Genome-wide identification, characteristics and expression of the prolamin genes in Thinopyrum elongatum

Background Prolamins, unique to Gramineae (grasses), play a key role in the human diet. Thinopyrum elongatum (syn. Agropyron elongatum or Lophopyrum elongatum), a grass of the Triticeae family with a diploid E genome (2n = 2x = 14), is genetically well-characterized, but little is known about its prolamin genes and the relationships with homologous loci in the Triticeae species. Results In this study, a total of 19 α-gliadin, 9 γ-gliadin, 19 ω-gliadin, 2 high-molecular-weight glutenin subunit (HMW-GS), and 5 low-molecular-weight glutenin subunit (LMW-GS) genes were identified in the Th. elongatum genome. Micro-synteny and phylogenetic analysis revealed dynamic changes of prolamin gene regions and genetic affinities among Th. elongatum, Triticum aestivum, T. urartu and Aegilops tauschii. The Th. elongatum genome, like the B subgenome of T. aestivum, only contained celiac disease epitope DQ8-glia-α1/DQ8.5-glia-α1, which provided a theoretical basis for the low gluten toxicity wheat breeding. The transcriptome data of Th. elongatum exhibited differential expression in quantity and pattern in the same subfamily or different subfamilies. Dough rheological properties of T. aestivum-Th. elongatum disomic substitution (DS) line 1E(1D) showed higher peak height values than that of their parents, and DS6E(6D) exhibited fewer α-gliadins, which indicates the potential usage for wheat quality breeding. Conclusions Overall, this study provided a comprehensive overview of the prolamin gene family in Th. elongatum, and suggested a promising use of this species in the generation of improved wheat breeds intended for the human diet. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-021-08088-x.


Background
Prolamins (glutenins and gliadins), comprising 80% of wheat endosperm protein, are the main component of glutens [1]. High-and low-molecular-weight glutenin subunits (HMW-GSs and LMW-GSs) form polymeric proteins by interchain disulfide bonds, imparting the elasticity of dough. Gliadins, divided into α-, β-, γ-, and ω-gliadins, are monomeric proteins of 30-78 kDa that determine the ductility and viscosity of dough [2][3][4][5]. These unique properties determine the quality of wheat flour in various technological processes and enable the manufacture of a wide range of products such as bread, pasta, noodles, cakes, and pastries [6,7]. Prolamin genes were reported to be closely linked and mainly located on a few chromosomes in common wheat [8]. The HMW-GS genes are encoded by the Glu-1 loci on the long arm of the first homologous group [9]. The γ-gliadin and ωgliadin genes are encoded by the Gli-1 loci closely linked to the Glu-3 loci encoding LMW-GS genes on the short arm of the same chromosome, on which HMW-GS genes are located [10]. The α-gliadin genes are encoded by the Gli-2 loci on the short arm of the sixth homologous group [11].
Prolamin genes belong to a large family. Comprehensive understanding of these genes is challenging, but essential for improving the end-use quality of wheat flour [7]. Generally, T. urartu (AA, 2n = 2x = 14) and Ae. Tauschii (DD, 2n = 2x = 14) are regarded as donors of the A and D genomes of common wheat [12]. Th. elongatum, closely related to wheat, has excellent performance in biotic and abiotic stress resistance [13]. At present, genome data of these Triticeae species have been published, but the relationship among their prolamin genes has not been well described, including gene features and molecular characteristics [13][14][15][16]. High pseudogene rate was associated with gliadin families and this was estimated to be 87% of 230 distinct α-gliadin gene sequences in several diploid wheat species [1]. Hence, it is necessary to understand the expression profile of these genes to clarify the mechanism of gene activation. However, current research is still insufficient, especially regarding species other than common wheat [17].
Gene clusters of a gene family are often prone to genetic variations in copy number, sequence polymorphism, and expression [18]. Comparisons of these homologous regions between different genomes of related species will provide insights into gene differentiation, as well as local rearrangements [19]. For example, a comparison of the Gli-2 loci between T. dicoccoides Korn and T. aestivum cv. Chinese Spring (CS) showed a large sequence difference between the two A subgenomes and a conservative region between the two B subgenomes [20]. To date, a detailed analysis of other genomes (except for the A, B, and D genomes) on the interval of these loci has not been reported.
Gluten is the most important source of proteins in common wheat for human beings. Unfortunately, prolamins of gluten are also responsible for certain intolerances, among which celiac disease (CD) is one of the most common wheat-related disorders [21]. CD is a chronic intestinal immune-mediated bowel disease, which occurs in genetically susceptible people and is caused by the intake of gluten [22]. α-gliadins are the main substance causing CD [18]. A breeding effort had been proposed to develop wheat with reduced immunoreactive epitopes while retaining baking functions [23]. Prolamin genes from wild relatives of wheat were reported to have low gluten toxicity and could be used to improve wheat quality by distant hybridization [24].
In this study, prolamin genes were obtained and comparative analysis were carried out between Th. elongatum and three related species (T. aestivum, T. urartu, and Ae. tauschii) in terms of gene numbers, molecular characteristics, micro-synteny, phylogenetic relationship, CD epitope content and dynamic expression pattern. Mixogram tests of disomic substitution (DS) line 1E(1D) was also performed. The results presented will provide helpful information of prolamin genes in Triticeae and future application in wheat breeding.

