Tree Genetics & Genomes (2012) 8:975990 DOI 10.

1007/s11295-012-0477-8

ORIGINAL PAPER

Genetic diversity, linkage disequilibrium, and association mapping analyses of peach (Prunus persica) landraces in China
Ke Cao & Lirong Wang & Gengrui Zhu & Weichao Fang & Changwen Chen & Jing Luo

Received: 6 May 2011 / Revised: 14 November 2011 / Accepted: 14 February 2012 / Published online: 16 March 2012 # Springer-Verlag 2012

Abstract The genetic diversity, population structure, and linkage disequilibrium (LD) of peaches are greatly important in genome-wide association mapping. In the current study, 104 peach landrace accessions from six Chinese geographical regions were evaluated for fruit and phenological period. The accessions were genotyped with 53 genome-wide simple sequence repeat (SSR) markers. All SSR markers were highly polymorphic across the accessions, and a total of 340 alleles were detected, including 59 private alleles. Of the six regions studied, the northern part of China as well as the middle and lower reaches of the Changjiang River were found to be the most highly diverse genetically. Based on population structure analysis, the peaches were divided into five groups, which well agreed with the geographical distribution. Of the SSR pairs in these accessions, 18.07% (P <0.05) were in LD. The mean r2 value for all intrachromosomal loci pairs was 0.0149, and LD decayed at 6.01 cM. The general linear model was used to calculate the genome-wide marker-trait associations of 10 complex traits. The traits include flesh color around the stone, red pigment in the flesh, flesh texture, flesh adhesion, flesh firmness, fruit weight, chilling requirement, flowering time, ripening time, and fruit development period. These traits were estimated by analyzing the 104 landraces. Many of the associated markers were located in regions where quantitative trait loci (QTLs) were previously identified. Peach association mapping is an effective approach for identifying QTLs and

may be an alternative to QTL mapping based on crosses between different lines. Keywords Genetic diversity . Linkage disequilibrium . Association mapping . Peach landrace

Introduction Peach (Prunus persica L.) was originally domesticated in China 4,0005,000 years ago (Faust and Timon 1995). Peach is still grown now as a delicious and healthy summer fruit in the Chinese provinces of Guangdong in the south to Heilongjiang in the north. According to topographic distribution, peach landrace cultivation in China is concentrated in seven main areas: the QinghaiTibetan Plateau, northwest China, the YunGui plateau, northeast China, northern China, the middle and lower reaches of the Changjiang River, and southern China (Wang and Zhuang 2001). The diverse resources in these regions have already contributed many useful alleles to the cultivated gene pool (especially fruit quality-related alleles) that have become indispensable in peach breeding. Understanding the molecular genetic control of phenotypic variations (such as yield and quality-related traits) is very essential and remains a major task in the genetic study of some important crops (Jin et al. 2010). Peach has a genome size of 220 Mb, which is approximately twice the size of Arabidopsis. Given this relatively small size, peach was used as the model species for studying genomics in Rosaceae. Ever since, peach has received increasing attention in the genetic dissection of simple or complex traits by linkage mapping. So far, 23 monogenic morphological traits associated with adaptation, flower color, fertility, leaf shape and color, plant habit, fruit quality, as well as pest resistance

Communicated by A. Abbott K. Cao : L. Wang (*) : G. Zhu : W. Fang : C. Chen : J. Luo Zhengzhou Fruit Research Institute, Chinese Academy of Agriculture Sciences, Zhengzhou 450009, Peoples Republic of China e-mail: wyandck@126.com

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have been described through linkage analysis. Quantitative trait loci (QTLs) have also been identified for 23 horticulturally important traits, including bloom and ripening time, fruit quality, storage life, freestone trait, as well as pest resistance (Hancock 2008). Traditional QTL mapping is an important tool in crop gene tagging. However, for the study of linkage, suitably designed crosses need to be performed. These crosses sometimes lead to the development of mapping populations or near-isogenic lines. Crossing is a serious limitation on the use of QTL mapping in some cases because the desired crosses are not applicable in all cases (e.g., sterility in distant hybridization). Mapping populations examined for this purpose are also sometimes too small (Pushpendra et al. 2005). Association mapping is another effective approach for connecting phenotype and genotype in plants when information on population structure and linkage disequilibrium (LD) is available (Thornsberry et al. 2001). Association mapping complements and enhances previous QTL information for marker-assisted selection in rice (Agrama et al. 2007), wheat (Tommasini et al. 2007), and maize (Yu and Buckler 2006). Many important crops have complex population structures that arose from a long domestication and breeding history (Flint-Garcia et al. 2003). Understanding these structures is important to avoid identifying spurious associations in association mapping. With the development of statistical methods, independent markers that are distributed throughout the genome can be successfully used to detect population structures (Pritchard et al. 2000). Previous studies on crop population structure as well as its effect on crop diversity and LD are abundant. Jin et al. (2010) detected 416 rice accessions, including landraces, and breeding lines collected mostly in China, using 100 genome-wide simple sequence repeat (SSR) markers. A model-based population structure analysis divided the rice materials into seven subpopulations. Of the SSR pairs in these accessions, 63% were in LD. The intrachromosomal LD average was 2550 cM for different subpopulations. Kwak and Gepts (2009) analyzed the genomewide genetic composition at 26, mostly unlinked microsatellite loci in 349 accessions of the wild and domesticated common bean from the Andean and Mesoamerican gene pools. Nine wild or domesticated populations, including four of Andean and four of Mesoamerican origins, were identified. In fruit trees, the genetic structure of sweet cherry was constructed on 207 of 211 wild cherry varieties. The structure results revealed three populations, namely, wild cherries, landraces, and modern varieties (Mariette et al. 2010). To date, only a single study has been conducted on 224 peach commercial varieties using 50 SSRs (Aranzana et al. 2010). LD analyses of three peach subpopulations therein revealed high levels of LD conservation in all populations extending up to 1315 cM, no association analysis was done in this paper. In the current paper, we examined the 104 landraces, except for improved varieties, from six geographical regions

in China with a set of 53 SSRs that cover the peach genome. These markers were used to analyze the genetic diversity, population structure, and LD extent between SSR marker pairs. Association mapping was also performed for several fruit and phenological periods of peach landrace accessions collected in China. The results of the current study provide molecular information for exploring the QTLs of important agronomic peach traits. The results will also help utilize and conserve Chinese peach landraces effectively.

Materials and methods Plant materials Considering that genetic recombination and artificial selection would interfere with LD analyses, commercial varieties were excluded from the dataset. A total of 104 peach landraces (Table 1) were selected from the National Clonal Germplasm Repository of Peach Centers (Zhengzhou, China). According to the classification established by Wang and Zhuang (2001), the accessions were divided into six different geographical populations (Fig. 1), such as northwest China (NWC), the YunGui plateau (YGC), northeast China (NEC), northern China (NC), the middle and lower reaches of the Changjiang River (MLCJ), and southern China (SC). No wild peach accession was used because a database of its fruit or phenological period traits is very difficult to obtain. Phenotypic data The resulting materials were grafted on the same rootstock (Prunus davidiana). They were planted in the National Clonal Germplasm Repository of Peach Center's orchard in Zhengzhou, China in the year 2000. The trees were trained in a Y shape and were planted at a spacing of 52 m. Hand thinning was carried out to reduce fruit load when required. The trees were grown under standard conditions of irrigation, fertilization, as well as pest and disease control. Fruit quality (red pigment in flesh, flesh color around the stone, flesh texture, stone adhesion to flesh, fruit weight, and flesh firmness without skin) and chilling requirement traits were evaluated in 2007, phenological period (flowering time, ripening time, and fruit development period) traits were evaluated over three consecutive years (20062009), and more integrate data (2007 and 2008) were chosen for analysis. Fruit quality traits from each accession were evaluated immediately after harvest. A fruit on a tree was considered ripe based on Celia et al. (2010). For the evaluation of fruit quality parameters, a representative sample consisting of 10 fruits per tree was selected. Color-card readings were recorded from the central section of the flesh to the flesh around the stone. To qualitatively score flesh color, six

