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Evaluation of the genetic diversity of Asian peach accessions using a selected set of SSR markers
Scientia Horticulturae 125 (2010) 622–629
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Evaluation of the genetic diversity of Asian peach accessions using a selected set of SSR markers Rangjin Xie a,d , Xiongwei Li a , Mingliang Chai a , Lijuan Song a,1 , Huijuan Jia a , Dajun Wu b , Miaojin Chen b , Keming Chen b , Maria Jose Aranzana c , Zhongshan Gao a,∗ a
Department of Horticulture, The State Agriculture Ministry Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Zhejiang University, Hangzhou 310029, China b Fenghua Honey Peach Institute, Xikou, Fenghua, Zhejiang Province, 315521, China c IRTA, Centre de Recerca en Agrigenòmica CSIC-IRTA-UAB, Carretera de Cabrils Km2; 08348 Cabrils (Barcelona), Spain d Citrus Research Institute, Chinese Academy of Agricultural Sciences, Chongqing 400712, China
a r t i c l e
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Article history: Received 16 March 2010 Received in revised form 5 May 2010 Accepted 17 May 2010 Keywords: Peach Melting peach Genetic diversity Allelic composition SSR
a b s t r a c t A total of 94 peach accessions from the Zhejiang province of China were analyzed using 34 polymorphic single-locus simple sequence repeat (SSR) markers. Genetic distance analysis divided the accessions into two major clusters, one with mainly local accessions from the Fenghua region. Preselected lines from some crosses were exclusively clustered with the local ones, possibly related to maintaining the taste quality of the fruit. The number of alleles per locus at most loci was two or three, with an average of 2.85. The value of observed heterozygosity varied from 0.05 to 0.84 (average of 0.48). Diversity within the introduced accessions was higher than that of the Fenghua local accessions. Of the accessions analyzed in this study, 94% were individually identified. Those that could not be differentiated were all derived from the ‘Yulu’ cultivar, being either mutations or of identical origin. Our results suggest that Fenghua accessions are derived from limited parental materials and inbreeding over a long period of time. They will be useful for breeders to better manage their pre-breeding materials and choice of parents for further crossings. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Peach (Prunus persica) is one of the most economically important deciduous fruit tree species in the Rosaceae family. China has the longest history of peach cultivation, of more than 4000 years (Cheng and Huang, 2009), and it is generally thought that it has the richest genetically diverse germplasm, with many natural mutants and chance seedlings (Huang et al., 2008). According to the Chinese traditional classification based on texture, shape and skin hair characters, peach cultivars have been divided into six groups, namely crispy peach (very firm melting), sweet peach (firm melting), honey peach (soft melting), yellow fleshed peach (non-melting), flat peach and nectarine (Wang and Zhuang, 2001). Honey peach, sweet, juicy and aromatic with a very soft texture, was initially found in the Zhejiang and Jiangsu provinces and planted in just a few places close to Shanghai. For the consumer market, peach cultivar identity is not precise, and the ‘Shanghaishuimi’ peaches sold on the Shanghai
∗ Corresponding author. Tel.: +86 571 86971172; fax: +86 571 86971630. E-mail address: [email protected] (Z. Gao). 1 Current address: Department of Horticulture, Wenzhou Polytechnics College, Wenzhou 325006, China. 0304-4238/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2010.05.015
market are a group of cultivars belonging to the honey peach group. They became more popular and were gradually introduced to different areas and other countries because of the good fruit quality and adaptability to various climate and soil conditions. Fenghua city (in the east of Zhejiang province, near Shanghai) is one of the four regions in China where honey peach originated and is produced. According to the records, Fenghua honey peach was developed from a pointed-tip honey peach introduced from Shanghai by Yinchong Zhang in 1883 (Wang and Zhuang, 2001). Over more than 120 years of selection, a number of closely related elite cultivars referred to as ‘Yulu’ were cultivated, and were also used as parents in breeding programs. ‘Yulu’ cultivars are still grown on nearly half of the production area in Fenghua, 25 thousand tons, annually. In the last 10 years, new cultivars have been introduced to replace the local ones for two main reasons: (1) A narrow genetic background and inbreeding has caused general degradation phenomena in local cultivars, such as small fruit size, lack of desired red skin color, susceptibility to pests and diseases and poor postharvest properties, and (2) The local cultivars ripen late and around the same time (most in early August). During the 1990s, cultivar evaluation was used to select the best local cultivars, introduce new ones and for small-scale crossing. As a result, more than 130 honey peach cultivars/selections were pre-
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served in a local peach germplasm repository, but the genotypes and phenotypes of these accessions have not been well documented. Here we used molecular tools, in collaboration with the university and local research institute, to bridge the gap between geneticists and breeders. Microsatellites or simple sequence repeats (SSR) have extensively proved to be highly efficient for systematic evaluation of genetic diversity and cultivar identification in highly homozygous species such as peach (Sosinski et al., 2000; Aranzana et al., 2002, 2003a; Dirlewanger et al., 2004; Wünsch et al., 2006; Bouhadida et al., 2007; Li et al., 2008; Cheng et al., 2009). To date, nearly 500 SSRs have been developed and mapped in the Prunus reference map (Aranzana et al., 2003b; Dirlewanger et al., 2004; Howad et al., 2005), making it a valuable tool for genetic analysis. There are two recent reports on genetic diversity of Chinese peach cultivars using SSR markers (Li et al., 2008; Cheng and Huang, 2009) with a limited number of accessions (51 and 32) and SSR markers (22 and 7, respectively). For the purposes of cultivar identification and studying diversity, full coverage of representative SSRs and more accessions are needed. Marker genotypes flanking the main oligogenic or monogenic and polygenic fruit traits (QTLs) can be used for association studies and subsequent selection (Infante et al., 2008). In this study, we used 34 polymorphic SSR loci to: (1) assess the genetic identity and diversity among the honey peach cultivars, and (2) analyze the genetic background of the selected cultivars and their genetic relationship. The results of this study provide guidelines and a scientific base to better explore honey peach germplasm resources and facilitate local cultivar improvement.
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mated by comparing to the pBR322 DNA-MspIDigest (New England BioLabs) ladder. All amplifications were scored as either a present (1) or absent (0) band. The alleles were coded A, B, C, etc. for each band, in decreasing size order. A single band in one accession was assumed to be homozygous. 2.3. Statistical analysis Molecular diversity was estimated according to the following parameters: (1) number of alleles per locus (Na) and number of effective alleles per locus (Ne), (2) polymorphism information content (PIC), (3) observed heterozygotes at a given loci (Ho), and (4) occurrence of unique and rare alleles. Alleles were considered rare if they occurred in less than 5% of the accessions (up to 4 accessions) and unique if they occurred in only one accession. Na, Ne and Ho were calculated with the program POPGENE version 1.31 developed by Yeh et al. (1999), and PIC with the PowerstatsV12.xls software (Brenner and Morris, 1990), which is freely available on the internet (http://www.promega.com/geneticidtools/powerstats/). Genetic distances between the 94 accessions were calculated using Nei’s coefficient index (Nei, 1972) with the software NTSYSpc 2.1 (Rohlf, 2000), and a dendrogram was constructed with the same software using the unweighted pair-group method with arithmetic mean (UPGMA). The fit of the UPGMA cluster to the original similarity indices was computed according to the Mantel test procedure (Mantel, 1967) by using MxComp of the software package NTSYSpc 2.1 (Rohlf, 2000). 3. Results
