Evaluation of Genetic Diversity in Chinese Wild Apple ... - Springer Link

Plant Mol Biol Rep (2012) 30:539–546 DOI 10.1007/s11105-011-0366-6

Evaluation of Genetic Diversity in Chinese Wild Apple Species Along with Apple Cultivars Using SSR Markers Qiong Zhang & Jing Li & Yongbo Zhao & Schuyler S. Korban & Yuepeng Han

Published online: 27 September 2011 # Springer-Verlag 2011

Abstract China, one of the primary centers of genetic diversity for the genus Malus, is very rich in wild apple germplasm. In this study, genetic diversity in 29 Malus accessions, including 12 accessions from 7 Chinese Malus species, 4 Chinese landraces, and 13 introduced apple cultivars, was assessed using a set of 19 single-locus simple sequence repeat (SSR) markers distributed across all 17 linkage groups of the apple genome. The number of alleles detected at each locus ranged from 2 to 11, with an average of 5.3 per SSR marker. In some accessions, 16 unique alleles were identified. Ten out of these 16 unique alleles (62.5%) were detected exclusively in wild species, indicating that these Chinese wild apple species have considerable genetic diversity and can be used in breeding programs to increase the genetic diversity of apple cultivars. Using 19 SSRs, an unweighted pair-group method with arithmetic average cluster analysis was conducted, and the Q. Zhang : J. Li : Y. Han (*) Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, People’s Republic of China e-mail: [email protected] Q. Zhang Graduate School, Chinese Academy of Sciences, Beijing 100039, People’s Republic of China Y. Zhao Changli Institute for Pomology, Hebei Academy of Agricultural and Forestry Sciences, Changli, Hebei 066600, People’s Republic of China S. S. Korban Department of Natural Resources and Environmental Sciences, University of Illinois, 1201 W. Gregory, Urbana, IL 61801, USA

resulting dendrogram revealed that all cultivars, except for EфpeMeBckoe, were clustered together in the same group. The Russian cultivar EфpeMeBckoe was closely related to the Chinese crabapple Baihaitang (M. prunifolia), with a high similarity coefficient value of 0.94. Of the two M. sieversii accessions used, one accession showed a close relationship to apple cultivars, while the other accession was closely related to wild apple species, suggesting the presence of a wider genetic diversity in Chinese M. sieversii species. The influence of SSR marker selection on genetic diversity analysis in this Malus collection was also discussed. Keywords Malus . Genetic diversity . SSR markers . Cluster analysis

Introduction Apple (Malus×domestica Borkh.) is one of the most economically important cultivated fruit crops that have been subjected to heavy selection and breeding. Except for a few selected apple breeding programs, such as the Purdue–Rutgers–Illinois program in the USA and some European programs wherein a few selected Malus species were used in breeding apple for disease resistance (Korban and Tartarini 2009), most of the parents used in breeding efforts have relied on a narrow genetic base, often involving crosses among popular commercial cultivars (Kumar et al. 2010). For example, several cultivars such as “Red Delicious,” “Golden Delicious,” and “Jonathan” have been frequently used in the parentages of large numbers of modern cultivars (Noiton and Alspach 1996). Moreover, the selection and release of mutants of popular cultivars have also accelerated the trend toward genetic uniformity in commercial apple cultivars (Brooks and Olmo

