Molecular characterisation of high molecular weight glutenin allele Glu-B1 h encoding 1Bx14+1By15 subunits in bread wheat (Triticum aestivum L.)
Lele Xiao A D , Ke Wang B D , Yanlin Liu A , Xingguo Ye B E , Wujun Ma C and Yueming Yan A E
+ Author Affiliations
- Author Affiliations
A Key Laboratory of Genetics and Biotechnology, College of Life Science, Capital Normal University, 100048 Beijing, China.
B Institute of Crop Sciences, Chinese Academy of Agricultural Sciences/National Key Facility for Crop Gene Resources and Genetic Improvement, 100081 Beijing, China.
C Australia–China Wheat Improvement Centre, Murdoch University/Australian Export Grains Innovation Centre, Perth, WA 6150, Australia.
D These authors contributed equally to this work.
E Corresponding authors. Emails: yexingguo@caas.cn; yanym@cnu.edu.cn
Crop and Pasture Science 65(3) 215-226 https://doi.org/10.1071/CP13409
Submitted: 27 November 2013 Accepted: 13 February 2014 Published: 14 April 2014
Abstract
In this study, the authentic high molecular weight glutenin (HMW-GS) allele Glu-B1 h encoding for subunits 1Bx14 and 1By15 from German bread wheat cultivars Hanno and Imbros was identified and cross-verified by a suite of established protein analysis technologies, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reversed-phase high-performance liquid chromatography, reversed-phase ultra-performance liquid chromatography, and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). The complete encoding sequences were isolated by allele-specific PCR, and consist of 2367 bp for 1Bx14 and 2151 bp for 1By15 and encode 789 and 717 amino acid residues, respectively. The deduced molecular masses of two subunit genes were 82 340.13 Da and 74 736.13 Da, corresponding well to those determined by MALDI-TOF-MS. The presence and authenticity of 1Bx14 and 1By15 subunits were further confirmed by liquid chromatography coupled to tandem mass spectrometry and heterologous expression in E. coli. Comparative analysis demonstrated that 1Bx14 possessed one deletion and 20 single-nucleotide polymorphism variations compared with seven other Glu-B1 x-type HMW-GS genes that mainly resulted from C–T substitutions, whereas compared with five other Glu-B1 y-type HMW-GS genes, 1By15 displayed few variations. Phylogenetic analysis based on the complete coding sequences of the published HMW-GS genes showed that 1Bx14 had a high divergence with other 1Bx subunit genes, whereas 1By15 displayed greater similarity with 1By20. A possible evolutionary route for 1Bx14 gene formation is proposed, which might have resulted from an intra-strand illegitimate recombination event that occurred ~1.32 million years ago.
Additional keywords: Glu-B1h, HMW-GS, molecular cloning, SNPs, wheat.
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