Tree Genetics & Genomes (2012) 8:975990 Table 1 Peach germplasm accessions included in the current study and their geographical locations in China
Geographic population Northwest China Origin (province) Sinkiang Sinkiang Sinkiang Sinkiang Sinkiang Sinkiang Sinkiang Sinkiang Sinkiang Sinkiang Sinkiang Sinkiang Sinkiang Sinkiang Sinkiang Sinkiang Sinkiang Sinkiang Sinkiang Gansu Gansu Gansu Gansu Gansu Gansu Gansu Gansu Gansu Gansu Gansu Gansu Gansu Shaanxi Shaanxi Shaanxi YunGui plateau Yunnan Yunnan Yunnan Yunnan Yunnan Yunnan Yunnan Guizhou Guizhou Guizhou Guizhou Northeast China Jilin Jilin Jilin No. Accession name Geographic regions Province of origin Beijing Beijing Beijing Beijing Beijing Beijing Beijing Beijing Tianjin Shanxi Hebei Hebei Hebei Hebei Hebei Hebei Henan Henan Henan Henan Henan Henan Henan Shandong Shandong Shandong Shandong Shandong Shandong Shandong The middle and lower reaches of the Changjiang River Sichuan Sichuan Hubei Hubei Hubei Anhui Anhui Jiangsu Jiangsu Jiangsu Jiangsu Jiangsu Shanghai Shanghai Shanghai Zhejiang Zhejiang Zhejiang Zhejiang No. Accession name

977

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

Kashi 1# Kashi 2# Kashi 3# Xinjiang Huang Rou Xinjiang Pan Tao Tian Ren Tao Tu-2 Tie 4-1 Bi Nan I Mi Yang Shan Moyu 8# Li He Tian Ren Hetian Huang Rou Yexian Huang Rou Tao Da Li He Huang Rou Kashi Huang Rou Li Guang Yilixian Huang Rou Tao Hong Li Guang Huang Li Guang Ying Chun Bai Sha Qi Tao Dunhuang Dong Tao Kashi 4# Zhao Shu Huang Gan Gaotai 1# Tugou 1# Zhang Huang 9# Lin Huang 9# Lin Bai 10# Zhang Bai 5# Zhang Bai Gan Xi Jiao 2# Xi Jiao 3# Qinling Dong Tao Qing Si Tao Huang Yan Bai Li Hu Bai Nian Hu Huang Nian He Bai Nian He Huo Lian Jin Dan Qing Tao Guizhou Shui Mi Guangyi Bai Hua Tao Wangmo Xiao Mi Tao Hun Chun Tao 8501 8601

Northern China

53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101

Wu Yue Xian Wu Yue Xian Bian Gan Shiwo Shui Mi Fei Tao Ju Hua Tao Yuan Yang Chui Zhi Hong Hua Bi Tao Bai Hua Shan Bi Tao Tian Jin Shui Mi Yangquan Rou Tao Ge Gu Hong Ya Zui Da Xue Tao Shen Zhou Shui Mi Shenzhou Bai Mi Shenzhou Li He Shui Mi Ji Zhui Bai Yexian Dong Tao Ren Mian Tao Wu Bao Tao Jiang Tao Hong Chui Zhi Sa Hong Tao Hei Bu Dai Da Guo Hei Tao Wu Hei Ji Rou Tao Shandong Si Yue Ban Qingzhou Hong Pi Mi Tao Fei Cheng Hong Li 6# Fei Cheng Bai Li 17# Jiu Yang Qing Tao Qing Mao Zi Bai Hua Liu Yue Bai Da Hong Pao Zao Chun Tao Ying Zui Diao Zhi Bai Fen Shou Xing Hong Shou Xing Ping Bei Zi Wan Pan Tao Lulin Shui Mi Bai Dan Ban Bai Mang Pan Tao Sha Hua Hong Pan Tao Yu Lu Pan Tao Fenghua Pan Tao Jiaqing Pan Tao Li He Pan Tao

978 Table 1 (continued)

Geographic population Origin (province) Jilin Jilin Jilin No. Accession name Geographic regions

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Province of origin Zhejiang

No.

Accession name

50 51 52

8701 8801 8903 Southern China

102 103 104

Chang Sheng Pan Tao Xian Tao Nanshan Tian Tao

Guangxi Guangdong

(05) color-card categories were used. Flesh texture (melting, M, or non-melting, NM) and flesh adhesion (freestone or clingstone) were scored using the rating scales appropriated for each quality by three researchers. These morphological descriptions and criteria are all summarized in Table 2. The average fruit weight (in grams per fruit) was determined using a BS200s electronic balance (Sartorius, Goettingen, Germany) for each seedling tree. Flesh firmness without skin measurements were performed on the opposite equatorial sides of each fruit on each tree using a FT-327 hand penetrometer (Breuzzi, Milan, Italy). Data are given in kilograms per square centimeter. In the current paper, the relative data for flowering and ripening times, compared with the oldest full bloom data (March 16) and the harvest maturity data (June 12), were recorded for each accession. The mean date of the 3 years was also calculated. The fruit development period was judged as the date of the first full bloom to the harvest maturity date. Chilling requirement was calculated similarly as reported by Fan et al. (2010). SSR marker genotyping Genomic DNA was extracted from leaf tissues using a DNA extract kit (Sangon, Shanghai, China). Fifty-three SSR

markers (average density 0 10 cM per marker) distributed on the eight linkage groups of the Prunus TE reference map (519 cM; Dirlewanger et al. 2004a) were selected (Table 3). The polymerase chain reaction, amplification, and detection were performed as described in Cao et al. (2011). All amplifications were scored as either a present (1) or an absent (0) band. The alleles were coded A, B, C, etc. in decreasing size order for each band. A single band in one accession was assumed to be homozygous. Genetic diversity and phylogenetic analyses The molecular diversity parameters of the six geographical populations, such as number of alleles per locus (NA), observed heterozygosity (HO), Shannons information index (I), number of private alleles (NPA), genetic distance (Nei et al. 1983), and pair-wise FST among the six geographical populations were all estimated with POPGENE version 1.31 developed by Yeh et al. (1999). Based on the genetic distances (Nei et al. 1983) of the six geographical populations, a neighbor-joining tree was constructed. Gene diversity (GD) and the polymorphism information content (PIC) of the six geographical populations were calculated with Powermarker version 3.25 (Liu and Muse 2005, http://statgen.ncsu.edu/powermarker/). Alleles were considered private if they occurred in less than 1% of the accessions. Neis genetic distances (Nei et al. 1983) among the 104 accessions were calculated with the Powermarker software. A dendrogram was constructed with the same software using the neighbor-joining method with arithmetic mean.

Table 2 Four characteristic descriptions and their coding

Character Flesh color around the stone Red pigment in flesh Classification No 0 0; little 0 1; moderate 0 2; much 0 3 No 0 0; little 0 1; moderate 0 2; much 0 3; very much 0 4 Non-melting 0 0; hard melting 0 1; soft melting 0 2; wooliness 0 3 Free 0 0; semi-free 0 1; cling 0 2

Fig. 1 Peach landraces were divided into six large geographic regions, namely, northwest China (NWC), the YunGui plateau (YGC), northeast China (NEC), northern China (NC), the middle and lower reaches of the Changjiang River (MLCJ), and southern China (SC)