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1994). Greater genetic diversity in apple breeding is highly desirable to develop new cultivars with disease resistance, good fruit quality, and high fruit productivity. Malus wild species are widely distributed throughout North America, Europe, Asia Minor, and Asia and serve as potential genetic resources for the development of new apple cultivars and/or rootstocks adapted to diverse environmental conditions (Hokanson et al. 2001). Knowledge of genetic diversity and of genetic relationships among breeding materials has a great impact on crop improvement as availability of a wide range of genetic diversity among parents is essential for breeding programs (Ganesh and Thangavelu 1995). China is one of the primary centers of genetic diversity of the genus Malus, and the west region of China such as Xinjiang Province is very rich in wild apple species. Evaluation of genetic diversity in Chinese wild Malus species is undoubtedly beneficial to apple breeding programs worldwide. Genetic diversity in crop species can be determined using morphological and agronomic characteristics, as well as biochemical and DNA marker analysis (Liu 1997). However, morphological and agronomic characteristics are greatly influenced by growth and environmental conditions. To overcome this limitation, DNA markers such as amplified fragment length polymorphisms and simple sequence repeats (SSRs) have been widely used in the analysis of genetic diversity. Among DNA markers, SSRs are highly polymorphic, multiallelic, co-dominant, reproducible, PCR-based, abundantly distributed in the genome, and easy to interpret (Crouch et al. 1999; Morgante et al. 2002; Tanya et al. 2011). Therefore, SSRs serve as valuable tools for genetic analysis. Recently, approximately 400 SSR markers have been developed and genetically mapped in apple and in other fruit crops (Guilford et al. 1997; Liebhard et al. 2002; Silfverberg-Dilworth et al. 2006; Han and Korban 2008; Celton et al. 2009; He et al. 2011). These SSR markers have been successfully used to assess genetic diversity and relationships among different apple germplasm (Laurens et al. 2004; Galli et al. 2005; Pereira-Lorenzo et al. 2008; Gasi et al. 2010; Han et al. 2011). To date, several studies have been reported on the genetic diversity of Chinese wild apple species. For example, Zhang et al. (2007a) investigated genetic diversity and relationships of 24 crabapple and 23 wild species of Malus in China. Gao et al. (2007) used 12 pairs of SSR markers to analyze genetic diversity of 59 Malus accessions in China, including 24 wild apple species. Chen et al. (2007) evaluated genetic relationships among Xinjiang wild apple (M. sieversii (Lebed.) (Roem.) using both qualitative and quantitative parameters of evaluation of volatile components. However, additional genetic information on indigenous apple germplasm available in China

Plant Mol Biol Rep (2012) 30:539–546

is still needed; moreover, a few studies have been reported on genetic relationships between Chinese wild apple species and modern apple cultivars. Recently, we have collected several wild apple species from northern regions of China. Therefore, the primary goal of this study is to investigate the genetic diversity among these wild apple species and assess their genetic relationships to the Xinjiang wild apple, M. sieversii. Moreover, the influence of SSR marker selection on the analysis of genetic diversity has also been evaluated.

Materials and Methods Plant Material A total of 29 accessions (Table 1), including 12 Chinese wild species and 17 popular commercial cultivars, maintained at the Changli Institute of Pomology (Hebei Province, People’s Republic of China), were analyzed for their genetic diversity. The wild apple accessions consisted of two M. Robusta, four M. prunifolia, three M. asiatica, two M. sieversii, and a single M. baccata species. In addition, 17 commercial cultivars introduced from North America, Japan, Britain, and Russia or native to China were also included in this study. Young and healthy leaves were collected from trees and stored at −70°C. DNA Extraction and SSR Analysis Total genomic DNA was extracted from 100-mg leaf tissue according to a modified CTAB DNA extraction protocol (Doyle 1991). The SSR markers selected were distributed across different linkage groups of the apple genome (Table 2). The PCR reaction was performed in a total volume of 10 μl containing approximately 30-ng genomic DNA, 1×PCR buffer (10-mM Tris-HCl, pH 8.3, 500-mM KCl), 1.5-mM MgCl2, 0.2 μM of each primer, 0.12 mM of each dNTP, and 0.5 U of Taq DNA polymerase. PCR amplification was carried out in an Applied Biosystems 9700 (Perkin-Elmer Applied Biosystems, Foster City, CA) using the following cycling protocol: 2 min and 30 s at 95°C (initial denaturation step), followed by 35 cycles consisting of 94°C for 45 s, 55°C for 45 s, 72°C for 1 min, and with a final extension of 72°C for 8 min. The PCR products were mixed with 5-μl loading buffer (98% formamide, 0.05% bromophenol blue, 0.05% xylene cyanol, and 10-mM NaOH), denatured at 95°C for 5 min and then separated on a 6% denaturing polyacrylamide gel for 1 to 1.5 h. Gels were stained with silver nitrate, and fragment sizes were determined by comparison to a 25 bp DNA ladder (Promega, Madison, WI) (Fig. 1).


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Table 1 The 29 Malus accessions used for the assessment of genetic diversity Cultivars

Wild species

No.

Accession

Origin

No.

Accession

Origin

1

Golden Delicious

USA

18

Bingzi (Malus asiatica var.rinki (koidz.) Asami)

Northern China

2 3

Jonathan Red Delicious

USA USA

19 20

Shaguo (Malus asiatica Nakai) Shandingzi (Malus baccata Borkh)

Northern China Northern China

4

Starkrimson

USA

21

Naizi (Malus asiatica Nakai)

Northern China

5

Ralls Janet

USA

22

Regunzihaitang (Malus prunifolia (Willd.) Borkh.)

