Molecular Characterization and Marker Development of the HMW-GS Gene from Thinopyrum elongatum for Improving Wheat Quality

The quality of wheat primarily depends on its storage protein quality, especially in regards to gluten content and high-molecular-weight glutenin subunits (HMW-GS). The number of HMW-GS alleles is limited in bread wheat (Triticum aestivum L.), whereas it is abundant in wheat relatives. Therefore, HMW-GS alleles from wheat relatives could provide a potential for improving quality in wheat breeding. Thinopyrum elongatum (EE) is one of the relatives of wheat. The E genome is closely related to the ABD genome in wheat; therefore, Th. elongatum is often used as an excellent exogenous gene donor for wheat genetic improvement. In this study, the high-molecular glutenin subunit gene was cloned and sequenced from Th. elongatum. A specific molecular marker for identifying the Glu-1Ey subunit gene was developed and applied to detected wheat-Th. elongatum alien introgression lines. Quality analysis indicated that the substitution and addition lines containing Th. elongatum alleles significantly (p < 0.05) increased grain protein content by 3.76% to 5.11%, wet-gluten content by 6.55% to 8.73%, flour 8-MW by 0.25% to 6.35%, and bread volume value by 33.77 mL to 246.50 mL, in comparing it with Chinese Spring. The GMP content and lactic acid SRC showed significant positive correlations with flour processing quality and might be used as indicators for wheat quality. The results were expected to provide a novel route for improving processing quality in wheat quality breeding.


Introduction
As the second-largest cereal crop in the world, wheat (Triticum aestivum L.) is an important source of food, fulfilling the dietary requirements of at least 70% of the global population. It serves as a key ingredient in the preparation of a wide range of food products, including bread, noodles, pastries, biscuits, cookies etc. The total amount of gluten and gliadin proteins in wheat endosperm accounts for approximately 85−90% of the total endosperm protein [1]. Their concentration, composition, and proportions have a crucial impact on gluten, dough function, and end-use products [2]. Wheat glutenin proteins are divided into high-molecular-weight wheat glutenin subunits (HMW-GS) and lowmolecular-weight wheat glutenin subunits (LMW-GS), which comprise 7.5% and 32.5% of the total endosperm protein, respectively [3]. Despite their low concentration in wheat grain, HMW-GS are recognized to critically affect the quality of wheat flour processing [4,5].
In wheat, HMW-GS are encoded by the Glu-1 loci on the long arm of the first homologous chromosome [6]. Each locus contains two closely linked genes, encoding the x-type high-molecular-weight subunit and the y-type low-molecular-weight subunit, respectively [7,8]. It was shown that different allelic variants of the HMW-GS gene in wheat urgent for molecular markers to assist identification in actual breeding populations, so as to improve the efficiency of wheat quality breeding.
To identify novel germplasm for quality breeding in wheat, we cloned an HMW-GS gene, developed specific molecular markers, and measured the contents of grain protein, wet gluten, glutenin macropolymer (GMP), and dough rheological properties, as well as solvent retention capacity (SRC), of the Chinese Spring (CS)-diploid Th. elongatum substitution and addition lines.

Analysis of the Relative Mobility of Th. elongatum HMW-GS
To investigate the types of HMW-GS in the CS-Th. elongatum lines, we analyzed the HMW-GS bands of CS, CS-Th. elongatum chromosome 1E addition line (DA1E), and substitution lines (DS1E/1A, DS1E/1B, DS1E/1D) by SDS-PAGE electrophoresis. As shown in Figure 1, the four bands in CS, from top to bottom, were identified as subunits 1Dx2, 1Bx7, 1By8, and 1Dy12. In comparing with Chinese Spring, a new band of HMW-GS appeared in the addition line: DA1E. The new band was between 1Bx7 and 1By8. Further analysis showed that the same bands appeared in the substitution lines, DS1E/1A, and the addition line, DA1E, since the 1A locus does not encode glutenin subunits in CS. As expected, the new band appeared in DS1E/1B, and DS1E/1D, whereas 1Bx7 and 1By8 subunits were not showed in DS1E/1B, and 1Dx2 + 1Dy12 subunits were not showed in DS1E/1D. Therefore, the new band was an HMW-GS from the 1E chromosome of Th. elongatum. Moreover, the combination of molecular marker research and breeding practice is not close enough, and there are few reports on the use of HMW-GS of Th. elongatum in wheat breeding. So, it is urgent for molecular markers to assist identification in actual breeding populations, so as to improve the efficiency of wheat quality breeding.
To identify novel germplasm for quality breeding in wheat, we cloned an HMW-GS gene, developed specific molecular markers, and measured the contents of grain protein, wet gluten, glutenin macropolymer (GMP), and dough rheological properties, as well as solvent retention capacity (SRC), of the Chinese Spring (CS)-diploid Th. elongatum substitution and addition lines.

