Introduction

Wheat is one of the most important food crops and can be processed into multiple types of food products (e.g. bread, noodles and cakes). Bread-making quality of wheat is determined to a large extent by the properties of the major storage proteins (Figueroa et al. 2011). Based on extraction and solubility in aqueous alcohol, the wheat storage proteins are classified as gliadins or glutenins (Shewry et al. 1986). These two protein types are important contributors for the rheological properties of dough. In general, gliadins are responsible for extensibility and viscosity of dough, while the glutenins contribute to elasticity (Anjum et al. 2007; Figueroa et al. 2011). According to categories based on electrophoretic behaviour and genetic control, the glutenins can be further divided into two subfamilies, high molecular weight glutenin subunits (HMW-GSs) and low molecular weight glutenin subunits (LMW-GSs) (Shewry 2009). The HMW-GSs are particularly important in determining dough elasticity (Anjum et al. 2007). In wheat, the allelic variation in the HMW-GS composition has been reported to account for \({\sim }70\%\) of the genetic variations of dough properties (Payne et al. 1988).

In hexaploid wheat, the HMW-GS genes are encoded at the Glu-1 loci (Glu-A1, Glu-B1 and Glu-D1) on the long arm of group-1 chromosomes (Payne et al. 1980; Shewry and Halford 2002). In each locus, there are two duplicated HMW-GS genes, designated as x-type and y-type subunits (Dong et al. 2017). Because of the allelic variation and gene inactivation, these loci are highly polymorphic in nature without environmental influence (Gale 2005). These variations frequently result in the expression of allelic x-type and y-type subunits differing in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), or the silencing of one or more HMW-GS genes in certain genotypes (Dong et al. 2017). In the 1980s, according to the SDS-PAGE allelic variation in HMW-GS, three (Glu-A1a, Glu-A1b and Glu-A1c), 11 (Glu-B1a and Glu-b-k) and seven (Glu-D1a and Glu-b-g) common alleles have been found in each locus (Payne et al. 1984; Anjum et al. 2007). To date, more alleles and HMW-GSs have been identified from wheat and its related species and have been studied (Gu et al. 2004; Jiang et al. 2012; Dong et al. 2013). In common wheat, x-type subunits at Glu-B1 and Glu-D1 loci, y-type subunits at Glu-D1 locus are always expressed, whereas x-type subunits at Glu-A1 and y-type at Glu-B1 are not always expressed (Izadi-Darbandi et al. 2010). Therefore, three to five HMW-GSs are usually expressed in wheat, with the HMW-GS composition often differing among different cultivars (Wang et al. 2017). It is known that the action of HMW-GSs in controlling wheat end-use quality is Glu-D1\({>}\)Glu-B1\({>}\)Glu-A1 (Lawrence et al. 1988; Uthayakumaran et al. 2002; Jiang et al. 2012; Yang et al. 2014). Further genetic studies have found that the subunit compositions of these proteins are also correlated with wheat end-use quality. For example, at the Glu-A1 locus, subunits Ax1 and Ax2* were better than those of the null allele having positive effects on bread-making quality (Payne 1987; Branlard et al. 2001). The subunits Bx7 (at the Glu-B1 locus) and Dx5 (at the Glu-D1 locus) have been proposed to be particularly important for loaf volume and dough quality (Wieser and Zimmermann 2000). Since the genetic diversity of HMW-GSs may be an important source of genes for quality improvement, uncovering the composition of HMW-GSs in different wheat varieties is essential for molecular design breeding in controlling wheat end-use quality.

In Hebei province, the study of genetic improvement of wheat quality has begun since 1980s, and some strong gluten wheat varieties have been cultivated, such as Shiluan 02-1, Gaoyou 2018 etc. However, the overall level is still not as good as those of the United States, Canada, Australia and so on. In this study, by means of the SDS-PAGE method, HMW-GSs were detected from 151 hexaploid wheat released by the Hebei Crops Varieties Approval Committee (1970s–2010s). The objectives of the studies are: (i) to elucidate the genetic basis and the law of evolution in wheat quality of Hebei province, further explore the main problems of wheat quality and corresponding countermeasures, and (ii) to screen some wheat varieties with excellent genetic basis for quality, providing support for breeders for good-quality wheat.

Materials and methods

Plant materials

In total, 151 winter wheat varieties (see table 1 in electronic supplementary material at http://www.ias.ac.in/jgenet/) suitable for sowing in the south of Hebei province were used. The wheat varieties were released from 1970s until now. We also included Shiluan 02-1 and Chinese spring cultivars as standard cultivars for identification of banding patterns of wheat HMW glutenin proteins.

