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A Practical Method for Peach-Related Species Identification and Hybrid

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A Practical Method for Peach-Related Species Identification and Hybrid J. AMER.SOC.HORT.SCI. 142(3):155–162. 2017. doi: 10.21273/JASHS03930-16 A Practical Method for Peach-related Species Identification and Hybrid Analysis Using Simple Sequence Repeat Markers Zuguo Cai Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, Zhengzhou 450009, People’s Republic of China; and College of Horticulture and Landscape Architecture, Henan Institute of Science and Technology, Xinxiang 453003, People’s Republic of China Wenfang Zeng, Liang Niu, Zhenhua Lu, Guochao Cui, Yunqin Zhu, Lei Pan, Yifeng Ding, and Zhiqiang Wang1 Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, Zhengzhou 450009, People’s Republic of China ADDITIONAL INDEX WORDS. microsatellite marker, DNA fingerprinting, SSR, Prunus persica, National Germplasm Repository of Peach in China, NGRPC ABSTRACT. Cultivated peach (Prunus persica) is an important fruit species worldwide. The wild relatives in Prunus,suchas P. mira, P. davidiana, P. kansuensis, P. ferganensis,andP. persica, are valuable for peach breeding, and early and accurate identification of parental and hybrid genotypes is critical. In this study, 20 representative accessions of peach germplasm from the National Germplasm Repository of Peach in China were used to select a set of 18 simple sequence repeat (SSR) markers for accurate species discrimination. Eight unknown peach samples were successfully identified using the SSR panel and species genotype database. Interspecific hybrid genotypes of P. persica · P. davidiana, P. persica · P. kansuensis, and P. persica · P. ferganensis were also analyzed reliably. The markers were amenable to high-throughput fluorescent labeling and capillary electrophoresis (CE) analysis, allowing rapid and efficient species identification. The practical method described in this study will facilitate peach breeding and germplasm management. Peach is one of the most important fruit species worldwide. nucleotide polymorphism (SNP) tools have been used for large- China, where peach originated, has the richest resource of species scale genetic analysis in peach cultivars and accessions (Cao related to cultivated peach. These species contributed to peach et al., 2014; Verde et al., 2012). domestication history over more than 4000 years, with some SSR (microsatellites) are tandem repeat DNA sequences with cultivars disseminating to Central Asia, the Mediterranean coast, a core unit of 1–6 bps, which are abundant in prokaryotic and Europe, America, and Japan (Layne and Bassi, 2008; Wang and eukaryotic genomes and are ubiquitously distributed in both the Zhuang, 2001). The main relatives of cultivated peach, namely P. protein-coding and noncoding regions (Guichoux et al., 2011; mira, P. davidiana, P. kansuensis,andP. ferganensis, are found Kalia et al., 2011). The high variability of microsatellites usually readily in southwestern and northwestern China. Fruit from manifests as different numbers of repeats in the region of the almost all of the related wild species exhibit poor eating quality, repeated motif, and short insertion/deletion events are also seen but these species could be valuable as sources of pest and disease (Decroocq et al., 2003). SSR markers are ideal for profiling as resistance traits or as rootstock (Cao et al., 2011; Moing et al., they are codominant and can display a large number of alleles per 2003). Increasing numbers of related species are being used to locus. In addition, assaying SSR markers is relatively simple and develop new peach cultivars. Efficient and accurate species reproducible and is accomplished using polymerase chain re- identification and hybrid analysis are necessary in peach breeding action (PCR)-based methods that are amenable to automation and germplasm management. (Kalia et al., 2011). SSR markers proved to be highly efficient for A range of practices were used previously to identify peach- genetic analysis in several fruit species, such as grape [Vitis related germplasm, including morphology (Wang and Zhuang, vinifera (Emanuelli et al., 2013)], jujube [Ziziphus jujuba (Ma 2001), cytology (Guo et al., 1996), palynology (Wang and Zhou, et al., 2012)], almond [Prunus dulcis (Dangl et al., 2009)], and 1990), isozymes (Mowrey et al., 1990), and DNA markers sweet cherry [Prunus avium (Lacis et al., 2009)]. SSR markers (Cheng et al., 2001; Yu et al., 2004). With the development of were developed previously in peach and used for genetic diversity sequencing technology, peach genome sequences have become assessment (Li et al., 2008), cultivar identification (Chen et al., available (Verde et al., 2013), and high-throughput single- 2011; Li et al., 2013), trait mapping (Lambert and Pascal, 2011; Liu et al., 2009), and phylogenetic studies (Cheng and Huang, Received for publication 23 Sept. 2016. Accepted for publication 2 Feb. 2017. 