ) ~ N ( P A P B + QA QB ) ] 2 + U b + c ) - n ( p A q B + q A P B ) ] 2  N ( PA PB + QA QB ) N ( PA QB + QA PB } with only one degree of freedom, since the i n i t i a l three are reduced by two causes of v a r i a t i o n , namely the segregation of a l l e l e s from the actual two l o c i studied. Recombination frequencies and t h e i r standard errors were estimated as follows: Recombination frequency (R) = — — (100) Standard error (S.E.) = / ^  n where: r = # of recombinant types observed, n = # of megagametophytes analysed. 51 Data were c o l l e c t e d from the same 27 trees that were studied f o r the mode of inheritance (Table 3). Results A t o t a l of 116 d i f f e r e n t two-locus combinations were a v a i l a b l e to test f o r non-random j o i n t segregation (linkage). Chi-square r e s u l t s for linkage analyses (as well as the recombination frequencies f o r s i g n i f i -cant ones) and t h e i r standard errors are presented i n Appendix 1 for each combination f or each i n d i v i d u a l tree separately. The linkage analysis generally assumed no deviation from the expected one-to-one r a t i o f o r the two a l t e r n a t i v e a l l e l e s within each two-locus combination. Therefore, another two chi-square tests (x2-£ a n d X2-^) were ca r r i e d out. However, f o r the equivalent double backcrosses, Bailey (1961) has shown that the chi-square test f o r the detection of linkage i s s t i l l v a l i d even i f only one, and not both, of the segregation r a t i o s deviated from the expected one-to-one r a t i o . For the a v a i l a b l e data only 14 combinations showed one s i g n i f i c a n t chi-square t e s t . None, however, showed s i g n i f i c a n t deviation from the expected r a t i o i n d i c a t i n g the absence of linkage. Only one combination had two s i g n i f i c a n t c h i -square tests and therefore t h i s combination (DIA-2:MDH-4) f o r tree # 3 was eliminated and 115 combinations were used to test for linkage. Results i n Appendix 1 can be grouped into three classes: A. Combinations without s i g n i f i c a n t deviation from the expected r a t i o (no l i n k a g e ) . This c l a s s contains 92 d i f f e r e n t combinations of paired l o c i , the number of trees within combinations varied between one and fourteen. No deviation from the expected r a t i o s was 52 observed i n any tree f o r any combinations. B. Combinations with s i g n i f i c a n t deviation from the expected r a t i o . A t o t a l of 23 d i f f e r e n t combinations are included i n t h i s c l a s s , and the number of trees within combinations varies between one and t h i r t e e n . A l l combinations included i n t h i s c l a s s showed s i g n i f i c a n t deviation from the expected r a t i o for at l e a s t one tree. This c l a s s can be further divided into two sub-classes: B . l S i g n i f i c a n t deviation from the expected r a t i o with only one tree present i n each combination (Table 19). TABLE 19: Test of linkage between polymorphic l o c i . Combination Tree # n. FF FS SF SS *\ X 2 x II X 2 * L Recomb. % S.E. AAT-2:PGI-2 123 68 1 34 33 0 0.059 0.000 6.4.059*** 1.471 1.460 AAT-3:SOD 20 58 6 24 21 7 0.069 0.276 17.503*** 22.414 5.476 DIA-2:MDH-3 3 55 12 23 14 6 4.091* 0.164 6.012* 32.727 6.327 MDH-1:SOD 29 58 18 11 10 19 0.000 0.069 4.414* 36.207 6.311 MDH-3:6PG-1 114 58 21 7 14 16 0.069 3.765 4.645* 36.207 6.311 Sig n i f i c a n c e : *, P<0.05; **, P<0.01; ***, P< 0.001. 53 B.2 Heterogeneous data. This group contains 18 d i f f e r e n t combinations with more than one tree within each combination. The re s u l t s f o r any combina-t i o n included s i g n i f i c a n t and/or not s i g n i f i c a n t deviations from the expected r a t i o . A combined chi-square test f o r each combination was conducted and the re s u l t s from the combined analyses can be divided into two groups. B.2.1* Combinations with no s i g n i f i c a n t deviation from the expected r a t i o f o r the combined data. A t o t a l of 14 d i f f e r e n t combinations were included i n t h i s group (Table 20). TABLE 20: Test of linkage between polymorphic l o c i from combined data. Combination # of trees n FF FS SF SS APH-2:G6P 9 528 115 139 140 134 1.7825 APH-2-.HA 9 526 130 117 129 150 2.1354 APH-2:IDH 6 365 92 79 90 104 1.9878 APH-2:PGM 9 526 114 137 141 134 1.7953 EST-1:HA 13 771 198 196 178 119 0.7112 EST-1:MDH-4 8 460 118 117 123 102 0.9039 EST-1 :ME 8 479 116 124 106 133 0.7592 EST-1:PGM 8 469 103 126 120 120 1.1817 G6P:MDH-1 4 231 56 67 57 51 1.2039 G6P:ME 6 354 90 86 75 103 2.8684 HA:IDH 5 287 69 72 84 62 2.1205 HA:ME 5 307 62 84 76 85 0.6861 IDH:PGM 2 135 38 38 24 35 1.1363 MDH-4:PGM 4 230 55 58 62 55 0.4287 54 The s i g n i f i c a n t deviation from the expected r a t i o between trees w i t h i n any combination may be caused by two possible f a c t o r s , namely the inversion polymorphism (Saylor and Smith, 1966) and/or the small number of megagametophytes analysed (sampling error) which could be the case i n these combinations. B.2.2 Combinations with s i g n i f i c a n t deviation from the expected r a t i o f o r the combined data. Only four d i f f e r e n t combinations were included i n t h i s group (Table 21). TABLE 21: Test of linkage between polymorphic l o c i from combined data. Combination # of trees n FF FS SF SS 2 X L Recomb. % S.E. AAT-2:MDH-3 2 125 37 15 35 38 5 .287* 40.000 4.382 EST-1:MDH-1 3 184 57 43 34 50 4 .948* 41.848 3.637 G6P:IDH 3 201 45 59 63 34 9 .424*** 39.303 3.445 MDH-4:ME 4 236 48 69 64 55 3 .839* 43.644 3.228 Sig n i f i c a n c e ; *, P < 0.05; ** , P < O.i 01; *** , P < 0. 001. Discussion From the chi-square analyses a t o t a l of nine d i f f e r e n t p a i r s of combinations showed s i g n i f i c a n t deviation from the expected r a t i o . The number of l o c i involved i n these combinations i s 13. The r e l a t i o n and the i n t e r a c t i o n between these l o c i have formed two n e t - l i k e groups and two s i n g l e p a i r combinations. 55 The f i r s t n e t - l i k e group involved f i v e d i f f e r e n t l o c i (AAT-2, DIA-2, MDH-3, PGI-2, and 6PG-1). Ten possible pair-wise combinations between these l o c i are presented i n Figure 6. Six combinations were av a i l a b l e to test for linkage. These combinations showed both non-s i g n i f i c a n t and s i g n i f i c a n t r e s u l t s . The non-significant r e s u l t s were for MDH-3:PGI-2 and PGI-2:6PG-1. The s i g n i f i c a n t r e s u l t s were f o r AAT-2:MDH-3, AAT-2:PGI-2, DIA-2:MDH-3, and MDH-3:6PG-1. Only one p a i r AAT-2:PGI-2 was found to be t i g h t l y l i n k e d with a recombination frequency of 1.5 percent. These two l o c i appear to be linked i n l o b l o l l y pine (Adams and J o l y , 1980), p i t c h pine (Guries j 2 t a l . , 1978), and ponderosa pine (0'Malley et a l . , 1979). Three combinations AAT-2:MDH-3, DIA-2:MDH-3, and MDH-3:6PG-1 were found to be loosely linked with recom-bin a t i o n frequencies of 40.0 percent, 32.7 percent, and 36.2 percent r e s p e c t i v e l y . These combinations were found i n only one tree, with the exception of the combination of MDH-3:6PG-1, which existed i n two trees. A linkage r e l a t i o n s h i p between MDH-3:6PG-1 was suggested for ponderosa pine by O'Malley et a l . (1979). The a v a i l a b l e combinations, however, did not allow f o r three-point linkage between these l o c i . I t seems premature to consider these f i v e l o c i to be within a si n g l e linkage group without the analysis of a d d i t i o n a l trees having these combinations. The second n e t - l i k e group involved four d i f f e r e n t . l o c i (AAT-3, EST-1, MDH-1, and SOD). A l l s i x possible pair-wise combinations are presented i n Figure 7. Five combinations were a v a i l a b l e to test f o r linkage. These combinations also showed both no n - s i g n i f i c a n t and s i g n i f i c a n t r e s u l t s . The non- s i g n i f i c a n t combinations were AAT-3:EST-1 and EST-1:SOD. S i g n i f i c a n t deviations were detected for AAT-3:SOD, 56 FIGURE 6: Pair-wise combinations between the f i v e l o c i included i n the f i r s t n e t - l i k e groupt AAT-2 DIA-2 MDH-3 PGI-2 6PG-1 AAT-2 - (2)* -DIA-2 - 1*. - -MDH-3 40.0 32.7 N.'Si.-, 1* PGI-2 1.5 - I N.S. 6PG-1 - - 36.2 I Significance; *, P<0.05; **, P<0.01; ***, P< 0.001. t The numbers above the diagonal represent the number of fa m i l i e s having the same combination and t h e i r l e v e l of s i g n i f i c a n c e . Numbers included i n parentheses represent combined data. Numerical values below the diagonal represent the recombination frequencies and I's i n d i c a t e the independence between l o c i . 57 FIGURE 7: Pair-wise combinations between the four l o c i included i n the second n e t - l i k e groupt AAT-3 EST-1 MDH-1 SOD AAT-3 N.S. - .'..1*** EST-1 I (3)* N.S. MDH-1 - 41.9 1* SOD 22.4 I 36.2 Sig n i f i c a n c e ; *, P<0.05; **, P<0.01; ***, P<0.001. t The numbers above the diagonal represent the number of fam i l i e s having the same combination and t h e i r l e v e l of s i g n i f i c a n c e . Numbers included i n parentheses represent combined data. Numerical values below the diagonal represent the recombination frequencies and I's i n d i c a t e the independence between l o c i . 58 EST-1:MDH-1, and MDH-1:SOD. One. p a i r (AAT-3:S0D) was found to be t i g h t l y linked with a recombination frequency of 22.4 percent. The other two combinations EST-1:MDH-1 and MDH-1:SOD were found to be loose l y l i n k e d , with recombination frequencies of 41.9 percent and 36.2 percent, respec-t i v e l y . The a v a i l a b l e combinations did not allow for three-point linkage between these l o c i . I t seems premature to consider these four l o c i to be i n another linkage group. The two si n g l e p a i r combinations were G6P:IDH and MDH-4:ME with recombination frequencies of 39.3 percent and 43.6 percent, r e s p e c t i v e l y . These combinations existed i n three trees only f o r G6P:IDH and four trees for MDH:ME. Summary and conclusion Linkage r e l a t i o n s h i p s among 19 d i f f e r e n t polymorphic l o c i were studied. A t o t a l of 115 of 171 possible two-locus combinations were tested. Out of the 115 combinations a t o t a l of 106 conform to the hypothesis of independence between l o c i i n d i c a t i n g that no linkage ex i s t s between these p a i r s . Of the remaining nine p a i r s , two were t i g h t l y linked with recombination frequencies of 1.5 percent ("AAT-2 :PGI-2) and 22.4 percent (AAT-3:S0D). The res t were lo o s e l y linked, with recombination frequencies varying between 32.7 percent and 41.9 percent. Two of these nine linked p a i r s have been detected i n other co n i f e r species. Three-point linkages were not studied due to the lack of appropriate combinations. 59 These r e s u l t s are the f i r s t reported for linkage i n Douglas-fir and represent a beginning i n chromosome mapping for t h i s species. To achieve accurate chromosome maps, however, a d d i t i o n a l studies are needed and they should involve increasing the number of trees, l o c i , and megagametophytes per tree. 3.3 Estimation of the outcrossing rate (t) Forest trees are known to predominantly exhibit outbreeding (Stern and Roche, 1974), but i t i s also known that t h i s parameter of the mating system i s subject to natural s e l e c t i o n (Kahler, et a l . , 1976). Therefore, an important p r e r e q u i s i t e for understanding and manipulating the genetic structure of a species i s a knowledge of i t s breeding system. The major d i f f i c u l t y i n estimating the o v e r a l l mating system of tree species has been the a v a i l a b i l i t y of s u i t a b l e genetic markers. The two most frequently used markers' - dominant and recessive - are i d e n t i f i e d by examining phenotypic appearances and r a t i o s i n the F2 and l a t e r genera-t i o n s . However, there are d i f f i c u l t i e s associated with the p r a c t i c a l a p p l i c a t i o n of these methods, e s p e c i a l l y i n forest trees with long l i f e c y c l e s . For example, i t i s impossible to d i f f e r e n t i a t e between heterozygous dominant and homozygous dominant i n d i v i d u a l s without progeny t e s t i n g . Furthermore, a large number of progeny i s usually required to i d e n t i f y homozygous recessive i n d i v i d u a l s . These problems can be over-come with the use of isozyme techniques. The codominant nature of allozymes enables one to i d e n t i f y not only the phenotype but also the genotype. Estimates from outcrossing and inbreeding f o r annual plant species based on allozyme polymorphisms were reported i n barley (Hordeum vulgare) 60 by A l l a r d et a l . (1972), i n corn (Zea maize) by Brown and A l l a r d (1970), and i n wild oat by Marshall and A l l a r d (1970). In contrast to annual plants, quantitative estimates of the mating system of forest trees using the a i d of allozyme polymorphism are rare. Brown et al_. (1975) reported that the outcrossing rate for four- Eucalyptus obliqua populations was 76 percent over three l o c i , Muller (1976) estimated an 85.3 percent outcrossing rate i n Norway spruce using a s i n g l e locus as a marker, and Rudin et; a l . (1977) found t h i s rate to be between 76 and 83 percent i n Scots pine over three l o c i . An attempt to estimate the outcrossing rate for a stand of Douglas-fir using isozyme polymorphisms i s described i n the following section. Data c o l l e c t i o n Isozyme data were c o l l e c t e d from the haploid megagametophyte and d i p l o i d embryo f o r each s i n g l e seed. Four l o c i (G6P, IDH, PGM, and 6PG-1) were used to estimate the outcrossing rate. These four l o c i were chosen because of the c l a r i t y of the zymograms when embryonic material was used as an enzyme source. The c l a r i t y greatly decreased the proba-b i l i t y of m i s i n t e r p r e t a t i o n of any of the phenotypes. A t o t a l of 36 trees were included i n t h i s study. Ten open-pollinated seeds from each tree were used for the enzyme assay. Data i n t e r p r e t a t i o n The maternal genotype has a major impact on the i d e n t i f i c a t i o n of the p o l l e n contribution. Two mutually exclusive p o s s i b i l i t i e s existed, assuming that there are three a l l e l e s ( A i , A2 and A3) i n the enzyme system: 61 1. The maternal genotype was homozygous for A^A^. Then i t s contribu-t i o n would be A} i n a l l cases. The possible embryo genotypes would either be AjA^, A^A 2 or A1A3. Embryos having an A^A 1 c o n s t i -t u t i o n would e x h i b i t a banding pattern described by a s i n g l e band having the same r e l a t i v e migration distance as the corresponding megagametophytes. For the heterozygous embryos the banding pattern would be a two-banded configuration f o r a monomeric enzyme ( i . e . PGM) or a three-banded configuration i n the case of dimeric enzymes ( i . e . G6P, IDH, and 6PG-1). 