Analysis of the Relative Mobility of Th. elongatum HMW-GS
To investigate the types of HMW-GS in the CS-Th. elongatum lines, we analyzed the HMW-GS bands of CS, CS-Th. elongatum chromosome 1E addition line (DA1E), and substitution lines (DS1E/1A, DS1E/1B, DS1E/1D) by SDS-PAGE electrophoresis. As shown in Figure 1, the four bands in CS, from top to bottom, were identified as subunits 1Dx2, 1Bx7, 1By8, and 1Dy12. In comparing with Chinese Spring, a new band of HMW-GS appeared in the addition line: DA1E. The new band was between 1Bx7 and 1By8. Further analysis showed that the same bands appeared in the substitution lines, DS1E/1A, and the addition line, DA1E, since the 1A locus does not encode glutenin subunits in CS. As expected, the new band appeared in DS1E/1B, and DS1E/1D, whereas 1Bx7 and 1By8 subunits were not showed in DS1E/1B, and 1Dx2 + 1Dy12 subunits were not showed in DS1E/1D. Therefore, the new band was an HMW-GS from the 1E chromosome of Th. elongatum.

Cloning and Analysis of the Th. elongatum HMW-GS Gene
The HMW-GS gene was amplified from the genomic DNA of the Th. elongatum chromosome 1E addition and substitution lines of CS, using the degenerative primers P1 and P2. Compared with the bands amplified from CS, the CS-Th. elongatum chromosome 1E addition and substitution lines presented an extra band of approximately 1500 bp ( Figure  2A). Sequencing of this band showed that the length of gene sequence was 1512 bp, beginning from the start codon, ATG, and ending with two consecutive stop codons, TGATAG without introns (Supplementary Figure S1)., Within the sequence, a total of 502 amino acid residues are encoded, including a signal peptide starting with 21 amino acid residues, followed by an N-terminal conserved region consisting of 105 amino acids, a central repeat region composed of hexapeptide (PGQGQQ) and nonapeptide (GYYPTSLQQ) units, and a C-terminal conserved region consisting of 42 amino acids ( Figure 2B) at the end. The central repeat region starts with a dodecapeptide (GYYPSVTSPQQG) and an undecapeptide (SYYPGQASPQQ), followed by the intermediate repeating units of hexapeptides and nonapeptides, and ends with a tripeptide (GYN). Besides the consistent sequence

