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Chinese cymbidiums (Cymbidium sp.) are important ornamental plants because of their foliage, flower shape, and fragrance. Well-known Chinese cymbidiums mainly include Cymbidium goeringii, Cymbidium faberi, Cymbidium ensifolium, Cymbidium kanran, and Cymbidium sinense. The population genetics of Chinese cymbidiums can be efficiently analyzed using small-scale marker panels with high discriminatory power. In this study, we tested several genic simple sequence repeats (SSRs) and built six genic SSR panels. The panels included several robust markers, which can rapidly assign Chinese cymbidium accessions to their source species. Fifty-three accessions of Chinese cymbidiums were analyzed using 25 markers, which exhibited polymorphism among five species. These markers were ranked according to their discriminatory scores (D scores). The program selected six markers to build an ‘‘overall’’ panel for all Cymbidium classifications and yielded 95.16% population assignment accuracy. Considering one species as the ‘‘critical’’ population and the four other species as one population, we built five genic SSR panels: C. ensifolium panel (four markers, 98.05% accuracy), C. faberi panel (six markers, 95.90% accuracy),C. goeringii panel (six markers, 95.15%accuracy),C. sinense panel (six markers, 96.35% accuracy), andC. kanran panel (five markers, 96.10% accuracy). Genetic distance matrices calculated using the ‘‘overall’’ panels and those derived with the 25 markers were compared. Results showed a high correlation (R = 0.807) with statistical significance (P = 0.042). Moreover, ‘‘all panels’’ revealed higher genetic variations among populations than ‘‘all markers.’’ Hence, the developed panels are suitable for efficient population classification of Chinese cymbidiums. Chinese cymbidiums refer to species and hybrids derived from Cymbidium goeringii, C. faberi, C. ensifolium, C. kanran, and C. sinense. These are terrestrial species and most cultivars of these species present superior flower shape, foliage, and fragrance (Li et al., 2014c; Schneitz, 1999). Chinese cymbidiums have been cultivated for several centuries in Asia, especially in China, Korea, and Japan, because of the high ornamental and economic value of these plants (Moea and Parka, 2012). To conserve the genetic resources of Chinese cymbidiums, scholars have focused on conserving the genetic diversity of these species. Genetic diversity and population structure have to be preliminarily characterized before germplasm conservation. The genetic diversity of Cymbidium species can be analyzed using DNA markers, including restriction enzyme polymorphism markers (Obara-Okeyo and Kako, 1998); random amplified polymorphic DNA markers (Choi et al., 2006; Obara-Okeyo and Kako, 1998); amplified fragment length polymorphism markers (Wang et al., 2004); polymorphisms of internal transcribed spacers of nuclear ribosomal DNA and plastids, as well as inter-SSR (Lu et al., 2011); and SSR (Capesius, 1976; Moe et al., 2010; Moea and Parka, 2012). Novel Cymbidium SSRs derived from genes (genic SSRs) are developed from genic regions and can be applied for genetic analysis (Li et al., 2013, 2014a). SSRs are ubiquitous, locus specific, codominant, highly polymorphic, and easily operated, thereby requiring lower cost compared with other molecular markers (Honma and Goto, 2001; Received for publication 20 Nov. 2015. Accepted for publication 30 Dec. 2015. This research was supported by the National Basic Research Program funded by the Nature Science Foundation of China (no. 31201648) and the Qianjiang Talents Project (no. 2013R10081). These authors contributed equally to this work. Corresponding author. E-mail: hufanfan1982815@gmail.com. J. AMER. SOC. HORT. SCI. 141(2):125–130. 2016. 125 Li et al., 2006; Schwarz-Sommer et al., 1990; Tsai et al., 2004; Venkateswarlu et al., 2006; Xu et al., 2006). In addition, genic SSRs exhibit high transferability across species. These features render genic SSRs as suitable for discriminating closely related species. The use of numerous marker loci in population genetic analysis is an expensive and inefficient method for genotyping large germplasm collections. As such, scholars developed informative panels by using a minimum number of marker locus combinations. The panel can be considered a DNA bar code and can be used for research on taxonomy and population genetics. Minimum combination of highly ranked loci for population assignment can be determined through two basic procedures. First, simulated test data are generated using the observed allele frequencies for