2. Materials and methods 2.1. Plant material A total of 94 accessions (Table 1) obtained from the Fenghua Honey Peach Research Institute were used to evaluate genetic diversity and identity. Of these accessions 48 were from local collections and selections, and 46 introduced from different places in China, Japan and Korea or were of unknown origin. Young, non-fully expanded leaves were collected from healthy trees, frozen in liquid nitrogen, and then stored at −40 ◦ C prior to DNA extraction. 2.2. DNA isolation and SSR analysis Genomic DNA was extracted from 1 g of ground leaf tissue using CTAB as described by Zhang et al. (2009). The DNA concentration was quantified by ultraviolet spectrophotometry (Beckman coulter DU800). Thirty-four SSRs (Fig. 1) chosen as the given clear polymorphic bands were used to assay the set of 94 accessions. The SSR loci selected were recommended by the Spain IRTA group, who used them for cultivar genetic diversity. We added 2–4 bases at the 5 end of the original reverse primer to form a GTTT pigtail sequence to allow addition of an A-tail to all PCR products for accurate scoring of fragments (Brownstein et al., 1996). PCR products were in a total volume of 20 l containing 40 ng of DNA, 0.25 M of each primer, 0.2 mM of each dNTP, 0.5 U Taq polymerase (TAKARA BIO INC.) and 1× PCR buffer mix. PCR amplifications were performed in an Eppendorf Mastercycler 5333/5331 thermocycler (Gradient No. 5331-41264, Germany) with the following programs: 2.5 min at 94 ◦ C, 35 cycles of 30 s at 94 ◦ C, 30 s at the appropriate annealing temperature, and 30 s at 72 ◦ C, followed by a 10 min final extension at 72 ◦ C. The PCR products were separated on 6% denatured polyacrylamide gel at 75 W (BioRad Electrophoresis system) for 2 h and then visualized by silver staining. The band sizes were esti-
3.1. Analysis of SSR loci diversity Ninety-four honey peach accessions were analyzed with 34 polymorphic SSRs, distributed on the 8 linkage groups of the Prunus T × E reference map (Fig. 1). The variability detected by each SSR locus is shown in Table 2. The total number of amplified alleles was 97, 93 of them (95.9%) being polymorphic in the 94 accessions studied. The number of alleles amplified per marker ranged from 2 to 5 (in CPPCT029 and UDP96-13), with an average of 2.85 alleles/locus. With a mean value of 0.48, the observed heterozygosity (Ho ) ranged from 0.05 in BPPCT028 to 0.84 in Pchgms3. The Ne and PIC varied from 1.05 in the BPPCT028 to 3.66 in UDP96013 (mean value of 2.04) and from 0.05 to 0.68 in UDP96-013 (mean value of 0.4). Based on the PIC, the most informative loci were UDP96-013 and BPPCT025, with values of 0.68 and 0.63, respectively, whereas the least informative were CPPCT028 with a PIC value of 0.05. Allelic frequencies ranged from 0.01 to 1.0. Among the 97 observed alleles, 6 were unique, and 7 rare (frequency ≤ 0.05). The 48 Fenghua local accessions had slightly lower genetic diversity than the 46 introduced. The number of effective alleles per locus ranged from 2 to 3 (mean value 1.90) in Fenghua accessions, and from 2 to 3.87 (mean value 2.05) in the introduced accessions. These findings were also reflected in the PIC, where the average for Fenghua cultivars was 0.34 and for the introduced cultivars, 0.46. None of the studied accessions were homozygous at all loci (Fig. 2). Fenghua accessions were less heterozygous than the introduced (Ho = 0.46 and Ho = 0.50, respectively): the number of heterozygous loci in Fenghua accessions ranged from 6 to 22 and from 11 to 27 in the introduced ones. One of the Fenghua accessions was the most homozygous, with only 6 out of 34 loci heterozygous (Fig. 2). The most heterozygous local accession was ‘X2-7 , while the introduced ‘Hujingmilu’ was the most heterozygous (27/34). Sixteen of the 94 accessions carried unique and/or rare alleles. Most of them (14) were found in the introduced cultivars ‘Bailu’, ‘Zhongqiumi’, ‘Lais-
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Table 1 The 94 peach accessions used for the genetic variability assay, sorted as in Fig. 3. Accession
Origin
Pedigree
Harvest time
Red skin color
Texture
Stone
Hakuho1 Hakuho2 Nkazu Hakuto Yuzora Yamanashi Hakuho Ohdama Hakuho Hikawa Hakuho Matsumori Akatsuki Datuanmilu Hakuri Yamane Hakuto Mibaekdo Xinhong Hujingmilu Qiukonga Dabaifeng Riaia Taoyan2 Zhongqiumi Laishanmi Yumyeong New Kawanakajima Hakutoa Hongtaowang Juwangtao Xueyulu Yuhualu Xiahui2 Zaohualu2 Kurakato wase Yanhong Takei Hakuho Nunome Zaotaimi Xuexianglu Sunago Wase Asama Hakuho Matsumor Xumi X2-5 X1-22 X2-14 X2-17 X1-8 X1-3 Kawanakajima Hakuto Yahata Hakuto Reiho Shangshanyou Anping Jinjun Jianfang Jianding Xiangtu Sanbao3 Xipu4 Linjia Shangshandayulu XiPu1 Sanbao4 Chiyulu Sanbao2 Linguochang Sanbao1 Laoyulu Yulu Taoyan1 Beijinga Zaoyulu Xinyu Chibaifeng X5-17 X2-7 X4-1 X1-19
Japan Japan Japan Japan Japan Japan Japan Japan Japan Shanghai-China Japan Japan Korea Shanghai-China Jiangsu-China Japan Fenghua Japan Fenghua Heilongjiang -China Shandong-China Korea Japan Liaoning-China Anhui-China Hangzhou-China Jiangsu-China Jiangsu-China Jiangsu-China Japan Beijing-China Japan Japan Unknown Jiangsu-China Japan Japan Japan Anhui-China Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Japan Japan Japan Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Beijing Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua
Selected from Hakuho Selected from Hakuho Hakuto seedling Hakuto × Akatsuki Selected from Hakuto Selected from Hakuho Hakuho mutation A sport from Hakuho Hakuto × Hakuho Shanghaidatuan seedling Okubo × Feichengtao Unknown Shimizu seedling Selected from Hakuho Selected from Hakuho Unknown Selected from Hakuho Unknown Selected from Yulu Unknown Shandonglaishan seedling Okubo × Numome Selected from Kawanakajima Hakuto Baitao × Hakuho Unknown Baihuashuimi × Yuhualu Baihua × Shanghaishuimi Zhaohui × Zhaoxia Yuhualu seedling Unknown Unknown Hakuho × Yamanehakuto Seedling of Hakuto or Okubo Unknown Baihua × Shanghaishuimi Okubo or Kamidama seedling Takayo Hakuto Seedling of Hakuto Selected from Baihua Shangshandayulu × Hujingmilu Shangshandayulu × Hujingmilu Shangshandayulu × Hujingmilu Shangshandayulu × Hujingmilu Shangshandayulu × Hujingmilu Shangshandayulu × Hujingmilu Shanghaishuimi × Hakuto Selected from Hakuto Unknown Selected from Yulu Selected from Yulu Selected from Yulu Selected from Yulu Selected from Yulu Selected from Yulu Selected from Yulu Selected from Yulu Selected from Yulu Selected from Yulu Selected from Yulu Selected from Yulu Selected from Yulu Selected from Yulu Selected from Yulu Selected from Yulu Local cultivar Selected from Shanghaishuimi Selected from Yulu Unknown Selected from Yulu Selected from Yulu Selected from Hakuho Xipu1 × Hujingmilu Shangshandayulu × Hujingmilu Xipu1 × Hujingmilu Shangshandayulu × Hujingmilu
Late June Early July Mid-July Early September Early July Early–mid-July Late June Late June Early–mid-July Late June Early–mid-August Late June Mid-July Late July Early–mid-July Unknown Mid-late July Mid-July Mid-late June Late August Late August Early August Mid-July Mid-August Mid-August Early June Late June Early June Early June Early June Late August Late June Early June Early June Early June Early June Early–mid-July Late June Late August Late June Early July Mid-July Mid-August Mid-July Mid-August Late July Mid-July Mid-July Early August Early August Early August Early August Early August Early August Early August Late July Early August Early August Late July Early August Mid-late August Early August Early August Early August Early–mid-August Early August Late July Late June Late July Mid-late July Early July Late July Mid-late July Mid-late July Early August
P P P P P F P F F P P P F F F P F P F F F F P F F P F P P P F P P P P P P P F F F F F F F F F F L L L L L L L L L L L L L L L L L L P F P F P F F P F
SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM FM FM NM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM FM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM
C C C C C C C C C C C C C C C C C C C C C C C C C F C F C C C C C F F C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C
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Table 1 (Continued. ) Accession
Origin
Pedigree
Harvest time
Red skin color
Texture
Stone
X2-2 X1-14 X1-7 X2-11 X3-4 X2-6 X1-4 X2-12 X3-3 X1-1 Renhea X2-10 X5-20 X2-18 X1-18 X2-19 X1-13 Bailua Zaoshanghaishuimi
Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Japan Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Japan Shanghai
Shangshandayulu × Hujingmilu Shangshandayulu × Hujingmilu Shangshandayulu × Hujingmilu Shangshandayulu × Hujingmilu Xipu1 × Hujingmilu Shangshandayulu × Hujingmilu Shangshandayulu × Hujingmilu Shangshandayulu × Hujingmilu Xipu1 × Hujingmilu Shangshandayulu × Hujingmilu Unknown Shangshandayulu × Hujingmilu Xipu1 × Hujingmilu Xipu1 × Hujingmilu Shangshandayulu × Hujingmilu Xipu1 × Hujingmilu Shangshandayulu × Hujingmilu Unknown Selected from Shanghaishuimi
Early August Mid-July Early August Early July Early August Early August Early August Early August Early August Late July Late July Early August Early August Mid-late July Early August Late July Early August Mid-late July Early July
F F F F F F F F P F F P F F F F F P P
SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM
C C C C C C C C C C C C C C C C C C C
Skin color: full blush coverage (F), partially red, about 25% coverage (P), very little red color on top or sutural line (L). Texture: soft melting (SM), firm melting (FM) and non-melting (NM). Stone: C-cling, F-free. a Local name record in Fenghua,
Fig. 1. The 34 mapped SSR loci on the eight peach linkage groups. The SSR markers developed by a Cipriani et al. (1999), b Aranzana et al. (2002), c Dirlewanger et al. (2002), d Sosinski et al. (2000), e Downey and Iezzoni (2000), f Testolin et al. (2000), g Mnejja et al. (2004), h Cantini et al. (2001), i Joobeur et al. (2000), and k EPPCU1090 and EPPCU1775 were mapped in bin G1:34 and G4:63 (Howad et al., 2005); l PTS1 is EST-SSR derived from an intron when genomic cloning based on DY639772 annotated as terpene synthase in NCBI, this marker was bin-mapped on G4:18 (unpublished).
Fig. 2. Distribution of the number of heterozygous loci per accession detected in Fenghua cultivars (white) and the introduced cultivars (black). A total of 34 SSR loci were tested.
