DISCOVERY OF A QTL FOR CHERRY LEAF SPOT RESISTANCE AND
VALIDATION IN TETRAPLOID SOUR CHERRY OF QTLS FOR BLOOM TIME AND
FRUIT QUALITY TRAITS FROM DIPLOID Prunus SPECIES

By

Travis Stegmeir

A DISSERTATION
Submitted to
Michigan State University
In partial fulfillment of the requirements
For the degree of
Plant Breeding, Genetics, and Biotechnology – Doctor of Philosophy
2013

ABSTRACT
DISCOVERY OF A QTL FOR CHERRY LEAF SPOT RESISTANCE AND VALIDATION IN
TETRAPLOID SOUR CHERRY OF QTLS FOR BLOOM TIME AND FRUIT QUALITY
TRAITS FROM DIPLOID Prunus SPECIES
By
Travis Stegmeir

With heterozygous polyploid species, detecting quantitative trait loci (QTL) can be an arduous
process, especially in segmental allopolyploids like sour cherry (2n=4x=32) where nonhomologous pairing is common. In our sour cherry breeding and genetics program at Michigan
State University, we have taken a QTL validation approach for identifying relevant QTLs,
whereby QTLs more easily discovered in related diploid species are tested for their association in
sour cherry. SNP markers on the Illumina 6K Infinium II array were used for genotyping sour
cherry plant materials included in the USDA-SCRI funded RosBREED project
(www.rosbreed.org). GenomeStudio polyploidy functionalities were used to score SNP
genotypes, including dosage. Previously identified QTLs/candidate genes for several
horticulturally important traits (fruit size, fruit flesh color, fruit acidity, fruit firmness and bloom
time) were identified from the peach (P. persica), almond (P. dulcis) and sweet cherry (P.
avium) literature. SNP markers spanning the target QTL intervals were identified based on
synteny with the peach genome sequence, and marker linkage phase was determined based on
sour cherry progeny segregation. The different haplotypes identified for these targeted regions
were then tested for haplotype trait association. Haplotypes with significant effect on phenotype
were identified for marker-assisted breeding. In certain cases, the SNP haplotype was
‘converted’ to an SSR marker to facilitate future genotyping. Not all regions found to be
significant in diploid relatives were significant in sour cherry, indicating either they are absent,

fixed or cannot be detected due to complexity of dosage and more allelic variants compared to
diploid species. This approach has been successful for QTLs with fairly large effects, which are
good targets for marker-assisted breeding. Since no QTL studies have been done previously
with cherry leaf spot (CLS) resistance, we utilized the Bayesian approach, implemented in
FlexQTLTM software which allowed us to follow important genotypic regions from multiple
populations through generations by including pedigreed parents and grandparents in the analysis.
By studying two populations, one with P. canescens derived CLS resistance, and one without,
we were able to locate a QTL on the top of linkage group (G)4 between SNP markers
ss490552303 and ss490552492 (between ~2.9-13.4 cM). When individuals with and without the
P. canescens haplotype at this region were compared, it was found the P. canescens haplotype
was significantly associated with disease resistance. The same was found in sour cherry, where
all individuals that were resistant to CLS had P. canescens haplotypes at this region. In both
sweet and sour cherry, however, individuals with the P. canescens segment were found that were
also susceptible, indicating that this is not the only region important for conferring CLS disease
resistance.

ACKNOWLEDEMENTS

I would like to thank my major professor, Dr. Amy Iezzoni, for her guidance, support and
editorial assistance, without which, I would not be where I am today. I especially appreciate her
willingness to let me partake in several aspects of her breeding program, where I got to use the
knowledge I gained throughout my PhD journey to design and implement crosses in the field.

I also appreciated the assistance of my other committee members, Dr. Jim Hancock, Dr. Dechun
Wang, and Dr. George Sundin for their valuable suggestions and helpful advice.

Special thanks are also given to past and present members of the Iezzoni lab, especially Audrey
Sebolt who was always available to help, and whose knowledge of the lab and procedures was
imperative to my success.

Thanks are given also to Mirko Schuster of the Julius Kühn-Institut, Dresden, Germany, for his
phenotyping of his sweet cherry populations for Cherry Leaf Spot resistance, and his allowing us
to collaborate with him in this project.

The NIFA-funded RosBREED project (www.rosbreed.org) was also pivotal in my research for
providing the funding and resources needed to carry out my research.

iv

TABLE OF CONTENTS
LIST OF TABLES……………………………………………………………………………....vii
LIST OF FIGURES……………………………………………………………………………...xi
CHAPTER 1: DISCOVERY OF A QTL FOR CHERRY LEAF SPOT RESISTANCE ...……..1
Introduction....................................................................................................................................2
Materials and Methods................…………………………………………………………….......3
Plant materials.....................................................................................................................4
Disease rating......................................................................................................................4
Pedigree confirmation in sweet cherry................................................................................5
SNP data and sweet cherry map construction.....................................................................5
QTL analysis and QTL allele identification in sweet cherry..............................................6
Results.............................................................................................................................................7
P. canescens-derived cherry leaf spot resistance in sweet cherry.......................................7
Haplotype construction and QTL allele identification........................................................9
P. canescens-derived cherry leaf spot resistance in sour cherry........................................10
Discussion......................................................................................................................................11
REFERENCES..............................................................................................................................53
CHAPTER 2: VALIDATION IN TETRAPLOID SOUR CHERRY OF QTLS FOR BLOOM
TIME AND FRUIT QUALITY TRAITS FROM DIPLOID Prunus SPECIES .56
Introduction...........…………………………………………………………………………........57
Materials and Methods................………………………………………………………..............60
Plant material and phenotyping.........................................................................................61
Genotyping and Haplotyping.............................................................................................62
Statistical analysis..............................................................................................................63
Results......…………………………………………………………………………………..........64
Phenotypic variation..........................................................................................................64
Haplotyping to identify different alleles for the target regions.........................................67
Haplotype analysis.............................................................................................................69
Bloom................................................................................................................................69
Fruit/pit size.......................................................................................................................71
Fruit firmness.....................................................................................................................73
Flesh color.........................................................................................................................75
Malic acid..........................................................................................................................77
Discussion.......…………………………………………………………………………………...77
Bloom.................................................................................................................................78
Fruit/pit size.......................................................................................................................79
Fruit firmness.....................................................................................................................82
Flesh color.........................................................................................................................83
Malic acid..........................................................................................................................84
Dosage...............................................................................................................................84
Linkage and breeding implications....................................................................................85
v

Conclusions....................................................................................................................................85
REFERENCES…………………………………………………………………………………141

vi

LIST OF TABLES

Table 1.1: Plant materials used in this study and cherry leaf spot disease evaluation scores for
2008 to 2011………………………………………………………...………………15

Table 1.2: SSR markers designed and used to validate the presence of P. canescens in the G4
disease resistant region in sour cherry………………………………………………18

Table 1.3: Pedigree confirmation of 34 individuals found to have P. canescens in their pedigree
via parent F5-18-167. P-values are calculated using the program KINGROUP
(Konovalov et. al., 2004) testing a null hypothesis that no parent offspring relation
between listed pair in rows vs. columns…………………………………………….19

Table 1.4: Pedigree confirmation of 38 progeny individuals found to not have P. canescens in
their pedigree. P-values are calculated using the program KINGROUP (Konovalov
et. al., 2004) testing a null hypothesis that no parent offspring relation between listed
pair in rows vs. columns…………………………………………………………….20

Table 1.5: Estimates of 2ln Bayes factors for the first replicate of the genome-wide analysis for
mean disease resistance identified using the sweet cherry plant material listed in
Table 1.1. The number of QTLs being compared in the models are separated by a
back slash (“/”)………………………………………………………………………22

Table 1.6: Estimates 2ln Bayes factors for the first replicate of the G4 analysis for mean disease
resistance identified using the sweet cherry plant material listed in Table 1.1. The
number of QTLs being compared in the models are separated by a back slash (“/”)..22
Table 1.7: Marker interval and name for the CLS resistance QTL found on G4………………..22

Table 1.8: Mean cherry leaf spot scores for the presence, or absence of the G4 P. canescens
haplotype. All sweet cherry progeny individuals are included, those with, and those
without P. canescens in their background (See Table 1.1). The means are
significantly different (P = 0.004) as denoted by different letters in the disease score
mean column…………………………………………………………………………23

Table 1.9: Mean cherry leaf spot score for the presence or absence of the G4 P. canescens
haplotype. Only individuals confirmed to have P. canescens in their background (See
vii

Table 1.3) are included. The means are significantly different (P < 0.0001) as
denoted by different letters in the disease score mean column………………………23

Table 1.10: Disease scores and the presence/absence of the G4 region from P. canescens in
sweet cherry progeny with confirmed P. canescens background (See Table 1.3).
Individuals in bold and underlined contain the P. canescens haplotype, but are
susceptible……………………………………………………………………………24

Table 1.11: Cherry leaf spot disease scores and the presence/absence of the G4 region from P.
canescens of sour cherry progeny (See Table 1.1). Individuals in bold and underlined
contain the P. canescens haplotype, but are disease susceptible……………...……..25

Table 2.1: SNP informativeness in sour cherry for the eight sets of chromosomes based on
whether the SNP was derived from polymorphism in sweet cherry or in one of the
two sour cherry subgenomes (i.e., avium or fruticosa)………………………………87

Table 2.2: Summary of all traits, QTLs and their locations that were validated in this study. The
species source and original QTL reference(s) are included…………………………88
Table 2.3: SSR markers used in this study and the original SSR reference……………………..90

Table 2.4: Number of progeny individuals from each bi-parental family for which the four
chromosome segments for the target QTL regions in each progeny individual could
be identified as haplotypes inherited from its parents. Individuals were not haplotyped
when SNPs were ambiguous for dosage, or if individual haplotypes could not be
determined…………………………………………………………………………...91

Table 2.5: Phenotypic means for bloom time in 2011 and 2012 for the presence or absence of the
G1 haplotypes in sour cherry progeny individuals from the five bi-parental families.
Parental genotypes for the G1 haplotypes are: ‘M172’ (ijkk), ‘25-02-29’ (abcd),
‘Montmorency’ (aceh), ‘25-14-20’ (adej), ‘UF’ (bfij), ‘Surefire’ (ahjj), ‘RS’ (acdi)
and ‘ET’ (egkl)……………………………………………………………………….92

Table 2.6: Phenotypic means for bloom time in 2011 and 2012 for the presence or absence of the
G2 G2SSR1566 haplotypes in all sour cherry individuals from the five bi-parental
families. Parental genotypes for the G2 haplotypes are: ‘M172’ (2447), ‘25-02-29’
(2467), ‘Montmorency’ (4488), ‘25-14-20’ (4489), ‘UF’ (2449), ‘Surefire’ (4458),
‘RS’ (4478) and ‘ET’ (4678)………………………………………………………...93
viii

Table 2.7: Phenotypic means for bloom time in 2011 and 2012 for the presence or absence of the
G4 haplotypes for all sour cherry individuals from the five bi-parental families.
Parental genotypes for the G1 haplotypes are: ‘M172’ (defh), ‘25-02-29’ (abcd),
‘Montmorency’ (dilo), ‘25-14-20’ (aghj), ‘UF’ (ahkn), ‘Surefire’ (giks), ‘RS’ (bdgi),
and ‘ET’ (amnp)……………………………………………………………………...94

Table 2.8: Phenotypic means for bloom time in 2011 and 2012 for the presence or absence of the
G4 haplotypes within individual bi-parental families. Only those populations and
haplotypes that were significant in one or both years are presented………………...95

Table 2.9: Phenotypic means for fruit, pit and mesocarp weights (g) in 2011 for the presence or
absence of the G2 G2SSR1566 haplotypes for all sour cherry individuals from all five
bi-parental families. Parental genotypes for the G2 region are: ‘M172’ (2447), ‘2502-29’ (2467), ‘Montmorency’ (4488), ‘25-14-20’ (4489), ‘UF’ (2449), ‘Surefire’
(4458), ‘RS’ (4478) and ‘ET’ (4678)………………………………………………..96

Table 2.10: Phenotypic means for fruit, pit and mesocarp weights (g) in 2011 for the presence or
absence of the G3 haplotypes for all sour cherry individuals for all five bi-parental
families. Parental genotypes for the G3 haplotypes are: ‘M172’ (degh), ‘25-02-29’
(abcd), ‘Montmorency’ (acjn), ‘25-14-20’ (adjk), ‘UF’ (ehjk), ‘Surefire’ (cjjk), ‘RS’
(acdj), and ‘ET’ (dgjl)………………………………………………………………..97

Table 2.11: Phenotypic means for fruit, pit and mesocarp weights (g) in 2011 for the presence or
absence of the G5 haplotypes for all sour cherry individuals for all five bi-parental
families. Parental genotypes for the G5 haplotypes are: ‘M172’ (dfhj), ‘25-02-29’
(abcd), ‘Montmorency’ (aceh), ‘25-14-20’ (bdfh), ‘UF’ (ddfn), ‘Surefire’ (ceil), ‘RS’
(bceh), and ‘ET’ (djkn)………………………………………………………………98

Table 2.12: Phenotypic means for fruit, pit and mesocarp weight (g) in 2011 for the presence or
absence of the G6 G6SSR2208 haplotypes for all sour cherry individuals for all five
bi-parental families. Parental genotypes for the G6 haplotypes are: ‘M172’ (3 5 null
null), ‘25-02-29’ (1 3 5 null), ‘Montmorency’ (1 4 5 null), ‘25-14-20’ (2 3 null null),
‘UF’ (1 3 3 null), ‘Surefire’ (1 3 5 null), ‘RS’ (1 2 5 null), and ‘ET’ (3 5 null null)...99
2

Table 2.13: Phenotypic means for fruit firmness (g/mm ) in 2011 for the presence or absence of
the G2 G2SSR1566 haplotypes for all sour cherry individuals from all five bi-parental
families. Parental genotypes for the G2 haplotypes are: ‘M172’ (2447), ‘25-02-29’

ix

(2467), ‘Montmorency’ (4488), ‘25-14-20’ (4489), ‘UF’ (2449), ‘Surefire’ (4458),
‘RS’ (4478) and ‘ET’ (4678)……………………………………………………….100
2

Table 2.14: Phenotypic means for fruit firmness (g/mm ) in 2011 for the presence or absence of
the G3 haplotypes for all sour cherry individuals for all five bi-parental families.
Parental genotypes for the G3 haplotypes are: ‘M172’ (efgh), ‘25-02-29’ (abcd),
‘Montmorency’ (ijno), ‘25-14-20’ (adjk), ‘UF’ (ehjk), ‘Surefire’ (jjop), ‘RS’ (adjo),
and ‘ET’ (fjlm)……………………………………………………………………..101
2

Table 2.15: Phenotypic means for fruit firmness (g/mm ) in 2011 for the presence or absence of
the G5 haplotypes for all sour cherry individuals for all five bi-parental families.
Parental genotypes for the G5 haplotypes are: ‘M172’ (dfhj), ‘25-02-29’ (abcd),
‘Montmorency’ (aceh), ‘25-14-20’ (bdfh), ‘UF’ (ddfn), ‘Surefire’ (ceil), ‘RS’ (bceh),
and ‘ET’ (djkn)……………………………………………………………………102
2

Table 2.16: Phenotypic means for fruit firmness (g/mm ) in 2011 for the presence or absence of
the G6 G6SSR2208 haplotypes for all sour cherry individuals for all five bi-parental
families. Parental genotypes for the G6 haplotypes are: ‘M172’ (3 5 null null), ‘2502-29’ (1 3 5 null), ‘Montmorency’ (1 4 5 null), ‘25-14-20’ (2 3 null null), ‘UF’ (1 3
3 null), ‘Surefire’ (1 3 5 null), ‘RS’ (1 2 5 null), and ‘ET’ (3 5 null null)…………103

Table 2.17: Phenotypic means for flesh color in 2011 for the presence or absence of the G3
haplotypes for all sour cherry individuals for all five bi-parental families. Parental
genotypes for the G3 haplotypes are: ‘M172’ (clnu), ‘25-02-29’ (nnop),
‘Montmorency’ (fhno), ‘25-14-20’ (bdhn), ‘UF’ (bcdn), ‘Surefire’ (efgh), ‘RS’
(khnp), and ‘ET’ (eglu)…………………………………………………………….104

Table 2.18: Phenotypic means for flesh color in 2011 for the presence or absence of the G3
haplotypes for all sour cherry individuals within individual families. Only haplotypes
with significant differences are presented………………………………………….105

Table 2.19: Phenotypic means for malic acid (mg/ml) in 2011 for the presence or absence of the
condensed G5 haplotypes for all sour cherry individuals for all five bi-parental
families. Parental genotypes for the G5 haplotypes are: ‘M172’ (1236), ‘25-02-29’
(2346), ‘Montmorency’ (2345), ‘25-14-20’ (1236), ‘UF’ (1236), ‘Surefire’ (1123),
‘RS’ (1234) and ‘ET’ (1146)……………………………………………………….106

x

LIST OF FIGURES

Figure 1.1: Pedigree of the incorporation of CLS resistant P. canescens into the sour cherry
background. Individual which are resistant are colored black, susceptible individuals
are colored white, and families which are segregating for resistance are grey………26

Figure 1.2: Images of cherry leaf spot disease ratings of 1 (a), 2 (b), 3 (c), 4 (d) and 5 (e) on the
disease scale used for sweet cherry and P. canescens derived diploid individuals….27

Figure 1.3: Linkage map used for QTL analysis. Large linkage groups were divided into multiple
sections (denoted by [ ]) to fit on the page. Marker cM distances were approximated
by multiplying marker peach physical map location in Mb by four. A total of 548
markers spanning the 8 linkage groups were used. Markers are a part of the NCBI’s
dbSNP repository (Sherry et al. 2001)……………………………………………….28

Figure 1.4: Genome Studio (Illumina Inc. 2011) SNP dosage calls were done for each marker.
Sweet cherry (yellow) individuals were included to help define the two homozygous
(AAAA and BBBB) classes and the balanced heterozygous class (AABB).
Determining dosage was necessary to build haplotypes………………………….....36

Figure 1.5: Frequency distribution of mean cherry leaf spot disease scores for all sweet cherry
individuals (See Table 1.1). The mean disease score for the P. canescens-containing
parent ‘F5-18-167’ and ‘Namati’ were 1.3 and 2.0, respectively……………………37

Figure 1.6: Expanded G4 linkage map used for QTL analysis. The G4 linkage group is broken up
into multiple sections (denoted by [ ]) to fit on the page. Marker cM distances were
approximated by multiplying marker peach physical map location in Mb by four. A
total of 241 markers spanning G4 were used. Markers are a part of the NCBI’s
dbSNP repository (Sherry et al. 2001)……………………………………………….38

Figure 1.7: a) Disease resistance QTL location on G4 between SNP markers ss490552323 and
ss490552500 (between 4.0-13.8 cM). b). Trace plot for the QTL region…………....43
Figure 1.8: Haplotype R is associated with P. canescens across the QTL region……………….45
Figure 1.9a: Sour cherry haplotypes for the G4 QTL region. Parents ‘23-23-13’ and ‘Ujfehertoi
Furtos’ with 5 progeny. Haplotype R is from P. canescens………………………....46
xi

Figure 1.9b: Parents ‘Montmorency’ and ‘23-23-13’ with 4 progeny individuals that were
haplotyped. Black regions indicate where a crossover took place, but due to identical
SNP markers in that area, the exact location of the crossover is unknown. Haplotype
R is from P. canescens…………………………………………………………….....47

Figure 1.10: Location of SSR markers used to tag the P. canescens haplotype in relation to the
CLSR_G4 QTL region of interest between SNP markers ss490552323 (4.0 cM, 1.0
Mb) and ss490552500 (13.8 cM, 3.46 Mb)……………………………………….....48

Figure 1.11: SSR fragments for sour cherry individuals (See Table 1.1) for four primer pairs in
the G4 cherry leaf spot QTL region: a) CLS004 and b) CLS005 c) CLS026 d)
CLS028 (See Table 1.2) run on sour cherry individuals for the G4 QTL region.
Arrows denote the location and fragment size of the P. canescens allele……….......49

Figure 1.12: Proposed two gene model for disease resistance in sweet cherry, where the
individuals are only resistant with dominant genes at both the P. canescens-associated
G4 “A”, and the unknown “B” second loci. Disease resistant parent F5-18-167 is
shown to be heterozygous at both loci (AaBb) where parent 2 is shown to be
homozygous recessive for the G4 “A” locus, and heterozygous for the proposed
second locus needed to warrant resistance. Squares highlighted in grey are those that
would be resistant…………………………………………………………………....51

Figure 1.13: Proposed two gene model for disease resistance in sour cherry, where the
individuals are only resistant with dominant genes at both the G4 “A”, and the
unknown “B” second loci. This model assumes preferential pairing within subgenomes, where sum-genomes are denoted by subscript numbers. Disease resistant
parent ‘23-23-13’ is shown to be heterozygous at both loci in only one sub-genome
(A1a1a2a2B1b1b2b2) where both ‘UF’ and ‘Montmorency’ are shown to be
homozygous recessive for the G4 “A” locus, and heterozygous in one sub-genome for
the proposed second locus needed to warrant resistance (a1a1a2a2B1b1b2b2). Squares
highlighted in grey are those that would be resistant…………………………….......52

Figure 2.1: Pedigrees of the five bi-parental families used in this study. Populations are colored
white with the progeny number below. Grandparents are colored grey, while parents
are colored black. If individuals are both parents and grandparents, they are colored
black………………………………………………………………………………...107
Figure 2.2: Washington State University flesh color card rating scale used to determine flesh
color rating for sour cherry individuals…………………………………………….108
xii

Figure 2.3: Genome Studio (Illumina Inc. 2011) SNP dosage calls were done for each marker.
Sweet cherry (yellow) individuals were included to help define the two homozygous
(AAAA and BBBB) classes and the balanced heterozygous class (AABB).
Determining dosage was necessary to build haplotypes…………………………....109

Figure 2.4: Reconstruction of a ~1.2 Mb region spanning the self-incompatibility S-locus and its
inheritance in (a) Sweet cherry, with four parental haplotypes (1–4) and (b) Sour
cherry, with eight parental haplotypes (1–8). Identical haplotypes have the same
background colors. Haplotypes are shown for five sweet cherry and two sour cherry
seedlings. Monomorphic SNPs within cross-over regions are highlighted in grey.
Genotypes indicated as “u” are for an unresolved polymorphic SNP in sour cherry
(Peace et al. 2013, Figure 4)………………………………………………………..110

Figure 2.5: Phenotypic distributions for bloom growing degree days (GDD) in 2011 for all
populations and each of the five bi-parental populations. Parental values are shown in
individual populations when data is available……………………………………...112

Figure 2.6: Phenotypic distributions for bloom growing degree days (GDD) in 2012 for all
populations and each of the five bi-parental populations. Parental values are shown in
individual populations when data is available……………………………………...113

Figure 2.7: Phenotypic distributions for fruit weight (g) in 2011 for all populations and each of
the five bi-parental populations. Parental values are shown in individual
populations……………………………………………………………………….....114

Figure 2.8: Phenotypic distributions for pit weight (g) in 2011 for all populations and each of the
five bi-parental populations. Parental values are shown in individual populations..115

Figure 2.9: Phenotypic distributions for mesocarp weight (g) in 2011 for all populations and each
of the five bi-parental populations. Parental values are shown in individual
populations………………………………………………………………………….116
2

Figure 2.10: Phenotypic distributions for fruit firmness (g/mm ) in 2011 for all populations and
each of the five bi-parental populations. Parental values are shown in individual
populations when data is available…………………………………………………117

xiii

Figure 2.11: Phenotypic distributions for flesh color based on Washington State University’s
flesh color rating in 2011 for all populations and each of the five bi-parental
populations. Parental values are shown in individual populations when data is
available…………………………………………………………………………...118

Figure 2.12: Phenotypic distributions for malic acid content (mg/ml) in 2011 for all populations
and each of the five bi-parental populations. Parental values are shown in individual
populations when data is available………………………………………………...119

Figure 2.13: Bloom and fruit trait QTLs from diploid Prunus (peach and sweet cherry) that were
targets of validation in tetraploid sour cherry…………………………………......120

Figure 2.14: The twelve haplotypes identified in sour cherry for the G1 region used to test for the
bloom time QTL…………………………………………………………………..121

Figure 2.15: The 21 haplotypes identified in sour cherry for the G2 region used to test for bloom
time and fruit size/fruit firmness QTLs. Twenty-one unique haplotypes were found,
so SSR marker G2SSR1566 was used to “condense” haplotypes based on marker
score to 7 haplotypes……………………………………………………………....122

Figure 2.16: The 17 haplotypes identified in sour cherry for the G4 region used to test for the
bloom time QTL. Haplotype designations q-r were not used………………….....127

Figure 2.17: The 16 haplotypes identified in sour cherry for the G3 region used to test for the
fruit size and firmness QTL. The number of haplotypes was “condensed” to eleven
based on the SNP calls for the region in bold and underlined. For the analysis,
haplotype i = a, o = c, f = d, m = g and p = k……………………………………...129

Figure 2.18: The twelve haplotypes identified in sour cherry for the G5 region used to test for
fruit size and firmness QTL. Haplotype designations g and m were not used….....132

Figure 2.19: The thirteen haplotypes identified in sour cherry for the G6 region between CNR20
and the S-locus. These haplotypes were “condensed” to five haplotypes based on
results of the CNR – linked SSR marker G6SSR2008. This region was used to test
for QTLs for fruit size and firmness……………………………………………......134

xiv

Figure 2.20: The thirteen haplotypes identified in sour cherry for the G3 region containing
MYB10. This region was used to test for the flesh color QTL. Haplotype
designations a, i, j, m, q, r, s and t were not used…………………………………..136

Figure 2.21: The 17 haplotypes identified in sour cherry for the G5 Malic acid QTL region. The
Seventeen haplotypes were condensed to just six using the region in bold and
underlined. Above the haplotypes are what haplotype group each haplotype belongs
to (1-6)……………………………………………………………………………...138

Figure 2.22: Within population mean comparisons of dark flesh haplotypes d, e, l, and p. Means
are significantly different (P< 0.05) if letters after the mean score are different. Colors
are representative of the rating………………………………………………….......140

xv

CHAPTER 1
DISCOVERY OF A QTL FOR CHERRY LEAF SPOT RESISTANCE

1

Introduction

Cherry leaf spot (CLS), caused by the fungal pathogen Blumeriella jaapii (Rehm) Arx
(anamorph Phloeosporella padi [Lib.] Arx), is a major disease in all humid cherry growing
regions worldwide. While sour cherries (Prunus cerasus) are generally more prone to infection
and the resulting leaf yellowing and defoliation, sweet cherries (P. avium) are also affected.
When not controlled, CLS can cause early leaf defoliation, which can result in fruit that are
poorly colored, soft, and low in soluble solids (Keitt et al., 1937). Premature defoliation can also
weaken the tree and reduce winter hardiness which can lead to flower bud freeze damage and
even tree death (Howell and Stackhouse, 1973). Studies have shown that fruit have priority over
other sinks in Prunus (Richards, 1986), so fewer leaves would produce fewer storage
carbohydrates for the following year’s growth.