2. The maternal genotype was heterozygous for A i A 2 . The possible embryo genotypes would vary according to the contr i b u t i o n of the female parent tree: , (a) When the maternal contribution was Aj, the embryo genotypes would either be AjA^, A^A 2 or A1A3. (b) When the maternal contribution was A 2, the embryo genotypes would either be A 2 A ^ A 2A 2 or A 2A3» S t a t i s t i c a l analyses A l l e l e frequencies f o r the four l o c i studied were computed from the genotypic frequency of 360 embryos. Conditional p r o b a b i l i t i e s f o r obtaining progeny of a p a r t i c u l a r genotype were used to estimate the out-crossing rate ( t ) . Conditional p r o b a b i l i t i e s were necessary because one-ha l f of the embryo genotype was known from the corresponding megagametophyte. Estimation procedures f o r the outcrossing rate (t) varied according to the maternal and progeny genotypes. The following formulae were used 62 estimate t : 1) Homozygous progeny from homozygous (A1A1) maternal tree: P ( A l A l ) = tp. + (1 - t) .'. t = P ( A l A i ) - 1 , 2) Heterozygous progeny from homozygous (A^Ai) maternal tree: P(AlA ±) = t p A t = p ( A l A i ) , 1 PA. l 3) Homozygous progeny from heterozygous maternal tree of given Aj a l l e l e : ± _ . 2{p(A 1A 1)} - 1 p ( A l A l ) = t p A i + - y - t = 2 p A i _ 1 , 4) Heterozygous progeny from heterozygous maternal tree of given A^ a l l e l e : •1 _ t 2{p(A iA 1)} - 1 p ( A l A i ) = tp + - y - .'. t = _ 1  where t = outcrossing rate. p = frequency of A, a l l e l e from the progeny information A| p = frequency of A. a l l e l e from the progeny information A ± I pCA^A^) = frequency of A-JAJ progeny from the t o t a l progeny of the AJAJ parent trees. PCA^JL) = frequency of A^A^ progeny from the t o t a l progeny of the AjA^ parent trees. 63 For each locus, t values were calculated f o r each condition, and a weighted t was computed. F i n a l l y an o v e r a l l t was calculated by averaging the d i f f e r e n t t values f o r each locus. Results and conclusion Data f o r the estimated outcrossing rate (t) are presented i n Table 22 for each locus separately and combined over four l o c i . TABLE 22: Estimated outcrossing rate. Locus t G6P 0.8427 IDH 1.0213 PGM 0.6233 6PG-•1 1.1267 Average (t) 0.9035 + 0.1103 The estimated outcrossing rate (t) averaged 0.9 over four l o c i . The resultant inbreeding rate was, therefore, 0.1. This i s i n good agreement with the previously reported value of seven percent for d i f f e r e n t Douglas-fir trees from western Oregon, using a recessive mutant as a genetic marker (Sorensen, 1973). Langner (1966) reported that an increase i n the c o e f f i c i e n t of inbreeding (F) of 0.1 i s accompanied by a height growth reduction of about f i v e percent i n several plant species. Therefore, the estimated value of F from the mathematical r e l a t i o n s h i p F = (1 - t ) / ( l + t)(Brown 64 et a l . 1975) y i e l d e d 0.051, suggesting an expected reduction i n height growth of approximately 2.64 percent. In other words, average stem height could be increased by about 2.64 percent by eliminating natural inbreeding. The d r a s t i c reduction of v i a b l e seed on inbred trees (Orr-Ewing, 1965) and higher mortality among inbred seedlings are possible s e l e c t i v e mechanisms against inbreds i n natural populations. On the other hand, Eriksson and Lindgren (1975) showed that under optimum nursery conditions, selfed plants have grown equally as well as seedlings which originated from outcrossing. Therefore, there ;is. l i m i t e d opportunity to rogue out the inbred o f f s p r i n g before outplanting. This increases the r i s k of l o s s i n wood p r o d u c t i v i t y . I t i s expected that inbreeding could occur i n seed orchards where randomization of ramets i s la c k i n g . In order to avoid possible inbreed-ing depression i n progeny obtained from seed orchards the ramets of each clone should not be planted i n close proximity. Through proper design, reduced p r o d u c t i v i t y of the next generation could be eliminated. 3.4 E f f e c t of sample s i z e on the p r e c i s i o n of the estimate of a l l e l e frequency. The genetic a r c h i t e c t u r e of natural populations can be described by estimates of a l l e l i c frequencies within and between populations. Allozyme frequencies have been extensively used by several authors to study population structure i n forest trees (Sakai and Park, 1971; Bergmann, 1973; Tigerstedt, 1973; Feret, 1974; Muhs, 1974; Rudin et a l . , 1974; Copes and Beckwith, 1977; Fowler and Morris, 1977; Guries and Ledig, 1977; Mitton et a l . , 1977; Yang et a l . , 1977; Bergmann, 1978; 65 O'Malley et a l . , 1979; Yeh and El-Kassaby, 1980; Yeh and Layton, 1979; and Yeh and O'Malley, 1980). In the above investigations the number of trees sampled from each population varied from one to 153. The number of seeds analysed from each tree also varied widely (Table 23). The determination of an adequate sample s i z e from the base popula-t i o n which would r e l i a b l y estimate a l l e l i c frequencies was not given proper att e n t i o n . I t must be emphasized that estimates obtained by any sample should be interpreted r e l a t i v e to the population from which the experimental material was taken. Considerable bias i n estimates of the population a l l e l i c frequencies occurs when a small number of trees i s sampled from the base population. The following section presents the in v e s t i g a t i o n of the e f f e c t of sample s i z e on the estimation of a l l e l e frequencies. Materials and methods The previously described 42 trees were again used i n t h i s a n a l y s i s . Their genotypes were determined by analysing eight megagametophytes from each tree (section 2.2). A l l e l i c frequencies f o r each enzyme system were estimated from genotypic frequencies. D i a l l e l i c data were obtained through a grouping procedure described by Brown «it a l . (1975) . A l l e l e s other than the 100 form were placed into a s i n g l e c l a s s . I t was not f e a s i b l e to do t h i s f o r 'EST-1 and APH-2. because the varia n t a l l e l e s exhibited s i m i l a r frequencies. Therefore, these two l o c i were excluded from t h i s section. Two procedures were used to f i n d the optimum number of trees required to obtain r e l i a b l e estimates of the a l l e l e frequency. The f i r s t method u t i l i z e d graphs of standard error estimates of a l l e l i c TABLE 23: Review of different sample sizes used for different species. Species If of trees 9 of seeds/ tree Enzyme source Reference Douglas-fir Japanese cedar (Cryptomerla japonlca) Lodgepole pine Norway spruce Ponderosa pine Pitch pine Red pine (Pinus reslnosa) Scots pine Sitka spruce Table mountain pine (Pinus pungens) 96 seedlings/provenance 10-20 trees/provenance Bulk collection of 120 seeds from <* 100 treea/provenance 50 trees/site 15 trees/stand 15-20 trees/population 18-25 trees/population 50 trees/population 70-90 trees/slope 4-5 trees/stand 61-153 trees/population 1-several trees/location 20-132 trees/location > 5 trees/stand or unknown number 20 trees/provenance 15-20 trees/stand 8 seeds 9 seeds 9 seeds 6 seeds 6-8 seeds 6-8 seeds Needles Germinants* Megagametophytes Leaves Kuhs (1974) Yang et a l . (1977) Yeh and O'Malley (1980) Sakai and Park (1971) Megagametophytes Yeh and Layton (1979) Megagametophytes Megagametophytes Megagametophytes Needles Megagame to phytes Megagametophytes Megagametophytes Needles Seedlings* Megagametophytes Needles and Megagametophytes Bergraann (1973) Bergmann (1978) Tigerstedt (1973) Mitton et a l . (1977) O'Malley et a l . (1979) Guries and Ledig (1977) Fowler and Morris (1977) Rudin et a l . (1974) Copes and Beakwith (1977) Yeh and El-Kassaby (1980) Feret (1974) * The whole germinant or seedling used as enzyme source. ON ON 67 frequencies (S.E.q) against sample s i z e s . By v i s u a l inspection, the point of maximum curvature was determined where there was no s u b s t a n t i a l change i n the p r e c i s i o n of the estimates of the a l l e l e frequencies as the number of trees increased. The second procedure u t i l i z e d t - t e s t s to determine the optimum number of trees when estimates of a l l e l e frequencies appeared to be r e l a t i v e l y s t a b l e . Since d i a l l e l i c data were provided, the methods f o r estimating the standard error for a l l e l e frequency (S.E.q) and t - t e s t f o r each sample s i z e were based on a binomial d i s t r i b u t i o n (Falconer, 1960; Spiess, 1977; and Mendenhall and Scheaffer, 1973). Separate analyses using sample groups of 5 to 42 trees were ca r r i e d out to check f o r the e f f e c t of sample s i z e on a l l e l i c frequency estimation. For each sample, trees were selected randomly only a f t e r those previously selected had been replaced ( i . e . sampling with replace-ment) . A l l e l e frequencies and t h e i r standard errors were estimated f o r each sample s i z e and a t - t e s t was conducted to compare the a l l e l e frequency estimates with the whole population estimates. Equations for c a l c u l a t i n g S.E.q and t-value were: S.E. ( q ) ± t. q. - q 1 o , with d.f. = 2n. + 2n - 2 1 o 68 where S.E. (q)^ = standard error estimate of a l l e l e frequency at n^ sample s i z e . t. = t-value at n. sample s i z e . 1 1 p_^  = frequency of the most common a l l e l e at n_ sample s i z e . q_^  = frequency of the other a l l e l e at n^ sample s i z e = 1 -n^ = # of d i p l o i d i n d i v i d u a l s sampled. P o = frequency of the most common a l l e l e for the 42 trees. q Q = frequency of the other a l l e l e f o r the 42 trees. n = 42 trees. o To check the r e p e a t a b i l i t y of the r e s u l t s , three d i f f e r e n t random runs (A, B, and C) were done for each locus separately. Based on the frequency of the rare a l l e l e , the 25 a v a i l a b l e l o c i were considered under the following four a r b i t r a r y categories. 1. The frequency of the rare a l l e l e (q) equal to 0.0 (monomorphic l o c u s ) . A t o t a l of nine l o c i f a l l into t h i s category. These were AAT-1, GAPDH, GDH, MPI-1, MPI-2, MDH-2, PEP-1, PEP-2, and PEP-3. Due to the monomorphic nature of these l o c i , no sampling work could be done and one or few trees could be a good representative sample of the population. 2. The frequency of the rare a l l e l e (q) equal to or less than 0.05. A t o t a l of s i x l o c i f a l l into t h i s category. These were AAT-3, DIA-1, 6PG-1, 6PG-2, PGI-2, and SOD. The PGI-2 locus was selected at random to represent t h i s category. 69 3. The frequency of the rare a l l e l e (q) between 0.1 and 0.2 i n c l u s i v e . This^category contains seven l o c i : AAT-2, ACO, IDH, MDH-1, MDH-3, ME, and PGM. The MDH-3 locus was selected at random to represent t h i s category. 4. The frequency of the rare a l l e l e (q) almost equal to the frequency  of the common a l l e l e (p) -0.5. The remaining three l o c i f a l l i nto t h i s category. These were HA, MDH-4, and G6P. The MDH-4 locus was selected at random to represent t h i s category. Results and discussion Data of a l l e l e frequencies and t h e i r standard error estimates including t-values for the d i f f e r e n t sample si z e s f o r each locus f o r the three random runs (A, B, and C) are presented i n Appendix 2 and Figures 8, 9, and 10. Interpretation for each locus w i l l be presented separately and a conclusion w i l l be proposed regarding the multilocus case. The PGI-2 locus: The graphs i n Figure 8 and the data i n Appendix 2 showed a sharp f l u c t u a t i o n i n the standard error of the a l l e l e frequencies estimates, i n p a r t i c u l a r f o r small sample sizes (5-32). This f l u c t u a t i o n could be due to the low frequency of the rare a l l e l e (q) which makes i t d i f f i c u l t to detect t h i s a l l e l e i n many of these sample s i z e s . Then the frequency of q. i n t h i s case w i l l be equal to zero and the S.E.q w i l l also drop to zero. For instance S.E.q estimates of zero were obtained i n sample sizes of 5, 6, 7, 11, and 13 i n the f i r s t random run (A); i n sample sizes of 6, 8, 11, 13, 18, 29, and 32 i n second run (B); and i n sample s i z e s of Standard error estimates of a l l e l i c frequencies for PGI-2, f o r d i f f e r e n t sample s i z e s . The three random runs are presented i n the A, B, and C graphs. 72 7, 8, 17, 19, 24, and 30 i n the t h i r d run (C). In contrast, the amount of f l u c t u a t i o n beyond a sample s i z e of 32 trees i n the second run (B) was not large and a p l a t e a u - l i k e condition was achieved. The stable conditions of the S.E.q estimates were pronounced i n the two other random runs (A and C). This indicates that with a rare a l l e l e (q < 0.05) a sample s i z e of 32 or more trees i s needed to detect t h i s a l l e l e i n the population. The t - t e s t r e s u l t s , on the other hand, were not s e n s i t i v e enough to detect the differences between a l l e l e frequencies among d i f f e r e n t sample s i z e s . This lack of s e n s i t i v i t y could be due to very small differences between a l l e l e frequencies as w e l l as to the reduction of the variance of a l l e l e frequencies as n^ increased. The MDH-3 locus: Only one incident of reaching f i x a t i o n f o r the most common a l l e l e (p = 1.0) and a subsequent zero value f o r S.E.q estimate was obtained. This was pronounced i n random run A when sample s i z e s of 5 and 8 were used (Figure 9,A). A sharp decline i n the S.E.q estimates was noticed as the number of trees increased. This decline was accompanied by some f l u c t u a t i o n i n the S.E.q estimates, and the pl a t e a u - l i k e condition was achieved a f t e r a sample s i z e of 35 trees i n run A; 30 trees i n run B; and 35 trees i n run C. The amount of f l u c t u a t i o n beyond the sample s i z e of 35 trees was n e g l i g i b l e . S i g n i f i c a n t t-values (a = 0.05) were obtained when the rare a l l e l e was l o s t (q = 0). The lack of s e n s i t i v i t y of the t - t e s t , on the other hand, was also present i n t h i s case. I t i s concluded that a sample s i z e of 35 trees or more i s required to obtain accurate estimates of a l l e l e frequencies when systems have rare a l l e l e frequencies l y i n g between 0.1 and 0.2. 73 FIGURE 9. Standard error estimates of a l l e l i c frequencies f o r MDH-3, for d i f f e r e n t sample s i z e s . The three random runs are presented i n the A, B, and C graphs. 75 The MDH-4 locus: Two d e c l i n i n g zones were observed i n Figure 10. The f i r s t zone was between a sample s i z e of 5 to 15 trees. The rate of decline i n t h i s zone was sharp and was accompanied by some f l u c t u a t i o n i n the S.E.q estimates. The second zone occurs beyond a sample s i z e of 15. This zone had a rate of decline of l e s s than that of the f i r s t zone but the range was greater. The plateau condition was not achieved due to the steady decline i n the S.E.q estimate. I t appears that i n an enzyme system with almost equal frequencies f o r two a l t e r n a t i v e a l l e l e s (p and q), a sample s i z e of more than 42 trees i s needed to get r e l i a b l e estimates of a l l e l e frequencies. Summary Unbiased estimations of a l l e l e frequencies and t h e i r respective standard error estimates were obtained for l o c i i d e n t i f i e d by Mendelian segregation i n haploid megagametophyte tissues from i n d i v i d u a l trees. By a minimum sampling variance c r i t e r i o n , the optimum sample s i z e (when eight megagametophytes per tree are used to give a 0.99 p r o b a b i l i t y estimate of the true genotype of the parent tree) i s not l e s s than 32 trees when the frequency of the rare a l l e l e i s equal to or l e s s than 0.05, not l e s s than 35 trees when the frequency of the rare a l l e l e i s between 0.1 and 0.2, and more than 42 trees when the enzyme system has almost equal frequencies for p and q. Furthermore, Guries and Ledig (1977) reported (after extensive sampling for four populations of p i t c h pine with sample sizes which va r i e d between 61 to 153 trees) that i n a large population with a wide outcrossing rate, predictable patterns of gene frequencies w i l l e x i s t , and therefore sampling should not be as intense. 