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Molecular genetic diversity and relationships among 86Chrysopogon aciculatus (Retz.) Trin. accessions were assessed using intersimple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) markers. Twenty-five ISSR markers generated 283 amplification bands, of which 266 were polymorphic. In addition, 576 polymorphic bands were detected from 627 bands amplified using 30 SRAP primers. Both marker types revealed a high level of genetic diversity, with ISSRmarkers showing a higher proportion of polymorphic loci (PPL; 94%) than SRAP markers (91.87%). The ISSR and SRAP data were significantly correlated (r = 0.8023). Cluster analysis of the separate ISSR and SRAP data sets clustered the accessions into three groups, which generally were consistent with geographic provenance. Cluster analysis of the combined ISSR and SRAP data set revealed fourmajor groups similar to those based solely on ISSR or SRAP markers. The findings demonstrate that ISSR and SRAP markers are reliable and effective tools for analysis of genetic diversity in C. aciculatus. The perennial grass genusChrysopogon is mainly distributed in tropical and subtropical regions of the world. Three species are indigenous to China: Chrysopogon echinulatus (Nees ex Steud.) Will. Watson, Chrysopogon orientalis A. Camus, and Chrysopogon aciculatus. C. aciculatus is the only member of the genus suitable for use as a turfgrass. The species is drought and shade tolerance, and shows low maintenance requirements. C. aciculatus is widely distributed in southern China and commonly used in lawns, sports fields, and shaded turf (Zheng et al., 2005). The development and application of DNAbased molecular markers has revolutionized the study of genetic diversity (Kalendar et al., 2011). DNA-based molecular markers are often simple to amplify and rapidly operable. Such markers have been widely used for research into interspecific and intraspecific genetic diversity among turfgrasses. Commonly used molecular marker technologies include random amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLPs), and simple sequence repeats (SSRs) (Kalia et al., 2011). Intersimple sequence repeat markers can be amplified simply and directly without knowledge of the flanking sequences, thus enabling easier development of specific primers compared with SSR markers. The development costs for ISSR markers are low, and ISSRmarkers show higher reproducibility and reliability compared with AFLP and RAPD markers (Barth et al., 2002; Huang et al., 2013; Zietkiewicz et al., 1994). ISSR technology has been widely used in genetic diversity analysis (Baliyan et al., 2014; Biswas et al., 2010; Dj e et al., 2010; Tao et al., 2014; Wang et al., 2013) and DNA fingerprinting (Huang et al., 2010; Kalpana et al., 2012; Reunova et al., 2010; Smolik et al., 2010; Zietkiewicz et al., 1994). The SRAP technique is based on polymerase chain reaction (PCR) amplification. SRAP primers are designed on the basis that exons are GC rich whereas promoters and introns are AT rich; the polymorphism reflects fundamental variation in the lengths of introns, promoters, and intergenic spacers, both among individuals and among species (Li and Quiros, 2001). The unique primer designs used in the SRAP technique allow direct amplification of open reading frames in the genome, regardless of flanking sequence (Budak et al., 2004a; Wang et al., 2009a). SRAP technology has been widely used in population genetic analysis, paternity testing, genome mapping, and marker-assisted selection. The technique has numerous advantages over other molecular marker technologies, such as abundant polymorphism, high reproducibility, easy amplification, and good stability (Babaei et al., 2014; Budak et al., 2004a; Cai et al., 2011; Li and Quiros, 2001; Uzun et al., 2009). Combination of ISSR and SRAP markers has been used for assessment of genetic diversity among diverse species, such asDianthus L. species and Salvia miltiorrhiza Bunge (Budak et al., 2004a; Li et al., 2014; Liu et al., 2008; Song et al., 2010; Yildiz et al., 2011; Yilmaza et al., 2012). Previous studies of variability within C. aciculatus have mainly focused on morphological characteristics (Clarkson, 1992; Liao et al., 2011; Zheng et al., 2005), with no information available on genetic diversity using molecular markers. In this study, we collected 86C. aciculatus accessions fromfive provinces in Chinawith the aim to evaluate the genetic diversity among the accessions and to assess the level of polymorphism detectable by the use of ISSR and SRAP markers. Materials and Methods Plant materials. Eighty-six C. aciculatus accessions were collected from the Hainan, Guangxi, Guangdong, Fujian, and Yunnan provinces of China (Table 1). The accessions were grown under field conditions at the Agronomy Garden, Hainan University, Haikou, Hainan, China (20 03# N, 110 19# E; 69.8 m above sea level). This area experiences a tropicalmonsoon climate. The average annual rainfall is 900–2200 mm and average annual