THESiS

iiiiiiiiiiiiiiiiiiii iii

3 1293 01766 8132

 

LIBRARY

Michigan State
University

 

 

 

This is to certify that the

dissertation entitled
R‘FL? mapping I QTL édwiifi‘wflm, amfl
C" Wefi‘c gum/(#411 1:” gp-W MMYg

presented by

Dec/tun WM?“

has been accepted towards fulfillment
of the requirements for

vle degree in ELO’M‘ Britt/£7 M“, ggflef’y’f

 

 

@/ K/flhflc’w"

L/ Wérofessor

 

[)ate

 

PLACE IN RHURN BOX to remove this chedcout from your record.
To AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

‘ ' #1:!!!ng
Q

 

 

SEP 0 6 201]

th‘

APR 0 5:94..

 

 

o3

‘

 

 

. 2’20
* asaoliffiai

APRZOHBIZOU'

 

 

 

 

 

 

 

 

 

RF LP MAPPING, QTL IDENTIFICATION, AND CYTOGENETIC
ANALYSIS IN SOUR CHERRY

By

Dechun Wang

A DISSERTATION

Submitted to
Michigan State University
in partial fulfilment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Horticulture
Plant Breeding and Genetics Program

1998

ABSTRACT

RFLP MAPPING, QTL IDENTIFICATION, AND CYTOGENETIC
ANALYSIS IN SOUR CHERRY

By

Dechun Wang

Three separate but related projects were carried out to establish a foundation for
the utilization of molecular markers and cytogenetic tools in the genetic study and
breeding of tetraploid sour cherry (Prunus cerasus L., 2n=4x=32).

In the first project, restriction fragment length polymorphism (RFLP) linkage
maps of two tetraploid sour cherry cultivars, Rheinische Schattenmorelle (RS) and Erdi
Botermo (BB), were constructed from 86 progeny from the cross RS x EB. The RS
linkage map consists of 126 single dose restriction fragment (SDRF, Wu et al. 1992)
markers assigned tol9 linkage groups covering 461.6 cM. The BB linkage map has 95
SDRF markers assigned to 16 linkage groups covering 279.2 cM. Fifiy-three markers
mapped in both parents were used as bridges between both maps and 13 sets of
homologous linkage groups were identified. F ifiy-nine of the markers on the linkage
maps were detected with probes used in other Prunus genetic linkage maps. Six of the
sour cherry linkage groups may be homologous with six of the eight genetic linkage
groups identified in peach and almond.

In the second project, the map locations and effects of quantitative trait loci
(QTLs) for eight flower and fruit traits in sour cherry were estimated using the RFLP
genetic linkage maps constructed in the first project. Eleven putatively significant QTLs
(LOD > 2.4) were detected for six characters (bloom time, ripening time, % pistil death,

% pollen germination, fruit weight and soluble solids concentration). The percentage of

phenotypic variation explained by a single QTL ranged from 12.9 % to 25.9 %. Fifty
percent of the QTLs identified for the traits in which the two parents differed significantly
had allelic effects opposite to those predicted from the parental phenotype. Three QTLs
affecting flower traits (bloom time, % pistil death, and % pollen germination) mapped to
a single linkage group, EBl. The RFLP closest to the bloom time QTL on E8] was
detected by a sweet cherry (P. avium L.) cDNA clone pSl4l whose partial amino acid
sequence was 81% identical to that of a Japanese pear (Pyrus pyrifolia Nakai

) stylar RNase.

In the final project, genomic in situ hybridization (GISH) was used to examine
meiotic pairing behavior and parental genomic contributions in the allotetraploid sour
cherry. Three sour cherry cultivars were studied: Montmorency, Rheinische
Schattenmorelle (RS), and Erdi Botermo (EB). GISH analysis suggested that EB may
have a higher genomic contribution from P. avium than P. fruticosa (the two putative
progenitor species). In contrast, GISH analysis only identified a relatively few number of
species-specific chromosomes and chromosome segments in RS, suggesting that
significant intergenomic recombination had occurred. In the meiotic analyses, in addition
to the normal bivalent pairing configuration, univalents, trivalents, and quadrivalents
were frequently observed in the pollen mother cells of the three cultivars. RS had the
most bivalents and the least number of quadrivalents. Montmorency and EB had
approximately the same numbers of bivalents and quadrivalents. RS had a bivalents to
non-bivalents ratio of 4.4:] while EB and Montmorency had a ratio of 3.5: 1. The ratio of
bivalents to non-bivalents may be an important factor in determining the proportion of

balanced and unbalanced meiotic products.

Copyright by
DECHUN WANG

1998

ACKNOWLEDGMENTS

I am very grateful to my major professor, Dr. Amy Iezzoni, for her guidance,
financial support, and editorial assistance.

I appreciate my committee members, Dr. Jim Hancock, Dr. David Douches, Dr.
Brian Diets, and Dr. Mitch McGrath, for their thoughtful reviews of my dissertation and
helpful advice during the course of my study here at Michigan State University.

I thank Dr. Joanne Whallon, Dr. Mitch McGrath, and Dr. Mirko Schuster (Federal
Center for Breeding Research on Cultivated Plants, Germany), for their advice on my
cytogenetic project. I appreciate Dr. Mitch McGrath for giving me the free access to his
fluorescence microscope which was critical in the GISH experiments.

I would like to express my gratitude to Renate Karle, Tom Brettin, Wendy Huss,
Rebecca Henry, Suzanne Downey, Chris Owens, and F atih Canli for their assistance in
various aspects of my projects. I would also like to thank Pete Callow and Terry Ball for
their permission to use their lab equipment.

Special thanks to my wife Cuihua for her constant support, patience, and love.

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................................... ix
LIST OF FIGURES ........................................................................................................... x
CHAPTER 1: GENETIC LINKAGE MAP IN SOUR CHERRY USING RFLP
MARKERS ................................................................................................ 1
ABSTRACT ....................................................................................................................... 2
INTRODUCTION .............................................................................................................. 3
MATERIAL AND METHODS ......................................................................................... 9
Mapping population and DNA isolation ................................................................ 9
Source of DNA probes ......................................................................................... 10
Southern analysis ................................................................................................. 14
X2 and linkage analysis ........................................................................................ 15
RESULTS AND DISCUSSION ...................................................................................... 16
REFERENCES ................................................................................................................ 29
CHAPTER 2: QTL ANALYSIS OF FLOWER AND FRUIT TRAITS
IN SOUR CHERRY ................................................................................ 31
ABSTRACT ..................................................................................................................... 32
INTRODUCTION ............................................................................................................ 33
MATERIAL AND METHODS ....................................................................................... 34
Plant material ....................................................................................................... 34
Traits measured .................................................................................................... 34
Bloom time ............................................................................................... 34
Ripening time ........................................................................................... 35
Flower bud death ...................................................................................... 35
Pistil death ................................................................................................ 35
Pollen germination ................................................................................... 36
Fruit set .................................................................................................... 36
Fruit weight and total soluble solids concentration ................................. 36
Molecular marker and QTL analysis ................................................................... 36
RESULTS AND DISCUSSION ...................................................................................... 39
Distribution of traits ............................................................................................. 39
Correlation of traits .............................................................................................. 43
Genetic linkage maps ........................................................................................... 43
QTL analysis ........................................................................................................ 44
REFERENCES ................................................................................................................ 54

vi

TABLE OF CONTENTS (continued)

CHAPTER 3: CYTOGENETIC ANALYSIS OF SOUR CHERRY USING

GENOMIC IN SIT U HYBRIDIZATION ................................................ 57

ABSTRACT ..................................................................................................................... 58
INTRODUCTION ............................................................................................................ 59
MATERIAL AND METHODS ....................................................................................... 61
Plant material ....................................................................................................... 61
Chromosome preparation ..................................................................................... 62

Probe preparation and in situ hybridization ......................................................... 62
RESULTS ........................................................................................................................ 64
Genomic in situ hybridization .............................................................................. 64
Pairing configuration at meiosis .......................................................................... 67
DISCUSSION .................................................................................................................. 70
REFERENCES ................................................................................................................ 76
APPENDIX ..................................................................................................................... 78

vii

LIST OF TABLES

Chapter 1

. Probes from other Prunus research groups which were unlinked or mapped to
one or more locations in sour cherry. RS and EB refer to the Rheinische
Schattenmorelle and Erdi Botermo linkage groups, respectively ............................... 11

. RFLP genetic analysis of progeny from Rheinische Schattenmorelle (RS) x Erdi
Botermo (EB) .............................................................................................................. 17

. Polymorphic markers that were present in one parent and did not segregate 1:1
in progeny .................................................................................................................. 18

. Polymorphic markers that were present in both parents and did not segregate 3:1
in progeny .................................................................................................................. 20

. Shared probes on which the assignment of linkage group number of sour cherry
maps were based ........................................................................................................ 26

. Distances of common pairs of markers which were mapped in sour cherry and
other Prunus species .................................................................................................. 26

Chapter 2

. Mean phenotypic values and standard deviations (SD) for the progeny and
parents (RS and EB), and the value range for the progeny ........................................ 4O

. QTLs detected for each trait. QTLs are named according to trait
abbreviations and a number is used to distinguish QTLs affecting the
same trait. Data were based on the analysis of trait means over three

years except for the trait percent pollen germination and the QTL pdl .................... 45
Chapter 3
. Mean number of chromosome pairing configurations per PMC at metaphase I ....... 70

viii

LIST OF TABLES (continued)

Appendix

. Information about all single dose restriction fragment (SDRF) markers that
fit the expected ratios ................................................................................................. 80

. Degree days (DD) for bloom and ripening for each progeny in the mapping
population for the years 1995, 1996, 1997 and the average ....................................... 84

. Fruit weight, soluble solids concentration for each progeny in the mapping
population for the years 1995, 1996, 1997 and the average ....................................... 86

. Pistil death, fruit set, pollen germination, flower bud death for each progeny
in the mapping population ......................................................................................... 88

ix

LIST OF FIGURES

Chapter 1

. RFLP maps for Rheinische Schattenmorelle (RS) and Erdi Botermo (EB).

Markers shown on the right are identified by the probe followed by a letter

when more than one marker is generated from a single probe. Correspondences
between anchor loci of RS and EB linkage groups are shown with dashed lines ...... 22

Chapter 2

. Frequency distributions for each character in the mapping population.
Means for the parents RS and EB are shown by arrows ............................................ 41

. LOD scores for bloom date on linkage groups EB 1 (blml) (A); and Group 2

(blm2) (B); pistil death (pd) on linkage groups EB 1 (C); RS 6 (D) and pollen
germination % (pg) on linkage group EBl (E). Peak LOD scores for each trait

are indicated by arrows. Linkage groups are shown below the x-axes. The

horizontal line indicates the level of significance at LOD = 2.4. Curves

represent results from individual years of 1995, 1996, 1997, and over years ............ 46

. LOD scores for ripening date on linkage groups RS4 (rpl) (A) and Group 6

(rp2) (B); soluble solids concentration on linkage groups EB7 (sscl) (E) and

RS6 (ssc2) (F), and fruit weight on linkage groups EB4 (fwl) (C) and Groups

2 (wa) (D). Peak LOD scores for each trait are indicated by arrows. Linkage

groups are shown below the x-axes. The horizontal line indicates the level of
significance at LOD = 2.4. Curves represent results from individual years of

1995, 1996, 1997, and over years .............................................................................. 47

. Alignment of the amino acid sequences of the sweet cherry cv. Emperor Francis
RNase (pSl41) with the pear (Pyrus pyrifolia) non-S- RNase (D49529)(Norioka

et al. 1996). The alignment maximizes homology at the nucleotide and amino

acid sequence levels. The conserved amino acids are indicated by asterisks ........... 50

Chapter 3

. Fluorescent micrographs of metaphase PMC chromosomes from the sour

cherry cultivar Montmorency afier genomic in situ hybridization using total

genomic DNA from P. fruticosa PF -HortFarm as the probe labeled with biotin

and detected with fluorescein. The hybridization signal appears as yellow-green
fluorescence while the unhybridized regions appear as orange-red with the
counterstain propidium iodide. Examples of different meiotic pairing

configurations are identified by roman numerals in the top micrograph as:

I - univalent, II - bivalent, III - trivalent, and IV — quadrivalent ................................. 65

LIST OF FIGURES (continued)

Chapter 3

. a-d. Fluorescent micrographs of metaphase PMC chromosomes from the sour

cherry cultivar Erdi Botermo after genomic in situ hybridization using total

genomic DNA from either P. avium (a, b), or P. fruticosa PF 26e1(36) (c, d) as

the probe. The probes were labeled with digoxigenin and detected with

fluorescein. The hybridization signal appears as yellow-green fluorescence

while the unhybridized regions appear as orange-red with the counterstain

propidium iodide. The arrow in micrograph a points to a bivalent formed by
homoeologous chromosomes ..................................................................................... 66

. a-d. Fluorescent micrographs of metaphase PMC chromosomes from the sour

cherry cultivar Rheinische Schattenmorelle after genomic in situ hybridization

using total genomic DNA from either P. fiuticosa PF26el(36) (a, b), or P. avium

(c, d), as the probe. The probes were labeled with digoxigenin and detected with
fluorescein. The hybridization signal appears as yellow-green fluorescence while

the unhybridized regions appear as orange-red with the counterstain propidium
iodide. The arrows in micrograph a point to bivalents formed by homoeologous
chromosomes ............................................................................................................. 68

. a, b Fluorescent micrographs of PMC chromosomes from sour cherry cultivar
Rheinische Schattenmorelle. The chromosomes were stained with the DNA-
specific dye DAPI and were shown by blue fluorescence. a Metaphase-I
chromosomes showing examples of different meiotic pairing configurations
identified by roman numerals as: l - univalent, II - bivalent, and IV -
quadrivalent. b Telophase-I chromosomes showing two univalents (arrows)
remain stationary at the equatorial plate when other chromosomes have reached

the poles ..................................................................................................................... 69
. Phase-contrast micrograph of metaphase PMC chromosomes from sour cherry
cultivar Erdi Botermo showing linear chain quadrivalents (arrows) ......................... 71
APPENDIX

. The consensus map of RS and EB maps (Chapter 1) constructed from the

combined data using JoinMap with a minimum LOD of 3.0 and a maximum
recombination frequency of 0.35. Markers in bold were present in EB only.

Markers indicated by asterisks were present in both RS and EB. All other

markers were present in RS only. .............................................................................. 79

xi

CHAPTER 1

GENETIC LINKAGE MAP IN SOUR CHERRY USING RF LP MARKERS

ABSTRACT

Restriction fragment length polymorphism (RFLP) linkage maps of two tetraploid
sour cherry (Prunus cerasus L., 2n=4x=32) cultivars, Rheinische Schattenmorelle (RS)
and Erdi Botermo (EB), were constructed from 86 progeny from the cross RS x EB. The
RS linkage map consists of 126 single dose restriction fragment (SDRF, Wu et al. 1992)
markers assigned to 19 linkage groups covering 461.6 cM. The EB linkage map has 95
SDRF markers assigned to 16 linkage groups covering 279.2 cM. F ifty—three markers
mapped in both parents were used as bridges between both maps and 13 sets of
homologous linkage groups were identified. Homoeologous relationships among the
sour cherry linkage groups could not be determined because only 15 probes identified
duplicate loci. F ifty-nine of the markers on the linkage maps were detected with probes
used in other Prunus genetic linkage maps. Six of the sour cherry linkage groups may be
homologous with six of the eight genetic linkage groups identified in peach and almond.
Twenty one fragments expected to segregate in a 1:1 ratio segregated in a 2:1 ratio. Three
of these fragments were used in the final map construction because they all mapped to the
same linkage group. Six fragments exhibited segregation consistent with the expectations

of intergenomic pairing and/or recombination.

INTRODUCTION

The sour cherry (Prunus cerasus L.) industry in the United States desperately
needs new improved cultivars to remain competitive in the world market and to reduce
pesticide and fungicide use. The sour cherry industry in the United States is a
monoculture of a 400 year-old cultivar from France, Montmorency. This cultivar must be
sprayed approximately 10-15 times a year to control various insects and diseases to
produce marketable fruit. It is also affected by numerous virus and mycoplasma diseases
that cannot be completely controlled, and yields are frequently reduced because flower
buds are killed by mid-winter cold temperatures or spring freezes.

A critical stage in the development of new cultivars is the selection of desired
individuals from breeding populations. Selections are traditionally based on the
phenotypic performance, which depends on the plant’s genetic potential and the
environment in which it grows. To distinguish the genetic component from the
environmental component of a phenotypic trait such as the yield, a resource-intensive
experiment has to be carried out. Moreover, in sour cherry breeding, selection for fruit
traits can not begin until seedlings pass a 3 - 5 years of juvenile stage.

With the advancement of molecular technology, genetic markers can be used to
start the selection as early as the seedling develops the first leaf (Beckman and Sollar
1983; Darvasi and Sollar 1994). Genetic markers are heritable entities, which can be
associated with economically important traits. The ideal genetic markers to be used in
marker-assisted selection (MAS) are those which are not influenced by the environment

and are tightly linked to the trait under selection (Staub et a1. 1996).

There are two general categories of genetic markers: phenotypic markers and
genotypic markers. Phenotypic markers include morphological traits and isozymes.
Morphological traits controlled by a single locus and reproducible over a range of
environments can be used as genetic markers. Isozymes, which are differently charged
protein molecules with the same activity, can also be used as genetic markers. Due to
their phenotypic nature, morphological traits and isozymes are influenced by the
environment (Staub et al. 1982) and the number of informative markers of both types is
limited. These two factors ofien restrict their utility (Staub et al. 1996). On the other
hand, genotypic markers are not limited in number and are not influenced by the
environment in which the plant grows. Genotypic markers include all DNA markers.

Restriction fragment length polymorphisms (RF LPs) were the first commonly
used DNA markers. RF LPs are revealed by cutting DNA with restriction enzymes and
using labeled DNA fragments as probes. Restriction enzymes cut DNA molecules at
specific nucleotide sequences (recognition sites), resulting in fragments of different sizes.
Mutations within restriction sites, as well as insertions or deletions of DNA fiagments
between two restriction sites, result in variation in sizes of restriction fragments. This
variation can be visualized with labeled probes by Southern blotting (Southern 1975).
Probes are usually genomic or cDNA fragments of 500 to 3000 base pairs. Different
species of the same genus oflen have sequence homology within the probe DNA
fragment, allowing RFLP probes to be shared among species. This makes RFLPs ideal
markers for comparative mapping. RFLPs are not subject to subtle changes in detection
procedure, therefore they are highly reproducible. The disadvantage of RF LPs is that they

require a large amount of sample DNA. Since the introduction of polymerase chain

reaction (PCR) (Mullis et al. 1986), RF LPs are gradually being replaced by PCR-based
DNA markers, which require a very small amount of sample DNA.

Many types of PCR-based markers have been developed. Among the commonly
used types are random amplified polymorphic DNA (RAPD), amplified fragment length
polymorphisms (AFLPs), and simple sequence repeats (SSRs). RAPD markers are
generated by PCR amplification of random genomic DNA segments with single primers
(usually 10 nucleotides long) of arbitrary sequence (Williams et al. 1990). AF LPs are
based on PCR amplification of restriction enzyme-digested DNA fragments with two
selective primers (Zabeau and Vos 1993). SSRs are tandem arrays of two or more
nucleotides. The polymorphisms result from variation in the number of repeats in a given
motif. SSRs are detected by PCR amplification of the repeat motif with two primers
designed from the sequenced regions flanking the repeat motifs.

Each of the three types of PCR-based markers has advantages and disadvantages.
RAPDs are technically the easiest to use among the three types. But extensive primer
testing is involved in generating RAPD markers. AF LPs can be generated in large
numbers with minimum primer testing. However, they are technically the most
complicated to use. SSRs are the most informative and reproducible. But extensive
initial cloning and sequencing are required to generate SSR markers. In addition to the
advantage of less sample DNA requirement, the analysis of PCR-based markers takes less
time than that of RF LPs. However, because PCR is error prone and sensitive to
contamination, PCR-based markers are less reproducible than RF LPs. The other
disadvantage of PCR-based markers as compared to RFLPs is that they are generally not

usable across species, not even across populations within species because the sequence

information revealed by a PCR-based marker is limited (Staub et al. 1996).

In order to use genetic markers in marker-assisted selection, the linkage
relationships among genetic markers and economic traits such as fruit quality must be
determined and presented in the form of genetic linkage maps. A genetic linkage map is
constructed from a mapping population in which the genetic markers are segregating.
Different types of mapping populations are typically used in map construction for diploid
species. Among the commonly used are F2, backcross (BC), recombinant inbred line
(RI), and doubled haploid populations. A completely classified F2 population provides
the maximum genetic information for map construction (Mather 1951). BC and doubled
haploid populations provide only half the genetic information of F2 population when
codominant markers are used (Mather 1951). When dominant markers are used, BC and
doubled haploid populations are more informative than F2 populations (Mather 1951).
R1 populations are less informative then backcross populations at low marker saturation
(Staub et al. 1996).

