Genetic diversity assessment of Vitis ficifolia Bge. populations from Henan province of China by SRAP markers

Eighteen sequence-related amplified polymorphism (SRAP) primer combinations were used to assess the genetic diversity of 126 individuals from five different geographical populations of Vitis ficifolia Bge. The numbers of bands scored per primer combination ranged from 8 to 27, with an average of 18.6 bands. At the population level, the percentage of polymorphic bands (PPB), Nei's gene diversity index (H) and Shannon's information index (I) were the highest in the Shihe (Xinyang) population (77.31%, 0.1987, 0.2805) and the lowest in the Linzhou (Anyang) population (55.82%, 0.1112, 0.1727). At the species level, PPB, H and I were 80.56%, 0.2129 and 0.3075, respectively. The genetic differentiation coefficient (G ST) was 0.2055 and the gene flow (Nm) was 1.9328, indicating strong intra-population genetic differentiation. Based on the unweighted pair group method based arithmetic average clustering diagram, the five studied populations may be divided into three groups. The clustering results were almost in accordance with the populations’ geographical distribution.


Introduction
The genetic diversity of plant species is a major concern for geneticists and plant breeders. [1] The current methods for measuring genetic diversity of populations and germplasm collections are often conducted through statistics calculated from molecular marker data. [2] Analysis of the genetic structure at an intra-specific level is important for better understanding of future adaptive changes or evolution, [3] and also for future breeding programmes.
Grapevine (Vitis spp.) is one of the most important fruit crops worldwide with more than 70 species of Vitis spp. in the world.[4À6] More than 38 Vitis species have their origin in China, making China one of the major gene centres of Vitis species origination. [5] Chinese wild grape species are of particular interest in grape breeding for their desirable characteristics, such as disease resistance, drought tolerance and cold hardiness genes. [5,7,8] V. ficifolia Bge. is a subspecies of V. heyneana Roem. et Schult, and is distributed mainly in the northern and eastern China, at altitudes between 100 and 1300 m, [5] especially in Henan Province in Central China. V. ficifolia has excellent tolerance to anthracnose, ripe rot diseases and winter cold, and is used both as table grape and in wine production.
One of the most powerful tools for analysis of genomes is molecular genetic markers. They allow for heritable traits to be associated with underlying genomic variation. [9] Sequence-related amplified polymorphism (SRAP) is a polymerase chain reaction (PCR)-based marker system targeting open reading frames (ORFs). [10] With reproducible results, high reliability, simple technology and low cost, SRAP has been applied in the assessment of the population structure and genetic diversity of many fruit species. [11À18] In this study, SRAP analysis was employed to investigate: (1) the levels of SRAP variations in V. ficifolia Bge. populations; (2) the levels of genetic diversity within and among populations and (3) the genetic relationships among populations.

Plant materials
A total of 126 V. ficifolia Bge. individuals of five different wild populations were selected from Henan Province of China. Their places of origin and distributions are shown in Table 1 and Figure 1.

DNA isolation
Total genomic DNA was isolated from fresh leaves following the procedure previously described by Liu et al. [16]. The quality and concentration of the DNA samples were checked in a biophotometer plus (Eppendorf, Germany) and a portion of the DNA was diluted to 50 ng/mL for subsequent SRAP analysis. Both the stock and diluted portions were stored at ¡20 C.

SRAP analysis
The SRAP analysis was carried out using 26 primers including 13 forward (Me1-13) and 13 reverse (Em1-13) SRAP primers 11-18, [10] for a total of 169 primer combinations. The primer sequences were synthesized by Shanghai Biological Engineering Technology and Service Co. Ltd. Each 10 mL PCR reaction mixture consisted of 30À50 ng of template DNA, 0.2 mmol/L of deoxynucleoside triphosphates (dNTPs), 1.0 mmol/L of each primer, 2.0 mmol/L of Mg 2C and 1 U Taq DNA polymerase (TaKaRa). The amplification reaction procedure was as follows: after denaturation at 94 C for 5 min, the reaction mixture was subjected to amplification for seven cycles consisting of 90 s at 94 C, 75 s at 35 C, 30 s at 72 C, followed by 30 cycles consisting of 60 s at 94 C, 60 s at 55 C and 90 s at 72 C, with a final extension at 72 C for 5 min.

Data analysis
Amplified bands were scored as either present (1) or absent (0). The data were entered into a binary matrix and, subsequently, POPGENE 32 software was used to compute the population genetic parameters, the percentage of polymorphic bands (PPB), Shannon's information index (I), Nei's gene diversity (H), genetic identity and genetic distance, etc. AMOVA 30 software was used to estimate the relative genetic differentiation coefficient (G ST ) and the level of gene flow (N m ). Cluster analyses were conducted on the similarity matrix with the unweighted pair group method based arithmetic average  (UPGMA), and the resulting clusters were expressed as dendrograms.