As many as seven fungicide applications can be needed each growing season on sour cherry to
manage CLS, resulting in substantial cost to growers and a significant amount of pesticides
released into the environment. There is also the threat that the pesticides currently being used to
control CLS may be removed from the market, jeopardizing the sustainability of the industry. B.
jaapii has also been found to develop resistance to site-specific sterol demethylation inhibitor
fungicides (DMIs) which have been used extensively to control CLS on cherry (Proffer et al.,
2006). This developed resistance to a major class of CLS-controlling fungicide increases the
need for new varieties that have genetic resistance to CLS. Many studies have shown there are
no sour cherry cultivars that have complete resistance to CLS (Sjulin et al., 1989; Schuster and

2

Tobutt, 2004; Budan et al., 2005); however, in all of these studies, there were some individuals
that displayed moderate resistance, indicating some polygenic resistance.

The susceptibility of all current cultivars in P. avium and P. cerasus germplasm to CLS
warranted the need to examine wild cherry species in an attempt to find resistance. One
promising candidate to introgress disease resistance was shown to be the wild diploid species P.
canescens (Wharton et al., 2003; Wharton and Iezzoni, 2005; Schuster and Tobutt, 2004). As a
result, P. canescens has been used in breeding for CLS resistance in both sweet and sour cherry
with diploid and tetraploid populations, respectively, segregating for disease resistance. With the
development of an Illumina Infinium® cherry SNP array (Peace et al., 2012), it was possible to
locate introgressed chromosome regions from P. canescens due to the high marker coverage
across all 8 linkage groups. The objective of this study was to determine the inheritance of P.
canescens-derived resistance to sweet cherry and identify the locations of gene(s) controlling this
resistance. Because of the simpler genetics of diploid cherry, the initial investigation was done
with P. canescens-derived materials from crosses with sweet cherry, followed by validation
using P. canescens-derived plant materials from sour cherry. In this study, we used the Bayesian
approach, implemented in FlexQTLTM software (Bink et al. 2002, Rosyara et al. 2013). This
allowed us to follow important genotypic regions from multiple populations through generations
by including pedigreed parents and grandparents in the analysis.

Materials and Methods

3

Plant materials

The sweet cherry plant materials for this study were developed and maintained at the Julius
Kühn-Institut (JKI) in Dresden, Germany and included 74 BC1 progeny individuals (Table 1.1).
P. canescens was used as a pollen parent and crossed with the sweet cherry selection (P. avium
M30). From this F1 population, the CLS-resistant individual F5-18-167 was selected due to its
disease resistance to CLS, and was used as a parent to transfer this resistance into one of the
populations in this study (Table 1.1).
In sour cherry, P. canescens was first incorporated from the triploid grandparent 148-1
(Schmidt and Gruppe, 1988). Seed from this triploid were planted, and from those, a CLS
resistant parent ‘23-23-13’ was shown to have sufficient fertility to be used in crosses with sour
cherry parents ‘Montmorency’ and ‘Újfehértói Fürtös’ (‘UF’) (Figure 1.1). The resulting 15 sour
cherry selections were used in this study and were maintained at Michigan State University’s
Clarksville Horticultural Experimental Station of Michigan State University in Clarksville, MI,
USA (Table 1.1).

Disease rating

The sweet cherry individuals were not sprayed with fungicides for the control of CLS in the
years 2008-2011. Selections were scored using the following rating scale modified from
Wharton and Iezzoni (2005), where a score of 2 or less was considered resistant:

1 – No chlorotic symptoms, small hypersensitive response at point of infection (Fig. 1.2a)

4

2 – Scattered pigmented lesions, chlorotic or necrotic points, no visible sporulation (Fig. 1.2b)
3 – Larger lesions, partly with aerial mycelium and stunted sporulating acervuli (Fig. 1.2c)
4 – Sporulating acervuli with chlorotic and necrotic lesions (Fig. 1.2d)
5 – Heavily sporulating acervuli (Fig. 1.2e)

For the sour cherry individuals, CLS was also not controlled in the orchard containing the plant
material used for this analysis, resulting in intense disease pressure. In 2010, CLS severity was
recorded using the same scale as with sweet cherry. In 2011, the trees were also left untreated
for CLS, however individuals were rated only as resistant or susceptible, with those considered
resistant if infection did not result in conidia formation and trees did not defoliate (disease score
of 2 or less). All individuals on which we observed conidia formation on leaves, or that
exhibited defoliation, or both were considered susceptible.

Pedigree confirmation in sweet cherry

P. canescens pedigree confirmation in sweet cherry was done using the SNP data described
below and the program KINGROUP (Konovalov et. al., 2004) with R-script. Individuals were
considered to have P. canescens in their background if the P-value comparing marker scores to
the parent F5-18-167, which contains P. canescens, was equal to or less than 0.05.

SNP data and sweet cherry map construction

5

For sweet cherry, two sources of P. canescens, (one from Michigan State, and one from
Germany), ‘Namati’, F5-18-167, and 72 individuals were genotyped using the RosBREED
Illumina Infinium® cherry SNP array of 5,696 SNP markers (Peace et. al., 2012). SNP
genotypes were determined using the Genotyping Module of GenomeStudio Data Analysis
software v2010.3 (Illumina Inc. 2010). A total 2,949 polymorphic SNPs were found, from
which 548 SNPs were selected manually to cover the 8 Prunus linkage groups (Figure 1.3).
Markers were selected to be spread across each chromosome as equally as possible based on
physical map distances previously determined in peach. As the number of true offspring was too
small for linkage map construction, the map used for QTL analysis was based on the peach
physical map positions (Verde et al. 2013) where the physical map was scaled to a genetic map
by conversion factor of 1 Mb = 4 cM.

For sour cherry, a separate GenomeStudio project was done where ‘UF’, ‘Montmorency’, ‘2323-13’, and 15 seedlings and other sour cherry founders and populations (384 individuals) were
genotyped the same as above; however, available SNP data from a diverse array of sweet cherry
selections and seedlings (105 individuals) were included to aid in the determination of dosage by
showing the two homozygous (AAAA and BBBB for sour cherry corresponding to AA and BB
in sweet cherry) and balanced heterozygous (AABB for sour cherry corresponding to AB in
sweet cherry) classes (Figure 1.4).

QTL analysis and QTL allele identification in sweet cherry
QTL mapping was done initially using the genome-wide set of 548 SNPs selected from those
markers found to be polymorphic to equally cover the 8 Prunus linkage groups, followed by an
6

analysis of a single chromosome with a dense map - once a QTL was located on G4, all available
polymorphic SNPs found for that linkage group, i.e. 241 markers, were run. The consistency in
marker order and distance was verified by comparing expected and observed double cross-over
frequency for both maps.
QTL mapping was done with mean disease score for 2008-2011. The parents, grandparents and
progenies were included in the pedigree for analysis. QTL analysis was done using a Markov
chain Monte Carlo (MCMC) based Bayesian analysis method (Bink et al. 2002, 2008)
implemented in the FlexQTLTM software as was done in Rosyara et al. (2013), but with a
simulation length of 100,000 iterations with a thinning value of 10, and a simulation length of
200,000 and a thinning value of 20 for all 8 chromosomes, and the dense G4 map respectively.
The haplotypes for the QTL identified were manually constructed for both sweet and sour cherry
using the SNP data where linkage phase of the markers could be determined. To determine the
effect of the QTL alleles identified, Student’s t-tests were performed with all sweet cherry
individuals comparing the presence/absence of the P. canescens haplotype, and then just within
the family with confirmed P. canescens pedigree. SSR markers were also used in sour cherry to
follow the P. canescens chromosome segment when haplotypes were unable to be constructed
based on ambiguous SNP calls (Table 1.2).

Results

P. canescens-derived cherry leaf spot resistance in sweet cherry
7

Thirty four of the 74 progeny individuals were found to have F5-18-167 as a likely parent as the
SNPs indicated a high likelihood of relatedness (P < 0.05, Table 1.3) confirming that these
individuals were derived from this P. canescens-containing parent. The other 38 individuals
were found to be completely unrelated to P. canescens, as marker data showed a low likelihood
of relatedness (P > 0.05, Table 1.4). These results indicated two distinct populations, one with
P. canescens ancestry, and one without.

In sweet cherry, the frequency distribution of mean disease scores with all progeny individuals
showed a continuous distribution (Figure 1.5). In the population of 34 individuals with
confirmed P. canescens in their background, and in the populations of 38 individuals without, a
bi-modal, and a continuous distribution were observed, respectively (Figure 1.5). The continuous
distribution indicated that there may be several genes influencing disease resistance. However,
in the population derived from P. canescens, the bi-modal distribution suggested one major gene
influencing disease score (Figure 1.5). No individuals had a disease score of 1 (hypersensitive
response, green leaf) for all of the 4 years of field susceptibility analysis to CLS (Table 1.1). In
some instances the fungus was able to infect and produce conidia for secondary infection,
resulting in scores of 3 or higher which was considered susceptible. Individuals with disease
scores of less than 2 were considered to be resistant.

The Genome-wide QTL analysis showed positive evidence for one QTL on G4 (Table 1.5).
Since the Bayes factors for the number and location of the QTL was consistent in the five
replications, only that of the first is presented. Once a QTL was located on G4, all 241
polymorphic SNPs found for this linkage group were used in the FlexQTLTM analysis (Figure

8

1.6). When the QTL analysis was run using all of the polymorphic G4 markers, there was
decisive evidence that one QTL was located on the top of G4 between SNP markers
ss490552323 and ss490552500 (between 4.0-13.8 cM) (Table 1.6, Figure 1.7a). At over 0.15 at
its highest peak, the intensity of this QTL is over the 0.10 threshold, indicating this is a
significant QTL. The traceplot for this QTL is also a good indicator that this as a valid QTL
(Figure 1.7b). Two new seeds were also used to confirm the first FlexQTLTM run with the same
results (data not shown). This QTL was named CLSR_G4 for CLS resistance found on G4
(Table 1.7).

Haplotype construction and QTL allele identification

To validate this major QTL in sweet cherry, haplotypes were constructed for the QTL region. As
P. canescens was homozygous at this region and had several unique SNPs, this haplotype was
easy to construct as linkage phase between the SNPs was known (Figure 1.8). When comparing
mean disease scores for all sweet cherry individuals containing the P. canescens haplotype at the
G4 QTL region with those that did not, a significantly lower (P = 0.004) mean disease score was
found for those with this P. canescens-derived haplotype than those individuals without the P.
canescens haplotype at G4, with mean disease scores of 2.3 and 3.2 for individuals with the P.
canescens haplotype, and those without it, respectively (Table 1.8).

When considering only those 34 individuals with confirmed P. canescens lineage (See Table
1.3), those with, and without the haplotype for this region had an even larger difference, with a

9

mean disease score of 2.3 and 4.1 for those with the P. canescens haplotype, and those without it,
respectively (Table 1.9).

Not all individuals with this haplotype from P. canescens were rated as resistant to CLS (Disease
score less than 2), as five of the 15 individuals with this haplotype were susceptible (Table 1.9).
No individuals in this family without the P. canescens allele at this region were found to be
resistant however, indicating that while this region is necessary for CLS resistance, there may be
other genes involved.

P. canescens-derived cherry leaf spot resistance in sour cherry

For sour cherry, of the 15 ‘23-23-13’-derived seedlings screened, 6 had susceptible ratings for
CLS, while the other 9 had resistant ratings (Table 1.1). Both ‘UF’ and ‘Montmorency’ were
susceptible, while the P. canescens-containing parent ‘23-23-13’ was resistant (Table 1.1).

Of the 18 sour cherry individuals genotyped, haplotypes for the G4 QTL region could be
determined for all parents (‘Montmorency,’ ‘UF’ and the P. canescens-derived ‘23-23-13’) and
nine of the 15 progeny (Figure 1.9 a and b). All of the discerned haplotypes from the progeny of
the cross between the resistant parent ‘23-23-13’ and either susceptible parent ‘Montmorency’ or
‘UF’ contained the P. canescens haplotype R that is associated with disease resistance in sweet
cherry. The six individuals for which haplotypes could not be reliably determined were due to
either undetermined or ambiguous SNP calls for this region.

10

To verify P. canescens haplotypes, and determine if P. canescens is present at the G4 QTL
region in those individuals where haplotypes were unable to be constructed, four SSR markers
situated within the haplotype and spanning the QTL region between SNP markers ss490552323
(4.0 cM, 1.0 Mb) and ss490552500 (13.8 cM, 3.46 Mb) were designed and run to confirm the
presence or absence of the P. canescens chromosome (Table 1.2, Figure 1.10). All markers had
a unique band representing the P. canescens chromosome, which was present in the resistant
parent ‘23-23-13’, and in 12 of the 15 seedlings (Figure 1.11 a-d). This allowed us to essentially
“tag” the P. canescens chromosome at this region, even when haplotypes were unable to be
constructed. Due to the unique bands from P. canescens, we were confident that no crossovers
took place within the QTL region.

The three seedlings which did not contain the P. canescens alleles were all susceptible to CLS.
There were, however, three individuals which had the P. canescens G4 allele, but were also
susceptible to CLS (Figure 1.11 a-d). This, as with the case of sweet cherry, shows that the mere
presence of this region does not guarantee that the tree will be resistant, but without it, trees are
likely to be susceptible.

Discussion
One major QTL controlling CLS resistance, named CLSR_G4, was identified on G4. This is in
agreement with the phenotypic data in sweet cherry which suggested a major gene effect from P.
canescens in the population verified to have P. canescens in its background. The G4 region has
been shown to be a major contributing factor to CLS disease resistance. It appears, however,
11

that a two gene model where both parents are heterozygous for the second gene may be a better
fit (Figures 1.12 and 1.13), as five of the 15 sweet cherry individuals with the P. canescens
haplotype for the QTL region were susceptible to CLS, and three of the 12 sour cherry seedlings
with this G4 P. canescens region were found to still be susceptible (Tables 1.10 and 1.11). If
only one gene were involved, all 15 sweet cherry, and all 12 sour cherry individuals with this G4
genomic region would be resistant. In sweet cherry, one-third of the individuals with this G4
haplotype from P. canescens were still susceptible. While this is close to the one-fourth
predicted by the model, since the other parent is unknown, it is possible that some of the
seedlings do not share this same parent, perhaps a parent without this second important gene.
This would slightly skew the expected ratio. In sour cherry, two-sevenths of the progeny from
the cross ‘Montmorency’ × ‘23-23-13’ with the G4 haplotype from P. canescens are susceptible,
and one-fifth of the progeny from the cross ’UF’ × ‘23-23-13’ with this haplotype are
susceptible. Both of these numbers are close to the expected one-fourth of those carrying the
resistance haplotype from P. canescens exhibiting susceptibility that would be predicted by the
suggested model.

Since we had the marker data spanning all 8 linkage groups, a bulked segregant analysis was
done in sweet cherry comparing individuals that had the G4 P. canescens region and were either
resistant or susceptible. No discernible region was determined to be absent only in those
individuals that were susceptible, while present in the resistant individuals (data not shown).

Finding individuals within the sweet cherry background without P. canescens but still resistant is
not surprising as a study on partial resistance to CLS indicated that there were gradients of

12

infection and subsequent defoliation which was not always associated with high infection rates
(Sjulin et. al., 1989). This variation is likely caused by polygenic genes for horizontal resistance
present in cherry.

The ambiguity of SNP calls for certain regions in sour cherry is likely due to the segmental allopolyploid nature of this species (Beaver and Iezzoni, 1993; Beaver et. al., 1995). Cytological
studies on sour cherry have revealed that multivalent and univalent formations at meiosis are not
uncommon (Schuster, 2000; Schuster and Wolfram, 2005). Any surviving seedling that resulted
from an abnormal number of any chromosome would therefore give results that would be
ambiguous from the expected results of 4 copies for that region. Due to the SNP dosage
ambiguity, the use of the SSR markers aided greatly in this study to allow us to follow the P.
canescens chromosome for this region. These markers can also be used in subsequent
generations in marker assisted breeding (MAB) which would allow the breeder to discard more
seedlings at an earlier stage in development to reduce field maintenance costs, and allow for the
planting of more superior seedlings for improved chances of CLS-resistant cultivar breeding
success. Since sour cherry is more susceptible to CLS than sweet cherry, it is likely that sour
cherry has less horizontal resistance either due to lack of the genes, or due to the polyploidy
nature of sour cherry, so it is imperative that this G4 region is present if resistance is desired.

Future work in CLS resistance would be important in a larger population to allow for the location
of other QTL that contribute to disease resistance. Additional QTL may also be identified for
horizontal resistance which would help maintain the integrity of the resistance. This would be

13

especially beneficial in sour cherry, which is generally more susceptible to CLS, and therefore is
likely to carry fewer horizontal resistance genes.

14

Table 1.1: Plant materials used in this study and cherry leaf spot disease evaluation scores for
2008 to 2011.

Plants
704010-003
704010-004
704010-008
704010-009
704010-010
704010-015
704010-019
704010-022
704010-025
704010-028
704010-029
704010-034
704010-037
704010-050
704010-057
704010-061
704010-062
704010-066
704010-072
704010-074
704010-078
704010-079
704010-083
704010-084
704010-085
704010-086
704010-087
704010-093
704010-099
704010-125
705012-005
705012-020
705012-025
704010-005
704010-007
704010-012

Parent 1
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
F5-18-167
Namati
unknown
Namati

Parent 2
op
op
op
op
op
op
op
op
op
op
op
op
op
op
op
op
op
op
op
op
op
op
op
op
op
op
op
op
op
op
op
op
op
op
op

Species
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet

15

Disease Score
2008 2009 2010 2011 Mean
5
5
4
4
4.5
2
1
2
2
1.8
4
2
4
4
3.5
3
5
4
4
4.0
4
5
4
4
4.3
2
2
2
2
2.0
4
4
4
4
4.0
3
4
4
4
3.8
2
1
1
1
1.3
4
5
5
5
4.8
4
4
2
4
3.5
4
5
5
4
4.5
4
5
5
5
4.8
5
5
4
4.7
5
5
5
4
4.8
2
2
2
2
2.0
2
2
2
2
2.0
2
1
2
2
1.8
2
2
2
2
2.0
4
3
3.5
4
5
5
5
4.8
4
3
2
4
3.3
2
3
5
3.3
4
5
2
4
3.8
2
1
1
2
1.5
3
1
2
4
2.5
2
2
2
2
2.0
2
1
2
2
1.8
4
5
4.5
5
5
5
4
4.8
5
5
5
4
4.8
4
4
2
4
3.5
5
5
5
5
5.0
3
2
2
3
2.5
4
3
4
4
3.8
4
2
2
2
2.5

Table 1.1 (cont’d)

Plants
704010-013
704010-014
704010-016
704010-017
704010-020
704010-024
704010-026
704010-030
704010-032
704010-033
704010-035
704010-036
704010-039
704010-040
704010-043
704010-044
704010-045
704010-047
704010-051
704010-053
704010-054
704010-060
704010-063
704010-064
704010-068
704010-069
704010-071
704010-077
704010-080
704010-081
704010-082
704010-091
704010-092
704010-097
704010-110
705012-002

Parent 1
Namati
Namati
unknown
Namati
unknown
Namati
unknown
unknown
unknown
Namati
unknown
unknown
Namati
Namati
unknown
unknown
Namati
unknown
Namati
Namati
unknown
Namati
Namati
Namati
Namati
unknown
unknown
Namati
unknown
Namati
unknown
Namati
unknown
unknown
Namati
Namati

Parent 2
op
op
op
op
op
op
op
op
op
op
op
op
op
op
op
op
op
op
op

Species
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet
Sweet

16

Disease Score
2008 2009 2010 2011 Mean
4
4
4
4
4.0
2
1
2
2
1.8
4
5
4
4
4.3
4
3
2
3
3.0
2
1
2
3
2.0
3
1
2
2
2.0
3
2
2
4
2.8
3
2
1
2
2.0
3
1
3
2
2.3
3
2
2
3
2.5
3
2
2
4
2.8
4
2
3
4
3.3
5
5
5
4
4.8
4
2
2
4
3.0
2
2
2
2
2.0
2
2
2
3
2.3
2
1
2
2
1.8
4
1
3
4
3.0
4
4
4
4
4.0
2
2
2
2
2.0
3
2
2
3
2.5
3
2
2
2
2.3
4
2
2
3
2.8
4
4
2
3
3.3
2
2
2
3
2.3
3
4
4
4
3.8
2
2
2
4
2.5
2
1
2
2
1.8
4
3
5
4
4.0
3
1
2
3
2.3
4
3
3
4
3.5
3
2
2
2
2.3
2
2
2
3
2.3
3
2
2
3
2.5
5
4
5
4
4.5
4
2
2
2
2.5

Table 1.1 (cont’d)

Plants
F5-18-167
Namati
GerP-can
P.canescens
148-1
23-23-13
24-32-17
24-32-18
24-32-20
24-32-21
24-32-23

Parent 1
M30
-

Parent 2
a
GerP-can
-

Sweet
Sweet
P.canescens
P.canescens

1

1

2

2

1.5

2
-

2
-

3
-

2.3
-

P.canescens
148-1
Montmorency
Montmorency
Montmorency
Montmorency

RS
op
23-23-13
23-23-13
23-23-13
23-23-13

Sour
Sour
Sour
Sour
Sour
Sour

-

-

2

-

-

2
2
2
2
2

23-23-13
23-23-13
23-23-13
23-23-13
23-23-13
23-23-13
23-23-13
23-23-13
23-23-13
23-23-13
23-23-13
-

Sour
Sour
Sour
Sour
Sour
Sour
Sour
Sour
Sour
Sour
Sour
Sour
Sour

-

-

3

-

-

5
3
5
2
2
3
5
2
2
4
-

2
b
R
R
R
R
R
R
c
S
S
S
S
R
R
S
S
R
R
S
S
S

Montmorency
24-32-24
Montmorency
24-32-25
Montmorency
24-32-26
Montmorency
24-32-27
Montmorency
24-32-37
Balaton
24-32-39
Balaton
24-32-40
Balaton
24-32-41
Balaton
24-32-43
Balaton
24-32-44
Balaton
Balaton
Montmorency
a
Prunus canescens from Germany
b
Resistant
c
Susceptible

Species

Disease Score
2008 2009 2010 2011 Mean

17

R
R
R
R
R
R
S
S
S
S
R
R
S
S
R
R
S
S
S

Table 1.2: SSR markers designed and used to validate the presence of P. canescens in the G4 disease resistant region in sour cherry.
Marker
Name

Sequence (5’ to 3')

Ta

P. canescens fragment size
(bp)

Scaffold 4 location
a
(bp)
1414429-1414449
1414644-1414663

CLS004-F
CLS004-R

TGGGCCAGTATTTTACAGGAG
TTGGCTGGTCTCTCACAAAA

54

230

CLS005-F
CLS005-R

AATTGTGCGGGAGCTACAAG
GCCATCATCAGGTAGCAATG

54

232

1359841-1359860
1359616-1359635

CLS026-F
CLS026-R

AGCCCAACGTCTCATTCACC
GGAGATGAAGCAAAAGAGATGC

b
Touchdown

180

3456175-3456194
3456412-3456391

CLS028-F
3334891-3334911
GAATGCAGTTGGGGAGTTACC
Touchdown
168
CLS028-R
3335078-3335058
CTTCTTGCACCAAAAACAACC
a
Distances according to the Peach v1.0 'dhLovell' genome assembly (International Peach Genome Initiative;
www.rosaceae.org/peach/genome (Verde et al. 2013)
b
Ta of 60°C for 45 seconds, with an extension at 72°C for 60 seconds. For the next 9 cycles, the Ta drops 1°C per cycle,
then for the last 24 cycles, Ta remains at 55°C for 45 seconds with the same extension time.

18

Table 1.3: Pedigree confirmation of 34 individuals found to have P. canescens in their pedigree
via parent F5-18-167. P-values are calculated using the program KINGROUP (Konovalov et.
al., 2004) testing a null hypothesis that no parent offspring relation between listed pair in rows vs
columns.
704010-003
704010-004
704010-008
704010-009
704010-010
704010-015
704010-019
704010-022
704010-025
704010-028
704010-029
704010-034
704010-037
704010-050
704010-061
704010-062
704010-066
704010-069
704010-071
704010-072
704010-074
704010-078
704010-079
704010-083
704010-084
704010-085
704010-086
704010-087
704010-093
704010-099
704010-125
705012-005
705012-020
705012-025
Namati
GerP-can

F5-18-167
0.00
0.00
0.00
0.00
0.00
0.00
0.05
0.00
0.00
0.00
0.00
0.05
0.05
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.05
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.03
1.00
0.00

P.canescens
0.18
0.00
0.05
0.02
0.06
0.17
0.28
0.00
0.07
0.05
0.00
0.16
0.09
0.00
0.04
0.01
0.00
0.01
0.02
0.02
0.05
0.17
0.02
0.09
0.07
0.02
0.17
0.03
0.17
0.08
0.13
0.00
0.02
0.09
1.00
0.00

19

Table 1.4: Pedigree confirmation of 38 progeny individuals found to not have P. canescens in
their pedigree. P-values are calculated using the program KINGROUP (Konovalov et. al., 2004)
testing a null hypothesis that no parent offspring relation between listed pair in rows vs. columns.
704010-005
704010-007
704010-012
704010-013
704010-014
704010-016
704010-017
704010-020
704010-024
704010-026
704010-030
704010-032
704010-033
704010-035
704010-036
704010-039
704010-040
704010-043
704010-044
704010-045
704010-047
704010-051
704010-053
704010-054
704010-057
704010-060
704010-063
704010-064
704010-068
704010-077
704010-080
704010-081
704010-082
704010-091
704010-092

P. canescens
1.00
1.00
1.00
0.99
0.99
1.00
0.99
1.00
1.00
0.84
0.99
0.96
1.00
1.00
1.00
1.00
0.99
0.93
0.99
1.00
0.98
0.99
1.00
1.00
0.99
0.99
1.00
1.00
1.00
1.00
1.00
0.99
1.00
1.00
1.00

20

Table 1.4 (cont’d)
704010-097
704010-110
705012-002
F5-18-167
Namati
GerP-can

P. canescens
1.00
1.00
0.90
0.00
1.00
0.00

21

a

Table 1.5: Estimates of 2ln Bayes factors for the first replicate of the genome-wide analysis for
mean disease resistance identified using the sweet cherry plant material listed in Table 1.1. The
number of QTLs being compared in the models are separated by a back slash (“/”).
Group
1/0
2/1
3/2
4/3
5/4
1
0.2
0.0
-0.0
NA
NA
2
0.0
0.0
NA
NA
NA
3
-0.1
-0.0
NA
NA
NA
4
4.5
0.2
0.0
NA
NA
5
-0.0
-0.0
NA
NA
NA
6
-0.1
-0.0
NA
NA
NA
7
0.5
0.0
0.0
NA
NA
8
-0.1
-0.0
NA
NA
NA
a
2ln Bayes factors for the comparison of two models.
Interpretation of the pairwise model comparison 2lnBF range
and evidence: 0-2 hardly any, 2-5 positive, 5-10 strong, > 10
decisive, NA not available due to insufficient MCMC draws
from one of the two models.

a

Table 1.6: Estimates 2ln Bayes factors for the first replicate of the G4 analysis for mean disease
resistance identified using the sweet cherry plant material listed in Table 1.1. The number of
QTLs being compared in the models are separated by a back slash (“/”).
Group
1/0
2/1
3/2
4/3
5/4
4
10.9
0.7
0.2
NA
NA
a
2ln Bayes factors for the comparison of two models.
Interpretation of the pairwise model comparison 2lnBF range
and evidence: 0-2 hardly any, 2-5 positive, 5-10 strong, > 10
decisive, NA not available due to insufficient MCMC draws
from one of the two models.

