Genetic diversity and population genetic structure of pomegranate (Punica granatum L.) in Iran using AFLP markers

Pomegranate (Punica granatum L.) is one of the oldest known edible fruits. It is native to Iran and spread from Iran to other areas. In this study amplified fragment length polymorphism (AFLP) was used to detect intra- and inter-population genetic diversity of pomegranate. A group of 67 accessions belonged to 4 populations from Iran was studied using eight primer combinations. A total of 221 scorable bands were amplified, of which, 118 (54.13%) were polymorphic. Resolving power (Rp) ranged from 5.70 to 9.21, and the average of polymorphism information content (PIC) per primer pair was 0.40. According to Nei's gene diversity and allelic statistics, Isfahan population had a highest genetic diversity (H = 0.3646, I = 0.5327, N_e = 1.6467). Coefficient of gene differentiation between populations (GST) was 0.124, indicated that mainly proportion of genetic variation (87.6%), was within populations and the remaining (12.4%) of the variation was among populations that, also supported by analysis of molecular variance (AMOVA). The gene flow (Nm) varied from 0.969 to 10.404 between pair-wise populations and was 3.504 among all of the populations. The Jaccard similarity coefficient between individuals ranged from 0.26 to 0.88. The UPGMA dendrogram clustered all 67 accessions into 6 groups. In some cases accessions from same region were grouped together but in most cases, there was gene exchange. To study the genetic relationships among populations, a principal coordinate analysis (PCoA) based on Nei's genetic distances was performed. Results of this study showed that AFLP marker can be a useful tool for investigating the genetic diversity of pomegranate genotypes.     

Evaluation of genetic diversity among Iranian soft-seed pomegranate accessions by fruit characteristics and RAPD markers

Soft-seedness in pomegranate is a desirable trait for fresh consumption of this valuable fruit. At the main Iran pomegranate collection, 21 pomegranate accessions gathered from different parts of Iran are registered as soft-seed genotypes. The aim of this research was to study these soft-seed pomegranate accessions using fruit morphopomological traits and DNA markers to reveal their relatedness. Thirty-six fruit characteristics were measured in these accessions together with applying 29 random decamer primers already reported to be polymorphic on pomegranate. Factor analysis on mean values of fruit characteristics determined 10 main factors and applied for grouping of the accessions using Ward's method. Also 14 of the random primers showed good amplification and polymorphism on these samples, and a total of 43 RAPD markers were produced. Estimates of genetic similarity, using Jaccard's similarity coefficient, ranged from 0.13 to 1.0 using the RAPD data. Grouping based on the fruit traits compared with that based on RAPD data did not produce a significant correlation (r = −0.36). Morphometric measurements and sensory evaluation confirmed that some accessions are hard or semi-hard seeded. This study showed that information based on fruit characteristics and RAPD markers are complementary for genetic discrimination in soft-seed pomegranate accessions. This might be due to the high level of similarity between soft-seed pomegranate accessions.     

Use of ISSR,SRAP, and RAPD markers to assess genetic diversity in Turkish melons

The genetic relationships among 63 melon (Cucumis melo L.) genotypes collected from various regions of Turkey were determined by comparing their molecular ISSR, SRAP, and RAPD markers with those of 19 foreign melon genotypes to investigate the taxonomic relationships and genetic variation of Turkish melon germplasm. Total 162 polymorphic markers (69, 18, and 75 obtained from ISSR, SRAP, and RAPD primers, respectively) were used to define the genetic similarity among the melon genotypes by dendrogram or two and three dimensional scalings. The average similarity (SM coefficient) between any two pairs of accessions examined as estimated by molecular variation was 0.73 ± 0.48. Within-group genetic similarities ranged between 0.46 and 0.96. Related genotypes or genotypes collected from similar regions were partitioned to similar clusters. Southeastern Anatolian genotypes were distinctly apart from group inodorus and group cantalupensis (sweet) genotypes. This reinforced the position of Turkey in the secondary genetic diversity center of melon. The genetic diversity among Turkish genotypes (H = 0.28 and I = 0.42) was only a little less than that of the world accessions (H = 0.30 and I = 0.45). On the other hand, the percentage of polymorphic loci among Turkish melon genotypes (90.7%) was even higher than that of the world accessions (87.6%).     