Normally, mapping populations are developed from crosses between relatively
homozygous inbred lines. In outcrossing species such as sour cherry, apple, and most
fruit tree species, homozygous inbred lines are not available due to inbreeding depression.
This precludes the use of regular backcross populations for genetic analysis in these
species. Instead, a pseudotestcross design is typically used in which the variety of interest
is crossed to a standard variety known to be homozygous recessive for the traits being
investigated. The segregation ratio for a single gene traits is either 1:1 or 1:0 in such a
population. A refinement of this approach is a double pseudotestcross in which genetic

analysis are performed on both parents in a controlled cross by keeping track of which

loci are heterozygous in each parent. Hemmat et al. (1994) employed this approach to
construct genetic maps for apple with each parent in such a double pseudotestcross.

Marker assisted selection would be especially advantageous for sour cherry
breeding. Sour cherry seedlings require a minimum of 3-5 years of growth before they
flower and fi'uit. If prior knowledge of linkage relationships between marker loci and
important flower and fruit characteristics were available, undesirable individuals could be
eliminated from progeny populations allowing more resources to be devoted to promising
individuals. Additionally, in whole genome BC selection using RFLP markers, it is
estimated that the recurrent parent genotype could be reconstructed and the introduced
gene maintained in three BC generations as opposed to the six BC generations required
without RF LP genotyping (Tanksley et al. 1989). Map-based BC selection is especially
attractive in sour cherry where a reduction in three BC generations could mean a saving
of 9-1 5 years.

Linkage map construction in sour cherry is complicated due to the species’
polyploid origin. The presumed progenitor species of the tetraploid sour cherry are the
diploid sweet cherry (P. A vium L, 2n=2x=16) and the tetraploid ground cherry (P.
fiuticosa Pall, 2n=4x=32)(Olden and Nybom 1968). Although sour cherry exhibits
primary disomic inheritance, there is evidence from allozyme segregation data for
occasional intergenomic pairing characteristic of a segmental allopolyploid (Beaver and
lezzoni 1993).

Construction of genetic linkage maps for polyploids is inherently more difficult
than for diploids for the following reasons: (1) a large number of genotypes is expected

for a single locus in a segregating population; (2) poorly characterized genome

constitution and/or chromosome pairing behavior are observed; (3) genome
characterization is complicated due to multiple fragments (Wu et al. 1992; Sorrells 1992).
To overcome these difficulty, several approaches have been employed, including:
construction of linkage maps for diploid relatives, using aneuploid stocks, taking
advantage of haploid or doubled haploid populations. However, one or more of these
approaches may not be feasible for certain species, including sour cherry. Moreover, the
gene order in the polyploid may have changed. One approach that is applicable to all
polyploid species is the use of single-dose restriction fragment (SDRF, Wu et al. 1992).
A SDRF is a fragment that is present in a single dose in a parent and that segregates in a
ratio of 1 :1 in the progeny.

Despite the potential utility of a genetic linkage map in the tetraploid sour cherry,
no linkage relationships have been reported. In Prunus, linkage maps are most advanced
in the diploid species. Genetic linkage maps have been constructed for: peach (P.
persica) (Chaparro et al. 1994; Rajapakse et al. 1995), almond (P. amygdalus, syn. P.
dulcis) (Viruel et al. 1995), sweet cherry (P. avium) (Stockinger et al. 1996), sweet cherry
x P. incisa, sweet cherry x P. nippom'ca (Boékovic’ et al. 1997), peach x almond (Foolad
et al. 1995) and peach x P. davidiana interspecific hybrid populations (Dirlewanger and
Bodo 1994). The markers used for these maps were predominantly RFLPs except for the
sweet cherry map constructed by Stockinger et al. (1996), for which RAPDs were used.

As in apple (Hemmat et al. 1994), the linkage mapping population in sour cherry
is a ‘pseudotestcross’ in which informative markers are those that are heterozygous in one
parent and homozygous recessive in the other parent and segregate 1 :1. However, in the

tetraploid sour cherry, if a band is present in one of the parents, the parental genotype can

be ++++, H+-, ++--, +-+-, or +---. Approximately 75 progeny are required to
conclusively identify the informative SDRF genotype (+---) based on 1:] segregation (Wu
et al. 1992).

The objective of this study was to construct low density RF LP linkage maps for
two sour cherry cultivars and compare these maps to previously constructed Prunus
RF LP maps. RF LP probes developed by other researchers were used to facilitate
comparative mapping; specifically the alignment of sour cherry linkage groups with the 8

linkage groups identified in peach and almond (Arr'rs, pers. comm; Bliss, pers. comm).

MATERIALS AND METHODS

Mapping population and DNA isolation

The mapping population consisted of 86 progeny from the cross between two sour
cherry cultivars, Rheinische Schattenmorelle (RS) x Erdi Botermo (EB). RS and EB
were chosen because they originated from different geographic areas (Germany and
Hungary, respectively) and differed in important horticultural traits such as bloom date.
cold hardiness, fruit quality and percent fruit set. The parents and progeny population are
maintained at the Michigan State University Clarksville Horticultural Experiment Station,
Clarksville, MI.

Young unfolded leaves were collected from 7-year-old trees and transported to the
laboratory in coolers filled with dry ice. The leaf samples were frozen at -80 C overnight
and then lyophilized for 48 - 72 hours. DNA isolation for Southern analysis followed the

procedure of Stockinger et al. (1996) with the following modifications: four hundred mg

of lyophilized leaves were placed in a 50-ml centrifuge tube together with five 4 mm
glass beads (Fischer Scientific, Pittsburgh, PA) and shaken vigorously for 4 minutes with
a paint shaker to grind the sample to a fine powder prior to the addition of extraction
buffer.
Source of DNA probes

DNA clones from the following sources were used to identify informative RF LP
markers (Table 1): (1) Plum genomic and peach cDNA clones (F. Bliss & S. Arulsekar;
Univ. of CA, Davis, CA), (2) peach genomic clones (S. Rajapakse & A. Abbott;
Clemson Univ., Clemson, SC), (3) peach cDNA clones (A. Callahan; USDA,
Kearneysville, WV), (4) almond genomic and cDNA clones (P. Arr’rs; IRTA, Barcelona,
Spain), (5) P511 genomic clones from the sweet cherry cultivar Emperor Francis, and (6)
cDNA clones from a stylar cDNA library from the sweet cherry cultivar Emperor Francis.

Sweet cherry genomic clones: A genomic library was constructed using size
fractionated Prunus avium cv. Emperor Francis DNA. Methylation sensitive Pstl
(Boehringer Mannheim, Indianapolis, IN) was used to digest genomic DNA which was
isolated as described (Stockinger et al. 1996) except that an additional CTAB-chlorofonn
extraction was performed followed by ethanol precipitation. The plasmid vector, pUC19,
was cut with PstI and dephosphorylated with calf intestinal alkaline phosphatase (Gibco
BRL, Gaithsburg, MD). Size selection of genomic DNA was achieved by fractionating
the digested DNA on a 1 % TAE agarose gel (Sambrook et al. 1989). Fragments 500bp to
2000bp were isolated from the gel by placing a piece of DEAE NA45 membrane
(Schleicher & Schuell, Keene, NH) into the gel and electrophoresing the appropriately

sized DNA into the membrane. The membrane was prepared and the DNA was

10

Table l. Probes from other Prunus research groups which were unlinked or mapped
to one or more locations in sour cherry. RS and EB refer to the Rheinische
Schattenmorelle and Erdi Botermo linkage groups, respectively.

 

 

CPM53

Probe“ Linkage group(s) in sour cherry map References
AC1 unlinked Viruel et al. 1995
AC6 unlinked
Pru2 RS 8
AC27 RS 2, EB 2
AG6 RS 12
AG8 EB 13
AGIO RS 7, BB 7
AG21 RS 2
AG40 RS 17, EB 17, R818, EB 18
Extl RS 8, BB 8
Old RS} e + ++ - - e e
B4G10 EB 6, RS 17 Rajapakse et al. 1995
B6D1 unlinked
B7H2 RS 16
3% . 38.19)“ - - e- - e _
CPM2 RS 5, BB 5 Bliss (pers comm);
CPM6 RS 12 Foolad et al. 1995
CPMIZ RS 1, BB 1
CPM20 RS 5, BB 5, RS 5', RS 6, EB 6
CPM23 RS 6, EB 6, EB 14
CPM30 RS 5
CPM39 RS 6, EB 6, RS 17, EB 17, RS 18,
CPM48 RS 7, EB 7, EB 7'

RS 4, EB 4

11

Table 1. (cont’d)

 

 

Probe“ Linkage group(s) in sour cheny map References

CPM57 RS 9, BB 9 Bliss (pers. comm.);
CPM58 RS 4, EB 4 Foolad et al. 1995
CPM59 RS 2

CPM64 RS 7, BB 7

CPM67 RS 7, EB 7

CPM70 EB 5, RS 5', RS 19

CPM90 RS 2

CPM104 RS 6, RS 6'

PLG10 unlinked

PLG86 RS 2, EB2

Hsp4 RS 2, EB 2 Callahan (pers. comm.)
pcth8 unlinked

pch202 RS 5, EB 5

pch205 RS 3

 

*AC = almond cDNA clones, Pru2 = cDNA for the seed protein Prunin (P. Arus,
personal comm.), AG = almond genomic clones, Extl = cDNA for Extensine, Olel =
cDNA for Oleosine, B- = peach genomic clones, CPM = peach mesocarp cDNA clones,
PLG = plum genomic clones, Hsp4 = peach cDNA for a heat shock protein, pch108 =
peach cDNA for chlorophyll A/B binding protein, pch202 = peach cDNA for a
thioredoxin, pch205 = peach cDNA for a water stress protein.

12

recovered according to the manufacturer’s instructions. The size selected DNA and
pUC19 DNA were concentrated in a Microcon concentrator (Amicon Inc., Beverly, MA),
heated to 65 C for 5 min, then ligated in a 10 ul reaction with T4 DNA ligase (Boehringer
Mannheim, Indianapolis, IN) as described by Sambrook et al. (1989). Recombinant
plasmid DNA was then transformed by electroporation into E. coli DHS-a
electrocompetent cells using the manufacturer’s protocol (Bio-Rad Laboratories,
Hercules, CA). White colonies were picked from LB plates containing ampicillin (125
ug/ml), X-gal (40 rig/ml), and IPTG (0.95 ugml), for further analyses. Inserts were
amplified by PCR using primers which flank the multiple cloning site of pUC 1 9
(Promega, Madison, WI). The size of amplified insert DNA was checked on a 1 %
agarose gel. The approximate copy number of cloned fragments was determined by dot
blotting. One hundred nanograms of insert DNA was blotted onto a Zeta-Probe GT
membrane (Bio-Rad Laboratories, Hercules, CA) with control DNAs which were known
to be low, medium, and high copy in the cherry genome. The dot blots were hybridized
with sour cherry genomic DNA labeled with 32P dCTP using a nick translation kit
(Boehringer Mannheim, Indianapolis, IN). Prehybridization and hybridization conditions
were as described by Stockinger et al. (1996). These genomic clones are identified by
“EF” referring to Emperor Francis and the clone number.

Sweet cherry cDNAs: RNA was isolated from approximately 1 g of stylar tissue
from the sweet cherry cultivar Emperor Francis by the method of Manning (1991) with
the following modifications: four phenol chloroform isoamylalcohol (25:24: 1) extractions
were performed and the [Na+] in the first butoxyethanol precipitation was adjusted to 100

mM. Stylar cDNA was prepared using a cDNA synthesis kit (Boehringer Mannheim.

l3

Indianapolis, IN) and a cDNA amplification protocol (Jepson et al. 1991). This stylar
cDNA was subsequently used in a PCR amplification with two degenerate primers,
ATNCA(T/C)GGN(C/T)TNTGGCC and (C/G)(A/1‘)(A/G)CANGTNCC(A/G)TG(T
/C)TT, designed to amplify ribonuclease sequences. Primer design was based on
conserved amino acids identified by aligning several S-allele and ribonuclease amino acid
sequences (T-H. Kao, personal communication). Four major bands resulting from
amplification with the degenerate primers were isolated from a 5 % native
polyacrylamide gel (Sambrook et al. 1989). These fragments were then reamplified,
cloned into pUC118, and copy number determined as described above for the sweet
cherry genomic clones. These probes were identified by “PS” for Prunus stylar tissue and
the clone number.

Southern analysis

DNA (6ug) of both parents and 12 progeny was digested with 20 - 30 units of one
of six restriction enzymes (BamHI, Dral, EcoRI, HindIII, Pstl, or Xbal; Boehringer
Mannheim Biochemicals, Indianapolis, IN) and separated on a 0.9 % agarose gel for 30 h
at 23V. Southern analysis was performed according to Stockinger et al. (1996) using
Hyborid-N+ membranes (Ambersham, Arlington Heights, IL).

Probe DNAs were prepared by PCR amplification of the inserts from pUC19 or
Bluescript plasmids (Stratagene, La Jolla, CA) using a pair of primers flanking the
cloning sites. Radiolabelling of probes with 32P-dCTP (DuPont, Boston, MA) was done
using the random priming method of Feinberg and Vogelstein (1983). Those enzyme and
probe combinations that identified useful polymorphisms from the two parents and 12

progeny were used to genotype the additional 74 progeny in the mapping population.

14

X2 and linkage analysis

Informative markers for a pseudotestcross mapping population are single-dose
restriction fragment (SDRFs) that differ between parents and segregate in a 1 :1
(presence:absence) ratio and SDRFs present in both parents that segregate in a 3:1 ratio
(Wu et al. 1992). Therefore, markers which differed between parents were tested for fit to
a 1:1 (presence:absence) ratio. Markers present in both parents were tested for fit to a 3:1
(presence:absence) ratio. Those markers which fit the appropriate ratios at the 5 % level
were used in the linkage analysis.

Markers present in one parent that did not fit to a 1:1 ratio were tested for fit to a
5:1 or 2:1 ratio. A 5:1 ratio would be expected for tetrasomic inheritance of a double dose
restriction fragment (DDRF, +-+- x ----; Wu et al. 1992). A ratio of 2:1 could probably
represent either (1) a skewed 1:1 ratio due to possibly gametophytic selection or a lethal
allele or (2) a skewed 3:1 ratio which would be expected for disomic inheritance of a
DDRF (+-+- x ----). Markers which fit a 2:1 ratio at the 5 % level were included in an
initial linkage analysis; however, only those 2:1 markers that exhibited linkage with
another 2:1 marker were included in the final linkage analysis. These linked 2:1 markers
may identify linkage groups which have been preferentially selected. The other 2:1
markers were not used because their genotype (DDRF or SDRF) could not be determined
based on the 2:1 ratio.

Linkage analyses were performed with JoinMap V2.0 (Stam 1993) using a
minimum LOD score of 3.0 and a maximum recombination fraction of 0.35. Distances
were calculated by the Kosambi function and expressed in centi-Morgans . Multiple loci

detected using the same probe were labeled with a letter after the probe designation.

15

Where possible, linkage groups were numbered based upon suspected homology with
previously constructed peach and almond linkage maps (Bliss, personal comm.; Viruel et

al. 1995).

RESULTS AND DISCUSSION

Two hundred sixty probes were tested to select informative probes. Ninety-nine
probes were found to detect polymorphic markers. Eighty-two of these probes were able
to identify SDRFs (Table 2).

Seventy-six percent of the polymorphic markers detected with the selected probes
fit the expected ratios for SDRFs. A total of 190 SDRFs were identified, of which, 1 10
SDRF markers fit a 1:1 ratio. RS and EB were heterozygous for 67 and 43 of these 1:1
markers, respectively. A total of 80 SDRF markers were present in both parents and fit a
3:1 ratio (Table 2).

Twenty seven segregating fragments present in one parent and absent in the other
parent did not fit a 1:1 ratio (Table 3). Of these fragments, 9 were present in RS and
absent in EB, and 19 were present in EB and absent in RS. Of the 9 RS fragments, 8
fragments fit 1:2 or 2:1 ratios and one fit a 5:1 (+,—) ratio . Of the 19 EB fragments, 13
fragments fit 1:2 or 2:] ratios, and 3 fragments fit a 5:1 (+,-) ratio. The other 3 fragments
had distorted presencezabsence ratios of 79:6, 81 :2 and 84:2.

Sour cherry is an allotetraploid originated from two distinct species, P. avium and
P fruticosa. Disomic inheritance is characteristic of an allotetraploid. In a cross between

two strict allotetraploids, if a band is present in one parent and absent in the other. the

16

Table 2 RFLP genetic analysis of progeny from Rheinische Schattenmorelle (RS) x

 

Erdi Botermo (EB).

Mapping population size 86
Number of probes tested 260
Number of polymorphic probes 99
Number of probes that identified SDRFs 82
Number of markers segregating 1:1 in RS 67
Number of markers segregating 1:1 in EB 43
Number of markers present in EB and RS segregating 3:1 80
Number of linkage groups in RS map 19
Number of linkage groups in EB map 16

Map units for RS map 461.6 cM
Map units for EB map 279.2 cM
Number of markers mapped in RS map 130
Number of markers mapped in EB map 100
Number of unlinked markers in RS 17
Number of unlinked markers in EB 23

 

17

Table 3 Polymorphic markers that were present in one parent and did
not segregate 1:1 in progeny.

 

 

Marker name Parent No. of individual tested ratio xj value
RS EB + -

EF146H1 + - 27 57 1:2 0.08
EF176EV3 + - 27 55 122 0.03
EF60EI + - 33 52 122 1.08
EF187EI4 + - 52 33 2:1 1.08
EF158H4 + - 51 30 2:1 0.46
B8A3X1 + - 49 28 2: 1 0.29
EF66E11 + - 54 30 2:1 0.19
AC27EV4 + - 55 23 2:1 0.59
EF48EV1 + - 66 16 521 0.36
PLG86E11 - + 28 52 122 0.09
EF 661312 - + 31 53 122 0.44
CPM6EV2 - + 31 51 l :2 0.68
EF176H3 - + 50 32 2:1 1.12
AG40H4 - + 52 33 2: 1 1.08
CPM53a - + 52 33 2:1 1.08
PS41EV2 - + 52 32 2:1 0.80
PLGI 0H3 - + 49 30 2:1 0.71
EF71 E12 - + 53 32 2:1 0.66
EF182a - + 54 30 221 0.19
CPM70E13 - + 55 30 2:1 0.13
EF156a - + 58 25 2:1 0.45
EF173X1 - + 55 22 221 0.87
EF187EV5 - + 64 18 521 1.42
EF156H4 - + 65 18 5: 1 1.29
EF132X4 - + 62 14 5:1 0.11
EF172EV4 - + 79 6 - -
PLG10H2 - + 81 2 - -
PLGlOHl - + 84 2 - -

 

l8

band will theoretically either be present in all progeny or segregates at a 1:1 (SDRF) or a
3 :1 (DDRF) ratio when there is no intergenomic recombination. The 1:2 ratios observed
in this study could be a skewed 1:1 ratio resulting from gametophytic selection. The 2:1
ratio could be either a skewed 1:1 ratio or a skewed 3:1 ratio as discussed above. The 5:1
ratio observed for the 4 markers could only be explained as the results of tetrasomic
inheritance from a cross +-+- x ----. The segregation ratios of 79:6, 81 :2 and 84:2 could
be explained as the results from loss of fixed heterozygosity. Skewed segregation ratios
and loss of fixed heterozygosity in sour cherry were also observed in a genetic study using
allozymes by Beaver and Iezzoni (1993). A 2:1 ratio was accepted and the expected 3:1
ratio was rejected for three out of nine inheritance ratios for three unlinked allozyme loci
(Beaver and Iezzoni 1993). Fifieen out of 308 progeny exhibited a loss of fixed
heterozygosity for 6-Pgd-2 (Beaver and Iezzoni 1993). The observations of a few
markers showing tetrasomic inheritance and loss of fixed heterozygosity indicate that
intergenomic chromosome pairings occur in a low frequence during meiosis of sour
cherry. Cytogenetic studies support the theory that some of the segregation results are
due to intergenomic recombination. Meiosis-I in sour cherry should result in the
formation of 16 bivalents. However, quadrivalents were frequently observed for the
mapping parents RS and EB (see Chapter 3).

Thirty-two fragments that were present in both parents and segregating in the
progeny did not fit a 3:1 ratio which was expected from segregation of a SDRF in each
parent (+--- x +---) (Table 4). Nine of these fragments fit a 2:1 ratio which could be a
skewed 3:1 ratio resulted from gametophytic selection or zygotic lethal genes. The other

fragments had segregation ratios ranging from 5:1 to 84: 1. In these cases, it is possible

19

Table 4 Polymorphic markers that were present in
both parents and did not segregate 3:1 in progeny.

