Characterization of the major fragance gene from an aromatic japonica rice and analysis of its diversity in Asian cultivated rice

In Asian cultivated rice (Oryza sativa L.), aroma is one of the most valuable traits in grain quality and 2-ACP is the main volatile compound contributing to the characteristic popcorn-like odour of aromatic rices. Although the major locus for grain fragrance (frg gene) has been described recently in Basmati rice, this gene has not been characterised in true japonica varieties and molecular information available on the genetic diversity and evolutionary origin of this gene among the different varieties is still limited. Here we report on characterisation of the frg gene in the Azucena variety, one of the few aromatic japonica cultivars. We used a RIL population from a cross between Azucena and IR64, a non-aromatic indica, the reference genomic sequence of Nipponbare (japonica) and 93–11 (indica) as well as an Azucena BAC library, to identify the major fragance gene in Azucena. We thus identified a betaine aldehyde dehydrogenase gene, badh2, as the candidate locus responsible for aroma, which presented exactly the same mutation as that identified in Basmati and Jasmine-like rices. Comparative genomic analyses showed very high sequence conservation between Azucena and Nipponbare BADH2, and a MITE was identified in the promotor region of the BADH2 allele in 93–11. The badh2 mutation and MITE were surveyed in a representative rice collection, including traditional aromatic and non-aromatic rice varieties, and strongly suggested a monophylogenetic origin of this badh2 mutation in Asian cultivated rices. Altogether these new data are discussed here in the light of current hypotheses on the origin of rice genetic diversity

The gene for fragrance in rice

The flavour or fragrance of basmati and jasmine rice is associated with the presence of 2-acetyl-1-pyrroline. A recessive gene (fgr) on chromosome 8 of rice has been linked to this important trait. Here, we show that a gene with homology to the gene that encodes betaine aldehyde dehydrogenase (BAD) has significant polymorphisms in the coding region of fragrant genotypes relative to non-fragrant genotypes. The accumulation of 2-acetyl-1-pyrroline in fragrant rice genotypes may be explained by the presence of mutations resulting in a loss of function of the fgr gene product. The allele in fragrant genotypes has a mutation introducing a stop codon upstream of key amino acid sequences conserved in other BADs. The fgr gene corresponds to the gene encoding BAD2 in rice, while BAD1 is encoded by a gene on chromosome 4. BAD has been linked to stress tolerance in plants. However, the apparent loss of function of BAD2 does not seem to limit the growth of fragrant rice genotypes. Fragrance in domesticated rice has apparently originated from a common ancestor and may have evolved in a genetically isolated population, or may be the outcome of a separate domestication event. This is an example of effective human selection for a recessive trait during domestication

Inactivation of an aminoaldehyde dehydrogenase is responsible for fragrance in rice

Rice (Oryza sativa) has two betaine aldehyde dehydrogenase homologs, BAD1 and BAD2, encoded on chromosome four and chromosome eight respectively. BAD2 is responsible for the characteristic aroma of fragrant rice. Complementary DNA clones of both BAD1 and BAD2 were isolated and expressed in E. coli. BAD2 had optimum activity at pH 10, little to no affinity towards N-acetyl-gamma-aminobutyraldehyde (NAGABald) with a Km of approximately 10 mM and moderate affinity towards gamma-guanidinobutyraldehyde (GGBald) and betaine aldehyde (bet-ald) with Km values of approximately 260 mu M and 63 mu M respectively. A lower Km of approximately 9 mu M was observed with gamma-aminobutyraldehyde (GABald), suggesting BAD2 has a higher affinity towards this substate in vivo. The enzyme encoded on chromosome four, BAD1, had optimum activity at pH 9.5, showed little to no affinity towards bet-ald with a Km of 3 mM and had moderate affinity towards GGBald, NAGABald and GABald with Km values of approximately 545, 420 and 497 mu M respectively. BAD1 had a half life roughly double that of BAD2. We discuss the implications of these findings on the pathway of fragrance generation in Basmati and Jasmine rice and the potential of rice to accumulate the osmoprotectant glycine betaine