An approach to DNA polymorphism screening in SBEIIa homeologous genes of polyploid wheat (Triticum L.)

Abstract

The non-transgenic manipulation of starch properties in common wheat (Triticum aestivum L.) generally implies combining mutant alleles of the particular gene copies in all three subgenomes (A, B and D). The redundancy of the hexaploid wheat chromosome set substantially complicates the identification of recessive mutations and breeding. Nevertheless, naturally occurring or induced genetic polymorphism has already been successfully exploited for the production of waxy (GBSSI-deficient) and elevated amylose (SSIIa-deficient) wheats. However, in order to achieve the amylose content above 50% of wheat endosperm starch, it may be necessary to inactivate the starch branching enzyme (SBEIIa) isoforms, as the RNAi repression results and gene expression data strongly suggest. The identification of null SBEIIa alleles and their combination in a single genotype is therefore a promising approach to the production of non-transgenic high-amylose wheat; however, wheat SBEIIa polymorphism has not been characterized as of yet. In order to develop an approach to SBEIIa mutation screening, we sequenced the SBEIIa central region (exons 9–12) from the three subgenomes of common wheat cv. Chinese Spring and the A genome of diploid einkorn T. monococcum. The genome-specific primers were developed that amplify the exons downstream from intron 11 selectively from each homeologous gene. Using a single-stranded DNA conformation polymorphism (SSCP) approach, we screened 60 wheat cultivars, landraces, and rare species for naturally occurring SNPs in exons 12, 13 and 14 of the three SBEIIa homeologs. In total, 13 SNPs were discovered in the A and B wheat genomes. Two of these SNPs affect the amino acid sequences of SBEIIa isoforms and may change the enzyme functional properties. The presence of restriction site polymorphism at SNP positions enables their easy genotyping with CAPS assays. Our results indicate that the mining for naturally occurring sequence polymorphism in starch biosynthesis genes of wheat can be successfully performed at the DNA level, providing the starting point for a search for SBEIIa mutants at a larger scale.