Cloning and Analysis of the Th. elongatum HMW-GS Gene
The HMW-GS gene was amplified from the genomic DNA of the Th. elongatum chromosome 1E addition and substitution lines of CS, using the degenerative primers P1 and P2. Compared with the bands amplified from CS, the CS-Th. elongatum chromosome 1E addition and substitution lines presented an extra band of approximately 1500 bp (Figure 2A). Sequencing of this band showed that the length of gene sequence was 1512 bp, beginning from the start codon, ATG, and ending with two consecutive stop codons, TGATAG without introns (Supplementary Figure S1). Within the sequence, a total of 502 amino acid residues are encoded, including a signal peptide starting with 21 amino acid residues, followed by an N-terminal conserved region consisting of 105 amino acids, a central repeat region composed of hexapeptide (PGQGQQ) and nonapeptide (GYYPTSLQQ) units, and a C-terminal conserved region consisting of 42 amino acids ( Figure 2B) at the end. The central repeat region starts with a dodecapeptide (GYYPSVTSPQQG) and an undecapeptide (SYYPGQASPQQ), followed by the intermediate repeating units of hexapeptides and nonapeptides, and ends with a tripeptide (GYN). Besides the consistent sequence PGQGQQ, the hexapeptide also contains repeat units, such as PGKWQE, SGQEKQ, IGKGKQ, PAQGQQ, QGQGQQ, IGQGQQ, PEQGQQ, PGQWQQ, LGQGQQ, SGQGQQ, and others. In addition to GYYPTSLQQ, many other types of repeat units, including GHYPASQQQ, GQIPASQQQ, GHYLASQQQ, and GYYPTSPQQ, are present in the nonapeptide. According to further analysis, the number of major occurrences of β-turning angle QPGQ, YPTS, SPQQ, and QQGY in the intermediate repeat sequences are 15, 11, 9, and 8, respectively. The number of glutamine (Q) residues present is 121, with an average molar percentage of 36.22%, and the number of tyrosine (Y) residues present is 29, with an average molar percentage of 8.68%. In addition, there are seven cysteine residues in Th. elongatum polymeric gluten, five of which are at positions 31, 43, 65, 66, and 76 in the N-terminus, one of which is at position 209 near the N-terminus, and one of which is at position 490 in the C-terminus. The phylogenetic analysis shows that the HMW gluten Glu-1E obtained in this study is most closely related to Glu-1Ey15 (AY299518.1), as identified through NCBI, and clustered with the y-type subunit of wheat and its relatives ( Figure 2C). Therefore, the sequence analysis suggests that the HMWG Glu-1E obtained in this study belongs to the y-type subunit of Th. elongatum and was named Glu-1Ey.  15, 11, 9, and 8, respectively. The number of glutamine (Q) residues present is 121, with an average molar percentage of 36.22%, and the number of tyrosine (Y) residues present is 29, with an average molar percentage of 8.68%. In addition, there are seven cysteine residues in Th. elongatum polymeric gluten, five of which are at positions 31, 43, 65, 66, and 76 in the Nterminus, one of which is at position 209 near the N-terminus, and one of which is at position 490 in the C-terminus. The phylogenetic analysis shows that the HMW gluten Glu-1E obtained in this study is most closely related to Glu-1Ey15 (AY299518.1), as identified through NCBI, and clustered with the y-type subunit of wheat and its relatives ( Figure  2C). Therefore, the sequence analysis suggests that the HMWG Glu-1E obtained in this study belongs to the y-type subunit of Th. elongatum and was named Glu-1Ey.  ; hexapeptide (pink); nonapeptides (red); C-terminal domain (black); cysteine residues (red arrow). (C) The tree topologies generated by maximum-parsimony (MP) analyses, derived from HMW-GS amino acid sequence data, conducted using heuristic search with TBR branch swapping.

Development of a Molecular Marker for Th. elongatum HMW Glu-1Ey
In general, an alien chromosome fragment could be detected by genome in situ hybridization or fluorescence in situ hybridization. However, these techniques are timeconsuming and laborious, and an alien chromosome cannot be detected when the translocation fragments are small. Specific molecular markers can detect the presence of exogenous genes and can be effectively applied in wheat breeding. In this study, we designed specific primers to detect the specific HMW-GS of Th. elongatum by comparing the DNA sequences between Glu-1Ey and other HMW-GS in wheat. The primer CMSGlu-1Ey was found to amplify the specific bands in the germplasm carried chromosome 1E of Th. elongatum, but not those of the different subunits found in common wheat ( Figure 3, Table 1). Indeed, the amplified specific bands were confirmed as partial sequences of Glu-1Ey, after having been recovered, sequenced, and compared with Glu-1Ey. Therefore, CMSGlu-1Ey can be used as a specific primer for the HMW gluten gene Glu-1Ey of Th. elongatum in future studies aimed at producing small fragment translocation lines.