 

 

Marker Name No. of Ratio of
+ - +/.
AG6E|2 47 35 1.3
EF127EV2 49 35 1.4
EF182EV1 50 34 1.5
EF61 EV 51 34 1.5
AC1 H 45 29 1.6
CPM12EI2 51 32 1.6
EF187H1 52 30 1.7
EFSODZ 54 30 1.8
OleoEl1 52 28 1.9
AG6E|3 71 13 5.5
EF180X 66 12 5.5
EF132X1 65 11 5.9
AC27EV3 70 10 7.0
pch205El1 73 10 7.3
EF87EV 75 10 7.5
EF191X1 69 9 7.7
EF187EV2 72 9 8.0
EF67EV2 76 9 8.4
EF77H3 77 8 9.6
CPM6EV1 75 6 12.5
EF187H6 77 6 12.8
EF187E|1 78 6 13.0
CPM104E|1 79 5 15.8
AG40H3 81 4 20.3
EF156H2 80 3 26.7
EF77H1 82 3 27.3
CPM20H6 80 2 40.0
EF48EV3 81 2 40.5
CPM43EI3 81 2 40.5
EF187EI5 83 2 41.5
CPM90H2 83 2 41.5
EF133H3 84 1 84.0

 

20

that one or both of the parents was double dose for the scored fragment ( +-+- x +--- or
+-+- x +-+-). However, the progeny size of 86 was too small to statistically distinguish
between these various segregation hypotheses.

Linkage analysis of the 21 markers segregating 2:1 or 1:2 revealed that only three
of these markers, EF156a, CPM53a, and EF182a, were linked. These three markers
were added to the data containing the 190 SDRFs for map construction. Two maps were
constructed separately, one for the parent RS and the other for the parent EB.

The RS linkage map consists of 130 markers assigned to 19 linkage groups
covering 461.6 cM (Fig. 1). Seventeen markers remained unlinked. Four redundant
markers were removed from the map because each of them was mapped to the same
location with another marker detected by the same probe. The longest linkage group in
RS map, RS8, is 71.8 cM while the shortest linkage group, RS7', is 5.8 cM. The average
length of all linkage groups is 24.3 cM. The longest distance between two adjacent
markers is 20.5 cM (RS3). The average distance between two adjacent markers is 4.3
cM.

The EB linkage map possesses 100 markers assigned to 16 linkage groups
covering 279.2 cM (Fig. 1). Twenty-three markers were unlinked. Five redundant
markers were removed from the map. The longest linkage group in EB map, EB7, is 35.5
cM while the shortest linkage group, EB6', is 0 cM. The average length of all linkage
groups is 17.5 cM. The longest distance between two adjacent markers is 20.9 cM
(EBl 7). The average distance between two adjacent markers is 3.5 cM.

Thirteen EB linkage groups homologous to the RS linkage groups were identified

using 53 bridging markers heterozygous in both parents. EB counterparts of RS linkage

21

RSI

 

EBl

 

 

RSZ

 

 

 

"1—161'1726 Tr—I‘ZHHC Apt-1.10%
3.0
4'8 — ~rrr7ra M
, ‘ ‘ —l-—EF12&a
I
-4'—EFI723 , 1x
5.5 .
ar—(rmo
23
6‘ . —(.?M12b _4,_ ACIII
’ "I -— r—(‘PMIIA
I
--—(‘i-Mr:h I ,0 EB 2
0'9 — u—(‘leh 4.0 --
Hrmb _ L
T H‘Ho -4 - H178 \ _‘ “1,.
\
7.3 4.2 3.8 \ 3.5
\ .
PLUKO
d—ersoc —"—Pl(186-—"f _
’10 09—1’_A(‘Z7b—-" fi-4A‘27b
— EFIO7d ’ ‘
o.o:_[. , _,’ -“r—EFISQd ”—~—r;m_rc ‘(3-3
11.9, 1.1 _4 L— .“ a
I 2.2 At-.. -*—-FFI.u
I -— HIM La
3.8 I ‘0—1 27 _‘__”‘M
-Hrtm-I--.‘ -h—u —-"
8'Q:_”""“” ”de1132.“ '04
ITTH'W’" ' ' i T leb
49
9.6
6-7 -—4--r-_Hsz
4,7
.2 7
_lh-EH6C —<>-—I3Frsxc
.2 rm - - "
12 l_r -ac
' -—~—orcr
no
15.4
—i*—t'PM59
19
—‘*-'EFl7lb
3.1
“Two“ —"-FH76a
RS5 EBS RSS'
-J—(‘PM304 -—1r— Hm.
'0 4'— ”6‘4 """ FFhZa
0" HI} 0"
H:— 0a —————— 05— ”130.
"“ 2'6 Hl‘il ------ ' ‘ H-|9|
‘Jl—EFMJ
'4'— EFMb 2) 43
26 ‘ s ' EF1-39h
\ _1 . ,
' _ ‘ "{(PM (In ----- _ >—<‘PM?0.
- -—l 1— II‘7KC ‘ \ ‘ [.5 [J ‘1?)
0* -‘*—('PM.‘b‘\ sq I "“ "it'wroc
l7 [inph “ ,’ ' -'lt-—F|'I)t 35
‘-—1y—-|. ‘ _-;\-~. 0.8... iinthH-h’h .
| r ””0“ , \A‘ 1.0 _ (‘i’Mia.(,PMZUh - - —1 ~— (Pmob
-1r—('PM20c’ ’ -,3 (mm
’ ‘ —-4 mm
25 I , r .
, -i>—('PMza 3,.
I. ..
‘7'— [+149- —l'—pch202t>
3.3
EFMC
'7 tzrisr.
3.0
jh— EF7Kh
0" "ptnzrm

 

RS3

4.11

20.5

—1

d

H46,-
HpchIOS

'— [SF-16d

)— H1326

 

1

RS

93

5.5

l

EB4

-‘r-H|56u

4 10.7

>—' PHSXb
2': Wm
- t‘PMSJb — — ‘ tPstnb
»— (‘PMSKA ~ ~ .17

—‘ *— (’PMSXa

-<r-Erlxla

 

32
— HI27 - ~ g
- -1f— Hm
H
I
l—Erlxm
'—EF7I

 

7

RS

10.)!

40

1.4

12
0.s_,

111

3.2

100

6

>— [r110
EB6'
(‘I'\lm-t
[FISZJ
I
I
I
, EB6
I
Hrszb , a
(Pure-s ll
4.1
1'
(‘I‘MZOt -
“1”le ‘ or #71:“le
,' \ ' ' 1 E17136":
1'16“! I I 3
HIl-fo“ \r, 3‘.
,_ (. Mirna Auk
HH-me I \ ‘ Hives,-
CPMDJ \
\1.0
Him - ‘ ""2“
(musk 10 ~',_ ' "‘M‘
H4010“
\
\
\ 3 r ,
4 (I'MMJ
4.0 -
—5‘ ( PMZOu
— (‘PMzod I
>—- l~Fl76j

 

Figure 1. RFLP maps for Rheinische Schattenmorelle (RS) and Erdi Botermo (EB).
Markers shown on the right are identified by the probe followed by a letter when more

than one marker is generated from a single probe. Correspondences between anchor loci

of RS and EB linkage groups are shown with dashed lines.

22

RS7

—« -—A(ilOa

11.5

I

I \

8.7 I '
I

I

I

._1 h—(‘PMMQ’
12.5
-—+ — Him
I
2.9 I
.— r527 - - .1 _
0.7 :1 »— Hum ,
2.0 l
7-1 — A01“: ’
87

-—1 — ('I’NIMJ

 

RSll

.1_.Fr72---_

123

—l’— 1.11061) ’ I

 

_l.1t

>—-CPM67a\ I
‘ I 4
\I
A

EB7

— -— (TI'M481:

--v—-EF185___-

._ _. (”PM-186 .. - ..
1.2 (- I'MM’b
0.0 : an.” - - -
()1, _l (‘1’Mb7a
'75 FFI7M‘

4 ‘— FFI76n

3.5

-—4>—A(ilm)

9.5

—( ~— (,‘PMMD
1.4

.1 _. AGIUC
2.5

w 1—- 1:1-187k
0.6 __‘ ,— P527

_1 _. than

 

EBll

—i L FFTZ

8.7

FFIOM
1 2 FFISHd
' —1 .— 1:1 1116b

 

Figure l (cont’d)

RS 7'

EH!“
4.4

('PMJRI)
I 4 1:17?

RS 12

-—1 — AUb
4.0

—-( — EPISKQ
5.9

— EPISRf

14

—- (‘PM6

6.6

—n-—F.F158c

 

RS8

—1 r-—PS41

16.9

— —-E1~156b
2.4

— y—Pml

19.0

--1 -—FFI43|
3 4

—4 v—FFl-Ub
6.4

fl h—EFl‘k - - -

9.6

—u—F.F76----

6.2

-¢v—Extlb - - _ _

—l r-I:.‘11I

1.2

EB8

9.7

6.3

3.9

3.0—J
>—F.F145b - " ‘ '

2.7
1.0

—F;Flll
-—EFI4Sa

1 l

 

23

— FF76

h—Exllb

»— 5171451)

 

RS9

—«F—EF17bk

47

1

r— FF176d

104

r— FFI761v ’ 7

1

-l '— CPMS'M

 

RS 10

E89

-4l-FFI766

114

-‘ i— (‘I‘MS'Ib

-ll_ FF176i

 

   

-‘l—F.F169a
9.4
—l—Errrsg
3" EB 10
-<--L~r-129 I
32 mm ---- ' rrm
”'7 l-'.Fl76c--- H trim
‘ -— —- u-mxa - - - " Hlusa

 

EB 13

— >-— I‘F4Sb
6.4

— ? 1:148:

Ar—AGR

 

 

EB 14

FF108b
FI‘IUH

—<

9.5

—< >-— CPMHc

 

 

 

 

 

 

 

RS 15 EB 15 RS 19
EB 17
RS 18
-—1 EI’187C -—4 )— EI‘1878 J '— [1 A3
2.. 7 R817 7...... EB 18 “ "
-—4 1—- E1487, 3'7 I 3‘9
,‘dt—EF'W RS 16 —-H-—AG406” —1—Ar.40a —l—uam
‘1 '-- —1»—1-.1-mb 1.3 ——B4(i|0c
—11—[1Ht17d‘ —.—r-;1~‘157
7.0
6.5
12.3 8.1 209 I44 —‘ 5' 1.14615
__, __ use 14.3
5.6
2 .1 ,— R7112.
" arms . .
fil—liflx‘ld -1_ —"-(.PM-06
1— (mm
_[—CPM396 \\‘ T (,9
_ -— (‘PM196
"13— 11167
l

 

Figure 1 (cont’d)

groups 3, 12, 16, and 19, were not identified. Conversely, RS counterparts of EB linkage

groups 13 and 14 were also not identified. Two EB linkage groups were homologous to

RS linkage group 6. The longer of the two was named EB6 and the shorter was named

EB6'. Two homologous RS linkage groups were identified for each of the two EB

linkage groups, 5 and 7. As for BB linkage group 6, RS5 and RS7 were used to name the

longer linkage groups, and RSS' and RS7' were used to name the shorter linkage groups

of RS linkage group 5 and 7, respectively. In all these cases, the two linkage groups

homologous to the same linkage group of the other parent may actually be two segments

of a single linkage group. When more markers are added to the map, the two linkage

groups may eventually become one linkage group.

The three markers that fit a 2:1 ratio were mapped to EB Group 4. All three

markers had an overabundance of the allele unique to EB, suggesting that the region

containing these alleles may have been preferentially selected.

Since sour cherry is a tetraploid with x=8, the ultimate goal is to identify 16

24

linkage groups and the homoeologous relationships among these linkage groups. For
example, Groups 17 and 18 may be homoeologous groups because markers identified
with probes AG40 and CPM39 mapped an average of 18.2 and 14.4 cM apart in both
linkage groups, respectively (Fig. 1). However, no other homoeologous segments could
be identified with the set of probes used in this analysis. The ideal probe for identifying
homoeologous linkage groups in a tetraploid is a probe that identifies 2 segregating bands
which map to different linkage groups. Of the 82 probes that identified mapped
fragments, only 15 probes met this criterion. F orty-six probes identified only one mapped
fragment, and 21 probes identified two or more fragments which were mapped to the
same linkage group.

Fifty-nine markers on the linkage maps were detected with probes placed on other
Prunus linkage maps. Based on these common probes (Table 5), linkage groups were
numbered according to suspected homology to the previously constructed almond x peach
map (Arus, personal com.) and the peach x almond map (Bliss, personal comm.) Six of
the sour cherry linkage groups share 2 or more probes with the corresponding linkage
groups in the almond x peach and the peach x almond maps (Table 5), suggesting that
they may be homologous to the corresponding linkage groups. The map distances
between markers detected by shared probes are generally consistent with those in the
almond x peach and peach x almond maps (Table 6). For example, group 2 markers
identified with the probes AG21 and Ole] mapped 25.6 cM apart in RS (Fig. 1) and 24
cM apart in almond x peach (Arus, personal comm.). Another example is that the map
distance between group 2 markers identified with the probes CPM90 and PLG86 is 11.1

cM in RS (Fig. 1) and 13.2 cM in peach x almond (Bliss, personal comm.) However.

25

Table 5 Shared probes on which the assignment of linkage group number of
sour cherry maps were based.

 

Group number Probes common with the corresponding linkage group in
the almond x peach and peach x almond linkage maps

 

1 CPM12

AC27, AG21, CPM59, CPM90, PLG86, Olel
CPM53, CPM58

CPM2, CPM20

CPM20, CPM23, CPM39

AGlO, CPM48, CPM64, CPM67

Pru2, Extl

OONONLlrhN

 

Table 6 Distances of common pairs of markers which were mapped in sour cherry and
other Prunus species.

 

Probe pair in Linkage Distance in sour Distance in other Prunus map
common group cherry map (cM) (cM)
number

 

RS EB almond x peach peach x almond

 

AG21 - Olel 2 25.6 - 24 -
AC27 - AG21 2 9.7, 12.1 - 13 -
AC27 - Olel 2 13.5, 15.9 - ll -
CPM90 - PLG86 2 11.1 - - 13.2
PLG86 - CPM59 2 24.8 - - 48.2
CPM53 - CPM58 4 1.5 3.7 - 27.9
Pru2 - Ext] 8 44.6 - 52 -

 

26

inconsistency in map distances between markers detected by shared probes was also
found (Table 6). For example, group 4 markers identified with the probes CPM53 and
CPM58 mapped 27.9 cM apart in peach x almond (Bliss, personal comm.) but just 1.5
cM and 3.7 cM apart in RS and EB, respectively (Fig. 1). The general consistency in
map distances of common markers between sour cherry linkage groups and the
corresponding linkage groups in the almond x peach and peach x almond maps provide
firrther support for the likelihood of homologous relationship between the corresponding
linkage groups. These associations, however, are preliminary until more alignment
comparisons can be made.

Sweet cherry, a diploid Prunus, is suspected to be an ancestral progenitor of sour
cheny. Unfortunately, it is not possible to compare the sour cherry map with the
previously published maps from sweet cherry, sweet cherry x P. incisa, and sweet cherry
x P. nippom'ca, because these diploid maps consist exclusively of RAPD and isozyme
markers (Boskovié et al. 1997; Stockinger et al. 1996)

The longest Prunus linkage map published is a peach x almond map consisting of
approximately 800 cM (F oolad et al. 1995). Given that sour cherry is a tetraploid, a map
of comparable coverage should be 1500 cM. Our current maps cover only one third of
this expected total length. The requirements for informative marker state in a tetraploid
make it more challenging to add more markers to the map than that for a diploid. A
project to develop and map simple sequence repeat (SSR) loci is currently underway in
our laboratory to determine if potentially higher levels of heterozygosity at SSR loci will
increase the likelihood of identifying informative markers and identifying homoeologous

linkage groups in sour cherry. Additionally, if SSRs are conserved among Prunus

27

species, they would be excellent markers for comparative mapping.

The maps constructed in this study are the first genetic linkage maps for sour
cherry. They form the base for firrther genetic studies of important traits such as fruit
quality and stress tolerance in sour cherry using molecular markers. The following
chapter will describe the first application of these maps to identify quantitative trait loci

(QTL) controlling flower and fruit traits in sour cherry.

28

REFERENCES

Beaver JA, Iezzoni AF (1993) Allozyme inheritance in tetraploid sour cherry (Prunus
cerasus L.). J Amer Soc Hort Sci 118:873-877

Beckmann J S, Sollar M (1983) Restriction fragment length polymorphisms in genetic
improvement: Methodologies, mapping and costs. Theor Appl Genet 67:35-43

Boékovic’ R, Tobutt KR, Nicoll FJ (1997) Inheritance of isoenzymes and their linkage
relationships in two interspecific cherry progenies. Euphytica 932129-143

Chaparro J S, Werner DJ, O'Malley D, Sederoff RR (1994) Targeted mapping and linkage
analysis of morphological, isozyme, and RF LP markers in peach. Theor Appl Genet
87:805-815

Darvasi A, Sollar M (1994) Optimum spacing of genetic markers for determining linkage
between marker loci and quantitative loci. Theor Appl Genet 89:351-357

Dirlewanger E, Bodo C (1994) Molecular genetic map of peach. Euphytica 77: 101-103

Fehr WR (1987) Principles of cultivar development, volume 1, Theory and Technique.
McGraw-Hill, Inc, New York

F einberg AD, Vogelstein G (1983) A technique for radiolabelling DNA restriction
fragments to high specific activity. Anal Biochem 13216-13

Foolad MR, Arulsekar S., Becerra V, Bliss FA (1995) A genetic map of Prunus based on
an interspecific cross between peach and almond. Theor Appl Genet 91 :262-269

Hemmat M, Weeden NF, Manganaris AG, Lawson DM (1994) Molecular markers
linkage map for apple. J Hered 85:4-11

J epson I, Bray J, Jenkins G, Schuch W, Edwards K (1991) A rapid procedure for the
construction of PCR cDNA libraries for small amounts of plant tissue. Plant Mol Bio
Rep 9:131-133

Manning K (1991) Isolation of nucleic acids from plants by differential solvent
precipitation. Anal Biochem 195:45-50

Mather K (1951) Measurement of genetic linkage in heredity. John Wiley & Sons, Inc,
New York

Mullis K, F aloona F, Scharf S, Saiki R, Horn G, Erlich H (1986) Specific enzymatic

amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb
Symp Quant Biol 51 :263-273

29

Olden EJ and Nybom N (1968) On the origin of Prunus cerasus L. Hereditas 59:327-345

Rajapakse S, Belthoff LE, He G, Estanger AE, Scorza R, Verde 1, Ballard RE, Baird WV,
Callahan A, Monet R, Abbott AG (1995) Genetic linkage mapping in peach using
morphological, RF LP, and RAPD markers. Theor Appl Genet 90:503-510

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual, 2nd
ed. Cold Spring Harbor, New York, Cold spring Harbor Laboratory Press

Sorrells ME (1992) Development and application of RFLPs in polyploids. Crop Sci
32:1086-1091

Southern EM (1975) Detection of specific sequences among DNA fragments separated by
gel electrophoresis. J Mol Biol 98:503-517

Stam P (1993) Construction of integrated genetic linkage maps by means of a new
computer package: JoinMap. The Plant Journal 3:739-744

Staub J E, Kuhns LJ, May B, Grun P (1982) Stability of potato tuber isozymes under
different storage regimes. J Amer Soc Hort Sci 107:405-408

Staub J E, Serquen FC, Gupta M (1996) Genetic markers, map construction, and their
application in plant breeding. HortScience 31 :729-741

Stockinger EJ, Mulinix CA, Long CM, Brettin TS, Iezzoni AF (1996) A linkage map of
sweet cherry based on RAPD analysis of a microspore-derived callus culture
population. J Hered 87:214-21 8

Tanksley SD, Young ND, Paterson AH, Bonierbale, MW. 1989. RF LP mapping in plant
breeding: New tools for an old science. Bio/Technology 7:257-264

Viruel MA, Messeguer R, de Vicente MC, Garcia-Mas J, Puidomenech P, Vargas F, Arus
P (1995) A linkage map with RFLP and isozyme makers in almond. Theor Appl
Genet 91 :964-971

Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV (1990) DNA
polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic
Acids Res 18:6531-6535

Wu KK, Bumquist W, Sorrells ME, Tew TL, Moore PH, Tanksley SD (1992) The
detection and estimation of linkage in polyploids using single-dose restriction

fragments. Theor Appl Genet 83:294-300

Zabeau M, Vos P (1993) Selective restriction fragment amplification: A general method
for DNA fingerprints. European Patent Application. Publ 0534858A1

30

CHAPTER 2

QTL ANALYSIS OF FLOWER AND FRUIT TRAITS IN SOUR CHERRY

31

ABSTRACT

The map locations and effects of quantitative trait loci (QTLs) were estimated for
eight flower and fruit traits in sour cherry (Prunus cerasus L.) using a restriction fragment
length polymorphism (RFLP) genetic linkage map constructed from a double pseudo-
testcross. The mapping population consisted of 86 progeny from the cross between two
sour cherry cultivars, Rheinische Schattenmorelle (RS) x Erdi Botermo (EB). The genetic
linkage maps for RS and EB were 398.2 cM and 222.2 cM, respectively, with an average
interval length of 9.8 cM. The RS/EB linkage map that was generated with shared
segregating markers consisted of 17 linkage groups covering 272.9 cM with an average
interval length of 4.8 cM. Eleven putatively significant QTLs (LOD > 2.4) were detected
for 6 characters (bloom time, ripening time, % pistil death due to freeze damage, %
pollen germination, fruit weight and soluble solids concentration). The percentage of
phenotypic variation explained by a single QTL ranged from 12.9 % to 25.9 %. Fifty
percent of the QTLs identified for the traits in which the two parents differed significantly
had allelic effects opposite to those predicted from the parental phenotype. Three QTLs
affecting flower traits (bloom time, % pistil death due to freeze damage, and % pollen
germination) mapped to a single linkage group, EBl. The RF LP closest to the bloom
time QTL on E8] was detected by a sweet cherry cDNA clone pS 141 whose partial

amino acid sequence was 81% identical to that of a Japanese pear stylar RN ase.