Flesh texture Flesh adhesion

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Population structure analysis The model-based software Structure version 2.3.3 (Pritchard and Wen 2004, http://pritch.bsd.uchicago.edu/software. html) was used to infer the population structure (testing from K 0 2 to K 0 10) using a burn-in of 10,000 and a run length of 100,000. Three independent runs yielded consistent results. A model-based clustering algorithm was applied to identify subgroups with distinctive allele frequencies. K was chosen in advance, but was varied for independent runs of the algorithm. The most likely number of clusters (K) was selected by comparing the logarithmized probabilities of data L(K) and K according to Evanno et al. (2005). LD and association analysis As P. persica is a heterozygous species, it is not possible to distinguish the two possible double heterozygotes AB/ab and Ab/aB when parentage is unknown (Barnaud et al. 2006). Therefore, LD and association analysis were measured using haplotypic data reconstructed by PHASE 2.1 software (Stephens et al. 2001) based on raw population genotypic data prior TASSEL 2.0.1 software (http://www. maizegenetics.net/). The haplotypic data within whole linkage group were inferred using a Bayesian method. Then LD was evaluated for each pair of SSR loci using the TASSEL 2.0.1. All accession clusters were inferred by the Structure software as a covariate. The pairs of loci were considered to have a significant LD if P was <0.1. The significance (P values) of r2 for each SSR pair was determined by 50,000 permutations. The LD decay was evaluated when r2 0 0.1, and the curve was performed using curve regression of SPSS software. The estimated genetic distance (in centimorgans) between loci was inferred from the genome database for Rosaceae (Washington State University 2010). The associations among marker alleles and different phenotype data in 2007 and 2008, respectively, were performed with the general linear model (GLM) method using the TASSEL 2.0.1 software. The P value determined whether a marker (QTL) was associated with the trait, and the r2 marker evaluated the magnitude of the QTL effects. Results Genetic diversity of all markers All 53 SSR markers distributed across the genome every 10-cM interval were used to evaluate the genetic diversity of the population. All of the markers were polymorphic and produced a total of 340 alleles from the 104 accessions. The average number of alleles per locus was 6.4, ranging from 2 (CPPCT008 on chromosome 6) to 21 (BPPCT008 on chromosome 6). The average genetic diversity was 0.567, ranging

from 0.038 (CPPCT008) to 0.865 (BPPCT008). The average PIC value was 0.533, ranging from 0.037 (CPPCT008) to 0.853 (BPPCT008). Genetic diversity of geographic populations Genetic diversity (such as the gene diversity PIC) and observed heterozygosity for each geographic population was assessed (Table 4). The number of alleles per locus varied from 1.7885 in SC to 3.8868 in the NC population. Observed heterozygosity ranged from 0.1584 in NWC to 0.3046 in the MLCJ population. The MLCJ population had the highest gene diversity of 0.5616, and the second highest I index of 0.8086, with 3.1887 alleles per locus. The NC population followed, with the second highest gene diversity and the highest I index at with 3.8868 alleles per locus. The PIC values varied from 0.2264 to 0.5202, with the NC and SC populations showing the highest and lowest, respectively. Among the 340 alleles detected in all six populations, 59 (17.35%) alleles were population-specific or private (NPA) in 26 loci of 34 accessions. The NC population had more private alleles (25 or 7.35%) than others. Among these private alleles, 12 unique private alleles were found in the Bai Hua Shan Bi Tao accession, and five private alleles were found in the Nanshan Tian Tao. In summary, the NC and MLCJ populations showed highest values for genetic variability, whereas the SC population exhibited the lowest level of diversity. Genetic relationships among geographic populations The overall FST revealed a considerable and statistically significant degree of differentiation among the peach populations in China. The FST for each locus ranged from 0.0005 (CPPCT008) to 0.4693 (CPPCT018), with an average FST of 0.1931. This result indicated that 19.31% of the total variation in allele frequency of the 104 accessions was caused by the genetic differences among the clusters. Pairwise comparison on the basis of the values of FST could be interpreted as the standardized population distances between two populations. FST values among pairs of populations were found to range from 0.0342 (between the NC and MLCJ populations) to 0.2558 (between the NEC and SC populations), with an overall average of 0.1273 (Table 5). This result indicated that genetic differentiation among the clusters was highest in the combination of the NEC and SC populations. The genetic distance data agreed with the FST estimates. Within the geographical populations, the average Neis genetic distance between all pairs of populations was 0.1927 (Table 4). The lowest distance value was obtained for the pair of populations NCMLCJ (0.0546), whereas the highest value was obtained for populations NECSC (0.3948).

980 Table 3 The 53 mapped SSR loci on the eight peach linkage groups

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Linkage group 1

SSR CPSCT008 CPPCT027 CPPCT026 BPPCT020 BPPCT016 EPDCU2862 BPPCT028

Location (cM) 9 23.1 33.9 52.6 55.2 66.5 77.4 12.5 23.6 27.8 27.8 38 43.9 48.6(GN) 92.8(GN) 11.2 18 28.4 36.4 36.4 46.4 1.8 7 10.4 16.8 47.4 112.2 (GN)

Linkage group 5

SSR CPSCT011 UDP97-401 BPPCT017 BPPCT037 BPPCT032 CPPCT025 CPSCT022 BPPCT014

Location (cM) 5.2 11 20.1 25.6 34.7 37.4 (GN) 40.7 44 8.7 30.1 35.8 49.7 (GN) 56.4 72 80.2 18.6 22.3 29.6 38.9 41.3 51.4 0 14.1 20.8 24.8 44.5

CPDCT044 CPSCT044 UDP96-013 UDP98-411 BPPCT030 pceGA34 BPPCT034 UDP98-406

CPPCT008 BPPCT008 CPPCT015 UDP98-407 BPPCT025 UDP98-412 CPPCT030

BPPCT007 CPPCT018 CPDCT008 CPDCT025 UDP96-008 CPDCT027

CPPCT022 UDP98-405 BPPCT029 CPPCT033 CPSCT042 pchcms2

CPSCT039 pchgms2 CPPCT005 CPDCT045 BPPCT023 BPPCT031

CPSCT018 BPPCT006 UDP96-019 CPPCT006 UDP98-409

A neighbor-joining tree of the seven geographical populations (Fig. 2) was constructed based on pair-wise Neis genetic distances calculated by the POPGENE software. Table 5 shows that these populations could be clustered into four groups. The first group, the SC population, was very distinct. The second group was the NEC population, the third group was the YGC population, and the fourth group was constituted by the NWC, NC, and MLCG populations. Among the

populations in the fourth group, NC and MLCG, which had the highest genetic differentiation, were classified into subgroups. The results showed that the SC population had the largest genetic distance from the others, followed by the NEC and the YEC populations. These results were in agreement with the geographical distances among these regions. A dendrogram based on the genetic similarity matrix divided the 104 landraces accessions into five clusters

Table 4 Summarized statistics of genetic diversity for the six geographical populations
Population NWC YGC NEC NC MLCJ SC Number 35 11 6 30 20 2 NA 3.5094 2.6038 2.1698 3.8868 3.1887 1.7885 Ho 0.1584 0.2200 0.1604 0.2170 0.3046 0.2212 I 0.7295 0.6631 0.5556 0.8730 0.8086 0.4655 GD 0.4714 0.4750 0.3648 0.5564 0.5616 0.3019 PIC 0.4382 0.4217 0.3183 0.5202 0.5115 0.2264 NPA 11 6 2 25 9 6

NWC northwest China, YGC the YunGui plateau, NEC northeast China, NC northern China, MLCJ the middle and lower reaches of the Changjiang River, SC southern China, NA the number of alleles per locus, Ho the observed heterozygosity, I Shannons information index, GD gene diversity, PIC polymorphism information content, NPA the number of private alleles

Tree Genetics & Genomes (2012) 8:975990 Table 5 Pair-wise estimates of FST and Neis 1972 unbiased genetic distances based on 53 SSR loci among the six geographical populations
Population NWC YGC NEC NC MLCJ SC NWC 0.0698 0.1253 0.0459 0.0539 0.2176 YGC 0.0967 0.1557 0.0689 0.0771 0.2407 NEC 0.1773 0.2160 0.0864 0.1121 0.2558 NC 0.0693 0.1011 0.1153 0.0342 0.1827 MLCJ 0.0907 0.1107 0.1586 0.0546 0.1828 SC 0.3759 0.3750 0.3948 0.2875 0.2667