Results
Identification of the prolamin genes in Th. Elongatum and error correction in the related species A total of 19 α-gliadin, 9 γ-gliadin, 19 ω-gliadin, 2 HMW-GS, and 5 LMW-GS genes were identified in the Th. elongatum genome while no δ-gliadin genes were detected (Additional file 1: Table S1). Prolamin genes in the related species were also collected and reannotated. In addition, some annotation errors were found in related species. For example, there were no sequences for AET6Gv20125500.1 and AET6Gv20126100.1 in the existing CDS and protein files of Ae. tauschii. By checking their positions provided in that study, we found an incorrect coordinate layout (Additional file 1: Table S2) [15]. Several δ-gliadins were also identified in T. aestivum genome (Additional file 1: Table S4). All prolamin genes found in this study were manually checked and corrected according to their structure information (Additional file 1: Table S3 and Table S4) [8,[25][26][27][28].
Characteristics and sequence analysis of the prolamin gene family All prolamin genes of four species were first named based on abbreviations of species names and chromosomal locations. Then, the isoelectric point (pI), molecular weight (MW) and other characteristics of putative functional prolamin genes were calculated (Additional file 1: Table S4). No obvious differences were found between Th. elongatum and other three species except for Tel_LMW_glutenin_1E_1, a special prolamin gene that has the highest MW and the longest protein sequence (Additional file 1: Table S4).
The prolamin gene numbers showed variable patterns among different ploidy species (Fig. 1a). Only HMW-GS gene subfamily, carrying one x-type and one y-type in each diploid genome, follow a conservative pattern in the process of evolution [7]. In contrast, no specific pattern was observed among gliadin subfamilies across the species. What is particularly noticeable is that Th. elongatum had the highest numbers in three gliadin subfamilies of the three diploid species, and the number of ω-gliadin genes of Th. elongatum was about 3-5 times than that of the other two diploid species. A common feature of gliadin genes in the four species was that the numbers of α-gliadin genes were the largest, followed by ω-gliadin genes, and finally γ-gliadin genes.
The pseudogene rates of each prolamin subfamilies were calculated according to the pseudogene number based on the inference from coding sequences to protein sequences (Additional file 1: Table S3). Th. elongatum had the highest pseudogene rates in α-gliadin (84.2%) and γ-gliadin gene families (77.8%), while no pseudogenes were found in γ-gliadin and LMW-glutenin gene families in T. urartu (Fig. 1b).

