Identification of gene for rice (Oryza sativa) seed lipoxygenase-3 involved in the generation of stale flavor and development of SNP markers for lipoxygenase-3 deficiency

Deterioration of rice grains and the development of stale flavor during storage are serious problems that can reduce the quality of stored rice. Lipoxygenase (LOX) catalyzes the peroxidation of lipids in rice grains, leading to the formation of volatile compounds. LOX activity in rice seeds is localized in the bran fraction, and LOX-3 is the major isozyme component. We previously found that the Thai variety Daw Dam lacks LOX-3 protein and that LOX-3 null rice has less stale flavor during storage than normal LOX-3 rice. Thus, introduction of the LOX-3 null phenotype in mature seeds would facilitate the retention of high quality during storage. Although the LOX-3 null phenotype is inherited as a single recessive trait, the widely used screening method for LOX-3 null strains involves Western blotting with a progeny test that requires considerable time and effort. To develop a convenient LOX-3 null strain detection method, we performed linkage analysis between the LOX-3 null phenotype and restriction fragment length polymorphism and simple sequence repeat markers using F, progeny from Daw Dam and Nipponbare, and mapped the LOX-3 null phenotype between RM6736 and RM6329 on chromosome 3 containing three putative LOX genes. LOX-3 protein was purified using an affinity column bound with anti-LOX-3 monoclonal antibodies, and its amino acid sequences were determined. These sequences matched only those of the protein encoded by Os03g0700400, one of the three putative LOX genes. In DNA sequencing analysis, the Daw Dam allele of Os03g0700400 showed transition Mutation from G to A on the seventh exon. This change resulted in a stop codon, implying a nonsense mutation in the Daw Dam allele. Based on these results, Os03g0700400 was confirmed to be the rice seed LOX-3 gene. Using these DNA sequence data, we developed a cleaved amplified polymorphic sequence and dot-blot-single nucleotide polymorphism analyses for screening and breeding LOX-3 null rice varieties. These methods are easy and cost-effective for screening LOX-3 null strains.