An approach to DNA polymorphism screening in <Emphasis Type="Italic">SBEIIa</Emphasis> homeologous genes of polyploid wheat (<Emphasis Type="Italic">Triticum</Emphasis> L.) |
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Authors: | F Konovalov A Shaturova O Mitrofanova A Kudryavtsev |
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Institution: | (1) Vavilov Institute of General Genetics, Moscow, Russia;(2) Moscow State University, Moscow, Russia;(3) Vavilov All-Russian Institute of Plant Industry, St. Petersburg, Russia |
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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. |
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