Introduction
In bread wheat (Triticum aestivum L.), gliadins and the glutenins serve as endosperm storage proteins (Payne et al., Reference Payne, Holt, Lawrence and Law1982). Glutenins are the largest molecular weight protein in grains, with high-molecular-weight glutenin subunits (HMW-GS) and low-molecular-weight glutenin subunits (Jackson et al., Reference Jackson, Holt and Payne1983; Verbruggen et al., Reference Verbruggen, Veraverbeke, Vandamme and Delcour1998; Pflüger et al., Reference Pflüger, D'Ovidio, Margiotta, Peña, Mujeeb-Kazi and Lafiandra2001; D'Ovidio and Masci, Reference D'Ovidio and Masci2004; Hailu et al., Reference Hailu, Johansson, Merker, Belay and Zeleke2006). HMW-GS proteins represent approximately 10% of glutenins, and are the major determinants of dough elasticity and bread-baking quality (Payne, Reference Payne1987; Chen et al., Reference Chen, Wang, Shen and Ji2009; Sarkar et al., Reference Sarkar, Singh, Ahlawat, Chakraborti and Singh2015; Zhang et al., Reference Zhang, Hu, Liu, Sun, Chen, Lv, Liu, Jia and Li2018). Many studies have confirmed the significant correlation of HMW-GS to wheat quality characteristics (Liu and Xu Reference Liu and Xu1988; Zhao and Lu Reference Zhao and Lu1994; Lu and Ma Reference Lu and Ma2000; Tang et al., Reference Tang, Yang, Tian, Li and Chen2008).
Alleles of HMW-GS are associated with flour-processing quality and are encoded by genes at the Glu-1 loci located on the long arm of homologous chromosomes (1A 1B and 1D) (Payne, Reference Payne1987; Shewry et al., Reference Shewry, Halford and Tatham1992). Each Glu-1 locus consists of two tightly linked genes: a large molecular weight x-type subunit and a small y-type subunit (Lafiandra et al., Reference Lafiandra, Tucci, Pavoni, Turchetta and Margiotta1997; Rodríguez-Quijano et al., Reference Rodríguez-Quijano, Nieto-Taladriz, Gómez, Vázquez, Carrillo, Bedö and Láng2001; Li et al., Reference Li, Zhang, Gao, Wang, Ji, He, Appels, Ma and Yan2007; Fang et al., Reference Fang, Liu, Zhang, Yang, Li, Shewry and He2008). D'Ovidio et al. (Reference D'Ovidio, Porceddu and Lafiandra1994) reported that HMW-GS genes are highly conserved in molecular structure with encoding sequences that contain no introns. Considerable efforts have been devoted to determining the amino acid sequences (AASs) of different HMW-GS genes, which exhibit high structural similarity: a signal peptide composed of 21 amino acid residues, a conserved N-terminal domain, a central repetitive domain and a C-terminal domain (Sun et al., Reference Sun, Wand, Li and Liu2000).
The x-type subunit possesses three cysteine (Cys) residues in the N-terminal domain and one Cys residue in the C-terminal domain, and the novel y-type subunit has five Cys residues in the N-terminal domain, one Cys residue in the C-terminal domain, as well as one Cys residue in the central tail of the repetitive domain. The central repetitive domain contains three kinds of oligopeptides: the tripeptide GQQ, the hexapeptide PGQGQQ and the nonapeptide GYYPTSP/LQQ. The tripeptide is only present in the x-type subunit (Shewry et al., Reference Shewry, Tatham, Barro, Barcelo and Lazzeri1995).
