Identification and characterization of genes involved in antioxidant traits in local Thai rice (Oryza sativa L.)

Developing rice (Oryza sativa L) cultivars with high antioxidant activities have become increasingly important since they have nutritional advantages for human health. Hence, the researchers must precisely elucidate the genetic bases controlling antioxidant-related traits in rice grains. In this study, we aimed to identify the genes involved in antioxidant traits in local Thai rice. The first strategy was conducting a genome-wide association analysis (GWAS) using 233 accessions of local Thai rice. A total of 119,541 single nucleotide polymorphic (SNP) markers was used as genotype data. In contrast, the five antioxidant assays, ABTS, DPPH, FRAP, TFC, and TFC, were used as phenotypic traits. The single-locus GWAS model (MLM) predicted 336 QTNs located in 93 loci. About 83 % of the predicted loci in this study were consistently mapped with the reported QTLs of antioxidant traits. There were 10 loci, including OsRc gene, found consistently co-associated in all phenotypic traits. Two genetic markers for polymerase chain reaction detection were developed in OsTT8 gene and promoter region of OsRc and both markers were positively correlated with the antioxidant properties. The second strategy to identify the genes related to antioxidant traits was performing GWAS with multiple models, which were a single-locus GWAS model (GLM, MLM, and CMLM) and multi-locus GWAS model (mrMLM, FASTmrMLM, and pKWmEB). A total of 1893 QTNs was significantly identified across six GWAS models, and 30 loci were co-detected by at least two GWAS models. Six selected loci, LOC_Os07g11070, predicted by single locus - GWAS model only, LOC_Os04g03860 and LOC_Os04g07200 predicted by multi-locus model only, and LOC_Os02g23960, LOC_Os04g47080 / OsRa and LOC_Os07g11020 / OsRc, predicted by both single-locus and multi-locus models to investigate the gene expression in the developing seeds of white rice and red-pigmented rice, HGD rice. All of the selected genes showed significantly different gene expression, compared between white rice and colored rice developing seeds, except LOC_Os07g11070 showing the similar gene expression level, suggesting that this gene may not be really related to antioxidant level in seeds. The third strategy, the protein-protein interaction of Rc protein was determined by a shotgun proteomic approach. Two Rc proteins (full length/Rc206 and truncated/Rc201) have been expressed in the E. coli vector. Then, these proteins were bound to rice seed protein extracts. The Q10GP0 (Putative B3 domain protein) from the white rice seeds was found to be the most significantly binding to the Rc protein from red-pigmented rice. This suggested that Q10GP0 may act competitively with Rc protein targets and inhibit Rc protein function in white rice leading to the no synthesis of pigments in white rice seeds.