Northern China

6

Ben Davis

USA

23

Xiaomianhaitang (Malus prunifolia (Willd.) Borkh.)

Northern China

7

Geneva Early

USA

24

Pingding Crab (Malus Robusta Rehd.)

Northern China

8 9

Malus Sparkler Fuji

USA Japan

25 26

Dayehaitang (Malus prunifolia (Willd.) Borkh.) Baihaitang (Malus prunifolia (Willd.) Borkh.)

Northern China Northern China

10

Crispin

Japan

27

Balenghaitang (Malus Robusta Rehd.)

Northern China

11 12

Orin Maypole

Japan Britain

28 29

Xinjiangyepingguo 12 (Malus sieversii (Lebed.) Roem.) Xinjiangyepingguo 23 (Malus sieversii (Lebed.) Roem.)

Western China Western China

13 14 15

EфpeMeBckoe Xiangyanghong Tianhuangkui

Russia China China

16 17

Kuihua Baifugao

China China

Data Analysis SSR polymorphic bands were scored as either present (1) or absent (0). Alleles were alphabetically coded (e.g., A, B, and C for each band) in decreasing size order. Accessions Table 2 SSR markers assayed in the characterization of apple accessions

a, b, and c indicate that these SSR markers were developed by Celton et al. (2009), Han and Korban (2008), and SilfverbergDilworth et al. (2006), respectively LG individual linkage group, Na number of alleles, Ne effective number of alleles, I Shannon’s information index, He expected heterozygosity, Ho observed heterozygosity, PIC polymorphism information content, EST-SSR expressed sequence tag–simple sequence repeat, G-SSR genomic simple sequence repeat

with single bands were assumed to be homozygous. The number of alleles per locus (Na), number of effective alleles per locus (Ne), Shannon index (I), observed heterozygosity (Ho), and expected heterozygosity (He) were calculated using the program POPGENE 32 (Yeh and Boyle 1997).

SSR locus

SSR type

LG

Na

Ne

I

Ho

He

PIC

NZmsEB119405a

EST-SSR EST-SSR EST-SSR EST-SSR G-SSR G-SSR G-SSR G-SSR G-SSR G-SSR G-SSR

2 14 7 6 16 11 15 3 8 12 16

5 5 3 8 6 8 6 9 3 2 3

2.403 3.311 1.898 5.066 2.604 5.588 4.043 5.160 2.470 1.399 1.736

1.152 1.317 0.756 1.809 1.296 1.857 1.565 1.824 0.974 0.460 0.671

0.586 0.828 0.483 0.793 0.587 0.798 0.931 0.517 0.414 0.345 0.552

0.594 0.710 0.482 0.817 0.655 0.862 0.766 0.820 0.606 0.290 0.431

0.544 0.644 0.389 0.778 0.627 0.836 0.719 0.780 0.510 0.245 0.347

G-SSR G-SSR G-SSR EST-SSR EST-SSR EST-SSR EST-SSR EST-SSR

10 4 15 9 17 5 1 13 5.263

3 2 6 7 4 6 3 11 3.167

1.474 1.708 2.661 5.340 2.998 2.982 1.456 5.881 1.217

0.597 0.605 1.281 1.781 1.203 1.408 0.544 2.021 0.561

0.379 0.586 0.724 0.483 0.962 0.724 0.379 0.621 0.622

0.327 0.422 0.635 0.827 0.680 0.676 0.319 0.845 0.616

0.293 0.329 0.589 0.787 0.608 0.635 0.274 0.810 5.263

NZms EB146613a NZms EE663789a NZms MDAJ1681a BACSSR10b BACSSR20b BACSSR51b BACSSR58b Hi20b03c Hi02b07c Hi08d09c Hi08g06c Hi08h03c Hi11a01c CN868471 CN876284 CN893277 CTG1066091 CTG1073738 Mean

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Fig. 1 Segregation of alleles of an SSR marker observed on 6% polyacrylamide gel in cultivars worldwide and the Chinese wild apple species. Lanes 1 to 29 correspond to accessions listed in Table 1

The polymorphism information content (PIC) was estimated using Power Stats V12.xls software (Brenner and Morris 1990). A dendrogram was constructed by NTSYS-pc 2.11 program (Exeter Software, Stauket, NY) using the unweighted pair-group method of arithmetic average (UPGMA) cluster analysis. Principal coordinate analysis (PCoA) and analysis of molecular variance (AMOVA) were also conducted.

