Development of genomic SSR markers in Gymnema sylvestre (Retz.) R.Br. ex Sm. using next generation DNA sequencing and their application in genetic diversity analysis

Abstract

Gymnema sylvestre (Retz.) R.Br. ex Sm. is one of the important medicinal plants widely used in traditional medicine due to its anti-diabetic properties. Due to its medicinal properties, huge pressure on its natural resources of G. sylvestre is under severe threat of diversity loss. Further, the lack of molecular markers in G. sylvestre is the limiting factor for the adoption of modern genomic studies. In the present study, DNA was isolated from G. sylvestre leaves which were used for NGS sequencing (Illumina HiSeq 2500 NGS platform). After de-novo assembly of raw reads, 77679 contigs were identified with the sum of contigs length 1, 246, 048, 78 bp. Among all the assembled contigs, 55859 contigs with SSR motifs were identified. Based on repeat sequences, we designed SSR markers using the MISA tool. The hexanucleotide SSR was the most prominent among other SSR types. Among various SSR motifs identified, AT/TA, AG/CT, and AC/GT repeats were most frequently found in dinucleotide repeats. A total of 100 SSR primer pairs were synthesized through the flanking sequences of SSR motifs using Primer3 in which 58 primers were amplified and 42 were not amplified. Novel genomic SSR markers were used for the genetic diversity analysis of 26 genotypes of G. sylvestre. Fifty-eight primer pairs produced alleles ranging from 1 to 6. The maximum number of alleles (6) were generated by Xdagsm10 which showed the highest polymorphic information content (0.602) and allelic diversity (0.662) followed by Xdagsm96 recorded maximum PIC (0.607) and allelic diversity (0.671). According to the UPGMA dendrogram, the genotypes are clustered into three major groups, and they are not formed according to geographical origin. Among all the genotypes, DGS10, DGS22, DGS16, DGS5, and DGS23 were found to be distinctly divergent which can be used in the future breeding programme. Primers Xdagsm10 and Xdagsm96 are highly efficient polymorphic markers that can be used to distinguish the genotypes of G. sylvestre, and are highly useful in the molecular breeding programme.