Comparative analysis of genetic diversity in Indian bitter gourd (Momordica charantia L.) using RAPD and ISSR markers for developing crop improvement strategies

A genetic analysis of 38 diverse Indian bitter gourd (Momordicacharantia var. charantia, and var. muricata) accessions was performed using 29 RAPD and 15 ISSR markers. RAPD primers yielded 208 amplicons of which 76 (36.5%) were polymorphic providing an average of 2.6 amplicons per primer. RAPD amplicons per primer ranged from 3 (OPE-19, OPW-09) to 15 (OPW-05), and varied in size from 200 bp to 3000 bp. Fifteen ISSR primers provided a total of 125 bands of which 94 (74.7%) were polymorphic. Polymorphic ISSR markers ranged from 0 (UBC-841) to 12 (UBC-890) providing a mean of 6.3 amplicons per primer that ranged in size from 150 bp to 2700 bp. Nevertheless, the concordance among bitter gourd accession groupings after cluster analysis was relatively high (r = 0.77), indicating that RAPD- and ISSR-based diversity assessments in this germplasm array were generally consistent. The M.charantia var. charantia (domesticated) and var. muricata (wild, free-living) accessions examined were genetically distinct, and these differences provided for the development of strategies for genetic analyses and crop improvement in this species.     

Use of ISSR markers to assess genetic diversity of African edible seeded Citrullus lanatus landraces

We used the twenty primers to evaluate the genetic variability of 80 individuals belonging to four accessions of edible seeded Citrullus lanatus originated from Côte d’Ivoire. Edible seeded C. lanatus, named “egusi” or “pistachio”, had a great importance in nutrition in West Africa. Nevertheless, due to its neglected status no study to our knowledge has been devoted to its genetic variability using DNA markers. The twenty ISSR primers generated 258 bands among which 252 were polymorphic (97.67%). On the whole, the bands generated revealed three types of profile sharply distinct from each other with minor differences within each type. One profile (P1) was most frequent with 65 individuals. Three accessions (NI084, NI127 and NI145) generated the three types of profile and had medium values of genetic diversity (GD = 0.246–0.275, respectively). On the opposite, the accession NI076 only contained individuals of the most represented type of profile (P1) and had the lowest genetic diversity (GD = 0.055 ± 0.017). The pairwise genetic distance between the 80 individuals varied from 0 to 0.61. The Factorial Component Analysis and the dendrogram clearly separated the 80 individuals into three clusters corresponding to the three types of profile. The results showed that clusters were well separated from each other whereas accessions were not. Our results suggest that high number of individuals should be taken into account for sampling missions and conservation strategies because accessions were not well differentiated from each other. Local agricultural practices consisting of frequent seeds exchanges between farmers and the conservation of harvested seeds for next year culture could be one explanation.     

Assessment of genetic diversity in cashew germplasm using RAPD and ISSR markers

Diversity and genetic relationship in 100 cashew germplasm accessions were analyzed by using RAPD and ISSR markers. Using 10 selected RAPD primers 60 bands were generated, of which 51 bands were polymorphic (85%), and with 10 selected ISSR primers 67 amplified bands were observed with 58 polymorphic bands (86.6%). Though both kinds of markers discriminated the accessions effectively, analysis of combined data of markers (RAPD + ISSR) resulted in better distinction of accessions. By combining markers, a total of 127 bands were detected, of which 109 bands (85.8%) were polymorphic and produced on an average of 5.45 polymorphic bands per primer. Primers with high polymorphic information content and marker index were identified for discriminating accessions. High percentage of polymorphism (>85%) observed with different markers indicated high level of genetic variation existing among the accessions. Genetic relationship estimated using similarity co-efficient (Jaccard’s) values between different pair of accessions varied from 0.43 to 0.94 in RAPD, 0.38 to 0.89 in ISSR and 0.43 to 0.87 with combined markers suggested a diversity (dissimilarity) ranging from 6 to 57%, 11 to 62% and 13 to 57% respectively and the diversity skewed around 50% indicated moderate diversity. The cluster analysis with UPGMA method separated the accessions broadly into 13 clusters and in that three into smaller clusters. Some correspondence between the molecular groupings and the morphological clusters were observed. Among the accessions, NRC-142 and NRC-12 were highly divergent and NRC-231 and NRC-232 were genetically similar.     