Chromosomal location and duplication of prolamin genes
Consistent with previous studies, the γ-gliadin, ω-gliadin and LMW-GS genes were distributed on the short arm, and the HMW-GS genes were located on the long arm of the first homologous group [10,29,30]. Most αgliadins were mainly distributed on the sixth homologous group (Fig. 2) [11]. It is interesting to note that a new α-gliadin cluster with four pseudogenes was detected on the short arm of chromosome 7E (109,202,365-109,312,140 bp). Both tandem duplication and segmental duplication are associated with gene production. The proportions of tandem duplication genes of α-, γ-, ω-gliadins and LMW-GSs in Th. elongatum were 73.7, 88.9, 68.4 and 50%, which supports the hypothesis that the major expansion of prolamin genes was through tandem duplication.
To better understand the evolutionary mechanism of γ-gliadin, ω-gliadin and LMW-GS gene subfamilies, an interval covering the first to the last prolamin gene on the short arm of the first homologous group was used for micro-synteny analysis among the four genomes (Fig. 3). The results showed that all these regions were located in collinear blocks with an inversion occurred between Glu-3 and Gli-1 loci on chromosome 1D of common wheat and one isolated ω-gliadin gene existed in Th. elongatum, A and B subgenomes of common wheat but lost in T. urartu, Ae. Tauschill and the D subgenome. Micro-synteny analysis of α-gliadins in the four studied species exhibited multiple duplicates in the loci expect for D genomes (Additional file 2). Although gene collinearity appears to be retained, four of six genes including the two paralogous HMW-GS genes were reversed in the homologous region of the T. urartu genome (Additional file 2) [31]. Changes in these gene loci between related species provided evidences for the dynamic evolution of the prolamin gene family.
All 66 α-gliadin genes were divided into 8 clades and genes from the same genome were generally clustered together (Fig. 4). It is interesting to note that 4 genes located on the 7E chromosome of Th. elongatum formed a separate clade (clade 8) with a high bootstrap value and had a closer genetic distance with the outgroup species, which indicates that these genes on chromosome 7E evolved earlier than those on chromosome 6E and formed as a result of different evolutionary trajectories in early stages. In HMW-GS gene subfamily, all 12 genes were well divided into 2 clades, representing y-type HMW-GSs (clade 1) and x-type HMW-GSs (clade 2) (Additional file 3) [14]. The topological structure of ytype HMW-GSs had some differences with x-type HMW-GSs, which might be related to the sequence variation of the C-terminal region of y-type HMW-GSs. The LMW-GS, γ-gliadin and ω-gliadin gene subfamilies showed similar phylogenetic relationship as α-gliadin
The epitope types in the D genome were the most abundant and all six types were identified. The E genome, like the B genome, also only contained one type epitope, DQ8-glia-α1/DQ8.5-glia-α1. 33-mer peptide was only detected in the D genome of common wheat, which was consisted with previous report [1]. DQ2.5-glia-α1b and DQ2.5-glia-α2 were also only found in the D genome and often existed in the form of multiple peptides, especially DQ2.5-glia-α2, and in contrast the other small peptides existed in the form of single peptides.

Expression pattern of prolamin genes in Th. Elongatum
The expression of prolamin genes in Th. elongatum were investigated with transcriptome data collected at six  Table  S6). Genes with predicted putative functions (Marked with asterisk) were tended to have a higher TPM value than pseudogenes, which indicted high accurate prediction of our study. Comparative analysis revealed that the expression pattern of prolamin genes in Th. elongatum were different in the same subfamily, which was one of the factors influencing grain prolamin content. For example, the expression of Tel_gamma_gliadin_1E_8 was about 5-6 times than that of Tel_gamma_gliadin_1E_2. Slight differences in gene expression were found in the α-gliadin, LMW-GS and HMW-GS gene subfamilies. In addition, different prolamin gene subfamilies had their own specific expression patterns. In α-gliadin and LMW-GS gene subfamilies, the expression level of these genes in the half-grain stage was higher than that in the grain stage, which indicated that expression of these genes increased at first and then decreased [27]. However, expression of γ-gliadin genes was increasing and we speculate that they will decline rapidly or slowly in the future due to reduced gene activity with the maturity of grains. In the HMW-GS gene subfamily, the expression of x-type HMW-GS genes was different from that of y-type. The expression of Tel_hmw_glutenin_1E_x in the half-grain stage was higher than that in the grain stage, while the expression of Tel_hmw_glutenin_1E_y increased slightly from the half-grain to the grain stage. In addition, 59 starch synthesis related genes were found in Th. elongatum by homologous sequences search of previous reported wheat starch metabolic related genes [34]. Expression of these genes at six grain development stages was investigated and profiled (Additional file 5). Of the 4 expressed aldolases (ALDs), obvious pattern was observed that these genes were expressed before ovary expansion, whose function were produce fructose-6-P through the gluconeogenic pathway and then convert to glucose-6-P by PGIs to maintain carbon flux toward starch formation [34]. AGPL1 and AGPS1 were expressed at the early stage of grain development, which was consisted with previous study [35]. GBSSI, a granule-bound starch synthase enzyme, expressed at the beginning of grain development, then reached the peak at the half grain stage, suggesting an important role in the seed development [36]. Nevertheless, a large number of starch synthesis-related genes were not expressed in Th. elongatum, which may give a explanation why its seeds is much smaller than common wheat. Overall, although some starch metabolic related genes follow similar express pattern as gluten genes in Th. elongatum, but most of them showed different patterns, how genes in protein and starch metabolic pathways are coordinated for the gliadins and glutenins accumulation need more research.

