Development and Validation of Multi-Locus GWAS-Based KASP Markers for Maize Ustilago maydis Resistance

Corn smut, caused by Ustilago maydis, significantly threatens maize production. This study evaluated 199 maize inbred lines at the seedling stage under greenhouse conditions for resistance to U. maydis, identifying 39 highly resistant lines. A genome-wide association study (GWAS) using the mrMLM model detected 19 significant single-nucleotide polymorphism (SNP) loci. Based on a linkage disequilibrium (LD) decay distance of 260 kb, 226 candidate genes were identified. Utilizing the significant loci chr1_244281660 and chr5_220156746, two kompetitive allele-specific PCR (KASP) markers were successfully developed. A PCR-based sequence-specific oligonucleotide probe hybridization technique applied to the 199 experimental lines and 60 validation lines confirmed polymorphism for both markers, with selection efficiencies of 48.12% and 43.33%, respectively. The tested materials were derived from foundational inbred lines of domestic and foreign origin. Analysis of 39 highly resistant lines showed that the advantageous alleles carrying thymine/cytosine (T/C) predominated at frequencies of 94.87% and 53.84%, respectively. The genotype TTCC conferred high resistance, while CCTT was highly susceptible. The resistance exhibited high heritability and significant gene-by-environment interaction. This work systematically dissects the genetic basis of common smut resistance in maize, identifies favorable alleles, and provides a novel KASP marker-based strategy for developing disease-resistant germplasm.