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. 2019 Sep 5;9(10):355. doi: 10.1007/s13205-019-1887-1

Enriching LMW-GS alleles and strengthening gluten properties of common wheat through wide hybridization with wild emmer

Lan Xiang 1,2,#, Lin Huang 2,✉,#, Fangyi Gong 2, Jia Liu 2, Yufan Wang 2, Yarong Jin 2, Yu He 2, Jingshu He 2, Qiantao Jiang 1,2,3, Youliang Zheng 1,2,3, Dengcai Liu 1,2,3, Bihua Wu 1,2,3,✉
PMCID: PMC6728113  PMID: 31501756

Abstract

Two advanced lines (BAd7-209 and BAd7-213) with identical high-molecular-weight glutenin subunit composition were obtained via wide hybridization between low-gluten cultivar chuannong16 (CN16) and wild emmer D97 (D97). BAd7-209 was better than BAd7-213, and both of them were much better than CN16 in a dough quality test. We found that BAd7-209 had more abundant and higher expression levels of low-molecular-weight glutenin subunit (LMW-GS) proteins than those of BAd7-213. Twenty-nine novel LMW-GS genes at Glu-A3 locus were isolated from BAd7-209, BAd7-213 and their parents. We found that all 29 LMW-GS genes possessed the same primary structure shared by other known LMW-GSs. Twenty-seven genes encode LMW-m-type subunits, and two encode LMW-i-type subunits. BAd7-209 had a higher number of LMW-GS genes than BAd7-213, CN16, and D97. Two wild emmer genes MG574329 and MG574330 were present in the two advanced lines. Most of the LMW-m-type genes showed minor nucleotide variations between wide hybrids and their parents that could be induced through the wide hybridization process. Our results demonstrated that the wild emmer LMW-GS alleles could be feasibly transferred and integrated into common wheat background via wide hybridization and the potential value of the wild emmer LMW-GS alleles in breeding programs designed to improve wheat flour quality.

Keywords: Wild emmer wheat, Wide hybridization, LMW-GS alleles, Flour quality

Introduction

Wheat (Triticum aestivum L.), one of the three major cereal crops worldwide, provides approximately 20% of calories and 25% of proteins in the human diet. The functional characteristics of wheat flour products are largely determined by the gluten proteins present in the seed endosperm, which consist of polymeric glutenins and monomeric gliadins (Shewry and Halford 2002). The polymeric glutenins can be subdivided into high-molecular-weight (HMW) and low-molecular-weight (LMW) glutenin subunits (GSs) according to their mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. Of which, LMW-GSs account for ~ 60% of the total glutenins and primarily determine dough strength and extensibility, thus playing a crucial role in flour processing quality (D’Ovidio and Masci 2004; Lee et al. 2016; Pogna et al. 1990; Tanaka et al. 2005).

LMW-GSs are encoded by Glu-A3, Glu-B3, and Glu-D3 loci on the short arms of the group 1 chromosomes, and these loci are linked to the Gli-1 loci, encoding gliadins (Anderson et al. 2009; Pogna et al. 1990). The general structure of a typical LMW-GS consists of four domains: (1) a conserved signal peptide of 20 amino acids, (2) a short N-terminal domain with 13 amino acids, (3) a central repetitive domain rich with glutamine, and (4) a C-terminal domain rich with cysteine and glutamine.

According to the molecular weight of the subunits, LMW-GSs can be classified into B-, C-, and D-type subunits (Payne et al. 1979), of which, B-type subunits are the main type of LMW-GSs with molecular weights that range from 42 to 51 kD and belong to alkaline proteins. The C-type subunits are mainly present in the overlap region of LMW-GSs and gliadins with molecular weights ranging from 20 to 40 kD, while the D-type subunits possess a low percentage of total LMW-GS and their molecular weights are similar to the ω-gliadins migrating in gliadin fractions (D’Ovidio and Masci 2004). Based on the first amino acid residue at the N-terminal region, LMW-GSs could be classified into three types: LMW-serine (s), LMW-methionine (m), and LMW-isoleucine (i) (Cloutier et al. 2001; D’Ovidio and Masci 2004). Recently, a new class of LMW-GS genes from Aegilops comosa, designated as LMW-leucine (l) type subunits, was reported by Huang et al. (2018) and Wang et al. (2011).

