Genetic diversity analysis of kiwifruit germplasm and identification of variant based on SRAP and SCoT markers

Abstract:【Objective】Kiwifruit species are diverse due to obivous interspecific and intraspecific hy- bridization. Therefore, investigation of the genetic background of 59 kiwifruit varieties/germplasm and identificationoftherelativesofkiwifruitmaterialwithfullyredflesh‘( H-16’)canprovideaneffective molecular marker-assisted breeding method for genetic background analysis and classification of kiwi- fruit germplasm and selection of new varieties.【Methods】The genomic DNA of kiwifruit leaves was extracted by DNA kit. Then, two kinds of molecular markers, SRAP and SCoT, were selected for genet- ic diversity analysis and variant identification. The SRAP marker was designed by designing a pair of special primers at both ends of the open reading frame, and the primer sequences included filler bases, specific bases and selective bases. While the SCoT marker was designed by designing a single primer based on the conserved sequences flanking the ATG translation start site, and the amplification pro- duced a dominant polymorphic marker biased toward the candidate functional gene region. The differ- ences in electrophoretic bands obtained from the two markers would reflect the differences between the genetic materials, so the germplasm identification could be constructed based on the differences in elec-trophoretic bands. A 0, 1 matrix was established based on the polymorphic amplified bands, and the number of effective alleles (Ne), genetic diversity (H), and Shannon information index (I) were calculat- ed using Popgene 1.31 software. The genetic distance and genetic similarity among the 59 kiwifruit germplasm resources were calculated using Ntsyspc 2.10 software, and the NJ adjacent cluster trees of 59 kiwifruit germplasm resources based on Nei’s genetic distance was constructed using MEGA 7.0 software. Finally, 169 pairs of SRAP primers and 57 SCoT primers were used for the molecular identifi- cation of the‘H-16’variant material.【Results】The screened 12 pairs of SRAP primers and 16 SCoT primers amplified 125 and 143 polymorphic bands in 59 germplasm resources, respectively. The aver- age numbers of amplified bands were 10.50 and 8.94, the average polymorphism ratios were 97.07% and 100%, and the average genetic similarity coefficients were 0.668 and 0.640. The genetic distance between the 59 kiwifruit germplasm resources obtained with the two kinds of markers were about 0.437-0.952 and 0.441-0.930, respectively. The high genetic similarity coefficients of 0.952 and 0.930 between H-16 and Hongyang indicated that the genetic backgrounds of them were highly consistent. From the clustering results, two kinds of molecular markers, SRAP and SCoT, divided the 59 kiwifruit germplasm resources into 4 and 8 groups, respectively. The SCoT marker provided more genetic varia- tion information than the SRAP marker and could distinguish kiwifruit species more effectively. Some of the A. chinensis clustered with most of the A. deliciosa germplasm, showing that the A. chinensis andA. deliciosa were more closely related interspecies and there were frequent gene exchanges between them, further confirming that A. deliciosa is a variety of A. chinensis. From the analysis of species ori- gin, among the A. arguta tested, Hongbaoshixing and Zhongxiahong both originated from the Kiwifruit Resource Collection of Zhengzhou Fruit Tree Research Institute, Chinese Academy of Agricultural Sci- ences, which belonged to the fully red A. arguta, both clustered together by the two kinds of markers, to a certain extent, indicating that the relatives and geographical distribution were related. The above ge- netic analysis based on SRAP and SCoT markers indicated that the genetic similarity coefficients be- tween Hongyang and‘H-16’were high, so it was assumed that‘H-16’was a variant of Hongyang. In order to investigate the genetic differences between them, the specific primers were screened from 57 SCoT primers and 169 SRAP primer pairs, and the results showed that the SP49 and SC68 primers of SCoT markers amplified differential bands between the two specimens with a difference ratio of 3.45%. The Me7-em11, Me9-em2 and Me13-em7 primer combinations of SRAP markers amplified specific dif- ferential bands with a difference ratio of 2.36%. This indicated that there was a difference in genetic ma- terial between‘H-16’and Hongyang. Combined with the clustering results, it was proved that‘H-16’was the mutant of Hongyang at the DNA level, and also indicated that both markers could be applied to the identification of mutant materials. In addition, 12 pairs of SRAP primers clustered all A. chinensislines of red-fleshed kiwifruit, which could be used as candidate SRAP primers for the screening of loci controlling red traits.【Conclusion】The SRAP and SCoT molecular markers classified 59 kiwifruit accessions into 4 and 8 groups effectively, indicating differences in their genetic backgrounds and a high level of genetic diversity, and the two kinds of markers were both available, with SCoT being more efficient. Both markers could be used for early identification of kiwifruit variant materials, and the success- ful identification of‘H-16’as a color variant (strain) of Hongyang would provide technical reference and theoretical basis for later germplasm resource evaluation and new germplasm selection.