National Library Bibliothèque nationale 1+1 of Canada du Canada Acquisitions and Direction des acquisitions et Bibliographie services Branch des services bibliographiques 395 wellington Strcel 395. rue Wellinglon Ottawa. Onlano onawa (On13OO) K1AON~ K1AQN4 ' ..... r.... \.>t',.,.. ,.~, .." .••

NOTICE AVIS

The quality of this microform is La qualité de cette microforme heavily dependent upon the dépend grandement de la qualité quality of the original thesis de la thèse soumise au submitted for microfilming. microfilmage. Nous avons tout Every effort has been made to fait pour assurer une qualité ensure the highest quality of supérieure de reproduction. reproduction possible.

If pages are missing, contact the S'il manque des pages, veuillez university which granted the communiquer avec l'université degree. qui a conféré le grade.

Some pages May have indistinct La qualité d'impression de print especially if the original certaines pages peut laisser à pages were typed with a poor désirer, surtout si les pages typewriter ribbon or if the originales ont été university sent us an inferior dactylographiées à l'aide d'un photocopy. ruban usé ou si l'université nous a tait parvenir une photocopie de : qualité inférieure.

Reproduction in full or in part of La reproduction, même partielle, = this microform is governed by de cette microforme est soumise the Canadian Copyright Act, à la Loi canadienne sur le droit R.S.C. 1970, c. C-30, and d'auteur, SRC 1970, c. e-30, et subsequent amendments. ses amendements subséquents.

Canada •

GENETIC STUDIES OF RED (TRIFOLIUM PRATENSE 1..)

USING MORPHOLOGICAL, ISOZVME AND

RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) MARKERS

PRASERT KONGKlATNGAM

Oepartment of Science

McGilI University

Montreal

July 1995

A thesis submitted to the Faculty of Graduate Studies and Research

in partial fulfilment of the reqùirements of the degree of Ooetor of Philosophy

ID Prasert Kongkiatngam, 1995 National Library Bibliothèque nationale 1+1 of Canada du Canada Acquisitions and Direction des acquisitions et Bibliographie Services Branch des services bibliographiques 395 Welli~ton Street 395. rue Welhnglon Ottawa. Oritano Ottawa (On,.roo) K1A0N4 K1AON4

The author has granted an L'auteur a accordé une licence irrevocable non-exclusive licence irrévocable et non exclusive allowing the National Ubrary of permettant à la Bibliothèque Canada to reproduce, loan, nationale du Canada de distribute or sell copies of reproduire, prêter, distribuer ou his/her thesis by any means and vendre des copies de sa thèse in any form or format, making de quelque manière et sous this thesis available to interested quelque forme que ce soit pour persons. mettre des exemplaires de cette thèse à la disposition des personnes intéressées.

The author retains ownership of L'auteur conserve la propriété du the copyright in his/her thesis. droit d'auteur qui protège sa Neither the thesis nor substantial thèse. Ni la thèse ni des extraits extracts from it may be printed or substantiels de celle-ci ne otherwise reproduced without doivent être imprimés ou hiS/11er permission. autrement reproduits sans son autorisation.

ISBN 0-612-08123-0

Canada •

Short Title:

Genetic Studies of Red Clover (Trifolium pratelL~e 1..)

Prasert Kongkiatngam • ABSTRACT Genctic variation within and between two cultivars of red clover (Tri/oliam pratense L), Essi from Europe and Ottawa from Canada was estimated using morphological, isozyme and randol11 amplified polymorphie DNA (RAPD) markers. A total of 21 enzyme-coding loci with 43 alleles was deteeted using twelve enzyme systems. The mean number of aileles per locus was 1.81 in Essi and 1.67 in Ottawa. Nine 10-mer primers were used to assay 20 individuals from eaeh cultivar for RAPD m:-:rkers. Eaeh primer gave from 7 to 20 amplified bands with an average of 14.8 bands per primer. High within-eultivar variation was observed in both cultivars using both isozyme and RAPD markers. The mode of inheritanee of seven isozyme loci: Aat-2, Amy-l, Est-4, Est-7, Pgd-1, Pgd-2 and Skd-1. in red clover was verified. The genetic basis of banding patterns for 16 other isozyme loci: Aat-3, Adlz-1, Dia-l, Dia­ 2, Dia-3, Est-l, Est-2, Gpi-2, ldlz-l, Mdlz-l, Mdlz-2, Mdlz-3, Mdlz-4, Me-l, Me-2 and Pgm-2, was also postulated, based on the segregation patterns observed within cultivars. Two pairs oflinked enzyme-coding loci, Est-4jEst-7 and Pgd-2/Skd-1, were found with joint segregation analysis. Estimates ofgenetic variability of lS red clover cultivars from three different origins indicated that within-cultivar variation was much higher than between-cultivar variation. Allele frquencies of these isozymes could discriminate the five North American cultivars assayed, but they could not differentiate cultivars from Europe and Japan. The use of RAPD markers obtained frum bulked samples was investigated for cultivar identification in red clover. Pooled samples were examined in order to minimize variation within cultivars. Twenty was found to be an appropriate number of red clover individuals per bulk for homogenizing genetic variation within cultivars. Fourteen 1D-mer primers were used to amplify genomic DNA from combined leafsamples of lS red clover cultivars from European, Japanese and North American origins. A total of 79 amplified bands, of which SS were polymorphie, was obtained. Cultivar-specifie bands were observed • with 13 primers. The amplification patterns obtained from two primers could distinguish ail 15 red c10ver cultivars. Cluster analysis b'lscd on Rogers' genctic • distances separated these 15 cultivars inw four groups. but each group cont.lins cultivars from more than one geogr;:phical origin. Six lllorpholugiC

• ii • RÉSUMÉ Les variations génétiques parmi et entre deux cultivars de trèfle rouge (Trifolium pratense L) ont eté étudiées. Les cultivars Essi provenant d'Europe et Ottaw-.t provenant du Canada ont été analysés aux niveaux morphologique, de l'étude des isozymes et de l'étude des marqueurs RAPD ("Random Amplified Polymorphie DNA"). Un total de 21 loci contenant 43 allèles codant pour les enzymcsont été détectés en utilisant douze différents systèmes d'enzymes. Le nombre moyen d'allèles par locus était de 1.81 pour le cultivar Essi et de 1.67 pour Ottawa. Neuf amorces de 10 nucléotides ont été utilisées sur 20 individus de chacun des deux cultivars afin d'identifier les marqueurs RAPD appropriés. Chacune des amorces permet de detecter de 7 à 20 bandes amplifiées, avec une moyenne de 14.8 bandes detectées par amorce. L'étude des isozymes et des marqueurs RAPD a permis de détecter une grande variation au sein du même cultivar et cela pour les 2 cultivars étudiés. La transmission génétique de sept loci d'isczymes: Aat-2, Amy-l, &t-4, &t-7, Pgd-1, Pgd-2 et Skd-1 du tréfle rouge a été étudiée. La base génétique des patror,s de bandes pour 16 autre loci d'isozymes:Aat-3, Adh-1, Dia-l, Dia-2, Dia­ 3, Est-l, &t-2, Gpi-2, Idh-1, Mdll-1, Mdll-2, Mdll-3, Mdh-4, Me-l, Me-2 et Pgm-2 a aussi été postulée, basé sur le schéma de ségrégation observé parmis les cultivars. Deux paires de loci codant pour des enzymes Est-4jEst-7 et Pgd-2jSkd-1 ont été découvert à l'aide d'une analyse de ségrégation. L'estimation de la variabilité génétique de 15 cultivars de trèfle rouge, de trois différentes origines, indique clairement que la variation génétique est beaucoup plus importante au sein du même cultivar qu'entre les différents cultivars. Dest possible d'utiliser la fréquence des allèles de ces isozymes pour distinguer les cinq cultivars américains testé. Par contre, il est impossible d'utiliser cette méthode pour différencier les cultivars en provenance d'Europe et du Japon. L'utilisation de marqueurs RAPD obtenus déchantillons combinés a été étudiée afin de pennetté l'identification des cultivars de trèfle rouge. Les échantillons ont été combinés afin de minimiser la variation parmis les cultivars. Vmgt individus semblait être un nombre approprié de plants de trèfles rouges par échantillon combiné, ceci afin d'avoir une variation génétique

• iü parmis les cultivars la plus homogène possible. Quatorze amorces de 10 nucléotides • ont été utilisées afin d'amplifier rADN génomique des échantillons comhinés contenant les feuilles de 15 cultivars de trèfle rouge. Ces cultivars étaient d'origine européenne. japonaise et nord-Américaine, Un total de 79 bandes ampIilïées ;1 été

obtenu desquelles 55 se sont révélées polymorphes. Des bandes spécifiques ;IUX cultivars ont été observées à raide de 13 amorces. Le patron d'amplific

supervision of my research and thesis preparation. A special thanlc goes to Dr.

Marc G. Fortin for his advice on RAPO research and thesis preparation. 1 also

would like to thank my original supervisor, Dr. Bruce E. CouIman, for his advice and

help throughout my study. 1 would also like to give my gratitude to Dr. Diane E.

Mather and Dr. Nick A Tinker for advice and help. The financial help from the

Canada-Egypt-McGill Agricultural Response Program for my scholarship is very

much appreciated. 1 would like to extend my deep gratitude to Chonticha Rukkrai

fOI' her love, understanding, patience, support and help. My thanks also go to Kathy

McClintock and Wendy Asbil for their advice, help and generosity, and to Helen

Rimmer for her photographie assistance. 1 also want to thank ail graduate students

-=in the Plant Science Department especially those in the genetics and plant

systematics labs for their he!p. Finally, 1 would like,to extend my gratitude to ail

m~mbers of my family, especially my parents, for their support.

• v • TABLE OF CONTENTS Abslracl

Résumé 111

Acknowledgmems \' List of Figures x

List of Tables XI

List of Appendices XII1 Description of Thesis Format xiv Chapter One General Introduction 1 Chapter Two General Literature Review 6 2.1. Economie Importance of Red Clover b 22. Origin, Distribution and of Red Clover 7

2.3. Morphology of Red Clover <) 2.4. Genelics and Breeding of Red Clover II 2.4.1. Morphological and Agronomie Traits Il 2.4.2. Pest Resistance 14 25. Seed Production in Red Clover 15 2.6. Applications of Isozymes in Plant Breeding and Genetics Hl 2.6.1. Discrimination of Crop Cultivars IS 2.62. Studies of Genetic Diversity in Plant Populations and 22 2.7. The Use of RAPD Markers in Plant Breeding and Genetks 25 ~7.1. Studies of Genetic Diversity and Relationships of Crop Germplasms 26 2.72. Identification of Crop Genotypes and Cultivars 29 Chapter Three Genetic Variation Within and Between Cultivars ofRed c10ver 31 3.1. SummaI)' 32 32. Introduction 33 3.3. Materials and Methods 35 3.3.1. Morphologica\ Markers 35 • vi 3.3.2. Isozyme Marker, 36 • 3.3.3. RAPD Markers 37 3.3.4. Data Analysis 38 3.4. Results and Discussion 40 3.4.1. Morphological Markers 40 3.4.2. Isozyme Markers 41 3.4.3. RAPD Markers 44 3.4.4. Comparisons of Morphological. Isozyme and RAPD Markers 47 Chapter Four Diversity and Genetics of Isozymes in Red c10ver 55 4.1. Abstract 56 4.2. Introduction 56 4.3. Materials and Methods 59 4.3.1. lsozyme Assay 59 4.3.2. Data Analysis 61 4.4. Results and Discussions 62 4.4.1. Polymorphism and Genetics of Isozymes in Red Clover 62 4.4.1.1. Aspartate Aminotransferase (AAT) 63 4.4.12. Amylase (AMY) 63 4.4.1.3. Esterase (EST) 64 4.4.1.4. 6-Phosphogluconate Dehydrogenase (PGD) 65 4.4.1.5. Shikimate Dehydrogenase (SKO) 65 4.4.1.6. A1cohol Dehydrogenase (ADH) 66 4.4.1.7. NADH Diaphorase (DIA) 66 4.4.1.8. GlucosepGosphate Isomerase (GPI) 67 4.4.1.9. Isocitrate Dehydrogenase (IDH) 67 4.4.1.10. Malate Dehydrogenase (MOH) 67 4.4.1.11. Malic Enzyme (ME) 68 4.4.1.12 Phosphoglucomutase (PGM) 69 4.4.1.13. linkage Analysis 69 4.42. Isozyme Diversity in Red Clover 69 • vii 4.4.3. Identitïcation and Genetic Rlationships • of Red Clover Cultivars 7~ Chapter Five Polymorphisms of KAPD Markers from Bulked Genomic DNA among Cultivars of Red Clo\'er 89 5.1. Abstract QO 5.2. Introduction 91 5.3. Materials and Methods 94 5.3.1. Bulked Genomic DNA E:maction and PurifiC'.ltion 94 5.3.2. DNA Amplification 95 5.3.3. Data Analysis 96 5.4. Results 9b 5.4.1. Determination of the Optimal Numberof Individuals in Bulked Samples 96 5.4.2. RAPD Polymorphism Between Red Clover Cultivars 97 55. Discussion 9~ 55.1. Determination of the Appropriate Number of Individuals in Bulked Samples 98 552. RAPD Polymorphism Between Red Clover Cultivars 99 55.3. Genetic Relatioships of Red Clover Cultivars from RAPD Data 101 Chapter Six EtTect ofThree Cycles of Natural Selection on Morphological Traits and lsozymes in Red Clover 109 6.1. Abstraet 110 6.2. Introduction 111 6.3. Materials and Methods 114 6.3.1. Morphological Traits 114 6.32. Isozyme Assay 115 6.3.3. Data Apalysis 117 6.4. Results and Discussion 117 6.4.1. Effeet on Morphological Traits 117 • vili 6.4.2. Effect on ABele Frequencies and Diversity of lsozyme Loci 119 • 6.4.3. Natural Selection and Seed Production in Red Clover 123 Contributions lo Knowledge 132 General Conclusions 134 Bibliography 136

• ix • LIST OF FIGURES

3.l. RAPD patterns obtained from individual genomie DNA samples

of red clover CVS. Essi and Ottawa with primer H-IS. S4

4.l. Sehematie diagrams of isozyme banding patlems and assignment

of loci and alleles in the polymorphie isozymes in red clover. 87

4.2. Banding patterns of EST, MDH and 6-PGD isozymes in red clover.

Genotypes are shown at the bottom. 88

5.1. RAPD patterns obtained from bulked genomie DNA samples

with different numbers of individuals in the bulks. 106

5.2. RAPD patterns generated with primer H-02 from bulked samples

of 15 red clover cultivars. 107

5.3. Clusters based on Rogers' genetie distances calculated from RAPD

data for 15 red clover cultivars. 108

e· x • LIST Of TABLES 3.1. Sequences and amplified products of nine arbitrary primers (Operon)

used te generate RAPD markers in Trifolium pracense L 56

3.2. Genetic variation detected by morphological, isozyme, and

RAPD markers in red clover cultivars Essi and Ottawa. 57

33. Allele frequencies at polymorphic loci eoding for isozymes and

morphological traits. 58

3.4. Comparisons of genetie variation within populations of different

Trifolium species based on isozymes. 60

4.1. List of red clover cultivars and their origins. 81

4.2 Segregation analysis of isozyme loci in red clover. 82

43. Joint segregation analysis of two-locus eombinations

of enzyme-coding loci in red clover 78

4.4. Allele frequencies at polymorphie loci in red clover. 80

4.5. Estimates of Mean of heterozygosity from direct eount (HœJ, Mean

of expected hetero~gosity (~), Mean number of alleles per locus (A)

and pereentage of polymorphie loci (P) from 21 isozyme loci in red clover. 84

4.6. G-statistics and genetie distances between 15 cultivars of red clover. 85

4.7. Mean genetie distane~ and U-statistics between groups of cultivars

from different origins. 86

• xi 5.1. RAPD patterns generated from bulked sampIes of 15 red dover

• cultivars from three geographical origins. 104

5.2. Sequences and number of amplified products of nine arbitrary primers

(Operon) used to generate RAPD markers from bulked DNA samples

in red clover (Trifolium pralense L). 105

6.1. Percentages of red clover plants \Vith sorne morphologie.lI traits

from different cycles of natural selection. 125

6.2. Allele frequencies of polymorphie isozyme loci in red clover

populations that have undergone three cycles of natural selection. 126

6.3. Contingency table x2 test V"alues of allele frequency heterogeneity

of red clover populations. 130

6.4. Estimates of mean of heterozygosity from direct count (Hobl;)'

mean of expeeted heterozygosity (Hcxp), mean number of aileles

per locus (A) and percentage of polymorphie loci (P) from

21 isozyme loci in red clover populations. 131

• xii • LIST OF APPENDICES

1. Estimates of total heterozygosity (Hr). within-cultivar heterozygosity

(Hs), coefficient of gene differentiation (Gsr) and between-cultivar

heterozygosity (Dsr) of polymorphie isozyme loci in red clover. 155

2. The RAPD data matrix generated from 14 arbitrary primers of

15 red clover cultivars. 156

3. Rogers' (1972) genetic distances between 15 cultivars of red clover

based on isozyme (b:low diagonal) and R<\PD (above diagonal) data. 157

• xiii • DESCRIPTION OF THESIS FORMAT In accordance with pan B. section 2 of the "Guideline Concerning Thesis

Preparation" from the Faculty ofGraduate Studies and Research. McGill University.

this thesis consislS of submitted manuscripts (papers).

The entire text of the guideline that applies to this format is quoted below:

" 21 Manuscripts and Autborsbip: Candidates have the option, subject to the approval oftheir Department, ofinc/uding, as part oftheir thesis, copies ofthe text ofa paper(s) submitted for publication, or the c/early- duplicated text ofa published paper(s), providing that these copies are bound as an integral part ofthe thesis. Ifthis option is chosen, connecting texts, providing logical bridges between the different papers, are mandatoty. The thesis must still confonn to ail other ~quirements ofthe "Guideline Conceming Thesis Preparation" and should be in a literary fonn that is more than a mere collection ofmanuscripts published or to be published. The thesis must include, as separate cllapters or sections: (1) a table ofcontents, (2) a general abstract in English and Frencll, (3) and introduction which c/early states the rationale and objectives ofthe study, (4) a comprehensive general n.'View of the background lilerature to the subject ofthe thesis, when this review is appropriate, and (5) a final overall conclusion and/orsummary. Additional material (proceduraJ and design data, as weil as descriptions ofequipment used) must be provided where appropriate and in sufficient detail (eg. in appendices) to aIlow a c/ear and precise judgement to be made oftlle importance and originality oftlle research reported in the tlzesis. In the , case ofmanuscripts Co-autllOred by the candidate andothers, the candidate is required to make an explicit statement in the thesis ofwho contributed to such work and to what extent; supervisors must attest to the accuracy ofsucll c/aims at the PII.D. Oral Defense. Since tlle task oftlle exominers is made more difficult in these cases, il is

• xiv in tlze candidates interest to make perfectly clear tlze responsibilities oftlze different • autlzors ofco-autlzored papers."

To conform with the above statemenl, this thesis is organized as fol1ows. The

thesis starts with a table of contents, fol1owed by abstraets in English and French.

Chapter one is a general introduction which gives the rationale and objectives of this

study. A general literature review in chapter IWO describes mainly the genetics and

breeding of red clover, the use of isozymes in plant genetics and breeding, and the

applications of RAl'D markers for plant germplasm evaluation and cultivar

identification. In chapters three to six, each chapter includes a separate manuscript

su;,mined for publication. These chapter~ begin with a statement about the

contributions of each author. The thesis finishes with a summary, a statement of

contributions to knowledge, and general conclusions. AlI references, including those

cited in the manuscripts, are included at the end of the thesis as a bibliography•

• CHAPTER ONE

• GENERAL INTRODUCTION

Red clover (Trifoiiun: pratense L) is recognised as one of the most important

forage legurnes in temperate regions of the world. Il c"•.m be grown in a wide range

of soil types, pH levels and environmental conditions (Smith et al. 1985) and gives

satisfaetory yield in areas that are not suitable for growing alfalfa because the soils

are too wet and/or too acid. Red clover is a significant forage legume grown in

eastern Canada and the northeastern United States. Through symbiotic nitrogen

fIXation, red clover also provides nitrogen for soils, companion crops and subsequent

crops. The range offlXed nitrogen was estimated to be from 125 to 220 kg ha·1year· 1

(LaRue and Patterson 1981; Rohweder et al. 1977). These qualities have made red

clover useful for hay, silage, pasture, intercropping and green manure in several

collntries (Smith et al. 1985; Taylor and Smith 1979).

Red clover is a cross-pollinated specie~ with a gametophytic self·

incompatibility system (Fergus and Hollowell 1960). Insects, for example honeybees

(Apis spp.), bumblebees (Bombus spp.), alkali bees (Nomia melonderi Ckll.), and leaf

cutter bees (Megaclùle rotumlata F.) are required for flower pollination (Taylor and

Smith 1979). Red clover is a short-lived perennial, persisting for two to three years,

depending on the cultivar and environmental conditions (Smith et al. 1985). AlI

cultivars of red clover in use today are heterogeneous populations consisting of

numerous heterozygous individuals (Taylor and Smith 1979). Most ofthese cultivars

have been developed by controlled mass selection and recurrent selection.

• 1 Currently, only a few morphological and agronomie traits, which are under • environmental influence, are available for use as genetic markers in genetic and breeding studies in rl"d clover (Fergus and Hollowell 1960; Quesenberry et al. 1991;

Taylor and Smith 1979). As more cultivars of red clover are developed, more

reliable markers are also needed for distinguishing them in cultivar development

programs, commercial seed production and seed certification programs. More

genetic markers, which are under simple genetic control, less influenced by

environmental conditions and easily assayed, are required. A problem of genetic

shift when seed of red clover cultivars is produced outside areas of their adaptation

is also present in red clover seed production. Therefore, it would be beneficial to

obtain information about genetic changes after several cycles of natural selection in

red c\over using morphological and isozyme markers.

Isozymes, discovered by Hunter and Markert (1957), are defined as different

fortn5 of enzymes present in the same individual that have identical or sirnilar

functions and share a common substrate but differ in eleetrophoretic mobility

(Markert and Moller 1959). Isozymes can be deteeted when tissue extraets are

suojeeted to eleetrophoresis and subsequenùy stained with enzyme-specific solutions.

Isozymes have been used extensively to study genetic structure and taxonomic

relationsbips of plant populations and species (Soltis and Soltis 1989), to distinguish

crop cultivars (for review, Tanksley and Orton 1983a, 1983b, Weeden 1989), and to

examine the effeet of natural and artificial selection on plant populations (Hamrick

1989).

• 2 Recently. molecular markers have also been used extensively in genetic and

• breeding studies of many crop species. Restriction fragment lenglh polymorphisms

(RFLPs), based on restriction enzymes and Southern hybridization. are useful

m~,rkers for constructing genetic linkage maps of crop plants (for review, Beckman

and Soller 1985). New molecular markers C".llled random amplitïed polymorphie

DNAs (RAPDs) have been developed recently (Williams et al. 1990). This technique

is based on the polymel"'.lSe chain reaction (peR) which had been developed earlier

(Mullis and Faloona 1987). The RAPD technique is faster and less laborious than

the RFLP technique and does not require work with radioactive compounds.

However, RAPD markers are dominant markers, therefore less information can be

obtained compared to RFLPs. Since the development of RAPD markeTS, they have

been used extensively to construct genetic linkage maps, to study genetic diversity and

taxonomie relationships of plant populations and species, and to differentiate crop

cultivars (for review, Bowditch et al. 1993; Williams et aL 1993).

ln this researeh projeet, isozymes and RAPD markers are examined for

genetie and breeding studies in red c1over. 1 investigate the potential use of these

new markers in red clover for estimating genetie diversity of germplasms,

distinguishing cultivars and studying the effect of natural selection. The specifie

objectives of this research project are as follows:

1. To compare morphological traits, isozymes and RAPD markers for

estimating genetie diversity within and between cultivars of red clo:ier.

2. To investigate the potential use ofisozymes for evaluating genetie diversity

• 3 of red clover germplasm and discriminating red c10ver cultivars. • 3. To confirm genetic control of sorne enzyme-coding loci in red c1over. 4. To examine the use of RAPD markers from bulked sampies for cultivar

identification in red c1over.

5. To demonstrate the genetic changes using morphological traits and

isozymes in red c10ver populations after three cycles of natura! selection.

First, the techniques for isozyme and RAPD assays in red c10ver were

established. These {wo assays were then used to examine genetic variation within

and between {wo red c10ver cultivars, Essi from Europe and Ottawa from Canada.

Comparisons of these {wo markers with morphological traits were also made. High

levels of genetic variation within cultivars were observed with both isozyme and

RAPD markers. Then, isozymes were used to estimate genetic diversity of 15 red

clover cultivars from three different ongins. The potential use of allele frequencies

at isozyme loci for cultivar identification in red clover was also investigated. ln

addition, genetic interpretation of these isozymes was obtained from controlled

crossing experiments and inference from isozyme polymorphisms. Since high

variation within cultivars was found and isozymes could not be used to discriminate

all 15 red clover cultivars, RAPD markers from bulked samples were examined for

cultivar identification in red clover. RAPD markers from buiked samples were ~ 1 1 shown to beeffective in discriminating all cultivars. Finally, morphological traits and

. isozymes were used to study the effect of three cycles of natura! selection in red

• 4 clover. This study has implications for seed production of red clover and contrihutcs

• to basic knowledge in plant population genetics.

• 5 CHAPTER TWO • GENERAL LlTERATURE REVIEW

1. Economie Importance of Red Clover

The over.lIl economic imponance of red c10ver 10 world agriculture is difficult

to estimate. Red c10ver is consumed by animais in the forms of hay. silage. or

pasture. and then it is consumed by human beings in the forms of meat, fibre. or

diary produets (Smith et al. 1985). Red c10ver is also used as a plowdown green

manure crop between cash crops and as a companion crop with cereal crops. 115

introduction to central and nonhern Europe and Nonh America was said to have a

profound impact on agriculture and civilization by steadilysupplying high quality feed

for livestock (Fergus and Hollowell 1960; Taylor and Smith 1979).

Red clover is normally grown in mixtures with grasses (Taylor and Smith

1979). It is used as a feed in beef cow-calf and stocker production, and it is also

utilised in many dairy. sheep. and horse production systems (Smith et al. 1985).

Under good growing conditions, herbage yields as high as 19 tons ha'! year'! dry

matter have been obtained (Bowley et al. 1984b). The accurate area, production,

and yield figures for re~ clover are difficu1t to obtain because data on many of the

minor crops are not compiled by crop reporting services (Taylor 1985). In 1985.

about 20 million hectares of red c10ver were grown worldwide and, approximately

seven million hectares in North America were sown to red clover for hay, silage,

• 6 pasture, and soil improvement (Smith et al. 1985). Annual seed production in thc

• USA in recent years has been about 11,300 to 13.600 t (Rincker and Rampton 1985).

ln Canada, the amount of red clover seed production in 1990 W:IS 5,640 tons which

was about a 50% increase from the five year average (1985-1989) of 3.750 tons

(Anonymous 1991).

2. Origin, Distribution and Taxonomy of Red Clover

Red clover is thought to have originated in southeastern Europe and Asia

Mmor surrounding the Mediterr..mean and Red seas, where many Trifolium species

and their relatives have been found (Fergus and Hollowell 1960; Taylor and Smith

1979). Red clover was introduced to England and to central and northern Europe

about 1650. It is believed to have been introduced to North America by the Dutch

colonists in 1625 (Fergus and Hollowell 1960).

Red clover will adapt best to areas where summer is moderately cool or warm

with best growth when the temperatures are between 21 to 24° C and moisture is

adequate throughout the growing season (Duke 1981; Taylor 1985). In Europe, red

clover is an impor+.ant forage legume in Scandinavia, England, Scotland, Wales, and

Ireland and it is also cultivated in other temperate regions which include Russia,

Ukraine, Hungary, Poland, former Yugoslavia, and most countries in western Europe

(Smith et al. 1985; Taylor and Smith 1979). In North America, red clover is grown - in the humid northeastem region which extends west into North and South Dakota,

• 7 Nebraska, and Kansas, north into Ontario and Quebec, and south into Tennessee and • North Carolina. At low elevations in the southeastern USA, it is used as a winter annual and in the Pacific Northwest, it is grown primarily under irrigation as a seed

crop. Red clover is also grown less extensively in other temperate regions of the

world such as Japan, South Africa, Argentina, Columbia, Mexico, Chile, New

Zealand, and Australia (Smith et al. 1985; Taylor 1985; Taylor and Smith 1979).

Red clover is in the genus Trifolium which contains approximately 240 species

and is divided into eight sections (Zohary and Helier 1984). The Trifolium L genus

is in the tribe ofthe subfamily Papilionoideae, family , along with

the genera Ononis L (ca. 75 species), Medicago L (ca. 50 species), Melilotus P. Mill.

(ca. 20 species), and Trigonella L (ca. 80 species). There are 72 species in the

section Trifolium which is divided into 17 subsections. Red clover is in subsection

Trifolium which includes two annual species, T. diffusum Ehrh. and T. pallidum

Waldst. & Kit. with the diploid chromosome number of 16, and two perennial

species, T. noricum Wulf. and T. mazanderanicum Rech. fil., with the diploid

chromosome number of 16 (Fergus and Hollowell 1960; Taylor and Smith 1979).

Cultivated red clover can be divided into two groups: early flowering or

medium or double-cut type, and late flowering or mammoth or single-cut type. The

double-cut type is the main type grown in North America while the single-cut type

is grown mainly in western Canada. Normally, the mammoth type is taller, more

winterhardy, flowers 10-14 days later, éIJld gives a higher first eut yield than the

medium type. The mammoth type will produce no flowers in the seeding year but

• 8 • will have a rosette plant instead. The medium type can usually be harvested several

times a year depending upon the length of growing season and moisture availability

(Smith et al. 1985: Taylor and Smith 1979).

3. Morphology of Red Clover

Red clover is an herbaceous plant with leafy stems growing from a crown.

The plant consists of hollow stems, branches, leaves. and flower heads when it is fully

grown (Bowley et al. 1984b). The stems have a trilacunar. three-trace nodal anatomy

and have about ten vascular bundles in cross-section (Devadas and Beck 1972). In

general, stems, petioles, and leaves of most North American cultivars are densely

pubescent whereas European and Chilean cultivars have short appressed hairs on

these organs (Fergus and Hollowell 1960). These nonglandular hairs have an

elongated terminal cell with a number ofshort basal cells (Bowley and Lackie 1989;

Metcalf and Chalk 1950). The number of nodes per flowering stem is related to

maturity: early flowering cultivars have fewer nodes than late flowering cultivars

(Jones 1974).