Table 1.7: Marker interval and name for the CLS resistance QTL found on G4.
Group

QTL name

Interval (cM)

Peak
position
(cM)

4

CLSR_G4

4.0-13.8

7.0

Physical map
a
interval (Mb)

ss490552323ss490552500

a

Marker interval

1.00-3.46

Mb distances according to the Peach v1.0 ‘dhLovell’ genome assembly (International Peach
Genome Initiative; www.rosaceae.org/peach/genome) Verde et al. 2013

22

Table 1.8: Mean cherry leaf spot scores for the presence, or absence of the G4 P. canescens
haplotype. All sweet cherry progeny individuals are included, those with, and those without P.
canescens in their background (See Table 1.1). The means are significantly different (P = 0.004)
as denoted by different letters in the disease score mean column.

G4 P. canescens
Haplotype

Number of
individuals

Disease
Score Range

Yes

16

1.3-4.5

Disease Score
Mean
a
2.3 A

No

58

1.6-5.0

3.2 B

a

The means are significantly different (P = 0.004) as denoted by different letters in the disease
score mean column.

Table 1.9: Mean cherry leaf spot score for the presence or absence of the G4 P. canescens
haplotype. Only individuals confirmed to have P. canescens in their background (See Table 1.3)
are included. The means are significantly different (P < 0.0001) as denoted by different letters in
the disease score mean column.

G4 P. canescens
Haplotype

Number of
individuals

Disease Score
Range

Yes

16

1.3-4.5

Disease Score
Mean
a
2.3 A

No

18

2.5-5.0

4.1 B

a

The means are significantly different (P < 0.0001) as denoted by different letters in the disease
score mean column.

23

Table 1.10: Disease scores and the presence/absence of the G4 region from P. canescens in
sweet cherry progeny with confirmed P. canescens background (See Table 1.3). Individuals in
bold and underlined contain the P. canescens haplotype, but are susceptible.
Genotype
704010-003
704010-004
704010-008
704010-009
704010-010
704010-015
704010-019
704010-022
704010-025
704010-028
704010-029
704010-034
704010-037
704010-050
704010-061
704010-062
704010-066
704010-069
704010-071
704010-072
704010-074
704010-078
704010-079
704010-083
704010-084
704010-085
704010-086
704010-087
704010-093
704010-099
704010-125
705012-005
705012-020
705012-025

Mean disease score
4.5
1.8
3.5
4.0
4.3
2.0
4.0
3.8
1.3
4.8
3.5
4.5
4.8
4.7
2.0
2.0
1.8
3.8
2.5
2.0
3.5
4.8
3.3
3.3
3.8
1.5
2.5
2.0
1.8
4.5
4.8
4.8
3.5
5.0

G4 P. canescens haplotype
No
Yes
Yes
Yes
No
Yes
No
No
Yes
No
No
Yes
No
No
Yes
Yes
Yes
No
Yes
Yes
No
No
No
Yes
No
Yes
No
Yes
Yes
No
No
No
No
No

24

Table 1.11: Cherry leaf spot disease scores and the presence/absence of the G4 region from P.
canescens of sour cherry progeny (See Table 1.1). Individuals in bold and underlined contain the
P. canescens haplotype, but are disease susceptible.
Genotype
24-32-17
24-32-18
24-32-20
24-32-21
34-32-23
24-32-24
24-32-25
24-32-26
24-32-27
24-32-37
24-32-39
24-32-40
24-32-41
24-32-43
24-32-44
148-1
23-23-13

Disease rating
Resistant
Resistant
Resistant
Resistant
Susceptible
Susceptible
Susceptible
Susceptible
Resistant
Resistant
Susceptible
Susceptible
Resistant
Resistant
Resistant
Resistant
Resistant

G4 P. canescens region
Yes
Yes
Yes
Yes
No
Yes
No
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes

25

Figure 1.1: Pedigree of the incorporation of CLS resistant P. canescens into the sour cherry
background. Individual which are resistant are colored black, susceptible individuals are colored
white, and families which are segregating for resistance are grey.

RS

P.canescens

148-1

O.P.

23-23-13

Ujfehertoi_Furtos

UF_x_23-23-13

Montmorency

M_x_23-23-13

N=6

N=9

26

Figure 1.2: Images of cherry leaf spot disease ratings of 1 (a), 2 (b), 3 (c), 4 (d) and 5 (e) on the
disease scale used for sweet cherry and P. canescens derived diploid individuals. For
interpretation of the references to color in this and all other figures, the reader is referred to the
electronic version of this dissertation.

a
.

b
.

d
.

c
.

27

e
.

Figure 1.3: Linkage map used for QTL analysis. Large linkage groups were divided into
multiple sections (denoted by [ ]) to fit on the page. Marker cM distances were approximated by
multiplying marker peach physical map location in Mb by four. A total of 548 markers spanning
the 8 linkage groups were used. Markers are a part of the NCBI’s dbSNP repository (Sherry et
al. 2001).

28

Figure 1.3 (cont’d)

29

Figure 1.3 (cont’d)

30

Figure 1.3 (cont’d)

31

Figure 1.3 (cont’d)

32

Figure 1.3 (cont’d)

33

Figure 1.3 (cont’d)

34

Figure 1.3 (cont’d)

35

Figure 1.4: Genome Studio (Illumina Inc. 2011) SNP dosage calls were done for each marker. Sweet cherry (yellow) individuals
were included to help define the two homozygous (AAAA and BBBB) classes and the balanced heterozygous class (AABB).
Determining dosage was necessary to build haplotypes.

36

Figure 1.5: Frequency distribution of mean cherry leaf spot disease scores for all sweet cherry
individuals (See Table 1.1). The mean disease score for the P. canescens-containing parent ‘F518-167’ and ‘Namati’ were 1.3 and 2.0, respectively.

37

Figure 1.6: Expanded G4 linkage map used for QTL analysis. The G4 linkage group is broken
up into multiple sections (denoted by [ ]) to fit on the page. Marker cM distances were
approximated by multiplying marker peach physical map location in Mb by four. A total of 241
markers spanning G4 were used. Markers are a part of the NCBI’s dbSNP repository (Sherry et
al. 2001).

38

Figure 1.6 (cont’d)

39

Figure 1.6 (cont’d)

40

Figure 1.6 (cont’d)

41

Figure 1.6 (cont’d)

42

Figure 1.7: a) Disease resistance QTL location on G4 between SNP markers ss490552323 and
ss490552500 (between 4.0-13.8 cM). b). Trace plot for the QTL region.

a.
43

Figure 1.7 (cont’d)

b.

44

Figure 1.8: Haplotype R is associated with P. canescens across the QTL region.

NCBI SS#

ss490552380
ss490552383
ss490552385
ss490552388
ss490548390
ss490552400
ss490552403
ss490552406
ss490548393
ss490548397
ss490552415
ss490552418
ss490552423
ss490552426
ss490548409
ss490552429
ss490548413
ss490552440
ss490552443
ss490552446
ss490548417
ss490552457
ss490559087
ss490552460
ss490552463
ss490548433
ss490552466
ss490552469
ss490548437
ss490552474
ss490552477
ss490548445

P. canescens
R R
RB_S_4_01793880 A A
RB_S_4_01831038 A A
RB_S_4_01871597 B B
RB_S_4_01910142 B B
RB_T_4_02018251 A A
RB_S_4_02071162 A A
RB_S_4_02108244 B B
RB_S_4_02151854 A A
RB_T_4_02170096 B B
RB_T_4_02258698 A A
RB_S_4_02273965 A A
RB_S_4_02314853 B B
RB_S_4_02394394 A A
RB_S_4_02427939 A A
RB_T_4_02451708 A A
RB_S_4_02472320 A A
RB_T_4_02604396 B B
RB_S_4_02633721 A A
RB_S_4_02656936 B B
RB_S_4_02682092 B B
RB_T_4_02689311 A A
RB_S_4_02821081 A A
RC1422-162_4_02841085 A A
RB_S_4_02872061 A A
RB_S_4_02901893 B B
RB_T_4_02960827 A A
RB_S_4_02961208 B B
RB_S_4_02991481 B B
RB_T_4_03068652 B B
RB_S_4_03072387 B B
RB_S_4_03146679 B B
RB_T_4_03188480 B B
45

Figure 1.9a: Sour cherry haplotypes for the G4 QTL region. Parents ‘23-23-13’ and ‘Ujfehertoi Furtos’ with 5 progeny. Haplotype R
is from P. canescens.
UF
23-23-13
24-32-37
24-32-39
24-32-41
24-32-43
24-32-44
NCBI SS#
w x R z
a b c d
b c x R
b d x R
a d w R
a d w R
b c w R
ss490552385 A A B B
A A B B
A B A B
A B A B
A B A B
A B A B
A B A B
ss490548390 A A A B
A A A B
A A A A
A B A A
A B A A
A B A A
A A A A
ss490552400 B B A A
B B A A
B A B A
B A B A
B A B A
B A B A
B A B A
ss490552406 A B A B
B B B A
B B B A
B A B A
B A A A
B A A A
B B A A
ss490548393 A B B A
A B B B
B B B B
B B B B
A B A B
A B A B
B B A B
ss490548397 B B A B
B B B A
B B B A
B A B A
B A B A
B A B A
B B B A
ss490552426 A A A B
A A A B
A A A A
A B A A
A B A A
A B A A
A A A A
ss490548409 B B A A
B B A A
B A B A
B A B A
B A B A
B A B A
B A B A
ss490548413 A A B B
A A B B
A B A B
A B A B
A B A B
A B A B
A B A B
ss490552446 B A B B
A A B B
A B A B
A B A B
A B B B
A B B B
A B B B
ss490548417 A B A A
B B A A
B A B A
B A B A
B A A A
B A A A
B A A A
ss490559087 B A A A
A A A A
A A A A
A A A A
A A B A
A A B A
A A B A
ss490548433 B B A A
B B A A
B A B A
B A B A
B A B A
B A B A
B A B A
ss490552474 A A B B
A A B B
A B A B
A B A B
A B A B
A B A B
A B A B
ss490548445 B B B A
B B A A
B A B B
B A B B
B A B B
B A B B
B A B B

46

Figure 1.9b: Parents ‘Montmorency’ and ‘23-23-13’ with 4 progeny individuals that were haplotyped. Black regions indicate where a
crossover took place, but due to identical SNP markers in that area, the exact location of the crossover is unknown. Haplotype R is
from P. canescens.
23-23-13
Montmorency
24-32-17
24-32-20
24-32-24
24-32-27
NCBI SS#
w x R z
e f c d
f c x R
c d x R
e c x R
f d w/x R
ss490552385 A A B B
A A B B
A B A B
B B A B
A B A B
A B A B
ss490548390 A A A B
A A A B
A A A A
A B A A
A A A A
A B A A
ss490552400 B B A A
B B A A
B A B A
A A B A
B A B A
B A B A
ss490552406 A B A B
A B B A
B B B A
B A B A
A B B A
B A A A
ss490548393 A B B A
A B B B
B B B B
B B B B
A B B B
B B A B
ss490548397 B B A B
B B B A
B B B A
B A B A
B B B A
B A B A
ss490552426 A A A B
A A A B
A A A A
A B A A
A A A A
A B A A
ss490548409 B B A A
B B A A
B A B A
A A B A
B A B A
B A B A
ss490548413 A A B B
A A B B
A B A B
B B A B
A B A B
A B A B
ss490552446 B A B B
B A B B
A B A B
B B A B
B B A B
A B A B
ss490548417 A B A A
A B A A
B A B A
A A B A
A A B A
B A B A
ss490559087 B A A A
B A A A
A A A A
A A A A
B A A A
A A A A
ss490548433 B B A A
B B A A
B A B A
A A B A
B A B A
B A B A
ss490552474 A A B B
A A B B
A B A B
B B A B
A B A B
A B A B
ss490548445 B B B A
B B A A
B A B B
A A B B
B A B B
B A B B

47

Figure 1.10: Location of SSR markers used to tag the P. canescens haplotype in relation to the
CLSR_G4 QTL region of interest between SNP markers ss490552323 (4.0 cM, 1.0 Mb) and
ss490552500 (13.8 cM, 3.46 Mb).

CLSR_G4

CLS005

CLS004 CLS028

CLS026

48

Figure 1.11: SSR fragments for sour cherry individuals (See Table 1.1) for four primer pairs in the G4 cherry leaf spot QTL region: a)
CLS004 and b) CLS005 c) CLS026 d) CLS028 (See Table 1.2) run on sour cherry individuals for the G4 QTL region. Arrows denote
the location and fragment size of the P. canescens allele.

a.

b.

49

Figure 1.11 (cont’d)

c.

d.

50

Figure 1.12: Proposed two gene model for disease resistance in sweet cherry, where the
individuals are only resistant with dominant genes at both the P. canescens-associated G4 “A”,
and the unknown “B” second loci. Disease resistant parent ‘F5-18-167’ is shown to be
heterozygous at both loci (AaBb) where parent 2 is shown to be homozygous recessive for the
G4 “A” locus, and heterozygous for the proposed second locus needed to warrant resistance.
Squares highlighted in grey are those that would be resistant.

F5-18-167 (AaBb)
AB

Parent 2 (aaBb)

Ab

aB

ab

aB

AaBB AaBb aaBB

aaBb

ab

AaBb Aabb aaBb

aabb

51

Figure 1.13: Proposed two gene model for disease resistance in sour cherry, where the individuals are only resistant with dominant
genes at both the G4 “A”, and the unknown “B” second loci. This model assumes preferential pairing within sub-genomes, where
sum-genomes are denoted by subscript numbers. Disease resistant parent ‘23-23-13’ is shown to be heterozygous at both loci in only
one sub-genome (A1a1a2a2B1b1b2b2) where both ‘UF’ and ‘Montmorency” are shown to be homozygous recessive for the G4 “A”
locus, and heterozygous in one sub-genome for the proposed second locus needed to warrant resistance (a1a1a2a2B1b1b2b2). Squares
highlighted in grey are those that would be resistant.

23-23-13 (A1a1a2a2B1b1b2b2)
A1a2B1b2

A1a2b1b2

a1a2B1b2

a1a2b1b2

UF & Montmorency

a1a2B1b2 A1a1a2a2B1B1b2b2 A1a1a2a2B1b1b2b2 a1a1a2a2B1B1b2b2 a1a1a2a2B1b1b2b2

(a1a1a2a2B1b1b2b2)

a1a2b1b2 A1a1a2a2B1b1b2b2 A1a1a2a2b1b1b2b2 a1a1a2a2B1b1b2b2 a1a1a2a2b1b1b2b2

52

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53

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Rosyara UR, Bink MCAM, van de Weg E, Zhang G, Wang D, Sebolt A, Dirlewanger E, QueroGarcía J, Schuster M, Iezzoni AF. (2013) Fruit size QTL identification and the prediction
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sweet cherry. Mol. Breeding doi 10.1007/s11032-013-9916-y
Schmidt, H. and Gruppe, W. 1988. Breeding dwarfing rootstocks for sweet cherry. HortScience
23(1):112-114
Schuster, M. 2000. Genome investigation of sour cherry, Prunus cerasus L. Acta Hort. 538:375379
Schuster, M. and Tobutt, K.R. 2004. Screening of cherries for resistance to Leaf Spot,
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the NCBI database of genetic variation. Mucleic Acids Res. 29(1):308-311
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spot in cultivars of sweet, sour, duke, and European ground cherry. Plant Dis. 73:56-61
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Ficklin S, Goodstein DM, Xuan P, Del Fabbro C, Aramini V, Copetti D, Gonzalez S,
Horner DS, Ralchi R, Lucas S, Mica E, Maldonado J, Lazzari B, Bielenberg D, Pirona R,
Miculan M, Barakat A, Testolin R, Stella A, Tartarini S, Tonutti P, Arús P, Orellana A,
Wells C, Main D, Vizzotto G, Silva H, Salamini F, Schmutz J, Morgante M, Rokhsar DS
(2013) The high-quality draft of peach (Prunus persica) identifies unique patterns of
genetic diversity, domestication and genome evolution. Nature Genetics 45:487-494
doi:10.1038/ng.2586
Wharton, P.S., Iezzoni, A., and Jones, A.L. 2003. Screening cherry germplasm for resistance to
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for resistance to Cherry Leaf Spot. Acta Hort. 667:509-514

55

CHAPTER 2

VALIDATION IN TETRAPLOID SOUR CHERRY OF QTLS FOR BLOOM
TIME AND FRUIT QUALITY TRAITS FROM DIPLOID Prunus
SPECIES

56

Introduction
Sour cherry (Prunus cerasus L.) is an allotetraploid (2n=4x=32) derived from the hybridization
of diploid (2n=2x=16) sweet cherry (P. avium L.) and tetraploid (2n=4x=32) ground cherry (P.
fruticosa Pall.) (Olden and Nybom, 1968; Hancock and Iezzoni, 1988). A high degree of
synteny between the two sub-genomes is expected, as Prunus species have been found to be
highly syntenic (Dirlewanger et al. 2004; Lambert et al. 2004; Arús et al. 2006; Dondini et al.
2007; Olmstead et al. 2008). This indeed, has proven to be the case, as inheritance of isozymes in
various crosses of sour cherry also indicated that sour cherry behaves as a segmental
allopolyploid (Beaver and Iezzoni, 1993; Beaver et al. 1995) meaning that while there seems to
be a preferential pairing of chromosomes within sub-genomes of sour cherry, crossovers, and
non-bivalent pairing between sub-genomes is not uncommon (Schuster and Wolfram, 2005). A
genetic example of unbalanced chromosomes can be seen when looking at the selfincompatability locus (S-locus) on G6 where sour cherry individuals such as ‘Rheinische
Schattenmorelle’ (RS) has three sweet cherry S-alleles, and one P. fruticosa allele (Hauck et al.
2006). The difficulty in constructing genetic linkage maps of segmental polyploids occurs
because marker segregation ratios need to be single-dose restriction fragments (SDRF) or
double-dose restriction fragments (DDRF) in order to map (Wu et al. 2002). Linkage mapping
in sour cherry showed several markers segregating in ratios other than SDRF or DDRF, which
could not be mapped (Wang et al. 1998). This makes genetic studies of sour cherry difficult as
alleles are frequently not sub-genome specific and the sub-genomes can be unbalanced.
Due to the mixed mode of inheritance hindering linkage map construction, the understanding of
the genetic control of trait variation in sour cherry has lagged behind that of other Prunus
57

species, in particular peach and sweet cherry. Key progress in diploid Prunus species has been
made in recent years.

Delayed bloom time is a key goal of sour cherry breeding, as the development of new cultivars
with later bloom would reduce the chance of pistil damage caused by spring freeze events
(Iezzoni 1996). The diversity of bloom times in the sour cherry background is significant
(Iezzoni and Mulinix, 1992), probably owing to the P. fruticosa background. Within Prunus, a
number of QTLs have been identified in several different species including almond (Ballester et
al. 2001; Silva et al. 2005), peach (Dirlewanger et al. 2012), apricot (Ruiz et al. 2010;
Dirlewanger et al. 2012) and sweet cherry (Dirlewanger et al. 2012) making bloom date QTLs a
perfect candidate to search for within the sour cherry background.

Knowledge of the genetic control of fruit size is also a major goal in cherry breeding. This,
fortunately, has seen some progress in the last year with the discovery that previously found fruit
and pit size QTL in sweet cherry (Zhang et al. 2010) are likely due to cell number regulator
(CNR) genes which have been found by dissecting the peach genome (De Franceschi et al.
2013). There are, however, several other CNR genes which have yet to be explored as to their
possible role in contributing to fruit size. Due to the 3-5 year juvenility period of cherry,
seedlings could be screened before planting to determine if they are likely to have adequate fruit
size if this important trait was even better understood. It would also allow for the more efficient
incorporation of small-fruited wild germplasm for traits such as disease resistance, and allow for
the selection for large fruit alleles to minimize the number of generations needed to recover
commercial sized fruit.

58

Fruit flesh color is mainly an industry driven trait for sour cherries in the US, where much of
their production is geared toward the industry standard amorello (clear-fleshed) variety
‘Montmorency.’ This is another area that is not well understood genetically in sour cherries, but
work in apple has found MYB10 as a causal gene for red apple color (Espley et al. 2007) and
sweet cherry has given us a clear place to start looking through work on QTL mapping of skin
and flesh color (Sooriyapathirana et al. 2010). If the industry keeps pushing for amorello
varieties, then a better understanding of fruit color will be needed to aid in selection for these
types. This is especially important, as colorless flesh and skin color are recessive in sweet cherry
(Fogle 1958; Hedtrich 1985; Schmidt 1998), indicating that it will likely be recessive in sour
cherry. Much of the sour cherry germplasm is morello (colored flesh), making the understanding
of this trait especially important as individuals with multiple dark flesh alleles would be unlikely
to give rise to the desired lighter fleshed progeny.

Like flesh color, fruit firmness is also an important trait for the industry. Blemishes from harvest
can cause juice loss, and therefore a reduction in harvest weight reducing the profit growers can
achieve. Softer, juicier fruit are also preferred by birds, causing an increase in predation to softer
cultivars. Utilization of wild germplasm for traits such as disease resistance are also likely to
bring with it the softer fruit trait, which warrants an understanding of fruit firmness to allow
breeders to bring fruit back to commercial standards. Preliminary data in sweet cherry has
indicated some co-localization of fruit weight and fruit firmness QTL, and a negative correlation
between these two traits (Quero-García et al. 2010).

59

The D-locus on the top of G5 in peach has been found to be associated with a variation of 83%
for malic acid content (Etienne et al. 2002; Boudehri et al. 2009). While the amount of malic
acid content is not a huge concern in breeding programs, if the large effect of the D-locus in
peach were the same in sour cherry, it would be a trait which could be easily controlled if it was
warranted in the future.

Because of the complexity of the chromosome pairing, and due to the high degree of synteny in
Prunus, our strategy to advance our understanding of sour cherry genetics is to test whether
QTLs for bloom and fruit quality traits previously identified in sweet cherry or other Prunus
species control trait variation in sour cherry and to determine if functional allelic variants which
exist in sour cherry could then be used in marker-assisted breeding (MAB). With the availability
of the cherry 6K Infinium® II SNP array as part of the RosBREED project (www.rosbreed.org)
(Peace et al. 2012), many markers can now be screened and dosage can be determined which
then allows us to determine the phase of closely linked markers and to follow each of the four
individual chromosome segments through inheritance data. This must be done since a
comprehensive linkage map is not available in sour cherry. We are also relying on using
multiple populations that represent multiple founders and a wide range of the diversity present
within breeding populations instead of focusing our attention on just one F1 population for
validation.

Materials and Methods

60

Plant material and phenotyping
A total of 338 cultivars and seedlings from 5 bi-parental populations including parents were used
in this study (Figure 2.1). Populations were as follows: ‘Újfehértói Fürtös’ (‘UF’) בSurefire’
(n=76), ‘M172’ × ‘25-02-29’ (n=111), ‘25-14-20’ × ‘25-02-29’ (n=67), ‘Montmorency’× ‘2502-29’ (53) and ‘Rheinische Schattenmorelle’ (‘RS’) × ‘Englaise Timpurii’ (‘ET’) (n=23).
These individuals are planted at the Michigan State University Clarksville Research Station,
Clarksville, Michigan.

Bloom time was taken in 2011 and 2012 and determined when 50% of the flowers were opened
on a tree. The day of blooming was converted to Growing Degree Days (GDD) with a base of
4.4 C, as done in Wang et al. (2000), with temperature data collected from Michigan State
University’s Weather Station “Enviro-weather” resource (www.agweather.geo.msu.edu).

Harvest of fruit was done twice on each tree to better determine maturity. Each harvest consisted
of the collection of 30 fruit (of which the best 25 went on to be evaluated), with an additional
collection of around 20 fruit on the second harvest to be frozen and later processed for malic acid
content.

2

Fruit firmness (g/mm ) was measured in 2011 as an average of 25 fruit per harvest using the
compression test of BioWorks’ FirmTech 2 (Wamego, KS). Compression was done from cheek
to cheek (perpendicular to the suture) when fruit were at room temperature.

61

Color rating for each individual was taken in 2011 and was defined according to the Sweet
Cherry Flesh Color Index from Washington State University (WSU) and The Flower Council of
Holland, Leiden, The Royal Horticultural Society, London. A visual rating was given from 1-5
representing clear to deeply pigmented color respectively (Figure 2.2).

Fruit size was measured as fruit weight, pit weight and mesocarp weight (mean fruit weight –
mean pit weight) in 2011. Fruit and pit weights were taken as the average of the 5 largest fruit
during the harvest to capture the maximum genetic potential.

Malic acid content (mg/ml) was evaluated in 2011 and was calculated using an automatic titrator,
coupled to an auto-sampler and control unit (Titroline 96, Schott, Germany). Fruit was collected
during the second harvest and frozen, then thawed before being strained through a Kim wipe.
Juice (10ml) was then placed in 100 ml of water and titrated to a pH of 8.2 using 0.1 N NaOH.

Genotying and Haplotype construction

Four hundred and two sour cherry individuals, including founders, seedlings, and all 338
individuals in the five bi-parental populations were genotyped using the 6K Infinium® II SNP
array as part of the RosBREED project (www.rosbreed.org) (Peace et al. 2012). The Illumina®
Genome Studio software was used to determine the SNP genotype. Available SNP data from a
diverse array of sweet cherry selections and seedlings (105 individuals) were included to aid in
the determination of dosage by showing the two homozygous (AAAA and BBBB for sour cherry

62

corresponding to AA and BB in sweet cherry) and balanced heterozygous (AABB for sour
cherry corresponding to AB in sweet cherry) classes (Figure 2.3).

SNP markers which were polymorphic but un-resolved were not used. For each parent and
progeny individual, four haplotypes (to represent the four chromosomes in a tetraploid) were
built for the target regions of the genome in an Excel spreadsheet by hand based on progeny
inheritance and segregation in each of the bi-parental populations. This is in contrast to just two
haplotypes which are built for each parent in a diploid species, which can be seen when
comparing the S-locus region between sweet cherry parents ‘NY54’ and ‘Emperor Francis’ and
sour cherry parents ‘Újfehértói Fürtös’ and ‘Surefire’ (Figure 2.4 a and b). When more than
three ambiguous SNP dosage calls were made in a region for an individual, haplotypes were not
built for that individual at that region. Multiple ambiguous SNP dosage calls for a region are
likely due to too few or too many chromosomes at that region, or poor quality DNA.

When a region of interest exhibited a large number of haplotypes, haplotypes were condensed
either through comparison of SNP calls in a smaller region surrounding the region of interest, or
through the use of SSR markers in close proximity with candidate genes/QTL. SSR markers
with “null” alleles, or alleles that were not represented with a band, were not analyzed.