Electrophoretic maps and kneading quality performance of disomic substitution lines
In order to investigate the potential usage of Th. elongatum prolamin family in wheat high quality breeding, we have speculated the protein expression and processing quality of T. aestivum-Th. elongatum Fig. 3 Micro-synteny analysis of ω-, γ-gliadin and LMW-GS genes. The relationship of these families is indicated by blue, red, and orange lines, respectively disomic substitution lines. Considering the distribution pattern of prolamin genes on chromosomes, the substitution lines of DS1E(1D) and DS6E(6D) were mainly studied, which have been identified by genetic markers and cytological approaches (Additional file 6) [37]. The results of A-PAGE electrophoretic map showed that most α-gliadins disappeared in the substitution line DS6E(6D) (Fig. 6a), and the two visible bands for β-gliadins became weakened. This result is consistent with the fact that less α-gliadins were expressed in Th. elongatum (Fig. 5), indicating its potential application for quality breeding to reduce CD causal epitopes. Interestingly, we found one ωgliadin band in wheat became silent in this substitution line, which may due to gene silencing during the process of chromosome engineering (Fig.  6a). Even a α-gliadin gene cluster have uniquely evolved on the chromosome 7E (Fig. 2), we found no significant difference in the A-PAGE map of DS7E(7D) compared with the control CS (Additional file 7), which was consistent with the transcriptome results that no genes in this cluster were abundantly expressed (Fig. 5). HWM-GS accounts for 16% percent of the total wheat gluten protein, but the quality of wheat or gluten quality has a decisive role [38]. Consist with previous reports, CS contained the HMW-GSs of 7 + 8 subunits (from B subgenome), and 2 + 12 subunits (from D subgenome), named as Glu-B1 and Glu-D1, respectively (Fig. 6b). In the substitution line of DS1E(1D), 2 + 12 subunites of Glu-D1 loci were proposed to be substituted by the HWM-GS from E genome, both Ex and Ey subunits were detected in DS1E(1D) (Fig. 6b). Next, kneading parameters were speculated to check the kneading performance of DS1E(1D). The protein content of DS1E(1D) was 3% higher than that of CS ( Fig. 6c and  d). There was no significant difference in width at 8 min between CS and DS1E(1D), but the peak height of DS1E(1D) increased, indicating that the introduction of 1E chromosome enhanced the kneading resistance of dough ( Fig. 6c and d). This is consistent with previous result that the HMW-GSs of E genome may be novel genetic resource for wheat dough quality improvement [28], and the short segment translocation lines can be further created to verify its breeding value.