The LMW-GS genes were estimated to have 10–20 (Harberd et al. 1985) or 30–40 copies in hexaploid wheat (Lee et al. 2016) due to extensive allelic variation present at Glu-3 loci. Previous studies have shown the differential effects of LMW-GS alleles on wheat flour processing quality. For example, Lee et al. (1999) reported that the LMW-E2 and LMW-E4 genes at Glu-A3 had inferior effects on dough resistance, while superior effects on dough elongation properties. Luo et al. (2001) found that the allelic variants at the Glu-3 significantly affect wholemeal flour protein content, SDS sedimentation volume, Pelshenke time, mid-line peak value, and the mid-line peak time of a mixograph. Zhang et al. (2012a) used a set of Aroona near-isogenic lines (NILs) to study the function of LMW-GS alleles in bread wheat, and found that Glu-A3e and Glu-B3c were inferior alleles with respect to bread-making quality, whereas Glu-A3d, Glu-B3b, Glu-B3g, and Glu-B3i were associated with superior bread-making quality. More recently, Wang et al. (2016) used a deletion line at Glu-B3 locus to study the function of Glu-B3h, and found that Glu-B3h affects protein body number and size and main quality parameters, including inferior mixing property, dough strength, loaf volume, and score.

Wild emmer wheat (Triticum turgidum ssp. dicoccoides, 2n = 4x = 28, AABB), the tetraploid progenitor of common wheat, has valuable residual genotypic variations in agronomic traits such as yield, quality of storage proteins, and resistance to biotic and abiotic stresses (Nevo et al. 2002; Huang et al. 2016, 2019). It shares the A and B genomes with common wheat, and introgression is thereby feasible due to occurrence of homologous recombination between the A and B genomes of wild emmer and modern wheat (Jiang et al. 2017; Liu et al. 2019; Nevo et al. 2002; Wang et al. 2018). Previous studies have demonstrated that wild emmer accessions show extensive allelic variations at Glu-3 loci (Ciaffi et al. 1991). However, the utilization of wild emmer LMW-GS alleles for wheat quality improvement has been less reported.

Our previous studies revealed that wild emmer accession D97 could be effectively utilized to enrich the genetic bases of the Glu-1A alleles of common wheat (Jiang et al. 2017; Wang et al. 2018). In the current study, two agronomically stable advanced wheat lines were developed via wide hybridization between low-gluten wheat cultivar CN16 and wild emmer D97. The two wheat lines show identical HMW-GS composition, but differ in flour processing quality. The objectives of current study were: (1) to investigate the composition of LMW-GSs in two advanced lines and compare them with those of their parents; (2) to characterize the sequence variations at Glu-A3 locus in advanced lines and their parents; (3) to evaluate the potential value of wild emmer LMW-GS genes for wheat quality improvement.

Materials and methods

Plant materials

The low-gluten wheat cultivar CN16 (T. aestivum, AABBDD, 2n = 6x = 42) was crossed as the female parent with a high-protein content wild emmer accession D97 (Jiang et al. 2017) as the male parent. The resulting pentaploid F1 was advanced to F12 to generate a number of stable 42 chromosome plants expressing desirable agronomic properties. Two sister lines, BAd7-209 and BAd7-213, were developed and maintained at the Triticeae Research Institute, Sichuan Agricultural University, China.

All of the materials were grown in field trials using a randomized complete block design with three replicates at the Wenjiang (30º43′9″N 103º52′17″ E) experimental stations. Nitrogen was applied at 150 kg N ha−1 before sowing.

