Chromosome-scale reference genome of an ancient landrace: unveiling the genetic basis of seed weight in the food legume crop pigeonpea (Cajanus cajan)

Abstract Pigeonpea (Cajanus cajan) is a nutrient-rich and versatile food legume crop of tropical and subtropical regions. In this study, we describe the de novo assembly of a high-quality genome for the ancient pigeonpea landrace 'D30', achieved through a combination of Pacific Biosciences high-fidelity (PacBio HiFi) and high-throughput chromatin conformation capture (Hi-C) sequencing technologies. The assembled 'D30' genome has a size of 813.54 Mb, with a contig N50 of 10.74 Mb, a scaffold N50 of 73.07 Mb, and a GC content of 35.67%. Genomic evaluation revealed that the 'D30' genome contains 99.2% of Benchmarking Universal Single-Copy Orthologs (BUSCO) and achieves a 29.06 LTR Assembly Index (LAI). Genome annotation indicated that 'D30' encompasses 431.37 Mb of repeat elements (53.02% of the genome) and 37,977 protein-coding genes. Identification of single nucleotide polymorphisms (SNPs), insertions/deletions (indels), and structural variations (SVs) between 'D30' and the published genome of pigeonpea cultivar 'Asha' suggests that genes affected by these variations may play important roles in biotic and abiotic stress responses. Further investigation of genomic regions under selection highlights genes enriched in starch and sucrose metabolism, with 42.11% of these genes highly expressed in seeds. Finally, we conducted genome-wide association studies (GWAS) to facilitate the identification of 28 marker–trait associations for six agronomic traits of pigeonpea. Notably, we discovered a calmodulin-like protein (CcCML) that harbors a dominant haplotype associated with the 100-seed weight of pigeonpea. Our study provides a foundational resource for developing genomics-assisted breeding programs in pigeonpea.