Discussion
Prolamin gene families play important roles in flour viscoelasticity, nutritional quality, and CD epitope content [14][15][16]39]. Currently, knowledge is still limited about prolamin genes in Th. elongatum, a well-known species for wheat distant breeding. In this study, we identified 19 α-gliadins, 9 γ-gliadins, 19 ω-gliadins, 2 HMW-GSs, and 5 LMW-GSs in the Th. elongatum genome. Comparison of prolamin genes among four Triticeae species revealed dynamic changes regarding to gene numbers and pseudogene rates, which indicated the complexity and variable of this gene family.
Detailed synteny analysis showed that although the order of ω-gliadin, γ-gliadin and LMW-GS genes on the first homologous group was maintained among the four studied species, local rearrangement and copy number variation were observed in this interval, revealing a dynamic change in this region (Fig. 3). Similar results were also found in α-gliadins and HMW-GS subfamilies. Further classification of duplication genes showed that each gene subfamilies was largely composed with tandem duplications (73.7% for α-gliadins, 88.9% for γ-gliadins, 68.4% for ω-gliadin and 50% for LMW-GSs). The tandem duplication should have driven fast evolution for these prolamin genes.
Phylogenetic analysis of prolamin genes among four studied species showed that genes from the same species were tended to be clustered together. Besides, some genes were not well clustered according to species, which may due to incomplete or large changes in the protein sequences, especially HMW-GS and ω-gliadin genes from common wheat, which all have a large proportion of gap regions (Additional file 1: Table S3). In addition, many of these genes have been lost or newly evolved after speciation, possibly due to unequal crossover or gene conversion (Fig. 4, Additional files 3 and 4) [40]. For instance, Th. elongatum has evolved 19 copies of ω-gliadin genes, while many copies in clade 2, clade 3 were apparently duplicated after diverging from the other species (Additional file 4). Similarly, many αgliadin genes in clade 1 to clade 4 are likely lost and resulting in fewer α-gliadins in Th. elongatum genome ( Fig. 4 and Fig. 6a). This fast evolution of prolamin family is accompanied with high rates of pseudogenes and large genetic diversity inner-or inter-species, and the richness of these seed-storage protein resources favor future wheat quality breeding.
According to previous reports of common wheat, the expression of LMW-GS genes began to express at the 5th day and reached the peak at the 10~14th day after anthesis, and then decreased with the maturity of seeds [27]. In Th. elongatum, the expression of three LMW-GS genes showed a consistent trend (Fig. 5). For the gliadin genes, their expression has genomic differences that genes from B and D subgenomes are early-expressed (highest level at 10 days after flowering), similar to the expression of LMW-GS genes, while those of the A genome are late-expressed genes (highest level at 20 days after flowering) [27]. The expression trend of α-gliadin genes is synchronous with that of LMW-GS genes in Th. elongatum, as early-expressed genes (Fig. 5). However, the expression of γ-gliadin genes increased from the half grain to the grain stage and reached its peak later than that of α-gliadin genes and LMW-GS genes, which demonstrated different roles of these prolamin genes in the grain development process.
Based on the putative functional α-gliadin sequences of four species, our results showed that CD peptides were genome-specific, which is consistent with the results of a previous study (Additional file 1: Table S5) [1]. Importantly, α-gliadins of the Th. elongatum genome contain only one type CD peptide, which is beneficial to low-CD breeding. Moreover, although α-gliadin genes were both found in the sixth homologous group of CS wheat and Th. elongatum, but the CD peptide of αgliadins in Th. elongatum genome is much fewer than that in D genome (Additional file 1: Table S5). The grain of DS6E(6D), which replaces the chromosome 6D genome with the chromosome 6E genome and has been verified through cytological study (Additional file 6), were speculated for protein content by A-Page, and the protein bands for α-gliadin genes did largely disappeared (Fig. 6a). This result indicates that the α-gliadins locus on D genome can be further translocated by short E genome fragment by distant hybridization, generating less toxic wheat varieties for CD patient population. Moreover, gene silencing may also favor such utility during engineering alien chromatin into wheat background [41]. In DS6E(6D), we did found one band of ω-gliadin was silenced (Fig. 6a).

Conclusions
Prolamins, the major protein component in cereal endosperm, play important roles for human diet. Here, we characterized and compared this gene family in Th. elongatum and three other wheat relatives. We explained the different expression pattern of prolamin gene subfamilies, found the dynamic changes of gene intervals, elaborated the evolutionary relationship among the Triticeae species used in this study. Our results showed that the HMW-GSs from Th. elongatum genome had highly potential for future improvement of wheat dough quality. Also, the Th. elongatum genome with low content of small CD peptides will be profitable genomic choice for the cultivation of wheat with low celiac disease.

Feature analysis
Putative functional genes were deduced according to whether the amino acid sequences of prolamins could produce proteins with complete structure. The pseudogene rate of the gene family was calculated with the number of pseudogenes divided by the total number of genes. Two genes were deleted because the sequences were not completed. All putative functional prolamin sequences were submitted to Expasy (https://web.expasy. org/compute_pi/) for calculation of molecular weight (MW) and theoretical isoelectric point (pI).

Distribution and duplication of prolamin genes
The distribution of prolamin genes on chromosomes was determined by the MapChart software [43]. Three roles were used to determine whether they were tandem repeat genes [44,45]: (a) The distance between two genes on the same chromosomal fragment was less than 100 kb; (b) the shorter aligned sequence covered > 70% of the longer sequence; (c) the similarity of aligned sequences was > 70%. If AB and BC are two pairs of tandem repeat genes (A, B, and C are three adjacent genes) but AC does not meet the tandem replication criteria, A and C are considered to be tandem repeat genes. The micro-synteny analysis was conducted using the JCVI software with appropriate parameters (cscore = 0.8) (https://github.com/tanghaibao/jcvi/wiki).

Phylogenetic analysis and classification of each prolamin subfamily
Phylogenetic trees were constructed using CDSs of the conservative domains derived from α-gliadin, γ-gliadin, LMW-GS, and HMW-GS genes. The evolutionary tree of ω-gliadins was constructed using gene conservative sequences. Multiple sequence alignments were first performed using the MUSCLE program with default parameters [46]. Phylogenetic trees were constructed using the MEGA X software with the maximum likelihood method [47]. Bootstrap was set as 1000 replicates. Based on model tests, the best models of these phylogenetic trees were set to be TN93 + G, K2 + G, K2 + I, and T92 + I, K2 + G respectively [48].

