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thesis entitled

ISOZYME INHERITANCE AND DIVERSITY IN CHERRY

presented by

James Allen Beaver

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ISOZYME INHERITANCE AND DIVERSITY IN CHERRY

BY

James Allen Beaver

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Horticulture

1993

ABSTRACT
ISOZYME INHERITANCE AND DIVERSITY IN CHERRY
BY

James Allen Beaver

Inheritance was studied at seven isozyme loci using
seeds produced from crosses involving four sour cherries and
one open-pollinated ground cherry. Three alleles at
64Pgd-1 and two alleles at Adh-l, Idh-z, Lap-1, Pgm-z,
Pgi-z, and 6-Pgd-2 accounted for the allozyme polymorphisms
observed at these loci. Idh-z, Pgm-z, 6-Pgd-1, and 6-Pgd-2
exhibited disomic inheritance confirming the allotetraploid
hypothesis for sour cherry. Inheritance mode could not be
determined at Adh-l, Lap-1, or Pgi-Z. Adh-l, Idh-z, Pgi-z,
6-Pgd-1, and 6-Pgd-2 were not linked. Linkage could not be
determined for Lap-1 or Pym-2.

Isozyme diversity was evaluated for 67 sour, six
ground, 26 sweet, and 12 interspecific hybrid cherries from
the MSU germplasm collection. Tetraploid cherries exhibited
78% heterozygosity across seven enzyme loci compared to 19%
for sweet cherry. Principal coordinate analysis based on
isozyme diversity separated diploid sweet cherries from
tetraploid cherries, but failed to separate sour and ground

cherries.

To my wife Hannah
To my mother Joyce
To my little girl Nona Mae

For
Perseverance

Wisdom
Curiosity

iii

ACKNOWLEDGMENTS

I am grateful for the guidance of my major advisor Dr.
Amy Iezzoni and for the suggestions of my committee members
Dr. Jim Hancock and Dr. Steve Tonsor. I also thank Dr.
Steve Krebs for his instruction on starch gel
electrophoresis and inheritance mode theory, Dr. Carl Ramm
for his technical expertise on principal coordinate

analysis, and Colleen Mulinix for her assistance.

iv

List of Tables . . . . . .

List of Figures . . . . . .

Chapter I. Allozyme Inheritance in Prunus cerasus
Introduction . . . . . .
Literature Review . . . . .
Methods . . . . . . .
Results . . . . . . .
Discussion . . . . . .

Chapter II.

TABLE OF CONTENTS

Comparative Isozyme Diversity in Prunus

cerasus, P. avian, and P. fruticosa

Introduction . . . . .
Literature Review . . . .
Methods . . . . . .
Results . . . . . .
Discussion . . . . .
Appendix A. Raw Diversity Data . .
Appendix B. Similarity Matrix . .
Appendix C. Inheritance Computer Program
Appendix D. Similarity Computer Program
Appendix E. Principal Coordinate Analysis
Computer Program . .
Literature Cited . . . . .

vi

viii

11
16

23

27
29
41
44
65
77

_82
89

105

111

114

Table

Table

Table

Table

Table

Table

Table

2.

3.

4.

5.

LIST OF TABLES

Origin of cultivars and selections used
in the inheritance study. . . .
Expected gamete and progeny class
frequencies for disomic and tetrasomic
inheritance. . . . . . .
Segregation and chi-square values at five
polymorphic loci in sour cherry where the
expected phenotypic ratios for disomic
and tetrasomic inheritance are the same.
Segregation and chi-square values at five
polymorphic loci in sour cherry where the
expected phenotypic ratios for disomic
and tetrasomic inheritance differ. . .
Summary of isozyme diversity in the

genus Prunus. . . . . . .
A list of isozyme phenotypes for

each of 111 cherry selections and
'Redhaven' peach. . . . . .
Number of sweet, sour, ground, and

hybrid cherry selections which possessed

polymorphisms for eight enzyme systems. .

vi

12

15

19

20

30

45

48

Table 8.

Table 9.

Table 10.

- Percent of diploid and tetraploid

genotypes heterozygous for each of the
enzyme loci listed. . . . . . 59
Percent variation accounted for by the
first five principal coordinates for
PC01 (Figure 6). . . . . . 59
Percent variation accounted for by the
first five principal coordinates for

PC02 (Figure 7). . . . . . 63

vii

Figure

Figure

Figure

Figure

Figure

Figure

1.

2.

LIST OF FIGURES

Allozyme phenotypes of sour cherry
demonstrate di-allelic segregation at
(A) Pgi-z, (B) Lap-1, (C) Adh-l, and
(D) Idh-Z; di-allelic fixed
heterozygosity at (E) Pym-2 and

(F) 6-Pgd-2 (alleles 60 and 48); and
tri-allelic segregation at (F) 6-Pgd-1.
(A) PGI, (B) MDH, (C) PGM, (D) 6-PGD,
(E) SKDH, and (F) PX isozyme patterns
in cherry. . . . . .
Isozyme phenotypes exhibited by cherry
selections and 'Redhaven' peach for (A)
Pgi-z, (B) rah-2, (C) Lap-1, (D) MDH,
and (E) Pgm-Z. . . . .
Isozyme phenotypes exhibited by cherry
selections and 'Redhaven' peach for (A)
6-Pgd-1 and 6-Pgd-2 and (B) Skdh-l.
Isozyme phenotypes exhibited by cherry
selections for cathodal PX. . .
Principal coordinate analysis for PCl
vs. PC2 for selected cherry genotypes
(PC01). . . . . . .

viii

17

50

52

55

57

61

Figure 7.

Principal coordinate analysis for PCI

vs. PC2 for only selections containing
Prunus cerasus in their pedigrees

(PC02) . . . . . . .

ix

64

Chapter I. Allozyme Inheritance in Prunus cerasus

INTRODUCTION

Controversy exists as to whether sour cherry (Prunus
cerasus L., 2n=4x=32) is an allotetraploid, autotetraploid,
or a segmental allotetraploid. Conclusions concerning the
type of polyploidy were largely based on cytogenetic and
morphological criteria. Although morphological and
cytological evidence is useful in understanding polyploidy,
these criteria can not be conclusively related to the type
of polyploidy. Both allo- and autotetraploids may exhibit
regular bivalent pairing and a lack of multivalent formation
(Krebs and Hancock, 1989; Soltis and Rieseberg, 1986).
Allelic segregation, specifically the determination of
disomic or tetrasomic inheritance, is the most definitive
way to distinguish polyploid type (Krebs and Hancock, 1989;
Soltis and Rieseberg, 1986).

Because of their codominant expression, isozyme loci
have been commonly used as genetic markers to study
inheritance in polyploids. However, allozymes have not been
used to determine polyploid type in sour cherry. Isozyme
studies in sour cherry to date have been limited to
zymograms, patterns, or descriptions of putative alleles
from different cultivars (Fernqvist and Huntrieser, 1988;
Hancock and Iezzoni, 1988; Kaurisch et al., 1988, 1991).

2

3
These studies did not conduct progeny tests. Based on
phenotypic segregation of allozyme patterns, genetic models
can be formulated consisting of true alleles and inheritance
type in tetraploid sour cherry.

An understanding of sour cherry inheritance would
clearly determine sour cherry’s evolutionary origin and aid
in predicting the likelyhood of desired genotypes from
crosses. Genotypic frequencies of progeny differ with
inheritance type.

The objectives of my research were to identify alleles
and loci of marker enzyme systems, and to use the
segregation patterns of these loci to determine polyploid

type in sour cherry.

LITERATURE REVIEW

The most widespread hypothesis for the origin of sour
cherry is that it arose from hybridization between the
diploid sweet cherry (P. avium L., 2n=2x=16) and the
tetraploid ground cherry (P. fruticosa Pall., 2n=4x=32).
Ground cherry is a spreading shrub, reaching a height of
about one meter, which has small leaves 20-50 mm long, small
white flowers, and small red-purple fruit (Bailey and
Bailey, 1976; Hillig and Iezzoni, 1988; Olden and Nybom,
1968). Sweet cherry is a tree growing 18 to 24 m tall with
leaves 60-150 mm long, white flowers larger than those of
ground cherry, and fruit ranging from small in wild types to
approximately 25 mm in diameter in some cultivars (Bailey
and Bailey, 1976; Hillig and Iezzoni, 1988; Olden and Nybom,
1968). Morphological data for sour cherry, ranging from
that of sweet cherry to ground cherry, suggests that sour
cherry is an interspecific hybrid between these two species
(Hillig and Iezzoni, 1988).

Ground cherry, considered the most cold hardy of the
cherry species, originated and exists in maximum diversity
in the former Soviet Union reaching as far north as the 60th
parallel (Kolesnikova, 1975). In contrast, sweet cherry,
less cold hardy than ground cherry, is found in greatest

concentration between and south of the Caspian and Black

5

Seas, but is also found wild thoughout Europe and into
southern Russia (Hedrick, 1915). The center of diversity
for sour cherry is eastern Europe and Russia, where the
habitat of sour cherry overlaps with that of sweet and
ground cherry on the southwest and northeast, respectively.

Morphology and chemotaxonomy suggest that sour cherry
is a polyploid hybrid of sweet and ground cherry.
Olden and Nybom (1968) hybridized ground cherry with several
varieties of sweet cherry in an attempt to resynthesize sour
cherry. Morphological characters (flowers, leaves, fruit,
and tree morphology), biochemical characters (fruit
anthocyanins and leaf phenolics), and disease responses were
examined in the progeny and compared to similar data
collected from the parents and sour cherry. Data collected
from the hybrids were intermediate to the parental data and
strikingly similar to the sour cherry data.

Hillig and Iezzoni (1988) studied morphological
traits of sour cherry with principal component analysis to
examine variation in sixteen cultivars. Data were collected
on leaf, flower, and fruit characters and assembled into
multivariate observations. These observations were then
plotted to create a three-dimensional scatter diagram whose
three axes each correspond to a different principal
component of character variance. The scatter diagram of the
sixteen sour cherry cultivars presented a gradation of
morphology between the two proposed progenitors, sweet and
ground cherry (Hillig and Iezzoni, 1988).

Additional studies support the hypothesis that sour

6
cherry is derived from sexual polyploidization between
ground cherry and an unreduced gamete from sWeet cherry.
Malate dehydrogenase bands from sweet and ground cherry were
expressed codominantly in the sour cherry cultivars tested
(Hancock and Iezzoni, 1988). Chloroplast RFLP’s tested to
date indicate that those from ground cherry are similar to
the sour cherry cultivar 'Montmorency’ and different from
sweet cherry (Iezzoni et al., 1989). Unreduced pollen is
produced in small quantities by numerous sweet cherry
cultivars (Iezzoni and Hancock, 1984).

Other evidence might support an autotetraploid or
segmental.allotetraploid origin of sour cherry. Several
landraces of sour cherry are self-incompatible;
autopolyploids would favor such a breeding system.
Additionally, meiotic analysis of sour cherry reveals a high
percentage of infertility due to unbalanced gametes. Even
quadrivalent formation occurs at a low frequency (Galletta,
1959; Hruby, 1939).

The hypothesis that sour cherry is an autotetraploid is
not inconsistent with morphological and isozyme data. Gene
flow between sour cherry and sweet and ground cherry may be
a significant evolutionary factor regardless of the
polyploid origin of sour cherry. Interspecific hybrids
between sour cherry and ground cherry have been reported in
regions where these species coexist. Additionally, if the
three cherry species in the Eucerasus section (sweet,
ground, and sour cherry) arose from a common diploid

ancestor as suggested by Raptopoulos (1941), they would be

7
expected to share morphological and isozyme homology. An
autopolyploid origin of sour cherry could be hypothesized if
sour cherry exhibited tetrasomic inheritance and if tri-

and/or tetra-allelic loci were identified.

Inheritance
Genetic data is essential in order to distinguish allo-

from autopolyploidy. Allopolyploids exhibit disomic
inheritance which may result in fixed heterozygosity, while
autotetraploids exhibit tetrasomic inheritance. These
different segregation ratios are dependent upon the
chromosomal pairing relationships defined by their genomic
origin.

Allotetraploids arise from interspecific hybridization
of two diploid genetic complements, one from each of two
divergent progenitors. Homoeologous chromosomes can
structurally differ to some degree. During meiosis I,
homologous chromosomes preferentially pair and segregate
independently of their homoeologous counterparts. However,
homoeologous chromosome pairings or heterogenetic
associations may occur infrequently (Stebbins, 1947).
Allotetraploids exhibit disomic inheritance, a two locus
model for duplicate gene segregation. True allotetraploids
rarely exhibit tetrasomic ratios and multivalent
associations (Stebbins, 1947); they possess disomic
inheritance and often exhibit fixed heterozygosity (Krebs
and Hancock, 1989; Soltis and Rieseberg, 1986).

Fixed heterozygosity is one possible result of disomic

8
inheritance. Roose and Gottlieb (1976) define fixed
heterozygosity as heterozygous phenotypes which do not
segregate at meiosis. This occurs when each of the two
ancestral genomes in an allotetraploid is homozygous for a
different allele. For example, aabb produces only ab
gametes and is therefore fixed heterozygous, while abab
produces aa, ab, and DD gametes in a 1:2:1 ratio and is not
fixed heterozygous.

However, a more inclusive definition for fixed
heterozygosity is that only heterozygous gametes are formed
at meiosis which may or may not be all the same genotype.
This would include the situation where one ancestral genome
in an allotetraploid is heterozygous and the other is
homozygous for a different allele resulting in a tri-allelic
genotype. For example, aabc produces only the heterozygous
ab and ac gametes and is therefore also considered fixed
heterozygous, while abac produces one-quarter homozygous aa
gametes and is not fixed heterozygous.

Autotetraploids inherit two diploid genetic complements
from a common progenitor or closely related progenitors.
Their chromosomes are structurally similar; therefore, the
four homologous chromosomes randomly pair during meiosis. A
gene on each of four homologues segregates randomly at a
single locus (Krebs and Hancock, 1989). Autotetraploids,
and to a lesser extent segmental allotetraploids, exhibit
tetrasomic inheritance and usually multivalent associations
(Stebbins, 1947).

Criteria for diagnosing segmental allotetraploids fall

9

in between that for true allotetraploids and autotetraploids
because the two ancestral genomes involved are less
divergent than in true allotetraploids. Chromosome pairing
is more random due to similarities between homoeologous
chromosomes. This results in frequencies of homoeologous
pairing and multivalent formation that are higher than
expected in true allotetraploids and lower than expected in
autotetraploids. The two ancestral genomes of a segmental
allotetraploid lose their identities as a result of partial
or complete homoeologous chromosome pairing and
recombination of this homoeologous genetic material
(Stebbins, 1947).

To use allozyme polymorphisms in an inheritance
study, the unit structure of marker enzymes must be
understood. Generally, this unit structure is conserved
throughout different species. Studies involving Prunus
species report leucine amino peptidase (LAP) (Byrne and
Littleton, 1988; Hauagge, Kester, Arulsekar, Parfitt, and
Liu, 1987), and phosphoglucomutase (PGM) (Byrne and
Littleton, 1988, 1989a, 1989b; Chaparro et al., 1987:
Hauagge, Kester, Arulsekar, Parfitt, and Liu, 1987; Hauagge,
Kester, and Asay, 1987) to be monomeric. Other enzymes are
reported to be dimeric: alcohol dehydrogenase (ADH)
(Kaurisch et al., 1991), isocitrate dehydrogenase (IDH)
(Kaurisch et al., 1991; Mowrey et al., 1990a),
phosphoglucose isomerase or glucose phosphate isomerase
(PGI, same as GPI) (Byrne and Littleton, 1988; Hauagge,

Kester, Arulsekar, Parfitt, and Liu, 1987; Hauagge, Kester,

10
and Asay, 1987; Parfitt et al., 1985), and
6-phosphogluconate dehydrogenase (6-PGD) (Byrne, 1989a,
1989b; Byrne and Littleton, 1989b; Chaparro et al., 1987;
Mowrey et al., 1990b).

Inheritance studies involving segregating progenies
diagnose the number of loci that encode isozymes and the
number of alleles per locus that encode allozymes of a
marker enzyme. By observing segregation, the number of
zones of activity can be determined, and the bands at each
zone or locus can be designated as allelic or heteromeric.
Genotypes of cultivars and other clones can thus be
determined for marker enzyme systems.

Allozymes are a useful tool to study tetraploid
inheritance because the alleles that encode them are
codominantly expressed and they can be used as genetic
markers to distinguish between disomic and tetrasomic
inheritance. Inheritance type has not been diagnosed in
sour cherry using the allozyme phenotypes of segregating

alleles at marker loci.

