The grain protein content (GPC) of rice is an important factor that determines its nutritional, cooking, and eating qualities. To date, although a number of genes affecting GPC have been identified in rice, most of them have been cloned using mutants, and only a few genes have been cloned in the natural population. In this study, 135 significant loci were detected in a genome-wide association study (GWAS), many of which could be repeatedly detected across different years and populations. Four minor quantitative trait loci affecting rice GPC at four significant association loci, qPC2.1, qPC7.1, qPC7.2, and qPC1.1, were further identified and validated in near-isogenic line F2 populations (NIL-F2), explaining 9.82, 43.4, 29.2, and 13.6% of the phenotypic variation, respectively. The role of the associated flo5 was evaluated with knockdown mutants, which exhibited both increased grain chalkiness rate and GPC. Three candidate genes in...

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