Development and application of microsatellite markers in subsp. Rousi ( L.) based on transcriptome sequencing.

subsp. Rousi is a cold- and drought-tolerant pioneer species with significant economic and ecological value. Evaluating its genetic diversity and population structure is of great importance for guiding the development and utilization of resources. In this study, a total of 41,804 SSRs were generated by transcriptome sequencing of subsp. Rousi. Among the different SSR motif types, mononucleotide repeats (26,972) were the most abundant, followed by trinucleotides, tetranucleotides, and pentanucleotides. 200 pairs of SSR primers were selected to detect polymorphisms, of which 15 pairs primers were selected as validated polymorphic SSRs used for genetic diversity and population structure analysis. A total of 63 alleles were identified with 15 pairs primers, with Nei's genetic diversity index ranged from 0.27 to 0.83 (average: 0.54), and the expected heterozygosity ranged from 0.16 to 0.73 (average: 0.46). The polymorphism information content ranged from 0.23 to 0.81 (average: 0.48). Genetic structure analyses showed that the 10 populations could be broadly categorized into two groups. AMOVA denoted that genetic variations primarily originated from within the populations, with minimal differences observed between the groups, accounting for only 7% of the total genetic variation. This implies that mutation in subsp. Rousi mainly occurred within the populations. The results showed that the 10 populations of subsp. Rousi are rich in genetic diversity, with low levels of population differentiation and a high degree of gene exchange, which should be taken into consideration for the future work of germplasm resource preservation and seedling breeding.