Novel Wx Gene Functional Markers for High-Resistant Starch Rice Breeding

<b>Background/Objectives</b>: 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 <i>Wx</i> gene to enable rapid and accurate genotype screening for RS content, thereby accelerating the development of high-RS rice varieties. <b>Methods</b>: Based on sequence alignment of the <i>Wx</i> 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-<i>Wx</i>9-RS1 and T-<i>Wx</i>9-RS2, were designed. These markers were used to genotype diverse rice varieties and F₄ segregating populations, with results validated against standard chemical assays. <b>Results</b>: 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. <b>Conclusions</b>: The developed molecular markers, T-<i>Wx</i>9-RS1 and T-<i>Wx</i>9-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.