Background/Objectives: Chemical methods for quantifying resistant starch (RS) in rice are labor-intensive, costly, and lack high repeatability, creating a bottleneck in breeding. This study aimed to develop specific, codominant molecular markers for the Wx gene to enable rapid and accurate genotype screening for RS content, thereby accelerating the development of high-RS rice varieties. Methods: Based on sequence alignment of the Wx gene in rice varieties with divergent RS content, a key single-nucleotide polymorphism was targeted. Two sets of tetra-primer amplification refractory mutation system polymerase chain reaction (ARMS-PCR) markers, T-Wx9-RS1 and T-Wx9-RS2, were designed. These markers were used to genotype diverse rice varieties and F4 segregating populations, with results validated against standard chemical assays. Results: Sequence analysis identified a critical T → C base mutation at position 202 of the ninth exon in high-RS varieties. The developed ARMS-PCR markers successfully and consistently distinguished all three possible genotypes (homozygous mutant, homozygous wild-type, and heterozygous). The genotyping results showed complete concordance with the phenotypes determined by chemical methods. Conclusions: The developed molecular markers, T-Wx9-RS1 and T-Wx9-RS2, provide a rapid, reliable, and cost-effective tool for marker-assisted selection of high resistant starch content in rice. Their implementation can significantly enhance screening efficiency and expedite the breeding pipeline for novel, nutritionally improved rice cultivars.
Ouyang et al. (2026) studied this question.