Due to gene inactivation, common wheat has only three to five types of HMW-GS. One subunit (1Ax) or no subunit at all is encoded at the Glu-A1 locus in y-type genes at the Glu-A1 locus, the other one or two types are encoded at the Glu-B1 locus and two types are encoded at the Glu-D1 locus (Huebner et al., Reference Huebner, Donaldson and Wall1974). Multiple alleles of Glu-D1 locus including Glu-D1a, b, c, d, e and f have been reported (Wrigley et al., Reference Wrigley, Asenstorfer, Batey, Cornish, Day, Mares, Mrva and Carver2009). 1Dx2 and 1Dy12 are Glu-D1a alleles, encoding 1Dx2 and 1DY12 subunits, respectively, and 1Dx5 and 1Dy10 are Glu-D1d alleles, encoding 1Dx5 and 1Dy10 subunits, respectively (Payne and Lawrence, Reference Payne and Lawrence1983; Payne et al., Reference Payne, Nightingale, Krattiger and Holt1987).
The contributions of different alleles to dough and baking quality are not consistent, but the 1Dx5 + 1Dy10 glutenin subunit combination is frequently associated with best baking quality and various dough strength indicators (Payne et al., Reference Payne, Nightingale, Krattiger and Holt1987). The HMW-GS genes are highly conserved in terminal domains, requiring the use of allele-specific polymerase chain reaction (PCR) for cloning. HMW-GS genes have also been cloned and sequenced in wheat-related species, such as wild emmer wheat (Triticum dicoccoides) (Jin et al., Reference Jin, Xie, Ge, Li, Jiang, Subburaj, Li, Zeller, Hsam and Yan2012; Zhang et al., Reference Zhang, He, Liang, Huang, Su, Li and Li2016; Zhang, Reference Zhang2019; Orlovskaya et al., Reference Orlovskaya, Yatsevich, Vakula, Khotyleva and Kilchevsky2020), spelt wheat (Triticum spelta L.) (An et al., Reference An, Li, Yan, Xiao, Hsam and Zeller2005; Dubois et al., Reference Dubois, Bertin and Mingeot2016), goat-grass (Aegilops tauschii Coss.) (Yan, Reference Yan2001), land races (Shao et al., Reference Shao, Ran, Yu, Li and Gao2015), commercial varieties (Xu et al., Reference Xu, Zhang and Dong2006; Li, Reference Li2017) and artificial materials (Liang, Reference Liang2019).
The disulphide bonds of Cys residues can affect flour-processing quality and baking qualities to play essential roles in dough elasticity (Zhao, Reference Zhao2002). The Cys residues in HMW-GS are usually conserved in number and position. Compared to other subunits, 1Dx5 may have improved function as it contains an extra Cys residue replacing a conserved serine (Ser) residue in its repetitive domain (Anderson et al., Reference Anderson, Greene, Yip, Halford and Shewry1989). The extra Cys residue is important for stronger dough elasticity and superior end-use qualities of bread and noodle processing (Lafiandra et al., Reference Lafiandra, D'Ovidio, Porceddu, Margiotta and Colaprico1993; Don et al., Reference Don, Lookhart, Naeem, MacRitchie and Hamer2005; Zhang et al., Reference Zhang, Ma, Yao and He2009). Different folded domains created by various repetitive domain sequences may impact the overall stability of the protein structure. The x-type subunits generally exhibit a broad unfolding pattern, with the detection of several conformational intermediates with increasing urea concentration (Lafiandra et al., Reference Lafiandra, Turchetta, D'Ovidio, Anderson, Facchiano and Colonna1999).
The main objectives of this work were to determine the sequence signature and differences of the 1Dx gene in nine wheat varieties with different flour-processing quality types and to characterize the heredity of the 1Dx gene in several strong gluten wheat cultivars using the pedigree system. This work should provide reference to improve wheat quality and facilitate the breeding of good quality wheat varieties.
Materials and methods
Plant material
A total of nine wheat varieties were investigated in this study. Different varieties were selected according to the Chinese standards, with four strong gluten wheat varieties: Xinmai 26, Gaoyou 8901, Xinmai 19 and Xinmai 28; three middle-gluten wheat varieties: Zhoumai 24, Aikang 58 and Xinmai 9 and two land races: Yumai and Jinbaoyin. All materials were provided and preserved by the Xinxiang Academy of Agricultural Sciences, Henan Province. Three accessions of known subunit composition were used as controls: Chinese Spring, Yumai 34 and Hengguan 35 with alleles ‘Null, 7 + 8, 2 + 12’, ‘1, 7 + 8, 5 + 10’ and ‘Null, 7 + 9, 2 + 12’, respectively.