Results and discussion SRAP polymorphism
Eighteen informative primers were selected due to their ability to produce clearly and repeatedly polymorphic and unambiguous bands among the accessions ( Table 2). As shown in Table 3 SRAP was initially developed for Brassica species, and was tested in other crops. In genetic diversity analysis, the information given by SRAP markers is concordant with the morphological variability. [16,19] The detection of high levels of polymorphism makes SRAP analysis a powerful tool for assessment of genetic diversity in many species. [15,20,21] To the best of our knowledge, the present study is the first report of genetic investigation of V. ficifolia Bge. using SRAP markers. The results indicated that SRAP markers could be used efficiently in the genetic diversity and genetic variability of V. ficifolia Bge.

Genetic diversity
Genetic polymorphism may be indicative of evolutionary adaptation which plays a key role for survival of a population in the changing environment. [22] Accurate estimates of genetic diversity are particularly useful for optimization of sampling strategies and for conservation and management of the genetic diversity of trees. [23] The estimates of the genetic diversity in each population are summarized in Table 3 Table 3.

Genetic differentiation and gene flow
Genetic differentiation and gene flow are important indices for evaluation of the genetic structure of a group. Based on the analysis by AMOVA 30 software, the relative genetic differentiation coefficient (G ST ) was 0.2055,  which showed that the major genetic variation (72.95%) originated from intra-population diversity, and the rest (20.55%) existed among populations. The level of gene flow (N m ) is a key factor that affects the genetic structure and genetic differentiation among populations. Based on the AMOVA analysis, the level of gene flow (N m ) among the five populations was 1.9328. The results obtained here showed that a high proportion of the variability was due to intra-population variability, and this high genetic variability was consistent with the previous results obtained by morphological analysis. [24] Gene flow is reversely correlated with the group genetic differentiation. Also, gene flow is very important for the dispersal and evolution of plant populations. In seed plants, gene flow is mainly achieved by seeds or pollen carrying foreign genes between groups. [25] When N m > 1.0, this indicates that the level of gene flow is higher; when N m > 5.0, the outcrossing rate is higher. [26] In this study, the gene flow among populations was high (N m D 1.9328). This indicated that high-level gene flow existed within V. ficifolia populations. This result was consistent with the previous results. [22,27À31] The reason for slightly higher G ST may lie in the morphological character of the wild V. ficifolia, with the vines growing up into tall plants, and the tall plants benefitting due to reduced resistance to pollen movement in the air. Moreover, a lack of effective geographic isolation may also contribute to the improvement of gene homogenization among populations. [32] Widely distributed plant species must adapt to a broad range of environmental conditions to maintain their large geographic distributions. [33] Consequently, many widespread species have high genetic diversity or exhibit considerable phenotypic plasticity. [34,35] V. ficifolia has wide distribution in northern and eastern China, especially in Henan Province. However, it should be noted that the V. ficifolia populations used in this study were sampled from a narrow eco-geographical area of distribution.

Genetic similarity
In genetic diversity analysis, there are various genetic distance measures for analysis of molecular marker data. Genetic identity is an important index for estimation of the genetic differentiation among populations. Based on our POPGENE analysis, the genetic identity and genetic distance between each two populations, respectively, ranged from 0.9171 to 0.9825 and from 0.0176 to 0.0866. This suggested that there was a high genetic similarity between the populations, indicating lower reproductive isolation among the populations ( Table 4). The minimum genetic identity occurred between SH and LZ; the maximum one occurred between LN and XA; and SH population constituted the farthest genetic relationship with the other four populations.

Cluster analysis
The dendrogram obtained with SRAP data is shown in Figure 2. The five populations may be divided into three groups. The populations located nearest to each other, NZ, XA and LN, were classified as the first group. Then, they clustered with population LZ, and population SH in southern Henan formed a separate group. The genetic relationships between populations of a widespread species often do not correspond to their geographical distance. [36,37] However, Ma et al. [33] found that most of the Elymus sibiricus populations studied by them clustered in accordance with the geographic distribution. In our study, the clustering results were almost in accordance with the geographical distribution.
The in situ method allows continuing evolution of the species in its natural habitat and then guaranteeing the maintenance of most of the species' genetic variation. In this study, population SH showed relatively high genetic diversity within V. ficifolia Bge., and should therefore be a priority site for in situ conservation.

Conclusions
The present study is, to the best of our knowledge, the first report of genetic investigation of V. ficifolia Bge., using SRAP markers. The results indicated that SRAP markers could be used efficiently in the study of genetic diversity and genetic variability of V. ficifolia. High-level gene flow existed within V. ficifolia populations. The clustering results based on SRAP were almost in accordance with the geographical distribution of the populations. In our study, the Shihe population (SH) showed relatively high  genetic diversity within V. ficifolia Bge., and should therefore be a priority site for in situ conservation.