Red clover has epigeal emergence. The germination pattern is the same aS

a typical leguminous pattern which. produces two cotyledonary leavei foUowed by

one true unifoliolate leaf. The rest of the leaves, usually a maximum offour or five

leaves on a primary shoot, are palmately trlfoliolate with a slender petiole and a

broadly triangular stipule. Leaf1ets are suborcular to sublanceolate and generally

• 9 have a marking. However, leaves of sorne plants may have no mark (Bowley et al. • 1984b). The inflorescence of red clover is a terminal capitulum or head which arises

from an axillary bud at the shoot apex (Aitken 1960). The head consists of50 to 275

sessile f10rets depending on environmental conditions. Each floret is a complete

legume f10wer with calyx, coroHa, ten stamens, and one pistil (Taylor and Smith 1979;

Taylor 1980). The calyx has five lobes; the coroHa has five petais: a standard, two

wings, and two fused keel petais. Ten stamens form a tube surrounding the pistil;

nine stamens are fused and one is free. Each ovary contains two ovules; however,

only one seed usuaHy develops. Seed set per head is between 37 to 65 seeds. Each

seed is about 2-3 mm long with an oval shape, a notch on one side and seeds differ

in colour witbin a seed lot. Seed size varies depending on environmental conditions

(Duke 1981; Stariing et al. 1950; Taylor 1985).

Red clover bas a tap root system with lateral roots originating from the upper

portion (Fergus and Hollowell 1960). The tap roots will norma\ly die and

disintegrate in the second year and the plants will depend on secondary roots for

tbeir survival (Montpetit and Coulman 1991a). Growth and expansion ofaxillary

buds of the cotyledons, primary shoots, and branches give rise to a large number of

axillary buds at the top of the tap root. This complex of buds at/or near the soil

surface is ca\led a crown (Bowley et ai. 1984b; Taylor 1985).

• 10 • 4. Genetics and Breeding of Red Clover

Genetics and breeding of red clover have already been reviewed in detail by

Fergus and Hollowell (1960). and Taylor and Smith (1979). therefore only literature

published after those {wo reviews will be presented. Red clover is a diploid species

with a chromosome number of 14. Tetraploid (211=28) types ofred clover that have

been developed and mosdy used in Europe are often more disease-resistant and may

give higher yields than the diploid cultivars (Smith et al. 1985). However. tetraploids

are not widely used in North America probably because of the difficulty in seed

production (Puri and Laidlaw 1983). Yield, pest resistance. regrowth potential.

persistence and winter-hardiness for northern atmosphere climatic conditions are the

characters that are emphasized in most recent red claver breeding programs (Smith

et al. 1985). Very few prograrns have attempted to improve red claver quality since

it is one of the best quality forage legumes.

4.1. Morphologieal, Pll)Isiologieal and Agronomie Traits

Split-leaflet was found ta be controUed by a single gene, designated si (Parrot

and Smith 1986). A single recessive gene, ln, contrais the absence of nonglandular

stem trichomes in red claver (Bowley and Lackie 1989). This gene does not affect

other epidermal characters such as the density of glandular trichomes or stomata.

Bowley et al. (1984a; 1984c) used six cycles of phenotypic recurrent selection to

increase stem length ofred claver plants 3.7 and 2.9 cm cycle-! at the first and second

• 11 harvests, respectively. This increase resulted from the increase of cell number per • internode and was associated with decreases in stem number per plant and persistence. A gene designated dig (developmental influencing gibberellin), which

has several pleiotropic effeets including suppressing the initiation of f10wering in

normally f1orally-inductive environments, has been identified in red c10ver (Jones

1991). These mutants will not f10wer under long days, but will do so when certain

gibberellins are applied (Jones 1993). Increases oftwo apical proteins are associated

with the switch from vegetative to floral development (Jones and Thomas 1994). A

daylength between 12 and 14 hours was critical for f10wer initiation in cv. Kenland

(Bowley et al. 1987). PetaI colour is conditioned by about 25 genes with complex

inheritance (Cornelius and Taylor 1981). However, purple-red flower colour is

controUed-bYa single gene which is designated rp (Parrot and Smith 1986). Petai

colour intensity of red clover flowers was changed from 3.1 to 8.2 on a scale of two

to nine (light pink to purple) by imposing seven cycles of phenotypic recurrent

selection (Cornelius and Taylor 1981). This selection method has also been used to

incre:l.Se the number ofhead pans in red clover with muIti-parted flower heads from

1 to 7.4 in six cycles with a regression coefficient of 0.92 head parts per generation

(Taylor et al. 1985a). An unusual flower type -designated rudimentary coroUa, in

which the petaIs are s~ortened and wrinkled, is controlled by a reeessive gene, r

(Taylor and Snead "1986). Plants with this phenotype are male sterile and are

reduced in femaIe fertiIity. Round pollen is conditioned by a gene p (Parrot and

Smith 1986). Growth type of red clover which is correlated with winter-hardiness

• 12 and persistence in both diploid and tetraploid cultivars has a moderate level of • additive genetic control with narrow-sense heritability between 0.56 to 0.62 (Choo 1984; Christie and Choo 1991; Coulman and Oakes 1%'7). The non-flowering types

(types 1 and 2) survived winter killing and persisted better than the flowering types

(types 3, 4 and 5) (Choo 1984). Plants that did not flower in the seedling year couId

give a forage and seed yield as high as flowering plants in the production years. The

flowering response is a useful selection criterion for persistence. The growth of

aèventitious roots which is associated with persistence and spring vigour in red clover

was found to have low narrow-sense heritability of 0.30 (Montpetit and Coulman

1991a; 1991b). This low heritability estimate suggested that progeny testing would

be required for successful selection of this trait.

Significant response to selection for NaCI tolerance in red clover was observed

(Ashraf et al. 1987). ReaJized and narrow-sense heritability estimates for NaCI

tolerance were 057 and 0.98, respectively. Four cycles of half-sib family selection

have been used effeetively to select red clover tolerant to 2,4-0 [(2,4­

dichlorophenoxy) acetic acid] herbicide (Taylor et al. 1989a). The narrow-sense

heritability of the 2,4-0 tolerance trait was near 50% and inbreeding depression was

minimal. In vitro selection bas a\so been employed to enhance 2,4-0 tolerance in red

clover because high correlation between in vivo and in vitro responses to 2,4-0 was

found (Taylor et al. 1989b). The tolerant callus \ines had 61-83% less 2,4-0 in their

tissues than the susceptible control tissues.

• 13 • 4.2 Pest Resistance Normally, only one or a few genes will determine disease resistance in red

c10ver (Taylor and Smith 1979). However, resistance of red c10ver to Aplzanomyces

eUleiciles Drechs., which is a root rot fungus, is inherited as a quantitative trait (Tofte

et al. 1991). Narrow-sense heritability of this character was found to be high enough

to improve red c10ver populations by using family recurrent selection. Tetraploid red

c10ver developed by asexual and sexual methods was found to be as susceptible to

A. euteiclzes as the diploid cultivars (Tofte and Smith 1992). By evaluating the diallel

cross progenies of red c1over, Pederson et al. (1981) found significant general

combining ability for length of rotted roots after inoculating with Fusarium roseum

(Lk.). The low heritability estimate of 0.075 for root rot severity was obtained, which

indicated that evaluation ofprogenies would be necessary for selection. Intercrossing

plants seleeted for Fusarium root rot resistance produced progenies with lower root

rot scores than the parental Swedish cultivar Hermes II (Rufelt 1985). Natura\

selection for root rot resistance was observed in red c10ver since root rot severity in

two local strains and two wild populations wàs lower than in cv. Hermes II. Progeny

testing using more than one isolate or species of the Fusarium fungus is necessary to

obtain effective selection for resistance to Fusarium root rot in red clover (Coulman

and Lambert 1995). By using six cycles of phenotypic recurrent selection, Taylor et

al. (1990) successfully increased the resistance to northem anthracnose of ten red

clover populations. They found that the mean disease severity index was reduced by

36% over six generations and rea\ized heritability averaged 20% per cycle.

• 14 Tolerance to root-knot nematodes was improved signitk.lOtly with five cycles of • restrieted recurrent selection and 1\\'0 cycles of half-sib family selection (Quesenberry et al. 1989). Germplasm resistant to stunting by the clover cyst nematode

(Heterodera tri/ol;; Goff.) has been released (Leath et al. 19S7b).

Different genes control different types of resistance to bean yellow !T\osaic

virus (BYMV) (Taylor et al. 1986). Resistance to BYMV in red clover is conveyed

by a hypersensitive reaetion. However, incorporation of the hypersensitivity reaction

into clones of the cultivar Kenstar did not increase resistance under spaced-plant

field conditions as compared to Kenstar polycross progenies. Germplasrns resistant

to BYMV have been released (Leath et al. 1987a; Taylor et al. 1985b). Resistance

to vein mosaic virus in red clover is conditioned by a single dominant gene (Khan et

al. 1978). Tolerance to white c10ver mosaic virus (WCMV) was found to be

determined by polygenes (Martin et al. 1990). Recurrent selection with progeny - testing was suggested for improving tolerance to WCMV in red clover.

5. Seed Production in Red Oover

Since details ofseed production in red clover has already been reviewed by

Rincker and Rampton (1985), only information reponed after that review is

presented here. To determine the optimUlÎl time to harvest red clover for seed, a

compromise between having sufficient time for the maximum number of seeds to

mature and avoiding losses due to over-ripening and adverse environmentaio _

• 15 • conditions in fall (Taylor and Smith 1979) is necessary. When Jess than 4% of the

inflorescences were still unripe was the optimum time to harvest seed of diploid cvs.

Sabtoron and SI23, and tetraploid cv. Hungaropoly (Puri and Laidlaw 1983). Seed

yield and number of inflorescences per unit area were lower in the cultivar

Hungaropoly than in CYS. Sabtoron and SI23 but the tetraploid had heavier seed.

The effects of cultivar and vegetative stage of cutting on red clover seed production

have been investigated (Belzile 1990). The total annual seed yield was different

within cultivars. A vegetative cutting prior to seed setting is necessary to obtain good

seed yields. The stage of development at which vegetative cutting is done affects

seed yields but has no influence on percentage of seed germination. Effects of

harvest time and the growth regulator Alar-8S on seed yield and seed yield

components in red clover were cxamined (Christie and Choo 1990). A higher yield

was obtained from the second crop (aftermath), both with and without Alar-85, in ail

cultivars; DoUard, F1orex, Prosperand Hungaropo\i. Alar-8S shortened coroUa tubes,

reduced plant height and increased seed yie1ds through increases in seed setting and

the number of heads per unit area. Soil types were also found to influence seed

production within the sarne climatic conditions (Belzile 1991). A light soiltype gave

a higher number of seeds per plant and seedccyield than a heavy soiL Water

requirements and irrip:tion timing for red clover seed production were investigated

by Oliva et al. (19943, 1994b). Crop water stress index (CWSI) for red clover seed

crop was evaluated. CWSI was found to be a useful indicator of red clover water

stress status and can be used to schedule irrigations under typical western Oregon

r • 16 • c1imatic conditions (Oliva et al. 1994a). The duration ofseason-long bud and flower

production. stem length. potential seed yield. and seed yield were reduced with

increased plant water stress (Oliva et al. 1994b). The reduction in seed yield resulted

primarily from a decrease in floral fenility. and from reductions in flower number per

unit area. Application of water at peak flowering increased seed yield more than

watering soon after haying (Oliva et al. 1994b).

Ail cultivars of red c10ver are heterogeneous populations consisted of many

heterozygous individuals (Smith et al. 1985). Genetic shifts due to selection pressures

can occur during seed increases. especially when seed is multiplied outside the area

of adaptation (Beard and Hollowell 1952; Bula et al. 1965; Dovan and Waldman

1969; Taylor et al. 1979). A significant change in the relative proportions of the

various growth type was observed on red c10ver plants grown from seed ofcv. Lasalle

harvested in the year of seeding (Steppler and Raymond 1954). This change was

undesirable and unacceptable for farmers who grew red c10ver for forage in eastern

Canada. Shifts toward more early flowering types and fewer winter-hardy plants were

observed for seed lots of Dollard red c10ver produced at two locations in California

(Bula et al. 1969). Genetic shift toward lower yield and less persistence took place

with advancing generations, and was more obvious in seed lots produced in the

southem locations (Ta~lor et al. 1979). The importance of adhering to certification

standards and canying out seed multiplication in the area of adaptation must be

emphasized (Taylor et al. 1979)•

• 17 • 6. Applications of Isozymes in Plant Breeding and Cenetics The applications of isozymes in plant breeding and genetics have been

reviewed thoroughly by Nielsen (1985) and Weeden (1989) and by several authors

in the volumes edited by Tanksley and Orton (1983a; 1983b) and Soltis and Soltis

(1989). Isozymes are defined as any distinguishable proteins that catalyze the same

biochemical reaetion (Humer and Markert 1957). The Most common method used

by plant geneticists and breeders to discriminate different isozymes is horizontal

starch gel eleetrophoresis (Weeden 1989). This method separates proteins on the

basis ofcharge and size. The applications of isozymes in plant breeding and genetics

are enormous. They can be used to estimate genetic variability of erop germplasms,

to identify erop cultivars, to eonfirm hybridity, tO mark monogenie and polygenie

(quantitative) tl"J.Ïts, to analyze aneuploids and polyploids, to study somaclonal

variation, to confirm the parentages of clonal cultivars, to develop linkage maps, and

to analyze wide crosses (Nielsen 1985; Soltis and Soltis 1989; Tanks1ey and Orton

1983a; 1983b; Weeden 1989). In this review, only the two applications of isozymes

in plant breeding and genetics that are relevant to this study will be presented.

6.1. Discrimination ofCrop Cultivars

Cultivar identification and techniques to assess cultivar purity are essential to

seed certification and commercial seed production of crop plants (Balley 1983).

Traditionally such identification has been achieved through the use of aeeurate

• 18 records and evaluated using a combinations of laboratory and field plot methods. • However. the continued development of new cultivars has increasingly demanded a larger number of discriminating morphological. physiological. and biochemical

characters be used 10 differentiate cultivars.

The use of isozymes in cultivar identification was proposed and applied over

20 years age (Almgard and Clapham 1975: Bassiri 1976). The progress in this

application has been reviewed by Bailey (1983). Nielsen (1985). and Weeden (1989).

lsozymes are polypeptides whose amine acid sequences are transcribed

directly from the nucleotide sequences of a gene. Therefore any consistent banding

variation between cultivar zymograms is ultimately attributable to genotypic

differences between cultivars under comparison (Weeden 1989). Although the planù

growing environment has no direct effect on the primary structure of a particular

enzyme, the environment can affect gene aetivity and determine the quality and

quantity of that enzyme produced in different organs or tissues (Weeden 1989).

However, isozyme instability has been found to be minimal (Aimgard and Clapham

1975; Bringhurst et al. 1981; Cherry and Ory 1973; Gates and Boultier 1979;

Kobrehel and Gautier 1974). Both qualitative and quantitative intervarietal variation

of many enzymes have been reported (Bailey, 1983). Qualitative variation occurs

when a particular isozyme band is present in the zymogram ofone cultivar but absent

in that ofanother cultivar. Such bands may be described as polymorphie and for any

one species the number of polymorphie bands deteetable largely determines the

potential value of that isozyme system in cultivar identification. Polymorphie bands

•• 19 are typically under the genetic control of codominant alleles and are inherited

• according to monogenic Mendelian ratios. The actual number of polymorphic bands observed in the phenotype is a function of the number of loci. number of alleles per

locus. and the quaternary structure of the enzyme system. Quantitative variation

occurs when a particular band is present in zymograms of two different cultivars with

different intensity of ~taining. However, quantitative variation does not give clear

and discrete differences as qualitative variation does. Thus, its potential use in

cultivar identification is limited, especially since quantitative variation is more

sensitive to the growth environment of the plants, and to ontogenetic differences

among tissues (Almgard and Clapham 1975, 1977; Gates and Boutier 1974; Kuhns

ar.d Fretz 1978: Siegal and Galston 1967). However, extreme quantitative differences

have sometimes been used to differentiate cultivars (Buttery and Buzzell 1968; Payne

and Koszykowski 1978).

The occurrence of dissimilar isozyme phenotypes among individuals of the

same cultivars represents intravarietal variation (Bailey 1983). Intravarietal variàt;~n

serves as a measure of the uniformity and stability of an isozyme phenotype within

the cultivar to be identified. This variation depends on the types of cultivars

evaluated. Clonai cultivars should have extremely uniform and stable isozyme

phenotypes with the absence of intra-varietal variation (Cousineau and Donnelly

1989). Pure-line cultivars of self-pollinated crops whicb are composed of severa!

morphologically similar lines may contain significant amounts of isozymic intra­

varietal variation (Collin et al. 1984; Fedak and Rajhathy 1972: Singh et al. 1973;

• 20 Weeden 1984). Whenever there is intra-varietal variation in isozyme phenotypes to • be used in cultivar identification. the bands of the zymograms which Sh0'" !:

varietal uniformity may still be useful to distinguish cultiv:lrs (Bailcy 191\3).

Cultivars of cross-pollinated crops arc heterogenellus consisting of numerous

heterozygous individuals. A high level of variation within cultivars is always present

(Bassiri 1977; Hamill and Brewbaker 1969: Weeden and Emmo 191\5). To use

isozymes for cultivar discriminaùon in these crops. therefore. is not as straightforward

as in homogeneous self-pollinated crops or clonai and hybrid cultivars. Comparisons

of allelic frequencies at isozyme loci can a1so be used to identify heterogeneous

cultivars (Hayward and McAdam 1977: Ostergaard and Nielsen 1981). Allele

frequencies at four isozyme loci, Got-3, Pgd-J, Pgi-2 and Pgm-2, were used to identify

15 cultivars ofperennial ryegrass (Lolium perenne) and six cultivars ofItalian ryegrass

(Lolium multiflorum) (Nielsen et al. 1985). The Got-3 and Pgi-2 loci could

discriminate perennial ryegrass culùvars fairly well, but the discrimination in ltalian

ryegrass was unsaùsfaetory at any single locus. The PGI-2 and ACP-l isozymes were

assayed for identificaùon of 298 diploid and tetraploid Lotium cultivars, belonging

to the species L. perenne, L. multiflorum and the interspecific hybrid L. x

boudleanum (Booy et al. 1993). Dendrograrns based on allele frequencies of these

enzymes were tised t~ classify these cu\ùvars into groups. Polyacrylamide gel

eleetrophoresis of threeenzyme systems (cathodal peroxidase, alkaline phosphatase

and alpha-amylase) was used to classify six rye (Secale cereale L) seed sampies

(Ramirez and Pisabarro 1985). Allele frequencies at nine isozyrne loci from six

• 21 • enzyme systems (ACP. GOT. MDH. PGD. PGl and PGM) were obtained from six

cultivars of forage rye (Ramirez et al. 1985). Genetic distances between these rye

cultivars were estimated and used for cluster analysis to obtain a phenograrn. The

technique of using only intra-varietally uniform zymograms has also been applied to

identify heterogeneous cultivars of Kentucky bluegrass (Weeden and Emmo 1985).

Isozymes have been used successfully to identify cultivars in more than 30

species of field crops. vegetable crops, fruit trees. and ornarnental plants (Weeden

1989). However, this techniGue has not been used extensively in forage crops. It has

been reported in alfalfa (Quiros 1980). Kentuck')' bluegrass (Weeden and Emmo

1985), forage rye (Ramirez et al. 1985), ryegrass (Booy et al. 1993; Nielsen et al.

1985), and subterranean clover (Collins et al. 1984).

6.2 Studies ofGenetic diversity ofPlant Population and Species

The application of gel electrophoresis of isozymes to studies of genetic

variation in plant populations has had a remarkable impact on research inpopulation

genetics, evolution, and systematics (Brown and Weir 1983; Gottlieb 1981a; Hubby

and Lewontin 1966). This technique can identify large numbers ofgene loci and the

proportion of p,'>lymorphic loci can he determined (Gottlieb 1981a). It cao aIso he

used to determine the proportion of heterozygous gene loci. The study of genetic

variation in the past dependèd on the use of rare recessive morphological mutants

that gave visible changes or on infrequent morphological polymorphisms such as

heterostyly or flower colour (Gottlieb 1981a). Such characters; however, represent

• 22 only a smail fraction of the genetic variation within plant populmions. Most • phenotypic characters are eontrolled by many gene loci and aÏfected by

environmental factors. Although isozyme assays can detect a higher level ofvariation

they cannot deteet ail variation within plant populations. Mutations which do not

cause amino acid changes, and amino add changes whieh do not C:lUse mobility

changes, are not detected by isozyme assays (Gottlieb 1981a). Sorne isozymes are

also expressed at different stages of development and under different environmental

conditions (Wendel and Weeden 1989).

The most common deseriptors of intrapopulational variation are the percent

ofpolymorphie loci, the number of alleles per locus, and the mean proportion of loci

heterozygous per individual (Erskine and Muehlbauer 1991; Gottlieb 1981a; Hamrick

1989). Other estimates that have been used -:re the number of alleles per

polymorphie locus, the observed proportion of loci heterozygous per individual, and

the genetie diversity index (Erskine and Muehlbauer 1991; Garcia et al. 1989;

Gottlieb 1981a; Hamriek 1989). The estimates used to measure variation among

populations are Wright's F statistics and Nei'sgene diversity satatistics (Erskine and

Muehlbauer 1991; Harnriek 19~9; Nei 1973; Wright 1951). Principal eomponent

analysis, compact linkage analysis, and classification and regression tree analysis have

also been utilized to estimate genetic variation within and among plant populations

(Brieman et al. 1984; Harris 1975; Knerr et al. 1989; Smith 1984; Sneath and Sokal

1973)•

• 23 • Normally, plant species that are widespread, long-lived, outcrossed, and have

high lifetime fertilities have higher levels of intrapopulational variation than species

with other combinations ofthese traits (Hamrick 1989). The distribution ofvariation

among populations results from the interactions of mutation, gene migration,

selection, and genetic drift which will operate within the evolutionary and biological

context ofeach plant species (Hamrick 1989; Loveless and Hamrick 1984). Selection

and genetic drift are expected to increase differentiation among populations. Species

with more pollen or seed movement should have less variation among populations

than species with restricted gene flow.

Isozymes have been used to study genetic diversity in many natural plant

populations (reviewed by Gottlieb 1981a; Hamrick 1989). The use of isozymes to

characterize genetic variation in crop plants is less extensive (reviewed by Tanksley

and Orton 1983; Weeden 1989). An understanding of the genetic variation, its

partitioning among and within populations and the population structure of crop

populations are required to maintain and exploit germplasm resources efficiently

(Brown and Weir 1983). Isozymes have been used to study genetic variation in

germplasms ofmore than 40 crop species (Weeden 1989). Since that review, the use

of isozymes to Study genetic variation has been reponed in several other species

including birdsfoot trefoil, Lotus cornicuJatus (Raelson and Grant 1989); Brassica

campestris (McGrath and Quïros 1992); cucumber, Cucurnis sativus L (Knerr et al.

1989); lentil, Lens cuJlinaris (Erskine and Muehlbauer 1991); Phaseolus vulgaris

(Chase et al. 1991)•

• 24 7. The Use of Random Amplilied Polymorphie DNA (RAPD) Markers in Plant • Breeding and Geneties

The use of RAPD markeTS was developed by Williams et al. (1990) as a too1

to construct genetic maps in eukaryotic species. At the same time. Welsh and

McClelland (1990) used arbitrary-primed polymerase chain reaction (AP-PCR) to

fingerprint plant genomes. These two similar techniques were based on the

polymer.lSe chain reaetion (PCR) that was developed earlier (Saiki et al. 1985; Mullis

and Faloona 1987). Both methods are based on the fact that a short oligonuc\eotide

of randomly chosen DNA sequence, when mixed with genomic DNA and

thermostable DNA polymerase, and subjected to temperature cyc\ing under

conditions resembling those of the PCR will prime the amplification ofseveral DNA

fragments (lnnis et al. 1990). The reaction products cao be separated on standard

agarose or acrylamide gels and visualized with ethidium bromide staining. The

nllture of the fragments that are amplified is highly dependent on the primer

sequence and on the genomic DNA sequence being assayed. PrimeTS differing by a

single nucleotide give rise to different amplified,bands, and genomic polymorphisms

at one or both priming sites result in the disappearance of the amplified band. A

primer normally amplifies severa! bands, each originating from a differeDt genomic

location (Rafalski et al. 1991). Conditions to optimize the reaction have been

discussed (Williams et al. 1990; 1993; Welsh and McClelland 1990). ln general, ten

nucleotide primers with at least 50% GC content are preferred. RAPD markers

• 25 have been applied in several crop species for evaluating gcrmplasms, distinguishing • gcnotypes and cultivars, constructing genetic linkage maps and marking qualitative and quantitative genes. In this review, only applications of RAPD markers for

evaluating genetic diversity and relationships of crop germplasms and discriminating

cultivars and genotypes will be presented.

7.1. Studies of Genetic Diversity and Relationslzips ofCrop Germplasms

RAPD markers were used to study the genetic diversity ofclosely related Iines

of common wheat (Triticum aestivum) (He et al. 1992), IWO diploid wheat species,

Triticum monococcum and T. uranu (2n=2x=14) (Vierling and Nguyen 1992), wiJd

emmer wheat (Triticum turgidum L ssp. dicoccoides) from Israel, Turkey and Jordan

and cultivated American, Turkish and Syrian durum wheats (Joshi and Nguyen 1993).

High levels of variation were observed both within and among 19 Hordeum species

and subspecies (Gonzaiez and Ferrer 1993) whiJe 57 percent of the variation

deteeted with RAPD markers was partitioned within ten populations of Hordeum

spontaneum from Israel (Dawson et al. 1993). Values of genetic distance computed

from the frequencies of RAPD polymorphism in pairs of spring barley (Hordeum

vulgare L) inbred Iines were compared to kinship coefficients between the same pairs

of !ines (Tinker et al. 1993). Cluster analysis showed that groups of inbred lines

based onkinship coefficient were simiJar to those based on genetic distance. Genetic

re!ationships of 24 accessions of hexaploid wild oat (Avena steriüs) were compared

using isozyme and RAPD markers (HellO et aL 1994). A higher level of

. ,'-., '.~.,--_/ ~~- • 26 • polymorphism was found between two subspecies of rice (O,:\';a satil'a L spp.

japonica and indica) than was observed between upland and lowland cultivars within

the indiea subspecies (Yu and Nguyen 1994). RFLP and RAPD markers were used

to investigate the genetic diversity of elite sorghum (Sorglzum bie%r) lines (Vierling

et al. 1994). A higher level of polymorphism was detected with RAPD markers.

Variation among four species of the genus Panicum (millets) and within the crop

species P. miLiaeeum and P. sumarrense was evaluated using RAPD markers (M'Ribu

and Hilu 1994).

A high level of RAPD polymorphism was observed among wild and cultivated

Araelzis species (Lanham et al. 1992). However, Halward and coworkers (1992) did

not find any RAPD polymorphisms among two cultivars and 25 unadapted

germplasm lines of peanut (Araclzis Izypogaea L). The degree of RAPD marker

vari..!:>ility between and within the Andean and Middle Arnerican gene pools of

common bean (PlzaseoLus vuLgaris L) was evaluated (Haley et al. 1994). Three levels

of polymorphism were observed: between gene pools > between races > within

races. Tomato germplasms including wild species and modern cultivars were

evaluated using RFLP and RAPD markers (Williams and St-Clair 1993). Rus­

Koetekaas et al. (1994) used RAPD markers to deteet genetic variation in the genus

Lycopersieon. Four seleeted RAPD primers were able to deteet polymorphic bands

among species at a frequency of 80% and among cultivars at a frequency of 44%.

The potential use of RAPD markers for taxonomie study of the cruciferous

species was investigated by usingBrassica, Sinapis and RapIUl1lUS taxa (Demeke et al:

• 27 • 1992). Principal coordinate analysis of the RAPD bands revealed the classical U­

triangle relationship between diploid and amphidiploid Brussiea taxa. RAPD markers

were used to evaluate genetic relationships among 18 accessions from six cultivated

Brussiea species and one accession from RapluUlus sarivus (Thormann et al. 1994),

Brussiea oleraeea genotypes (Kresovich et al. 1992; dos Santos et al. 1994), twelve

Indian mustard [Brussiea juneea (L) Czen and Coss) and eleven exoùc B. juneea

genotypes (Jain et al. 1994), six clonai cultivars of sweet potato (Connolly et al.

1994), and birdsfoot trefoil (Lotus spp.) (Campos et al. 1994).

Kazan et al. (1993a) applied RAPD markers to examine geneùc variaùon in

four agronomically important species of the genus Stylosanl/zes: S. seabra, S. lzamara,

S. guianensis and S. /zumilis. Relatively low levels of polymorphism were observed

within each species, while polymorphism between species was much higher. Genetic

variaùon of 4S accessions in the five taxonomie groups of the Stylosant/zes guianensis

(Aubl.) Sw. complex was aIso investigated using RAPD markers (Kazan et al. 1993b).

None or little variation was observed within these accessions but higher levels of

variation were found both within and between the five taxa. RAPD markers were

used to investigate genetic variation in heterogeneous, outcrossing, natural

populations of buffa10grass [Buellloe daetyloides (Nutt.) Engelm.] (Huff et al. 1993).

There was considerable variation within each of the four populations studied, and

every individual \VllS found to be geneticaJJy distinct. The use of RAPD markers

obtained from bulked genomicDNAsampiesto estimate genetic relationships among

heterogeneous populaùons of alfalfa (Medicago saliva L) was demonstrated by Yu

• 28 • and Pauls (1993b). Bulked samples of perennial ryegrass (Lolium perenne L) were

also used to discriminate t\VO synthetic cultivars (Sweeney and Danneberger 1994).