2009). However, to date, reliable and efficient SSRs for discrim- The research was financially supported by the Agricultural Science and ination of peach-related species have not been developed, and no Technology Innovation Program (ASTIP) (CAAS-ASTIP-2017-ZFRI), the database of species genotypes has been established. A universal National Key Technology Support Program of China (2013BAD02B03-2; database of SSR marker profiles from peach-related species 2014BAD16B04). We thank Gengrui Zhu, Mingliang Yu, and Quan Jiang providing peach-related would facilitate peach breeding and germplasm management. species samples. The NGRPC, located in Zhengzhou (central China), Nanjing 1Corresponding author. E-mail: [email protected]. (eastern China), and Beijing (northern China), is the most J. AMER.SOC.HORT.SCI. 142(3):155–162. 2017. 155 important germplasm repository of peach, nectarine, and related Table 1. Species, sources, and characteristics of the 29 peach-related species in China. The NGRPC preserves original and represen- samples in Prunus. tative accessions of species related to peach and is the core Codez Prunus species Sourcey Characteristic collection of such species for China and the world. Here, we 1 P. davidiana NGRPC, Zhengzhou Red flower describe a practical SSR-based method for discriminating 2 P. davidiana NGRPC, Zhengzhou White flower species related to peach and for identifying interspecific 3 P. davidiana NGRPC, Nanjing Red flower hybrids. The aims of this study were to 1) screen SSRs and 4 P. davidiana NGRPC, Beijing White flower develop a set of markers for efficient identification of peach- 5 P. kansuensis NGRPC, Zhengzhou Red root related species, 2) establish a universal genotype database for 6 P. kansuensis NGRPC, Zhengzhou White root peach-related species using fluorescent-labeled SSR markers, 7 P. kansuensis NGRPC, Nanjing and 3) demonstrate the practical utility of the SSR set for 8 P. kansuensis NGRPC, Beijing Red root unknown genotype identification and interspecific hybrid 9 P. ferganensis NGRPC, Zhengzhou Kashi no.1 analysis. 10 P. ferganensis NGRPC, Zhengzhou Kashi no.2 11 P. ferganensis NGRPC, Nanjing Datianren Materials and Methods 12 P. ferganensis NGRPC, Beijing 13 P. mira NGRPC, Zhengzhou Linzhi, Tibet, China PLANT MATERIAL. Twenty-nine Prunus samples were used in 14 P. mira NGRPC, Zhengzhou Aba, Sichuan, China the study (Table 1). Among them, 20 samples (codes 1–20) 15 P. mira NGRPC, Nanjing were obtained from three NGRPCs (Zhengzhou, Nanjing, and 16 P. mira NGRPC, Beijing Beijing). These 20 accessions were representative samples of 17 P. persica NGRPC, Beijing Hubei, China five species (P. persica, P. davidiana, P. kansuensis, P. 18 P. persica NGRPC, Beijing Guizhou, China ferganensis, and P. mira) that were established on the core 19 P. persica NGRPC, Beijing Sichuan, China collection during initial investigations in China in the last 20 P. persica NGRPC, Beijing Hebei, China century. Some of these representative accessions were de- 21 unknown ZFRI, CAAS Chance seedling scribed using classical morphological characteristics, regional 22 unknown ZFRI, CAAS Chance seedling origin, and landrace names. Eight samples (codes 21–28) were 23 unknown ZFRI, CAAS Chance seedling unknown accessions awaiting species identification, and one 24 unknown ZFRI, CAAS Chance seedling sample (code 29) was a cultivar that was used as a maternal 25 unknown ZFRI, CAAS Chance seedling parent for hybridization. Three interspecific hybridizations 26 unknown ZFRI, CAAS Chance seedling were performed by the Zhengzhou Fruit Research Institute, 27 unknown ZFRI, CAAS Chance seedling Chinese Academy of Agricultural Sciences (ZFRI, CAAS), as 28 unknown ZFRI, CAAS Chance seedling follows: P. persica (code 29) · P. davidiana (code 1), P. 29 P. persica ZFRI, CAAS Cultivar (Dajiubao) persica (code 29) · P. kansuensis (code 5), and P. persica (code z · Twenty-nine samples including 20 representative accessions of five 29) P. ferganensis (code 10). Three F1 hybrid populations species (codes 1–20) in Prunus, eight unknown samples awaiting were obtained and used alongside their parents for hybrid species identification (codes 21–28), and one cultivar (code 29). analysis. yNational Germplasm Repository of Peach in China (NGRPC), DNA EXTRACTION. Fresh young leaves were collected and Zhengzhou Fruit Research Institute (ZFRI), Chinese Academy of rapidly dried at room temperature using solid silica gel pellet Agricultural Sciences CAAS). desiccant. Genomic DNA was isolated using the modified cetyltrimethylammonium bromide protocol described by Cheng et al. (1997). DNA was quantified using a spectropho- sample combination at least twice to identify an appropriate tometer (BioPhotometer plus;
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