Genetic Diversity For New Strains Of Vitis Vinifera Cabernet Gernischt

Grape(Vitis vinifera L.)have long cultivated history and extensive fruit area.It also has important economic value.Cabernet Gernischt(Vitis vinifera L.cv.),a chinese characteristic cultivars,is the most widely grown red grape in Jiaodong Peninsula of Shandong province,and is one of the main raw grape and wine production bases in China.For more than 100 years,the botanical characters of Cabernet Gernischt have shown obvious differences in different areas,and several new strains have been formed,but the problem of degeneration has gradually appeared.Therefore,the genetic diversity of wine grape play an important role in breed selection and germplasm resources protection and management.In this paper,the morphological methods were used to describe the varieties of grapevine,and the genetic diversity of wine grape was analyzed by SSR,ISSR,ITS and other molecular markers.The results are as follows:(1)According to ampelography,a number of indexes were chosen as the morphological basis.Shape of base of upper sinuses,shape of base of petiole sinus,bunch weigh,bunch density,thickness of bloom and soluble solid content show the diversity of different level.Cabernet Gernischt E04(Xingfu)has the most economic value.Used the method of Galet's grape leaf shape structure numerical analysis method,the result show that The results showed that the genetic relationship between E05(Laishan)and E06(Longkou)was the closest.(2)14 SSR primers were screened with clear bands and good polymorphism.A total of 139 bands were amplified from 14 SSR primers,of which 69 were polymorphic loci.Effective number of alleles(Ne)of the SSR primers ranged between 1.0216(VMC4F3)and 1.5901(VrZAG25);Nei's gene diversity(H)from 0.0195(VMC4F3)to 0.3220(VrZAG25);Shannon's Information index(I)from 0.0405(VMC4F3)to 0.4673(VrZAG25).The genetic similarities coefficient between cultivars was from 0.5677 to 0.9744.Eight new strains of “Cabernet Gernischt” were divided into 2 groups based on UPGMA.The first cluster had only Cabernet Gernischt E06,and the second cluster contained the other seven strains.(3)11 ISSR primers selected generated a total of 96 clear DNA fragments.The mean similarity index of all cultivars was 0.8256,and ranged from 0.7347 to 0.9388.Effective number of alleles(Ne)of the ISSR primers ranged between 1.0721(UBC817)and 1.5848(UBC807);Nei's gene diversity(H)from 0.0628(UBC826)to 0.3351(UBC835),the average of 0.1858;and Shannon's Information index(I)from 0.0902(UBC826)to 0.5057(UBC835),the average of 0.2754.The clustering dendrogram was constructed by UPGMA,Cabernet Gernischt E02(North of Qixia)and E03(South of Qixia)classified into one group,genetic distance in accord with geographic distance,E05 and E08(Development Zone)classified into one group,There were significant differences between E01(Penglai),E04,E06 and E07(Development Zone).(4)Length of the Vvmyb A1 gene sequence of 11 cultivars was 905-914 bp.The GC content varied from 42.1% to 43.5%.The VvmybA1 gene sequence of 11 cultivars showed a low level of diversity(Hd=0.673,Pi=0.00221).The analysis of VvmybA1 gene fragments sequence alignment shows that there is only a deletion in codingregions,but five base substitutions were found in non-codingregions.The results of three neutralities indicated that there is a non depature from neutrality expectation for gene VvmybA1 of 11 cultivars,which reflected between equilibrium bwteen drift and selectively neutral mutation.According to the phylogenetic tree of the gene sequence,analysis of all polymorphisms did not provide a consensus tree depicting the geographical partitioning of the species.(5)ITS2 of DNA barcoding was used to study genetic polymorphism of Cabernet Gernischt eight new strains.The sequence of ITS2 was 425bp~459bp.The GC content varied from 66.6% to 67.7%.The maximum intraspecific genetic distance was 0.0037,and the maximum species genetic distance was 0.0114.Cabernet Gernischt E01 and E02 classified into one group,E04 and E07 classified into one group,E05 and E08 classified into one group.(6)The carboxylesterase gene fragments were amplificed and sequenced.Analysis of the differences among Carboxylesterase gene fragments was done to investigate the genetic diversity in Cabernet Gernischt and other wine grape cultivars.The alignments showed that there were different degrees of base substitutions between the samples in the coding and non-coding regions.A total of 87 SNPs were detected in the sequenced fragments,a SNP locus was found on every 74 bp sequence on average.Nucleotide diversity(Pi)with a value of 0.05076(±0.01149),and Haplotype diversity(Hd)with 1.000(±0.039)give a high leve1 of polymorphism.Three neutrality tests had been used to compare the patterns of sequence variations,which indicated that there was no departure from neutrality expectations.The clustering dendrogram was then constructed by Neighbour Joining(NJ).It showed that Cabernet Gernischt E02 and E03 were classified into one group,and E07 and E08 were classified into another group,suggesting that genetic distance is in accord with geographic distance.With this work,several mutations found in the Carboxylesterase gene fragments might be selected to be the molecular marker to distinguish and identify Cabernet Gernischt eight new strains and other wine grape varieties.