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

in HortScience

Molecular genetic diversity and relationships among 86 Chrysopogon 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 ISSR markers 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 four major 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.

Abstract

Molecular genetic diversity and relationships among 86 Chrysopogon 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 ISSR markers 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 four major 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 genus Chrysopogon 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 DNA-based 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 ISSR markers 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; Djè 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 as Dianthus 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 86 C. aciculatus accessions from five provinces in China with 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 tropical monsoon 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.

Table 1.

Eighty-six Chrysopogon aciculatus samples collected from different locations in China and used in the study.

Table 1.

For each accession, 40 healthy stolons with two internodes of one accession were transplanted into separate 0.25-m2 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, 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·µL−1 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 PCR mixture (total volume 20 µL) consisted of 2 µL 10 × buffer [100 mm Tris-HCl (pH 8.3), 500 mm KCl, and 15 mm MgCl2], 2.5 mm dNTPs (Invitrogen), 0.2 µm primer (Invitrogen), 0.2 µL Taq DNA polymerase (Takara Biotechnology, Dalian, China), 14 µL 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 100-bp 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).

Table 2.

Twenty-five primers sequences used for intersimple sequence repeat (ISSR) amplification and number of bands for each primer.

Table 2.

SRAP analysis.

A total of 400 SRAP primer 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 µL containing 2 µL 10× buffer [100 mm Tris-HCl (pH 8.3), 500 mm KCl, and 15 mm MgCl2], 2.5 mm dNTPs, 0.2 µm primer, 0.2 µL Taq DNA polymerase, 13.3 µL 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 step of 7 min at 72 °C. PCR amplicons were 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).

Table 3.

Forward and reverse sequence-related amplified polymorphism primers used in this study.

Table 3.
Table 4.

Amplification results of 30 sequence-related amplified polymorphism primer combinations used in this study.

Table 4.

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).

Results

Polymorphism analysis.

Twenty-five ISSR primers amplified 283 scorable bands, with an average of 11.32 amplified fragments per primer pair (Table 2; Fig. 1). The number of bands ranged from 8 (ISSR827) to 15 (ISSR850 and ISSR858) bands per primer, with an average of 10.92 bands showing polymorphism. The percentage of PPL of the 25 primers tested ranged from 84.62% for primer ISSR895 to 100% for primers ISSR808, ISSR816, ISSR823, ISSR824, ISSR827, ISSR835, ISSR842, SSR859, ISSR866, and ISSR890 (Table 2). Of the 627 bands amplified by 30 SRAP primer pairs, 576 bands were polymorphic (91.87%). Polymorphisms of all loci ranged from a maximum of 100% (Me03-Em13, Me04-Em10, Me04-Em16, Me05-Em10, Me06-Em16, Me07-Em18, Me16-Em09, Me16-Em16, and Me19-Em09) to a minimum of 66.67% (Me08-Em19). An average of 20.9 bands was amplified per primer, and 19.2 bands were polymorphic (Table 3; Fig. 2).

Fig. 1.
Fig. 1.

Intersimple sequence repeat amplified with primer ISSR842 in 86 Chrysopogon aciculatus DNA of C. aciculatus samples. The size standard is a 100-bp DNA ladder and the sample number is shown in Table 1.

Citation: HortScience horts 51, 8; 10.21273/HORTSCI.51.8.972

Fig. 2.
Fig. 2.

Sequence-related amplified polymorphism amplified with primer combination Me19-Em09 in 86 Chrysopogon aciculatus DNA of C. aciculatus samples. The size standard is 100-bp DNA ladder and the sample number is shown in Table 1.

Citation: HortScience horts 51, 8; 10.21273/HORTSCI.51.8.972

Genetic diversity.

Genetic variation accessed using ISSR and SRAP markers for all 86 C. aciculatus accessions are shown in Tables 2 and 4. The average values for PPL, He, and I obtained with the ISSR markers were 94%, 0.3, and 0.29, respectively. Genetic diversity identified using the SRAP markers was similar to that of the ISSR markers, as indicated by the average values for PPL, He, and I of 91.87%, 0.29, and 0.26, respectively. The genetic similarity coefficients (GSCs) calculated using the UPGMA clustering method based on ISSR polymorphism ranged from 0.52 to 0.93. Accessions from the same geographic location (CA01 and CA10) in Haikou, Hainan Province, showed a GSC of 0.93. The GSC values derived from the SRAP data set ranged from 0.56 to 0.91. The largest GSC was between accessions CA21 (Dingan, Hainan Province) and CA24 (Sanya, Hainan Province). These results suggested that a high level of genetic diversity was exhibited among all samples.

Cluster analysis.

