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Development and applicability of GBS approach for genomic studies in

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DOI: 10.1080/14620316.2018.1543559

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Development and applicability of GBS approach

for genomic studies in Japanese plum (Prunus
salicina Lindl.)

Juan Alfonso Salazar, Igor Pacheco, Claudia Silva, Patricio Zapata, Paulina
Shinya, David Ruiz, Pedro Martínez-Gómez & Rodrigo Infante

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THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY
https://doi.org/10.1080/14620316.2018.1543559

Development and applicability of GBS approach for genomic studies in

Japanese plum (Prunus salicina Lindl.)
Juan Alfonso Salazar a, Igor Pachecob, Claudia Silvab, Patricio Zapataa, Paulina Shinyaa, David Ruizc,
Pedro Martínez-Gómezc and Rodrigo Infantea
a
Departamento de Producción Agrícola, Universidad de Chile, Santiago, Chile; bInstituto de Nutrición y Tecnología de los Alimentos
(INTA), Universidad de Chile, Santiago, Chile; cDepartamento de Mejora Vegetal, CEBAS-CSIC, Murcia, Spain

ABSTRACT ARTICLE HISTORY

Genotyping by sequencing (GBS) provides a large quantity of useful data suitable for the Accepted 29 October 2018
identification of single nucleotide polymorphisms (SNPs), facilitating accurate genomic stu-
KEYWORDS
dies in plant species. In this study, GBS-based SNPs were used to characterise 11 Japanese Japanese plum; breeding;
plum cultivars and to explore their natural allelic diversity in relation to the most important genetic diversity; phenology;
phenology events (flowering date, ripening date and fruit development period) and fruit fruit quality; Prunus salicina;
quality traits (weight, shape, skin and flesh colour, over colour, skin and flesh chlorophyll SNP; sequencing
index, flesh firmness and soluble solids concentration). GBS-based SNPs were shown to be
a powerful tool for genetic diversity and other genomic studies where SNP markers were
related to several traits, particularly for flowering date, ripening date, fruit development
period, skin chlorophyll degradation, flesh chlorophyll degradation and flesh colour. These
results represent a preliminary approach using GBS as a possible breeding tool in current and
new Japanese plum breeding programmes.

Introduction 2012a; Gurcan, Teber, Ercisli, & Yilmaz, 2016; Khadivi-

Khub, Yarahmadi, Jannatizadeh, & Ebrahimi, 2015;
Over the years, the study of phenology and fruit quality
Meng & Peng, 2015; Zeinalabedini et al., 2014). The
traits in stone fruit species including Japanese plum
most widely used molecular markers include amplified
(Prunus salicina Lindl); also known as Asian plum or
fragment length polymorphisms (AFLPs) and microsa-
Chinese plum has been and still is of great interest for
tellites, also called simple sequence repeats (SSRs)
both the fresh market and the canning industry.
(Salazar et al., 2014). More recently, researchers have
Previous studies have been focused on classical genetic
begun to use SNPs with greater frequency (Martínez-
improvement, heritability and trait transmission in pro-
García et al., 2012; Salazar et al., 2017; Zeballos et al.,
genies. The general aim of these studies has been to
2016). Given their considerable genomic abundance
provide a solid guide for selecting individuals within
and bi-allelic codominance, SNPs allow us to obtain
breeding programmes, including how to obtain geno-
higher marker saturation within maps and cover larger
types that are better adapted to biotic and abiotic stres-
areas of the plant genome.
ses (Pacheco et al., 2014; Rousselin et al., 2016) and
The development of genetic maps together with
varieties with exceptional organoleptic quality
deep phenotyping has provided valuable information
(Infante, Reginato, & Hinrichsen, 2011a; Ruiz et al.,
for identifying the QTLs of different traits in the
2010; Salazar et al., 2017).
Prunus genus in species such as almond, apricot,
During recent years, molecular biology has provided
peach and cherry. These tools have also helped us
more robust tools for more deeply studying the genetic
identify candidate genes that provide relevant and
base that determines and controls several phenological
useful information for selection based on molecular
traits in different Prunus species, including flowering
markers (Salazar et al., 2014). In the case of Japanese
date (Castède et al., 2014); the fruit development period
plum, however, there is very little information on
(Salazar et al., 2016); and fruit quality traits like acidity
genetic mapping and QTLs (Salazar et al., 2017).
and sugar content (Desnoues et al., 2016; Wang et al.,
Another recent tool, genotyping by sequencing
2016). Over the last 15 years, the trend has been to
(GBS), provides a great wealth of information that
study variability and genetic diversity as well as genetic
make it possible to identify thousands of SNPs,
map construction and quantitative trait locus (QTL)
which, after adequate filtering, allow us to carry out
identification through molecular markers (Cao et al.,
very precise genetic diversity studies (Gurcan et al.,