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Table 2 Variability identified using 34 SSRs in 94 peach cultivars. Linkage groups
SSR locus
No. of alleles (effective alleles) per locus
Polymorphism information content (PIC)
Observed Heterozygosity
No. of rare (unique) alleles
G1
UDP96-018 UDP96-005 EPPCU1090 CPPCT026 Pchgms3 BPPCT020 CPPCT029 BPPCT028
4(2.76) 3(2.60) 2(1.87) 4(3.0) 3(2.17) 2(1.80) 5(1.32) 2(1.05)
0.57 0.55 0.36 0.60 0.44 0.35 0.23 0.05
0.53 0.45 0.44 0.32 0.84 0.59 0.16 0.05
0(1) 0(0) 0(0) 1(0) 0(0) 0(0) 1(2) 1(0)
G2
CPPCT044 BPPCT001 UDP96-013 Pchgms1 PceGA34
3(1.88) 4(1.65) 5(3.66) 2(1.70) 3(2.86)
0.38 0.33 0.68 0.33 0.57
0.32 0.45 0.49 0.56 0.69
1(0) 0(1) 1(0) 0(0) 0(0)
G3
BPPCT007 UDP96-008
3(1.94) 3(2.73)
0.37 0.56
0.46 0.55
0(0) 0(0)
G4
CPPCT005 UDP98-024 PTS1 BPPCT015 EPPCU1775
2(1.61) 3(2.19) 3(2.88) 2(1.77) 3(1.42)
0.31 0.47 0.57 0.34 0.28
0.49 0.41 0.82 0.57 0.16
0(0) 0(0) 0(0) 0(0) 0(0)
G5
CPPCT040 BPPCT017 CPSCT006 PceGA25 CPPCT013 BPPCT014
4(2.08) 2(1.83) 3(2.01) 2(1.92) 2(1.50) 2(1.68)
0.43 0.35 0.38 0.36 0.28 0.32
0.55 0.46 0.70 0.54 0.42 0.52
1(1) 0(0) 0(1) 0(0) 0(0) 0(0)
G6
CPPCT015 Pchgms5 BPPCT025 CPPCT030
2(1.53) 2(1.98) 4(3.16) 3(2.38)
0.29 0.38 0.63 0.52
0.38 0.51 0.68 0.71
0(0) 0(0) 0(0) 0(0)
G7
CPPCT022 CPPCT017
2(1.88) 4(1.57)
0.36 0.33
0.30 0.31
0(0) 1(0)
G8
CPPCT006 UDP98-409
2(1.30) 2(1.90)
0.24 0.36
0.16 0.76
0(0) 0(0)
2.85(2.04)
0.40
0.48
Total Mean
7(6)
hanmi’, ‘Zaohualu’ and ‘Yanhong’, while for the Fenghua accessions, only ‘Xiangtu’ and ‘X1-13’ amplified. 3.2. Genetic relationship and pedigree A UPGMA dendrogram derived from the similarity matrix (Fig. 3), grouped the accessions into two major clusters, according to their origin. Mantel test (Mantel, 1967) showed a very good fit of the unweighted pair-grouped method with arithmetic average (UPGMA) cluster to the original genetic distance matrix (r = 0.978, p < 0.001). The first cluster (from ‘Hakuho1 to ‘Sunago wase’ in Fig. 3) included 34 introduced and two local cultivars. The second cluster (from ‘Asama Hakuho’ to ‘Bailu’ in Fig. 3), contained 57 accessions, 48 from Fenghua, and 9 introduced. The new Fenghua selections (names starting with X) were distributed into two groups, one clustering together with ‘Xumi’ from China, and several cultivars from Japan including ‘Renhe’. ‘Linjia’, ‘Shangshandayulu’ and ‘Xipu1 shared the same SSR genotype. These three accessions were all selected from ‘Yulu’ and are considered identical or with small morphological differences. Two accessions related to ‘Yulu’ (‘Jinjun’ and ‘Jianfang’) were also not distinguished by any of the 34 SSRs. Other local cultivars have more than 95% identical alleles. For example, ‘Laoyulu’ (which means “old Yulu”) differed from ‘Yulu’ at two SSR loci (UDP96-018 and CPPCT022).
The new Fenghua selections are descendants of two different recorded pedigrees (‘Shangshandayulu’ × ‘Hujingmilu’ and ‘Xipu1 × ‘Hujingmilu’). SSR data was used to validate the pedigree information of these accessions. We found that most selections carried the allele from both parents, consistent with their pedigree, while a few new selections were not derived from the original crosses ‘Shangshandayulu’ × ‘Hujingmilu’ and ‘Xipu1 × ‘Hujingmilu’. The introduced accessions ‘Reiho’, ‘Yahata Hakuto’ and ‘KawanakaJima Hakuto’ had very similar SSR genotypes. ‘KawanakaJima Hakuto’ is a progeny from ‘Shanghaishuimi’ × ‘Hakuto’, differing from ‘Reiho’ at one locus (PST1) and from ‘Yahata Hakuto’ at CPPCT022. ‘Yahata Hakuto’, derived from ‘Hakuto’ as recorded (Agricultural and Fishing Village Cultural Association, 2000), differed from ‘Reiho’ at the PST1 and CPPCT022 loci. These three Japanese cultivars are very close to Fenghua local cultivars (Fig. 3, sub-cluster 2.1). 4. Discussion 4.1. SSR polymorphism In this study, the average number of alleles was 2.85, which was lower than the 5 as previously reported by Li et al. (2008) in a sample of 51 Chinese cultivars (including honey peach, nectarine, flat peach, crispy peach and yellow fleshed peach) analyzed with 22
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Fig. 3. UPGMA dendrogram of 94 accessions based on their variation at 34 loci. (♦) Fenghua cultivars; () introduced cultivars.
polymorphic markers, the 7.3 observed by Aranzana et al. (2003a) using 16 polymorphic single-locus microsatellites (9 SSR loci are in common with this study) to evaluate a set of 212 occidental peach cultivars, the 4.2 detected by Dirlewanger et al. (2002) in 27 peach cultivar using 41 SSRs, the 3.5 by Wünsch et al. (2006) in a set of 85 local Spanish peach genotypes with polymorphic SSRs and the 3.1 observed by Cheng et al. (2009). While, it was higher than the 2.6 detected by Sosinski et al. (2000) in 28 scion peach cultivars with 10 SSRs and the 2.3 observed by Bouhadida et al. (2007) in 30 peach accessions using 20 polymorphic SSRs.
Our results suggest that the honey peach cultivars collected by the Fenghua Honey Peach Research Institute had a low level of genetic diversity. This was consistent with that observed by Li et al. (2008) in the subsample of 11 honey peach cultivars included in their study. There is some agreement in the results from Aranzana et al. (2003a) and Li et al. (2008) with respect to allelic variation we found at certain loci which amplified a higher (CPPCT029, BPPCT001) or lower number of alleles (CPPCT002), while we found a relatively low level of polymorphism for other loci (CPPCT022, CPPCT030)
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compared to the literature. This suggests that the ability of a marker to detect polymorphism may vary depending on the sample analyzed. 4.2. Comparative analysis of genetic diversity between the Fenghua and the introduced accessions In the present study, our results show that the introduced cultivars had a higher level of genetic diversity than that of the Fenghua local cultivars. This may be explained by the origin of the samples; the introduced cultivars were collected from different places while the Fenghua accessions are derived from limited parental materials and are a result of inbreeding over a long period of time. More than 90% of alleles were identical in local cultivars, and in one accession 28 out of 34 loci were homozygous. Preselected lines from a few crosses were exclusively clustered with the local ones, possibly related to maintaining the quality and characteristics of the old varieties. The results also indicate the need to make full use of the introduced cultivars as parental materials to enlarge the genetic diversity in breeding programs. Recent gene maps of fruit quality (Ogundiwin et al., 2009) enable closer analysis of the linked SSR alleles in the tested accessions. By combining the phenotypic traits and SSR marker data from this study, it would be possible to improve the crosses for specific variations. 4.3. Genetic relationship, cultivar identification and pedigree The UPGMA dendrogram obtained with our SSR data separates the accessions into two clusters. Most of the introduced cultivars were clearly separated from the Fenghua old local cultivars, indicating that the Fenghua group has been seed-propagated for a long period of time, with very little exchange with other cultivars. While it is generally accepted that Chinese cling peaches from Shanghai are the founders of modern cultivars, their pedigree is not well documented. ‘Shanghaishuimi’ is not likely to be a single cultivar, but a group of cultivars marketed in Shanghai. The two distinct clusters imply that the original ‘Shanghaishuimi’ was also comprised of two groups. It is unlikely that Fenghua ‘Yulu’ has been commonly used in modern peach breeding programs in Japan, since most Japanese cultivars are in the first cluster (Fig. 3). Analysis of the most representative cultivars and ‘Shanghaishuimi’, from different places in the world, with common polymorphic SSR marker is necessary to confirm whether this is true in western countries. In the present study, only 5 of the 94 accessions studied could not be differentiated: ‘Jinjun’, ‘Jianfang’, ‘Linjia’, ‘Shangshandayu’ and ‘Xipu1 . These are all selections of ‘Yulu’, possibly sports. In order to identify peach cultivars and/or analyze pedigree relationship in future more efficiently and low-costly, we selected a subset of 12 SSRs (EPPCU1090, BPPCT020, CPPCT029 and BPPCT028 on G1, UDP96-013 on G2; BPPCT007 on G3; PTS1 on G4, CPPCT040 and PceGA25 on G5; CPPCT015 on G6, CPPCT022 on G7 and UDP98-409 on G8), which have the same effectiveness of cultivar identification as the 34 SSRs used in this study. Six of these markers belong to the genotyping set proposed by Aranzana et al. (2003b). With the data from the 12 representative SSR markers, we largely confirmed the recorded pedigree of our tested materials. In two cases there were two loci with unexpected alleles, indicating that some of the discrepancies may also have been caused by SSR mutations, as has been reported previously (Aranzana et al., 2003a). With the availability of SSR genotypes of these peach accessions, a detailed systematic recording of agronomic traits is necessary for association studies of a number of important fruit traits, based on recent publications of the genetics of fruit quality traits (Peace et al., 2005; Ogundiwin et al., 2008, 2009). It is also possible to