76 FIGURE 10. Standard error estimates of a l l e l i c frequencies for MDH-4, for d i f f e r e n t sample s i z e s . The three random runs are presented i n the A, B, and C graphs. 78 Since most information on enzyme systems could be obtained from protein extraction of an i n d i v i d u a l seed, r e s u l t s of t h i s study suggested that a sample s i z e of more than 42 trees (between 42 to 60 trees) i s needed to obtain r e l i a b l e estimates for allozyme frequencies. These estimates w i l l f u r n i s h base material f o r the study of genetic a r c h i t e c t u r e of natural populations. 3.5 Gene d i v e r s i t y within the " G r i f f i t h " population. Natural populations contain large amounts of v a r i a b i l i t y f o r both quantitative and q u a l i t a t i v e characters. Quantitative characters are generally affected by both genetic and environmental f a c t o r s . On the other hand, the v a r i a t i o n i n q u a l i t a t i v e characters i s almost e x c l u s i v e l y determined by genetic f a c t o r s . The extent of genetic v a r i a b i l i t y i n natural populations has been extensively studied with respect to quantita-t i v e characters using mainly quantitative genetic methods. But these studies could not give much i n s i g h t into v a r i a t i o n at the s i n g l e gene l e v e l since the.relationship between phenotypes of these characters and genes i s so complicated (Nei, 1975). Development of starch gel e l e c t r o -phoresis, however, provided a valuable t o o l by which genetic heterogeneity of proteins and isozymes can e a s i l y be detected. Nei (1973) devised a set of s t a t i s t i c s that describes the amount and structure of genetic v a r i a b i l i t y i n subdivided populations. These measures enable t o t a l genetic v a r i a b i l i t y to be p a r t i t i o n e d within and between sub-populations. Electrophoretic r e s u l t s also can be compared with quantitative data from f i e l d t r i a l s . These two d i f f e r e n t types of information can also be used to complement each other (Allendorf and Utter, 1978). 79 Several species showed close agreement between the e l e c t r o -phoretic and the quantitative data for the estimation of v a r i a b i l i t y between and within populations. There i s as yet l i t t l e information a v a i l a b l e on these aspects. Douglas-fir Yeh and O'Malley (1980) reported that three percent of genie v a r i a t i o n i n coastal Douglas-fir was due to interpopulation gene dif f e r e n c e s . They also extended the analysis of gene d i v e r s i t y to the work of Yang e_t a l . (1977) on isozyme v a r i a t i o n i n another c o l l e c t i o n of coastal Douglas-fir, and the r e s u l t s were s t r i k i n g l y s i m i l a r . On the other hand, Fashler (1979) reported that the majority of the genetic v a r i a t i o n i n height growth of j u v e n i l e Douglas-fir trees i n the International Union of Forestry Research Organization (I.U.F.R.O.) provenance-progeny test could be a t t r i b u t e d to within provenance e f f e c t s for two seed zones. However, t h i s trend was reversed f o r two other seed zones. The apparent c o n t r a d i c t i o n may be explained by d i f f e r e n t adaptation responses of the d i f f e r e n t provenances to the progeny test s i t e . Lodgepole pine Yeh and Layton (1979) pointed out that four percent of the detected genie v a r i a t i o n was due to inter-population gene d i f f e r e n c e s . This high l e v e l of w i t h i n population isozyme v a r i a t i o n i n t h i s species was p a r a l l e l e d by observations on general p h y s i o l o g i c a l functions (Perry and Lotan, 1977). 80 Ponderosa pine O'Malley ej^ a l . (1979) estimated that approximately 12 percent of genetic v a r i a t i o n detected e l e c t r o p h o r e t i c a l l y was associated with genie differences between stands. This large d i f f e r e n c e within stands was also reported by 'Madsen and Blake, 1977. They a t t r i b u t e d 71.5 percent of v a r i a b i l i t y i n 2-year height growth to within- and among-family v a r i a t i o n , and the remaining 28.5 percent to among-stand v a r i a t i o n . Sitka spruce Yeh and El-Kassaby (1980) estimated that eight percent of t o t a l genetic v a r i a t i o n existed between populations by using isozyme data from ten I.U.F.R.O.. provenances of S i t k a spruce. Illingworth (1978), using the same provenances, reported that 11 percent of t o t a l v a r i a t i o n f o r 3-year height growth of seedlings was a t t r i b u t e d to among population e f f e c t s . The above studies indicated a r e l a t i v e l y high amount of genetic v a r i a t i o n within populations. In contrast, genetic uniformity was detected i n red pine for both e l e c t r o p h o r e t i c (Fowler and Morris, 1977) and quantitative data (Fowler and Lester, 1970 and Thielges, 1972). These studies, thus, indicated that the technique of g e l e l e c t r o -phoresis could complement t r a d i t i o n a l studies i n estimating the amount and extent of genetic v a r i a b i l i t y i n f o r e s t tree species. Nei's (1975) analysis of gene d i v e r s i t y was used f o r a l l of the previously described studies which considered the whole range of the species as a population and the provenance as a l o c a l or sub-population. The following section i s a presentation of a d i f f e r e n t approach which was employed to study both electrophoretic and quantitative data within a 81 s i n g l e population with respect to four d i f f e r e n t e l e v a t i o n a l classes or sub-populations [A:366 - 381 m (1200-1250'), B:381 - 396 m (1250-1300'), C:396 - 412 m (1300-1350'), and D: >412 m (1350')] i n an attempt to study micro-genetic d i f f e r e n t i a t i o n and to compare electrophoretic and quanti-t a t i v e data obtained from a s i n g l e population. Data used f o r gene d i v e r s i t y analysis and analyses of variances are c o l l e c t e d i n sections 2.2 and 2.3 r e s p e c t i v e l y . Tree number 28 was not included i n the analyses of variances because of a l i m i t e d number of f i l l e d seeds (15). Gene d i v e r s i t y analysis Levels of genie v a r i a b i l i t y and i t s h i e r a r c h i c a l organization can be quantified (Nei, 1975). This analysis permits genetic v a r i a t i o n to be p a r t i t i o n e d w i t h i n d i f f e r e n t h i e r a r c h i c a l l e v e l s of population s t r u c -ture [within versus between sub-populations ( e l e v a t i o n s ) ] . The basis for the analysis i s the c a l c u l a t i o n of expected or t h e o r e t i c a l heterozy-gosity per locus (h): where x^ i s the i a l l e l e at the locus i n question. The average heterozygosity of the population, which i s the average of h over r l o c i sampled, i s estimated by: h = 1 -th r H = Organization of gene d i v e r s i t y within the ' G r i f f i t h " population (stand) can be described by the r e l a t i o n s h i p H T " Hs + D: ST 82 where t o t a l gene d i v e r s i t y (H^) i s p a r t i t i o n e d into gene d i v e r s i t y w i t h i n four sub-populations (H ) and that between sub-populations (D ). These O J 1 values are calculated.for each locus separately and averaged over a l l l o c i (Table 25). Tota l genie v a r i a t i o n (H^ ,) i s a function of mean a l l e l e frequencies and i s estimated as H T = 1 - Z x 2 ± — th where x_^  i s the weighted mean frequency of i a l l e l e over a l l sampled sub-populations. The average amount of genetic v a r i a t i o n maintained within any sub-population i s estimated by H^. Since four sub-populations are . examined, average gene d i v e r s i t y w i t h i n any sub-population at a p a r t i c u l a r locus i s : where h^ i s the expected heterozygosity i n the k.1"*1 sub-population. The l e v e l of d i f f e r e n t i a t i o n between sub-populations i s calculated by D g T, and i s given by: DST = H T " H S If a l l sub-populations are members of a si n g l e large panmictic unit and there i s no s e l e c t i o n for l o c a l adaptation, then a l l a l l e l e s w i l l be equally d i s t r i b u t e d over the en t i r e range of the population and w i l l equal H . However, i n nature t h i s i s not the case because natural populations tend to d i f f e r e n t i a t e over time into sub-populations due to genetic processes of mutation, s e l e c t i o n , random d r i f t , and r e s t r i c t e d 83 gene flow. Therefore, H w i l l be a subset of "-.H •.,. The greater the l e v e l of sub-division within a stand, the l a r g e r the value of Dg^-The r e l a t i v e amount of genetic d i f f e r e n t i a t i o n between sub-populations (Ggrr.) can be estimated by: G = °ST T This value varies from zero to one and i s c a l l e d the c o e f f i c i e n t of gene d i f f e r e n t i a t i o n . Sampling variance and standard error of Gg T value [V(Gg T)] were calculated following Chakraborty (1974), to test f o r s i g n i -ficance at the stand sub - d i v i s i o n assessed. The equation f o r c a l c u l a t i n g V(G g T) i s as follows: v r r >^ r2 ( V ( D S T > V ( R T ) 2 Cov.(H T,D S T) ) v<^ S T; „ ^ S T i + i ( D 2 s t H 2 t D s t . H T ) Analysis of variance The analysis f o r estimation of the v a r i a t i o n within and between elevations was based on the following l i n e a r model: Y..,. = u + E. + T... . + S + e .... i j l k i ( i ) j ( i j ) l ( i j l ) k where Y..., • := mean measurement of the k ^ r e p l i c a t i o n i n the l * " * 1 i j l k v t h seedling i n the j tree i n the i elevation. u = mean of a l l r e p l i c a t i o n s over a l l seedlings, trees, and elevations. t i l E. = e f f e c t of the i elevation. 1 T.. v. =^ e f f e c t of the i * " ^ 1 tree within the i * " * 1 elevation. ( i ) j 84 t i l ttl S,..,. = e f f e c t of the 1 seedling within the j tree within (xj)p t h the i elevation. : ( i j l ) k = random experimental e r r o r . Sources of v a r i a t i o n s , degrees of freedom, and expected mean squares f or analysis of variance are given i n Table 24. TABLE 24: Analysis of variance table. Degrees of Source of v a r i a t i o n . , Expected mean squares freedom _ Among elevations t-1 a 2 g + ^ a 2 , ^ + Kka + K 6 a 2 E Between trees/elevation n-t ° 2 & + K 2 ° S / T + ^ ^ T / E Between seedlings/tree/ m-n °\+ "^3<^s/T elevation Error p-m a 2 where t = number of elevation ttl n = En., where n. = number of trees i n i elevation l l t i l m = Elm... where m.. = number of seedlings i n the j tree i n the i ^ elevation p = EEEp.., > where p.., = number of r e p l i c a t i o n s i n the IJ K 1J K seedling i n the j t n tree i n the i t n elevation a 2 e = r e s i d u a l variance CT 2S/T = variance between seedlings within tree a 2 T / E = variance between trees within elevation a2-g = variance among elevations Ki - Kg = c o e f f i c i e n t s of the variance components. 85 As the experimental design was unbalanced due to varying numbers of trees within elevations and missing seedlings due to mortality, the s t a t i s t i c a l method described by Sokal and Rohlf (1969) was used to c a l c u l a t e values of the c o e f f i c i e n t s of variance components (K\ - K 5 ) . This method involves computation of seven d i f f e r e n t basic quantities (A - G). Methods f o r c a l c u l a t i n g these quantities and the c o e f f i c i e n t s are described below: EEEn 2.., A = ... l j k x j l J EEn 2.. B = . . xj En 2. C = . i 1 EEEn..., D = .., l j k E E ^ - ^ " . . En. ., i j 1 i j k EEn 2. ., • i !jk . EEn.., 1 j l l j k En 2.. j 1 J E J  K 7 En. . 1 j 1 3 F - (A/D) 1 _ t-1 E - F T>2 = n-t 86 K 3 = ^ ° m-n „ _ G - (B/D) D - G K 5 = n-1 _ D - (C/D) Appropriate c a l c u l a t i o n s f o r each of the variance components ( c 2 e , CT2g/i>> a 2 x / E ' a n c ^ ° 2 E^ synthesized new mean squares to be used as denominators of F r a t i o s . The value of each of these factors was then determined. F i n a l l y Duncan's multiple-range test f o r unequal number of groups was used to compare between d i f f e r e n t elevations (Steel and T o r r i e , 1960). Results and discussion Gene d i v e r s i t y analysis Analysis of gene d i v e r s i t y i n the " G r i f f i t h " population f o r d i f f e r e n t e l e v a t i o n a l classes i s presented f o r each locus separately and combined over a l l l o c i i n Table 25.and Appendix 3. Data i n Table 25 showed considerable v a r i a t i o n f o r both and H^. The value of G_T also varied considerably between l o c i , ranging from 0.0 to 0.216 for the 27 l o c i investigated. The mean value of Gg^ was 0.068, so about seven percent of detected genie v a r i a t i o n i n the stand was due to i n t e r - e l e v a t i o n a l gene d i f f e r e n c e s . Thus, the majority of genie v a r i a b i l i t y (93 percent) resided within elevation. The standard error of 0.014 f o r G.^ [given by the square root of the expression i n TABLE 25: Analysis of gene d i v e r s i t y and degree of d i f f e r e n t i a t i o n at 27 l o c i between d i f f e r e n t elevations. Proportion of Gene d i v e r s i t y i n t e r e l e v a t i o n Locus To t a l gene within gene d i v e r s i t y elevations d i f f e r e n t i a t i o n (H T) (H s) <6ST> AAT-1 0.000 0.000 0.000 AAT-2 0.233 0.229 0.019 AAT-3 0.091 0.088 0.029 ACO 0.262 0.228 0.130 APH-2 0.697 0.644 0.076 DIA-2 0.024 0.023 0.042 EST-1 0.690 0.677 0.019 GAPDH 0.000 0.000 0.000 GDH 0.000 0.000 0.000 G6P 0.523 0.444 0.151 HA 0.540 0.511 0.054 IDH 0.216 0.203 0.060 MDH-1 0.156 0.152 0.024 MDH-2 0.000 0.000 0.000 MDH-3 0.251 0.197 0.216 MDH-4 0.561 0.511 0.089 ME 0.212 0.204 0.039 MPI-1 0.000 0.000 0.000 MPI-2 0.000 0.000 0.000 PEP-1 0.000 0.000 0.000 PEP-2 0.000 0.000 0.000 PEP-3 0.000 0.000 0.000 PGI-2 0.091 0.089 0.019 PGM 0.406 0.400 0.014 6PG-1 0.047 0.046 0.020 6PG-2 0.069 0.067 0.030 SOD 0.070 0.067 0.043 Combining a l l 0.190 0.177 0.068 27 l o c i Standard error 0.044 0.041 0.014 88 V(G„ )] however, indicates that t h i s amount of subdivision i n t h i s ST stand was s t a t i s t i c a l l y s i g n i f i c a n t . These data suggested that the micro-genetic d i f f e r e n t i a t i o n i n the stand was not strongly pronounced at the random sample of l o c i examined. This indicates that there was genetic continuity within these e l e v a t i o n a l classes. This lack of d i f f e r e n t i a t i o n on the micro-genetic l e v e l was i n agreement with the previously l i s t e d e l e c t rophoretic data on the macro-genetic l e v e l i n coniferous species which declared that the majority of gene d i v e r s i t y existed w i t h i n populations. This high l e v e l of within elevation and/or w i t h i n population i n isozyme v a r i a t i o n i s , perhaps, a r e f l e c t i o n of the species' (Douglas-fir) e c o l o g i c a l ampli-tude, i t s breeding system, and lack of e f f e c t i v e b a r r i e r s to gene flow between sub-populations. This high degree of genetic v a r i a b i l i t y which has been noted by numerous authors, has been expressed by Larsen (1937) i n the following way: "... one has to t r a v e l very widely throughout the natural range of Douglas-fir i n order to get an impression of differences i n geographical type, but standing on one place one can, without moving a foot, see many i n d i v i -duals d i f f e r i n g widely i n t h e i r structure; i t i s often more d i f f i c u l t to pick out those that resemble one another. Although they belong to the same species and the same geographical type, yet they can be widely divergent i n respect of t h e i r economic value i n f o r e s t r y . . This can .be observed elsewhere. I t does not matter i f one chooses i n C a l i f o r n i a a s i t e i n the Coast Range or i n the S i e r r a Nevada, passes through Oregon and Washington, or i n B r i t i s h Columbia sel e c t s a place on Vancouver Island or i n the Rocky Mountains; everywhere one i s bound to be impressed by the great i n d i v i d u a l v a r i a t i o n of t h i s tree species." 