32

INTRODUCTION

An important goal in sour cherry (Prunus cerasus L.) breeding is to develop
cultivars with improved fi'uit quality, delayed bloom time to avoid spring freezes, and a
range of ripening dates. Therefore, many flower and fruit traits such as bloom date,
percent pistil death due to freeze damage, ripening date, fruit weight and fruit soluble
solids concentration are important for selection in a sour cherry breeding program.
Unfortunately, direct selection for these traits can not be carried out until the seedlings
flower and fruit after a minimum of 3-5 years of growth. If prior knowledge of linkage
relationships between marker loci and important flower and fruit characteristics were
available, undesirable individuals could be eliminated from progeny populations with
marker-assisted selection as early as when the seedlings develop the first few leaves.

Linkage relationships between molecular markers and agronomically important
traits have been extensively studied in many crop plants for over a decade (Edwards et al.
1987; Stuber et al. 1987, 1992; Paterson et al. 1988, 1990; Keim et a1. 1990; Heyes et al.
1993; Wang et a1. 1994; Toroser et al. 1995; Grandillo and Tanksley 1996; Rebai et al.
1997; Pilet et al. 1998). In tree fruit crops, a QTL analyses was reported for growth and
development traits in apple (Conner et al. 1998) and QTL analyses for fruit size and fruit
sugar content are underway in peach (A. Abbott, pers. comm). In contrast, no QTL
analyses have been reported in sour cherry. The delay has been due to the difficulties in
the construction of a molecular linkage map for sour cherry because of the species’
polyploid origin and mixed patterns of inheritance (disomic and tetrasomic) (Beaver and

Iezzoni 1993; Wang et al. 1998) . Recently, we constructed the first molecular linkage

33

maps in sour cherry using RFLP markers (Wang et al. 1998) and in this report we
describe the first QTL analysis in sour cherry. Our objectives were to estimate the

locations and effects of QTLs affecting flower and fruit traits in sour cherry.

MATERIAL AND METHODS

Plant material

The mapping population utilized in this study is a double pseudo-testcross
population (Lawson et al. 1995) which consisted of 86 progeny from the cross between
two sour cherry cultivars, Rheinische Schattenmorelle (RS) x Erdi Botermo.(EB). RS and
EB were chosen because they are from different geographic areas (Germany and Hungary,
respectively) and differ for important horticultural traits. The trees are planted at the
Michigan State University Clarksville Horticultural Experiment Station, Clarksville, MI.
A total of 8 traits were evaluated for each progeny individual and the two parents. Five
traits were evaluated over 3 years and three traits were evaluated in one year. Details of

trait evaluations are given below.

Traits measured
Bloom time

The bloom date of an individual was recorded as the day when approximately
50% of the flowers were open. Hourly temperature readings were available from an
automated weather station at the Clarksville Horticultural ~Experiment Station. Time to

bloom was expressed as degree days (DD) from January 1 with a base temperature of 4.4

34

°C. Daily heat unit accumulation was calculated by summing the positive differences of
hourly temperature readings minus 4.4 °C and then dividing by 24. On the day of bloom,
heat unit accumulation was calculated to hour 10, which was the approximate time the
data were recorded. Bloom time was evaluated over 3 years (1995 - 1997).
Rip_ening time

The ripening time of an individual was recorded as the first day when the fruits
could be easily pulled off the stems. Time to ripening was expressed as degree days (DD)
following the same calculation as for bloom time except that the ripening date was used
as the ending date. Time to ripening was evaluated for 3 years (1995 - 1997).
Flower bud death

Flower bud death due to freeze damage is common in Michigan when the buds
start swelling in the early spring. Following a spring freeze to -10 °C on the night of April
5, 1995, flower bud death was evaluated from the swelled buds. About twenty flower
buds from each individual were cut open to determine bud death, which was expressed as

the percentage of dead buds. The data in percentage were angular transformed (i.e.

arcsin «[17 transformed) to normalize the distribution of the data for QTL analysis.

Pistil death

Pistil death was evaluated during the bloom periods of 1995, 1996, and 1997
following natural freezing events. Ten flowers were randomly selected for evaluation
from each of the four sides (north, south, west, and east) of a tree. The dead pistils were
counted to calculate the percentage of dead pistils in 40 flowers. The percentage data
were angular transformed in the same way as for flower bud death data before QTL

analysis.

35

Pollen germination

Percent pollen germination was evaluated in 1996. Pollen was collected from
flowers at anthesis, dried at room temperature overnight, and then germinated in two
separate experiments on Brewbaker & Kwack medium (1963) at room temperature.
Pollen germination was determined under a light microscope afler 3 hours. The number
of pollen grains germinated from a total of 100 pollen grains was recorded. The mean
pollen germination percentage from the two experiments for each individual was used for
QTL analysis. The data were angular transformed in the same way as for flower bud
death before QTL analysis.

ELu_i_t_§§_t.

Fruit set, calculated as the percent of flowers that set fruit, was measured in 1998
when the flowers had no apparent cold damage due to mild winter and spring
temperatures. Two branches from opposite sides (east and west sides) of each tree were
selected so that all branches had similar vigor. Each branch bore approximately 300
flowers.

Fruit weight and total soluble solids concentration

Fruit weight (g) and percent soluble solids were evaluated for five ripe fruits from
each parent and the progeny. Percent soluble solids was measured with a refractometer as
° Brix. The average of the five fruits was used for QTL analysis. These data were

collected over 3 years (1995-1997).

Molecular marker and QTL analysis

RFLP markers were used to construct linkage maps for each parent of the

36

mapping population (Wang et a1. 1998). All markers used were single dose restriction
fragments (SDRFs, Wu et al. 1992) which were either: (1) present in one but not both of
the parents and fit a 1:1 (presence:absence) segregation ratio, or (2) present in both
parents and fit a 3:1 (presence:absence) segregation ratio. A total of 190 SDRF markers
were used, of which 110 were present in one parent (67 and 43 markers in RS and EB,
respectively) and 80 markers were present in both parents.

Our previous sour cherry linkage map (Wang et al. 1998) was generated by
JoinMap (Stam 1993) which is able to determine linkage relationships between markers
segregating 1:1 and markers segregating 3:1 in a pseudo-testcross. Since QTL-
CARTOGRAPHER (Basten et al. 1997) can not analyze data containing both 1:1 markers
and 3:1 markers simultaneously from a pseudo-testcross mapping population, it was
necessary to generate three linkage maps for QTL analysis. The three linkage maps
constructed were the EB and RS maps using the 1:1 markers segregating in EB and RS,
respectively, and an RS/EB map using the 3:1 markers. Linkage analyses were performed
using MAPMAKER (Lander et al. 1987) and the Kosambi (1944) mapping function with
a minimum LOD score of 3.0 and a maximum recombination fraction of 0.30. Linkage
group numbers assigned were the same as previously used (Wang et al. 1998).

Means, standard deviations, and skewness of trait distribution were calculated for
each trait. T-tests for significance of differences between means of parents and progeny
were carried out for each trait and correlations among traits were also calculated. All
these analyses were accomplished using the analysis tools of Microsoft Excel 7.0.

QTL mapping was performed using composite interval mapping (CIM) (Zeng

1994; Jansen and Stam 1994) which is an extension of interval mapping (Lander and

37

Botstein 1989). Interval mapping calculates the likelihood score for a putative QTL
placed in any position within an interval flanked by two adjacent markers. CIM extends
this method by fitting the most significant markers outside the interval into the model,
allowing more precise and efficient mapping of QTLs (Zeng 1994).

QTL analysis was carried out with the program QTL-CARTOGRAPHER (Basten
et al. 1997). CIM was run with model 6 of the program and a window size of 10 cM for
all analyses. The number of markers for the background control was set to 5, which
means that the 5 most significant markers outside the interval under analysis were fitted
to the model. The markers used for the background control were detected through
forward and backward stepwise regression. The likelihood value of the presence of a QTL
was expressed as LOD score log,0(L,/L0), where L1 is the maximized likelihood of the
model with the putative QTL and L0 is the maximized likelihood of the model without the
QTL. The threshold of the LOD score for declaring a putative QTL significant was
chosen to be 2.4, which is approximately equivalent to applying a significance level of
0.001 for any single test. The estimate of the QTL position is the point where the
maximum LOD score was found in the region under consideration. A one-LOD support
interval was constructed for each QTL as described by Lander and Botstein (1989).

The phenotypic variance explained by a single QTL was estimated by the square
of the partial correlation coefficient (R2). Estimates of the R2 value and the additive
effect of a single QTL at its peak LOD position were obtained from the output of QTL
analysis using the program QTL-CARTOGRAPHER (Basten et al. 1997).

For traits evaluated over three years, each year was considered as a different

environment. Therefore, the data from each year were analyzed separately. When a

38

putative QTL was detected in more than one year, the mean of the three years was

analyzed and the results were reported as the generalized results for the QTL.

RESULTS AND DISCUSSION

Distribution of traits

All traits evaluated exhibited continuous variation which is typical of quantitative
or polygenic inheritance (Fig. 1). The two parents, RS and EB, differed significantly (P <
0.05) for 5 traits, including bloom time, ripening time, fruit weight, percent flower bud
death, and percent pollen germination (Table 1). There were no significant differences
between the two parents for soluble solids concentration, percent pistil death, and percent
fruit set (Table 1). Transgressive segregation was observed for all traits analyzed (Table
1; Fig. 1).

The progeny distribution for bloom time was normal (Fig. 1) and the mean was
similar to the mid parent value of 395 (Table 1). The difference in bloom time for the
two parents (66 degree days) was statistically significant (P < 0.05). However, parental
values were not the two extremes; 22% of the progeny bloomed later than the late parent
and 13% of the progeny bloomed earlier than the early parent (Fig. 1).

The difference in ripening time between the two parents (61] degree days) was
statistically significant (P < 0.05) (Table 1; Fig. 1). The progeny mean was not
statistically different from the average of the two parents and the distribution was normal.
Seventy three percent of the progeny values fell into the range defined by the values of

the two parents. Six percent of the progeny ripened later than the late parent and 21% of

39

Table 1 Mean phenotypic values and standard deviations (SD) for the progeny and
parents (RS and EB), and the value range for the progeny.

 

 

 

Mean 3: SD Progeny range
Trait RS EB Progeny Min. Max.
Bloom time (DD) 428.1 3: 362.2 :t 398.4 :t 317.8 516.2
22.9 16.0 33.8
Ripening time (DD) 2474.9 3: 1863.9 :1: 2084.8 1 1465.0 2712.0
262.7 85.9 233.3
Pistil death (%) 11.3 d: 12.4 41.7 i 30.0 23.8 :1: 15.3 0.0 55.0
Fruit set (%) 16.0 :t 0.2 13.4 :1: 1.3 6.8 :t 6.7 0.0 34.4
Fruit weight (g) 5.5 i 0.5 7.4 i 0.8 4.7 i 1.2 2.3 8.8
Soluble solids (0 Brix) 16.3 :t 1.3 17.2 :t 0.5 15.9 :1: 2.0 9.8 20.1
Pollen germination (%) 18.5 i 0.7 8.0 :t 1.4 5.6 d: 7.0 0.0 34.0
Flower bud death (%) 0.0 :t 0.0 55.0 :t 17.7 33.4 i 26.3 0.0 100.0

 

the progeny ripened earlier than the early parent.

The two parents differed significantly (P < 0.001) for percent flower bud death,
and EB had 55% more damage than RS (Table 1). The distribution of progeny values was
significantly skewed towards a smaller percent of death (Fig.1 ); however, the progeny
mean was not significantly different from the average of the two parents and only 48% of
the progeny had a lower percent flower bud death than the mid parent. Twenty-three

percent of the progeny had a higher percent flower bud death than EB (Fig. l).

40

25 2D

 

 

 

 

 

 

 

 

 

 

 

S ”—— RS

3‘ 20 ‘i ‘F '5 " E8
5 15h EB 1 '0 ¢
3 n
3 m..

6 up
i 5+

0 I I’ I I - a I U 1

 

 

 

 

1465
1604

318 310 362 381 606 £28 £60 £12 £96 516

Eiéfgii

N
,_
5 R u 8 R

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Tim e to bloom [degree day] Tim e to ripening [degree day]
20 25
RS
A w.. 20. EB
3 1
15.
g m.t R A
g l — EB ‘u ‘D
t 5» I l s"
n i I I Y I I T I u T T T T U
23 an 31 15 52 53 65 73 an as g g g g g 3 3 g g g
'- v- v- v- v- v- e- e- N
Fruit which! to) Soluble solid conc. (' Brill]
w
20
RS an"

15.. ¢ EB
20 ‘ EB RS

1 .
7 i T ¢
5.. F—7 ‘"~~ ‘
u . 0 . . a

[1.0 6.3 8.6 12.9 11.2 21.8 25.8 30.1 34.4

 

Frequency
3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0. "'. N. 0". V. 0. h. 0). 0. O
O O N 00 V O O N 8 O
F N m m c 0 F it 1'16
Plstlldulhlm '9 “ l l
m
an R8 F—-
25.
“ EB 10., EB
g 20 ¢ RS i
g m
s.
3 m.
t
s
n "—17 n . . CE
7 I r I T '7 if D '— N m v o h Q o o
o 0 rs- : 1'9 g a a 8 3) v- N m 3 3 8 I: 8 '8
Pollen germination [96] Flower bud death [91:]

Figure 1. Frequency distributions for each character in the mapping population. Means for
the parents RS and EB are shown by arrows.

41

RS had a lower percent pistil death than EB; however, the difference was not
significant (Fig. 1, Table 1). The progeny distribution was skewed toward the lower
values; however, angular transformation of the percentage data reduced the skewness
from 0.35 to 0.02.

RS and EB had similar values for percent fruit set (Table 1). The distribution of
progeny values was skewed toward lower values with 84% of the progeny having reduced
percent fruit set than the average of the two parents (Fig. 1). The skewness was reduced
from 1.50 to 0.37 after angular transformation of the percentage data. One progeny plant
had over two times higher percent fi'uit set than the mid parent.

EB had significantly (P < 0.05) lower pollen germination percent than RS. Low
percent pollen germination was more prevalent among the progeny than high percent
pollen germination (Fig. 1). Seventy five percent of the progeny had lower percent pollen
germination than EB. The progeny mean was significantly (P < 0.001) lower than the
mean of RS but not significantly different from the mean of EB (Table 1). Although low
percent pollen germination was more prevalent, 5% of the progeny had higher percent
pollen germination than that of the RS parent.

Mean fruit weight of EB was significantly larger than that of RS (P < 0.05).

Mean fruit weight for the progeny was significantly (P < 0.05) smaller than the mean of
RS, the small fruited parent (Table 1; Fig. 1). Progeny fruit weight ranged from 2.3 to 8.8
g. Small fruit weight appeared to be dominant with 77% of the progeny having fruits
smaller than those of the small fruited parent. However, one progeny individual had
fruits over 6 standard deviations larger than EB, the large fruited parent.

Fruit from RS and EB had similar percent soluble solids. The progeny distribution

42

ranged from 9.8 to 20.1 % soluble solids and was skewed towards the higher parental

values (Fig. 1).

Correlation of traits

Three significant correlations were found among the traits analyzed. A significant
(P < 0.05) negative correlation was observed between bloom time and percent pistil death
( r = - 0.25). Early flowering was also found associated with pistil freeze damage in
almonds (Viti et al. 1994). Presumably, the earlier the flowers open, the more likely their
pistils would be exposed to freezing temperatures. A significant (P < 0.0001) negative
correlation was found between bloom time and fruit weight (r = - 0.45). This correlation
may be associated with the polyploid origin of sour cherry. The two presumed progenitor
species of the allotetraploid sour cherry are sweet cherry (P. avium L.) and ground cherry
(P. fiuticosa Pall). Sweet cherry is early blooming and large fruited compared to ground
cherry which is late blooming and small fruited. Additionally, a significant (P < 0.05)
positive correlation was observed between percent pistil death and fruit soluble solids

concentration ( r = 0.24). The basis for this last correlation is unclear.

Genetic linkage maps

The RS and EB linkage maps identified 23 linkage groups. Fifteen linkage groups
were a subset of the 19 linkage groups of the RS map and the other 8 linkage groups were
a subset of the 16 linkage groups of the EB map described previously (Wang et al. 1998).
The RS and EB maps covered 398.2 cM and 222.2 cM, respectively, with an average

interval length of 9.8 cM. The RS/EB map consisted of 17 linkage groups covering

43

272.9 cM with an average interval length of 4.8 cM.

QTL analysis

Eleven QTLs were identified for 6 traits: bloom time, % pistil death, % pollen
germination, ripening time, fruit weight and soluble solids concentration (Table 2, Fig. 2
and Fig. 3). No QTLs were identified for flower bud death and % fruit set.

Two QTLs were identified for bloom time on two different linkage groups, EBl
(blml) and Group 2 (blm2) (Fig. 2, A and B). The QTL, blml, explained 19.9 % of the
phenotypic variation. This QTL had the effect predicted by the parental phenotype, with
an allele from the early blooming parent, EB, reducing bloom time by 27.8 degree days.
This QTL was the only QTL identified in this study that was consistently detected in each
of the three years analyzed. The QTL blm2 explained 22.3% of the phenotypic variance
and was detected in 2 of the 3 years and in all three years when the data were combined.
The stabilities of the bloom time QTLs are likely due to the ease of scoring for this trait
plus the conversion of the calendar day data to a heat accumulation value which reduces
the variation among years. As a result, the bloom time data for all three years had the
lowest average coefficient of variation (3.0 %) of all the quantitative traits analyzed.

Two QTLs were detected for percent pistil death on linkage groups EBl (pdl) and
RS8 (de) (Fig. 2, C and D). The QTLs pdl and de explained 12.9 % and 14.3 % of the
phenotypic variance, respectively. Both QTLs had effects in the direction opposite to
those predicted by the phenotype of the parents. An EB allele of pdl reduced the percent
pistil death by 2.1 % while a RS allele of de increased percent pistil death by 1.5 %.

The QTLs pd 1 and de were both detected with the threshold LOD score in only one of

44

Table 2 QTLs detected for each trait. QTLs are named according to trait abbreviations and
a number is used to distinguish QTLs affecting the same trait. Data were based on the
analysis of trait means over three years except for the trait percent pollen germination and

 

 

the QTL pd].

. Linkage Interval LOD peak Nearest Max. 2 Genetic
Trait QTL group ‘ length (cM) position (cM) marker LOD R effect: ab
Bloom (degree blml EB] > 21.5 81.1 pSl4l 3.6 19.9 -27.8
d3” blm2 RS/EB 2 > 20.1 32.1 PLG86 3.3 22.3 -101

. pdl EBl 28.8 14.8 EF194c 2.6 12.9 -2.1
Pistil death (%)

de RS8 >l4.7 0.0 EF156b 2.7 14.3 1.5
Po'le“ pgr EB] > 14 0 4.0 EFl46 3.0 17.0 1.4
germination (%) '
Ripe (degree rpl RS4 > 10.0 0.0 EF158b 4.1 21.5 197.5
daY) rp2 RS/EB6 > 8.7 4.5 cmzoe 3.7 25.9 156.2
fwl EB4 26.5 10.01 EF1823 2.3 13.7 0.9
Fruit weight (g)

. RS/EB 2 > 20.1 32.1 PLGS6 2.5 15.5 0.6
Soluble solids sscl EB7 > 6.0 0.0 AGIOb 3.2 16.5 l.9
concentration (0
Brix) ssc2 RS6 25.8 23.1 EF159a 2.5 13.1 -1 .5

 

" Linkage groups as assigned in Wang et al. (1998)
b a = additive value of the QTL

45

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4 “31"»; 4
I

3* I ,_ 3
{7"

D I ~ D
82 g 32
l l
’4
.z.

0- 0 - ~ .-.
— 0 2O 40 60 80 CM 0 10 20 30 40 CM
60.: i 4. r i Hfi "r: ‘rH.
e é . z. z. s. .2; U 6 .2'1-

.. i r: 5; :5 2:: :: “5'5

la. - "' - "

.. :3 a g a a: a as}.
C D

3 3
Q
02 82
....l ._.r

l‘ l

O 0""

....o m,020 4o 60 80cM

”:4 ] ml ii if 4r ii 1

m a n It I It
fig as; .7. =.=
... 3: 2 E:
a. mu. 1.x, mm
ill WEI-I III

E

3 -kpg
Q 1.

OZ '.

A I

1‘ E

0’...

0 ‘41 7* v ‘M
— 0 20 4O 60 80cM
are; a as a 4::J

:2 es: s as
1.1 :3 a '5: 8' :1:

Fig. 2. LOD scores for bloom date on linkage groups EB 1 (blml) (A); and Group 2
(blm2) (B); pistil death (pd) on linkage groups EB 1 (C); RS 6 (D) and pollen
germination % (pg) on linkage group EB] (E). Peak LOD scores for each trait are
indicated by arrows. Linkage groups are shown below the x-axes. The horizontal line
indicates the level of significance at LOD = 2.4. Curves represent results from individual
years of 1995 ( --- ), 1996 ( ----- ), 1997 ( — - - ), and over years ( ).