Glutenin extraction, SDS-PAGE and two-dimensional electrophoresis (2-DE)

Glutenin extraction was performed using the methods described by Duan and Zhao (2004) and Wan et al. (2000) with minor modifications. In brief, 50 mg of white flour was used for protein extraction. The sample was initially treated with 70% ethanol (v/v) and 50% isopropanol (v/v) to remove albumins, globulins, and gliadins. Then, the total glutenin subunits were isolated with extraction buffer [50% isopropanol (v/v), 0.08 mol/L Tris–HCl, pH 8, and 1% (w/v) dithiothreitol (DTT)], and precipitated with 80% (v/v) acetone. The pellet was resuspended in buffer [0.063 mol/L Tris–HCI, pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 3% (v/v) β-mercaptoethanol]. A 4 µL aliquot of the extraction was separated by SDS-PAGE according to the method described by Gorg (2000) with 4.5% concentration gel and 15% separation gel and electrophoresed at 40 mA for 2.5 h.

The 2-DE was employed to identify LMW-GS using an IPGphor isoelectric focusing electrophoresis apparatus (BIO-RAD, USA) for isoelectric focusing (IEF). 2-DE was performed according to the method of Zhang et al. (2017) with minor modifications. After electrophoresis, the 2-DE gels were stained within colloidal CBB R-250/G-250 (4:1) and analyzed using Imagemaster2D platinum software version 6.0 (Amersham Bioscience) according to Ikeda et al. (2006).Three biological replicates were performed.

DNA extraction and PCR amplification

Seeds of D97, CN16, BAd7-209, and BAd7-213 were germinated at room temperature for 2 weeks. The seedlings genomic DNA was extracted using a Plant Genomic DNA Kit (Tiangen Biotech, Beijing, China). A pair of primers (LMW-F: 5′-GCCTTTCTTGTTTACGGCTG-3′; LMW-R: 5′-TCAGATTGACATCCACACAAT-3′) was used to amplify the complete open reading frames (ORFs) of the LMW-GS genes at Glu-A3 locus (Fang et al. 2008). A high-fidelity ExTaq polymerase (Takara, Dalian, China) was used for PCR amplifications. The PCR consisted of 35 cycles of 94 °C for 30 s, 58 °C for 40 s, and 72 °C for 1 min and 20 s, with an initial denaturation of 94 °C for 5 min, and a final extension of 72 °C for 10 min using a VeritiTM 96-Well Fast Thermal Cycler (Applied Biosystems, USA). The PCR fragments were separated using 1.0% agarose gel electrophoresis.

Molecular cloning and sequencing of LMW-GS genes

The PCR products of the expected length were recovered, purified, and further ligated into the pMD19-T vector (TaKaRa, Dalian, China). The ligation mixtures were transformed into cells of Escherichia coli strain DH5a. Positive clones were selected by colony PCR. DNA sequencing was performed by the Sangon Biotechnology Company (Chengdu, China). The final nucleotide sequence for each LMW-GS gene was determined from the sequencing results of three independent clones.

Sequence alignment and phylogenetic analysis

Multiple sequence alignment of LMW-GS nucleotide and protein sequences was performed using BioEdit (Hall 2007).The phylogenetic tree was constructed based on the full-length coding sequences using the neighbor-joining (NJ) algorithm in the MEGA 6.0 software (Tamura et al. 2013). Bootstrap values were estimated based on 1000 replications.

Flour processing quality determination

Mature seeds were harvested for flour processing quality analyses. Seeds of BAd7-209, BAd7-213, and CN16 were conditioned to 14% moisture and milled using the Chopin CD1 AUTO (Renault, Boulogne-Billancourt, France) (Wang et al. 2018). An equal amount of white flour for each material was used to measure the grain protein content (0.5 g), SDS sedimentation value (0.5 g), wet gluten content (10 g), and farinograph parameter (10 g). Grain protein content was measured following the American Association of Cereal Chemists (AACC 2000) method AACC 39-10. SDS sedimentation value was determined according to AACC 44-15A. Wet gluten content was determined following the AACC 38-12A. Farinograph parameter, including dough development time and stability, was measured following the AACC 54-21. All of the experiments were repeated three times.

