Validation of single nucleotide polymorphism markers associated with hydrogen cyanide, total carotenoid and dry matter contents in cassava (Manihot esculenta Crantz).

Enhancing total carotenoid content (TCC) and dry matter content (DMC) while simultaneously reducing hydrogen cyanide (HCN) are key objectives in cassava breeding. Although numerous SNP markers have been identified through genome-wide association studies (GWAS) as promising tools for accelerating improvement of quality traits, they have not been widely adopted in marker-assisted selection (MAS) due to limited validation across diverse genetic backgrounds. To address this gap, we validated nine previously reported single nucleotide polymorphism (SNP) markers associated with TCC, DMC, and HCN using 435 genotypes from two independent cassava breeding populations: a HarvestPlus Seedling Nursery (SN) population from IITA and Advanced Yield Trial (AYT) at University of Ibadan (UIC). The markers were evaluated using Kompetitive allele-specific PCR (KASP) genotyping across a subset of 360 genotypes drawn from HarvestPlus SN population, its progenitors, and selected UIC lines. The markers demonstrated high genotyping efficiency, with call rates exceeding 96% and clear allelic discrimination. Trait relationships varied across populations, with TCC and DMC showing a negative correlation in AYT (r = -0.26**) and a positive correlation in SN (r = 0.37***). Marker S1_24155522 showed a strong and significant association with TCC, while S8_25598183 exhibited a moderate effect. For DMC, marker S1_24197219 displayed limited segregation, with the unfavorable allele appearing fixed in the SN population. For HCN, markers S14_6050078 and S16_773999 showed associations with variation in cyanogenic potential. Overall, S1_24155522 emerged as the most promising validated candidate marker for MAS targeting carotenoid biofortification. However, the limited and population-dependent performance of markers associated with DMC and HCN indicates that these traits are governed by more complex genetic architectures and require further validation across diverse breeding populations and environments before routine deployment in marker-assisted selection. These findings provide valuable insights for improving the efficiency of molecular breeding strategies aimed at enhancing nutritional quality and food safety in cassava.