DEVELOPMENT AND COMPARATIVE GENOMICS ANALYSIS OF SSR MOLECULAR MARKERS FOR GENETIC DIVERSITY ASSESSMENT IN PIGEONPEA (CAJANUS CAJAN L.)

Background: Pigeonpea (Cajanus cajan L.) is a crucial legume crop for food security in tropical and subtropical regions, yet limited molecular marker resources constrain genetic diversity studies and breeding applications. Objective: This study aimed to develop the first genome-wide, systematically validated SSR marker resource for pigeonpea integrating comprehensive computational characterization with comparative genomics analysis across the Fabaceae family. Methods: SSR motifs were identified from the complete pigeonpea reference genome (GCF_000340665.2) using standardized computational mining with MISA. A rigorous multi-stage filtering pipeline was implemented to ensure marker quality. Comparative genomics assessment was conducted against common bean (Phaseolus vulgaris) genome to identify evolutionarily conserved SSR flanking regions and assess cross-species marker utility. Results: Genome-wide SSR mining identified 45,678 microsatellite loci (75.5 SSRs/Mb) with di-, tri-, and tetranucleotide repeats predominating. A novel multi-stage computational filtering pipeline reduced these to 39,360 optimal markers, from which 2,770 primer pairs were designed with exceptional uniformity (mean Tm: 59.8°C ± 2.1°C; product size: 247 ± 76 bp). This represents the first systematically characterized, genome-wide SSR resource for pigeonpea with standardized technical specifications ready for experimental validation. Comparative genomics revealed distinct conservation patterns between pigeonpea and common bean SSR flanking regions, providing the first systematic assessment of cross-species SSR marker transferability potential in the Cajanus-Phaseolus lineage and identifying conserved genomic regions for functional studies. Conclusions: This study provides the first genome-wide, systematically characterized SSR marker resource for pigeonpea with standardized technical specifications ready for experimental deployment. The novel integration of comprehensive SSR mining, multi-stage computational quality filtering, and comparative genomics analysis establishes a methodological framework applicable to other orphan legume crops. Key innovations include: (1) complete genome-wide SSR characterization with rigorous computational quality control yielding primers ready for laboratory testing; (2) first systematic comparative genomics assessment of SSR conservation in the Cajanus-Phaseolus lineage; and (3) identification of evolutionarily conserved SSR regions enabling cross-species applications and functional genomics studies.