HMW glutenin analysis
To study the HMW-GS constitution types, HMW-GS were extracted and analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as described by Singh et al. (Reference Singh, Shepherd and Cornish1991). Subunits were named according to the system proposed by Payne and Lawrence (Reference Payne and Lawrence1983).
DNA isolation and PCR amplification
Genomic DNA samples were extracted from single wheat grain using a sodium lauroylsarcosine method described by Chen et al. (Reference Chen, Gao, Zhang, Zuo and Cui2013). According to the HMW-GS gene sequences reported on the NCBI website (https://www.ncbi.nlm.nih.gov/), a pair of primers were designed by using Primer 5.0 software: F: 5′-ATCATCACCCACAACACCG-3′ and R: 5′-GCTGAGACATGCAGCACATA-3′. The forward primer was −61 bp relative to the start site and the reverse primer adjoined the end site of the C-terminal domain, so amplified PCR products should contain the entire sequence of the HMW-GS gene. Primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.
PCR amplification reactions were performed in a final volume of 20 μl using 1 unit of Taq DNA polymerase (Roche), 20 mmol/l Tris-HCl (pH 8.4), 20 mmol/l KCl, 200 μmol/l dNTPs, 1.5 mmol/l MgCl2, 50 ng DNA and the upstream and downstream primers at 8 pmol each, as described by Chen et al. (Reference Chen, Gao, Zhang, Zuo and Cui2013) and Lou et al. (Reference Lou, Li, Li, Pu, Shoaib, Liu, Sun, Zhang and Yang2016). PCR amplification reactions were conducted according to the following programme: 94°C for 5 min denaturation followed by 35 cycles of 1 min at 94°C, 1 min at 61°C and 2.5 min at 72°C, followed by a final extension at 72°C for 10 min (Wan et al., Reference Wan, Wang, Shewry and Halford2002).
PCR products were analysed by electrophoresis using a 1.0% agarose gel in Tris-acetate-EDTA (TAE) buffer followed by staining with ethidium bromide (Chandima et al., Reference Chandima, Ariyarathna, Oldach and Francki2016). Marker III (Tiangen Biochemical Technology (Beijing) Co., Ltd.) was used as a DNA molecular weight standard.
DNA cloning and sequencing
The amplification products were purified from agarose gels using SanPrep Spin Column & Collection Tube (Shanghai Sangon Biotechnology Co., Ltd.). Next, the purified products were ligated into the pMD18-T vector (TaKaRa Biotechnology, Dalian, China) overnight at 16°C in 1× ligase buffer with 150 U of T4 DNA ligase (TaKaRa Biotechnology, Dalian, China). The products were transformed into Escherichia coli DH5α competent cells and the transformed cells were plated on Luria–Bertani agar containing 100 mg/l ampicillin, 4 μl 1 mol/l IPTG and 40 μl 20 mg m/l X-gal. Three white clones containing fragments of the expected size were selected randomly and DNA sequences were obtained by two directional sequencing performed by Shanghai Sangon Biotechnology Co., Ltd.
Multiple alignment and phylogenetic analysis
Sequence alignment was performed using the blastn program (http://www.ncbi.nlm.nib.gov/BLAST/). Multiple alignments of nucleotide and AASs were completed using DNAMAN. The MEGA 2 program (Kumar et al., Reference Kumar, Tamura, Jakobsen and Nei2001) was used to construct phylogenetic trees based on the clustering of AASs using neighbour-joining method. Phylogenetic trees were constructed and figures were prepared using ITOL (https://itol.embl.de/).
