Establishment and application of SSR molecular identification system in grapevine

Abstract:【Objective】A set of SSR (Simple Sequence Repeat) molecular markers adapting to the identi- fication standards of grape cultivar was screened to construct a DNA fingerprint database of grape cultivar to provides technical support for protection of new grape cultivar, registration of cultivar and maintenance of market rights in China.【Methods】Based on the grape SSR markers reported by the predeces- sors, six grape cultivars representing different species, ploidies and consumptions were used for PCR amplification. The clear and stable SSR markers were initially selected under detection by agarose gel electrophoresis. SSR markers were checked by detecting 8% non-denaturing polyacrylamide gel electro- phoresis using PCR amplification of 24 representative grape cultivars. Then, combining with fluorescent capillary electrophoresis detection technology, the final SSR markers were selected according to the PCR amplification of 52 new grape cultivars bred recently in China. We selected‘Cabernet Sauvignon’‘Chardonnay’and‘Red Globe’as test materials, the selected markers were sent to Beijing Microread Genetics Technology Co., Ltd and Suzhou Genewiz Biotechnology Technology Co., Ltd for capillary electrophoresis detection (Model: ABI 3730 XL) to check the consistency and stability of the markers. In order to correct and eliminate the errors caused by different instruments, different test batch- es, several common and representative reference cultivar with less variation were identified. We used GeneMapper ID v3.2 software to read the fragment size, exported the data in Excel format, and manual- ly analyzed and corrected the fluorescence data obtained. The peaks were recorded as“1”, the non- peaks were recorded as“0”, and the missing data was recorded as“999”to form a“1/0”matrix. Excel 2019 was used to calculate the number of variant alleles, genotypes, and frequency of occurrence in the population. PIC-CALC Version 0.6 software was used to calculate polymorphism information content (PIC). Using the software NTSYS-pc 2.10 and the unweighted group average method (UPGMA), the detected data were clustered and compared with 52 new grape cultivars. The pedigree relationship was evaluated for the accuracy and reliability of the selected markers, and finally the Chinese grape SSR mo- lecular marker system were established【. Results】At first, using the DNA template of the six cultivars of‘Chardonnary’‘Red Globe’‘Summer Black’‘Kyoho’‘Concord’‘Beta’, 137 grape SSR markers were amplified by PCR, and 84 markers were selected as clear and stable markers. Secondly, 38 SSR markers with clear stability and high polymorphism were selected from 24 representative cultivars. Fi- nally, 30 SSR markers with stable amplification results, high polymorphism, relatively consistent an- nealing temperature and uniform distribution on chromosomes were selected from 52 representative cul- tivars, in which the optimal annealing temperatures of 22 SSR markers and other 8 SSR markers were 56 °C and 58 °C, respectively. The stability and reliability of the 30 markers were verified using 3 culti- vars‘Cabernet Sauvignon’‘Chardonnay’and‘Red Globe’, then‘Chardonnay’and‘Red Globe’were identified as reference varieties and the size of standard variant fragments was determined. Analy- sis of genetic diversity showed that the 30 markers obtained PIC values of not less than 0.40 in the 24 material populations and the 52 material population, among which 9 commonly used markers had PICs greater than 0.50 in different populations. The PIC values had a range of 0.40-0.88, with an average value of 0.75 in the 52 material populations, in which VrZAG79 marker had the highest PIC value. A total of 309 alleles were obtained in the 52 material populations, ranging from 4 to 16, and VVMD28 marker was found with the most amplified alleles, followed by VVS2 and VMC1C10 markers, and Vchr17a marker with the least amplified alleles. The clustering results showed that the genetic similarity coefficient among the 52 varieties was 0.76-0.96, and when the genetic similarity coefficient was 0.76, the 52 cultivars could be divided into two categories. It was considered that the set of SSR markers could dis- tinguish diploids and polyploidys. Besides, V. vinifera were closely related to V. vinifera-V. labrusca, while Chinese wild grapes were more distantly related to other grape species. The cluster analysis map was relatively consistent with the pedigree maps of the 52 cultivars. In particular, several groups of sis- ter breeds were well clustered together, but there were differences. Just 9 markers that are used interna- tionally could distinguish 43 cultivars with the identification efficiency of 82.7%, while 25 cultivars could be distinguished by only using VMC4F3-1 marker. Using the five marker combinations VMC4F3- 1, VVS2, VrZAG79, Vchr9b and Vchr4a, 49 cultivars could be distinguished with the identification efficiency of 94.2%. We used up 8 marker combinations (VMC4F3-1, VVS2, VrZAG79, Vchr9b, Vchr4a, VrZAG62, VVMD27, and Vchr13b) to completely distinguish the 52 cultivars.【Conclusion】30 grape SSR molecular markers were screened out of 137 markers, and a set of grape SSR molecular marker system was constructed. The core markers of the system were VVMD28, VVMD32, VVMD27, VrZAG79, VVMD7, VrZAG62, VVMD25, VVS2, VVMD5 which could ensure the identification efficiency and international cultivar identification at the same time.