CONTACT Juan Alfonso Salazar jasalazar@cebas.csic.es

Supplemental data for this article can be accessed here.
© 2018 The Journal of Horticultural Science & Biotechnology Trust
2 J. A. SALAZAR ET AL.
2016). GBS has also made possible to use thousands
of SNPs in the search for marker-trait associations in
Prunus (Bielenberg et al., 2015; Guajardo et al., 2015;
Salazar et al., 2017). Genome-wide association
(GWAS) is another powerful strategy based on gen-
ome sequencing using thousands of SNPs from any
plant species for genomic association studies (Kujur
et al., 2015). Despite the fact that GBS provides major
SNP coverage, this strategy has not yet been applied
in many fruit species. This approach has been suc-
cessfully applied, however, in grain crops (Jia et al.,
2013), where 916 varieties have been evaluated in five
different environments, and 512 loci related to 47
agronomic traits have been identified.
In peach, GWAS has been applied for identifying
genes related to diseases such as PPV (Mariette et al.,
2016) and genes related to agronomic (Cao et al.,
2016b) and fruit quality traits (Cao et al., 2016c;
Wang et al., 2016). Nevertheless, the combination of
GBS-based SNPs and GWAS has not yet been used in
any Prunus species studies. GWAS offers an alterna-
tive approach to using the 9K SNP array developed
by the International Peach Genome Initiative (IPGI)
(Verde et al., 2012). This SNP array was used to
provide candidate genes related to fruit size in
peach (Cao et al., 2016c).
The objective of this work was to apply GBS-based
SNPs for genetic diversity and preliminary marker-
trait association analysis of plant phenology and fruit
quality traits in Japanese plum varieties.
Materials and methods
Plant material
The plant material used in this trial consisted of 11
Japanese plum cultivars maintained in the germplasm
collection of the University of Chile, at Santiago.
Fruit samples were randomly harvested from three
plants per each genotype. Most of these genotypes
were developed by Californian breeding
programmes (‘Larry Ann’, ‘Black Splendor’, ‘Friar’,
‘Owen T’, ‘98–99’, ‘September King’, ‘September
Queen’ and ‘Angeleno’), while ‘Golden Kiss’, ‘Sun
Dew’ and ‘Sapphire’ were developed in South Africa
(ARC-Infruitec/Nietvoorbij), Stellenbosch.
Evaluation of phenology traits
The phenological traits of interest (flowering date,
ripening date and fruit development period) were
determined throughout the years 2016, 2017 and
2018. The flowering date was evaluated every 4 days
and expressed in Julian days (days after 1 July) until
50% of the flowers were completely opened (F50).
The ripening date was determined at commercial
maturity stage when the skin chlorophyll index
(IAD) was close to 1 and was also expressed in
Julian days. Finally, the fruit development period
was calculated as the difference between flowering
date and ripening date. Descriptive phenology data
was summarised using INFOSTAT v16.
Evaluation of fruit quality traits
The following nine fruit quality traits were evaluated:
fruit weight, measured with a digital scale in grams;
fruit shape [elongated (1), heart-shaped (2), spherical
(3) or oblate (4)]; skin colour, determined by visual
observation [green (1), yellow (2), red (3), purple (4) or
black (5)]; flesh colour, determined by visual observation
[white (1), green (2), yellow (3) or red (4)]; skin and flesh
colour, (5 and 6) by colorimeter, over colour (7), skin and
flesh chlorophyll index (8 and 9) measured with
a Minolta Chroma Meter (CR-300, Minolta, Ramsey,
NJ, USA; over-colour, determined by visual observation
[25% (1), 50% (2), 75% (3) or 100% (4)]; and skin
chlorophyll index (SIAD) and flesh chlorophyll index
(FIAD), both measured with a DA-meter device
(Bonora, Stefanelli, & Costa, 2013; Gottardi et al., 2009;
Infante, Rubio, Contador, Noferini, & Costa, 2011b).
Flesh firmness was measured with a texture-analyser
(TA.XT plus, Texture Technologies, Hamilton, MA)
using a 7.9 mm diameter plunger (N), and the soluble
solids concentration was measured with an ATAGO®
hand-held refractometer (%). The skin chlorophyll
index, flesh chlorophyll index, firmness and soluble
solids were evaluated at two maturity stages: at harvest
(1) and 4 days after harvest (2). Ten fruits were evaluated
for each cultivar in 2015. Descriptive fruit quality data
was also analysed by INFOSTAT v16.
Genotyping using GBS-based SNPs
Young leaves for each accession were collected and
lyophilised for DNA extraction, which was carried
out according to the CTAB procedure described by
Doyle and Doyle (1987). DNA samples were sent to
the Biotechnology Resource Center (BRC) at Cornell
University (New York, NY). Genotype by sequen-
cing (GBS) technology was used to generate DNA
fragment libraries from each genotype by Illumina
next-generation sequencing platform. ApeKI restric-
tion enzyme was used for cutting the most repetitive
fractions of the genome. DNA samples, common
adaptors and barcode adaptors were digested using
this restriction enzyme for 2 hours at 75°C. The
adapters were ligated to the ends of DNA fragments
by the addition of ligand buffer with ATP and T4
ligase. Samples were incubated at 22°C for 1 h and
then heated at 65°C for 30 min to inactivate the
ligase. Finally, restriction fragments from each
library were amplified by PCR. These fragments
amplified constituted the sequencing library which
 THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY 3

were considered suitable for sequencing with at least
128 bp (Elshire et al., 2011).
FASTQ files (raw sequences), TOPM files (tags on
a physical map), VCF files (an alternative format for
holding SNPs) and filtered Hapmap files (SNP-calling)
were generated as described by Salazar et al. (2017)
using peach v1 as a reference genome. In that work,
the authors obtained an initial set of 42,909 variants
after SNP calling. In this work, tags from GBS were
aligned to peach genome v2 (Verde et al., 2017) obtain-
ing 43.34% of tags aligned to unique position, 52.86% of
tags not aligned and 3.8% of tags aligned to multiple
positions; these results being very similar to previous
alignment to peach v1 (Supplementary Table S3). In
order to perform diversity and association analysis with
highly robust markers, we selected individual SNP calls
(the genotype of each SNP on each accession) generated
from a minimum of six reads. In addition, we selected
homozygous genotypes generated from reads corre-
sponding 100% to the same allele, and heterozygous
genotypes generated with a maximum of 75% of the
reads corresponding to one of the alleles. From the
resulting dataset, we selected polymorphic SNPs with
a missing data rate of less than 10%.
Genetic diversity evaluation and preliminary
marker-trait association
The heterozygosity (H) of each SNP marker and Japanese
plum cultivar was calculated as the number of heterozy-
gous genotypes divided by all the genotypes analysed. The
power of discrimination (PD) of each SNP marker was
calculated as PD = 1 – ∑ g2i, where gi is the frequency of
the ith genotype. The genetic diversity study, including
the distance matrix between genotypes and the clado-
gram, was calculated by TASSEL v5 using
neighbour joining (NJ) as a clustering method. Marker-
trait association analyses between SNPs and agronomic
traits were performed by TASSEL v5 using the general
lineal model (GLM). Intersections between phenotypic
and genotypic data together with a principal component
analysis (PCA) from the genotypic data were used to
calculate phenotype-genotype association by GLM
using a reference p-value of 0.05 and a test of
1000 permutations.
Results and discussion
Phenotypic evaluation of Japanese plum cultivars
This small variety collection is characterised for their
high heterogeneity and variability as to ripening date,
fruit quality or post-harvest performance. Thus, we
can find early or mid-early ripening varieties such as
‘Black Splendor’, ‘Owen T’, ‘Sun Dew’, ‘Sapphire’,
´98–99’, ‘Golden Kiss’, ‘Friar’ and ‘Larry Ann’ or
late ripening varieties such as ‘Angeleno’,
‘September King’ and ‘September Queen’. In addi-
tion, most of them have a good fruit quality, espe-
cially ‘Golden Kiss’, which is characterised by its
yellow skin and flesh colour, or ‘Larry Ann’, ‘Friar’
and ‘Angeleno’, which are characterised by their great
resistance to commercial handling conditions and are
well-suited for cold storage. Results showed great
variability and segregation in the Japanese plum cul-
tivars for all the phenology and fruit quality traits
evaluated (Tables 1 and 2). Among the phenological
traits evaluated, for the flowering date there weren’t
significant differences between genotypes; however,
there were differences between years, while for ripen-
ing date and fruit development period there were
differences between genotypes, but not between
years (Supplementary Table S1 and Figure 1). For
the rest of the fruit quality traits evaluated in 2016,
there were significant differences between the geno-
types (Supplementary Tables S1 and S2).
The flowering date ranged from that of the early
flowering cultivars, such as ‘Black Splendor’ and
‘Golden Kiss’ (51 and 52 Julian days, respectively)
to that of the late flowering cultivars, ‘98–99’ and
‘September King’ (67 and 68 Julian days, respec-
tively), which bloomed over 2 weeks later, on average
(Table 1). The ripening date range was even greater,
with a difference of over 2 months (60 Julian days)
between early ripening cultivars like ‘Black Splendor’
and late ripening cultivars like ‘September King’ and