establish core collections that will be of further use in breeding programs. In conclusion, we found that the Fenghua local cultivars are isolated descendents of Shanghaishuimi with very narrow genetic backgrounds. Their good taste quality traits are worth exploring for use in breeding programs. With the genetic information of peach germplasm, local breeders will have better knowledge of their breeding materials, leading to more efficient crossing combinations and selections. Acknowledgements This study has been carried out with financial support from the Natural Science Foundation of China (30771496), the Science and Technology Department of Zhejiang Province (2007C22068) and Ningbo (2006C100029). We thank Sun Qinan for his assistance in collecting plant materials and Chen Lin for some PCR work. The Institute of Horticulture of the Jiangsu Academy of Agricultural Sciences provided some plant materials. We thank Dr. Luud Gilissen for critical reading of this manuscript and Dr. Yinghui Li from CAAS for her advice on data analysis. References Agricultural and Fishing Village Cultural Association, 2000. Fruit Gardening Encyclopedia. Agricultural and Fishing Village Cultural Association, p. 70 (in Japanese). Aranzana, M.J., Garcia-Mas, J., Carbó, J., Arús, P., 2002. Development and variability analysis of microsatellite markers in peach. Plant Breeding 121, 87–92. Aranzana, M.J., Carbo, J., Arús, P., 2003a. Microsatellite variability in peach [Prunus persica (L.) Batsch]: cultivar identification, marker mutation, pedigree inferences and population structure. Theor. Appl. Genet. 106, 1341–1352. Aranzana, M.J., Pineda, A., Cosson, P., Dirlewanger, E., Ascasibar, J., Cipriani, G., Ryder, C.D., Testolin, R., Abbott, A., King, G.J., Iezzoni, A.F., Arús, P., 2003b. A set of simplesequence repeat (SSR) markers covering the Prunus genome. Theor. Appl. Genet. 106, 819–825. Bouhadida, M., Casas, A.M., Moreno, M.A., Gogorcena, Y., 2007. Molecular characterization of Miraflores peach variety and relatives using SSRs. Scientia Horticulture 111, 140–145. Brenner, C., Morris, J., 1990. Paternity index calculations in single locus hypervariable DNA probes: validation and other studies. http://www.promega.com/ geneticidtools/powerstats. Brownstein, M.J., Carpten, J.D., Smith, J.R., 1996. Modulation of non-templated nucleotide addition by Taq DNA polymerase: Primer modifications that facilitate genotyping. Biotechniques 20, 1004–1010. Cantini, C., Iezzoni, A.F., Lamboy, W.F., Boritzki, M., Struss, D., 2001. DNA fingerprinting of tetraploid cherry germplasm using simple sequence repeats. J. Am. Soc. Hort. Sci. 126, 205–209. Cheng, Z.P., Huang, H.W., 2009. SSR fingerprinting Chinese peach cultivars and landraces (Prunus persica) and analysis of their genetic relationships. Scientia Horticulturae 120, 188–193. Cipriani, G., Lot, G., Huang, W.G., Marrazzo, M.T., Peterlunger, E., Testolin, R., 1999. AC/GT and AG/CT microsatellite repeats in peach [Prunus persica (L) Batsch]: isolation, characterisation and cross-species amplification in Prunus. Theor. Appl. Genet. 99, 65–72. Dirlewanger, E., Cosson, P., Tavaud, M., Aranzana, M.J., Poizat, C., Zanetto, A., Arús, P., Laigret, F., 2002. Development of microsatellite markers in peach [Prunus persica (L.) Batsch] and their use in genetic diversity analysis in peach and sweet cherry (Prunus avium L.). Theor. Appl. Genet. 105, 127–138. Dirlewanger, E., Graziano, E., Joobeur, T., Garriga-Calderé, F., Cossonm, P., Howad, W., Arús, P., 2004. Comparative mapping and marker assisted selection in Rosaceae fruit crops. Proc. Natl. Acad. Sci. U.S.A. 101, 9891–9896. Downey, S.L., Iezzoni, A.F., 2000. Polymorphic DNA markers in black cherry (Prunus serotina) are identified using sequences from sweet cherry, peach and sour cherry. J. Am. Soc. Hort. Sci. 125, 76–80. Howad, W., Yamamoto, T., Dirlewanger, E., Testolin, R., Cosson, P., Cipriani, G., Monforte, A.J., Georgi, L., Abbott, A.G., Arus, P., 2005. Mapping with a few plants: using selective mapping for microsatellite saturation of the Prunus reference map. Genetics 171, 1305–1309. Huang, H.W., Cheng, Z.P., Zhang, Z.H., Wang, Y., 2008. History of cultivation and trends in China. In: Layne, D.R. (Ed.), The Peach: Botany, Production and Uses (Chapter 2). CABI, Wallingford, Oxfordshire, UK. Infante, R., Martínez-Gómez, P., Predieri, S., 2008. Quality oriented fruit breeding: Peach [Prunus persica (L.) Batsch]. J. Food Agric. Environ. 6 (2), 342–356. Joobeur, T., Periam, N., de Vicente, M.C., King, G.J., Arús, P., 2000. Development of a second generation linkage map for almond using RAPD and SSR markers. Genome 43, 649–655. Li, T.H., Li, Y.X., Li, Z.C., Zhang, H.L., Qi, Y.W., Wang, T., 2008. Simple sequence repeat analysis of genetic diversity in primary core collection of peach (Prunus persica). J. Integrative Plant Biol. 50 (1), 102–110.
R. Xie et al. / Scientia Horticulturae 125 (2010) 622–629 Mantel, M., 1967. The detection of disease clustering and generalized regression approach. Cancer Res. 27, 209–220. Mnejja, M., Garcia-Mas, J., Howad, W., Badenes, M.L., Arús, P., 2004. Simple-sequence repeat (SSR) markers of Japanese plum (Prunus salicina Lindl) are highly polymorphic and transferable to peach and almond. Mol. Ecol. Notes 4, 163–166. Nei, M., 1972. Genetic distance between populations. The Am. Naturalist 106, 283–292. Ogundiwin, E.A., Peace, C.P., Nicolet, C.M., Rashbrook, V.K., Graziel, T.M., Bliss, F.A., Parfitt, D., Crisosto, C.H., 2008. Leucoanthocyanidin dioxygenase gene (PpLDOX): a potential functional marker for cold storage browning in peach. Tree Genet Genomes 4, 543–554. Ogundiwin, E.A., Peace, C.P., Graziel, T.M., Parfitt, D.E., Bliss, F.A., Crisosto, C.H., 2009. A fruit quality gene map of Prunus. BMC Genomics 10, 587. Peace, C.P., Crisoto, C.H., Gradzeil, T.M., 2005. Endopolygalacturonase: a candidate gene for Freestone and Melting flesh in peach. Mol. Breed. 16, 21–31. Rohlf, F.J., 2000. NTSYS-pc. Numerical Taxonomy and Multivariate Analysis System, Version 2.1. Exeter Software, Setauket, New York. Sosinski, B., Gannavarapu, M., Hager, L.D., Beck, L.E., King, G.J., Ryder, C.D., Rajapakse, S., Baird, W.V., Ballard, R.E., Abbott, A.G., 2000. Characterization of microsatel-
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lite markers in peach [Prunus persica (L.) Batsch]. Theor. Appl. Genet. 101, 421– 428. Testolin, R., Marrazzo, T., Cipriani, G., Quarta, R., Verde, I., Dettori, M., Pancaldi, M., Sansavini, S., 2000. Microsatellite DNA in peach [Prunus persica (L.) Batsch] and its use in fingerprinting and testing the genetic origin of cultivars. Genome 43, 512–520. Wang, Z.H., Zhuang, E.J., 2001. Records of Chinese Fruit Trees, Peach volume. China Forestry Publishing, pp. 86–87, p. 195 (in Chinese). Wünsch, A., Carrera, M., Hormaza, J.I., 2006. Molecular characterization of local Spanish peach [Prunus persica (L.) Batsch] germplasm. Genet. Resour. Crop Evol. 53, 925–932. Yeh, F.C., Yang, R.C., Boyle, T., 1999. POPGENE Microsoft windows-based software for Population genetic analysis. A joint project development by Fancis C. Yeh and Rong-Cai Yang, University of Alberta and Tim Boyle, Center for International Forestry Research, Bogor, Indonesia. Zhang, S.M., Xu, C.J., Gao, Z.S., Chen, K.S., Wang, G.Y., 2009. Development and characterization of microsatellite markers for Chinese bayberry (Myrica rubra Sieb. & Zucc.). Conserv. Genet. 10, 1605–1607.