 

46

 

 

 

 

 

4 ' 4
{—002
3‘ 3 4x
a a :7 " ' ’
32 32 '
1 1
o - . . .v o “'3"‘:’".".~.-=2'-'-:-'a‘-~- ,
o 5 10 15 20cm 0 2 4 6 8 lOcM

 

 

 

Q
all 1

EF158b“
EF182b-
HF“ {ji-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

b 0 5 lOcM \o 0 10 20 30 40cM
“ti i] 2L i iii: i in
H‘F r I I II I I I
e i 2 'éé‘ § 3‘3
c: 2 E §;;§ U a
< 8 "‘ suit-:6 § In:

Figure. 3. LOD scores for ripening date on linkage groups RS4 (rpl) (A) and Group 6
(rp2) (B); soluble solids concentration on linkage groups EB7 (sscl) (E) and RS6 (ssc2)
(F), and fruit weight on linkage groups EB4 (fwl) (C) and Groups 2 (fw2) (D). Peak
LOD scores for each trait are indicated by arrows. Linkage groups are shown below the x-
axes. The horizontal line indicates the level of significance at LOD = 2.4. Curves
represent results from individual years of 1995, 1996, 1997, and over years ( See the

legend for Fig. 2 for details).

47

the three years analyzed and were identified in different years, 1995 and 1996,
respectively.

Since pistil death in 1995 and 1996 was caused by freezing events that occurred at
different stages of flower development, it is not surprising that different QTLs were
identified for the different years. In 1995, the only damaging freezing event after bud
break was -1 O “C which occurred 21 days before bloom. In contrast, there were two
damaging freezing events in 1996. The first freezing event occurred 12 days before the
population started blooming when the temperature lowered to - 2.6 °C for 11 hours. The
second freezing event was 4 days afier the population started blooming when the air
temperature was below - 1.5 °C for 3 hours. Consequently, the average percent pistil
death of the progeny population was larger in 1996 than in 1995, 40.9 % and 8.7%,
respectively.

One QTL, pg, was found for percent pollen germination on linkage group EB]
(Fig. 2B). This QTL explained 17.0 % of the phenotypic variance. It had an effect
opposite to that predicted by the phenotype of the parent, with an EB allele increasing the
pollen germination rate by 1.4 %.

Two QTLs were identified for ripening time on two different linkage groups, RS4
(rpl) and Group 6 (rp2)(Fig. 3, A and B). The QTL rpl was detected in two of the three
years analyzed and was responsible for 21.5% of the phenotypic variance. This QTL had
the effect predicted by the parental phenotype, with an allele from the late ripening
parent, RS, increasing ripening time by 197.5 degree days. The QTL rp2 was detected in
one of three years and was responsible for 25.9% of the phenotypic variance.

Two QTLs were identified for fruit weight on two different linkage groups, EB4

48

(fwl) and Group 2 (fw2)(Fig. 3, C and D). The two QTLs were both detected with the
threshold LOD score in only one of the three years analyzed and were identified in the
same year, 1997. The QTLsfwl and fw2 were responsible for 13.7 % and 15.5 % of the
phenotypic variance, respectively. The effect of the QTwal was in the direction
predicted by the phenotype, with an allele from the large fruited parent, EB, increasing
fruit weight by 0.9 g.

Two QTLs were identified for soluble solids concentration on two different
linkage groups, EB7 (ssc) and RS6 (ssc2) (Fig. 3, E and F). The QTL sscl was detected
with the threshold LOD score of 2.4 using 1995 data and the average data of 1995, 1996,
and 1997. The QTL ssc2 was detected with the critical LOD score of 2.4 using the data of
1995, 1996, and the average data of 1995, 1996, and 1997. The QTLs sscl and ssc2
explained 16.5 % and 13.1 % of the phenotypic variance, respectively. The two QTLs
were from different parents and had opposite effects. An EB allele of sscl increased
percent soluble solids by 1.9 ° Brix while a RS allele of ssc2 decreased percent soluble
solids by 1.5 ° Brix.

Previous QTL studies on other plant species have identified regions of the genome
that seem to contain clusters of QTLs (Edwards et al. 1987, Fulton et al. 1997). In tomato
for example, a 25-cM region of linkage group 1 contained QTLs for many fruit quality
traits (Fulton et al. 1997). In our study, QTLs affecting three flower traits, bloom time,
pollen germination percent, and pistil death in 1996, mapped to linkage group EB] (Fig.
2; Table 2). Two QTLs, pg and pdl , mapped at the lower end of the linkage group. The
positions of the peak LOD scores for QTLs pg and pd] were 10.8 cM apart; however, the

intervals for the two QTLs overlapped. The third QTL, blml, mapped to the other end of

49

the linkage group closest to the RF LP marker pSl4l. Since pSl41 is a clone derived
from sweet cherry stylar cDNA (Iezzoni and Brettin 1998), partial sequence was obtained
to determine if this RFLP identified a putative gene. Following a BLAST search
(Altschul et al. 1990) using 185 nucleotides, the closest nucleotide and amino acid
similarity to pSl4l was a non-S-allele RNase identified from pear stylar cDNA (Norioka
et al. 1996). The pear RNase and p814] have 81% amino acid homology, suggesting that
pSI4l also identifies a non-S-allele stylar RNase (Fig. 4). With the putative
identification of pSl41 as identifying a stylar RNase, 4 genes affecting floral traits

mapped to EBl.

 

 

Pa LGFRP NYKDG SYPSN CDPDS VFDKS EISEL MSNLE KNWPS LXCPS XNGFR
Pp HGLWP NYKDG GYPSN CDPDS VFDKS QISEL LTSLN KNWPS LSCPS SNGYR

* * ***** **** ***** ***** **** * ***** * ** **~k *

Pa rwsua WEKHG TC
Pp rwsne WEKHG TC

***** ***** **

Figure 4. Alignment of the amino acid sequences of the sweet cheny cv. Emperor
Francis RNase (pSl41) with the pear (Pyrus pyrifolia) non-S- RNase (D49529)(Norioka
et al. 1996). The alignment maximizes homology at the nucleotide and amino acid
sequence levels. The conserved amino acids are indicated by asterisks.

 

In this study, 50% of the QTLs identified for the traits in which the two parents
differed significantly had allelic effects opposite to those expected from the parental
phenotype. Such a high percentage of QTLs with allelic effects opposite to those
predicted from the parent may explain the common transgressive segregation observed
for all traits analyzed. Each parent was likely to possess both favorable and unfavorable
alleles of different QTLs affecting the same trait. Recombinations of favorable alleles as

well as unfavorable alleles from both parents would most likely generate transgressive

50

 

phenotypes. QTLs with effects opposite to those expected from parental phenotypes have
been reported to be responsible for transgressive segregation in an interspecific tomato
cross, where 36% of the QTLs bad effects opposite to those predicted by the parental
phenotypes and these QTLs were directly related to the appearance of transgressive
individuals in the F2 (de Vicente et al. 1993).

The QTLs detected for each individual trait explained from 17 % to 47.4 % of the
phenotypic variance with an average of 32.1%. These values are comparable to those
from a QTL analysis of horticultural traits in tomato, where the cumulative action of all
QTLs detected for each trait accounted for 12 - 59 % of the phenotypic variation
(Grandillo and Tanksley 1996). The extent of the phenotypic variance explained in our
analysis is encouraging given the theoretical limitations of QTL mapping in a pseudo-
testcross and a polyploid crop plus the present limited length of the sour cherry map.

For example, both sour cherry analyses were done with pseudo-testcross mapping
populations. Since both parents in a pseudo-testcross can be heterozygous (QIQ2Q3Q4 +
Q5Q6Q7Q8 for sour cherry), QTL identification in a pseudo-testcross population would
theoretically be less likely than in a backcross-inbred population used in tomato since the
effect of an individual allelic substitution would have to be sufficiently large to be
identified in a segregating heterozygous background (Conner et al. 1998).

Additionally, identification of major QTL alleles is theoretically more difficult in
a polyploid mapping population because in order to detect a QTL allele it would have to
meet the same segregation requirement as a molecular marker, i.e. segregate as a single
dose restriction fragment (Wang et al. 1998). The simplest case meeting this

requirement could be diagramed as Q,Q2Q2Q2 x Q2Q2Q2Q2_ Given this requirement

51

which favors the detection of a unique QTL allele (i.e. Ql), it is not unexpected that half
of the QTL alleles identified in sour cherry contrasted to the parental phenotype. This
requirement also makes it theoretically more difficult to identify the QTL allele
contributing to the parental phenotype if this allele is present in at least 2 copies (i.e. Q).
There is. some speculation in allotetraploid cotton, that this may be the case. In cotton,
major QTL alleles donated from the high value parent were not detected presumable
because they are present in more than one dose (J iang et al. 1998). It is important to note
however, that a QTL locus can still be identified by mapping the allele that is present in a
single dose.

Improved map coverage should increase our ability to identify QTLs and estimate
their location. The RS and EB linkage maps used in the QTL analysis represent only
approximately one third of the estimated total sour cherry linkage map distance (Wang et
al. 1998). In addition, the marker density in certain regions of the linkage maps was
relatively low. For traits that exhibited little variation among years such as bloom and
ripening time, additional QTLs might have been identified if a more complete linkage
map were available. Additionally, the One-LOD support interval lengths could not be
determined for five of the QTLs (rpl , blml, sscI , pd2, and pg), because these QTLs
mapped to the ends of the linkage groups (Table 1, Figs. 2 and 3). Despite the
limitations discussed above, the results confirm that significant QTLs can be identified
for important flower and fruit traits in sour cherry.

It has been demonstrated in other plants that QTLs can be conserved among
species and even across genera (Paterson et al. 1995). If QTLs were conserved within

Prunus and then between Prunus and Malus, it might be possible to predict regions in

52

other species that might be homologous to QTL regions in sour cherry. The sour cherry
linkage Groups 2, 4 and 7 which contain QTLs for boom date, ripening date, fruit weight,
and soluble solids, are suspected to be homologous to the peach and almond linkage
Groups 2, 4 and 7 based on shared RF LP markers (Wang et al. 1998). Ongoing QTL
analyses in peach for fruit size and soluble solids (A. Abbott, per comm.) should provide
data for QTL comparison between sour cherry and peach.

Unfortunately, the peach-almond homologue for the sour cherry linkage Group 1
that appears to have bloom related traits, has not been identified. Due to the year to year
stability in bloom time measurements and the universal importance of this trait in
Rosaceous crops, bloom time would be an appropriate quantitative trait for QTL

comparison among Prunus species and between Prunus and Malus.

53

REFERENCES

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment
search tool. J Mol Biol 215:403-410.

Basten CJ, Weir BS, Zeng Z-B (1997) QTL CARTOGRAPHER, reference manual and
tutorial for QTL mapping. North Carolina State University, anonymous ftp:
esssjp.stat.ncsu.edu/pub/qtlcart

Brewbaker JL, Kwack BH (1963) The essential role of the calcium ion in pollen
germination and pollen tube growth. Amer J Bot 50: 859-865.

Conner PJ, Brown SK, Weeden NF (1998) Molecular-marker analysis of quantitative
traits for growth and development in juvenile apple trees. Theor Appl Genet 96:
1027-1035

de Vicente MC, Tanksley SD (1993) QTL analysis of transgressive segregation in an
interspecific tomato cross. Genetics 134:585-596

Edwards MD, Stuber CW, Wendel JF (1987) Molecular-marker—facilitated investigations
of quantitative-trait loci in maize. 1. Numbers, genomic distribution and types of
gene action. Genetics 116:113-125

Fulton TM, Beck-Bunn T, Emmatty D, Eshed Y, Lopez J, Petiard V, Uhllig J, Zamir D,
Tanksley SD (1997) QTL analysis of an advanced backcross of Lycopersicon
peruvianum to the cultivated tomato and comparisons with QTLs found in other wild
species. Theor Appl Genet 95: 881-894

Grandillo S, Tanksley SD (1996) QTL analysis of horticultural traits differentiating the
cultivated tomato from the closely related species Lycopersicon pimpinellifolium.
Theor Appl Genet 92:93 5-951

Hayes PM, Blake TK, Chen TNN, Tragoonrung S, Chen F, Pan A, Liu B (1993)
Quantitative trait loci on barley (Hordeum valgare) chromosome 7 associated with

winter hardiness. Genome 36:66-71

Iezzoni AF, Brettin TS (1998) Utilization of molecular genetics in cherry. Acta
Horticulturae 468155-62.

Jansen RC, Stam P (1994) High resolution of quantitative traits into multiple loci via
interval mapping. Genetics 136:1447-1455

J iang CX, Wright RJ, El-Zik KM, Paterson AH (1998) Polyploid formation created
unique avenues for response to selection in Gossypium (coton). Proc Nat Acad Sci

54

95: 4419-4424.

Keim P, Diers BW, Olson TC, Shoemaker RC (1990) RFLP mapping in soybean:
association between marker loci and variation in quantitative traits. Genetics
126:735-742

Kosambi DD (1944) The estimation of map distance from recombination values. Ann
Eugen 12:172-175

Lander ES, Botstein D (1989) Mapping Mendelian factors underlying quantitative traits
using RFLP linkage maps. Genetics 121:185-199

Lander ES, Green P, Abrahamson J, Barlow A, Daly MJ, Lincoln SE, Newburg L (1987)
MAPMAKER: an interactive computer package for constructing primary genetic
linkage maps of experimental and natural populations. Genomics 1:174-l8l

Lawson DM, Hemmat M, Weeden NF (1995) The use of molecular markers to analyze
the inheritance of morphological and developmental traits in apple. J Amer Soc Hort
Sci 120: 532-537.

Norioka N, Norioka S, Ohnishi Y, Ishimizu T, Oneyama C, Nakanishi T, Sakiyama F
(1996) Molecular cloning and nucleotide sequences of cDNAs encoding S-allele
specific stylar RNases in a self-incompatible cultivar and its self-compatible mutant
of Japanese pear, Pyrus pyrifolia Nakai. J Biochem 120: 335-345

Paterson AH, Lander ES, Hewitt JD, Peterson S, Lincoln SE, Tanksley SD (1988)
Resolution of quantitative traits into Mendelian factors by using a complete linkage
map of restriction fragment length polymorphisms. Nature 335:721-726

Paterson AH, DeVema J W, Lanini B, Tanksley SD (1990) Fine mapping of quantitative
trait loci using selected overlapping recombinant chromosomes, in an interspecies
cross of tomato. Genetics 1242735-742

Paterson AH, Lin YR, Ki Z., Schertz KF, Doebley JF, Pinson SRM, Liu SC, Stansel J W,
Irvine J E (1995) Convergent domestication of cereal crops by independent mutations
at corresponding genetic loci. Science 269: 1714-1718

Pilet ML, Delourme R, F oisset N, Renard M (1998) Identification of loci contributing to
quantitative field resistance to blackleg disease, causal agent Leptosphaeria
maculans (Desm.) Ces. et de Not., in winter rapeseed (Brassica napus L.). Theor
Appl Genet 96:23-30

Rebai A, Blanchard P, Perret D, Vincourt P (1997) Mapping quantitative trait loci

controlling silking date in a diallel cross among four lines of maize. Theor Appl
Genet 95:451-459

55

Stam P (1993) Construction of integrated genetic linkage maps by means of a new
computer package: J oinMap. Plant Jour 3: 739-744

Stuber CW, Edwards MD, Wendel J F (1987) Molecular-marker-facilitated investigations
of quantitative-trait loci in maize. II. Factors influencing yield and its component
traits. Crop Sci 27:639-648

Stuber CW, Lincoln SE, Wolff DW, Helentjaris T, Lander ES (1992) Identification of
genetic factors contributing to heterosis in a hybrid from two elite maize inbred lines
using molecular markers. Genetics 132:823-839

Toroser D, Thormann CE, Osborn TC, Mithen R (1995) RF LP mapping of quantitative
trait loci controlling seed aliphatic-glucosinolate content in oilseed rape (Brassica
napus L.). Theor Appl Genet 91 :802-808

Viti R, Bartolini S, Giorgelli F, Barbera G (1994) Effect of low temperatures on flower
buds of several almond cultivars. First international congress on almond, Agrigento,
Italy. Acta Horticulturae 373: 1 93-199

Wang D, Karle R, Brettin TS, Iezzoni AF (1998) Genetic linkage map in sour cherry
using RF LP markers. Theor Appl Genet 97:(in press)

Wang G, Mackill DJ, Bonman J M, McCouch SR, Charnpoux MC, Nelson RJ (1994)
RF LP mapping of genes conferring complete and partial resistance to blast in a
durably resistant rice cultivar. Genetics 136: 1421-1434

Wu K, Burnquist W, Sorrells ME, Tew TL, Moore PH, Tanksley SD (1992) The
detection and estimation of linkage in polyploids using single-dose restriction

fragments. Theor Appl Genet 83:294-300

Zeng Z-B (1994) Precision mapping of quantitative trait loci. Genetics 136:1457-1468

56

CHAPTER 3

CYTOGENETIC ANALYSIS OF SOUR CHERRY USING
GENOMIC IN SI T U HYBRIDIZATION

57

ABSTRACT

Genomic in situ hybridization (GISH) was used to examine meiotic pairing
behavior and parental genomic contributions in the allotetraploid sour cherry (P. cerasus).
Three sour cherry cultivars were studied: Montmorency, Rheinische Schattenmorelle
(RS), and Erdi Botermo (EB). GISH analysis suggested that EB might have a higher
genomic contribution from P. avium than P. fiuticosa. However, GISH analysis only
identified a relatively few number of species specific chromosomes and chromosome
segments in RS suggesting that significant intergenomic recombination has occurred. In
the meiotic analyses, in addition to the normal bivalent pairing configuration, univalents,
trivalents, and quadrivalents were frequently observed in the pollen mother cells of the
three cultivars. RS had the most bivalents and the lowest number of quadrivalents.
Montmorency and EB had approximately the same numbers of bivalents and
quadrivalents. RS had a bivalents to non-bivalents ratio of 4.4:] while EB and
Montmorency had a bivalents to non-bivalents ratio of 3.521. The ratio of bivalents to
non-bivalents may be an important factor in determining the proportion of balanced and

unbalanced meiotic products.

58

INTRODUCTION

The MSU sour cherry (Prunus cerasus L.) germplasm collection is one of the
largest in the world with material collected thoughout the species range. Individuals in
this germplasm collection possess many important fi'uit quality and disease resistance
traits that are important for future gains in cultivar breeding. However, use of superior
individuals in the breeding program is severely limited because approximately 95% of the
individuals in the germplasm collection are highly infertile with fruit set frequently
between 0.1 and 10%; approximately 30% fi'uit set is needed to produce a commercial
crop.

Even commercial cultivars and progeny resulting from commercial cultivars
exhibit a high level of sterility. In the sour cherry cultivar Montmorency, 25 % to 40 % of
the embryo sacs were non-functional (F urukawa and Bukovac 1989). In the progeny from
the cross Rheinische Schattenmorelle x Erdi Botermo, pollen germination rate ranged
from 34 % to 0 % (Table 1 in Chapter 2).

Sour cherry is a polyploid (2n=4x=32) presumed to be derived from sexual
polyploidization between sweet cherry (P. avium L.; 2n=2x=16) and ground cherry (P.
fruticosa Pall.; 2n=4x=32) (Olden and Nybom 1968). Low fertility is hypothesized to
result from lack of complete bivalent pairing between homologues. Evidence for this is
derived from several sources. Isozyme segregation consistent with occasional
intergenomic pairing has been observed (Beaver and Iezzoni 1993). Segregation of seven
restriction fragment length polymorphism (RF LP) markers in sour cherry revealed

intergenomic pairing and recombination (Wang et al. 1998). Pollen mother cell meiosis

59

in sour cherry resulted in the frequent formation of aneuploid gametes (Murawski and
Endlick 1962; Kotoman and Krylova 1977). Megasporogenesis exhibited abnormalities
similar to microsporogenesis which resulted in the degeneration of the embryo sac (Leach
and Tylus 1983; Murawski and Endlick 1962; Potemkina 1973; Manesu et al. 1980).
Sour cherry, like other Prunus, has just two ovules per flower, one of which degenerates.
Therefore, the degeneration of the other ovule due to aneuploidy will translate into
reduced fruit set.

Lack of complete bivalent pairing between homologues in sour cherry could be
explained by two theories: (1) homology between chromosomes of the progenitor species
resulting in pairing of homoeologous chromosomes, and/or (2) unbalanced genomes due
to introgression with progenitor species resulting in meiotic pairing irregularity. Since the
generation of a viable allopolyploid requires some level of phylogenetic relatedness
between the parental species, it is expected that some homology will exist between the
two parental genomes. The resulting irregularities in pairing or segregation in the
allopolyploids can lead to unbalance gametes and infertility (Heiser 1973). In this first
case, one would suspect that there would be selection for a mechanism which would
restrict bivalent pairing to homologues. In support of the second theory, sour Chen)I is
not reproductively isolated from sweet and ground cherry, its presumed progenitor
species (Olden and Nybom 1968), and crossing with its progenitor parent is prevalent
(Hruby 1962). Unbalanced genomes can result from repeated introgression by one of the
progenitor species and retard any move towards diploidization (Stebbins 1947).

