Phylogenetic Analysis and Development of SSR Markers Based on RAD-seq in Passiflora
TIAN Qing-Lan1, LIU Jie-Yun1, HUANG Wei-Hua1, XIA Xiu-Zhong2, YANG Xing-Hai2, QIN Liu-Yan1, ZHANG Ying-Jun1, MOU Hai-Fei1, WU Yan-Yan1,*
1 Biotechnology Research Institute, Guangxi Academy of Agricultural Sciences, Nanning 530007, China; 2 Rice Research Institute, Guangxi Academy of Agricultural Sciences, Nanning 530007, China
Abstract：Passion fruit (Passiflora) is an important fruit tree in southern China, but the evolutionary relationship among Passiflora species is still unclear, and there is a lack of universal molecular markers to distinguish between different species. In this study, the phylogeny, SSR loci and marker versatility of 10 germplasms from 6 passion fruit species were investigated based on restriction site-associated DNA sequencing (RAD-seq). The results showed that EcoRⅠ was a suitable enzyme for simplified sequencing of passion fruit genome. According to the number of remaining reads/read lengths, the phylogenetic tree was constructed based on the 46 451 high-quality SNPs detected in RAD-seq, the partially assembled sequence of 'Purple fruit No. 7' (P. edulis)(P4) was used as the reference genome. The results showed that the blue-crowned passion fruit (P. caerulea)(P6), safflower passion fruit (P. coccinea)(P7), Banna passion fruit (P. xishuangbannaensis)(P8), and quadrangularis passion fruit (P. quadragularis)(P9) were on the same phylogenetic branch, green-skin passion fruit (P. edulis)(P5) and Colombian passion fruit (P. ligularis)(P10) were on the same branch, and Jinling purple fruit (P. edulis)(P1), guava golden fruit (P. edulis var. flavicarpa)(P2), and Yunnan yellow fruit native species (P. edulis var. flavicarpa)(P3) were closer to 'Purple fruit No. 7' in genetic relationship. When using 12 452 high-quality SNPs to construct the phylogenetic tree and using Banna passion fruit as the reference genome, the Yunnan yellow fruit native species, 'Purple fruit No. 7', safflower passion fruit, Banna passion fruit, quadrangularis passion fruit, and Colombian passion fruit were all on separate phylogenetic branches, Jinling purple fruit and guava golden fruit were on the same branch; and green-skin passion fruit and blue-crowned passion fruit were on the same branch. A total of 2 614 SSRs were identified from the 10 passion fruit germplasms, and their core motifs were AT, GA and AAG, etc., with 2~6 bases in length, and the repeat number was 4~16 times; 2 515 pairs of SSR primers were successfully developed accordingly. 50 pairs of SSR markers were used to evaluate the rate of shared markers among different passion fruit species, and the rate was 54.22%; notably, the shared rate in cultivar passion fruit (P1~P5) was significantly higher than the other 5 species (P6~P10), while the shared rate of wild Banna passion fruit in Yunnan, China was 0. This might be due to the distant genetic relationship between Banna passion fruit and other germplasms, and there were large differences in genome sequences. This study establishes the genetic relationships among different Passiflora species, provides a theoretical basis for the genetic improvement of passion fruit, and developes a useful panel of SSR markers for molecular marker-assisted selection of passion fruit.
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