Genetic relationships of 15 improved cultivars and four wild accessions of

rabbiteye blueberry (Vaccinium asllei) (Aruna et al. 1993), different Malus species

(Harada et al. 1993), the genus Colfea (Orozco-Castillo et al. 1994). 25 apple

rootstocks (Landry et al. 1994), and different species of the genus PopullL~

(Castiglione et al. 1993) were investigated with RAPD markers. Genetic diversity

of22 cultivars ofcranberry [Vaccinium macrocarpon (Ait.) Pursh] (Novy et al. 1994).

cocoa (Tlzeobroma cacoa) germplasms (Russell et al. 1993; Wilde et al. 1992).22

families of oil palm (Elaeis guineensis) germplasm collected from Africa (Shah et al.

1994), leguminous trees Gliricidia sepium and G. maculata from seven countries

(Chalmers et al. 1992), and trembling and bigtooth aspens (Uu and Fumier 1993)

was also evaluated with RAPD markers.

7.2 Identification ofCrop Cultivars

RAPD markers have been used for cultivar identification in severa! crop

species. They 'Vere used to discriminate five Taiwanese rice cultivars (Pang et al.

1992), 37 AustraIian rice cultivars (Ko et al. 1993), 13 upland and lowland rice

cultivars (Yu and Nguyen 1994),23 cultivars ofrapeseed (Brassica napus L) seleeted

from severa! breeding programs (Mailer et al 1994), 14 broccoli and 12 caU1iflower

(Brassica o[eracea L) cultivars (Hu and Quiros 1991), accessions and clones ofvetiver

grass (Vetiver zizannioïdes) (Kresovich et al. 1994), 25 breeding lines of buffalograss

• 29 • (Wu and Lin 1994), five rose (Rosa spp.) cultivars (Torres et al. 1993), apple cultivars

(Koller et al. 1993; Landry et al. 1994; Mulcahy et al. 1993), ten cultivars of plum

(Prunus dornestieu) (Gregor et al. 1994), eleven olive tree cultivars (Bogani et al.

1994), 15 raspberry (Rubus spp.) cultivars of the Quebec certification program

(Parent et al. 1993), Musa germplasm (Howell et al. 1994), and ten cocoa clones

(Wilde et al. 1992).

" ..,:.

•,' "c: .

. ,-, ;."

30

, ,- ~\~':~~~ CHAPTER THREE • GENETIC VARIATION WlTHIN AND BE1WEEN 'IWO CULTIVARS

OF RED CLOVER:

COMPARISONS OF MORPHOLOGICAL, ISOZYME AND RAPD MARKERS

P. Kongkiatngam, MJ. Waterway, M.G. Fortin, and B.E. Coulman Euphytica (In press)

P. Kongkiatngam planned the experiments, performed aU laboratory work and data analysis described in this manuscript and is the first author of the manuscript. M,J. Waterway assisted in planning the isozyme experiments, provided laboratory facilities and research materials and supervised laboratory work for isozyme experiments, gave valuable advice and suggestions throughout the projecl, and contributed significantly towards editing and revising the manuscript. M.G. Fortin assisted in planning the RAPD experiments, provided laboratory facilities and research materials and supervised laboratory work for RAPD experiments, gave valuable advice and suggestions throughout the projecl, and contributed significaittly towafds editing and revising the manuscript. B.E. Coulman assisted in planning the field experi~ents, provided facilities and materials and supervised all field experiments, gave valuable advice and suggestions throughout the project, andhelped in editing and revising the manuscript.

Acknowledgements: We would Iike to thank the Canada-Egypt-McGiIl Agricultura1 Response Program for scholarship support to PK. This study was supported in part by grants from the National Science and Engineering Research Couneil of Canada to MJW and MGF. ~

• 31 • 3.1. Summary Morphological, isozyme and random amplified polymorphie DNA (RAPD)

markers were used to estimate genetie variation within and between cultivars of red

clover (Trifolium praJense L), an important temperate forage legume. Two cultivars

of red elover, Essi from Europe and Ottawa from Canada, were evaluated. Six

monogenie morphologica\ eharaeters were observed for 80 plants from eaeh of these

two cultivars. Ali six morphologica\ loci were polymorphie in the cultivar Essi

whereas only four loci were polymorphie in the cultivar Ottawa. Forty plants from

eaeh cultivar were assayed for isozyme markers. A total of 21 enzyme-eoding loci

with 43 alleles was deteeted using twelve enzyme systems. Thineen and nine ofthese

loci were polymorphie in Essi and Ottawa, respeetively. The mean number of alleles

per locus was 1.81 in Essi and 1.67 in Ottawa. Seventeen random lQ-mer primers

were screened for RAPD markers. Nine primers which gave clear and consistent

amplified produets were used to assay 20 individuals from each cultivar. Each primer

gave from 7 to 20 amplified bands with an average of 14.8 bands per primer. One

hundred and eight of 116 putative loci were polymorphie in Essi and 90 of 98 loci

were polymorphie in Ottawa. High within-cultivar variation was observed in both

cultivars using both isozYrne and'RAPD markers. This high p01ymorphism makes

these markers usefu1 for germplasm charaeterization and genetie studies in red

clover.

'. 32 • 3.2. Introduction

Red clove:r. Trifa/iuln pratellse L. is recognised as one of the: most important

forage legume:s in temperate regions of the: world. It can be: grown in a wide: range

of soi! types. pH levels, and environmental conditions and give:s satisfactory yidds in

areas that are not suitable for growing alfalfa due to problems of high soil acidity or

excessive moisture (Smith et al. 1985). Red clover is a diploid (211 = 14), cross­

pollinated species with a gametophytic self-incompatibility system (Taylor & Smith

1979). Most of the cultivars in use today have been developed by using sorne form

of eontrolled mass selection (Smith et al. 1985), and thus are heterogeneous

populations consisting of heterozygous individuals.

Genetie variation within and between populations of crop species is a major

interest of plant breeders and population geneticists. An estimate of the extent of

variation within and between populations of a species is useful for analyzing the

genetie structure of crop germplasms (Hayward & Breese 1993), monitoring

germplasms during the maintenance phase (Moore & Collins 1983), and predicting

"0 potentia! genetie gain in a breeding program (Moreno-Gonzalez & Cubero 1993).

Morphological, phenologica.l, and agronomie characteristics are often used for

estimating genetie variation. However, these traits are often polygenie and

influenced by environmental conditions. An additiona! problem in red clover isthat

onIy a few morphologicaI charaeteristics that are under simple genetie control have

been identified (Quesenberry et al. 1991).

• 33 Isozymes have proven to be reliable genetic markers in breeding and genetic • studies of plant species (reviewed by Soltis & Soltis 1989; Tanksley & Orton 1983). Within the genus Trifolium. isozymes have been used to study genetic variation of the

colonizing species Trifolium hinum All. (rose clover) in California (Molina-Freaner

& Jain 1992) and to identify cultivars and genetic relationships ofsubterranean clover

(Trifolium subte"aneum L) (Collins et al. 1984). The only previous study of isozymes

in red clover was reported by Hickey et al. (1991). In that study ten enzyme systems

were assayed to compare IWO naturalized populations of red clover from Ohio with

introduced alsike clover (Trifolium hybridum L) and with IWO endangered native

species: running buffalo clover (Trifolium stoloniferum Muhl.). and buffalo clover

(Trifolium reflexum L).

Moleeular markers, such as random amplified polymorphie DNAs (RAPDs),

have been developed and used in genetic and breeding studies in many plant species

(reviewed by Williams et al. 1993). RAPD markers are DNA fragments amplified

by the polymerase chain reaction (PCR) using short arbitrary primers (Williams et

al. 1990). Compared to restriction fragment length polymorphism (RFLP) markers,

RAPD can generate markers more rapidly but sorne loss of information may oecur

because RAPD markers are usually dominant rather than codominant as are RFLP

markers. Previous studies in legumes using RAPD markers have shown a wide

range ofvariability. For example, a high level ofvariation within cultivars was found

with RAPD markers in alfalfa (Medicago:sativa L) which is a tetraploid outcrossing

species (Yu & Pauls 1993b) while a three-tiered pattern of RAPD polymorphisms

34 was observed in gene pools of common bean (Phaseoills l'lIlgaris L): between gent: • pools > between races > within races ( Haley et al. 1994). No variation was deteeted among cultivars and germplasm Iines ofcultivated peanut (Arachis hypogaea

L). whereas the wild Arachis species were differentiated with nearly every individual

primer screened (Halward et al. 1992). Little or no variation was found within each

accession of the Srylosanthes guianensis (Aubl.) Sw. species complex, which includes

several diploid pasture legumes widely used in many parts of the tropies and

subtropies (Kazan et al. 1993a). However. only four individuals were sampled from

each accession and the outcrossing !"'

(Miles 1985). Few studies have been done with cross-pollinated, heterogeneous

forage legumes Iike red c1over. The objective of this study was to compare

morphological. isozyme and RAPD markers for estimating genette variability within

and between two heterogeneous red c10ver populations.

3.3. Materials and methods

3.3.1. Morph%gicaI markers

.' Two red c10ver cultivars, Ottawa, a standard cultivar in eastem Canada, and

Essi, which originated in Europe, the center of origin of red c10ver (Taylor & Smith

1979), were used in this study. Six monogenic morphologica1 charaeters previously

described' by Quesenberry et al. (1991) were monito:ed: l_eaf mark/no leaf mark,

stem hair (spreading,hair/appressed haïr), petiole haïr (spreading haïr/appressed

35 • hair), basal internode (hairy/glabrous), stipule (hairy/glabrous) and f10wer colour

(pink/white). FortYfield-grown plants and 40 greenhouse-grown plants were scored

for each of the [WO cultivars. Actual sample sizes for sorne characters were slightly

lower than this because sorne plants remained at the rosette stage and did not

produce stems and f1owers.

3.3.2 Iso::yme markers

Young leaves from 40 plants each of Essi and Ottawa red c10ver cultivars

gl\)wn in the greenhouse were ground in grinding buffer [1 mM EDTA, 10 mM KCI,

10 mM MgCI:!, 7% (w/v) PYP, 100 mM Tris-CI pH 7.5, 3% sucrose (w/v), 0.2% (v/v)

2-mercaptoethanol) (modified from Gottlieb 1981b) and the leaf extraet was

absorbed onto Whatman #3 filter paper wicks. These samples were assayed for

twelve isozyme syslems which gave c1ear and weil resolved bands on 10% starch gels

run \Vith three buffer systems as descnbed by Wendel and Weeden (1989).

Diaphorase (DIA, EC. 1.6.99.-); glucosephosphate isomerase (GPI, E.C. 5.3.1.9);

isocitrate dehydrogenase (IDH, E.C. 1.1.1.42) and phosphoglucomutase (PGM, E.C.

5.4.2.2) were assayed with histidine-citrate buffer, pH 6.5. Alcohol dehydrogenase

(ADH,E.C. 1.1.1.1); rnalate dehydrogenase (MOR, E.C. 1.1.1.37); 6-phophogluconate

dehydrogenase (PGD, EC. 1.1.!.44) ~d shikimate dehydrogenase(SKD, E.C.

1.1.1.25) were resolved using a morpholine-citrate buffer, pH 6.1. Aspartate

aminotransferase (AAT, E.c. 2.6.1.1); amylase (AMY, E.C. 3.2.1.1 and EC. 3.2.1.2);

esterase (EST, E.C. 3.1.1.-) and malic enzyme (ME, E.C. 1.1.1.40) \Vere resolved with

36 lithium-borate/Tris-citrate buffer. pH 8.3. Assays for ail enzyme systems. except for

• esterase in which N-propanol was substituted for acetone. were the same as thuse

described by Wendel & Weeden (1989). Loci and aileles were sequentially

numbered and lettered. respectively. beginning with the most anod:ll form.

3.3.3. RAPD markers

DNA was extraeted from leaf tissue of 20 plants from each cultivar using :1

hexadecyltrimethylammonium bromide (crAB) method (Doyle & Doyle 1987). Five

grams offrozen red clover leaves from each plant were ground in 15 ml of buffer [1.4

M NaCl, 0.2% (v/v) 2-mercaptoethanol, 20 mM EDTA, 100 mM Tris-Cl pH 8.0] in

a Waring blender. Five millilitres ofcrAB buffer [the above buffer plus 2% (w/v)

crAB (Sigma)] were then added to the ground tissue, and this mixture was

incubated at 60 oC for 30 min with occasional swirling. DNA was e:'Ctraeted with one

volume of chIoroform-isoamyl alcohol (24:1) then centrifuged at 3500 gs for 5 min.

DNA was precipitated by the addition of2/3 volume of cold isopropanol, storage at

-20 oC for 1 br and centrifugation. The DNA pellet was washed with wash buffer (10

mM ammonium acetate in 76% ethanol) and dried under vacuum. The DNA pellet

was resuspended in 200 p.l of deionizedH20 and purified with phenol-chloroform-

isoamyl alcohol (25:24:.1) (Ausubel et al. 1989).

Polymerase chain reactions were performed in 25 ",1 containing80 mMTris-CI

pH 9.0, 20 mM

(pharmacia), 35 mM Mg02> 400 DM ofasingle primer, 2 U ofTOIJ DNA polymerase

• 37

'., .\ • (BRL) and approximately 25 ng of red clover genomic DNA. The reactions were

performed in a thermal cycler (Hyhaid) with the following temperature conditions:

94 oC for 5 min. followed by 35 cycles of 92 oC for 1 min. 36 oC for 1 min, 72 oC for

2 min, and ending with 72 oC for 6 min. PCR produetS were visualized with ethidium

bromide after eleetrophoresis in a 1.4% agarose gel. Nine primers (Operon

Technologies) were used to amplify individual genomic DNA (Table 3.1). RAPD

bands were scored with GPTools v. 3.0 (BioPhotonics). A locus was considered to

be polymorphie if the band was present in sorne individuals and absent in others and

monomorphic if the band was present among ail individuals in the same cultivar.

Two alleles were assigned for a polymorphie locus and one allele for a monomorphic

locus. The most intense monomorphic band from each cultivar amplified with each

primer was used as reference to C"

present. When there were no monomorphic bands, bands with the highest frequency

in each cultivar were used for calibmtion. Within each lane, bands were scored as

present if their intensity was at least ten percent that of the monomorphie reference

band within that lane (Figure 3.1).

3.3.4. Data analysis

Data analysis for isozyme markers was performed using BIOSY5-I(Swofford

& Selander 1981). Four estimates of genetie variation were obtained for each

population. They werethe pereêmage of polymorphie loci, mean number of alleles

per loCUS, mean observed heterozygosity based on a direët COllOt and mean expeeted

• 38 heterozygosity based on the unbiased estimale of Nei (1978). A locus was considered • polymorphie when more than one allele was found in a population. The chi-square: test was performed to test for signitïcant deviation l'rom Hardy-Weinberg equilibrium

with the expected frequency adjusted for small sample size using Levene's (1949)

correction factor. Wright's fixation index (Wright 1951) was calculated for each

polymorphic locus in each cultivar.

The percentage of polymorphic loci and mean number of aileles per locus

were also estimated for morphologiC'J.1 and RAPD markers. Observed heterozygosity

could not be obtained for morphologiC'J.l and RAPD data because these markers are

dominant. For these markers. Hardy-Weinberg equilibrium was assumed allowing

the frequency of each recessive morphological allele to be estim:tted as the square

root of the frequency of the reces.~ive homozygous genotype. The frequency of a

RAPD recessive allele was also determined as the square root of the frequency of

individuals lacking the band These ailele frequencies were then used to estimate

expeeted heterozygosity. The Shannon information measure. 1, = -E [Pi log2 pa,

where Pi is the frequency of the i'h allele of a locus, was also calculated per locus for

morphological, isozyme and RAPD markers (Lewc.ntin 1972). This statistis.widely

used in ecology to evaluate diversity in plant commùnities (Pielou 1969), always has

values greater than zero, with higher values indicating greater diversity. This

measure has the additive property and is useful forhierarchical analysis of diversity

of germplasm resources (Jaïn et al. 1975)•

• 39 • 3.4. Results and discussion 3.4./. MorpllOlogical markers

Ali six morphologicalloci were polymorphie in cv. Essi whereas only four loci

were polymorphie in cv. Ottawa (Tables 3.2 and 3.3). The loci controlling spreading

hairs on the petiole and pink f10wer eolour were monomorphic in cv. Ottawa.

Natural selection for resistance to potato leafhopper injury, which is more severe in

North America than in Europe, has made cv. Ottawa more pubescent than cv. Essi

(Fergus & Hollowell 1960). The cultivar Ottawa was developed by mass selection

for winter hardiness, high yield and for adaptation to conditions of eastern Canada

whereas cv. Essi originated from Europe (Fergus & Hollowell 1960). A higher

percentage of plants with spreading hairs on stems, hairy basal internodes and hairy

stipules was observed in cv. Ottawa. Ali plants in cv. Ottawa have spreading hairs

on petioles and 78 (out of 79) plants have spreading hairs on stems. In contrast,

most of the Essi plants have appressed hairs on stems and petioles (7Land 67 out

of 73 and 79, respectively). Leaf mark, which is not under natura! or artificial

selection. showed high variation in both cultivars (Table 3.3). Only one (out of50)

white-flowered plant was found in cv. Essi whereas none was observed in cv. Ottawa.

There may be natura! selection for pink f10wers which are more attractive than white

flowers .to bumble bees (Bombus spp.) and honey bees (Apis spp.), the prevalent

poUinators in eastern North America (Cornelius & Taylor 1981). Since red clover

bas a gametophytic self-incompaubility system and needs inseClS for pollination. a

• 40 reeessive al1ele (c) controlling white flower eolour tends to be lost gradual1y l'rom

• populations (Taylor & Smith 1979). Although cv. Essi had a higher number of polymorphie loci, al1ele distribution within polymorphie loci in cv. Ottawa was more

even than in cv. Essi resulting in a higher expeeted heterozygosity and Shannon

diversity index in Ottawa (Tables 3.2 and 3.3).

3.4.2 IsoZJllne markeTS

A total of 21 isozyme loci with 43 al1eles was deteeted using twelve enzyme

systems in these two red clover cultivars. Isozymes AMY-l, DIA-l, DIA-2, EST-!,

EST-2, EST-4, EST-7, FGM-2, and SKO-1 were interpreted as monomers while

AAT-2, AAT-3, ADH-1, DIA-3, GPI-2, IDH-1, MDH-1, MDH-2, MDH-3, PGD-l,

and PGD-2 were eonsidered dimerie enzymes. An ME-1 enzyme was found to be

monomorphie with a two-banded pattern. Three malate dehydrogenases were found

to form three heterodimers giving six bands in a homozygous genotype

(Kongkiatngam et al. in press, ehapter 4). Many strong and variable loci ofesterases

were found in these red clover plants but only the four loci that eonsistently gave

clear and well-resolved bands were seored. The homozygous genotype ofEST-? (aa)

showed a two-banded pattern that segregated according to Mendelian inheritanee

(Kongkiatngam et al. in press, ehapter 4).

More variation with isozyme markers was found in cv. Essi thali in cv. Ottawa

(Tables 3.2 and 3.3). The cultivar Essi had more polymorphie loci (13 out of 21)

than cv. Ottawa (9 out of21) as well as having a higher mean number of aIleles per

• 41 • locus. Seven loci were monomorphic in both cultivars: Dia-2, Esr-2, Idlz-I, Mdlz-l,

Mdlz-2, Mdlz-] and Me-I. Aat-2, Adlz-I, Dia-l, Dia-] and Pgd-I were polymorphic

only in cv. Essi whereas the Aat-] locus was polymorphic only in cv. Ottawa. Amy-l,

Est-J, Est-4, Est-7, Gpi-2, Pgd-2, Pgm-2 and SIal-J were polymorphic in both cultivars.

Amy-J, Est-J and Pgd-2 were more variable in cv. Ottawa but Est-4, Est-7, Pgm-2 and

Slal-I were more variable in cv. Essi (Table 33). Observed heterozygosity was

similar in both cultivars but expected heterozygosity and the Shannon diversity

measure were slightly higher in cv. Essi (Table 32).

Observed genotype frequencies deviated significantly (p < 0.05) from those

expected under Hardy-Weinberg equilibria at four of the 13 polymorphie loci - (30.8%) in cv. Essi (Adh-J, Amy-l, Dia-3, and Gpi-2 with fixation indices of 0359,

0.786, 0.653 and 0380, respectively) and at one of the nine polymorphie loci (11.1%)

in cv. Ottawa (Est-7 with a fixation index of 0307). These positive fixation indices

indicated a deficiency ofheterozygotes at those loci. Ifthese loci are linked to genes

eontrolling morphological or agronomie eharacteristics which are under natura! or

artificial selection, they may be more likely to be homozygous than heterozygous.

Levels of variability in this study were comparable to those found in two

naturalized populations of red clover from Ohio (Hickey et al. 1991) (Table 3.4).

Tuese high levels ofvariation within populations are not swprising since red clover

is a perenniaI, outcrossing species. Hamrick (1989) has reviewed many studies that

used isozymes to investigate the genetie structure ofplant species and concluded that

long-lived and outcrossing species maintained higher levels of variation within

• 42 populations than annual or selfing species. Extensive isozyme polymorphism within • cultivars was also observed in ryegrass which is a cross-pollinated pasture grass (Ostergaard et al. 1985). In contrast, ail naturally occurring populations of self-

fertilizing subterranean clover (TrifoliulIl subte"aneulIl L) were found to be

isozymically homogeneous whereas three out offive of its cultivars were polymorphie

fOl' at least one locus (Collins et al. 1984). As a result, isozyme patterns could be

used to differentiate among most of the 15 subterranean clover cultivars. In rose

clover (Trifolium hirtum All.), only eight (out of 23) loci analyzed in 48 populations " from various areas were polymorphie (Molina-Freaner & Jain 1992). They suggested

that these populations had a selfing or mixed mating system.

Using isozymes as genetic markers for cultivar identification in red clover

would not be as straightforward as in homogeneous, selfing species because of high

variation within cultivars. Isozyme loci that show cultivar-specifie alleles could be

used to differentiate between cultivars but only if they were present at reasonably

high frequency. Although cultivar-specifie alleles were found for ail polymorphie loci

except Est-l, Est-7 and Pgd-2 (Table 3.3), ail of them were present at low frequency

(less than 7%). These low frequencies for cultivar-specifie alleles make tbem less

usefui for cultivar identification in red clover sinee a large number ofplants must be

assayed to obtain accurate results. AlI polymorphie loci had at least one ~mmon .

al1ele in the two cultivars. Differences in al1ele frequencies at isozyme loci could

also be used to discriminate red clover cultivars as suggested for other beterogeneous

species, but large sample sizes would be essential to obtain accurate estimates of

• 43 allele frequencies (Booy et al. 1993). Although only two red clover cultivars were • examined in this study, they should represent most red clover cultivars because they were from the most important regions of red clover diversity (Taylor & Smith 1979).

European and North American cultivars are always used for developm~nt of red

clover cultivars for other growing areas.

3.4.3. RAPD markeTS

Nine ofthe seventeen arbitrary primers that were screened for RAPD markers

produced clear and consistent amplification produets. A total of 133 bands (putative

loci) were generated by these primers in the assay of20 individual DNA sarnples for

each cultivar. Each primer gave 7 to 20 amplification produets with an average of

14.8 bands per primer (Table 3.1). High variation of amplification products within

cultivars was observed (Figure 3.1). ln cv. Essi, 108 of 116 loci were polymorphie

while 90 of98 loci were polymorphic in cv. Ottawa. Eight monomorphic bands were

obtained in each of the twO cultivars; one each from primers G-3, G-17, G-18, H-4,

H-9, H-l3 and twO from H-15 in cv. Essi, one each from primers G-3, G-17, G-18,

H-4, H-7, H-9 and twO from H-15 in cv. Ottawa. Five monomorphic bands were the

sarne in bath cultivars but one monomorphic band each from primers G-18, H-13 and

H-15 in cv. Essi was polymorphic in cv. Ottawa and one monomorphic band each

from G-18, H-7 and H-15 in cv. Ottawa was polymorphic in cv. Essi. Many cultivar­

specific RAPD bands from aIl primers were observed in these twO cultivars, but with

low frequencies. The estimates of genetic variation obtained with RAPD markers

• 44 • were about the same in both populations with slightly higher values in cv. Ottawa

(Table 3.2).

High variation within cultivars of red clover has also been observed using

ehloroplast DNA RFLP markers (Milligan 1991). These high levels of variation

within populations using molecular markers are consistent with observations on other

outcrossing plant species, sueh as alfalfa (Yu & Pauls 1993b) and perennial ryegras.~

(Sweeney & Danneberger 1994). However. in both those studies variation among

cultivars was low as it was in our study.

Both nuclear and organellar DNA can serve as template for RAPD

amplification, but polymorphie RAPD markers are usually inherited in a Mendelian

fashion in many erop species (Kazan et al. 1993b; Yu & Pauls 1993a). indicating that

they are of nuclear origin. Amplification from organellar DNA is likely to produce

amplification produets that are not polymorphie and therefore not retained for

analysis. Thus, this type of marker eould be applied in red clover breeding and

genetie studies. RAPD markers eould be very valuable in studies that require a high

number of polymorphie loci. sueh as eonstrueting a genetie linkage map or marking

a single gene (Miehelmore et al. 1992).

High genetie variation within populations of red clover found with RAPD

markers makes them less useful as markers for cultivar identification than they are

in self-pollinated and clonally propagatedspecies (He et al. 1992; Wu & Lin 1994).

RAPD markers from bulked DNA samples should be investigated for use in cultiV".l.r·

identification in redclover since RAPD patterns obtained from bulked DNA samples

• 45 • have been demonstratetl to represent plant populations or cultivars (Michelmore et

al. 1992; Yu & Pauls 1993b).

Two factors should be taken into consideration in using RAPD markers to

estimate genetic variation of cross-pollinated species like red clover. The first one

is the assumption that each band is a locus with IWO alleles. At least sorne RAPD

markers have been shown to be codominant {Tinker et al. 1993; Williams et al.

1990). Three to four percent of the total number of RAPD polymorphisms were

found to be codominant in their studies. If the codominance was not known and all

bands were assumed to be dominant. each band would be scored as a different locus

rather than as different alleles of the same locus. as for a codominant locus. These

errors in scoring would resuit in an overestimation ofthe number ofpolymorphie loci

and an underestimation of the Mean number of alleles per locus. The second factor

is the dominant nature of Most RAPD markers. The only monomorphic loci

observed with dominant markers are those ofdominant alleles (presence ofa band),

not recessive alleles (absence of a band). However, monomorphie loci for Most

al!eles are deteeted with codominant isozyrne and RFLP markers. The oniy allele(s) cC-

whieh are deteeted in RAPD analysis are those that allow amplification; allelie

variants involving various point mutations, insenions and deletions within the

amplified segment are unlikely to be observed. Thus, the percentage ofpolymorphie

•~.: RAPD loci, lTIean number of alleles per locus. and expeeted heterozygosity May be

._~-- overestimated. ln our study, 92 to 93 percent of RAPD loci were polymorphie

whereas only 43 to 62 percent of i.~ozyme loci were polymorphie. The estimates of

46 expected heterozygosity and the Shannon diversity index were almost douhled with • RAPD markers.

3.4.4. Comparïsolls of/lIorplzological, isoZ)'me alld RAPD markers

The number of loci detected with RAPD markers was much higher than that

detected with morphological traits and isozymes (Table 3.2). Mean nLlmber ofalleles

per locus and percentage of polymorphic loci were higher with RAPD markers than

with isozymes in both cultivars. The estimates of expected heterozygosity and the

Shannon diversity measure obtained with morphological and RAPD markers were

about the same in these !wo cultivars and almost doubled those detected with

isozymes. Genetic variability of these IWO red clover cultivars may be overestimated

with morphological traits since only a small number of morphological loci (six loci)

were e.xamined and these loci were chosen because they are often polymorphic.

Heterozygotes ofsorne morphological loci under selection, such as those controlling

hairs on stems, petioles and stipules, may be less frequent than could be expected

under Hardy-Weinberg equilibrium. Estimates of genetic variation obtained with

RAPD markers may also he higher than those aetually existing because of the

'..>minant nature and one-band/one-Iocus assumption of these markers previously

discussed. They may also be higher due to the fuet that RAPD markers can deteet

both coding and non-coding sequences in the genome. The non-coding sequences

have been shown to be more variable than the cading sequences (Ohla 1992)..

RAPD markers also gave slighùy higher estimates ofgenetic diversity than isozymes

• 47 in trembling aspen (Populus Iremuloides Michx.) and bigtaoth aspen (Populus • grandidentata Michx.) which are outcrossing tree species widely distributed in North

America (Liu & Fumier 1993). On the other hand, estimates based on isozyme

polymorphism may underestimate overallieveis of genetic variation because they are

sampling only coding regions that may be conserved ta maintain the function of the

enzymes (Gottlieb 1982).

Isozymes have been shown to be more useful as genetic markers for studies

of plant populations than morphol<.'gicaJ charaeters. More information can be

obtained from isozymes than from morphologicaJ markers since more loci can be

detected and isozymes are codominant markers. Genetic inheritance of isozymes is

also easier to detennine and allele frequencies can be caJculated direct1y. Isozymes

have also been found to be independent ofenvironmental influence (McMillin 1983).

The primary advantage that RAPD markers have over isozymes is the potential to

deteet polymorphism at iÎlany more loci. In terms of time and resources, isozyme

assays are less expensive and faster than RAPD assays.

This study bas shown thatiso~me and RAPD markers could be used - -.-- effectively to estimate genetic variability within and between cultivars of red clover.

. Compared with morphologicaJ charaeteristics, better estimates of genetic variation

,~ , . could be obtained using these two types of markers. Isozymes and RAPD markers

'.: willprovide_useful tools for germplasm charaeterization, conservation and utilization,

as wèn as for genetic and breeding studies in red claver.

,'. ~, .