Previously characterized genes, KJ144185 from wheat, JX173939 from goat-grass, JX173949 from spelt wheat, KF381059 from intermediate wheatgrass (Thinopyrum intermedium) and KC167176 from wild emmer wheat, were compared to the newly identified sequences. Other published sequences of HMW-GS genes available in GenBank were also included in the phylogenetic analysis: 1Ax1 (CAA43331), 1Bx7 (JF736013), 1Bx13 (CAW30791), 1Bx14 (KF733216), 1Bx17 (KC254854), 1Bx20 (CAD24586), 1Bx23 (AAS60207), 1By8 (JF736014), 1By9 (X61026), 1By15 (DQ086215), 1By16 (EF540765), 1By18 (KF430649), 1Dx1.5 (ADF32930), 1Dx2 (X03346), 1Dx5 (X12928), 1Dy10 (ABX89298) and 1Dy12 (X03041).
HMW-GS protein secondary structures were predicted by SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html).
Results
HMW-GS compositions in different wheat varieties
To determine the composition of HMW-GS subunits, the nine selected wheat varieties and three varieties with defined HMW-GS compositions were subjected to SDS-PAGE analysis (Fig. 1(a), Table 1). As shown in Table 1, the varieties exhibited three classes of HMW-GS composition for Glu-D1: 5 + 10, 2 + 12 and 4 + 12. Gaoyou 8901, Xinmai 28, Xinmai 19, Xinmai 26 and Jinbaoyin all contained 1Dx5; Zhoumai 24, Xinmai 9 and Yumai all contained 1Dx2; and Aikang 58 contained the only 1Dx4 subunit.
Fig. 1. HMW-GS composition and PCR product of nine wheat varieties. (a) Analysis of HMW-GS composition of nine wheat varieties and (b) the PCR product of nine wheat varieties separated on agarose gel. M1: Chinese Spring, M2: Yumai 34, M3: Hengguan 35, M: Tiangen Biotech (Beijing) Co., Ltd. Marker III, and 1–9 were Xinmai 26, Gaoyou 8901, Zhoumai 24, Aikang 58, Xinmai 9, Xinmai 19, Xinmai 28, Jinbaoyin and Yumai, respectively.
Table 1. HMW-GS compositions of nine wheat varieties
Isolation, cloning and sequencing of 1Dx subunit genes
Upstream and downstream designed primers were used to amplify the total grain DNA from the nine wheat varieties based on the ends of the conserved gene sequence. The resulting PCR products were then analysed by electrophoresis using a 1.0% agarose gel. As shown in Fig. 1(b), all nine wheat varieties showed two bands of about 2500 and 1900 bp in agarose gels, corresponding to the x-type and y-type HMW-GS subunits, respectively. The ~2500 bp band was cut out from the agarose gels, and then extracted, purified and directly sequenced in both directions using the designed primers. The DNA sequences of 1Dx-type HMW-GS from the nine wheat varieties were 2523–2550 bp in length (online Supplementary Fig. S1). Sequences cloned from Gaoyou 8901, Xinmai 28, Xinmai 19, Xinmai 26 and Jinbaoyin were named 1Dx-GY8901, 1Dx-XM28, 1Dx-XM19 and 1Dx-XM26, respectively, with all sequences 2550 bp in length and encoding 848 amino acids. Sequences cloned from Aikang 58, Zhoumai 24, Jinbaoyin, Yumai and Xinmai 9, were named 1Dx-AK58, 1Dx-ZM24, 1Dx-JBY, 1Dx-YM and 1Dx-XM9, respectively, with all sequences 2523 bp in length and encoding 839 amino acids.
Two sequential stop codons (TGATAG) were found in all nine sequences. The predicted protein translation results showed typical structural characteristics of wheat HMW-GS gene sequences, including signal peptide, N-terminal conserved region, C-terminal conserved region and repeat region. The signal peptide consisted of 21 amino acids, there were three Cys residues found in the N-terminal conserved region and one Cys residue in the C-terminal region, and the repeat region contained GQQ tripeptides. The shared features suggested that the nine cloned fragments were all 1Dx genes of HMW-GS.
