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Genetic Diversity of Vitis Davidii Accessions Revealed Using CONSERVATION AND RESTORATION HORTSCIENCE 53(3):283–287. 2018. https://doi.org/10.21273/HORTSCI12506-17 and Luo, 2006). Polymorphic SRAPs were abundant and demonstrated genetic diversity Vitis davidii among closely related cultivars (Budak et al., Genetic Diversity of 2004). SSR and SRAP have also been used in Accessions Revealed Using genetic diversity analyses of the Chinese wild grape resources. Liu et al. (2012a) reported Microsatellite and Sequence-related on the relationship of 15 Chinese wild grape species based on the 10 microsatellite markers and 12 SRAP combinations. Fan Amplified Polymorphism Markers et al. (2015) assessed genetic polymorphisms Xiu Cai Fan, Hai Sheng Sun, Ying Zhang, Jian Fu Jiang, Min Li, of 126 individuals from five different geo- and Chong Huai Liu1 graphical populations of wild Vitis ficifolia Bge. Zhang et al. (2012) analyzed the genetic Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural diversity of 40 individuals of wild V. davidii Sciences, 450009 Zhengzhou, Henan Province, People’s Republic of China collected from Huangshan region, Anhui province, by eight SSR markers. Additional index words. wild thorn grapes, genetic polymorphisms, genetic relationship, In this study, we used 11 microsatellite molecular genetic markers markers and 12 SRAP combinations in 48 wild Abstract. In this study, simple sequence repeat (SSR) and sequence-related amplified V. davidii accessions to investigate genetic polymorphism (SRAP) markers were used to analyze the genetic diversity of 48 wild Vitis polymorphisms and possible relationships. davidii accessions. A total of 78 distinct alleles were amplified by 11 SSR primers, and the average allele number was 8.8. The average observed heterozygosity (Ho) and expected Materials and Methods heterozygosity (He) values were 0.785 and 0.814, respectively. The effective allele numbers ranged from 3.92 to 9.61. The average polymorphism information content Plant materials. Forty-eight accessions (PIC) was 0.798. Twelve of 169 SRAP primer combinations were selected for SRAP were collected from Hunan, Jiangxi, Zhejiang, analysis. A total of 188 bands were produced, and the average was 15.7 bands per primer and Anhui provinces in China. These mate- combination; the average percentage of polymorphic bands was 84.0%. The average PIC rials were planted at the National Grape was 0.76. The results of the clustering analysis based on SSR markers showed that the 48 Germplasm Repository, Zhengzhou Fruit Re- wild V. davidii accessions could be classified into five main clusters and had a genetic search Institute at the Chinese Academy of similarity coefficient level of 0.68. The dendrogram obtained from the SRAP data showed Agricultural Sciences (lat. 34°42#47.52$N, that 48 wild V. davidii accessions could be classified into five main clusters and had long. 113°42#3.19$E). Information on the a genetic similarity coefficient of 0.72. SSR and SRAP markers differentiated all materials is listed in Table 1. accessions studied including those with a similar pedigree. We speculated on the origin DNA extraction. Genomic DNA was of Ciputao 0941$, Ciputao 0940#, and Fu’an-ci-01 using SSR markers and used both SSR extracted from fresh young leaves using the and SRAP markers to resolve homonymy. The result will be valuable for further cetyltrimethylammonium bromide method and management and protection of V. davidii germplasm resources. purified in accordance with the method re- ported by Liu et al. (2012b), with minor modifications. This protocol was much faster Grapevine (Vitis spp.) is one of the most davidii is the only east Asiatic species that has and required only two to four shoot tips. The important fruit crops in the world. More than large berries, and this species has excellent quality and concentration of the DNA samples 70 species of Vitis exist, which are mostly tolerance to anthracnose, ripe rot disease, and were checked using a BioPhotometer Plus found in the temperate zones of the Northern hot climate (He, 1999; Shi et al., 2002). People (Eppendorf, Hamburg, Germany), and a portion Hemisphere. These species are distributed who live in the mountainous areas of Hunan, of the DNA was diluted to 30–50 ng·mL–1 for mostly in three regions: Europe and western Anhui, Jiangxi, and Shanxi provinces usually use in SSR and SRAP analyses. Both the stock Asia, North America, and eastern Asia plant this wild species in their courtyard as and diluted portions were stored at –20 °C. (Alleweldt and Possingham, 1988; Mullins a table grape. In the recent years, V. davidii and SSR analysis. Eleven polymorphic micro- et al., 1992). China is one of the major gene their hybrids have been used for making wines satellite loci were analyzed. Two loci (VVS2 centers of origin of the Vitis species; more and juice in China (Bao et al., 2010; Qin et