Results SSR Polymorphism A total of 19 single-locus SSRs distributed across all 17 apple linkage groups were selected to assess genetic diversity in 29 apple accessions, including 17 cultivars and 12 wild accessions. Of the 19 SSRs, nine with prefixes of either “CTG/CN” or “BAC” were recently developed based on either expression sequence tags (ESTs) or BAC end sequences, respectively. As a result, 100 alleles were detected at these 19 SSR loci, with an average of 5.3 alleles per locus (Table 2). The number of alleles detected for each locus ranged from 2 for both Hi02b07 and Hi08h03 to 11 for CTG1073738. The effective number of alleles per locus ranged from 1.4 for Hi02b07 to 5.9 for CTG1073738, with an average of 3.2 alleles per locus. The Shannon index ranged from 0.460 to 2.021, with an average of 1.217. The expected heterozygosity ranged from 0.290 for Hi02b07 to 0.845 for CTG1073738, with an average value of 0.616. The observed heterozygosity varied between 0.345 for Hi02b07 and 0.962 for CN876284, with an average value of 0.622. To measure the informativeness of these markers, the polymorphism information content (PIC) for each SSR locus was calculated. The PIC value varied from 0.245 to 0.836, with an average of 0.561. Based on the PIC value, the most informative locus was BACSSR20, which had the highest value of 0.836 (Table 2). Allelic frequencies showed wide variations, ranging from 0.017 to 0.828. The most frequent alleles were detected at three SSR loci, i.e., Hi02b07, Hi08g06, and CTG1066091, with high frequencies of more than 0.8. Out of all 100 alleles detected, 14 exhibited frequencies between 0.3 and 0.8, and 22, including 16 unique alleles, showed low frequencies of

less than 0.05. The AMOVA results demonstrated that 83% of the total diversity was retained within either cultivars or wild species groups, while 17% of the variance was attributed to differences among groups of accessions. Genetic Relationships Based on SSR Data The pair-wise genetic similarity coefficients between accessions ranged from 0.207 (between M. baccata and “Jonathan”) to 0.946 (between “Baihaitang” and “EфpeMeBckoe”). Twenty-nine accessions were clustered into two groups based on UPGMA analysis using the similarity matrix (Fig. 2). Group 1 consisted of 12 introduced cultivars from USA, Japan, and Britain; four Chinese local cultivars; and a single Chinese wild species, M. sieversii Roemer 12. Group 2 was composed of a single introduced cultivar, “EфpeMeBckoe,” from Russia and 11 wild apple accessions from China. The cultivar EфpeMeBckoe showed the closest relationship with the Chinese wild species “Baihaitang” (M. prunifolia). Three most polymorphic loci, including BACSSR20, CTG1073738, and CN868471, were also selected to estimate genetic relationships among all 29 apple accessions. The UPGMA dendrogram generated from the three SSR markers resulted in distinct separation of all 29 accessions (Fig. 3). However, this clustering was not completely consistent with the resulting dendrogram based on all 19 SSR markers. All apple accessions were divided into two groups. Group I contained two subgroups, namely, A and B. Subgroup A consisted of nine wild accessions and two introduced cultivars Golden Delicious and Ralls Janet, along with a single local cultivar Tianhuangkui. Subgroup B consisted of 12 cultivars and the Chinese wild apple “Baihaitang” (M. prunifolia), which had a very close relationship with the Russian cultivar EфpeMeBckoe. Group II consisted of only four accessions, including a single introduced cultivar Maypole from Britain, a single Chinese local cultivar Baifugao, and two Chinese wild species M. sieversii Roemer 23 and Naizi (M. asiatica). Principal coordinate analysis was also performed to visualize genetic relationships among these 29 apple accessions. Similar to UPGMA cluster analysis, PCoA also indicated that these apple accessions could be roughly divided into two groups. One consisted of most of the cultivars, while another contained most of the wild species (Fig. 4). However, three accessions, including


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Fig. 2 An UPGMA dendrogram showing genetic relationships among 29 genus Malus accessions (listed in Table 1) generated from 19 SSR markers

“EфpeMeBckoe,” “Baihaitang,” and the crabapple Malus Sparkler, were closely related to each other, but separated from all other cultivars or wild species. These three accessions could not be unambiguously assigned to either cultivar or wild species groups.