A UPGMA dendrogram derived from the ISSR data were generated to explore the genetic relationships among the 86 accessions. The accessions were divided into three main clusters (I, II, and III) (Fig. 3). Cluster I comprised of 19 samples from Hainan (18) and Guangxi (1) provinces. Cluster II included 54 accessions, which were further divided into two subclusters: subcluster II-1 contained eight accessions from Hainan (1), Guangxi (3), Guangdong (2), Fujian (1), and Yunnan (1) provinces; and subcluster II-2 contained 36 accessions from Guangxi (18), Hainan (12), Guangdong (4), and Fujian (2) provinces. Cluster III comprised 14 samples from Hainan (8), Yunnan (3), Guangdong (2), and Guangxi (1) provinces. Accessions from the same region were generally grouped within the same cluster.

Fig. 3.
Fig. 3.

Unweighted pair-group method with arithmetic averages clustering for 86 Chrysopogon aciculatus accessions based on intersimple sequence repeat markers.

Citation: HortScience horts 51, 8; 10.21273/HORTSCI.51.8.972

The dendrogram (Fig. 4) derived from the SRAP data grouped the 86 accessions into three main clusters (IV, V, and VI). Cluster V was divided into two subgroups, V-1 and V-2. Cluster IV was similar to cluster III. Cluster V was similar to cluster II, and cluster VI was similar to cluster I. CA04 was placed in cluster I, and CA22 and CA23 were grouped in cluster II in the ISSR analysis; in contrast, CA04, CA22, and CA23 were assigned to cluster IV in the SRAP analysis.

Fig. 4.
Fig. 4.

Unweighted pair-group method with arithmetic averages dendrogram based on sequence-related amplified polymorphism data of 86 Chrysopogon aciculatus accessions.

Citation: HortScience horts 51, 8; 10.21273/HORTSCI.51.8.972

In addition, a dendrogram derived from a combined ISSR and SSR data set was constructed, for which the GSCs ranged from 0.56 to 0.88 (Fig. 5). In the combined data analysis, the 86 accessions were classified into four major clusters (VII, VIII, IX, and X). The groups were similar to those obtained in the ISSR analysis. Cluster I was similar to cluster X, except that CA01, CA04, CA06, CA07, and CA10 placed in cluster I based on the ISSR data (Fig. 3) were grouped in cluster VII (Fig. 5). CA20, CA22, CA23, and CA27 placed in subcluster II-1 (Fig. 3) were grouped in cluster X (Fig. 5). The accessions placed in subcluster II-2 and cluster III were identical to those grouped in clusters VIII and IX, respectively.

Fig. 5.
Fig. 5.

Unweighted pair-group method with arithmetic averages cluster for 86 Chrysopogon aciculatus accessions based on intersimple sequence repeat and sequence-related amplified polymorphism markers.

Citation: HortScience horts 51, 8; 10.21273/HORTSCI.51.8.972

Discussion

Previous studies demonstrate that ISSR and SRAP markers are valuable for marker-assisted selection, analysis of genetic diversity, and population genetic analysis (Budak et al., 2004a, 2004b, 2004c; Carvalho et al., 2009; Gupta and Varshney, 2000). Both marker types have been successfully used for genetic analyses of warm-season turfgrasses, such as Zoysia japonica Steud. (Chen et al., 2009; Xie et al., 2012), Cynodon dactylon (Huang et al., 2010; Li et al., 2011; Ling et al., 2010; Wang et al., 2009b, 2013; Yi et al., 2008), Cynodon arcuatus J. Presl (Huang et al., 2013), and Eremochloa ophiuroides (Munro) Hack. (Zheng et al., 2009, 2013). The present study is the first assessment of the molecular genetic diversity among C. aciculatus accessions using ISSR and SRAP markers.

Evaluation of genetic diversity among a sample of accessions provides an important foundation for conservation of genetic resources and marker-assisted breeding. In the present study, ISSR and SRAP data revealed high genetic diversity among the 86 C. aciculatus accessions. The 25 ISSR markers yielded a higher PPL (94%) than the 30 SRAP markers (91.87%). The values of He and I generated by the ISSR markers (0.3 and 0.29, respectively) were slightly higher than those generated using SRAP markers (0.29 and 0.26, respectively), which indicated that ISSR markers provided more informative data than SRAP markers. Differences in the results obtained with the ISSR and SRAP markers probably reflect the different genomic regions amplified by the two marker types. Similar results were obtained for Auricularia auricula-judae Quel. (Tang et al., 2010), Pogostemon cablin Benth. (Wu et al., 2010), and Prunella vulgaris L. (Liao et al., 2012) using ISSR and SRAP molecular markers.

The Mantel matrix correspondence test was used for an overall comparison of matrices of cophenetic values produced from dissimilarity matrices generated in the UPGMA cluster analysis (Mantel, 1967). The Mantel’s test indicated that the ISSR and SRAP cophenetic values were significantly correlated (r = 0.8023), thus the ISSR and SRAP markers revealed similar patterns of genetic diversity in C. aciculatus accurately.