SDS-PAGE analysis

Two seeds were selected randomly from each wheat variety. The whole seed was crushed and extracted into 200 \(\mu \hbox {L}\) SDS-PAGE sample buffer, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.002% bromophenol blue, 62.5 mM Tris-HCl pH 6.8 and placed in a refrigerator at \(4{^{\circ }}\hbox {C}\) for 12 h. After centrifugation, the supernatant was analysed using 10% SDS-PAGE and a mini-cell apparatus (Liuyi, Beijing, China) at 20 mA for 3.5 h. Then, gels were stained overnight with 12% (w/v) trichloroacetic acid solution containing 0.05% Coomassie Brilliant Blue R250 in the absolute ethanol (5% w/v) and destained with 25% methanol and 10% acetic acid. A gel documentation system (Bio-Rad, California, USA) was used to scan the gel.

Results and discussion

Allelic variation at Glu-1 loci for HMW glutenin subunits

The results obtained from this study demonstrated as HMW glutenin subunit compositions and allele frequencies in 151 winter wheat varieties are described in table 1 and table 1 in electronic supplementary material. The allelic variability in part wheat varieties at 10% gel is presented in figure 1. A total of 13 different allele variants were detected for HMW-GSs: three at Glu-A1, seven at Glu-B1 and three at Glu-D1 (table 1).

Table 1 Allelic variation and frequency at the Glu-1 loci.
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Fig. 1
figure 1

Electrophoretic patterns of HMW-GS in part of wheat varieties tested. 1, 71-3; 2, Shi 5093; 3, Zhongmai 9; 4, Han 4564 9; 5, Henong 85-9; 6, 5099; 7, 8901-11; CK, Shiluan 02-1.

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For the Glu-A1 locus, 1 or 2* subunits have positive effects on bread-making quality whereas the null allele appears inferior (Luo et al. 2001). The frequencies of the two active types 1 (Glu-A1a) and 2* (Glu-A1b) were 43.0 and 31.8%, respectively, the rest (25.2%) were null-type gene Glu-A1c (table 1). The sum of the frequencies (74.8%) of subunits 1 and 2* was far higher than those obtained in the wheat varieties released from other wheat regions of China. For example, a low proportion, 37.5, 37.2 and 30.7%, contains the Glu-A1, 1 or 2* alleles, respectively, in Hubei, Gansu and Xinjiang province of China (Hao et al. 2004; Liu et al. 2004; Cao et al. 2009). However, a high proportion had been found in Argentinean (97.5%), Spanish (98.4%) and Moroccan (85.0%) wheat varieties (Caballero et al. 2004; Lerner et al. 2009; Henkrar et al. 2017). This implies that targeting for continuing to improve frequency of 1 or 2* alleles at the Glu-A1 locus would be a strategy for promoting quality for the national wheat.

At the Glu-B1 locus, seven alleles were detected, 2.0, 64.9, 23.2, 2.6, 0.7, 6.0 and 0.7%, accounting for 7 (Glu-B1a), 7+8 (Glu-B1b), 7+9 (Glu-B1c), 6+8 (Glu-B1d), 13+16 (Glu-B1f), 14+15 (Glu-B1h) and 17+18 (Glu-B1i), respectively. In Hebei province, the most frequent alleles were 7+8 and 7+9, which were also the most predominant in the bread wheat varieties of China, France and Argentina (Branlard et al. 2003; Song et al. 2006; Lerner et al. 2009). Subunit 6+8 is very common in synthetic hexaploids and durum wheat but its frequency is very low in bread wheat (Rasheed et al. 2012) and 14+15 is the unique subunit type of wheat in China (Liu et al. 2007). In fact, the Glu-B1 locus possesses a significantly greater diversity of alleles than theGlu-A1 and Glu-D1 loci in hexaploid wheats (Li et al. 2004). Four types of subunits at the Glu-B1 locus, 20 (Glu-B1e), 13+19 (Glu-B1f), 21 (Glu-B1j) and 22 (Glu-B1k) were observed in the previous study (Payne 1987) that are no longer found in the current study, but were present in other province wheat varieties of China at low frequency (Liang et al. 2008; Zhang et al. 2008; Hua et al. 2009).

At the Glu-D1 locus, three subunits 2+12, 5+10 and 5+12 encoded by alleles Glu-D1a, Glu-D1d and Glu-D1h, respectively, were found. Subunit 2+12 was found most frequently in 113 (74.8%) genotypes followed by subunit 5+12 in 23 (15.2%) genotypes while subunit 5+10 was observed in 15 (9.9%) genotypes. Subunit 5+10 frequently confers superior end-use qualities to commercial wheat varieties (Dong et al. 2013). Yet synthetic hexaploids (\(Triticum~turgidum\times Aegilops~tauschii\); \(2n=6x=42\)) with subunit 5+12 showed better overall quality characteristics and bread loaf volume compared with any other Glu-D1 encoded glutenin subunits (Peña et al. 1995). Subunits 5+12 are common in synthetic hexaploids (Peña et al. 1995; Rasheed et al. 2012). These results showed that subunits 5+12 have been successfully introduced into bread wheat through the excellent crossability of synthetic wheats with conventional bread wheats in China. On the other hand, 32.9% of the entries contain subunits 5+10 or 5+12, compared with 80.6% from the previous study of cultivars released from America, indicating that subunits 5+10 or 5+12 is not in great use in the Hebei province of China.

