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by Robert Henry and Daniel Waters November 2006
RIRDC Publication No 06/123 RIRDC Project No USC-6A
The flavour and fragrance of Basmati and Jasmine style rice have been associated with increased levels of the compound, 2-acetyl-1-pyrroline (2AP). Rice breeders require a simple, accurate and inexpensive method to distinguish fragrant from non-fragrant rice within a breeding program if they are to efficiently develop fragrant rice varieties and take advantage of the premium consumers are willing to pay for fragrant rice. A number of methods have been utilised to assist breeders in selecting fragrant rice but they have limitations when processing large numbers of samples. A perfect molecular marker is a marker that is within the gene that codes for a trait. Workers at Southern Cross University found that an eight base pair deletion and three SNPs in a gene encoding a betaine aldehyde dehydrogenase 2 (BAD2) homolog was the likely cause of fragrance in Jasmine and Basmati style rice. Non-fragrant rice varieties possess what appears to be a fully functional copy of the gene encoding BAD2 while fragrant varieties possess a copy of the gene encoding BAD2 which contains the deletion that presumably disables the BAD2 enzyme. This polymorphism provided an opportunity for the construction of a perfect marker for fragrance in rice. A competitive allele specific PCR assay for the polymorphism was developed and accurately predicted the fragrance status of each of the individuals within a population of 168 plants derived from a cross of fragrant (Kyeema) and nonfragrant (Gulfmont) parents which were segregating for fragrance. The assay also accurately predicted the fragrant status of 88 fragrant and non-fragrant rice varieties. The assay can be used to detect heterozygous individuals and mixed populations.
Rice starch has a semi-crystalline structure which is disrupted by cooking, transforming it into a softer edible gel like material. The temperature at which rice starch gelatinises is an important component of rice eating quality because it is associated with the cooking time and texture of cooked rice. Although rice starch gelatinisation temperature (GT) is genetically determined, it also displays a high level of variability in response to the influence of the environment in which the plant is grown. It was known the major gene that controls rice amylopectin structure and starch GT codes for soluble starch synthase IIa (SSIIa), it was not known how many versions of the gene were found in commercial rice varieties and which were responsible for high or low GT starch. DNA sequence analysis of 70 rice varieties that differed by GT allowed identification of DNA differences which led to amino acid changes that were associated with two statistically significant GT classes. The high GT class (average GT of 78 oC) was genotype G/GC only, while the low GT class (average GT of 70 oC) was either genotype A/GC or G/TT. A competitive allele specific PCR was chosen as the assay for this trait. The nature of the mutation and the kinetics of each PCR meant each possible DNA difference was assayed independently. The markers detect all possible genotypes which impact upon GT. The parents within a breeding program can be screened in order to identify which versions of SSIIa each carry. Using the information derived from this process, rice breeders will be able to identify undesirable genotypes to be removed from the breeding program.
Based on the experience of the Californian rice industry which suffered its first blast outbreak in 1996 after being blast free for 85 years and experience with other crops, breeding rice varieties not carrying any resistance to internationally important diseases is undesirable. If a disease was introduced to the Australian rice industry, the consequences could be serious. For those diseases which do not currently exist in the Australian rice industry, use of molecular markers tightly linked to resistance genes avoids the need for selection using a pathogen challenge, a technique which would expose the industry to obvious risk because the pathogen would be introduced into the country. Molecular markers also offer other benefits including allowing the rice breeder to identify plants in the breeding program which have broken the linkage between disease resistance genes and undesirable genes, determining if a resistant plant derives its resistance from one or more resistance genes, and enabling more than one disease resistance gene to be efficiently incorporated into a single line of rice. This is particularly useful as incorporation of several resistance genes has been shown to reduce the likelihood of pathogens overcoming resistance. It has been determined that blast poses the greatest threat to the Australian rice industry. Under blast favourable conditions, outbreaks have caused crop losses of up to 60%. Because of this risk, varieties which carry a range of genes for blast resistance were procured by the rice breeding program as the first step in initiating a program of pre-emptive rice blast disease resistance breeding. Rice varieties BL14 and BL24 were chosen as donors of resistance as they carry blast resistance genes which are well characterised at the molecular level and hence allow accurate application of molecular marker technology.
Hybrid rice lines are used widely in China because they yield up to 20% higher than traditional inbred rice lines. The type of hybrid rice system most suited to the Australian rice growing regions is the cytoplasmic male sterile or three line, system. For the system to work efficiently it is important no B line rice contaminates the A line as the yield will be reduced due to the non-hybrid self fertilised low yielding B line diluting the high yielding hybrid seed. A simple laboratory test has been developed which distinguishes between the A and B lines so A line purity can be checked without having to grow the rice. We have checked and found the laboratory test that distinguishes between the A and B lines works with 12 hybrid rice lines that were imported from China.
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