• 48 • Table 3.1. Sequences and amplified producls of ni ne arbilrary primers (Operon)

used 10 generale RAPD markers in Trifolium prarenst! L

Primer Sequence Number of bands

G-03 5'-GAGCCCfCCA-3' 16

G-17 5'-ACGACCGACA-3' 20

G-18 5'-GGCfC.A..TGTG-3' 16

H-02 5'-TCGGACGTGA-3' 15

H-Q4 5'-GGAAGTCGCC-3' 7

H-07 5'-CfGCATCGTG-3' 14

H-Q9 5'-TGTAGCfGGG-3' 12

H-13 5'-GACGCCACAC-3' 17

H-l5 5'-AATGGCGCAG-3' 16

:

• 49 .. , "F.! r" l," '} )1 ' , '.' •

il Table 3.2. Genetic variation detected by morphological, isozyme, and RAPD markers in red clover cultivars Essi If " l, . and Ottawa. Estimates are mean number of nlleles per locus (A), percentage of polymorphic loci (P). menn l, . " . , ,' i," observed heterozygosity (Ho), mean expected heterozygosity (HJ and menn of the Shannon diversity measure

(1.)." Standard., errors are in parentheses.

l,; Essi OUawa (, Estimate Mprphology Isozyme RAPD Morphology Isozyme RAPD

" plants 79 39 20 80 40 20

loci 6 21 116 6 21 98

A 2.00 (.00) 1.81 (.16) 1.93 1 (.02) 1.67 (.21) 1.67 (.20) 1.92 1 (.03)

P 100.0 61.9 93.1 66.7 42.9 91.8

Ho . 0.10 (.03) - . 0.10 (.03)

" H. 0.19 (.06) 0.12 (.03) 0.20 (.01) 0.26 (.09) 0.10 (.03) 0.22 (.02) " 1. 0.46 (.11) 0.29 (.08) 0.46 (.03) 0.55 (.17) 0.24 (.05) 0.50 (.03)

1 The proportion of alleHc bands was not determined.

50 Table 3.3. Allele frequeneies al polymorphie loci eoding for isozymcs and • morphologieal traits. Allele frequency Helerozygosily Loeus A1lele Essi Ottawa Essi Ottawa

Aar-2 a 0.013 0 0.026 0.000 b 0.987 1.000

Aat-3 a 0.000 0.025 0.000 0.100 b 1.000 0.950 e 0.000 0.025

Adlz-] a 0.936 1.000 0.077 0.000 b 0.064 0.000

Amy-] a 0.064 0.063 0.026 0.150 b 0.936 0.925 e 0.000 0.013

Dia-] a 0.026 0.000 0.051 0.000 b 0.974 1.000

Dia-3 a 0.038 0.000 0.026 0.000 b 0.962 1.000

Est-] a 0.936 0.800 0.128 0.350 b 0.064 0.200

Est-4 a' 0.038 0.000 0.590 0.500 b 0.538 0.700 c 0.423 0.300

Est-7 a 0.756 0.825 0.282 0.200 b 0.244 0.175

Gpi-2 a 0.051 0.000 0.179 0.075 b 0.833 0.962 c 0.155 0.038 "

• .51 • Table 3.3. Continued. Pgd-J a 0.987 1.000 0.026 0.000 b 0.013 0.000

Pgd-2 a 0.154 0.213 0.410 0.425 b 0.795 0.700 e 0.051 0.087

Pgm-2 a 0.000 0.013 0.051 0.025 b 0.974 0.988 e 0.026 0.000 Skd-J a O.1C3 0.013 0.308 0200 b 0.821 0.900 e 0.077 0.075 d 0.000 0.013 Leafmark M 0.663 0.706 0.450 0.421 No leaf mark m 0.338 0.287 Spreading stem hair He 0.014 0.886 0.027 0203 Appressed stem haïr he 0.986 0.114 Spreading petiole hair Hp 0.076 1.000 0.141 0.000 Appressed petiole haïr hp 0.924 0.000 Hairy intemode Hb 0.125 0.627 0220 0.471 Glabrous internode hb 0.875 0.373 Hairy stipule Hs 0.031 0.356 0.061 0.462 Glabrous stipule hs 0.969 0.644 Pink flower C 0.860 1.000 0243 0.000 - White flower e 0.140 0.000

• 52 • •

Table 3.4. Comparisons of genetic variation within populations of different TrifoliulII species based on isozymes.

P is the percentage of polymorphic loci and A is the mean number of alleles per locus.

Species Breeding system P A Reference

T. prafellse outcrossed perennial 53.3 1.93 Hickey el al. 1991

T. prafellse oulcrossed perennial 61.9 1.81 This sludy

T. hybridlllll outcrossed perennial 53.3 1.73 Hickey et al. 1991

T. Sf%llifen/III outcrossed perennial 15.0 1.10 Hickey el al. 1991

T. refleJ.11111 selfing annual 0.0 1.0 Hickey el al. 1991

T. "irtlllll selfing or ntixed maling 0.Q..23.8 1.02-1.18 Molina·Freaner and Jain 1992 annual

53 •

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 M

1636

1018

510 344 220

Figure 3.1. RAPD patterÎ1s obtained from individual genomic DNA samples ofred

clover cvs. Essi (lane 1-9) and Ottawa (lane 10-18) with primer H-15. Marker lanes

(M) are 1 kb DNA ladders. The monomorphic bands of 370 bp were used to

calibrate for amount ofDNA present. On lane 11, four bands: 1175 bp (11% ofthe intensity ofthe reference band), 955 bp (21%), 363 bp (the monomorphic reference :

bl:lnd), and 251 bp (11%), were scored and two bands: 513 bp (7%) and 446 bp (9%),

were too faint to be counted.

• 54 CHAPTER FOUR • DIVERSI1Y AND GENETICS OF ISOZYMES IN RED CLOVER

P. Kongkiatngam, B.E. Coulman, and MJ. Waterway

Submitted to Journal of Heredity (March 15, 1995: provisional acceptu.nce pending

revision, June 12, 1995)

P. Kongkiatngam planned the experiments, performed ail laboratory work and data analysis described in this manuscript and is the first author of the manuscript. M.J. Waterway assisted in planning the experiments, provided laboratory facilities and research materials and supervised laboratory work for ail experiments, gave valuable advice and suggestions throughout the projec!, and contributed significantly towards editing and revising the manuscript. B.E. Coulman assisted in planning the crossing experiments, provided greenhouse facilities and plant materials, gave valuable advice and suggestions throughout the projec\, and helped in editing and revising the manuscript. -">-..., ~

AcknoWîedgements: We would like to thank the Canada-Egypt-McGill Agricultural Response Program for scholarship support to PK. This study was supported in part by grants from the National Science and Engineering Research Couneil of Canada to MJW. We also would like to thank Helen Rimmel' for her photographie assistance.

• 55 • 4.1. Abstrdct

We examined isozyme variability in 15 cultivars of red clover (Trifolium

pratense L), an important temperate forage legume, from three different

geographic-dlorigins. We crossed individuals with different allelomorphs to verify the

genetic control of seven isozyme loci: Aat-2, Amy-l, Est-4, Est-7, Pgd-l, Pgd-2 and

Skd-l. We also postulated the geneùc basis ofbanding patterns for 16 other isozyme

loci: Aat-S, Adh-l, Dia-l, Dia-2, Dia-S, Est-l, Est-2, Gpi-2, ldh-l, Mdh-l, Mdll-2, Mdll-

S, Mdh-4, Me-l, Me-2 and Pgm-2, based on the segregation patterns observed within

cultivars. Estimates ofgenetic variability for these red clover cultivars indicated that

within-cultivar variation was much higher than between-cultivar variation. We also

exarnined the potential use of allele frequencies at enzyme-coding loci for cultivar

identification in red clover. We found that they could discriminate one North

American and one Japanese cultivar but they could not differ<,ntiate the other

cultivars from Europe, Japan and North America.

4.2. Introduction

Red clover, Trifoüum pratense L, is one ofthe most important forage legumes

in the temperate regions of the world. It is used for hay, silage, pasture, green

manure and cover crops and is widely grown in humid northeastem North America

and most areas of Europe. It is also cultivated to sorne extent in Japan, temperate

• 56 South America. AU5tralia. New Zealand and South Africa. Compared 10 alfalf'l. • which requires well-drained fertile soils. red clover has greater adaptability and can adjust to wet and acid soils (Smith et al. 1985).

Red clover is a diploid (2n =14). cross-pollinated perennial species wilh a

gametophytic self-incompatibility system (Taylor and Smith 1979). Telraploid

cultivars (2n =28) of red clover, with higher yields and greater disease resistance than

diploid forros, have been developed in Europe. However. these cultivars are nol

widely used in North America because of low seed yields and lack of fomge yield

advantage (Smith et al. 1985). Most cultivars of red clover in use today have been

developed through controlled mass selection (Smith et al. 1985) and are

heterogeneous populations consisting of heterozygous individuals. Red clover

breeding is difficult and time-consuming due to the growth habit and because only

a few qualitative markers have been described and genetically confirmed

(Quesenberry et al. 1991).

Isozyme polymorphisms may be useful genetic markeTS, but only one previous

study of isozymes in red clover has been reported (Hickey et al. 1991). Two

naturaIized populations of red clover from Ohio were examined for ten enzyme

systems to com!,are with IWO native species: an endangered species Trifo/ium sloloni-

ferum MuhI. (running buffalo clover) and Trifo/ium ref1exum L (buffalo clover), and

another introduced species Trifoüum Ilybridum L (alsike clover). Eight polymorphie

loci (Aar-l, Gdll-l, Gpi-2, Lap-l, Md/z-l, Pgd-2, Pgm-l and Tpi-2), and seven

monomorphic loci (Ad/z-l, Gpi-l, Id/z-l, Md/z-2, MdIt-3, Pgd-l and Tpi-l) were

• 57 • resolved in red c1over, but crossing experiments to confirm the genetic basis of the

banding patterns were not included in that study (Hickey et al. 1991).

Identification of crop cultivars is fundamental to commercial seed production

and certification prograrns. Three basic criteria for any potential charaeter to be

used in cultivar identification are environmental stability, distinguishable between­

cultivar variation and minimal within-cultivar variation (Bailey 1983). Traditionally,

red c10ver cultivars have been discriminated on the basis of morphological and

agronomic characteristics. Although these charaeters have been genetically

confirmed (Fergus and Hollowell 1960; Quesenbeny et al. 1991; Taylor and Smith

1979), they are limited in number and subject to environmental and ontogenetic

influences. As the number of red clover cultivars increases from breeding programs

in several countries, it becomes more difficult to differentiate all cultivars using these

charaeters. Markers that are objective, genetically weil charaeterized, and

conveniently assayed are needed for this purpose. Isllzymes have been shown to be

reliable genetic markers for cultivar identification in severa! species (reviewed by

Nielsen 1985 and Weeden 1989) and could also be very useful genetic markers for

breeding and genetic studies in red clover. Because cultivars of red clover in use

today are heterogeneous populations, high variation within cultivars is retained

.(Kongkiatngam et al. in press; Milligan 1991; Nelke et al. 1993). Successfu1 cultivar

discrimination using allelicand genotypic frequencies has been demonstrated inother

cross-pollinated species with high variation within cultivars, sueb as ryegrass, Lolium

•• 58 spp. (Booy et al. 1993; Nielsen et al. 1985; Ostergaard et al. 19R5) and rye. Seca!I'

• cereale L (Adam et al. 1987; Ramirez and Pisabarro 1985; Ramirez et al. 1985). In tbis paper. wc evaluate isozyme diversity in red clover cultivars. confirm the

genetic inheritance of several isozymes in red clover. ;md explore the potential

applications of these isozymes for cultivar identification and genctic studies.

4.3. MateriaIs and Methods

4.3.1. Isozyme assay

Fifteen cultivars ofred clover from three different geographical origins (Table

4.1) were examined in our study. Forty plants each of cultivars E.o;si and Ottawa, and

20 plants each from the other cultivars were assayed. Young leaves from five-week

old plants were ground in extraction buffer [0.001 M EDTA, 0.010 M KCl, 0.010 M

MgCI2> 7% (w/v) PVP, 0.10 M Tris-HCl buffer, pH 75, 3% sucrose (w/v)] to which

02% (v/v) 2-mercaptoethanol was added before use (modified from Gottlieb 1981).

Extraets were absorbed onto Whatman #3 filter paper wicks and stored in

microcentrifuge tubes at -80 oC until eleetrophoresis. These sampIes were assayed for

twelve enzyme systems which gave clear and welI resolved bands on 10% starch gels

run with three gel-buffer systems as described by Wendel and Weeden (1989).

NADH diaphorase (DIA, E.e. 1.6.99.-); glucosephosphate isomerase (GP!, E.e.

5.3.1.9); isocitrate dehydrogenase (IOH, E.e. 1.1.1.42) and phosphogJucomutas~

(PGM, B.e. 5.422) were assayed with histidine-cltrate buffer, pH 65. Alcohol

• 59 dehydrogenase (ADH, E.C. 1.1.1.1); malate dehydrogenase (IvI-DH, E.C. 1.1.137); 6- • phosphogluconate dehydrogenase (PGO, E.C. 1.1.1.44) and shikimate dehydrogenase (SKO, E.C. 1.1.1.25) were resolved by using morpholine-citrate buffer, pH 6.1.

Aspartate aminotransferase (MT, E.C. 2.6.1.1); amylase (AMY, E.C. 3.2.1.1 and

E.C. 3.2.1. 2); esterase (EST, E.C. 3.1.1.-) and malie enzyme (ME, E.C. 1.1.1.40) were

resolved with Iithium-bor.t.tejTris-citr.ue buffer, pH 8.3. Ali gels were run at a

constant current of 35 mA, the histidine-citrate system for 4 hours, the morpholine-

citrate system for 4 hours and the lithium-borate system for 5 hours. The assays for

ail enzyme systems were the same as those described by Wendel and Weeden (1989),

except esterase for which N-propanol was used instead of acetone to dissolve the

substrate. Loci and alleles were sequentially numbered and lettered, respeetively,

beginning with the most anodal forro.

Red clover plants with known genotypes from the cultivar screening were used

for our crossing assays. .A total of twelve parents were available for crossing

experiments to analyze segregation at seven isozyme loci; Aar-2, Amy-], Est-4, Est-7,

Pgd-]. Pgd-2 and Skd-] (Table 4.2). Emasculation was not necessary ÏJ1,red clover

because of the~ametopbyticself-incompatibility system (Taylor and Smith 1979).

Controlled pollination was obtained by using a toothpick method, in which a

" '.''':,'' :- - ': '. ::'.:': .; -" toothpick covered with cotton fibre was used to transfer pollen from a male flower

to a female flower.

60 4.3.2 Dara ana/ysis • Chi-square statistics were calculated to test goodncss-of-fit to expected ratios for single-locus segregation of enzyme aileles (Table 4.2). Chi-square tests of

independence were used to examine linkages belWeen these enzyme loci. Signilicant

deviations from Hardy-Weinberg equilibrium of observed allozyme frequencies were

tested by the chi-square test adjusted for small sample size using Levene's (1949)

correction factor as implemented in BlOSYS-I (Swofford and Selander 1981). Nei's

(1973) gene diversity statistics were calculated using GENESTAT (Whitkus 1988).

The estimates of Nei's (1972) modified genetic distances belWeen cultiV".lrs were

obtained from the SIDGEND program (Satder and Hilburn 1985). This program

combines Nei's (1978) modification for smalt sample size, Mueller and Ayala's (1982)

jack-knife routine to reduce the bias from sampling a small number of loci, and

Hillis' (1984tmodification to reduce the bias resulting from unequal rates of amino

acid substitution at ail loci (Sattler and Hilburn 1985). SIDGEND was a150 used to

calculate U-statistics and their confidence intervals (MueUer and Ayala 1982), where

U = the average genetic distance between groups (Da> - the average genetic distance

within groups (Dw), to evaluate whether there was significant difference belWeen

groups of cultivars from cach geographjc regioÎL AU-value significantly different

from zero indicates a:-significant difference belWeen groups. The PRINCOMP

procedure of SAS (SAS Institute, Ine. 1985) was wied to perform principal

component analysis using the variance-covariance matrix derived from allele

frequencies. e· 61 The: cultivar, were compare:d IWO by IWO by likelihood ralio leSts for • homogeneily of the allele frequencies al each locus and al several loci (Nielsen el al. 1985: Sakai and Rohlf 1981). G-slalislics were eSlimaled as follows:

where Xij is the absolule frequency of the r allele in the i'h cultivar (i = 1. 2) at a

given locus with j = 1.2•..•01 aileles. and n = :E xii" The G-values are X2-distributed

with degrees offreedom e'lual ta the number of alleles minus one (01-1). For several

independent loci. the sum of single-locus G-values is x2-distributed with the degrees

of freedom equal to lhe sum of the single-locus degrees of freedom. Significant

values of G be(Ween (wo cultivars mean that the (wo cultivars differ in their allele

frequencies at those loci.

4.4. Results and Discussions

4.4.1. Polymorphism and genetics of isozymes in red clover

ln this paper. we present segregation data from crossing experiments for seven

isozyme loci: Aat-2, Amy-l, Est-4, Est-?, Pgd-l, Pgd-2 and Skd-l. For the other 16

loci: Aat-3, Adlz-l. Dia-l, Dia-2, Dia-S, Est-l, Est-2, Gpi-2, Idlz-l, Mdii-l, Mdll-2, Mdll-

3, Mdll-4, Me-l, Me-2, and Pgm-2. p?lymorphism was tao low or FI progeny coold not

• 62 be obtained so we report only isozyme patterns from ;\ssays of individual red clovcr • plants from those cultivars listed in Table 4.1.

4.4.1.1. Aspanate aminotransferase (MT)

There were three distinguishable areas of AAT activity on gels. Thus. the

AAT isozymes in red clover appeared to be specified by three loci. Aat-l. Aat-2 and

Aut-3. However. only two loci, Aat-2 and Aat-3. consistently gave clear and well­

resolved bands. Both the AAT-2 and the AAT-3 enzymes were controlled by single

loci, Aat-2 and Aut-3, respeetively, which segregated independently (Table 42).

Three alleles were observed at each of these loci (Figure 4.1). Both the AAT-2 and

the AAT-3 enzymes showed dimeric banding patterns, as has been found in other

legumes (Mahmoud et al. 1984; Mancini et al. 1989; Weeden and Marx 1984; Zamir

and Ladizinsky 1984). Three AAT isozymes were observed in Viciafaba (Mancini

et al. 1989), while four MT isozymes were found in pea (Weeden and Marx 1987).

Both three (Zamir and Ladizinsk-y 1984) and four (Muehlbauer et al. 1989) MT

isozymes have been found in lentil.

4.4.1.2 Amylase (AMY)

We found only one locus with three alleles controlling AMY-1 in red clover.

Crosses between plants with different alleles gave hybrids showing both alleles, as

expeeted for a monomeric enzyme. The segregation ratio of AMY-1 \Y3.S that • expeeted ifthere is monogenic control ofAmy-l (Table 42). Mahmoud etal. (1984) • also found a single locus controlling monomeric AMY-l in pea but only twO alleles

were observed.

4.4.1.3. Eslerase (EST)

We could detect seven esterases from red clover on the gels, but only four

enzymes, EST-l, EST-2, EST-4 and EST-?, were clear and well-resolved. The EST-2

isuzyme was monomorphic across ail red clover cultivars. Both Est-1 and Est-4 loci

showed simple segregation ratios (Table 42). EST-1 and EST-4 were monomeric

enzymes with two and three variants, respectively (Figure 4.1). An EST-? enzyme

was also a monomer and the Est-7 locus with 2 al1eles, segregated according to

Mendelian rdtios (Figure 4.1 and Table 42). A two-banded pattern was observed for

the slow variant of the EST-? enzyme (Figure 42). Nul1 homozygotes of the Est-7

locus were found in ail cultivars examined, except Hamidori. Weeden (1984)

observed only two loci controlling EST isozymes, Est-1 and Est-2, in white-seeded

bean. However, Weeden and Marx (1984) found one cathodal and three anodal

zones ofesterase aetivity from young leaf tissue ofpea, one ofwhich was interpreted

as a dimer with two variants while the others were monomeric. Mahmoud et al.

(1984) also found the cathodal esterase tO be a monogenic monomer with two­

banded homozygotes and three-banded heterozygotes in pea.

• 64 • 4.4.1.4. 6-P/ZOSpllOgluconare de/zydrogenase {PGD}

We observed {wo overlapping zones of PGO activity i.: red claver (Figure 4.2).

Bùth PGO-1 and PGO-2 enzymes were dimers, each comrolled by single loci. Pgd-J

and Pgd-2. respectively, which segregated independent\y (Table 4.2). We found two

and four alleles at the Pgd-1 and Pgd-2 loci. respectively (Figure 4.\). Two loci

controlling dimeric PGO isozymes were also found in buffalo claver. running buffalo

claver and alsike claver (Hickey et al. 1991). cowpea (Vaillancourt et al. 1993).

birdsfoot trefoil (Raelson and Grant 1989). lentil (zamir and Ladizinsk-y 1984). and

pea (Weeden and Marx 1984).

4.4.1.5. Slzikimate delzydrogenase (SKD)

We saw {wo zones of SKD activity in red claver with the morpholine-citl".lte

buffer system but only the anodal form was clear and well-resolved. SKD-l wa.~ a

monomer which was controlled by a single gene. Skd-1. with four alleles (Figure 4.1

and Table 4.2). We observed shadow bands above and below the SKD-1 band across

the gels. Two areas of SKD aetivity were also observed in pea but only the more

anodal zone was well-resolved and clear (Weeden and Gottlieb 1980). SKD-1 was

found ta be a monomeric enzyme in pea and lentil (Muehlbauer etal. 1989; Weeden

and Gottlieb 1980).

• 6S 4.4.1.6. Aico/IOI dehydrogenase (ADH) • We observed only one area of ADH activity among ail red clover plants evaluated. This ADH-1 enzyme was polymorphie in five cultivars and exhibited a

dimerie banding pattern in sorne individuals (Figure 4.1). The total number ofADH

enzymes in red clover may be more than one because they are inducible under

anaerohic conditions. We assumed that we detected only one locus, Adh-1, with IWO

a11eles in rc<' c10ver leaves under the growing conditions in this study. Only one

dimeric ADH enzyme controlled by a single gene was found in rose clover (Trifolium

hirtum AIl.) (Molina-Freaner and Jain 1992) and lentil (Lens culinaris L) (Zamir and

Ladizinsky 1984) but IWO dimeric enzymes, ADH-1 and ADH-2, were observed in

pea (Weeden and Marx 1987) and three ADH isozymes were found in cotyledons of

chickpea (Gomes et al. 1982).

4.4.1.7. NADH diaphorase (DIA)

We observed three distinct areas of DIA activity in red clover on gl'Is from

three gel-buffer systems. Based on their independent segregation within populations,

we assumed that there were three loci, Dia-l, Dia-2 and Dia-3, controlling DIA

isozymes. The loci Dia-1 and Dia-3 were polymorphie but the locus Dia-2 was

'. monomorphic. We postulated that the DIA-1 enzyme was a monomer with two

variants and the DIA-3 isozyme was a dimer with IWO variants. Weeden (1984) aIso

found three zones of DIA activity in white-seeded bean (Phaseolus vulgaris 1..)•

• 66 • 4.4.1.8. Glucoseplzosplzate isomerase (GPI)

We observed two distinct zones of GPI activity but only the GPI-2 enzyme was

clear and well-resolved. This enzyme \Vas dimeric and controlled by a single gene.

Gpi-2. \Vith three allelcs (Figure 4.1). T\Vo areas of GPI activity were also seen in

other legume species (Collins et al. 1984; Hickey et al. 1991; Molina-Freaner and

Jain 1992; Muehlbauer et al. 1989; Raelson and Grant 1989; Vaillancourt et al. 1993;

Weeden et al. 1989).

4.4.1.9. Isocitrare delrydrogenase (IDH)

We assumed tbat a single locus, IdIr-1, controlled this enzyme in red clover

since there was only one zone of IDH aetivity among ail red clover plants examined.

The IDH enzyme in red clover was a dimer \Vith !Wo variants a~ bas been found in

pea (Weeden and Marx 1984) and lentil (Zamir and Ladizinsky 1984). One zone of

IDH aetivity was also seen in cowpea (Vaillancort et al. 1993), lentil (Zamir and

Ladizinsky 1984), pea (Weeden and Marx 1984), and rose clover (Molina-Freaner

and Jain 1992). However, three IDH isozymes were observed in subterranean clover

(Collins et al. 1984) and four loci controlling IDH isozymes were confmned in

soybean (Kiang and Gorman 1985).

4.4.1.10. Ma/are delrydrogenase (MDH)

Resolution~and banding patterns of MDH were clear and consistent with the

morpboline-citrate running buffer and staining procedure used. MDH isozymes bave

• 67 • a dimeric bandmg pattern in other species (Weeden and Wendel 1989), but genetic

control of these enzymes has been found to be complex (Goodman et al. 1980;

Arulsekar et al. 1986). We observed six intensely-staining bands of MDH isozymes

from most gcnotypes (Figure 4.1). Crossing of these genotypes gave progenies with

identical six-banded phenotypes, confirming our assumption of their homozygosity.

Because heterodimeric bands are often found in MDH (Goodman et al. 1980), we

eonservatively assigned three putative loci to this phenotype, Mdh-1, Mdh-2 andMdh­

3 (Fil;ure 42), and postulated that the other three bands were heterodimeric as has

been observed in maize (Goodman et al. 1980). Another weak band, always found

near the origin and therefore designated MDH-4, was monomorphie among all

individuals assayed. We found polymorphism only at the Mdh-3 locus, resulting in

eight bands for polymorphie individuals.

4.4.1.11. Malic enzyme (ME)

We observed two-banded aetivity ofthe ME-1 enzyme in a11 red clover plant"

assayed. The strength ofthese two bands was almost the same. When the genotypes

with this two-banded pattern were crossed, all progeny had the two-banded pattern.

This result suggested that there could be two ME isozymes. Two zones of ME

activity were also observed in lentil (Erskine and Muehlbauer 1991; zamir and

Ladizinsk'Y 1984), b~t only one zone of ME activity was seen in eowpea (Vaillaneourt

et al. 1993), white-seeded bean (Weeden 1984), birdsfoot trefoil (Raelson and Grant

1989) and rose clover (Molina-Freaner and Jaïn 1992).

• 68 • 4.4.1.12 PhospllOglucollllICase (PGM)

We ohserved two distinct zones of POM activity using the histidine-citrate

buffer system. However. only the POM-2 enzyme was clear and well-resolved

enough to be scored. It was a monomerie enzyme. apparently controlled by a single

locus, Pgm-2, with three variants in red clover (Figure 4.1). Two monomeric POM

isozymes comrolled by single genes were also observed in lentil (zatr.ir and

Ladizinsky 1984), and pea (Weeden and Oottlieb 1980).

4.4.1.13. Linkage analysis

From the data set of Tahle 4.2, 15 IWo-locus combinations of loci could be

tested for evidence of non-random joint segregation (Table 4.3). Two pairs of linked

2 enzyme loci were found: Est-4/Est-7 (X 11dl = 75.26, P < 0.001) and Pgd-2/Skd·]

2 (X 7dl = 24.71, P < 0.001). The.~ was no evidence of linkages beIWeen any of the

other thirteen pairs of loci (Table 4.3).

4.4.2. Isozyme diversity in red c10ver

An unden;tanding of genetic structure of a crop species is useful for c:

germplasm conservation and utilization (Moore and Collins 1983) and can be used

to make practical decisions regarding genetic resource management. Isozymes have

been demonstrated to be valuable genetic markers for obtaining estimates of the

genetic structure of plant species (Brown and Weir 1983). Fifty a\leles were

• 69 observed at the 23 enzyme-coding loci in the 15 cultivars of red clover assayed in this • study. Allele frequencies at each polymorphic locus for ail cultivars are shown in Table 4.4. The Aut-2, Amy-l, Est-], Est-4, Est-7, Gpi-2, Pgd-2 and SI«1.-] loci showed

high degrees of genetic variability whereas moderate levels of variation were

observed for Aut-3, Adll-], Mdll-3, Pgd-] and Pgm-2. In contras!, Dia-], Dia-3 and

ldll-] were each found to be polymorphic in only one cultivar, and seven isozyme loci

(Dia-2, Est-2, MdII-l, Mdh-2, Mdlz-4, Me-] and Me-2) were monomorphic for all

cultivars and populations surveyed. Even for the most polymorphic loci, a common

aHele was found in all cultivars (Table 4.4). For most of these isozyme loci, the

genotypic frequencies within culùvar samples did not differ from those expeeted

under Hardy-Weinberg equilibrium. Significant deviations from Hardy-Weinberg

equilibrium were only found for three loci, Aut-2, Amy-] and Est-7. At the locusAat­

2, only cv. Sapporo, which included an individual homczygous for a rare allele,

showed significant deviation from Hardy-Weinberg equilibrium. The presence of a

null allele at the Est-7locus may explain the significant deviaùons of the genotypic

frequencies from Hardy-Weinberg equilibrium at this locus. Null homozygotes could

be scored correcùy, but the null beterozygotes could be scored either as aa or bb

genotypes.

Esùmates of geneùc variability within cultivars (Table 4.5) indicate that red

clover cultivars from Europe had a higher level of isozyme diversity than those from

North America and Japan, which had similar levels of isozyme diversity. The mean

expeeted heterozygosity over all European cultivars was 0.115, while for North

• 70

= American and Japanese cultivars. mean expected heterozygosities were 0.101 and • 0.102. respectively. H.;;her values for the European cultivars are not surprising because red clover originated in Europe and has been grown there for centuries

(Fergus and Hol1owel1 1960). Later. it was introduced to other parts of the world

including Nonh America and Japan. Mean estim:!tes of total heterozygosity (Hor).

within-cultivar heterozygosity (Hs). and the coefficient of gene differentiation (Gl>ï')

were 0.130. 0.126 and 0.032, respectively (Appendix 1). Thus. a greater proponion

of the total variation in red clover was due to within-cultivar rather than beIWeen­

cultivar variation. as expected for an outbreeding species with a gametophytic self­

incompatibility system (Hamriek 1989: Taylor and Smith 1979). A high level of

variation within populations was also observed in IWO natunllized populations of red

clover in Ohio (Hiekey et al. 1991). The percentage of polymorphie loci and the

mean number of alleles per locus in their study were 53.3 and 1.93. respectively.

slightly higher than those of our study (Table 4.5). However, their estimate of

average expeeted heterozygosity was 0.085, lower than the ovenlll heterozygosity but

aImost the same as the mean estimates of heterozygosity for the Nonh American

cultivars in our study (Table 4.5). Ten of the 15 loei examined in their study (AcU-2,-=:C

Adh-I. Gpi-2, ldh-I. Mdh-I. Mdh-2, Mdlz-3. Pgd-I. Pgd-2 and Pgm-2) were the same

as those in our study. High diversity within cultiV'

outcrossing erop species. For example. Adam et al. (1987) obtained estimates of

0.295 and 0.274 for HT and Hg, reSpeetively, for eight winter rye cultivars using eight

polymorphie isozyme loci.