ISSR polymorphism in Indian wild orange (Citrus indica Tanaka,Rutaceae) and related wild species in North-east India

Inter simple sequence repeats (ISSR) polymorphism in Citrus indica Tanaka (Rutaceae), an endemic and threatened wild species, was examined along with three other closely related wild taxa (C. medica L., C. latipes (Swingle) Tanaka, and C. sp. ‘Memang athur’) by analyzing 53 representative accessions sampled from North-east India. Jaccard's similarity values among 53 accessions of Citrus ranged from 0.46 to 0.97 (average = 0.75). Genetic similarity values among all the 34 accessions of C. indica were found in the range of 0.82 to 0.97 with an average of 0.90. Heterozygosity (Ht = 0.123) and Shannon's information index (I = 0.188) values estimated for C. indica revealed significantly low level of genetic variation within the species. UPGMA dendrogram grouped all 53 accessions of Citrus into four major clusters: Cluster I – C. latipes; Cluster II – C. medica; Cluster III – ‘Memang athur’ and Cluster IV – C. indica. The dendrogram placed all the 34 accessions of C. indica in five sub-clusters under Cluster IV. The placement of C. indica accessions in various sub-clusters and groups in the dendrogram was based on molecular differentiation of individual accessions rather than their geographical origin. Very low genetic diversity and destruction of its natural habitat pose serious threat to C. indica even in the Citrus Gene Sanctuary in Nokrek Biosphere Reserve (NBR) in Meghalaya. Low genetic variability, heterozygosity and Shannon's information index in C. medica, C. latipes and ‘Memang athur’ are also concerns that need to be addressed for developing appropriate strategies to conserve the genetic diversity extant in these valuable genetic resources.     

Comparative analysis of genetic diversity in Tunisian apricot germplasm using AFLP and SSR markers

Eighty-one accessions representing apricot germplasm in Tunisia were collected from different areas of cultivation and fingerprinted using amplified fragment length polymorphism (AFLP) and microsatellites (SSR) markers. A total of 339 polymorphic markers were revealed using 5 AFLP primers combinations and 24 SSR loci. AFLP and SSR markers expressed a high level of polymorphism allowing the distinction of the accessions with an efficiency coefficient of discrimination of 100% for AFLP and 97% for SSR markers. Genetic diversity structure was assessed with AFLPs and SSRs markers separately then with combined matrix data by the help of hierarchical clustering elaborated using Wards method based on Nei and Li (1979) distances. Comparison of the obtained dendrograms revealed a phylogeographic structure into two major groups with significant conservation between the observed subgroups in relation with the geographic origin of the accessions. The relative efficiency of the markers in determining the genetic relationships among apricot accessions has been assessed and a combination of AFLPs and SSRs markers was the most effective. In addition, Mantel test based on genetic distances indicated highly significant correlation between AFLP-SSR data and each of the AFLP and SSR ones, with Pearson correlation values of r = 0.873 and r = 0.692, respectively, revealing the higher efficiency of the combination of both molecular techniques (AFLP and SSR) to estimate the levels of genetic variability among apricot germplasm.     

Genetic divergence among gerbera accessions evaluated by RAPD

Genetic diversity was evaluated by RAPD markers and morpho-agronomic characters for a total of 42 accessions of Barberton daisy (Gerbera jamesonii) consisting of 29 commercial and 13 wild accessions. A total of 74 polymorphic bands were obtained employing a set of 12 primer pairs. The average genetic similarity coefficient for the 42 accessions, evaluated by Jaccard index was 0.55 ranging from 0.28 to 1.00. The genetic structure found among Barberton daisy accessions was evaluated by hierarchic classification analyses and UPGMA modeling, revealing six clusters of genotypes where two of them include the wild accessions and the remaining four including commercial material, except for wild genotype number 9. Shannon (H′) index was calculated using the molecular markers to investigate the genetic variation among the Gerbera accessions and showed higher values for the commercial cluster in comparison to the values obtained for the individuals from the non-commercial cluster, namely 0.34 versus 0.27, respectively. Therefore, both calculated indices (Jaccard and Shannon) indicated the presence of higher genetic variation among commercial accessions in comparison to the cluster representing non-commercial accessions, suggesting that genetic breeding programs may focus on commercial accessions to recombine interesting genotypes with commercially important and marketing-desired characteristics.     