Statistical analysis

ANOVA calculations to determine if the target genomic regions were significantly associated
with traits were done using a linear additive model test with a user defined design matrix to

63

consider each haplotype and scores for the presence or absence of those haplotypes as well as the
number of times each haplotype is present (to account for dosage), using a modified R-script
(version 2.15.1). When a crossover took place within the region of interest, the haplotype was
represented as missing data since it was unclear which haplotype would be contributing to the
trait.

To confirm the ANOVA calculations, and determine if allele haplotype had a positive, or
negative effect on the trait, Student’s t-tests were performed comparing groups with, and those
without each of the haplotypes that were significant in the linear additive model. Individuals
with crossovers between two haplotypes were left out of t-tests comparing the presence or
absence of haplotypes only if the crossover took place with a haplotype that was being analyzed.
The first t-test determined if haplotypes were significant across all five populations, and an
additional t-test was done to determine if haplotypes were also significant within individual
families.

For flesh color, the Proc Mixed least squares means statement in SAS (SAS Institute version 9.2)
was used to determine if the means of the different haplotypes were significantly different.

Results

Phenotypic variation

64

Bloom time in 2011 for individuals of all five populations ranged from 256 to 440 GDD (Figure
2.5). All populations exhibited a normal distribution for bloom time except for ‘M172’ × ‘2502-29’ which had a narrow bloom time with the bulk of individuals blooming between 256 and
348 growing degree days. Bloom time in 2012 for all populations ranged from 275 to 488 GDD
(Figure 2.6). In 2012 there was less of a spread in bloom time GDD. In both years, the
populations ‘UF’ × ‘Surefire’, ‘Montmorency’ × ‘25-02-29’, and ‘RS’ × ‘ET’ tended to be more
late blooming than the populations ‘M172’ × ‘25-02-29’ and ‘25-14-20’ × ‘25-02-29’. The
correlation between these two years was quite high, with R2 = 0.81.

Fruit weights ranged from 1.6 to 13.0 grams when all populations were considered together
(Figures 2.7). The phenotypic distributions for fruit weight in all populations and within
individual populations were normally distributed. With the exception of ‘M172’ × ‘25-02-29’,
transgressive segregation with individuals having larger fruit than either parent were found, most
notably in the ‘RS’ × ‘ET’ population, with one individual far larger than either parent. Pit
weight, like bulk weight, was also normally distributed with evidence of transgressive
segregation within all families for larger pits, with the exception of the lack of small pit weights
among progeny of ‘RS’ × ‘ET’ (Figure 2.8). The correlation between pit weight and fruit weight
2

was moderate, with R = 0.56. Mesocarp fruit weight showed phenotypic distributions similar to
fruit weight (Figure 2.9) which is to be expected as the correlation between these was very high
2

(R = 0.99). All populations and the combination of all five populations were normally
distributed with all showing some individuals with larger mesocarp size than either parent, with
the exception of ‘M172’ × ‘25-02-29’. The correlation between fruit weight and mesocarp

65

2

weight was very high with R = 0.998 and the correlation between mesocarp weight and pit
2

weight was similar to that of pit weight and fruit weight (R = 0.52).

2

Fruit firmness ranged from 103 (soft) to 234 (firm) g/mm and was normally distributed in all
individual populations, but when all populations were combined there were higher numbers on
the softer end of the scale (e.g. smaller values) (Figure 2.10). The populations with the most firm
fruited progeny individuals were populations ‘M172’ × ‘25-02-29’ and ‘RS’ × ‘ET’, with all
populations having individuals that were firmer, and softer than the measured parents. The
2

correlation between fruit size and fruit firmness was not significant (R = 0.01).

The phenotypic distributions for flesh color ranged from 1 (no red color in the flesh) to 5 (very
dark red-purple flesh) but did not show a normal distribution in any of the populations, or when
considering all populations together (Figure 2.11). When all populations were considered
together, there were fewer individuals in the middle rankings (2, 3 and 4) with higher numbers
on both ends (1 and 5). Within individual populations, ‘UF’ × ‘Surefire’, ‘25-14-20’ × ‘25-0229’, and ‘RS’ × ‘ET’ tended to skew toward the darker flesh haplotypes, while ‘Montmorency’ ×
‘25-02-29’ had a slight skew to lighter flesh. ‘M172’ × ‘25-02-29’ had a distribution similar to
the combination of all populations, with higher distributions on the light, and dark ends of the
scale.

Malic acid content ranged from 0.40 to 2.80 mg/ml when the progeny individuals from all
populations were considered together. The phenotypic distributions of malic acid content also
66

showed a normal distribution (Figure 2.12). Each population had individuals with mean values
in excess of either parent. The populations ‘UF’ × ‘Surefire’ and ‘RS’ × ‘ET’ tended to have
more individuals with the highest concentration of malic acid, while progeny of ‘Montmorency’
× ‘25-02-29’ and ‘M172’ × ‘25-02-29’ tended to have lower concentrations of malic acid.

Haplotyping to identify different alleles for the target regions

A total of 2058 of the SNP markers evaluated were of sufficient quality for genotyping and
polymorphic in the sour cherry materials (Table 2.1). These SNP markers provided the set from
which SNPs were chosen to build haplotypes for the regions of interest. Haplotypes were built
for all parents and progeny of the 5 bi-parental populations when ambiguous genotypic scores
were not encountered. Eight different regions of various sizes were looked at on 6 different
chromosomes based on where QTL had been previously found for the traits studied (Table 2.2).
Four different SSR markers were used for the construction of haplotypes in on G2 and G6 or as
proxy haplotypes to condense haplotypes (Table 2.3). With the exception of few regions in the
‘RS’ × ‘ET’ population, all of the populations at all of the regions haplotyped had a small
number of individuals which were unable to be haplotyped (Table 2.4). These haplotypes
covered eight separate regions on six different chromosomes targeting the QTL regions
previously found in Prunus studies for the traits of interest (Figure 2.13).

Three different regions were targeted for bloom on G1, G2 and G4. On G1, twelve unique
haplotypes were found, and were designated as haplotype ‘a’ to ‘l’. (Figure 2.14). On G2, 21
unique haplotypes were found (Figure 2.15) facilitating the need for an SSR marker

67

(G2SSR1566) in the region to be used as a proxy haplotype to narrow down the number of
haplotypes to just seven haplotypes (2, and 4-9, where haplotypes 1 and 3 were skipped as they
were previously classified in sweet cherry in De Franceschi et al. (2013). Dosage for these
haplotypes was inferred based on the original haplotypes since dosage could not be determined
based solely on SSR banding on the polyacrylamide gel. Seventeen haplotypes were found for
the G4 bloom haplotype designated as ‘a’ to ‘p’ and ‘s’, where ‘q’ and ‘r’ were found to be
equivalent to other haplotypes already designated (Figure 2.16).

Haplotypes targeting fruit size and firmness were built for four different regions on G2, G3, G5,
and G6. The G2 region was the same as the G2 bloom region, where 21 haplotypes were found,
but narrowed down to seven based on a proxy SSR marker, where seven unique fragment sized
were found, enabling us to essentially break down the number of proxy haplotypes to seven
(Figure 2.15). Sixteen haplotypes (designated ‘a’ to ‘p’) were found for the G3 fruit size/firmness
region, but this number was reduced to eleven when haplotypes were compared in a narrower
region centered around the CNR16 region (Figure 2.17). In condensing the haplotypes, a=i, c=o,
d=f, g=m, and k=p. On G5, centered around CNR18 and CNR19, 12 haplotypes were found and
designated as ‘a’ to ‘f’, and ‘h’ to ‘l’ and ‘n’ with ‘g’ and ‘m’ being skipped as they were found
to be equivalent to previously designated haplotypes (Figure 2.18). The G6 fruit size/firmness
region had 13 unique haplotypes designated as ‘a’ to ‘j’ with ‘d’, ‘e’, and ‘e2’, but an SSR
marker (G6SSR2206) close to the CNR20 gene was used to condense the number of haplotypes
down to just five (1-5) with two null alleles found, which were not analyzed (Figure 2.19).

68

For flesh color and malic acid content, only one region was targeted for each trait on G3 and G5,
respectively. Thirteen unique haplotypes were found centered around three MYB10 homologs on
the G3 flesh color region with haplotypes designated as ‘b’ to ‘u’ skipping ‘i’, ‘j’, ‘m’, ‘q’, ‘r’,
‘s’, and ‘t’ due to combining of similar haplotypes (Figure 2.20). On the top of G5 the location
of the D-locus in peach, 17 haplotypes were found, designated as ‘b’ to ‘y’ (with ‘c’, ‘d’, and ‘t’
to ‘x’ being skipped due to combining of like haplotypes) but that number was reduced to just six
(1-6) when just the top of the chromosome that spanned the likely location of the D-locus was
considered (Figure 2.21).

Haplotype analysis

Bloom

On G1, the presence or absence of seven of the twelve haplotypes were associated with
significant differences in bloom when looked at for all five populations (Table 2.5). The
presence of haplotypes c, d, and k lead to earlier bloom, while the presence of f, g, h, and l had
mean bloom times which were later than individuals without those haplotypes. All of these
haplotypes were significant in both 2011 and 2012 with the exception of haplotype g which was
only significant in 2012. None of these haplotypes were significant when looked at within
individual families. ‘M172’ and ’25-02-29’ each have two G1 haplotypes significantly
associated with early bloom [‘M172’ = ijkk; ‘25-02-29’ = abcd] and no haplotypes significantly
associated with late bloom. This finding is consistent with the ‘M172’ × ‘25-02-29’ progeny
exhibiting the earliest bloom time and absence of late blooming individuals.

69

Due to the high number of haplotypes for the G2 region for bloom, the SSR marker G2SSR1566
was used to define proxy haplotypes. The presence vs. the absence of all seven haplotypes on
G2 was associated with significant differences in bloom over all five populations for both 2011
and 2012 (Table 2.6). Earlier bloom was associated with haplotype 2, 6 and 7, while later bloom
was associated with haplotype 4, 5, 8 and 9. When the presence vs. the absence of the
haplotypes was compared within individual families, none were significant. As with G1, ‘M172’
and ‘25-02-29’ each have two haplotypes significantly associated with early bloom [‘M172’ =
2447; ‘25-02-29’ = 2467] which is consistent with the earlier blooming associated with this
family. Haplotype 2 being an early blooming haplotype in sour cherry makes sense as this
haplotype is also found in sweet cherry (De Franceschi et al. 2013). Sweet cherry tends to bloom
earlier than sour cherry, so it would be expected that a sweet cherry haplotype would be
associated with earlier blooming.

Eleven of the 15 haplotypes for G4 were found to be associated with significant differences in
bloom time when evaluated across all five populations (Table 2.7). All but one, haplotype h,
produced significant differences in both 2011 and 2012, while h was just significant in 2011.
Haplotype c, d, e, f, h, and j lead to earlier bloom, while haplotype g, i, k, n, and s lead to later
bloom. The largest difference was seen with haplotype k, where the mean of individuals with k
was 36 and 35 growing degree days later blooming than those without k in 2011 and 2012
respectively. Both ‘UF’ and ‘Surefire’ have this very late k haplotype [‘UF’ = ahkn; ‘Surefire’ =
giks] which is consistent with this population having a high proportion of later blooming
individuals.

70

When haplotypes were compared within individual families for G4, seven haplotypes were found
to significantly affect bloom time (Table 2.8). In the ‘UF’ × ‘Surefire’ population, two
haplotypes were found to be significant. Haplotype k, when tested over all five populations, led
to later bloom in both 2011 and 2012. This haplotype is likely from the founder ‘Pandy 38’, as
‘UF’ arose from a mutation from ‘Pandy 38’ (Figure 1.1). The haplotype is also in Surefire,
indicating that ‘Pandy 38’ may also be in its background. Haplotype a, on the other hand, was
found to lead to earlier bloom in just 2012. This haplotype was not found to be significant when
tested across all five populations. Two haplotypes were also found to be significant in the
population ‘25-14-20’ × ‘25-02-29’. Haplotype b was found to lead to later bloom within this
family only in 2011, while haplotype c was found to lead to earlier bloom only in 2012. Only
one haplotype, haplotype e, in the population ‘M172’ × ‘25-02-29’ was found to significantly
affect bloom time. In both 2011 and 2012, the presence of haplotype e led to later bloom. This
is the opposite effect that haplotype e had when compared over all five populations. For the
populations of ‘Montmorency’ × ‘25-02-29’ and ‘RS’ x ‘ET’, only one haplotype in each
population was found to be significant, and only in 2012. In ‘Montmorency’ × ‘25-02-29’,
haplotype i was associated with later bloom, while in ‘RS’ × ‘ET’, haplotype m was found to be
associated with earlier bloom.

Fruit/pit size

For the fruit size region on G2, two haplotypes, 6 and 8, were found to be significantly
associated with fruit and mesocarp weight, while only haplotype 8, was significantly associated

71

with pit weight (Table 2.9).

Haplotype 6 was found to be significantly associated with lower

fruit and mesocarp weight, but not pit weight. Only haplotype 8 was significantly associated
with fruit, pit and mesocarp weight, where it was associated with lower weights when present.
None of the haplotypes produced significant variation within individual families.

On G3, seven of the eleven haplotypes were significantly associated with fruit size and six were
associated with pit size (Table 2.10). Haplotypes a, b, c, and n were significantly associated with
smaller fruit, while haplotypes e, g, and h were significantly associated with larger fruit. All of
these haplotypes were significant for both mesocarp weight and fruit weight. Pit weight was not
significantly associated with haplotypes a and e as were fruit and mesocarp weight, but had a
significant association with haplotype k, with an average pit weight less when k was present.
Haplotypes b, c, n, g, and h had the same associations with pit weight as with fruit and mesocarp
weight. No haplotypes were significant within individual families for fruit, mesocarp, or pit
weights. ‘Montmorency’ and ‘25-02-29’ each have three small-fruit haplotypes, and no large
fruit haplotypes [‘Montmorency’ = acjn; ‘25-02-29’ = abcd]. This is consistent with this
population being mostly small-fruited individuals.

Only three of the 13 haplotypes built for the G5 fruit/pit size region were significantly associated
with fruit size, and two shared significant haplotypes and one unique one being significantly
associated with pit size (Table 2.11). Haplotypes f and j were significantly associated with larger
fruit, and haplotype a was significantly associated with smaller fruit. Haplotypes a and j were
also significantly associated with pit weight in the same direction they were with fruit and
mesocarp weight. Haplotype n was also significantly associated with larger pit weight. None of

72

the haplotypes were significantly associated with fruit, mesocarp or pit weights within individual
populations. The three populations that have the largest individuals in them (‘UF’ × ‘Surefire’,
‘M172’ × ‘25-02-29’ and ‘RS’ × ‘ET’), each have one parent that has two large fruit haplotypes:
‘UF’ = ddfn; ‘M172’ = dfhj; and ‘ET’ = djkn. The populations with smaller fruit size
(‘Montmorency’ × ‘25-02-29’ and ‘25-14-20’ × ‘25-02-29’) have zero and one parent with large
haplotypes, which is consistent with the phenotypes observed in these families (Figure 2.7).

For the G6 region, only two haplotypes were found to be significantly associated with
fruit/mesocarp and pit size, with an additional third for pit size (Table 2.12). Haplotypes 3 and 4
were significantly associated with larger and smaller fruit, mesocarp and pit weights
respectively. Haplotype 1 was also associated with smaller pits, but not significantly for fruit
and mesocarp weight. None of these haplotypes were significant within individual families.

Fruit firmness

Since fruit firmness was found to be correlated with fruit size in sweet cherry (Quero-García et
al. 2010), the haplotypes built for the fruit size regions were also tested for association with fruit
firmness. For the G2 region, three haplotypes were associated with fruit firmness (Table 2.13).
The three haplotypes significantly associated with firmness were different haplotypes than those
associated with fruit size. Haplotypes 5 and 9 were associated with fruit that were less firm than
those without the haplotypes, while haplotype 7 was associated with firmer fruit. No haplotypes
were significant within individual families. The two populations with the firmest individuals
were the two populations where both parents have haplotype 7: ‘M172’ × ‘25-02-29’ [‘M172’ =

73

2447; ‘25-02-29’ = 2467] and ‘RS’ × ‘ET’ [‘RS’ = 4478; ‘ET’ = 4678]. Neither of these families
have the haplotypes 5 or 9 which are associated with softer fruit.

The G3 region had three haplotypes which were significantly associated with fruit firmness
(Table 2.14). The, haplotypes j and k, were associated with softer fruit while haplotype d was
associated with firmer fruit. Haplotypes j and d were not significantly associated with fruit or pit
weights, however k was associated with smaller pits as well. No haplotypes were significant
within individual families.

For the fruit size CNR region on G5, haplotypes b and j were significantly associated with firmer
fruit while haplotypes e, i, and l were significantly associated with softer fruit (Table 2.15). Of
those five haplotypes that were significantly associated with fruit firmness, only one of them was
also associated with fruit or pit size. Haplotype j was significantly associated with larger fruit
and pit weights in addition to being associated with firmer fruit. No haplotypes produced
significant variability within individual families. ‘M172’ × ‘25-02-29’ again, like for G2, have
only firm haplotypes that were significantly associated with firmness, b in ‘25-02-29’ and j in
‘M172’: [‘M172’ = dfhj; ‘25-02-29’ = abcd] which is consistent with having progeny with firmer
fruit. Surefire, on the other hand, has three soft-associated haplotypes at this region [‘Surefire’ =
ceil] which is consistent with the ‘UF’ × ‘Surefire’ population have a large number of individuals
which are soft fruited, as ‘UF’ also has no haplotypes that are associated with either firm, or soft
fruit [‘UF’ = ddfn].

74

Only one haplotype in the G6 region was found to be significantly associated with fruit firmness
(Table 2.16). Haplotype 1 was associated with softer fruit when compared to individuals without
this haplotype. This haplotype was not significantly associated with fruit or pit weights. No
haplotype were significant within individual families.

Flesh color

When evaluated across all five bi-parental populations, ten of the thirteen G3 flesh color
haplotypes were found to be associated with flesh color (Table 2.17). Haplotypes b, d, e, k, l,
and p were significantly associated with darker flesh, while haplotypes c, f, n, and o were
associated with lighter flesh.

Within individual families, six haplotypes were found to be significant (Table 2.18). In the ‘UF’
× ‘Surefire’ population, three haplotypes were significantly associated with flesh color.
Haplotype d and c from ‘UF’ were associated with darker and lighter flesh respectively. From
‘Surefire’, haplotype e was associated with dark flesh. In the ‘25-14-20’ × ‘25-02-29’
population, there were also three haplotypes which were significantly associated with flesh color.
One dark flesh haplotype was from each parent, haplotype d from ‘25-14-20’, and haplotype p
from ‘25-02-29’. The light flesh haplotype o from ‘25-02-29’ was also significant in this
population. In ‘M172’ × ‘25-02-29’, the dark flesh haplotype p, and the light flesh haplotype o
from ‘25-02-29’ are significant again, as well as the dark flesh haplotype l from the ‘M172’
parent. Haplotype p and o from ‘25-02-29’ are once again significant in the ‘Montmorency’ ×
‘25-02-29’ population, with haplotype p being a darker flesh haplotype, and haplotype o a lighter

75

flesh one. Haplotype o is also present in ‘Montmorency’. Only dark flesh haplotypes were
found to be significant in the ‘RS’ × ‘ET’ population. Haplotype p is from the parent ‘RS’,
while haplotype l comes from ‘ET’.

Since each parent carried only one dark flesh haplotype (with the exception of ‘ET’ which had
two) we wanted to investigate if dosage was important in this region. To test this, means were
compared within individual families (Figure 2.22). For ‘UF’ × ‘Surefire’, individuals with both
the d haplotype and the e haplotype have a darker mean flesh score (4.8) than those with only
haplotype e (3.0) or with no dark flesh haplotypes (1.9). The mean score of those with both d and
e, and those with only d are not significantly different. In the ‘M172’ × ‘25-02-29’ population,
the dark flesh haplotype l came from ‘M172’ and the dark flesh haplotype p came from ‘25-0229’. In each comparison in this family, the mean scores were significantly different from each
other. Those with both dark flesh haplotypes had the darkest mean flesh score (4.2), followed by
those with only the p haplotype (3.6), those with only the l haplotype (3.2) and lastly those with
no dark flesh haplotypes (1.3). In the population ‘25-14-20’ × ‘25-02-29’, dark flesh haplotype d
from ‘25-14-20’ was compared with the dark flesh haplotype p in ‘25-02-29’. Individuals in this
population with both d and p had the darkest mean flesh score (4.9) which was significantly
different from individuals with just the p haplotype (4.2), and those with no dark flesh
haplotypes (1.6). Individuals with just the d haplotype (mean score of 4.4) were not significantly
different from those with both p and d, or individuals with only p (4.9 and 4.2 respectively). In
the ‘Montmorency’ × ‘25-02-29’ population, only one dark flesh haplotype was present. The
presence of the p haplotype from ‘25-02-29’ in individuals (mean score of 3.9) was significantly

76

different from individuals with no dark flesh haplotypes (1.5). The family ‘RS’ × ‘ET’ was too
small to do a comparison, as there were too few individuals in all of the classes to compare.

Malic acid

On G5, three haplotypes were found to be associated with significant differences in the amount
of malic acid (Table 2.19). Haplotype 1 was significantly associated with a higher amount of
malic acid. When individuals with two copies of haplotype 1 were compared to those with only
one, there was also a significant difference, where multiple copies of haplotype 1 had an even
higher concentration of malic acid than those with only one copy. Haplotype 4 and haplotype 5
also had significant differences between those with, and those without the haplotype, with those
with 4 and 5 having a lower concentration of malic acid. None of these haplotypes were
significant within individual families. The families ‘UF’ × ‘Surefire’ and ‘RS’ × ‘ET’ have the
most individuals with higher malic acid. Each of these families has one parent with two high
malic acid haplotypes, and the other parent has one: [‘UF’ = 1236; ‘Surefire’ = 1123; ‘RS’ =
1234; ‘ET’ = 1146].

Discussion

In this study, we used multiple sour cherry populations to validate QTL that have been found in
other Prunus species. Due to the relatively few sour cherry founders, these populations share
many common ancestors, and therefore are inter-related (Figure 2.1). This made comparisons

77

between the families easier due to the reduced number of unique haplotypes present in this
tetraploid species.

In some regions, however, there were still a large number of haplotypes found, such as the G2
region for bloom, fruit size and firmness (Figure 2.15). It was considered that the SNP diversity
may over represent the number of functional haplotypes. Therefore the use of the polymorphic
SSR marker in this region was important in order to condense the haplotypes to what might
better approximate the number of functional haplotypes. Due to the number of important traits in
this one narrow region, it is expected that there were so many haplotypes found, with selections
taking place in this region, it is likely that unique crossovers in this region to get desired
combinations of these traits produced the variety of haplotypes found in this region. SSR
markers in all regions could be used in the future to condense all of the regions evaluated.

Bloom

All three of the regions studied were validated by locating haplotypes that were associated with
bloom time. The G4 region found in almond (Silva et al. 2005), sweet cherry and peach
(Dirlewanger et al. 2012), was expected to have a bloom time QTL in sour cherry. The G4
bloom time QTL was found to explain from 24 to 47 percent of the variance in the sweet cherry
population studied (Dirlewanger et al. 2012). It is likely due to the high impact of this region on
the bloom phenotype that not only was this region significant when looked at across all
populations, but even within individual populations. One haplotype, haplotype e, found only in
the early blooming parent ‘M172’, was significantly associated with early blooming when

78

looked at across all populations, but within ‘M172’ × ‘25-02-29’ it was found to be associated
with later bloom (Tables 2.7 and 2.8). A likely explanation of this finding is that while overall
haplotype e is an earlier blooming haplotype, within an early blooming family like ‘M172’ ×
‘25-02-29’ it is relatively late blooming compared to the other haplotypes present in that family.
Of all the significant haplotypes found for the G4 region, haplotype k from ‘UF’ and ‘Surefire’
had the highest impact on bloom time within individual populations. This haplotype was
associated with a mean delay in bloom of 39 and 33 GDD in 2011 and 2012, respectively. With
such a large effect on bloom time for one haplotype, it is likely that later bloom is dominant to
early bloom. Sweet cherry tends to bloom earlier than sour cherry (Iezzoni et al. 1990), which
would indicate that sweet cherry-derived haplotypes would also be associated with earlier bloom.
This is what was found on G2, where the haplotype 2 and the corresponding haplotype is
associated with earlier bloom, and is the only haplotype that is known to be equivalent in sweet
cherry (De Franceschi et al. 2013).

For breeding purposes, where later bloom would help individuals avoid freeze damage in the
spring, it would be beneficial to focus mostly on the G4 bloom region, as this region has the most
impact, keeping in mind that other regions do play a role in contributing to bloom time. For
example, on G1 in populations like ‘M172’ × ‘25-02-29’, each parent has two early bloom
haplotypes, and no haplotypes that are significantly associated with later bloom. It is unlikely,
therefore, for this cross to produce individuals with late bloom.

Fruit/pit size

79

All four regions that targeted previously identified QTLs for fruit/pit size were validated in sour
cherry. Fruit weight and mesocarp weight were always significantly associated with the same
haplotypes in all regions studied; however, pit weight was not always found to have the same
significance, indicating that not all haplotypes effect fruit weight and pit weights similarly.

On G2, both haplotypes 6 and 8 were found to be negatively associated with fruit size. The two
largest fruited parents, ‘UF’ and ‘M172’, are the only two parents that do not have either of these
negatively associated haplotypes. This could be a contributing factor in that the populations
‘UF’ × ‘Surefire’ and ‘M172’ × ‘25-02-29’ are the two populations with larger-fruited
individuals. The haplotype 2 for this region in sour cherry is equivalent to the PavCNR12
genotype 2 in sweet cherry, where it was found to be associated with smaller fruit in
domesticated sweet cherry (De Franceschi et al. 2013). In sour cherry, which are generally
smaller than sweet cherry, there was a tendency toward larger fruit (mean fruit weight values of
5.64g vs. 5.30g with and without haplotype 2 respectively) when haplotype 2 was present,
however this only significant at the P = 0.07 level. While this is opposite to what is found in
sweet cherry, the size difference between sweet and sour cherries could account for a negatively
associated haplotype in sweet cherry still contributing to larger fruit in sour cherry. This same
comparison of sweet cherry haplotypes contributing to larger fruit in sour cherry can also be seen
on G6, where the haplotype 3 which is significantly associated with larger fruit (Table 2.12) has
also been found to be equivalent to the haplotypes found in sweet cherry (De Franceschi et al.
2013).

80

The almost total lack of transgressive segregation for fruit size in ‘M172’ × ‘25-02-29’ may
partially be explained by the G3 haplotypes, where ‘M172’ has three large-fruit associated
haplotypes (e, g and h), and ‘25-02-29’ has three small-fruit associated haplotypes (a, b, and c).
No combinations of haplotypes would allow for individuals in this population to have equivalent
or greater numbers of either small-, or large-associated haplotypes in this cross, which would not
allow for progeny to exceed the parental means in this region.

