Identification of a Seed Phospholipase D Null Allele in Rice ( Oryza sativa L.) and Development of SNP Markers for Phospholipase D Deficiency

Rice (Oryza sativa L.) bran usage is severely limited by the rapid degradation of rice bran oil and rapid development of hydrolytic rancidity. Because phospholipase D (PLD) disintegrates phospholipid membranes of oil bodies to dete- riorate bran quality, we isolated a PLD-null rice mutant, '03-s108', whose PLD-null phenotype is inherited as a single recessive trait. To develop a convenient DNA marker-assisted selection method for the PLD-null trait, we fi rst per- formed a linkage analysis between the PLD-null phenotype and simple sequence repeat (SSR) markers by using F 2 progeny from 03-s108 × 'Koshihikari' crosses, and we mapped the PLD- null genotype to chromosome 1 near a putative PLD gene sequence, PLDα1. DNA sequencing of the 03-s108 allele of PLDα1 revealed a tran- sition mutation (G to A) in the third exon. This change resulted in a stop codon, indicating the presence of a nonsense mutation in the 03-s108 allele. Using this DNA sequence information, we then developed methods of cleaved ampli- fi ed polymorphic sequence (CAPS) and dot-blot single-nucleotide polymorphism (SNP) detec- tion for this mutation, and we confi rmed that the sequence polymorphism was predictive of the presence or absence of the seed PLD-null phe- notype. These CAPS and dot-blot-SNP methods are easy and cost-effective means for identify- ing seed PLD-null strains, to enable screening and breeding of PLD-null rice varieties. National Agriculture and Food Research Organization (NARO), Insti- tute of Crop Science (NICS), Kannondai, Tsukuba, Ibaraki 305-8518, Japan. K. Shirasawa, current address: Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan. Received 20 Dec. 2010. *Corresponding author (suzuyasu@aff rc.go.jp). Abbreviations: CAPS, cleaved amplifi ed polymorphic sequence; FFA, free fatty acid; LOX-3, lipoxygenase-3; PCR, polymerase chain reac- tion; PLD, phospholipase D; SNP, single-nucleotide polymorphism; SSR, simple sequence repeat; TAG, triacylglycerol.