Are rhododendron hybrids distinguishable on the basis of morphology and microsatellite polymorphism?

Sequence Tagged Microsatellite Sites (STMSs) and morphological trait markers were used to evaluate 33 rhododendron germplasm for genetic diversity assessment and discrimination power. The average genetic diversity estimates were 0.724 (morphological traits) and 0.174 (STMSs) marker datasets. The Shannon index was higher for morphological traits (1.797) than STMS (0.302). The correlation coefficients obtained by the Mantel matrix correspondence test, which was used to compare the cophenetic matrices for the two markers, showed that estimated values of relationships given for morphological and STMS were not significantly related (p > 0.05). The dataset from STMS, supported by the total probability of identity (1.13 × 10⁻⁹) and total paternity exclusion probability (0.9999), allowed all accessions to be uniquely identified. In summary, STMS marker proved to be an efficient tool in assessing the genetic variability among old broad leaf rhododendron genotypes. The pattern of variation appeared to be consistent, and it can be used for germplasm conservation and management for restoration of historical genetic resources.     

Assessment of genetic diversity in common bean (Phaseolus vulgaris L.) germplasm using amplified fragment length polymorphism (AFLP)

AFLP technique was applied to assess genetic diversity among 44 common bean accessions that included 6 exotic accessions, 15 Indian land races and 23 released varieties. Eight AFLP primer pairs were used that produced 820 products of which 698 were polymorphic (85.12%). Wide variations were observed among all the accessions for the number of amplification products, percent polymorphism and average polymorphism information content (PIC). The Jaccard's similarity indices (J) based on the AFLP profiles were subjected to UPGMA cluster analysis. The dendrogram generated revealed seven major groups. Seventeen out of 23 released varieties were restricted to clusters VI and VII. The value of r = 0.934 in Mantel's test for cophenetic corrlelation applied to the cluster analysis indicated the high fitness of the accessions to a group. The germplasm used in the present study had narrow genetic base, although moderate to high genetic diversity was observed. The details of diversity analysis and the potential use of Indian common bean accessions in common bean breeding programme are provided in the present study.     

A comparative analysis of the genetic diversity between inbred lines of Zinnia elegans using morphological traits and RAPD and ISSR markers

Genetic diversity within Zinnia elegans is key to the genetic improvement of this important ornamental species. Here, morphological traits and RAPD and ISSR molecular markers were used to assess levels of polymorphism across 20 inbred lines. Thirty-four morphological traits were scored and also 147 RAPD marker-fragments, as amplified by 12 arbitrary primers, and 128 ISSR marker-fragments as generated by 9 primers. The number of polymorphic loci, the percentage of polymorphic loci, Shannon's Information index (I) and the effective number of alleles (Ne) were calculated from the RAPD data as 100, 68.03%, 0.3559 and 1.4169, respectively. From the ISSR data, these respective statistics were calculated as 97, 76.38%, 0.4013 and 1.4728. Thus, ISSR markers were considered slightly superior to RAPD markers for assessing genetic diversity between the accessions; however, Mantel's test indicated significant correlation (R = 0.733) of the RAPD and ISSR results. By contrast, the morphological matrix showed low correlation with both RAPD and ISSR data matrices (R = 0.3814 and 0.3765, respectively). Cluster analysis showed that groupings of the accessions according to all three methods correlated well with their geographic region of origin, but flower color was not strongly associated with the genetic classification of these inbred lines of Z. elegans.     

Inter- and intra-specific genetic diversity among cyclamen accessions investigated by RAPD markers

Random amplified polymorphic DNA (RAPD) analysis was used to characterize genetic diversity of 26 Cyclamen persicum and Cyclamen com accessions. Eighty-four arbitrary primers tested, among which nine primers showed reliable polymorphic banding patterns and yielded 104 polymorphic markers. Jaccard's similarity coefficient among accessions ranged from 0.99 to 0.08. At a similarity of 68%, accessions were divided into three clusters. The cophenetic correlation coefficient between the similarity matrix and the dendrogram was relatively high (r = 0.9) showing the goodness of fit of the dendrogram. The RAPD analysis offered a rapid and reliable tool for the estimation of inter- and intra-specific variability in cyclamens. The wide genetic variation observed for cyclamens within Iran guarantees a promising future of breeding.     

