Gene mapping and identification of candidate genes controlling carotenoid accumulation of yellow kernels in foxtail millet

Kernel color is an important characteristic of foxtail millet (Setaria italica) associated with its market ability, quality, and nutritional value, which is mainly due to the accumulation of carotenoids. Despite its importance, the genetic basis of carotenoid variation in foxtail millet remains largely unexplored. Herein, the molecular mechanisms governing carotenoid accumulation in the kernel of foxtail millet were investigated by an exhaustive methodology encompassing Genome-Wide Association Study (GWAS), Bulk Segregant Analysis sequencing (BSA-seq), and integrated transcriptomic and metabolomic analyses. The total carotenoid content in kernels across 201 foxtail millet germplasms showed a spectrum of variations, which indicated that the kernel color is a quantitative genetic trait controlled by multiple genes. Using GWAS on these germplasms and BSA-seq on an F6 generation Recombinant Inbred Line (RIL) population derived from the GBS (yellow kernel) and NMB (white kernel) cross, we identified genome regions linked with total carotenoid content in foxtail millet kernels. Integrated transcriptomic and metabolomic profiling during grain filling in both yellow and white varieties pinpointed SiPSY1 and SiCCD1 as key genes controlling carotenoid accumulation. Notably, the SNP (G/A) at 364 bp and the Indel (29 bp insertion) at 856 bp within the SiPSY1 promoter predominantly contributed to the variance in promoter activity. These variations markedly affected SiPSY1 expression levels, ultimately determining the phenotypic difference between yellow and white kernels. These findings provide crucial genetic insights for the molecular mechanisms involved in carotenoid metabolism and lay a solid foundation for millet color breeding in foxtail millet.