The dendrograms generated from the ISSR and SRAP data sets showed strong similarity in topology. Accessions with the same provenance were generally clustered in the same group. Accessions from the same geographical area that were clustered into different groups may reflect gene flow from introduced species or introgression (Li et al., 2011). Conversely, accessions from different regions that clustered together may be explained by the outcrossing breeding system of C. aciculatus or by exchange of germplasm resources between regions (Farsani et al., 2012). Genetic overlap occurs in neighboring regions (Li et al., 2011; Yi et al., 2008). In the present study, the 86 C. aciculatus accessions originated from five provinces, of which Fujian, Guangdong, Guangxi, and Yunnan provinces are adjacent to each other. C. aciculatus shows strong vegetative reproduction, and human activities may have aided dispersal, with subsequent vegetative spread and sexual reproduction leading to a high level of genetic diversity.

In this study, we combined the ISSR and SRAP data sets to examine the degree of agreement between the individual marker types. Although combining data remains controversial (Mohammadi and Prasanna, 2003), combining the ISSR and SRAP data sets allowed a more comprehensive analysis of genetic diversity and provided greater information for future marker-assisted selection in the breeding of C. aciculatus. The 86 C. aciculatus accessions were clustered into four groups based on the combined data sets. The clusters generally reflected the geographical distribution of the accessions, similar to that based on separate analyses of the ISSR and SRAP data sets. In addition, we measured five indices of plant growth, namely chlorophyll content, maximum leaf length, withering rate, quality evaluation, and quality of dry matter, with the aim of assessing the shade tolerance of C. aciculatus. A clustering analysis diagram was generated based on the growth parameter data (X.Y. Zhang, Z.Y. Wang, and L. Liao, unpublished data). From these two cluster analyses, it was determined that cluster VII is characterized by excellent shade tolerance. The growth parameters of clusters VIII and IX are generally intermediate to those of clusters I and X, with the growth parameters of cluster IX slightly lower than those of cluster VIII. Accessions grouped in cluster X showed opposing characteristics to those of cluster VII. Accessions placed in clusters VII and VIII may prove useful turfgrasses for planting. Thus, the information provided by the combined marker analysis may prove useful for selection of parents in the breeding of improved shade-tolerant accessions of C. aciculatus.

In conclusion, this study assessed molecular genetic diversity and relationships among 86 C. aciculatus accessions using ISSR and SRAP markers. Both ISSR and SRAP markers are reliable and effective tools for analysis of genetic diversity in C. aciculatus, with high levels of genetic diversity detected among the 86 populations sampled. ISSR markers show a higher PPL (94%) than SRAP markers (91.87%). Cluster analysis of the ISSR and SRAP data independently yielded similar results. The C. aciculatus accessions were clustered into three groups. The clusters were generally consistent with the geographic provenances of the accessions. Cluster analysis of the combined ISSR and SRAP data sets generated similar groupings to those derived from separate analyses of the ISSR and SRAP data sets. Indices of shade tolerance differ among each group of accessions. Thus, ISSR and SRAP markers are informative for evaluation of genetic diversity in C. aciculatus, and may be useful in the conservation of genetic resources, development of biodiversity conservation strategies, and identification of wild germplasm resources. The markers may also contribute to future molecular marker-assisted breeding programs.

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  • WangZ.LiaoL.YuanX.GuoH.GuoA.LiuJ.2013Genetic diversity analysis of Cynodon dactylon (bermudagrass) accessions and cultivars from different countries based on ISSR and SSR markersBiochem. Syst. Ecol.46108115

    • Search Google Scholar
    • Export Citation
  • WangZ.Y.YuanX.J.GuoH.L.LiuX.L.ZhouZ.F.2009aOptimization of ISSR-PCR system on Zoysia sppActa Pratacult. Sin.174851

  • WangZ.YuanX.ZhengY.LiuJ.2009bMolecular identification and genetic analysis for 24 turf-type Cynodon cultivars by sequence-related amplified polymorphism markersSci. Hort.122461467

    • Search Google Scholar
    • Export Citation
  • WuY.G.GuoQ.S.HeJ.C.LinY.F.LuoL.J.LiuG.D.2010Genetic diversity analysis among and within populations of Pogostemon cablin from China with ISSR and SRAP markersBiochem. Syst. Ecol.386372

    • Search Google Scholar
    • Export Citation
  • XieY.LiuL.FuJ.LiH.2012Genetic diversity in Chinese natural zoysiagrass based on inter-simple sequence repeat (ISSR) analysisAfr. J. Biotechnol.1176597669

    • Search Google Scholar
    • Export Citation
  • YiY.J.ZhangX.Q.HuangL.K.LingY.XiaoM.A.2008Genetic diversity of wild Cynodon dactylon germplasm detected by SRAP markersHereditas3094100