Sequence analysis of identified 1Dx genes from different wheat varieties
To study the sequence conservation of 1Dx genes in the different wheat varieties, DNAMAN was used to perform multiple sequence alignment using the nine cloned 1Dx gene sequences, 1Dx5, and 1Dx2. As shown in online Supplementary Fig. S1, the four sequences 2550 bp in length were similar to the 1Dx5 gene and the five sequences 2523 bp in length were similar to the 1Dx2 gene. A total of 27 single-nucleotide polymorphisms (SNPs) (s1–s27) and five insertion–deletion (InDel) sites (ID1–ID5) were found in the 2586 bp sequences. There was no variable site in the signal peptide region, s1 was detected in the N-terminal region (64–330 bp), s2–s23 were detected in the repeat region (331–2454 bp) and s24–s27 were detected in the C-terminal region. The InDel sites were all located in the repeat region. The nine 1Dx subunit gene sequences had 97.85% nucleotide sequence similarity with each other and 77.11–77.36% sequence similarity to the five previously known HMW-GS from common wheat, goat-grass, spelt wheat, intermediate wheatgrass (T. intermedium) and wild emmer wheat (Table 2).
Table 2. Sequence similarity (%) of cloned sequences with several other known HMW-GS cds
The sequence similarity among the predicted AASs from the nine 1Dx subunit sequences was 97.29%. Due to the presence of repeated units, SNP sites and InDel sites, the nucleotide sequence and deduced AAS were matched by appropriate readjustments to define variable sites. As shown in online Supplementary Fig. S2, 21 single amino acid mutation sites (S1–S21) and five InDel peptide sites were found in the 848 AASs, as six of 27 SNP sites were silent mutations and the other 21 were non-synonymous mutations.
No amino acid mutations were identified in the signal peptide region, S1 was detected in the N-terminal region, S2–S18 were detected in the repeat region and S19–S21 were detected in the C-terminal region. The five InDel peptide sites (ID1–ID5) were all detected in the repeat region. For the S2 mutation, a Ser residue is changed to Cys, so the four sequences containing this mutations, 1Dx-XM19, 1Dx-XM26, 1Dx-XM28 and 1Dx-GY8901, have an extra Cys in their repeat regions. ID1–ID5 changed the repetitive unit number, causing the final variation of AAS. ID1 and ID5 were caused by hexapeptide InDel, ID2 and ID4 were caused by nonapeptide InDel, and ID3 was caused by a tripeptide InDel.
The deduced AASs of 1Dx-YM, 1Dx-AK58, 1Dx-GY8901 and 1Dx-XM9 contained some specific changes that differed from the other sequences. S7, S14, S17 and S18 were only detected in 1Dx-YM; S11, ID5 and S14 were only detected in 1Dx-AK58; S8 and S9 were only detected in 1Dx-GY8901. S6 was only detected in 1Dx-XM19, corresponding to a substitution of glutamate (Glu) to alanine (Ala), consistent with the sequence of 1Dx5. The deduced AASs of 1Dx-ZM24 and 1Dx-XM9 were completely identical, with the only difference to the nucleotide sequence a C–T transition resulting in a nonsense mutation.
Phylogenetic analysis
To explore the genetic relationships of the identified genes, the deduced AAS of cloned 1Dx genes were compared with previously reported AASs of HMW-GS genes from Glu-1 (total of 17 sequences) and phylogenetic trees were constructed (Fig. 2(a)). Phylogenetic analysis showed that 1Dx-XM26, 1Dx-XM28, 1Dx-GY8901 and 1Dx-XM19 were classified into a group with 1Dx5 and 1Dx-YM, 1Dx-AK58, 1Dx-JBY, 1Dx-XM9 and 1Dx-ZM24 were classified into a group with 1Dx2 and 1Dx2.2. All nine cloned 1Dx sequences were out of the 1Ax and 1Bx branches on the phylogenetic tree.