each locus, and then the number of times each test genotype is correctly assigned to the appropriate source population is determined and markers are then ranked by comparison of individual marker scores. Second, another round of iterations yield ranked loci by increasing one locus at each run until the assignment score reaches or exceeds the accuracy criterion. This strategy is applied for locus selection in numerous species during population assignment; such species include chinook salmon [Oncorhynchus tshawytscha (Greig et al., 2003)], asian seabass [Lates calcarifer (Yue et al., 2012)], trematomids [Trematomus sp. (Van de Putte et al., 2009), humans [Homo sapiens (Rosenberg et al., 2003)], switchgrass [Panicum virgatum (Okada et al., 2011)], and rice [Oryza sativa (Agrama et al., 2012)]. In the present study, we aim to develop informative panels by using a minimum number of genic SSRs. The panels can be used to assign Chinese cymbidiums to five populations with high accuracy. The proposed strategy could be an efficient tool for population classification. Materials and Methods Plant samples Fifty-three accessions from C. goeringii, C. faberi, C. ensifolium, C. kanran, and C. sinense were used for testing genetic markers (Table 1). Fifty-three accessions were selected from 105 accessions (Supplemental Fig. 1) based on their ancestries derived from STRUCTURE (Pritchard et al., 2000), which had maximum ancestry index higher than 0.6. The plants were grown and maintained in a greenhouse under natural light. Fresh leaves were collected from two or three seedlings of each accession for genomic DNA extraction. Genotyping Genomic DNA was extracted from leaf samples as previously described (Li et al., 2007). Twenty-five genic SSRs were selected from the genic SSR collection (Li et al., 2014a) for panel development. Polymerase chain reaction (PCR) primers were synthesized by Life Technologies (ABI and Invitrogen, Shanghai, China). PCR experiments were conducted, and the products were separated using polyacrylamide gel electrophoresis gel (Li et al., 2007). The gel was silver stained according to the procedure reported by Li et al. (2001), and the results were recorded using a scanner. The genotype was determined by analyzing band patterns (Li et al., 2014a). The accessions were labeled by 1, 2, 3, and so on, according to the number and position of bands, and then the data were converted into binary matrix using ‘‘compute frequency’’ procedure in PowerMarker version 2.7 (Liu and Muse, 2005). Statistical analysis POLYMORPHISM OF MARKERS. Genetic distances were calculated using Nei’s distance (Nei and Takezaki, 1983). Phylogenetic reconstruction was performed based on neighbor-joining (NJ) method (with 1000 bootstrap replicates) and implemented in PowerMarker version 2.7. The phylogenetic tree was visualized in MEGA version 4 (Tamura et al., 2007). PowerMarker was also used to calculate the average number of marker alleles and polymorphism information content (PIC). ANALYSIS OF POPULATION STRUCTURE. Population structure was initially analyzed using the software STRUCTURE as described in previous studies (Li et al., 2010, 2011, 2012, 2014b). Genetic variations within and among populations were then calculated by analysis of molecular variance (AMOVA) in Arlequin V2.000 (Schneider and Excoffier, 1999). Genetic distances among species were calculated using Nei’s distance (Nei and Takezaki, 1983). A phylogenetic tree was constructed by NJ method. Both calculations were performed in PowerMarker version 2.7. The phylogenetic tree was visualized using MEGA version 4 (Tamura et al., 2007). The correlation of genetic distances based on total markers or panels was assessed using Mantel test in PowerMarker. Table 1. Fifty-three Chinese cymbidium accessions for genetic analysis. Cymbidium ensifolium, Cymbidium faberi, Cymbidium sinense, Cymbidium goeringii, and Cymbidium kanran had 10, 10, 10, 12, and 11 accessions, respectively. Species Name Species Name C. ensifolium Bailian C. goeringii Chunjiansuxin Baodao Hexingmei Jinhe Hongmeigui Minnanlvdie Huangmei Qingshanyuquan Jiuxianmudan Shisantaibao Puchunhong Tiegusu Silan Yandongsu Songmei Yinsimawei Wenyi Zhongshanmei Yipin C. faberi Dayipin Yuhudie Duanhuimei Yumei Jiepeimei C. kanran Changuihong Laoranzi Danxia LvlanA Dongmingmei LvlanB Jihanmei LvlanC Jinwan Panlv Mengzhongmeiren Xianhejipin Xianhe Xianlv Yingcaihe C. sinense Baimo Yipinhong Damo Yisudie Fuguiminglan Zhizunhongyan Piaoyi Qiebang Qihei Taoji Wuzicui Yingbianmolan

Development of Genic Simple Sequence Repeat Panels for Population Classification of Chinese Cymbidium Species