This study is the first to report on the association between haplotypes across the CNR18 and
CNR19 region on G5 and fruit weight. Three of the 4 haplotypes that were significantly
associated with fruit size for this region were associated with larger fruit, indicating that this
could be a good region to start accumulating positive haplotypes for fruit size. Considering that
even the small-fruited parent ‘ET’ (4.11g) has two of the large-associated haplotypes (the same
number of large-associated haplotypes as the large-fruited parents ‘M172’ and ‘UF’ with 8.27g
and 7.75g fruit weight respectively), it shows that while this may not be a major QTL, progeny
‘27-12-12’ (12.96g) and ‘27-12-13’ (8.09g), the two largest progeny in the ‘RS’ × ‘ET’
population, each have no negative-associated haplotypes for the G5 region, and one and two of
the positive associated haplotypes respectively for this region.

Haplotype 6 on G2, haplotype e on G3 and haplotype f on G5 are all significantly associated
with fruit size, but not pit size. Conversely, haplotype k on G3, haplotype n on G5 and haplotype
1 on G6 are all significantly associated with pit size but not fruit weight. This indicates that not
all haplotypes contribute to both pit and fruit weight in the same manner. When associations
between both fruit weight and pit weight do occur from the same haplotype, they are always in

81

the same direction, where a haplotype negatively associated with fruit weight is also negatively
associated with pit weight.

For breeding purposes, large fruit with small pits are desired. To achieve this goal, the
accumulation of the larger fruited haplotypes in all the regions validated here, or the exclusion of
the smaller fruited ones, would be a good strategy. This, however, may also lead to larger pit
sizes. By also focusing haplotypes that only effect pit size, such as small pit-associated
haplotypes like k from G3, or haplotype 1 from G6 may help reduced the pit size without
adversely affecting fruit size.

Fruit firmness

Fruit firmness was validated for the G2 region, and was discovered to be associated with the
other three regions also examined for fruit/pit size on G3, G5, and G6. Of the 14 different
haplotypes found to be associated with fruit size for this region, only haplotype k from G3 and
haplotype j from G5 were also associated with fruit firmness. In the case of haplotype k from
G3, it was linked to softer, smaller fruit, while the opposite effect was found for haplotype j from
G5. All other haplotypes significantly associated with fruit firmness were haplotypes that were
not significantly associated with fruit size, indicating that these two traits are not affected by the
same genetic components. No other regions tested besides these four presented, were important
for determining fruit firmness, indicating that there may be other regions of greater importance
for this trait, or that many regions affect fruit firmness, so it may be difficult to pin down.

82

Flesh color

Four haplotypes in total were found to be associated with dark flesh color in sour cherry,
validating the QTL in sweet cherry (Sooriyapathirana et al. 2010). Perhaps most surprising is
how few dark-flesh haplotypes are present in these 5 populations that cause a range in color from
light red to dark mahogany. Haplotype d found in ‘UF’, and ‘25-14-20’ was found to be
associated with the darkest coloration in the two populations it was present in. In both
populations, with just this dark flesh haplotype and none of the others, an average color score of
4.4-4.5 was found to be similar to individuals with both d and e in the ‘UF’ × ‘Surefire’
population, or with both d and p in the ‘25-14-20’ × ‘25-02-29’ population. This indicates that
with just this one haplotype, individuals will be mostly dark fleshed. All of the other dark flesh
haplotypes tend to be more moderate in coloration, from a mean color score of 3 for individuals
with just haplotype e, to a mean color score of 3.6-4.2 for individuals with one copy of haplotype
p. It is unknown how multiple copies of the same haplotype would influence flesh color, as no
populations studied had more than one copy of any dark flesh haplotype. Due to the fact that for
the moderate flesh color haplotypes e, l and p tend to have an additive affect when found
together in the same genotype, it could be inferred that two copies of the same dark flesh
haplotype would have a similar result.

Breeding decisions could easily be made to select against dark flesh haplotype d, and select for
single copies of the more moderate haplotype l and e if flesh color scores of around 3 were
desired. If light-fleshed, industry-standard ‘Montmorency’-type flesh color were desired, then
selecting against these four dark flesh haplotypes would be the breeding recommendation, as

83

individuals without any of these four dark flesh haplotypes ranged from 1.3 in the ‘M172’ × ‘2502-29’ population to 1.9 in the ‘UF’ × ‘Surefire’ population (Figure 2.22).

Malic acid

The D-locus region responsible for variation of malic acid content in peach (Bouderhi et al.
2009) has been validated to also be associated with malic acid content in sour cherry. The
families ‘UF’ × ‘Surefire’ and ‘RS’ × ‘ET’ have the most individuals with high malic acid.
These also are the families that have the most haplotypes classed as 1 in this study (Figure 2.21).
It is likely that since this class of haplotypes had the most individuals (6 of the 17 haplotypes
found) that these are haplotypes that belong to the P. fruticosa subgenome of sour cherry, rather
than the P. avium subgenome. Sweet cherry is not known for their high acidity, but P. fruticosa
is known to be acidic (Iezzoni et al. 1990). Future studies comparing haplotypes found in sweet
cherry and haplotypes found in sour cherry for this region could confirm this.

Dosage

While more targeted crosses would need to be undertaken to study the full effects of dosage for
all of the traits validated in this study, there were a few notable results that confirmed that dosage
can play a role in trait variation in tetraploid sour cherry. Flesh color in the family that had the
more moderate flesh colors (‘M172’ × ‘25-02-29’) had increased flesh color when both were
present in the same family as opposed to each of haplotype p or haplotype l separately. In this
case, flesh color appears to be additive when moderate flesh colors are present. Dosage with

84

haplotypes with very dark haplotype d makes little difference, as just one copy of d results in
very dark fleshed fruit. The effects of dosage can also be seen with malic acid, where multiple
copies of haplotype class 1 give rise to an average malic acid content greater than if just one
copy of this haplotype were present. In peach where the D locus controls fruit acidity, it was
found that low acidity is partially dominant (Boudehri et al. 2009). In sour cherry, this would
mean that an accumulation of more high-acid haplotypes would help offset low acid ones.

Linkage and breeding implications

G2 was found to be associated with bloom, fruit size, and fruit firmness. Given that the desired
cultivar would be a late blooming tree with firm and large fruit, careful consideration is needed
when selecting for or against certain haplotypes for this region. For example, haplotype 8 is
associated with later bloom, but small fruit. Two of the other haplotypes associated with late
bloom, 5 and 9, are also associated with soft fruit. The only haplotype in this region that is
associated with later bloom, but has no negative associating with fruit size or fruit firmness is
haplotype 4. Haplotype 6 could be selected against, as it is associated with both early bloom and
small fruit. As more regions are found to be associated with multiple traits, these kinds of
considerations will need to take place in order to avoid negative linkage drag.

Conclusions

Several QTL found in diploid Prunus species have been validated in the background of
tetraploid sour cherry. While the methods used here of comparing the presence or absence of

85

haplotypes has proven to be useful as a means of simplifying the genetics of this species, best
results seem to be found when looking at regions with a high contribution to phenotypic
variation such as flesh color on G3, and bloom time on G4. In using multiple populations with
inter-connected pedigrees which still represent the diverse germplasm of sour cherry, we have
been able to determine how individual haplotypes perform in different backgrounds. Future
studies with targeted crosses to see how dosage plays a role in some of these populations could
provide better understanding to these traits in the future.

86

Table 2.1: SNP informativeness in sour cherry for the eight sets of chromosomes based on whether the SNP was derived from
polymorphism in sweet cherry or in one of the two sour cherry subgenomes (i.e., avium or fruticosa).
Unresolved
a
b
SNPs chosen
Chromosome SNP source
Failed
Monomorphic
Polymorphic
Polymorphic
Sweet
902 (50)
8 (0)
364 (11)
211 (8)
319 (31)
1
Sour
164/161
1/2
14/18
68/62
81/79
Sweet
557 (21)
10 (0)
199 (5)
166 (5)
182 (11)
2
Sour
92/83
2/0
12/20
40/26
38/37
Sweet
434 (18)
4 (0)
165 (4)
107 (6)
158 (8)
3
Sour
87/74
1/2
13/12
33/35
40/25
Sweet
479 (26)
9 (0)
176 (0)
141 (9)
153 (17)
4
Sour
89/73
1/0
8/15
45/28
35/30
Sweet
489 (33)
4 (0)
208 (8)
128 (7)
149 (18)
5
Sour
66/84
0/0
4/16
30/36
32/32
Sweet
508 (32)
13 (0)
188 (3)
145 (11)
162 (18)
6
Sour
108/100
0/0
8/14
43/31
57/55
Sweet
453 (22)
6 (0)
184 (5)
113 (3)
150 (14)
7
Sour
71/75
2/0
2/16
22/29
45/30
Sweet
392 (19)
14 (0)
130 (4)
140 (6)
108 (9)
8
Sour
75/80
3/1
9/17
28/36
35/26
68 (0)
Total Sweet
4214 (221)
1614 (40)
1151 (55)
1381 (126)
10/5
Total Sour
752/730
70/128
309/283
363/314
83
1743
Grand Total
5696
1812
2058
a

Numbers of SNPs for the subgenomes of sour cherry are split (/) between avium and fruticosa.

b

Numbers in parentheses are totals for RosCOS SNPs derived from sweet cherry and are included in the first number

87

Table 2.2: Summary of all traits, QTLs and their locations that were validated in this study. The species source and original QTL
reference(s) are included.
Linkage
Group
1

Haplotype
region built
a

(Mb)

45.02-46.75
14.93-22.08

2

14.93-22.08

14.93-22.08

Markers
used
28 SNPs
122
SNPs, 3
SSRs
122
SNPs, 3
SSRs
122
SNPs, 3
SSRs

Trait

Validation
region/marker

No. of significant
alleles/No. of
alleles

Bloom
(GDD)

45.02-46.75 Mb

7 /12

Fruit
Firmness

SSR marker
(G2SSR1566)

3 /7

Fruit size

SSR marker
(G2SSR1566)

2 /7

Bloom
(GDD)

SSR marker
(G2SSR1566)

7 /7

b

P. avium/Dirlewanger et
al. 2012

b

P. avium/Quero-García
et al. 2010

b

P. avium/Zhang et al.
2010

b

P. avium/Dirlewanger et
al. 2012

b

1.14-7.57

4

75 SNPs

Fruit size

2.74-4.76 Mb

8 /11

1.14-7.57

75 SNPs

Fruit
Firmness

2.74-4.76 Mb

3 /11

9.73-15.46

3

47 SNPs

Flesh
Color

10.68-13.41 Mb

4 /13

7.01-10.83

44 SNPs

Bloom
(GDD)

7.31-9.15 Mb

6 /15

88

QTL Source/reference

b

c

c

P. avium/Quero-García
et al. 2010; Rosyara et al.
(in review)
P. avium/Quero-García
et al. 2010
P. avium/
Sooriyapathirana et al.
2010
P. dulcis/Silva et al. 2005
Sanches-Perez et al.
2007; P. avium
/Dirlewanger et al. 2012

Table 2.2 (cont’d)

Trait

Validation
region/marker

54 SNPs

Acidity
(Malic Acid
content)

0.69-1.46 Mb

3 /6

b

P. persica/Boudehri et al. 2009

16.13-18.10

42 SNPs

Fruit size

16.72-17.76 Mb

4 /13

b

P. persica/De Franceschi et al.

16.13-18.10

5

Markers
used

0.69-5.43

Linkage
Group

No. of
significant
alleles/No. of
alleles

42 SNPs

Fruit
Firmness

16.72-17.76 Mb

5 /13

Haplotype region
built (Mb)

a

22.12-27.52
6
22.12-27.52

69 SNPs,
2 SSRs
69 SNPs,
2 SSRs

Fruit
Firmness
Fruit size

SSR marker
(G6SSR2208)
SSR marker
(G6SSR2208)

QTL Source/reference

2013

b

-

b

d

b

P. avium/Zhang et al. 2010

3 /5

e

Trait values for these alleles were significantly different when all five families were considered together
Trait values for these alleles were significantly different within families
Candidate gene was used instead of a QTL region
QTLs for firmness had not previously been reported but were tested in this study

89

e

-

Mb distances according to the Peach v1.0 ‘dhLovell’ genome assembly (International Peach Genome Initiative;
www.rosaceae.org/peach/genome) Verde et al. 2013
c

e

1 /5

a

b

d

Table 2.3: SSR markers used in this study and the original SSR reference.

SSR
G2SSR1566
G6SSR2208
CPSCT038
BPPCT034

Species
Origin
P. persica
P. persica
P. salicina
P. persica

Peach physical map location
a

(Mb)
Scaffold 2 (15.66)
Scaffold 6 (22.08)
Scaffold 2 (15.05)
Scaffold 2 (16.49)

a

Reference
De Franceschi et al. 2013
De Franceschi et al. 2013
Mnejja et al. 2004
Dirlewanger et al. 2002

Mb distances according to the Peach v1.0 ‘dhLovell’ genome assembly (International
Peach Genome Initiative; www.rosaceae.org/peach/genome) (Verde et al. 2013)

90

Table 2.4: Number of progeny individuals from each bi-parental family for which the four chromosome segments for the target QTL
regions in each progeny individual could be identified as haplotypes inherited from its parents. Individuals were not haplotyped when
SNPs were ambiguous for dosage, or if individual haplotypes could not be determined.
Linkage
group
1
2
3
3
4
5
5
6

b

c

Region (Mb)

UF x Surefire
(n=76)

RS x ET
(n=23)

45.02-46.75
14.93-22.08
1.14-7.57
9.73-15.46
7.01-10.83
0.69-5.43
16.13-18.10
22.12-27.52

74
72
73
70
74
66
70
71

23
22
23
23
22
22
23
21

a

d

M172 x 25-02-29
(n=111)

25-14-20 x 25-02-29
(n=67)

Montmorency x
25-02-29 (n=53)

110
100
100
101
108
105
100
106

66
62
56
61
58
63
64
60

48
45
46
46
48
49
51
49

a

Mb distances according to the Peach v1.0 ‘dhLovell’ genome assembly (International Peach
Genome Initiative; www.rosaceae.org/peach/genome) (Verde et al. 2013)
b
c
d
Újfehértói Fürtös, Rheinische Schattenmorelle, Englaise Timpurii

91

Table 2.5: Phenotypic means for bloom time in 2011 and 2012 for the presence or absence of the
a
G1 haplotypes in sour cherry progeny individuals from the five bi-parental families. Parental
genotypes for the G1 haplotypes are: ‘M172’ (ijkk), 25-02-29 (abcd), ‘Montmorency’ (aceh),
‘25-14-20’ (adej), ‘UF’ (bfij), ‘Surefire’ (ahjj), ‘RS’ (acdi) and ‘ET’ (egkl).

Bloom 2011 (GDD)
N
G1 haplotype

c

Means

d

b

Bloom 2012 (GDD)

P value

N

Means

P value

e
A

a/no a

182/122 318A /315

b/no b

133/170

317 /317

c/no c

124/173

309 /322

A

A

A

0.406

A

A

0.557

A

B

<0.0001

0.491

187/127 368 /365

A

A

0.956

137/175 366 /368

A

B

0.0006

131/176 357 /373

138/164

A

311 /321

B

0.007

146/166

60/249

A

316 /317

A

0.891

63/256

42/269

A

339 /313

B

<0.0001

42/279

12/309

A

336 /316

A

0.06

12/321

h/no h

51/252

A

333 /312

B

0.0003

50/265

i/no i

79/235

317 /317

A

A

0.953

81/245

j/no j

153/160

317 /316

A

A

0.735

158/165

k/no k

100/210

300 /324

A

B

<0.0001

103/217

A

B

0.025

10/322

d/no d
e/no e
f/no f
g/no g

l/no l

10/310

339 /316

A
B
361 /372
A
A
362 /368
A
B
390 /363
A
B
384 /366
A
B
382 /362
A
A
372 /365
A
A
368 /366
A
B
356 /371
A
B
385 /366

0.0009
0.14
<0.0001
0.046
0.0006
0.105
0.579
<0.0001
0.024

a

Peach physical map distance 45,021,181-46,751,928 bp (see Figure 2.14 for descriptions of
the haplotypes).
b
c
d
e

Growing Degree Days (GDD) with a base of 4.4 °C
Number of individuals
Means with the same letter within a row are not significantly different (P>0.05)
The allelic state significantly associated with the increased trait value is identified in bold

92

Table 2.6: Phenotypic means for bloom time in 2011 and 2012 for the presence or absence of the
a
G2 G2SSR1566 haplotypes in all sour cherry individuals from the five bi-parental families.
Parental genotypes for the G2 haplotypes are: ‘M172’ (2447), ‘25-02-29’ (2467),
‘Montmorency’ (4488), ‘25-14-20’ (4489), ‘UF’ (2449), ‘Surefire’ (4458), ‘RS’ (4478) and ‘ET’
(4678).

Bloom 2011 (GDD)
N
G2 haplotypes

c

Means

d

<0.0001

150/154 359 /372

A

B

0.0009

284/19

367 /354

A

B

0.0002

32/272

390 /363

A

B

A

B

A

B

A

B

273/19

317 /297

5/no 5

32/261

340 /313

9/no 9

Means

B

4/no 4

8/no 8

N

A

142/151 308 /324

7/no 7

P value

Bloom 2012 (GDD)
P value

e

2/no 2

6/no 6

b

127/166 309 /322
137/156 307 /324
142/151 328 /305
70/223

325 /313

0.0008
<0.0001
<0.0001
0.014

A

B

<0.0001

A

B

0.0005

A

B

<0.0001

A

B

A

B

<0.0001

A

B

<0.0001

A

B

0.02

134/166 360 /371
144/160 359 /372
145/159 375 /358
75/229

373 /363

0.001

a

Peach physical map location 15,666894-15,667,139 bp (See Figure 2.15 and Table 2.3 for
haplotype region and SSR marker information)
b
c
d
e

Growing Degree Days (GDD) with a base of 4.4 °C
Number of individuals
Means with the same letter within a row are not significantly different (P>0.05)
The allelic state significantly associated with the increased trait value is identified in bold

93

Table 2.7: Phenotypic means for bloom time in 2011 and 2012 for the presence or absence of the
a
G4 haplotypes for all sour cherry individuals from the five bi-parental families. Parental
genotypes for the G1 haplotypes are: ‘M172’ (defh), ‘25-02-29’ (abcd), ‘Montmorency’ (dilo),
‘25-14-20’ (aghj), ‘UF’ (ahkn), ‘Surefire’ (giks), ‘RS’ (bdgi), and ‘ET’ (amnp).

Bloom 2011 (GDD)
N
G4 haplotypes

c

Means

d

b

Bloom 2012 (GDD)

P value

N

Means

P value

e
A

A

0.99

153/145 366 /367

A

A

0.13

123/175 363 /370

A

B

<0.0001

112/189 355 /375

144/147

A

308 /325

B

56/250

A

303 /319

B

50/248

A

294 /321

B

58/235

A

329 /313

B

h/no h

112/182

A

312 /320

B

0.04

i/no i

79/226

332 /310

A

B

<0.0001

78/235

382 /361

j/no j

30/273

308 /317

A

B

0.04

33/278

356 /368

k/no k

55/253

346 /310

A

B

<0.0001

55/261

395 /360

18/296

A

311 /316

A

8/313

A

319 /317

A

47/269

A

341 /312

B

41/275

A

B

a/no a

150/141

316 /316

b/no b

121/169

313 /319

c/no c

108/186

306 /324

d/no d
e/no e
f/no f
g/no g

l/no l
m/no m
n/no n
s/no s

334 /314

<0.0001
<0.0001
<0.0001
0.006

0.43
0.84
<0.0001
0.002

A

A

0.62

A

A

0.07

A

B

<0.0001

A

B

57/257

A

361 /368

B

0.03

52/254

A

349 /370

B

<0.0001

59/240

A

B

0.01

A

A

0.28

A

B

<0.0001

A

B

0.0001

A

B

<0.0001

17/309

A

359 /367

A

8/325

A

373 /367

A

0.42

47/281

A

390 /362

B

<0.0001

41/287

A

B

0.0008

149/150 358 /375

376 /365

119/183 364 /368

384 /364

<0.0001

0.25

a

Region analyzed defined by peach physical map location 7,309,282-9,148,953 bp (see Figure
2.16 for descriptions of the haplotypes)
b
c
d
e

Growing Degree Days (GDD) with base of 4.4 °C
Number of individuals
Means with the same letter within a row are not significantly different (P>0.05)
The allelic state significantly associated with the increased trait value is identified in bold

94

Table 2.8: Phenotypic means for bloom time in 2011 and 2012 for the presence or absence of the
a
G4 haplotypes within individual bi-parental families. Only those populations and haplotypes
that were significant in one or both years are presented.
b
UF x Surefire
Bloom 2012 (GDD)
Bloom 2011 (GDD)
(ahkn x giks)
LG4 haplotypes

N

c

Means

d

P value

N

Means

P value

e
A

B

0.0002

55/18

396 /363

A

A

0.07

35/37

379 /394

k/no k

55/18

347 /308

a/no a

35/37

328 /344

25-14-20 x 25-02-29
(aghj x abcd)
G4 Haplotypes
b/no b

32/21

317 /302

c/no c

29/21

305 /317

M172 x 25-02-29
(defh x abcd)
G4 Haplotypes
e/no e
Montmorency x 25-0229
(dilo x abcd)
G4 Haplotypes
i/no i
RS x ET
(bdgi x amnp)
G4 Haplotypes
m/no m

Bloom 2011 (GDD)
N
Means
P value

A

B

0.05

B

0.03

34/24

361 /355

A

A

0.14

31/24

351 /365

A

303 /289

N

B

Means

28/20

329 /314

8/14

0.0001

Bloom 2012 (GDD)
N
Means
P value

0.0007

A

A

A

0.23

A

B

0.02

57/51

A

361 /344

A

A

B

N

Means

0.13

28/20

374 /354

0.06

<0.0001

Bloom 2012 (GDD)

P value

Bloom 2011 (GDD)
Means
P value
319 /350

A

Bloom 2012 (GDD)
N
Means
P value

Bloom 2011 (GDD)

N

B

A

Bloom 2011 (GDD)
N
Means
P value
56/48

A

A

P value
B

0.04

Bloom 2012 (GDD)
N
Means
P value
8/14

A

373 /397

B

0.05

a

Region analyzed defined by peach physical map location 7,309,282-9,148,953 bp (See Figure
2.16 for a description of the G4 haplotypes.)
b
c
d
e

Growing Degree Days (GDD) with a base of 4.4 degrees C
Number of individuals
Means with the same letter within a row are not significantly different (P>0.05)
The allelic state significantly associated with the increased trait value is identified in bold
95

Table 2.9: Phenotypic means for fruit, pit and mesocarp weights (g) in 2011 for the presence or absence of the G2 G2SSR1566
a
haplotypes for all sour cherry individuals from all five bi-parental families. Parental genotypes for the G2 region are: ‘M172’ (2447),
‘25-02-29’ (2467), ‘Montmorency’ (4488), ‘25-14-20’ (4489), ‘UF’ (2449), ‘Surefire’ (4458), ‘RS’ (4478) and ‘ET’ (4678).

N
G2 haplotypes

b

Fruit Weight 2011
c
P value
Means
A

A

A

A

A

A

A

B

A

A

A

B

A

A

128/146 5.64 /5.30

4/no 4

249/17

5.41 /5.52

30/236

6/no 6
7/no 7
8/no 8
9/no 9

Mesocarp Weight 2011
N
Means
P value

d

2/no 2
5/no 5

N

Pit Weight 2011
Means
P value

5.55 /5.55

116/158 5.24 /5.62
122/152 5.45 /5.47

139/135 5.11 /5.38
70/196

5.21 /5.50

A

A

A

A

A

A

A

A

A

A

A

B

A

A

0.07

126/145 0.34 /0.34

0.74

245/17

0.34 /0.34

32/239

0.89
0.05
0.94
0.0002
0.20

0.32 /0.34

116/155 0.34 /0.34
122/149 0.35 /0.33

137/134 0.33 /0.35
71/200

0.32 /0.35

A

A

A

A

0.89

A

A

0.52

A

B

0.03

A

A

A

B

0.0003

A

A

0.24

0.62

126/145 5.32 /4.98

0.85

245/17

5.14 /5.18

32/239

0.19
0.51
0.11
0.007
0.07

5.32 /5.11

116/155 4.91 /5.31
122/149 5.10 /5.17

137/134 4.81 /5.47
71/200

4.95 /5.21

a

Region analyzed defined by peach physical map location 15,666894-15,667,139 bp (See Figure 2.13 and Table 2.3 for
haplotype region and SSR marker information) Number of individuals
b
c
d

Number of individuals
Means with the same letter within a row are not significantly different (P>0.05)
The allelic states significantly associated with the increased trait value for at least one trait are identified in bold

96

0.06

0.73

a

Table 2.10: Phenotypic means for fruit, pit and mesocarp weights (g) in 2011 for the presence or absence of the G3 haplotypes for all
sour cherry individuals for all five bi-parental families. Parental genotypes for the G3 haplotypes are: ‘M172’ (degh), ‘25-02-29’
(abcd), ‘Montmorency’ (acjn), ‘25-14-20’ (adjk), ‘UF’ (ehjk), ‘Surefire’ (cjjk), ‘RS’ (acdj), and ‘ET’ (dgjl).

