Identification and molecular characterization of mutant line deficiency in three waxy proteins of common wheat (Triticum aestivum L.)

Starch is the main component of wheat (Triticum aestivum L.) grain and a key factor in determining wheat processing quality. The Wx gene is the gene responsible for amylose synthesis. An ethyl methanesulfonate (EMS) mutagenized population was generated using common wheat cv. Gao 8901, a popular and high-quality cultivar in China. A waxy mutant (Wx-null) was isolated by screening M3 seeds with KI-I(2) staining of endosperm starch. No obvious waxy proteins in Wx-null line were detected using Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). DNA sequencing revealed three SNPs and a 3-bp InDel in the first exon, and a 16-bp InDel at the junction region of the first Wx-A1 intron from the Wx-null line. Six SNPs were identified in Wx-B1 gene of Wx-null line compared to the wild-type Gao 8901, including four missense mutations. One nonsense mutation was found at position 857 in the fourth exon, which resulted in a premature stop codon. Expression levels of Wx genes were dramatically reduced in the Wx-null line. There were no detectable differences in granule size and morphology between Wx-null and wild-type, but the Wx-null line contained more B-type starch granules. The amylose content of the Wx-null line (0.22%) was remarkably lower compared to the wild-type Gao 8901 (24.71%). Total starch is also lower in the Wx-null line. The Wx-null line may provide a potential waxy material with high agronomic performance in wheat breeding programs.