Development of novel genome‑wide simple sequence repeats (SSR) markers in Bunium persicum

Bunium persicum (Boiss.) Fedtsch or Kala jeera is an economically important medicinal spice that grows in cold temperate regions of Central Asia and Northern India. It is mainly used as spice and condiment in various food preparations. However, its high aroma and richness in essential oils make it industrially important. The bioactivity and pharmacological activities of its phytochemicals have been well elucidated. Due to limited cultivation and huge harvest pressure on natural resources, this species is under severe threat of diversity loss. Further, genetics and molecular breeding studies are lagging for this important crop plant due to a dearth of efficient genome wide molecular markers. In the present study, genomic resources for B. persicum have been generated for the first time through de novo sequencing. In addition, novel genomic SSRs from these resources are identified for their use in future diversity analysis and breeding programs. A total of 177029 perfect and 5915 compound SSR motifs were identified in 212585 assembled sequences. Mononucleotide repeats were the most abundant (55.18%) followed by di- (32.35%), tri- (7.21%), tetra- (3.15%) and hexa- nucleotide (0.76%) repeats. Total 101519 and 2649 primers were successfully designed for sequences containing perfect and compound SSR repeat motifs, respectively. For validation, 88 primer pairs were synthesized for perfect SSR motifs, from which 68 pairs gave scorable amplification. From these, 16 primer pairs were found monomorphic and 52 were polymorphic. These 68 primer pairs altogether yielded 101 alleles with an average of 1.71 alleles per loci. Overall average allele frequency (0.7908), PIC value (0.213), heterozygosity value (0.2995), and gene diversity (0.2680) was recorded for the evaluated Bunium accessions. UPGMA tree constructed based on Nei genetic distances successfully distinguished the evaluated B. persicum accessions. Low genetic diversity recorded in the present study emphasizes the need for restoration and maintenance of effective B. persicum population sizes in situ for its future sustenance.