11

METHODS

El§n§_M§£§Ii§l

Seeds for this study were obtained from crosses using
four sour cherry parents and one open-pollinated ground
cherry parent (Table 1). Standard self— and cross-
pollination techniques for cherry were used to produce the
seeds (Fogle, 1975).

Horizontal starch gel electrophoresis was performed on
extracts from young leaves and dormant vegetative buds of
parent trees. Isozyme data on progeny were obtained from
individual seeds prior to germination. Leaves, buds, and
seeds were stored at approximately 2 C with moist paper
towels in sealed plastic bags to prevent desiccation until
they were used. Seeds were removed from their exocarps and
mesocarps and treated with a fungicide suspension prior to
storage. Endocarps were removed just prior to extraction.
All material was macerated on ice the day of electrophoresis
using the procedures of Krebs and Hancock (1989) with slight
modification. The extraction buffer was maintained at pH
7.5 rather than adjusted to pH 8.0 and nylon screens were

not used during extraction.

12

Table 1. Origin of cultivars and selections used in the inheritance

 

 

study.
Cultivar/Selection Origin
Montmorency local selection from France
Meteor Montmorency x Vladimir
I 24(41) P. fruticosa open-pollinated
II 13(36) Cigany Meggy open-pollinated
II 15(4) Montmorency x M63 (Pandy x Nagy

Gobet)

 

13
W

Phosphoglucose isomerase (PGI, E.C.5.3.1.9), alcohol
dehydrogenase (ADH, E.C.1.1.1.1), isocitrate dehydrogenase
(IDH, E.C.1.1.1.42), phosphoglucomutase (PGM, E.C.5.4.2.2),
and 6-phosphogluconate dehydrogenase (6-PGD, E.C.1.1.1.44)
were resolved by six hours of electrical current on
morpholine-citrate pH 6.1 gels (Clayton and Tretiak, 1972).
Electrical current was maintained at 50 mA during elution,
the first 30 minutes of electrophoresis, and as close as
possible to 65 mA without exceeding 300 V for the rest of
electrophoresis. Leucine aminopeptidase (LAP, E.C.3.4.11.1)
was resolved by five hours of current (50 mA, not exceeding
300 V) on tris-citrate/lithium-borate pH 8.3 gels
(Scandalios, 1969). Gels consisted of 12% hydrolyzed potato
starch.

Stain recipes were prepared at one-half (50 ml) the
volume reported per gel slice. IDH was assayed as described
by Soltis et al. (1983). All other enzymes were assayed
with slight modification as reported by Arulsekar and
Parfitt (1986). The LAP substrate, leucyl-naphthyl amide
HCl, was dissolved in 2.5 m1 of N,N-dimethyl formamide per
gel slice before adding it to the stain solution. Tris-HCl
buffer pH 8.5 rather than pH 8.0 was used in the 6-PGD
assay.

Relative mobilities were calculated for isozymes using
the ratio of the particular isozyme's migration distance in
mm from the origin to that of the most anodal isozyme of the

enzyme system (Mowrey and Werner, 1990). Each isozyme was

14
named by multiplying its relative mobility by 100. The most
anodal isozyme of each enzyme system was referred to as 100,
while others of the same system were named as a fraction of
this number. Loci of an enzyme system were numbered
progressively beginning with 1 in the most anodal position.
Letters representing alleles in Tables 2, 3, and 4 are used

for convenience only and are not the assigned allelic names.

5! !' !' J E 2
The chi-square goodness-of-fit test was used to compare
observed progeny phenotypes to expected classes (Table 2)
fer each inheritance mode. Phenotype rather than genotype
was scored since it does not require a subjective
determination of gene dosages based on visual assessment of
differential band staining intensity among heterozygotes.
For those loci which did not fit the expected 3:1 ratio, a
2:1 ratio was tested. The Inheritance Computer Program
(Appendix C) written by me was used to aid in proposing
genetic models and in calculating chi-square values.
Linkage was studied using the chi-square test of

independence.

5
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16

RESULTS

Seven loci resolved well and exhibited good activity
using horizontal starch gel electrophoresis: Pgi-z, Lap-1,
Adh-l, Idh-z, Pym-2, 6-Pgd-1, and 6-Pgd-2 (Figure 1).
Activity slightly anodal to Idh-Z and Pym-2 was observed but
was not studied due to inconsistent resolution. Kaurisch et
a1. (1988) designated these anodal bands Idh-l and
Pgm-l .

Two of the crosses for Pgi-Z (Table 3) segregated in a
1:1 phenotypic ratio and 'Meteor’ selfed fit a 3:1 ratio,
supporting the existence of two alleles. 'Montmorency'
selfed fit a 2:1 segregation ratio for Pgi-z, rather than
the expected 3:1. Two Lap-1 phenotypes (Table 3) segregated
in expected 3:1 ratios indicating the presence of two
alleles.

Segregation for two alleles at the Adh-l locus (Table
3) fit expected 3:1 ratios for 'Montmorency’ selfed and
’Meteor’ selfed. Insufficient progeny were available to
reject either mode of inheritance for the crosses 'Meteor' x
I 24(41) and I 24(41) x II 13(36) (Table 4). However,
progeny were all heterozygous for the 100 and 56 alleles at
Adh-l for I 24(41) x II 13(36) because II 13(36) exhibited

fixed heterozygosity at this locus.

 

Figure 1.

17

Allozyme phenotypes of sour cherry demonstrate
di-allelic segregation at (A) Pgi-Z, (B) Lap-1,
(C) Adh-l, and (D) Idh-Z: di-allelic fixed
heterozygosity at (E) Pgm-2 and (F) 6-Pgd-2
(alleles 60 and 48): and tri-allelic segregation
at (F) 6-Pgd-l. PGI, ADH, IDH, and 6-PGD are
dimeric enzymes producing intralocus heterodimers
for heterozygous genotypes. LAP and PGM are
monomeric.

 

A V
--100
' I ‘ L...

 

D

19

Table 3. Segregation and chi-square values at five polymorphic loci in sour cherry where the
expected phenotypic ratios for disomic and tetrasomic inheritance are the same.

 

 

Cross Observed Progeny
Locus Cross Type' Phenotypes Ratios Tested' (1!" )'
ab a
Pgi-Z Montmorency selfed 1 162:76 3:1 (6.10") 2:1(0.20)
Meteor selfed 1 181:73 3:1 (1.90)
Meteor x I 24(41) 2 75:85 1:1 (0.63)
Meteor x II 15(4) 2 86:62 1:1 (3.89')
ab a
Lap-1 Montmorency selfed 1 83:24 3:1 (0.38)
Meteor selfed 1 26: 6 3:1 (0.67)
a ab b
Adh-I Montmorency selfed 1 42:165 1:3 (2.45)
Meteor selfed 3 133:43 3:1 (0.03)
a ab
[db-2 Montmorency selfed 1 55:152 1:3 (0.27)
Meteor selfed 1 79:175 1:3 (5.04') 2:1 (0.55)
Meteor x I 24(41) 2 73: 84 1:1 (0.77)
Meteor x II 15(4) 1 43:105 - 1:3 (1.30)
cd d
6-Pgd-2 Montmorency selfed 12 153:85 3:1 (14.57') 2:1 (0.62)

 

'Cross types and expected phenotypic ratios are defined in Table 2.

'Yate's Correction for Continuity was used for chi-square tests with 1 degree of freedom.

(tabs - e :- 0.5)2
I": of;

" Significance at 5‘ for deviation from expected ratio.

20

Table 4. Segregation and chi-square values at five polymorphic loci in sour cherry where the
expected phenotypic ratios for disomic and tetrasomic inheritance differ.

 

 

 

Cross Observed Progeny x5
Locus Cross Type’ Phenotypes Disomic’ Tetrasomic‘
a ab b
Adh-I I 24(41) x II 13(36)" 4 O: 75: 0 0 4.41
Meteor x I 24(41) 5 145:15 1.43 0.23
a ab
Idh-Z I 24(41) x II 13(36)" 6 2:73 -- 10.58'll
a ab b
Pym-2 Montmorency' selfed 7 O:122:0 0 7.18’
Meteor" selfed 7 O:128:0 0 7.53'
a ac ab abc bc b
6-Pgd-l Montmorency selfed 8 13: 206: 19 1.40 30.96'
Meteor selfed 9 10: 48:137:40:18 4.48 25.14'
I 24(41) x II 13(36) 10 6: 29: 32: 6: 2 3.25 9.35
Meteor x I 24(41) 9 ll: 26: 76:33:12 1.53 25.76'
Meteor 8 II 15(4)" 11 22: 32: 79: O 1.99 21.91°
c cd d
6-Pgd-2 Meteor" selfed 13 0:254: 0 0 14.94'
I 24(41) x II 13(36)“ 14 75: O O 6.79'
Meteor“ x I 24(41) 15 156: 4 -- 7.13'
Meteor” x II 15(4) 16 137:11 -- 9.09°

 

'Cross types and expected phenotypic ratios for disomic and tetrasomic
in Table 2.

inheritance are defined

'Yate's Correction for Continuity was used for chi-square tests with 1 degree of freedom.

tabs - I - 0.5 3
12.: I as; )

" Significance at 54 for deviation from expected ratio

”Exhibits a fixed heterozygous genotype

21

Three of the Idh-z crosses (Table 3) fit the proposed
3:1 or 1:1 models. 'Meteor' selfed fit a 2:1 alternate
ratio. Tetrasomic inheritance was rejected at Idh-z (Table
4) for the cross I 24(41) x II 13(36). Goodness-of-fit
could not be tested for disomic inheritance due to the
observation of two unexpected homozygotes resulting in an
undefined chi-square equation. The two homozygotes may be
the result of pollen contamination or heterogenetic
associations in II 13(36). Without these two homozygotes,
the data fit the proposed model for fixed heterozygosity in
II 13(36).

All Pym-2 and 6-Pgd-1 crosses (Table 4) fit the
proposed models for disomic inheritance involving two and
three alleles per locus, respectively. ’Montmorency' and
'Meteor' exhibited fixed heterozygosity at Pym-2.
Tetrasomic inheritance was rejected for all crosses except
I 24(41) x II 13(36) at 6-Pgd-1. Insufficient progeny were
obtained to reject either mode of inheritance for this
cross.

None of the crosses tested segregated for Pym-2.
However, all of the 26 sweet cherry cultivars studied had
only the Pym-2’” allele and three of four ground cherry
clones studied had only the Pym-2” allele (Chapter II).
Therefore, the two bands of Pym-2 in 'Montmorency' and
'Meteor’ were considered allelic with both cultivars
exhibiting fixed heterozygosity.

'Montmorency' selfed data for 6-Pgd-2 (Table 3) fit a

2:1 instead of the 3:1 ratio expected with both disomic and

22

tetrasomic inheritance. The 6-Pgd-2 locus (Table 4) in
'Meteor' selfed and in I 24(41) x II 13(36) exhibited fixed
heterozygosity due to disomic inheritance. The final two
crosses involving 'Meteor’ were again potentially diagnostic
of disomic inheritance if the homozygotes are considered to
be the result of pairing among homoeologous chromosomes.
Tetrasomic inheritance was rejected for all 6-Pgd-2 crosses.

PGI, ADH, IDH, and 6-PGD are dimeric in cherry as
indicated by the presence of heteromeric bands (Figure 1).
LAP and PGM did not exhibit heteromers and are thus monomers
in cherry.

Linkage between loci was tested using five segregating
loci in selfed 'Montmorency’ progeny. Lap-1 and Pym-2 were
not tested for linkage because they were assayed from
different seeds than the other loci and because Pgm-Z was
fixed heterozygous in 'Montmorency' and 'Meteor.’ None of
the loci examined were linked, indicating that they may be
located on different chromosomes or chromosome arms in the

sour cherry genome.

23

DISCUSSION

Polymorphisms and putative alleles presented by
Kaurisch et al. (1991) for 6-Pgd-2, Idh-z, Pym-2, Adh-l, and
Lap-1 for ‘Montmorency' and ‘Meteor' are similar to my
results. However, in my analysis, ‘Montmorency’ and
‘Meteor' have three bands corresponding to two alleles for
Pgi-Z rather than one allele as proposed by Kaurisch et al.
(1991). Their Pgi-Z band appears to correspond to my
Pgi -2’°° .

In my analysis, ‘Meteor' has five bands for 6-Pgd-1
representing three alleles and three intralocus
heterodimers. Kaurisch et a1. (1991) presented only three
bands which most likely correspond to my 6-Pgd-Im’and 1“
alleles and the heterodimer at 1“. Fernqvist and Huntrieser
(1988) only presented two bands for ‘Meteor' at the putative
6-Pgd—1 locus. These discrepancies could either be caused
by variation due to different buffer systems or differences
between the clones used. However, unlike the previous
studies, progeny segregation was used in my study to
diagnose true alleles and reliably determine the allele
dosage of the parents at all the loci presented.

Segregation data for ‘Meteor' at the Pgi-Z and 6-Pgd-1 loci
were consistent with two and three segregating alleles,

respectively.

24

The skewed 2:1 ratios for Pgi-z, Idh-Z, and 6-Pgd-2 may
have resulted from gametophytic selection. These ratios
only occurred when the progeny were produced by selfing
'Montmorency’ and 'Meteor.’ ’Montmorency’ and ’Meteor' have
been shown to be partially self-incompatible (Lansari and
Iezzoni, 1990) which is defined as a majority of the pollen
grains stopping tube growth prematurely in the style,
resembling gametophytic incompatibility. This partial
self-incompatibility may have caused the progeny class
frequencies to deviate from the expected models.
Additionally, the 2:1 ratios could be caused by zygotic
lethality due to inbreeding depression.

Enzyme unit structure in cherry is monomeric for LAP
and PGM and dimeric for ADH, IDH, PGI, and 6-PGD. This
confirms the results of other Prunus studies and
demonstrates that enzyme unit structure is conserved in the
genus Prunus.

Segregation data confirmed the existence of two alleles
at the Pgi-z, Lap-1, Adh-l, Idh-Z, Pgm-Z, and 6-Pgd-2 loci
and three alleles at the 6-Pgd-1 locus. Linkage to Lap-1
and Pym-2 was not testable, while the other loci were found
to independently assort from one another. Disomic
inheritance has been clearly shown to occur at Idh-z,

Pym-2, 6-Pgd-1, and 6-Pgd-2, consistent with an
allotetraploid origin of sour cherry.

The possibility remains that sour cherry could be a
segmental allotetraploid exhibiting occassional homoeologous

chromosome pairing. Infrequent homoeologous pairing could

25
account for the low level of homozygous offspring for Idh-Z
from the cross I 24(41) x II 13(36) and for 6-Pgd-2 from the
crosses Meteor x I 24(41) and Meteor x II 15(4). However,
true allotetraploids can also exhibit heterogenetic
associations (Stebbins, 1947).

A maximum of four nonhomologous chromosomes in sour
cherry (x=8) have been clearly demonstrated to undergo
disomic inheritance during meiosis. The molecular data
presented herein support the previously published
morphological, chemotaxonomical, and geographical evidence

that sour cherry is an allotetraploid.

Chapter II. Comparative Isozyme Diversity in
Prunus cerasus, P. avium, and P. fruticosa

INTRODUCTION

Sour cherry is an allotetraploid based on allozyme
inheritance data from Chapter I, with sweet and ground
cherry as its proposed progenitor species based on shared
isozyme, chloroplast RFLP, and morphological homology
between the three cherry species.

Unlike self-pollinating allopolyploids such as wheat,
the two ancestral genomes in sour cherry are heterozygous at
many loci. In Chapter I, some genotypes clearly exhibited
fixed heterozygosity characterized by homozygosity within
the ancestral genomes and others exhibited assortment in
agreement with disomic inheritance due to heterozygous
genomes at four isozyme loci.

The mating behavior in cherry, regulated by a
gametophytic self-incompatibility system in sweet cherry and
commonly a self-incompatibility system in sour cherry,
suggests that sour cherry would exhibit a high level of
heterozygosity due to outcrossing. Therefore, sour cherry
could potentially have four different alleles at a locus.

To assess isozyme diversity in sour cherry, it is
necessary to screen a very diverse collection. The MSU sour
cherry germplasm collection includes material collected in
Yugoslavia, Bulgaria, Hungary, Romania, Poland, and portions

27

28
of the former Soviet Union.

The objectives were twofold. Can sour cherry
individuals be identified which exhibit a tri- or tetra-
allelic condition at various isozyme loci? How does the
isozyme diversity in sour cherry compare to that identified

in the limited sweet and ground cherry collection?

