Analysis of grape rootstocks by SSR markers

Astract:【Objective】The rootstock varieties of grape commonly used in China are mostly selected fromEurope and America, some rootstock varieties have been confused during the course of introduction.More-over, many grape rootstock varieties have similar genetic background and similar morphology, which aredifficult to identify by traditional morphological characters.Therefore, the correct identification of graperootstock varieties is of great significance for efficient utilization and protection of the rootstock resources.Simple sequence repeat (SSR) is a molecular genetic marker based on PCR technology.SSR marker hasbeen widely used in genetic identification, relationship analysis, genetic map construction, and pedigreereconstruction of grape germplasm resources for its advantages of high polymorphism, good repeatability, codominant inheritance and strong specificity.This study was carried ou to analyze the genetic relation-ship and genetic diversity of grape rootstocks.【Methods】21 grape rootstocks and 3 varieties of Vitis vinifera varieties'Cabernet Sauvignon''Chardonnay''Syrah'were amplified by 17 pairs of SSR primers, such as VVS2, VVMD5, VVMD7, VVMD27, Vrzag62, Vrzag79 and so on.Genomic DNA of grape leafsamples was extracted by modified CTAB method.Genomic SSR-PCR was amplified using the followingamplification systems and procedures:The system included 2 μL, 10×PCR buffer, 1 U Taq enzyme, 0.2 mmol·L-1 d NTP, 1.5 mmol·L-1 Mg2+, 0.4 μmol·L-1 primer (each) , 50 ng DNA template, and dd H2 O supple-mentation to 20 μL.The PCR reaction was carried out on the Prime G02 PCR instrument produced byBibby Scientific.The running amplification reaction program was as follows:an initial denaturation 94 ℃for 5 min, denaturation at 94 ℃ for 30 s, annealing at 50-60 ℃ for 30 s, extension at 72 ℃ for 40 s, reac-tion of 35 cycles, last extension at 72 ℃ for 10 min.The same template was repeated twice.SSR-PCR am-plification products were analyzed by 8% denatured polyacrylamide gel electrophoresis and amplificationproducts were subjected to capillary electrophoresis to determine the fragment size of each locus.【Results】17 pairs of SSR primers showed higher genetic polymorphism, a total of 195 alleles were amplifiedin 21 rootstock varieties and 3 Vitis vinifera varieties, and the number of alleles amplified by each primerwas 5-17.VVMD27 and Vrzag47 amplified 17 alleles and VVS3 amplified 5 alleles.The fragment sizeswere 87 bp (VVIV52) -265 bp (Vrzag79) , the average number of effective alleles (Ne) of 17 amplified lociwere 7.21.Among the primers used, VVS29 and VVMD27 ampified 2.27 and 12.26 effective alleles, re-spectively.The observed heterozygosity (Ho) was 0.46-0.96, and the average observed heterozygosity (Ho) of each primer was 0.78.The expected heterozygosity (He) was 0.57-0.94, and average expected heterozy-gosity (He) was 0.83, The He value of VVS29 was the lowest, and the He value of VVMD27 was the high-est.The Shannon diversity index (I) was 1.10-2.62, and the average Shannon diversity index was 2.04 andthe percentage of polymorphic loci was 100%.The I value of UDV-058 was the lowest, and the I value ofVrzag47 was the highest.Distinctive alleles contributed greatly to the identification of grape varieties, ex-cept for primers VVS4 and VVII52, the other primers amplified distinctive alleles in 21 rootstock varietiesand 3 Vitis vinifera varieties, among the primers used, VMC8 G6 amplified 7 distinctive alleles.The genet-ic polymorphism of the selected primers was high and suitable for genetic diversity analysis of grape root-stock varieties.In single primer identification, VVMD5 was the most efficient and could identify 19 variet-ies.VVS3 and UDV-058 had the lowest efficiency, and only 2 varieties could be identified by them.2 pairs of primer combinations could greatly improve the efficiency of identification.The identification effi-ciency of the combination of primers VVMD27 & UDV-058, VVS2 & VVMD5 is 83.3%.The combina-tion of VVMD5 & VVS4, VMC1 B11 & VVS4 could be used to identify all the tested rootstock germplasmresources, and the identification efficiency was 100%.SSR markers showed high efficiency in grape root-stock variety identification.Cluster analysis of the amplified results showed that the genetic similarity co-efficients among the 24 grape varieties were 0.74-0.99.The grape rootstocks and wine grape varietiesstudied were separated into two clusters by UPGMA with the similarity index of 0.74.The 21 stock variet-ies could be divided into 2 subgroups with the genetic similarity coefficient of 0.752.The first subgroupcontained 4 rootstock varieties, and the second subgroup contained 17 rootstock varieties.The clusteringresults were consistent with the behaviors of grape rootstocks in breeding.【Conclusion】SSR markers caneffectively identify the variety of grape rootstock and use for genetic diversity analysis.