89 The present analysis, even with t h i s small number of trees, has substantiated the previous statement. However, Allendorf et^ a l . , (.1977) and Nei (1975) reported that including a large number of l o c i i n the analysis of gene d i v e r s i t y i s more important than the number of in d i v i d u a l s i n acquiring a general p i c t u r e of gene d i f f e r e n t i a t i o n among sub-populations. Analysis of variance Due to the d i f f e r e n t number of trees i n each of the four sub-populations ( e l e v a t i o n a l classes A, B, C, and D) and to missing seedlings due to mortality, an unbalanced set of data was a v a i l a b l e f o r the analyses. Therefore, two attempts were made to evaluate the e f f e c t of elevation on the response of seven d i f f e r e n t characters. The f i r s t attempt involved random elim i n a t i o n of some trees from e l e v a t i o n a l classes with large numbers of trees i n order to balance the number of trees within each elevation c l a s s . This resulted i n four trees i n each c l a s s . The second attempt included a l l of the trees which existed i n each e l e v a t i o n a l class (18 trees f o r A, 14 trees f o r B, 4 trees f o r C, and 5 trees f o r D). Variance component estimates ( ° 2 E » ^ T / E ' ^ S / t / E ' a n d a 2R/S/T/E^ as a percentage of t o t a l v a r i a t i o n f o r the balanced and unbalanced designs are presented as well as a combined within elevation v a r i a t i o n ( a 2W = G 2 T / E + a 2 S / T / E + 0 2R/S/T/E ) ± N T A B L E 2 6 F ° R S 6 V E N C H A R A C T E R S ' The amount of v a r i a t i o n due to d i f f e r e n t e l e v a t i o n a l classes varied from zero (number of needles and length of e p i c o t y l and hypocotyl) to 9.62 percent (number of cotyledons) i n the balanced set. V a r i a t i o n between elevations was s t a t i s t i c a l l y s i g n i f i c a n t with respect to number TABLE 26: Estimates of the variance components and their l e v e l of significance for the different seven characters for both analyses (balanced and unbalanced) Equal t of trees/elevation Unequal // of trees/elevation Character ° T / E ° 2S /T/E 0 R/S/T/E ° 2E " T / E ° S / T / E °R/S / T/E °2w // of cotyledons 9.62**% 10.51**% 0.0 % 79.87% 90.38% 6.25**% 9.93**% 0.0 % 83.82% 93.75% # of needles 0.0 % 28.60**% 1.31% 70.09% 100.0 % 0.0 % 21.60**% 3.0 % 74.80% 100.0% Total height 0.57 % 20.40**% 0.0 % 79.03% 99.43% 0.0 % 17.74**% 0.0 % 82.26% 100.0% Length of epicotyl 0.0 % 20.56**% 4.15% 75.30% 100.0 % 0.0 % 20.18**7. 0.55% 79.27% 100.0% Length of hypocotyl 0.0 % 21.57**% 0.0 % 78.43% 100.0% 0.0 % 18.01**% 0.0 % 81.99% 100.0% Root dry weight 0.72* % 8.26**% 0.0 % 91.02% 99.28% 0.11**% 5.86**% 0.0 % 94.03% 99.89% Shoot dry weight 0.85* % 18.74**% 0.0 % 80.41% 99.15% 0.0 % 18.61**% 0.0 % 81.39% 100.0 % Significance; *, P <0.05, **, P < 0.01. 91 of cotyledons and root and shoot dry weight, but not s t a t i s t i c a l l y s i g n i f i c a n t f o r number of needles, t o t a l height, and length of e p i c o t y l and hypocotyl. V a r i a t i o n between trees within elevations, on the other hand, was s i g n i f i c a n t f or a l l seven characters. These varied from 8.26 percent (foot dry weight) to 28.60 percent (number of needles). The amount of v a r i a t i o n which existed within and between elevations showed that the within e l e v a t i o n v a r i a t i o n was s i g n i f i c a n t for a l l of the seven characters, varied from 90.30 (number of cotyledons) to 100 percent (number of needles and length of hypocotyl and e p i c o t y l ) . Strong agreement was noticed between the balanced and unbalanced data sets. This showed that analysis based on equal or unequal numbers of trees within elevation w i l l probably y i e l d the same outcome. A s l i g h t decrease i n the amount of v a r i a t i o n between and within elevations was noticed i n unbalanced a n a l y s i s . This probably r e s u l t e d from a reduction of a r t i f i c i a l v a r i a t i o n which might have existed when a small number of trees was used. The d i f f e r e n t response of the phenotypic characters which showed v a r i a t i o n between elevations could be due to the uniform condition i n the growth chamber. Therefore, the only character (number of cotyledons) which was not affected by the environmental condition showed i d e n t i c a l r e s u l t s compared to the isozyme data which dealt with the f i r s t product of the gene-protein which i s the r e a l genetic differences.., Summarizing and comparing the r e s u l t s from phenotypic and electrophoretic data, i t appears that these two independent sources of information are i n good agreement. Electrophoretic data, therefore, could be used to complement r e s u l t s obtained from conventional methods 92 f o r estimating v a r i a b i l i t y w i t h i n and between sub-populations. The use of the electrophoresis approach to assess genetic v a r i a b i l i t y and i t s h i e r a r c h i c a l l e v e l s can provide a r e l i a b l e yet rapid i n d i c a t i o n of the d i s t r i b u t i o n of v a r i a t i o n i n natural populations at micro- and macro-d i f f e r e n t i a t i o n l e v e l s . These aspects have p r a c t i c a l implications f o r tree improvement programs; i n addition the importance of w i t h i n popula-t i o n v a r i a t i o n should not be overlooked. 3.6 Association between isozyme genotypes and quantitative characters. Quantitative genetic theory has r e l i e d h e avily during i t s develop-ment on biometrical techniques. However, these r e s u l t s give a s t a t i c d e s c r i p t i o n of population a r c h i t e c t u r e and more d e t a i l e d information i s needed, e s p e c i a l l y at the chromosome and gene l e v e l s , to reveal genetic control of population v a r i a b i l i t y . Studies with plant (Wehrhahn and A l l a r d , 1965) and animal populations (Thody, 1961) seem to refute the assumption of biometrical genetics that a l l quantitative t r a i t s are c o n t r o l l e d by many genes each having a small and equal e f f e c t . These studies also implied that the number of major genes which control quantitative v a r i a t i o n for some t r a i t s i s rather l e s s than previously thought. I f these l o c i could be defined, then i t would be f e a s i b l e to manipulate major genes i n order to control quantitative t r a i t s into desired combinations through breeding programs. The electrophoresis technique has provided a powerful t o o l f o r i d e n t i f y i n g some biochemical l o c i (gene markers). These markers could have a possible a s s o c i a t i o n with economically important t r a i t s . In forest tree genetics, e l e c t r o -phoretic techniques have been l i m i t e d to the study of isozyme v a r i a t i o n s 93 within and between natural populations (section 3.5). The lack of intensive e f f o r t s to r e l a t e v a r i a t i o n s at the biochemical l e v e l with quantitative v a r i a t i o n s of economically important t r a i t s i s due to a c o n f l i c t of opinion regarding the f e a s i b i l i t y of t h i s approach as w e l l as to problems a r i s i n g mainly from experimental manipulation. Further-more, the establishment of an e f f i c i e n t experimental design coupled with s k i l l e d laboratory techniques to screen large numbers of l o c i i s necessary to e f f e c t i v e l y deal with t h i s question. Limited information has been reported regarding the c o r r e l a t i o n between isozyme genotypes and quantitative characters. For example, Copes (1975) was able to detect isozyme differences between Douglas-fir seedlings with dwarf yew-like, twisted, and normal needles. These differences were not only represented by changes i n the frequency of the most common a l l e l e s i n the isozyme system studied, but also by the presence or absence of some bands ( a l l e l e s ) . Hamrick and A l l a r d (1975) provided evidence that the observed v a r i a t i o n i n several quantitative characters (such as flowering time, seed maturation time, height, and number of t i l l e r s ) i n natural populations of w i l d oat has a s i g n i f i c a n t genetic component of isozyme d i f f e r e n c e s . Singh and Zouros (1978) reported that there was a strong c o r r e l a t i o n between the degree of heterozygosity (for 0, 1, 2, 3 or 4 l o c i ) and weight of i n d i v i d u a l s i n American oyster (Cr.assostrea v i r g i n i c a ) . They also reported 1 that an a l l e l e of glutamate oxalo-acetate transaminase was associated with a f a s t e r growth rate. Powers et a l . (1979) found p h y s i o l o g i c a l c o r r e l a t i o n between l a c t a t e dehydrogenase genotypes and haemoglobin function i n K i l l i f i s h (Fundulus h e t e r o c l i t u s ) . 94. In the following section, the as s o c i a t i o n between the mother trees' isozyme genotypes and the performance of t h e i r h a l f - s i b f a m i l i e s w i l l be discussed. The quantitative t r a i t s of the eight-week-old seedlings under i n v e s t i g a t i o n were the number of cotyledons, number of needles, length of hypocotyl, length of e p i c o t y l , t o t a l height, and root and shoot dry weight. Materials and methods The previously described 41 trees (section 3.5) and the quantitative data from the growth chamber progeny test (section 2.3) were used i n t h i s study. The s t a t i s t i c a l analysis used was i d e n t i c a l to that previously employed (section 3.5), except that v a r i a t i o n between genotypes was substituted f o r v a r i a t i o n among elevation classes. Where s i g n i f i c a n t v a r i a t i o n existed between genotypes, Duncan's multiple range test was performed to compare the d i f f e r e n t genotypes. A second analysis was performed to compare the d i f f e r e n t l e v e l s of heterozygosity and t h e i r e f f e c t on quantitative characters. Trees were divided into d i f f e r e n t classes according to t h e i r genotypes or t h e i r l e v e l of heterozygosity. The number of trees within d i f f e r e n t classes varied between one and t h i r t y . Eight enzyme systems and eight heterozygosity l e v e l s were examined. Nomenclature of enzymes and t h e i r d i f f e r e n t genotypes was based on migration distances (section 2.2) and i s as follows: (a) ACO with three genotypes 77/77, 100/100, and 77/100. (b) APH-2 with eight genotypes 72/72, 87/87, 100/100, null/72, null/100, 72/87, 72/100, and 87/100. (c) EST-1 with eight genotypes 92/92, 100/100, 110/110, 85/92, 85/100, 92/100, 92/110, and 100/110. 95 (d) G6P with f i v e genotypes 90/90, 100/100, 80/90, 80/100, and 90/100. (e) HA with s i x genotypes 100/100, 140/140, 170/170, 100/140, 100/170, and 140/170. (f) MDH-3 with three genotypes 100/100, 84/100, and null/100. (g) MDH-4 with f i v e genotypes 75/75, 100/100, 129/129, 75/100,rand 100/129. (h) PGM with four genotypes 100/100, 94/100, 94/105, and 100/105. Eight d i f f e r e n t heterozygosity l e v e l s were used and the number of heterozygous l o c i per tree varied between one and eight from the sample of 27 l o c i studied. Results and discussion A t o t a l of 63 d i f f e r e n t analyses of variance were conducted to compare the d i f f e r e n t genotypes wi t h i n each of the isozyme systems and di f f e r e n t l e v e l s of heterozygosity. Of the analyses, 58 d i f f e r e n t com-parisons showed no s i g n i f i c a n t d i f f e r e n c e (a = 0.05) while the remaining f i v e comparisons showed s i g n i f i c a n t differences (a = 0.05) between d i f f e r e n t genotypes. A summary of these analyses i s presented i n Table 27. The 58 non-significant comparisons indi c a t e that these isozyme systems are not involved or associated with any of the possible major genes c o n t r o l l i n g these characters. Another attempt was also made to compare the monomorphic and heteromorphic genotype classes within these enzyme systems (56 ad d i t i o n a l d i f f e r e n t comparisons). This analysis was made to determine whether heterozygous genotypes and homozygous genotypes d i f f e r e d . Results from these comparisons y i e l d e d no s i g n i f i c a n t d i f f e r -ences between these two classes with respect to the e f f e c t of the TABLE 27: A summary of the 63 d i f f e r e n t analyses made to compare d i f f e r e n t genotypes within each isozyme system, and also between d i f f e r e n t l e v e l s of heterozygosity. L o c i ACO APH-2 EST-1 G6P HA MDH-3 MDH-4 PGM # of heterozygous l o c i Character Number of cotyledons x x Number of needles Length of hypocotyl Length of e p i c o t y l T o t a l height Root dry weight Shoot dry weight ( - ): not s i g n i f i c a n t ( a = 0 . 0 5 ) ( x ) : s i g n i f i c a n t ( a = 0 . 