• 71 • 4.4.3. Identification and genetic relationship~ of red clover cultivars

The genetic distances (Nei 1972) and G-statistics (Nielsen et al. 1985; $okal

and Rohlf 1981) between pairs of cultivars are shown in Table 4.6. The G-statistics

were estimated using eleven modennely and highly polymorphic isozyme loci to

obtain the highest discrimination power whereas the genetic distances were

calculated using ail loci assayed. Genetic distances between most cultivars were very

low, suggesting that these cultivars may be closely related. Cultivars Do11ard

(Canada) and Hamidori (lapan) showed the highest genetic distances with other

cultivars. Based on isozyme variation, ail European cultivars were closely related

with pairwise genetic distances of zero or close to zero (Table 4.6). The estimates

of genetic distance showed a similar pattern to the combined G-statistics which

evaiuated the distinctness of cultivars (Table 4.6). Principal components analysis

using a variance-covariance matrix of the allele frequencies could not di<:inguish

these cultivars into three groups according to their geographical origins (data not

shown). Instead, the first and second components, which accounted for 50% of the

variation, separated these cultivars into four groups. Cultivar DoUard was placed in

a distinct group separated from all other cultivars while the other three groups each

/:::- consisted of cultivars from heterogeneous origins. In red clover breeding programs, : it is not unusu.~l to use thousands of plants from different cultivars to establish a

nursery field for :releetion and crossing to obtain a new cultivar. Therefore, it is

possible for these cultivars to have sorne common ancestors. C'ultivar DoUard was

• 72 developed by maternal-line selection from cvs. Silesian and Orel at Macdon:lld • Campus (Fergus and Hollowell 1960) and was selected for tolerance to a long period ofwinter dormancy and low temperatures. It has been grown under natural selection

for a long period of time in the cold climate of Canada. The set of Japanese

cultivars was found to be distinct from the European cultivars (Tahle 4.7) hut lhey

are similar to the North American cultivars. These Japanese cultivars may h:lve a

different set of parents from the European cultivars assayed because the climatic

conditions in Japan are different from those European areas where red clover is

grown.

Due to the low level of allozyme variability among cultivars observed in red

clover, their potentiai to distinguish among red clover cultivars is not promising.

However, sorne of the cultivars could be differentiated by G-statistics (Table 4.6).

Interestingly, ail cultivars ofNorth American origin could be discriminated from eaeh

other but ofthese, only cv. DoUard could be distinguished from ail 14 other cultivars

assay-::d. Most of the European cultivars were not distinguishable from eaeh other.

If more individuaIs were assayed for highly polymorphie isozymes. better

discrimination among cultivars might be obtained. In ryegrass and winter rye, whieh

are aIso outcrossing species, it was neeessary to assay more than 100 individuals of

each cultivar to achieve cultivar identification (Adam et al. 1987; Neilsen et al. 1985; . ~ Ostergaardet aI. 1984). Other marke_cs, such as RAPD markers from bulked

samples, could be more efficient than isozymes Jr:; cultivar identification in red

• 73 c10ver as weil as other heterogeneous, cross-pollinated crops (Kongkiatngam et al. • unpublished data, chapter 5). ln conclusion. we have confirmed the mode of inheritance of seven enzyme-

coding loci in red c1over. These seveli isozyme loci are useful new genetic markers

for red c10ver breeding and genetic studies such as constructing a genetic linkage

map, estimating outcrossing rates and marker-assisted selection for qualitative and

quantitative traits. In addition, the inheritance of 16 other enzymes were also

postulated from their segregation among red clover plants within cultivars. Two pairs

of linked genes controlling four enzymes were found. Genetic diversity of red clover

cultivars was found to be high, with most of the variation found within cultivars

rather than between them. Allele frequencies at enzyme-coding loci were not able

to discriminate ail cultivars of red claver examined, but they could differentiate the

five North American cultivars, examined.

• 74 • Table 4.1. List of red clover cultivars and their origins.

Cultivar Origin Cultivar Origin

Hamidori (HM) Japan Es~i (ES) Europe

Hayakita (HY) Japan Hermes II (HE) Europe

Sapporo (SP) Japan Kuhn (KU) Europe

DoUard (DL) Canada Marino (MN) Europe

Ottawa (OT) Canada Mistral (MT) Europe

Marathon (MR) U.S.A. Napoca (NP) Europe

Persist (PS) U.S.A. Stan (ST) Europe

Prosper (PP) U.S.A.

• 7S • " •

Table 4.2. Segregation analysis of isozyme loci in red clover.

EnZY\lle locus Cross· Parental genotypes Offspring genotypes Expecled ratio 1.2 P Aat-2 " HY-16 x HY-06 be x bb hb(2):be(4) 1:1 0.66 0.30 - 0.50 AIIIY-] OT-02 x ES-18 aa xab aa(t2):ab(11) 1:1 0.04 0.70 - 0.90

,1 ES-18 x OT-02 ab x aa aa(22):ab(18) 1:1 0.40 0.50·0.70 Pooled aa(34):ab(29) 1:1 0.40 0.50 - 0.70 OT-20 x OT-34 ab x aa aa(7):ab(8) 1:1 0.07 n.70 - 0.90 Est·4 OT-02 x ES·18 be x ae ab(5):ae(3):be(8):ee(7) 1:1:1:1 2.56 0.30 - 0.50 ES-18 x OT-02 ae xbe ab(9):ac(9):be(13):ce(8) 1:1:1:1 1.51 0.50 - 0.70 Pooled ab( 14):ae(12):be(21 ):ee(15) 1:1:1:1 2.90 0.30 - 0.50 OT-20 x OT-34 bb x be hh(8):be(7) 1:1 om 0.70 - 0.90 OT-ll x ES-12 be x be bb(6):be(9):ec(5) 1:2: 1 0.30 0.70 - 0.90 HM-18 x HM-06 be x ab ab(5):bb(4):be(5):ae(5) 1:1:1:1 0.16 0.95 - 0.99 Es/-7 OT-02 x ES-18 ab x ab aa(6):ab( 12):bb(5) 1:2: 1 0.13 0.90 - n.95 ES-18 x OT-02 ab x ab aa(12):ab( 19):bb(8~ 1:2:1 0.85 0.50 • n.70 Pooled aa( 18):ab(31):bb(3) 1:2: 1 0.81 n.7(; - 0.90 OT-1l x ES-12 ab x aa aa( 10):ab( 10) 1:1 0.00 1.00 HM-18 x HM·06 aa x ab aa(9):ab(10) 1:1 0.24 0.50 - 0.70 HM-06 x HM-18 ab xaa aa(2):ah(3) 1:1 0.20 0.50 - 0.70 " 76 • '.> •

Table 4.2. Continued.

Pooled aa(II):ab(13) 1: 1 0.17 0.:\0 - 0.50 Pgd-J OT-02 x ES-18 aa x ab aa(II):ab(IZ) 1: 1 0.04 0.70 - 0.90 ES-18 x. OT-OZ ab x aa aa(ZZ):ab( 17) 1: 1 0.64 0.30 - 0.50 Pooled aa(33 ):ab(Z9) 1: 1 0.26 0.50 - 0.70 Pgd-2 OT-02 x ES-18 be x bb 1JIJ( 16):be(7) 1:1 :\.52 Il.05 - 0.10 ES-18 x OT-02 bb x be bb(ZI):/Jc(18) 1:1 0.23 0.50 - 0.70 Poo1ed bb(37):bc(Z5) 1:1 2.32 0.10 - 0.20 HY-16 x HY-06 be x be 1JIJ(4):/Jc(3) 1: 1 0.14 0.50 - n.70 OT-ZO x OT-34 bb x be bb(8):be(7) 1: 1 0.07 0.70 - n.90 OT-ll x ES-IZ aa x bl> ab(ZO) Skd-J OT-02 x ES-18 bd x be 1JIJ(5):be(8):ed(6):/u/(4) 1:1:1:1 1.52 0.50 - n.7n ES-18 x OT-OZ be x bd bb(8):bc(11 ):ec/( 13):/u/(7) 1:1:1:1 2.33 0.50 - 0.70 Pooled bb( 13):bc( 19):cd(19):1Jd(11) 1:1:1:1 3.Z9 0.30 - n.so OT-20 x OT-34 bb x be bb(8):bc(7) 1:1 0.07 0.30 - 0.50 HY-16 x HY-06 ab x bb ab(3):bb(4) 1:1 0.14 0.50 - 0.70

• The first parent is the female parent. Pooled indicates combined data for the two reciproca1 crosses just above.

77 Table 4.3. Joint segregation analysis of two-locus combinations ofcnzymc-coding loci • in red clover. , Locus combination Cross x- df P

Amy-I/Esc-4 E-IS x 0-02 4.32 7 0.70 - 0.90

0·20 x 0-34 0.20 3 0.90 - 0.95

Amy-I/Est-7 E-18 x 0-02 1.42 5 0.90 - 0.95

~ Amy-IjPgd-I E-18 x 0-02 3.16 .:> 0.30 - 0.50

~ Amy-I/Pgd-2 E-18 x 0-02 7.03 .:> 0.05 - 0.10

~ 0-20 x 0-34 0.20 .:> 0.90 - 0.95

Amy-I/Skd-l E-18 x 0-02 5.63 7 0.50 - 0.70

0-20 x 0-34 020 3 0.90 - 0.95

E.st-4/Est-7 0-02 x E-18 75.26 11 < 0.001

0-11 x E-12 8.00 5 0.10 - 0.20

Est-4/Pgd-l 0-02 x E-18 3.55 7 0.70 - 0.90

Est-4jPgd-2 0-02 x E-18 7.68 7 0.30 - 0.50

0-20 x 0-34 0.20 3 0.90 - 0.95

Est-4/Skd-l 0-02 x E-18 18.51 15 0.20 - 0.30

0-20 x 0-34 0.20 3 0.90 - 0.95

Est-7jPgd-l 0-02 x E-18 1.42 5 0.90 - 0.95

Est-7jPgd-2 0-02 x E-18 4.00 5 0.50 - 0.70

• 78 Table 4.3. Continuel!. • , Locus combination Cross le df P

Est-?/Skd-I 0-02 x E-H! 13.51 11 0.20 - 0.30

Pgd-I/Pgd-2 0-02.x E-HI 2.64 3 0.30 - 0.50

Pgd-I/Skd-I 0-02 xE-Il! 3.55 7 0.70 - 0.90

Pgd-2/Skd-1 0-02 x E-18 24.71 7 < 0.001

0-20 x 0-34 1.80 5 0.10 - 020

'-~-:-::::-­ '<::-, ,--,

• 79 • ) •

80 • • Table 4.4. Conlinued.

1'1 l", f •.•• Cultivar Locus Allele DL ES HM HY HE KU MR MN MT NP DT PS PP SP ST

Dia·] a 0.00 0.03 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

b 1.00 0.97 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

Dia·3 a 0.00 0.04 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

b 1.00 0.96 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

Est·], ',' a 0.93 0.94 0.98 0.95 0.95 0.98 0.95 0.78 0.88 0.93 0.80 1.00 O.SO 0.90 0.90 IJ 0.07 0.06 0.02 0.05 0.05 0.02 0.05 0.22 0.12 0.07 0.20 0.00 0.20 0.10 0.10

Est·4 a Q.OO 0.04 0.32 0.02 0.02 0.10 0.00 0.07 0.05 0.00 0.00 0.00 0.00 0.10 O.OS

b 0.55 Il.54 0,45 0.68 0.50 0.70 0.65 0.60 0.78 0.63 0.70 0.70 0,4'i 0.55 0.58

c 0,45 0.42 0.23 0.30 0,48 0.20 0.35 0.33 0.17 0.37 0.30 0.30 0.53 0.35 0.37

Est·7 a 0.38 0.65 0.88 0.55, 0,43 0,40 0.63 0.50 0.25 0.83 0.64 0.78 0.83 0,48 0.53 " b 0.27 0.22 0.12 0.30 0.32 0.35 0.12 0.25 0.50 0.07 0.16 0.07 0.02 0.27 0.32

c 0.35 0.13 0.00 0.15 0.25 0.25 0.25 0.25 0.25 0.10 0.20 0.15 0.15 0.25 0.15

81 • •

Table 4.4. Continued.

Cultivar Locus Allele DL ES HM HY HE KU MR MN MT NP OT PS PP SP ST

Gpi-2 Cl 0.00 0.05 0.00 0.00 0.00 0.00 0.02 0.00 0.00 0.02 0.00 0.00 0.00 0.00 0.00

b 1.00 0.83 1.00 0.98 0.85 0.83 0.83 0.80 0.85 0.73 0.96 0.85 0.83 0.90 O.XS

c 0.00 0.12 0.00 0.02 0.15 0.17 0.15 0.20 0.15 0.25 0.04 0.15 0.17 0.10 0.13

el 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.02

IcI"-1 Cl 1.00 1.00 1.00 1.00 0.98 1.00 1.00 \.00 1.00 \.00 \.00 1.00 1.00 1.00 1.00

b 0.00 0.00 0.00 0.00 0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Md"-3 Cl 0.93 1.00 \.00 0.95 0.95 0.98 \.00 0.95 1.00 1.00 1.00 0.95 \.00 \.00 LOO

b 0.07 0.00 0.00 0.05 0.05 0.02 0.00 0.05 0.00 0.00 0.00 0.05 0.00 0.00 IWO

Pgel-] Cl 1.00 0.99 1.00 1.00 1.00 1.00 0.93 0.98 1.00 0.98 1.00 1.00 1.00 1.00 O.9X

b 0.00 0.01 0.00 0.00 0.00 0.00 0.07 0.02 0.00 0.02 0.00 0.00 0.00 0.00 O.lJ2

82 • •

83

• •

Table 4.6. G·statistics (above diagonal) and genetic distances (below diagonal) between 15 cultivars of ft'd c1over. - Cv. DL OT MR PS pp HM HY SP ES HE KU MN MT NP ST DL 71.9 56.1 52.5 48.1 53.3 42.5 36.2 71.0 41.1 48.8 59.0 55.3 53.5 4ll.7 01' .009 48.0 53.3 51.0 70.4 23.0" 39.7 49.0 29.7a 59.5 42.6 34.4 35.5 53.2 MR .007 .003 40.6 33.7 49.2 28.9" 35.1 21.68 20.4- 33.S 33.3 27.5- 19.3- 29.03 \\ PS .013 .003 .005 33.3 60.8 28.9- 31.1' 45.7 21.1- 20.0" 26.1- 43.S 45.4 27.3 3 PP .003 .007 .004 .008 65.0 44.9 20.68 39.1 18.9- 28.9- 22.3' 36.4 31.8 ZO.7- HM .015 .012 .009 .ot5 .017 34.0 31.4 50.3 41.7 38.6 56.7 40.1 56.0 36.4 HY .007 .000 .000 .000 .007 .OOS 20.68 25.88 15.88 26.3- 27.2- 23.2- 31.5 3ll.9 3 SP .005 .000 .000 .000 .001 .005 .000 28.0" 18.3- 14.0" 20.68 21.8- 28.7 3 16.S- ES .005 .003 .000 .003 .000 .008 .000 .000 26.3 8 31.0" 36.4 32.0 28.6a 26.5a HE .004 .004 .000 .004 .000 .010 .019 .000 .000 19.0" 16.3- 23.68 21.Z- 1ll.0" '1 KU .013 .002 .001 .000 .009 .008 .000 .000 .003 .005 18.2- 17.7- 37.9 16.23 MN .010 .000 .002 .000 .001 .011 .000 .000 .000 .001 .000 24.8- 34.7 19.0- ··MT .013 .001 .001 .004 .013 .009 .000 .002 .005 .007 .000 .002 26.1- 22.0- ,NP .006 .002 .000 .004 .003 .Olt .001 .000 .000 .000 .002 .000 .001 37.6 ST .004 .001 .000 .000 .000 .007 .000 .000 .000 .000 .000 .000 .003 .000

8 shows non-significant difference at 0.05 level.

85

" • •

Table 4.7. Mean genetic distances (below diagonal) and U-statistics (above diagonal) between groups of cultivars

from different origins.

" Group NA Europe Japan NA+E NA+J E+J

North America (NA) ++ 0.0003 0.0000 - . 0.0009'

Europe 0.0033 ++ 0.0016' - -0.0006

Japan 0.0059 0.0033 ++ 0.0012

North America and Europe (NA+ E) - . 0.0044 ++

North America and Japan (NA+J) - 0.0033 - - ++

Europe and Japan(E+J) 1 0.0041 - - - - ++

• shows significant difference at 0.05 level.

86 ((

• " •

ANODE AAT ADH EST GPI MDH 6PGD PGM SKD 1 ---- 1 " -- 2 2 2 - --• - 1 -- -.- 4 --• - •• --• - -- 2 3 ----• • ••- • ------1 --- -- . ---• -• ------.-- - 1 ---• ------7 -- lOCUS 2 .a ab bb bo 10 CC 1 la ab bb t Il ab bb 2 .. ab bb be cc le bd 1 aa aa 1 ., ab 2 ab bb be 1811 ab bb be cc cd bd ad dd 3.bbbbo 4 ...bbb be cc le 2 Il aa 2 aa ab bb be cc le 7 .. ab bb 3 III ab

Figure 4.1. Sehematic diagrams of isozyme banding patterns and assignment of loci and aile les in the

polymorphie isozymes in red elover.

1\ 87

.'. •

lAA lAB =ST-4< 2AA 2AB ~1BB '2BB

ORIGIN

,. bb bc bc cc cc bb aa nn aa ab

Figure 4.2. Banding patterns of EST, MDH and 6-PGD isozymes in red clover.

Genotypes are shown at the bottom.

• 88 CHAPTER FIVE • POLYMORPHISMS OF RAPD MARKERS FROM BULKED GENOMIC DNA AMONG CULTIVARS OF RED CLOVER

Prasert Kongkiatngam, Marcia J. Waterway, Bruce E. CouIman & Marc G. Fortin

Submitted to Euphytica (June 20. 1995)

Prasert Kongkiatngam planned the e.xperiments, performed aIl laboratory work and data analysis described in this manuscript and is the first author of the manuscript. Man: G. Fortin assisted in planning the experiments, provided laboratory facilities and researd. materiais and supervised the laboratory work, gave vaiuable advice and suggestions throughout the project, and contributed significantly towards editing and revising the manuscript. Man:ia J. Watenvay assisted in planning the experiments, gave vaiuable advice and suggestions throughout the projeet, and contributed significantly towards editing and revising the manuscript. Bruce E. Coulman provided~greenhouse facilities and plant materiais, gave vaiuable advice and suggestions throughout the projeet, and helped in editing and revising the manuscript.

Acknowledgements: We thank the Canada-Egypt-McGilI AgriculturaI Response

, Program for scholarship support to PK. This study was supported in part by a grant

from the Nationai Science and Engineering Researeh Council of Canada to MGF.

.• We also thank Helen Rimmer for photographie assistance.

89 • Key words: Cultivar identific-.ltion. genetie relationships. RAPD markcrs. red dovcr.

Trifolium pralense L

5.1. Abstraet

The use of random amplified polymorphie DNA (RAPD) markers obtained

from bulked samples was investigated for cultivar identification in red clover. Pooled

samples were examined in order to minimize variation within cultivars. To

determine the appropriate number of individuals to include in the bulked samples

represenüng each cultivar, DNA samples from two, three, four. five, ten and twenty

individuals were pooled. Twenty was found to be an appropriate number of red

clover individuals per bulk for homogenizing genetic variation within .::ultivars.

Founeen 1Q-mer primers were used to amplify genomic DNA from combined leaf

samplesof 15 red clover cultivars from European, Japanese and Nonh Arnerican

origins. A total of 79 amplified produets, of which 55 were polymorphie, was

obtained. Culüvar-specific bands were observed with 13 primèrs. The amplificaüon

patterns obtained from two primers couId distinguish aU 15 red clover cultivars.

Rogers' genetic distances for all 105 pairwise comparisons were. calculated to

evaluate relationships among these cultivars. Cluster analysis based on these geneüc

distances could separate these 15 cultivars into four groups.

• 90 • 5.2. Introduction

Identification of crop cultivars is fundamental to commercial seed production

and certification programs. The phenotypes used for cultivar identification must

provide readily distinguishable differences among cultivars with little within-cultivar

variation, in addition to being stable in various environrnents (Bailey 1983).

Traditionally, cultivars have been discriminated on the basis of morphological and

agronomie chardcteristics. Although such characters are genetically weil

characterized, they are limited in number and May be subject to environrnental and

ontogenetic influences. Isozymes have also been used extensively as genetic markers

for cultivar identification in crop species (reviewed by Weeden 1989). However, the

use of isozymes to distinguish cultivars of crossed-pollinated, heterogeneous crops is

not as straightforward as in homogeneous cultivars ofselfing or clonally propagated

crops or hybrid cultivars since allele frequencies of isozymes from a large number of

individuals are required (Adam et al. 1987; Ostergaard et al. 1985). Molecular

markers, such as restriction fragment length polymorphisms (RFLPs), random

amplified polymorphie DNAs (RAPDs), minisatellite or microsatellite DNA, have

.been used in genetic studies in many plant species (reviewed by Beckmann & Soller

19S5; Beekmann & Soller 1989; Bowditch et aL 1993; Tautz 1989 and Williams et aL

1993). RAPD markers are DNA· fragments amplified by the polymerase chain

reaetion (PCR) using short arbitrary primers (Williams et aL 1990). These genetic

markers segregate according to Mendelian inheritance and give consistent results

• 91 • irrespective of environmental conditions or tissue sampled since genomic DNA is

used to obtain a marker phenotype (Echt et al. 1992: Heun & Helentjaris 1993:

Kazan et al. 1993c; Yu & Pauls 1993a). Recently. RAPD markers have becn used

extensively to differentiate cultivars of several crops (He et al. 1992: Hu & Ouiros

1991; Koller et al. 1993; Wilde et al. 1992: Wu & Lin 1994).

Red clover, Trifolium pratense L, is a cross-pollinated diploid species (211 =14)

with a gametophytic self-incompatibility system (Taylor & Smith 1979). Cultivars of

red clover in use today are composed of heterogeneous individuals obtained through

mass selection. The expected high levels of variation within cultivars have been

observed (Kongkiatngam et al. in press, chapter 3; Milligan 1991; Nelke et al. 1993).

Currently, red clover cultivars are discriminated by using genetically eonfirmed

morphological and agronomie charaeteristics. The number ofsueh markers is Iimited

(Quesenberry et al. 1991), while the number of red clover cultivars generated by

breeding programs increases. Additional markers are thus required, whieh should

ideally be able to deteet more genetie variation, while being applicable for large-scale

genotyping. Isozymes have been evaluated for that purpose (Kongkiatngam et

al. in press, chapter 4) but they were found to have more variation within cultivars

than between them. Allele frequencies at eleven polymorphie isozyme loci could

differentiate all North American and Japanese cultivars tested. However, several

European cultivars eould not be distinguished using these isozymes. Nelke and

coworkers (1993) were able to distinguish individual genotypes in seven cultivars of

red clover using DNA fingerprints generated by probing with human minisatellite

• 92 • DNA probes (Jeffreys et al. 1985a, 1985b), which detected variable numbers of

tandem repeats (VNTRs) in minisatellites. However, high variation within cultivars

in red clover was also Closerved with these probes, making them less useful for

cultivar identification. RAPD markers were also found to be variable within and

between two cultivars of red clover (Kongkiatngam et al. in press, chapter 3).

Similarly, high variation within cultivars has a1so been observed in both cross­

pollinated a1falfa (Yu & Pauls 1993b) and perennial ryegrass (Sweeney &

Danneberger 1994) using RAPD markers. Bulked genomic DNA from severa!

individuals was used to generate RAPD markers in these two crops in order to

homogenize the geneticvariation between individuals and rctain only cultivar-specifie

lod. The pooled samples produced amplification patterns that represented the

homogenized charaeteristics ofheterogeneous populations (Sweeney & Danneberger

1994; Yu & Pauls 1993b). ln this paper, we evaluate the use of RAPD markers

amplified from bulked genomic DNA samples for cultivar identification in red clover

and test the effeet of pooling different numbers of individuals in the bulk samples.

We also demonstrate the reproducibility of the method, as this is an important

criterion for the application of a biochemica1 or molecular technique (Bailey 1983).

:

93 • 5.3. Materials and Methods

5.3.1. Bulked genomic DNA extraction and purification

Fifteen cultivars of red clover from three different geographic origins werc

assayed (Table S.l). DNA was extracted using a hexadecyltrimcthylammonium

bromide (CfAB) method (Doyle & Doyle 1987). For each cultivar, bulked samples

were obtained by collectir.g approximately 250 mg of fresh leaves from each of 20

individuals grown in the greenhouse. Two different sets of bulked samples were

collected for assay. Five grams of bulked frozen leaves were ground in lS ml of

buffer [1.4 M NaCl, 02% (v/v) 2-mercaptoethanol, 20 mM EDTA. 100 mM Tris-Cl

pH 8.0) in a Waring blender. Five ml of CfAB buffer [the above buffer plus 2%

(w/v) CfAB (Sigma») was added to the ground tissue, and the mixture was incubated

at 600.C for 30 min with occasional swirling. The preparation was extraeted with one

volume of chloroform-isoamyl alcobol (24:1) tben centrifuged at 3500 gs for 5 min.

DNA was precipitated by the addition of2/3 volume of cold isopropanol, stored at­

200 C for 1 hr and centrifuged. The DNA pellet was washed with wasb buffer (10

mM ammonium acetate in 76% ethanol) and dried under vacuum. The DNA pellet

was resuspended in 200 l'lof deionized H20 and purified with phenol-chloroform­

isoamyl alcohol (25:24:1) (Ausubel et al. 1989).

DNAsamples for testing the optimal number of individuals to be pooled into

each bulk sample were obtained by combining equal amounts of genomie DNA

• 94 • purified from each individual from two, three, four, five, ten or 20 individuals using

the same prolocol (Kongkiatngam et al. in preSS, chapter 3).

5.3.2 DNA amplification

PolymerdSe chain reactions were performed in 25 !LI containing 80 mMTris-CI

pH 9.0, 20 mM (NH4)2S04' 100 mM each of dATP, dCTP, dGTP and dTfP

(Pharmacia), :;5 mM MgCI2' 400 nM of a single primer, 2 U ofTaq DNA polymerase

(BRL) and approximately 25 ng of red clover genomic DNA The reaction was

performed in a thermal cycler (Hybaid) with the following temperature conditions:

94° C for 5 min, followed by 35 cycles of 92° C for 1 min, 36° C for 1 min, 72° C for

2 min, and ending with 72" C for 6 min. PCR produets were visualized with ethidium

bromide after elecl1'ophoresis in a 1.4% agarose gel. Thirty-three IG-mer primers

(Operon Technologies) were screened for RAPD markers. Fourteenprimers (Table

52) which gave clear and consistent amplification produets were used to amplify a

bulked sample of each cultivar. In order to test reproducibility of the bulk method .

for cultivar identification, another set ofgenomic ONA samples from al115 cultivars ':=C was prepared usirig bulked leaf material from new groups of 20 individuals of each

cultivar for amplification with five primers. The amplified bands were scored as

present (1) or absent (0). Faintly stained bands and bands that were not well

resolved were not considered.

• 95 • 5.3.3. Data analysis

The phenotypes were considered as binary characters and a data matri.x of lOs

and O's was constructed from the 79 bands scored (Appendi.x 2). A pairwise Rogers'

(1972) genetic distance matrix (Appendi.x 3) was constructed based on the

commonality of the amplified bands. i.e., the number of nonshared amplified

fragments between two cultivars divided by the total number of amplified fragments

in the data set. Cluster analysis based on these genetic distances to show genetic

relationships among cultivars was performed using the AVERAGE method in the

CLUSTER procedu.e of Statistical Analysis Software (SAS lnstitute, lnc. 1989).

Each cultivar was considered as a separate cluster by the AVERAGE method of

SAS, then clusters were joined sequentially based on increasing average distance

between clusters.

5.4. Results

5.4.1. Determination oftlze optimal number ofindividuals in bulked sumples

The RAPD amplification patterns obtained from combining samples

containing genomic DNAfrom two, three, four, five, ten or 20 red clover individuals

of cv. Essi are shown in Figure 5.1. With two, three, four, five or ten individuals in

the bulks, variation within cultivars from different sets of individuals could still be

observed (Figure 5.1). When the number of individuals in the bulks was increased

to 20, the same RAPD patterns were ob'~ined from tWo different sets of samples.

• 96 • Sorne amplification bands disappeared when the number of inùividuals in the bulks

wa~ increased from five to ten indiviùuals (Figure 5.1). The same results were

obtained when RAPD patterns were generated using bulked samples from cultivar

Ottawa (data not shown). These results indicated that bulks of 20 individuals

h{'lmogenized intra-eultivar variation in red clover.

5.4.2 RAPD polymorphism between red clover cultivars

Thirty-three lo-mer primers were sereened for DNA amplification of bulked

genomie DNA in red clover. Fourteen primers gave clear and consistent

amplification produets and were used to amplify bulked DNA samples from all 15

cultivars. A total of 79 amplification bands was obtained using these 14 arbitrary

primers. From one to eleven amplification bands were obtained using eaeh primer

with an average of 5.6 bands per primer from these bulked DNA samples (e.g~

Figure 5.2). Ali primers exeept one (H-09) produeed polymorphie bands among

these 15 cultivars of red clover. The size of the amplified produets ranged from 320

bp to 1700 bp. Fifty-five polymorphie markers (70% of the total bands) were found

among these cultivars, an average of 3.9 polymorphie bands per primer.

Cultivar-specifie bandsuseful for cultivar identificationwere observed for mast

primers (Figure 5.2). At least two primers were required to produee RAPD patterns

that will differêntiate all 15 red clover cultivars (Table 5.1). However, only one

primer ~ necessary to generate RAPD patterns that eould discriminate all cultivars

from North American and Japanese origins (Table 5.1). RAPD patterns generated

• 97 • from bulked samples from a second set of bulks of individuals of each cultivar were

found to be consistent with the first set of sampies. The only exception was cu ltivar

Hayakita, which gave different amplification patterns with one of the live primers

tested.

The 79 amplification bands from bulked genomic DNA of the 15 red c10ver

cultivars were used to calculate Rogers' (1972) genetic distances for an 105 pairwise

comparisons (Appendix 3). Values of genetic distances ranged from 0.101 to 0304

with an average of 0200 (Appendix 3), and were used for c1uster analysis which

separated these 15 red c10ver cultivars into four groups (Figure 53). The Japanese

cultivars Hamidori and Hayakita were clearly distinguished from other cultiV'.lrs.

