A functional PCR-CTPP marker targeting an intronic SNP in OsLAC11 gene for detecting kernel smut resistance in rice

Rice kernel smut, caused by the fungus Tilletia horrida Tak., is an emerging disease of rice. The pathogen infects germ cells at the early flowering stage, yet the plant remains asymptomatic until maturity. The rice plant may recognize it as a stress-inducible factor, triggering the expression of various stress-responsive genes and pathways. However, researchers are attempting to identify kernel smut-resistant genes in cell-surface proteins (elicitors), secretory proteins, glycoside hydrolase family proteins, and effector proteins. Based on annotated effector proteins and integrated analyses of available gene-expression datasets, this study aims to identify allelic variation in a putative candidate gene to develop a functional marker for marker-assisted identification of both resistant and susceptible rice lines. The gene OsLAC11 was found to be strongly associated with kernel smut resistance and is expressed on the first day of flowering in rice, which aligns with the infection window and supports its role in resistance. Sanger sequencing of the OsLAC11 gene across ten rice germplasms with varying levels of kernel smut resistance identified two single nucleotide polymorphisms (SNPs) in intronic regions. The first SNP (G/T allele) was used to develop a functional marker using Polymerase Chain Reaction (PCR) with the Confronting Two-Pair Primers (CTPP). This marker effectively differentiated kernel smut-resistant rice lines from susceptible ones and was validated through phenotypic disease ratings. The molecular marker developed in this study represents a significant advancement in breeding for kernel smut resistance, providing a robust tool to accelerate genetic gains and streamline marker-assisted selection in rice improvement programs.