N
G3 haplotypes

b

Fruit Weight 2011
c
P value
Means
A

B

A

B

A

B

A

A

91/175

A

B

6.07 /5.09

39/240

A

B

5.85 /5.29

79/197

A

B

A

A

A

A

A

A

116/146 5.04 /5.72

b/no b

84/176

5.18 /5.59

25/258

d/no d
e/no e
g/no g
h/no h

N

P value

Means

Mesocarp Weight 2011
Means
N
P value

d

a/no a
c/no c

Pit Weight 2011

4.31 /5.53

140/146 5.43 /5.45

6.12 /5.04

j/no j

135/128 5.37 /5.49

k/no k

73/190

5.12 /5.53

l/no l

9/280

6.42 /5.36

n/no n

25/258

4.31 /5.53

0.0003
0.04
<0.0001
0.92
<0.0001
0.009
<0.0001

A

A

115/147 0.33 /0.34
85/175

0.32 /0.35

A

B

25/258

A

B

A

A

94/172

A

0.35 /0.33

A

39/239

A

B

0.39 /0.33

79/196

A

B

A

A

0.29 /0.34

139/126 0.35 /0.33

0.37 /0.32

0.54

134/129 0.33 /0.34

0.07

72/191

A

0.28

9/280

0.38 /0.34

B

<0.0001

25/258

0.29 /0.34

A

B

A

B

0.03

A

B

4.03 /5.21

<0.0001

A

A

0.84

91/173

A

B

5.72 /4.78

<0.0001

39/238

A

B

5.46 /4.98

0.02

79/195

A

B

5.76 /4.74

<0.0001

A

A

0.69

A

A

A

A

0.29

A

B

<0.0001

0.27

115/145 4.72 /5.39

0.02

84/174

4.86 /5.27

25/255

0.0004
0.23
0.23
<0.0001
0.0001

139/125 5.10 /5.14

0.39

133/128 5.08 /5.15

0.31 /0.35

A

B

0.001

71/190

4.87 /5.18

A

A

0.11

9/277

6.04 /5.05

A

B

0.0004

25/255

4.03 /5.21

a

Region analyzed defined by peach physical map location 2,738,097-4,755,490 bp (See Figure 2.15 for descriptions of the
haplotypes)
b
c
d

Number of individuals
Means with the same letter within a row are not significantly different (P>0.05)
The allelic states significantly associated with the increased trait value for at least one trait are identified in bold

97

0.0002

0.15

a

Table 2.11: Phenotypic means for fruit, pit and mesocarp weights (g) in 2011 for the presence or absence of the G5 haplotypes for all
sour cherry individuals for all five bi-parental families. Parental genotypes for the G5 haplotypes are: ‘M172’ (dfhj), ‘25-02-29’
(abcd), ‘Montmorency’ (aceh), ‘25-14-20’ (bdfh), ‘UF’ (ddfn), ‘Surefire’ (ceil), ‘RS’ (bceh), and ‘ET’ (djkn).
Fruit Weight 2011
b
N

Means

a/no a

80/195

b/no b

132/137 5.37 /5.46

G5 haplotypes

c/no c
d/no d
e/no e
f/no f
h/no h

c

P value

N

Means

P value

4.91 /5.60

A

B

0.0002

80/194

0.31 /0.35

A

B

0.0009

A

A

0.67

A

A

0.53

A

A

A

A

A

A

A

A

A

A

A

A

A

B

A

A

A

A

A

A

A

A

A

A

Mesocarp Firmness 2011
Means
N
P value

d

161/114 5.24 /5.62
193/84
69/206

5.44 /5.30
5.37 /5.41

115/160 5.65 /5.21
103/172 5.18 /5.53

i/no i

29/246

5.79 /5.35

j/no j

58/217

k/no k

8/267

6.57 /5.37

l/no l

25/250

5.84 /5.36

43/232

n/no n

Pit Weight 2011

0.06
0.47
0.87
0.03
0.07

132/137 0.34 /0.34
159/115 0.34 /0.34
193/83
70/204

0.34 /0.33
0.34 /0.34

113/161 0.34 /0.34
103/171 0.34 /0.34

0.28

29/245

0.34 /0.34

5.85 /5.28

A

B

0.003

58/216

A

A

0.28

8/266

0.39 /0.34

A

A

0.18

26/249

0.33 /0.34

A

A

43/232

5.87 /5.31

0.08

0.59
0.46
0.91
0.85
0.67

4.60 /5.28

A

B

0.0002

A

A

0.61

A

A

0.07

A

A

0.52

A

A

A

B

0.01

A

80/192

A

0.06

A

A

0.18

131/136 5.04 /5.13
158/114 4.93 /5.29
192/82
68/203

5.12 /4.99
5.03 /5.10

112/160 5.36 /4.88
102/170 4.86 /5.21

0.79

0.78

28/244

5.55 /5.02

0.37 /0.33

A

B

0.0001

57/215

5.48 /4.97

A

B

0.006

A

A

0.12

8/264

6.17 /5.05

A

A

0.29

A

A

0.55

25/247

5.51 /5.04

A

A

0.17

A

B

43/229

A

A

0.10

0.37 /0.33

0.02

5.50 /5.00

a

Region analyzed defined by peach physical map location 16,125,708-18,100,331 bp (See Figure 2.16 for descriptions of the
haplotypes)
b
c
d

Number of individuals
Means with the same letter within a row are not significantly different (P>0.05)
The allelic states significantly associated with increased trait values for at least one trait are identified in bold
98

Table 2.12: Phenotypic means for fruit, pit and mesocarp weight (g) in 2011 for the presence or absence of the G6 G6SSR2208
a
haplotypes for all sour cherry individuals for all five bi-parental families. Parental genotypes for the G6 haplotypes are: ‘M172’ (3 5
null null), ‘25-02-29’ (1 3 5 null), ‘Montmorency’ (1 4 5 null), ‘25-14-20’ (2 3 null null), ‘UF’ (1 3 3 null), ‘Surefire’ (1 3 5 null),
‘RS’ (1 2 5 null), and ‘ET’ (3 5 null null).

N
G6 haplotypes
1/no 1
2/no 2
3/no 3
4/no 4
5/no 5

b

Fruit Weight 2011
c
P value
Means

Pit Weight 2011
N

P value

Means

Mesocarp Weight 2011
Means
N
P value

d
A

A

A

A

A

B

A

B

A

A

82/168 5.21 /5.63
40/215 5.09 /5.51

188/60 5.66 /4.98
20/245 4.19 /5.53
174/75 5.47 /5.55

0.06
0.15
0.0008
<0.0001
0.71

A

B

A

A

A

B

A

B

A

A

81/168 0.33 /0.35
39/215 0.33 /0.34

188/59 0.35 /0.31
20/244 0.29 /0.34
174/74 0.34 /0.35

0.03
0.33
0.0004
0.0001
0.11

a

A

A

A

A

0.19

A

B

0.0006

A

B

<0.0001

A

A

81/166 4.91 /5.30
39/213 4.81 /5.19

186/59 5.34 /4.67
20/242 3.90 /5.21
172/74 5.15 /5.23

0.07

Region analyzed defined by peach physical map location 15,666894-15,667,139 bp (See Figure 2.17 and Table 2.3 for
descriptions of the haplotypes and SSR marker information)
b
c
d

Number of individuals
Means with the same letter within a row are not significantly different (P>0.05)
The allelic state significantly associated with the increased trait value is identified in bold

99

0.72

2

Table 2.13: Phenotypic means for fruit firmness (g/mm ) in 2011 for the presence or absence of
a

the G2 G2SSR1566 haplotypes for all sour cherry individuals from all five bi-parental families.
Parental genotypes for the G2 haplotypes are: ‘M172’ (2447), ‘25-02-29’ (2467),
‘Montmorency’ (4488), ‘25-14-20’ (4489), ‘UF’ (2449), ‘Surefire’ (4458), ‘RS’ (4478) and ‘ET’
(4678).

N
G2 haplotypes

b

Fruit Firmness 2011
c
P value
Means

d
A

A

0.87

A

A

0.47

A

B

0.02

114/154

A

142 /141

A

0.65

120/148

A

145 /139

B

0.02

134/134

A

A

0.10

A

B

2/no 2

125/143

141 /142

4/no 4

251/17

141 /145

5/no 5

31/237

135 /142

6/no 6
7/no 7
8/no 8
9/no 9

71/197

139 /144

137 /143

0.02

a

Region analyzed defined by peach physical map
location 15,666894-15,667,139 bp (See Figure 2.13
and Table 2.3 for haplotype region and SSR marker
information)
b

Number of individuals

c

Means with the same letter within a row are not
significantly different (P>0.05)
d

The allelic states significantly associated with the
increased trait value are identified in bold

100

2

Table 2.14: Phenotypic means for fruit firmness (g/mm ) in 2011 for the presence or absence of
a

the G3 haplotypes for all sour cherry individuals for all five bi-parental families. Parental
genotypes for the G3 haplotypes are: ‘M172’ (efgh), ‘25-02-29’ (abcd), ‘Montmorency’ (ijno),
‘25-14-20’ (adjk), ‘UF’ (ehjk), ‘Surefire’ (jjop), ‘RS’ (adjo), and ‘ET’ (fjlm).

Fruit Firmness 2011
c
P value
N
Means
b

G3 haplotypes

d
A

A

0.08

A

A

0.14

A

A

0.94

A

B

0.03

93/174

A

141 /142

A

0.75

38/241

A

144 /141

A

0.34

79/197

A

A

0.83

A

B

A

B

0.004

A

A

0.70

A

A

a/no a

115/148 144 /139

b/no b

85/176

c/no c
d/no d
e/no e
g/no g
h/no h

144 /140

150/115 142 /142

138/128 144 /139

142 /141

j/no j

136/128 137 /146

k/no k

75/189

137 /144

l/no l

8/284

146 /141

n/no n

25/261

142 /142

0.001

0.97

a

Region analyzed defined by peach physical map
location 2,738,097-4,755,490 bp (See Figure 2.15
for descriptions of the haplotypes)
b

Number of individuals

c

Means with the same letter within a row are
not significantly different (P>0.05)
d

The allelic states significantly associated with
increased trait values are identified in bold

101

2

Table 2.15: Phenotypic means for fruit firmness (g/mm ) in 2011 for the presence or absence of
a

the G5 haplotypes for all sour cherry individuals for all five bi-parental families. Parental
genotypes for the G5 haplotypes are: ‘M172’ (dfhj), ‘25-02-29’ (abcd), ‘Montmorency’ (aceh),
‘25-14-20’ (bdfh), ‘UF’ (ddfn), ‘Surefire’ (ceil), ‘RS’ (bceh), and ‘ET’ (djkn).

Fruit Firmness 2011
c
P value
N
Means
b

G5 haplotypes

d
A

A

A

B

A

A

0.80

195/84

A

141 /144

A

0.43

e/no e

72/205

A

B

0.01

f/no f

114/163 141 /143

A

A

0.35

h/no h

103/174 144 /141

A

A

32/245

A

134 /143

B

0.02

58/219

A

B

0.008

7/270

A

164 /142

A

26/251

A

134 /143

B

0.003

44/233

A

A

0.16

a/no a
b/no b
c/no c
d/no d

i/no i
j/no j
k/no k
l/no l
n/no n

80/197

142 /142

132/139 146 /139
160/117 143 /142

137 /144

150 /140

138 /143

0.97
0.01

0.33

0.16

a

Region analyzed defined by peach physical map
location 16,125,708-18,100,331 bp (See Figure
2.16 for descriptions of the haplotypes)
b

Number of individuals

c

Means with the same letter within a row are not
significantly different (P>0.05)
d

The allelic states significantly associated with
increased trait values are identified in bold

102

2

Table 2.16: Phenotypic means for fruit firmness (g/mm ) in 2011 for the presence or absence of
a

the G6 G6SSR2208 haplotypes for all sour cherry individuals for all five bi-parental families.
Parental genotypes for the G6 haplotypes are: ‘M172’ (3 5 null null), ‘25-02-29’ (1 3 5 null),
‘Montmorency’ (1 4 5 null), ‘25-14-20’ (2 3 null null), ‘UF’ (1 3 3 null), ‘Surefire’ (1 3 5 null),
‘RS’ (1 2 5 null), and ‘ET’ (3 5 null null).

Fruit Firmness 2011
c
P value
N
Mean
b

G6 haplotypes

d
A

B

0.006

A

A

0.99

A

A

0.32

A

A

0.35

A

A

0.59

1/no 1

82/169 136 /143

2/no 2

39/218 141 /141

3/no 3

189/60 141 /138

4/no 4
5/no 5

20/247 138 /142
176/74 140 /142

a

Region analyzed defined by peach physical
map location 15,666894-15,667,139 bp (See
Figure 2.17 and Table 2.3 for descriptions of the
haplotypes and SSR marker information)
b

Number of individuals

c

Means with the same letter within a row are
not significantly different (P>0.05)
d

The allelic state significantly associated with
the increased trait value is identified in bold

103

a

Table 2.17: Phenotypic means for flesh color in 2011 for the presence or absence of the G3
b

haplotypes for all sour cherry individuals for all five bi-parental families. Parental genotypes
for the G3 haplotypes are: ‘M172’ (clnu), ‘25-02-29’ (nnop), ‘Montmorency’ (fhno), ‘25-14-20’
(bdhn), ‘UF’ (bcdn), ‘Surefire’ (efgh), ‘RS’ (khnp), and ‘ET’ (eglu).

Flesh color 2011
N
G3 Flesh color haplotypes

c

Mean

d

P value

e
A

B

84/206

A

B

2.71 /3.43

0.0002

d/no d

53/217

A

B

4.65 /2.94

<0.0001

e/no e

36/233

A

B

4.06 /3.15

<0.0001

f/no f

65/219

A

B

2.87 /3.36

0.02

g/no g

40/250

3.13 /3.24

A

A

0.66

h/no h

84/197

3.06 /3.32

A

A

k/no k

14/272

A

B

4.14 /3.20

0.01

l/no l

48/220

A

B

<0.0001

n/no n

235/34

A

B

3.20 /3.79

0.03

o/no o

98/177

A

B

2.41 /3.75

<0.0001

p/no p

97/172

A

B

4.19 /2.76

<0.0001

u/no u

107/256

A

A

0.59

b/no b
c/no c

a

49/236

3.63 /3.13

3.96 /3.13

3.21 /3.30

0.04

0.17

Washington State University color card rating (See Figure 2.2)

b

Region analyzed defined by peach physical map location
10,675,150-13,406,263 bp (See Figure 2.20 for the descriptions
of the haplotypes)
c

Number of individuals

d

Means with the same letter within a row are not significantly
different (P>0.05)
e

The allelic states significantly associated with increased trait
values are identified in bold

104

a

Table 2.18: Phenotypic means for flesh color in 2011 for the presence or absence of the G3
b

haplotypes for all sour cherry individuals within individual families. Only haplotypes with
significant differences are presented.
Flesh color 2011
c
d
e
P value
G3 haplotypes
N
Means
UF x Surefire
(bcdn x efgh)
d/no d

27/42

e/no e

26/43

c/no c
25-14-20 x 25-02-29
(bdhn x nnop)
d/no d

37/32

p/no p

29/29

o/no o
M172 x 25-02-29
(clnu x nnop)
p/no p

27/31

l/no l

35/42

o/no o
Montmorency x 25-02-29
(fhno x nnop)
p/ no p

39/39

o/no o
RS x ET
(hknp x eglu)
p/ no p

33/8

13/11

l/no l

13/11

a

A
B
4.63 /2.43
A
B
3.92 /2.91
A
B
2.62 /4.06

A
B
4.68 /2.97
A
B
4.52 /2.90
A
B
2.96 /4.35

<0.0001

A
B
4.62 /3.36
A
B
4.62 /3.36

20/22

<0.0001

A
B
3.90 /1.63
A
B
2.33 /4.13

37/41

<0.0001

A
B
3.92 /2.20
A
B
3.71 /2.48
A
B
2.13 /3.90

25/33

<0.0001

0.03

0.002
<0.0001

<0.0001
0.0003

<0.0001
<0.0001

0.002

0.02

Washington State University color card rating (See Figure 2.2)

b

Region analyzed defined by peach physical map location 10,675,150-13,406,263
bp (See Figure 2.20 for the descriptions of the haplotypes)
c

The allelic states significantly associated with increased trait values are identified
in bold
d
e

Number of individuals
Means with the same letter within a row are not significantly different (P>0.05)
105

Table 2.19: Phenotypic means for malic acid (mg/ml) in 2011 for the presence or absence of the
a
condensed G5 haplotypes for all sour cherry individuals for all five bi-parental families.
Parental genotypes for the G5 haplotypes are: ‘M172’ (1236), ‘25-02-29’ (2346),
‘Montmorency’ (2345), ‘25-14-20’ (1236), ‘UF’ (1236), ‘Surefire’ (1123), ‘RS’ (1234) and ‘ET’
(1146).

Malic Acid (mg/ml) 2011
b
c
P value
N
Means
G5 haplotypes

d

99/68

1.55 /1.29

A

B

0.001

23/76

A

B

1.84 /1.46

0.0004

125/42

A

1.48 /1.34

A

0.13

34/91

A

A

0.36

126/41

A

1.43 /1.49

A

28/98

A

1.30 /1.47

A

0.16

4/no 4

78/89

A

B

1.29 /1.58

0.0003

Two 4's/one 4

10/68

1.31 /1.29

A

A

0.88

5/no 5

13/154

1.06 /1.48

A

B

0.0003

6/no 6

110/57

1.47 /1.39

A

A

0.34

17/93

A

A

0.31

1/no 1
Two 1's/one 1
2/no 2
Two 2's/one 2
3/no 3
Two 3's/one 3

Two 6's/one 6

1.41 /1.50

1.37 /1.49

0.54

a

Region analyzed defined by peach physical map location
689,941-1,463,960 bp (See Figure 2.21 for descriptions of the
haplotypes)
b

Number of individuals

c

Means with the same letter within a row are not significantly
different (P>0.05)
d

The allelic states significantly associated with increased trait
values are identified in bold

106

Figure 2.1: Pedigrees of the five bi-parental families used in this study. Populations are colored white with the progeny number
below. Grandparents are colored grey, while parents are colored black. If individuals are both parents and grandparents, they are
colored black.

107

Figure 2.2: Washington State University flesh color card rating scale used to determine flesh
color rating for sour cherry individuals.

108

Figure 2.3: Genome Studio (Illumina Inc. 2011) SNP dosage calls were done for each marker. Sweet cherry (yellow) individuals
were included to help define the two homozygous (AAAA and BBBB) classes and the balanced heterozygous class (AABB).
Determining dosage was necessary to build haplotypes.

109

Figure 2.4: Reconstruction of a ~1.2 Mb region spanning the self-incompatibility S-locus and its inheritance in (a) Sweet cherry, with
four parental haplotypes (1–4) and (b) Sour cherry, with eight parental haplotypes (1–8). Identical haplotypes have the same
background colors. Haplotypes are shown for five sweet cherry and two sour cherry seedlings. Monomorphic SNPs within cross-over
regions are highlighted in grey. Genotypes indicated as “u” are for an unresolved polymorphic SNP in sour cherry (Peace et al. 2013,
Figure 4).

110

Figure 2.4 (cont’d)

111

Figure 2.5: Phenotypic distributions for bloom growing degree days (GDD) in 2011 for all
populations and each of the five bi-parental populations. Parental values are shown in individual
populations when data is available.

Bloom (GDD) 2011 All Populations
100
0
256-274275-293294-311312-329330-348349-366367-384385-403404-421422-440

UF x Surefire
UF

50
0

256-274275-293294-311312-329330-348349-366367-384385-403404-421422-440

M172 x 25-02-29

M172 & 25-02-29
50
0

256-274275-293294-311312-329330-348349-366367-384385-403404-421422-440

25-14-20 x 25-02-29

50

25-14-20 & 25-02-29

0
256-274275-293294-311312-329330-348349-366367-384385-403404-421422-440

Montmorency x 25-02-29

20

25-02-29

Montmorency

0
256-274275-293294-311312-329330-348349-366367-384385-403404-421422-440

RS x ET

RS

10
0

256-274275-293294-311312-329330-348349-366367-384385-403404-421422-440
112

Figure 2.6: Phenotypic distributions for bloom growing degree days (GDD) in 2012 for all
populations and each of the five bi-parental populations. Parental values are shown in individual
populations when data is available.

Bloom (GDD) 2012 All Populations
200
0
275-296 297-318 319-339 340-360 361-381 382-403 404-424 425-445 446-466 467-488

UF x Surefire
UF

50
0

275-296 297-318 319-339 340-360 361-381 382-403 404-424 425-445 446-466 467-488

M172 x 25-02-29
100

M172 & 25-02-29

0
275-296 297-318 319-339 340-360 361-381 382-403 404-424 425-445 446-466 467-488

25-14-20 x 25-02-29
25-14-20 & 25-02-29
50
0
275-296 297-318 319-339 340-360 361-381 382-403 404-424 425-445 446-466 467-488

Montmorency x 25-02-29
50

25-02-29

Montmorency

0
275-296 297-318 319-339 340-360 361-381 382-403 404-424 425-445 446-466 467-488

RS x ET
RS

10
0

275-296 297-318 319-339 340-360 361-381 382-403 404-424 425-445 446-466 467-488
113

Figure 2.7: Phenotypic distributions for fruit weight (g) in 2011 for all populations and each of
the five bi-parental populations. Parental values are shown in individual populations.

Fruit Weight (g) 2011 All Populations
100
0
1.6-2.8

2.9-3.9

4.0-5.0

5.1-6.2

6.3-7.3

7.4-8.4

8.5-9.6 9.7-10.7 10.8-11.8 11.9-13.0

UF x Surefire
Surefire

20

UF

0

1.6-2.8

2.9-3.9

4.0-5.0

5.1-6.2

6.3-7.3

7.4-8.4

8.5-9.6 9.7-10.7 10.8-11.8 11.9-13.0

M172 x 25-02-29
25-02-29

50

M172

0
1.6-2.8

2.9-3.9

4.0-5.0

5.1-6.2

6.3-7.3

7.4-8.4

8.5-9.6 9.7-10.7 10.8-11.8 11.9-13.0

25-14-20 x 25-02-29
25-14-20 & 25-02-29

50
0
1.6-2.8

2.9-3.9

4.0-5.0

5.1-6.2

6.3-7.3

7.4-8.4

8.5-9.6 9.7-10.7 10.8-11.8 11.9-13.0

Montmorency x 25-02-29
Montmorency & 25-02-29
20
0
1.6-2.8

2.9-3.9

4.0-5.0

5.1-6.2

6.3-7.3

7.4-8.4

8.5-9.6 9.7-10.7 10.8-11.8 11.9-13.0

7.4-8.4

8.5-9.6 9.7-10.7 10.8-11.8 11.9-13.0

RS x ET
RS & ET

10
5
0

1.6-2.8

2.9-3.9

4.0-5.0

5.1-6.2

6.3-7.3
114

Figure 2.8: Phenotypic distributions for pit weight (g) in 2011 for all populations and each of the
five bi-parental populations. Parental values are shown in individual populations.

Pit Weight (g) 2011 All Populations
100
0
0.10-0.150.16-0.200.21-0.250.26-0.300.31-0.360.37-0.410.42-0.460.47-0.510.52-0.560.57-0.61

UF x Surefire
50

Surefire

UF

0
0.10-0.15 0.16-0.20 0.21-0.25 0.26-0.30 0.31-0.36 0.37-0.41 0.42-0.46 0.47-0.51 0.52-0.56 0.57-0.61

M172 x 25-02-29

25-02-29

M172

20
0
0.10-0.15 0.16-0.20 0.21-0.25 0.26-0.30 0.31-0.36 0.37-0.41 0.42-0.46 0.47-0.51 0.52-0.56 0.57-0.61

25-14-20 x 25-02-29
50

25-14-20 & 25-02-29

0
0.10-0.15 0.16-0.20 0.21-0.25 0.26-0.30 0.31-0.36 0.37-0.41 0.42-0.46 0.47-0.51 0.52-0.56 0.57-0.61

Montmorency x 25-02-29

20

25-14-20 & 25-02-29

0
0.10-0.15 0.16-0.20 0.21-0.25 0.26-0.30 0.31-0.36 0.37-0.41 0.42-0.46 0.47-0.51 0.52-0.56 0.57-0.61

RS x ET
10

RS

ET

0
0.10-0.15 0.16-0.20 0.21-0.25 0.26-0.30 0.31-0.36 0.37-0.41 0.42-0.46 0.47-0.51 0.52-0.56 0.57-0.61
115

Figure 2.9: Phenotypic distributions for mesocarp weight (g) in 2011 for all populations and
each of the five bi-parental populations. Parental values are shown in individual populations.

Mesocarp Weight (g) 2011 All Populations
100
0
1.56-2.65 2.66-3.73 3.74-4.82 4.83-5.90 5.91-6.99 7.00-8.07 8.08-9.16 9.17-10.2 10.2-11.3 11.3-12.4

UF x Surefire

Surefire

UF

20
0

1.56-2.65 2.66-3.73 3.74-4.82 4.83-5.90 5.91-6.99 7.00-8.07 8.08-9.16 9.17-10.2 10.2-11.3 11.3-12.4

M172 x 25-02-29
50

25-02-29

M172

0
1.56-2.65 2.66-3.73 3.74-4.82 4.83-5.90 5.91-6.99 7.00-8.07 8.08-9.16 9.17-10.2 10.2-11.3 11.3-12.4

25-14-20 x 25-02-29
25-14-20 & 25-02-29
50
0
1.56-2.65 2.66-3.73 3.74-4.82 4.83-5.90 5.91-6.99 7.00-8.07 8.08-9.16 9.17-10.2 10.2-11.3 11.3-12.4

Montmorency x 25-02-29
20

Montmorency & 25-02-29

0
1.56-2.65 2.66-3.73 3.74-4.82 4.83-5.90 5.91-6.99 7.00-8.07 8.08-9.16 9.17-10.2 10.2-11.3 11.3-12.4

RS x ET
20

RS & ET

0
1.56-2.65 2.66-3.73 3.74-4.82 4.83-5.90 5.91-6.99 7.00-8.07 8.08-9.16 9.17-10.2 10.2-11.3 11.3-12.4
116

2

Figure 2.10: Phenotypic distributions for fruit firmness (g/mm ) in 2011 for all populations and
each of the five bi-parental populations. Parental values are shown in individual populations
when data is available.

Fruit Firmness (g/mm2) 2011 All Populations
100
0
103-117 118-130 131-143 144-156 157-169 170-182 183-195 196-208 209-221 222-234

UF x Surefire
50

Surefire

UF

0
103-117 118-130 131-143 144-156 157-169 170-182 183-195 196-208 209-221 222-234

M172 x 25-02-29
50

M172 25-02-29

0
103-117 118-130 131-143 144-156 157-169 170-182 183-195 196-208 209-221 222-234

25-14-20 x 25-02-29

25-14-20 & 25-02-29
20
0

103-117 118-130 131-143 144-156 157-169 170-182 183-195 196-208 209-221 222-234

Montmorency x 25-02-29
20

Montmorency 25-02-29

0
103-117 118-130 131-143 144-156 157-169 170-182 183-195 196-208 209-221 222-234

RS x ET
RS

10
0

103-117 118-130 131-143 144-156 157-169 170-182 183-195 196-208 209-221 222-234
117

Figure 2.11: Phenotypic distributions for flesh color based on Washington State University’s
flesh color rating in 2011 for all populations and each of the five bi-parental populations.
Parental values are shown in individual populations when data is available.

Flesh Color Rating 2011 All Populations
100
0
1

2

3

4

5

UF x Surefire
UF & Surefire

50
0
1

2

3

4

5

M172 x 25-02-29
M172 & 25-02-29

50
0
1

2

3

4

5

25-02-29

25-14-20

4

5

25-14-20 x 25-02-29
50
0
1

2

3

Montmorency x 25-02-29
Montmorency

20

25-02-29

0
1

2

3

4

5

RS x ET
RS

20
0
1

2

3
118

4

5

Figure 2.12: Phenotypic distributions for malic acid content (mg/ml) in 2011 for all populations
and each of the five bi-parental populations. Parental values are shown in individual populations
when data is available.

Malic Acid (mg/ml) 2011
50
0

0.40-0.64 0.65-0.88 0.89-1.12 1.13-1.36 1.37-1.60 1.61-1.84 1.85-2.08 2.09-2.32 2.34-2.56 2.57-2.80

UF x Surefire
UF

20

Surefire

0
0.40-0.64 0.65-0.88 0.89-1.12 1.13-1.36 1.37-1.60 1.61-1.84 1.85-2.08 2.09-2.32 2.34-2.56 2.57-2.80

M172 x 25-02-29
20

M172 & 25-02-29

0
0.40-0.64 0.65-0.88 0.89-1.12 1.13-1.36 1.37-1.60 1.61-1.84 1.85-2.08 2.09-2.32 2.34-2.56 2.57-2.80

25-14-20 x 25-02-29
20

25-02-29

25-14-20

0
0.40-0.64 0.65-0.88 0.89-1.12 1.13-1.36 1.37-1.60 1.61-1.84 1.85-2.08 2.09-2.32 2.34-2.56 2.57-2.80

Montmorency x 25-02-29
10

Montmorency & 25-02-29

0
0.40-0.64 0.65-0.88 0.89-1.12 1.13-1.36 1.37-1.60 1.61-1.84 1.85-2.08 2.09-2.32 2.34-2.56 2.57-2.80

RS x ET

RS

10
0
0.40-0.64 0.65-0.88 0.89-1.12 1.13-1.36 1.37-1.60 1.61-1.84 1.85-2.08 2.09-2.32 2.34-2.56 2.57-2.80
119

Figure 2.13: Bloom and fruit trait QTLs from diploid Prunus (peach and sweet cherry) that were targets of validation in tetraploid
sour cherry.