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Evaluation of the genetic diversity of Asian peach accessions using a selected set of SSR markers Rangjin Xie a,d , Xiongwei Li a , Mingliang Chai a , Lijuan Song a,1 , Huijuan Jia a , Dajun Wu b , Miaojin Chen b , Keming Chen b , Maria Jose Aranzana c , Zhongshan Gao a,∗ a
Department of Horticulture, The State Agriculture Ministry Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Zhejiang University, Hangzhou 310029, China b Fenghua Honey Peach Institute, Xikou, Fenghua, Zhejiang Province, 315521, China c IRTA, Centre de Recerca en Agrigenòmica CSIC-IRTA-UAB, Carretera de Cabrils Km2; 08348 Cabrils (Barcelona), Spain d Citrus Research Institute, Chinese Academy of Agricultural Sciences, Chongqing 400712, China
a r t i c l e
i n f o
Article history: Received 16 March 2010 Received in revised form 5 May 2010 Accepted 17 May 2010 Keywords: Peach Melting peach Genetic diversity Allelic composition SSR
a b s t r a c t A total of 94 peach accessions from the Zhejiang province of China were analyzed using 34 polymorphic single-locus simple sequence repeat (SSR) markers. Genetic distance analysis divided the accessions into two major clusters, one with mainly local accessions from the Fenghua region. Preselected lines from some crosses were exclusively clustered with the local ones, possibly related to maintaining the taste quality of the fruit. The number of alleles per locus at most loci was two or three, with an average of 2.85. The value of observed heterozygosity varied from 0.05 to 0.84 (average of 0.48). Diversity within the introduced accessions was higher than that of the Fenghua local accessions. Of the accessions analyzed in this study, 94% were individually identified. Those that could not be differentiated were all derived from the ‘Yulu’ cultivar, being either mutations or of identical origin. Our results suggest that Fenghua accessions are derived from limited parental materials and inbreeding over a long period of time. They will be useful for breeders to better manage their pre-breeding materials and choice of parents for further crossings. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Peach (Prunus persica) is one of the most economically important deciduous fruit tree species in the Rosaceae family. China has the longest history of peach cultivation, of more than 4000 years (Cheng and Huang, 2009), and it is generally thought that it has the richest genetically diverse germplasm, with many natural mutants and chance seedlings (Huang et al., 2008). According to the Chinese traditional classification based on texture, shape and skin hair characters, peach cultivars have been divided into six groups, namely crispy peach (very firm melting), sweet peach (firm melting), honey peach (soft melting), yellow fleshed peach (non-melting), flat peach and nectarine (Wang and Zhuang, 2001). Honey peach, sweet, juicy and aromatic with a very soft texture, was initially found in the Zhejiang and Jiangsu provinces and planted in just a few places close to Shanghai. For the consumer market, peach cultivar identity is not precise, and the ‘Shanghaishuimi’ peaches sold on the Shanghai
∗ Corresponding author. Tel.: +86 571 86971172; fax: +86 571 86971630. E-mail address: [email protected] (Z. Gao). 1 Current address: Department of Horticulture, Wenzhou Polytechnics College, Wenzhou 325006, China. 0304-4238/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2010.05.015
market are a group of cultivars belonging to the honey peach group. They became more popular and were gradually introduced to different areas and other countries because of the good fruit quality and adaptability to various climate and soil conditions. Fenghua city (in the east of Zhejiang province, near Shanghai) is one of the four regions in China where honey peach originated and is produced. According to the records, Fenghua honey peach was developed from a pointed-tip honey peach introduced from Shanghai by Yinchong Zhang in 1883 (Wang and Zhuang, 2001). Over more than 120 years of selection, a number of closely related elite cultivars referred to as ‘Yulu’ were cultivated, and were also used as parents in breeding programs. ‘Yulu’ cultivars are still grown on nearly half of the production area in Fenghua, 25 thousand tons, annually. In the last 10 years, new cultivars have been introduced to replace the local ones for two main reasons: (1) A narrow genetic background and inbreeding has caused general degradation phenomena in local cultivars, such as small fruit size, lack of desired red skin color, susceptibility to pests and diseases and poor postharvest properties, and (2) The local cultivars ripen late and around the same time (most in early August). During the 1990s, cultivar evaluation was used to select the best local cultivars, introduce new ones and for small-scale crossing. As a result, more than 130 honey peach cultivars/selections were pre-
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served in a local peach germplasm repository, but the genotypes and phenotypes of these accessions have not been well documented. Here we used molecular tools, in collaboration with the university and local research institute, to bridge the gap between geneticists and breeders. Microsatellites or simple sequence repeats (SSR) have extensively proved to be highly efficient for systematic evaluation of genetic diversity and cultivar identification in highly homozygous species such as peach (Sosinski et al., 2000; Aranzana et al., 2002, 2003a; Dirlewanger et al., 2004; Wünsch et al., 2006; Bouhadida et al., 2007; Li et al., 2008; Cheng et al., 2009). To date, nearly 500 SSRs have been developed and mapped in the Prunus reference map (Aranzana et al., 2003b; Dirlewanger et al., 2004; Howad et al., 2005), making it a valuable tool for genetic analysis. There are two recent reports on genetic diversity of Chinese peach cultivars using SSR markers (Li et al., 2008; Cheng and Huang, 2009) with a limited number of accessions (51 and 32) and SSR markers (22 and 7, respectively). For the purposes of cultivar identification and studying diversity, full coverage of representative SSRs and more accessions are needed. Marker genotypes flanking the main oligogenic or monogenic and polygenic fruit traits (QTLs) can be used for association studies and subsequent selection (Infante et al., 2008). In this study, we used 34 polymorphic SSR loci to: (1) assess the genetic identity and diversity among the honey peach cultivars, and (2) analyze the genetic background of the selected cultivars and their genetic relationship. The results of this study provide guidelines and a scientific base to better explore honey peach germplasm resources and facilitate local cultivar improvement.
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mated by comparing to the pBR322 DNA-MspIDigest (New England BioLabs) ladder. All amplifications were scored as either a present (1) or absent (0) band. The alleles were coded A, B, C, etc. for each band, in decreasing size order. A single band in one accession was assumed to be homozygous. 2.3. Statistical analysis Molecular diversity was estimated according to the following parameters: (1) number of alleles per locus (Na) and number of effective alleles per locus (Ne), (2) polymorphism information content (PIC), (3) observed heterozygotes at a given loci (Ho), and (4) occurrence of unique and rare alleles. Alleles were considered rare if they occurred in less than 5% of the accessions (up to 4 accessions) and unique if they occurred in only one accession. Na, Ne and Ho were calculated with the program POPGENE version 1.31 developed by Yeh et al. (1999), and PIC with the PowerstatsV12.xls software (Brenner and Morris, 1990), which is freely available on the internet (http://www.promega.com/geneticidtools/powerstats/). Genetic distances between the 94 accessions were calculated using Nei’s coefficient index (Nei, 1972) with the software NTSYSpc 2.1 (Rohlf, 2000), and a dendrogram was constructed with the same software using the unweighted pair-group method with arithmetic mean (UPGMA). The fit of the UPGMA cluster to the original similarity indices was computed according to the Mantel test procedure (Mantel, 1967) by using MxComp of the software package NTSYSpc 2.1 (Rohlf, 2000). 3. Results
2. Materials and methods 2.1. Plant material A total of 94 accessions (Table 1) obtained from the Fenghua Honey Peach Research Institute were used to evaluate genetic diversity and identity. Of these accessions 48 were from local collections and selections, and 46 introduced from different places in China, Japan and Korea or were of unknown origin. Young, non-fully expanded leaves were collected from healthy trees, frozen in liquid nitrogen, and then stored at −40 ◦ C prior to DNA extraction. 2.2. DNA isolation and SSR analysis Genomic DNA was extracted from 1 g of ground leaf tissue using CTAB as described by Zhang et al. (2009). The DNA concentration was quantified by ultraviolet spectrophotometry (Beckman coulter DU800). Thirty-four SSRs (Fig. 1) chosen as the given clear polymorphic bands were used to assay the set of 94 accessions. The SSR loci selected were recommended by the Spain IRTA group, who used them for cultivar genetic diversity. We added 2–4 bases at the 5 end of the original reverse primer to form a GTTT pigtail sequence to allow addition of an A-tail to all PCR products for accurate scoring of fragments (Brownstein et al., 1996). PCR products were in a total volume of 20 l containing 40 ng of DNA, 0.25 M of each primer, 0.2 mM of each dNTP, 0.5 U Taq polymerase (TAKARA BIO INC.) and 1× PCR buffer mix. PCR amplifications were performed in an Eppendorf Mastercycler 5333/5331 thermocycler (Gradient No. 5331-41264, Germany) with the following programs: 2.5 min at 94 ◦ C, 35 cycles of 30 s at 94 ◦ C, 30 s at the appropriate annealing temperature, and 30 s at 72 ◦ C, followed by a 10 min final extension at 72 ◦ C. The PCR products were separated on 6% denatured polyacrylamide gel at 75 W (BioRad Electrophoresis system) for 2 h and then visualized by silver staining. The band sizes were esti-