Table 2 HMW-GS combination frequencies and quality scores of tested wheat.
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Table 3 Frequency and composition of HMW-GS of wheat varieties in different decades from 1970 to 2010.
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HMW-GS composition in hexaploid wheat

Twenty-two allelic combinations were observed in the survey of 151 Hebei province wheat varieties (table 2) of which 5.3% genotypes possessed the combination of subunits 1, 7+8 and 5+10; 2.0% genotypes had subunit composition of 1, 7+9 and 5+10; 8.6% genotypes had subunit composition of 1, 7+8 and 5+12; 2.6% genotypes had subunit composition of 1, 7+9 and 5+12; 0.7% genotypes had subunit composition of 1, 13+16 and 5+12; 0.7% genotypes had subunit composition of 1, 14+15 and 5+10 and 0.7% genotypes had subunit composition of 1, 17+18 and 5+10. All the above combinations’ quality score with 9 corresponded to good bread-making quality according to the Glu-1 score of Payne (1987). The high potency of Glu-D1 over Glu-A1 and Glu-B1 in promoting the incorporation of HMW-GSs and LMW-GSs into glutenin macropolymers (GMPs) had also been suggested by prior studies using a new series of deletion mutants lacking one or two of the three Glu-1 loci (Glu-A1, Glu-B1 and Glu-D1) specifying HMW-GSs (Wang et al. 2017). Whereas, subunits 2+12 encoded by alleles Glu-D1a are considered deleterious for bread-making quality but favourable for biscuit making (Payne et al. 1987). In this study, the wheat varieties with a high-quality score do not contain 2+12 subunits (table 2). The x–x and x–y dimers of HMW-GSs were found, but those consisting of solely y-type HMW-GSs to form the backbone of the GMPs by the interchain disulphide bonds were not found (Wieser 2007). It is likely that the number and distribution of cysteine residues differ within the subunits which influence the structure of glutenin polymers (Lindsay et al. 2000). Distinguishing from other x-type subunits, 1Dx5 subunit containing an additional cysteine in the central repetitive domain might be the major factor which explains why this gluten subunit is associated with good quality (Ribeiro et al. 2013). Further, the high abundance of 1Dx+1Dy in insoluble glutenin preparation (relative to that of 1Bx+1By or 1Ax) is also an important factor for the functional dominance of Glu-D1 (Wang et al. 2017). The results suggest that the majority of the wheat cultivars in Hebei province possess 5+10 or 5+12 subunits in their Glu-D1 locus and 1 or 2* subunits in the Glu-A1 locus and consequently relatively higher quality assessment score was found to be one of the key strategies to hold for improved wheat quality in our breeding programmes.

Historical evolution of Glu-1 locus

Our results showed that the frequencies of 1 subunit, 7+8 subunits, 5+10 subunits and 5+12 subunits have increased from 0 to 54.8, 33.3 to 71.0, 0 to 16.1 and 0 to 22.6%, respectively, in Hebei province winter wheat varieties over the last 40 years (table 3). While subunit 2* declined from 66.7 of 1970s entries to 22.6% of 2010 entries, and 2+12 subunits declined from 100 to 61.3% of entries in the same period (table 3). Although subunit 2* declined about 40%, the combined percentages of subunits 2* and 1 increased by 11.8% over the past 40 years. Subunits 2* and 1 have equivalent effects on bread-making quality according to Payne’s observation (Payne et al. 1987), while subunit 1 appears to have increased in frequency at the expense of subunit 2* which is similar to the situation in American wheat, where subunit 2* declined from 84.2 of 1970–1990 entries to 76.1% of 1991–2005 entries, and subunit 1 increased from 13.4 to 21.2% of entries in the same period (Shan et al. 2007). The tendency of variation differences between the subunits 2* and 1 may also contribute to the choice of breeding parents or other functional dominance. Further work is needed to validate this possibility. At the end of the last century, 17+18 subunits, 14+15 subunits and 13+16 at the Glu-B1 locus began to appear at a low frequency (table 3). The functional dominance was ranked in the order \(17{+}18{>}15{+}14{>}7{+}8{>}7{+}9\) subunits based on data on the secondary structure of gluten using a series of near-isogenic lines. This allelic richness for the Glu-B1 locus is compatible with hybridized among different wheat varieties and can be transferred through standard breeding procedures. The increased frequency of both subunits 5+10 and 5+12 and the reduced frequency of subunits 2+12 are consistent with the important contribution of the Glu-D1d and Glu-D1h alleles to dough strength and greater attention was paid to end-use quality in the region’s wheat breeding programmes.