Analysis on genetic diversity of wine grape ‘Cabernet Gernischt’ (Vitis vinifera) new strains by SSR, ISSR and VvmybA1

Abstract: 【Objective】‘Cabernet Gernischt’(Vitis vinifera L.) is one of the most widely grown red grapefor making wine in Jiaodong Peninsula of Shandong province The aim of this study is to investigate geneticdiversity of eight new strains of‘Cabernet Gernischt’(E01-E08) for screening new excellent clone(s).【Methods】Young leaves of‘Cabernet Gernischt’(E01-E08),‘Cabernet Sauvignon’‘Cabernet Franc’and‘Pinot Noir’were collected from Changyu garden. The total genomic DNA was extracted by usingEzup Column Plant Genomic DNA Purification Kit. 14 SSR (simple sequence repeats) primers and 11ISSR (inter-simple sequence repeat) primers were employed according to previous study. SSR and ISSRdata were scored with presence (1), absence (0) and each band were regarded as a locus. Jaccard similari⁃ty matrix was constructed by NTSYS-pc 2.1 software. Based on the similarity matrix, a dendrogram showing the genetic relationships between the strains and cultivars was constructed by using the unweightedpair group method with arithmetic average (UPGMA). The number of loci (N), effective number of alleles(Ne), Nei’s gene diversity (H), Shannon’s information index (I) and percentage of polymorphic loci (P)were calculated by using POPENE1.32. VvmybA1 product was amplified by using primers of K and S.PCR amplification products that yielded single, well-defined band were harvested for sequencing. The pu⁃rification was performed with SanPrep Column DNA Gel Extraction Kit. The VvmybA1 sequences werealigned with sequence data obtained by using BioEdit version 7.0.5 and ClustalX 2.0. Phylogenetic treesbased on Neighbor-Joining methods were created by using MEGA 5.0. Three neutrality tests were con⁃structed with DnaSP 5.0.【Results】A total of 139 bands were amplified from 14 SSR primers, of which 69were polymorphic loci. The effective number of alleles (Ne) of the SSR primers ranged from 1.021 6(VMC4F3) to 1.590 1 (VrZAG25). The Nei’s gene diversity (H) ranged from 0.019 5 (VMC4F3) to 0.322 0(VrZAG25). Shannon’s information index (I) ranged from 0.040 5 (VMC4F3) to 0.467 3 (VrZAG25). Thegenetic similarity coefficients between stains and cultivars ranged from 0.567 7 to 0.974 4. Eight strains of‘Cabernet Gernischt’were divided into 2 groups based on UPGMA. The first cluster had only‘CabernetGernischt’E06, and the second cluster contained seven strains. A total of 96 clear DNA fragments wereamplified from 11 ISSR primers. The mean similarity index of all strains and cultivars was 0.825 6, andranged from 0.734 7‘( Cabernet Gernischt’E04 and‘Point Noir’) to 0.938 8‘( Cabernet Gernischt’E02and‘Cabernet Gernischt’E03). The effective number of alleles (Ne) of the ISSR primers ranged from1.072 1 (UBC817) to 1.584 8 (UBC807). The Nei’s gene diversity (H) ranged from 0.062 8 (UBC826) to0.335 1 (UBC835), and Shannon’s information index (I) ranged from 0.090 2 (UBC826) to 0.505 7(UBC835). The cluster dendrogram was constructed by UPGMA.‘Cabernet Gernischt’E02 (north of Qix⁃ia) and E03 (south of Qixia) classified into one group. The genetic distance between them accorded withthe geographic distance. E05 (Laishan) and E08 (Development zone) were classified into one group, Therewere significant differences among E01, E04, E06 and E07. The lengths of the VvmybA1 gene sequence of11 strains and cultivars were 905-914 bp, the GC contents varied from 42.1% to 43.5%. The VvmybA1gene sequence of 11 strains and cultivars showed a low level of diversity (Hd=0.673, Pi=0.002 21). Theanalysis of VvmybA1 gene fragment sequence alignment showed that there was only a deletion in coding regions, but there were five base substitutions in non-coding regions. The results of three neutralities indi⁃cated that there was a non depature from neutrality expectation for gene VvmybA1 of 11 strains and cultivars According to the phylogenetic tree of the gene sequence, analysis of all polymorphisms did not pro⁃vide a consensus tree depicting the geographical partitioning of the species. By analyzing the differencesamong VvmybA1 gene fragments for investigating the genetic diversity of‘Cabernet Gernischt’, it was concluded that VvmybA1 genes of‘Cabernet Gernischt’E01, E02, E04, E05, E06 and E07 had close relationship, which could be clustered into one group. VvmybA1 genes of‘Cabernet Gernischt’E03 and E08 weredifferent from those of the other six strains.【Conclusion】The results revealed that ISSR could be a goodtool for evaluation of genetic diversity among the grape strains and cultivars. It was suggested that the ge⁃netic distance between the different strains should be firstly considered rather than the geographic distance for identifying the different strains of‘Cabernet Gernischt’. The VvmybA1 gene sequences of 11strains and cultivars showed a low level of diversity, and should not be used as genetic markers for strainidentification of‘Cabernet Gernischt’.