Fig. 2. Phylogenetic tree of Glu-1 HMW-GS gene and prediction of secondary structure of HMW-GS proteins from 1Dx-XM26. (a) The phylogenetic tree generated from the Glu-1 HMW-GS gene and (b) the prediction of secondary structure of HMW-GS proteins from 1Dx-XM26.
Prediction of secondary structure of HMW-GS proteins
Structural information can provide insight into HMW-GS function. Thus, the secondary structures of the HMW-GS proteins from nine wheat varieties were predicted. The results show similar structures, with a representative one, 1Dx-XM26, shown in Fig. 2(b). The positions of α-helix, extended strand, β-turn and random coil structure were almost the same among the nine HMW-GS proteins (Table 3), confirming the high conservation of wheat HMW-GS. The percentages of α-helix, extended strand, β-turn and random coil were 13.33–13.59, 4.77–5.78, 7.08–9.18 and 72.35–73.94%, respectively.
Table 3. Secondary structure states' proportions of nine 1Dx sequences
1Dx-XM19 and 1Dx-XM26 exhibited the lowest percentages of α-helix and 1Dx-JBY prediction exhibited the highest. 1Dx-AK58 and 1Dx-GY8901 had the lowest and the highest percentages of extended strand, respectively, with the opposite trends for β-turn. For the percentages of different types of secondary structure, Dx-XM9 and 1Dx-ZM24 were identical and 1Dx-XM19 and 1Dx-XM26 were identical.
Discussion
Many wheat germplasmic resources are widely distributed in China, and more than 40,000 accessions have been preserved in the National Crop Gene Bank (Li et al., Reference Li, Zheng, Yang, Li, Liu and Song1998). The identification of new HMW-GS genes in wheat genetic resources (e.g. local land races) or wheat relatives (e.g. spelt wheat and goat-grass) can broaden the genetic basis of wheat breeding and could be utilized in efforts to improve wheat quality. Dong et al. (Reference Dong, Yang, Li, Zhang, Lou, An, Dong, Gu, Anderson, Liu, Qin and Wang2013) deduced that the Glu-D1 haplotype containing Glu-D1d was possibly differentiated in the ancestral hexaploid wheat about 10,000 years ago, and was subsequently introgressed into domesticated common wheat and common spelt wheat. Guo et al. (Reference Guo, Guo, Li, Yang and Li2010) cloned two novel Glu-D1-encoded subunits, 1Dx1.5* and 1Dy12.2*, from a wheat landrace Jiuquanjinbaoyin, and showed that 1Dx1.5* was more similar with HMW-GS from the goat-grass than from that from the bread wheat varieties (T. aestivum L.). Similar results were obtained in this study, with higher sequence similarity between the cloned 1Dx subunit gene sequences of nine wheat varieties and the HMW-GS gene sequences of common spelt wheat (T. spelta L.) or wild goat-grass than that of common wheat. Two 1Dx genes were successfully cloned from two wheat land races, Yumai and Jinbaoyin, respectively, with four species-specific SNP loci in Yumai, the most abundant among the nine varieties. These results provide a foundation to explore new candidate glutenin alleles for Glu-D1 loci in wheat land races and wheat relative species.
Xie et al. (Reference Xie, Zhu, Sun, Liu, Chen, Ren, Cheng, Xue, Chang and Zhan2016) detected the subunit composition of HMW-GS of Aikang 58 as ‘1, 7 + 8, 5 + 12’. However, Wang et al. (Reference Wang, Liu, Chen, Feng, Zhang and Ma2015, Reference Wang, Gao and Zhao2018) reported the composition as ‘1, 7 + 8, 4 + 12’, which we confirmed here. Additionally, our sequence analysis revealed that there was no Ser–Cys mutation in S1 of 1Dx-AK58. Thus, the cloned subunit gene is not a 1Dx5 gene. Wang et al. (Reference Wang, Liu, Chen, Feng, Zhang and Ma2015) detected the HMW-GS subunit composition of Xinmai 26 as ‘1, 7 + 9, 5 + 12’, but our results showed a composition of ‘1, 7 + 8, 5 + 10’. This difference suggests the samples of Xinmai 26 used as experimental materials differed for the two studies.