Pairing between homoeologous chromosomes and genome balance can be

investigated using the genomic in situ hybridization (GISH) technique (Schwarzacher et

60

al. 1994). GISH has been successfully used to distinguish chromosomes from different
progenitor species in allopolyploid species (reviewed by Jiang and Gill 1994). To
distinguish between two species, genomic DNA from one species was labeled and used as
the probe, while unlabeled DNA from the other species was applied at a much higher
concentration as a block (Anamthawat-Jénsson et al. 1990).

The objective of this research was to study the meiotic pairing behavior and

determine the parental genome contributions for three sour cherry cultivars.

MATERIAL AND METHODS

Plant material

Three sour cherry cultivars, Montmorency, Rheinische Schattenmorelle (RS), and
Erdi Botermo (EB), were chosen for the meiotic analysis. Montmorency is the only
commercially grown cultivar in the United States. RS and BB were the two mapping
parents for which genetic linkage maps have been constructed (Chapter 1). The three
cultivars originated from different geographic regions. Montmorency originated from
France and RS and EB originated from Germany and Hungary, respectively.

The two progenitor species of sour cherry, P. avium (sweet cheny) and P.
fruticosa (ground cherry), were used in the GISH analysis of the meiotic chromosomes of
the three sour cherry cultivars. The sweet cherry used in the GISH analysis was cultivar
Emperor Francis. Two genotypes of ground cherry, PF -HortFarm and PF26el(36), were
used. Since ground cherry is distinct from sweet cheny and sour cherry in tree size, PF-

HortF arm and PF26el(36) were chosen based on their tree sizes. PF-HortFarm has the

61

largest tree size (about 2 m tall) while PF26el(36) has the smallest tree size (about 0.5 m

tall) in the P. fiuticosa collection at Michigan State University.

Chromosome preparation

Meiotic chromosomes of pollen mother cells (PMC) were prepared from anthers
of Montmorency, RS, and EB. Branches bearing flower buds were collected in February,
1998 when the plants were still in dormancy and had received enough chilling. The
branches were kept at room temperature and the progress of meiosis was monitored.
Flower bud samples were collected twice a day (in the morning and in the afternoon) until
pollen grains were observed. Flower buds were fixed in ice-cold 3:1 methanol : acetic
acid immediately after removal from branches and stored in the fixative at 4 ° C until use.
The fixed anthers at first metaphase of meiosis were soaked in 45% acetic acid for three
hours before chromosome preparation. Slides were prepared by placing the fixed anthers
in a drop of 45% acetic acid on a pre-cleaned slide; the pollen mother cells were
squeezed from the anthers and the anthers discarded. A cover-glass was added and the
preparation was gently squashed beneath filter paper. The slides were observed using
phase-contrast microscopy and the selected slides were frozen and stored at - 80 ° C until

use.

Probe preparation and in situ hybridization
Total genomic DNAs of P. fiuticosa PF-HortF arm and PF 26e1(36) and P. avium
cv. Emperor Francis were extracted as described by Stockinger et al. (1996) with the

following modifications: 400 mg of lyophilized leaves were placed in a 50-ml centrifuge .

62

tube together with five 4 mm glass beads (Fischer Scientific, Pittsburgh, PA) and shaken
vigorously for 4 minutes with a paint shaker to grind the sample to a fine powder prior to
the addition of extraction buffer. Subsequent procedures were the same as those described
by Stockinger et al. (1996).

Total genomic DNAs of P. fruticosa and P. avium were separately labeled with
either digoxigenin-1 l-dUTP (Boehringer Mannheim, Indianapolis, IN) or biotin-l4-dATP
(GibcoBRL, Gaithersburg, MD) by nick translation for use as the in situ probes.
Unlabeled total genomic DNA from P. fruticosa or P. avium was fragmented to pieces of
100 - 600 bp long by autoclaving for 5 min and then used as blocking DNA. When
labeled DNA from P. fiuticosa was used as the in situ probe, the unlabeled DNA from P.
avium was used as blocking DNA . Conversely, when labeled DNA from P. avium was
used as the probe, the unlabeled DNA from PF26el(36), the small-sized genotype of P.

fruticosa was used as blocking DNA .

The protocols followed for pretreatment of slide preparations, in situ
hybridization, and detection of digoxigenin or biotin labeled probe were essentially those
described by Schwarzacher et al. (1994). Chromosome preparations were treated with
RNase A, pepsin and paraformaldehyde (Sigma, St. Louis, MO) as described in the
protocols. The slide preparations were denatured in preheated solution of 70 %
forrnamide, 2x SSC at 75 °C for 3 minutes and dehydrated in a cold (-20 °C ) ethanol
series (70 %, 95 %, and 100 %, 2 minutes each). Twenty ul of hybridization mixture
containing 100 ng of labeled probe DNA, 1.5 to 10 pg of unlabeled blocking DNA, and
10 ug of sheared fish sperm DNA in 50 % formamide, 10 % dextran sulfate was applied

to each slide. The hybridization mixture was denatured at 75 °C for 5 minutes, chilled on

63

ice for 5 minutes, and then pre-annealed at 37 °C for 5 minutes before application to the
slide. The hybridization was allowed to occur at 37 °C for 16 hours. After hybridization,
slides were washed in three changes of 50 % forrnamide, 2x SSC at 45 °C for 5 minutes
each. Both the digoxigenin and biotin hybridization sites were visualized with the
appropriate fluorescence conjugates. All preparations were counterstained with
propidium iodide and/or DAPI (Sigma, St. Louis, MO). The preparations were mounted
in Vectashield antifade (Vector Laboratories, Burlingame, CA), examined using a
Olympus BX6OF fluorescence microscope, and photographed using Kodak Ektachrome

400HC slide film.

RESULTS

Genomic in situ hybridization

All Montmorency chromosomes were labeled when the preparations were
hybridized with labeled total DNA from P. fruticosa PF-HortFarm and blocked with
unlabeled total DNA from P. avium at a blocking ratio of 50:1 (blockzlabel) or less (Fig.
1). When the blocking ratio was above 50:1, the hybridization signal was either not
present or very weak.

The EB chromosomes showed differential signals of hybridization when labeled
total DNA from either P. avium or P. fruticosa PF26el(36) was used as the probe and
unlabeled total DNA from the other progenitor species was used as the blocking DNA
with a blocking ratio of 50:1 (Fig. 2). When P. avium was used as the probe DNA, about

1 1 pairs of EB chromosomes showed a strong hybridization signal while the other 5 pairs

64

 

Figure 1. Fluorescent micrographs of metaphase PMC chromosomes from the sour
cherry cultivar Montmorency after genomic in situ hybridization using total genomic
DNA from P. fruticosa PF—HortFarm as the probe labeled with biotin and detected with
fluorescein. The hybridization signal appears as yellow-green fluorescence while the
unhybridized regions appear as orange-red with the counterstain propidium iodide.
Examples of different meiotic pairing configurations are identified by roman numerals in
the top micrograph as: I — univalent, H - bivalent, III - trivalent, and IV — quadrivalent.

65

 

Figure 2 a-d. Fluorescent micrographs of metaphase PMC chromosomes from the sour
cherry cultivar Erdi Botermo after genomic in situ hybridization using total genomic
DNA from either P. avium (a, b), or P. fruticosa PF26e136 (c, d) as the probe. The
probes were labeled with digoxigenin and detected with fluorescein. The hybridization
signal appears as yellow-green fluorescence while the unhybridized regions appear as
orange-red with the counterstain propidium iodide. The arrow in micrograph a points to a
bivalent formed by homoeologous chromosomes.

66

showed a very weak or no hybridization signal (Fig. 2 a and b). In contrast, when P.
fruticosa was used as the probe DNA, more than two thirds of the EB chromosomes
showed no or very weak hybridization signal while a few chromosomes showed strong
hybridization signals (Fig. 2 c and (1).

Most RS chromosomes displayed hybridization when labeled and probed in both
directions; i.e. total DNA from either P. avium or P. fruticosa PF 26c] (36) was used as
the probe and unlabeled total DNA from the other progenitor species was used as the
blocking DNA with a blocking ratio of 50:1 (Fig. 3). However, the strength of the signal
varied among chromosomes (Fig. 3).

When EB and RS chromosomes were hybridized with labeled total DNA from P.

fruticosa PF-HortFarm and blocked with unlabeled total DNA from P. avium at a
blocking ratio of 50:1 (block:1abel) or less, all chromosomes showed a signal of

hybridization.

Pairing configuration at meiosis

Univalents, bivalents, trivalents, and quadrivalents were observed in PMCs at
metaphase-I of the three sour cherry cultivars (Figs. 1 and 4a). Table 1 shows the average
number of pairing configurations per PMC at metaphase I. RS had the most bivalents
(12.9) and univalents (1.7) and the least number of quadrivalents (0.9). During anaphase
I to telephase I, the univalents may not be included in the telophase nuclei (Fig. 4b).
Montmorency and EB had approximately the same numbers of bivalents and
quadrivalents. However, the quadrivalent configurations of Montmorency and EB

differed. Most quadrivalents observed in the Montmorency PMCs were in ring or other

67

 

Figure 3 a-d. Fluorescent micrographs of metaphase PMC chromosomes from the sour
cherry cultivar Rheinische Schattenmorelle after genomic in situ hybridization using total
genomic DNA from either P. fruticosa PF26el36 (a, b), or P. avium (c, d), as the probe.
The probes were labeled with digoxigenin and detected with fluorescein. The
hybridization signal appears as yellow-green fluorescence while the unhybridized regions
appear as orange-red with the counterstain propidium iodide. The arrows in micrograph a
point to bivalents formed by homoeologous chromosomes.

68

 

Figure 4 a, b Fluorescent micrographs of PMC chromosomes from sour cherry cultivar
Rheinische Schattenmorelle. The chromosomes were stained with the DNA-specific dye
DAPI and were shown by blue fluorescence. a Metaphase-I chromosomes showing
examples of different meiotic pairing configurations identified by roman numerals as: I -
univalent, H - bivalent, and IV - quadrivalent. b Telophase-I chromosomes showing two
univalents (arrows) remain stationary at the eqUatorial plate when other chromosomes
have reached the poles.

69

 

parallel configurations (Fig. l) as diagrammed by Kuspira et al. (1985), whereas most
quadrivalents observed in BB PMCs were in a linear chain configuration (Fig. 5). The

quadrivalents observed in RS PMCs were in ring or open-ring configurations (Fig. 4a).

Table 1 Mean number of chromosome pairing configurations per PMC at metaphase I.

 

 

Cultivar No. of PMCs Uni- Bi- Tri- Quadri- Bivalent:
analyzed valent valent valent valent nonbivalent
RS 36 1.7 12.9 0.3 0.9 4.4 : 1
EB 20 0.6 10.9 0.0 2.5 3.5 : 1
Montmorency l3 0-9 11.2 0.2 2.1 3.5 : l

 

Non-bivalent configurations were found in most of the PMCs analyzed. Non-
bivalent configurations were observed in 92.3 %, 80.6 % and 100 % of the PMCs of
Montmorency, RS, and EB, respectively.

Bivalents formed by homoeologous chromosomes were observed with the GISH
labeling. In some bivalent configurations, only one of the two chromosomes was labeled

by the P. avium probe DNA (Fig. 2a) or by the P. fruticosa probe DNA (Fig. 3a).

DISCUSSION

One of our objectives was to use GISH analysis to discriminate the ancestral
parental chromosomes and/or chromosome segments in three sour chen'y cultivars. For
GISH analysis to be effective, the blocking DNA from one progenitor species must
presumably hybridize to sequences in common between the blocking DNA and the

labelled probe. Then mainly species specific sequences would remain as sites for probe

70

   

Figure 5 Phase-contrast micrograph of metaphase PMC chromosomes from sour cherry
cultivar Erdi Botermo showing linear chain quadrivalents (arrows).

71

 

hybridization (Anamthawat-Jonsson et al. 1990). It follows that if sour cherry were a
recent allopolyploid between P. avium and P. fruticosa, ideally 8 chromosomes would be
identified as being derived from each of the two progenitor species.

GISH analysis of EB appeared to identify species specific chromosomes when
probed with either P. avium or P. fiuticosa PF26el(36) DNAs; however, the parental
contributions were not equal. The relative abundance of chromosomes hybridizing to P.
avium suggests that the EB genome consists primarily of P. avium derived chromosomes.
This observation is consistent with the pedigree of EB. BB is derived from a cross
between two tetraploid cherries, Pandy 38 and Nagy Angol, both of which are considered
to be natural hybrids with sweet cherry (Apostol and Iezzoni 1992).

GISH analysis of RS using P. avium and P. fiuticosa PF26el(36) as the probe
DNAs only identified a few chromosomes and chromosome regions that appeared to be
derived solely from P. avium or P fruticosa. Both P. avium and P. fiuticosa PF26el (36)
DNAs hybridized to most of the chromosomes. Since the GISH technique was able to
discriminate species specific chromosomes in EB, we felt that the GISH protocol was
reliable. RS is an old landrace sour cherry variety from Germany and it is possible that
continual recombination between the chromosomes from the two ancestral genomes has
resulted in our inability to identify more species specific chromosomes. Our
identification in RS of bivalents between presumably homoeologous chromosomes
supports this theory (Fig. 3a).

When Montmorency chromosomes were probed with P. fiuticosa PF -HortF arm
and blocked with P. avium DNA, all the chromosomes exhibited hybridization signal.

However, the GISH results with Montmorency are difficult to interpret since the converse

72

experiment was not done, i.e. P. avium was not used as the probe. Additionally, only P.
fiuticosa PF -HortF arm, and not P. fruticosa PF 26c] (36) was used as the probe. For EB
and RS, only PF26el(36) was able to distinguish the two progenitor genomes of sour
cherry. The failure of PF -HortFarm to distinguish the two progenitor genomes could be
due to possible introgression of P. avium into its genome. P. fiuticosa and P. avium are
not reproductively isolated and the two species coexist in the wild (Olden and Nybom
1968). Trees of P. avium are typically tall and trees of P. fruticosa are typically very
short. PF-HortFarm has the largest tree size in our P. fruticosa collection, indicating
possible introgression by P. avium.

The meiotic analyses support our hypothesis that sour cherry is not completely
diploidized with the expectedl6 bivalents at meiosis. Instead the three cultivars analyzed
all exhibited meiotic irregularities. RS had the highest number of bivalents and the least
number of quadrivalents at metaphase 1, suggesting that RS may be the most diploidized
among the three cultivars. GISH analyses revealed that most RS chromosomes hybridized
to DNA probes from both progenitor species, indicating that the two genomes in RS have
undergone significant intergenomic exchange. However, the presence of quadrivalents
and trivalents at metaphase I indicates that the process of diploidization in RS is not
completed. In contrast to RS, EB had the least number of bivalents and the most number
of quadrivalents at metaphase 1, suggesting that EB was the least diploidized among the
three cultivars. GISH analyses revealed that EB had unbalanced genomes of the two
progenitor species. Over two thirds of the EB chromosomes hybridized to the P. avium
DNA probe and only less than one third of chromosomes hybridized to the P. fiuticosa

DNA probe.

73

The homology between the two progenitor species of sour chen'y was significant
enough to cause pairing between homoeologous chromosomes. Trivalents and
quadrivalents were frequently observed in this study. Bivalents formed by homoeologous
chromosomes were also found in this study.

The commonly observed non-bivalent pairing configurations in this study may
explain the high level of sterility in sour chen'y. Pollen mother cells with univalents,
trivalents, and quadrivalents are the source of aneuploid gametes (Kuspira et al. 1985).
During the disjunction of chromosomes at anaphase I to telophase I, univalents may
remain more or less stationary at the equatorial plate (Fig. 4b) and fail to be included in
either telophase nuclei (Singh 1993). The disjunction of chromosomes in a trivalent
generally result in a 2:1 split of the three chromosomes to the opposite poles (Singh
1993). The disjunction of chromosomes in a quadrivalent may result in unequal split of
the four chromosomes to the telophase nuclei (Kuspira et al. 1985). Most PMCs of the
three sour cultivars analyzed in this study contained at least one of the three non-bivalent
configurations. This is consistent with the reports that meiosis in PMCs of sour cherry
resulted in the frequent formation of aneuploid gametes (Murawski and Endlick 1962;
Kotoman and Krylova. 1977). Aneuploid gametes could be responsible for the low
average pollen germination rate of 5.6 % for the 86 progeny from the cross RS x EB
(Table 1 in Chapter 2).

The ratio of bivalents to non-bivalents may be an important factor in determining
the proportion of balanced and unbalanced meiotic products and ultimately in
determining the proportion of fertile and sterile gametes. RS had a higher ratio of

bivalents to non-bivalents (4.4 : 1) than that of EB (3.5 : 1) (Table 1). As expected, RS

74

had a higher pollen germination rate (18.5 %) than that of EB (8.0 %) (Table 1 in Chapter
2).

In conclusion, GISH analysis failed to identify balanced parental genomic
contributions in the sour cherry cultivars. Instead, the GISH evidence suggests that higher
fertility levels may be associated with ancestral chromosomes that have undergone
significant intergenomic recombination. Additionally, the relatively high number of
PMCs exhibiting meiotic irregularities suggest that these meiotic disturbances may be

contributing to low fertility in sour cherry.

75

REFERENCES

Anamthawat-Jonsson K, Schwarzacher T, Leitch AR, Bennett MD, Heslop-Harrison J S
(1990) Discrimination between closely related T riticeae species using genomic DNA
as a probe. Theor Appl Genet 79:721-728.

Apostol J and Iezzoni A (1992) Sour cherry breeding and production in Hungary. Fruit
VarJ 46: 11-15.

Beaver J, Iezzoni AF (1993) Allozyme inheritance in tetraploid sour cherry (Prunus
cerasus L.). J Amer Soc Hort Sci 118:873-877.

F urukawa Y, Bukovac MJ (1989) Embryo sac development in sour cherry during the
pollination period as related to fruit set. HortScience 24: 1005-1008.

Heiser CB (1973) Introgression re-examined. Bot Rev 39: 347-366.

Hruby K (1962) Chromosome behaviour and phylogeny of cultivated Cerasus. Preslia 34:
85-97

J iang J, Gill BS (1994) Nonisotopic in situ hybridization and plant genome mapping: the
first 10 years. Genome 37:717-725

Kotoman EM, Krylova VV (1977) Cytoembryological study of some sour cherry
varieties (in Russian). Selektsiza i sortoizuch. plodov. i orekhoplodn. kul'tur. 7: 95-
100. [Plant Breed. Abstr. 49: 5104; 1979]

Kuspira J, Bhambhani RN, Shimada T (1985) Genetic and cytogenetic analyses of the A
genome of Triticum monococcum. I. Cytology, breeding behavior, fertility, and
morphology of induced autotetraploids. Can J Genet Cytol 27:51-63

Leach W, Tylus K (1983) Pollination, fertilization and fruit setting of some sour cherry
varieties. Acta Hort 139: 33-39

Manescu C, Radulescu-Mitroiu N, Simionescu M (1980) Investigation into some
embryological causes of infertility in sour cherries. Lucrari Stiintifice, Institutul
Agronomic "N. Balcescu", Horticultura 6: 37-42. [Plant Breed. Abstr. 52: 5177;
1982]

Murawski H, and Endlich J (1962) Contributions to research on breeding cherries. II.
Investigations of the biology of fertilization and embryology in the sour cherry
variety Koroser Weichsel. Arch Gartenb 10:616-646

Olden EJ, Nybom N (1968) On the origin of Prunus cerasus L. Hereditas 59: 327-345

Potemkina GA (1973) Features of the formation of the female gametophyte in sour cherry

76

varieties showing female sterility (in Russian). Tsitogenetich. i tsitoembriol. metody
v selektsii plodov. i yagodn. kul'tur. [Plant Breed. Abstr. 47: 1594, 1977]

Stebbins GL (1947) Types of polyploids: Their classification and significance. Adv.
Genet 1: 403-429.