Discussion Polymorphic Information Content of Different Types of SSR Markers The apple SSR markers used in this study consist of both genomic SSR (G-SSR) and expressed sequence tag-derived SSR (EST-SSR). A total of nine EST-SSRs and ten G-SSRs have been used to estimate the genetic diversity in a collection of Malus species and apple, as well as crabapple cultivars. Generally, EST-SSRs display lower levels of polymorphism than genomic SSRs as expressed sequences are more likely to be more conserved than non-coding regions (Eujayl et al. 2002; Wen et al. 2010). However, in Fig. 3 An UPGMA dendrogram of 29 genus Malus accessions (listed in Table 1) generated from the three most polymorphic SSR markers

this study, the levels of polymorphisms of EST-SSRs are higher than that observed for G-SSRs. For example, the average number of alleles per EST-SSR locus (5.78) is higher than that of alleles per G-SSR locus (4.80). The EST-SSR NZms MDAJ1681 has shown a high level of polymorphism, resulting in a high PIC value of 0.778. Thus, the findings in this study have clearly demonstrated that highly polymorphic SSR markers could be derived from expressed sequences. However, both genomic and genic microsatellite markers can provide an accurate indication of genetic diversity in apple and in other plant species (Martin et al. 2010). Microsatellite markers can also be divided into singlelocus SSRs and multiple-locus SSRs based on the number of amplified loci. The apple genome has a polyploid origin, and thus, many multiple-locus SSRs have been developed (http://users.unimi.it/hidras/). In this study, 19 single-locus SSRs have been used to assess the genetic diversity and relationships among apple cultivars and wild Malus species. The results have indicated that the average number of alleles per SSR locus is 5.263, which is significantly lower

544


Fig. 4 Principal coordinate analysis of 29 Malus accessions (listed in Table 1) based on the genetic similarity matrix derived from 19 polymorphic SSR markers

than that reported in previous studies. For example, Gasi et al. (2010) have selected ten genomic SSRs (one mutiple locus and nine single locus) to assess genetic diversity in 39 apple accessions, including 24 traditional cultivars from Bosnia and Herzegovina and 15 modern popular cultivars, and they have reported that the average number of alleles per SSR marker is 10.4. Gao et al. (2007) have investigated 59 Malus accessions using 12 genomic SSRs (six multiple locus and six single locus), and an average of 14.7 alleles per SSR marker has been detected. In this study, all SSRs used are single locus, while both single-locus and multiple-locus SSRs have been used to assess genetic diversity in the aforementioned previous studies. The average value of expected heterozygosity in this study is 0.616, which is very similar to that reported (0.6156) by Gao et al. (2007). Therefore, the higher average number of alleles per SSR marker reported in previous studies may be primarily attributed to the selection of multiple-locus SSRs. However, genotypes may also influence the number of alleles detected at each SSR locus. Overall, single-locus SSRs provide more reliable scoring of genotypes compared to multiple-locus SSRs. Thus, for genetic diversity analysis, single-locus SSRs offer more advantages than multiple-locus SSRs. Genetic Diversity and Relationships Among Apple Cultivars and Chinese Wild Malus Species In this study, a total of 16 unique alleles were identified in 29 apple accessions using 19 SSR markers. Of these 16 unique alleles, ten (62.5%) were exclusively present in Chinese wild apple species. Moreover, the UPGMA dendrogram indicated that the Chinese wild apple species

were separated from cultivars. These results clearly suggested that the Chinese wild apple species had wider genetic diversity and would serve as valuable resources for apple breeding efforts. The genetic diversity of apple cultivars would certainly be enhanced by including Chinese wild Malus species in apple breeding programs (Hokanson et al. 2001). Six Chinese crabapples were used in this study, including two M. Robusta accessions “Banenghaitang” and “Pingding Crab” and four M. prunifolia accessions, namely, “Dayehaitang,” “Regunzihaitang,” “Baihaitang,” and “Xiaomianhaitang.” Based on the UPGMA dendrogram developed using all 19 SSR markers, these six Chinese crabapples clustered together and the two M. Robusta accessions showed a very close relationship (Fig. 2). These findings were consistent with previous taxonomic classification of Chinese crabapples, supporting these results of genetic diversity analysis. These Chinese crabapples not only have tolerance to adverse conditions such as drought and salt, but they also have resistance to diseases such as Botryosphaeria (bot) rot caused by Botryosphaeria dothidea (Liu et al. 2011). Moreover, the UPGMA cluster analysis showed separation of Chinese crabapples from apple cultivars (Fig. 2). Therefore, these Chinese crabapples would also serve as valuable resources for breeding for disease resistance in apple. In this study, the UPGMA dendrogram has revealed that the introduced cultivar “EфpeMeBckoe” from Russia is closely related to the Chinese crabapple “Baihaitang,” with a high similarity coefficient value of 0.94. The wild apple Baihaitang is widely distributed in the Hebei Province and in China’s western region. As China borders Russia on the north end, it is reasonable to speculate that the Chinese wild