Statistical analysis

All of the processing quality parameters were subjected to analysis of variance and differences among genotypes were tested using Tukey’s test, which were carried out using SPSS version 19.0 software (SPSS lnc. Chicago, IL USA).

Results

Separation and characterization of LMW-GSs

The glutenin composition of BAd7-209, BAd7-213, and their parents was separated by SDS-PAGE (Fig. 1a). BAd7-209 and BAd7-213 had the same HMW-GS composition at the Glu-1 loci (1Ax1.2, 1Bx7 + 1By8, 1Dx5 + 1Dy10) (Jiang et al. 2017). At the Glu-3 loci, all the materials tested had B-, C-, and D-type subunits. However, obvious differences were determined among the wheat lines. BAd7-209 and BAd7-213 had the same B-type subunits as female parent CN16. Although the C-type subunits were blurred and patterns were almost the same, the expression levels of BAd7-209 were higher than those of BAd7-213. The two advanced lines had the same D-type subunits (D1 and D2) as male parent D97, but the expression levels were extremely low.

Fig. 1.

Fig. 1

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Separation of glutenin proteins from CN16, D97, BAd7-209, and BAd7-213 by SDS-PAGE (a) and 2-DE (b). a The D1- and D2-type subunits were marked by diamonds and triangles. b Circles highlight the B-type protein spots. The protein spots showed differences among wheat lines were marked by arrows

2-DE analysis revealed that the expression levels of B- and C-type protein spots in D97 were higher than those of in CN16 and their progenies (Fig. 1b). BAd7-209 had more abundant protein spots than BAd7-213, whereas both had the same B-type spots as female parent CN16. Most of the C-type spots in advanced lines were identical to CN16. However, the C3 and C4 spots of D97 were only present in BAd7-209. Notably, BAd7-213 and D97 had only one D-type protein spot (D4), whereas BAd7-209 had five D-type spots (D5-D9), of which, three spots were the same as CN16 and two were newly generated (D8 and D9).

Isolation and characterization of LMW-GS genes

A total of eight, eight, ten, and nine LMW-GS genes have been isolated from D97, CN16, BAd7-209, and BAd7-213, respectively. All obtained sequences had ORFs varied from 696 to 1053 bp. GenBank database comparison indicated that the ORFs of these genes were different from known LMW-GS genes in Triticum species, thus, all these 29 genes were novel. The complete nucleotide sequences of these 29 LMW-GS genes were deposited in GenBank with the accession numbers MG574321 to MG574349. These 29 sequences encoding two LMW-i-type and 20 LMW-m-type proteins begin with the amino acids isoleucine and methionine, respectively (Table 1). Similar to other LMW-GS genes, the coding regions of these genes reported hereby were all terminated by double stop codons.

Table 1.

