Twelve new LMW-GS genes were characterized from bread wheat (Triticum aestivum L.) cultivar Zhongyou 9507 and five Aegilops species by AS-PCR. These genes belong to the LMW-m type and can be classified into two subclasses designated as 1 and 2, with the latter predominant in both wheat and related wild species. Genes in the two subclasses were significantly different from each other in SNPs and InDels variations. In comparison to subclass 1, the structural features of subclass 2 differs in possessing 21 amino acid residue substitutions, two fragment deletions (each with 7 amino acid residues), and a double-residue deletion and two fragment insertions (12 and 2-5 residues). Phylogenetic analysis revealed that the two subclasses were divergent at about 6.8 MYA, earlier than the divergence of C, M, N, S(s) and U genomes. The S(s) and B genomes displayed a very close relationship, whereas the C, M, N and U genomes appeared to be related to the D genome of bread wheat. The presently characterized genes ZyLMW-m1 and ZyLMW-m2 from Zhongyou 9507 were assigned to the D genome. Moreover, these genes were expressed successfully in Escherichia coli. Their transcriptional levels during grain developmental stages detected by quantitative real-time PCR (qRT-PCR) showed that both genes started to express at 5 days post-anthesis (DPA), reaching the maximum at 14 DPA after which their expressions decreased. Furthermore, the expression level of ZyLMW-m2 genes was much higher than that of ZyLMW-m1 during all grain developmental stages, suggesting that the expression efficiency of LMW-GS genes between the two subclasses was highly discrepant.

Two x-type high molecular weight glutenin subunits (HMW-GS) in Aegilops tauschii, 1Dx3(t) and 1Dx4(t) were identified by SDS-PAGE and MALDI-TOF-MS. Their complete coding sequences were isolated by AS-PCR. 1Dx3(t) and 1Dx4(t) genes consist of 2535 bp and 2508 bp and encode 845 and 836 amino acid residues, respectively. The deduced molecular masses of 1Dx3(t) and 1Dx4(t) gene products are 87655.26 Da and 86664.24 Da, respectively, well corresponding to the molecular masses measured by MALDI-TOF-MS. A total of 18 SNPs were identified between 1Dx3(t) and 1Dx4(t). Comparing with 1Dx5 subunit, 1Dx3(t) had a six amino acid insertion at 146-151 while the 1Dx4(t) had a nine amino acid deletion when compared with 1Dx3(t) subunit. The authenticity of the cloned 1Dx3(t) and 1Dx4(t) genes were confirmed by successful expression of their ORFs in E. coli. Comparison and phylogenetic tree based on the amino acid and nucleotide sequences confirmed that 1Dx3(t) was most closely related to 1Dx5 subunit that is widely accepted as a superior subunit for bread-making property. The secondary structure prediction demonstrated that 1Dx3(t) subunit has significantly high α-helix and β-strand contents, suggesting it might have positive effects on dough quality.

Two new low-molecular-weight glutenin subunits (LMW-GS) genes from Triticum timopheevii (Zhuk.) were cloned by reverse-transcription PCR (RT-PCR). The complete open reading frames of 897 and 894 bp, corresponding to the size of LMW-GS genes were designated as TTLMW-m1 and TTLMW-m2, respectively. Multiple sequence alignment analysis indicated that these two genes showed high similarity with known LMW-m type genes, albeit possessing distinct sequence variations. A total of 10 single-nucleotide polymorphisms in the TTLMW-m1 and TTLMW-m2 genes were identified, including five non-synonymous mutations. The predicted secondary structure of the deduced mature glutenin subunits of TTLMW-m1 and TTLMW-m2 genes showed that both subunits possessed more a-helixes and b-strands than the control subunit AY263369, suggesting a more viscoelastic dough structure and a positive effect on quality. The homology tree and phylogenetic analysis showed that the two LMW-m type genes were most likely located at the Glu-G3 locus and that they diverged from the LMW-i type genes about 13 million years ago (MYA). The G genome in T. timopheevii appears to be closely related to the D and B genomes of Triticum aestivum.

A new x-type HMW glutenin subunit, designated as 1Dx1.6 t from Aegilops tauschii was identified and characterized by SDS-PAGE and MALDI-TOF-MS. This subunit is located between 1Dx2 and 1Dx1.5 t and possesses a molecular mass (M r ) of 88565.8 Da. Its complete coding sequence was amplified via allele-specific PCR (AS-PCR), and the amplified product was cloned and sequenced. The authenticity of the cloned 1Dx1.6 t gene was confirmed by successful expression of its open reading frame in Escherichia coli. The molecular characterization of 1Dx1.6 t gene showed that its coding region consisted of 2541 bp encoding a polypeptide of 845 amino acid residues. Sequence comparison to previously characterized 1Dx1.5 t subunit which is related to good dough quality of bread wheat indicated that the 1Dx1.6 t subunit displayed high homology, but possesses 14 residue substitutions and a nonapeptide insertion. A total of 12 singlenucleotide polymorphisms (1 per 212 bp) was identified in the 1Dx1.6 t allele (11 in repetitive domain and 1 in the C-terminal domain), which could facilitate the design of AS-PCR markers.