29

LITERATURE REVIEW

Prunus species have been analyzed for isozyme
polymorphism using horizontal starch gel electrophoresis by
numerous authors. The number of polymorphisms observed and
a genetic description of the diversity are presented for
each enzyme system studied (Table 5). The symbol "-/-"
denotes that the reference did not indicate the number of
alleles per locus and the number of loci encoding the enzyme
system, most likely due to the lack of diagnostic
segregation data, and often in the case of MDH, the complex
nature of its inheritance. Enzymes evaluated include acid
phosphatase (APS), aconitase (ACON), ADH, aspartate amino
transferase or glutamate oxaloacetate transaminase (AAT,
same as GOT), catalase (CAT), diaphorase (DIA), esterase
(EST), glutamate dehydrogenase (GDH), glutathione reductase
(GRD), IDH, LAP, malate dehydrogenase (MDH), peroxidase
(PX), PGM, PGI, 6-PGD, shikimate dehydrogenase (SKDH), and
triose phosphate isomerase (TPI).

Results for ten enzyme systems have been reported in
almond (P. amygdalus) leaf tissue and eight enzyme systems
involving pollen (Table 5). AAT, IDH, LAP, PGM, and PGI
were studied in the sporophytic and gametophytic

generations. The number of polymorphisms varied from one

 

 

Table 5. Summary of isozyme diversity in the genus Prunus.
Number of Number of
W EnzwmmusmulleleuLMs—References:
Subgenus Anygdalus
dulcis or APS’ -/1 1
asygdalus’
(ALMOND) ADH’ -/l, -/2, -/3 l
AAT 2/1, 2/2 2, 3, 4, 19
AAT’ 2/1 1
cm" -/1‘ 1
IDH 3/1 19
IDH’ -/l', -/2 1
up 3‘/1, 2/2 2, 3, 4,
13, 19
LAP’ 2/1 1
non 2/1, 2/2 2, 13
PX 1/1, 1/2‘ l3, l9
PGI 1/1, 2/2 2, 3, 4,
13, 19
PGI’ 1/1, 3/2 1
PGM 2/1, 3/2 2, 3, 4.
13, 19, 20
PGM’ 2/1, 2/2 1
31(1):: 1/1 19
6-PGD 1/1, 2/2 2, 4, 13,
19, 20
TPI 1/1, 1/2 13
argentea AAT 1/1, 1/2 4
PGI 1/1, 1/2 4

Table 5 (cont'd).

31

 

 

. Number of

fiDfiQlfifi, EDZYEE

bucharica AAT 1 1/1,
PGI 1 1/1,

davidiana AAT 1 1/1,
IDH 1 2/1
LAP 2 1/1:
9x 2 2/1.
PGI 2 1/1.
PGM 2 2/1:
SKDH 2 2/1
6-PGD 1 1/1,

kansuensis AAT 2 1/1,
IDH 1 1/1
LAP 1 1/1,
PX 1 1/1,
PGI 1 1/1,
PGM 1 1/1.
SKDH 1 1/1
6-PGD 1 1/1.

mire AAT 1 1/1,
IDH 1 1/1
LAP 1 1/1,
PX 1 1/1,
PGI 1 1/1,

Number of
Polvmgrph1Sm§_____Allsl3§_£_LQQH§—————R§£§I§DQQ§

1/2

1/2

1/2

2/2

1/2‘

2/2

1/2

1/2

2/2

1/2

1/2‘

1/2

1/2

1/2

1/2

1/2

1/2‘

1/2

19

19

19

19

19

19

19

19

19

19

19

19

19

19

19

19

19

19

19

19

19

Table 5 (cont'd).

32

 

PGM

SKDH

6-PGD

persica ACP’
(PEACH)

EST
EST’
GDH’

GRD

MDH’
PX

PGI

PGI’

Number of Number of
1 1/1, 1/2
1 1/1
1 1/1, 1/2
2 2/1
1 -/-
1 1/1, 1/2
1 -/-
3 2/1
1 -/-
3 3/1
1 -/-
3 2/1
1 -/—
1 -/-
3 2/1
3 -/1’, 2/2, -/35
1 1/1, 1/2
1 -/-
6 3/1
4 -/l, -/2, -/3, -/4‘
3 1/1, 2/2‘
1 1/1, 1/2
1 -/-

19
19

19

17
17
2, 9, 19
17
14

17

17

17

9, 15, 19

2, 9, 13,

17
9, 13, 19

2, 9, 13,
19, 21

17

Table 5 (cont’d).

33

 

5nsciea_______Emzxme_____29lxmcrnhisma_____Al1eles_L_Locus_____Befsrence§

persica
ssp.
ferganensis

tangutica

PGM

PGM’
SKDH
SKDH’

6-PGD

6-PGDJ

TPI

AAT

IDH

SKDH

6-PGD

AAT

PGI

Subgenus Cerasus

aviue

(SWEET CHERRY)

ACON

ADH

IDH

Number of Number of
1 1/1. 1/2
1 -/-

2 2/1
1 -/-
1 1/1. 1/2
1 -/-
1 1/11 1/2
1 1/1. 1/2
1 1/1
1 1/1, 1/2
1 1/1, 1/2‘
1 1/lr 1/2
1 1/1, 1/2
1 1/1
1 1/1. 1/2
1 1/1, 1/2
1 1/1, 1/2
4 1/1, 3/2
1 1/1
3 1/1, 2/2
1 1/1

2, 9,
19, 20

17
17

2. 9.
19, 20

17

13

19
19
19
19
19
19
19

19

6, 18
18
6, 18

18

13,
, 21

19

13,

34

Table 5 (cont'd).

 

Number of Number of

5necisa_______Enzxme_____29lxm9znhisns_____Allele§_2_Locus_____neference§

non 1 -/- 7

per 2 1/1, 2/2 ' 6, 18

pan 3 1/1, 3/2 18

6-PGD 6 2/1, 2/2 6, 8, 18
canescens ACON 1 1/1, 1/2 6

IDH 1 1/1, 1/2 6

MDH’ 1 -/- 7

PGI 1 1/1, 1/2 6

6-PGD 1 1/1, 1/2 6
cerasus ACON 4 1/1, 3/2 6, 18
(SOUR CHERRY)

non 2 2/1 12, 18

IDH 2 1/1, 2/2 6, 12, 18

LAP 2 2/1 12, 18

non 1 -/- 7

PGI 3 1/1, 3/2 6, 12, 18

PGM 4 1/1, 3/2 12, 18

6-PGD 5-6 3/1, 2/2 6, 8, 12,

18

fruticosa ACON 1 1/1, 1/2 6
(GROUND CHERRY)

“D” 1 -/- 7

per 1 1/1, 1/2 6

6-PGD 1 1/1, 2/2 6

incisa MDH’ 2 -/- 7

35

Table 5 (cont'd).

 

Number of Number of
E . E E J 1' E1] 1 I I E E
eahaleb MDH’ 1 -/- 7
subhirtella ACON 1 1/1, 2/2 6
MDH' 2 -/- 7
PGI 1 1/1, 1/2 6
6-PGD 1 1/1, 1/2 6
Subgenus Prunus
areaniaca LAP 1 7 1/1, 1/2 5, 13, 22
sandshurica
(APRICOT) MDH 5 2/1, 2/2 5, l3, 16,
22
RX 1 1/2‘ 5, 13
PGI 1 -/l’, 1/2 5, 13, 22
pan 5 2/1, 3/2‘ 5, 13, 16,
22
6-PGD 2 1/1, 2/2 5, 13, 16,
22
TPI 1 1/1, 1/2 5. 13
aeericana LAP 4 3/1, 1/2 11, 13, 22
angustifolia -
cerasifera MDH 3 3/1, 1/2 11, 13, 22
hortulana
lunsoniana PX 2 -/1‘, 2/2‘ 11, 13
salicina
silonii PGI 4 1/1, 3/2 11, 13, 21,
(PLUM) 22
PGM 6-8 2/1, 2/2 ll, 13, 21,
22
6-PGD 1 1/1, 1/2 11, 13, 22

TPI 1 1/1, 1/2 11, 13

36

Table 5 (cont'd).

deueuue—

Key to references follows.

Synonymous

Activity was evaluated from pollen.

Cathodal activity

Inconsistent resolution

One of the alleles is a null allele.

Activity was evaluated from Open-pollinated progeny.

Kc¥_to_References
1) Cerezo et al. (1989)
2) Arulsekar, Parfitt, a Kester (1986)
3) Hauagge, Kester, Arulsekar, Parfitt, & Liu (1987)
4) Hauagge, Kester, a Asay (1987)
5) Byrne 8 Littleton (1989a)
6) Kaurisch et al. (1988)
7) Hancock 5 Iezzoni (1988)
8) Fernqvist & Huntrieser (1988)
9) Durham et a1. (1987)
10) Arulsekar, Parfitt, Bares, & Hansche (1986)
11) Byrne & Littleton (1988)
12) Chapter I
13) Byrne (1989a)
14) Werner (1992)
15) Mowrey et al. (1990a)
16) Byrne (1989b)
17) Messeguer et al. (1987)
18) Kaurisch et al. (1991)
19) Mowrey et al. (1990b)
20) Chaparro et al. (1987)
21) Parfitt et al. (1985)
22) Byrne 6 Littleton (1989b)

37
for PX, SKDH, and TPI to eight for PGM. Two loci were
reported for each enzyme in leaf tissue except for IDH and
SKDH which only exhibited one. The number of alleles per
enzyme in leaf tissue varied from one in SKDH to five for
LAP and PGM. One locus was expressed by pollen for APS,
AAT, CAT, and LAP, while two loci were expressed for IDH,
PGI, and PGM, and three for ADH. P. argentea, P. bucharica,
and P. tangutica are all homozygous at two loci for AAT and
PGI for alleles also found in P. amygdalus.

Eight out of the 14 enzyme systems studied in leaf
tissue of peach clones were monomorphic (Table 5). All loci
were homozygous in leaf tissue except Cat-1, Idh-l, Px-z,
and Skdh-l and Dia-l and Mdh-l which exhibit two and three
alleles each, respectively. Px-z shows cathodal activity.
MDH was expressed at four loci in pollen as compared to only
two in leaf tissue.

Mowrey et al. (1990b) studied isozyme polymorphisms in
'Redhaven’ peach and several other species also in subgenus
Amygdalus. Leaf tissue from P. persica ssp. ferganensis
expressed the same results as 'Redhaven' peach leaf tissue.
P. davidiana exhibited one different putative allele at
Aat-l, Pgi-Z, Idb-l, Lap-1, Lap-2, 6-Pgd—2, Pgm-z, and
Skdh-l and two different putative alleles at Px-l and Pgm-l
as compared to 'Redhaven’ peach. P. kansuensis expressed
one different putative allele at Aat-l, Aat-z, Lap-1,
6-Pgd-2, and Pgm-l in comparison with 'Redhaven' peach.

P. mira exhibited one different putative allele at Aat-l,

Aat-z, Idh-l, Lap-2, 6-Pgd-2, Pym-2, and Skdh-l as compared

38
to 'Redhaven’ peach.

In sour cherry two alleles were identified at Adh-l,
Idh-z, Lap-1, and 6-Pgd-2 and three alleles at Acon-z,
Pgm-Z, Pgi-Z, and 6-Pgd-1 (Table 5). MDH bands of sweet and
ground cherry, sour cherry's presumed progenitors, were
codominantly expressed in sour cherry (Hancock and Iezzoni,
1988). No genetic description for MDH is presented for
cherry species due to the complex nature of MDH inheritance
and the lack of segregation data. Data from open-pollinated
progeny of P. canescens, P. incisa, P. mahaleb, and
P. subhirtella suggests, however, that MDH is dimeric in
cherry and that segregation is occurring for two alleles at
one cytosolic locus for P. incisa and P. subhirtella.

P. canescens exhibits two homozygous loci per enzyme
studied, not considering MDH. P. subhirtella and ground
cherry are monomorphic for all enzyme systems except for MDH
in P. subhirtella: however, they exhibit at least one
heterozygous locus. One-half of the sweet cherry loci
studied exhibit only one allele.

Santi et a1. (1990) studied inheritance and linkage of
isozyme loci in 286 wild sweet cherries. Santi and Lemoine
(1990) found diversity at eight isozyme loci in 198 wild
sweet cherries which allowed them to devise an
identification key for the sweet cherries. Santi and
Lemoine (1990) also studied 33 sour and duke cherries and
found only three to ten additional isozyme bands which might
characterize the ground cherry genome from that of sweet

cherry. Santi et al. (1990) and Santi and Lemoine (1990)

39
are not included in Table 5 because they used PAGE and
isoelectric focusing techniques to collect isozyme data.
These data do not resemble and are not comparable to that
from the other studies.

Apricot Clones exhibited activity at two loci for each
enzyme system except PX (Table 5). PX cathodal activity
encoded by one locus was resolvable. LAP, PX, PGI, 6-Pgd-1,
and TPI loci were monomorphic. Two alleles were reported at
Mdh-l, Mdh-z, Pgm-l, and 6-Pgd-2 and three at Pgm-z.

The plum clones studied generally involve several
species in their pedigrees. The species involved in the
clonal parentage precede the heading "Plum" (Table 5).
Plums exhibit from one polymorphism for TPI and 6-PGD up to
a minimum of six polymorphisms for PGM. All enzyme systems
exhibit two loci. Two alleles were observed at Px-2 and
both PGM loci and three at Lap-1, Mdh-l, and Pgi-Z. All
other loci studied were monomorphic for one allele.

The Prunus species that exhibit the most isozyme
polymorphism are almond, sour Cherry, and plum. Almond’s
extensive isozyme variability has been attributed to
outcrossing due to high levels of self-incompatibility
(Arulsekar, Parfitt, and Kester, 1986). Plums also are
self-incompatible and have complex pedigrees involving
numerous species and ploidy levels (Byrne and Littleton,
1988). Sour cherry is also a polyploid with self-
incompatibility prevalent in the species.

The high degree of isozyme homozygosity in peach could

have resulted from inbreeding (self-incompatibility is rare)

40
and a limited gene pool. The peach cultivars in the United
States trace back to just a few introductions (Arulsekar,
Parfitt, and Kester, 1986). Apricots exhibit intermediate
isozyme variability compared to other Prunus species and
consist of inbreeding and outcrossing populations (Byrne and

Littleton, 1989a).

41

METHODS

El§n£_M§l§Ii§l

A total of 26 sweet, 67 sour, six ground, and 12
interspecific hybrid cherry genotypes and ’Redhaven' peach
were evaluated for their isozyme patterns. All of the plant
material was from the MSU collection at the Clarksville
Horticultural Experiment Station, Clarksville, Michigan or
the Horticultural Research Center, East Lansing, Michigan.

Two principal coordinate analyses were performed.
Thirty-six selections were analyzed for all enzyme systems
except PX. These 36 sweet, sour, ground, and interspecific
hybrid cherries made up the first comparison (PC01) based on
a total of 44 isozyme bands. Fifty-seven tetraploid
selections with sour cherry in their pedigrees were analyzed
for all enzyme systems except PX and PGM. These were
compared in the second analysis (PC02) using 41 isozyme

bands.

IfiQZ¥m§_EIQ£§QQI§§

Starch gel electrophoresis was performed on extracts
from young leaves and dormant vegetative buds. Enzyme
systems studied include PGI, IDH, PGM, 6-PGD, and LAP as
well as shikimate dehydrogenase (SKDH, E.C.1.1.1.25), malate

dehydrogenase (MDH, E.C.1.1.l.37), and peroxidase (PX,

42
E.C.1.11.1.7). Electrophoresis and staining procedures were
the same as for Chapter I. ADH did not exhibit sufficient
activity from leaves and buds and so was not used in the
diversity study. SKDH and MDH were resolved on morphiline
citrate pH 6.1 gels (Clayton and Tretiak, 1972). PX was
resolved on the cathodal (bottom) section of
tris-citrate/lithium-borate pH 8.3 gels (Scandalios, 1969).
SKDH, MDH, and PX were assayed according to Arulsekar and
Parfitt (1986), Vallejos (1983), and Soltis et al. (1983),
respectively. Hydrogen peroxide was added just prior to PX
activity staining.

Isozymes were named based on their mobilities relative
to the 100 band as in Chapter I. However, a few cherry
genotypes exhibited rare isozymes anodal to 100 for PGI (110
and 105) and SKDH (120). The most anodal of these rare
isozymes were not named 100 because of their scarcity and
they were discovered after this nomenclature was all ready
in use. 'Montmorency' or ’Meteor’ controls were run on most
gels during this germplasm diversity study to aid in band
identification. Both cultivars possess the 100 band for all

enzyme systems except SKDH.

5! !° !° 1 E i

A '0' or a '1’ was entered for each isozyme band using
the Similarity Computer Program (Appendix D) indicating its
absence or presence for every genotype analyzed. The matrix
of 36 genotypes x 44 isozymes utilized in the first

principal coordinate analysis (PCO) is presented in Appendix

43
A. The second analysis used a raw data matrix of 57
genotypes x 41 isozymes. The isozyme bands utilized are
defined in Figures 3 and 4.