Xinmai 9, Xinmai 19, Xinmai 26 and Xinmai 28 were bred by the Xinxiang Academy of Agricultural Sciences. Xinmai 26 and Xinmai 28 shared the same female parent, Xinmai 18, with the HMW-GS subunit composition of ‘1, 7 + 9, 5 + 10’. Xinmai 26 has a male parent, Jimai 17, with the HMW-GS subunit composition of ‘1, 7 + 8, 5 + 10’ and Xinmai 28 has a male parent, Shanyou 225, with the subunit composition of ‘1, 14 + 15, 2 + 12’. Dong et al. (Reference Dong, Wang, Yang and Zhao2011) concluded that Xinmai 26 has potential for parental complementary effect and accumulative super parent heterosis. Based on the pedigrees (Fig. 3) and the results of this study, the quality of Xinmai 26 likely benefited from the selection of ‘1, 7 + 8, 5 + 10’ type HMW-GS subunit composition by generation selection in the pedigree system. Xinmai 9 was the male parent of Xinmai 19 but analysis revealed 17 different loci between 1Dx-XM9 and 1Dx-XM19, which accounting for 54.8% of the total 31 variations. Xinmai 19 differs in quality significantly from Xinmai 9, suggesting that Xinmai 19 may have reserved more quality characteristics from its maternal parent in the pedigree system. Further breeding of Xinmai wheat cultivars will benefit from this characterization of genetic relationships and the analysis of the HMW-GS genes.
Fig. 3. Pedigree of several strong gluten wheat varieties.
Xiang et al. (Reference Xiang, Wang, Liu and Zhang2015) cloned the 1Dx gene from wheat line 2004s-47 and determined it was highly homologous to the 1Dx2.1t gene in Ae. tauschii, with two amino acid changes in the repetitive domain. In this study, 17 amino acid changes and five InDels were detected in the repetitive domain, further confirming the variations in this domain. Both ends of the HMW-GS genes are highly conserved (D'Ovidio et al., Reference D'Ovidio, Masci and Porcedu1995). Dong et al. (Reference Dong, Yang, Li, Zhang, Lou, An, Dong, Gu, Anderson, Liu, Qin and Wang2013) found that 20 deduced AASs of 1Dx from goat-grass and spelta wheat were identical to 1Dx5 and 1Dx2 from wheat in the C-terminal regions, but two mutations were found in this region when comparing five deduced AASs of 1Dy from goat-grass and spelta wheat with 1Dy10 and 1Dy12 from wheat. In this study, all nine cloned sequences were x-type submits with significant polymorphism in the C-terminal region.
According to the phylogenetic tree analysis, 1Dx-XM26, 1Dx-XM28, 1Dx-GY8901, 1Dx-XM19 and 1Dx5 belonged to one clade, with all of their HMW-GS ‘5 + 10’ on Glu-D1 loci. Genes 1Dx-YM, 1Dx-AK58, 1Dx-XM9, 1Dx-ZM24, belonged to the same clade as 1Dx2 and 1Dx2.2, with all HMW-GS ‘2 + 12’ or ‘4 + 12’ on Glu-D1 loci. Interestingly, 1Dx-JBY appeared to be ‘5 + 10’ on Glu-D1 loci but was not included in the clade with 1Dx5, suggesting that further analysis of its HMW-GS combination and related gene sequences is needed. Xinmai 26 and Gaoyou 8901 are both strong gluten wheat cultivars and have the same HMW-GS composition of 1, 7 + 8 and 5 + 10, however there were two different SNP sites and two amino acid mutation sites at 1Dx5. The detection of different loci can facilitate the development of simple sequence repeats (SSR) or SNP markers for authenticity identification of varieties but the location of loci in the repetitive domain may make it difficult to develop markers. The relationships between different loci and quality should be further studied in the future.