3.1. Analysis of SSR loci diversity Ninety-four honey peach accessions were analyzed with 34 polymorphic SSRs, distributed on the 8 linkage groups of the Prunus T × E reference map (Fig. 1). The variability detected by each SSR locus is shown in Table 2. The total number of amplified alleles was 97, 93 of them (95.9%) being polymorphic in the 94 accessions studied. The number of alleles amplified per marker ranged from 2 to 5 (in CPPCT029 and UDP96-13), with an average of 2.85 alleles/locus. With a mean value of 0.48, the observed heterozygosity (Ho ) ranged from 0.05 in BPPCT028 to 0.84 in Pchgms3. The Ne and PIC varied from 1.05 in the BPPCT028 to 3.66 in UDP96013 (mean value of 2.04) and from 0.05 to 0.68 in UDP96-013 (mean value of 0.4). Based on the PIC, the most informative loci were UDP96-013 and BPPCT025, with values of 0.68 and 0.63, respectively, whereas the least informative were CPPCT028 with a PIC value of 0.05. Allelic frequencies ranged from 0.01 to 1.0. Among the 97 observed alleles, 6 were unique, and 7 rare (frequency ≤ 0.05). The 48 Fenghua local accessions had slightly lower genetic diversity than the 46 introduced. The number of effective alleles per locus ranged from 2 to 3 (mean value 1.90) in Fenghua accessions, and from 2 to 3.87 (mean value 2.05) in the introduced accessions. These findings were also reflected in the PIC, where the average for Fenghua cultivars was 0.34 and for the introduced cultivars, 0.46. None of the studied accessions were homozygous at all loci (Fig. 2). Fenghua accessions were less heterozygous than the introduced (Ho = 0.46 and Ho = 0.50, respectively): the number of heterozygous loci in Fenghua accessions ranged from 6 to 22 and from 11 to 27 in the introduced ones. One of the Fenghua accessions was the most homozygous, with only 6 out of 34 loci heterozygous (Fig. 2). The most heterozygous local accession was ‘X2-7 , while the introduced ‘Hujingmilu’ was the most heterozygous (27/34). Sixteen of the 94 accessions carried unique and/or rare alleles. Most of them (14) were found in the introduced cultivars ‘Bailu’, ‘Zhongqiumi’, ‘Lais-
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Table 1 The 94 peach accessions used for the genetic variability assay, sorted as in Fig. 3. Accession
Origin
Pedigree
Harvest time
Red skin color
Texture
Stone
Hakuho1 Hakuho2 Nkazu Hakuto Yuzora Yamanashi Hakuho Ohdama Hakuho Hikawa Hakuho Matsumori Akatsuki Datuanmilu Hakuri Yamane Hakuto Mibaekdo Xinhong Hujingmilu Qiukonga Dabaifeng Riaia Taoyan2 Zhongqiumi Laishanmi Yumyeong New Kawanakajima Hakutoa Hongtaowang Juwangtao Xueyulu Yuhualu Xiahui2 Zaohualu2 Kurakato wase Yanhong Takei Hakuho Nunome Zaotaimi Xuexianglu Sunago Wase Asama Hakuho Matsumor Xumi X2-5 X1-22 X2-14 X2-17 X1-8 X1-3 Kawanakajima Hakuto Yahata Hakuto Reiho Shangshanyou Anping Jinjun Jianfang Jianding Xiangtu Sanbao3 Xipu4 Linjia Shangshandayulu XiPu1 Sanbao4 Chiyulu Sanbao2 Linguochang Sanbao1 Laoyulu Yulu Taoyan1 Beijinga Zaoyulu Xinyu Chibaifeng X5-17 X2-7 X4-1 X1-19
Japan Japan Japan Japan Japan Japan Japan Japan Japan Shanghai-China Japan Japan Korea Shanghai-China Jiangsu-China Japan Fenghua Japan Fenghua Heilongjiang -China Shandong-China Korea Japan Liaoning-China Anhui-China Hangzhou-China Jiangsu-China Jiangsu-China Jiangsu-China Japan Beijing-China Japan Japan Unknown Jiangsu-China Japan Japan Japan Anhui-China Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Japan Japan Japan Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Beijing Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua
Selected from Hakuho Selected from Hakuho Hakuto seedling Hakuto × Akatsuki Selected from Hakuto Selected from Hakuho Hakuho mutation A sport from Hakuho Hakuto × Hakuho Shanghaidatuan seedling Okubo × Feichengtao Unknown Shimizu seedling Selected from Hakuho Selected from Hakuho Unknown Selected from Hakuho Unknown Selected from Yulu Unknown Shandonglaishan seedling Okubo × Numome Selected from Kawanakajima Hakuto Baitao × Hakuho Unknown Baihuashuimi × Yuhualu Baihua × Shanghaishuimi Zhaohui × Zhaoxia Yuhualu seedling Unknown Unknown Hakuho × Yamanehakuto Seedling of Hakuto or Okubo Unknown Baihua × Shanghaishuimi Okubo or Kamidama seedling Takayo Hakuto Seedling of Hakuto Selected from Baihua Shangshandayulu × Hujingmilu Shangshandayulu × Hujingmilu Shangshandayulu × Hujingmilu Shangshandayulu × Hujingmilu Shangshandayulu × Hujingmilu Shangshandayulu × Hujingmilu Shanghaishuimi × Hakuto Selected from Hakuto Unknown Selected from Yulu Selected from Yulu Selected from Yulu Selected from Yulu Selected from Yulu Selected from Yulu Selected from Yulu Selected from Yulu Selected from Yulu Selected from Yulu Selected from Yulu Selected from Yulu Selected from Yulu Selected from Yulu Selected from Yulu Selected from Yulu Local cultivar Selected from Shanghaishuimi Selected from Yulu Unknown Selected from Yulu Selected from Yulu Selected from Hakuho Xipu1 × Hujingmilu Shangshandayulu × Hujingmilu Xipu1 × Hujingmilu Shangshandayulu × Hujingmilu
Late June Early July Mid-July Early September Early July Early–mid-July Late June Late June Early–mid-July Late June Early–mid-August Late June Mid-July Late July Early–mid-July Unknown Mid-late July Mid-July Mid-late June Late August Late August Early August Mid-July Mid-August Mid-August Early June Late June Early June Early June Early June Late August Late June Early June Early June Early June Early June Early–mid-July Late June Late August Late June Early July Mid-July Mid-August Mid-July Mid-August Late July Mid-July Mid-July Early August Early August Early August Early August Early August Early August Early August Late July Early August Early August Late July Early August Mid-late August Early August Early August Early August Early–mid-August Early August Late July Late June Late July Mid-late July Early July Late July Mid-late July Mid-late July Early August
P P P P P F P F F P P P F F F P F P F F F F P F F P F P P P F P P P P P P P F F F F F F F F F F L L L L L L L L L L L L L L L L L L P F P F P F F P F
SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM FM FM NM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM FM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM
C C C C C C C C C C C C C C C C C C C C C C C C C F C F C C C C C F F C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C
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Table 1 (Continued. ) Accession
Origin
Pedigree
Harvest time
Red skin color
Texture
Stone
X2-2 X1-14 X1-7 X2-11 X3-4 X2-6 X1-4 X2-12 X3-3 X1-1 Renhea X2-10 X5-20 X2-18 X1-18 X2-19 X1-13 Bailua Zaoshanghaishuimi
Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Japan Fenghua Fenghua Fenghua Fenghua Fenghua Fenghua Japan Shanghai
Shangshandayulu × Hujingmilu Shangshandayulu × Hujingmilu Shangshandayulu × Hujingmilu Shangshandayulu × Hujingmilu Xipu1 × Hujingmilu Shangshandayulu × Hujingmilu Shangshandayulu × Hujingmilu Shangshandayulu × Hujingmilu Xipu1 × Hujingmilu Shangshandayulu × Hujingmilu Unknown Shangshandayulu × Hujingmilu Xipu1 × Hujingmilu Xipu1 × Hujingmilu Shangshandayulu × Hujingmilu Xipu1 × Hujingmilu Shangshandayulu × Hujingmilu Unknown Selected from Shanghaishuimi
Early August Mid-July Early August Early July Early August Early August Early August Early August Early August Late July Late July Early August Early August Mid-late July Early August Late July Early August Mid-late July Early July
F F F F F F F F P F F P F F F F F P P
SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM SM
C C C C C C C C C C C C C C C C C C C
Skin color: full blush coverage (F), partially red, about 25% coverage (P), very little red color on top or sutural line (L). Texture: soft melting (SM), firm melting (FM) and non-melting (NM). Stone: C-cling, F-free. a Local name record in Fenghua,
Fig. 1. The 34 mapped SSR loci on the eight peach linkage groups. The SSR markers developed by a Cipriani et al. (1999), b Aranzana et al. (2002), c Dirlewanger et al. (2002), d Sosinski et al. (2000), e Downey and Iezzoni (2000), f Testolin et al. (2000), g Mnejja et al. (2004), h Cantini et al. (2001), i Joobeur et al. (2000), and k EPPCU1090 and EPPCU1775 were mapped in bin G1:34 and G4:63 (Howad et al., 2005); l PTS1 is EST-SSR derived from an intron when genomic cloning based on DY639772 annotated as terpene synthase in NCBI, this marker was bin-mapped on G4:18 (unpublished).
Fig. 2. Distribution of the number of heterozygous loci per accession detected in Fenghua cultivars (white) and the introduced cultivars (black). A total of 34 SSR loci were tested.