Data were analyzed by calculating similarity matrices
(Appendix B) again using the Similarity Computer Program.
The similarity statistic used by this program is the
Marczewski and Steinhaus Similarity statistic:

S = w / (a + b - w), where ’a' and 'b’ are the number of
isozyme bands observed in the two individuals being compared
and ’w' is the number of isozyme bands the two individuals
share in common (Angus et al., 1988). The similarity
matrices were subjected to PCO. SAS (SAS Institute, Inc.,
Cary, N.C.) was used on the IBM0 mainframe at Michigan State
University to run the PCO Program written by Dr. Carl Ramm
(Appendix E).

Scores for the first two principal coordinates were
imported into 123° version 2.01 for editing and for
compatibility with PlotIt’. PlotIto version 1.5 was then
used to graph the data in two dimensions. Individual points
on the graphs were labelled with the corresponding
genotype's identity. The graphs were then scrutinized for

possible ordinations.

44

RESULTS

W

The cherry genotypes studied exhibited three
polymorphisms for Pgi-z (Tables 6 and 7, Figures 2 and 3)
encoded by three alleles at one locus. Results from progeny
testing reported in Chapter I indicate that the isozyme
bands 82 and 100 represent alleles while 91 is heteromeric.
All of the cherry species and species hybrids exhibited
polymorphisms 1 and 2. Putative allele 110 and the
heteromeric band 105 were rare among the germplasm studied,
being only exhibited by a NR3F2 open-pollinated mutant (0).
'Redhaven' peach exhibited the 100 allele.

Three polymorphisms were discovered for Idh-z, two of
which were exhibited by cherry clones while polymorphism 3
was exhibited by ’Redhaven' peach (Tables 6 and 7, Figure
3). Bands 100 and 64 were diagnosed as alleles and 82 as
their intralocus heterodimer (Chapter I). Nearly all sweet
cherries were homozygous for the 100 allele, while all but
one of the ground cherries possessed the heterozygous
polymorphism 2. Sour cherry and the species hybrids
exhibited polymorphisms 1 and 2.

Five polymorphisms were diagnosed for Lap-1 (Tables 6
and 7, Figure 3). Polymorphism 3 was unique to 'Redhaven'

peach. Bands 100 and 95 represent alleles (Chapter I).

45

 

 

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48

Table 7. Number of sweet, sour, ground, and hybrid cherry selections which
possessed polymorphisms for eight enzyme systems.

 

 

 

80:25:19.:
8112er Prunus Prunus Prunus P. cerasus P. cerasus X
I E 1 I . . E I . E . E E
Pgi-Z 1‘ A , 1o 39 2 2 6
2 11 27 3 3 1
3 - 1 - - -
Idh-z 1 18 23 1 2 6
2 2 44 4 3 1
3’ - - - - -
Lap-l 1 - 46 2 4 6
2 - 1 2 - -
31 - - - - -
4 21 - - 1 -
5 - - 1 - -
MDH 1 - 4 3 - -
2 - 43 2 3 5
3 25 1 - 1 -
4 1 3 - 1 1
5 - 1o 1 - 1
6 - 2 - - -
7 - A A -
8‘ - E - - -
9 - 2 - - -
Pgm-z 1 - 1o 3 3 1
2 26 - - - -
3 - - 3 - -
4l - - - - —
6-PGD 1 - 1o 3 - -
2 - 20 - 2 s
3 11 9 - - 1
4 - 9 2 - -
5 - 4 - - -
6 - 6 - 1 -
7 11 - - -
8 - - - 1 1
9 - 1 - - -
1o 4 - - 1 -
11 - 1 - - -
12 - 2 - - -
13 - 1 - - -
14 - 1 - - -
15 - 2 - - -
16‘ - - - - -
17 - - 1 - -

49

Table 7 (cont'd).

 

 

 

595821.95

Enzyme Prunus Pru nus Prunus P . cerasus P . cerasus X

Skdh-l 1 - 18 1 1 6
2 - 1 - - -
3 - 4 - - -
4 - 1 9 2 - 1
5 - 1 5 - 3 -
6 - 1 - - -
7 1 8 4 - - -
8 - 1 - 1 -
9 8 1 - - -
1 o - 1 - - -
1 1 - 2 - - -
1 2‘ - - - - -
1 3 - - 2 - -
1 4 - - 1 - -

PX 1 - 6 - - 1
2 - 2 - - 1
3 2 1 - - -
4 - 3 - - -
5 - - - - 2
6 - 1 - - -
7 - 4 - - -

 

‘Polymorphism exhibited by 'Redhaven' peach

 

Figure 2.

50

(A) PGI, (B) MDH, (C) PGM, (D) 6-PGD, (E) SKDH,
and (F) PX isozyme patterns in cherry. The
origin is at the bottom of each photograph.

The bottom zone of activity in A was due to
6-Pgd-2. Anodal activity was observed in A - E
and cathodal activity in F. Mobilities are given
for bands which represent true and putative
alleles in A and C - E. Mobilities are presented
for all isozymes in B and F because genetic
control of these enzymes is not understood in
cherry species. Band 63 in B is the
mitochondrial form of MDH in cherry. In B and C,
lanes 1 - 6 are sweet cherries, 7 - 9 and 11 are
ground cherries, 10 is a sour x sweet cherry
hybrid, and 12 - 16 are sour cherries.

 

 

52

 

 

 

 

 

 

 

 

 

 

Isozyme phenotypes exhibited by cherry selections
(B) 1611-2,

and 'Redhaven' peach for (A) Pgi-z,
The origin is

(C) Lap-1, (D) MDH, and (E) Pym-2.

c110 o
105 ..
8100 o o o 8100 o
91 o 82 0
b 82 o b 64 0
2 3 2
l\ 13
100 o I»¢b«o e
95 o» a» o
88 0 11491. 0 CD
81 o
75 ‘0 ch «9 0 crap 0
57
63 1o o Iii. o eȢp o
57
51 O .1 o .11} o
43 o
35 o o
28 o o
1 2 £3 4 5 (5‘7 9
I)
Figure 3.

 

 

 

 

 

105 o
a 100 _ o o
97 b 95 A C! 3
83 o
1 2 £3 4 5
(3
146 .

8100 01c.

b 75 db 1.

 

 

 

1 2 :3 4

E3

at the bottom of each diagram.

 

53
Putative allele 97 was only diagnosed in one ground cherry
clone. All ground cherries possessed the 95 allele. All
sweet cherries that were studied for LAP were homozygous for
the 100 allele. Sour cherry genotypes predominantly
exhibited polymorphism 1, which is comprised of both the 100
and 95 alleles. Four out of the five genotypes studied with
sour and sweet cherry in their pedigrees also exhibit
polymorphism 1. All six of the genotypes studied for LAP
that contain sour and ground cherry in their pedigrees and

the one open-pollinated ground cherry studied exhibited

 

polymorphism 1.

A total of nine polymorphisms were discovered for MDH
(Tables 6 and 7, Figures 2 and 3). Polymorphism 8 was only
recorded for ’Redhaven’ peach. All but one of the sweet
cherries possessed polymorphism 3, while the other exhibited
polymorphism 4. Both polymorphisms have bands 100, 88, and
63. Band 63 was the largest and most intense of the MDH
bands. Sometimes additional bands were resolved at 67 and
57 when gels exhibited excellent resolution, suggesting
possible comigration with 63. Polymorphism 4 also has band
75. The six ground cherries possessed polymorphisms 1, 2,
and 5 which contain the bands possessed by the sweet cherry
as well as three unique bands: 95, 51, and 35. Sour
cherries possess all of the polymorphisms except number 8.
Bands 81, 43, and 28 were unique to sour cherry, although
'Redhaven' peach also possessed band 35. Progeny testing
(data not presented) using the parental sour cherries from

Chapter I was unsuccessful at determining a genetic model

54

for MDH, although a general lack of segregation indicated
possible fixed heterozygosity in these genotypes. MDH may
be dimeric in cherry due to the large number of bands
comprising many of the polymorphisms and the spatial
symmetries among the bands.

Four polymorphisms were diagnosed for Pgm-z (Tables 6
and 7, Figures 2 and 3). Polymorphism 4 was only found in I
'Redhaven’ peach. Bands 100 and 75 were found to be alleles

(Chapter I). Sweet cherries were all homozygous for the 100

 

allele, while three out of four ground cherries (f) were

ol-\n .r ‘——‘—

homozygous for the 75 allele. All other genotypes tested l
for PGM were heterozygous.

Seventeen polymorphisms encoded by nine alleles at two
loci were discovered for 6-PGD (Tables 6 and 7, Figures 2
and 4). Progeny testing (Chapter I) indicated that bands
100, 88, and 76 at 6-Pgd-1 and bands 60 and 48 at 6-Pgd-2
are alleles and bands 94 and 82 at 6-Pgd-1 and band 54 at
6-Pgd-2 are heteromeric. Band 88 is an intralocus
heterodimer for polymorphisms l, 5, and 17. Band 82 at
6-Pgd-1 is an intralocus heterodimer for polymorphisms 4 and
6, while it is a putative allele at 6-Pgd-2 for
polymorphisms 12 and 15. Band 72 at 6-Pgd-2 is a putative
allele for polymorphisms 11 and 16 and an intralocus
heterodimer for polymorphism 15. Bands 66 and 43 at
6-Pgd-2 are intralocus heterodimers. Band 38 at 6-Pgd-2 is
a putative allele for only polymorphism 14, and an
intralocus heterodimer for polymorphisms 9, 13, and 17.

Band 28 is a putative allele at 6-Pgd-2. The middle band of

55

 

 

 

 

:fl100 o A... o IDID 0 ID 01 19 .119 o .119 o
94 o 111! o o 19 o cnqo1o o
If 88 O 1119 O a) o 1149 0 114910 0
a2 82 . . . .
c9 76 o o «D o
tfi 72 o 19 o
66 1o 0 0
<9 60 0 A» o o 1» o o o» o
54 o a» o o A» e 0 up 0
d2 43 . . g o O O Q 0 O O O O O O O
43 o o o
12 38 0 1D 0 o
é’28 O o o
A 1 2 3 4 6 7 8 91011121314151617
‘QO 0 o
100 1o o 0 lb «9 o
95 O o 10 at «o o o
77 o «n o .1
71 O 1.10 .1 1o 0

 

 

 

1234567891011121314

 

Figure 4. Isozyme phenotypes exhibited by cherry selections
and 'Redhaven’ peach for (A) 6—Pgd-1 and 6-Pgd-2
and (B) Skdh-l. The origin is at the bottom of

each diagram.

56

five-banded patterns at both 6-PGD loci represents
comigration between an intralocus heterodimer and a
homodimer representing an allele.

Sweet cherries possess one or both of alleles 100 and
88 at 6-Pgd-1 and were all homozygous for 6-Pgd-2“. All of
the ground cherries possess 6-Pgd-1" in addition to one or
both of the sweet cherry alleles at this locus. In addition
to 6-Pgd-2“, the ground cherry clones possess allele 60 and
one also possesses putative allele 28 at this locus. Sour
cherry germplasm exhibited all of the polymorphisms for
6-PGD except 7, 8, 10, 16, and 17. Sour cherry possesses
all of the 6-PGD alleles and putative alleles found in sweet
and ground cherry. Only 'Redhaven' exhibited pattern 16.

Fourteen polymorphisms encoded by five putative alleles
were found for Skdh-l (Tables 6 and 7, Figures 2 and 4).
Polymorphism 12 was found only in ’Redhaven' peach. Sweet
cherries exhibit either polymorphism 7 or 9 consisting of
the putative 100 or 100 and 95 alleles, respectively. The
ground cherries exhibit polymorphisms 1, 4, 13, and 14 and
all of the putative alleles except Skdh-l". Sour cherries
exhibit the first eleven polymorphisms and have all of the
putative alleles found in sweet and ground cherry and also
putative allele 77. The structure of SKDH appears to be
monomeric in cherry.

Seven polymorphisms were found for PX (Tables 6 and 7,
Figures 2 and 5). PX was difficult to resolve consistently;
therefore, data exists for only a few genotypes and it was

not used in the principal coordinate analyses. PX appears

2' ‘1 4'

 

57

 

-100 0.00. o
-92 o o o
-81 a.

-64 o. o o.
'30 O. Q
-15 0......

 

 

 

1234567

Figure 5. Isozyme phenotypes exhibited by cherry selections
for cathodal PX. The origin is at the bottom of'

the figure.

 

58
to be monomeric in structure and encoded by a minimum of two
cathodal loci in cherry. Polymorphism 7 exhibits five bands
which is one too many for one locus in tetraploid sour
cherry assuming each band was encoded by a unique allele.
Polymorphism 3 has three bands which is one too many for one
locus in diploid sweet cherry. The only two sweet cherries
for which data are available exhibit polymorphism 3. Data
for ground cherries are unavailable. Sour cherry exhibits
all of the PX polymorphisms except number 5 which was
expressed by two species hybrids.

None of the diploid sweet cherry genotypes were
heterozygous for Lap-1, 6-Pgd-2, or Pym-2 (Table 8). Only
ten percent were heterozygous for Idh-z and 42% and 52% were
heterozygous for 6-Pgd-1 and Pgi-z, respectively.

Tetraploid cherry genotypes exhibited heterozygosity for all
of the isozyme loci presented. Total heterozygosity ranged
from 42 percent for Pgi-z to 96 percent for 6-Pgd-1. Most
of the heterozygous tetraploid genotypes were di-allelic,
some were tri-allelic, and none were tetra-allelic. A much
greater percentage of tetraploid genotypes were heterozygous
than were diploid genotypes for all isozyme loci except
Pgi-z. Average heterozygosity over the loci was 78% for the

tetraploids compared to 19% for the diploids.

2091
The first two principal coordinates from the PCO of 36
clones and seven enzyme systems, PC01, accounted for 53.8%

of the isozyme variation (Table 9). A two-dimensional plot

59

Table 8. Percent of diploid and tetraploid genotypes
heterozygous for each of the enzyme loci

 

 

 

 

listed.
Ploidy
. .1 u-Tetraploid’ ._' .
Idh-Z 10 62 62 0
Lap-1 o 94 94 o
Pgi-Z 52 42 42 o
Pym-2 o 85 85 o
Skdh-l 31 90 62 28
6-Pgd-1 42 96 75 21
6-Pgd-2 o 74 67 7

 

1Prunus avium
2Prunus cerasus, P. cerasus x P. avium, P. cerasus x
P. fruticosa, and P. fruticosa

Table 9. Percent variation accounted for by the first five
principal coordinates for PC01 (Figure 6).

 

Principal
3° ! E ! . !°
1 41.1
2 12.7
3 9.7
4 6.2
5 5.5

 

Total 75.2

60
separated the diploid sweet cherry selections from the sour
cherry, ground cherry, and sour x ground cherry tetraploids
along the first axis (Figure 6). Idh-Z“, Lap-1”, Pgm-z”,
6-Pgd-1", 6-Pgd-2“, putative allele Skdh-l“, and MDH bands
75 and 51 are primarily responsible for this separation.
These alleles and bands were generally present in the
tetraploids on the positive end of axis 1 and were generally
absent in the sweet cherry diploids on the negative end of
axis 1. Most other alleles and bands were present in
diploids and tetraploids.

Sweet cherry genotypes 6 and 7, 'Germersdorf' and
'Italia,’ respectively, lie closer to the tetraploids on the
first axis than the other diploids. 'Germersdorf' and
’Italia,’ unlike the other sweet cherries, exhibit Idh-z
polymorphism 2 consisting of alleles 100 and 64 as do the
tetraploids except 30 and 31. 'Italia,’ again unlike the
other sweet cherries, also has MDH band 75 as do all of the
tetraploids excluding genotype 30.

The 'Montmorency' x 'Angela' (sour x sweet) tetraploids
30 and 31 resembled the other tetraploids for Pym-2 and
Skdh-l and the sweet cherries for Idh-z. Genotype 30
resembled the sweet cherries for MDH and 6-PGD and the
tetraploids for Lap-1, while genotype 31 was just the
opposite. Therefore, both 30 and 31 lie near the center of
axis 1 in between the diploid sweet cherries and the
tetraploids.

The presence or absence of Pgi-z“ separated both the

sweet cherry diploids and the sour cherry, ground cherry,

61

 

 

0.7-
0.5-
0.3_ oz. 4. 9.16. 20
1 Q18 01‘ .6 036°."
0 1 .10. 12.19 ‘27 D35
. "' +30 822
N .23.25
.. +31 2‘
Q)
0— -O.1" 05
‘ 08,13 .7 35:13 “33426
_0°3- 911.21
- “"5'” D FRUTICOSA and F Op
_0 5_ u C and F in pedigree
' + C and A in pedigree
- A CERASUS
._Q'7 o .NWUM
o 1 l r _' V l I I I l r l I I
-0.7 -0.5 -0.3 -0.1 0.1 0.3 0.5 0.7

PC 1

Figure 6. Principal coordinate analysis for PC1 vs. PC2
for selected cherry genotypes (PC01).
Identities of clones are presented in Table 6.