0 5 ) 97 quantitative t r a i t s , Five isozymes which exhibited s i g n i f i c a n t influences on quanti<-t a t i v e t r a i t s were APH-2 and PGM f o r number of cotyledons, G6P and MDH-4 for shoot dry weight, and PGM f o r length of hypocotyl. Duncan's multiple range test f o r the differences between the APH-2 genotypes and number of cotyledons (Figure 11,A) showed that the null/72 genotype produced seedlings with the la r g e s t number of cotyledons (7.48). This value was not s i g n i f i c a n t l y d i f f e r e n t from the values obtained from 87/87, 72/87, and 87/100, but d i f f e r e d s i g n i f i c a n t l y from the other genotypes. These r e s u l t s showed that there was no preference for heterozygous or homozygous genotypes since both types were present i n each group. The 87 a l l e l e was found most frequently i n the homogeneous group containing the highest number of cotyledons. This may in d i c a t e the p o s s i b i l i t y of an association between t h i s a l l e l e and a large number of cotyledons. However, the null/72 genotype had the highest cotyledon number. At the G6P locus, homozygous 100/100 and heterozygous 90/100 genotypes had the la r g e s t value f o r shoot dry weight (0.06 g). The homozygous (100/100) and heterozygous (90/100) genotypes shared the largest value (0.06 g). This value d i f f e r e d s i g n i f i c a n t l y from the averages of 0.04 g and 0.05 g f o r the other three genotypes (Figure 11,B). Detection of a s p e c i f i c a l l e l e or genotype associated with the heaviest shoot dry weight was not possible because of the presence of both 90 and 100 a l l e l e s i n most of the other genotypes. FIGURE 11: A summary of the five Duncan's multiple range tests. A. APH-2 genotypes and number of cotyledons 100/100 null/100 72/100 • 72/72 82/100 72/87 87/87 null/72 6.91 7.06 7.16 7.16 7.29 7.44 7.46 7.43 B. G6P genotypes and shoot dry weight (0.01 g) 80/100 80/90 90/90 90/100 100/100 0.04 0.050 0.053 0.06 0.06 C. MDH-4 genotypes and shoot dry weight (0.01 g) 75/100 129/129 100/100 100/129 75/75 0.04 0.05 0.05 0.06 0.06 D. PGM genotypes and number of cotyledons 100/105 100/100 94/100 94/105 7.10 7.16 7.39 7.82 E. PGM genotypes and length of hypocotyl (1.0 mm) 94/100 100/105 94/105 100/100 24.13 24.46 26.66 26.94 99 At the MDH-4 locus, homozygous 75/75 and heterozygous 100/129 genotypes exhibited the highest value of shoot dry weight (0.06 g). This mean value d i f f e r e d s i g n i f i c a n t l y from the other three genotypic means which resulted i n two s i g n i f i c a n t l y d i f f e r e n t groups. The homozy-gous condition f o r the 75 a l l e l e y i e l d e d the highest shoot dry weight while the heterozygous case (75/100) with the 100 a l l e l e y i e l d e d the lowest weight. Therefore, p r e d i c t i o n of the a l l e l e or genotype which might be associated with t h i s character was not possi b l e . PGM genotypes had s i g n i f i c a n t differences i n two quantitative characters , number of cotyledons and length of hypocotylC The heterozygous genotype (94/105) showed the largest number cotyledons -with a value of 7.82 (Figure 11,D). This value d i f f e r e d s i g n i f i c a n t l y from the mean values of the other three genotypes. Increasing d i f f e r -ences i n the biochemical l e v e l at the enzyme sub-unit structure seemed to have a pronounced e f f e c t of increasing the number of cotyledons. The 94/105 combination which had the greatest d i f f e r e n c e i n e l e c t r o -phoretic m o b i l i t y ( i . e . migration distance) between i t s two a l l e l e s had a l a r g e r number of cotyledons than the 94/100 combination. In turn, the l a t t e r combination had a larger number of cotyledons than e i t h e r the homozygous (100/100) or heterozygous (100/105) a l l e l e combinations. The homozygous genotype (100/100) showed the largest hypocotyl length (26.94 mm). This value was not s i g n i f i c a n t l y d i f f e r e n t from the value obtained from the 94/105 genotype, but d i f f e r e d s i g n i f i c a n t l y from the other two genotypes (Figure 11,E). However, the r e l a t i o n s h i p regarding the electrophoretic mobility differences was reversed i n t h i s case and the homozygous genotype (100/100) performed equally as w e l l as the heterozygous 100 genotype (94/105), 'Two-way combinations between d i f f e r e n t isozyme genotypes f o r APH-2 and PGM with number of cotyledons and between G6P and MDH-4 geno-types with shoot dry weight were not possible because of the large number of genotypic combinations and the l i m i t e d number of trees studied. Summary and conclusions Associations were found between maternal genotypes and l e v e l s of heterozygosity with seven quantitative t r a i t s i n t h e i r h a l f - s i b f a m i l i e s . A t o t a l of 63 combinations were tested. F i f t y - e i g h t (51 f o r comparing d i f f e r e n t genotypes and seven f or comparing d i f f e r e n t l e v e l s of heterozygosity) showed no s i g n i f i c a n t differences between d i f f e r e n t allozyme combinations within any enzyme system or between d i f f e r e n t l e v e l s of heterozygosity. Another attempt was made to compare the homozygous and heterozygous genotypes f o r each of the isozymes studied with the seven quantitative characters, and no s i g n i f i c a n t differences were detected. The remaining f i v e s i g n i f i c a n t comparisons were discussed. It was d i f f i c u l t to detect the contribution of a c e r t a i n a l l e l e or geno-type to any of the quantitative t r a i t s studied. Maternal influence appeared to be minimal on the quan t i t a t i v e t r a i t s studied. This may be due to the fac t that the female parent represents only one-half of the t o t a l genetic composition of the progeny. The other h a l f i s contributed by the male parent and thus could not be studied i n t h i s experiment since open p o l l i n a t e d seeds were used. Furthermore, i t i s also possible to assume that those eight enzyme 101 systems were not associated at a l l with those quantitative t r a i t s investigated. It i s suggested that c o n t r o l l e d p o l l i n a t i o n should be c a r r i e d out on the 41 trees studied to elucidate the po l l e n parent c o n t r i b u t i o n to the progeny. Also, increasing the number of l o c i and quantitative t r a i t s w i l l probably increase the chance of detecting possible associations between isozyme genotype and quantitative t r a i t s . 102 4. SUMMARY AND CONCLUSIONS Dif f e r e n t aspects of isozyme patterns f o r a selected group of trees sampled from 'Griffith's''(1968) Douglas-fir population (located at the Univ e r s i t y of B r i t i s h Columbia Research Forest, Haney, B.C.) were studied. The r e s u l t s are summarized as follows: 1. The mode of inheritance of 14 polymorphic isozyme systems encoded by 21 l o c i was studied using haploid megagametophytes and d i p l o i d embryos from an open-pollinated seed c o l l e c t i o n . E l e ctrophoretic variants segregated i n megagametophytes i n a co-dominant fashion with d i s t i n c t simple Mendelian expression. The enzymes' sub-unit structure showed that two l o c i were monomers and nine l o c i were dimers. The remaining l o c i were ei t h e r monomorphic or d i f f i c u l t to study using these methods. 2. The linkage r e l a t i o n s h i p s among 19 d i f f e r e n t polymorphic l o c i y ielded nine d i f f e r e n t linked p a i r s . Two of them were t i g h t l y l i n k e d with recombination frequencies of 1.5 percent (AAT-2:PGI-2) and 22.4 percent (AAT-3:SOD). The remaining seven were loos e l y linked, with recombination frequencies varying between 32.7 percent (DIA-2:MDH-3) and 41.9 percent (EST-1:MDH-1). 3. Conditional p r o b a b i l i t i e s were used to estimate the outcrossing rate (t) from information derived from megagametophytes and embryos f or four d i f f e r e n t enzyme systems. The estimated outcrossing rate was 0.90 + 0.11. The inbreeding rate, therefore, was calculated to be 0.1. The elimination of inbreeding i n seed orchards w i l l possibly r e s u l t i n a 103 2.64 percent increase i n height growth of the progeny. 4. The optimum sample s i z e needed to sample the base population was determined by a minimum sampling variance c r i t e r i o n . In order to obtain r e l i a b l e estimates for the a l l e l i c frequencies, i t was estimated that between 42 to 60 trees would be needed for any enzyme system. 5. The analysis of gene d i v e r s i t y was conducted with respect to d i f f e r e n t e l e v a t i o n a l classes. Approximately 93 percent of the t o t a l v a r i a t i o n resided within e l e v a t i o n a l classes and the remaining seven percent was due to between-class e f f e c t s . The r e s u l t s from the analysis of variance for seven quantitative characters showed s t r i k i n g agreement with the r e s u l t s obtained from the gene d i v e r s i t y a n a l y s i s . This con-firmed that the majority of v a r i a t i o n existed w i t h i n and not between sub-populations. 6. The a s s o c i a t i o n between mother trees' genotypes and the performance of t h e i r h a l f - s i b f a m i l i e s f o r seven quantitative t r a i t s showed that maternal influences were minimal. I t was recommended that the genetic contribution of the p o l l e n parent should be considered to e f f e c t i v e l y investigate these associations. Based on published s c i e n t i f i c r e s u l t s and the findings of t h i s study, i t i s apparent that more information on linkage and chromosome mapping can be obtained when both the number of parent trees and a v a i l a b l e l o c i are increased. The association between isozymes genotypes and quantitative t r a i t s should be studied with f u l l - s i b s to elucidate paternal contribution. 104 LITERATURE CITED Adams, W.T. and R.J. J o l y . 1980. Linkage r e l a t i o n s h i p s among twelve allozyme l o c i i n l o b l o l l y pine. J . Hered. (In p r e s s ) . A l l a r d , R.W., A.L. Kahler, and M.L. Clegg. 1975. Isozyme i n plant population genetics. In: Isozymes IV. Genetics and Evaluation ( C L . Markert ed.). Acad. Press, N.Y. 261-272. A l l a r d , R.W., A.L. Kahler, and B.S. Weir. 1972. The e f f e c t of s e l e c t i o n on esterase allozymes i n a barley population. Genetics 72: 489-503. A l l e n , G.S. and W. Bientjes. 1954. Studies on coniferous tree seed at the University of B r i t i s h Columbia. For. Chron. 30: 184-196. Allendorf, F.W., N. M i t c h e l l , N. Ryman, and G. Stahl. 1977. Isozyme l o c i i n brown trout (Salmo t r u t t a L . ) : detection and i n t e r p r e t a -t i o n from population data. Hereditas 86: 179-190. Allendorf, F.W. and F.M. Utter. 1978. Population genetics. F i s h P h y s i o l . 8: 407. Ayala, F.J. 1974. B i o l o g i c a l evolution: Natural s e l e c t i o n or random walk? American S c i e n t i s t 62: 692-701. Bailey, J.T.J. 1961. Introduction to the mathematical theory of genetic linkage. Oxford University Press, London. 298 p. B a r t e l s , H. 1971. Genetic control of multiple esterases from needles and macro-gametophytes of Picea abies. Planta (Berl.) 99: 283-289. Baur, E.W. and R.T. Schorr. 1969. Genetic polymorphism of tetrazolium oxidase i n dogs. Science 166: 1524-1525. Bergmann, F. 1973. Geographic pattern of genetic v a r i a t i o n of four isozyme l o c i i n the Finnish spruce population (Picea abies). I.U.F.R.O. Joi n t Workshop and Symp. on "Norway spruce provenances", Biri/Norway 2.02.11. 6 p. Bergmann, F. 1974. The genetics of some isozyme systems i n spruce endosperm (Picea a b i e s ) . Genetika 6: 353-360. Bergmann, F. 1978. The a l l e l i c d i s t r i b u t i o n at an acid phosphatase locus i n Norway spruce (Picea abies) along s i m i l a r c l i m a t i c gradients. Theor. Appl. Genet. 52: 57-64. Brewer, G.J. 1970. An introduction to isozyme techniques. Academic Press, N.Y., San Francisco, London. 186 p. 105 Brown, A.H.D. and R.W. A l l a r d . 1970. Estimation of mating system i n open-pollinated maize populations using isozyme polymorphisms. Genetics 66: 133-145. Brown, A.H.D., A.C. Matheson, and K.G. Eldridge. 1975. Estimation of the mating system of Eucalyptus obliqua L'H£rit by using allozyme polymorphisms. Aust. J . Bot. 23: 931-949. Chakraborty, R. 1974. A note on Nei's measure of gene d i v e r s i t y i n a substructured population. Humangenetik 21: 85-88. Cheng, M., T. Yamauchi, and C.S. Hacker. 1977. Genetic polymorphis of i s o c i t r a t e dehydrogenase i n the Culex pipiens complex. Biochemical Genetics 15: 903-907. Clayton, J.W. and D.N. Treti a k . 1972. Amine-citrate buffers for pH contr o l i n starch gel electrophoresis. J . F i s h e r i e s Res. Board Can. 29: 1169-1172. Cleary, B.D., R.D. Greaves, and R.K. Hermann. 1978. Regeneration Oregon's Forests, a guide f o r the regeneration f o r e s t e r . Oregon State U n i v e r s i t y Extension Service, C o r v a l l i s , Oregon. 286 p. Clegg, M.T., R.W. A l l a r d , and A.L. Kahler. 1972. Is the gene the unit of selection? Evidence from two experimental plant popula-t i o n s . Proc. Nat. Acad. S c i . (U.S.A.). 69: 2474-2478. Conkle, M.T. 1971. Inheritance of alcohol dehydrogenase and leucine aminopeptidase isozymes i n knobcone pine. Forest Science 17: 190-194. Copes, D.L. 1975. Isoenzyme study of dwarf and normal Douglas-fir trees. Botanical Gazette 186: 347-352. Copes, D.L. and R.C. Beckwith. 1977. Isoenzyme i d e n t i f i c a t i o n of Picea glauca, _P. s i t c h e n s i s , and P_. l u t z i i populations. Botanical Gazette 138: 512-521. Dobzhansky, Th. 1970. Genetics of the Evolutionary Process, Columbia University Press, N.Y. Dobzhansky, Th. and F.J. Ayala. 1973. Temporal frequency changes of enzyme and chromosomal polymorphisms i n natural populations of Drosophila. Proc. Nat. Acad. S c i . (U.S.A.). 70: 680-683. Eriksson, G. and D. Lindgren. 1975. Nagra genetiska r e f l e x i o n e r kring plant-sortering-Sveriges Skogsforbunds Tidskr. Arg. 73, Heft 3:287-294. Falconer, D.S. 1960. Introduction to quan t i t a t i v e genetics. The Ronald Press Co., N.Y. 365 p. 106 Fashler, A.K. 1979. I n t r a s p e c i f i c v a r i a t i o n i n non-selected natural populations of Douglas-fir (Pseudotsuga menziesii (Mirb.). Franco). M.S.F. Thesis. Univ. of B.C. 74 p. Feret, P.P. 1974. Genetic differences among three small stands of Pinus pungens. Theor. Appl. Genet. 44: 173-177. Feret, P.P. and F. Bergmann. 1976. Gel electrophoresis of proteins and enzymes. In: Modern methods i n fo r e s t genetics. Springer Verlag. Berlin-Heidelberg, N.Y. 49-77. Feret, P.P. and G.R. S t a i r s . 1971. Peroxidase inheritance i n Siberian Elm. Forest Science 17: 472-475. Feret, P.P. and M.S. Witter. 1977. GOT Macrogametophyte isozymes of V i r g i n i a pine. In: Proc. The Fourteenth Southern Forest Tree Improvement Conference. G a i n e s v i l l e , F l o r i d a . 225 p. F i l d e s , R.A. and H. H a r r i s . 1966. Genetically determined v a r i a t i o n of adenylate kinase i n man. Nature 209: 261-263. Fowler, D.P. and D.T. Lester. 1970. Genetics of red pine. U.S.D.A. For. Serv., Res. Pap. W0-8. Fowler, D.P. and R.W. Morris. 1977. Genetic d i v e r s i t y i n red pine: evidence f o r low genie heterozygosity. Can. J . For. Res. 7: 343-347. G r i f f i t h , B.G. 1968. Phenology, growth, and flower and cone production of 154 Douglas-fir trees on the University Research Forest as influenced by climate and f e r t i l i z e r , 1957-1967. U.B.C, Faculty of Forestry, B u l l e t i n No. 6. Guries, R.P. and F.T. Ledig. 1977. Analysis of population structure from allozyme frequencies. Southern Forest Tree Improve. Comm. Publ. 36: 219-226. Guries, R.P. and F.T. Ledig. 1978. Inheritance of some polymorphic isoenzymes i n p i t c h pine (Pinus r i g i d a M i l l . ) . Heredity 40: 27-32. Guries, R.P., S.T. Friedman, and F.T. Ledig. 1978. A megagametophyte analysis of genetic linkage i n p i t c h pine (Pinus r i g i d a M i l l . ) . Heredity 40: 309-314. Hamrick, J.L. and R.W. A l l a r d . 1975. Correlations between quantitative characters and enzyme genotypes i n Avena barbata. Evolution 29: 438-442. Ha r r i s , H. and D.A. Hopkinson. 1976. Handbook of enzyme electrophoresis i n human genetics. North-Holland Publishing Company, Amsterdam, Oxford. 107 Illingworth, K. 1978. Sit k a spruce provenance t r i a l s , three years a f t e r planting i n B r i t i s h Columbia. In: Proc. I.U.F.R.O. Joint Meeting (Vancouver, B.C., Canada). S2.02-12 ( i n press). J a i n , S.K. 1979. Estimation of outcrossing rates: some a l t e r n a t i v e procedures. Crop Science 19: 23-26. Johnson, G.B. 1973. Enzyme polymorphism and blosystematics. The hypothesis of s e l e c t i v e n e u t r a l i t y . Ann. Rev. Eco l . System. 4: 93-116. Kahler, A., M.C. Clegg, and R.W. A l l a r d . 1975. Evolutionary changes i n mating system of an experimental population of barley (Hordeum vulgare L . ) . Proc. Nat. Acad. S c i . (U.S.A.) 72: 943-946. Kimura, M. 1969. The rate of molecular evolution considered from the stand point of population genetics. Proc. Nat. Acad. S c i . (U.S.A.). 