Conclusions
In this study, we analysed the composition of HMW-GS in Glu-D1 in nine wheat varieties. Based on SDS-PAGE analysis, three types were identified: 1Dx5 (5 + 10, Gaoyou 8901, Xinmai 28, Xinmai 19, Xinmai 26 and Jinbaoyin), 1Dx2 (2 + 12, Zhoumai 24, Xinmai 9 and Yumai) and 1Dx4 (4 + 12, Aikang 58). The 1Dx-type genes of 1Dx-GY8901, 1Dx-XM28, 1Dx-XM19 and 1Dx-XM26, on Glu-D1 loci were cloned by PCR. The complete coding frames of these genes are 2550 bp in length, similar to the 1Dx5 gene and encoding 848 amino acids. Genes 1Dx-AK58, 1Dx-ZM24, 1Dx-JBY, 1Dx-YM and 1Dx-XM9 are 2523 bp in length, similar to the 1Dx2 gene and encoding 839 amino acids. The nine 1Dx subunit gene sequences exhibit sequence similarity of 97.85%, with conserved structural characteristics. Alignment of the deduced AASs showed their N-terminal signal peptides were conserved, there was subtle variation in the C-terminal regions, and there was significant variation in the repeat region. Five InDel sites were identified, and these were all located in the repeat region. In the 2586 bp sequences, 21 of 27 SNPs (sites s1–s21) were non-synonymous mutations, and five InDel sites ID1–ID5 were detected in the repeat region. Variety-specific sites were also identified. A Ser to Cys mutation was identified at the position corresponding to S2, leading to an extra Cys residue in the repeat regions in the 1Dx-XM19, 1Dx-XM26, 1Dx-XM28 and 1Dx-GY8901 genes. Analysis revealed that 1Dx-XM26, 1Dx-XM28, 1Dx-GY8901, 1Dx-XM19 and 1Dx5 were clustered on one branch, and 1Dx-YM, 1Dx-AK58, 1Dx-JBY, 1Dx-XM9, 1Dx-ZM24 1Dx2 and 1Dx.2.2 were clustered on another branch. The numbers and locations of α-helix (13.33–13.59%), extended strand (4.77–5.78%), β-turn (7.08–9.18%) and random coil (72.35–73.94%) were almost the same among the nine cloned HMW-GS proteins, confirming strong conservation of wheat HMW-GS. The HMW-GS subunit composition for several strong gluten wheat varieties, e.g. Xinmai 9, Xinmai 19, Xinmai 26 and Xinmai 28 were clarified with the pedigree system. In conclusion, we identified sequences and differences of 1Dx genes in nine wheat varieties, and determined the heredity of the 1Dx gene in several strong gluten wheat cultivars in the pedigree system.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S1479262122000211.
Data
The datasets generated during and/or analysed in this study are available from the corresponding author on reasonable request.
Acknowledgements
We thank Professor Rachel M. (University of Wisconsin) for their contributions in revising the manuscript. This work was supported by the Modern Technology System of Agricultural Industry (CARS-3-2-34), the Science and Technology Major Project of Henan Province (181100110200-0104) and the Key Scientific and Technological Program of Henan Province (182102110172).
Author contributions
L.-J. Yang, H.-P. Ma and Z.-K. Jiang conceived and designed the manuscript. L.-J. Yang and Y.-Q. Dong wrote the manuscript. X.-F. Zhang, Y.-T. Zheng, J.-X. Zhang, Y.-L. He and X.-F. Tan participated in the experiment design, data collection, analysis of data and preparation of the manuscript. All authors read and approved the final manuscript.
Conflict of interest
The authors declare that they have no conflict of interest.
