Analysis of SSR site characteristics of Trifolium repens transcriptome and primer development

The main study aim was to comprehensively understand the SSR loci information and sequence characteristics of white clover (Trifolium repens), develop polymorphic SSR primers, and distinguish 20 white clover varieties. An SSR site search was conducted using the sequencing data of the petals transcriptome of different colors of T. repens, and primers were designed in batches. Sixty pairs of primers were randomly selected for polymorphism verification, and primers with high polymorphism were selected for fingerprint construction. A total of 24960 SSRs were obtained, with a frequency of 13.25%, and an average of one SSR locus appeared at approximately 5.29 kb. Six SSR repeating base types were found in the transcriptome of white clover, including the main repeation types: trinucleotides (33.70%), dinucleotides (28.26%), and mononucleotides (25.02%). The other base repeating types were lower, accounting for only 9.01% of the total SSR. The main repeating motifs were A/T (24.85%), AG/CT (16.48%), and AAG/CTT (8.89%) from mononucleotide to trinucleotide, respectively. The number of repetitions of each primitive from mononucleotide to hexanucleotide concentrated between 4 and 21 times, accounting for 98.11% of the total, and the sequence length varied between 12 and 35 bp, accounting for 96.43% of the total sequence length. A total of 18594 primers were yielded from 24960 SSRs, accounting for 74.50%. Among the 60 pairs of random primers, 52 and 17 pairs of could amplify bands and polymorphic primers, accounting for 86.7% and 32.7%, respectively. The fingerprint map of white clover constructed with five pairs of primers with high polymorphism could effectively distinguish the 20 different white clover varieties. The SSR loci of the white clover transcriptome in this study were showed high frequency and rich distribution, with high polymorphism, demonstrating considerable potential for application in the analysis of the genetic diversity, molecular-marker-assisted breeding, and fingerprint profile construction of white clover.