Development of a Molecular Marker for Th. elongatum HMW Glu-1Ey
In general, an alien chromosome fragment could be detected by genome in situ hybridization or fluorescence in situ hybridization. However, these techniques are time-consuming and laborious, and an alien chromosome cannot be detected when the translocation fragments are small. Specific molecular markers can detect the presence of exogenous genes and can be effectively applied in wheat breeding. In this study, we designed specific primers to detect the specific HMW-GS of Th. elongatum by comparing the DNA sequences between Glu-1Ey and other HMW-GS in wheat. The primer CMSGlu-1Ey was found to amplify the specific bands in the germplasm carried chromosome 1E of Th. elongatum, but not those of the different subunits found in common wheat ( Figure 3, Table 1). Indeed, the amplified specific bands were confirmed as partial sequences of Glu-1Ey, after having been recovered, sequenced, and compared with Glu-1Ey. Therefore, CMSGlu-1Ey can be used as a specific primer for the HMW gluten gene Glu-1Ey of Th. elongatum in future studies aimed at producing small fragment translocation lines.

Influence of Th. elongatum 1E Chromosome on Grain Quality
Wheat HMW-GS content correlates to wheat processing quality, gluten quality, and gluten strength. Inbred wheat germplasm contains more HMW-GS variant types, which have improved wheat processing quality. In this study, the wheat lines with substitution and addition of Th. elongatum chromosome 1E were employed to investigate the effect of diploid chromosome 1E on wheat quality. The wheat containing diploid Th. elongatum 1E chromosome showed significantly higher grain protein and wet gluten than CS, with the DS1E/1D line having the highest content of all these components. However, in terms of storage protein components, GMP content was lowest in the DS1E/1D line, at 34.56%, and highest in the DS1E/1A line, at 48.93%. The ratio of wheat glutenin to gliadin (Glu/Gli) was 1.26 in the DS1E/1A line, significantly higher than that of lines DS1E/1B, DS1E/1D, and DA1E. In addition, introducing chromosome 1E considerably enhanced grain hardness, whereas substituting chromosomes from the original homologous chromosomes of wheat with chromosome 1E reduced the hardness to varying degrees ( Table 2).

Influence of Th. elongatum 1E Chromosome on Grain Quality
Wheat HMW-GS content correlates to wheat processing quality, gluten quality, and gluten strength. Inbred wheat germplasm contains more HMW-GS variant types, which have improved wheat processing quality. In this study, the wheat lines with substitution and addition of Th. elongatum chromosome 1E were employed to investigate the effect of diploid chromosome 1E on wheat quality. The wheat containing diploid Th. elongatum 1E chromosome showed significantly higher grain protein and wet gluten than CS, with the DS1E/1D line having the highest content of all these components. However, in terms of storage protein components, GMP content was lowest in the DS1E/1D line, at 34.56%, and highest in the DS1E/1A line, at 48.93%. The ratio of wheat glutenin to gliadin (Glu/Gli) was 1.26 in the DS1E/1A line, significantly higher than that of lines DS1E/1B, DS1E/1D, and DA1E. In addition, introducing chromosome 1E considerably enhanced grain hardness, whereas substituting chromosomes from the original homologous chromosomes of wheat with chromosome 1E reduced the hardness to varying degrees ( Table 2).   (Table 3). SRC is the ability of wheat flour to retain solvent under specific centrifugal forces. In addition, it is closely related to wheat quality and is one of the key indicators when evaluating soft wheat. We measured the SRC of the CS-Th. elongatum chromosome 1E addition and substitution lines, revealing that the water, sodium carbonate, and sucrose SRCs of the substitution lines DS1E/1A and DS1E/1B were comparable to those of CS, but significantly lower than those of the substitution line DS1E/1D and addition line DA1E. Additionally, the lactic acid SRC of the substitution line DS1E/1D was the lowest of all the lines at only 70.28%, similar to that of CS. In contrast, the lactic acid SRC of the substitution line DS1E/1A was significantly higher than that of the other lines, reaching 107.03% (Table 4).