63: 1181-1188. Kimura, M. and T. Ohta. 1971. Protein polymorphism as a phase of molecular evolution. Nature 229: 467-469. Kimura, M. and T. Ohta. 1974. On some p r i n c i p l e s governing molecular evolution. Proc. Nat. Acad. S c i . (U.S.A.) 71: 2848-2852. King, J.L. and T.J. Jukes. 1969. Non-Darwinian evolution. Science 164: 788-798. Langley, C.H., Y.N. Tobari, and K. Kojima. 1974. Linkage d i s e q u i l i b r i u m i n natural populations of Drosophila melanogaster. Genetics 78: 921-936. Langner, W. 1966. Ubr Fehlbeurteilungen von Saatguternte-bestanden nach dem .phanotyp. Forstpflanzen-Forstsamen, Heft. 3: 25-28, 30-36. Larsen, C S . 1937. Genetics i n s i l v i c u l t u r e . Translated by H.L.-Anderson. 1956. O l i v e r and Boyd, Edinburgh. 224 p. Lever, K.G. and J . Burley. 1974. The a p p l i c a t i o n of biochemical methods i n f o r e s t r y . In: Proceedings from Tenth Commonwealth Forestry Conference. 20 p. Lewontin, R.C. 1973. Population genetics. Ann. Rev. Genet. 7: 1-17. L i p p i t t , B. and I. F r i d o v i c h . 1973. Tetrazolium oxidase and superoxide dismutase: Evidence f o r i d e n t i t y . Archives of Biochemistry and Biophysics 159: 738-741. Lorimer, N. 1979. Genetics of superoxide dismutase i n the fo r e s t tent c a t e r p i l l a r and other organisms. J . Hered. 70: 199-204. 108 Lundkvist, K. 1974. Analysis of linkage i n Picea abies by means of isozyme studied. In: Proc. I.U.F.R.O. Meeting. S.02.04. 1-3, Stockholm. 468 p. Lundkvist, K. 1975. Inheritance of acid phosphatase isozymes i n Picea abies. Hereditas 79: 221-226. Madsen, J.L. and G.M. Blake. 1977. E c o l o g i c a l genetics of Ponderosa pine i n the northern Rocky Mountains. Silvae Genet. 26: 1-8. Markert, C.L. and F. Moller. 1959. M u l t i p l e forms of enzymes: ti s s u e , onto-genetic and species s p e c i f i c patterns. Proc. Nat. Acad. S c i . (U.S.A.). 45: 753-763. Marshall, D.R. and R.W. A l l a r d . 1969. Genetics of electrophoretic variants i n Avena. I. The esterase E i ^ , Eg, E^Q, phosphatase P 5 , and anodal peroxidase A P X 5 l o c i i n A. barbata. J . Hered. 60: 17-19. Marshall, D.R. and R.W. A l l a r d . 1970. Maintenance of isozyme polymor-phisms i n natural population of Avena barbata. Genetics 66: 393-399. Mendenhall, W. and R.L. Scheaffer. 1973. Mathematical s t a t i s t i c s with a p p l i c a t i o n s . Wadsworth Publishing Co., Inc. Belmont, C a l i f o r n i a 561 p. Mitton, J.B., Y.B. Linhart, J.L. Hamrick, and J.S. Beckman. 1977. Observation on the genetic structure and mating system of Ponderosa pine i n the Colorado Front Range. Theor. Appl. Genet. 51: 5-13. Mitton, J.B., Y.B. Linhart, K.B. Sturgeon, and J.L. Hamrick. 1979. Allozyme polymorphisms detected i n mature needle t i s s u e of Ponderosa pine. J . Hered. 70: 86-89. Muhs, Hans-J. 1974. D i s t i n c t i o n of Douglas-fir provenances using peroxidase-isoenzyme-patterns of needles. Silvae Genet. 23: 71-76. Muller, G. 1976. A simple method of estimating rates of s e l f - f e r t i l i z a -t i o n by analyzing isozymes i n tree seeds. S i l v a e Genet. 25: 15-17. Nei, M. 1973. Analysis of gene d i v e r s i t y i n subdivided population. Proc. Nat. Acad. S c i . (U.S.A.). 70: 3321-3323. Nei, M. 1975. Molecular population genetics and evolution. North-Holland, Amsterdam. Oxford. 288<p. Nevo, E. 1978. Genetic v a r i a t i o n i n natural populations: Patterns and Theory. Theo. Pop. B i o l . 13: 121-177. O'Malley, D.M., F.W. Allendorf, and G.M. Blake. 1979. Inheritance of isozyme v a r i a t i o n and heterozygosity i n Pinus ponderosa. Biochemical Genetics 17: 223-250. 109 Orr-Ewing, A.L. 1965. Inbreeding and s i n g l e crossing i n Douglas-fir Forest Science 11: 279-290. Perry, D.A. and J.E. Lotan. 1977. V a r i a t i o n i n lodgepole pine (Pinus  contorta var. L a t i f o l i a ) : greenhouse response of wind p o l l i n a t e d f a m i l i e s from f i v e populations to day length and temperature-soil. Can. J . For. Res. 8: 81-89. Powers, D.A., G.S. Greaney, and A.R. Place. 1979. P h y s i o l o g i c a l c o r r e l a t i o n between l a c t a t e dehydrogenase genotype and haemo-globin function i n k i l l i f i s h . Nature 277: 240-241. Prakash, S., R.C. Lewontin, and J.L. Hubby. 1969. A molecular approach to the study of genie heterozygosity i n natural populations. IV. Patterns of genie v a r i a t i o n s i n Central, Marginal, and Isolated Populations of Drosophila pseudoobscura. Genetics 61: 841-858. Ridgway, G.J., S.W. Sherburne, and R.D. Lewis. 1970. Polymorphisms i n the esterases of A t l a n t i c herring. Trans. Am. F i s h e r i e s Soc. 99: 147-151. Rudin, D. 1975. Inheritance of glutamate-oxalate-transaminases (GOT) from needles and endosperms of Pinus s y l v e s t r i s . Hereditas 80: 296-300. Rudin, D. 1976. Biochemical genetics and s e l e c t i o n , a p p l i c a t i o n of isozymes i n tree breeding. In: Proc. I.U.F.R.O. Joint Meeting on Advanced Generation Breeding (Bordeaux, France, June 14-18, 1976). p. 145-164. Rudin, D. and I. Ekberg. 1978. Linkage studies i n Pinus s y l v e s t r i s L. -using macrogametophyte allozymes. Silvae Genet. 27: 1-12. Rudin, D., G. Eriksson, I. Ekberg, and M. Rasmuson. 1974. Studies of a l l e l e frequencies and inbreeding i n Scots pine populations by the a i d of the isozyme technique. S i l v a e Genet. 23: 10-13. Rudin, D., G. Eriksson, and M. Rasmuson. 1977. Inbreeding i n a seed tree stand i n Northern Sweden. A study by the aid of the isozyme technique. Ph.D. t h e s i s . Department of Genetics, University of Umea, Sweden. Sakai, K. and Y. Park. 1971. Genetic studies i n natural populations of f o r e s t trees. I I I . Genetic d i f f e r e n t i a t i o n within a f o r e s t of Cryptomeria japonica. Theor. Appl. Genet. 41: 13-17. Saylor, L.C. and B.W. Smith. 1966. Meiotic i r r e g u l a r i t y i n species and i n t e r s p e c i f i c hybrids of Pinus. Am. J . Bot. 53: 453-468. 110 S i c i l iano, M.J. and C.R. Shaw. 1976. Separation and v i s u a l i z a t i o n of enzymes on gels. Chromatographic and electrophoretic techniques (Ed. Smith, I . ) . 2: 185-209. Heinemann, London. Simonsen, V. and H. Wellendorf. 1975. Some polymorphic isoenzymes i n the seed endorsperm of Sit k a spruce (Picea s t i c h e n s i s (Bong.) Carr. ) . Forest Tree Improvement 9: 5-20. Singh, S.M. and E. Zouros. 1978. Genetic v a r i a t i o n associated with growth rate i n the American oyster Crassostrea v i r g i n i c a . Evolution 32: 342-353. Slaughter, C.A., D.A. Hopkinson, and H. H a r r i s . 1975. Aconitase polymorphism i n man. Ann. Hum. Genet. 39: 193. Snow, A.G. and J.W. D u f f i e l d . 1940. Genetics i n Forestry. J . of Forestry 38: 404-408. Sokal, R.R. and F.J. Rohlf. 1969. The p r i n c i p l e s and p r a c t i c e of s t a t i s t i c s i n b i o l o g i c a l research. W.H. Freeman and Company. San Francisco. 776 p. Sorensen, F.C. 1973. Frequency of seedlings from natural s e l f -f e r t i l i z a t i o n i n Coastal Douglas-fir. Silvae Genet. 22: 20-24. Spiess, E.B. 1977. Genes i n populations. John Wiley and Sons, Inc. N.Y. Santa Barbara. London. Sydney. Toronto. 780 p. Ste e l , R.G.D. and J.H. T o r r i e . 1960. P r i n c i p l e s and procedures of s t a t i s t i c s . McGraw-Hill Book Company, Inc. N.Y. 481 p. Stern, K. and L. Roche. 1974. Genetics of forest ecosystems. B e r l i n -Heidelberg-N.Y.: Springer. 330.p. Thielges, B.A. 1972. I n t r a s p e c i f i c v a r i a t i o n i n f o l i a g e polyphenols of pinus (Subsection S y l v e s t r e s ) . Silvae Genet. 2: 114-119. Thody, J.M. 1961. The l o c a t i o n of polygenes. Nature 191: 368-370. Tigerstedt, P.M.A. 1973. Studies on isozyme v a r i a t i o n i n marginal and centr a l populations of Picea abies. Hereditas 75: 47-60. Wehrahahn, C. and R.W. A l l a r d . 1965. The defection and measurement of the e f f e c t s of i n d i v i d u a l genes involved i n the inheritance of a quantitative character i n wheat. Genetics 51: 109-119. Yang, J.-Ch., T.M. Ching, and K.K. Ching. 1977. Isoenzyme v a r i a t i o n of Coastal Douglas-fir. I. A study of geographic v a r i a t i o n i n three enzyme systems. S i l v a e Genet. 26: 10-18. I l l -Yeh, F.C. and Y.A. E l - Kassaby. 1980. Enzyme v a r i a t i o n i n natural populations of S i t k a spruce (Picea s i t c h e n s i s (Bong.) Carr . ) . I. Genetic v a r i a t i o n patterns among trees from ten I.U.F.R.O. Provenances. Can. J . For. Res. ( i n press). Yeh, F.C. and C. Layton. 1979. The organization of genetic v a r i a b i l i t y i n c e n t r a l and marginal populations of lodgepole pine (Pinus  contorta spp. L a t i f o l i a ) . Can. J . Genet. Cytol. 21: 487-503. Yeh, F.C. and D.M. O'Malley. 1978. Detecting geographic v a r i a t i o n i n coastal and i n t e r i o r Douglas-fir populations by biochemical method. B.C.F.S., Research D i v i s i o n . Research Memo. No:31. Yeh, F.C. and D.M. O'Malley. 1980. Enzyme v a r i a t i o n i n natural popula-tions of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) from B r i t i s h Columbia. I. Genetic v a r i a t i o n patterns i n coastal populations. Silvae Genet. (In press). Zouros, E. 1976. Hybrid molecules and the s u p e r i o r i t y of the heterozygote. Nature 262: 227-229. Zouros, E., C.B. Kaimbas, S. Tsakas, and M. Lonkas. 1974. Genie versus chromosomal v a r i a t i o n i n natural populations of Drosophila subobscura. Genetics 78: 1223-1244. 112. APPENDIX 1. Chi-square r e s u l t s for linkage analyses. Combination A A T - 2 A A T - 2 AAT-2 AAT-2 AAT-2 AAT-2 AAT-3 AAT- 3 A A T - 3 Tree # N FF FS SF SS 20 58 17 13 13 1 5 02 63 12 15 22 19 128 5fl 15 17 11 15 : ACO 33 58 16 14 12 16 : ACO 92 68 14 13 18 23 : ACO 128 58 17 15 18 8 0 .06897 2 . 8 8? 3 5 0 .62069 0 .06897 2 .88235 0. 62069 0 .06897 0 .0 0 .62069 0 .0689T 0 .23529 2 .48276 Recomb. % 0.60650 0.52941 0. 11843 0 .63504 0 . 39411 1.47314 A A T - 2 AAT-2 AAT-2 A A T - 2 : APH-2 33 58 14 16 15 13 : APH-2 > 68 68 15 20 13 20 : APH-2 92 68 11 16 20 21 : APH-2 123 68 18 17 20' 13 0 .06897 0 .05882 2 .88235 0 .05882 0.0 2 .11765 0.52941 0 .94118 0.27536 0.03142 0 . 40320 0 . 57176 A A T - 2 AAT-2 AAT-2 A A T - 2 A A T - 2 E S T - l E S T - 1 EST- 1 E S T - 1 E S T - 1 20 33 68 123 142 58 58 68 68 58 16 17 22 22 12 14 13 13 13 14 L5 14 16 18 1 5 13 14 17 15 17 0 .06897 0 .06397 0.05882 0.05882 0 .62069 0 .27586 0 .27586 0 .94118 2 .11765 0. 27586 0.00033 0 .25717 1.43221 0.46398 0 .00295 A A T - 2 AAT-2 A A T - 2 AAT-2 A A T - 2 A A T - 2 AAT-2 AAT-2 AAT-2 AAT-2 A A T - 2 AAT-2 AAT-2 AAT-3 AAT-3 : G6P 68 68 15 20 1 5 18 : G6P 123 68 18 17 14 I 9 : HA 20 58 22 8 18 10 : HA 123 68 17 18 16 17 HA 128 58 14 18 15 11 : HA 142 58 9 17 15 17 : IDH 92 68 12 15 17 24 : MOH-3 92 68 17 10 21 20 : MDH-3 142 57 20 5 14 IS : ME 123 68 15 20 14 1 9 : PG I -2 123 68 1 34 33 0 : PGM 123 68 17 13 18 1 5 : 6PG-2 68 69 22 13 14 19 : son 20 58 12 13 15 13 : ACO 9? 68 15 19 17 17 : ACO 123 58 15 11 2 0 12 0 .05382 0 .05882 0 .06397 0 .05882 0 . 62069 0 .62069 2 .38235 0 .62069 0. 05882 0.05382 0.05382 0 .0538? 0 .06897 0. 0 0 .62069 0 .46154 0 .0 3 .34433 0 .05882 0 .0 1.72414 0 .94118 2 .12231 0.04530 0 . 55038 0 .47374 0 .00005 1.10345 0 .85344 2 .38235 1.61333 0 .0554? 0.86047 7.27031 33 .33333 6.24390 1.47059 0 .00127 0 .0 54 .05384 1.47059 1.45973 0 ,05123 0 .24227 0 .20513 2.33413 0 .27586 t .06573 0 .23529 0.23529 2.432 76 0 .1312? Combination Tree # N FF FS SF SS 4 AAT-2 : AAT-3 20 53 1 7 13 13 1 5 0 .06897 AAT-2 : AAT- 3 97 63 1? 15 22 19 2 . 8 8? 3 5 AAT-2 : AAT-3 128 58 15 17 11 15 0 .67069 AAT-2 : ACO 33 58 16 1 4 12 16 0 .06397 AAT-2 : ACO 9 2 68 1* 13 13 2 3 7 . 3 82 3 5 AAT-2 : Acn 123 58 17 15 18 8 0 .62069 AAT-2 : APH-2 33 58 14 16 15 13 0 .06997 AAT-2 : APH-2 > 69 68 15 20 13 20 0 .05382 AAT-2 : APH-2 92 68 11 16 20 21 2 .38235 AAT-2 : APH- 2 1 23 68 18 17 20' 13 0 .05982 A A T - 2 : E S T - I 20 58 16 14 15 13 0 .06997 AAT-2 : E S T - 1 33 58 17 13 14 14 0 .06397 AAT-2 : FST- 1 68 68 22 13 16 17 0.05882 AAT-2 : E S T - 1 123 68 27 13 1 8 15 0 .05882 AAT-2 : E S T - 1 142 58 12 14 1 5 17 0 .62069 A A T - 2 : G6P 68 58 15 20 1 5 18 0 .05987 AAT-2 : G6P 123 68 18 17 14 1 9 0 .05892 AAT-2 : HA 20 58 22 8 18 10 0 .06997 AAT-2 : HA 123 68 17 18 16 17 0 .05982 AAT-2 : HA 128 58 14 18 15 11 0 .62069 AAT-2 : HA 1*7 58 9 17 15 17 0 .62069 AAT-2 : IDH 92 68 12 15 17 24 2 .98235 AAT-2 : MDH-3 92 68 17 10 21 20 2 .98235 AAT-2 : MDH-3 1*2 57 20 5 14 13 0 .62069 AAT-2 : ME 123 68 15 20 14 1 9 0. 05832 AAT-2 : PG I -2 123 68 1 34 33 0 0 .05387 AAT-2 : PGM 1 73 63 17 19 18 I 5 0 .05382 AAT-2 : 6PG-2 68 63 22 13 14 19 0 .0598? AAT-7 : SOD 20 58 12 1 9 15 1 3 0 .06397 AAT-3 : AM 97 68 15 19 17 17 0 .0 AAT-3 : ACO 129 58 15 I I 2 0 12 0 .62069 0.05 997 0.0 0.62069 0.06897 0 .23529 2 .48275 0. 0 2. 11765 0.52941 0 .94119 0 .27586 0 .27586 0 .94118 2 .11765 0 .77586 0 .46154 0 .0 Recomb. % 0.60650 0.57941 0. 11843 0.63504 0. 3941 1 1.4M14 0.77596 0.03147 0.40320 0.57176 0.00033 0.25717 1.40221 0.46398 0 .00795 0.04530 0.55038 3 .34493 0.47374 0 .05982 0.00005 0. 0 1.10345 1. 72414 0.35344 1.61333 0.05547 941 19 0.86047 12231 7.27001 33 .33333 6 .74390 1.47059 0.00127 0 .0 54.05894 1.47059 1.45973 0 .05123 0 .24277 0 .20513 2.33413 77586 1.06573 0. 23529 0.23579 432^6 0.13127 Combination A A T - 3 A A T - 3 A A T - 3 APH-2 APH-2 Tree ft 66 92 E S T - 1 20 68 63 58 20 12 16 13 22 18 19 SS 1 7 1 5 0.05382 0. 0 0 .94113 0. 52941 0.57176 2.38235 l 4 i s 13 0 .06397 0.27586 0.00033 Recomb. % A A T - 3 A A T - 3 HA HA 20 128 58 58 20 11 10 15 20 18 3 14 0.06397 0 .62069 8 .34433 0 .0 0 .13123 1.10345 A A T - 3 A A T - 3 IOH IDH 66 92 63 68 12 16 21 18 18 13 I 7 21 0 .0538? 0 .0 0 .82051 1.41333 1.54063 0 . 52941 AAT-3 MDH-3 92 17 17 13 0 .0 0 .94118 0.94118 AAT-3 : MOH-4 AAT-3 SOD 6 6 .63 18 15 13 17 0.05882 0 .23529 0 .06595 2 0 5 8 6 24 21 7 0 .06897 0 .27536 17.50342 22 .41379 5 .47565 ACO ACD ACO ACO ACO ACO APH-2 APH-2 APH-2 APH-2 APH- 2 APH-2 1 10 33 53 55 92 47 57 58 58 56 68 14 8 12 10 13 12 12 18 16 15 11 20 10 15 17 14 14 19 11 16 13 19 18 17 0.53191 0 .43860 0 .06897 1.10345 0 .85965 0 .23529 0 .02128 1.72414 0 .0 1.72414 0. 07143 0 . 5 ? 9 4 l 0. 17815 1. 74259 1.10345 0.03282 0 . 53310 1. 57627 ACO ACO ACO ACO ACO ACO ACO E S T - l 10 57 13 13 9 22 EST- 1 33 58 15 13 16 14 E S T - l 53 58 12 13 15 18 E S T - 1 55 57 17 8 16 16 G6P 1 47 10 16 9 12 G6P 10 55 1 I 14 I 4 16 G6P 53 58 11 14 18 15 0 .43860 0 . 06897 1. 10345 0 .85965 0 . 53191 0 .43360 1.10345 2 .48276 0.2 7586 0 .27586 1.10345 2.12281 0 .13846 0. 05332 2 .46856 0.00033 0.03617 I .79218 0.03880 0 . 03847 0 .62069 ACO ACO ACO ACO ACO ACO ACO ACO ACO ACO HA 10 57 11 1 5 17 14 0 .43860 HA 53 58 1 1 14 13 20 1.10345 HA 55 57 9 16 17 15 0 .35965 HA 128 58 20 1 5 9 14 2 .48276 IDH 10 57 16 10 14 17 0. 43360 IDH 92 68 14 I 8 15 21 0 .23529 MDH- 1 I 47 14 12 8 13 0.53191 MQH- •j 1 47 14 12 8 13 0.53191 MDH- 3 55 57 12 13 10 22 0 .85965 MDH- 3 92 68 19 13 19 17 0 .23529 0 .05897 1.72414 0 .27536 0. 0 0.52941 1.S1333 0.19149 0 .19149 2.432 76 0 .94118 0.38133 0 . 11848 1.62176 1.72414 1. 