Identification of rice seed phospholipase D null gene and characterization of seed phospholipase D null rice

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Rice bran could be a valuable source of nutrition because of its high content of fat, protein, and crude fiber, but its use for human consumption is severely limited by the rapid degradation of rice bran oil and more rapid development of hydrolytic rancidity than occurs in other vegetable oils. Triacylglycerols (TAGs), which are the primary lipids contained in rice bran, occur in oil bodies with phospholipid membranes. These membranes are disintegrated by phospholipase D (PLD) and the released TAGs are degraded by lipase to free fatty acids, resulting in poor bran quality. Since PLD serves as a trigger to initiate the lipid degradation and the deterioration of bran quality, we isolated a PLD-null rice mutant, "03-s108" by using anti-PLD polyclonal antibodies. Although PLD null phenotype is inherited as a single recessive trait, the screening method for PLD null strains involves western blotting with progeny test that consumes time and effort. To develop a convenient PLD null strain detection method, we performed a linkage analysis between PLD null phenotype and SSR markers using F2 progeny from "03-s108" and "Koshihikari" and mapped PLD null phenotype near RM3453 on chromosome 1 carrying one putative PLDs genes (AB001920).In the DNA sequencing analysis, the "03-s108" allele of AB001920 showed a transition mutation from G to A on the third exon. This change resulted in a stop codon, implying a nonsense mutation in the "03-s108" allele. Using this DNA sequence data, we developed cleaved amplified polymorphic sequence and dot-blot-single nucleotide polymorphism analyses for screening and breeding of PLD null rice varieties. These methods are easy and cost-effective for screening PLD null strains.