62
and sour x ground cherry tetraploids into two groups along
axis 2. Individuals of both groups at the positive end of
axis 2 exhibited polymorphism 2 for PGI consisting of

alleles 100 and 82. Those at the negative end of axis 2

were homozygous for Pgi-zmfl.

1"

EQQZ

1 ”km.”

The first two principal coordinates from the PCO of 57

sour cherries and six enzyme systems, PC02, accounted for

only 30.2% of the isozyme variation (Table 10). A two-

! ’ . .‘ 1.5.27.7

dimensional plot (Figure 7) did not reveal any natural
clusters in the data. Additionally, the sour cherry, sour
cherry x sweet cherry, and sour cherry x ground cherry
clones did not aggregate.

The three isozyme loci contributing to the separation
in the first two dimensions were Idh-z, Pgi-Z, and 6-Pgd-1.
Idh-z“ was more frequent at the positive end of the first
axis. Pgi-z” was very frequent at the negative end of axis
2 and rare at the positive end of axis 2. 6-Pgd-1" was more
prevalent towards the positive end of axis 2. None of these

alleles were found exclusively at one end of an axis.

63

Table 10. Percent variation accounted for by the first five
principal coordinates for PC02 (Figure 7).

 

Principal
coordinate Percent yariatign
1 15.8
2 14.4
3 11.0
4 10.1
5 7.3

 

Total 58.6

65

DISCUSSION

W

SKDH polymorphisms are probably encoded by genetic
variation at one locus in cherry. No more than three bands
were reported for tetraploid cherries and two bands for
diploid sweet cherries (Figure 4) which lends support for a
one locus hypothesis. Asymmetrical polymorphisms and the
apparent lack of heterodimeric bands indicate that SKDH is
most likely a monomeric enzyme in cherry. Segregation data
for peach indicated that SKDH is encoded by one locus and
that SKDH is a monomer in peach (Mowrey et al., 1990a).

Data for almond and other species in subgenus Amygdalus
(Table 5) also suggest that SKDH is encoded by one locus in
these species.

No other PX studies utilizing horizontal starch gel
electrophoresis exist for cherry species. In peach,
inheritance data has confirmed the existence of two alleles
at one cathodal locus (Durham et al., 1987). However, in my
study, three bands and up to five bands were observed on the
cathodal section of gels for sweet and sour cherry,
respectively. Assuming that PX is monomeric and that each
band is encoded by a unique allele as was the case at the
peach cathodal locus (Durham et al., 1987), a minimum of two

cathodal loci is needed to genetically explain the observed

66

PX polymorphisms in sweet and sour cherry.

The genetic basis for the banding patterns observed for
MDH in the three cherry species studied is not known.
However, the polymorphisms presented in my diversity study
and data on the genetic control of MDH in other Prunus
species do provide some clues. MDH polymorphisms observed
in almond, peach, apricot, and plum leaf tissue are encoded
by alleles at two different loci (Table 5). In almond,
apricot, and plum, both loci are polymorphic exhibiting
typical banding patterns for a dimeric enzyme (one-banded
homozygotes and symmetrical three-banded heterozygotes). In
peach, the polymorphisms at Mdh-l are often complex due to
single alleles producing two protein products which can
interact to produce homodimers and heterodimers for a
homozygous genotype. nah-2 encodes the mitochondrial form
of MDH and is monomorphic for one band in peach (Mowrey et
al., 1990a). Likewise in cherry, MDH polymorphisms are
quite complex, but may also be accounted for by genetic
variation at a minimum of two loci. Hancock and Iezzoni
(1988) diagnosed the mitochondrial form of MDH in sour
cherry which corresponds to the 63 band in my study. This
MDH band is the only one found in every genotype analyzed in
my diversity study. Therefore, two loci can be
hypothesized. Putative Mdh-z is probably monomorphic for
the mitochondrial band 63, and the other isozyme bands may

be accounted for by alleles at Mdh-l.

67
Comparisgns with cher Cherry Studies

The MDH polymorphisms presented by Hancock and Iezzoni
(1988) for sour and sweet cherry compare well with those
diagnosed in my diversity study. Even though different gel
and electrode buffers were used, their patterns for sweet
and sour cherries correspond to polymorphisms 3 and 2,
respectively (Figure 3) in my study. In addition to these
polymorphisms, one out of 26 sweet cherries was found to
exhibit polymorphism 4 and sour cherries were found to
exhibit polymorphisms 1 through 7 and 9. Fifty-one out of
79 genotypes with sour cherry in their pedigrees exhibited
MDH polymorphism 2. Hancock and Iezzoni (1988) studied 19
sour cherries and six sweet cherries and so subsampled the
set of diversity found in my larger study for sour and sweet
cherry.

The MDH polymorphism presented by Hancock and Iezzoni
(1988) for ground cherry was similar to two of the three
that my study diagnosed with two exceptions. Polymorphism 1
has the most anodal band at 95 rather than theirs which
corresponds to band 88 in my study and polymorphism 2 has
band 100 in addition to 88 which they did not find. Both
studies may have analyzed the same ground cherry clones as
such germplasm is limited in the Michigan State University
collection. They analyzed four ground cherries and my study
analyzed six.

Kaurisch et al. (1988) assayed a ground cherry genotype
for 6-PGD and PGI. The six ground cherries assayed in my

study were more heterozygous than the genotype they studied.

68
Their clone was homozygous for my 6-Pgd-Im’and Pgi--2°2
alleles and heterozygous for my 60 and 48 alleles at
6-Pgd-2. The ground cherries in my study exhibited the 88
and 76 alleles at 6-Pgd-1, the 28 allele at 6-Pgd-2, and the
100 allele for Pgi-Z as well as those present in the
genotype studied by Kaurisch et al. (1988).

Fernqvist and Huntrieser (1988) presented 6-PGD
polymorphisms for cultivated and wild sweet cherry. These
polymorphisms generally agreed with my results in sweet
cherry except that some of the patterns seem to be missing
bands. Fernqvist and Huntrieser (1988) present some two-
banded polymorphisms corresponding to my 100 and 94 and 54
and 48 bands for 6-Pgd-1 and 6-Pgd-2, respectively, which
are unlikely due to the dimeric nature of 6-PGD. Another
pattern that they present for 'Fanal' sweet cherry is
suspect due to its highly asymmetric pattern at 6-Pgd-1.

Isozyme inheritance and diversity data presented by
Santi et al. (1990) and Santi and Lemoine (1990) is not
directly comparable to my study due to different
electrophoretic methods. They used PAGE and isoelectric
focusing. Also, expression of SKDH isozymes in their study
was dependent on the physiological state of the clones.
Banding patterns did not phenotypically vary with date of
sampling in my study.

Kaurisch et al. (1991) studied IDH, LAP, 6-PGD, PGI,
and PGM polymorphisms in 65 sweet cherry cultivars and 45
sour cherry cultivars. Their results were similar to those

of my study with a few exceptions. The sour cherry

69
collection analyzed by me exhibited more diversity for
6-Pgd-1 and 6-Pgd-2. This is probably due to two reasons:
1) the Michigan State University sour cherry germplasm
collection is very diverse (Hillig and Iezzoni, 1988) and
includes specimens collected throughout sour cherry's center
of diversity, and 2) a large number of sour cherry
genotypes, 66, were analyzed for 6-PGD in my study.

Kaurisch et al. (1991) presented a two-banded pattern
for 6-Pgd-1 in sour cherry which corresponds to my 100 and
88 bands. Band 94 should be present also since inheritance
data in Chapter I demonstrate that 6-PGD is dimeric at both
lOci in cherry. 6-PGD is also dimeric in other Prunus
species.

For Idh-Z, Kaurisch et a1. (1991) found sweet cherry
homozygotes for my 64 allele. I did not observe the
Idh-Z“"‘ genotype in sweet cherry. For Lap-1, they found
sour cherries homozygous for my 100 allele, while I did not.
I did find one sour cherry that was homozygous for Lap-1”,
while they did not observe this polymorphism. For Pgm-z,
Kaurisch et al. (1991) found more polymorphisms for sweet
and sour cherry probably because they assayed many more
specimens. Their Pym-2 polymorphism 3 in sweet cherry
appears to be the same as polymorphism 1 in my study. My
polymorphism 1 was only found in sour and ground cherry
genotypes.

PGI exhibited only one locus of activity in my study,
while Kaurisch et a1. (1988, 1991) identified two PGI loci

for cherry. My diversity data for Pgi-Z appears to be the

70
same as that presented by Kaurisch et al. (1988, 1991) for
sweet and sour cherry. Cherry genotypes assayed by Kaurisch
et al. (1988, 1991) were found to be monomorphic for one
band at Pgi-l. Assuming only one-banded activity for
Pgi-l, I may not have observed Pgi-l activity due to its
comigration with the Pgi-z’” band present in all cherry
selections in my study. I used a different buffer system
for PGI electrophoresis than did Kaurisch et al. (1988,
1991). Another possibility for the lack of Pgi-l activity
in my study is that Pgi-l isozymes were inactive under my
assay conditions.

Usually, the minimum number of loci encoding an
enzyme and the subcellular compartmentalization of its
isozymes are conserved in plants (Gottlieb, 1982). Studies
involving other Prunus species (Table 5) also report two

loci of activity for PGI.

: . '!l :!l E S .

Isozyme polymorphisms were studied for 'Redhaven’ peach
for all enzyme systems except PX in order to make the cherry
diversity data more easily comparable with studies in other
Prunus species. Mowrey et al. (1990b) studied various
species in the subgenus Amygdalus and included 'Redhaven'
also. They studied all of the enzyme systems that I did and
used the same gel and electrode buffers for IDH, LAP,
6-PGD, and SKDH.

'Redhaven' is homozygous for Pgiezm’and Idh-z“ in my

study which most likely correspond to the Pgi-z‘ and Idh-l’

71
alleles of Mowrey et al. (1990b). In my study, ’Redhaven'
exhibits LAP bands 105 and 83 which are not present in the
cherry clones examined. These two bands correspond to their
Lap-1’ and Lap-2’ alleles.

For MDH, 'Redhaven' was found to possess the 75, 63,
51, and 35 bands, polymorphism 8, in my study. The first
three bands correspond to the Mdh-I’adlele of Mowrey et al.
(1990b) and the last band is from the mitochondria and
corresponds to their monomorphic locus Mdh-z. In peach,
homozygous genotypes for Mdh-l produce two homodimers and
one heterodimer (Mowrey et al., 1990a).

'Redhaven’ possesses PGM band 146 in my study. It most
likely corresponds to Pgmaz'in Mowrey et al. (1990b) as
their Pgm-l alleles produce two-banded phenotypes. In my
study 'Redhaven' exhibits 6-Pgd-1””“ and putative allele
6-Pgd-2”. In their study 'Redhaven' exhibits 6-Pgd-1’ and
6-Pgd-22 6-Pgd-1 is problematic because ’Redhaven' is
heterozygous in my study and homozygous in theirs. Results
for 6-Pgd-2 correspond in these two studies. ’Redhaven’
exhibits polymorphism 12 and is homozygous for Skdh-l" in my
study. This corresponds to Skdh-I’in Mowrey et al.

(1990b). A standard such as 'Redhaven' peach should be
included in all Prunus isozyme work to help eliminate

uncertainties when comparing data from different studies.

WW
Sour cherry has been shown to exhibit fixed

heterozygosity or segregation also consistent with digenic-

72
disomic inheritance at several isozyme marker loci (Chapter
I). This confirms an allotetraploid origin for sour cherry.
Olden and Nybom (1968) hypothesized that ground and sweet
cherry are the parents of sour cherry. The diversity data
for sour, sweet, and ground cherry do suggest that ground
cherry is a parent of sour cherry but are inconclusive as to
whether sweet cherry is the other parent. All alleles or
bands found in ground cherry are expressed in sour cherry
except Lap-1”. Many of these ground cherry alleles and
bands are not expressed in sweet cherry. However, all sweet
cherry alleles or bands are not only expressed in sour
cherry, but are also expressed in ground cherry. Because
all discovered sweet cherry alleles or bands were also found
in ground cherry, the diversity data does not clearly
demonstrate that sweet cherry is a parent of sour cherry.
The data does not discredit this hypothesis either.

Hancock and Iezzoni (1988) concluded that their data
for MDH supported both sweet and ground cherry as parents of
sour cherry. The unique bands they found for sweet and
ground cherry were codominantly expressed in sour cherry.

My diversity study surveyed a larger number of sweet and
sour cherry genotypes than did Hancock and Iezzoni (1988)
and at least as many ground cherry genotypes. My study
found additional MDH polymorphism for sweet and ground
cherry resulting in a lack of unique MDH bands for sweet
cherry.

My diversity data may even suggest that some of the

sweet cherry gene pool has been introgressed into ground

73
cherry. Interspecific hybridization could have occurred in
sympatric ground cherry and sweet cherry populations
resulting in allotetraploid sour cherry. Sour cherry could
have then backcrossed with ground cherry, introgressing
sweet cherry alleles into the ground cherry gene pool. Any
of the alleles or bands that sweet and ground cherry share
in common may be evidence for such a process. However,
these shared alleles may have also existed in both species
before sour cherry evolved.

Whichever the case, whether sweet cherry genes have
been introgressed into ground cherry via sour cherry or the
shared diversity existed in both species before the
evolution of sour cherry, hybridizing surely and frequently
occurs between sour and ground cherry. They have the same
ploidy levels. There is no evidence of any reproductive
isolation between sour and ground cherry; they can be
crossed easily and this probably occurs frequently in
nature. Both of the species share much of the diversity
found in the enzyme systems studied. Multivariate
statistics using morphological characteristics (Hillig and
Iezzoni, 1988) and these enzyme systems were unable to
separate sour and ground cherry graphically (Figure 6).

The reverse example of introgression, ground cherry
alleles into sweet cherry, probably would occur much less
frequently. Many of the progeny resulting from a sweet
cherry (2n=2x) x sour cherry (2n=4x) cross would be expected
to be triploid and therefore infertile due to abnormal

segregation of chromosomes to their gametes. This is called

74

segregational hybrid sterility (Stebbins, 1977) and is one
form of postzygotic reproductive isolation. Also, sweet
cherry is monomorphic for Lap-1, Pgm-Z, and 6-Pgd-2 and
lacks many of the tetraploid alleles and bands for MDH,
6-PGD, SKDH, and PX demonstrating that such introgression
into sweet cherry is probably limited. Santi and Lemoine
(1990) do propose a possible slight introgression of sour
cherry into three out of 286 wild sweet cherry clones.

Due to their system of self-incompatibility, diploid
sweet cherries were expected to be highly heterozygous.
However, the 26 diploid cherries exhibited no heterozygosity
or polymorphism at three out of seven isozyme loci and only
a small percentage exhibited heterozygosity at another
locus. A much higher percentage of tetraploid sour and
ground cherries were heterozygous at each locus except one,
keeping in mind the low number of diploid cherries studied.
My study, like many others (Lack and Kay, 1986; Roose and
Gottlieb, 1976; Soltis and Rieseberg, 1986; Soltis and
Soltis, 1989), demonstrates that polyploids can maintain
higher levels of heterozygosity than their diploid
relatives.

Soltis and Rieseberg (1986) state that unlike
autopolyploids, allopolyploids maintain heterozygosity
through fixed heterozygosity. While examples of fixed
heterozygosity for most isozyme loci studied in
allotetraploid sour cherry were found (Chapter I), other
sour cherry selections did not exhibit fixed heterozygosity

at these same loci. Therefore, fixed heterozygosity and

75
outcrossing together most likely maintain heterozygosity in
allotetraploid sour cherry. Self-incompatibility commonly
occurs in sour cherry (Lansari and Iezzoni, 1990) which
would encourage outcrossing.

Tetraploid cherries exhibited more enzyme multiplicity
than diploid cherries for all enzyme systems studied except
PGI and IDH. Many studies have suggested that enzyme
multiplicity and increased heterozygosity (Adams and Allard,
1977; Lack and Kay, 1986; Roose and Gottlieb, 1976; Soltis
and Rieseberg, 1986; Soltis and Soltis, 1989) may provide
polyploids with an adaptive advantage over diploid
ancestors. In the case of sour cherry, interspecific
hybridization with ground cherry, in addition to enzyme
multiplicity and increased heterozygosity, may also confer

such an advantage over diploid sweet cherry.