sunlight hours exceed 2000 h. The temperature ranges from 9.4 to 38.7 C, with an annual average of 23.8 C. Detailed information for each collection provenance is provided in Table 1. For each accession, 40 healthy stolons with two internodes of one accession were transplanted into separate 0.25-m plot, with 50-cm spacing between each plot. Each plot was trimmed weekly to prevent interplot contamination. The plots were frequently mowed to a height of 50 mm. In accordance with the methods of Zheng et al. (2005), routine irrigation, fertilization, and fungicide application were performed to ensure active and healthy growth of the accessions. DNA extraction. Genomic DNA of each accession was extracted from healthy, fresh, Received for publication 26 May 2016. Accepted for publication 23 June 2016. This work was supported by the National Natural Science Foundation of China (31260489). We thank three anonymous reviewers for useful comments and suggestions to improve the manuscript. Corresponding author. E-mail: wangzhiyong7989@ 163.com. 972 HORTSCIENCE VOL. 51(8) AUGUST 2016 young leaf tissue using the cetyltrimethylammonium bromide method (Doyle and Doyle, 1987). DNA concentrations were determined by electrophoresis in 1.5% agarose gel by comparison with a sample of known concentration. Genomic DNA was diluted to a final concentration of 30 ng·mL and stored at –20 C until required. ISSR analysis. Twenty-five ISSR primers were chosen from 100 primers supplied by Invitrogen Biotech Co. (Shanghai, China) to amplify polymorphisms in the 86 C. aciculatus accessions. Detailed primer information is given in Table 2. The amplification procedure followed the protocol established by Wang et al. (2009a). Each PCRmixture (total volume 20 mL) consisted of 2 mL 10 · buffer [100 mM Tris-HCl (pH 8.3), 500mMKCl, and 15 mM MgCl2], 2.5 mM dNTPs (Invitrogen), 0.2 mM primer (Invitrogen), 0.2 mL Taq DNA polymerase (Takara Biotechnology, Dalian, China), 14 mL ddH2O, and 60 ng template DNA. Amplifications were carried out in a PTC-200 Thermal Cycler (MJ Research, Watertown, MA) using the following PCR program: initial denaturation at 95 C for 5 min; 45 cycles of 45 s degeneration at 94 C, 1 min annealing at 52–61 C, and 90 s elongation at 72 C; and a final elongation step of 7 min at 72 C. Following amplification, PCR fragments were electrophoresed in 1.5% agarose gel using a Hoefer vertical gel apparatus (JY-SCZ6; Beijing Liuyi Biotechnology Co., Ltd., Beijing, China) at 120 V for 2.5 h in 1· Tris/borate/EDTA buffer. A 100bp DNA ladder (Dye Plus; Takara Biotechnology) was used as a reference for estimation of allele sizes. After electrophoresis, the gels were stained with ethidium bromide and photographed under ultraviolet light, as described by Wang et al. (2009a). SRAP analysis.A total of 400 SRAPprimer combinations were used for PCR amplification (Table 3). Thirty primer combinations were selected (Table 4) for further analysis. PCR amplification was based on the technique previously described for Cynodon dactylon (L.) Pers. (Wang et al., 2009b) with modifications. SRAP reactions were conducted in a total volume of 20 mL containing 2 mL 10· buffer [100 mM Tris-HCl (pH 8.3), 500 mM KCl, and 15 mM MgCl2], 2.5 mM dNTPs, 0.2 mM primer, 0.2 mL Taq DNA polymerase, 13.3 mL ddH2O, and 30 ng template DNA. Amplifications were performed in a PTC-200 Thermal Cycler using the following PCR program: 4 min denaturation at 94 C, 1 min degeneration at 94 C, 1 min annealing at 37 C, and 10 s elongation at 72 C. In the following 35 cycles, the annealing temperature was increased to 50 C, with a final elongation stepof7minat 72 C.PCRampliconswere fractionated on 8.0% nondenatured polyacrylamide gels using a Hoefer vertical gel apparatus at 200 V for 2.5 h in 1· Tris/borate/EDTA buffer. A 100-bp DNA ladder (Dye Plus) was used as a reference for estimation of allele sizes. After electrophoresis, the gel was subjected to rapid silver staining for detection of DNA bands (Wang et al., 2009b). Data analysis. The presence or absence of each SRAP or ISSR fragment was coded as 1 or 0, respectively. Popgene version 1.32 was used to calculate Nei’s gene diversity (He) (Nei, 1973) and Shannon’s information index (I) (Shannon and Weaver, 1949), which were used to compute Nei’s standard genetic distance coefficients (Nei and Li, 1979) for the 86 accessions. The resulting distance matrix was used to construct a dendrogram with the unweighted pair-group method with arithmetic averages (UPGMA) (Sneath and Sokal, 1973) using NTSYS-pc version 2.10s software. The correlation between similarity matrices generated from the ISSR and SRAP data sets was estimated using the COPH and MXCOP modules in NTSYS-pc (Mantel, 1967). The COPH and MXCOMP programs were used to calculate the goodness of fit between the cluster analysis and the original distance matrix for three data sets (ISSR, SRAP, and ISSR + SRAP).

Analysis of Genetic Diversity in Chrysopogon aciculatus Using Intersimple Sequence Repeat and Sequence-related Amplified Polymorphism Markers