Stockinger EJ, Mulinix CA, Long CM, Brettin TS, Iezzoni AF (1996) A linkage map of
sweet cherry based on RAPD analysis of a microspore-derived callus culture
population. J Hered 87:214-21 8

Schwarzacher T, Leitch AR, Heslop-Harrison J S (1994) DNAzDNA in situ hybridization
- methods for light microscopy. In: Plant cell Biology: a practical approach. Eds
Harris N and Oparka KJ. Oxford: Oxford University Press

Singh RJ (1993) Plant cytogenetics. Boca Raton, Florida: CRC Press, Inc

Wang D, Karle R, Brettin TS, Iezzoni AF (1998) Genetic linkage map in sour cherry
using RFLP markers. Theor Appl Genet 97: (in press)

77

APPENDIX

78

Group I Group 3 Group 6 Group 8 Group 9 Group 13 Group 17

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

emit- L l
. ,, _ __ ,- .
U -I l— EH -b 4mm, - — (:PMJI): ~ PS4! I— I mm _ __ A“ _‘.P_ “Mb,
—1 I— Iznm 19 "3 —I 5 34mm
‘ " [mm 5.7
3.6 _‘ (want--
. . , , _ _ l 3 LI I59a
- I— thu EHM - .. HlJ-IT’M‘ _I
2 2 , 0 H162: I— enm-
p— R0010 .' (Pugh! 2
—l HI Tbm' '6
2.6 2.5 -— —- (‘PMS‘Ib' Is 4
-I >- ('I‘MIZb' o 7 — — EI‘I 7w ”‘9
1.6 ' — — cwzw I74
I— (mm.
2.8
7.9
3.6 -I -- (‘PMJ9d'
Llama I II
"‘ mm. 204 1 — Kris»
I5 I-IISHI' -— — FF 7w m *- "if“?
_I J . . I
45 0,7 __, Humour I—FFISbb
- arm. a 4 _l
" 3'5 s 3
— - - 37 i —1 7 O
H — H 1590 — MI I I __4 __ m7“ »—- ( PMJQh
'1 '— EI‘IS‘M‘ .— ('PMIOd -I *— FFITOk J _ Eli“
I.7
1’ -— — El~l7oj
—‘ I— EFIW‘
L7
,
-+ -— WW -I — 5“ Lb Group 10 Group 14 Group 18
l.5 _4 um:
"lfihwb' .
I.9 . . mm
j— Ll I67: _ l- ” ”‘9" —I -I — Ah-IOII‘
Group 4 I90 III III
‘ —l crusw H
I. .
Group 2 — ”66a Group 7 9.5
Is
— — Hm.
-w--£Hoob ~r—Amo. I44
Io ‘ " — — u I763 J
. J fl ,_’;':|43C *— CPM23¢
-— 1+th a — FFIZ?‘ 3.4 3'9
2.3 I— 5
‘ 4| _[ ._H I43b -‘ ”“9
T >— t M490 2.2 u» I 70' —l — cmnoc'
2.3 — — FFIIIzb L55 H _‘ IiI‘IThc‘
-—4 I- A02: 2 o J Em 6.4 ’ -— [— 1:1-1081'
€ng-
1 I
5-0 — — ('PMSIb'
- r—EFIUC’ Group 15
— »- mm-
Group I 1
H 7.4 — I— (‘me -- — arm.
. 2.9 9 6 -I l- EF'W" 2.8
o» '1" like": ‘— ”I”. I 2 El luna- __J ”W
—«I— 7 ' -- - ‘ . I— -
. « z I on -
'3 _lI— lfHJIc T " "'5‘“ [I 584 I2 d _ In."
I I ‘lI— M27... 3 7 L ‘ -« -— emu:
J ‘ 0
2.7 05 ogH—‘szm d U") g9
.- , ~ (‘ 4%. ~
-l 1'— "Sp" [,2 d EFF-n. 6.l
|.2 CPM”! 0.2
49 — »- IfFlSHb H ‘1 r :rmt _I .
~ ‘ —I ,_ Err". _4 EF72. ’—’ I‘] '37!
--1 —I‘: l o 2‘
—l — EI-IZRI'I '2 u b
59 —- rtailn "‘ ’- EH57}
47 Group 5 i 3 II
3.0 '
. - Frmbo
. __ . -I l— I1I‘187h T “ — EFIIITd‘
- —II——oIcI —I—A0I0c *' “m
H __ ..
5,, —I —- arm.- l~° _ .—~I:|~HSI
L3 -H—PSZ7‘ —--—AG¢.
IIo
— — new ‘0
2,2 FF'IKC‘ . .
-4 - ('PMS‘) 0‘" “ "1tl’512b‘ Group l6
l9 I,4__‘I;H30Ia-
—. —- Ifl-‘|73b (’PM20e’ 5'9 — - Imm-
3.5 I 2 ’
3’, , — — 87l12a'
_i I. _ .I “53" —1 — EFISM
EFonc ‘ ‘ (1PM2n‘ 2,0
-.. — >— (Pm
-I — [EH-491'
III
3.I
- — rm» M
l.9—‘HF}.MC "FEFIS.’
EFSJa
Him
.0 I r
—- I— FFm
H — I— M202!

 

Figure 1. The consensus map of RS and EB maps (Chapter 1) constructed from the
combined data using JoinMap with a minimum LOD .of 3.0 and a maximum
recombination frequency of 0.35. Markers in bold were present in EB only. Markers
indicated by asterisks were present in both RS and EB. All other markers were present in
RS only.

79

Table 1. Information about all single dose restriction fragment (SDRF) markers

that fit the emeted ratios

 

 

 

Marker Probe Restriction Parent genotype‘ Expected Observed X7
lame mme RS EB ratio ratio

Pru2 A012 Xbal H A 1 : 1 35 : 43 0.83
A027a A027 Eco RV H H 3 : 1 57 : 26 1.62
A027b A027 Eco RV H H 3 : 1 64 : 19 0.27
AG10a AG10 Eco RI H A 1 : 1 41 :42 0.02
AG10b AG10 Eco RI A H 1 : 1 42 :41 0.02
AG1OC AG10 Eco RI H H 3: 1 63:21 0.02
AG21 AG21 Eco RV H A 1 : 1 46: 39 0.59
AG40a AG40 Hind III H H 3 : 1 69 : 16 1.91
AG40b AG40 Hind Ill H H 3: 1 64 : 21 0.03
AG6 AG6 Eco RI H A 1 : 1 36 :48 1.73
AG8 A08 Hind III A H 1 : 1 41 :43 0.06
B4G10a B4010 Xbal H H 3: 1 47 : 16 0.02
B4G10b B4010 Xbal H A 1 : 1 35: 29 0.58
B4610c B4010 Xbal H A 1 : 1 36:42 0.47
8601 BGD1 Hind Ill H H 3: 1 51 : 18 0.03
B7H2a B7H2 Eco RV H H 3 : 1 59 : 24 0.59
B7H2b B7H2 Eco RV H H 3 : 1 66 : 16 1.48
B8A3 B8A3 Xbal H A 1 : 1 34 :40 0.50
CPM104 P0104 Eco RI H H 3 : 1 59 : 25 0.90
CPM12a P012 Eco RI H H 3: 1 56:27 2.33
0PM12b P012 Eco RI H H 3: 1 61 :22 0.08
CPM20e P020 Hind III A H 1 : 1 43:29 2.74
0PM20b P020 Hind Ill H H 3: 1 61 :21 0.02
0PM20c P020 Hind Ill H H 3: 1 56: 25 1.35
CPM20d P020 Hind III H A 1 : 1 50 : 33 3.49
CPM20e P020 Hind Ill H H 3 : 1 67: 15 2.16
0PM23a P023 Eco RI H A 1 : 1 36 :48 1.73
CPM23b P023 Eco RI A H 1 : 1 49 : 35 2.35
0PM23c P023 Hind III A H 1 : 1 49:35 2.35
0PM23d P023 Hind Ill H H 3: 1 57:27 2.11
0PM2a P02 Eco RI H H 3 : 1 59 : 24 0.59
CPM2b P02 Eco RI H H 3: 1 62 : 18 0.35
0PM30a P030 Hind Ill H A 1 : 1 47 : 38 0.96
0PM30b P030 Hind III A H 1 : 1 39 :46 0.59
0PM39a P039 Eco RV H A 1 : 1 38 :46 0.77
0PM39b P039 Eco RV H H 3: 1 61 : 23 0.21
CPM39c P039 Eco RV H H 3 : 1 66 : 18 0.68
0PM39d P039 Eco RV H H 3 : 1 67 : 17 1.16
0PM43 P043 Eco RI H A 1 : 1 44: 39 0.31
0PM45a P045 Eco RI A H 1 : 1 36 :49 2.00
0PM45b P045 Eco RI A H 1 : 1 41 :44 0.12
0PM48a P048 Hind Ill H H 3: 1 63 : 22 0.03
CPM48b P048 Hind Ill H H 3: 1 69: 16 1.91
0PM53b P053 Eco RI H H 3: 1 68 : 17 1.28
0PM57a P057 Xbal H A 1 : 1 40: 36 0.22
CPM57b P057 )0); I H H 3: 1 62 : 15 1.42

 

* H = presence of the marker; A = absence of the marker

Table 1. (cont’d)

 

 

 

Marker Probe Restriction Parent genotype Expected Observed X2
Dime E__nzy_me RS EB ratio ratio

0PM58a P058 Hind III H H 3 : 1 65: 17 0.93
0PM58b P058 Hind III H H 3 : 1 55:27 2.55
CPM59 P059 Hind Ill H A 1 : 1 46: 39 0.59
0PM64a P064 Eco RI H A 1 : 1 48 : 37 1.44
0PM64b P064 Eco RI A H 1 : 1 43 : 42 0.02
CPM67a P067 Eco RV H H 3 : 1 52 : 27 3.32
0PM67b P067 Eco RV A H 1 : 1 41 : 36 0.34
CPM6 P06 Eco RV H A 1 : 1 36:46 1.23
0PM70a P070 Eco RI H H 3 : 1 66 : 18 0.68
0PM70b P070 Eco RI H H 3 : 1 63 : 22 0.03
0PM90 P090 Hind III H A 1 : 1 43:42 0.02
EF1063 EF106 Dra I H H 3: 1 68: 14 2.98
EF106b EF106 Dra I H H 3 : 1 62 : 20 0.05
EF108a EF108 Xbal H H 3 : 1 52 : 21 0.47
EF108b EF108 Xbal A H 1 : 1 42: 31 1.67
EF1080 EF108 Xbal H A 1 : 1 44: 31 2.27
EF110 EF110 Eco RV H A 1 :1 36:43 0.63
EF111 EF111 Eco RV H A 1 : 1 45: 38 0.60
EF126 EF126 Eco RV A H 1 : 1 38 :45 0.60
EF127 EF127 Eco RV H H 3 :1 65 :20 0.15
EF128a EF128 Xbal H A 1 : 1 40: 33 0.68
EF128b EF128 Xbal H A 1 : 1 40: 34 0.50
EF128c EF128 Xbal A H 1 : 1 38: 37 0.03
EF129 EF129 Hind III H A 1 : 1 51 :34 3.41
EF130a EF130 Hind Ill H H 3 : 1 64 : 22 0.02
EF130b EF130 Hind III H H 3: 1 68 : 18 0.88
EF132a EF132 Xba I H H 3: 1 52 :23 1.15
EF1325 EF132 Xbal H A 1 : 1 43: 33 1.33
EF133a EF133 Hind III H H 3 : 1 57 :28 2.66
EF133b EF133 Hind III H H 3 : 1 68 : 17 1.28
EF133c EF133 Hind III H A 1 : 1 45 :40 0.31
EF143a EF143 Eco RV H A 1 : 1 41 :43 0.06
EF143b EF143 Eco RV H A 1 : 1 46 : 38 0.77
EF143¢ EF143 Eco RV H H 3 : 1 69: 15 2.49
EF143d EF143 Xbal H H 3: 1 67 : 13 3.52
EF143a EF143 Xbal H A 1 : 1 41 :39 0.06
EF1453 EF145 Eco RI H A 1 : 1 46: 35 1.51
EF145b EF145 Eco RI H H 3 : 1 67 : 14 2.79
EF146 EF146 Hind III A H 1 : 1 43:41 0.06
EF149a EF149 Eco RV H H 3 : 1 58: 24 0.70
EF149b EF149 Eco RV A H 1 : 1 38:46 0.77
EF152a EF152 Xbal A H 1 : 1 41 :41 0.01
EF152b EF152 Xbal H H 3 :1 67:15 2.16
EF156b EF156 Hind III H A 1 : 1 41 :43 0.06
EF157 EF157 Eco RI H A 1 : 1 50 : 33 3.49
EF158a EF158 Eco RI H A 1 : 1 48: 34 2.40
EF158b EF158 Eco RI H A 1 : 1 44: 37 0.62
EF1580 EF158 Eco RI H A 1 : 1 39 :44 0.31

 

81

Table 1 . (cont’d)

 

 

 

Marker Probe Restriction Parent genotype Expected Observed X7
LI_a_me Enzyme RS EB ratio ratio

EF158d EF158 Eco RI A H 1 I 1 42 I 41 0.02
EF158a EF158 Hind III H H 3 I 1 55 I 22 0.45
EF158f EF158 Hind III H A 1 I 1 35 I 43 0.83
EF1589 EF158 Hind III H A 1 Z 1 36 2 42 0.47
EF1598 EF159 ECO RV H A 1 I 1 36 I 49 2.00
EF15QD EF159 ECO RV A H 1 I 1 41 :44 0.12
EF1590 EF159 ECO RV H H 3 I 1 60 2 25 0.78
EF159d EF159 ECO RV H H 3 I 1 64 2 21 0.03
EF1628 EF162 Dra I H A 1 I 1 36 Z 48 1.73
EF162b EF162 Dra I A H 1 I 1 37 Z 47 1.20
EF167a EF167 Xba I A H 1 I 1 39 I 40 0.03
EF167b EF167 Xba I A H 1 I 1 33: 46 2.15
EF167C EF167 Xba I H A 1 I 1 43 I 37 0.46
EF167d EF167 Xba I H A 1 I 1 40 I 40 0.01
EF1698 EF169 ECO RI H A 1 I 1 48 I 35 2.05
EF169b EF169 ECO RI H A 1 I 1 45 I 38 0.60
EF1728 EF172 ECO RV H H 3 I 1 61 I 24 0.40
EF172b EF172 E00 RV H A 1 I1 41 :44 0.12
EF172C EF172 ECO RV A H 1 2 1 45 I 40 0.31
EF173a EF173 Xba I A H 1 I 1 44 Z 33 1.58
EF173b EF173 Xba I H A 1 I 1 442 34 1.29
EF174 EF174 Xba I A H 1 I 1 35 I 42 0.65
EF176a EF176 ECO RV H H 3 I 1 55 I 26 2.00
EF1760 EF176 ECO RV A H 1 I 1 45 I 37 0.79
EF1760 EF176 ECO RV H H 3 2 1 592 24 0.59
EF176d EF176 ECO RV H A 1 I 1 35 2 46 1.51
EF1768 EF176 ECO RV H H 3 I 1 54: 27 2.79
EF176f EF176 Eco RV A H 1 Z 1 40 2 41 0.02
EF1769 EF176 ECO RV H A 1 Z 1 44 I 37 0.62
EF176I’I EF176 Hind III A H 1 2 1 48 I 35 2.05
EF1761 EF‘I76 Hind III H H 3 I 1 652 19 0.33
EF176j EF176 Hind III H A 1 I 1 43 :40 0.12
EF176k EF176 Hind III H A 1 I 1 42 I 41 0.02
EF176| EF176 Hind III H H 3 2 1 65 I 17 0.93
EF176m EF176 Hind III H H 3 I 1 57 2 26 1.62
EF176n EF176 Hind III A H 1 I 1 40 2 42 0.06
EF178 EF178 ECO RV H H 3 I 1 57 I 26 1.62
EF179 EF179 ECO RI H H 3 I 1 63 i 20 0.08
EF182b EF182 ECO RV H A 1 I 1 45 I 37 0.79
EF185 EF185 Xba I H H 32 1 62: 15 1.42
EF187a EF187 ECO RI H H 3 I 1 692 16 1.91
EF187b EF187 ECO RI A H 1 2 1 47 I 37 1.20
EF187C EF187 ECO RV H A 1 I 1 43 Z 36 0.63
EF187d EF187 ECO RV H H 3 I 1 57 2 26 1.62
EF187e EF187 ECO RV H A 1 I 1 49 I 33 3.13
EF187I EF187 ECO RV A H 1 I 1 46 I 36 1.23
EF187g EF187 Hind III A H 1 Z 1 46 2 37 0.99
EF187I'I EF187 Hind III H A 1 I 1 44 I 39 0.31

 

82

Table 1. (cont’d)

 

 

 

 

Marker Probe Restriction Parent genotype Expected Observed X2
n_ame Egvme RS EB ratio ratio

EF187i EF187 Hind III A H 1 : 1 39:44 0.31
EF187] EF187 Hind III H A 1 : 1 50: 33 3.49
EF187k EF187 Hind III H H 3: 1 67: 16 1.62
EF187| EF187 Hind III A H 1 : 1 47 : 36 1.47
EF191 EF191 Xba I H H 3: 1 58: 20 0.02
EF194D1 EF194 Dral H A 1 : 1 46: 38 0.77
EF19402 EF194 Dral A H 1 : 1 42:42 0.01
EF194D3 EF194 Dral A H 1 : 1 41 :43 0.06
EF19404 EF194 Dral A H 1 : 1 41 :43 0.06
EF19405 EF194 Dra I H H 3: 1 56: 27 2.33
EF19406 EF194 Dral H A 1 : 1 47 : 36 1.47
EF194a EF194 Hind III H H 3: 1 63:21 0.02
EF194b EF194 Hind III H H 3: 1 64:20 0.11
EF194c EF194 Hind III A H 1 : 1 42:42 0.01
EF194d EF194 Hind III H H 3: 1 57:28 2.66
EF46a EF46 Xbal H A 1 : 1 37:41 0.22
EF46b EF46 Xba I H H 3: 1 63 : 15 1.56
EF46c EF46 Xbal H A 1 : 1 41 :36 0.34
EF46d EF46 Xba I H A 1 : 1 39 : 39 0.01
EF48a EF48 Eco RV H H 3 : 1 62 : 20 0.05
EF48b EF48 Eco RV A H 1 : 1 40:42 0.06
EF50 EF50 Dra I H H 3 : 1 70: 14 3.35
EF533 EF53 Xbal H A 1 : 1 41 : 39 0.06
EF53b EF53 Xbal A H 1 : 1 36 :44 0.81
EF62a EF62 Xba I H H 3 : 1 57 : 20 0.03
EF62b EF62 Xba I H H 3: 1 62 : 16 0.97
EF64a EF64 Hind III A H 1 : 1 38 :46 0.77
EF64b EF64 Hind III H H 3: 1 65: 20 0.15
EF64c EF64 Hind Ill H A 1 : 1 41 :43 0.06
EF67 EF67 Eco RV H A 1 : 1 36 :49 2.00
EF71 EF71 Eco RI H A 1 : 1 44:41 0.12
EF72 EF72 Eco RV H H 3 : 1 63 : 20 0.08
EF76 EF76 Eco RV H H 3 : 1 66 : 17 1.04
EF77 EF77 Hind III H H 3 : 1 70: 15 2.66
EF78a EF78 Dral A H 1 : 1 35:42 0.65
EF78b EF78 Dral H A 1 : 1 44: 36 0.81
EF78c EF78 Dra I H H 3 : 1 63 : 22 0.03
Ext1a Extensine Eco RV H A 1 : 1 50 : 35 2.66
Exth Extensine Eco RV H H 3 : 1 68 : 17 1.28
Hsp4 Hsp4 Eco RV H H 3: 1 61 :21 0.02
Oleo Oleosine Eco RI H A 1 : 1 48: 34 2.40
pch202a pch202 Hind III H A 1 : 1 46: 38 0.77
pch202b pch202 Hind III A H 1 : 1 40 : 44 0.20
pch205 pch205 Eco RI H H 3 : 1 57 : 26 1.62
PLGB6 PLG86 Eco RI H H 3 : 1 57 : 26 1.62
P8141 P8141 Eco RI A H 1 : 1 37:43 0.46
P827 P827 Eco RV H H 3: 1 62 : 22 0.05
P841 P841 Eco RV H A 1 : 1 42 :40 0.06

 

83

Table 2. Degree days (DD) for bloom and ripening for each progeny in the mapping
population for the years 1995, 1996, 1997 and the average.