Table 1. Summary of phenology traits for 11 Japanese plum cultivars in 2016, 2017 and 2018: flowering date (FD), ripening date
(RD) and fruit development period (FDP) in Julian days.
Flowering date Ripening date Fruit development period
Genotype 2016 2017 2018 Average SD 2016 2017 2018 Average SD 2016 2017 2018 Average SD
98–99 76 61 64 67.0 7.9 212 200 206 206.0 6.0 136 139 142 139.0 3.0
Angeleno 63 50 58 57.0 6.6 237 221 223 227.0 8.7 174 171 165 170.0 4.6
September Q 72 58 62 64.0 7.2 259 253 264 258.7 5.5 187 195 202 194.7 7.5
September K 74 61 69 68.0 6.6 259 250 262 257.0 6.2 185 189 193 189.0 4.0
Owen T 64 49 51 54.7 8.1 198 194 195 195.7 2.1 134 145 144 141.0 6.1
Friar 77 53 62 64.0 12.1 219 200 213 210.7 9.7 142 147 151 146.7 4.5
Sapphire 72 43 55 56.7 14.6 204 190 200 198.0 7.2 132 147 145 141.3 8.1
Black Splendor 69 39 45 51.0 15.9 198 180 185 187.7 9.3 129 141 140 136.7 6.7
Larry Ann 72 52 56 60.0 10.6 219 200 206 208.3 9.7 147 148 150 148.3 1.5
Golden Kiss 69 39 47 51.7 15.5 219 197 202 206.0 11.5 150 158 155 154.3 4.0
Sun Dew* 70 — — 70.0 — 200 — — 200.0 — 130 — — 130.0 —
* Data from commercial orchards.
 4
J. A. SALAZAR ET AL.

Table 2. Summary of fruit quality traits for 11 Japanese plum cultivars in 2016: fruit weight (FW) in grams; shape (Shape): elongated (1), heart-shaped (2), spherical (3) or oblate (4); skin colour by
visually (SKC): green (1), yellow (2), red (3), purple (4) or black (5); flesh colour by visually (FLSC): white (1), green (2), yellow (3) or red (4); skin colour (SKC(H°)); flesh colour (FLSC(H°)); over
colour (OVC) by visually: 25% (1), 50% (2), 75% (3) and 100% (4); skin chlorophyll index (SIAD): at harvest (1) or 4 days later (2); flesh chlorophyll index (FIAD): at harvest (1) or 4 days later (2); firmness
(Fmax) in Newton: at harvest (1) or 4 days after (2) and soluble solids content (SSC) in %: at harvest (1) or 4 days after (2).
98–99 Angeleno September Q September K Owen T Friar Sapphire Black Splendor Larry Ann Golden Kiss Sun Dew*
Trait Average SD Average SD Average SD Average SD Average SD Average SD Average SD Average SD Average SD Average SD Average SD
FW 137.3 11.96 76.54 12.87 119.76 11.72 150.59 14.68 85.96 8.63 116.71 14.1 120.37 14.19 82.89 7.27 146.17 11.79 69.13 6.84 65.00 —
Shape 2 — 4 — 3 — 3 — 3 — 3 — 3 — 3 — 3 — 1 — 3 —
SKC 3 — 5 — 4 — 3 — 4 — 5 — 4 — 5 — 4 — 2 — 2 —
FLSC 4 — 3 — 2 — 3 — 3 — 3 — 4 — 4 — 3 — 3 — 3 —
SKC (H°) 15.69 5.17 9.22 4.79 17.5 5.50 24.27 6.94 18.24 3.03 13.78 6.07 12.72 3.91 44.32 106.75 17.73 5.44 94.63 2.53 — —
FLSC (H°) 13.68 3.67 93.27 2.16 105.92 1.43 63.35 14.76 97.33 2.83 101.51 3.89 35.36 6.44 21.11 3.12 85.78 4.36 80.11 2.40 — —
OVC 3.9 0.32 4 — 3.60 0.52 3.50 0.53 4 — 4 — 4 — 4 — 2.90 0.32 4 — 4 —
SIad_1 0.84 0.08 1.27 0.18 1.44 0.09 0.97 0.12 1.26 0.19 1.37 0.18 1.39 0.11 1.58 0.18 1.07 0.13 1.11 0.17 — —
SIad_2 0.52 0.10 1.08 0.17 1.43 0.18 0.86 0.12 1.13 0.09 1.37 0.11 1.22 0.19 1.38 0.12 0.79 0.21 1.07 0.10 — —
FIad_1 0.32 0.17 0.4 0.16 0.94 0.10 0.46 0.09 0.38 0.20 0.45 0.12 0.63 0.14 0.67 0.16 0.31 0.15 0.43 0.19 — —
FIad_2 0.13 0.04 0.52 0.12 0.84 0.19 0.35 0.12 0.32 0.14 0.41 0.15 0.34 0.15 0.45 0.18 0.23 0.18 0.37 0.12 — —
Fmax_1 46.58 7.96 46.61 5.69 49.82 5.11 45.16 3.81 26.32 5.11 34.04 6.19 16.65 3.02 13.8 1.69 48.31 4.99 33.19 4.57 — —
Fmax_2 20.93 12.78 43.9 3.67 48.89 6.20 41.85 3.85 22.68 10.73 30.19 5.57 3.54 5.28 10.18 3.31 43.74 4.56 33.09 2.96 — —
SSC_1 16.36 0.59 18.53 0.58 15.27 1.62 15.78 1.51 16.92 3.09 12.26 1.13 17.04 1.75 13.28 1.09 15.31 0.87 21.53 0.82 — —
SSC_2 16.36 1.59 18.71 1.29 15.95 1.14 15.5 1.09 18.35 1.20 12.33 1.16 17.82 1.83 15.2 1.51 17.34 0.65 21.81 0.90 — —
* Data from commercial orchards.
 THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY 5

