‘蛇龙珠’葡萄新株系遗传多样性的SSR、ISSR和VvmybA1基因片段分析

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

【目的】探究‘蛇龙珠’8个新株系的亲缘关系和遗传多样性,选育出具有推广价值的‘蛇龙珠’新株系。【方法】以‘蛇龙珠’8个新株系(E01~E08)为样品,近缘品种‘品丽珠’‘赤霞珠’等为对照,利用SSR、ISSR分子标记和VvmybA1基因序列进行遗传多样性和亲缘关系分析。【结果】利用14对SSR引物对10份葡萄材料进行基因组DNA的扩增,通过聚类分析得出只有‘蛇龙珠’E06与其他7个株系有差异,另外7个株系聚为一类。利用11个ISSR引物进一步研究得出,‘蛇龙珠’E02(栖霞北)和E03(栖霞南)聚为一类,遗传距离与地理距离一致,E05(莱山)和E08(开发区)聚为一类,E01、E04、E06、E07之间差异显著。分析VvmybA1基因片段的差异探讨‘蛇龙珠’8个新株系的遗传差异,得出‘蛇龙珠’E01、E02、E04、E05、E06、E07的VvmybA1基因亲缘关系较近,聚为一支。‘蛇龙珠’E08和E03的VvmybA1基因分别单独聚为一支。【结论】利用SSR和ISSR可以显示‘蛇龙珠’8个新株系的亲缘关系,在区别‘蛇龙珠’8个株系时首先考虑遗传距离而不是地理距离。这些葡萄样品的VvmybA1基因片段的遗传多样性较低,在近期进化史上VvmybA1基因较稳定,在过去一段历史未发生种群扩张。

【Objective】‘Cabernet Gernischt＇（Vitis vinifera L.） is one of the most widely grown red grape for making wine in Jiaodong Peninsula of Shandong province The aim of this study is to investigate genetic diversity 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 using Ezup 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 ISSR data were scored with presence（1）, absence（0） and each band were regarded as a locus. Jaccard similarity 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 purification was performed with San Prep Column DNA Gel Extraction Kit. The VvmybA1 sequences were aligned with sequence data obtained by using Bio Edit version 7.0.5 and Clustal X 2.0. Phylogenetic trees based on Neighbor-Joining methods were created by using MEGA 5.0. Three neutrality tests were constructed with Dna SP 5.0.【Results】A total of 139 bands were amplified from 14 SSR primers, of which 69 were polymorphic loci. The effective number of alleles（Ne） of the SSR primers ranged from 1.021 6（VMC4F3） to 1.590 1（Vr ZAG25）. The Nei＇s gene diversity（H） ranged from 0.019 5（VMC4F3） to 0.322 0（Vr ZAG25）. Shannon＇s information index（I） ranged from 0.040 5（VMC4F3） to 0.467 3（Vr ZAG25）. The genetic 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‘Cabernet Gernischt＇E06, and the second cluster contained seven strains. A total of 96 clear DNA fragments were amplified from 11 ISSR primers. The mean similarity index of all strains and cultivars was 0.825 6, and ranged 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 Qixia） and E03（south of Qixia） classified into one group. The genetic distance between them accorded with the geographic distance. E05（Laishan） and E08（Development zone） were classified into one group, There were 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 VvmybA1 gene sequence of 11 strains and cultivars showed a low level of diversity（Hd=0.673, Pi=0.002 21）. The analysis 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 indicated 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 provide a consensus tree depicting the geographical partitioning of the species. By analyzing the differences among 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 were different from those of the other six strains.【Conclusion】The results revealed that ISSR could be a good tool for evaluation of genetic diversity among the grape strains and cultivars. It was suggested that the genetic 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 11 strains and cultivars showed a low level of diversity, and should not be used as genetic markers for strain identification of‘Cabernet Gernischt＇.