A Genetic Research On The Relationshipe Of Chinese Grape Germplasam

Vitis vinifera L. is one of the oldest plants in the world, being prevalent and having lotsof varieties. There are over70species existing on the earth, distributing in Eurasia, NorthernAmerica, and East Asia. As one of the most important exporting countries, China hasabundant wild grape resources native to China, accounting for60%of the total number of theworld, including39species, one subspecies, and thirteen varieties. We herein analyzed therelationships of twenty-four species of Vitis germplasm resources, up to thirty nine accessions(including twenty two Chinese wild species,36accessions). The main conclusions werelisted as following,1. The genetic relationships of twenty two species of Chinese indigenous grapes,totally36accessions, were analyzed by Sequence-Related Ampified Polymorphism(SRAP)markers,with Pinot Noir and Thomson of Vitis vinifera L. treated as referenced genetypes.25SRAPmarkers, capable of amplifying clear, reproducible and polymorphic bands, were screenedout from72pairs of SRAP universal primer. A total number of135sites were produced bythe25SRAP markers, among which106were polymorphic with variation of78.3%. And5.52sites were produced by each pair primer with4.32polymorphism loci. Based on theamplified profiles of SRAP, the genetic similarity coefficients (SM) of39grape accessionsvaried from0.453to0.991using NTSYS pc2.10e software, which showed abundantpolymorphisms of Chinese wild grapes. According to the clustering dendrogram generated bythe set of25SRAP markers, all grape accessions in this study were clustered into21classesand clearly differentiated from each other when SM was0.83, and also these clusteringresults were consistent with the morphological classification results of grapes reported before.Besides, in our study, V. heynean subsp. ficifolia and V. baihensis were classified into V.heyneana and V. bashanica, separately, and also we questioned whether V. davidiivar.cyanocarpa was a variety of V. davidii.2.18cpDNA non-coding regions from39accessions of the genus Vitis were amplified with cpDNA universal primers, and only single bands were produced from all targetedregions except for trnTUGU-trnLUAAspacer regions. And then the amplified PCR products of15out of20intergenic spacer regions were separately digested with several restrictionendonucleases, which showed the results as following: Low polymorphisms were presentedby PCR-RFLP that only13of80components of primer/restriction identified variable loci incpDNA of grape accessions in this study. By the analysis of UPGMA,39accessions of Vitiswere classified into two main clusters, and only a few interspecific and even intraspecificdifferences were identified among several genotypes of Chinese wild grapes. A small numberof variable sites represented the conservation in cpDNA of grape and limitations on theapplication of PCR-RFLP to identify relationships of the genus Vitis on genus and specieslevel.10primer sets, aimed to amplify the special cpDNA regions of grapes, were developedin this study, among which4intergenic spacer regions showed polymorphism after beingdigested by restriction endonucleases.3. Sequenced trnTUGU-trnLUAAspaces from39genotypes of the genus Vitis revealedthat nucleotide sequences of39genotypes were between837bp and1005bp in length, and theconsensus length was1008bp after aligning by MEGA5.0package. By comparing nucleotidesequences with each other, the average nucleotides comprised with A(32.4%), T(39.7%),C(16.4%) and G(11.6%), and94.4%of the nucleotide sequences from39accessions wereconsensus.55variable sites were identified, including19singleton sites(S) and36parsimonyinformative(Pi) sites, and the nucleotide polymorphism was0.02069. Among39Vitisindividuals,19insertions and deletions (InDel) sites were found with polymorphism (Hd) of1.776. The Tajima's D test (0.6747) was unremarkable and so rejected neutrality assumptionin the whole studied sample. And also we found19haploid with polymorphism of0.818. Thedendrogram, generated with Neighbor-Joining by MEGA5.0package based on the variablesites in trnTUGU-trnLUAAspaces, showed that all the grape accessions formed two clusters.Except for V. qinlingensis having distant genetic relationships with others, the remainingmaterials could not be discriminated with each other for their low bootstrap support values.4.31univeral mtDNA primers were applied to amplify the non-coding regions inmtDNA from39accessions of Vitis, and the results were listed as below:19pairs mtDNAprimers just produced single bands, and the rest primers of31pairs generated59bands,among which43were informative with polymorphic rate of71.2%and an average number of3.5bands produced by each pair of primer.The results of genetic similarity analysis thatsimilarity coefficients of Chinese wild grape resources varied between0.627and0.98indicated apparent genetic diversities existing in this grape group, which was probably due torampant gene recombination during the process of their evolution. Dendrogram based on the data of mtDNA was greatly consensus with the one based on SRAP markers, which all coulddistinguished most varieties of Chinese wild grape and identified V. riparia has the leastrelationship with the remaining studied grape accessions. Besides, the two dendrogramsshowed another similar result that both V. davidii var.ningqiangensis and V. davidiivar.cyanocarpa had a little relationship with V. davidii. While the differences were thatdendrogram based on mtDNA discriminated V. qinlingensis as a kind of older species inChinese wild grapes and did not disdinguished the relationship between V. heyneana andV. heyneana subsp. ficifolia.