120

Figure 2.14: The twelve haplotypes identified in sour cherry for the G1 region used to test for
the bloom time QTL.

LG1
NCBI SS#

Peach physical

a
a b c d e f g h i j k
map position
ss490548534
45021181
A B B B A A B B A B B
ss490548538
45028492
A B B B A A A B A B B
ss490558944
45077993
A A A B A A B A A B A
ss490548551
45163766
B B A A B B A A B B A
ss490546931
45169388
B B B B A A B B B B B
ss490548555
45210413
B B B B B B B B B A B
ss490559090
45226245
A A A A A A A B A A B
ss490546935
45299782
B B B B A A B B B B A
ss490548559
45322930
A B A A A A B B A B A
ss490548567
45402154
B B B B B B B A B B B
ss490546939
45418879
B A A A B B A A B A A
ss490559081
45469715
A B A A A A A B A A A
ss490548571
45473214
B B A A A A A B B A A
ss490548575
45535084
B A B B B A A A B A A
ss490548589
45633267
B B B A B B A A B A B
ss490548593
45680542
B A A B B B A A B A A
ss490559189
45682217
A B B A A A B B A B B
ss490546951
45748141
B A A A B B A A B A A
ss490548610
45823056
B B A A B B A A B A A
ss490548614
45924398
A A B B A A A B A A A
ss490546967
46207321
B B B B A A B B B B B
ss490548639
46237075
B A A A B B A A B A A
ss490548643
46277304
A B B B A A B B A B B
ss490548655
46402818
B A B B B B B A B A A
ss490546979
46512070
B B B B A A B B A B B
ss490548667
46530908
B A B A B B A B B A B
ss490548680
46635504
B A A A B B A A B A A
ss490548692
46751928
B A A A B B A A B A A
a
distances according to the Peach v1.0 ‘dhLovell’ genome assembly
(International Peach Genome Initiative; www.rosaceae.org/peach/genome)
(Verde et al. 2013)

121

l
B
B
A
B
B
B
A
B
B
B
A
A
A
A
A
A
B
A
A
A
B
A
B
B
B
A
A
A

Figure 2.15: The 21 haplotypes identified in sour cherry for the G2 region used to test for bloom time and fruit size/fruit firmness
QTLs. Twenty-one unique haplotypes were found, so SSR marker G2SSR1566 was used to “condense” haplotypes based on marker
score to 7 haplotypes.
LG2 Marker/ Peach physical
NCBI SS#
ss490549138
CPSCT038
ss490549172
ss490549184
ss490549187
ss490549192
ss490549196
ss490549216
ss490549227
ss490549238
ss490549254
b

(CNR12)
ss490549270
G2SSR1566
ss490549287
ss490549295
ss490549311
ss490547191
ss490549319
ss490549323
ss490549331
ss490549335
ss490547200
ss490547204

a

map distance (bp) d b
14926622
A A
15057199
204 185
15084429
A A
15127760
A B
15129278
B A
15162260
B A
15172649
B A
15337787
B A
15372418
B B
15492297
B B
15598480
B A

l

e

h

o

k

r

s

m

a

A
185
A
B
A
A
A
A
B
B
A

A
190
A
B
A
A
A
A
B
B
A

A
190
A
B
A
A
A
A
B
B
A

A
190
A
B
A
A
A
B
B
B
A

A
192
A
B
A
A
A
B
B
B
A

A
A
B
A
A
A
A
B
B
A

A A A
- null 185
A A A
B B B
A A A
A A A
A A A
B B A
B B B
B B B
A A A

A
4
A
B
B
A
A
A
A
B
B
A

B
4
A
A
B
B
B
B
A
A
A
B

B
4
A
B
B
A
A
A
A
B
A
A

B
4
A
B
B
A
A
A
A
B
A
A

B
4
A
B
B
A
A
A
A
B
A
A

A
4
A
B
B
A
A
A
A
B
B
A

B
4
A
B
B
A
A
A
A
B
A
A

q

c

g

p

j

n

t

u

f

i

A
192
A
B
A
A
A
A
B
B
A

A
192
A
B
A
B
B
A
B
A
B

A
192
B
A
B
B
B
A
B
A
B

A
190
B
A
B
B
B
B
B
A
B

A
192
B
A
B
B
B
B
B
A
B

A
192
B
A
B
B
B
B
B
A
B

A
B
A
B
B
B
B
B
A
B

A B A
- 190 null
B B A
A A A
B B B
B B B
B B B
B B B
B A B
A B B
B B B

A
7
A
B
B
A
A
A
A
B
A
A

B
7
A
A
B
B
B
B
B
A
A
B

B
7
A
A
B
B
B
B
B
A
A
B

B
8
B
A
B
B
B
B
B
A
A
B

B
8
B
A
B
B
B
A
A
A
A
B

B
8
A
A
B
B
B
B
B
A
A
B

B
8
B
A
B
B
B
A
A
A
A
B

B
8
B
A
B
B
B
A
A
A
A
B

15647989
15658996
15667139
15747822
15778222
15846482
15863936
15873315
15873418
15894385
15894441
16005866
16111179

B
2
A
A
B
B
B
B
B
A
A
B

A
4
A
B
B
A
A
A
A
B
B
A

122

B
5
A
B
B
A
A
A
A
B
A
A

A
6
A
B
B
A
A
A
A
B
B
A

B
9
B
A
B
B
B
B
B
A
A
B

B
9
A
A
B
B
B
B
A
A
A
B

Figure 2.15 (cont’d)
ss490549379
ss490549383
ss490549411
BPPCT034
ss490547212
ss490549435
ss490549443
ss490549447
ss490549451
ss490549455
ss490549474
ss490549482
ss490549494
ss490549498
ss490549506
ss490549514
ss490549525
ss490549529
ss490549537
ss490547231
ss490549549
ss490549561
ss490549565
ss490549569
ss490549573
ss490549590
ss490547235
ss490549615

16118423
16142700
16229065
16491740
16519837
16530061
16550340
16581454
16583654
16585549
16644104
16657611
16671049
16689292
16732373
16738223
16779535
16801115
16827404
16840994
16842231
16874016
16875339
16879478
16885395
16918304
16926980
17026524

d
B
B
B
235
A
B
B
B
B
A
A
A
A
B
A
B
B
B
A
B
B
A
A
B
B
B
A
B

b
A
A
A
218
A
B
B
A
A
B
A
B
B
B
A
B
B
A
B
B
A
B
B
A
A
A
B
B

l
A
A
A
241
A
A
B
A
A
B
A
B
B
A
B
B
A
A
B
A
A
B
B
A
A
A
B
A

e
B
A
B
228
A
B
B
A
A
B
B
B
A
B
A
B
B
B
A
B
B
A
A
B
B
B
A
B

h
A
A
A
206
B
B
B
A
A
B
A
B
B
B
A
A
B
A
B
B
A
B
B
A
A
A
B
B

o
A
A
A
206
B
B
B
A
A
B
A
B
B
B
A
B
B
A
B
B
A
B
B
A
A
A
B
B

k
A
A
A
206
B
B
B
A
A
B
A
B
B
B
A
B
B
A
B
B
A
B
B
A
A
A
B
B

123

r
A
A
A
A
A
B
A
A
B
A
B
B
A
B
B
A
A
B
A
A
B
B
A
A
A
B
A

s m a
A A A
A A A
A A A
- 206 210
B B A
B B A
B B B
A A A
A A A
B B B
A A A
B B B
B B B
B B A
A A B
B A B
B B A
A A A
B B B
B B A
A A A
B B B
B B B
A A A
A A A
A A A
B B B
B B A

q
A
A
A
235
A
A
B
A
A
B
A
B
B
A
B
B
A
A
B
A
A
B
B
A
A
A
B
A

c
B
A
B
237
A
B
A
B
B
A
B
A
A
B
A
B
B
B
A
B
B
A
B
A
B
B
A
B

g
B
A
B
237
A
B
A
B
B
A
B
A
A
B
A
B
B
B
A
B
B
A
B
A
B
B
A
B

p
B
A
B
225
A
B
B
B
B
A
B
A
A
B
A
B
B
B
A
B
B
A
A
B
B
B
A
B

j
B
A
B
237
A
B
A
B
B
A
B
A
A
B
A
B
B
B
A
B
B
A
B
A
B
B
A
B

n
B
A
B
237
A
B
A
B
B
A
B
A
A
B
A
B
B
B
A
B
B
A
B
A
B
B
A
B

t
B
A
B
A
B
A
B
B
A
B
A
A
B
A
B
B
B
A
B
B
A
B
A
B
B
A
B

u f
i
B A B
A B A
B B B
- 255 228
A A A
B B B
A A B
B B A
B B A
A A B
B B B
A A B
A A A
B B B
A A A
B B B
B B B
B B B
A A A
B B B
B B B
A A A
B A A
A B B
B B B
B B B
A A A
B B B

Figure 2.15 (cont’d)
ss490549619
ss490549623
ss490547239
ss490549642
ss490549646
ss490547242
ss490549670
ss490549674
ss490549677
ss490549681
ss490547246
ss490549685
ss490549709
ss490549720
ss490549724
ss490549756
ss490549760
ss490549809
ss490549837
ss490549849
ss490547270
ss490549857
ss490549861
ss490558996
ss490558999
ss490549869
ss490549873
ss490547281

17039549
17047675
17207142
17207160
17244146
17257558
17425495
17426363
17469838
17470794
17473563
17476462
17560969
17571610
17574355
17731307
17736916
17931590
18149310
18371629
18433815
18522250
18588411
18681412
18681519
18683588
18702609
18708318

d
A
A
A
A
A
A
A
B
A
B
A
B
A
A
B
A
B
B
B
A
A
B
B
B
A
A
A
B

b
B
B
A
B
B
B
A
A
B
B
B
A
B
B
A
B
A
B
A
B
B
A
A
A
A
B
B
A

l
B
B
B
B
B
A
B
A
B
A
B
A
B
A
A
B
A
A
A
B
B
A
A
A
A
B
A
A

e
A
B
A
A
A
A
A
A
B
B
B
A
A
A
A
B
B
B
B
A
A
B
B
A
A
B
B
B

h
B
B
A
B
B
B
B
A
B
A
B
A
B
A
A
B
A
B
A
B
B
A
A
A
A
B
B
A

o
B
B
A
B
B
B
A
A
B
B
B
A
B
B
A
B
A
B
A
B
B
A
A
A
A
B
B
A

k
B
B
A
B
B
B
A
A
B
B
B
A
B
B
A
B
A
B
A
B
B
A
A
A
A
B
B
A

124

r
B
B
B
B
B
A
B
A
B
A
B
A
B
A
A
B
A
A
A
B
B
A
A
A
A
B
A
A

s
B
B
A
B
B
B
A
A
B
B
B
A
B
B
A
B
A
B
A
B
B
A
A
A
A
B
B
A

m
B
B
A
B
B
B
B
A
B
A
B
A
B
B
A
B
A
B
A
B
B
A
A
A
A
B
B
A

a
B
B
B
B
B
A
B
A
B
A
B
A
B
A
A
B
A
A
A
B
B
A
A
A
A
B
A
A

q
B
B
B
B
B
A
B
A
B
A
B
A
B
A
A
B
A
A
A
B
B
A
A
A
A
B
A
A

c
A
A
A
A
A
A
B
B
B
B
A
B
B
A
B
B
B
B
A
A
A
B
A
A
A
B
A
B

g
A
A
A
A
A
A
B
B
B
B
A
B
B
A
B
B
B
B
A
B
B
A
A
A
A
B
B
A

p
A
A
A
A
A
A
A
B
A
B
A
B
B
A
B
B
A
B
A
A
A
B
B
B
A
A
A
B

j
A
A
A
A
A
A
A
B
A
B
A
B
A
A
B
B
A
B
A
A
B
B
A
A
A
A
B
B

n
A
A
A
A
A
A
B
B
B
B
A
B
B
A
B
B
B
B
A
A
A
B
A
A
A
B
A
B

t
A
A
A
A
A
A
A
B
A
B
A
B
A
A
B
B
A
B
A
A
B
B
A
A
A
A
B
B

u
A
A
A
A
A
A
A
B
A
B
A
B
A
A
B
B
A
B
A
A
B
B
A
A
A
A
B
B

f
A
A
A
A
A
A
A
B
A
B
A
B
A
A
B
A
B
B
B
A
A
B
B
B
B
A
A
B

i
A
B
A
A
A
A
A
A
B
B
B
A
A
A
A
B
B
B
B
A
A
B
B
A
A
B
B
B

Figure 2.15 (cont’d)
ss490547285
ss490549881
ss490549889
ss490549897
ss490547297
ss490549916
ss490549920
ss490549924
ss490549932
ss490547304
ss490549959
ss490549971
ss490549975
ss490549979
ss490549987
ss490547316
ss490547320
ss490550011
ss490550015
ss490550019
ss490550027
ss490550051
ss490547331
ss490550063
ss490550074
ss490550082
ss490550097
ss490547351

18753870
18755669
18871385
18897454
18963376
18988718
18997401
19027747
19061737
19138888
19308510
19364459
19384216
19422560
19477390
19597895
19629831
19643557
19664779
19684679
19724366
19869360
20041486
20170303
20340464
20371321
20458193
20463267

d
A
A
B
B
B
B
A
A
B
A
A
A
B
A
A
B
B
A
A
A
A
A
A
A
A
B
B
A

b
A
B
A
A
A
A
B
B
A
A
B
B
A
B
A
A
A
B
B
B
B
B
A
B
B
A
A
A

l
B
B
A
A
B
A
B
B
A
B
B
B
A
B
A
A
A
B
B
B
B
A
B
B
B
A
A
B

e
A
A
B
B
B
B
A
A
A
A
A
B
A
A
A
B
B
B
A
A
A
A
A
A
A
B
B
A

h
A
B
A
A
A
A
B
B
A
A
B
B
A
B
A
A
A
B
B
B
B
B
A
B
B
A
A
A

o
A
B
A
A
A
A
B
B
A
A
B
B
A
B
A
A
A
B
B
B
B
B
A
B
B
A
A
A

k
A
B
A
A
A
A
B
B
A
A
B
B
A
B
A
A
A
B
B
B
B
B
A
B
B
A
A
A

125

r
B
B
A
A
B
A
B
B
A
B
B
B
A
B
A
A
A
B
B
B
B
A
B
B
B
A
A
B

s
A
B
A
A
A
A
B
B
A
A
B
B
A
B
A
A
A
B
B
B
B
B
A
B
B
A
A
A

m
A
B
A
A
A
A
B
B
A
A
B
B
A
B
A
A
A
B
B
B
B
B
A
B
B
A
A
A

a
B
B
A
A
B
A
B
B
A
B
B
B
A
B
A
A
A
B
B
B
B
A
B
B
B
A
A
B

q
B
B
A
A
B
A
B
B
A
B
B
B
A
B
B
A
A
B
B
B
B
A
B
B
B
A
A
B

c
A
A
B
B
B
A
A
A
A
A
A
B
A
A
A
B
A
A
A
A
A
A
A
B
A
A
B
A

g
A
B
A
A
A
A
B
B
A
A
B
B
A
B
A
A
A
B
B
B
B
B
A
B
B
A
A
A

p
A
A
B
B
B
B
A
A
B
A
A
A
B
A
A
B
B
A
A
A
A
A
A
B
A
A
B
A

j
A
A
B
B
B
B
A
A
A
A
A
B
A
A
A
B
A
A
A
A
A
A
A
B
A
A
B
A

n
A
A
B
B
B
A
A
A
A
A
A
B
A
A
A
B
A
A
A
A
A
A
A
B
A
A
B
A

t
A
A
B
B
B
A
A
A
B
A
A
B
B
A
A
B
A
A
A
B
A
A
A
A
A
B
B
A

u
A
A
B
B
B
B
A
A
A
A
A
B
A
A
A
B
A
A
A
A
A
A
A
B
A
A
B
A

f
A
A
B
B
B
A
A
A
A
A
A
A
B
A
A
B
B
A
A
A
B
A
A
A
A
B
B
A

i
A
A
B
B
B
B
A
A
A
A
A
B
A
A
A
B
B
B
A
A
A
A
A
A
A
B
B
A

Figure 2.15 (cont’d)

ss490547354
ss490559384
ss490550125
ss490550133
ss490550140
ss490550148
ss490547382
ss490550156
ss490550173
ss490547401
ss490550213
ss490547405
ss490547413
ss490547416
ss490547420
ss490550244
ss490547428
ss490547432
ss490550273
ss490547439

20470247
20616108
20694575
20724836
20758268
20799111
20808955
20834183
20925443
21131678
21264736
21290638
21399347
21611064
21616635
21670225
21905412
21952248
21965305
22081401

d
B
B
A
B
A
A
B
A
B
B
A
B
B
A
A
B
B
A
B
B

b
B
A
A
A
B
B
A
B
B
A
B
A
A
B
A
A
A
A
B
B

l
B
A
A
A
B
B
B
B
B
A
B
A
A
B
B
A
A
B
B
A

e
A
B
A
B
B
A
B
A
B
B
A
B
B
A
A
B
B
A
B
B

h
B
A
A
A
B
B
A
B
B
A
B
A
A
B
A
A
A
A
B
B

o
B
A
A
A
B
B
A
B
B
A
B
A
A
B
A
A
A
A
B
B

k
B
A
A
A
B
B
A
B
B
A
B
A
A
B
A
A
A
A
B
B

r
B
A
A
A
B
B
B
B
B
A
B
A
A
B
B
A
A
B
B
A

s
B
A
A
A
B
B
A
B
B
A
B
A
A
B
A
A
A
A
B
B

m
B
A
A
A
B
B
A
B
B
A
B
A
A
B
A
A
A
A
B
B

a
B
A
A
A
B
B
B
B
B
A
B
A
A
B
B
A
A
B
B
A

a

q
B
A
A
A
B
B
B
B
B
A
B
A
A
B
B
A
A
B
B
A

c
B
B
A
B
B
A
B
A
A
B
A
B
B
A
A
B
B
A
A
B

g
B
A
A
A
B
B
A
B
B
A
B
A
A
B
A
A
A
A
B
B

p
B
B
A
B
B
A
B
A
A
B
A
B
B
A
A
B
B
A
A
B

j
B
B
A
B
A
A
B
A
B
B
A
B
B
A
A
A
B
A
B
B

n
B
B
A
B
B
A
B
A
A
B
A
B
B
A
A
B
B
A
A
B

t
A
B
A
B
A
A
B
A
B
B
A
B
B
A
A
B
B
A
A
B

u
B
B
A
B
A
A
B
A
B
B
A
B
B
A
A
A
B
A
B
B

f
B
B
B
B
A
A
B
A
B
B
A
B
B
A
A
A
B
A
B
B

distances according to the Peach v1.0 ‘dhLovell’ genome assembly (International Peach Genome Initiative;
www.rosaceae.org/peach/genome)
b

CNR16 location reported in De Franceschi et al. 2013 (Verde et al. 2013)

126

i
A
B
A
B
B
A
B
A
B
B
A
B
B
A
A
B
B
A
B
B

Figure 2.16: The 17 haplotypes identified in sour cherry for the G4 region used to test for the
bloom time QTL. Haplotype designations q-r were not used.
LG4
Peach physical

NCBI SS# map distance (bp)
ss490548584
7010787
ss490552724
7040511
ss490552727
7065857
ss490548587
7070007
ss490548591
7147868
ss490552741
7309282
ss490548603
7398453
ss490552750
7429956
ss490548607
7434938
ss490552764
7666480
ss490552767
7706352
ss490552770
7731253
ss490548615
7813828
ss490552776
7835311
ss490548619
7861540
ss490548623
7862326
ss490548646
8425845
ss490548650
8495239
ss490552805
8500917
ss490552808
8538987
ss490548658
8893154
ss490552840
8950735
ss490548662
8955576
ss490545355
9145953
ss490552856
9148953

a

a
A
B
B
A
B
A
B
A
A
A
B
A
A
A
B
A
A
A
A
A
B
B
A
B
A

b
B
B
A
B
B
B
A
A
B
B
A
B
B
B
A
B
A
A
B
B
B
A
A
B
B

c
B
B
B
A
B
A
A
B
B
B
A
B
B
B
A
B
A
B
B
B
B
A
B
B
A

d
B
B
B
A
B
B
B
B
B
B
A
B
B
B
A
B
A
B
B
B
B
A
B
B
A

e
A
B
B
A
B
A
B
A
A
B
B
A
A
A
B
B
B
A
B
A
A
B
A
B
A

127

f
B
B
A
B
B
B
A
B
B
B
B
B
B
B
A
B
A
B
B
B
B
A
B
B
A

g
A
A
B
A
A
A
B
A
A
A
B
A
A
A
B
A
A
A
A
A
B
B
A
B
A

h
B
B
B
A
B
B
B
B
B
B
B
B
B
B
A
B
A
B
B
B
B
A
A
A
B

i
A
A
B
A
B
A
B
A
B
B
B
A
A
A
B
B
B
A
B
A
A
B
A
B
A

j
B
B
B
A
B
B
B
B
B
B
A
B
B
B
A
B
A
A
B
B
B
A
A
B
B

k
A
A
B
A
B
A
B
A
B
B
B
A
A
A
B
B
B
A
B
A
A
B
A
B
A

l
B
B
B
A
B
B
A
A
B
B
A
B
B
B
A
B
A
B
B
B
B
A
B
B
A

m
A
A
B
A
A
A
B
A
A
A
B
A
A
A
B
A
A
A
A
A
B
B
A
B
A

n
B
B
B
A
B
A
A
B
B
B
B
A
A
A
A
B
A
B
B
A
B
B
B
B
B

o
A
B
B
A
B
A
B
A
A
A
B
A
A
A
B
A
A
A
A
A
B
B
A
B
A

p
B
B
A
B
B
B
B
B
B
B
B
B
B
B
A
B
A
B
A
B
B
A
B
B
A

s
B
B
A
B
B
B
A
A
B
B
A
B
B
B
A
B
A
A
B
B
B
A
A
B
A

Figure 2.16 (cont’d)

ss490552868
ss490548682
ss490552880
ss490559401
ss490559398
ss490552883
ss490552889
ss490559054
ss490548706
ss490552912
ss490548714
ss490552931
ss490552933
ss490548726
ss490552936
ss490548730
ss490548734
ss490548738
ss490552959

9660471
9732651
10145480
10183945
10184012
10230259
10402945
10403896
10832168
11034104
11135825
11510521
11588410
11651018
11651543
11979679
12520610
12532690
12567325

a
A
B
A
A
B
B
B
B
A
A
A
B
B
B
B
A
B
B
A

b
B
B
B
B
B
B
A
A
B
B
B
A
B
B
B
B
A
A
B

c
B
B
A
B
B
A
A
A
B
B
B
A
B
B
B
B
A
A
B

d
B
B
A
B
B
A
A
B
B
B
B
A
B
A
A
B
A
A
B

e
A
B
B
A
B
B
B
B
B
A
B
B
B
B
B
A
B
B
A

a

f
B
B
B
B
B
B
A
B
B
B
B
A
B
A
A
B
A
A
B

g
A
B
B
A
B
B
B
B
B
A
B
B
B
B
B
A
B
B
A

h
B
B
B
B
B
B
A
A
B
B
B
B
B
A
A
B
A
A
B

i
A
A
B
A
B
B
B
B
B
A
B
B
B
B
B
A
B
B
A

j
B
B
B
B
B
B
A
A
B
B
B
A
B
B
B
B
A
A
B

k
A
B
B
A
B
B
B
B
B
A
B
B
B
B
B
A
B
B
A

l
B
B
A
B
B
A
A
A
B
B
B
A
B
B
B
B
A
A
B

m
A
B
A
A
B
B
B
B
A
A
A
B
A
B
B
A
B
B
A

n
B
B
A
B
A
A
A
A
B
A
B
A
B
A
A
B
A
A
A

o
A
B
A
A
B
B
B
B
A
A
A
B
A
B
B
A
B
B
A

p
B
B
A
B
B
A
A
B
B
B
B
A
B
A
A
B
A
A
B

s
B
B
A
B
B
A
A
A
B
B
B
A
B
A
A
B
A
A
B

distances according to the Peach v1.0 ‘dhLovell’ genome assembly (International
Peach Genome Initiative; www.rosaceae.org/peach/genome) (Verde et al. 2013)

128

Figure 2.17: The 16 haplotypes identified in sour cherry for the G3 region used to test for the
fruit size and firmness QTL. The number of haplotypes was “condensed” to eleven based on the
SNP calls for the region in bold and underlined. For the analysis, haplotype i = a, o = c, f = d, m
= g and p = k.
LG3
Peach physical
a a b c d e f g h i j k l m n o p
NCBI SS#
map location (bp)
1137810
A B B B A A B B A A B B B B B B
ss490547685
1182045
A B B B A B B B A B B B B B B B
ss490547689
1257143
B B B B B B A B B B B B B A B B
ss490550887
1409656
A A B B A A B A A A A B B B B A
ss490547697
1414476
B A A A B A A A B A A A A A A A
ss490550895
1439124
A B B B A A B B A A B B B B B B
ss490550899
1476283
A B B B A A B B A A B B B B B B
ss490550906
1487618
B A A A B B A A B B A A A A A A
ss490547699
1494588
A B B B A A B B A A B B B B B B
ss490550910
1496302
B A A A B A A A B A A A A A A A
ss490547703
1544427
A B B B A A B B A A B B B B B B
ss490550917
1841799
A B B B A A B B A A B B B B B B
ss490547720
1933543
B A A A B B A A B B A A A A A A
ss490550975
1953269
A A A A A B A A A B A A A A A A
ss490547728
2129632
A B B B A B B B A A B B B B B B
ss490547732
2181990
B A A A B A A A B B A B A B A A
ss490551023
2200082
B A A A B A A A B B A A A A A A
ss490551026
2203771
A B B B A B B B A B B B B B B B
ss490547736
2262982
B A B B B B A A B A A A A A B A
ss490551030
2265477
A B B B A B B B A A B B B B B B
ss490547744
2307153
A B B B A B B B A B B B B B B B
ss490551038
2387092
B A A A B A A A B B A A A A A A
ss490551054
2431726
A B B B A B B B A A B A B A B B
ss490551062
2449907
A B A A A A B B A A B B B B A B
ss490551066
2512839
B A A A B A B A B B A B B B A A
ss490551074
2532841
B B B B B B B B B B B B B B B B
ss490551078
2598588
A B B B A B B B A A B B B B B B
ss490547752
2615230
B A A A B A A A B A A A A A A A
ss490551094
2679794
B A A A B A A A B B A A A A A A
ss490551106
2698079
A B B B A B B B A A B B B B B B
ss490547760
2699030
B A A A B A B B B B A A B A A A
ss490551110
2735639
A B B B A B B B A A B B B B B B
ss490547764