Correlational Analysis of Quality Traits of CS-Th. elongatum Chromosome 1E Substitution and Addition Lines
Pearson's bivariate correlation analysis was conducted to evaluate the quality traits of the Th. elongatum chromosome 1E substitution and addition lines of common wheat. In terms of grain quality, the results shown in Figure 1 indicate a significant, positive correlation between protein content, wet gluten content, and unit weight (R > 0.8, p < 0.001), and a weak, positive correlation between GMP and grain hardness (R = 0.53, p < 0.05) ( Figure 4A). In addition, GMP had a significant, positive correlation with PH and 8-MW (R > 0.7, p < 0.01), and a very significant, positive correlation with PW, bread volume, total bread score, and lactic acid SRC (R > 0.8, p < 0.001) ( Figure 4B,C). However, GMP had a weak, negative correlation with sodium carbonate SRC (R = −0.5, p < 0.05) ( Figure 4C). For flour processing quality, PH, 8-MW, PW, bread volume, and total bread score, all showed significant positive correlations with each other (R > 0.8, p < 0.001) ( Figure 4D), as well as with lactic acid SRC (R > 0.7, p < 0.01). However, only MT and PW showed weakly negative correlations with water, sucrose, and sodium carbonate SRCs (R < −0.5, p < 0.05) ( Figure 4E). In addition, within SRCs, apart from a significant, weak, negative correlation (R = −0.64, p < 0.01) between the sodium carbonate and lactic acid SRCs, all others showed a significant, positive correlation (R > 0.7, p < 0.01) ( Figure 4F).

Correlational Analysis of Quality Traits of CS-Th. elongatum Chromosome 1E Substitution and Addition Lines
Pearsonʹs bivariate correlation analysis was conducted to evaluate the quality traits of the Th. elongatum chromosome 1E substitution and addition lines of common wheat. In terms of grain quality, the results shown in Figure 1 indicate a significant, positive correlation between protein content, wet gluten content, and unit weight (R > 0.8, p < 0.001), and a weak, positive correlation between GMP and grain hardness (R = 0.53, p < 0.05) (Figure 4A). In addition, GMP had a significant, positive correlation with PH and 8-MW (R > 0.7, p < 0.01), and a very significant, positive correlation with PW, bread volume, total bread score, and lactic acid SRC (R > 0.8, p < 0.001) ( Figure 4B,C). However, GMP had a weak, negative correlation with sodium carbonate SRC (R = −0.5, p < 0.05) ( Figure 4C). For flour processing quality, PH, 8-MW, PW, bread volume, and total bread score, all showed significant positive correlations with each other (R > 0.8, p < 0.001) ( Figure 4D), as well as with lactic acid SRC (R > 0.7, p < 0.01). However, only MT and PW showed weakly negative correlations with water, sucrose, and sodium carbonate SRCs (R < −0.5, p < 0.05) ( Figure  4E). In addition, within SRCs, apart from a significant, weak, negative correlation (R = −0.64, p < 0.01) between the sodium carbonate and lactic acid SRCs, all others showed a significant, positive correlation (R > 0.7, p < 0.01) ( Figure 4F).