981

Population structure Association mapping requires population structures to be taken into account to avoid identifying spurious associations (Yu et al. 2006). The estimated likelihood values for a given K in three independent runs yielded consistent results using the structure program. However, the distribution of log Pr (X/K) increased continuously with increased K values. To overcome difficulties in determining the real value of K, another quantity criterion (K) was used (Evanno et al. 2005). In the current study, the value of K for the 104 peach accessions was highest at K 0 5 (Fig. 3). This result suggested that these peach accessions can be grouped into five populations, herein denoted as POP1, POP2, POP3, POP4, and POP5, respectively (Fig. 4, Table 6). Therefore, the respective Q matrix outputs of the five population runs for the structure-based association analysis were used. According to the membership pattern, when K 0 5, the 104 accessions were classified into five groups. POP2 contained 20 accessions, of which 10 originated from the NC population. POP5 contained 21 accessions, mainly including the NWC population (Fig. 4). POP1, POP3, and POP4 had more than one ancestral background, defined as an admixture, and these clusters included accessions from the YGC, NC, NWC, NEC, and MLCJ populations. The highly significant association (Table 6, 2 0 97.65 0.01, 2 20 0 37.57) between the model-based clusters and the geographical populations was found after analyzing the relativity of all accessions cluster, revealing that the model-based cluster has a geographical foundation. So, it will be more precise to use the model-based cluster parameter as a covariant in analyzing association than using the dendrogram based on genetic similarities parameter. LD An analysis of the extent and evolution of LD was a foundation for detecting the true associations within the mapping population. LD extent of was assessed among all 1,378 pairs of the SSRs loci for all accessions. Across all accessions, as
300 250 200

Neis 1972 unbiased genetic distance estimates appear above the diagonal, and pair-wise FST appears below the diagonal NWC northwest China, YGC the YunGui plateau, NEC northeast China, NC northern China, MLCJ the middle and lower reaches of the Changjiang River, SC southern China

(Fig. 4a, b). All accessions from the SC region, 5 of 11 from the YGC region, 13 of 30 from the NC region, and 8 of 20 from the MLCG region were placed in the G1 cluster. Most accessions of the NWC and NEC populations were placed in the G5 and G3 clusters, respectively. However, a few accessions displayed as admixtures in the different clusters. For example, the typical accessions of the NEC population, such as Hun Chun Tao, 8501, 8601, 8701, and 8801, were assigned to the G3 cluster, but one accession (8903) of the NEC population was placed in the G1 cluster in the dendrogram (shown in dark red in Fig. 2b). The 2 test showed that these clusters revealed genetic relationships fairly consistent with the geographic origin of most accessions (Table 5).

150 100 50 0 2 3 4 5 6 7

Fig. 2 Unrooted neighbor-joining trees of the six geographical populations based on Neis genetic distances

Fig. 3 Values of K, with the modal values used to detect the true K of five groups (K 0 5)

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Fig. 4 Diversity analysis and population structure of 104 peach landraces using 53 SSRs loci. a Dendrogram based on genetic distance. b The geographic origin of landraces: dark blue NEC population, light blue YGC population, dark red NEC population, light red NC

population, dark green MLCJ population, and light green SC population. c Population stratification for K 0 5 (each color represents a different subpopulation)

many as 249 (18.07%) of the total marker pairs were in LD (based on r2, P <0.05) after Bonferroni correction. The number of LD for the marker pairs from the different chromosomes was 214, higher than the markers (35) on the same chromosomes.

At the whole population level, the r2 values among all the SSR pairs ranged from 0.001 to 0.7525. The mean r2 value for all intrachromosomal loci pairs was 0.0250, and the r2 values for the interchromosomal pairs ranged from 0 to 0.2017, with an average of 0.0149. At

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Table 6 Associations among the dendrogram based on genetic similarities, the model-based clusters, and the geographical populations in peach landraces
Geographical populations Total Dendrogram based on genetic similarities G1 NWC YGC NEC NC MLCJ SC Total 35 11 6 30 20 2 104 7 5 1 13 8 2 36 G2 9 1 0 3 2 0 15 G3 1 1 5 13 2 0 22 G4 1 1 0 1 7 0 10 G5 17 3 0 0 1 0 21 2 0 78.63 P <0.01 0.01, 202 0 37.57 2 Model-based clusters POP1 3 7 0 8 1 0 19 POP2 1 3 1 10 5 0 20 POP3 11 0 4 6 3 1 25 POP4 0 0 1 6 11 1 19 POP5 20 1 0 0 0 0 21 2 0 97.65 P <0.01 0.01, 202 0 37.57 2

the highly significant threshold of r2 >0.1, 0.8% (11) of the marker pairs remained in LD, and the loci pairs mostly distributed among different chromosomes. Some unlinked SSR markers on different chromosomes had higher r 2 values. For example, the r 2 values were 0.2018 (CPSCT022 on chromosome 5 and CPPCT005 on chromosome 4), 0.2004 (BPPCT014 on chromosome 5 and CPPCT005 on chromosome 4), 0.1786 (pchcms2 on chromosome 7 and pchgms2 on chromosome 4), and 0.1174 (BPPCT029 on chromosome 7 and BPPCT037 on chromosome 5). Figure 5 showed the distribution of the r2 values of the interchromosomal pairs for the whole population. About 95.51% of the r2 values were below 0.05, and 3.43% ranged from 0.05 to 0.10. LD decay was investigated using the r2 (P <0.1) values of the SSRs pairs in the same chromosomes of the three main geographic populations. As shown in Fig. 6, in NC population, the LD decays with distance, whereas in NWC and MLCJ populations, high levels of LD extend were not found between pairs of SSRs with near linkage distance. And in whole samples, the intrachromosomal LD was very common for distances of 40 cM. Occasionally, LD occurred between SSR loci that were further apart, and LD extended to 70 cM in most cases. Data point distributions in the plot of LD (r2) decay against genetic distance (in centimorgans) within the eight chromosomes, followed by the equation y 0 0.0579 ln (x)+0.2038, showed that LD was not a simple monotonic function of the distance between markers. The r2 between the linked markers was mainly <0.1. LD extent for a particular region can be estimated from an LD decay plot generated using the dataset obtained from a region of interest. When such an LD decay plot is generated, the usual practice is to look for the distance point where the LD value (r2) decreases below 0.1 or for the half strength of D (D 0 0.5) based on the curve of the nonlinear logarithmic trend line (Ibrokhim and Abdusattor 2008). In the present study, the LD across all the populations decayed below the critical r2 value of 0.1 within 6.01 cM.

Association mapping based on structured population An association map (P <0.01) of SSR loci with fruit quality and phenological period traits from the peach landraces accessions was constructed. A GLM model implemented in TASSEL was used for the mapping. Out of the 53 polymorphic SSR markers used for the association mapping, 9 (17.0%) were associated with flowering time in 2007, 10 (18.9%) associated with that

0.8 0.7 0.6

Frequency

0.5 0.4 0.3 0.2 0.1 0

0.05

0.15

0.25 r2

0.35

0.45

100 80

95.51

Frequency (%)

60 40 20
3.43 0.25 0.08 0.16

0 0-0.05 0.05-0.10 0.10-0.15 0.15-0.20 0.20-0.25

r2

Fig. 5 Distribution of linkage disequilibrium r2 values for interchromosomal SSR pairs

984 Fig. 6 Pattern of linkage disequilibrium indicating the correlation of allele frequency (r2, P <0.1) values against genetic distance (in centimorgans) between all loci pairs in the same chromosome in NWC, NC, and MLCJ populations and whole samples

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in 2008, and 8 (15.1%) were associated with chilling requirement traits in 2007. Only five association markers, which had highest r2 values, were selected in the present study (Table 7). The four SSR markers were significantly associated with the red pigment in the flesh explained between 8.1 and 14.5% phenotypic variations. One marker, CPPCT018 on chromosome 3 was associated with flesh color around the stone, explained 14.6% phenotypic variations. The gene which controlled the trait was Cs, which was located on chromosome 3 previously (Yamamoto et al. 2001). BPPCT023 on chromosome 4 and BPPCT028 on chromosome 1 were associated with the flesh adhesion trait, and the corresponding gene, F, reported by Yamamoto et al. (2001) was located on chromosome 4. Two markers were associated with flesh texture traits, and five with chilling requirement. Six, 5, 10, 4, and 3 markers were associated with flesh weight, flesh firmness without skin, flowering time, ripening time, and fruit development period in 2007 and 2008, respectively. We also found that some loci were associated simultaneously with two or more traits. For example, CPDCT045 on chromosome 4 was associated with three traits (red pigment in flesh, ripening time, and fruit development period), and BPPCT008 on chromosome 6 was associated with three traits (red pigment in flesh, fruit weight, and flowering time). Furthermore, nine markers were associated with two traits. The details of the association mapping results for each trait are listed in Table 7.