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Table 2 Variability identified using 34 SSRs in 94 peach cultivars. Linkage groups
SSR locus
No. of alleles (effective alleles) per locus
Polymorphism information content (PIC)
Observed Heterozygosity
No. of rare (unique) alleles
G1
UDP96-018 UDP96-005 EPPCU1090 CPPCT026 Pchgms3 BPPCT020 CPPCT029 BPPCT028
4(2.76) 3(2.60) 2(1.87) 4(3.0) 3(2.17) 2(1.80) 5(1.32) 2(1.05)
0.57 0.55 0.36 0.60 0.44 0.35 0.23 0.05
0.53 0.45 0.44 0.32 0.84 0.59 0.16 0.05
0(1) 0(0) 0(0) 1(0) 0(0) 0(0) 1(2) 1(0)
G2
CPPCT044 BPPCT001 UDP96-013 Pchgms1 PceGA34
3(1.88) 4(1.65) 5(3.66) 2(1.70) 3(2.86)
0.38 0.33 0.68 0.33 0.57
0.32 0.45 0.49 0.56 0.69
1(0) 0(1) 1(0) 0(0) 0(0)
G3
BPPCT007 UDP96-008
3(1.94) 3(2.73)
0.37 0.56
0.46 0.55
0(0) 0(0)
G4
CPPCT005 UDP98-024 PTS1 BPPCT015 EPPCU1775
2(1.61) 3(2.19) 3(2.88) 2(1.77) 3(1.42)
0.31 0.47 0.57 0.34 0.28
0.49 0.41 0.82 0.57 0.16
0(0) 0(0) 0(0) 0(0) 0(0)
G5
CPPCT040 BPPCT017 CPSCT006 PceGA25 CPPCT013 BPPCT014
4(2.08) 2(1.83) 3(2.01) 2(1.92) 2(1.50) 2(1.68)
0.43 0.35 0.38 0.36 0.28 0.32
0.55 0.46 0.70 0.54 0.42 0.52
1(1) 0(0) 0(1) 0(0) 0(0) 0(0)
G6
CPPCT015 Pchgms5 BPPCT025 CPPCT030
2(1.53) 2(1.98) 4(3.16) 3(2.38)
0.29 0.38 0.63 0.52
0.38 0.51 0.68 0.71
0(0) 0(0) 0(0) 0(0)
G7
CPPCT022 CPPCT017
2(1.88) 4(1.57)
0.36 0.33
0.30 0.31
0(0) 1(0)
G8
CPPCT006 UDP98-409
2(1.30) 2(1.90)
0.24 0.36
0.16 0.76
0(0) 0(0)
2.85(2.04)
0.40
0.48
Total Mean
7(6)
hanmi’, ‘Zaohualu’ and ‘Yanhong’, while for the Fenghua accessions, only ‘Xiangtu’ and ‘X1-13’ amplified. 3.2. Genetic relationship and pedigree A UPGMA dendrogram derived from the similarity matrix (Fig. 3), grouped the accessions into two major clusters, according to their origin. Mantel test (Mantel, 1967) showed a very good fit of the unweighted pair-grouped method with arithmetic average (UPGMA) cluster to the original genetic distance matrix (r = 0.978, p < 0.001). The first cluster (from ‘Hakuho1 to ‘Sunago wase’ in Fig. 3) included 34 introduced and two local cultivars. The second cluster (from ‘Asama Hakuho’ to ‘Bailu’ in Fig. 3), contained 57 accessions, 48 from Fenghua, and 9 introduced. The new Fenghua selections (names starting with X) were distributed into two groups, one clustering together with ‘Xumi’ from China, and several cultivars from Japan including ‘Renhe’. ‘Linjia’, ‘Shangshandayulu’ and ‘Xipu1 shared the same SSR genotype. These three accessions were all selected from ‘Yulu’ and are considered identical or with small morphological differences. Two accessions related to ‘Yulu’ (‘Jinjun’ and ‘Jianfang’) were also not distinguished by any of the 34 SSRs. Other local cultivars have more than 95% identical alleles. For example, ‘Laoyulu’ (which means “old Yulu”) differed from ‘Yulu’ at two SSR loci (UDP96-018 and CPPCT022).
The new Fenghua selections are descendants of two different recorded pedigrees (‘Shangshandayulu’ × ‘Hujingmilu’ and ‘Xipu1 × ‘Hujingmilu’). SSR data was used to validate the pedigree information of these accessions. We found that most selections carried the allele from both parents, consistent with their pedigree, while a few new selections were not derived from the original crosses ‘Shangshandayulu’ × ‘Hujingmilu’ and ‘Xipu1 × ‘Hujingmilu’. The introduced accessions ‘Reiho’, ‘Yahata Hakuto’ and ‘KawanakaJima Hakuto’ had very similar SSR genotypes. ‘KawanakaJima Hakuto’ is a progeny from ‘Shanghaishuimi’ × ‘Hakuto’, differing from ‘Reiho’ at one locus (PST1) and from ‘Yahata Hakuto’ at CPPCT022. ‘Yahata Hakuto’, derived from ‘Hakuto’ as recorded (Agricultural and Fishing Village Cultural Association, 2000), differed from ‘Reiho’ at the PST1 and CPPCT022 loci. These three Japanese cultivars are very close to Fenghua local cultivars (Fig. 3, sub-cluster 2.1). 4. Discussion 4.1. SSR polymorphism In this study, the average number of alleles was 2.85, which was lower than the 5 as previously reported by Li et al. (2008) in a sample of 51 Chinese cultivars (including honey peach, nectarine, flat peach, crispy peach and yellow fleshed peach) analyzed with 22
R. Xie et al. / Scientia Horticulturae 125 (2010) 622–629
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Fig. 3. UPGMA dendrogram of 94 accessions based on their variation at 34 loci. (♦) Fenghua cultivars; () introduced cultivars.
polymorphic markers, the 7.3 observed by Aranzana et al. (2003a) using 16 polymorphic single-locus microsatellites (9 SSR loci are in common with this study) to evaluate a set of 212 occidental peach cultivars, the 4.2 detected by Dirlewanger et al. (2002) in 27 peach cultivar using 41 SSRs, the 3.5 by Wünsch et al. (2006) in a set of 85 local Spanish peach genotypes with polymorphic SSRs and the 3.1 observed by Cheng et al. (2009). While, it was higher than the 2.6 detected by Sosinski et al. (2000) in 28 scion peach cultivars with 10 SSRs and the 2.3 observed by Bouhadida et al. (2007) in 30 peach accessions using 20 polymorphic SSRs.
Our results suggest that the honey peach cultivars collected by the Fenghua Honey Peach Research Institute had a low level of genetic diversity. This was consistent with that observed by Li et al. (2008) in the subsample of 11 honey peach cultivars included in their study. There is some agreement in the results from Aranzana et al. (2003a) and Li et al. (2008) with respect to allelic variation we found at certain loci which amplified a higher (CPPCT029, BPPCT001) or lower number of alleles (CPPCT002), while we found a relatively low level of polymorphism for other loci (CPPCT022, CPPCT030)
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compared to the literature. This suggests that the ability of a marker to detect polymorphism may vary depending on the sample analyzed. 4.2. Comparative analysis of genetic diversity between the Fenghua and the introduced accessions In the present study, our results show that the introduced cultivars had a higher level of genetic diversity than that of the Fenghua local cultivars. This may be explained by the origin of the samples; the introduced cultivars were collected from different places while the Fenghua accessions are derived from limited parental materials and are a result of inbreeding over a long period of time. More than 90% of alleles were identical in local cultivars, and in one accession 28 out of 34 loci were homozygous. Preselected lines from a few crosses were exclusively clustered with the local ones, possibly related to maintaining the quality and characteristics of the old varieties. The results also indicate the need to make full use of the introduced cultivars as parental materials to enlarge the genetic diversity in breeding programs. Recent gene maps of fruit quality (Ogundiwin et al., 2009) enable closer analysis of the linked SSR alleles in the tested accessions. By combining the phenotypic traits and SSR marker data from this study, it would be possible to improve the crosses for specific variations. 4.3. Genetic relationship, cultivar identification and pedigree The UPGMA dendrogram obtained with our SSR data separates the accessions into two clusters. Most of the introduced cultivars were clearly separated from the Fenghua old local cultivars, indicating that the Fenghua group has been seed-propagated for a long period of time, with very little exchange with other cultivars. While it is generally accepted that Chinese cling peaches from Shanghai are the founders of modern cultivars, their pedigree is not well documented. ‘Shanghaishuimi’ is not likely to be a single cultivar, but a group of cultivars marketed in Shanghai. The two distinct clusters imply that the original ‘Shanghaishuimi’ was also comprised of two groups. It is unlikely that Fenghua ‘Yulu’ has been commonly used in modern peach breeding programs in Japan, since most Japanese cultivars are in the first cluster (Fig. 3). Analysis of the most representative cultivars and ‘Shanghaishuimi’, from different places in the world, with common polymorphic SSR marker is necessary to confirm whether this is true in western countries. In the present study, only 5 of the 94 accessions studied could not be differentiated: ‘Jinjun’, ‘Jianfang’, ‘Linjia’, ‘Shangshandayu’ and ‘Xipu1 . These are all selections of ‘Yulu’, possibly sports. In order to identify peach cultivars and/or analyze pedigree relationship in future more efficiently and low-costly, we selected a subset of 12 SSRs (EPPCU1090, BPPCT020, CPPCT029 and BPPCT028 on G1, UDP96-013 on G2; BPPCT007 on G3; PTS1 on G4, CPPCT040 and PceGA25 on G5; CPPCT015 on G6, CPPCT022 on G7 and UDP98-409 on G8), which have the same effectiveness of cultivar identification as the 34 SSRs used in this study. Six of these markers belong to the genotyping set proposed by Aranzana et al. (2003b). With the data from the 12 representative SSR markers, we largely confirmed the recorded pedigree of our tested materials. In two cases there were two loci with unexpected alleles, indicating that some of the discrepancies may also have been caused by SSR mutations, as has been reported previously (Aranzana et al., 2003a). With the availability of SSR genotypes of these peach accessions, a detailed systematic recording of agronomic traits is necessary for association studies of a number of important fruit traits, based on recent publications of the genetics of fruit quality traits (Peace et al., 2005; Ogundiwin et al., 2008, 2009). It is also possible to