50540 0.02931 1.13934 1.13934 1.55255 0.29393 116 e* or a —. o — o o . - . x s - t c o c o ^ f f ^ c r . c o i r © 0 0 - J 0 - J O f \ . O O O f v O O f\jOfllAC0i"<lfrf\JfV O - J O O O N N O - 1 ui o fv: * »t ir, ir **-• • • • • • • • • CNl^OOC-OO if\ rr, r-~ cr «e ru r- r-, ^  —< u~ o o m *o m ty-(T O O ITi N 1^  fX' o cc ^  —« f1 f\j o »* o o o so r*- a CP r*. eo —* >e *o • • • • • • • • • • • • * • o n < M O O O o m o — I O O M O • • • • • • • • • (MOOOOCOfAO O O fV CD Ifi If. «*• If i or ^ «fl «t O t\' f\i ^ ^ O (\.'C<VOOO-^ 00 an —i w m ^ * U*\ O K N O C C lA H H tC o o r \ i v C r<-. e-—- or o in a vf —« o — CC —I MD ^  <0 »C •-• —* IT, f\J r-- O * f- f\J CO tA O f\J r\j f- ^  00 «tf wc->tH-• o r- o —i r~ a o O O - M O ^ OOHOfNj-OO f\jrvccOr\|--r\jrM^ -• • • • • • • • • OsD %t 0" ir\ **• m cc CCO H CD <t © ir- ^ r— »t .—> or- (\i IA c N ^ • • • • • • « • * (NlOO—'O—'-^OO O sC ^  i(> o CO O tt w <}• C H O IT« (*. 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Q. *r <: <• <r «j <J Combination Tree # N FF FS SF SS APH-2 : I OH 92 68 14 17 15 22 APH-2 : MOH- 1 1 47 13 1 1 9 14 APH-2 : MDH-1 14 58 14 21 10 13 APH-? : MDH-3 I 47 9 1 5 13 10 APH-2 : MDH-3 3 55 10 12 16 17 APH-2 : MDH-3 4 58 1 1 20 14 13 APH-7 : MDH-3 18 57 13 14 12 18 APH-2 : MDH-3 55 56 12 15 10 19 APH-2 : MDH-3 92 68 18 13 20 17 APH-2 : MDH-4 3 55 7 15 13 20 APH-2 : MDH-4 4 58 17 14 I 7 10 APH-7 : MDH-4 53 58 14 10 17 17 APH—2 : MDH-4 55 48 11 13 12 12 APH-7 : MDH-4 66 68 19 19 17 13 APH-2 : MDH-4 118 58 9 15 18 16 APH-2 : MDH-4 121 58 1 3 16 10 14 APH-2 : MOH-4 127 56 13 15 14 14 APH-2 : ME 1 47 12 12 10 13 APH-2 : ME 10 58 10 14 13 21 APH-2 : ME 14 58 13 17 12 11 APH-2 : ME 18 57 13 14 15 15 APH-2 : ME 118 58 10 14 20 14 APH-2 : ME 123 68 14 24 15 15 APH-2 : ME 127 56 9 19 15 13 APH—2 : PG 1-2 18 57 16 11 15 15 APH-2 : PGI-2 118 59 14 10 20 14 APH-2 : PGI-2 123 68 20 18 14 16 , 52941 02128 48276 0.02128 2.20000 0.27586 0.15789 0.07143 0.52941 20000 27586 ,72414 ,0 7143 ,941 18 ,72414 ,72414 ,0 0.02128 1.72414 2.48276 0.15789 1.72414 0.94118 0.0 0.15789 1.72414 0.94118 1.61333 0.19149 1.72414 0.19149 0.15364 1.10345 1.10345 2.43276 0.94118 4.09091 1.72414 0.27586 0.0 0.23529 0.27596 0.06897 0.37143 0.19149 7.48276 0.06897 0.06397 0.06897 1 .47059 1.14296 0.62069 1.72414 0.0 L 0.14296 1.05166 0.06437 1.69905 0.04655 1.53929 0.37636 0.55433 0.10769 0.29173 0.37924 0. 37924 0.09333 0.29393 1.30208 0.69411 0.07143 0.19972 0.06437 0.00295 0.01944 1.60734 1.14576 2.57143 0.49599 0.00131 0.23529 Re comb. % APH-2 APH-2 APH-2 APH-2 APH-2 APH-2 APH-2 APH-2 APH-2 APH-2 APH-2 APH-2 0IA-1 PGM PGM PGM PGM PGM PGM PGM PGM PGM 6P5-1 6PG-2 5P G-7 EST- 1 5 58 11 19 17 12 0.0 0.52941 10 57 7 16 1 7 17 1.72414 0.73134 18 55 13 13 11 18 0.15789 1.51515 73 53 12 13 15 19 1.10345 0.2352 9 53 58 1 1 13 15 19 1.7241 4 0.94118 113 58 6 18 20 14 1.72414 0.52941 121 53 19 15 1 3 1 1 1.72414 1.84615 123 68 17 21 19 12 0.94118 0.05123 127 56 19 10 15 13 o.o 1.51515 118 53. 10 14 15 19 1. 72414 1 .47059 3 55 9 13 1 8 15 2.20000 0.01538 69 68 15 13 2 1 19 2.11765 0.2051.3 I 19 58 18 16 13 11 1.72414 0.27586 2.48275 2.02433 0.79641 0.03517 0.01603 6.74874 0.01608 1.54063 0.64286 0.03282 0.94255 0.00733 0.00820 34.48274 6.74115 Combination Tree It N FF 9 DIA-1 : ».DH-l DIA-1 : PGM DIA-1 : SOD 29 49 29 49 9 29 49 10 FS SF 10 15 10 13 9 14 SS 15 17 2 .46939 0 .0 0.03060 Recomb. * 2 . 46939 2 .33235 0.0719S 2 .46939 0 .06897 0.15722 D IA-2 : HA 55 21 14 12 4 .09091 2 .23000 0.00000 DIA-2 DIA-2 OIA-2 IDH MDH-3 6PG-2 3 55 20 3 55 12 3 55 19 15 23 14 16 12 12 4 .09091 0 .01533 1.39496 4 .09091 0 .16364 6.01136 32 .72726 6 .32693 4 .09091 0 .01538 0.96170 E S T - l EST-1 E S T - 1 E S T - 1 E S T - 1 E S T - 1 E S T - l E S T - 1 E S T - 1 G6P G6° G6P G6P G6P G6P G6P G 6 D G6P 10 14 23 53 68 114 117 118 123 55 58 58 58 68 58 68 58 68 15 17 14 15 20 23 12 20 14 14 13 13 23 14 14 14 20 17 16 15 15 15 13 15 19 12 16 1 3 13 16 15 11 16 13 16 2 .48276 0 .0 0 .06997 0 .27586 0.94118 1.72414 0 .52941 0 .62069 2.1 1765 0 .13846 0 .52941 0. 9*113 0 .05882 0 .46154 0. 05882 0 .92051 0 .94113 0. 0 1.16402 0.06996 0 .05544 0 .06897 0 . 73299 0.11848 1.27044 0.99230 0 . 32570 E S T - 1 E S T - 1 E S T - 1 E S T - 1 E S T - 1 E S T - l EST-1 E S T - 1 E S T - 1 EST-1 E S T - 1 E S T - l EST-1 E S T - 1 E S T - l E S T - 1 E S T - 1 E ST-1 EST-1 EST-1 EST-1 E S T - 1 HA 4 58 14 1 2 12 20 HA 5 58 17 12 18 11 HA 10 58 8 15 20 15 HA 18 58 14 14 13 1 7 HA 20 58 24 7 16 11 HA 23 58 1 7 13 10 18 HA 53 58 14 1 3 10 21 HA 55 58 13 20 14 11 HA 1 14 58 13 2 1 14 10 HA 117 63 17 20 15 16 HA 123 68 19 21 14 14 HA 124 55 18 11 8 18 HA 142 53 10 1 7 14 17 IDH 4 58 14 12 16 16 IDH 10 58 13 10 18 I 7 IDH 23 5 8 15 15 19 9 I DH 37 58 12 10 2 I 15 I DH 1 14 58 1 5 19 15 9 MDH- l 1 4 58 1 2 17 12 17 MOH- 1 114 53 19 15 12 12 MOH- 1 I 17 68 26 1 1 10 21 MDH- 3 4 58 11 15 14 18 0 .62069 0 .0 2. 48276 0 .06997 0.27586 0 .06897 0 . 27586 1.10345 1. 7241 4 0 .52941 2 .11765 0 .16364 0 .27586 0 .62069 2 .48276 0 .06897 3.37931 1.72414 0 . 0 1. 7241 4 0.52941 0 .62069 0 .62069 2 .49276 0. 05897 0 .27536 8 .34483 0 .27586 1.72414 0. 27586 0 .27596 0 .23529 0 .05382 0 .16364 1.72414 0.0 0 .52941 2 .11765 2 .11765 0 .05382 1.72414 0 .27586 0 .23529 1.10345 1. 51692 0 .06897 2.65707 0 .25717 1.89593 2.54017 2.20590 1.53928 2.20590 0 .03989 0 .03939 5.35623 0.37924 0 .03397 0.13788 1.94515 0 .07389 1.94516 3 4 . 5 4 5 4 4 6 .41186 0.00000 0 . 13897 9. 67339 30 .88234 0.01191 5 .60267 4 Combination E S T - 1 E S T - 1 E S T - l E S T - 1 MDH-3 MDH-3 MOH- 3 MDH-3 Tree # N FF FS SF SS IS 59 I 1 17 14 16 55 59 11 22 12 13 114 5B 19 15 9 15 14? 57 17 10 17 13 0 .06997 1.10345 1.72414 0 .27536 1 .10345 2 .43276 0 .06897 2 .12291 Recomb. % 0. 31523 1.70161 1.84516 0.22474 E S T - l E S T - l E S T - 1 E S T - l E S T - 1 E S T - l E S T - 1 EST-1 MDH-4 MDH-4 MDH-4 MDH-4 MDH-4 MDH-4 MDH-4 MDH-4 4 53 55 114 117 118 124 127 58 •58 50 58 68 58 54 56 16 15 18 19 17 6 16 1 1 10 12 10 15 20 20 13 17 18 16 7 15 19 2 1 1 1 16 14 15 15 9 12 11 14 12 0 .62069 0 .27586 I .10345 1.72414 0.52941 0 .62069 0. 16364 0 .0 1.72414 0 .27586 0. 0 1.72414 0 .23529 0. 27586 0 .0 0 .07143 0.15881 0.09930 5.12000 0 . 23933 1. 5762 7 10. 2 7696 0 .66667 1.73571 33 .99998 6 .69925 29 .31033 5 .97689 E S T - 1 ME 10 58 9 E S T - 1 ME 14 58 15 E S T - 1 MC 18 58 14 E S T - 1 ME 117 63 16 E S T - 1 ME 118 58 14 E S T - l ME 123 68 22 E S T - 1 ME 124 55 16 E S T - l : ME 127 56 10 14 14 14 21 12 1 8 13 19 14 15 14 16 16 7 10 14 21 14 16 15 16 2 I 16 14 2 .48276 0 .0 0 .06897 0.52941 0 .62069 2 .11765 0 . 16364 0 .0 2 .48276 0 .06897 0. 06897 0 .23529 0 .05897 1 .47059 0 .16364 I .14286 0.00403 0 .00000 0 .06429 0. 46397 0 .08397 5.74363 1.52678 1.14286 36 .76469 5 .84710 E S T - 1 E S T - 1 E S T - 1 E S T - 1 EST-1 EST-1 E S T - 1 E S T - 1 E S T - 1 E S T - 1 E S T - 1 E S T - 1 E S T - 1 E S T - l EST-1 G6P G6P G6P G6P G6P G6P G6P P G I - 2 18 58 14 14 1 8 12 0 .06897 PG1- 2 118 58 15 I I 19 13 0 .62069 PG1- 2 123 63 19 21 15 13 2 .11765 PGM 5 58 13 16 15 14 0 .0 PGM 10 57 11 12 1 3 21 2 .48276 PGM 18 56 11 15 13 17 0 .06397 PGM 23 58 10 20 1 7 11 0 .06897 PGM 53 58 10 17 16 15 0.2 75 86 PGM 119 58 8 I 3 13 14 0 .62069 PGM 123 68 23 1 7 12 16 2.1 1765 PGM 127 56 1 7 11 16 12 0.0 6PG- I 114 58 22 12 13 I 1 1.72414 6 ° G - I 118 58 11 I 5 14 13 0 .62069 6PG- 2 63 68 2 3 ^ 15 13 17 0 .94118 SOD ?0 58 13 1 8 14 13 0.2 7596 HA 10 55 14 1 1 13 17 0.13346 HA 23 58 14 1 8 13 13 0 .94118 HA 53 58 13 I 6 I I 18 0.05392 HA 11 4 58 16 1 7 1 I 14 0 .0 53 82 HA 1 17 69 19 19 13 17 0. 92051 HA 123 68 14 18 19 17 0 .0 IDH 10 65 9 22 26 8 0 .13346 0. 62069 1.72414 0. 0 0 .52941 0 .73134 1.51515 0 .23529 0 .94113 0 .52941 0 .05128 1.51515 3.76471 1 .47059 0 .20513 0.2 7586 0 .06897 0 .27536 1.72414 0 .27586 0 .23529 0.05832 0.57363 0.01608 0 .23529 0.27586 0.43643 0 .00583 4 .33805 1.22061 3.68601 1.36866 0.07143 0.60728 0.01131 1.95491 0.54494 0.84792 0.22175 0.27536 0 . 11227 0.2939 3 0. 55018 36 .20689 6 .31056 0 . 5 ? 9 4 l 14.55551 26.1 5384 5 .45098 Combination Tree # N FF FS SF G6P IDH 23 68 16 22 24 G6P IDH 1 14 68 20 1 5 13 G6P MDH- 1 1 47 5 14 17 G6P MDH- 1 14 58 12 19 12 G6P MDH-1 1 14 58 20 13 11 G6P MDH- 1 117 68 22 16 14 G6P MDH-3 1 47 8 11 14 G6P MDH-3 114 58 15 18 13 G6P MDH-4 53 58 16 13 15 G6P MDH-4 1 14 58 20 13 14 G6P MDH-4 117 68 19 19 17 G6P MDH-4 1 18 58 1 7 14 10 G6P MDH-4 121 58 15 11 13 G6P ME 1 47 11 8 1 I G6P ME 10 55 11 14 1 1 G6P ME 14 58 21 10 9 G6P ME 117 68 19 19 13 G6P ME 118 58 16 15 14 G6P ME 123 68 12 20 17 G6P PGI -2 118 58 20 11 14 G6P PGI -2 123 68 15 17 19 G6P PGM 10 65 14 17 16 G6P : PGM 23 68 17 21 15 G6P PGM 53 68 13 22 17 G6P : PGM 1 18 68 20 18 11 G6P PGM 121 78 20 11 25 G6P PGM 123 78 18 21 20 G6P : 6PG-1 1 14 68 1 8 17 24 G6P : 6PG-1 118 68 16 22 13 G6P : 6PG-2 68 78 1 6 20 25 HA : IDH 3 55 13 20 15 HA : IDH 4 58 10 16 20 HA : IDH 10 58 16 12 15 HA : IDH 23 58 15 12 19 HA : IDH 114 58 15 12 I 5 HA : MDH-1 1 14 58 14 13 1 7 HA : MDH- 1 117 68 16 16 20 HA : MDH-3 3 55 13 20 13 SS X 2 *I x 2r X2 6 0 .94118 ?. 1 l 765 9 .50053 20 0 .0538? 0 . 05832 2. 13946 1 1 2.12281 0. 19149 5. 16171 15 0 . 52941 1. 72414 0 .18897 14 0.05382 0 . 27586 1.53928 16 0.82051 0. 23529 0.83375 14 2. 12281 0 . 19149 0.27139 12 0.05882 0 . 06397 0. 23913 14 0 .05882 0. 27586 0 .06897 I 1 0 .05382 1. 72414 0 .11848 13 0.82051 0. 23529 0 .29393 17 0 .94119 0 . 27536 1.82061 19 3 .28205 0 .06897 1.65355 17 2 .12281 0 . 19149 1.51064 19 0 . 13346 2 . 48276 0 .29100 18 0.52941 0 . 06897 6 .80180 17 0.82051 0. 23529 0 .29393 13 0.94118 0 . 05897 0 .00033 19 0 .0 1. 47059 0 .63836 Recomb. % 32 .35294 S . E . 5 .67319 34 .04254 6 .91184 32 .75861 6 .16264 13 0 .94118 I .72414 0 .92153 17 0 .0 0 .0 0 .23529 18 0 .13846 0 .73134 0.. 02330 15 0 .94118 0. 23529 0. 13320 16 0 .05882 0 .94113 1.40222 19 0.941 18 0. 52941 1.68571 22 3 .28205 1.84615 0. 91884 19 0 .0 0 .05123 0 .20513 9 0 .05882 3 .76471 3.07953 17 0.9<,l 18 1 .47059 0.00998 17 0 .46154 0 .20513 1.75272 7 2 .20000 0 .01533 4 .20079 12 0 .62069 0. 0 3.23021 1 5 0 .06397 0.52941 0.29521 12 0 .27586 2 .11765 0.13896 16 0 .27586 0 .05882 0.29521 14 16 0 .27586 0 .23529 0. 77536 0 .23529 0 .05175 0. 70943 36.36362 6 .48647 h-1 o 2 .20000 0 .16364 1.96679 Combination HA HA HA HA HA HA HA HA HA HA HA HA MDH- 3 MDH- 3 MDH-3 MDH-3 MDH-3 MDH-4 MDH-4 MDH-4 MDH-4 MDH-4 MD H- 4 MDH-4 Tree # N FF FS SF SS 4 4 58 9 17 16 16 0. 62069 18 58 1 3 14 12 19 0 .27596 55 58 11 16 12 1 9 0 .27596 114 58 12 15 16 15 0 .27586 142 57 12 12 22 11 1.72414 3 55 15 19 5 17 2 .20000 4 58 19 3 16 16 0 .62069 53 58 14 10 1 7 17 I. 72414 55 50 14 11 11 14 0 .27586 I 14 58 19 9 16 15 0 .27586 1 1 7 68 17 I 5 19 1 7 0 .23529 124 54 14 12 13 15 0. 16364 1. 10345 I .10345 2 .43276 0 .04897 2 .12281 4 .09091 1.72414 0.2 7586 0 .0 1.72414 0 .23529 0 .0 L 1.34393 0 .51134 0 .02370 0.29521 1.50677 2 .62599 2.09997 0.37924 0.72000 1.30209 0. 00081 0.29630 Recomb. % HA HA HA HA HA HA HA ME MF ME ME ME P G I - 2 P G I - 2 10 58 12 1 6 11 19 0 .06897 2 .48276 0 .22175 18 5 8 16 11 12 19 0 . ' 7 5 8 6 0 .06897 2.42603 117 68 9 23 23 13 0 .23529 0. 23529 8.63761 123 68 12 21 17 13 0 .05882 1 .47059 1.01167 124 55 13 13 13 16 0 . 16364 0 .16364 0 .14627 1 3 58 13 9 14 17 0 .27586 0 .62069 2 .65707 123 68 16 17 18 17 0 .05882 0 .0 0 .05392 32 .35294 5 .67318 HA : PGM 5 58 17 18 1 I 12 2 .48276 HA : PGM 10 57 11 16 13 17 0 .06897 HA : PGM 18 56 12 13 12 19 0 .27536 HA : PGM 23 58 12 15 15 16 0 .27586 HA : PGM 53 58 10 14 16 13 1. 72414 HA : PGM 123 68 16 17 I 9 16 0 .058B2 HA 6PG- 1 1 14 58 16 11 19 12 0 .27586 HA 6PG- 2 3 55 16 1 7 1 I 11 2 .20000 HA sno 20 58 20 20 7 11 8 .34483 IDH MDH- 1 114 58 19 11 12 16 0 .05882 IDH M D H -3 3 55 13 15 13 14 0 .01538 IDH MDH- 3 4 53 14 16 1 I 17 0.0 IDH : MDH-3 92 63 17 12 21 19 1.61333 IDH MDH- 3 1 14 58 15 1 5 13 15 0 .05387 IDH : MDH- 4 3 55 10 I 8 10 17 0 .01533 IDH MDH- 4 4 58 19 1 1 15 1 3 0.0 IDH : MDH- 4 66 68 13 17 23 15 0.82051 IDH : MDH- 4 1 14 58 15 15 19 9 0 .05832 IDH : ME 10 58 16 15 7 20 0.52941 0 .52941 0 .73134 1.51515 0 .23579 0 .94118 0 .05128 3. 76471 0 .01533 0 .27586 0.27536 0 . 16364 1.10345 0 .94119 0 .06397 4.09091 1.77414 0. 23529 1 . 72414 0.00295 0 .03910 0.47241 0. 08930 0 . 15381 0.22342 0 .02370 0.01164 0 .52513 2 .42603 0 .01625 0.31523 0.14943 0.07390 0.00962 0. 55147 1.95490 1.34515 7.48276 3.79141 Combination Tree IDH IDH IDH IDH POM PGM 10 23 6PG-1 114 6PG-2 3 MDH-1 : MDH-3 1 MDH-1 : .MDH-3 114 MDH-1 MDH-1 MDH-1 MDH-1 MDH-1 MDH-1 : MDH-1 : MDH-1 : MDH-3 : MDH-3 : MDH-3 : MDH-3 : MDH-3 MDH-3 MDH-4 MDH-4 ME ME ME PGM 6PG-1 S00 MDH-4 MOH-4 MDH-4 WDH-4 ME ME MDH-3 : MDH-3 : MDH-3 : MDH-3 : MDH-4 : MDH-4 : M.DH-4 : MOH-4 : PGI-2 PGM 6PG- 1 6PG-2 MF Mr ME V F 114 117 1 14 117 29 114 29 3 4 55 114 I 18 18 18 1 14 1 17 1 18 124 1 27 N FF FS SF SS x2 X 2 x2 Recomb. % S.E. 67 63 14 24 77 16 16 8 1 5 20 0.52941 2.11765 0.73134 0.23529 1.07275 6.30558 35.29411 5 .79520 68 21 12 21 14 0.05382 3.76471 0.03977 65 14 19 13 14 0.01538 0.01533 1.24190 47 58 11 14 11 17 11 14 14 13 0.19149 0.27586 0.19149 0.06897 0.16733 0.25717 58 63 1 7 19 14 1 7 17 17 10 15 0.27586 0.23529 1.72414 0.23529 0.37924 0.00031 47 58 68 8 13 20 14 11 16 14 17 12 11 17 20 0. 19149 1.72414 0.23529 0.19149 0. 06897 0.23529 1.79755 0.09430 2.20153 58 11 1 8 15 14 0.0 2.38235 I.10345 53 17 1 4 1 8 9 0.27586 3.76471 0.80389 58 18 11 10 19 0.0 0.06897 4.41379 36.20689 6.31056 55 58 50 58 10 14 10 17 16 11 12 11 10 20 15 17 19 13 13 13 0.16364 1.10345 2.48276 0.06897 4.09091 1.72414 0. 0 1. 72414 0.03657 0.11848 0.32000 0.09430 47 58 10 11 12 14 12 17 13 16 0.19149 1.10345 0.19149 0.05897 0.03071 0.31523 58 13 12 19 14 I.10345 0.62069 0.17356 56 7 17 17 15 1.10345 1.51515 3.03533 58 21 7 14 16 0.06897 3.76471 4.64529 36.20689 6.31056 55 10 16 17 12 0. 16364 0.01533 2.22137 68 58 54 56 14 10 15 9 22 1 7 12 18 1 3 20 I I 15 14 1 I 16 14 0.23529 0.27586 0.0 0.07143 0.23529 0.06397 0.16364 1.14236 2.03 544 4.33305 1.19513 1.33926 36.20689 6.31055 ro Combination MDH-4 MDH-4 MDH-4 MDH-4 MDH-4 MDH-4 MDH-4 MDH-4 ME ME ME ME ME ME ME ME ME PGI -2 PGI -2 PGI -2 PGI -2 PGM PGM Tree ft N FF FS SF SS PGI -2 118 58 16 1 1 1 8 13 PGM 53 58 13 18 13 14 PGM 113 58 15 . 12 1 1 20 PGM 121 58 1 1 1 7 21 9 PGM 127 56 16 1 I 17 12 6PG-1 114 58 22 12 13 11 6PG-1 113 58 10 17 15 16 6PG- 2 3 55 8 12 19 16 P G I - 2 19 58 18 10 14 16 P G I - 2 118 58 20 10 14 14 PGI -2 123 68 14 15 20 19 PGM 10 57 7 15 17 18 PGM 18 56 11 16 13 16 PGM 118 58 13 1 7 13 15 PGM 123 68 16 13 19 20 PGM 127 56 16 8 17 15 6PG-1 118 58 16 14 9 19 PGM 18 56 13 18 1 1 14 PGM 118 58 16 18 10 14 PGM 123 68 19 15 16 1 3 6PG- I 118 58 16 18 9 15 6PG-1 118 63 1 3 19 16 21 SOD 29 58 1 1 1 5 1 7 15 0 .27586 0. 27586 0. 27586 0 .06897 0 .07143 1.72414 0 .27586 0 .06897 0 .06897 1.47059 2 .48276 0 .06397 0 .06897 1.47059 1.14286 0 .67069 1.72414 0.0 1.72414 0.52941 2 .88235 *1I 1.72 414 0 .94113 0.52 941 1.84615 1.51515 3.76471 1 . 47059 Ij 0.00320 Recomb. % 0 .62069 1.72414 0.0 0 .73134 1.51515 0.52941 0 .05128 1.51515 1.51515 0.57941 0 .05128 1.47059 1 . 47059 0. 06 39 7 0 .22175 2 . 31 46 1 5.45860 34 .48274 0.00228 0 .60723 0 .74015 6 .24115 4 .09091 0 .01538 0 .96170 1.79624 1.60734 0.05832 1.43953 0. 09330 0.05544 0 .27117 0. 98606 0 .06397 1.47059 2 .59828 0 .02333 0. 15331 0.52941 0 .49919 0 .01145 0 . 66424 124 APPENDIX 2. A l l e l e frequencies, standard error estimates, and t-values for d i f f e r e n t sample sizes f or PGI-2, MDH-3, and MDH-4 l o c i for the three random runs (A, B, and C). RUN NUMBER A LOCUS i PGI-2 ' ***************** SAMPLE SIZE 5 6 7 8 9 1 0 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 23 29 30 31 3 2 33 34 35 36 37 38 39 4 0 41 42 000 000 000 93 9 399 0. 950 000 0 .875 000 0 .929 0. 967 0. 938 0 . 912 0 .917 0 .974 0 .950 0 .952 0. 932 0 .956 0. 979 0. 960 0 .942 0 .982 0 .964 0 .933 0 .983 0.952 0 .984 • 0. 985 0 .971 0.971 0 .972 0 . 