In silico mining and validation of microsatellite markers in Phlebotomus argentipes (Diptera: Psychodidae)

Phlebotomus argentipes is the primary vector of Leishmania donovani , the causative agent of leishmaniasis. This study aimed to expand genomic resources for P. argentipes by in silico mining and characterization of simple sequence repeats (SSRs) from a scaffold-level genome assembly retrieved from GenBank, followed by wet-lab validation of selected SSR markers. A total of 22 SSR loci were identified from intergenic regions and used for primer design. Laboratory validation using PCR amplification of genomic DNA from laboratory-reared flies confirmed successful amplification across all loci. Based on amplification quality and verification of SSR motifs, 11 primer pairs were selected for fragment analysis and allelic characterization in field-collected P. argentipes ( n = 20) from Ambalantota (Geo 1) and Polpithigama (Geo 2). All selected loci exhibited polymorphism, with allelic diversity ranging from 1 to 8 alleles per locus. Observed heterozygosity (H O ) ranged from 0.00 to 0.75, expected heterozygosity (H E ) from 0.29 to 0.85, and polymorphism information content (PIC) from 0.26 to 0.81, with five markers (IC27, IC02, IC51, IC68, IC74). Genetic differentiation between populations (F ST ) ranged from 0.03 to 0.10, while F IS ranged from 0.10 to 1.00, indicating heterozygote a deficit across loci, except for IC21, which showed heterozygote excess, supporting the utility of these SSR markers for population genetic studies.