These !wo cultivars showed distance estimates of 0.190 to 0304 from other cultivars

and formed their own group. The c\uster analysis of genetic distances l'rom RAPD

data did not separate these 15 cultivars into groups according to their geographic

origins (Figure 53). However, most of the European cultivars (Hermes, Kuhn,

Marino and Stan) were placed in the same group.

5.5, Discussion

5.5.1. Determination oftlle appropriate number ofindividuals in bulked samples

The ability of molecular markers to deteet more genetic variation, compared

to isozyme or morphological markers (Kongkiatngam et al. in press, chapter 3), has

the drawback that more iDtra-cu1tivar variation is now deteeted as well. In the

• 98 • context of cultivar identification in cross-pollinated crops, this sensitivity to DNA

~equence variation needs to be reduced by diluting rare alleles. The allelic variation

detected in pools of samples will then be the result of several individuals sharing the

same alleles (Michelmore et al. 1991). Bulked samples consisting of 20 individuals

ofeach red clover cultivar were able to generate RAPD patterns that could remove

intra-cultivar variation (Figure 5.1). Variation within cultivars was eliminated with

the disappearance of sorne polymorphie bands when the number of individuals was

increased from two to 20 (Figure 5.1). ln comparison, the appropriate number of

individuals pooled to produce RAPD markers that could represent the RAPD

patterns observed in larger samples of the same populations in a1falfa (Medicago

Saliva L) was seven (Yu & Pauls 1993b). However, variation within populations

between different bulked samples was not examined in that study. In perennial

ryegrass (Lotium perenne L), leaves from 30 individuals were used for bulked

genomic DNA extraction to generate RAPD markers (Sweeney & Danneberger

1994) which could be used for cultivar identification. Consistent RAPD patterns

were obtained from three different seed lots of the same cultivars using bulked

genomie DNA samples but eomparisons between different numbers of individuals

were not ineluded in that study.

5.5.2 RAPD polymorplzism between red clover cultivars

The average number ofpolymorphie bands per primer of3.9 obtained in red

claver is relatively high compared to that observed in other species (Ko et al. 1994;

• 99 • Tinker et al. 1993; Yu & Pauls 1993b). but is lower than those observed in assays of

individual plants in red clover (Kongkiatngam et al. in press. ch:lpter 3) and

buffalograss [Buclzloe dacryloides (Nutt.) Engelm.] (Huff et al. 1993). The number

of polymorphic RAPD b::nds relative to the total number of amplification products

observed in red clover (700/c) was also higher than in other species: for example

25.6% in Srylosanthesguianensis spp.guianensis (Kazan et al. 1993b). and 67% in rice

(Ko et al. 1994).

A1though allele frequencies of isozymes have been used to differentiate

cultivars of sorne heterogeneous, cross-pollinated crops, for example, ryegrass

(Ostergaard et al. 1985) and rye (Adam et al. 1987), they could not discriminate the

15 red clover cultivars used here (Kongkiatngam et al., in press, chapter 4). Only

five cultivars from North America and three cultivars from lapan could be

distinguished using isozymes. Use of isozymes to discriminate cultivars of cross-

pollinated, heterogeneous crops requires a large number of individuals (> 100) to

obtain accurate results (Adam et al 1987; Ostergaard et al. 1985). The cost of using

RAPD markers from bulked genomic DNA samples for cultivar identification of

heterogeneous crop species would be comparable to or even lower than that of

isozymes in that context. Molecular genotyping methods that use non-PCR-based

markers will require larger DNA amounts for analysis. This May become a limiting

/ factor when a large number ofsamples need to be analysed. Markers'cbat detect too

much genetic variation cao identi..~ sequence variation within the same individual

(Bell & Ecker 1994), confounding the analysis. RAPD assays with bulked DNA

• 100 • samples could he a quick and powerful 1001 for distinguishing red clover cultivars in

commercial seed production and seed certification programs. Red clover seedlings

could be used for bulked DNA extraction instead of leaf tissue because only a small

amount of DNA is required for RAPD assays. RAPD markers obtained from bulked

genomic DNA may aiso be useful for other genetic and breeding studies in red

clover, for example to mark monogenic traits (Michelmore et al. 1991) and

quantitative trait loci (QTI..s) (Wang & Paterson 1994), and might be useful to assess

relationships among species within the genus Trifolium.

5.5.3. Genetic relationslzips ofred clover cultivars fram RAPD data

Knowing genetic distances between cultivars is useful in a breeding program

because it al10ws efficient "sampling and utilization of germplasm resources. A

breeder may choose cultivars that are distantly related to obtain transgressive

segregation for a quantitative trait (e.g. yield). The genetic distances obtained with

RAPD markers among the clover cultivars studied here are higher than the Rogers'

distances computed from the data reponed in Kongkiatngam et al. (in press. chapter

4) on al1ele frequencies at 23 isozyme loci. The Rogers' genetic distances ranged

from 0.032 to 0.107, compared to the distances of 0.101 to 0.304 in this study

(Appendix 3). The cluster analysis based on 79 amplified bands separated these 15

red clover cultivars into four groups but it did not group them according to their

three different geographical origins (Figure 5.3). Pedigree data for these cultivars

is incomplete, since some ofthem are from private breeding programs, and could not

• 101 be used for comparison with the cluster analysis. Interestingly. cultivars Dollard. • Marathon. and Persist l'rom North America are placed in the same group (Figl.lre 5.3). Cultivar Dollard was developed for adaptation to long and cold winters of

eastern Canada l'rom two red claver cultivars l'rom Northern Europe (Fergus &

Hollowell 1960). Cultivar Dollard was used in the improvement of most rel! clover

cultivars adapted ta north-eastern North Americo..l. One of the Japanesc cultivars.

Sapporo, was placed with the North AmeriCO..ln cultivars (Figure 5.3) while two

Japanese cultivars, Hamidori and Hayakita, were separated l'rom the other cultivars

and placed together in their own group (Figure 5.3). Principal component an:llysis

of isazymes could not distinguish these 15 cultivars into three separate groups

according to their origins either (Kongkiatngam et al. in press. chapter 4). This may

be because thousands ofindividuals l'rom different cultivars are always a.'lSembled for

selection and crossing in red claver breeding progr..lrns (Taylor & Smith 1979),

resulting in a diverse genetic background in the improved cultivars obtained from

the-.se prograrns.

In conclusion, RAPD patterns of bulked genomic DNA samples l'rom 20

individuals were found to be good representatives of heterogeneous red c10ver

cultivars. A high level of polymorphism among the 15 cultivars was observed.

Cultivar-specifie amplification bands were found with the 14 arbitrary lQ-mer primers

scored. At least (wo primers were required to differentiate ail 15 red c10ver cultivars.

The cIuster analysis based on genetic distances computed from these RAPD data

• 102 separated these 15 cultivars inlO four groups. RAPD analysis is therefore a powerfu1 • tool for cultivar identification and for genotyping purposes.

,-

• 103

•

M 1 1 2 2334 4 5 5 W W ~~ M

Figure 5.1. RAPD patterns obtained from bulked genomic DNA samples with

different numbers,of individuals in the bulks. Numbers designate the number of

individuaIs in each bulk. Lanes with the same number are samples of different

groups of individuaIs from the same cultivar (Essi). Lane Misa 1 Kb l ladder

marker (BRL).

106 •

fi) ~o'" w:E > jZ u: ~ c.- c. fl)c.~ :Ew Q:::~:E:E:EZc.c.fI)fI):E

.. .

Figure 5.2. RAPD patterns generated with primer H-02 from bulked samples of 15

red clover cultivars. Lane Misa 1 Kb À ladder marker (BRL).

..

107 • DOLl-ARD ~ MISTRAL SAPPORO PERS/ST MARATHON

HERMES ~ r- KUHN I MARINO 1 l- PROSPER START l-- ESSI NAPOCA - 07TAWA HAMIDORI HAYAKITA

o 0.1 0.2 0.3 .' Rogers' genetic distance

Figure 5.3. Qusters based on Rogers' genetic distances for 15 red clover cultivars.

O1ltivars typed in bold are from Japan, those in italics are frOID North America and

the rest are frOID Europe.

• lOS CHAPTER SIX

• EFFECf OF THREE CYCLES OF NATURAL SELECfION

ON MORPHOLOGICAL TRAITS AND ISO:lYMES IN RED CLOVER

Prasert Kongkiatngam, Bruce E. Coulman and Marcia J. Waterway

Submitted to the Canadian Journal of Plant Science (July 10, 1995)

P. Kongkiatngam planned all experimems, perfonned all field and laboratory work and data analysis described in this manuscript and is the first author of the manuscript. B.E. Coulman assisted in planning the field experiments, provided greenhouse and field facilities and plant materia1s, gave valuable advice and suggestions throughout the projeet, and helped in editing and revising the manuscript. M,J. Waterway assisted in planning the isozyme experiments, provided laboratory facilities and research materials and supervised laboratory work for all experiments, gave valuable advice and suggestions throughout the projeet, and contributed significantly towards editing and revising the manuscript.

Acknowledgments: We would Iike to thank the Canada-Egypt-McGill Agricu1tural

Response Program for scholarship support to PK. This study was supported in part

by a grant from the National Science and Engineering Research Council of Canada

to MJW. We also wish to thank Wendy Asbil for assistance with the field wode.

• 109 • 6.1. Abstract

Six morphological traits and 23 enzyme-coding loci were used to compare the

genetic changes in IWO groups of red clover populations, one very diverse population

and the other with a much narrower genetic background, that have undergone three

cycles of natural selection. The diverse population, Mee 124. was formel! by mixing

equal quantities of seed of 20 cultivars originating from North America and Europe

while the narrowly-based population, Mee 125, was a polycross ofapproximately 15

Fusan"um resistant clones largely from the cultivar Ottawa, but including plants from

an ItaHan and a Spanish introduction. The percentages ofplants with spreading hairs

on stems and petioles, and with hairy internodes increased dramatically after the first

cycle of natural selection in the Mee 124 populations. The percentage ofplants with

leaf marks did not change in either group of populations. The percentage of plants

with flowering stems in the first year of growth increased after the first cycle of

selection in the Mee 124 populations while the percentage ofplants flowering in the

first year decreased slowly in the Mee 125 populations. These changes in

morphological characters related to environmental adaptation indicated that natural

selection had occurred in these red clover populations. Ten, !WO, and eleven

enzyme-coding loci were monomorphie, nearly monomorphic and polymorphie,

respectively, in the Mee 124 populations while !Welve, one, and ten loci were

monomorphie, nearly monomorphic and polymorphie, respeetively in the Mee 125

populations. The alIele frequencies of most polymorphie loci did not change after

• 110 • three cycles of selection. In the MCC 124 populations. the estimates of average

expected heterozygosity decreased slightly but the percentage ofpolymorphie loci and

the mean number of a11eles per locus were almost the same after three cycles of

selection. In the MCC 125 populations, ail of these estimates decreased slightly.

6.2. Introduction

Red clover (Trifoüum pratense L) is a significant forage legume in the

temperate regions of the world. It is believed to have originated in southeastem

Europe and Asia Minor (Taylor and Smith 1979) and is a leading crop in forage

production in central, northem and western Europe, and northeastem North America

(Smith et al. 1985). Red c10ver is also grown in Japan, South Africa, Mexico,

Columbia, Argentina, Chile, Australia and New Zealand (Smith et al. 1985). It is a

diploid (211 =14), cross-pollinated species with a gametophytie self-incompatibility

system (Taylor and Smith 1979). It is a short-Iived perennial, persisting for 2-3 yr,

depending on the cultivar and on environmental conditions (Smith et al. 1985) and

it can adapt to different soil types, soil pH levels, and c\imatie conditions (Smith et

al. 1985). This genetie diversity ofred clover has contnbuted to its wide distnbution

(Fergus and Hollowell 1960). Red clover exhibits genetie shifts in response to

different environments in relatively few generations under natura! and/or artificial

selection (Akerberg 1974; Beard and Hollowell 1952). New ecotypes can develop

through natura! selection in response to selective factors in new environments

• 111 • (Fergus and HolloweIl1960). For example. after the Tennessee anthracnose-resistant

cultivar was grown continuously for six generations at one location in Oregon. the genotypes. determined from persistence and forage yield. had shifted from those of

the original cultivar. becoming similar to the naturalized cultivars of the new

environment (Beard and Hollowell 1952). An understanding of the effect of natural

selection is necessary for defining breeding objectives (Fergus and Hollowell 1960).

The genetic shift poses a serious problem for commercial seed production in red

clover (Bula et al. 1965; Taylor et al. 1966). In northeastern North America. seed

of red clover cultivars is produced outside their areas of origin, usually in western

USA and western Canada, because environmental conditions are more favourable

for seed production and seed yields are more reliable (Taylor and Smith 1979).

Natural selection is believed to act on phenotypes, not on genotypes, and it

functions on the overail phenotypes as determined by many loci and severa!

environrnental factors (Hartl 1980). However, to examine the consequences of

natura! selection, it is easier to look at changes in allele frequency at a single locus.

Sorne morphological traits, for example, growth type, and pubescence on stems,

petioles, basal intemodes and stipules, in red clover were found to be associated with

adaptation to environmental conditions (Choo 1984; Fergus and Hollowell 1969;

Kusmiyati 1995; Smith 1957; Taylor and Smith 1979). Ali ofthese characters, except

growth type, are controlled by single genes (Quesenberry et al. 1991). These

morphological traits cao be used as genetic markers to study the effect of ndtura!

se:ection in red clover. Isozymes have also been shown to be useful markeTS for .

• 112 • documenting genetic changes over time and for describing patterns of microhabitat

adaptation in several plant species (Clegg et al. 1972; Ennos 1989). Two hypotheses

have been proposed for isozymic changes after natural selection. The first hypothesis

suggesL~ that natural selection is the major factor in the differentiation of al10zymes

in plant populations (Hamrick et al. 1979; Nevo 1978; Schall 1974) but the neutral

hypothesis argues that the molecular evolution of proteins and DNA is mostly

neutral, being maintained in populations by mutations and random fixation (Kimura

1983). Significant changes in al1ele frequencies of isozymes were observed over

several generations in two barley (Hordeum vulgare L) populations which were

constructed by crossing al1 combinations of several cultivars (Clegg et al. 1972). A

rapid increase in gametic phase disequilibrium was also found. The same multilocus

combinations in these two independently derived composite crosses of barley were

favoured by natura! selection. Selection was believed to act either direct1y on the

allozyme character or on the chromosomes that these isozyme loci marked (Clegg et

al. 1978). Ina cross-pollinated species, Liatris cylindracea Micbx. (Asteraceae), Schall

(1974) found marked local genetic differentiation, and allozyme frequencies varied

as much as 20% between adjacent quadrats. Schall and L.evin (1978) aIso obtained

a positive relationship betweenindividual heterozygosityandfecundity, longevity, and

speed of development in this species.

Isozymes have been found to be very variable both within and between

cultivars of red clover (Kongkiamgam et al in press, chapters 3 and 4). The

objective of this study was to examine changes in morphological traits and isozyme

• 113 • loci in red clover populations al'ter three cycles of natural selection. Two sets of

populations, MCC 124 and MCC PS, that have undergone three cycles of n;ltural

selection are used in this study. MCC 124 was formed by mLxing equal quantities of

seed of 20 cultivars originating from North America and Europe. Thus it is a broad­

based population consisting of adapted and unadapted germplasm. MCC 125 is a

polycross of approximately 15 Fusarium resistant clones largely from the cultivar

Ottawa, but including plants from an Italian and a Spanish introduction. Thus it has

a much narrower genetic base.

6.3. Materials and Methods

6.3.1. Morphological Traits

In the first group ofpopulations. MCC 124, seed ofthe MCC 124-0 population

was planted in 1984 and seed of the first cycle, MCC 124-1, was harvested in 1985.

Seed of MCC 124-1 and MCC 124-2 was planted in 1986 and 1987, and seed

harvested from these IWO populations in 1987 and 1988 was MCC 124-2 and MCC

124-3, respeetively. Seed of populations MCC U4-2P and MCC 124-3P was

harvested two years after seeding from the fields of MCC 124-1 and MCC 124-2,.

respeetively. In the second group of populations, the original population, MCC 125­

0, was planted in 1984 and seed of MCC 125-1 was harvested in 1985. Seed of MCe

125-1 and MeC 125-2 was planted in 1986 and 1987, and seed harvested from these

two populations in 1987 and 1988 was MeC 125-2 and Mec 125-3, respeetively•

• 114 • Seed of Mee 125-3P was harvested two years after seeding from the field of the

Mee 125-2 population. Seed of the Mee 125-0 population was not available for

evaluation. Seed of these ten populations of red clover. Mee 124-0. Mee 124-1.

Mee 124-2. Mee 124-2P, Mee 124-3. Mee 124-3P, Mee 125-1, Mee 125-2, Mee

125-3 and Mee 125-3P, was germinated and seedlings were grown in the greenhouse

in the winter of 1992. Forty seedlings from each of these populations were

transplanted to the field in the spring of the same year. These seedlings were space­

planted approximately 35 cm apart. AIl field experiments were condueted at the

EA Lods Agronomy Reseach Center of McGill University, Ste-Anne-de-Bellevue,

Quebec (latitude 450 26' N). Morphological traits (Table 6.1) were scored in the

summer of the same year. Red clover plants in the field were classified into one of

five growth types based on rosette-forming and flowering patterns (Bird 1948) in

August 1992. Plants with growth types 1 and 2 (non-flowering) do not flower in the

seedling year of growth but those with growth types 3, 4 and 5 (flowering) will

produce flowering stems (Bird 1948). In addition, 100 plants from each of these ten

populations grown in the greenhouse were scored for leaf mark and types of hairs on

petioles.

6.3.2 Isozyme Assay

Seed from the ten red clover populations were germinated and seedlings were

. grown in the greenhouse. Six-week old seedlings were used for isozyme assays.

Forty plants each of all populations were assayed fur ten enzyme systems (AAT,

• 115 • ADH. DIA, GPI. IDH. MDH. ME. 6PGD. PGM and SKO) and 100 plants each of

all populations were assayed for two enzyme systems (AMY and EST). Young leaves

were ground in extraction buffer [0.001 M EDTA, 0.010 M KCl. omo M MgCl~. 7%

(w/v) PVP. 0.10 M Tris-HCl buffer. pH 7.5. 3% sucrose (w/v)] to which 0.2% (v/v)

2-mercaptoethanol was added before use (modified l'rom Gottlieb 1981b). Extracts

were absorbed omo Whatman #3 filter paper wicks and stored in microcentrifuge

tubes at -80" C until electrophoresis. These samples were assayed for twelve enzyme

systems which gave clear and weil resolved bands on 10% starch gels run with three

gel-buffer systems as described by Wendel and Weeden (1989). NADH diaphor..lSe

(DIA. E.C 1.6.99.-); glucosephosphate isomerase (GPI, E.C. 5.3.1.9); isocitrate

dehydrogenase (IDH, E.C 1.1.1.42) and phosphoglucomutase (PGM, E.C. 5.4.2.2)

were assayed with histidine-citrate buffer, pH 65. Alcohol dehydrogenase (ADH,

E.C. 1.1.1.1); malate dehydrogenase (MDH, E.C. 1.1.1.37); 6-phosphogluconate

dehydrogenase (PGD, E.C 1.1.1.44) and shikimate dehydrogenase (SKD, E.c.

1.1.1.25) were resolved by using morpholine-citrate buffer, pH 6.1. Aspanate

aI!1Înotransferase (MT, E.C 2.6.1.1); amylase (AMY, E.C 3.2.1.1 and E.c. 3.2.1. 2);

esterase (EST, E.C3.1.1.-) and malic enzyme (ME, E.C 1.1.1.40) were resolved with

lithium-borate/Tris-citrate buffer, pH 8.3. Ali gels were run at a constant current of

35 mA, the histidine-citrate system for 4 hours, the morpholine-citrate system for 4

hours and the lithium-borate system for 5 hours. The assays for ail enzyme systems

were the same as those described by Wendel and Weeden (1989), except esterase for

which N-propanol was used instead of acetone to dissolve the substrate. lAci and

• 116 • aileles werc se4ucmially numbered and lettered. respectively. beginning with the

most anodal form.

6.3.3. Data Ana/ysis

Significant deviations ofobserved allozyme frequencies from Hardy-Weinberg

equilibrium were tested by the chi-square test adjusted for small sample size using

Levene's (1949) correction factor as implememed in BIOSYS-I (Swofford and

Selander 1981). ln order to test the differences of allele frequencies between

populations. contingency table 1.2 tests (Weir 1990) were also obtained from

BIOSYS-I. Estimates of observed heterozygosity from direct coum (HooJ, expected

heterozygosity (H"'I')' mean number of alleles per locus (A) and percentage of

polymorphic!oci (P) were also obtained from BIOSY5-I.

6.4. Results and Discussion

6.4.1. Effect on Morp/zo/ogical Traits

The percentages ofplants from the Mee 124 populations with spreading hairs

01" stems, petioles, and intemodes increased dramatically after the fust cycle of

natura! selection from 62% to 900/0, 69% to 880/0, and 59% to 770/0, respectively

(Table 6.1), after which the levels were almost the same for the next cycles.

However, the percentage of plants with hairy stipules did not increase after three ~ cycles of natura! selection (Table 6.1). Data for these morphological characters in

• 117 the MeC 125-0 population were not available. The percentage of plants \Vith • spreading hairs on stems and petioles and with hairy stipules in the three Mee 125 populations after natural selection were almost the same. but the percentage of

plants with hairy internodes decreased grJ.dually (Table 6.1). The percentage of

plants with leaf marks did not change in either group of populations (Table 6.1).

The percentage of plants with growth types 3. 4 and 5. which flowered in the first

year of growth. increased after the first cycle of natural selection from 65% to 85%.

and then became almost the same for the next cycles in the Mee 124 populations

(Table 6.1). However. the percentage of flowering plants decreased slowly in the

Mee 125 populations (Table 6.1).

Spreading hairs on stems and petioles, and pubescence ofbasal internodes and

stipules has been associated with insect resislance in red clover. particularly to potato

leafhoppers (Empoascafabae Harris) (Fergus and Hollowell 1969; Kusmiyati 1995).

These insects are a serious problem in areas where red clover is grown in North

America (Fergus and Hollowell 1960). Cultivars that are adapted to North America

are more pubescent than those of European origins (Fergus and Hollowell 1960;

Kongkiamgam et al. in press, chapter 3). These pubescence characters have been

found to be under simple genetic control in red clover (Quesenberry et al. 1991). A

higher percentage ofplants with appressed hairs on stems and petioles was observed

in the Mee 125 populations suggesting that genes controlling appressed haies on

stems and petioles may be linked to a gene conferring Fusarium resistan.ce in red

clover. Leaf mark, which is under monogenic con,troland not related to adaptation

• 118 • (Ciuesenberry et al. 1991; Taylor and Smith 1979), did not change over generations

in either the Mee 124 or the Mee 125 populations. Growth types, which are

believed to be under polygenic control, were also affected by natural selection (Table

6.1). Growth types of red clover have been found to be associated with winter

survival and persistence (Choo 1984; Smith 1957). Plants which do not f10wer in the

seedling year (plants with growth types 1 and 2) are more persistent and winter-

hardy. Flowering of red clover plants in the seedling stand is also dependent upon

heredity, daylength, date ofseeding and other environmental condiùons (Keller and

Peterson 1950; Ludwig et al. 1953; Smith 1957). Bula and co-workers (1965, 1969)

also found that changes in DoUard red clover as measured by growth type and winter

survival were of much smaUer magnitude from the second to the third generation of

seed increase than those of the first to the second geueration of increase. A greater

shift toward more f10wering types and fewer winter-hardy plants was observed in

plants grown from seed produced in California but changes were toward the opposite

direction with seed produced at Prosser, Washington (Bula et al 1965).

6.4.2 Elfeel on Allele Frequencies and Diversity ofIsozyme Loci

Ofthe 23 loci examined, ten loci (43.5%) were monomorphic (Dia-l, Dia-2,

Dia-3, Est-2, ldlt-l, Md/l-l, Md/I-2, Md/I-4, Me-l and Me-2); twO loci (8.7%) were

nearlYè monomorphic (Ad/l-l and Pgd-l); while the remaining eleven loci (47.8%) .-, were polymorphie (Aat-2, Aat-3,Amy-l, Est-l, Est-4, Est-7, Gpi-2, Mdh-3, Pgd-2, PgTiz-

2 and Skd-l) in the MCC 124 populations (Table 6.2). In the MCC 125-populations,

• 119 twelve loci (52.2%) were monomorphic (Adh-l. Dia-l. Dia-2. Dia-3. Est-2, Mdll-l.

• Mdll-2, Mdh-4, Me-l, Me-2, Pgd- 1 and Pgm-2): one locus (4.3%) WOlS nearly monomorphic (Aat-3); and ten loci (43.5%) were polymorphie (Aat-2, Amy-l. Est-l.

Est-4, Est-?, Gpi-2, ldll-l, Mdll-3, Pgd-2 and Skd-l) (Table 6.2). The mean number

of alleles per polymorphie locus was 2.77 and 2.64 in the MeC 124 and MeC 125

populations, respeetively. The contingency X! test values for allele frequency

heterogeneity are shown in Table 6.3. The frequencies of the common alleles ofEst­

] and Mdll-3 increased significantly (P < 0.05) after three cycles of naturai selection

in the MeC 124 populations (Tables 6.2 and 6.3). However, the allele frequencies

at most (eight) polymorphie loci did nllt change (Tables 6.2 and 6.3). ln the MeC

125 populations, the frequency of the common allele atAmy-] increased significantly

(P < 0.01) but the majority ofpolymorphie loci (nine) exhibited no changes (Tables

6.2 and 6.3). Rare alleles (frequency <: 0.05) were observed at severalloci (Aat-2,

A ....t-3. Adlz-I. Gpi-2, Mdll-3. Pgd-I and Pgm-2) in these two groups of populations

(Table 6.2).

The summary of genetic variability in these ten populations based on 22

isozyme loci is shown in Table 6.4. The Est-710cus was not included in this analysis

because a null allele was observed in red clover (Kongkiatngam et al. in press,

chapter 3 and 4). Because ofthe presence ofthe nu1l allele, aa and bb homozygotes

could Dot be distinguished from heterozygotes with null alleles. In the MeC 124

populations, the estimates ofaverage expeetèd heterozygosity decreased slightly from

0.119 to 0.105 but the percentage of polymorphie loci and the mean number of

• 120 aHeles per locus were the same after three cycles of natural selection (Table 6.4). • In the Mee 125 populations, the expected heterozygosity, the percentage of polymorphie loci and the mean number of alleles per locus decreased from 0.092 to

0.070,47.62 to 42.86 and 1.76 to 1.57, respeetively (Table 6.4). The genetic diversity

estimates for the Mee 124-2P and Mee 124-3P populations, which were harvested

from the second year fields of the Mee 124-1 and Mee 124-2 populations,

respectively, were not different from those of the Mee 124-2 and Mee 124-3

populations (Table 6.4). The genetic variability of the Mee 12S-3P population was

also the same as that of the Mee 125-3 population (Table 6.4). Genotypic

frequencies at sorne loci deviated from Hardy-Weinberg equilibrium in a few

populations (e.g. Amy-! in all populations, except Mee 125-1 and 12S-3P; Aat-2 in

Mee 124-1 and 124-2P), but in general, all populations could be considered as being

in Hardy-Weinberg equilibrium.

The changes in morphological traits related direetly to adaptation (Table 6.1)

illustrate the effeet of natural selection on red clover populations. However, changes

in aIlele frequencies at enzyme-coding loci are not evident (Tables 6.2 and 6.3). The

changes at sorne isozyme loci may result from linkage to morphological traits

influenced by natural selection. Since there were no changes at the majority of

isozyme loci (Table 6.3), one could assume that natural selection was not acting

direetly on these isozymes (Kimura 1983). In Liatris cylindracea. which is a cross­

pollinated, perennial herb found in dry, undisturbed climax prairies, gene frequencies

ofonly two isozyme loci were found to besignificantly correlated to adaptive factors,

• 121 but 13 loci showed no relationship to varying environmental factors (Schaal 1974). • Isozyme markers could detect changes in diversity belWeen these two sets of populations. The estimates of diversity (average heterozygosity. percentage of

polymorphie loci. and Mean number of alleles per locus) decreased slightly in both

groups (Table 6.4). After three cycles of natural selection. the estimate of expected

heterozygosity (Hcxp) of the MCC 124 populations was 0.105 which is comparable to

those of red clover cultivars adapted to growing conditions in North America

(Kongkiatngam et al. in press. chapter 4). In the MCC 125 populations. however. the

estimate of Hc:."P was 0.070 which is much lower than that of MCC 124 populations.

These populations originated from plants selected artificially for Fusarium resistance

from only one cultivar. Ottawa, and should. therefore. be less diverse than the MCC

124 populations. This can be seen from the estimates of Hcxp (0.114 in MCC 124-1

vs. 0.092 in MCC 125-1) in these populations after one cycle ofselection. A1though

the levels of heterozygosity of these red clover populations decreased after three

cycles of natural selection. they are still considered to have a high level of diversity

compared to other crossed-pollinated species (Harnrick 1989). This decreasing

heterozygosity may be caused by both natura! selection and inbreeding. Harvested

seed was not contnbuted from al! established plants since sorne plants did notsurvive

until harvesting and sorne of them did not flower in the fust year. A1though red

clover has a self-incompanbility system, selfing was found to occur to sorne extent

depending on environmental conditions and genetic background (Kongkiatngam et

• 122 al. unpublished data; Taylor and Smith 1979). Sib-mating within these populations • could also lead to inbreeding.

6.4.3. Natural selection and seed production in red clover

In North America, red clover is tbe most widely grown among the true

(Taylor 1985). Thus, the demand for red clover seed is highest, and annual seed

production in recent years has been about Il 300 to 13 600 t in the U.SA and 5 000

t in Canada (Rincker and Rampton 1985). Approximate1y 50-55% ofthis production

is from the second crop after harvesting for forage in the red clover growing areas.

