Submitted to: Rice Technical Working Group Meeting Proceedings
Publication Type: Proceedings
Publication Acceptance Date: December 1, 2003
Publication Date: June 1, 2004
Citation: Fjellstrom, R.G., Chen, M., Bergman, C.J., McClung, A.M. 2004. Single nucleotide polymorphism markers at the rice Alk locus controlling alkali spreading value. In: Proceedings of the 30th Rice Technical Working Group Meeting. 2004 CDROM. Technical Abstract: Alkali spreading value (ASV) is a standard assay used to classify the processing and cooking quality of rice cultivars. The ASV measurement is a rating of grain dispersion in a 1.5 or 1.7% KOH solution after 16-24 hours and provides a simple means of classifying rice into high, intermediate and low gelatinization temperature types. Classifying rice varieties according to gelatinization temperature is useful in determining ones that are appropriate for use in parboiling, quick cooking, puffing, extruding, and other rice cooking and processing technologies. ASV has been reported to be primarily controlled by the action of one or two genes. The Alk locus on rice chromosome 6 has often been cited as a major gene controlling ASV and has recently been found to encode soluble starch synthase IIa (SSSIIa). Our research aimed at analyzing sequence variation in the Alk gene across a wide range of rice germplasm in order to identify possible DNA markers associated with ASV in US cultivars and international rice accessions. Sequence variation analysis of the Alk gene was initiated by searching public database information for genomic sequences that had high similarity with starch synthase genes and mapped near the Alk locus on rice chromosome 6. DNA sequence information from the cultivars Nipponbare and 93-11, being sequenced by the Japanese Rice Genome Project and the Beijing Genomics Institute, respectively, were compared to identify candidate Alk gene polymorphisms resulting in amino acid changes in the encoded Alk protein. These functional single nucleotide polymorphisms (SNPs) could be identified by restriction endonuclease digestion of PCR amplification products, which was used to determine the degree of association between Alk gene SNPs and ASV in 190 rice accessions. Sequencing of the Alk gene exon regions in eight unrelated medium and long grain international cultivars (Lemont, L-202, M-202, Mars, Te-Qing, Khao Dawk Mali 105, Basmati 370, and Phudugey) was also performed to identify any additional sequence polymorphisms not found in Nipponbare and 93-11. Genetic linkage of functional SNPs in the Alk gene with ASV was analyzed in F3 families from two crosses: 1) Panda/M-104, where both parents have low amylose, but have low and high ASV scores, respectively; and 2) Lemont/Jasmine-85, where the parents have low ASV and intermediate amylose and high ASV and low amylose, respectively. A soluble starch synthase II isoform sequence (GenBank entry AF419099) from an unidentified rice cultivar was found to reside on a BAC (GenBank entry AP003509) located in proximity to the Alk locus. As we proceeded in our analysis of this gene, it was subsequently determined by Japanese researchers to be the Alk gene encoding the SSSIIa enzyme. Comparing the Nipponbare and 93-11 sequences of the Alk gene indicated the presence of only two DNA polymorphisms resulting in amino acid changes between these two rice accessions. The presence or absence of these changes resulted in the identification of three Alk alleles (sequence haplotypes): the Nipponbare haplotype, having a mutation at nucleotide 2412 of GenBank entry AF419099; the 93-11 haplotype, having a mutation at nucleotide 2514, and the Lemont haplotype, having no mutations. We have coded these alleles as the NPBR, M202 (which shares the same mutation as 93-11), and LMNT haplotypes, respectively. No other sequence differences resulting in amino acid changes were identified in Alk genes of the eight unrelated cultivars that were analyzed. We found that most Japanese varieties with high ASV scores (ASV = 6.0 to 7.0, corresponding to low gelatinization temperature) carry the NPBR haplotype. The M202 haplotype was found in medium grain cultivars grown in California and the southern US, which typically have high ASV scores. The M202 haplotype was the more common high ASV haplotype in international germplasm. Cultivars with either low or intermediate ASV scores (ASV = 2.0 to 5.0), which includes typical southern US long grain varieties, possessed the LMNT haplotype. So far, very low ASV scores of 2.0 to 3.0 have only been identified in genotypes having low amylose content (< 20%) with the LMNT haplotype for Alk. Waxy rices that had intermediate ASV values all carried the LMNT haplotype and those that had high ASV values commonly carried the M202 or, less commonly, the NPBR haplotypes (as was observed in the non-waxy cultivars). In genetic analyses, for the Panda/M-104 cross the Alk SNP marker explained over 91% of the variance in ASV. In the Lemont/Jasmine-85 cross, over 61% of the variation in ASV was explained by the Alk SNP marker. An additional 4.1% (p<0.0001) of the ASV variation in this cross was explained by a Waxy gene marker (RM190), which controlled 74% of the amylose content variation. Evidently, factors like amylose content or environmental effects can alter ASV levels to varying degrees in different crosses, which we are continuing to investigate. Conclusively, the Alk SNP markers are successful in explaining most of the variation in ASV. Because of this strong genetic association, we have developed allele specific oligonucleotide primers that allow the direct detection of Alk SNPs for use in marker aided selection of ASV in breeding populations.