Analysis of EST-SSR characteristics of prickly ash transcriptome and development of molecular markers

To explore the genetic diversity and relationships among prickly ash germplasm resources in China, the number, distribution, and repeat types of simple sequence repeats (EST-SSR) loci were analyzed using the MISA software system based on the transcriptome data of prickly ash. Thirty-five prickly ash germplasms from nine provinces were selected, and 30 pairs of EST-SSR primers were randomly designed for polymorphism screening. The obtained polymorphic primers were used for genetic diversity assessment, to provide genomics theoretical support for the construction of molecular breeding system and sustainable utilization of germplasm resources of prickly ash.The results showed that from 245,286 Unigene sequences, 36,656 sequences contained 20,271 EST-SSR loci. The frequency of occurrence of EST-SSR was 14.94% and the EST-SSR distribution frequency was 8.26%. Among the repeat types, the number of single-nucleotide repeats was the highest, accounting for 44.32% of all EST-SSR loci. After trinucleotide and dinucleotide repeats, pentanucleotide repeats were the least common, accounting for only 0.57%. Among the repeat motifs, A/T was the highest, accounting for 44.11% of all EST-SSR loci. AG/CT was the dominant repeat motif in the dinucleotide repeat motif, accounting for 11.35% of the total EST-SSR loci. Among the 30 pairs of randomly selected primers, 22 pairs showed polymorphisms, with an amplification efficiency of 73.33% and polymorphism rate of 90.99%. The average values for the 22 primer pairs, concerning the observed alleles (Na), effective alleles (Ne), Nei's genetic diversity metric (H), and Shannon Information Index (I), stood at 1.91, 1.44, 0.27, and 0.41, to be precise. UPGMA cluster analysis divided the 35 germplasm lines into four groups, and PCoA further verified the classification results. Analysis of molecular variance (AMOVA) showed that 79.80% of the total genetic variation was within individuals, 12.40% among populations and 7.80% among individuals within populations.Therefore, in this study, the development of EST-SSR markers and genetic diversity analysis of prickly ash transcriptome provide a scientific basis for the evaluation of germplasm resources, molecular-assisted selection, core germplasm screening and excellent variety cultivation of prickly ash, and respond to the needs of multi-omics-driven breeding innovation.