Leaf shape is an important factor of plant architecture and the primary contributor to photosynthesis in cereal crops. However, the genetic and molecular mechanism underlying flag leaf phenotypes remains largely unknown. In this research, 139 RILs derived from hybrid rice Xieyou9308, the parents of which are genetically different in leaf phenotype, were used to identify genetic variants for flag leaf length (FLL), width (FLW), and rolling rate (FLR) with high-density single nucleotide polymorphism (SNP) genotyping data. GWAS was carried out using 262,224 SNPs by modeling them as a quantitative phenotype. In the analysis implemented with QTXNetwork, 21 significant SNPs were observed (8 SNPs for FLL, 5 SNPs for FLW, 8 SNPs for FLR) with accumulated explanatory heritability ranging from 53.29–89.25%. Furthermore, 16 significant associated peaks (2 SNPs for FLL, 11 SNPs for FLW, 3 SNPs for FLR) were detected by EMMAX with explanatory effects ranging from − 8.138 to 37.15. To further validate our findings, relative expression analysis was conducted for 13 candidate genes and 6 known marker genes, revealing that 18 genes are differentially expressed in parents. Among them, the expression level of LOC_Os03g52510 is over 11,000 times in ZH9308 than that in XB, which may positively regulate FLL; the expression level of LOC_Os12g39000 is lower in ZH9308 than that in XB, which is potentially regulating FLW. LOC_Os03g52470, LOC_Os08g29809, and LOC_Os03g21660 had extremely differential expressions in parents. So far, these three genes have not been reported to regulate leaf morphology, indicating that they may be novel genes that regulate FLL, FLW, and FLR. These results may lay a theoretical basis and provide genetic resources for ideal plant architecture breeding in rice.
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