Comparison of the mature protein sequences of LMW-GSs in the current study

GenBank accession Subunit types Protein types Nucleotide number (amino acid number) N-terminal sequence Wheat lines Number of cysteine residues
Signal N-terminal domain Repetitive domain C-ter domain I C-ter domain II C-ter domain III Total
MG574331 LMW-i P1 696(231) ISQQQQPP D97 0 0 0 6 1 1 8
MG574334 LMW-i P2 720(239) ISQQQQPP D97 0 0 0 6 1 1 8
MG574332 LMW-m P3 915(304) METRCIPG D97 0 1 0 5 1 1 8
MG574333 LMW-m P4 924(307) METRCIPG D97 0 1 0 5 1 1 8
MG574335 LMW-m P5 924(307) METRCIPG D97 0 1 0 5 1 1 8
MG574321 LMW-m P6 924(307) METRCIPG CN16 0 1 0 5 1 1 8
MG574322 LMW-m P7 924(307) METRCIPG CN16 0 1 0 5 1 1 8
MG574323 LMW-m P8 924(307) METRCIPG CN16, D97, BAd7-209, BAd7-213 0 1 0 5 1 1 8
MG574324 LMW-m P9 924(307) METRCIPG CN16 0 1 0 5 1 1 8
MG574325 LMW-m P10 924(307) METRCIPG CN16 0 1 0 5 1 1 8
MG574326 LMW-m P11 924(307) METRCIPG CN16 0 1 0 5 1 1 8
MG574327 LMW-m P8 924(307) METRCIPG CN16 0 1 0 5 1 1 8
MG574328 LMW-m P12 1053(350) METSRVPG CN16 0 0 1 5 1 1 8
MG574329 LMW-m P13 924(307) METRCIPG D97, BAd7-213 0 1 0 5 1 1 8
MG574330 LMW-m P8 924(307) METRCIPG D97, BAd7-209, BAd7-213 0 1 0 5 1 1 8
MG574336 LMW-m P14 924(307) METSRVPG BAd7-209 0 1 0 5 1 1 8
MG574337 LMW-m P15 924(307) METSRVPG BAd7-209 0 1 0 5 1 1 8
MG574338 LMW-m P16 924(307) METRCIPG BAd7-209 0 1 0 5 1 1 8
MG574339 LMW-m P8 924(307) METRCIPG BAd7-209 0 1 0 5 1 1 8
MG574340 LMW-m P8 924(307) METRCIPG BAd7-209 0 1 0 5 1 1 8
MG574341 LMW-m P17 924(307) MEARCIPG BAd7-209 0 1 0 5 1 1 8
MG574342 LMW-m P8 924(307) METRCIPG BAd7-209 0 1 0 5 1 1 8
MG574343 LMW-m P18 924(307) METRCIPG BAd7-209 0 1 0 5 1 1 8
MG574344 LMW-m P19 924(307) METRCIPG BAd7-213 0 1 0 5 1 1 8
MG574345 LMW-m P20 924(307) METRCIPG BAd7-213 0 1 0 5 1 1 8
MG574346 LMW-m P21 924(307) METRCIPG BAd7-213 0 1 0 5 1 1 8
MG574347 LMW-m P13 924(307) METRCIPG BAd7-213 0 1 0 5 1 1 8
MG574348 LMW-m P22 924(307) METRCIPG BAd7-213 0 1 0 5 1 1 8
MG574349 LMW-m P8 924(307) METRCIPG BAd7-213 0 1 0 5 1 1 8
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The sizes of wild emmer-specific genes MG574331 and MG574334 were 696 bp and 720 bp, which encoded 231 and 239 amino acid residues, respectively. The gene MG574328 was 1053 bp long, which encoded 350 amino acid residues and was specific in CN16. For the remaining 26 genes, 25 genes were 924 bp long, which encoded 307 amino acid residues and one gene MG574332 was 915 bp in length, which encoded 304 amino acid residues. MG574323 was present in all wheat lines, whereas MG574329 and MG574330 were specific in wild emmer and hexaploid progenies (Table 1).

Amino acid sequence analysis revealed that all 29 LMW-GSs shared four main structural domains with previously characterized LMW-GSs (Cassidy et al. 1998), including a signal peptide (20 amino acid residues), a short N-terminal region (deleted in LMW-i-type), a repetitive domain and a C-terminal domain (Fig. 2). The C-terminal domain can be further classified into three sub-regions, including a cysteine-rich region (I), a glutamine-rich region (II), and a C-terminal conserved region (III) (Fig. 2; Huang et al. 2018). Most of LMW-m-type proteins begin with METRCIPG-, whereas MG574328 and MG574341 begin with METSRVPG- and MEARCIPG-, respectively. The two LMW-i-type proteins lack the typical deletion of the N-terminal region, following the signal peptide domain is the ISQQQQ- (Huang et al. 2018). The number and position of cysteine residues were as expected and conserved as typical LMW-m-type subunits except MG574328 has the first cysteine located in the repetitive domain (Table 1; Fig. 2). All eight cysteine residues of the two LMW-i-type subunits were located in the C terminus, consistent with previous reports (Huang et al. 2018; Wang et al. 2011).