Discussion Genetic diversity in peach germplasm The narrow genetic base of modern crop cultivars is a serious obstacle in sustaining and improving crop productivity. The reason for this is the rapid vulnerability of genetically uniform cultivars with potentially new biotic and abiotic stresses (Esbroeck et al. 1999). The efficient exploitation of the genetic diversities among these plant germplasm resources in their origin centers is vital. This process helps overcome problems associated with the narrowness of the modern cultivar genetic base. However, identifying the genetic variants underlying quantitative crop trait variations is a challenging task for plant breeders. Linkage mapping, and more recently, association or LD mapping, have been applied to elucidate the genetic bases of natural variation in important quantitative traits. Germplasm choice is critical to the success of association mapping. The group of individuals should be selected from a natural population or germplasm collection with a wide genetic diversity. Peach landraces have evolved from their wild progenitors by natural and human selection, leading to the maintenance of high genetic diversity. In the current study, a set of 104 landraces from the National Clonal Germplasm Repository of Peach Center (Zhengzhou) that consisted of six ecotypes was used. The abundant allelic variation per locus is 6.4. This amount is similar to a previous report of 6.36 alleles per locus (Aranzana et al.

Tree Genetics & Genomes (2012) 8:975990 Table 7 Associations between the microsatellite and the 10 quantitative traits in peach landraces' trait
SSR markera Chromosome Map position from the top (cM) 18 16.8 30.1 40.7 36.4 27.8 77.4 47.4 77.4 11 27.8 33.9 72 29.6 11 11.2 8.7 20.8 24.8 30.1 10.4 44 49.7d 51.4 35.8 49.7d 10.4 44 51.4 56.4 35.8 18.6 12.5 30.1 27.8 20.1 27.8 43.9 16.8 27.8 43.9 27.8 16.8 27.8 r2 (%)b P value Known loci and gene

985

Flesh color around the stone Red pigment in flesh

CPPCT018 CPDCT045 BPPCT008 CPSCT022 CPDCT025

3 4 6 5 3 2 1 4 1 5 2 1 6 7 5 3 6 8 8 6 4 5 6 7 6 6 4 5 7 6 6 7 2 6 2 5 2 2 4 2 2 2 4 2

14.6 14.5 14.1 8.3 8.1 13.1 12.8 21.2 12.5 22.8 20.6 23.4 15.6 14.1 24.3 21.3 20.4 17.4 14.9 23.0 25.1 20.9 20.6 18.4 17.8 49.9 49.9 49.8 41.4 30.1 21.9 18.0 17.0 15.6 15.5 32.1 22.8 19.2 10.9 11.0 16.2 16.1 12.9 10.8

0.0039 4.3106 0.0029 6.8104 0.0037 0.0029 0.0011 2.1106 6.9104 0.0041 0.0085 6.110 0.0089 0.0065 7.7104 0.0024 0.001 0.0031 0.0078 0.0052 8.9105 6.5104 0.0053 5.5104 0.0068 1.71010 6.11012 6.71012 4.41010 2.4105 4.6106 5.9104 1.6104 0.0025 1.4104 3.4105 0.0015 0.0029 0.003 0.0062 0.0065 0.0067 7.4104 0.0043
4

Cs (Yamamoto et al. 2001)

MYB10 (Wang et al. 2010)

Flesh texture Flesh adhesion

UDP96-013 BPPCT028 BPPCT023 BPPCT028 UDP97-401.2007 UDP98-411.2007 CPPCT026.2008 UDP98-412.2008 BPPCT029.2008

F (Yamamoto et al. 2001); endoPG (Peace et al. 2005)

Flesh firmness without skin

PME1 (Illa et al. 2010) PME7 (Illa et al. 2010)

Fruit weightc

UDP97-401.2007 BPPCT007.2007 CPPCT008.2007 UDP96-019.2007 CPPCT006.2007 BPPCT008.2008

QTL (Abbott et al. 1997) QTL (Abbott et al. 1997)

Chilling requirementc

CPPCT005 BPPCT014 UDP98-407 pchcms2 CPPCT015

Flowering timec

UDP98-407.2007 CPPCT005.2007 BPPCT014.2007 pchcms2.2007 BPPCT025.2007 CPPCT015.2008 CPPCT022.2008 CPDCT044.2008 BPPCT008.2008 UDP96-013.2008

QTL (Fan et al. 2010); QTL (Etienne et al. 2002)

QTL (Fan et al. 2010) QTL (Verde et al. 2002) QTL (Etienne et al. 2002)

Ripening time

BPPCT017.2007 UDP98-411.2007 pceGA34.2007 CPDCT045.2007 UDP98-411.2008 pceGA34.2007 UDP98-411.2007 CPDCT045.2007 UDP98-411.2008

Fruit development period

QTL (Etienne et al. 2002)

Only SSR markers with a significant marker-trait association are reported (P <0.01) r indicates the percentage of the total variation explained Loci position in the GN linkage map (Dirlewanger et al. 2004b) Five association markers that had the highest r2 values were selected

b 2 c d

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2010) using peaches selected from Europe and America, and even exceeded another report of 2.85 alleles per locus (Xie et al. 2010) using improved varieties from Fenghua (China) local accessions. These results may be an indication of a high level of genetic diversity among the Chinese landraces. Few studies on peach landraces of different geographical populations exist. Cheng and Huang (2009) reported an average I value of 0.319 (ranging from 0.087 to 0.520) in 32 cultivars and landraces from China. Xie et al. (2010) evaluated the genetic diversity and identity of 94 accessions, including Fenghua local accessions and introduced cultivars. The average PIC value was 0.34 for the Fenghua cultivars and was 0.46 for the introduced cultivars. The Fenghua accessions were also less heterozygous than the introduced ones (Ho 0 0.46 and 0.50, respectively). These results suggested that the honey peach cultivars collected in the Fenghua area had a lower level of genetic diversity than the 46 introduced ones. The results of the present study showed that the PIC values of the studied geographical populations varied from 0.2264 to 0.5202. The NC and SC populations showed the highest and lowest values, respectively. The SC population had the highest I (0.8730), whereas the NC population had the lowest value (0.4655). Compared with a previous study, a higher genetic diversity was shown in the present study based on the higher I and PIC values because more landraces were used. Higher genetic diversities were found among the NC and MLCJ populations than among the NWC and YGC populations. Among the peach geographical populations in China, NC and MLCJ attracted much attention given more landraces and very widely divergent phenotypes and genotypes. This may at least be helpful in selecting high differentiation parents for crossbreeding. This is the first report on the diversity of peach landraces selected from different geographical populations. The higher genetic diversity in our report indicated that huge phenotypic variations increased detection power and allowed the quantification of more allelic effects. The results showed that these alleles are more suitable for association mapping. The region with the highest genetic diversity is generally considered as the center of origin of a species (Vavilov 1951). Among the six populations, NC and MLCJ in central China, where the climate was suitable for peach cultivation and had the highest genetic diversity, are hence presumed as the landrace origin. However, Wang and Zhuang (2001) believed that the NWC and YGC peach populations, which differentiated from another wild species of peach, Prunus mira, were the landrace center of origin. The high genetic diversity within NC and MLCJ may be the result of several factors, including larger area and higher phenotype diversity. For example, more red flesh peach (Tian Jin Shui Mi, Hei Bu Dai, Da Guo Hei Tao, and Wu Hei Ji Rou Tao) and ornamental peach (Ren Mian Tao, Wu Bao Tao, Jiang Tao,