 

 

 

 

 

Progeny Bloom (degee day_s) Ri (deglee days)
— ._.1.9.2§__.J

2(02) - 516.2 - 516.2 - - - -

2(03) 438.6 441.7 411.5 430.6 2149.5 1957.7 2107.8 2071.7
2(04) 343.7 410.9 369.3 374.6 1443.4 - 1486.5 1465.0
2(05) 354.0 393.1 396.3 381.1 2437 2109.8 2164.0 2236.9
2(06) 343.7 371.4 371.4 362.2 2605.6 2109.8 2311.1 2342.2
2(07) 354.0 410.9 396.3 387.1 2149.5 1900.8 1844.9 1965.1
2(08) 328.5 361.1 335.1 341.6 1789 1900.8 1880.4 1856.7
2(09) 393.6 393.1 371.4 386.0 1789 2109.8 1844.9 1914.6
2(10) 354.0 371.4 369.3 364.9 2711.9 2192.4 2395.8 2433.4
2(11) 343.7 364.9 357.4 355.3 2711.9 2351.1 2395.8 2486.3
2(12) 393.6 410.9 408.6 404.4 1628.2 2109.8 1844.9 1861.0
2(13) 366.5 393.1 369.3 376.3 - - 2395.8 2395.8
2(14) 381.8 393.1 402.8 392.6 1789 2109.8 1844.9 1914.6
2(15) 414.2 410.9 402.8 409.3 2711.9 - 2520.4 2616.1
2(16) 354.0 393.1 369.3 372.1 1789 1900.8 1844.9 1844.9
2(17) 414.2 410.9 402.8 409.3 2711.9 2109.8 1955.8 2259.2
2(18) 393.6 410.9 371.4 392.0 1987.7 1957.7 2107.8 2017.7
2(19) 414.2 410.9 402.8 409.3 1859.4 - - 1859.4
2(20) 414.2 410.9 396.3 407.1 2149.5 2109.8 2164.0 2141.1
2(22) 354.0 410.9 398.9 387.9 1815.6 2109.8 1844.9 1923.4
2(23) 438.6 441.7 408.6 429.6 1987.7 2109.8 2164.0 2087.2
2(24) 414.2 410.9 396.3 407.1 1987.7 2109.8 2164.0 2087.2
2(25) 438.6 441.7 466.5 448.9 - 2109.8 2164.0 2136.9
2(27) 328.5 361.1 357.4 349.0 1568.8 2109.8 1955.8 1878.1
2(28) 454.1 379 466.5 433.2 - - 1955.8 1955.8
2(29) 313.8 361.1 335.1 336.7 1723.9 1900.8 1714.4 1779.7
2(30) - - 408.6 408.6 - - - -

2(32) 354.0 393.1 369.3 372.1 2210.3 2192.4 2164.0 2188.9
2(33) 393.6 393.1 402.8 396.5 - - - -

2(34) 414.2 410.9 398.9 408.0 1859.4 - - 1859.4
2(35) 438.6 410.9 411.5 420.3 1628.2 2109.8 1844.9 1861.0
2(36) 438.6 441.7 411.5 430.6 - 2109.8 2107.8 2108.8
2(37) 438.6 441.7 408.6 429.6 2210.3 2109.8 2107.8 2142.6
2(38) 438.6 441.7 411.5 430.6 - 2109.8 2107.8 2108.8
2(39) 393.6 410.9 402.8 402.4 - 2109.8 2311.1 2210.5
2(40) 438.6 441.7 411.5 430.6 - - 2164.0 2164.0
2(41) 393.6 410.9 398.9 401.1 2711.9 2109.8 1955.8 2259.2
2(42) 414.2 441.7 419.3 425.1 2210.3 - 2107.8 2159.1
2(43) 414.2 441.7 419.3 425.1 2149.5 2109.8 1844.9 2034.7
2(44) 393.6 410.9 396.3 400.3 - - 2107.8 2107.8
2(45) 414.2 379 455.7 416.3 - 2109.8 1844.9 1977.4
2(46) - - 455.7 455.7 - - - -

2(47) 414.2 441.7 411.5 422.5 1915.6 - 1844.9 1880.3
2(48) 366.5 393.1 381.5 380.4 2711.9 2109.8 - 2410.9
2(49) 438.6 441.7 455.7 445.3 2711.9 - 1844.9 2278.4

 

84

Table 2. (cont’d)

 

 

 

 

 

Progeny Bloom (degree days) Ri (degree days)
2(50) 438.6 441.7 411.5 430.6 - 2109.8 1844.9 1977.4
2(51) 393.6 410.9 402.8 402.4 2149.5 2109.8 2395.8 2218.4
2(52) 381.8 393.1 381.5 385.5 - 2109.8 2164.0 2136.9
2(53) 343.7 393.1 369.3 368.7 - - 1844.9 1844.9
2(54) 381.8 410.9 381.5 391.4 2711.9 — - 2711.9
2(55) 454.1 441.7 440.4 445.4 1987.7 - 1844.9 1916.3
2(56) 381.8 410.9 396.3 396.3 1628.2 1957.7 1844.9 1810.3
2(58) 366.5 393.1 369.3 376.3 1723.9 1900.8 1844.9 1823.2
2(59) 381.8 410.9 396.3 396.3 2711.9 2192.4 2107.8 2337.4
2(60) 354.0 393.1 381.5 376.2 - - 2520.4 2520.4
2(62) 366.5 371.4 357.4 365.1 - 2109.8 2164.0 2136.9
2(63) 393.6 441.7 411.5 415.6 1987.7 - - 1987.7
2(64) 328.5 371.4 357.4 352.4 - - 2311.1 2311.1
2(65) 354.0 393.1 369.3 372.1 1568.8 2109.8 1844.9 1841.2
2(66) 438.6 410.9 411.5 420.3 1885.8 2109.8 2107.8 2034.5
3(02) - 379 455.7 417.3 - 2351.1 - 2351.1
3(03) 343.7 382.3 369.3 365.1 1885.8 1957.7 1844.9 1896.1
3(04) 454.1 441.7 419.3 438.4 2437 1957.7 2107.8 2167.5
3(05) 414.2 441.7 440.4 432.1 2711.9 1957.7 2395.8 2355.1
3(06) 454.1 441.7 440.4 445.4 2210.3 2192.4 1844.9 2082.5
3(07) 354.0 410.9 396.3 387.1 2679 - 2107.8 2393.4
3(08) 393.6 393.1 371.4 386.0 2776.3 2109.8 1844.9 2243.7
3(09) 354.0 382.3 369.3 368.5 - 2109.8 1955.8 2032.8
3(10) 381.8 393.1 369.3 381.4 - - 1844.9 1844.9
3(13) 343.7 371.4 357.4 357.5 2711.9 2192.4 2107.8 2337.4
3(14) 381.8 410.9 371.4 388.0 2711.9 2109.8 2273.0 2364.9
3(16) 393.6 410.9 408.6 404.4 1628.2 1601.2 1642.6 1624.0
3(18) 393.6 410.9 396.3 400.3 2711.9 2109.8 1844.9 2222.2
3(20) 438.6 441.7 411.5 430.6 1628.2 - 1844.9 1736.6
3(21) 414.2 410.9 396.3 407.1 1885.8 1957.7 1844.9 1896.1
3(22) 438.6 441.7 411.5 430.6 2645.2 - 2311.1 2478.2
3(24) 303.3 326.3 323.8 317.8 1654.7 2109.8 2311.1 2025.2
3(25) 414.2 410.9 408.6 411.2 2744.3 2109.8 2107.8 2320.6
3(27) 474.8 - - 474.8 2210.3 - - 2210.3
3(28) 381.8 393.1 381.5 385.5 2210.3 1957.7 - 2084.0
3(29) 328.5 361.1 335.1 341.6 1789 1900.8 1844.9 1844.9
3(31) 343.7 361.1 357.4 354.1 2210.3 2109.8 1844.9 2055.0
3(32) 328.5 345.6 335.1 336.4 - 1957.7 1844.9 1901.3
3(34) 328.5 364.9 369.3 354.2 1885.8 - 1844.9 1865.4
3(35) 343.7 393.1 369.3 368.7 1885.8 1900.8 1880.4 1889.0
3(37) 414.2 441.7 407.7 421.2 - - 1844.9 1844.9

 

85

Table 3. Fruit weight, soluble solids concentration for each progeny in the

 

 

 

 

 

mapping pulation for the years 1995, 1996, 1997 and the average.
Progeny F rUIt welght (g) soluble SOlldS concentration (°Br1x)
_WW&

2(02) - - - - - - - -

2(03) 3.43 3.53 4.06 3.67 18.63 16.66 18.04 17.84
2(04) 4.25 - 4.86 4.56 17.50 - 12.34 14.92
2(05) 5.17 4.60 6.56 5.44 19.23 15.76 17.48 17.49
2(06) 6.46 5.10 6.46 6.00 13.57 13.86 14.6 14.01
2(07) 4.59 4.05 4.60 4.41 17.45 13.28 16.52 15.75
2(08) 5.78 6.25 5.40 5.81 16.95 17.8 18.24 17.66
2(09) 5.13 - 5.02 5.08 9.50 20 22.72 17.41
2(10) 4.54 3.79 5.44 4.59 15.50 15.9 18.88 16.76
2(11) 6.66 7.37 5.56 6.53 20.27 18.52 16.92 18.57
2(12) 3.75 4.78 4.36 4.30 15.20 20.9 18.84 18.31
2(13) - - 4.56 4.56 - - 9.76 9.76
2(14) 4.88 6.18 5.68 5.58 15.85 13.6 15.52 14.99
2(15) 7.46 - 8.38 7.92 14.67 - 15.52 15.09
2(16) 4.90 5.22 4.52 4.88 15.90 15.8 17.44 16.38
2(17) 5.48 5.90 6.04 5.81 14.28 12.6 17 14.63
2(18) 4.40 4.55 4.18 4.38 17.40 18.24 18.88 18.17
2(19) 3.89 - - 3.89 13.38 - - 13.38
2(20) 6.37 6.74 6.38 6.49 15.04 14.3 15.28 14.87
2(22) 3.80 3.27 3.78 3.62 15.03 14.2 17.56 15.60
2(23) 6.09 4.12 6.02 5.41 15.40 16.3 19.68 17.13
2(24) 5.79 6.09 7.36 6.41 13.85 14.9 15.6 14.78
2(25) - 3.00 3.60 3.30 - 11.5 13.5 12.50
2(27) 3.56 4.45 4.78 4.26 13.93 14.6 14.74 14.42
2(28) - - 5.50 5.50 - - 17.48 17.48
2(29) 4.69 5.40 4.28 4.79 16.60 14 15.72 15.44
2(30) - - - - - - - -

2(32) 4.95 4.45 5.14 4.85 16.27 14.28 16.44 15.66
2(33) - - - - - - - -

2(34) 4.53 - - 4.53 15.08 - - 15.08
2(35) 4.13 5.08 4.64 4.61 14.50 18.5 18.56 17.19
2(36) - - 2.90 2.90 - 14.5 19.36 16.93
2(37) 2.72 2.67 3.70 3.03 20.82 15.6 19.8 18.74
2(38) - 4.48 5.22 4.85 - 19.9 15.56 17.73
2(39) - 4.53 5.74 5.14 - 15 14.48 14.74
2(40) - - 5.98 5.98 - - 16.68 16.68
2(41) 4.21 3.32 4.12 3.88 12.70 18 16.84 15.85
2(42) 2.52 - 2.14 2.33 14.37 - 14.52 14.44
2(43) 5.25 5.26 5.52 5.34 17.90 15 16.36 16.42
2(44) - - 5.58 5.58 - - 18.52 18.52
2(45) - 3.25 2.92 3.09 - 14.64 16.72 15.68
2(46) - - - - - - - -

2(47) 3.54 - 3.38 3.46 15.43 - 15.44 15.44
2(48) 6.13 4.77 - 5.45 21.30 17.56 - 19.43
2(49) 4.97 - 4.00 4.49 - - 17.92 17.92
2(50) - 3.73 2.80 3.27 - 13.52 13.16 13.34
2(51) 6.52 4.82 6.08 5.80 14.30 15.08 14.88 14.75

 

86

Table 3. (cont’d)

 

 

 

 

 

Progeny Fruit weight (g) soluble solids concentration (°Brix)
2(52) - 3.54 5.10 4.32 - 14.14 14.96 14.55
2(53) - - 3.14 3.14 - - 16.16 16.16
2(54) 3.81 - - 3.81 11.65 - - 11.65
2(55) 3.99 - 3.54 3.76 14.90 - 14.52 14.71
2(56) 3.60 4.36 4.46 4.14 15.00 15.72 17.08 15.93
2(58) 5.61 6.30 4.86 5.59 15.37 15.2 16.64 15.74
2(59) 3.72 3.44 3.54 3.57 11.33 12.9 14.8 13.01
2(60) - - 4.00 4.00 - - 9.96 9.96
2(62) - 7.04 7.24 7.14 - 15.96 16.92 16.44
2(63) 3.25 - - 3.25 14.97 - - 14.97
2(64) - - 4.00 4.00 - - 16.6 16.60
2(65) 3.25 4.96 5.40 4.54 14.67 15.04 14.24 14.65
2(66) 6.45 5.28 6.28 6.00 14.57 15.52 16.88 15.66
3(02) - 3.23 - 3.23 - 13.68 - 13.68
3(03) 6.78 5.27 6.06 6.04 14.87 14.84 15.52 15.08
3(04) 4.83 4.52 4.00 4.45 20.00 18.6 16.76 18.45
3(05) 4.31 5.29 4.44 4.68 16.15 18.84 15.68 16.89
3(06) 2.86 2.30 3.06 2.74 19.25 21 18.12 19.46
3(07) 7.05 - 6.38 6.71 16.87 - 16.96 16.91
3(08) 5.89 5.51 4.78 5.39 18.00 16.84 17 17.28
3(09) — 5.38 4.38 4.88 - 19.5 20.56 20.03
3(10) - - 2.30 2.30 - - 15.72 15.72
3(13) 5.84 5.32 6.36 5.84 12.43 13.44 14.96 13.61
3(14) 3.26 5.77 4.18 4.40 16.00 15.52 16.4 15.97
3(16) 4.92 4.34 5.66 4.97 15.12 13.2 17 15.11
3(18) 4.56 4.46 4.92 4.65 12.80 13.88 15.12 13.93
3(20) 3.06 - 3.72 3.39 14.04 - 16.52 15.28
3(21) 5.08 5.23 4.12 4.81 15.20 15.94 15.76 15.63
3(22) 4.09 - 4.48 4.29 14.27 - 14.76 14.51
3(24) 7.49 9.00 9.86 8.78 14.30 17.2 14.34 15.28
3(25) 5.31 4.67 5.66 5.21 12.20 13.27 15.72 13.73
3(27) 2.93 - - 2.93 17.96 - - 17.96
3(28) 5.39 4.98 - 5.18 19.08 18.58 - 18.83
3(29) 5.56 5.31 5.76 5.54 16.23 14.2 14.56 15.00
3(31) 4.14 3.75 5.32 4.40 19.05 19 17.72 18.59
3(32) - 7.05 6.16 6.60 - 19.86 17.64 18.75
3(34) 3.57 - 4.44 4.01 11.97 - 14.04 13.00
3(35) 5.30 5.16 5.00 5.15 15.94 16 16 15.98
3(37) - - 3.80 3.80 - - 20.08 20.08

 

87

Table 4. Pistil death, fruit set, pollen germination, flower bud death for each progeny in

 

 

 

 

 

the mapping population.

P Pistil death (%) Fruit Wen Flower bud
rogeny 1925 1225 1222 a: IRE“: o ' o 0
2(02) 15 0 15. 0 0.0 0.0 0.0
2(03) 5.0 30.0 25.0 20.0 3.7 4.5 6.7
2(04) 15.0 90.0 45.0 50.0 2.4 6.5 0.0
2(05) 0.0 17.5 32.5 16.7 15.2 1.0 7.1
2(06) 12.5 25.0 30.0 22. 5 2.3 4.0 44.4
2(07) 2.5 0.0 17.5 6. 7 0.4 8.5 12.5
2(08) 7.5 85.0 20.0 37. 5 9.6 0.5 40.0
2(09) 0.0 22.5 50.0 24.2 3.4 5.5 56.3
2(10) 7.5 52.5 40.0 33.3 17.6 11.0 76.9
2(11) 17.5 97.5 20.0 45.0 14.7 2.5 50.0
2(12) 5.0 95.0 55.0 51.7 9.9 0.0 23.1
2(13) 10.0 30.0 2.5 14.2 1.8 3.5 72.2
2(14) 25.0 75.0 57.5 52.5 5.4 0.0 66.7
2(15) 0.0 82.5 12.5 31.7 4.9 3.5 7.1
2(16) 5.0 30.0 45.0 26.7 6.6 1.5 35. 7
2(17) 0.0 20.0 40.0 20.0 2.0 4.0 30.0
2(18) 2.5 10.0 37.5 16.7 16.0 18.0 21.4
2(19) 10.0 90.0 27.5 42.5 2.1 1.0 40.0
2(20) 0.0 12.5 12.5 8.3 - 7.0 12.0
2(22) 12.5 70.0 57.5 46. 7 5.2 1.0 81.3
2(23) 5.0 92.5 0.0 32. 5 8.7 3.0 14.3
2(24) 17.5 12.5 12.5 14. 2 0.4 2.0 19.0
2(25) 0.0 2.5 - 1. 3 17.6 10.0 5.3
2(27) 10.0 60.0 2.5 24. 2 8.1 0.0 0. 0
2(28) - 20.0 - 20.0 - 0.0 25. 0
2(29) 15.0 75.0 27. 5 39. 2 34.4 6.0 18.8
2(30) - - o. 0 0 0 - - -
2(32) 10 0 65.0 20. 0 31 7 13.7 18.0 10.0
2(33) 7. 5 20.0 13.8 - 3.5 40.0
2(34) 12. 5 12.5 22.5 15 8 0.4 10.5 64.3
2(35) 0.0 90.0 12.5 34.2 0.8 0.0 0.0
2(36) 0.0 17.5 27. 5 15. 0 0.5 3.0 0. 0
2(37) 0. 0 45.0 2. 5 15.8 0.6 1.5 20. 0
2(38) 15. 0 5.0 47. 5 22.5 1.2 0.0 68.0
2(39) 2.5 2.5 12. 5 5.8 1.9 0.0 0.0
2(40) 2.5 12.5 5. 0 6.7 4.1 0.0 34.8
2(41) 0.0 15.0 12 5 9 2 3.0 4.0 0 0
2(42) 0. 0 40.0 7. 5 15. 8 0.5 0.0 20. 0
2(43) 15. 0 95.0 30. 0 46.7 2.8 1.5 42.1
2(44) 15.0 10.0 12.5 12.5 2.0 0.0 66. 7
2(45) 12.5 5.0 0.0 5.8 4.2 17.5 20. 0
2(46) - - 0. 0 0. 0 - -

2(47) 12.5 60.0 55. 0 42. 5 0.0 0.0 13.3
2(48) 25.0 10.0 57.5 30.8 3. 7 17.0 63.2
2(49) 10.0 42.5 42.5 31.7 15. 2 25.0 35.7
2(50) 5.0 17.5 7.5 10.0 11.1 12.0 46.2

 

 

 

88

Table 4. (cont’d)

 

 

 

 

 

 

Progeny 1 Pistil de‘ath (%) Fruoit Pollen 0 Flower bud
2(51) 12.5 5.0 50.0 22.5 6.9 0.0 28.6
2(52) 5.0 0.0 0.0 1.7 2.6 . 1.5 57.9
2(53) 5.0 12.5 20.0 12.5 4.2 2.5 25.0
2(54) 7.5 30.0 5.0 14.2 5.8 2.5 47.1
2(55) 0.0 0.0 2.5 0.8 1.7 17.0 5.0
2(56) 17.5 25.0 70.0 37.5 23.3 0.0 58.8
2(58) 5.0 62.5 50.0 39.2 19.3 0.0
2(59) 0.0 5.0 2.5 2.5 10.1 5.0 6.3
2(60) 20.0 17.5 22.5 20.0 7.5 5.5 100.0
2(62) 2.5 2.5 27.5 10.8 3.8 1.0 72.0
2(63) 15.0 100.0 7.5 40.8 5.4 4.0 33.3
2(64) 32.5 90.0 37.5 53.3 7.3 1.5 20.0
2(65) 0.0 10.0 7.5 5.8 11.0 10.5 6.3
2(66) 0.0 5.0 30.0 11.7 18.6 15.5 20.0
3(02) - 10.0 2.5 6.3 1.4 0.0 0.0
3(03) 0.0 0.0 27.5 9.2 21.8 4.5 20.0
3(04) 0.0 37.5 10.0 15.8 13.7 2.5 0.0
3(05) 2.5 20.0 27.5 16.7 13.2 20.0 16.7
3(06) - 42.5 32.5 37.5 2.2 0.0 0.0
3(07) 2.5 15.0 27.5 15.0 0.0 1.5 0.0
3(08) 2.5 5.0 10.0 5.8 2.2 15.0 10.0
3(09) 7.5 47.5 45.0 33.3 16.1 3.5 66.7
3(10) 22.5 52.5 42.5 39.2 0.3 0.0 92.9
3(13) 2.5 45.0 2.5 16.7 7.2 8.5 63.2
3(14) 7.5 17.5 60.0 28.3 6.6 13.5 -
3(16) 17.5 100.0 32.5 50.0 8.1 16.5 10.0
3(18) 0.0 15.0 10.0 8.3 3.0 0.0 41.2
3(20) 0.0 57.5 17.5 25.0 2.3 3.5 45.0
3(21) 2.5 22.5 7.5 10.8 0.0 8.5 56.5
3(22) 0.0 90.0 57.5 49.2 7.2 3.5 45.5
3(24) 17.5 75.0 50.0 47.5 0.3 3.5 47.1
3(25) 0.0 20.0 0.0 6.7 0.6 0.0 38.5
3(27) - - - - - - 30.8
3(28) 22.5 7.5 30.0 20.0 19.3 24.5 70.6
3(29) 12.5 50.0 42.5 35.0 4.4 0.5 81.3
3(31) 12.5 72.5 22.5 35.8 6.3 5.0 33.3
3(32) 27.5 65.0 72.5 55.0 5.3 2.0 52.9
3(34) 32.5 25.0 60.0 39.2 2.5 4.5 64.7
3(35) 15.0 60.0 32.5 35.8 10.1 34.0 60.0
3(37) 0.0 57.5 20.0 25.8 5.4 6.0 0.0

 

89

nrcurcnn sran UNIV. LIBRARIES
llllllllllIIIIIIIIIIIIIIIlllllllllllllllllllll
31293017668132