Fig. 2.

Fig. 2

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Multiple alignments of the deduced amino acid sequences of LMW glutenin genes including LMW-m and LMW-i type subunits. Signal represents signal peptide. I, N-terminal domain; II, repetitive domain; C-terminal domains were divided into three sub-regions (I–III). The first amino acid residue of the mature proteins was highlighted by magenta. The cysteine residue was marked by gray shading. Identical sequences and deletions were indicated by dots and dashes, respectively

Phylogenetic analysis of LMW-GS genes

A phylogenetic tree was constructed using the coding sequences of 29 LMW-GS genes in the current study and other 18 representative LMW-GS genes retrieved from GenBank to understand the phylogenetic relationships among the LMW-GS genes at the Glu-3. These genes included EF190882, EF190885, KC136285, KC136286, U86028, AY695380, JF339155, AB06287, AY453158, AY453160, and AY542896 from T. aestivum; FJ461690, FJ461691, EF188290, AY748826, KF562511, KF562513, and KF562514 from T. dicoccoides. The results are presented in Fig. 3.

Fig. 3.

Fig. 3

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Phylogenetic relationships of the 29 newly cloned and LMW-GS sequences retrieved from GenBank. The nucleotide sequences were specified by their corresponding GenBank accession numbers. Bold, nucleotide sequences were detected in more than two wheat lines

The phylogenetic tree was separated into two clear clades, with alleles of LMW-m-type genes at the top and LMW-i-type genes at the bottom. For the LMW-m-type clade, two different subgroups were apparently separated. The LMW-m-type gene MG574328 was clustered with several published LMW-m-type genes in one subgroup, while the remaining 26 LMW-m-type genes obtained in the current study were located in another subgroup. The genes MG574331 and MG574334 were located in the LMW-i-type clade, further confirming that they are LMW-i-type subunit genes.

Processing quality parameters

Flours milled from BAd7-209, BAd7-213, and CN16 were compared with respect to their five processing quality parameters: grain protein content, SDS sedimentation value, wet gluten content, dough development time, and dough stability. All quality parameters scored were significantly higher (p < 0.05) for the BAd7-209 and BAd7-213 flours than for those of CN16. The quality parameters of BAd7-209 flour were also significantly higher than those of the flour milled from BAd7-213 except grain protein content (not significant) (Table 2). These results demonstrated that introgression lines BAd7-209 and BAd7-213 with the traits integrated from wild emmer D97 had better flour processing properties than CN16.

Table 2.

Main quality parameters of the two introgression lines and their female parent CN16

Genotype Grain protein content (%) SDS sedimentation value (mL) Wet gluten content (%) Development time (min) Stability (min)
CN16 11.06a 32.73c 19.64c 1.71bc 2.46c
BAd7-209 13.51b 59.23a 31.83a 4.64a 5.29a
BAd7-213 12.72bc 49.20b 26.39b 2.14b 3.29b
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The small letters behind the average values indicated the differences at 0.05 (p < 0.05)

Discussion

Wild emmer wheat is the progenitor of common wheat and harbors an abundant LMW-GS composition (Ciaffi et al. 1991; Li et al. 2008; Qin et al. 2015). In the present study, two introgression lines with identical HMW-GS composition were obtained from common wheat CN16 as female parent crossed with wild emmer D97 as male parent through successive selfing. We have investigated the composition of LMW-GSs and characterized the LMW-GS genes at Glu-A3 locus in two advanced wheat lines and their parents. We found that the two advanced lines with the traits integrated from wild emmer D97 had improved processing quality in relation to CN16.