Discussion
HMW-GS consist of various subunits, which individually and cooperatively affect the processing quality of wheat. Isolation and characterization of the HMW subunit genes in bread wheat and its wild relatives provide new ideas for in-depth research on how to improve wheat quality. All HMW-GS have similar primary structures, typically consisting of three distinct structural domains: The N-terminal region, the C-terminal region, and the repeat region, located in the middle of the peptide chain. A 63-bp sequence, downstream of the start codon, ATG, encodes a signal peptide consisting of 21 amino acids, while two closely linked stop codons, TGATAG, are located at the end of the C-terminus [33]. The HMW-GS sequence obtained from Th. elongatum in this study is entirely consistent with that of other common HMW-GS, indicated by the central repeat region consisting of hexapeptide (PGQGQQ) and nonapeptide (GYYPTSLQQ) units with no tripeptide (GQQ) repeat module ( Figure 2B). The phylogenetic analysis shows such sequences clustered with the y-type subunit ( Figure 2C), demonstrating the y-type subunit gene of Th. elongatum HMW-GS. According to the literature, the amino acids in the middle repeat region form a reverse β-fold. Based on different amino acid lengths and compositions of the subunits, the formed β-folds can have varying effects on the dough strength and viscoelasticity [34]. The number of β-fold-forming repeats QPGQ, YPTS, SPQQ, QQGY, as well as the average number and molar percentages of glutamine (Q) and tyrosine (Y) in the amino acid sequence of Glu-1Ey obtained in this study, are all significantly higher than those found in the common wheat. The cross-linking of tyrosine (Y) may also play a role in the structure and functionality of wheat glutenin. While the percentage concentration of tyrosine (Y) may be linked to the significantly higher dough-processing quality of the Th. elongatum chromosome 1E substitution and addition lines compared to CS, further investigation is required. In addition, cysteine, a particular amino acid found in HMW-GS, contains sulfur elements that construct the disulfide bonds necessary to form GMP. In contrast to the usual y-type subunit of six cysteine residues, some high-quality subunits, such as 1By9 and 1Dy10, have an extra cysteine residue in the central repeat region near the C-terminal region [33]. This excess cysteine residue may be involved in forming intermolecular disulfide bonds during the formation of GMP, which further improves the viscoelasticity of the dough [35]. Thus, the quantity and distribution of cysteine residues are strongly related to the number of disulfide bonds in the dough, resulting in the formation of intermolecular crosslinks by inter-and intra-chain disulfide bonds. This, in turn, affects the viscoelasticity of the dough [36]. The Glu-1Ey subunit obtained in this study contains seven cysteine residues, of which five are located at positions 31, 43, 65, 66, and 76 in the N-terminus, one at position 209, close to the N-terminus, and one at position 490 in the C-terminus. Consequently, the Glu-1Ey subunit shows potential value for wheat breeding because of its advantages in disulfide bond formation and dough elasticity.
Over the past few decades, some molecular markers, such as simple sequence repeats (SSR), restriction fragment length polymorphism (RFLP), amplified fragment length (AFLP), random amplified polymorphic DNA (RAPD), and diversity arrays technology (DArT), have been used to construct the molecular marker maps to identify QTL associated with important traits [37]. However, because these genetic markers have a certain distance from the target genes, their predictive value depends on the degree of linkage between the markers and the target genes in a specific population. With the increase of reproductive algebra and genetic distance, the probability of breaking the linkage relationship will gradually increase, and the accuracy of molecular marker-assisted selection will decrease. In contrast, functional markers formed by developing functional gene sequences associated with phenotypes have become a hot spot in DNA molecular markers, which are dominant markers and ideal markers for marker-assisted selection in molecular breeding. For example, Liu et al. [38] cloned the SAI gene (SAI-1) from sweet sorghum, and developed molecular markers for SAI molecular research and development of new varieties with high sugar in sweet sorghum through allelic variation analysis. Azmach et al. [39] developed functional markers of CrTRB1-5'TE and CRTRB1-3'TE using three polymorphic loci of the crtRB1 gene, which encodes β-carotene hydroxylase, to accelerate the breeding efficiency of maize varieties with high carotene. Wei et al. [40] developed a pair of dominant functional markers, POD-3A1 and POD-3A2, based on two allelic variants of the TaPod-A1 gene (TaPod-A1a and TaPod-A1b). Pod-3a1 can expand bands in materials containing TaPod-A1a, which is associated with low POD activity, and POD-3A2 can expand bands in materials containing TaPodA1b type, which is associated with high POD activity. Therefore, functional markers POD-3A1 and POD3A2 can be effectively used in the genetic improvement of POD activity in wheat. Chang et al. [41] developed three molecular markers based on three polymorphic loci in