‘September Queen’. ‘Black Splendor’ and ‘98–99’ The genotypes ‘Owen T’ and ‘Friar’ were also located
showed the shortest fruit development period over in the same cluster, in agreement with the findings of
an average of 3 years, as opposed to ‘September Minas et al. (2015).
King’ and ‘September Queen’, which showed the Genetic matrix distances among the 11 plum cul-
longest fruit development period, which correlated tivars studied, calculated by TASSEL, reached
with the ripening date (Table 1). a maximum value of 0.430 and a minimum value of
Regarding fruit colour, skin colour ranged from 0.253 (Supplementary Table S4). ‘Sun Dew’ was the
yellow (2) in ‘Golden Kiss’ to black in ‘Black genotype with the greatest genetic distance with
Splendor’ (5). Flesh colour ranged from yellow (3) respect to all the others. The cultivars ‘Owen T’ and
to red (4) (Figure 2). In addition, significant chlor- ‘Friar’ showed the lowest genetic distance (0.25),
ophyll degradation in the skin and flesh, measured indicating they are genetically close to each other
indirectly by the Chlorophyll absorption index (IAD), (Supplementary Table S4), as has also been reported
were particularly observed in ‘98–99’ and ‘Sapphire’, by Minas et al. (2015). This result makes sense given
respectively (Table 2). the fact that both cultivars were released by the same
USDA breeding programme in Fresno, CA. In gen-
eral, the genetic distances observed among the culti-
Evaluation of the genetic diversity of Japanese vars tested confirmed the phylogenic relationships
plum genotypes using GBS-based SNPs shown in Figure 2.
To study the genetic diversity of Japanese plum gen- The genetic distances found in this study never-
otypes, we used raw sequences from a plum GBS theless suggest a low level of genetic diversity in
(Salazar et al., 2017). These sequences were aligned comparison to the findings in other studies per-
to the new peach genome v2 (Verde et al., 2017). formed in Prunus salicina. For example, in a study
Furthermore, 7429 SNPs were filtered from calling using AFLP and SSR markers, Tamarzizt, Mustapha,
SNPs above six reads and genotype errors and com- Baraket, Abdallah, and Salhi-Hannachi (2015) found
pletely homozygous and heterozygous SNPs were a genetic distance ranging from 0.18–0.79. The low
discarded. diversity found in the current study may be due to
The heterozygosity of the Japanese plum genotypes the fact that SNPs have a lower level of heterozygosity
assayed ranged from 0.003–0.547 and the SNP het- than other markers like SSRs, as reported in other
erozygosity ranged to 0.09–0.90, while the power of species such as Chinese cherry (Chen et al., 2016).
discrimination (PD) of SNPs reached an average However, the most feasible explanation for this dis-
value of 0.40. The lowest heterozygosity value was tinct result is the fact that we chose a set of cultivars
obtained for ‘Sun Dew’ (0.003), while ‘Angeleno’ based on their importance in the current market,
had the highest score (0.547) (Table 3). Similar results rather than looking specifically for wide genetic
were previously obtained in apricot genotypes using diversity as other researchers have done (Tamarzizt
SNPlex technology (Salazar et al., 2015). et al., 2015). In our case, the results show the poor
Genotypic relationships between cultivars are genetic base of cultivated genotypes, confirming the
shown in the Figure 2 cladogram. The South need to obtain new varieties with different genetic
African varieties ‘Sun Dew’ and ‘Golden Kiss’, the backgrounds in order to be better prepared for cli-
only yellow-skinned genotypes, were placed together, mate change and biotic and abiotic challenges.
and the purple-skinned ‘Sapphire’, also from South
Africa, was located at the opposite end of the same Marker-trait-association using GBS-based SNPs
cluster, which implies a different genetic background.
In the genotype–phenotype association analysis, dif-
ferent markers were linked to several important traits,
Table 3. Heterozygosity of SNP markers and the 11 assayed
Japanese plum cultivars. especially flowering date (FD), ripening date (RD),
SNPs Average Max Min fruit development period (FDP), skin and flesh chlor-
Heterozygosity 0.35 0.90 0.09 ophyll degradation (SIAD_ 1–2 and FIAD_ 1–2) and
Power of discrimination 0.40 0.67 0.17 visually-assessed flesh colour (FLSC) (Table 4).
Genotypes Heterozygosity For flowering date, the most important markers
Golden Kiss 0.436 were located at the end of LG1 and around the mid-
Sun Dew 0.003
Black Splendor 0.362 dle region of chromosome 7, where early flowering
Friar 0.377 seems to be related to CT alleles as opposed to TT
Larry Ann 0.445
98–99 0.345 alleles, which appear to be related to later flowering
Owen T 0.427 (Tables 4 and 5). In addition, LGs 4, 6 and 8 also
Sapphire 0.334
September King 0.385 seem to be related to flowering date (Supplementary
September Queen 0.398 Table S5). Using the whole genome sequencing
Angeleno 0.547
approach by Illumina, Zhebentyayeva et al. (2014)
6 J. A. SALAZAR ET AL.