Previous studies have reported that the wild emmer HMW-GS alleles can be feasibly transferred and integrated into common wheat background through wide hybridization (Jiang et al. 2017; Wang et al. 2018). In the current study, we found that the wild emmer LMW-GS alleles could be transferred and integrated into the common wheat background. For instance, the D-type subunits (D1 and D2), C-type spots (C3 and C4), and D4 spot of D97 were observed in its hexaploid progenies. At Glu-A3 locus, the two advanced lines had a higher number of LMW-GS genes than their parents. Two wild emmer genes MG574329 and MG574330 were present in the two introgression lines, while absent in CN16. Therefore, our results demonstrated that wild emmer wheat is also beneficial to enriching the genetic bases of the Glu-3 loci of common wheat.

In this study, two novel LMW D-type protein spots (D8 and D9) were present in BAd7-209. In addition, many LMW-m-type sequences of Glu-A3 had minor nucleotide variations in between wide hybrids and their parents (data not shown). These genes were estranged to most published LMW-m-type genes in the phylogenetic tree. It is well known that wide hybridization can produce rich variations in different cross-parents in addition to the parental type (Acquaah 2012). For example, Yuan et al. (2014) found that wide hybridization of common wheat with rye induces novel DNA variation. Wang et al. (2018) identified 14 SNPs at the 1Ay allele between wild emmer and its wide hybrid. It has been demonstrated that repeat sequences are easily changed during wide hybridization process (Zhang et al. 2013). Therefore, the repetitive domain of LMW-GS could be easily affected by the wide hybridization that induced nucleotide variations. The genetic variations between LMW-GS genes of introgression lines and their parents could be produced by mechanisms including illegitimate recombination, genomic asymmetry, and unequal crossing-over through the wide hybridization (Jiang et al. 2017; Wang et al. 2018).

It is known that LMW-GSs are closely associated with wheat processing quality, which is mainly related to the number and composition of LMW-GSs (D’Ovidio and Masci 2004; Lee et al. 2016; Tanaka et al. 2005). In the current study, the processing quality of the introgression line BAd7-209 was better than BAd7-213, and both of them were much better than that of female parent CN16. A previous study demonstrated that the presence of Glu-1Ax1.2 should be responsible for the better dough quality of BAd7-209 than that of BAd7-210 (Jiang et al. 2017). In our study, the BAd7-209 and BAd7-213 had identical HMW-GSs but differed in LMW-GSs. As evidence from SDS-PAGE and 2-DE, BAd7-209 had either higher expression levels (SDS-PAGE) or more abundant protein spots (2-DE) of LMW-GSs than those of BAd7-213. Previous studies revealed that the expression levels of glutenin subunits are closely related to gluten quality (Butow et al. 2003). The Glu-A3 locus was considered to be a major contributor to the wheat processing quality (Zhang et al. 2012b; Zhen et al. 2014). In this study, the BAd7-209 had one more LMW-GS gene than BAd7-213 at Glu-A3 locus. Therefore, our results suggested that introgression of the LMW-GSs from wild emmer could increase the wheat gluten properties. That in turn can be co-related to some extent with the number and composition as well as expression levels of LMW-GSs.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (No. 31571668; No. 31801360), the National Key Research and Development Program of China (No. 2017YFD0100900), the Science & Technology Department of Sichuan Province (No. 2019YJ0435), and the Education Department of Sichuan Province (No. 18ZA0392).

Author contributions

BHW, LH, and LX designed the experiments. FYG, JL, YFW, YRJ, YH, and JSH carried out experiments. LX analyzed the data and drafted the manuscript. QTJ and YLZ provided their constructive comments and suggestions. LH, BHW, and DL revised the manuscript. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that there are no conflicts of interest in the reported research.

Contributor Information

Lin Huang, Email: lhuang@sicau.edu.cn.

Bihua Wu, Email: wubihua2017@126.com.

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