946 0. 961 0.961 0. 962 0.951 0 .952 Q 0 .0 0 . 0 0 . 0 0 .063 0 . 11 1 0 .050 0 .0 0 .125 0 . 0 0.071 0 .033 0 .063 0 .098 0 . 033 0 .026 0 .050 0 .04 8 0 .058 0 .043 0.021 0 .040 0 .058 0.01 9 0 .036 0 . 017 0 .017 0 .048 0. 016 0 .015 0 .029 0.02 9 0.02 3 0 .054 0. 039 0 .038 0 .039 0. 049 0 .048 NDARO ERP.33 T O . F . 0. 0 - 2 . 0 4 9 9? 0 .0 - 2 . 0 4 9 94 0 .0 - 2 . 0 4 9 96 0.05 1 0 .229 98 0 .074 0.813 100 0 .049 0 .044 102 0 .0 - 2 . 0 4 9 104 0 .063 1.084 106 0 .0 - 2 . 0 4 9 103 0. 049 0.442 110 0 .033 - 0 . 3 5 6 112 0 .043 0 .306 114 0 .049 0 .753 116 0 .046 0.692 118 0 .026 - 0 . 6 1 1 120 0 .034 0 .057 122 0 .033 0 .0 124 0 .033 0.462 126 0. 030 - 0 . 1 0 9 128 0.021 - 0 . 8 6 3 130 0.023 - 0 . 2 1 1 132 0.032 0 .253 134 0 .013 - 0 . 9 3 3 136 0 .025 - 0 . 3 5 0 133 0.01 7 - 1 . 0 5 3 140 0 .017 - 1 . 0 8 5 14? 0.027 0.021 1 44 0 .015 - 1 . 1 4 5 146 0 .015 - 1 . 1 7 3 148 0.020 - 0 . 5 3 3 150 0 .020 - 0 . 6 2 3 152 0.019 - 0 . 6 5 6 154 0. 026 0 . 183 156 0.022 - 0 . 2 5 3 158 0.022 - 0 . 2 3 3 160 0.021 - 0 . 3 2 ? . 162 0.024 0 .035 164 0 .023 0 .0 166 *UN NUMBER A LOCUSI MDH-3 > ***************** SAMPLE SI7E P 0 5 I.000 0.0 6 0.917 0.093 7 0. 857 0. 143 B 1 .000 0.0 9 0. 833 0. 167 10 0.900 0. 100 11 0. 909 0.091 12 0.792 0. 208 13 0. 923 0.077 14 0.993 0. 107 15 0.833 0. 167 16 0. 844 0. 156 17 0.823 0. 177 13 0. 861 0. 139 0. 863 0. 132 20 0. 825 0. 175 21 0. 333 0. 167 22 0.795 0.205 23 0. 870 0.130 24 0.854 0. 146 25 0. 860 0. 140 26 0. 846 0. 154 27 0. 970 0.130 29 0.857 0. 143 29 0. 845 0.155 30 0. 817 0.193 31 0.897 0.113 32 0. 859 0. 141 33 0. 909 0. 09 1 34 0. 933 0. 162 35 0. 871 0. 129 36 0. 375 0. 125 37 0. 973 0. 122 38 0. 895 0. 105 3 9 0. 897 0. 103 40 0.875 0.125 41 0. 873 0. 122 42 0. 369 0. 131 NOW) ERRH T O.F. 0.0 -3.553 92 0. 090 -0.543 • 94 0. 094 0.113 96 0. 0 -3.558 93 0.033 0.375 100 0. 06 7 -0.405 102 0.04 1 -0.561 104 0.033 0.852 106 0.052 -0.846 108 0.053 -0.345 110 0.04 3 0.461 112 0.064 0.342 114 0. 065 0.606 116 0.053 0.115 118 0.055 0.009 120 0.060 0.624 122 0.053 0.523 124 0. 061 1.034 126 0.050 -0.010 128 0.051 0.235 130 0.049 0.147 132 0.050 0.367 134 0.046 -0.024 136 0.047 0.200 138 0.04B 0.402 140 0.050 0.843 142 0.040 -0.332 144 0.043 0.169 146 0. 035 -0.785 148 0.045 0.532 150 0.040 -0.044 152 0.039 -0.112 154 0. 033 -0.173 156 0.03 5 -0.504 158 0.034 -0.564 160 0.037 -0..115 142 0.036 -0.174 1 64 0. 037 0.0 1 66 PUN NUMBER A LOCUS ( MDH-4 I * * * * * * * * * * * * * * * * * SAMPLE S IZE 5 6 7 R 9 10 11 1? 13 14 15 16 17 18 19 20 21 22 2 3 24 25 26 27 2 9 29 30 31 32 33 34 35 36 37 39 39 40 41 42 P 0. 600 0 . 500 0. 786 0 .813 0. 555 0 .600 0 .636 0 . 5 0 0 0 .539 0.643 0 . 5 33 0. 656 0. 533 0 .533 0. 605 0 .525 0 .571 0 .568 0 .544 0.521 0 .500 0 .553 0 .593 0. 519 0 . 500 0 .517 0. 516 0. 594 0 .530 0 . 529 0 . 5 5 7 0.542 0 .500 0. 566 0 . 564 3 .563 0. 537 0 . 524 0 3 0 . 400 0 . 500 0 .214 0 . 198 0 .444 0 . 400 0 .364 0. 500 0.461 0 . 357 0 .467 344 0 .412 0.41 7 0 .395 0 .475 0 .429 0 .432 0 .456 0 .479 0 .500 0 .442 0 .407 0.432 0 .500 0 .483 0 .484 0 .406 0 .470 0.471 0 .443 0 .458 0. 500 0 .434 0 . 436 0.43 8 0 . 46 3 0 .476 STANDARD ER^O* 0. 155 0. 144 0 .110 0.098 0 .117 0 . 109 0. 10 3 0.102 0 .099 0.091 0.091 0 .09 4 0.084 0 .092 0 .079 0. 079 0.076 0 .075 0 .073 0.072 0.071 0 .059 0 .067 0.05 7 0 .066 0 .065 0 .063 0.05 1 0.061 0.061 0. 059 0 . 059 0 .058 0 .057 0 .056 0 .055 0 .055 0. 054 T - 0 . 4 6 4 0 .154 - 2 . 1 3 9 - 2 . 5 8 3 - 0 . 2 4 6 - 0 . 6 2 3 - 0 . 9 7 0 0 .206 - 0 . 1 3 1 - 1 . 1 2 7 - 0 . 0 9 0 - 1 . 3 2 3 - 3 . 6 4 1 - 0 . 6 0 4 - 0 . 8 4 7 - 0 . 0 1 3 - 0 . 5 0 7 - 0 . 4 3 0 - 0 . 2 1 5 0.033 0 .267 - 0 . 3 8 6 - 0 . 7 9 8 0.068 0 .279 0 .084 0.097 - 0 . 8 5 1 - 0 . 0 7 9 - 0 . 0 6 9 - 0 . 4 1 3 - 0 . 2 2 3 0.299 - 0 . 5 3 3 - 0 . 5 1 5 - 0 . 4 9 8 - 0 . 1 6 5 0 .0 D .F . 92 94 96 98 100 102 104 106 1 08 110 112 114 116 113 120 122 124 126 128 130 132 134 136 138 140 142 144 146 148 150 152 154 156 159 160 162 164 166 RUN NUM9ER B LCCUSI PGI-2 ) ***************** SAMPLE SIZE P 0 5 0. 900 0. 100 5 1.000 0.0 7 0. 857 0.143 8 1.000 0.0 9 0.944 0.056 10 0.950 0.050 11 1.000 0.0 12 0.917 0. 09 3 13 1.000 0.0 14 0. 964 0.036 15 0.967 0.033 16 0.969 0.031 17 0.941 0.059 13 I. 000 0. 0 19 0.921 0.079 20 0.925 0.075 71 0.976 0. 07 4 22 0.932 0.068 2 3 0. 956 0.043 24 0.953 0.042 25 0. 960 3.040 26 0.981 0.019 27 0.982 0.019 28 0.98 2 0.018 29 I.000 0. 0 30 0. 967 0.033 31 0.968 0.032 32 I.000 0. 0 33 0.985 0.01 5 34 0.971 0.029 35 3.971 0. 029 36 0. 972 0.02 8 37 0. 973 0.077 38 0.947 0.053 39 0. 949 0.051 40 0. 962 0.038 41 0. 951 0. 049 42 0.952 0.048 STANDARD E R W T O . F . 0. 095 0.536 92 0.0 -7.049 94 0.094 0.989 96 0.0 -2.049 98 0.054 0.135 100 0.049 0.044 102 0.0 -2.049 104 0.056 0.585 106 0.0 -2.049 108 0.035 -0.283 110 0.033 -0.356 112 0.031 -0.425 114 0. 040 0.240 116 0.0 -2.049 118 0. 044 0.637 120 0.047 0.574 122 0.024 -0.720 124 0.033 0.462 126 0.030 -0.109 128 0.029 -0.161 130 0.078 -0.211 132 0. 019 -0.945 134 0.013 -0.983 136 0.013 -1.019 138 0.0 -2.049 140 0.023 -0.436 142 0.022 -0.475 144 0.0 -2.049 146 0.015 -1.173 149 0.020 -0.588 150 0.070 -0.623 152 0.019 -0.656 154 0.019 -0.633 156 0.076 0.145 158 0.075 0.107 160 0.071 -0.32? 162 0.074 0.035 164 0. 07 3 0.0 166 RUM NUMBER B LOCUS i MDH-3 i ***************** S AMBLE SIZE 5 6 7 8 9 10 11 17 13 14 15 16 17 18 19 20 21 22 23 24 25 26 77 23 29 30 31 32 33 34 35 36 37 38 39 40 41 42 P 0.800 3.833 0.857 0. 813 0. 77B 0.900 0.818 0. 917 0. 885 0. 893 0.833 0. 344 0.353 0.861 0. 316 0.950 0. 357 0.864 0. 84R 0. 854 0.920 0. 365 0. 339 0. 393 0. 379 0. 350 0. 355 Oi 8 59 0. 364 0.368 0.836 0.361 0. 865 0. 895 0. 335 0. 875 0.878 0. 869 0 0. 200 0. 147 0. 143 0. 138 0.22? 0. 100 0.18? 0.033 0.115 0. 107 0. 167 0.156 0. 147 0. 139 0. 134 0.050 0. 143 0. 136 0.152 0. 146 0.030 0.135 0. U 1 0. 107 0. 121 0. 150 0. 145 0.141 0.136 0.132 0.114 0. 139 0.135 0. 105 0.115 0. 125 0. 12? 0.131 STANDARD ER*}* 0.127 0. 103 0.094 0. 093 0.093 0.057 0.03? 0.056 0. 053 0.053 0.05 3 0.064 0.061 0.058 0.063 0.034 0.054 0. 052 0.053 0.051 0.038 0.047 0.043 0.041 0.043 0.046 0.045 0.043 0. 042 0.041 0.033 0.041 0.040 0.035 0.036 0.037 0.036 0.037 T 0.524 0.314 0.113 0.542 0.871 -0.405 0.564 -0.703 -0.215 -0.346 0.461 0.342 0.227 0.115 0.730 -1.606 0. 132 0.085 0.329 0.235 -0.959 0.060 -0.353 -0.432 -0.183 0.322 0.245 0. 169 0.096 0.025 -0.316 0.144 0.076 -0.504 -0.302 -0.115 -0.174 0.0 D.F. 92 94 96 98 100 102 104 106 108 110 112 114 116 118 120 122 124 126 128 130 132 134 136 138 140 142 144 146 148 150 152 154 156 158 160 162 164 166 Locust mm i * * * * * * * * * * * * * * * * * RUN NUMBER B SAMPLE S IZE 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 2? 23 24 25 26 27 28 29 30 31 3 2. 33 34 35 36 37 33 39 40 41 42 P 0. 700 0 . 583 0 . 7 1 4 0. 588 0 .556 0. 500 0 .773 0.542 0 . 6 5 4 3 .607 0 .633 0 .563 0. 598 0 . 578 0 . 579 0 . 5 5 0 3. 524 0.591 0 .500 0 . 521 9 . 500 0 .500 0 . 537 0. 519 0. 569 0 . 500 0 . 500 0 .563 0 . 515 0 .529 0. 514 0 . 542 3. 527 0. 526 0 .539 0 . 550 0 . 537 0 . 524 3 0 .300 0 .417 0 .286 0 .313 0 .444 0. 500 0 .227 0 .458 0. 346 0 .393 0 .367 0 .438 0.412 0.472 0.421 0 .450 0 .476 0 .409 0 .500 0 .479 0 .500 0 .500 0. 463 0.432 0 . 431 0 .500 0 .500 3 .438 0 .435 0.471 0 .436 0. 458 0 .473 0 .474 0.461 0 .450 0 .463 0 .475 STANDARD 0 . 145 0. 147 0.121 0 .116 0.117 0.112 0. 039 0.102 0. 093 0.092 0.098 0 .033 0.03 4 0 .08 3 0 .080 0 .079 0.077 0 .074 0.074 0.072 0.071 0.069 0. 068 0 .067 0.065 0.065 0 .063 0.062 0 .06? 0.061 0.060 0 . 059 0 .058 0.057 0 .056 0.056 0. 055 0 .0 54 T - 1 . 1 3 3 - 0 . 3 9 0 - 1 . 4 3 8 - 1 . 2 7 8 - 0 . 2 4 6 0.191 - 2 . 3 7 8 - 0 . 1 5 5 - 1 . 2 0 3 - 0 . 7 7 7 - 1 . 0 5 8 - 0 . 3 7 5 - 0 . 6 4 1 - 0 . 0 4 3 - 0 . 5 6 9 - 0 . 2 7 4 0 .0 - 0 . 7 2 9 0 .260 0.033 0.267 0 .770 - 0 . 1 5 2 0.068 - 0 . 5 3 3 0.282 0.234 - 0 . 4 6 9 0 .105 - 0 . 0 6 9 0.113 - 0 . 2 2 3 - 0 . 0 4 3 - 0 . 0 3 2 - 0 . 137 - 0 . 3 3 6 - 0 . 1 6 5 0 .0 D . F . 92 94 96 98 100 102 104 106 108 110 112 114 116 118 120 122 124 126 128 130 132 134 136 138 140 142 144 146 148 150 152 154 156 159 160 162 164 166 LO O RUN NUMBER C LOCUS! PGI-2 ) ***************** SAMPLE SIZE P 3 5 0 .900 0 . 130 6 0 .917 0 . 093 7 1.000 0 .0 8 1 .000 0 . 0 9 0 .889 0.111 10 0. 95 0 0 .050 11 0 .955 0 .045 12 0. 958 0 .042 13 0. 923 0 .077 14 0 .929 0.071 1 5 0. 967 0 .033 1 6 0 .969 0 .031 17 I . 000 0. 0 IB 0 .917 0 .083 19 1.000 0 . 0 20 0 . 950 0. 050 21 0 .929 0.071 22 0. 955 0 .045 23 0 .935 0 .065 24 1.000 0 . 0 25 0 .940 0 .050 26 0.981 0 . 019 27 0 .932 0 .019 23 1 . 000 0 .0 29 0 .993 0. 017 30 1 .000 0 .0 31 0 . 934 0. 016 32 0 .934 0 .016 33 0. 935 0 .015 34 0.971 0 .029 35 0 .957 0 .043 36 0 .972 0 .028 37 0 .973 0 .027 38 0 .947 0 .053 39 0.961 0 .033 40 0. 962 0. 038 41 0 .951 0 .049 42 0. 952 0 .048 NOARO ERR03 T O . F . 0.095 0 .536 92 0 .080 0 .430 94 0 .0 - 2 . 0 4 9 96 0 .0 - 2 . 0 4 9 98 0 .074 0.818 100 0 .049 0 .044 102 0. 044 - 0 . 0 4 3 104 0.041 - 0 . 1 2 7 106 0.052 0.512 108 0 .049 0.442 110 0 .033 - 0 . 3 5 6 112 0.031 - 0 . 4 2 5 114 0 .0 - 2 . 0 4 9 116 0 .046 0.692 118 0 .0 - 2 . 0 4 9 120 0 .034 0.057 122 0 .040 0 .517 124 0.031 - 0 . 0 5 6 126 0 .036 0 .409 128 0 .0 - 2 . 0 4 9 130 0. 034 0 .303 132 0 .019 - 0 . 9 4 5 134 o . o i a - 0 . 9 8 3 136 0 .0 - 2 . 0 4 9 138 0 .017 - 1 . 0 5 3 140 0. 0 - 2 . 0 4 9 142 0 .016 - 1 . 1 1 6 144 0 .016 - 1 . 1 4 5 146 0 .015 - 1 . 1 7 3 148 0. 020 - 0 . 5 8 9 150 0 .024 - 0 . 1 4 2 152 0. 019 - 0 . 6 5 6 154 0 .019 - 0 . 6 9 8 156 0 .026 0 .145 158 0.022 - 0 . 2 8 9 160 0.021 - 0 . 3 2 2 162 0. 02 4 0.035 164 0 .023 0 .0 166 RUN NUMBER C LOCUSI MDH-3 ) SAMPLE SIZE P 0 5 3 .800 0 .200 6 0 .917 0 .083 7 0 .786 0 . 2 1 4 8 0. 750 0 .250 9 0. 778 0 . 222 10 0 . 850 0 . 150 11 0. 364 0 . 136 12 0 .917 0 . 0 9 3 13 0. 885 0 .115 14 0 .786 0 . 2 1 4 15 0. 833 0 . 167 16 0 . 844 0. 156 I 7 0 .912 0 .038 18 0 . 961 0 .139 19 0 .842 0 .158 20 0. 850 0 . 150 21 0 . 833 0 . 167 22 0. 886 0 .114 23 0 .848 0 . 152 24 0 . 354 0 . 146 25 0. B90 0 . 120 26 0 .904 0 .096 27 0 .907 0 . 093 28 0 .946 0 .054 29 0. 879 0 . 121 30 0 .883 0 . 117 31 0. 839 0 . 161 32 0. 875 0 . 125 33 0. 849 0.151 34 0. 887 0 .118 35 3.871 0 . 129 36 0 .889 0. I l l 37 0 . 865 0 .135 38 0. 895 0 . 105 39 3 . 935 0. 115 40 0. 887 0 .113 41 0. 878 0 . 122 4? 0 . 869 0.131 NDARO ERR3* T O.F . 0 . 127 0.524 92 0.030 - 0 . 5 4 3 94 0. 110 0 .720 96 0.139 1.041 93 0.099 0.871 100 0.030 0 .216 102 0 .073 0 .066 104 0 .056 - 0 . 7 0 8 106 0 .063 - 0 . 2 1 5 108 0 .073 0.970 110 0.063 0.461 112 0 .064 0.342 114 0 .040 - 0 . 7 0 1 116 0 .058 0 .115 118 0 .059 0 .386 120 0.056 0.282 122 0. 05 3 0 .523 124 0 .043 - 0 . 2 3 8 126 0 .053 0 .329 128 0.051 0 .235 130 0 .046 - 0 . 1 8 7 132 0. 041 - 0 . 6 3 3 134 0 .039 - 0 . 7 1 2 136 0 .030 - 1 . 6 2 9 138 0.043 - 0 . 1 8 3 140 0.041 - 0 . 2 5 8 1 42 0.047 0 .510 144 0.041 - 0 . 1 0 8 146 0 .044 0 .357 148 0.039 - 0 . 2 5 0 150 0 .040 - 0 . 0 4 4 152 0.03 7 - 0 . 3 8 1 154 0 .040 0.076 t56 0.035 - 0 . 5 0 4 158 0.036 - 0 . 3 0 2 160 0.03 5 - 0 . 3 6 3 162 0.036 - 0 . 1 7 4 164 0 .037 0 .0 166 ho RUN NUMBER C LOCUSI MDH-4 ) ***************** SAMPLE SIZE P Q 5 0. 700 0 .300 6 0 . 593 0 .417 7 0 .643 0. 357 8 0 .563 0 .438 9 0. 556 0 . 444 10 0 .700 0 .300 11 0 .636 0 .364 12 0 .583 0 .417 1 3 0. 577 0 .423 14 0 . 500 0 . 500 I 5 0 .533 0 .467 16 0. 594 0 . 406 17 0 .529 0.471 18 0 .611 0 .339 19 0 . 605 0 .395 20 0. 500 0 .500 21 0 . 5 2 4 0 .476 22 0.591 0 .409 23 0 . 544 0 .456 24 0 .583 0 .417 25 0 .620 0 .380 26 0 . 558 0 .442 27 0 .513 0 .482 2 8 0. 536 0 . 4 6 4 79 0 . 60 3 0 .397 30 0 .517 0 . 493 31 0 .548 0 .452 32 0 . 531 0 .469 33 0 . 576 0 .424 34 0 .515 0 .43 5 35 0 . 529 0.471 36 0. 500 0 .500 37 0 . 500 0 . 500 38 0 .539 0.461 39 0. 5 26 0 .474 40 0 .550 0 .450 41 0. 549 0. 451 42 0 . 524 0 .476 STANDARD E R R H T D . F . 0 .145 - 1 . 1 3 8 92 0 .142 - 0 . 3 9 0 94 0 .123 - 0 . 8 5 6 96 0 .124 - 0 . 2 8 6 98 0 .117 - 0 . 2 4 6 100 0 . 103 - 1 . 5 1 3 102 0 . 103 - 0 . 9 7 0 104 0.101 - 0 . 5 2 0 106 0 .097 - 0 . 4 7 8 108 0 .094 0 .218 110 0 .09 1 - 0 . 0 9 0 112 0 .087 - 0 . 6 8 2 114 0. 036 - 0 . 0 5 5 116 0.031 - 0 . 8 9 2 118 0 .079 - 0 . 8 4 7 120 0 .079 0.248 122 0 .077 0 .0 124 0 .074 - 0 . 7 2 9 126 0. 073 - 0 . 2 1 5 128 0.071 - 0 . 6 6 4 130 0 .059 - 1 . 0 9 8 132 0. 069 - 0 . 3 8 6 134 0.06 3 0.061 136 0 . 057 - 0 . 1 3 3 138 0 .064 - 0 . 9 4 5 140 0. 065 0 .094 142 0. 063 - 0 . 2 9 5 144 0 .052 - 0 . 0 8 9 146 0.061 - 0 . 6 3 7 148 0.061 0 . 112 150 0. 060 - 0 . 0 5 9 152 0 .059 0 .297 154 0. 058 0 .299 156 0 .057 - 0 . 1 9 9 158 0.05 r - 0 . 0 7 3 160 0. 056 - 0 . 3 3 6 162 0.055 - 0 . 3 7 3 164 0 .054 0 .0 166 OJ 134 APPENDIX 3. A l l e l i c v a r i a t i o n of 27 l o c i i n the four e l e v a t i o n a l classes. 0 Locus A l l e l e A E l e v a t i o n a l Classes B C D AAT-1 100 1.000 h 0.000 AAT-2 82 0.000 100 0.868 112 0.132 h 0.229 AAT-3 100 0.974 130 0.026 h 0.051 ACO 77 0.132 100 0.868 h 0.229 APH-2 n u l l 0.079 72 0.184 87 0.342 100 0.395 h 0.687 DIA-2 79 0.026 100 0.974 h 0.051 EST-1 85 0.079 92 0.368 100 0.342 110 0.211 h 0.697 GAPDH 100 1.000 h 0.000 GDH 100 1.000 h 0.000 G6P 80 0.053 90 0.684 100 0.263 h 0.460 1^000 1.000 1.000 0.000 0.000 0.000 0.000 0.125 0.100 0.893 0.875 0.800 0.107 0.000 0.100 0.191 0.219 0.340 0.964 0.875 0.900 0.036 0.125 0.100 0.069 0.219 0.180 0.071 0.125 0.500 0.929 0.875 0.500 0.132 0.219 0.500 0.071 0.000 0.100 0.357 0.250 0.100 0.107 0.375 0.700 0.464 0.375 0.100 0.641 0.656 0.480 0.000 0.000 0.000 1.000 1.000 1.000 0.000 0.000 0.000 0.000 0.000 0.100 0.321 0.375 0.500 0.321 0.250 0.300 0.357 0.375 0.100 0.666 0.656 0.640 1.000 1.000 1.000 0.000 0.000 0.000 1.000 1.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.357 0.375 0.200 0.643 0.625 0.800 0.459 0.469 0.320 136-E l e v a t i o n a l Classes Locus A l l e l e D HA 100 0.605 140 0.342 170 0.053 h 0.514 IDH 65 0.079 90 0.079 100 0.842 122 0.000 h 0.279 MDH-1 90 0.079 100 0.868 105 0.053 h 0.238 MDH-2 100 1.000 h 0.000 MDH-3 n u l l 0.000 84 0.184 100 0.816 h 0.300 MDH-4 75 0.105 100 0.605 129 0.289 h 0.539 ME 85 0.079 100 0.921 130 0.000 h 0.146 MPI-1 100 1.000 h 0.000 MPI-2 100 1.000 h 0.000 PEP-1 100 1.000 h 0.000 PEP-2 100 1.000 h 0.000 0.571 0.875 0.600 0.143 0.125 0.200 0.286 0.000 0.200 0.572 0.219 0.560 0.000 0.125 0.000 0.000 0.125 0.200 1.000 0.750 0.800 0.000 0.000 0.000 1.000 0.406 0.320 0.036 0.000 0.000 0.929 1.000 0.000 0.036 0.000 1.000 0.134 0.000 0.000 1.000 1.000 1.000 0.000 0.000 0.000 0.000 0.000 0.200 0.107 0.000 0.000 0.893 1.000 0.800 0.069 0.000 0.320 0.000 0.375 0.000 0.464 0.625 0.400 0.536 0.000 0.600 0.497 0.469 0.480 0.179 0.000 0.100 0.786 1.000 0.900 0.036 0.000 0.000 0.349 0.000 0.180 1.000 1.000 1.000 0.000 0.000 0.000 1.000 1.000 1.000 0.000 0.000 0.000 1.000 1.000 1.000 0.000 0.000 0.000 1.000 1.000 1.000 0.000 0.000 0.000 13? E l e v a t i o n a l Classes Locus A l l e l e ~ D PEP-3 100 1.000 h 0.000 PGI-2 100 0.947 125 0.053 h 0.100 PGM 94 0.184 100 0.711 105 0.105 h 0.450 6PG-1 85 0.026 100 0.974 107 0.000 h 0.051 6PG-2 100 0.974 122 0.026 h 0.051 SOD 33 0.026 85 0.026 100 0.947 h 0.102 1.000 1.000 1.000 0.000 0.000 0.000 0.929 1.000 1.000 0.071 0.000 0.000 0.132 0.000 0.000 0.107 0.125 0.100 0.750 0.875 0.800 0.143 0.000 0.100 0.406 0.219 0.340 0.000 0.000 0.000 0.964 1.000 1.000 0.036 0.000 0.000 0.069 0.000 0.000 0.964 0.875 1.000 0.036 0.125 0.000 0.069 0.219 0.000 0.000 0.000 0.000 0.000 0.125 0.000 1.000. 0.875 1.000 0.000 0.219 0.000 

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