Almost half of red clover seed is produced as a seed crop in irrigated areas outside

its growing areas. Seed production of red clover cultivars in regions where they are

not adapted can result in genetic shifts (Smith et al. 1985). This problem has been

confirmed by our study. The morphological characters that are related to

environmental adaptation (i.e. pubescence and growth type) showed changes after

three cycles of natural selection (Table 6.1). A higher percentage of plants with

pubescence and with flowering stems was observed. Plants, that produce flowering

stems in the seedling year (growth types 3, 4 and 5), are less persistent and less

winter-hardy than those that remain in rosettes (growth types 1 and 2) (Choo 1984;

Smith 1957). The problem ofgenetic shift could be more severe ifseed is produced

in the areas that are in more southem latitudes (Buia et al. 1965; 1969). Genetic

structure of these cultivars, for example heterozygosity, could a\so be different from

the original seed lots (Table 6.4). Seed obtained from this production would be

• 1"23 more comparable to naturalized populations rather than ta the original cultivars. • Genetic identity of the original cultivars could be assured by several me:lsures. such as a limited generation system. limiting the number of seed crops harvested from a

stand. specifying the area or latitude of seed production. and prohibiting seed

production in the year of planting (Rincker and Rampton 1985).

In summary. changes in red claver populations occurred after three cycles of

naturdl selection. Morphological characters that are related ta environmental

adaptation. e.g.• hairs on stems, petioles, basal internodes and stipules. and growth

types, changed dramatically after the first cycle of selection whereas there was no

ch::l.nge in the number of plants with leaf mark. There were no obvious changes in

allele frequencies of isozymes. Most isozyme loci were in Hardy-Weinberg

equilibrium. However. heterozygosity decreased after three cycles of natural

selection to the level of commonly grown adapted cultivars.

• 124 • • Table 6.1. Percentages of red clover plants with sorne morphological traits (N) from different cycles of natural selection.

Mee 124 population Mee 125 population Trait Allele 4-0 4-1 4-2 4-2P 4-3 4-3P 5-1 5-2 5-3 5-3P

Hairs on stems Spreading 62 90 92 93 88 96 45 40 49 55

(40) Appressed 38 10 8 7 12 4 55 60 51 45

Hairs on petioles Spreading 69 88 92 95 94 97 76 67 73 67

(140) Appressed 31 12 8 5 6 3 24 33 27 33

Hairs on basal Hairy 59 77 67 80 75 79 65 55 50 55

internodes (40) G1abrous 41 23 33 20 25 21 35 45 50 45

Hairs on stipules Hairy 41 46 28 35 40 36 17 17 16 13

(40) G1abrous 59 54 62 65 60 64 83 83 84 87

Leaf mark Present 75 69 65 62 62 66 78 77 80 76

(140) Absent 25 31 35 38 38 34 22 23 20 24

Growth types Flowering 65 85 90 90 83 93 100 95 85 84

(40) Non.f1owering 35 15 10 10 17 7 0 5 15 16

125 ••, .. •

Table 6.2. Allele frequencies of polymorphie isozyme loci in red clover populations that have undergone three cycles of natural selection.

Mee \24 population Mee \25 population Locus Alle\e 4·0 4-\ 4-2 4-21' 4-3 4-31' 5-\ 5-2 5·3 5-31'

Aal-2 a 0.05 0.05 0.00 0.04 0.0\ 0.00 0.00 0.00 0.00 0.00

b 0.94 0.86 0.99 0.94 0.89 0.96 0.94 \.00 0.98 0.96

c 0.01 0.09 0.0\ 0.02 0.\0 0.04 0.06 0.00 0.02 0.04

Aal-3 a 0.00 0.00 0.01 0.0\ 0.01 0.0\ 0.00 0.00 0.00 0.00

b 0.99 0.98 0.96 0.95 0.96 0.98 0.99 1.00 1.00 0.98

c 0.01 0.02 0.03 0.04 0.03 om 0.01 0.00 0.00 0.02

Ad"·J a \.00 \.00 \.00 0.99 \.00 1.00 1.00 1.00 1.00 \.00

b 0.00 0.00 0.00 0.0\ 0.00 0.00 0.00 0.00 0.00 0.00

AillY-J a 0.07 0.05 0.08 0.03 0.06 0.03 0.06 0.04 0.04 0.00

b 0.87 0.90 0.89 0.94 0.90 0.95 0.89 0.95 0.95 0.99

c 0.06 0.05 0.03 0.03 0.04 0.02 0.05 0.0\ 0.0\ 0.01

\26

i E • • Table 6.2. Continued.

MCC 124 population MCC 125 population Locus Allele 4·0 4·1 4-2 4-2P 4·3 4·3P 5·1 5·2 5·3 5·3P

Est-} a 0.79 0.86 0.82 0.88 0.89 0.90 0.92 0.95 0.95 0.92

b 0.21 0.14 0.18 0.12 0.11 0.10 0.08 0.05 0.05 0.08

Est-4 '. a 0.02 0.01 0.02 0.02 0.02 0.04 0.03 0.07 0.03 0.09

b 0.60 0.58 0.58 0.65 0.45 0.56 0.62 0.53 0.57 0.46

c 0.38 0.41 0.40 0.33 0.53 0.40 0.35 0.40 0.40 0.45

Est-7 a 0.50 0.65 0.50 0.61 0.65 0.51 0.66 0.48 0.52 0.52

b 0.27 0.14 0.25 0.22 0.16 0.23 0.17 0.41 0.33 0.24

c 0.23 0.21 0.25 0.17 0.19 0.26 0.17 0.11 0.15 0.24

Gpi-2 a 0.01 0.02 0.00 0.02 0.01 0.00 0.01 0.00 0.00 0.00

b 0.87 0.88 0.91 0.85 0.88 0.90 0.96 0.95 0.98 0.99

c 0.11 0.10 0.09 0.13 0.11 0.10 0.03 0.05 0.02 0.01

d 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1).00 0.00

127 fi • •

Table 6.2. Continued.

MCC 124 population MCC 125 population Locus Allele 4·0 4-1 4-2 4-2P 4-3 4-3P 5-\ 5-2 5-3 5-3P

Idll·) a 1.00 1.00 1.00 1.00 1.00 1.00 0.96 0.94 0.98 1.00

b 0.00 0.00 0.00 0.00 0.00 0.00 0.04 0.06 0.02 0.00

Mdll-3 a 0.90 1.00 0.95 0.99 0.98 0.99 0.98 0.98 0.98 0.98

b 0.10 0.00 0.05 0.0\ 0.02 0.0\ 0.02 0.02 0.02 0.02

Pgd·) a 1.00 1.00 . 1.00 0.99 1.00 0.99 1.00 1.00 1.00 1.00

b 0.00 0.00 0.00 0.01 0.00 0.0\ 0.00 0.00 0.00 0.00

Pgd.2 a 0.20 0.25 0.21 0.29 0.22 0.19 0.21 0.18 0.21 0.19

b 0.76 0.73 0.75 0.69 0.74 0.75 0.73 0.76 0.71 0.79

c 0.04 0.02 0.04 0.02 0.04 0.06 0.06 0.06 0.08 0.02

Pgm·2 a 0.05 0.05 0.01 0.02 0.01 0.02 0.00 0.00 0.00 0.00

b 0.95 0.95 0.99 0.98 0.99 0.98 1.00 1.00 1.00 1.00

128 • •

Table 6.2. Continued.

Mee 124 population Mee 125 population Locus Allele 4·0 4·1 4·2 4·21' 4·3 4·31' 5·1 5·2 5·3 5·31'

Skd·J a 0.10 0.11 0.05 0.08 0.09 0.11 0.04 0.04 0.00 0.01

b 0.80 0.75 0.88 0.82 0.82 0.80 0.84 0.89 0.95 0.96

c 0.04 0.05 0.02 0.02 0.04 0.06 0.10 0.07 0.04 0.03

(/ 0.06 0.09 0.05 0.08 0.05 0.03 0.02 0.00 0.01 0.00

129 • Table 6.3. Contingency tablc: x~ test values of allele frequency heterogeneity of reù

c10ver populations.

MCC 124 MCC 125

Locus , , x- df P x- dl' P

Aat-2 22.842 10 0.011 5.36~ 3 0.147

Aar-] 3.616 10 0.963 3.701 3 0.295

Adh-J 5.010 5 0.415

Amy-J 13.990 10 0.173 23.052 6 0.001

Est-J 14.828 5 0.011 3.455 3 0.327

Est-4 24.125 10 0.007 9.157 6 0.165

Gpi-2 9.893 15 0.826 5.176 6 0521

Id/I-J 5.368 3 0.147

Mdlz-3 16.810 5 0.005 0.000 3 1.000

Pgd-] 4.017 5 0547

Pgd-2 4.746 10 0.907 2.809 6 0.832

Pgm-2 4.120 5 0532

Skd-] 8.946 15 O.SSO 13203 9 0.154

"iota! 132.944 105 0.034 71290 48 0.016

• 130 • Tahle 6.4. Estimates of mean of heterozygosity from direct count (Ho"',). mean of

expcctcd hClcrozygosity (He,p). mean number of alleles per locus (A) and percentage

of polymorphie loci (P) from 21 isozyme loci in red clover populations. Standard

errors in parentheses.

Cultivar Hot» Hcxp P A

MCC 124-0 0.104 (0.032) 0.119 (0.035) 47.62 1.86 (0.23)

MCC 124-1 0.109 (0.037) 0.114 (0.036) 42.86 1.76 (0.22)

MCC 124-2 0.086 (0.030) 0.096 (0.033) 47.62 1.76 (021)

MCC 124-2P 0.094 (0.032) 0.104 (0.033) 57.14 1.95 (021)

MCC 124-3 0.100 (0.032) 0.105 (0.034) 47.62 1.86 (O.")

MCC 124-3P 0.086 (0.030) 0.094 (0.033) 52.38 1.81 (020)

MCC 125-1 0.086 (0.031) 0.092 (0.032) 47.62 1.76 (021)

MCC 125-2 0.072 (0.032) 0.077 (0.032) 38.10 1.57 (0.18)

MCC 125-3 0.060 (0.028) 0.Q70 (0.031) 42.86 1.62 (0.19)

MCC 125-3P 0.063 (0.029) 0.066 (0.031) 42.86 1.57 (0.16)

• 131 • CONTRIBUTIONS TO KNOWLEDGE

1. The genetic control ofseven enzyme-coding loci: Aat-2. Amy-l. E~t--I. Est- Î,

Pgd-I. Pgd-2 :md Skd-I. in red clover was confirmed for the first time. The genetic

basis of banding patterns for 16 other isozyme loci: Aat-3. Adlz-I. Dia-I. Dia-2. Dia-3.

Est-l, Est-2, Gpi-2 Id/z-I, Mdlz-I, Mdlz-2. Mdlz-3. Mdlz-4. Me-I. Me-2 and Pgm-2. was

also postulated. based on the segregation patterns observed within cultivars (Chaptcr

4). These isozymes will be useful genetic markers for genetic and breeding studies

in red clover.

2. The first study of isozyme diversity of red clover ct;ltivars l'rom three

different origins is presented (Chapters 3 and 4). A high level of variation within

cultivars was observed. This will have applications for germplasm evaluation and

utilization in red clover breeding progrJ.ms.

3. The first study on the use of RAPD markers in red clover is reported

(Chapters 3 and 4). Thi3 type of marker will have wide applications in red clover

which has a small number of morphological. agronomie and physiological markers

under simple genetie control. Because of their variability, they will be useful for

several applications in red clover genetie and breeding studies. for example to mark

g~nes controlling disease and inseet resistanœ, quantitative trait loci (QTLs), to

conStruct a genetie linkage map, and 'ô fingerprint individ!1aI plants. , .' 4. The eompansons among rr.'>rph?logicaI traits, isozyme loci and RAPD

markers for estimating genetie variability within and ':letween cultivars of red clover

• ]32 • arc also shown (Chapter 3). A higher level of genetic variation was detected with

isozyme and RAPD markers. These two types of markers will be useful for red

clover germplasm charactrization.

5. The potential use of isozymes for cultivar identification in red clover is

investigated (Chapter 4) for the first time. Allele frequencies of these isozymes could

discriminate two cultivars (DoUard and Hamidori) from aU Olher cultivars and

severdl cultivar combinations tested. This technique may have applications in

commercial seed production and seed certification programs of red c1over.

6. The first use of RAPD markers from bulked samples to distinguish

cultivars ofred clover is described (Chapter 5). The amplification patterns obtained

from only two primers could distinguish the 15 cultivars examined from Europe.

North America. and Japan. This will have a direct application in commercial seed

trade and seed certification programs of red clover. The technique is simple and fast

which is desirable for cultivar identification. RAPD markers from bulked samples

could also have other applications in red c10ver breeding, for example to mark

mOllogenic traits and quantative trait loci (QTLs).

7. The first study of genetic changes after three cycl,;:s of natural selection in

red clover using morphological traits and isozymes is presented (Chapter 6). This

wiU have implications for commercial seed pl'oduetionand cultivar improvement in

red clover.

• 133 • GENERAL CONCLUSIONS Isozyme and RAPD markers have been shown to be effective for estimating

genetic variability within and between cultivars of red c1over. Comparc:d with

morphological characteristics, better estimates ofgenetic variation could be obtained

using these two types of markers. Isozymes and RAPD markers will provide usefuI

tools for germplasm characterization. conservation and utilization. as weil as for

genetic and breeding studies in red c1over.

The inheritance of seven enzyme-coding loci in red c10ver was confirmed.

These seven isozyme loci are useful new genetic markers for red c10ver breeding and

genetic studies such as construeting a genetic linkage map, estimating outcrossing

rates and marker-assisted selection for qualitative and quantitative traiL~. In addition.

16 other enzymes that behaved the same as in other legumes were also observed.

Two pairs of Iinked genes controlling four enzymes were found. Genetic diversity

of red clover cultivars \VUS found to be high, with most of the variation found within

\.'Ultivars rather than between them. A1lele frequencies at enzyme-coding loci were

not able to discriminate ail cultivars of red c10ver examined, but they could

differentiate ail North American cultivars.

RAPD patterns of bulked genomic DNA samples from 20 individuals were

found to be good representatives of heterogeneous red c10ver cultivars. A high level

ofpolyrnorphism amongthe 15 cultivars \vas observed. Cultivar-specifie amplification

bands were found with 13 arbitrary lo-~er primers screened. At lcast !wo primers

• 134 were required lU give RAPD patterns that could discriminate all 15 red clover • cultivars. The cluster analysis based on Rogers' genetic distances computed from RAPD data separated these 15 cultivars into four groups. RAPD analysis is

therefore a powerful tool for cultivar identification and for genotyping purposes in

red clover.

Changes in IWO composite red clover populations that differed in their levels

ofdiversity occurred after three cycles of natural selection. Morphological characters

that are related to environmental adaptation, i.e. hairs on stems.. petioles, basal

internodes and stipules, and growth types, changed dramatically after the first cycle

of selection whereas there was no change in the number of plants ....ith leaf mark.

There were no obvious changes in allele frequencies of isozymes. Most isozyme loci

were in Hardy-Weinberg equilibrium. A higher level of genetic diversity was

observed in the MCC 124 populations than that in the MCC 125 populations.

However. heterozygosity decreased in both populations after three cycles of natural

selection, to the level of commonly grown adapted cultivars in the case of the MCC

12~ populations.

• 135 BIBLIOGRAPHY • Adam D. 5imonsen V. Loeschcke V (1987) Allozyme variation in rye. Sccale cen'ale L 2. Commercial varieties. Theor Appl Genet 74: 560-565

Aitken Y (1960) Axial origin of the terminal inflorescence in the genus Trifolium. Nature 187: 622-623

Akerberg E (1974) Red clover - an interesting species for genecological studies. Hereditas 77: 177-82.

Almgard G, Clapham D (1975) Isozyme variation distinguishing 18 Avena cultivars grown in 5weden. 5weden J Agric Res 5: 61-67

Anonymous (1991) Canadian legume seed forecast. Seed World 129(2): 26

AruIsekar 5, Parfitt DE, Berès W, Hansche PE (1986) Genetics of malate dehydrogenase isozymes in the peach. J Heredity 77: 49-51

Aruna M. Ozias-Akins P, Austin ME (1993) Genetic relatedness among rabbiteye blueberry (Vaccinium aslzei) cultivars determined by DNA amplification using single primers of abitrary sequence. Genome 36: 971-977

Ashraf M. McNeilly T, Bradshaw AD (1987) Selection and heritability of tolcrance to sodium chloride in four forage species. Crop Sei 27: 232-234

Ausubel FM. Brent R, Kingston RE, Moore DO, Seidman JG, Smith JA, Struhl K (eds) (1989) Short Protocols in Molecular" Biology. John Wiley & Sons, New York ·CC.

Bailey DC (1983) Isozymic variation and plant breeders' rights. In: 5D Tanksley, TJ Orton (eds), Isozymes in Plant Breeding and Genetics, Part A, pp 425-440. Elsevier Science Publishers BV, Amsterdam, The Netherlands

Bassiri A (1976) Barley cultivars identification by use of isoenzyme eleetrophoretic patterns. Can J Plant Sei 56: 1-6

Bassiri A (1977) Identification and polymorphism of cultivars and wild ecotypes of safflower based on isozyme patterns. Euphytica 26: 709-719

Beard OF, Hollowell EA (1952) The effect on performance when seed of forage crop varieties is grown under different environmental conditions. Proc6th IntI Grassland Congr, pp 860-866. Pennsylvania State College, State College, PA

• 136 Beckmann JS, Soller M (1985) Restriction fragment length polymorphisms and • genelic improvement of agricultural species. Euphytica 35: 111-124 Beckmann JS, Soller M (1989) Toward a unified approach to genetic mapping of eukaryotes based on sequence tagged microsatellite sites. Biotechnol 8: 930­ 932

Bell CJ, Ecker JR (1994) Assignment of 30 microsatellite loci to the linkage map of Arabidopsis. Genomics 19: 137-144

Belzile L (1990) Influence des cultivars et des stades de prelevement de la premiere pousse sur la production de semence de trefle rouge. Can J Plant Sci 70: 1071-1080

Belzile L (1991) Effet des types de sol sur la production de semence du trefle rouge. Can J Plant Sei 71: 1039-1046

Bird JN (1948) Early and late types of red clover. Sci Agrie 28: 444-453

Bogani P, Cavalier D, Petruccelli R, Polsinelli 1., Roselli G (1994) Identification of olive cultivars by using random amplified polymorphie DNA. Acta Hort 356: 98-101

Booy G, van Dreven F, Steverink-Raben A (1993) Identification of ryegrass varieties (Lalium spp.) using allele frequencies of the PGI-2 and ACP-1 isozyme systems. Plant Varieties Seeds 6: 179-196

Bowditeh BM, Albright DG, Williams JGK, Braun Ml (1993) Use of randomly amplified polymorphie DNA markers in comparative genome studies. Methods Enzymol 224: 294-309

Bowley SR, Laekie SM (1989) Genetie of nongIanduIar stem trichomes in red clover (Trifolium pratense L). J Heredity 80: 472-474

Bowley SR. Taylor NI., Cornelius PL(1984a) Phenotypie recurrentselection for stem length in 'Kenstar' red clover. Crop Sei 24: 578-582 -

Bowley SR, Taylor NI., Dougherty CT (1984b) Physiology and morphology of red clover. Adv Agron 37: 317-347 .

Bowley SR, Taylor NI., Cornelius Pl., Dougherty CT (1984c) Response to selection for stem length in red clover evaluated at wide and narrow spacings. Can J Plant Sei 64: 925-936

• 137 Bowley SR. Taylor NI., Dougherty cr(1987) PhOloperiodic response and heritahility of the pre-flowering interval of two red clover (Trifolium pra({"IISC L) • populations. Ann Appl Biol 111: 455-461 Brieman 1., Friedman JH. Olshen RA. Stone U (1984) Cla.~sification and regres.~ion trees. Wadsworth. Monterey

Bringhurst RS. Aru\sekar S. Hancock JF Jr, Voth IV (1981) Electrophoretic characterization of strawberry cultivars. J Amer Soc Hort Sei 106: 684-687

Brown ABD. Weir BS (1983) Measuring genetic variability in plant populations. In: DE Soitis, PS Soltis (eds), Isozymes in plant biology, pp 219-239. Dioscorides Press, Portland, Oregon

Bula RJ, May RG, Garrison CS, Rincker CM, Dean JG (1965) Comparisons of floral response of seed lots of DoUard red clover. Trifolium pratense L Crop Sei 5: 425-428

Bula RJ, May RG, Garrison CS, Rincker CM, Dean JG (1969) Flom\ response, wintersurvivaI, and leafmark frequency ofadvanced genemlionseed inereases of 'DoUard' red clover, Trifolium pratense L Crop Sei 9: 181-184

Bunery BR, Buzzell RI (1968) Peroxidase activity in seeds ofsoybean varieties. Crop Sei 8: 722-725

Campos LP, RaeIson JV, Grant WF (1994) Genome relationships among Lotus species based on random amplified polymorphie DNA (RAPD). Theor App\ Genet 88: 417-422 .

Castiglione S, Wang G, Damiani G, Bandi C, Bisoffi S, Sala F (1993) RAPD fngerprints for identification and taxononùe studies of eHte pop\ar (Populus spp.) clones. Theor Appl Genet 87: 54-59

Chalmers 10, Waugh R, SprentJI, Simons N, Powell W (1992) Detection ofgenetie variation between andwithin populations ofGüricidia sepium and G. macuJala using RAPD markers. Heredity 69: 465-472

Chase CD, Onega VM, VaIIejos CE (1991) DNA restriction fr.:tgment \ength polymorphism oorreIate with isozyme diversity in PhoseO/US vulgaris L Theor Appl Genet 81: 806-811

CherryJP, Ory RL (1973)E\eetroporetie characterization ofsix selected enzymes of peanut cuItivars.Phytochem 12: 283-289 • -,~ Choo TM (1984) A~soeiation between growth habit and persistence in red clover. • Euphytica 33: 177-185 Christie BR, Chuu TM (1990) Effects of harvest time and A1ar-85 on seed yield of red clover. Can J Plant Sei 70: 869-871

Christie BR, Choo TM (1991) Morphological characteristics assoeiated with winter survival of five growth types of tetraploid red clover. Euphytica 54: 275-278

Clegg MT, A1lard RW, Kahler AL (1972) Is the gene the unit ofselection? Evidence from (Wo experimental plant populations. Proe Natl Acad Sei USA 69: 2474­ 2478

Clegg MT. Kahler AL, A1lard RW (1978) Estimation of life cycle components of sp.lection in an experimental plant population. Genetics 89: 765-792

Co1lin WJ, Rossiter Re, Haynes Y. Brown AHD. Marshall DR (1984) Identification of subtermnean clover cultivars and their genetic relationships by isozyme analysis. Aust J Agrie Res 35: 399-411

Connolly AG. Godwin ID. Cooper M. DeLacy IH (1994) Interpretation of randomly amplified polymorphic DNA marker data for fingerprinting ~weet potato (/pomoea batatas L) genotypes. Theor Appl Genet 88: 332-336

Cornelius PL, Taylor NL (1981) Phenotypie recurrent selection for intensity ofpetai color in red clover. J Heredity 72: 275-276

Coulman BE, Lamben M (1995) Selection for resistance to root rot caused by Fusarium spp. in red clover (Trifolium pratense L). Cao J Plant Sci 75: 141­ 146

Coulman BE, Oakes B (1987) Inheritance and agronomie significance ofgrowth type in red clover. Cao J Plant Sci 67: 276-277

Cousineau Je, Donnelly DJ (1989) Identification of raspberry cultivars in vivo and in vitro using isoenzyme analysis. HonSci 24: 49D-492

Dawson II<, Chalmers KJ. Waugh R, Powell W (1993) Detection and analysis of genetievariaion inHordeum spomaneum populations from Israel using RAPD markers. Mol Eco12: 151-159

Demeke T. Adams RP, Chibbar R (1992) Potential taxonomie use of random amplified DNA (RAPD): a case study inBrassica. Theor Appt Genet84: 990­ 994 • 139 • Devadas C. Beck CB (1972) Comparative morphology of the primary vascular systems in sorne species of Rosaceae and Leguminosae. Amer J Bot 59: 557­ 567

Dovrat A, Waldman M (1969) Differentiai seed production of northern alsike anù red clovers at southern latitude. Crop Sci 9: 544-547

Doyle JJ, Doyle JL (1987) Isolation ofplant DNA from fresh tissue. Phytochem Bull 19: 11

Duke JA (1981) Handbook of legumes ofworld ecinomic importance. Plenum Pres.~. New York

Echt CS, Erdahl LA, McCoy TJ (1992) Genetic segregation of random amplified polymorphie DNA in diploid cultivated alfalfa. Genome 35: 84-87

Ennos RA (1989) Detection and measurement of selection: genetic and ecological approaches. In: AHD Brown, MT Clegg, AL Kahler, BS Weir (eds), Plant population genetics, breeding, and genetic resources, pp 200-214. Sinauer Assodates Ine, Sunderland, MA

Erskine W. Muehlbauer FJ (1991) Allozyme and morphological variability, outcrossing rate and core collection formation in lentil germplasm. Theor Appl Genet 83: 119-125

Fedak G, Rajhathy T (1972) Esterase isozymes in Canadian barley cultivars. Can J Plant Sci 52: 507-516

Fergus EN, Hollowell EA (1960) Red clover. Adv Agron 12: 365435

Garda P, Vences FJ, Perez de la Vega M, Allard RW (1989) Allelic and genotypic composition ofancestral 5panish and colonial Californian gene pools ofAvena barbata: evolutionary implications. Genetics 122: 687-694

Gates P, Boultier D (1979) The use ofseed isoenzymes as an aidto the breeding of field beans (Vicia/aba L). New Phytol83: 783-791

Gomes J, Jadric 5, Winterhalter M. BrIdc 5 (1982) Alcohol dehydrogenase isoenzymes in chickpea cotyledons. Phytochem 21: 1219-1224

Gonzalez JM. Ferrer E (1993) Random amplified polymorphic DNA analysis in Hordeum spedes. Genome 36: 1029-1031

• 140 Goodman MM, Stuber LW, Lee CN, Johnson FM (1980) Genetic control of malate • dehydrogenase isozymes in maize. Genetics 94: 153-168 Gottlieb LD (1 9l:1 la) Electrophoretic evidenee and plant populations. Progr Phytoehem 7: 1-46

Gottlieb LD (1981 b) Gene numbers in species of Astereae that have different chromosome numbers. Proc Nat! Acad Sci USA 78: 3726-3729

Gottlieb LD (1982) Conservation and duplication ofisozymes in plants. Science 216: 373-380

Gregor D. Hartmann W. Stosser R (1994) Cultivar identification in Prunus domestica using random amplified polymorphie DNA markers. Acta Hon 359: 33-40

Haley SD, Miklas PN. Afanador L., Kelly JD (1994) Random amplified polymorphic DNA (RAPD) marker variability between and within gene pools of common bean. J Amer Soc Hon Sci 119: 122-125

Halward T. Stalker T, LaRue E, Kochen G (1992) Use of single-prLner DNA amplifications in ge'1etic studies of peanut (Araclzis Izypogaea L). ?lant Mol Biol 18: 315-325

Hamill DE, Brewbaker JL (1969) Isoenzyme polymorphism in flowering plants. IV the peroxidase isoenzymes of maize (Zea mays). Physiol Plant 22: 945-958

Hamrick JL (1989) Isozymes and the analysis of genetic structure in plant populations. In: DE Soltis, PS Soltis (eds), Isozymes in Plant Biology, pp 87­ 105. Dioscorides Press, Ponland, Oregon

Hamrick JL., Linhan YB, Mitton JB (1979) Relationships between life history characteristics and electrophoretica\ly deteetable genetic variation in plants. Annu Rev Ecol Syst 10: 173-200

Harada T. Matsukawa K, Sato T. Ishikawà R, Niizeki M, Saito K (1993) DNA­ RAPDs detect genetic variation and patemity in Malus. Euphytica 65: 87-91

Harris RJ (1975) A primer of multivariate statistics. Academic Press, New York

Hart! DL (1980) Principles of population genetics. Sinauer Associates Ine, Sunderland, MA

• 141 • Hayward MD. Breese EL (1993) Population structure and variability. In: MD Hayward. NO Bosemark. 1 Romagosa (eds). Plant Breeding: Principles and Prospects. pp 17-29. Chapman & Hall. London

Hayward MD, McAdam NJ (1977) Isozyme polymorphism as a mea.~ure of distinetiveness and stability in cultivars of Lotium perenne Z Pflanzenzucht 79: 59-68

He S. Ohm H. Mackenzie S (1992) Detection of DNA sequence polymorphisms among wheat varieties. Theor App\ Genet 84: 573-578

Heun M, Helentjaris T (1993) Inheritance of RAPDs in FI hybrids of corn. Theor Appl Genet 85: 961-968

Heun M, Murphy JP, Phillips TD (1994) A comparison of RAPD and isozyme analysis for determining the genetic relationships among Avenu sren1is L accessions. Theor Appl Genet 87: 689-696

Hickey RJ, Vincent MA, Guttman SI (1991) Genetic variation in running buffalo clover (Trifoüum sroloniferum, Fabaceae). Conserv Biol 5: 309-316

HHlis DM (1984) Misuse and modification of Nei's genetic distance. Syst Zool 33: 238-240

Howell C, Newbury fiJ, Swennen RI., Withers LA, Ford-Lloyd BV (1994) The use of RAPD for identifying and classifying Musa germplasm. Genome 37: 328­ 332

Hu J, Quiros CF (1991) Identification of broccoli and cauliflower cultivars with RAPD markers. Plant Cell Rep 10: 505-511

Hubby Ji., Lewontin RC (1966) A molecular approach to the study of genic heterozygosity in natural populations 1. The number of alleles at different loci in Drosoplzila pseudoobscura. Genetics 54: 577-594

Huff DR, Peakall R, Smouse PE (1993) RAPD variation within and among natura! populations of outcrossing buffalograss [Buchloe duceyloides (Nutt.) Engelm.]. Theor Appl Genet 86: 927-934

Humer Ri., Marken CL (1957) Histochemical demoDstratioD ofenzymes separated . by zone eleetrophoresis in starch gels. Science 125: 1294-1295

Innis MA, Gelfond OH, Sninsky JJ, White TJ (1990) PCR protocols. In: A guide to methods and applications. Academic Press, New York • 142 Jain A, Bhatia S, Banga SS, Prakash S, Laksmikumaran M (1994)Potential use of random amplified polymorphie DNA (RAPD) technique to study the genetic • diversity in Indian mustard (Brussiea juneea) and it~ relationship to heterosis. Theor Appl Genet 88: 116-122

Jain SK, Qualset CO, Bhatt GM, Wu KI< (1975) Geographical patterns of phenotypic diversity in a world collection of durum wheats. Crop Sei 15: 700­ 704