: . H'!!' S :1

FCC did not identify different clusters of sour cherry
clones within the collection. The germplasm evaluated was
sufficiently diverse by origin to be considered
representative of the species. In contrast, Hillig and
Iezzoni (1988) also using multivariate statistics, found
that sour cherry is morphologically very diverse
representing gradations between morphologies exhibited by
sweet and ground cherry.

This lack of ordination indicates that sour cherry
populations from which selections were made are relatively

homogeneous for the diversity presented in my study. Gene

76

flow may exist between these sour cherry populations and
also ground cherry populations resulting in the presence of
this diversity throughout the center of diversity for sour
cherry. Also, certain alleles or isozyme bands may be
selectively neutral over environments and therefore are not
more frequent in germplasm from different environments.

Examples of tri-allelism were found in sour cherry for
6-Pgd-1 and 6-Pgd-2 and possibly Skdh-l, PX, and MDH. No
sour cherries were tetra-allelic for the enzyme loci
studied. Self-compatible sour cherries do tolerate some
inbreeding and may be more inbred than expected. Also,
sweet cherry, an obligate outcrosser, frequently exhibited
only one allele per locus. Therefore, the sweet cherry
genome contributed to sour cherry may not be highly
heterozygous, setting an upper limit of three alleles per

locus.

 

h .. afu.

" ‘fiy. A H“ «‘I‘Q.'I~ 'tIFW

 

APPENDICES

APPENDIX A

RAW DIVERSITY DATA

The raw data matrix of 36 genotypes x 44 isozymes
presented in this appendix is a printout from the Similarity
Computer Program (Appendix D). This is the raw binary data
used to calculate the similarity matrix presented in
Appendix B, and subsequently the POD which is graphed in
Figure 6.

The row of numbers across the top of each section
represent individual genotypes for which binary data is
presented in the columns directly beneath. The left-hand
column of each section is the abbreviated enzyme band names.
The first letter or number represents the enzyme system and
the following numbers represent the particular band's
relative mobility. The absence of a particular band for
each genotype is represented by a '0,’ while its presence is

indicated by a '1.’

77

78

MMDMAWWMX

10

00111100101001000010000000010000100001000010

00111100101001000011100000010000100001000010

001001001010010000001..00000010000100001000010

00100111101011000011100000010000110001000010

O0111111101001000011100000010000110001000010

00100100101001000011100000010000100001000010

00111100101001000011100000010000100001000010

00100100101001000010000000010000110001000010

001111.00101001000011100000010000100001000010

00100100101011100011111001110000000101010011

050 O 0 7 0 00 50 60
10012024058153135804826260483882057100753405
11198186198876543219887766544321197711998117
GGGGGIIIMMHHMHHHHMG6666666666GGSSSSSLLLLLPPP

20

m

N

U

M

15

H

D

H

n

00111

00111..

00111

00100

00111..

00100

00111

00100

00111..

00100
050

10012
11198
GGGGG

79

100101001000011100000010000100001000010

.10010.1001000010000000010000100001000010

100101001000000100000010000100001000010

100101001000010000000010000110001000010

10010100100001.1100000010000100001000010

1001010010000.10000000010000110001000010

100101001000011100000010000110001000010

100101001000000100000010000100001000010

100101001000010000000010000100001000010

100101001000010000000010000100001000010

L100
P146
P100
W5

w

B

28

N

%

25

H

a

u

H

00.111100101001000

00100111101011100

00100111101011100

00111111010011111

00100111101011100

00111111101011100

00111111101011000

00111111101011.1100

00111100010011100

00.100100101001000

050 0 0 7

10012024058153135
11198186198876543
GGGGGIIIMMMHMMMHM

80

000.100000010000110101010011

011111000010000010101010011

011100001110000110101010011

110101001110000100101010011

011111001110000010101010011

011100001110000010101010011

011111001110000010101010011

011100001110000010101010011

010101001110000010101010011

010000000010000100001000010

L”
L”
P146
P100
P”

n

35

M

n

n

M

00111111010011100011111001110

00111111010011100010101001111

00111111010011100010101001110

00100111101011100010101001110

00100100101011100011100001110

00111100101011100010000001110

050 0 0 7 0

10012024058153135804826260483
11198186198876543219887766544
GGGGGIIIMHMMMMHMMH66666666666

81

000100100110001

110010100010001

001000100010001

000000101010011

000010101010011

000110101000011

638
628
$120
8100
S95
877
L105
L100
L97
L95
L83
P146
P100
P75

82

APPENDIX B

SIMILARITY MATRIX

The 36 x 36 similarity matrix presented in this
appendix was calculated from the raw data matrix presented
in Appendix A by the Similarity Computer Program (Appendix
D). This similarity matrix was subjected to PCO and graphed
(Figure 6).

The numbers in the top row and left column of each
section refer to the individual genotypes being compared. A
’0' in the matrix body means that the two genotypes being
compared are completely different for the enzyme systems
studied, a '1' means that they are the same, while numbers
in between indicate increasing similarity with increasing

size.

83

MARCZEWSKI AND STEINHAUS SIMILARITY MATRIX

1 2 3 4 5 6 7 8 9 10
1 1
2 .478 1

3 .409 .667 1

4 .478 1 .667 1

5 .524 .857 .769 .857 l

6 .423 .824 .647 .824 .706 1

7 .5 .667 .688 .667 .75 .833 1

8 .429 .714 .75 .714 .833 .588 .625 1

9 .478 1 .667 1 .857 .824 .667 .714 1

10 .391 .857 .769 .857 .714 .706 .556 .692 .857 1
11 .429 .714 .909 .714 .833 .588 .625 .818 .714 .833

12 .391 .857 .769 .857 .714 .706 .556 .692 .857 1

13 .429 .714 .75 .714 .833 .588 .625 1 .714 .692
14 .458 .933 .733 .933 .8 .882 .722 .667 .933 .8

15 .409 .667 1 .667 .769 .647 .688 .75 .667 .769
16 .478 1 .667 1 .857 .824 .667 .714 1 .857
17 .409 .667 1 .667 .769 .647 .688 .75 .667 .769
18 .391 .857 .643 .857 .714 .706 .556 .833 .857 .846

19 .391 .857 .769 .857 .714 .706 .556 .692 .857 1

20 .478 1 .667 1 .857 .824 .667 .714 1 .857
21 .429 .714 .909 .714 .833 .588 .625 .818 .714 .833
22 .667 .417 .348 .417 .333 .423 .385 .304 .417 .391
23 .72 .542 .417 .542 .458 .667 .625 .375 .542 .458
24 .76 .52 .4 .52 .44 .64 .6 .36 .52 .44

25 .72 .542 .417 .542 .458 .667 .625 .375 .542 .458

26

27

28

29

30

31

32

33

34

35

36

10

11

12

13

14

15

16

17

.87
.552
.75

.783

.625
.857
.818
.538
.483

.593

11

.833
.818
.667
.909
.714

.909

.423

.393

.458
.667
.571
.5

.417
.308
.276

.37

12

.692
.8

.769
.857

.769

.417

.286

.455
.588

.579

.409
.192
.214

.214

13

.667
.75
.714

.75

.423

.393

.458
.667

.571

.417
.308
.276

.37

14

1
.733
.933

.733

84

.458
.321
.545
.5
.556
.476
.55
.455
.231
.207

.296

15

1
.667

1

.538
.448
.625
.583
.65

.565

.48

.37

.379

.429

16

.667

.625
.414
.727
.682
.524
.522
.591
.565
.333
.345

.393

17

1

.375
.296
.455
.409

.625

18

.423

.393

.458
.667
.571
.5

.417
.308
.276

.37

19

.346
.37

.417
.375
.647
.632
.409
.391
.28

.25

.296

20

18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35

36

.692
.833

.714

.304
.375
.36
.375
.375
.296
.455
.409
.529
.526
.45
.429
.2
.179

.222

21

.846

.857
.833
.391
.458
.44

.458
.346
.37

.417
.375
.647
.632
.409
.391
.28

.25

.296

22

.833
.692
.714
.818
.304
.375
.36

.375
.375
.296
.455
.409
.625
.45

.45

.429

.179

.222

23

.933

O 667

.458

.583

.56

.583

.462

.379

.542

.722

.619

.545

.296

.31

.357

24

85

.643
.769
.667
.909
.348

.417

.417
.417
.286
.5

.455
.588
.579
.5

.409
.192
.214

.214

25

.857

.857

.714
.417
.542
.52
.542
.423
.393
.5
.458
.667

.571

.417
.308
.276

.37

26

.643
.769
.667
.909
.348
.417
.4

.417
.417
.286
.5

.455
.588
.579
.5

.409
.192
.214

.214

27

.846
.857
.692
.391
.458
.44
.458
.346
.37
.417
.375
.75
.55
.409
.391
.28
.25

.296

28

.857
.833
.391
.458
.44

.458
.346
.37

.417
.375
.647
.632
.409
.391
.28

.25

.296

29

.714
.417
.542
.52
.542
.423
.393
.5
.458
.667

.571

.417
.308
.276

.37

30

10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35

36

.304

.375

.36

.375

.375

.296

.455

.409

.529

.526

.45

.429

.179

.222

31

.72

.692
.72

.654
.731
.615
.577
.565
.696
.696
.667
.739
.72

.654

32

.88

.84

.655

.875

.76

.625

.75

.826

.792

.654

.643

.643

33

.88

.633

.769

.6

.654

.72

.76

.63

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34

86

1
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.655
.875
.76
.625
.75
.826
.792
.654
.643

.643

35

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.875

.913

.615
.826
.87

.593
.586

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36

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.533
.464
.571
.517
.667
.731
.655

.714

.792
.583
.708
.864
.826
.556
.552

.607

.542
.538
.739
.783
.519
.517

.571

.667

.591

.385

.393

.393

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

87

31

32

33

34

35

36

.727

.625

.667

.593

.593

.792

.792

88

1

.704

89

APPENDIX C
INHERITANCE COMPUTER PROGRAM

This computer program was written by the author using
QuickBasic on an IBM0 compatible 486 computer with a SVGA
monitor. It can be easily modified to accommodate slower
computers and those with other monitor types. The program
was written to aid with calculating tetraploid disomic and
tetrasomic genetic models, testing these models using the
chi-square statistic, and double checking these
calculations. The program will print a hard copy of the
calculations if desired. Use the subprograms in the
following order: Disomic or Tetrasomic Inheritance, Chi-
Square Analysis, Print Data, and Erase Data.

When calculating disomic progeny classes and
frequencies, the order of the four alleles is important.
Segregation or fixed heterozygosity can be achieved with the
Disomic Inheritance Subprogram based upon the allele order
in the parental genotype. For example, the genotype abab
will result in aa, ab, and bb gametes in a 1:2:1 ratio.
However, if the order is aabb, the result will be only ab

gametes (fixed heterozygosity) .

9O

DECLARE SUB title ()

DECLARE SUB cprint (matings, diexp$(), ex!(), dichil,
obspro!, start!, paras, parb$, obs(), defree)

DECLARE SUB chi (diexp$(), difreq!(), start, obspro, dichi,
ex(), obs(), defree)

DECLARE SUB tetragam (a$(), b$(), asort!(), bsort!(),
atotgam$(), btotgam$(), atotfreq!(), btotfreq!(),
acounter, bcounter, start, progeny$(), profreq!(),
prosort!(), diexp$(), difreq(), matings, paras, parb$,
agam$(). bgam$(). afreq(), bfreq())

DECLARE SUB mate (atotgam$(), btotgam$(), atotfreq!(),
btotfreq!(), asort!(), bsort!(), acounterl, bcounterl,
progeny$(), profreq!(). prosort!(). dieXp$(). difreq(),
start)

DECLARE SUB diqam (a$(), b$(), asort(), bsort(), atotgam$(),
btotgam$(), atotfreq(), btotfreq(), acounter, bcounter,
start, progeny$(), profreq!(), prosort!(), diexp$(),
difreq(). matings. paras. parb$, agam$(). bgam$().
afreq(). bfreq())

DECLARE SUB mogene (a$(), b$(), matings, paras, parb$)

DECLARE SUB datagone (prosort(), agam$(), bgam$(),
progeny$(). profreq(). diexp$(), difreq(), ex(), obs(),
start, a$(), b$(), atotgam$(), btotgam$(), atotfreq(),
btotfreq(), paras, parb$, asort(), bsort(), afreq(),
bfreq())

DIM a$(10), b$(10), asort(lO), bsort(lO), atotgam$(10),
btotgam$(10), afreq(lO), bfreq(10), ex(36), obs(36)

DIM atotfreq(lO), btotfreq(lO), progeny$(100), profreq(100),
prosort(100), agam$(10), bgam$(10), diexp$(100),
difreq(lOO)

SCREEN 9

CLS 0

LOCATE 4, l: PRINT "TETRAPLOID INHERITANCE"

LOCATE 7, 1: PRINT "By James A. Beaver"

LOCATE 24, l

PRINT "Press ’enter' to continue. Use this command"
throughout the program."

LOCATE 10, 1: PRINT "This program calculates expected"
progeny classes and frequencies from a cross,"

PRINT "compares these expected disomic and tetrasomic values"
to your observed values,"

PRINT "and calculates chi-squared values for statistical"
analysis of the results."

LOCATE 25, 1

INPUT cont$

12 CLS 0

PRINT : PRINT , "MAIN MENU"

LOCATE 5, 1

PRINT , "1", "Disomic Inheritance"

PRINT : PRINT , "2", "Tetrasomic Inheritance"
PRINT : PRINT , "3", "Chi-Square Analysis"
PRINT : PRINT , "4", "Print Data"

91

PRINT : PRINT , "5", "Erase Data"

PRINT : PRINT , "9", "Exit"

LOCATE 25, 5

INPUT "What is the number of your topic of interest"; topic

IF topic = 1 THEN CALL digam(a$(), b$(), asort(), bsort(),
atotgam$(), btotgam$(), atotfreq(), btotfreq(), acounter,
bcounter, start, progeny$(), profreq!(), prosort!(),
diexp$(), difreq(), matings, paras, parb$, agam$(),
bgam$(). afreq(), bfreq())

IF topic = 2 THEN CALL tetragam(a$(), b$(), asort!(),
bsort!(), atotgam$(), btotgam$(), atotfreq!(),
btotfreq!(), acounter, bcounter, start, progeny$(),
profreq!(), prosort!(), diexp$(), difreq(), matings,
paras. parb$, agam$(). bgam$(), afreq(), bfreq())

IF topic = 3 THEN CALL chi(diexp$(), difreq!(), start,
obspro, dichi, ex(), obs(), defree)

IF topic = 4 THEN CALL cprint(mating$, diexp$(), ex!(),
dichil, obsprol, startl, paras, parb$, obs(), defree)

IF topic = 5 THEN CALL datagone(prosort(), agam$(), bgam$(),
progeny$(), profreq(). diexp$(), difreq(), eX(). obs().

astart, a$(), b$(), atotgam$(), btotgam$(), atotfreq(),
btotfreq(), paras, parb$, asort(), bsort(), afreq(),
bfreQ())

IF topic = 9 THEN END

GOTO 12

END

SUB title

S 0
= 1
= 640
= 350

NEXT x

END SUB

1 T0 135

LINE (1, a)-(640, a)
LINE (b, 1)-(b, 350)
LINE (640, c)-(l, c)
LINE (a, 350)-(a, 1)
a +
b ..
c
mycolor = INT(x / 9)
COLOR mycolor

FOR y = 1 TO 50
NEXT y

F'Hrd

a
b
c

92

93

SUB cprint (matings, diexp$(), ex(), dichi, obspro, start,
paras, parb$, obs(), defree)

CALL title

LOCATE 13, 35: PRINT "PRINT DATA”

LOCATE 25, 1

INPUT cont$

CLS 0

PRINT : IF matings = "s" THEN INPUT "Please enter the
identification of the selfed parent.", idl$

IF matings <> "3" THEN PRINT "Please enter the
identification of each of the two parents. Press ’enter’ after each."

IF matings <> "s" THEN INPUT id1$

IF matings <> ”s" THEN INPUT id2$

IF matings = "" THEN PRINT "Please enter the genotype of
each of the two parents. Press 'enter' after each."

