Genotyping of the fragrance allele in rice

We have previously determined that fragrance in rice, a recessive trait, is due to a large deletion (8bp) and 3 SNP’s in a gene on chromosome 8 which encodes a putative betaine aldehyde dehydrogenase 2 (BAD2). This mutation results in the formation of a truncated BAD2 enzyme because of the creation of an in-frame termination signal 800bp before that of the wild type. Because this truncated BAD2 is missing key binding domains, it is unlikely that it is capable of acting upon the target substrate and this leads to an accumulation of the principal fragrant molecule, 2-acetyl-1-pyrroline. Here we utilise single tube allele specific amplification (STASA) as a simple, low-cost, perfect assay to discriminate between fragrant and non-fragrant rice varieties in addition to homozygous fragrant, homozygous non-fragrant and heterozygous non-fragrant individuals in a population segregating for fragrance. Two external primers generate a 580bp fragment as a positive control for each sample. Internal primers in conjunction with their corresponding external primer pair produce a 355bp fragment from a non-fragrant allele and a 257bp fragment from a fragrant allele, allowing analysis on agarose gels.