Figure 1. Gantt diagrams for flowering date (purple bars), ripening date (orange bars) and fruit development period (red bars)
from minimal to maximum values in Julian days for 11 Japanese plum cultivars in 3 years of phenotyping.

studied a peach F2 population segregating for flower-
ing date and placed the most significant locus in LGs
1, 4 and 7, but the major QTL signal was located at
the lower end of LG1. In any case, it is widely known
that the flowering date in Prunus species, including
Japanese plum, is highly dependent on the climatic
conditions (Liverani, Giovannini, Versari, Sirri, &
Brandi, 2008). Late or early flowering is not always
related to an earlier or later harvest, which depends
much more on the fruit development period, as we
will discuss below. For example, ‘Angeleno’ is the
earliest flowering genotype within this set, but this
same cultivar is almost the latest variety harvested.
In terms of ripening date, the most important
markers linked to this trait were S3_20966457 and
S4_11138979, with a percentage of variance explained
(R2) between 0.71–0.85. However, we pay special
attention on the marker S4_11138979, where the
homozygous allele (GG) is related to early maturity
(180–219 JD, January), while the heterozygous allele,
present in ‘Angeleno’, ‘September King’ and
‘September Queen’, is related to late harvesting
(221–264 JD, end of February) (Tables 4 and 5).
This relationship is quite relevant, because several
major QTLs linked to ripening date have been
reported in LG4 in stone fruits such us peach, apricot,
 THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY 7

Figure 2. Cladogram of genetic diversity by neighbour-joining trees using cluster analysis based on the mean character
difference distances among 11 Japanese plum cultivars assayed.

Table 4. Marker-trait association by General Linear Model (GLM) in TASSEL v5 for flowering date (FD), ripening date (RD) and
fruit development period (FDP) in 2016, 2017 and 2018 and skin chlorophyll degradation (SIad_1–2), flesh chlorophyll
degradation (FIad_1–2) and flesh colour by visually (FLSC).
Trait Marker Chr Position (Mbp) marker_F marker_df p-value R2
FD_16 S1_42831517 1 42,831,517 9.590 2 0.019 0.771
FD_18 S1_42831517 1 42,831,517 7.117 1 0.044 0.407
FD_16 S1_47165263 1 47,165,263 9.964 2 0.018 0.769
FD_18 S1_47165263 1 47,165,263 6.980 1 0.046 0.324
FD_16 S7_13781041 7 13,781,041 28.246 2 0.002 0.882
FD_17 S7_13781041 7 13,781,041 7.159 1 0.044 0.327
RD_16 S3_20966457 3 20,966,457 19.805 2 0.002 0.773
RD_17 S3_20966457 3 20,966,457 41.660 2 0.001 0.851
RD_18 S3_20966457 3 20,966,457 32.351 2 0.001 0.821
RD_16 S4_11138979 4 11,138,979 30.622 1 0.001 0.725
RD_17 S4_11138979 4 11,138,979 34.731 1 0.001 0.769
RD_18 S4_11138979 4 11,138,979 24.928 1 0.002 0.713
RD_16 S6_18854841 6 18,854,841 24.220 1 0.002 0.698
RD_17 S6_18854841 6 18,854,841 26.326 1 0.003 0.753
RD_18 S6_18854841 6 18,854,841 20.465 1 0.006 0.714
FDP_16 S3_20966457 3 20,966,457 51.201 2 0.000 0.860
FDP_17 S3_20966457 3 20,966,457 33.557 2 0.001 0.863
FDP_18 S3_20966457 3 20,966,457 19.432 2 0.004 0.819
FDP_16 S4_11138979 4 11,138,979 68.205 1 0.000 0.826
FDP_17 S4_11138979 4 11,138,979 33.261 1 0.001 0.786
FDP_18 S4_11138979 4 11,138,979 20.716 1 0.004 0.717
FDP_16 S6_18854841 6 18,854,841 51.294 1 0.000 0.802
FDP_17 S6_18854841 6 18,854,841 19.604 1 0.006 0.713
FDP_18 S6_18854841 6 18,854,841 20.478 1 0.006 0.732
SIad_1-2_16 S6_22121851 6 22,121,851 10.062 2 0.018 0.594
SIad_1-2_16 S6_23309668 6 23,309,668 10.062 2 0.018 0.594
FIad_1-2_16 S4_9698928 4 9,698,928 9.5856 1 0.021 0.487
FLSC_16 S3_8499544 3 8,499,544 16.240 1 0.010 0.574
FLSC_16 S6_29514112 6 29,514,112 49.257 2 0.002 0.897

cherry and plum (Dirlewanger et al., 2012; Eduardo
et al., 2011; Salazar et al., 2017), all of them reaching
a very high phenotypic variability explanation so that
even in a small variety collection it could be possible
to detect important genetic associations. Our results
indicate that the most important ripening date QTL
is located in a region of chromosome 4 that is com-
mon among all Prunus species, suggesting the
opportunity to design molecular markers useful for
marker-assisted selection in Prunus breeding. The
S4_11138979 marker is also related to FDP just like
RD where the GT allele from ‘Angeleno’, ‘September
King’ and ‘September Queen’ coincides with a longer
FDP, with at least 2 weeks of difference between
different allelic classes (Table 5). However, there is
another important marker in LG6 (S6_18854841)
 8
J. A. SALAZAR ET AL.

Table 5. Allelic class of each Japanese plum genotype related to flowering date and ripening date in Julian days for 2016, 2017 and 2018.
Flowering date Ripening date Fruit development period
Marker Year Marker Year Year
Genotype S1_42831517 S7_13781041 2016 2017 2018 Genotype S3_20966457 S4_11138979 S6_18854841 2016 2017 2018 2016 2017 2018
Angeleno CT CT 63 50 58 Black Splendor AG GG — 198 180 185 129 141 140
Owen T CT CT 64 49 51 Owen T AG GG GG 198 194 195 134 145 144
Golden Kiss — CT 69 39 47 Sun Dew AA GG GG 200 — — 130 — —
Black Splendor CT CT 69 39 47 Sapphire AG GG GG 204 190 200 132 147 145
Sun Dew CC CC 70 — — 98–99 AG GG GG 212 200 206 136 139 142
Larry Ann CT CT 72 52 56 Golden Kiss GG GG AG 219 197 202 150 158 155
Sapphire CT CT 72 43 55 Friar AG GG GG 219 200 213 142 147 151
September Queen TT TT 72 58 62 Larry Ann AG GG AG 219 200 206 147 148 150
September King TT TT 74 61 69 Angeleno AA GT AG 237 221 223 174 171 165
98–99 TT TT 76 61 64 Septemb. K AA GT AG 259 250 262 185 189 193
Friar TT — 77 53 62 Septemb. Q AA GT AG 259 253 264 187 195 202
 THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY 9

