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Stigma exsertion and panicle enclosure of male sterile lines are two key determinants of outcrossing in hybrid rice seed production. Based on 43,394 single nucleotide polymorphism markers, 217 cytoplasmic male sterile lines were assigned into two subpopulations and a mixed-group where the linkage disequilibrium decay distances varied from 975 to 2,690 kb. Genome-wide association studies (GWAS) were performed for stigma exsertion rate (SE), panicle enclosure rate (PE) and seed-setting rate (SSR). A total of 154 significant association signals (P < 0.001) were identified. They were situated in 27 quantitative trait loci (QTLs), including 11 for SE, 6 for PE, and 10 for SSR. It was shown that six of the ten QTLs for SSR were tightly linked to QTLs for SE or/and PE with the expected allelic direction. These QTL clusters could be targeted to improve the outcrossing of female parents in hybrid rice breeding. Our study also indicates that GWAS-base QTL mapping can complement and enhance previous QTL information for understanding the genetic relationship between outcrossing and its related traits.

Outcrossing or cross hybridization is a potential concern in herbicide-resistant crop management strategies such as in the ClearWeld™ rice system. Recent studies have shown that the mutated acetolactate synthase (ALS) gene that confers resistance to imazethapyr (Newpath) herbicide can be transferred from ClearWeld rice cultivars via cross pollination under Weld conditions to weedy red rice. Resistance of commercial ClearWeld rice cultivars to imazethapyr is due to the presence of two point mutations in the ALS gene that result in amino acid substitutions from serine to asparagine (S to D) and glycine to glutamic acid (G to E). We report here development of a DNA-based method that involves application of allele-speciWc PCR assays to distinguish herbicide-susceptible and resistant ALS alleles in either homozygous or heterozygous genotypes produced from natural outcrosses between ClearWeld varieties CL121, CL141, CL161 and weedy red rice. PCR assays that can distinguish between the homozygous and heterozygous imazethapyr-resistant S 653 D and G 654 E SNP alleles of the rice ALS gene were developed and evaluated. A total of 483 individual red rice plants were successfully screened for the presence of S 653 D SNP and another 145 F 2 individuals from natural red rice £ CL121 hybridizations were screened for the presence of the G 654 E SNP. The PCR-based assays produced during this study are simple, rapid, inexpensive, reproducible and require only standard PCR and electrophoretic instruments that can be applied toward outcrossing evaluation and eVective weed management strategies for the ClearWeld crop system.

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