Fine Mapping and Candidate Gene Identification of ORUFILM03g000096 Gene in Weedy Rice LM8: Insights into Grain Length Regulation

Although the yield and resistance of cultivated rice have been significantly enhanced since domestication, its genetic basis remains narrow, with many beneficial genes having been lost. Weedy rice is an important source of genetic material for improving rice and provides valuable germplasm resources for identifying excellent genes. In our study, we constructed an F₂ genetic population by crossing the weedy rice variety LM8, which has extremely small grains, with cultivated rice. High-density genetic maps were constructed using whole-genome sequencing. Five grain shape traits were observed in the F₂ population, including grain length, grain width, grain thickness, length width ratio and thousand grain weight. The phenotype data were found to be consistent with a normal distribution. Through linkage analysis of the phenotype and genetic maps, a total of 14 QTL loci were identified, one of which was associated with grain length. Grain length values ranged from 5.32 to 11.14 mm. The associated locus was located at 18.81-18.87 Mb on chromosome 3, and accounted for 19.88% of the total phenotypic variation. The BC₁F₂ population was constructed using Bulked Segregant Analysis (BSA) sequencing to locate the grain length gene. The ORUFILM03g000096 gene, which is homologous to the Os03g0427300 gene of Nipponbare, was identified as the grain length gene. According to the annotation of the known genes, the ORUFILM03g000096 gene belongs to the Glutelin (GLU) gene family, which is generally associated with rice quality. A systematic investigation of the expression profile of ORUFILM03g000096 gene using the Rice Expression Profile (RiceXPro) database showed high expression in young panicles. Evolutionary tree analysis revealed a closer evolutionary relationship with the Japanese gene sequence. Hplotype analysis of the gene in the hybrid offspring indicated that a non-synonymous mutation (G-A), located 861 bp downstream of the ATG site in the third exon of the candidate gene, affected the grain length. It is hypothesized that this gene plays a crucial role in grain length growth and enhances rice quality.