related to FDP, in which a longer FDP (more than
150 days) is related to AG alleles, and a shorter FDP
(less than 150 days) is related to GG alleles. In
another recent association study performed in peach
(Cao et al., 2016b, 2016c), SNPs related to fruit
weight in LG6 were identified that may be associated
with longer or shorter fruit development periods. It,
therefore, seems interesting to study the FW, FDP
and RT traits together to analyse how they behave
between the different cultivars and in segregating
populations as well.
Regarding chlorophyll degradation, this process
enables us to make an indirect estimation of the phy-
siological maturity of the plant, which it is highly
correlated to fruit senescence (Azoulay Shemer et al.,
2008; Hörtensteiner, 2006; Singh, Singh, & Swinny,
2012) at both the skin and flesh level. In the current
study, we found several molecular markers linked to
this trait in LG6 and LG4. However, the most impor-
tant relationship was found for S4_9698928, in which
the heterozygous allele (AG) is related to a lower flesh
chlorophyll difference between day 1 and 4 (FIAD_1–2
below 0.10) and the GG allele is related to a higher
difference, which means a higher chlorophyll degrada-
tion (FIAD_1–2 above 0.10) and fruit senescence as
well (Tables 4 and 6 and S6). This difference is likely
minimal because a period of only 4 days between
maturity states may not be enough to show significant
chlorophyll degradation (Contador et al., 2016).
Finally, as for FLSC, this trait seems to be related
to LGs 3 and 6, as has been reported in other QTL
studies (Tables 4 and 6 and S6). In LG3, the signifi-
cant marker was S3_8499544, in which yellow fleshy
genotypes are linked to the CC allele, while the red-
dish fleshy varieties are related to the CT allele.
However, the behaviour of ‘Black Splendor’, ‘98–99’
and ‘Sapphire’ was very different, because ‘Black
Splendor’ showed completely red flesh, while ‘98–99’
and ‘Sapphire’ are often classified as yellow-fleshed.
Nevertheless, when ‘Sapphire’ and ‘98–99’ plums are
close to ripeness for consumption, their flesh
colour turns from yellow to red. It could, therefore,
be interesting to study when the anthocyanin and
carotenoid synthesis starts in different genotypes,
since those compound families are responsible for
colour of plant tissues in Rosaceae.
The putative QTLs we identified as being linked to
phenology and fruit quality traits in this variety col-
lection are similar to the results in previous studies
(Salazar et al., 2017). Determining the QTLs in
broader germplasm collections may help us establish
whether QTLs remain significantly stable in the same
genomic region between different cultivars within the
species. The assayed cultivars in this study can serve
as model populations from the appropriate parents
that provide recombinant seedlings in the potential
QTL regions.
Conclusions
Our results show that GBS-based SNPs are a powerful
methodology for developing SNP markers for genetic
diversity studies and markers potentially linked to rele-
vant agronomic and pomological traits of P. salicina.
Thus, GBS provides a large quantity of useful data sui-
table for the identification of single nucleotide poly-
morphisms (SNPs), which could allow more accurately
and easier marker-trait associations for upcoming asso-
ciation studies in a wider range of varieties and progenies
of P. salicina. In this study, we described different SNP
markers that were related to several important traits,
especially flowering date, ripening date and fruit

