Identification of the gene responsible for fragrance in rice and characterisation of the enzyme transcribed from this gene and its homologs

The flavour or fragrance of Basmati rice is associated with the presence of 2-acetyl-1- pyrroline. This work shows that a gene with homology to betaine aldehyde dehydrogenase (BAD) has significant polymorphisms in the coding region of fragrant genotypes relative to non fragrant genotypes. Accumulation of 2-acetyl-1-pyrroline in fragrant rice genotypes may be explained by the presence of mutations resulting in loss of function of the fgr gene product. The fgr gene corresponds to the gene encoding BAD2 in rice while BAD1 is encoded by a gene on chromosome 4. Development of an allele specific amplification (ASA) based around the deletion in the gene encoding BAD2 allows, perfect, simple and low cost discrimination between fragrant and non-fragrant rice varieties and identifies homozygous fragrant, homozygous non-fragrant and heterozygous non-fragrant individuals in a population segregating for fragrance. The cDNAs transcribed from rice chromosomes 4 and 8, each encoding an enzyme with homology to betaine aldehyde dehydrogenase were cloned and expressed in E. coli. The enzyme responsible for fragrance, encoded from chromosome 8, had optimum activity at pH 10, showed low affinity towards betaine aldehyde (bet-ald) with Km value of approximately 63ìM but a higher affinity towards -aminobutyraldehyde (GABald) with a Km value of approximately 9ìM. The enzyme encoded from chromosome 4 had optimum activity at pH 9.5 and showed generally lower affinity towards most substrates compared to the enzyme encoded from chromosome 8, substrate specificities suggest that the enzymes have higher specificity to aminoaldehydes and as such both should be renamed as an aminoaldehyde dehydrogenase (AAD). The inactivation of AAD2 (BAD2) in fragrant rice varieties likely leads to accumulation of its main substrate GABald which then cyclises to Δ¹-pyrroline the immediate precursor of 2AP

Generation of simple, perfect markers for cereal quality traits

Commercially important traits including fragrance and starch gelatinisation temperature (GT) are difficult phenotypes for breeders to asses. We recently identified polymorphisms that control these key rice quality traits. The annotated rice genome sequence used in combination with re-sequencing by PCR facilitated the discovery of these polymorphisms and the design of perfect molecular markers for each of these traits. In the absence of a genome sequence, larger mapping populations, a genome library and more sequencing would have been necessary. Although these traits result from different classes of polymorphism, fragrance is due to an eight base pair deletion while gelatinisation temperature variation results from a polymorphism at a single nucleotide in combination with another polymorphism at two adjacent bases elsewhere in the gene, both have proven to be amenable to the development of a competitive allele specific PCR assay. Both are simple, cheap, robust, perfect genotyping assays for these difficult to measure and complex traits. Understanding the link between these polymorphisms and the resultant phenotype could be extended to other cereals and used for the identification and or generation of valuable phenotypes in these cereals. Similar assays could then be easily developed for use in breeding programs

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

Fragrance of rice is associated with an altered form of BAD2 on chromosome 8

Fragrance is a desirable trait of Basmati and other rice varieties associated with a recessive gene on chromosome 8. Sequencing of chromosome 8 of rice in the region of the fgr locus has identified an allele of a betaine aldehyde dehydrogenase (BAD) gene common to all fragrant genotypes. The gene encoding BAD was found in the region of the BAC clone identified as most likely to contain the fgr gene by mapping with SSR and SNP markers. The alleles from fragrant varieties all have common indels and SNPs relative to non fragrant genotypes demonstrating that the fgr gene in all fragrant genotypes tested was derived from a common ancestor and suggesting that fragrant rice results from a separate domestication event or has evolved in isolation from non fragrant genotypes

Discovery of genes for quality and nutritional traits in rice

The appeal of rice to human consumers is influenced by the flavour or fragrance, and the texture of the cooked rice. Genes for both these traits have now been discovered. The recent availability of a complete high quality rice genome sequence has simplified the task of gene discovery in rice. The genetic basis of key quality traits may now be investigated by analysis of differences in the sequence of genes or genome regions associated with the traits. Fragrance of rice is very important in Basmati (Indian) and Jasmine (Thai) style rices. This attribute had been associated with a gene on chromosome 8. Sequencing of the genes in this chromosome region revealed fragrance is caused by a deletion of 8 bp in a gene encoding an aldehyde dehydrogenase resulting in the loss of function (Bradbury et al., 2005a). Fragrance in rice appears to be the result of a common mutation event in all fragrant varieties. Routine and robust selection tools for this trait are now available (Bradbury et al., 2006b). The gelatinization temperature of rice was known to be linked to a region encoding a starch synthase gene. DNA sequence variations resulting in two different amino acid substitutions in the encoded protein both result in a gelatinization temperature decrease of about 8 C (Waters et al., 2006). Selection for cooking quality in the domestication of rice may have contributed to the selection of rice varieties with a higher GI. New molecular selection tools allow high through put screening of rice lines for a wide range of important traits (Kennedy et al., 2006)

Functional analysis of rice genes determining fragrance

The fragrance of rice has been associated with a deletion resulting in the apparent loss of function of a gene annotated as a betaine aldehyde dehydrogenase (BAD2) on chromosome 8. We have now compared the levels of expression of BAD2 and BAD1 (chromosome 4) from fragrant and non fragrant genotypes. Expression of BAD2 predominates in non fragrant genotypes but is reduced in genotypes with the fragrance allele. Expression of BAD1 and BAD2 proteins in bacteria was used to test the specificity of the reactions catalysed by these enzymes. This analysis suggests that BAD2 encodes a 4-aminobutyraldehyde dehydrogenase. Analysis of transgenic rice plants over-expressing these genes is in progress

Rice genome sequence accelerates the discovery of commercially important genes and polymorphisms

Traditionally, identification of genes which control important traits has been labour intensive and time consuming. We have demonstrated the annotated rice genome sequence used in combination with re-sequencing by PCR greatly facilitates the discovery of both genes and polymorphisms within genes, which control commercially important traits. Identification of the gene which controls fragrance was achieved using a relatively small mapping population of 168 F2 individuals. Analysis of the recombination data and the relatively large tract (385 kbp) of annotated genome sequence between the flanking markers revealed a candidate gene in this region which plausibly explained the known biochemistry of fragrance. Re-sequencing the gene in a fragrant variety found a mutation which was in accord with the known genetics of fragrance. In the absence of a genome sequence, a much larger mapping population, a genome library and more sequencing would have been necessary. Likewise, availability of the rice genome sequence greatly simplified the task of re-sequencing the SSIIa encoding gene which allowed us to identify single nucleotide polymorphisms (SNP) in soluble starch synthase IIa which explain gelatinisation temperature (GT). This important quantitative trait seems to be determined by two SNP in the 3’ end of the coding sequence. Because starch bio-synthetic genes display high levels of interspecific conservation, it is likely this knowledge will have utility in other species