the TaSAP1-A1 promoter region, namely T7AM5, T7AM2606, and T7AM39. Association analysis showed that the three molecular markers were significantly associated with agronomic traits such as spike length, peduncle length, spikelet number per spike, grain number per spike, and 1000-grain weight. It can be seen that molecular marker technology has the advantages of simplicity, economy, speed, and high efficiency, and is the preferred means to speed up the process of crop breeding and to improve breeding efficiency. However, compared with many traits, the number of molecular markers for identifying wheat HMW-GS genes is still very little, and only a few subunits can be accurately and quickly identified. In this study, we developed a dominant molecular marker, CMSGlu-1Ey, using cloned y-type HMW-GS gene sequence of Th. elongatum, which can effectively track and detect the y-type HMW-GS gene of Th. elongatum under the wheat background. It will play a great role in improving the quality of wheat by using HMW-GS of Th. elongatum.
Additionally, quality traits are one of the critical factors involved in the selection and breeding of high-quality wheat varieties. The presence of HMW-GS is strongly related to the processing quality of wheat flour and plays a unique and vital role in gluten characteristics. A total deficiency of HMW-GS has been shown to considerably reduce gluten strength, PH, PW, tensile resistance, and other parameters of wheat [42]. Furthermore, the simultaneous deletion of Glu-1A and Glu-1D increased the diameter, crispness, and height of resulting cookies [43]. However, of the three, Glu-lA, Glu-1B, and Glu-1D loci, the deletion of the Glu-1D locus has the worst negative impact on dough quality with regards to strength and elasticity [44]. It has been reported that all species in the wheat family have HMW-GS. Despite the common wheat's low genetic diversity, the HMW-GS of close wheat relatives are highly varied, offering valuable genetic resources for the improvement of wheat quality [45]. For example, 45 HMW-GS gene haplotypes have been identified in Aegilops tauschii to date, of which the subunits 1Dx3t and 1Dx4t can improve the processing quality of wheat [46,47]. In addition, Glu-1U-encoded HMW-GS can also improve wheat quality. For example, Glu-1U g of Ae. geniculata significantly improved the rheological properties of dough [48]; Glu-1U u of Ae. umellulata increased the GMP content [49,50]; Glu-1U b of Ae. biuncialis increased the protein content, Zeleny sedimentation, grain hardness, and wet gluten content [51]; and Glu-1U k from Ae. kotschyi increased SDS sedimentation in wheat [52]. Furthermore, Dasypyrum villosum HMW-GS Glu-1V can improve protein content, Zeleny sedimentation, wet gluten content, and rheological properties of small fragment translocation line NAU425, which shows potential in wheat quality improvement [53]. In the present study, we analyzed the effect of Th. elongatum chromosome 1E on wheat quality and found that its presence significantly raised the protein, wet gluten, hardness, and bulk density of the grain. In terms of bread processing quality, the addition line, DA1E, and substitution lines, DS1E/1A and DS1E/1A, were superior in quality to the substitution lines, DS1E/1D, and control, CS. Indeed, in substitution line DS1E/1D, the substitution of the Th. elongatum HMW-GS in DS1E/1D by the high-quality subunit 2 + 12 in CS may cause a reduction in quality. Previous studies have found a significant correlation between GMP content and wheat flour processing quality. In the present study, we also found that the HMW-GS of the DS1E/1D line strongly increased the grain GMP content and was positively correlated with bread volume and overall bread score (R = 0.93 and 0.84, respectively; Figure 1B). Wheat flour lactic acid SRC reflects its gluten properties and are often positively correlated with gluten strength, with higher values indicating better gluten properties in soft wheat. Here, we found that Th. elongatum HMW-GS remarkably increased the lactic acid SRC and had significant, positive correlations with the GMP, MT, PH, PW, 8-MW, bread volume, and overall bread score ( Figure 4C,E). Therefore, to improve wheat quality using Th. elongatum HMW-GS, the GMP and lactic acid SRC content can be employed to select the lines with excellent bread-processing quality for future wheat breeding.
The HMW-GS carried by Th. elongatum was different from that of common wheat. Compared with the control Chinese Spring, the high-molecular-weight glutenin subunits of Th. elongatum significantly increased grain protein content by 3.76% to 5.11%, wet-gluten content by 6.55% to 8.73%, and unit weight by 25.88 g to 40.75 g; further, it increased flour 8-MW by 0.25% to 6.35% and bread volume value by 33.77 mL to 246.50 mL. Moreover, the solvent retention capacity of lactic acid and sucrose was increased by more than 19% and 14%, respectively, thereby contributing remarkably to overall dough quality. Therefore, in future studies, the HMW-GS of Th. elongatum can be studied in wheat with various genetic backgrounds via small fragment translocation lines to determine the effects on wheat and flour processing quality. In conclusion, intermediate materials can be produced for wheat breeding and can provide germplasm resources for wheat quality improvement.