Hong Chui Zhi, and Sa Hong Tao) are found in NC compared with NWC. Therefore, our results indicated that new characters will be formed in plant evaluation. Population structure and LD in peach germplasm Understanding the population structure is important to avoid identifying spurious associations between phenotypes and genotypes in association mapping (Pritchard et al. 2000). The genetic structure of peach had previously been analyzed. Aranzana et al. (2010) detected six major groups within a set of 50 SSRs in 224 peach varieties, but this analysis used improved commercial accessions from Spain and the USA. Using the Structure software with K 0 5, the 104 peach landraces from China were significantly differentiated into five groups. Moreover, several accessions had partial ancestry in more than one background. For example, POP5 contained 21 accessions, and 20 of them originated from the NWC region. However, one accession, Bai Nian Hu, belonged to the YGC population and was designated in POP5. This accession probably had a complex breeding history that involved intercrossing and introgression among germplasms from diverse backgrounds (Mather et al. 2004). Model-based analyses of population structures may be helpful in providing information for association mapping analyses. The number of markers needed for genome-wide LD scanning depends on the LD level. This requirement is based on a preliminary estimate of 420440 kb/cM for the correspondence between genetic and physical distances in peach (http://www.rosaceae.org/peach/genome). In the present study, r2 values decreased to 0.1 within 6.01 cM, approximately 2,5242,644 kb. Thus, peach LD extended farther than grape (Barnaud et al. 2006) and oat (Newell et al. 2011) LDs, where the r2 values reached 0.1 within 1,3002,160 kb and 2.5 cM, respectively. Nonetheless, this LD value is smaller than that of cotton (Abdurakhmonov et al. 2008) and durum wheat (Maccaferri et al. 2005), which had LD values approximately 10 cM and larger than 3040 cM at r2 0 0.1. Hence, peach LD extent, as measured by the r2 decline, seems to be moderate compared with other species. The moderate LD blocks in peach suggested the potential for conducting an effective LD mapping of complex traits with a fewer number of markers than that required for the grape genome. Considering that the peach genome has a total recombinational length of approximately 520 cM (Dirlewanger et al. 2004a) and the LD block distance of 6.01 cM such a mapping would require about 86 polymorphic markers distributed uniformly across the genome for a minimum coverage of LD blocks in the genome of various germplasms. LD level may vary across genomes because of different recombination rates, selective pressures, mating systems

Tree Genetics & Genomes (2012) 8:975990

987

(selfing vs. out-crossing), effective population sizes, and so on. Several reports suggested a longer size of LD blocks in other narrow-based germplasm crop groups than in broadbased germplasm groups (Remington et al. 2001; Gupta et al. 2005). As a selfing species, peach landraces are supposed to have a higher LD level than improved varieties because of few past recombination events. However, in the present study, the LD across the whole population of peach landraces decayed below the critical r2 value of 0.1 within 6.01 cM, which was less than that in the improved peach variety (Aranzana et al. 2010). The r2 values larger than 0.1 therein were maintained to up to 13.3 cM (M peach) and 15.2 cM (NM peach). These results are the same for cotton, in which the genome-wide LD block size averages at the r2 0.1 threshold were less than 10 cM in the landrace germplasm, and more than 30 cM in the improved variety germplasm (Abdurakhmonov et al. 2008). However, our estimate of genome-wide averages for LD extent inferred by the SSR markers in peach was larger than those inferred by the single nucleotide polymorphism reported in Arabidopsis (25250 kb; Nordborg et al. 2002) and maize (200 400 bp; Tenaillon et al. 2001). Consequently, LD patterns in the specific regions or population groups in peach may not be adequately reflected. Additional LD quantification in each targeted region or population group is required for an effective association mapping (Abdurakhmonov et al. 2008). Association mapping in peach Prunus fruit development, growth, ripening, and senescence all include major biochemical and sensory changes in texture, color, and flavor. The genetic dissection of these complex processes has important applications in crop improvement. A structure-based association mapping was performed for peach fruit and phenological period traits using a genome-wide association map based on haplotypic data. A total of 27 marker-trait associations were selected to be analyzed using 53 different SSR markers (Table 7). These trait-associated SSR markers from our study were compared with other reported SSR markers from QTL mapping analyses of various experimental populations. Among these associations, some were in regions where QTLs associated with the given trait had previously been identified. However, some associations were not consistent with the results of other published linkage maps because different marker systems were used (Abdurakhmonov et al. 2008). Considering that the genome parallels with reference genetic map, every linkage groups in the genetic map indicate the corresponding chromosomes. In the present study, one SSR marker, CPPCT018 (18 cM on the G3 of TE map) on chromosome 3 (Yamamoto et al. 2001) was associated with

the flesh color around the stone, also associated with Cs (17 18 cM on G3 of TE map) gene, reported priviously by QTL mapping. Four SSR markers associated with the red pigment in fruit flesh were distributed in chromosomes 3, 4, 5, and 6. Because the expression of MYB10 genes correlated with fruit anthocyanin levels (Wang et al. 2010), hence, MYB10 was identified as the candidate gene for the red pigment in the flesh. After blasting with the peach genome database (http:// www.rosaceae.org/peach/genome), the MYB10 (GenBank accession: EU155160) was found to be located in about 29.4 cM on chromosome 3. So, CPDCT025 (36.4 cM on the G3 of TE map) may be an association locus around the MYB10 gene in the present study. The M and the NM traits of peach are two opposite phenotypes of flesh texture (Bailey and French 1932). The M texture shows a prominent softening in the last stage of ripening before a complete melting. The lack of softening in the NM phenotype is related to the loss of endopolygalacturonase (endoPGase) activity, the enzyme responsible for cleaving pectins (polygalacturonic acid chains) from the cell wall in the M fruits (Lester et al. 1996). Moreover, the flesh adhesion to stone, according to Bailey and French (1932) is controlled by the freestone locus, where the freestone (F) allele is dominant over the clingstone (ff). Bailey and French (1932) suggested that the flesh adheres to the stone and the flesh texture gene is linked on the same chromosome. From recent studies on progenies segregated for endocarp adherence and flesh texture (M and NM), four alleles for the endoPGase enzyme were found responsible for the three flesh phenotypes: freestone and clingstone-M and clingstone-NM endoPGase were mapped to the linkage group G4, as previously reported (Peace et al. 2005). Subsequently, the endoPGase gene of peach was cloned (Morgutti et al. 2006, GenBank accession: DQ659241) and was located in chromosome 4 blasted with peach genome. In the present study, three SSR markers in chromosomes 1, 2, and 4 were associated with flesh texture and flesh adhesion to stone traits. Among them, BPPCT023 (physical location: 14731772 on chromosome 4) associated with endoPGase (DQ659241, physical location: 22684623 22686568 on chromosome 4) was found in the same region hosting F. F is the major gene controlling flesh texture and flesh adhesion to the stone. BPPCT028 associated with flesh texture on chromosome 1 explained 12.8% phenotype variation and also associated with the flesh color around the stone. This finding indicated that a common factor influencing the two traits exists. Peaches are characterized by a rapid loss of firmness at the end of the ripening process. Cell wall changes are particularly important in this phenomenon. Such cell wall changes include the dismantling of its structure, the degradation of the polymers of which it is composed, and the loss of turgor pressure in the fruit (Brummell 2006). These

988

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changes correlated with the concerted action of two proteins, endoPGases and pectin methylesterases (PMEs). PME isoforms detected in cell walls are encoded by a multigene family. The main PME function is polyuronide demethylation so that polyuronide can be degraded by endoPGase. Recently, Murayama et al. (2009) identified PpPME2 upregulation by PpPG2 during peach fruit softening. The endoPGase was located on chromosome 4, as described previously, but PME was located in a different linkage group in different papers. Ogundiwin et al. (2009) located two PME genes, PME1 on G1 (approximately 50 cM on G1 of the TE map) and PME5 on G7 (approximately 56 cM on G7 of the TE map). Soon after, Illa et al. (2010) located PME1 on G2 (45 cM) and PME7 on G1 (50 cM). Our results showed that five markers associated with flesh firmness were distributed in chromosomes 1, 2, 5, 6, and 7. Among these markers, CPPCT026.2008 which is located in chromosome 1 explained the higher phenotype variation and indicated that the PME gene maybe was essential to fruit firmness. Fruit weight is a complex trait that follows a quantitative inheritance depending on fruit shape and development period. Dirlewanger et al. (1999) considered that near the S gene, which is a dominant gene controlling fruit shape (i.e., flat or round). This finding is in agreement with the fact that flat fruits are generally lower in weight than round fruits. Based on QTL mapping, Abbott et al. detected the QTLs of fruit weight in groups 3 and 5 of peach. Dirlewanger et al. (1999) and Etienne et al. (2002) detected these QTLs in group 6. CPPCT008.2007 and BPPCT008.2008 were found in chromosome 6 associated with fruit weight. BPPCT007.2007 and UDP97-401.2007 were found in chromosomes 3 and 5, respectively. These results were similar with that of Abbott et al. (1997). Chilling requirement refers to the duration of low temperatures required for the release of temperate trees from endodormancy. Fan et al. (2010) constructed a linkage map used by a peach F2 population of 378 genotypes developed from two genotypes with contrasting chilling requirements for QTL mapping. The study detected five QTLs of chilling requirement. Among these QTLs, qCR1a and qCR7 showed very prominent effects and were declared to be the major QTLs. qCR1a explained 40.544.8% of the phenotypic variance, and qCR7 explained 17.824.9%. In comparison, the TE map showed that qCR1a was located in G1 at approximately 70 cM, and qCR7 in G7 was at approximately 38 cM. The two QTLs not only facilitate markerassisted breeding for low chill cultivars, but also pave the way for future fine mapping and map-based cloning of genes controlling the chilling requirement. Especially, the major QTL (qCR1a) spans only 2 cM, which overlaps with the peach EVG region. The evergreen trait, controlled by the EVG gene, has been described as a trait that did not have a

