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The wheat NAM-B1 and NAM-A1 genes are positively associated with grain protein content (GPC) in wheat. We conducted molecular characterisation of the NAM-1 genes in 51 Australian wheat varieties (Triticum aestivum L.), with the aim of improving GPC and nitrogen-usage efficiency in Australian wheat. In summary, the wild type NAM-B1 gene, which originated from Israel, was identified in two Australian wheat varieties. Five varieties contained a deletion allele, whereas the majority (43) harboured a non-functional NAM-B1 allele and one variety contained both functional and non-functional alleles. Twenty-six Australian wheat varieties contained the NAM-A1a haplotype, which was similar to its well-characterised homoeolog NAM-B1 wild type and associated with high GPC. The NAM-D1 gene in the 51 wheat varieties was also characterised, and no gene variation in the exon regions was noted; only two single-nucleotide polymorphisms in introns 1 and 2 were found among the 51 varieties.

In this study, the authentic high molecular weight glutenin (HMW-GS) allele Glu-B1h 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 2367bp for 1Bx14 and 2151bp for 1By15 and encode 789 and 717 amino acid residues, respectively. The deduced molecular masses of two subunit genes were 82340.13Da and 74736.13Da, 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 ~1.32million years ago.