species and/or interspecific hybrids in western China have spread to Russia, and after undergoing periods of domestication, they may have contributed to the development of local cultivars, such as EфpeMeBckoe. It is well known that M. sieversii is a wild progenitor species of the domesticated apple (Juniper et al. 1996). In this study, two Chinese M. sieversii accessions “Xinjiangyepingguo 12” and “Xinjiangyepingguo 23” have been used. “Xinjiangyepingguo 12” has shown close relationships with local and imported apple cultivars (Fig. 2). This finding supports the hypothesis that Chinese M. sieversii species must have spread to Europe via the old Silk Road trade route, which connected western China to the outside world, and it has played an important role in the evolution of the cultivated apple (Harris et al. 2002). M. sieversii has been subjected to domestication in Europe, leading to the evolution of the domesticated apple (Juniper et al. 1996). Interestingly, “Xinjiangyepingguo 12” is separated from “Xinjiangyepingguo 23,” which is closely related to wild Malus species (Fig. 2). This observed genetic divergence between the two M. sieversii accessions is consistent with results reported in previous studies. For example, Zhou (1999) has reported that there are more than 40 different types of wild species within the M. sieversii population found in the Xingjiang Autonomous Region of China (Zhou 1999). Earlier, Zhang et al. (2007b) has performed UPGMA cluster analysis based on eight SSR markers and has reported that there is a wide genetic diversity in the Chinese M. sieversii population. Influence of SSR Marker Selection on Assessment of Genetic Diversity To date, there are many reports on the assessment of genetic diversity in apple germplasm using SSR markers (Gasi et al. 2010; Guarino et al. 2006; Pereira-Lorenzo et al. 2008; Ramos-Cabrer et al. 2007). The haploid chromosome number of Malus species is 17. It has been suggested that DNA markers used to assess plant genetic diversity should be randomly distributed across chromosomes (Aranzana et al. 2003; Garkava-Gustavsson et al. 2008). However, the number of SSR markers used in all previous studies of apple genetic diversity has been less than 17. In this study, we have assessed the influence of SSR marker selection on the assessment of genetic diversity. First, we have selected three highly polymorphic SSRs to assess the genetic diversity in 29 Malus accessions. The UPGMA dendrogram obtained from these three SSRs has indicated that five Chinese crabapples are not clustered together, while a single Chinese crabapple, “Baihaitang,” is closely related to the introduced apple cultivars (Fig. 3). In contrast, the UPGMA cluster analysis generated using all 19 SSR markers has demonstrated that all six Chinese crabapples

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are clustered together. Second, the apple genome is derived from a polyploid origin, and the pairs of homoeologous chromosomes are as follows: 1–7, 1–10, 2–15, 3–11, 4–12, 5–10, 6–14, 8–15, 9–17, and 13–16 (Velasco et al. 2010). We have assigned these 19 SSR markers used in this study into two different sets. The first set consist of nine SSR markers along chromosomes 1, 2, 3, 4, 5, 6, 8, 9, and 13, while the second set consist of ten SSR markers along chromosomes 7, 10, 11, 12, 14, 15, 16, and 17, respectively. The UPGMA dendrogram generated based on the first set of SSR markers is different from the dendrograms generated from either the second set of SSR markers or all 19 SSR markers (data not shown). Therefore, it is clear that the selection of SSR markers has a significant influence on the assessment of genetic diversity in target germplasm. Acknowledgements This project was supported by the National Science Foundation of China under Grant No. 30971987 and 100 Talents program of the Chinese Academy of Sciences. We would like to thank Fenjie Su and Xingzhong Zhang for the assistance in revising the manuscript.

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