Submitted to: Rice Technical Working Group Meeting Proceedings
Publication Type: Proceedings
Publication Acceptance Date: December 1, 2003
Publication Date: June 1, 2004
Citation: Chen, M., Bergman, C.J., Fjellstrom, R.G. 2004. Waxy locus genetic variation associated with amylose content in international rice germplasm. In: 30th Proceedings of the Rice Technical Working Group Meeting. CDROM. Technical Abstract: Rice end-use quality is strongly impacted by amylose content. The Waxy gene on chromosome 6 encodes the granule-bound starch synthase enzyme (GBSS) and controls much of the variation in rice apparent amylose (AA). A microsatellite in the non-coding region of the Waxy exon 1 containing a CT simple sequence repeat has been identified, and displays variation associated with the different amylose classes, i.e. high, intermediate, and low AA. A genetic marker for this CT repeat has been developed and is being used by the rice breeders for progeny selection in varietal development. This marker accelerates the breeding process because progeny can be selected at any developmental stage, and also enables accurate varietal selection at the genetic level thus minimizing the analysis of grain AA, which is influenced by the environment. Further, a single nucleotide substitution from G to T (G/T SNP) at the first intron 5' splice site was identified that is associated with low AA varieties. This mutation reduces the splicing efficiency of the leader intron of GBSS, and is associated with differential temperature sensitivity during grain development of low AA varieties. In addition, a sequence alignment of two high- and one intermediate-AA varieties shows a single nucleotide difference in exon 6 (ex6 SNP), which alters the amino-acid codon, and provides separation of the two amylose classes. The objective of this study is to examine the linkage between the CT repeats, the G/T SNP at the leader intron splice site, and the ex6 SNP, and the association of these polymorphic combinations with AA using rice germplasm of diverse origin. The genomic DNA of rice varieties was extracted from leaf tissues. The CT repeat was determined using the Waxy microsatellite marker. The G/T SNP was analyzed by AccI cleavage after PCR amplification. The sequence variation in exon 6 was detected by the dideoxyfingerprinting method, a hybrid between single-strand conformation polymorphism and the dideoxy sequencing method, and was scored against a Nipponbare control. Of the 150 non-glutinous rice accessions tested, nine CT microsatellite alleles were identified that explained a large portion of the variation in AA. The CT repeats of 17, 18, and 19, which associate with the rice accessions of AA ranging from low- to intermediate-AA classes, can be subdivided into low-amylose haplotypes of 17T, 18T and 19T (combined CT repeats and a T at the G/T SNP), and intermediate-amylose haplotypes of 17G, 18G, and 19G. The high-amylose CT repeats of 8, 10, and 11, and the intermediate-amylose CT repeats of 14, 16, and 20, all have Gs at the G/T SNP. Together, these 12 Waxy CT-G/T SNP haplotypes explained more of the variation in AA than the CT microsatellite alone. By itself, the G/T SNP is unable to discriminate the intermediate- from the high-AA class of rice accessions. The ex6 SNP grouped the 150 non-glutinous rice accessions into two alleles: the Nipponbare allele (N-allele), which associates with high- and low-amylose classes of the rice accessions, and non-Nipponbare allele (M-allele), which associates with all the intermediate amylose rice accessions. By combining the G/T SNP and ex6 SNP, the rice accessions were grouped into three haplotypes: GN (a G at the G/T SNP and N-allele at ex6 SNP), the high-amylose haplotype; GM, the intermediate-amylose haplotype; and TN, the low-amylose haplotype. Together, these three haplotypes explained a comparable amount of the variation in AA as did the Waxy CT-G/T SNP haplotypes. These combined G/T and ex6 SNP haplotypes were able to further discriminate the high amylose rice accessions with a CT 20 allele, such as Jodon, L202, A201, from the other intermediate-amylose CT 20 rice accessions. A genetic marker, via allele-specific oligonucleotide PCR, to this ex6 SNP has been developed and is able to discriminate the high- or low-amylose class from the intermediate-amylose class. In conclusion, a high association was found between both CT-G/T SNP and G/T-ex6 SNP haplotypes and AA across a diverse set of international germplasm. The linkage of these sequence variations on the Waxy gene and their association with AA provides breeders with marker choices that can accurately select between amylose classes in diverse genetic backgrounds.