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-I2 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.

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

Background: Starch is the main component of wheat (Triticum aestivum L.) grain and a key factor in determining wheat processing quality. The Wx gene encodes the granule bound starch synthase I (GBSS I) and is the sole gene responsible for amylose synthesis. Results: A waxy mutant (Wx-null) was isolated by screening M3 seeds derived from 1.0% EMS mutagenized materials with I2-KI staining of endosperm starch. SDS-PAGE electrophoresis confirmed that the Wx-null line lacked all three waxy proteins. 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. Nonsense-mediated mRNA decay (NMD) may be triggered to degrade the non-functional Wx mRNA. There were no detectable differences in granule size and morphology between Wx-null and wild-type, but the Wx-null line contained a larger proportion of B-type starch granules. The amylose content of the Wx-null line (0.22%) was remarkably lower compared to the wild-type Gao 8901 (20.82%). Total starch is also lower in the Wx-null line. Conclusions: All three waxy proteins were non-functional in the Wx-null line. NMD may be the cause for reduced expression levels of Wx genes in the Wx-null line. The Wx-null line exhibited more B-type starch granules, dramatically lower amylose content, and decreased total starch. The Wx-null line may provide a potential waxy material with high agronomic performance in wheat breeding programs.

Comparative non-destructive classification of partial waxy wheats using near-infrared and Raman spectroscopy

Waxy proteins play a key role in amylose synthesis in wheat. Eight lines of common wheat (Triticum aestivum L.) carrying mutations in the three homoeologous waxy loci, Wx-A1, Wx-B1 and Wx-D1, have been classified by near-infrared (NIR) and Raman spectroscopy combined with chemometrics. Sample spectra from wheat seeds were collected by using a NIR spectrometer in the wave rage 1600–2400 nm, and then Raman spectrometer in the wave range 700–2000 cm–1. All samples were split randomly into a calibration sample set containing 284 seeds (~35 seeds per line) and a validation sample set containing the remaining 92 seeds. Classification of these samples was undertaken by discriminant analysis combined with principal component analysis (PCA) based on the raw spectra processed by appropriate pre-treatment methods. The classification results by discriminant analysis indicated that the percentage of correctly identified samples by NIR spectroscopy was 84.2% for the calibration set and 84.8% for the validation set, and by Raman spectroscopy 94.4% and 94.6%, respectively. The results demonstrated that Raman spectroscopy combined with chemometrics as a rapid method is superior to NIR spectroscopy in classifying eight partial waxy wheat lines with different waxy proteins.

Polymorphism of waxy proteins in Spanish hulled wheats

Hulled wheats are neglected crops that have potential in plant breeding programmes of modern durum and common wheat. Among these wheats, three species were widely cultivated in Spain until the mid 20th century: Triticum monococcum ssp. monococcum (einkorn), Triticum turgidum ssp. dicoccum (emmer) and Triticum aestivum ssp. spelta (spelt). One important aspect of wheat grain quality is starch composition, which is related to the action of waxy proteins. A collection of 536 accessions of Spanish hulled wheats was analyzed for waxy protein composition using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Polymorphism was found for the Wx-A1, Wx-B1 and Wx-D1 proteins, including new and null alleles in the three species. An allelic variant with an electrophoretic mobility not previously described was found in einkorn wheat. In emmer and spelt, some alleles with different mobility were also found. A Wx-B1 null allele was detected in emmer wheat, and null alleles for Wx-A1, Wx-B1 and Wx-D1 were found in spelt wheat. The variations found could be used to enlarge the gene pool available to breeders, and to design new cultivars with different levels of amylose content.