Genome-wide Identification and Comparative Analysis of Intron-spanning Markers (ISM) in Pigeonpea (Cajanus cajan) and Related Legumes

Background: Pigeonpea (Cajanus cajan) is the sixth most important grain legume globally, serving as a vital source of dietary protein. Despite available genomic resources, comprehensive molecular marker resources remain limited. Intron-spanning markers (ISM) offer advantages due to conserved exonic primer sites and polymorphic intronic regions. This study presents the first genome-wide computational identification of ISM candidate loci in pigeonpea, providing a foundational resource for ILP marker development. Methods: A computational pipeline was developed using pandas and BioPython packages to identify ISM candidate loci from the NCBI RefSeq pigeonpea genome annotation (GCF_000340665.2). Introns were extracted from protein-coding genes, deduplicated by collapsing identical genomic coordinates across transcript isoforms and filtered by size (50-5000 bp) for PCR feasibility. Result: A total of 139,873 unique non-redundant ISM candidate loci were identified from 23,948 intron-containing genes (of 29,045 protein-coding genes) 80,849 (57.8%) are anchored to the across 11 chromosomes and remainder lie on unplaced scaffolds. Pigeonpea exhibited 5.84 introns per gene with mean intron length of 491 bp. Among identified candidate loci, 68,547 (49.0%) fall within optimal PCR range (200-2000 bp). Fifty candidates targeting stress-responsive genes were prioritized for future experimental PCR validation.