Rhodoleia Champion ex Hooker is one of the most primitive relict genera of Hamamelidaceae, a key family exploited to understand the origin and early evolution of flowering plants. Genomic simple sequence repeats (SSRs) were developed for R. championii to perform genetic diversity, phylogeographical structure or even systematic evolution studies of the genus. Among the 278,743 contigs (105,758,242 bps) de novo assembled from the low-coverage whole genome sequencing of R. championii, a total of 9106 SSRs were detected in 8370 contigs, and SSR primer pairs were successfully designed for 6677 SSRs. Among the 110 selected primer pairs, 41 were amplified successfully in the preliminary test of SSR screening. Further amplification of these 41 primer pairs across the 122 individuals collected from six populations of the three Rhodoleia species showed that 32 and 40 SSR markers can be amplified in Vietnam and Jinping populations of R. parvipetala, 41, 33, and 41 SSR markers in Boluo, Hongkong and Xinyi populations of R. championii, 25 SSR markers in Fugong population of R. forrestii, and 20 SSR markers demonstrated to be polymorphic across the three species. Genetic analysis for these 20 polymorphic SSRs showed that Allele number (A) ranged from four to 13 and polymorphic information content (PIC) ranged from 0.479 to 0.876 across the three species. At the population level, observed heterozygosity (H) ranged from 0.000 to 1.000, and expected heterozygosity (H) ranged from 0.091 to 0.851. In the present study, we provided the first whole-genome sequencing database for the species R. championii, identified ample SSR loci with designed primers, and revealed that 20 of the 110 selected SSRs were polymorphic across three Rhodoleia species. These provide valuable resources for future studies on genetic study, species delimitation, phylogeography, and conservation of this genus.
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