Plant Materials
The four varieties of Th. elongatum and CS used in this study were planted in the Liuhe wheat-planting trial base of Jiangsu Academy of Agricultural Sciences (Nanjing, Jiangsu, China). The field was planted in sequential arrangement, with a row length of 1.5 m and row spacing of 20 cm, using three groups of replications and manually sowing approximately 30 seeds in each row. The fertility of the trial field was even and moderate under the local conventional field management. Two replicates of seeds were harvested, packaged separately, and numbered by location to identify and analyze the subunit composition of wheat glutenin. The grains harvested in the same year from various lines were used for experimental evaluation of quality traits. The harvested grains were fumigated and stored for approximately 1 month before being ground and left at 24 • C for over a month. In addition, three diploid lines Th. elongatum (WZ 218, PI 531717, PI 531718) and the common wheat with different high-molecular-weight gluten subunit types (Neimai 1505, Taimai 1918, Yumai 9, Yumai 13, Yumai 18, Zhenmai 12, Shannong 23, Fielder, Chinese Spring) were all planted in the greenhouse at Yangzhou University.

Protein Extraction from Grain and SDS-PAGE
Glutenin extraction from non-embryonic half-wheat grains followed the procedure described by Garg et al. [54], with minor modifications. Briefly, 10 µL of samples were added to a sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel, consisting of 8% gradient separation gel (pH 8.5) and 4% stacking gel (pH 6.8), and electrophoresed at a constant current of 10 mA for 10 h. After electrophoresis, the gels were run at a constant current of 0.5 mA. Next, the gels were stained with 0.1% w/v Komas Brilliant Blue R-250 (A610037, Sangon Biotech, Shanghai, China), 25% w/v isopropanol (A600918, Sangon Biotech, Shanghai, China) and 10% w/v acetic acid (A501931, Sangon Biotech, Shanghai, China) for 10 h. The gels were then rinsed several times with deionized water and left submersed until the bands became visible.

Determination of Wheat Quality
The determination method of wheat quality mainly refers to the article by Liu et al [55]. Wheat grain hardness was determined using an SKCS 4100 analyzer (Perten, Zürich, Switzerland), which provides a hardness index of wheat, based on the systemdefined formula.

DNA Isolation and PCR Amplification
Genomic DNA was extracted from wheat leaves by the CTAB method [56]. Degenerative primers P1 and P2 (P1: ATGGCTAAGCGGYTRGTCCTCTTTG, P2: CTATCACTG-GCTRGCCGACAATGCG) were used to amplify the high-molecular-weight gluten gene sequence of Th. elongatum. PCR amplification was carried out in a 25 µL reaction, containing 1 µL genomic DNA (100 ng/µL), 12.5 µL 2× Phanta Master Mix (Vazyme Biotech Co., Nanjing, China), 1 µL 10 µmol/L of each primer, and 9.5 µL double-distilled water. The PCR procedure was as follows: 94 • C for 5 min, followed by 35 cycles of 94 • C for 45 s, appropriate anneal temperature (65 • C) for 45 s, 72 • C for 2 min, and a final extension for 10 min at 72 • C. Amplified PCR products were separated on 1% agarose gels at 130 V for 20 min, stained with ethidium bromide, and visualized using ultraviolet (UV) light. The amplified DNA product was purified by the Universal DNA Purification Kit (Tiangen Biotech Co., Ltd., Beijing, China) and cloned by the 5-min TA/Blunt-Zero Cloning Kit (Vazyme Biotech Co., Nanjing, China).

Phylogenetic Analyses
ClustalX in MEGA6 (6.06) was used for multiple sequence alignments with the default parameters. Multiple sequence alignments were adjusted manually to minimize gaps [57]. Multiple sequences from each accession were first compared to identify distinct copies of sequences by ClustalX. The high-molecular-weight glutenin subunit sequences of wheat and its related species were downloaded from the NCBI website for phylogenetic analysis (Supplementary Table S1). Phylogenetic analysis, using the maximum-parsimony (MP) method, was performed with the computer software MEGA6 (6.06). The MP trees were constructed by performing a heuristic search using the Tree Bisection-Reconnection (TBR), with Mul-Trees on, and 10 replications of random addition sequences, with the stepwise addition option. Overall character congruence was estimated by the consistency index (CI) and the retention index (RI). To test the robustness of clades, bootstrap (BS) values with 1000 replications were calculated by performing a heuristic search, using the TBR option with Mul-Tree on [58].