Development of three allele-specific co-Dominant PCR markers suitable for marker-assisted selection of amylose class and paste viscosity of rice

Authors: Chen, Ming Hsuan; Fjellstrom, Robert; Christensen, Eric; BERGMAN, CHRISTINE - University Of Nevada

Submitted to: Rice Technical Working Group Meeting Proceedings
Publication Type: Proceedings
Publication Acceptance Date: 1/20/2010
Publication Date: 2/22/2010
Citation: Chen, M., Fjellstrom, R.G., Christensen, E.F., Bergman, C.J. Development of three allele-specific co-Dominant PCR markers suitable for marker-assisted selection of amylose class and paste viscosity of rice. Poster Presentation, Rice Technical Working Groups, Biloxi, MS, Feb. 22, 2010.

Interpretive Summary:

Technical Abstract: Most rice is consumed as whole kernel cooked rice, and the consumer preferences for cooked rice texture and other sensory properties differ among regions of the world. Rice is also used as an ingredient in a multitude of foods by food-processing companies across the globe. These sensory and functional properties of rice are predominantly associated with its apparent amylose content (AAC). Thus, rice breeders around the world developing breeding lines with specific end-use properties often select for specific AAC. However, AAC and other processing related properties, such as starch paste viscosity, are traits known to be variably affected by environmental factors. Both AAC and pasting properties have been genetically mapped to the Waxy gene, encoding the granule bound starch synthase enzyme that is active in the developing rice endosperm. Recent studies identified three functional single nucleotide polymorphisms (SNPs) in the Waxy gene. These SNPs had a strong association with AAC and pasting properties. These findings provide opportunities for the selection of desired end-use quality traits using molecular genetic technologies. The methods reported in the original identification of these SNPs and their associations are either low-throughput genotyping methods or require special instrumentation along with skills not typically found in molecular-breeding programs. The development of a high-throughput method with a simple protocol for analyzing these SNPs would greatly enhance their usefulness as part of a marker assisted selection scheme. We have successfully developed three co-dominant allele-specific PCR (AS-PCR) markers to genotype these three functional SNPs in the rice Waxy gene. Each marker contained two allele-specific primers and one common primer. For each marker, the two allele-specific primers differed by one base at the 3'-end to provide discrimination of the SNP alleles, and were labeled with unique fluorescence probes. An additional mismatched base, the third base from the 3'-end, was inserted in some allele-specific primers to increase selectivity. Thus, for each AS-PCR marker, the alleles were differentiated or detected by color and by length of the amplified products. These co-dominant markers selectively identify and amplify target SNP alleles from genomic DNA in a single step by including both allele-specific primers and one common primer. The PCR-amplified product can be detected by capillary electrophoresis and fluorescence using the same instrumentation as those for microsatellite markers. The assay is as simple as those for microsatellite markers. With these AS-PCR markers, rice breeders are now able to select the desired quality traits in their breeding lines based on genetics, thus eliminating the need to routinely phenotype the same breeding lines in multiple locations. These markers would be useful in selecting homozygous breeding lines and diverse germplasm and in verifying varieties and cultivars with respect to their amylose classes and paste viscosities.