establish core collections that will be of further use in breeding programs. In conclusion, we found that the Fenghua local cultivars are isolated descendents of Shanghaishuimi with very narrow genetic backgrounds. Their good taste quality traits are worth exploring for use in breeding programs. With the genetic information of peach germplasm, local breeders will have better knowledge of their breeding materials, leading to more efficient crossing combinations and selections. Acknowledgements This study has been carried out with financial support from the Natural Science Foundation of China (30771496), the Science and Technology Department of Zhejiang Province (2007C22068) and Ningbo (2006C100029). We thank Sun Qinan for his assistance in collecting plant materials and Chen Lin for some PCR work. The Institute of Horticulture of the Jiangsu Academy of Agricultural Sciences provided some plant materials. We thank Dr. Luud Gilissen for critical reading of this manuscript and Dr. Yinghui Li from CAAS for her advice on data analysis. References Agricultural and Fishing Village Cultural Association, 2000. Fruit Gardening Encyclopedia. Agricultural and Fishing Village Cultural Association, p. 70 (in Japanese). Aranzana, M.J., Garcia-Mas, J., Carbó, J., Arús, P., 2002. Development and variability analysis of microsatellite markers in peach. Plant Breeding 121, 87–92. Aranzana, M.J., Carbo, J., Arús, P., 2003a. Microsatellite variability in peach [Prunus persica (L.) Batsch]: cultivar identification, marker mutation, pedigree inferences and population structure. Theor. Appl. Genet. 106, 1341–1352. Aranzana, M.J., Pineda, A., Cosson, P., Dirlewanger, E., Ascasibar, J., Cipriani, G., Ryder, C.D., Testolin, R., Abbott, A., King, G.J., Iezzoni, A.F., Arús, P., 2003b. A set of simplesequence repeat (SSR) markers covering the Prunus genome. Theor. Appl. Genet. 106, 819–825. Bouhadida, M., Casas, A.M., Moreno, M.A., Gogorcena, Y., 2007. Molecular characterization of Miraflores peach variety and relatives using SSRs. Scientia Horticulture 111, 140–145. Brenner, C., Morris, J., 1990. Paternity index calculations in single locus hypervariable DNA probes: validation and other studies. http://www.promega.com/ geneticidtools/powerstats. Brownstein, M.J., Carpten, J.D., Smith, J.R., 1996. Modulation of non-templated nucleotide addition by Taq DNA polymerase: Primer modifications that facilitate genotyping. Biotechniques 20, 1004–1010. Cantini, C., Iezzoni, A.F., Lamboy, W.F., Boritzki, M., Struss, D., 2001. DNA fingerprinting of tetraploid cherry germplasm using simple sequence repeats. J. Am. Soc. Hort. Sci. 126, 205–209. Cheng, Z.P., Huang, H.W., 2009. SSR fingerprinting Chinese peach cultivars and landraces (Prunus persica) and analysis of their genetic relationships. Scientia Horticulturae 120, 188–193. Cipriani, G., Lot, G., Huang, W.G., Marrazzo, M.T., Peterlunger, E., Testolin, R., 1999. AC/GT and AG/CT microsatellite repeats in peach [Prunus persica (L) Batsch]: isolation, characterisation and cross-species amplification in Prunus. Theor. Appl. Genet. 99, 65–72. Dirlewanger, E., Cosson, P., Tavaud, M., Aranzana, M.J., Poizat, C., Zanetto, A., Arús, P., Laigret, F., 2002. Development of microsatellite markers in peach [Prunus persica (L.) Batsch] and their use in genetic diversity analysis in peach and sweet cherry (Prunus avium L.). Theor. Appl. Genet. 105, 127–138. Dirlewanger, E., Graziano, E., Joobeur, T., Garriga-Calderé, F., Cossonm, P., Howad, W., Arús, P., 2004. Comparative mapping and marker assisted selection in Rosaceae fruit crops. Proc. Natl. Acad. Sci. U.S.A. 101, 9891–9896. Downey, S.L., Iezzoni, A.F., 2000. Polymorphic DNA markers in black cherry (Prunus serotina) are identified using sequences from sweet cherry, peach and sour cherry. J. Am. Soc. Hort. Sci. 125, 76–80. Howad, W., Yamamoto, T., Dirlewanger, E., Testolin, R., Cosson, P., Cipriani, G., Monforte, A.J., Georgi, L., Abbott, A.G., Arus, P., 2005. Mapping with a few plants: using selective mapping for microsatellite saturation of the Prunus reference map. Genetics 171, 1305–1309. Huang, H.W., Cheng, Z.P., Zhang, Z.H., Wang, Y., 2008. History of cultivation and trends in China. In: Layne, D.R. (Ed.), The Peach: Botany, Production and Uses (Chapter 2). CABI, Wallingford, Oxfordshire, UK. Infante, R., Martínez-Gómez, P., Predieri, S., 2008. Quality oriented fruit breeding: Peach [Prunus persica (L.) Batsch]. J. Food Agric. Environ. 6 (2), 342–356. Joobeur, T., Periam, N., de Vicente, M.C., King, G.J., Arús, P., 2000. Development of a second generation linkage map for almond using RAPD and SSR markers. Genome 43, 649–655. Li, T.H., Li, Y.X., Li, Z.C., Zhang, H.L., Qi, Y.W., Wang, T., 2008. Simple sequence repeat analysis of genetic diversity in primary core collection of peach (Prunus persica). J. Integrative Plant Biol. 50 (1), 102–110.
R. Xie et al. / Scientia Horticulturae 125 (2010) 622–629 Mantel, M., 1967. The detection of disease clustering and generalized regression approach. Cancer Res. 27, 209–220. Mnejja, M., Garcia-Mas, J., Howad, W., Badenes, M.L., Arús, P., 2004. Simple-sequence repeat (SSR) markers of Japanese plum (Prunus salicina Lindl) are highly polymorphic and transferable to peach and almond. Mol. Ecol. Notes 4, 163–166. Nei, M., 1972. Genetic distance between populations. The Am. Naturalist 106, 283–292. Ogundiwin, E.A., Peace, C.P., Nicolet, C.M., Rashbrook, V.K., Graziel, T.M., Bliss, F.A., Parfitt, D., Crisosto, C.H., 2008. Leucoanthocyanidin dioxygenase gene (PpLDOX): a potential functional marker for cold storage browning in peach. Tree Genet Genomes 4, 543–554. Ogundiwin, E.A., Peace, C.P., Graziel, T.M., Parfitt, D.E., Bliss, F.A., Crisosto, C.H., 2009. A fruit quality gene map of Prunus. BMC Genomics 10, 587. Peace, C.P., Crisoto, C.H., Gradzeil, T.M., 2005. Endopolygalacturonase: a candidate gene for Freestone and Melting flesh in peach. Mol. Breed. 16, 21–31. Rohlf, F.J., 2000. NTSYS-pc. Numerical Taxonomy and Multivariate Analysis System, Version 2.1. Exeter Software, Setauket, New York. Sosinski, B., Gannavarapu, M., Hager, L.D., Beck, L.E., King, G.J., Ryder, C.D., Rajapakse, S., Baird, W.V., Ballard, R.E., Abbott, A.G., 2000. Characterization of microsatel-
629
lite markers in peach [Prunus persica (L.) Batsch]. Theor. Appl. Genet. 101, 421– 428. Testolin, R., Marrazzo, T., Cipriani, G., Quarta, R., Verde, I., Dettori, M., Pancaldi, M., Sansavini, S., 2000. Microsatellite DNA in peach [Prunus persica (L.) Batsch] and its use in fingerprinting and testing the genetic origin of cultivars. Genome 43, 512–520. Wang, Z.H., Zhuang, E.J., 2001. Records of Chinese Fruit Trees, Peach volume. China Forestry Publishing, pp. 86–87, p. 195 (in Chinese). Wünsch, A., Carrera, M., Hormaza, J.I., 2006. Molecular characterization of local Spanish peach [Prunus persica (L.) Batsch] germplasm. Genet. Resour. Crop Evol. 53, 925–932. Yeh, F.C., Yang, R.C., Boyle, T., 1999. POPGENE Microsoft windows-based software for Population genetic analysis. A joint project development by Fancis C. Yeh and Rong-Cai Yang, University of Alberta and Tim Boyle, Center for International Forestry Research, Bogor, Indonesia. Zhang, S.M., Xu, C.J., Gao, Z.S., Chen, K.S., Wang, G.Y., 2009. Development and characterization of microsatellite markers for Chinese bayberry (Myrica rubra Sieb. & Zucc.). Conserv. Genet. 10, 1605–1607.