terminal growth (i.e., no terminal bud formation) unless killed by frost (Rodriguez et al. 1994). The EVG gene is located in G1 (Wang et al. 2002). Table 7 shows that the most highly significant association found was CPPCT005 on chromosome 4, which explained 25.1% of the phenotypic variation. And another association, pchcms2 may be associated with qCR7 (Fan et al. 2010). However, no marker-trait association was found in region around that qCR1a and EVG gene. Flowering time depends on the chilling requirement necessary to fulfill rest and on the growing degree hour accumulation to reach full bloom. QTL mapping results for flowering times in various genomic regions in Prunus have been reported. Using the TE Prunus reference map of linkage groups, four QTLs on G1, G4, G6, and G7 were detected by Joobeur (1998) in an almondpeach F2 population. Two QTLs on G2 and G7 were found by Dirlewanger et al. (1999) in a peach F2 population. One QTL on G4 was determined by Verde et al. (2002) in a peach backcross (BC1) population. Fan et al. (2010) detected four QTLs in chromosomes 1, 2, 4, and 7. Among these QTLs, qBD1a (G1, 70 cM) and qBD7a (G7, 35 cM) had very prominent effects. In conclusion, several QTLs were detected in chromosomes 1, 2, 4, 6, and 7. In the present study, 10 QTLs were found in chromosomes 2, 4, 5, 6, and 7, and the location of trait-marker associations in chromosomes 2, 4, and 7 was close to the results reported by Fan et al. (2010). Furthermore, five markers associated with chilling requirement were also found be associated with flowering time. These results showed that the same genes may affect two traits in Peach. In the current study, four SSR markers were associated with ripening date and were mainly distributed on chromosomes 2, 4, and 5. The location of some QTLs is similar with previous results (Verde et al. 2002; Etienne et al. 2002). In 2008, only one marker, UDP98-411, was associated with ripening time. It was also found in 2007, which exhibits a good repetitiveness. No relationship was found between flowering and ripening times, same results as described by Layne and Bassi (2008). Fruit development is regulated by complex interactions between physiological and environmental factors. Few studies on the QTLs of fruit development period exist. Three markers on chromosomes 2 and 4 which contribute for fruit development period were found in the current study. Because fruit development period was calculated from flowering and ripening time, same markers were found in association with the traits of fruit development period and ripening time. Identifying the genetic variants that underlie complex traits was very essential to plant genetics. Two main approaches are available for mapping the relevant genes and identifying the variants associated with these complex traits: linkage mapping in families and population-based

Tree Genetics & Genomes (2012) 8:975990

989 Dirlewanger E, Graziano E, Joobeur T, Garriga-Caldere F, Cosson P, Howard W, Arus P (2004a) Comparative mapping and marker assisted selection in Rosaceae fruit crops. PNAS 101:98919896 Dirlewanger E, Cosson P, Howad W, Capdeville G, Bosselut N, Claverie M, Voisin R, Poizat C, Lafargue B, Baron O, Laigret F, Kleinhentz M, Arus P, Esmenjaud D (2004b) Microsatellite genetic linkage maps of myrobalan plum and an almond-peach hybrid location of root-knot nematode resistance genes. Theor Appl Genet 109 (4):827838 Esbroeck GA, Bowman DT, May OL, Calhoun DS (1999) Genetic similarity indices for ancestral cotton cultivars and their impact on genetic diversity estimates of modern cultivars. Crop Sci 39 (2):323328 Etienne C, Rothan C, Moing A, Plomion C, Bodenes C, Dumas LS, Cosson P, Pronier V, Monet R, Dirlewanger E (2002) Candidate genes and QTL for sugar and organic acid content in peach (Prunus persica (L.) Batsch). Theor Appl Genet 105:145159 Evanno G, Regaut S, Goudet J (2005) Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol Ecol 14:26112620 Fan SH, Bielenberg DG, Zhebentyayeva TN, Reighard GL, Okie WR, Holland D, Abbott AG (2010) Mapping quantitative trait loci associated with chilling requirement, heat requirement and bloom date in peach (Prunus persica). New Phytol 185:917930 Faust M, Timon B (1995) Origin and dissemination of peach. Hortic Rev 17:331379 Flint-Garcia SA, Thornsberry JM, Buckler ES (2003) Structure of linkage disequilibrium in plants. Annu Rev Plant Biol 54:357374 Gupta PK, Rustgi S, Kulwal PL (2005) Linkage disequilibrium and association studies in higher plants: present status and future prospects. Plant Mol Biol 57:461485 Hancock JF (2008) Temperate fruit crop breeding: germplasm to genomics. Springer, Heidelberg Ibrokhim YA, Abdusattor A (2008) Application of association mapping to understanding the genetic diversity of plant germplasm resources. Int J Plant Genomics 2008:118 Illa E, Eduardo I, Marc Audergon J, Barale F, Dirlewanger E, Li XW, Moing A, Lambert P, Dantec LL, Gao ZS, Possel JL, Pozzi C, Rossini L, Vecchietti A, Aru P, Howad W (2010) Saturating the Prunus (stone fruits) genome with candidate genes for fruit quality. Mol Breed 28(4):667682 Jin L, Lu Y, Xiao P, Sun M, Corke H, Bao JS (2010) Genetic diversity and population structure of a diverse set of rice germplasm for association mapping. Theor Appl Genet 121:475487 Joobeur T (1998) Construccon de un mapa de marcadores moleculares y anlisis gentico de caracteres agronnicos en Prunus. PhD thesis, Universtat de Lleida, Spain Kwak M, Gepts P (2009) Structure of genetic diversity in the two major gene pools of common bean (Phaseolus vulgaris L., Fabaceae). Theor Appl Genet 118:979992 Layne DR, Bassi D (2008) The peach: botany, production and uses. CAB, Cambridge Lester DR, Sherman WB, Atwell BJ (1996) Endopolygalacturonase and the melting flesh (M) locus in peach. J Am Soc Hortic Sci 121:231235 Liu K, Muse SV (2005) PowerMarker: integrated analysis environment for genetic marker data. Bioinformatics 21:21282129 Maccaferri M, Sanguineti MC, Noli E, Tuberosa R (2005) Population structure and long-range linkage disequilibrium in a durum wheat elite collection. Mol Breed 15:271289 Mariette S, Tavaud M, Arunyawat U, Capdeville G, Millan M, Salin F (2010) Population structure and genetic bottleneck in sweet cherry estimated with SSRs and the gametophytic self-incompatibility locus. Genetics 11:77 Mather DE, Hyes PM, Chalmers KJ, Eglinton J, Matus I, Richardson K, Von Zitzewitz J, Marquez-Cedillo L, Hearnden P, Pal N (2004)

genetic association studies (Agrama et al. 2007). In theory, genetic association mapping is more powerful than linkage studies in identifying variants with weak effects that may contribute risks to common complex traits (Risch and Merikangas 1996). As an example, except for BPPCT023 linked with F gene, another marker, BPPCT028, which was not reported before showed a weaker effect associated with flesh adhesion. Our results have shown that LD studies are efficient means of dissecting complex agronomic characters. However, further studies are necessary to confirm the association results in specific biparental crossing populations and consequently avoid identifying spurious associations.