Genetic relatedness (diversity) and cultivar identification by randomly amplified polymorphic DNA (RAPD) markers in teasle gourd (Momordica dioica Roxb.)

Genetic relationship and variation of 29 accessions of teasle gourd (Momordica dioica Roxb.) and 1 accession of Momordica cochinchinensis Spreng. (wild relatives of teasle gourd) were examined by RAPD analysis using 44 dodecamer oligonucleotide primers. A total of 496 fragments were produced by 44 primers of which 95% bands were polymorphic. Using presence or absence of specific RAPD markers or combination of primers, 23 out of 30 accessions were identified. The genetic relatedness or genetic distance based on Nei and Li's genetic similarity varied from 0.86 to 0.65 with an average of 0.74 among 29 M. dioica accessions (when M. cochinchinensis excluded). In the phenetic dendrogram developed from cluster analysis using UPGMA method, M. cochinchinensis was out grouped as single accession, while others showing relatively weak grouping formed four groups. Clustering pattern did not demonstrate any relationship between geographical origin and genetic diversity. A DNA extraction method has been standardized. This is the first report of using RAPD techniques in teasle gourd. It was concluded that RAPD analysis is a useful tool for genotypic identification and estimation of genetic similarity in teasle gourd.     

Genetic characterization of different demes in Prunus persica revealed by RAPD markers

DNAs of 180 accessions in 10 demes in Prunus persica were amplified with twenty-two, 10-base primers selected from 200 arbitrary primers using Randomly Amplified Polymorphic DNA (RAPD) technology. One hundred and eighty loci were observed and recorded. With statistical analyses of the data from the study, genetic diversity of the demes was expressed as follow: yellow peach group > honey peach group > flat peach group > red leaf peach group > crisp peach group > bitao group and juicy peach group > nectarine group > shouxingtao group > weeping peach group. Genetic variations among and within groups by AMOVA analyses were 11.9, 88.1%, respectively. Demes clustered by UPGMA modified from NEIGHBOR procedure of PHYLIP Version 3.5, the edible peaches of which were combined as a section, while the ornamental species were classified into separate sections. Through analyses of genetic diversity and genetic structure, the results could provide molecular biological evidence for conservation and utilization of P. persica germplasm.     

High-frequency embryogenesis,regeneration of broccoli (Brassica oleracea var. italica) and analysis of genetic stability by RAPD

High-frequency somatic embryogenesis and shoot regeneration of broccoli (Brassica oleracea var. italica) were achieved. Cotyledon and hypocotyl explants from four varieties of broccoli were cultured on MS and modified MS media (mMS, supplemented with PG-96 organic components) with different combinations of growth regulator. The effects of genotypes, different explants, growth regulator combinations, organic components and AgNO₃ on induction of calli and shoots were evaluated. The optimal media for inducting calli/shoots and roots were mMS medium containing 3% (w/v) sucrose and 0.8% (w/v) agar supplemented with NAA at 0.5 mg l⁻¹, 6-BA at 3.0 mg l⁻¹, AgNO₃ at 4.0 mg l⁻¹ and MS medium containing 3% sucrose and 0.8% (w/v) agar supplemented with NAA at 0.2 mg l⁻¹, respectively. The callus induction percentages were over 90% in all four varieties; shoot induction percentage was 92.5% and the average number of shoot per explant was 4.1 from cotyledon explant in variety Bishan. In this study, we established high-efficient embryogenesis and shoot regeneration system of broccoli and analyzed genetic stability of regenerants at DNA level using RAPD molecular marker. Out of 62 arbitrary primers screened using PCR amplification, 79 polymorphic bands were amplified from 20 primers. The results demonstrated the genetic stability of regenerants from the same variety.     

Genetic diversity in Indian ash gourd (Benincasa hispida) accessions as revealed by quantitative traits and RAPD markers

In present study, an attempt has been made to assess the genetic diversity among 34 accessions of Benincasa hispida using quantitative traits and RAPD data. Variability was observed for characters like days to first female flower, fruit weight, equatorial and polar diameter. Cluster analysis based on quantitative traits revealed a high degree of diversity among the accessions. RAPD-based dendrogram showed dissimilarity values 0.64–0.943 suggesting that the accessions represent a genetically diverse population. A non-significant correlation was observed between the clustering based on quantitative traits and RAPD markers. But the accessions like IVAG-107 and IVAG-81 clearly separated from rest of the accessions based on the quantitative traits and as well as RAPD primers. Both the dendrogram revealed that the accessions from northeastern region of India are diverse from the accessions of other parts of India, as they cluster together in both the dendrogram. The information generated could be utilized in developing cultivars/hybrids for specific traits.     