IF matings = "" THEN INPUT paras

IF matings = ”” THEN INPUT parb$

PRINT : INPUT "What locus or enzyme system is being
studied": locus$

PRINT : PRINT : INPUT "Please be sure your printer is ready
and then press enter.”, pri$

LPRINT : LPRINT locuss

IF mating$ = "s" THEN LPRINT id1$, "selfed"

IF mating$ <> ”s” THEN LPRINT idl$, "x”, id2$

LPRINT paras, "x", parb$

LPRINT “Observations =": dichi

 

 

LPRINT "
I!
LPRINT : LPRINT "Progeny classes and frequencies"
LPRINT : LPRINT : LPRINT "CLASS", , , "EXP", "OBS": LPRINT
x = start

DO UNTIL diexp$(x) = "0"
IF diexp$(x) <> ”1" AND diexp$(x) <> "0" THEN LPRINT
diexp$(x)
IF diexp$(x) <> "1" AND dieXP$(x) <> "0" AND ex(x)
<> 0 THEN LPRINT , , , ex(x), obs(x)
x = x + l
LOOP

LPRINT "

 

IF start <> 1 AND start <> 5 THEN LPRINT : LPRINT
"Chi-square =”: obspro

IF start = 1 THEN LPRINT "DISOMIC chi-square =": obspro

IF start = 5 THEN LPRINT "TETRASOMIC chi-square -n; obspro

LPRINT "Degrees of freedom =": defree

LPRINT : LPRINT : LPRINT

END SUB

 

94

SUB chi (diexp$(), difreq(), start, dichi, obspro, ex(),
obs(), defree)

CALL title

LOCATE 13, 30: PRINT "CHI-SQUARE ANALYSIS"

LOCATE 25, 1

INPUT cont$

CLS O

DIM term(36)

-INPUT "How many progeny have you observed": obspro

IF diexp$(start) <> "" THEN GOTO 180

INPUT "How many progeny classes do you expect": exclass

PRINT "Enter each progeny class and its expected frequency
in decimal."

PRINT "Press 'enter' after the progeny class and after its
frequency."

PRINT

FOR x = 1 TO exclass
INPUT "Class"; diexp$(x)
INPUT "Frequency": difreq(x)
PRINT

NEXT x

defree = exclass - 1
exclass = exclass + 1
diexp$(exclass) = "O"
b = 1
CLS 0

180 PRINT : PRINT
IF b <> 1 THEN b = start

X l
y 0
DO UNTIL diexp$(b) = "0"
ex(b) = difreq(b) * obspro
IF diexp$(b) <> "1" AND ex(b) <> 0 THEN PRINT :
TAB(x): b: SPC(2); diexp$(b): SPC(2): ex(b)
x = x + 40
IF diexp$(b) <> "" AND ex(b) <> 0 THEN y = y + 1
b = b + 1
LOOP

defree = y - 1

VIEW PRINT 21 TO 25

INPUT "Would you like to sum any of the expected classes
(y or n)": m$

IF m$ = "n" THEN GOTO 251

95

232 INPUT "How many classes would you like to sum together":
sum

defree = (defree - sum) + 1

PRINT : PRINT "Enter the numbers of the classes to be summed
together. Press enter after each class.”

summers = ""

sumfreq = 0

FOR x = 1 TO sum
INPUT sumclass
IF x = 1 THEN y = sumclass
summers - summers + diexp$(sumclass) + " "
diexp$(sumclass) = "1”
sumfreq = sumfreq + ex(sumclass)
ex(sumclass) = 0

NEXT x

diexp$(y) = summers

ex(y) = sumfreq

PRINT : INPUT ”Would you like to sum another (y or n)": m$
IF m5 = ”y" THEN GOTO 232

CLS o ,

x = start

PRINT : PRINT "EXPECTED PHENOTYPIC CLASSES AND FREQUENCIES"
PRINT : PRINT

DO UNTIL diexp$(x) = "0"
IF diexp$(x) <> "1” AND ex(x) <> 0 THEN PRINT :
diexp$(x): TAB(65): ex(x)
x = x + 1
LOOP

251 x = start

CLS 0

VIEW PRINT 21 To 25
dichi = 0

DO UNTIL diexp$(x) = ”0"
IF diexp$(x) <> "1" AND ex(x) <> 0 THEN PRINT ”Enter
the observed frequency for progeny class", diexp$(x)
IF diexp$(x) <> "1" AND ex(x) <> 0 THEN INPUT obs(x)
IF diexp$(x) <> "1" AND ex(x) <> 0 AND defree <> 1
THEN term(x) = CSNG(CSNG(obs(x) - ex!(x))) A 2 / ex!(x)
IF diexp$(x) <> "1" AND ex(x) <> 0 AND defree = 1
THEN term(x) = CSNG(CSNG(ABS(obs(x) - ex!(x)) - .5)) A 2 / ex!(x)
IF diexp$(x) <> "1" AND ex(x) <> 0 THEN dichi = dichi + term(x)
x = x + 1
PRINT
LOOP

PRINT : PRINT

96

IF start = 1 THEN statements = " DISOMIC INHERITANCE"
IF start = 5 THEN statements = " TETRASOMIC INHERITANCE"
IF start <> 1 AND start <> 5 THEN statements = " the NULL

HYPOTHESIS”
PRINT "The chi-square value for": statements; " is": dichi

PRINT "Degrees of freedom =”: defree
LOCATE 25, 1

INPUT cont$

VIEW PRINT

CLSO

END SUB

97

SUB tetragam (a$(), b$(), asort(), bsort(), atotgam$(),
btotgam$(), atotfreq(), btotfreq(), acounter, bcounter,
start, progeny$(), profreq!(), prosort!(), diexp$(),
difreq(), matingS. paras, parb$, aqam$(). bgam$(),
afreQ(). bfreq())

CALL title

LOCATE 13, 29: PRINT "TETRASOMIC INHERITANCE"

LOCATE 25, 1

-INPUT cont$

CALL mogene(a$(), b$(), matings, paras, parb$) 'Allows entry
of genetic model for each parent.

CLS 0

PRINT : PRINT

aqam$(5) = a$(1) + a$(2)

asort(S) = VAL(a$(1)) A 2 + VAL(a$(2)) A 2
afreq(S) = 1 / 6

bgam$(5) = b$(1) + b$(2)

bsort(5) = VAL(b$(1)) A 2 + VAL(b$(2)) A 2
bfreq(5) = 1 / 6

agam$(6) = a$(1) + a$(3)

asort(6) = VAL(a$(1)) A 2 + VAL(a$(3)) A 2
afreq(6) = 1 / 6

bgam$(6) = b$(1) + b$(3)

bsort(6) = VAL(b$(1)) A 2 + VAL(b$(3)) A 2
bfreq(6) = l / 6

agam$(7) = a$(1) + a$(4)

asort(7) = VAL(a$(1)) A 2 + VAL(a$(4)) A 2
afreq(7) = 1 / 6

bgam$(7) = b$(1) + b$(4)

bsort(7) = VAL(b$(1)) A 2 + VAL(b$(4)) A 2
bfreq(7) = 1 / 6

agam$(8) = a$(2) + a$(3)

asort(8) = VAL(a$(2)) A 2 + VAL(a$(3)) A 2
afreq(8) = 1 / 6

bgam$(8) = b$(2) + b$(3)

bsort(8) = VAL(b$(2)) A 2 + VAL(b$(3)) A 2
bfreq(8) = 1 / 6

agam$(9) = a$(2) + a$(4)

asort(9) = VAL(a$(2)) A 2 + VAL(a$(4)) A 2
afreq(9) = 1 / 6

bgam$(9) = b$(2) + b$(4)

bsort(9) = VAL(b$(2)) A 2 + VAL(b$(4)) A 2
bfreq(9) = 1 / 6

aqam$(10) = a$(3) + a$(4)

asort(lO) = VAL(a$(3)) A 2 + VAL(a$(4)) A 2
afreq(lO) = 1 / 6

bgam$(10) = b$(3) + b$(4)

bsort(lO) = VAL(b$(3)) A 2 + VAL(b$(4)) A 2
bfreq(lO) = 1 / 6

n = 5

PRINT : PRINT "Tetrasomic gametic classes and frequencies of
the first parent are"

98

FOR X = 5 TO 10

atotgam$(x) = agam$(x)
atotfreq(x) = afreq(x)
btotgam$(x) = bgam$(x)
btotfreq(x) = bfreq(x)

FOR y = x + 1 To 10

IF asort(x) = asort(y) THEN agam$(y) = "0"
IF asort(x) = asort(y) THEN atotfreq(x) =
atotfreq(x) + afreq(y)

IF asort(x) - asort(y) THEN afreq(y) = 0

IF bsort(x) bsort(y) THEN bgam$(y) = "0"

IF bsort(x) bsort(y) THEN btotfreq(x) =
btotfreq(x) + bfreq(y)

IF bsort(x) = bsort(y) THEN bfreq(y) = 0

NEXT y

IF atotgam$(x) <> "0" THEN PRINT

IF atotgam$(x) <> "0" THEN PRINT atotgam$(x)
IF atotfreq(x) <> 0 THEN PRINT atotfreq(x)
IF atotgam$(x) <> "0" THEN acounter = x

NEXT X

LOCATE 25, 1

INPUT cont$

CLS 0

LOCATE 3, 1: PRINT "Tetrasomic gametic classes and
frequencies of the second parent are"

FOR x = 5 TO 10
IF btotgam$(x) <> "0" THEN PRINT
IF btotgam$(x) <> ”0" THEN PRINT btotgam$(x)
IF btotfreq(x) <> 0 THEN PRINT btotfreq(x)
IF btotgam$(x) <> "0" THEN bcounter = x
NEXT x

start = 5

LOCATE 25, 1

INPUT cont$

CLS 0

CALL mate(atotgam$(), btotgam$(), atotfreq!(), btotfreq!(),
asort!(), bsort!(), acounterl, bcounterl, progeny$(),
profreq!(), prosort!(), diexp$(), difreq(), start)

END SUB

99

SUB mate (atotgam$(), btotgam$(), atotfreq(), btotfreq(),
asort(), bsort(), acounter, bcounter, progeny$(),
profreq(), prosort(), diexp$(), difreq(), start)

REM Calculates progeny classes and frequencies.

2 = start

PRINT : IF start = 1 THEN PRINT "The expected disomic
progeny classes and frequencies are"

IF start = 5 THEN PRINT "The expected tetrasomic progeny
classes and frequencies are"

PRINT : PRINT

'FOR x = start TO acounter
FOR y = start TO bcounter
profreq(z) = atotfreq(x) * btotfreq(y)
IF profreq(z) <> 0 THEN prosort(z) =
asort(x) + bsort(y)
IF profreq(z) <> 0 THEN progeny$(z) =
atotgam$(x) + btotgam$(y)
IF profreq(z) <> 0 THEN z = z + 1
NEXT y
NEXT x

num = start

FOR x = start TO 2
FOR y = x + 1 TO 2

IF prosort(x) = prosort(y) THEN progeny$(y)
= non

IF prosort(x) = prosort(y) THEN profreq(x)
profreq(x) + profreq(y)

IF prosort(x) = prosort(y) THEN profreq(y)
= 0

NEXT y
IF progeny$(x) <> "0" THEN diexp$(num) = progeny$(x)
IF progeny$(x) <> "0" AND profreq(x) <> 0 THEN
difreq(num) = profreq(x)
PRINT diexp$(num): IF difreq(num) <> 0 THEN PRINT
difreq(num): PRINT
IF progeny$(x) <> "0" THEN num = num + 1
NEXT x

IF diexp$(num) <> "0" THEN num = num + 1
diexp$(num) = ”0"
INPUT cont$

END SUB

100

SUB digam (a$(), b$(), asort(), bsort(), atotgam$(),
btotgam$(), atotfreq(), btotfreq(), acounter,
bcounter, start, progeny$(), profreq!(), prosort!(),
diexp$(), difreq(), matings. paras, parb$, agam$().
bgam$(), afreq(), bfreq())

CALL title

LOCATE 13, 30: PRINT "DISOMIC INHERITANCE"

LOCATE 25, 1

INPUT cont$

CALL mogene(a$(), b$(), matings, paras, parb$)’Allows entry
of genetic model for each parent.

CLS 0

LET agam$(1) = a$(1) + a$(3) 'Beginning of calculating
parent 'a' gametes

LET asort(l) VAL(a$(1)) A 2 + VAL(a$(3)) A 2

LET agam$(2) a$(2) + a$(4)

LET asort(2) VAL(a$(2)) A 2 + VAL(a$(4)) A 2

LET agam$(3) a$(1) + a$(4)

LET asort(3) VAL(a$(1)) A 2 + VAL(a$(4)) A 2

LET agam$(4) a$(2) + a$(3)

LET asort(4) VAL(a$(2)) A 2 + VAL(a$(3)) A 2

uh

FOR w = 1 TO
afreq(w) = .25
NEXT w

PRINT : PRINT "Disomic gametic classes and frequencies of
the first parent are"

atotgam$(1) agam$(1) 'Beginning of sorting ’a' gametes

atotfreq(l) afreq(l)

FOR X = 2 TO 4

IF asort(l) = asort(x) THEN atotfreq(l) = afreq(x) +
atotfreq(l)

IF asort(l) = asort(x) THEN agam$(x) =

IF asort(l) = asort(x) THEN afreq(x) = 0

non

NEXT X

PRINT : PRINT atotgam$(l): PRINT atotfreq(l): PRINT
acounter = 1
atotgam$(2

) = agam$(2)
atotfreq(2) =

afreq(2)

FOR y = 3 To 4

IF asort(2) = asort(y) THEN atotfreq(2) = afreq(y) +
atotfreq(2) .

IF asort(2) = asort(y) THEN agam$(y) =

IF asort(2) a asort(y) THEN afreq(y) = 0

«on

NEXT y

IF agam$(2) <> "0" THEN PRINT atotgam$(2)
IF agam$(2) <> "0" THEN PRINT atotfreq(2): PRINT

101

IF agam$(2) <> "0" THEN acounter = 2

atotgam$(3) = agam$(3)

atotfreq(3) = afreq(3)

IF asort(3) = asort(4) THEN atotfreq(3) = afreq(4) +
atotfreq(3)

IF asort(3) = asort(4) THEN agam$(4) = "0"

IF asort(3) = asort(4) THEN afreq(4) = 0

IF agam$(3) <> ”0" THEN PRINT atotgam$(3)

IF agam$(3) <> "0" THEN PRINT atotfreq(3): PRINT
IF agam$(3) <> "0" THEN acounter = 3

atotgam$(4) = agam$(4)

' atotfreq(4) = afreq(4)

IF agam$(4) <> "0“ THEN PRINT atotgam$(4)

IF agam$(4) <> "0" THEN PRINT atotfreq(4): PRINT
IF agam$(4) <> "0" THEN acounter = 4

LOCATE 25, 1
INPUT cont$
CLS 0

LET bgam$(1) - b$(1) + b$(3) ’Beginning of parent ’b' gamete
calculation
LET bsort(l)
LET bgam$(2)
LET bsort(2)
LET bgam$(3)

VAL(b$(1)) A 2 + VAL(b$(3)) A 2
b$(2) + b$(4)
VAL(b$(2)) A 2 + VAL(b$(4)) A 2
b$(1) + b$(4)

LET bsort(3) VAL(b$(1)) A 2 + VAL(b$(4)) A 2
LET bgam$(4) b$(2) + b$(3)
LET bsort(4) VAL(b$(2)) A 2 + VAL(b$(3)) A 2

FOR w = 1 TO 4
bfreq(w) = .25
NEXT w

PRINT : PRINT

PRINT "Disomic gametic classes and frequencies of the second
parent are"

btotgam$(1) = bgam$(1) 'Beginning of sorting b gametes

btotfreq(l) = bfreq(l)

FOR x = 2 TO 4

IF bsort(l) = bsort(x) THEN btotfreq(l) = bfreq(x) +
btotfreq(l)

IF bsort(l) = bsort(x) THEN bgam$(x) =

IF bsort(l) = bsort(x) THEN bfreq(x) = 0

"on

NEXT X

PRINT : PRINT btotgam$(1): PRINT btotfreq(l): PRINT
bcounter = 1

btotgam$(2) = bgam$(2)

btotfreq(2) = bfreq(2)

102

FOR y = 3 To 4

IF bsort(2) = bsort(y) THEN btotfreq(2) = bfreq(y) +
btotfreq(2)

IF bsort(2)

IF bsort(2)

"0"

0

bsort(y) THEN bgam$(y)
bsort(y) THEN bfreq(y)

NEXT y

IF bgam$(2) <> "0" THEN PRINT btotgam$(2)
IF bgam$(2) <> "0" THEN PRINT btotfreq(2): PRINT
IF bgam$(2) <> "0" THEN bcounter = 2

btotgam$(3) = bgam$(3)

btotfreq(3) = bfreq(3)

.IF bsort(3) = bsort(4) THEN btotfreq(3) = bfreq(4) +
btotfreq(3)

IF bsort(3) bsort(4) THEN bgam$(4) = "0"

IF bsort(3) bsort(4) THEN bfreq(4) = 0

IF bgam$(3) <> "0" THEN PRINT btotgam$(3)

IF bgam$(3) <> "0” THEN PRINT btotfreq(3): PRINT

IF bgam$(3) <> "0" THEN bcounter = 3

btotgam$(4) = bgam$(4)

btotfreq(4) = bfreq(4)

IF bgam$(4) <> "0" THEN PRINT btotgam$(4)

IF bgam$(4) <> "0" THEN PRINT btotfreq(4): PRINT

IF bgam$(4) <> "0" THEN bcounter = 4

start = 1

LOCATE 25, 1

INPUT cont$

CLS 0

CALL mate(atotgam$(), btotgam$(), atotfreq!(), btotfreq!(),
asort!(), bsort!(), acounter!, bcounterl, progeny$(),
profreq!(), prosort!(), diexp$(), difreq(), start)

END SUB

103

SUB mogene (a$(), b$(), matings, paras, parb$) 'Allows entry
of genetic model for each parent.

