Genome-wide association studies reveal genetic variants associated with antineoplastic monoterpenoid indole alkaloid accumulation in Catharanthus roseus.

Catharanthus roseus produces pharmacologically important monoterpenoid indole alkaloids (MIAs), yet their natural accumulation is low, limiting therapeutic exploitation. To dissect the genetic basis of natural variation in MIA accumulation, we integrated phenotypic, chemotypic, and genomic analyses of 93 C. roseus accessions sampled from six locations across India, including New Delhi, Lucknow, Jodhpur, Bangalore, and two locations in Gujarat: Navsari and Bardoli. Morphological characterization showed limited differentiation among locations, whereas accessions from Gujarat tended to be taller compared to other locations and more frequently white-flowered. Quantitative HPLC profiling revealed substantial accession- and location-dependent variation in total indole alkaloid levels, with Gujarat accessions showing the highest accumulation, largely driven by vindoline and catharanthine. Genotyping-by-sequencing generated 10,801 high-quality variants comprising 10,087 SNPs and 714 InDels corresponding to an average density of 19.34 variants per Mbp of the genome, revealing three genetic subgroups with overall admixed ancestry and weak geographic stratification. Genome-wide association study (GWAS) using five benchmark models identified 47 variants potentially associated with catharanthine, vindoline, and vinblastine content. These putative candidate loci were located near genes implicated in hormone signaling, mitochondrial function, nitrogen metabolism, and RNA processing, suggesting complex regulatory control of MIA biosynthesis. Notably, two missense variants in a carboxylesterase-like gene were associated with vindoline accumulation, and highly significant intergenic SNP clusters suggested putative regulatory hotspots for vinblastine biosynthesis. These results provide GWAS-based insights into the genetic architecture of MIA metabolism in C. roseus and nominate candidate variants for precision breeding and metabolic engineering to enhance pharmaceutical alkaloid production.