Investigation of physico-chemical properties and antioxidant activity of twenty Iranian pomegranate (Punica granatum L.) cultivars

Pomegranate is one of the native fruits of Iran which contains high genetic resources, but there are insufficient information regarding properties of the fruit. The objective of the present study was to investigate the physcio-chemical characteristics and antioxidant activity of twenty pomegranate cultivars grown in Iran. This study showed that there were significant differences among the cultivars in all measured factors except the length/diameter ratio of fruit. The fruit weight, skin percentage, aril percentage and juice percentage were within the range of 196.89–315.28 g, 32.28–59.82%, 37.59–65% and 26.95–46.55%, respectively. The total soluble solids content varied from 11.37 (°Brix) to 15.07 (°Brix), pH values from 3.16 to 4.09, titratable acidity content from 0.33 g 100 g⁻¹ to 2.44 g 100 g⁻¹ and total sugars content from 13.23 g 100 g⁻¹ to 21.72 g 100 g⁻¹. The results also showed that the values of ascorbic acid ranged from 9.91 mg 100 g⁻¹ to 20.92 mg 100 g⁻¹. The total anthocyanins content was observed in pomegranate cultivars between 5.56  mg 100 g⁻¹ and 30.11 mg 100 g⁻¹. The level of total phenolics was varied from 295.79 mg 100 g⁻¹ to 985.37 mg 100 g⁻¹. The antioxidant activity of pomegranate cultivars was found between 15.59 and 40.72%. These data demonstrated that the cultivar was the main parameter which influences the physico-chemical properties and antioxidant activity in pomegranates.     

Fingerprinting of three typical macrosperma Italian lentil (Lens culinaris Medik.) landraces using fluorescence-based AFLP markers

Italian lentil landraces are principally cultivated for self or local consumption. Most of them are disappearing, particularly macrosperma types by being less required by the market. A pre-requisite for the conservation and the efficient use of genetic resources is the better understanding of the extent and the distribution of the existing genetic variation, useful for future breeding programmes. Our study was undertaken to analyse and quantify the genetic diversity within and among three macrosperma Italian lentil landraces (Onano, Altamura and Villalba), using fluorescent AFLP markers. AFLP markers generated information to differentiate among closely related genotypes and group within the same cluster individuals belonging to the same landrace. The total genetic diversity (H_T), the genetic diversity within population (H_S) and the extent of differentiation between populations (D_ST) were 0.198, 0.155 and 0.043, respectively. The fixation index (G_ST = 0.219) showed that about 78% of the observed total genetic variation can be attributed to within population differences and around 22% is due to differences among populations. The gene flow estimate (N_m = 1.774) and the mean genetic distance value (0.077) suggested narrow genetic base among the analysed populations, confirming the tendency of Italian lentil landraces to group together. The present study showed that fluorescence-based AFLP technique is a biotechnological tool that can provide significant insights for research in genetic diversity of lentil landraces and their subsequent conservation and utilization in breeding programs.     

Genetic diversity of Chinese natural bermudagrass (Cynodon dactylon) germplasm using ISSR markers

Bermudagrass (Cynodon dactylon L.) is an important warm-season turfgrass. Although it is widely distributed in China, studies on the genetic variation and relationship among the large-scale indigenous bermudagrass were relatively insufficient, especially at molecular level. The purpose of this study was to assess the molecular variation and relationship among one commercial cultivar ‘Tift3’ and 95 wild bermudagrass accessions collected from 11 provinces in China by ISSR marker technique. The results indicated that 29 ISSR primers generated a total of 248 bands among which 242 (97.6%) were polymorphic bands. The genetic similarity coefficients among accessions ranged from 0.51 to 0.97 with an average of 0.74. All accessions could be clustered into 11 groups with UPGMA method. Accessions from the same or adjacent regions generally were clustered into the same group or subgroups. A few accessions, however, were greatly different from the majority of all accessions. The results suggested that ISSR marker is an effective tool for the study of genetic variation in Chinese natural bermudagrass.