CLS O

PRINT "Define the genetic model."

PRINT : INPUT ”Did the progeny result from a self or a cross
(5 or c)": matings

PRINT : PRINT "Enter the genotype of the first parent. Type"
a number first and"

PRINT "then a letter to indicate an allele. Always use the"
same number with the"

'PRINT "same letter to designate a unique allele. Note the"
following examples:"

PRINT "1A, 2B, 3C, and so on. Press return after entering"
each allele.”

PRINT

paras = ""

FOR X = 1 TO 4
INPUT a$(x)
IF matings = "s" THEN b$(x) = a$(x)
paras = paras + a$(x)

NEXT X

IF matings = "s" THEN parb$ = paras
IF matings = ”s" THEN GOTO 100

PRINT : PRINT "Enter the genotype of the second parent."
parb$ = ""
FOR x = 1 TO 4
INPUT b$(x)
parb$ = parb$ + b$(x)
NEXT x

100 PRINT "The genotype of the first parent is "; paras
PRINT

PRINT "The genotype of the second parent is ": parb$
LOCATE 25, 1

INPUT cont$

CLS 0

END SUB

104

SUB datagone (prosort(), agam$(), bgam$(), progeny$(),
profreq(), diexp$(), difreq(). ex(), obs(), start. a$().
b$(), atotgam$(), btotgam$(), atotfreq(), btotfreq(),
paras, parb$, asort(), bsort(), afreq(), bfreq())

CALL title

LOCATE 13, 35: PRINT "ERASE DATA"
LOCATE 25, 1

INPUT cont$

CLS 0

PRINT : PRINT :

FOR x = 1 TO 3
LOCATE 3, 1
SOUND 100, 12
SOUND 250, 3
SOUND 50, 6
PRINT ”WARNING: THIS SUBPROGRAM ERASES YOUR DATA!"
FOR y = 1 To 100000
NEXT y
CLS 0
FOR y = 1 TO 30
NEXT y
NEXT x

PRINT : PRINT : PRINT "Would you like to erase your data
(y or n)? If you have not made"

PRINT "a hard copy of your data and would like to do so,
press ’n'."

INPUT gone$

IF gone$ <> "y" THEN PRINT : PRINT "Your data is safe."

IF gone$ <> "y" THEN GOTO 559

ERASE as, b$, asort, bsort, atotgam$, btotgam$, ex, obs,
atotfreq, btotfreq, progeny$, profreq, prosort, agam$,
bgam$, diexps, difreq, afreq, bfreq

0

start =

paras = "": parb$ = ""

PRINT : PRINT

PRINT "Your data has been erased."

559 LOCATE 25, 1
INPUT cont$
CLS 0

END SUB

105

APPENDIX D

SIMILARITY COMPUTER PROGRAM

I created this program by using QuickBasic to greatly
modify code written by Angus et al. (1988). The program
will run on IBM0 and other compatible computers. It can be
used to input binary data indicating the presence or absence
of enzyme bands for each genotype: edit, append, save, and
print raw data files; and calculate similarity matrices from
the binary data. Similarity matrices can also be saved and
printed.

Before entering data, the program asks for the number
of enzyme bands studied, the four-character alphanumeric
names of the enzyme bands, and the number of genotypes being
studied. After data entry is complete, the program will
create a matrix of similarity values calculated using the
Marczewski and Steinhaus Similarity statistic (Angus

et al., 1988).

106

DIM bandname$(1 TO 45), rawdata(l TO 115, 1 TO 45)

DIM similar(1 TO 115, 1 TO 115)

REM Major modifications by James Beaver, January 12, 1991

CLS 0

LOCATE 10, 1: PRINT "Similarity program"

LOCATE 12, 1: PRINT "This program calculates Marczewski and"
Steinhaus Similarity between genotypes."

100 LOCATE 19, 1: INPUT "Would you like to enter new data or
recall data from a disk file (e or r)"; cont$

' IF cont$ = "r" THEN GOTO 300

LOCATE 21, 5: INPUT "How many individuals are you studying":
indiv

LOCATE 22, 5: PRINT "What is the total number of different"
enzyme bands that you have observed"

INPUT : pronum

LOCATE 25, 1: INPUT "Please press 'enter' to continue. Use
this command throughout the program.": cont$

CLS 0

PRINT "Please label the bands. Use four characters or less"
for a name.”

LOCATE 6, 1

VIEW PRINT 6 TO 23

FOR x = 1 To pronum
PRINT : PRINT x: INPUT bandname$(x)
NEXT X

VIEW PRINT
LOCATE 25, 1: INPUT cont$
counter = 1

249 CLS 0

PRINT "Please enter the protein band data for each"
individual. Use '0' for absence of a band and '1’ for
presence of a band."

FOR x = counter TO indiv
SOUND 300, 4: SOUND 150, 2
LOCATE 4, 1: PRINT "Group ": x: PRINT : PRINT
VIEW PRINT 6 TO 23
FOR y = 1 TO pronum
PRINT bandname$(y)
275 INPUT rawdata(x, y): PRINT
IF rawdata(x, y) <> 0 AND rawdata(x, y) <> 1
THEN BEEP: GOTO 275
NEXT y
CLS
VIEW PRINT
NEXT x

107

LOCATE 25, 1: INPUT : cont$

299 CLS 0

INPUT "Would you like to create a disk file for your raw"
"data (y or n)": cont$

IF cont$ = "n" THEN GOTO 350

PRINT "Name of the raw data file to create or append"
(x:xxx.bas)": files

var = FREEFILE

OPEN files FOR OUTPUT AS #Var

FOR X = 1 TO indiv
FOR y = 1 TO pronum
WRITE #var, x, y, rawdata(x, Y).
bandname$(y)
NEXT y
NEXT X
GOTO 350

300 INPUT "Name of disk file of raw data to recall
(x:xxx.bas)": files

var = FREEFILE

OPEN files FOR INPUT AS #var

DO UNTIL EOF(var)
INPUT #var, x, y, rawdata(x, y), bandname$(y)
LOOP

CLOSE #var

counter = x + 1

pronum = y

INPUT "Would you like to add data on new individuals to the
recalled file (y or n)": cont$

IF cont$ <> "y" THEN indiv = x

IF cont$ <> ”y" THEN GOTO 340

INPUT "How many more individuals are you studying": add

indiv = x + add

GOTO 249

340 INPUT "Would you like to edit your data in the recalled
file (y or n)”: asks
IF ask$ <> "y” THEN GOTO 350

DO UNTIL stops = "n"
INPUT "What is the location of the datum to be
edited (x,y)": x, y
PRINT "Current value is ", rawdata(x, y)
INPUT "Change value to "3 new
rawdata(x, y) = new

108

INPUT "Would you like to edit another datum
(Y or U)": stOPS
LOOP

GOTO 299

350 CLS 0

INPUT "Would you like to make a hard copy of your raw data
(y or n)": cont$

IF cont$ = "n" THEN GOTO 400

IF indiv < 10 THEN k = indiv

IF indiv > 9 THEN k = 10

w = 1

LPRINT : LPRINT : LPRINT "RAW DATA MATRIX"

DO
tops = I! "
FOR 2 = j To k
IF 2 < 10 THEN tOpS = tOpS + " " + STR$(2)
+ I! It
IF 2 > 9 AND 2 < 100 THEN top$ = tops + " "
+ STR$(z) + " "
IF 2 > 99 THEN tops = tops + STR$(z) + "
NEXT 2

LPRINT : LPRINT
LPRINT tops
LPRINT
FOR x = 1 To pronum
IF LEN(bandname$(x))
bandname$(x) + " "
IF LEN(bandname$(x))
bandname$(x) + " "
IF LEN(bandname$(x)) = 1 THEN bandname$(x)
bandname$(x) + " "

3 THEN bandname$(x)

2 THEN‘bandname$(x)

enters = bandname$(x) + " "
FOR y = j TO k
enters = enters + " " +
STR$(rawdata(y, x)) + " "
NEXT y
LPRINT enters
NEXT X
j = j + 10
m = k
IF m + 10 <= indiv THEN k = k + 10
IF m + 10 > indiv THEN k = indiv
w = w + 1

LOOP UNTIL w > indiv / 10 + 1

400 CLS 0
PRINT : INPUT "Would you like to calculate Marczewski and"

Steinhaus Similarity between individuals (y or n)": cont$

ll

109

IF cont$ = "n" THEN END
PRINT : PRINT "Calculating Marczewski and Steinhaus"
Similarity between individuals"

FOR x = 1 TO indiv
FOR y = 1 TO indiv
IF x = y THEN similar(x, y) = 1: GOTO 500
w = 0: a = 0: b = 0
FOR m = 1 TO pronum
IF rawdata(x, m) <> 0 THEN a
IF rawdata(y, m) <> 0 THEN b
IF rawdata(x, m) <> 0 AND
rawdata(y, m) <> 0 THEN w = w + 1

a + 1
b + 1

NEXT m
IF w = 0 THEN similar(x, y) = 0: GOTO 500
similar(x, y) = w / (a + b - w)
similar(x, y) = INT((similar(x, y) + .0005)
* 1000) / 1000
500 NEXT y
NEXT x

CLS 0

INPUT ”Would you like to create a similarity matrix disk
file (y or n)": disks

IF diSRS = "n" THEN GOTO 600

INPUT "Name of similarity matrix file to create
(x:xxx.bas)":rmyfile$

var = FREEFILE

OPEN myfiles FOR OUTPUT AS ivar

FOR x = 1 TO indiv
data$ = ""
FOR y = 1 TO indiv
data$ = data$ + STR$(similar(x, y))
NEXT y
WRITE #var, data$
NEXT X

600 PRINT

INPUT "Would you like to make a hard copy of your similarity
matrix. "; cont$"

IF cont$ "n" THEN END

X = 1

IF indiv < 10 THEN y = indiv

IF indiv > 9 THEN y = 10

z = 1

LPRINT : LPRINT : LPRINT : LPRINT "MARCZEWSKI AND STEINHAUS"
SIMILARITY MATRIX"

DO
heads = " "
FOR I = x To y - 1

110

IF I < 10 THEN heads = heads + STR$(I) +
I! II
IF I > 9 AND I < 100 THEN heads = heads +
STR$(I) + " "
IF I > 99 THEN heads = heads + STR$(I) +
II II
NEXT I
heads = heads + STR$(y)
LPRINT : LPRINT : LPRINT
LPRINT head$: LPRINT
FOR k = 1 To indiv

data$ = ""
FOR j = x TO y
w$ = STR$(similar(j, k))
sp = 7 - LEN(ws)
in$ = nu
FOR q = 1 To sp
in$ = in$ + " "
NEXT q
IF k >= j THEN data$ = data$ +
STR$(similar(j, k)) + in$
NEXT j
LPRINT
IF k < 10 THEN LPRINT " "; k; " ": data$
IF k > 9 AND R < 100 THEN LPRINT " ": k;
n n; data$
IF k > 99 THEN LPRINT k: " ": data$
NEXT k
X = X + 10
P = Y

IF p + 10 <= indiv THEN y = y + 10
IF p + 10 > indiv THEN y = indiv
z = z + 1

LOOP UNTIL z > indiv / 10 + 1

LPRINT : LPRINT : LPRINT
CLOSE
END

111

APPENDIX E

PRINCIPAL COORDINATE ANALYSIS COMPUTER PROGRAM

This program subjects values in similarity matrices to
principal coordinate analysis (PCO). It was written by Dr.
Carl Ramm of the Department of Forestry at Michigan State
University. The program was run using SAS (SAS Institute,
Inc., Cary, N.C.) on the IBM0 mainframe at Michigan State
University. Print out from the program includes the percent
of genetic variation accounted for by each principal
coordinate, PCO scores in the first eight dimensions
(coordinates used for graphing), and two-dimensional graphs
of all combinations of the first three dimensions as axes.
However, the graphs should be recreated using other
software, such as PlotIt”, that can produce graph axes of

equal length.

112

CMS FILEDEF DATAI DISK NSALLI DATA A1;
DATA DI;
INFILE DATAI;

INPUT PLOTID 3 51-512;
RUN:
CMS FILEDEF DATA2 DISK NSALL2 DATA A13
DATA 02;
INFILE DATA2:

INPUT PLOTID s 513-524;
RUN:
CMS FILEDEF DATA3 DISK NSALL3 DATA A13
'DATA 03;
INFILE DATA3;

INPUT PLOTID s 325-536;
RUN;
DATA FULLSET3

MERGE D1 D2 03;
BY PLOTID;

PROC IML3
USE FULLSET;
READ ALL INTO A (IROWNAME=PLOTID COLNAME=COLS|);
PRINT A [FORMAT=10.4];
NN . NROW(A);
--------------- CONVERSION OPTION 1 ---------------—-------;
------- CHATFIELD & COLLINS PAGE 201 CHANGE SIMILARITY TO DISTANCE

8'8:

* D = 1(1‘) - A $0 HAVE ZERO DIAGONAL MATRIX

* THEN SQUARES DISTANCES (DIJ‘Z) AND RUN ANALYSIS AS USUAL

* SONE = J(NN,NN,1);

* DIST - SONE - A;

* S = (-1/2) * (DIST # DIST):

0 ---------------------------------------------------------- ;

t -------------- CONVERSION OPTION 2 ----------------------- ;

* ------- DIGBY & KEMPTION PAGE 83 ------------------------ ;

* IF AIJ - SIMILARITY 0<=AIJ<=1, USE A ;
S . A;

4 -------------------------------------------------------- ;

PRINT 5 [FORMAT=8.4] ;

RESET NONAME3
ONEN=J(NN,1,1);

I 8 DIAG(ONEN);

H I I - (1/NN)*ONEN*(ONEN‘);

I

USE H TO DOUBLE-CENTER THE MATRIX AT ORIGIN 3
RESULTS IN LOSS OF ONE DIMENSION 3

=H*S*(H');

$3118.8-

I
CALL EIGEN(D,L,E);

3

SAS/IML PRODUCES ORTHONORMAL EIGEN VECTORS 1!!
L'L = I

FORM THE MATRIX X = L *+ SQRT(D) WHERE L IS THE
MATRIX OF EIGEN VECTORS, D THE DIAGONAL MATRIX
OF EIGEN VALUES. THE NEW COORDINATES FOR THE N
POINTS ARE THE ROWS - REPEAT ROWS - OF X

08-88-88-8-

‘O ‘0 ‘0 ‘0 ‘0 ‘0

‘0 ‘0 ‘0

113

* SEE KRZANOWSKI. PAGE 106 + 3
* ( IF L'L 8 D THEN ROWS OF L ARE THE NEW COORDINATES 3
* 3

DP 8 100 * D/(SUM(D))3

RESET NAME3

PRINT ”EIGEN VALUES & PERCENT VARIATION”3

HVAR . D II DP;

HH - ("EIGEN VALUES- "s VARIATION");

PRINT HVAR (ICOLNAME=HH FORMAT=20.4I)3

I. - L[,1:4];

D . D[1:4'];

DD = DIAG(D);

DD . SQRT(DD);

PTSCORE = L*DD3

RESET NONAME;

PRINT 'PCO SCORES IN FIRST FOUR DIMENSIONS'3
PRINT PLOTID [FORMAT=10.4] PTSCORE [FORMAT=10.4];
RNAME = PLOTID;

R1=PTSCORE[,1]; R2=PTSCORE[,2];

R3=PTSCORE[,3]; R4-PTSCORE[,4];

RSCORE1=Rll|R23
RSCORE2=R2 R33

CALL PGRAF(RSCORE1,RNAME,”PCOl','PCO2',”PRINCIPAL COORDINATE ANALYSIS”);
CALL PGRAF(RSCORE2,RNAME,"PC02”,"PC03”,'PRINCIPAL COORDINATE ANALYSIS”);
RSCORE3=R1 | | R3 3

CALL PGRAF(RSCORE3.RNAME,"PC01",'PCO3".”PRINCIPAL COORDINATE ANALYSIS");

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HICHIGRN STQTE UNIV LIBRQRIE

I|I1III IIIIII3 IIIIII 8I I7|I|||I|III| II II