| Summary: | High-molecular-weight glutenin subunits (HMW-GSs) encoded by alleles at the <i>Glu-A1</i>, <i>Glu-B1</i>, and <i>Glu-D1</i> loci confer unique end-use quality properties of common wheat (<i>Triticum aestivum</i> L.). Wheat accessions with the high-quality HMW-GSs combination of Ax2*/Bx7<sup>OE</sup>/Dx5 usually exhibit strong gluten characteristics. In order to stack these three high-quality subunit genes by molecular markers in strong gluten wheat breeding, an agarose gel-based multiplex PCR marker for these high-quality HMW-GSs and two agarose gel-based multiplex PCR markers detecting the homozygosity of Ax2* and Bx7<sup>OE</sup> subunits were developed. These markers were verified in an F<sub>2</sub> segregating population from a cross between a medium-gluten winter wheat cultivar with the HMW-GSs combination of Ax null/Bx7 + By8/Dx4 + Dy12 and a strong-gluten spring wheat cultivar with the HMW-GSs combination of Ax2*/Bx7<sup>OE</sup> + By8*/Dx5 + Dy10. By integrating the newly established multiplex PCR markers and a published co-dominant PCR marker of the Dx5 subunit, a complete molecular marker selection system was established. After multiple rounds of molecular marker-assisted selection with the system, 17 homozygous winter wheat lines that stacked the three high-quality HMW-GSs were generated. The gluten strength of these homozygous lines was comparable to their strong-gluten parent, but significantly higher than that of their medium-gluten parent by measuring their lactic acid-sodium dodecyl sulfate solvent retention capacities of whole wheat meal. The multiplex PCR systems established in the present study can be used for molecular marker-assisted selection of strong gluten wheats.
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