Efficient Marker-Assisted Pyramiding of Xa21 and Xa23 Genes into Elite Rice Restorer Lines Confers Broad-Spectrum...

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major threat to global rice productivity. Although hybrid rice breeding has significantly enhanced yields, persistent genetic vulnerabilities within restorer lines continue to compromise BB resistance. This study addresses this challenge by implementing functional marker-assisted selection (FMAS) to pyramid two broad-spectrum resistance (R) genes, Xa21 and Xa23, into the elite, yet BB-susceptible, restorer line K608R. To enable precise Xa23 genotyping, we developed a novel three-primer functional marker (FM) system (IB23/CB23/IR23). This system complements the established U1/I2 markers used for Xa21. This recombination-independent FMAS platform facilitates simultaneous, high-precision tracking of both homozygous and heterozygous alleles, thereby effectively circumventing the linkage drag limitations typical of conventional markers. Through six generations of marker-assisted backcrossing followed by intercrossing, we generated K608R2123 pyramided lines harboring both R genes in homozygous states, achieving a recurrent parent genome recovery rate of 96.93%, as determined by single nucleotide polymorphism (SNP) chip analysis. The pyramided lines exhibited enhanced resistance against six virulent Xoo pathogenic races while retaining parental yield performance across key agronomic traits. Our FMAS strategy overcomes the historical trade-off between broad-spectrum resistance and the preservation of elite phenotypes, with the developed lines exhibiting resistance coverage complementary to that of both introgressed R genes. This integrated approach provides breeders with a reliable molecular tool to accelerate the development of high-yielding, disease-resistant varieties, demonstrating significant potential for practical deployment in rice improvement programs. The K608R2123 germplasm represents a dual-purpose resource suitable for both commercial hybrid seed production and marker-assisted breeding programs, and it confers synergistic resistance against diverse Xoo races, thereby providing a pivotal breeding resource for sustainable BB control in epidemic regions.