129

Figure 2.17 (cont’d)

ss490547768
ss490547771
ss490547775
ss490551130
ss490551138
ss490551142
ss490547779
ss490551171
ss490551175
ss490551183
ss490547791
ss490551199
ss490547799
ss490551218
ss490547803
ss490551226
ss490551234
ss490547811
b

(CNR16)
ss490559277
ss490547822
ss490551275
ss490551292
ss490551296
ss490551305
ss490547829
ss490547833
ss490551321
ss490551325
ss490551337
ss490559471
ss490547837
ss490551349

2738097
2804457
2820043
2845006
2887683
2910993
2912959
3136656
3161286
3196604
3241000
3300749
3318355
3466624
3501764
3530651
3593732
3644850

a
A
B
A
B
B
A
A
B
A
A
A
A
A
A
B
B
B
B

b
B
B
A
B
A
B
B
A
A
B
B
A
A
A
B
A
B
B

c
B
A
A
A
A
B
B
B
B
B
B
B
B
A
A
B
A
A

d
B
B
A
A
A
B
B
B
A
B
B
B
B
A
A
B
A
A

e
A
B
B
B
B
A
A
B
A
A
A
A
A
A
B
A
B
B

f
B
B
A
A
A
B
B
B
A
B
B
B
B
A
A
B
A
A

g
B
B
A
A
A
B
B
B
B
B
B
B
B
A
A
B
A
A

h
B
A
A
A
A
B
B
A
B
B
B
B
B
A
A
B
B
A

i
A
B
A
B
B
A
A
B
A
A
A
A
A
B
B
B
B
B

j
B
B
A
B
A
B
A
B
A
B
A
A
A
A
B
B
B
B

k
B
B
A
B
A
B
B
A
A
B
B
B
B
A
A
B
B
A

l m n
B B B
A B B
A A A
A A B
A A A
B B B
B B B
A B A
B B A
B B B
B B B
A B B
B B B
A A A
A A A
B B B
B A B
A A A

o
B
A
A
A
A
B
B
B
B
B
B
B
B
A
A
B
A
A

p
B
B
A
B
A
B
B
A
A
B
B
B
B
A
A
B
B
A

A
A
B
B
B
A
A
A
B
A
A
B
B
A

A
A
B
B
B
A
A
A
B
A
A
B
B
A

A
B
B
A
A
B
B
B
B
B
A
B
B
B

A
B
B
A
A
B
B
B
B
B
A
B
B
B

A
A
B
B
B
A
A
A
B
A
A
B
B
A

A
B
B
A
A
B
B
B
B
B
A
B
B
B

B
B
B
A
A
B
B
B
A
A
B
B
B
B

A
B
B
A
A
B
B
B
A
A
B
B
B
B

A
A
B
B
B
A
A
A
B
A
A
B
B
A

A
A
B
B
B
A
A
A
B
A
A
B
A
B

A
B
A
A
A
A
B
B
A
A
A
B
B
B

B
B
A
A
A
B
B
B
B
A
A
B
B
B

A
B
B
A
A
B
B
B
B
B
A
B
B
B

A
B
A
A
A
A
B
B
A
A
A
B
B
B

3774129
3792552
3988721
4016810
4123843
4145092
4197714
4224596
4298083
4301861
4334085
4468725
4571814
4633547
4755490

130

B
B
B
A
A
B
B
B
A
A
B
B
B
B

A
B
A
A
A
B
B
B
A
A
A
B
B
B

Figure 2.17 (cont’d)

ss490559004
ss490551365
ss490551378
ss490547854
ss490551390
ss490547862
ss490551403
ss490547873
ss490551411
ss490558935
ss490551446

5359437
5440880
6031878
6273459
6477319
6515224
6738267
6783562
6851920
7295951
7572277

a
A
B
A
B
B
B
B
B
A
B
B

b
A
B
A
B
B
B
B
B
A
B
B

c
A
B
B
A
A
A
A
B
B
B
A

d
A
A
B
A
B
A
A
B
B
B
A

e
A
B
A
B
B
B
B
B
A
B
B

a

f
A
A
B
A
B
A
A
B
B
B
A

g
B
A
B
A
A
A
A
B
B
B
A

h
A
B
B
A
A
A
A
B
B
B
A

i
A
B
A
B
B
B
B
B
A
B
B

j
A
B
A
B
B
B
B
B
A
B
A

k
A
A
B
A
A
A
A
A
B
B
A

l
B
A
A
A
A
A
B
B
B
B
A

m
B
A
B
A
A
A
A
B
B
B
A

n
B
A
B
A
A
A
A
B
B
B
A

distances according to the Peach v1.0 ‘dhLovell’ genome assembly (International Peach
Genome Initiative; www.rosaceae.org/peach/genome) (Verde et al. 2013)
b

CNR16 location reported in De Franceschi et al. 2013

131

o
A
B
B
A
A
A
A
A
B
A
B

p
A
B
A
A
A
A
A
B
B
B
A

Figure 2.18: The twelve haplotypes identified in sour cherry for the G5 region used to test for
fruit size and firmness QTL. Haplotype designations g and m were not used.

LG5

Peach physical map

NCBI SS#
ss490554588
ss490549377
ss490554594
ss490554600
ss490549381
ss490554609
ss490559465
ss490549388
ss490549396
ss490549400
ss490549408
ss490549412
ss490554652
ss490559264
ss490559261
ss490554664
ss490554677
ss490554680
ss490549417
ss490554713

distance (bp)
16125708
16216710
16228253
16320573
16330689
16375840
16429681
16439498
16580379
16585374
16712208
16720675
16768627
16803495
16803595
16812132
16882667
16907514
16911194
17082034

(CNR18)

b
b

(CNR19)
ss490549429
ss490549433
ss490549437
ss490554738
ss490554744
ss490549445
ss490554756

a

a
A
A
B
A
B
B
A
B
A
A
B
B
B
A
B
B
A
B
A
A

b
A
A
B
A
B
B
A
B
A
A
B
B
B
A
B
B
A
B
B
A

c
B
B
B
B
A
B
A
A
B
B
B
B
A
A
A
B
B
A
A
A

d
B
B
B
B
A
B
A
A
B
B
B
B
A
B
B
B
A
A
A
A

e
A
A
B
A
B
A
A
A
B
A
B
A
B
A
B
A
A
B
A
A

f
A
A
B
A
B
B
A
B
A
A
B
B
B
A
B
B
A
B
B
A

h
B
B
B
B
A
B
B
A
B
B
B
B
A
A
A
B
B
A
A
A

i
B
B
A
B
A
B
A
A
B
B
B
B
A
A
A
B
B
A
A
A

j
B
B
B
B
A
B
B
A
B
B
B
B
A
B
B
B
B
A
A
A

k
A
A
B
A
B
B
A
B
A
A
B
B
B
A
B
B
A
B
B
A

l
A
A
B
A
B
A
A
A
B
A
B
A
B
A
B
A
A
B
A
A

n
A
A
B
A
B
A
A
A
B
A
A
A
B
A
B
A
A
B
A
A

A
B
A
A
B
A
B

A
B
A
A
B
A
B

B
A
B
B
B
B
A

B
A
B
B
A
B
A

A
B
A
A
B
A
B

A
B
A
A
A
A
B

B
A
B
B
B
B
A

B
A
B
B
B
B
A

B
A
B
B
B
B
A

A
B
A
A
B
A
B

A
B
A
A
B
A
B

A
B
A
A
B
A
B

17128673
17130224
17134242
17175943
17224386
17229379
17255447
17279418
17328363

132

Figure 2.18 (cont’d)

ss490554768
ss490549456
ss490549460
ss490554798
ss490549472
ss490554819
ss490554837
ss490559381
ss490559292
ss490549480
ss490554871
ss490554877
ss490549496
ss490549500
ss490554898

17389247
17389482
17404543
17518546
17523276
17647878
17757729
17759765
17779312
17805694
17956179
17977057
17997178
18035286
18100331

a
B
A
B
A
A
B
A
B
B
B
B
A
A
A
A

b
A
A
A
B
B
B
A
B
B
B
B
A
A
B
A

c
A
B
A
B
B
A
B
B
A
A
B
B
B
B
A

d
A
B
A
B
B
A
B
B
B
A
B
B
B
B
B

e
A
A
A
B
B
B
A
B
B
B
B
A
A
B
A

f
B
A
B
A
A
B
A
B
B
B
B
A
A
A
A

h
A
A
A
B
B
A
B
B
A
A
B
B
B
B
B

i
A
A
A
B
B
A
B
B
B
A
B
B
B
B
A

j
A
A
A
B
B
A
B
B
A
A
B
B
B
B
A

k
B
A
B
A
A
B
A
B
B
B
B
A
A
A
A

l
A
A
A
B
B
B
A
B
B
B
B
A
A
A
A

a

n
A
A
A
B
B
B
A
B
B
B
B
A
A
B
A

distances according to the Peach v1.0 ‘dhLovell’ genome assembly
(International Peach Genome Initiative; www.rosaceae.org/peach/genome)
(Verde et al. 2013)
b

CNR18 and CNR19 locations reported in De Franceschi et al. 2013

133

Figure 2.19: The thirteen haplotypes identified in sour cherry for the G6 region between CNR20
and the S-locus. These haplotypes were “condensed” to five haplotypes based on results of the
CNR – linked SSR marker G6SSR2008. This region was used to test for QTLs for fruit size and
firmness.
Peach physical map
LG6
NCBI SS#
b

(CNR20)
G6SSR2208

ss490550132
ss490556003
ss490550135
ss490556011
ss490556014
ss490556018
ss490550143
ss490558923
ss490556027
ss490556030
ss490550163
ss490550167
ss490556045
ss490550171
ss490556048
ss490550179
ss490556080
ss490556083
ss490550183
ss490550187
ss490556092
ss490550192
ss490550196
ss490550200
ss490559356
ss490556117
ss490550208
ss490559289
ss490556126
ss490556129
ss490550216
ss490556147

distance (bp)

a

a

b

c

d

3
A
B
B
B
B
A
B
A
A
B
B
A
B
B
B
A
B
B
A
B
A
B
B
B
A
B
A
B
A
A
B
B

3 null 1
A B B
B A A
B A A
B A A
B B B
A B B
B B A
A A A
A B B
B A A
B A B
A B B
B A B
B A A
B B B
A B B
B A A
B A A
A B B
B B A
A B B
B A A
B A A
B A B
B A A
B B A
A A B
A B B
A B B
A A A
B A A
B A A

d' e

e' e2 f

3
A
B
B
B
B
A
B
A
A
B
B
A
B
B
B
A
B
B
A
B
A
B
B
B
B
B
A
A
A
A
A
A

5
A
B
B
B
A
A
B
B
A
B
B
A
B
B
A
A
B
B
A
B
A
B
B
B
B
B
A
B
A
A
B
B

g

h

i

j

4 null 2
A B A
B A B
B A B
B A B
A B A
A B A
B B B
B A B
A B A
B A B
B A B
A B A
B A B
B A B
A B A
A B A
B A B
B A B
A B A
B B B
A B A
B A B
B A B
B A B
B A B
B B B
A A A
B B B
A B A
A A A
B A B
B A B

3
A
B
B
A
A
A
B
A
A
B
B
A
B
B
A
A
A
B
A
B
A
B
B
B
A
B
A
B
B
B
B
B

1
B
A
A
A
B
B
A
A
B
A
B
B
B
A
B
B
A
A
B
A
B
A
A
B
A
A
B
B
B
A
A
A

22070836
22083802
22122175
22194565
22481221
22483330
22578566
22682473
22731299
22953307
22969579
23001313
23103755
23117929
23121232
23125937
23138881
23368978
23460054
23466387
23491156
23515096
23550234
23617261
23656729
23717104
23740186
23776068
23799947
23810925
23851662
23925194
24251557
24311905

134

5
A
B
B
B
A
A
B
B
A
B
B
A
B
B
A
A
B
B
A
B
A
B
B
B
B
B
A
B
A
A
B
B

5
A
B
B
B
A
A
B
B
A
B
B
A
B
B
A
A
B
B
A
B
A
B
B
B
B
B
A
B
A
A
B
B

Figure 2.19 (cont’d)

ss490559115
ss490550220
ss490556163
ss490556173
ss490550235
ss490556176
ss490556182
ss490550239
ss490550243
ss490556190
ss490556194
ss490558886
ss490558890
ss490550250
ss490556207
ss490550254
ss490556210
ss490550263
ss490556216
ss490556220
ss490550267
ss490556239
ss490556242
ss490556245
ss490556251
S-locus
ss490556260
ss490559322
ss490556263
ss490550286
ss490550290
ss490550294
ss490556278

24324785
24433974
24593485
24757894
24770664
24822456
24914294
25019161
25034869
25113415
25146440
25325556
25325607
25413648
25429330
25441080
25528480
25606798
25631852
25713330
25761628
26089443
26149926
26206403
26322018
~26447808
26484157
26505790
26537935
26634643
26800908
26801537
26816058

a
B
B
A
A
A
B
B
B
A
A
B
B
B
B
A
B
A
B
A
B
B
B
B
B
B

b
A
A
A
A
A
B
B
B
A
A
B
B
B
B
B
B
A
B
A
B
B
B
A
B
B

1' 4
B
B
B
B
A
A
A

A
A
B
B
A
A
A

c
A
A
B
B
A
A
B
A
A
A
B
B
A
A
A
B
A
B
B
B
B
B
A
B
B

d d' e e' e2
A A B B B
A A B B B
B B A A A
B B A A A
B B A A A
A A B B B
B B A A A
A A B B B
B B A A A
A A B B B
B B A A A
B B B B B
A A B B B
A A B B B
A A B B B
A A B B B
A A A A A
A A B B B
B B A A A
B B A A A
A A B B B
B B A A A
A A B B B
B B A A A
A A A A A
36b 35 35 13' 13' 13m
A A A B B B
A A A A A A
A B B B B B
A A A B B B
A B B A B A
B B B A B A
B B B A B A

135

f
B
B
A
A
A
B
A
B
A
B
A
B
B
B
B
B
A
B
A
A
B
A
B
A
A

g
A
A
B
B
A
A
B
A
A
A
B
B
A
A
A
B
A
B
B
B
B
B
A
B
B

h
B
B
A
A
A
B
A
B
A
B
A
B
B
B
B
B
A
B
A
A
B
A
B
A
A

i
B
B
A
A
A
B
A
B
A
B
A
B
B
B
A
B
A
B
B
B
B
B
A
A
B

j
A
A
B
B
B
A
B
A
B
A
B
B
A
A
A
A
A
A
B
B
A
B
A
B
A

6 36a 6 14 26
B
B
B
B
A
A
A

A
A
A
A
A
B
B

B
B
B
B
A
A
A

A
A
B
B
A
A
A

A
A
B
A
B
B
B

Figure 2.19 (cont’d)

ss490556284
ss490550302
ss490550306
ss490550310
ss490556318

a
A
A
B
B
B

26929208
27072416
27138059
27359089
27518176

b
A
A
B
B
B

c
B
B
B
B
A

d
B
B
A
A
A

d'
B
B
A
A
A

e
A
A
B
B
B

e'
B
B
A
A
A

e2
A
A
B
B
B

f
A
A
B
B
B

g
B
B
B
B
A

h
A
A
B
B
B

i
A
A
B
B
B

j
B
B
A
A
A

a

distances according to the Peach v1.0 ‘dhLovell’ genome assembly (International Peach
Genome Initiative; www.rosaceae.org/peach/genome) (Verde et al. 2013)
b

CNR20 location reported in De Franceschi et al. 2013

Figure 2.20: The thirteen haplotypes identified in sour cherry for the G3 region containing
MYB10. This region was used to test for the flesh color QTL. Haplotype designations a, i, j, m,
q, r, s and t were not used.
Peach physical map
LG3
a

distance (bp)
NCBI SS#
ss490551540
9729116
ss490547928
9782875
ss490551552
10022424
ss490551556
10105783
ss490551560
10162979
ss490551563
10264563
ss490551577
10573974
ss490547944
10590166
ss490551581
10626205
ss490551584
10675150
ss490551593
10822211
ss490547952
10908880
ss490547960
12115409
ss490551635
12383977
ss490551642
12474678
ss490551648
12500413
ss490547972
12503462
ss490547976
12539794
3 MYB10 homologs (12.84-12.91 Mb)
ss490551672
12944437
ss490551678
12987920
ss490551684
13025963
ss490547992
13063792

b
B
A
B
A
B
A
A
A
A
A
A
A
A
B
B
A
A
B

c
B
A
B
B
A
A
B
B
B
B
B
B
B
A
B
B
B
B

d
B
B
B
B
A
B
B
B
B
B
B
B
B
A
B
B
B
B

e
B
A
B
A
A
A
A
A
A
A
A
A
A
B
B
A
A
A

f
B
A
B
A
A
A
A
A
A
A
A
A
A
B
B
A
A
A

g
B
B
A
B
A
B
A
B
B
B
B
B
B
A
B
B
B
B

h
B
B
A
B
A
B
B
B
B
B
B
B
B
A
B
B
B
B

k
B
A
B
A
A
A
A
A
A
A
A
A
A
B
B
A
A
A

l
B
B
B
B
A
B
B
B
B
B
B
B
B
A
B
B
B
B

n
A
A
B
A
A
A
A
A
B
A
A
A
A
B
A
B
A
B

o
B
B
B
B
A
B
B
B
B
B
B
B
A
A
B
B
B
B

p
B
A
A
B
A
B
B
B
B
A
A
B
B
A
B
B
B
B

u
B
A
B
B
A
A
B
B
B
B
B
B
B
B
B
B
B
B

A
B
A
A

B
A
A
B

B
B
A
B

A
B
A
A

A
B
A
A

B
A
B
B

B
A
B
B

A
B
A
A

B
B
B
B

A
B
A
A

B
A
B
B

B
A
A
B

B
A
B
B

136

Figure 2.20 (cont’d)

ss490551699
ss490551705
ss490547996
ss490551720
ss490551723
ss490551730
ss490551739
ss490551746
ss490551749
ss490551771
ss490551778
ss490551784
ss490559450
ss490548016
ss490551803
ss490551812
ss490551824
ss490548032
ss490551830
ss490551837
ss490548055
ss490548059
ss490551869
ss490551872
ss490551878

13144730
13208005
13369328
13406263
13433848
13466702
13520194
13563908
13567593
13724726
13754793
13795019
13878008
13881088
14024780
14146853
14316165
14442011
14521488
14599590
15171728
15305145
15309954
15357433
15455662

b
B
B
A
A
B
B
B
A
A
A
B
B
A
A
A
B
B
B
B
B
A
B
A
B
A

c
B
B
A
B
A
B
A
B
B
B
A
A
A
A
B
B
A
A
A
A
B
A
B
A
B

d
B
A
A
B
B
B
B
B
B
B
A
A
B
B
A
B
A
B
A
B
B
A
B
A
B

a

e
B
B
A
A
B
B
B
A
A
A
B
B
A
A
A
B
B
B
B
B
A
B
A
B
A

f
B
B
A
B
B
B
B
A
A
A
B
B
A
A
A
B
B
B
B
B
A
B
A
B
A

g
A
A
A
A
A
A
A
B
B
B
A
A
A
A
A
B
A
A
A
A
B
A
B
B
B

h
A
A
A
A
A
A
A
B
B
B
A
A
B
B
A
B
A
A
A
A
B
A
A
A
A

k
B
B
A
B
B
B
B
A
A
A
B
B
A
A
B
A
B
B
B
B
B
B
A
B
A

l
B
A
A
A
A
A
A
B
B
B
A
A
A
A
B
B
A
A
A
A
B
A
B
A
B

n
B
B
B
B
B
B
B
A
A
A
B
B
A
A
B
A
B
B
B
B
B
B
A
B
A

o
A
A
A
A
A
A
B
B
A
B
A
A
A
A
A
B
B
A
A
B
B
A
B
B
A

p
B
B
A
B
B
A
B
B
B
B
B
A
A
A
A
B
A
A
A
A
B
A
B
A
B

distances according to the Peach v1.0 ‘dhLovell’ genome assembly (International Peach
Genome Initiative; www.rosaceae.org/peach/genome) (Verde et al. 2013)

137

u
A
A
A
A
A
A
A
B
B
B
A
A
B
B
A
B
A
A
A
A
B
A
A
A
A

Figure 2.21: The 17 haplotypes identified in sour cherry for the G5 Malic acid QTL region. The
Seventeen haplotypes were condensed to just six using the region in bold and underlined. Above
the haplotypes are what haplotype group each haplotype belongs to (1-6).
Peach physical map
1
2
3
4
6
5
LG5
NCBI SS#
ss490553644
ss490553647
ss490553668
ss490553674
ss490548963
ss490553677
ss490553680
ss490553683
ss490548967
ss490553686
ss490548971
ss490553696
ss490553708
ss490553720
ss490548999
ss490553732
ss490553738
ss490549009
ss490553772
ss490549017
ss490553790
ss490553808
ss490553814
ss490553817
ss490549028
ss490553823
ss490553829
ss490549036
ss490553838
ss490553844
ss490553847

distance (bp)
689941
710199
857660
934368
935896
949123
975724
987941
990328
1005418
1031051
1121958
1221714
1451354
1463960
1607433
1645289
1871057
2236592
2404347
2722408
3142214
3248658
3299986
3394531
3464400
3492263
3513593
3557553
3631504
3644242

a

b
B
A
A
B
A
A
B
B
A
B
A
B
B
B
B
A
A
A
B
B
B
A
A
B
A
A
B
B
B
A
A

i
B
A
A
B
A
A
B
B
A
B
A
B
B
B
B
A
B
A
B
B
B
A
A
B
A
A
B
B
B
A
A

e
B
A
A
B
A
A
B
B
A
B
A
B
B
B
B
A
A
A
B
B
B
A
A
B
A
B
A
B
B
A
A

f
B
A
A
B
A
A
B
B
A
B
A
B
B
B
B
A
A
A
B
B
B
A
A
B
A
A
B
B
B
A
A

k
B
A
A
B
A
A
B
B
A
B
A
B
B
B
B
A
A
A
B
B
B
A
A
B
A
B
A
B
B
A
A

138

y
B
A
A
B
A
A
B
B
A
B
A
B
B
B
B
A
A
A
B
B
B
A
A
B
A
A
B
B
B
A
A

s
B
B
B
A
B
B
B
A
B
A
B
A
A
A
A
B
A
B
A
A
B
B
A
B
B
B
A
B
B
A
B

n
B
B
B
A
B
B
B
A
B
A
B
A
A
A
A
B
A
B
A
A
B
B
A
B
B
B
A
B
B
A
B

r
B
A
B
A
B
B
A
A
B
A
B
A
A
A
A
A
A
B
A
B
B
B
B
A
B
B
A
A
A
A
B

g
B
A
B
A
B
B
A
A
B
A
B
A
A
A
A
A
A
B
A
B
B
B
B
A
B
B
A
A
A
A
B

l
B
A
B
A
B
B
A
A
B
A
B
A
A
A
A
A
A
B
A
B
B
B
B
A
B
B
A
A
A
A
B

j
A
A
A
A
A
B
B
B
A
B
A
B
B
B
B
A
B
A
B
B
B
A
A
B
A
A
B
B
B
A
A

p
A
A
A
A
A
B
B
B
A
B
A
B
B
B
B
A
B
A
B
B
B
A
A
B
A
A
B
B
B
A
A

o
A
A
A
A
A
B
B
B
A
B
A
B
B
B
B
A
B
A
B
B
B
A
A
B
A
A
B
B
B
A
A

m
A
A
A
B
A
A
B
B
A
B
A
B
A
B
B
A
A
A
B
B
B
A
A
B
A
A
B
B
B
B
B

h
A
A
A
B
A
A
B
B
A
B
A
B
B
B
B
A
B
A
B
B
B
A
A
A
A
A
B
B
B
A
A

q
A
A
A
B
A
A
B
B
A
B
A
B
B
B
B
A
B
A
B
B
B
A
A
A
A
A
B
B
B
A
A

Figure 2.21 (cont’d)

ss490553853
ss490553856
ss490553865
ss490553868
ss490549055
ss490553871
ss490553874
ss490553877
ss490549059
ss490553898
ss490553901
ss490553907
ss490549067
ss490553910
ss490553922
ss490553929
ss490553932
ss490553942
ss490549078
ss490553948
ss490549082
ss490553960
ss490553963

3695755
3731884
3767786
3864042
3909319
3917338
4005643
4028824
4181905
4345439
4357749
4413731
4415391
4486238
4755463
4897952
4994245
5143453
5238673
5242696
5354555
5409519
5429352

b
A
A
A
A
B
A
A
A
A
A
A
A
A
B
A
B
A
B
A
A
B
B
A

i
B
A
B
A
B
A
A
A
A
B
A
B
A
B
A
B
A
B
A
A
B
B
A

e
B
B
A
A
B
A
A
B
A
A
B
A
A
B
A
A
A
B
A
A
B
A
B

f
B
B
A
A
B
A
A
B
A
A
B
B
A
B
A
B
A
B
A
A
B
A
B

k
B
B
A
B
B
A
A
B
A
A
B
A
A
B
A
A
A
B
A
A
B
A
B

a

y
A
A
A
A
B
A
A
A
A
A
A
A
A
B
A
A
B
A
A
A
A
A
B

s
B
B
A
B
B
B
B
B
B
B
B
A
B
A
B
A
B
A
A
A
A
A
B

n
B
B
A
B
B
B
B
B
B
B
B
A
B
A
B
A
B
A
B
B
A
A
B

r
B
B
A
B
A
B
B
B
B
B
B
A
A
A
B
A
B
A
B
B
A
A
B

g
B
B
A
B
A
B
B
B
B
B
B
A
A
A
B
A
B
A
B
B
A
A
B

l
B
B
A
A
A
B
B
B
B
B
B
A
A
A
B
A
B
A
B
B
A
A
B

j
A
A
B
B
B
A
A
A
A
A
B
B
A
B
A
B
A
B
A
A
B
B
A

p
A
A
B
A
B
A
A
A
A
A
B
B
A
B
A
B
A
B
A
A
B
B
A

o
A
A
B
A
B
A
A
A
A
A
B
B
A
B
A
A
A
B
A
A
B
A
B

m
B
B
A
A
B
A
A
A
A
A
B
B
A
B
A
B
A
B
A
A
B
B
A

h
B
A
A
A
B
A
A
A
A
A
B
B
A
A
B
B
A
B
A
A
B
B
A

q
B
A
A
A
B
A
A
A
A
A
B
B
A
A
B
B
A
B
A
A
B
A
B

distances according to the Peach v1.0 ‘dhLovell’ genome assembly (International Peach Genome
Initiative; www.rosaceae.org/peach/genome) (Verde et al. 2013)

139

Figure 2.22: Within population mean comparisons of dark flesh haplotypes d, e, l, and p. Means
are significantly different (P< 0.05) if letters after the mean score are different. Colors are
representative of the rating.

140

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141

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