    • Search Google Scholar
    • Export Citation
  • YildizM.EkbicE.KelesD.SensoyS.AbakK.2011Use of ISSR, SRAP, and RAPD markers to assess genetic diversity in Turkish melonsSci. Hort.130349353

    • Search Google Scholar
    • Export Citation
  • YilmazK.U.Paydas-KargiS.DoganY.KafkasS.2012Genetic diversity analysis based on ISSR, RAPD and SSR among Turkish Apricot Germplasms in Iran Caucasian eco-geographical groupSci. Hort.138138143

    • Search Google Scholar
    • Export Citation
  • ZhengY.GuoH.ZangG.LiuJ.2013Genetic linkage maps of centipedegrass [Eremochloa ophiuroides (Munro) Hack.] based on sequence-related amplified polymorphism and expressed sequence tag-simple sequence repeat markersSci. Hort.1568692

    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
  • ZietkiewiczE.RafalskiA.LabudaD.1994Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplificationGenomics20176183

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    • Export Citation

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Contributor Notes

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.

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    Intersimple sequence repeat amplified with primer ISSR842 in 86 Chrysopogon aciculatus DNA of C. aciculatus samples. The size standard is a 100-bp DNA ladder and the sample number is shown in Table 1.

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    Sequence-related amplified polymorphism amplified with primer combination Me19-Em09 in 86 Chrysopogon aciculatus DNA of C. aciculatus samples. The size standard is 100-bp DNA ladder and the sample number is shown in Table 1.

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    Unweighted pair-group method with arithmetic averages clustering for 86 Chrysopogon aciculatus accessions based on intersimple sequence repeat markers.

  • View in gallery

    Unweighted pair-group method with arithmetic averages dendrogram based on sequence-related amplified polymorphism data of 86 Chrysopogon aciculatus accessions.

  • View in gallery

    Unweighted pair-group method with arithmetic averages cluster for 86 Chrysopogon aciculatus accessions based on intersimple sequence repeat and sequence-related amplified polymorphism markers.

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    • Search Google Scholar
    • Export Citation
  • WangZ.Y.YuanX.J.GuoH.L.LiuX.L.ZhouZ.F.2009aOptimization of ISSR-PCR system on Zoysia sppActa Pratacult. Sin.174851

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    • Search Google Scholar
    • Export Citation
  • WuY.G.GuoQ.S.HeJ.C.LinY.F.LuoL.J.LiuG.D.2010Genetic diversity analysis among and within populations of Pogostemon cablin from China with ISSR and SRAP markersBiochem. Syst. Ecol.386372

    • Search Google Scholar
    • Export Citation
  • XieY.LiuL.FuJ.LiH.2012Genetic diversity in Chinese natural zoysiagrass based on inter-simple sequence repeat (ISSR) analysisAfr. J. Biotechnol.1176597669

    • Search Google Scholar
    • Export Citation
  • YiY.J.ZhangX.Q.HuangL.K.LingY.XiaoM.A.2008Genetic diversity of wild Cynodon dactylon germplasm detected by SRAP markersHereditas3094100

    • Search Google Scholar
    • Export Citation
  • YildizM.EkbicE.KelesD.SensoyS.AbakK.2011Use of ISSR, SRAP, and RAPD markers to assess genetic diversity in Turkish melonsSci. Hort.130349353

    • Search Google Scholar
    • Export Citation
  • YilmazK.U.Paydas-KargiS.DoganY.KafkasS.2012Genetic diversity analysis based on ISSR, RAPD and SSR among Turkish Apricot Germplasms in Iran Caucasian eco-geographical groupSci. Hort.138138143

    • Search Google Scholar
    • Export Citation
  • ZhengY.GuoH.ZangG.LiuJ.2013Genetic linkage maps of centipedegrass [Eremochloa ophiuroides (Munro) Hack.] based on sequence-related amplified polymorphism and expressed sequence tag-simple sequence repeat markersSci. Hort.1568692

    • Search Google Scholar
    • Export Citation
  • ZhengY.Z.XiJ.B.YangZ.Y.2005Studies on distribution and morphological variation of wild grass Chrysopogon aciculatus collected from the tropics and subtropics of ChinaActa Agrestia Sin.13117122

    • Search Google Scholar
    • Export Citation
  • ZhengY.Q.ZongJ.Q.XueD.D.ChenX.LiuJ.X.2009Application of SRAP markers to the identification of Eremochloa ophiuroides (Munro) Hack hybridsActa Agrestia Sin.17135140

    • Search Google Scholar
    • Export Citation
  • ZietkiewiczE.RafalskiA.LabudaD.1994Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplificationGenomics20176183

    • Search Google Scholar
    • Export Citation
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