al., and VVS5) were identified by Thomas and than 38 Vitis species originate in China (Kong, 2008; Zhou et al., 2008). Scott (1993) and Thomas et al. (1994). Two 2004). China is a vast country with complex Molecular genetic markers constitute one additional loci (VVMD5 and VVMD7) were geographical environments that greatly differ of the most powerful tools for analyzing also identified in the laboratory of C.P. in climate, soil, and topography. Under these genomes and enable the association of herita- Meredith (Bowers et al., 1996, 1999). Other conditions, there is an abundance of Vitaceae ble traits with underlying genomic variation loci also included VMC4H6, VMC4C6 (Di species and Vitis spp. germplasm resources. (Duran et al., 2009). SSRs, whose advantages Gaspero et al., 2000; Techera et al., 2004), The common characteristics of the Vitis spe- include codominance, high allele diversity, UDV123 (Di Gaspero et al., 2005), VrZAG25, cies native to China include small clusters high stability, and simple operation, have been VrZAG62, VrZAG79 (Sefc et al., 1999), and and berries, low sugar content, high acidity, widely used in genetic diversity analyses of VVIb66 (Merdinoglu et al., 2005). The SSR andhightannincontents(Kong,2004).Vitis grape resources (Adam-Blondon et al., 2004; amplifications were performed in 20 mLsolu- Emanuelli et al., 2013; Martínez et al., 2006; tions that contained 30–50 ng of template Rakonjac et al., 2014; Wang et al., 2015) DNA, 0.1 mM dNTPs, each primer at 0.5 mM, and in other fields. Sequence-related ampli- 2mM Mg2+, and 1 U of Taq DNA polymerase Received for publication 22 Sept. 2017. Accepted fied polymorphisms constitute a polymerase (TaKaRa, Otsu, Japan), and the amplifications for publication 8 Jan. 2018. chain reaction (PCR)-based marker system were optimized based on the methods of Di This work was supported by the China Agriculture that targets open reading frames (Li and Gaspero et al. (2005). The amplification re- Research System (CARS-29), the Agricultural Quiros, 2001). Because of their reproducible action procedure was as follows: denaturation Science and Technology Innovation Program results, high reliability, simple technology, at 94 °C for 5 min; 35 cycles at 94 °Cfor30s, (CAAS-ASTIP-2017-ZFRI), and the Crop Re- ° ° sources Protection Program of China Ministry of and low cost, SRAPs have been applied to annealing at 50–57 C for 30 s, and 72 Cfor Agriculture (2130135-34). assess the genetic diversity of many fruit 45 s; and a final extension at 72 °Cfor7min. 1Corresponding author. E-mail: liuchonghuai@ species (Abedian et al., 2012; Ahmad et al., The amplification programs varied slightly caas.cn. 2004; Amar et al., 2011; Fan et al., 2015; Guo only in annealing temperature because of the HORTSCIENCE VOL. 53(3) MARCH 2018 283 Table 1. Vitis davidii accessions used for analysis. No. Accessions Source No. Accessions Source 1 Zi luolan Hunan province 25 HJ-ci-04 Hongjiang, Hunan province 2 Zi luolan Hunan province 26 HJ-ci-10 Hongjiang, Hunan province 3 Fu’an-ci-01 Fu’an, Fujian province 27 HJ-(HY)-ci-01 Hongjiang, Hunan province 4 LC-ci-03 Luocheng, Guangxi province 28 HJ-(HY)-ci-02 Hongjiang, Hunan province 5 HT-ci-01 Huitong, Guangxi province 29 HJ-(HY)-ci-03 Hongjiang, Hunan province 6 HT-ci-02 Huitong, Guangxi province 30 HJ-(YY)-ci-05 Hongjiang, Hunan province 7 Gaoshan No. 1 Jiangxi province 31 HJ-(TM)-ci-06 Hongjiang, Hunan province 8 Gaoshan No. 2 Jiangxi province 32 HJ-(TM)-ci-07 Hongjiang, Hunan province 9 Gaoshan No. 3 Jiangxi province 33 HJ-ci-09 Hongjiang, Hunan province 10 Tangwei 0942 Yushan, Jiangxi province 34 HJ-ci-11 Hongjiang, Hunan province 11 Tangwei seedling 1 Yushan, Jiangxi province 35 ZJ-ci-01 Zhijiang, Hunan province 12 Tangwei seedling 2 Yushan, Jiangxi province 36 ZJ-ci-07 Zhijiang, Hunan province 13 WH-ci-01 Wuhan, Hubei province 37 ZJ-(SX)-ci-08 Zhijiang, Hunan province 14 TMS-ci-02 Tian mushan, Zhejiang province 38 ZJ-(SX)-ci-13 Zhijiang, Hunan province 15 TMS-ci-03 Tian mushan, Zhejiang province 39 ZJ-(BMP)-ci-01 Zhijiang, Hunan province 16 Zhejiang-ci-01 Zhejiang province 40 HH-ci (Green) Huaihua, Hunan province 17 Zhejiang-ci-03 Zhejiang province 41 HH-miputao (sweet) Huaihua, Hunan province 18 Ciputao 0940# Unknown 42 HH-miputao (Green) Huaihua, Hunan province 19 Ciputao 0941$ Unknown 43 HH-Jiaputao Huaihua, Hunan province 20 Black pearl Hunan province 44 HN-ci-01 Hunan province 21 Xiang zhenzhu (Tongmu) Tongmu, Hunan province 45 HN-ci-02 Hunan province 22 Xiang zhenzhu Hunan province 46 Ciputao seedling 1 Hunan province 23 Xiang zhenzhu (Red) Hunan province 47 Ciputao seedling 2 Hunan province 24 Xiang zhenzhu (Green) Hunan province 48 Gaoshan mutation Hunan province different primers used. The amplification SSRs. The variability detected by each SSR Table 2. Number of alleles (Na), effective number products were separated using 8% poly- locus is shown in Table 2.
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