Table 6. Allelic class of each Japanese plum genotype related to skin chlorophyll degradation (SIAD_1–2), flesh chlorophyll
degradation (FIAD_1–2) and flesh colour (FLSC by visually and FLSC (H°)).
Skin chlorophyll degradation Flesh chlorophyll degradation Flesh color
Marker Marker Marker
FLSC
Genotype S6_22121851 S6_23309668 SIad_1–2 Genotype S4_9698928 FIad_1–2 Genotype *S3_8499544 S6_29514112 FLSC (H°)
Friar TT TT 0.00 Angeleno AG 0.00 September CC CC 2 105.95
Q.
September CT CT 0.01 Friar AG 0.04 Golden Kiss CC GC 3 80.11
Q.
Golden Kiss CC CC 0.04 Golden Kiss AG 0.06 Sun Dew – GG 3 —
September CT CT 0.11 Owen T AG 0.06 Friar CC GC 3 101.51
K.
Owen T CT CT 0.13 Larry Ann AG 0.08 Larry Ann CC GC 3 85.78
Sapphire CT CT 0.17 September AG 0.10 Owen T – GC 3 97.33
Q.
Angeleno CT CT 0.19 Septemb K. GG 0.11 September CC GC 3 63.35
K.
Black CT CT 0.20 98–99 GG 0.19 Angeleno CC GC 3 93.27
Splendor
Larry Ann CT CT 0.28 Black GG 0.22 Black CT GG 4 21.11
Splendor Splendor
98–99 CT CT 0.32 Sapphire GG 0.29 98–99 CT GG 4 13.68
Sun Dew CC CC — Sun Dew GG — Sapphire CT GG 4 35.36
* SNP position from Peach Genome v1.
10 J. A. SALAZAR ET AL.
development period during 3 consecutive years.
However, we must assume that the results obtained for
only 1 year of phenotyping should be validated in a wider
collection of varieties or in segregating populations by
QTL analysis with at least 2 years of phenotyping, espe-
cially for skin and flesh chlorophyll degradation. As for
the visual flesh colour, we must take into account it’s
a more Mendelian trait, which means that the fruit
colour doesn’t vary between years, so it would not be
necessary to quantify it if there is no intermediate inheri-
tance. However, it would be interesting to validate this
marker-trait association with a wider variety collection.
Nevertheless, these results show that the applicability of
GBS as an approach for genomic studies represent
a promising beginning as a genomic support for using
Molecular Assisted Selection in current and new
Japanese plum breeding programmes.
Acknowledgements
This research has been sponsored by the University of
Chile and supported by the National Commission for
Scientific and Technological Research (CONICYT) of the
government of Chile through FONDECYT Postdoctoral
fellowship No. 3160080. IP was supported by
FONDECYT Start into Research No. 11150662 and the
Program of Insertion Into Academy No. 79140020.
Data archiving statement
SNP data used will be submitted on Genome Database for
Rosaceae (www.rosaceae.org).
Disclosure statement
No potential conflict of interest was reported by the
authors.
ORCID
Juan Alfonso Salazar http://orcid.org/0000-0003-0380-
076X
References
Azoulay Shemer, T., Harpaz-Saad, S., Belausov, E.,
Lovat, N., Krokhin, O., Spicer, V., … Eyal, Y. (2008).
Citrus chlorophyllase dynamics at ethylene-induced fruit
color-break: A study of chlorophyllase expression,
post-translational processing kinetics and in-situ intra-
cellular localization. Plant Physiology, 148, 108–118.
doi:10.1104/pp.108.124933
Bielenberg, D.G., Rauh, B., Fan, S., Gasic, K., Abbott, A.G.,
Reighard, G.L., & Wells, C.E. (2015). Genotyping by
sequencing for SNP-based linkage map construction
and QTL analysis of chilling requirement and bloom
date in peach [Prunus persica (L.) Batsch]. PLoS One,
10, e0139406.
Bonora, E., Stefanelli, D., & Costa, G. (2013). Nectarine
Fruit Ripening and Quality Assessed Using the Index of
Absorbance Difference (IAD). International Journal of
Agronomy, no.242461, 9. doi:10.1155/2013/242461
Cao, K., Wang, L., Zhu, G., Fang, W., Chen, C., & Luo, J.
(2012a). Genetic diversity, linkage disequilibrium, and
association mapping analyses of peach (Prunus persica)
landraces in China. Tree Genetics & Genomes, 8, 975–990.
Cao, K., Zhao, P., Zhu, G., Fang, W., Chen, C., Wang, X., &
Wang, L. (2016b). Expansin genes are candidate markers
for the control of fruit weight in peach. Euphytica, 210,
441–449.
Cao, K., Zhou, Z., Wang, Q., Guo, J., Zhao, P., Zhu, G., …
Wang, L. (2016c). Genome-wide association study of 12
agronomic traits in peach. Nature Communications, 7,
13246.
Castède, S., Campoy, J.A., García, J.Q., Le Dantec, L.,
Lafargue, M., Barreneche, T., … Dirlewanger, E.
(2014). Genetic determinism of phenological traits
highly affected by climate change in Prunus avium:
Flowering date dissected into chilling and heat
requirements. New Phytologist, 202, 703–715.
Chen, T., Huang, X., Zhang, J., Chen, Q., Liu, Y., Tang, H.,
& Wang, X. (2016). Genetic diversity and population
structure patterns in Chinese Cherry (Prunus pseudocer-
asus Lindl) Landraces. Plant Molecular Biology Reporter,
34, 440–453.
Contador, L., Díaz, M., Millanao, M., Hernández, E.,
Shinya, P., Sáenz, C., & Infante, R. (2016). A proposal
for determining the flesh softening of peach and nectar-
ine in postharvest through simplified targeted modeling.
Scientia Horticulturae, 19, 47–52.
Desnoues, E., Baldazzi, V., Génard, M., Mauroux, J.B.,
Lambert, P., Confolent, C., & Quilot-Turion, B. (2016).
Dynamic QTLs for sugars and enzyme activities provide
an overview of genetic control of sugar metabolism
during peach fruit development. Journal of
Experimental Botany, 67, 3419–3431.
Dirlewanger, E., Quero-García, J., Le Dantec, L.,
Lambert, P., Ruiz, D., Dondini, L., & Arús, P. (2012).
Comparison of the genetic determinism of two key phe-
nological traits, flowering and maturity dates in three
Prunus species: Peach, apricot and sweet cherry.
Heredity, 109, 280–292.
Doyle, J.J., & Doyle, J.L. (1987). A rapid isolation proce-
dure for small quantities of fresh leaf tissue.
Phytochemical Bulletin, 19, 11–15.
Eduardo, I., Pacheco, I., Chietera, G., Bassi, D., Pozzi, C.,
Vecchietti, A., & Rossini, L. (2011). QTL analysis of fruit
quality traits in two peach intraspecific populations and
importance of maturity date pleiotropic effect. Tree
Genetics & Genomes, 7, 323–335.
Elshire, R.J., Glaubitz, J.C., Sun, Q., Poland, J.A.,
Kawamoto, K., Buckler, E.S., & Mitchell, S.E. (2011).
A robust, simple genotyping-by-sequencing (GBS)
approach for high diversity species. PLoS One, 6.
doi:10.1371/journal.pone.0019379
Gottardi, F., Noferini, M., Fiori, G., Barbanera, M.,
Mazzini, C., & Costa, G. (2009). The index of absor-
bance difference (IAD) as a tool for segregating peaches
and nectarines into homogeneous classes with different
shelf-life and consumer acceptance. In: Proceedings of
the 8th Pangborn Sensory Science Symposium. Firenze,
Italy.
Guajardo, V., Solís, S., Sagredo, B., Gainza, F., Muñoz, C.,
Gasic, K., & Hinrichsen, P. (2015). Construction of high
density sweet cherry (Prunus avium L.) linkage maps using
microsatellite markers and SNPs detected by
genotyping-by-sequencing (GBS). PLoS One, 10, e0127750.
 THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY
Gurcan, K., Teber, S., Ercisli, S., & Yilmaz, K.U. (2016).
Genotyping by Sequencing (GBS) in apricots and genetic
diversity assessment with GBS-derived Single-Nucleotide
Polymorphisms (SNPs). Biochemical Genetics, 54, 854–885.
Hörtensteiner, S. (2006). Chlorophyll degradation during
senescence. Annu Rev Plant Biol, 57, 55–77.
Infante, R., Reginato, G., & Hinrichsen, P. (2011a). “Andes-
1”: An early-maturing clingstone peach cultivar for can-
ning and fresh market. HortScience, 46, 499–500.
Infante, R., Rubio, P., Contador, L., Noferini, M., &
Costa, G. (2011b). “Determination of harvest matur-
ity of D’Agen plums using the chlorophyll absor-
bance index. Ciencia e Investigación Agraria, 38,
199–203.
Jia, G., Huang, X., Zhi, H., Zhao, Y., Zhao, Q., Diao, X., &
Han, B. (2013). A haplotype map of genomic variations
and genome-wide association studies of agronomic traits
in foxtail millet (Setaria italica). Nature Genetics, 45,
957–961. doi:10.1038/ng.2673
Khadivi-Khub, A., Yarahmadi, M., Jannatizadeh, A., &
Ebrahimi, A. (2015). Genetic relationships and diversity
of common apricot (Prunus armeniaca L.) based on
simple sequence repeat (SSR) markers. Biochemical
Systematics and Ecology, 61, 366–371.
Kujur, A., Bajaj, D., Upadhyaya, H.D., Das, S., Ranjan, R.,
Shree, T., … Parida, S.K. (2015). Employing genome-wide
SNP discovery and genotyping strategy to extrapolate the
natural allelic diversity and domestication patterns in
chickpea. Frontiers in Plant Science, 6, 162.
Liverani, A., Giovannini, D., Versari, N., Sirri, S., &
Brandi, F. (2008). Japanese and European plum cultivar
evaluation in the Po Valley of Italy: Yield and climate
influence. IX International Symposium on Plum and
Prune Genetics, Breeding and Pomology. Acta
Horticulturae, 874, 327–335.
Mariette, S., Wong, F., Roch, G., Barre, A., Chague, A.,
Decroocq, S., … Decroocq, V. (2016). Genome-wide
association links candidate genes to resistance to Plum
Pox Virus in apricot (Prunus armeniaca). New
Phytologist, 209, 773–784.
Martínez-García, P.J., Parfitt, D.E., Ogundiwin, E., Fass, J.,
Chan, H.M., Ahmad, R., & Crisosto, C.H. (2012). High
density SNP mapping and QTL analysis for fruit quality
characteristics in peach (Prunus persica L.). Tree Genetics
& Genomes, 9, 19–36.
Meng, F., & Peng, M. (2015). Amplified fragment length
polymorphism analysis of genetic diversity and relation-
ships of wild and cultivated peach (Prunus persica L.).
HortScience, 50, 44–50.
Minas, I.S., Font I Forcada, C., Dangl, G.S., Gradziel, T.
M., Dandekar, A.M., & Crisosto, C.H. (2015).
Discovery of non-climacteric and suppressed climac-
teric bud sport mutations originating from
a climacteric Japanese plum genotype (Prunus salicina
Lindl.). Frontiers in Plant Science, 6, 316. doi:10.3389/
fpls.2015.00316
Pacheco, I., Bassi, D., Eduardo, I., Ciacciulli, A., Pirona, R.,
Rossini, L., & Vecchietti, A. (2014). QTL mapping for
brown rot (Monilinia fructigena) tolerance in an intras-
pecific peach (Prunus persica L. Batsch) F1 progeny. Tree
Genetics & Genomes, 10, 1223–1242.
Rousselin, A., Sauge, M.H., Jordan, M.O., Vercambre, G.,
Lescourret, F., & Bevacqua, D. (2016). Nitrogen and
water supplies affect peach tree-green peach aphid
interactions: The key role played by vegetative
growth. Agricultural and Forest Entomology, 18,
367–375.
View publication stats
11
Ruiz, D., Lambert, P., Audergon, J.M., Dondini, L.,
Tartarini, S., Adami, M., … Testolin, R. (2010).
Identification of QTLs for fruit quality traits in apricot.
XIV International Symposium on Apricot Breeding and
Culture. Acta Horticulturae, 862, 587–592.
Salazar, J.A., Pacheco, I., Shinya, P., Zapata, P., Silva, C.,
Aradhya, M., … Infante, R. (2017). Genotyping by sequen-
cing for SNP-based linkage analysis and identification of
QTLs linked to fruit quality traits in Japanese Plum
(Prunus salicina Lindl.). Frontiers in Plant Science, 8, 476.
Salazar, J.A., Rubio, M., Ruiz, D., Tartarini, S., Martínez-
Gómez, P., & Dondini, L. (2015). SNP development for
genetic diversity analysis in apricot. Tree Genetics &
Genomes, 11, 15.
Salazar, J.A., Ruiz, D., Campoy, J.A., Sánchez-Pérez, R.,
Crisosto, C.H., Martínez-García, P.J., … Rubio, M. (2014).
Quantitative Trait Loci (QTL) and Mendelian Trait Loci
(MTL) Analysis in Prunus: A breeding perspective and
beyond. Plant Molecular Biology Reporter, 32, 1–18.
Salazar, J.A., Ruiz, D., Campoy, J.A., Tartarini, S.,
Dondini, L., & Martínez-Gómez, P. (2016).
Inheritance of reproductive phenology traits and
related QTL identification in apricot. Tree Genetics &
Genomes, 12, 71.
Singh, S.P., Singh, Z., & Swinny, E.E. (2012). Climacteric
level during fruit ripening influences lipid peroxidation
and enzymatic and non-enzymatic antioxidative systems
in Japanese plums (Prunus salicina Lindell). Postharvest
Biology & Technology, 65, 22–32.
Tamarzizt, H., Mustapha, B.S., Baraket, G., Abdallah, D., &
Salhi-Hannachi, A. (2015). Assessment of genetic diver-
sity and relationships among wild and cultivated
Tunisian plums (Prunus sp) using random amplified
microsatellite polymorphism markers. Genetics and
Molecular Research, 14, 1942–1956.
Verde, I., Bassil, N., Scalabrin, S., Gilmore, B., Lawley, C.T.,
Gasic, K., … Peace, C. (2012). Development and evalua-
tion of a 9K SNP array for peach by internationally
coordinated SNP detection and validation in breeding
germplasm. PLoS One, 7, e35668.
Verde, I., Jenkins, J., Dondini, L., Micali, S., Pagliarani, G.,
Vendramin, E., … Schmutz, J. (2017). The Peach v2.0
release: High-resolution linkage mapping and deep rese-
quencing improve chromosome-scale assembly and
contiguity. BMC Genomics, 18, 225.
Wang, Q., Wang, L., Zhu, G., Cao, K., Fang, W., Chen, C.,
& Wang, X. (2016). DNA marker-assisted evaluation of
fruit acidity in diverse peach (Prunus persica)
germplasm. Euphytica, 210, 413–426. doi:10.1007/
s10681-016-1709-z
Zeballos, J.L., Abidi, W., Giménez, S., Monforte, A.J.,
Moreno, M.A., & Gogorcena, Y. (2016). Mapping QTLs
associated with fruit quality traits in peach [Prunus persica
(L.) Batsch] using SNP maps. Tree Genetics & Genomes, 12,
3. doi:10.1007/s11295-016-0996-9
Zeinalabedini, M., Dezhampour, J., Majidian, P.,
Khakzad, M., Zanjani, B.M., Soleimani, A., & Farsi, M.
(2014). Molecular variability and genetic relationship
and structure of Iranian Prunus rootstocks revealed by
SSR and AFLP markers. Scientia Horticulturae, 172,
258–264. doi:10.1016/j.scienta.2014.04.006
Zhebentyayeva, T.N., Fan, S., Chandra, A., Bielenberg, D.
G., Reighard, G.L., Okie, W.R., & Abbott, A.G. (2014).
Dissection of chilling requirement and bloom date QTLs
in peach using a whole genome sequencing of sibling
trees from an F2 mapping population. Tree Genetics &
Genomes, 10, 35–51.