In silico mining of microsatellites from the Phlebotomus argentipes (Diptera: Psychodidae) scaffold-level genome assembly and validation of eleven genotyping markers

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 (Ho) ranged from 0.00 to 0.75, expected heterozygosity (He) from 0.29–0.85, and polymorphism information content (PIC) from 0.26–0.81, with four markers (IC27, IC51, IC68, IC74). Genetic differentiation between populations (F _ST ) ranged from 0.03–0.10, while F _IT ranged from 0.05–0.45, indicating heterozygote deficit across loci, except for IC21, which showed heterozygote excess, supporting the utility of these SSR markers for population genetic studies.