Identifying Heat Adaptability QTLs and Candidate Genes for Grain Appearance Quality at the Flowering Stage in Rice

High temperature significantly impacts grain appearance quality, yet few studies have focused on identifying new quantitative trait loci (QTLs)/genes related to these traits under heat stress during the flowering stage in rice. In this study, a natural population of 525 rice accessions was used to identify QTLs and candidate genes associated with grain appearance quality using a Genome-Wide Association Study under heat stress. We identified 25 QTLs associated with grain length (GL), grain width (GW), and grain chalkiness (GC) under heat stress across 10 chromosomes in the three rice populations (full, indica, and japonica). Notably, three sets of overlapping QTLs were identified (set 1: qHTT-L3 and qHTT-XL3; set 2: qHTT-C5 and qHTT-XC5; set 3: qHTT-L11.1 and qHTT-GL11), located on chromosomes 3, 5, and 11, respectively. Haplotype analysis indicated that Hap1 is the superior haplotype, and pyramiding more than two superior alleles improved rice grain appearance quality (longer GL, wider GW, and lower GC) in high-temperature environments. Based on RNA-seq, qRT-PCR and functional annotations analysis, LOC_Os05g06920, LOC_Os05g06970, and LOC_Os11g28104 were highly expressed, identifying them as the high-priority candidate genes for QTLs linked to grain appearance quality (GL, GW, and GC) under heat stress. Expression analysis revealed that LOC_Os05g06920, which encodes a relA-SpoT-like protein RSH4, and LOC_Os11g28104, which encodes a protein kinase with a DUF26 domain, were highly expressed in seeds, leaves, and shoots. And LOC_Os05g06970, encoding a peroxidase precursor, exhibited high expression levels in roots. Compared to the wild-type (WT) plants, the mutants of LOC_Os05g06920, LOC_Os05g06970, and LOC_Os11g28104 exhibited increased GL and grain length-to-width ratio, but reduced GW under both natural and heat stress conditions, while the LOC_Os05g06970 and LOC_Os11g28104 mutants significantly increased the chalky grain rate and grain chalkiness degree under natural conditions. Furthermore, the LOC_Os05g06920, LOC_Os05g06970, and LOC_Os11g28104 mutants showed a lower decline in grain appearance quality traits than the WT after high-temperature treatment. These findings suggest that LOC_Os05g06920, LOC_Os05g06970, and LOC_Os11g28104 play crucial roles in regulating both grain development and heat tolerance under heat stress at anthesis, thus affecting grain appearance quality in rice. Our results provide a promising genetic resource for improving rice grain appearance quality under heat stress.