Acknowledgments We thank Maria Jose Aranzana Civit, IRTA (Spain) for useful suggestions during manuscript editing. We also acknowledge the financial support of the Fundamental Research Fund of the Central Institute of the Chinese Academy of Agricultural Sciences (0032011017) and Modern Agro-industry Technology Research System (nycytx-31-1-2).

References
Abbott AG, Rajapakse S, Sosinski B, Lu ZX, Sossey-Alaoui K, Gannavarapu M, Reighard G, Ballard RE, Baird WV, Scorza R, Callahan A (1997) Construction of saturated linkage maps of peach crosses segregating for characters controlling fruit quality, tree architecture and pest resistance. Acta Horticult 465:4149 Abdurakhmonov IY, Kohel RJ, Yu JZ, Pepper AE, Abdullaev AA, Kushanov FN, Salakhutdinov IB, Buriev ZT, Saha S, Scheffler BE, Jenkins JN, Abdukarim A (2008) Molecular diversity and association mapping of fiber quality traits in exotic G. hirsutum L. germplasm. Genomics 92:478487 Agrama HA, Eizenga GC, Yan W (2007) Association mapping of yield and its components in rice cultivars. Mol Breed 19:341356 Aranzana MJ, Abbassi EK, Howad W, Arus P (2010) Genetic variation, population structure and linkage disequilibrium in peach commercial varieties. Genetics 11:69 Bailey JS, French AP (1932) The inheritance of certain characters in the peach. Proc Am Soc Hortic Sci 29:127130 Barnaud A, Lacombe T, Doligez A (2006) Linkage disequilibrium in cultivated grapevine, Vitis vinifera L. Theor Appl Genet 112:708 716 Brummell DA (2006) Cell wall disassembly in ripening fruit. Funct Plant Biol 33(2):103119 Cao K, Wang LR, Zhu GR, Fang WC, Cheng CW, Zhao P (2011) Construction of a linkage map and identification of a resistance gene analog markers for root-knot nematode in wild peach, Prunus kansuensis. J Am Soc Hortic Sci 136:190197 Celia M, Gogorcena CY, Moreno MA (2010) Phenotypic diversity and relationships of fruit quality traits in peach and nectarine [Prunus persica (L.) Batsch] breeding progenies. Euphytica 171:211226 Cheng ZP, Huang HW (2009) SSR fingerprinting Chinese peach cultivars and landraces (Prunus persica) and analysis of their genetic relationships. Sci Hortic-Amsterdam 120:188193 Dirlewanger E, Moing A, Rothan C, Svanella L, Pronier V, Guye A, Plomion C, Monet R (1999) Mapping QTL controlling fruit quality in peach [Prunus persica (L.) Batsch]. Theor Appl Genet 98:1831

990 Use of SSR marker data to study linkage disequilibrium and population structure in Hordeum vulgare: prospects for association mapping in barley. In: International barley genetics symposium, Brno, Czech Republic, pp 302307 Morgutti S, Negrini N, Nocito FF, Ghiani A, Bassi D, Cocucci M (2006) Changes in endopolygalacturonase levels and characterization of a putative endo-PG gene during fruit softening in peach genotypes with nonmelting and melting flesh fruit phenotypes. New Phytol 171:315328 Murayama H, Arikawa M, Sasaki Y, Cin VD, Mitsuhashi W, Toyomasu T (2009) Effect of ethylene treatment on expression of polyuronidemodifying genes and solubilization of polyuronides during ripening in two peach cultivars having different softening characteristics. Postharvest Biol Tec 52(2):196201 Nei M, Tajima FA, Tateno Y (1983) Accuracy of estimated phylogenetic trees from molecular data. J Mol Evol 19:153170 Newell MA, Cook D, Tinker NA, Jannink JL (2011) Population structure and linkage disequilibrium in oat (Avena sativa L.): implications for genome-wide association studies. Theor Appl Genet 122:623632 Nordborg M, Borevitz JO, Bergelson J, Berry CC, Chory J, Hagenblad J, Kreitman M, Maloof JN, Noyes T, Oefner PJ, Stahl EA, Weigel D (2002) The extent of linkage disequilibrium in Arabidopsis thaliana. Nat Genet 30:190193 Ogundiwin EA, Peace CP, Gradziel TM, Parfitt DE, Bliss FA, Crisosto CH (2009) A fruit quality gene map of Prunus. BMC Genomics 10:587 Peace CP, Crisosto CH, Gradziel TM (2005) Endopolygalacturonase: a candidate gene for Freestone and Melting flesh in peach. Mol Breed 16:2131 Pritchard JK, Wen W (2004) Documentation for STRUCTURE software. The University of Chicago Press, Chicago Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155:945959 Pushpendra KG, Sachin R, Pawan LK (2005) Linkage disequilibrium and association studies in higher plants: present status and future prospects. Plant Mol Biol 57:461485 Remington DL, Thornsberry JM, Matsuoka Y, Wilson LM, Whitt SR, Doebley J, Kresovich S, Goodman MM, Buckler ES IV (2001) Structure of linkage disequilibrium and phenotypic associations in the maize genome. PNAS 98:1147911484 Risch N, Merikangas K (1996) The future of genetic studies of complex human diseases. Science 273:15161517 Rodriguez AJ, Sherman WB, Scorza R, Wisniewski M, Okie WR (1994) Evergreen peach, its inheritance and dormant behavior. J Am Soc Hortic Sci 119:789792 Stephens M, Smith NJ, Donnelly P (2001) A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 68:978989

Tree Genetics & Genomes (2012) 8:975990 Tenaillon MI, Sawkins MC, Long AD, Gaut RL, Doebley JF, Gaut BS (2001) Patterns of DNA sequence polymorphism along chromosome 1 of maize (Zea mays ssp. mays L.). PNAS 98:91619166 Thornsberry JM, Goodman MM, Doebley J, Kresovich S, Nielsen D, Buckler ESIV (2001) Dwarf8 polymorphisms associate with variation in flowering time. Nat Genet 28:286289 Tommasini L, Schnurbusch T, Fossati D, Mascher F, Keller B (2007) Association mapping of Stagonospora nodorum blotch resistance in modern European winter wheat varieties. Theor Appl Genet 115:697708 Vavilov NI (1951) The origin variation immunity and breeding of cultivated plants. Chron Bot Ronald, New York (translation by K Star Chester) Verde I, Quarta R, Cerdrola C, Dettori MT (2002) QTL analysis of agronomic traits in a BC1 peach population. Acta Horticult 592:291297 Wang ZH, Zhuang EJ (2001) Fruit monograph for peach in China. China Forest Press, Beijing Wang Y, Georgi LL, Reighard GL, Scorza R, Abbott AG (2002) Genetic mapping of the evergrowing gene in peach [Prunus persica (L.) Batsch]. J Hered 93:352358 Wang KL, Bolitho K, Grafton K, Kortstee A, Karunairetnam S, McGhie TK, Espley RV, Hellens RP, Allan AC (2010) An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae. BMC Plant Biol 10:50 Washington State University (2010) Marker search. Department of Horticulture and Landscape Architecture. Pullman. Washington. US. 10 June 2010. <http://www.rosaceae.org/bio/content/?title0&url0/cgibin/gdr/gdr_marker_search.cgi&style0width:950px;height:1024px> Xie RJ, Li XW, Chai ML, Song LJ, Jia HJ, Wu DJ, Chen MJ, Chen KM, Aranzana MJ, Gao ZS (2010) Evaluation of the genetic diversity of Asian peach accessions using a selected set of SSR markers. Sci Hortic-Amsterdam 125:622629 Yamamoto T, Shimada T, Imai T, Yaegaki H, Haji T, Matsuta N, Yamaguchi M, Hayashi T (2001) Characterization of morphological traits based on a genetic linkage map in peach. Breed Sci 51:271278 Yeh FC, Yang RC, Boyle T (1999) POPGENE Microsoft windowsbased 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 Yu J, Buckler ES (2006) Genetic association mapping and genome organization of maize. Curr Opin Biotech 17:155160 Yu JM, Pressoir G, Briggs WH, Bi IV, Yamasaki M, Doebley JF, McMullen MD, Gaut BS, Nielsen DM, Holland JB, Kresovich S, Buckler ES (2006) A unified mixed-model method for association mapping that accounts for multiple levels of relatedness. Nat Genet 38:203208