Jeffreys AJ, Wilson V, Thein SL (1985a) Hypervariable 'minisatellite' regions in human DNA Nature 314: 67-73

Jeffreys AJ, Wilson V, Thein SL (1985b) Indi~idual-speeific 'fingerprints' of human DNA N:lture 316: 76-79

Jones TWA (1974) The effect of leaf number on the sensitivity of red clover seedlings to photoperiodie induction. J Br Grassl Soe 29: 25-28

Jones TWA (1991) Inheritanee of a mutation which suppresses flowering in red clover. J Exp Bot 42: 1589-1594

Jones TWA (1993) Flowering and gebberellins in a mutant red clover (Trifolium pratense L). Plant Growth Regul 12: 11-16

Jones TWA, Thomas AM (1994) Changes in gene produets associated with flowering in red clover (Trifolium pratense L). J Exp Bot 45: 517-519

Joshi CP, Nguyen HT (1993) Application ofthe random amplified polymorphie DNA technique for the detection of polymorphîsm among wild and cultivated tetraploid wheats. Genome 36: 602-609

Kazan K, Manner JM, Cameron DF (1993a) Genetie variation in agronomically important speeies of Stylosanthes determined using random amplified polymorphie DNA markeTS. Theor Appl Genet 85: 882-888

Kazan K, Manner JM, Cameron DF (1993b) Genetie relationships and variation in the Stylosanthes guianensis speeies complex assessed by random amplified polymorphie DNA Genome 36: 43-49'

Kazan K, Manner JM, Cameron DF (1993e) Inheritance of random amplified polymorphie DNA markeTS in an interspecifie cross in the genus Stylosanthes. Genome 36: 50-56

143 • Keller ER, Peterson ML (1950) Effect of photoperiod on red clover and timothy strains grown in association. Agron J 42: 598·603

Khan rvlA, Maxwell DP, Smith RR (1978) Inheritance of resistance to red clover vein mosaic virus in red clover. Phytopathology 68: 1084·1086

Kiang YT, Gorman MB (1985) Inheritance ofNADP-active isocitrate dehydrogenase isozymes in soybeans. J Heredity 76: 279-84

Kimura M (1983) The neutral theory of molecular evolution. Cambridge University Press, London

Knerr LO, Staub JE, Holder DJ, May BP (1989) Genetic diversity in Cucumi~ sativus L assessed by variation at 18 allozyme coding loci. Theor Appl Genet 78: 119-128

Ko HL, Cowan De, Henry RJ, Graham Ge, Blakeney AB, Lewin LG (1994) Random amplified polymorphic DNA analysis ofAustralian rice (Oryza Saliva L) varieties. Euphytica 80: 179-189

Kobrehel K, Gautier MF (1979) Variability in peroxidase isozymes in wheat and related ~pecies. Can J Bot 52: 755-759

Koller B, Lehmann A, McDermotl JM, Gessler C (1993) Identification of apple cultivars using RAPD markers. Theor Appl Genet 85: 901-904

Kongkiatngam P, Waterway MJ, Fortin MG, Coulman BE (1995) Genetic variation within and between cultivars of red' clover (Trifolium pratense L): Comparisons of morphological, isozyme and RAPD markers. Euphytica (in press)

KIesovieh S, Williams JGK, McFerson JR, Routman EJ (1992) Charaeterization of genetie identities and relationships of Brassica oleracea L via a random amplified polymorphie DNA assay. Theor Appl Genet 85: 190-196

KIesovich S, Lamboy WF, Li R, Ren J, Szewc·McFadden AI(, Bliek SM (1994) Application of molecular methods and statistical analysis for discrimination of accessions and clones ofvetiver grass. Crop Sei 34: 805-809

Kuhns U, Fretz TA (1978) Distinguishing rose cultivars by polyacrylamide gel eleetrophoresis. II. Isozyme variation among cultivars. J Amer Soc Hortie Sei 103: 509-516

• 144 Kusmiyati F (1995) .Pubescence in red clover: its inheritance and its relationship to potato leafhopper resistance. M Sc Thesis, McGill University, Montreal, • Canada Landry BS, Li RQ, Cheung WY, Granger RL (1994) Phylogeny analysis of 25 apple rootstocks using RAPD markers and tactical gene tagging. Theor Appl Genet 89: 847-852

Lanham PG, S. Fennell S, Moss JP, Powell W (1992) Detection of poltmorphic loci in Arac1lis germplasm using random amplified polymorphie DNA Genome 35: 885·889

LaRue TA, Patterson TG (1981) How much nitrogen do legumes flX? Adv Agron 34: 15-38

Leath KT, Romaine CP, Hill RR, Smith RR (1987a) Registration of red clover germplasm VR·l. Crop Sci 27: 1095-1096

Leath KT, Bloom JR, Hill RR, Smith RR (1987b) Registration of red clover germplasm line CCNR-l. Crop Sci 27: 1316

Levene H (1949) On a matching problem arising in genetics. Ann Math Stat 20: 31­ 48

Lewontill RC (1972) The apportionment of human diversity. ln: T Dobzhansky (ed), Evolutionary Biology, Vol 6, pp 381-398. Appleton-Century-Crofts, New York

Uu Z, Fumier GR (1993) Comparison of isozyme, RFLP, and RAPD markers for revealing genetie variation within and between trembling aspen and bigtooth aspen. Theor Appl Genet 87: 97-105

Loveless MD. Harnriek JL (1984) Ecological detreminants of genetic structure:in plant populations. Ann Rev Eeol Syst 15: 65-95

, -~ Ludwig RA, Barrales HG, Steppler H (1953) Studies on the effeet of light o;;:'ffie growth and development of red clover. Cao J Agric Sei 33: 274-287

Mahmoud SR, Gatehouse JA, Boulter D (1984) Inheritanceo'and mapping of isoenzymes in pea (Pisum sativum 1..). Theor Appl Genet 68,; 559-566

Mailer RJ, Scarth R, Fristensky B (1994) Discrimination among cultivars ofrapeseed (Brassica napus 1..) using DNA polymorphisms amplified from arbitrary primers. TheorAppl Genet 87: 697-704

• 145 • Mancini R. de Pace C. Scarascia Mugnozza GT. Delre V. Vittori T (1989) Isozyme gene markers in Vicia taba L Theor Appl Genet 77: 657-667

M:irkert CL Moller F (1959) Multiple forms of enzymes: tissue. ontogenetic. and species specific patterns. Proc Nat! Acad Sci USA 45: 753-763

Martin PH. Coulman BE, Peterson JF (1990) Genetics of tolerance to white clover mosaic virus in red clover. Crop Sci 30: 1191-1194

McG;ath JM, Quiros CF (1992) Genetic diversity at isozyme and RFLP loci in Brassiea eamprestTÏS as related to crop type and geographical origin. Theor Appl Genet 83: 783-790

McMillin DE (1983) Plant isozymes: a historical perspective. In: SD Tanksley, TJ Orton (eds), Isozymes in Plant Breeding and Geneties, Part A, pp 3-13. Elsevier Science Publishers BV, Amsterdam, The Netherlands

Metcalf CR. Chalk L (1950) Anatomy of the dicotyledonc, Vol I. Oxford Univ Press. London

Michelmore RW, Paran I, Kesseli RV (1991) Identification of markers Iinked to disease resistance genes by bulked segregant analysis: A r.lpid method to detect markers in specifie genomie regions using segregating populations. Proe Nat! Acad Sei USA 88: 9828-9832

Miles JW (1985) Evaluation of potential genetie marker traits and estimation of outcrossing rate in Srylosantlzes guianensis. Aust J Agrie Res 36: 259-265

Milligan BG (1991) Chloroplast DNA diversity withinand among populations of Trifolium pratense. Curr Genet 19: 411-416

Molina-Freaner F, JainSK (1992) Isozyme variation in Californian and Turkish populations of the colonizing species Trifolium lùrtum. J Heredity 83: 423-430

Montpatit JM, Coulman BE (1991a) Adventitious root development in red clover .(Trifoüum peratense L) and its inheritartce. Can J PlantSci 71: 749-754

Montpetit JM, Coulman BE (1991b) Responses to divergent selection for adventitious root growth'in red clover (Trifolium pratense L)~ Euphytica 58: 119-127

Moore GA, Collins GB (1983) New challenges confronting plant breeders. In::SD Tanksley, TJ Orton (eds), Isozymes in Plant Breeding aÎld Geneties, Part A, pp 25-58. Elsevier Science Publishers BV, 'Amsterdam, The Netherlands • 146 Morer.o-Gonzalez J, Cubero JI (1993) Selection strategies and choice of breeding materials. In: MD Hayward, NO Bosemarl<~ 1 Romagosa (eds), Plant • Breeding: Principles and Prospects, pp 281-313. Chapman & Hall, London M'Ribu HK, Hilu KW (1994) Detection of interspecific and intraspecific variation in Panicwn millets through random amplified polymorphie DNA. Theor Appl Genet 88: 412-416

Muehlbauer FJ, Weeden NF, Hoffm<1l1 DL (1989) Inheritance and linkage relationships of morphological and isozyme loci in lentil (Lens Miller). J Heredity 80: 298-303

Mueller LD, Ayala FJ (1982) Estimation and interpretation of genetic distance in empirical studies. Genet Res 40: 127-137

M

Mullis KB, Faloona FA (1987) Specifie synthesis of DNA in vitro via a polymerase­ catalyzed chain reaction. Methods Enzymol 155: 335·350

Nei M (1972) Genetie distance betwecn populations. Amer Nat 106: 283-292

Nei M (1973) Analysis of gene diversity in subdivided populations. Proc Natl Acad Sei USA 70: 3321-3323

Nei M (1978) Estimation ofaverage heterozygosity and genetie distance from a sma1l number of individuals. Geneties 89: 583-590

Nelke M, Nowak J, Wright JM, McClean NL (1993) DNA fingerprinting of red clover (TrifOÜU1n pratense L) with Jeffreys probes: detection of somaclonal variation and other applications. Plant Cell Rep 13; 72-78

Neva E (1978) Genetie variation in natural populations: patterns and theory. Theor - Pop Biol 13: 121-177 -

Nielsen G (1985) The use of isozymes as probes ta identify and labéfplant varieties and cultivars.:pp.1-32. In: MC Rattazzi, JG Scandalios, GS Whitt, (eds), c lsozymes: current topies in biologica1 arid:medica1 research. vol 12. Alan R Liss Ine. New york

• 147 • Nielsen G. Ostergaard H. Johansen H (1985) Cultivar identification by mcans of isoenzymes Il. Genetic variation at four enzyme loci in diploid ryegrass. Z Pflanzenzuchtg 94: 74-86

Novy RG, Kokak C, Goffreda J. Vorsa N (1994) RAPDs identify varietal misclassification and regional divergence in cranberry (VaccillilUn macrocarpOlI (Ait.) PurshJ. Theor Appl Genet 88: 1004-1010

Ohta T (1992) Tale n::arly neutr

Oliva RN, Steiner JJ, Young WC, III (1994a) Red clover seed production: I. Crop water requirements and irrigation timing. Crop Sei 34: 178-184

Oliva RN, Steiner 3J, Young WC, III (1994b) Red clover seed production: Il. Plant water status on yield and yield components. Crop Sei 34: 184-192

Orozco-Castillo C, Chalmers KJ. Waugh R, Powell W (1994) Detection of genetic diversity and selective gene introgression in coffee using RAPD markers. Theor Appl Genet 87: 934-940

Ostergaard H, Nielsen G (1981) Cultivar identification by me.ans of isozymes. 1. Ger.otypical survey ofthe PGI-210cus in tetr

Ostergaard H, Nielsen G,Johansen H (1985) Geneticvariation in cultivars ofdiploid ryegrass. Lolirun perenne and L multiflorum, at five enzyme systems. Theor Appl Genet69: 409-421

Pang J, Chen L, Chen LO, Chen SG (1992) DNA polymorphisms gener

ParentJG, Fortin MG, Page D (1993) Identification of raspberry cultivars by random amplified polymorphie DNA (RAPD) analysis. Can J Plant Sei 73: 1115-1122

Parrot WA, Smith RR (1986) Description and inheritance of new genes in red clover. J Heredity 77:355-358

Payne Rc, Koszykowski TJ-(1978) Esterase isozyme differences in seéd extraets among soybean cultivars. Crop Sei 18: 5S7-59

Pederson GA, Smith RR Jr, Leath KT (1981) Host-pathogen variability for Fusarium-caused root rot in red clover. Crop Sei 20: 787-789

148 -- ,~

1: Pielou EC (1969) An Introduction to Mathematical Ecology. John Wiley & Sons, • New York Puri KP, Laidlaw AS (1983) The effect of time of harvest on seed production of three red c10ver cultivars. Grass Forage Sci 39: 221-228

Ouesenberry KH, Baltensperger DO. Dunn RA, Wilcox Cl, Hardy SR (1989) Selection for tolt:rance to root-knot nematodes in red c1over. Crop Sci 29: 62­ 65

Ouesenberry KH, Smith RR, Taylor NI., Baltensperger DO, Parrott WA (1991) Geneùc nomenclature in c10vers and speeial-purpose legumes: I. red clover and white clover. Crop Sei 31: 861-867

Ouiros CF (1980) Identification ofalfalfa plants by enzyme electrophoresis. Crop Sei 20: 262-264

Raelson JV, Grant WF (1989) An isoenzyme study in the genus Lotus (Fabaceae). 1. Experimental protocols and genetic basis of eleetrophoretic phenotype. Theor Appl Genet 77: 595-607

Rafalski JA, Tingey SV, Williams JGK (1991) RAPD markers - a new technology for geneùc mapping and plant breeding. AgBiotech News Inform 3: 645-648

Ramirez L and Pisabarro G, 1985. Isozyme eleetrophoreùc patterns as a tool to charaeterize and classify rye (Secale cereale L) seed samples. Euphytica 34: 793-799. •

Ramirez I., Pisabarro G, Perez de la Vega M (1985) Isozyme geneùc similarity among rye (Secale cereale L). J Agric Sei Camb 105: 495-500

Rincker CM, Rampton HH (1985) Seed producùon. In: NL Taylor (ed), Clover science and technology, pp 417-443. ASA-es5A-SSSA, Madison, WI

Rogers JS (1972) Measures ofsimilarity and genetic distance. Studies in genetics VIT. University of Texas Publ7213, pp 145-153

Rohweder DA, Shrader WO, Templeton WC Jr (1977) Legumes, what is their place in today's agriculture? Crop Soils 29: 11-15

Rufelt S (1985) Selecùon for Fusarium root rot resistance in red clover. Ann appl Biol 107: 529-534

• 149 • Rus-Kor,ekaas W. Smulders MJM. Arens P. Vosman B (1994) Direct comparion of levels of genetic variation in tomato detected by a GACA-containing microsatellite probe and by random amplified polymorphic DNA. Genome 37: 375-381

Russell JR, Hosein F. Johnson E. Waugh R, Powell W (1993) Genetic differentation of cacoa (Theobroma cacao L) populations revealed by RAPD analysis. Mol Ecol 2: 89·97

Saiki RI<. $charf S. Faloona F. Mullis KB. Horn GT. Erlich HA. Arnheim N (1985) Enzymatic amplification of B-globin genome sequences and restricticn site analysis for diagnosis of sickle cell anemia. Science 230: 1350-1354

Santos JB dos, Nienhuis J. Skroch p. Tivang J. Slocum MK (1994) Comparison of RAPD and RFLP genetic markers in determining genetic similarity among Brassica oleracea L genotypes. Theor Appl Genet 87: 909-915

SAS Institute Inc (1985) SAS/SfATguide for personal computers. SAS Institute Ine. Cary, NC

SAS Institute Inc (1989) SAS/SfATguide for personal computers, version 6 ed. SAS Institute Ine. Cary, NC

Sattler PW, Hilburn LR (1985) A program for calculating genetic distances, and iL~ use in determining significant differences in genetic similarity between two groups of populations. J Heredity 76: 400

Schall BA (1974) Isolation by distance in Liatris eyündracea. Nature 252: 703

Schall BA, Levin DA (1978) The demographic genetics ofLiatris cylindracea Michx. (Compositae). Amer Nat 110: 191-206 , ShahFH, Rashid 0, Simons AJ, Dunsdon A (1994) The utility ofRAPD markers for the determination of genetic variation in oil palm (Elaeis guineensis). Theor Appl Genet 89: 713-718

Siegal BZ, GaIston AW (1967) The isoperoxidases ofPisum sativum. Plant Physiol 42: 221-226

Singh RS, Jaïn SK, Qualset CO (1973) Proteineleetrophoresis as an aid to oat variety identification. Euphytica 22: 98-105:

Smith JSC (1984) Genetic variability within U.S. hybrid maize: multivariate anaIysis of isozyme data. Crop Sei 24: 1041-1046 • 150 Smith D (1957) Flowering respon~e and winter survival in seedling stands of medium • c1over. Agron J 49: 126-129 Smith RR, Taylor NL, Bowley SR (1985) Red c1over. ln: NL Taylor (ed), Clover science and technology, pp 457-469. ASA-CSSA-SSSA, Madison, WI

Sneath PHA, Sokal RR (1973) Numerical taxonomy. Freeman and Co, San Francisco

$okal RR, Rohlf FJ (1981) Biometry. 2nd ed. WH Freeman and Co, San Francisco

$oltis DE, Soltis PS. eds (1989) lsozymes in plant biology. Dioscordes Press, Ponland. Oregon

Starling TM, Wilsie CP, Gilben NW (1950) Corolla tube length studies in red clover. Agron J 42: 1-8

Steppler HA, Raymond LC (1954) Note on the management of red clover for seed production. Can J Agric Res 34: 2"?-224

Sweeney PM, Danneberger TK (1994) Random amplified polymorphie DNA in perennial ryegrass: a comparison of bulked samples vs. individuals. HonSci 29: 624-626

Swofford DL, Selander RB (1981) BlOSYS-l: a Fonran program for the comprehensive analysis of eleetrophoretie data in population genetics and systematics. J Heredity 72: 281-283

Tanksley SD and Onon TJ (eds), 1983a. Isozymes in plant genetics and breeding Pan A, ,Elsevier Scientifie Publishing Co, Amsterdam, The Netherlands

Tanksley SD and Onon TJ (eds), 1983b. Isozymes in plant genetics and breeding Pan B. Elsevier Scientifie Publishing Co, Amsterdam, The Netherlands

Tautz D (1989) Hypervariability of simple sequences as a generaI source of polymorphie DNA markers. Nucleie Acids Res 17: 6463-6471

Taylor NL (1980) Oovers. In: WR Fehr, HH Hadiey (ed). Hybridization of crop plants, pp 261-272. ASA-CSSA, Madison, WI

Taylor NL (1985) Red clover. In: ME Heath, RF Barnes, DS MetcaIfe (eds), . Forages, pp 109-117. Iowa State University, Ames, Iowa . . Taylor NI.. Smith RR (1979) Red clover breeding and genetics. Adv Agron 31: 125- 153 : • 151 Taylor NL, Dade E. Garrison CS (1966) Factors involved in seed production of red clover clones and the polycross progenies at two diverse locations. Crop Sci • 6: 535-538 Taylor NL, May RG. Decker AM. Rincker CM. Garrison CS (1979) Genetic stability of 'Kenland' red clover during seed multiplic-.uion. Crop Sci 19: 429-434

Taylor NL, Cornelius PL, Long MG (1985a) Phenotypic recurrent selection for multiple-paned flower heads in red clover. Crop Sei 25: 4S9-494

Taylor NL, Diachun S. Ghabrial SA (1985b) Registration of red clover germpla.~m resistant to bean yellow mosaic virus. Crop Sci 25: 714-715

Taylor NL, Ghabrial SA, Diachun S. Cornelius PL (1986) Inheritance and backcross breeding of the hypersensitive reaction to bean yellow mosaic virus in red clover. Crop Sei 26: 68-74

Taylor NL, Smith RR. Anderson JA (1990) Selection in red clover for resistance to northern anthracnose. Crop Sei 30: 390-393

Taylor SG. Baltensperger DD. Quesenberry KH (1989a) Recurrent half-sib family selection for 2,4-D tolerance in red clover. Crop Sei 29: 1109-1114

Taylor SG. Shilling DG. Quesenberry KH (1989b) In vitro selection for 2,4-0 tolerance in red clover. Theor Appl Genet 78: 265-270

Thormann CE, Ferrira ME, Camargo LEA, TIvang J(':; Osborn TC (1994) Comparison of RFLP and RAPD markers for stimating genetic relationships within and among cruciferous species. Theor Appl Genet 88: 973-980

TInker NA, Fortin MG. Mather DE (1993) Random amplified polymorphie DNA and pedigree relationships in spring barley. Theor Appl Genet 85: 976-984

Tüfte JE, Smith RR (1992) Reaction of red clover to Aplzanomyces euteiches. Plant Dis 76: 39-42

Tofte JE, Smith RR, Grau CR (1991) Selection for resistance to Aphanomyces euteidles in red clover. Crop Sci 31: 1141-1144

Torres AM, MiIlan T, Cubero JI (1993) Identifying rose cultivars using random amplified polymorphie DNA markers. HonSci 28: 333-334

Vaillancourt RE, Weeden NF, Barnard J (1993) Isozyme diversity in the cowpea species complex. Crop Sci 33: 606-613 • 152 • VierIing RA, Nguyen HT (1992) Use of RAPD markers 10 determine the genetic diversity of diploid, wheat genotypes. Theor Appl Genet 84: 835-838

VierIing RA, Xiang Z, Joshi CP, Gilbert ML, Nguyen HT (1994) Genetic diversity arnong eIite Sorglzum Iines revealed by restriction fragment length polymorphisms and random arnplified polymorphie DNAs. Theor Appl Genet 87: 816-820

Wang GL, Peterson AH (1994) Assessment of DNA pooling strategies for mapping QTL

Weeden NF (1984) Distinguishing among white seeded bean cultivars by means of allozyme genotypes. Eup".ytica 33: 199-208

Weeden NF (1989) Applications of isozymes in plant breeding. Plant Breeding Rev 6: 11-54

Weeden NF, Emmo AC (1985) Isozyme charaeterization of Kentuck-y bluegrass cultivars. Can J Plant Sci 65: 985-94

Weeden NF, Gotùieb LD (1980) The genetics ofchloroplast enzymes. J Heredity 71: 392-396

Weeden NF, Marx GA (1984) Chromosomal locatic~'. of twelve isozyme loci in Pisum sativum. J Heredity 75: 365-370

Weeden NF, Marx GA (1987) Further genetie analysis and linkagé relationsbips of isozyme loci in the pea. J Heredity 78: 153-159

Weeden NF, Wendel JF (1989) Genetics of plant isozymes. In: DE Soltis DE, PS Soltis (eds), Isozymes in plant biology, pp 46-72. Dioscorides Press, Portland, Oregon

Weeden NF, Doyle JJ, Lavin M (1989) Distribution and evolution of a glucosephosphate isomerase duplication in the Leguminosae. Evolution 43: 1637-1651

Weir BS (1990) Inttaspecific differentiation. In DM Hillis, C Moritz eds, Molecular systematics, pp 373-410. Sinauer Associates Ine, Sunderland, Massachusetts

Welsh J, McClelland M (1990) Fmgerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res 18: 7213-7218

• 153 Wendel JF. Weeden NF (1989) Visualization and interpretation of plant isozymes. In: DE Soltis. PS Soltis (eds). Isozymes in Plant Biology. pp 5-45. Dioseorides • Press. Ponland. Oregon Whitkus R (1988) Modified version of GENESTAT: A program for eomputing genetie statistics from allelie frequency data. Plant Genet Newsletter 4: 10

Wilde J. Waugh R. Powell W (1992) Genetie fingerprinting of Theobroma clones using randomly amplified polymorphie DNA markers. Theor Appl Genet 83: 871-877

Williams CE. St-Clair DA (1993) Phenetie relationships and levels of variability detected by restriction fragment length polymorphism and random amplified polymorphie DNA analysis of cultivatd and wild accessions of Lycoprsicoll esculenrum. Genome 36: 619-630

Williams JGK, Kubelik AR, Livak J. Rafalski A. Tingey SV (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetie markers. Nucleic Aeids Res 18: 6531-6535

Williams JGI<, Hanafey MI<, RafaIski A. Tingey SV (1993) Genetic analysis using randomamplified polymorphie DNAmarkers. Methods Enzymo1218: 704-740

Wright S (1951) The genetical structure of populations. Ann Eugen 15: 323-354

Wu 1., Un H (1994) Identifying buffalograss [Buc/I/oe dacty/oides (Nutt.) Engelm.] cultivar breeding lines using random amplified polymorphie DNA (RAPD) markers. J Amer Soc Hon Sei 119: 120-130

Yu 1<, Pauls KP (1993a) Segregation of random amplified polymorphie DNA markers and strategies for molecular mapping in tetraploid a1falfa. Genome 36: 844-851

Yu 1<, Pauls KP (1993b) Rapid estimation of genetie relatedness among heterogeneous populations of alfalfa by random amplification of bulked genomie DNA samples. Theor Appl Genet 86: 788-794

Yu LX, Nguyen HT (1994) Genetievariation detected with RAPD markers among-:~ upland and lowland rice cultivars (Oryza sativa L).TheorAppl Genet87: 668­ 672

zamiT D, Ùldizinsky G (1984) Geneties of allozyme variants and linkage grciups in lentiI. Euphytica 33: 329-336

Zohary M, Heller D (1984) The genus Trifolium. The Israel Academy of Sciences • and Humanities, Jelusaiem, Israel 154 Appendix 1. Estimates uf total heterozygusity (Hr), within·cultivar heterozygosity • (Hs), coefficient of gene differentiation (Gsr) and between-cultivar heterozygosity (Dsr) of polymorphie isozyme loci in red clover.

Locus HT Hs Gsr DST

Aat-2 0.104 0.095 0.086 0.009

Aar-3 0.028 0.Q28 0.004 0.000

Adh-] 0.022 0.022 0.D10 0.000

Amy·] 0.117 0.108 0.077 0.009

Dia-] 0.004 0.004 0.000 0.000

Dia-3 0.005 0.005 0.020 0.000

Est-] 0.162 0.156 0.036 0.006

Est-4 0515 0500 0.028 O.Q1S

Gpi-2 0.230 0224 0.026 0.006

/dh-] 0.003 0.003 0.000 0.000

Mdh-3 0.038 0.037 0.014 0.001

Pgd·] 0.D18 0.ûl8 0.012 0.000

Pgd-2 0.344 0.333 0.031 0.011

"- Pgrn-2 0.019 0.019 0.000 0.000

Skd·/ 0.343 0.337 0.017 0.006

Mean 0.130 0.126 0.032 0.004 • 1SS Ir, o • c o =: =: o - o c =: o 8 -o -o C - ...... ::: - o o o o Cl o- o- o - o o -o o- =: ...... c o g o o 8 - • • o 8 8 o § o 8 8 § ::: - =: o - - - - o o ... c o o 8 o- o- -8 - o- - o... N - - - ... - - ­• ~ o - o- o 8- 8 ­ o 8- o- - 8- o- - o - - ... - = -o -o o o -o -o 8 o o o- o- o -o .,.,;... ="c E ± - - - - .;: c.. - - o ...... ~ o- -o 8- o o o- - o- o o- - - - ... g - - - - o- o- 8 8 o c o o Cl o o o o o o :.s >0 - 8 ... Cl - - ... C - - - - - o -o o - o o o 0 o o o o o o o o ­• 8 8 8 o o o o ------o - - -... - -8 8- - 8 -o 8- § § - - - - - g § ... - - - o- g - §- §- § 8- - 8- ~ - - - - mo- ~ - - - o- -o -o -o o o o -o o- - o ... o... - o- - - - 8- o­ 8- 8- -o 8- 8- § 8- 8- - 8- 8- o...------...- - ...- - o ------8 g g- o 8 8- § 8 § 8 § 8- 8 - - ...­ ------...- - - ...- o- o o o- -o ------o- -o ------o- 8 8 8 8 8 8 8 8 8 8- 8 -o 8- - ...l .,.~ z c. • Q - ~ Z • •

Appendlx 3. Rogers' (1972) genetic distances between 15 cultivars of red c10ver based on isozymc (below diagonal)

and RAPD (above diagonal) data.

Cv. DL ES HM HY HE KU MR MN MT NP OT PS pp SP ST DL 0.202 0.266 0.253 0.177 0.177 0.152 0.202 0.114 0.164 0.190 0.126 0.215 0.126 0.D9 ES 0.080 0.240 0.228 0.228 0.152 0.278 0.152 0.215 0.139 0.139 0.228 0.215 0.152 0.177 HM 0.073 0.072 0.291 0.240 0.240 0.291 0.266 0.215 0.278 0.202 0.266 0.253 0.240 0.2hh HY 0.075 0.059 0.064 0.304 0.228 0.304 0.253 0.266 0.240 0.190 0.304 0.2M 0.202 0.2711 HE 0.070 0.033 0.073 0.052 0.177 0.190 0.126 O. 190 0.240 0.240 0.202 0.19(J 0.215 n.190 KU 0.103 0.057 0.079 0.059 0.058 0.253 0.101 0.177 O.IM (J.IM 0.202 O.IM 0.101 n.12l! MR 0.085 0.044 0.077 0.063 0.045 0.056 O.IM 0.190 0.240 0.240 0.202 0.190 0.177 0.202 MN 0.106 0.063 0.107 0.064 0.057 0.054 0.06:> 0.190 0.164 0.215 0.228 O.IM 0.152 0.177 MT 0.101 0.058 0.084 0.067 0.063 0.045 0.0(10 0.069 0.202 0.202 0.215 0.202 0.114 O.IM NP 0.083 0.050 0.080 0.063 0.049 0.071 0.049 0.071 0.064 0.139 0.215 0.228 0.139 0.190 DT 0.091 0.065 0.079 0.035 0.068 0.074 0.072 0.055 0.060 0.063 0.240 0.177 0.139 (J.177 PS 0.098 0.061 0.094 0.046 0.049 0.042 0.058 0.047 0.068 0.076 0.061 0.215 0.177 0.152 PP 0.093 0.058 0.104 0.094 0.049 0.061 0.069 0.057 0.069 0.069 0.075 0.082 0.164 0.139 SP 0.074 0.046 0.Q70 0.050 0.048 0.052 0.062 0.053 0.058 0.062 0.055 0.055 0.058 0.152 ST 0.084 0.032 0.Q75 0.067 0.044 0.049 0.048 0.055 0.050 0.054 0.Q70 0.060 0.046 0.039

157