Development of EST-SSR markers and their application in the genetic diversity of persimmon (Diospyros kaki Thunb.)

Abstract

SSRs related to parent selection of D. kaki were validated by transcriptome sequencing, and genetic diversity of these markers among 76 persimmon germplasm resources was additionally investigated. The selection of hybrid parents is critical in cross-breeding plants for germplasm innovation and cultivating improved varieties. This study used a published RNA-Seq database of a persimmon cultivar ‘Zenjimaru’ to develop simple sequence repeat (SSR) markers to assist efficiently selection of appropriate persimmon cultivars for cross-breeding. A total of 154,741 unigenes with 41,999 SSR loci at a frequency of one per 2.30 kb were identified. Of the different SSR motif types, mononucleotide repeats (47.63%) were the most abundant, followed by dinucleotide repeats (38.23%). The main repeat motifs were A/T (42.26%) and AG/CT (24.72%). A total of 17,111 SSR primer pairs were designed, from which 100 selected loci were synthesized and preliminary screened with capillary electrophoresis by testing PCR products from 15 persimmon germplasm resources. The effectiveness of 13 primer pairs that produced products with distinct peaks at the predicted sizes was subsequently determined using 76 persimmon germplasm resources. A total of 111 alleles were identified by using 13 SSR markers, ranging from 2 to 17 with an average of 8.54 bands per locus. Additionally, different hybridized combinations could be selected according to a dendrogram based on these newly developed 13 SSR markers. And two androecious persimmons (“Yunjia-9” and “Luotian Yeshi-1”) were genetically closely related to Chinese pollination-constant non-astringent (C-PCNA) persimmons, which was significant for the cultivation of PCNA offspring. The identified SSR markers will not only promote persimmon cross-breeding programs by increasing the efficiency of parent selection but also assist in genetic resource identification and conservation.