Transcriptome analysis
In order to analyze the expression patterns of prolamin genes in Th. elongatum, we sampled tissues at six stages with three biological replicates: young spike, preflowering, flowering, ovary expansion, half-grain, and grain stages. Total RNA was extracted for each sample using TRIzol® Reagent (Invitrogen) according to the manufacturer's instructions. A total of 18 samples were paired-end sequenced on the Illumina HiSeq 4000 platform. The quality of all samples was checked by fastqc (v0.11.9) and controlled by fastp (v0.20.0) with default parameters [49,50]. Hisat2 (v2.2.1) was used to map clean reads to the genome of Th. elongatum [51]. The transcripts per kilobase of exon model per million mapped reads (TPM) value for each gene was calculated by StringTie (v2.1.2) and the heatmap was constructed by TBtools (v1.047) [52,53].

Protein extraction and gel electrophoresis
The method for glutenins Single milled seed was suspended in 0.5 mL mixture extraction liquid and then incubated at 65°C for 2 h. The components of the extracted liquid mixture were as follows: 25 mL Tris-HCl (0.5 M, pH 6.8), 20 mL glycerol, 4 g sodium dodecyl sulfate (SDS), 30 g bromophenol blue, and 1 g dithiothreitol (DTT). The total volume of above extracted liquid mixture was then made to 100 mL with deionized water. The glutenin subunits were separated by a discontinuous sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) system. The 100 mL 15% separating gel included 25 mL deionized water, 50 mL 30% Acryl/Bis solution (29:1), 25 mL 4X separation gel buffer, 1 mL 10% (m/v) ammonium persulfate solution and 800 uL TEMED. The 80 mL 5% stacking gel included 45.6 ml deionized water, 13.6 mL 30% Acryl/Bis solution (29:1), 20 mL concentrated rubber buffer, 800 uL 10% (m/v) ammonium persulfate solution and 65 uL TEMED. Gels were run at a constant current (12 mA) for 20 h.

The method for gliadins
Single milled seed was suspended in 0.3 mL 70% ethanol and then incubated at 37°C for 2 h. Acid gel composition was as follows: 0.5 g ascorbic acid, 0.01 g FeSO 4 , 50 g acrylamide, and 2.5 g Bis-Tris. The total volume of each sample was then made to 500 mL with deionized water. The separating gel included 40 mL acid gel liquid, 0.3 mL formic acid, and 30 uL 0.6% H 2 O 2 . The stacking gel included 10 mL acid gel liquid and 0.1 uL 0.6% H 2 O 2 . Gels were run at a constant voltage in two stages (150 V for 30 min, then 350 V for 4 h).
Coomassie brilliant blue dye liquor included 1 g coomassie brilliant blue R-250, 100 mL glacial acetic acid, 250 mL isopropanol and 650 mL deionized water. The decolorizing solution included 50 mL anhydrous ethanol, 100 mL glacial acetic acid and 850 mL deionized water. Both gels were stained with a Coomassie brilliant blue dye liquor for 4 h and destained overnight.
Fish FISH was performed with the probes oligo-psc119.2-1 from Secale cereal and oligo-pta535-1 from T. aestivum. Hybridization solution was prepared according to the number of samples prepared. The two probes were mixed at a ratio of 1:1 before hybridization. The specific methods used were performed according to previous method [54]..

Dough rheological properties test
Mature grains were milled into flour using a mill for further testing. In this study, the protein and water contents of flour were determined by a DA7200 multifunction near infrared analyzer. By referring to the formula of the "AACC54-40A" method, the main mixing parameters, such as the mixing time, middle peak height, middle peak time, middle peak at 8 min, and width at 8 min, were determined with a 10 g mixograph.
Additional file 1: Table S1. Prolamin genes in Th. elongatum genome. Table S2. Error correction of prolamin gene annotations in T. aestivum, Ae. tauschii and T. urartu. Table S3. Coding sequences for the prolamin genes in the four studied species. Table S4. Molecular characteristics of prolamin genes. Table S5. The distribution of CD epitopes in the genomes of T. aestivum, T. urartu, Ae. tauchii and Th. elongatum. Table  S6. Transcriptome data of Th. elongatum at different development stages. Additional file 4: Phylogenetic tree of ω-gliadin gene family. FJ600500 was set as outgroup.
Additional file 5: Expression profile of 59 starch synthesis related genes in Th. elongatum. Genes were named according to names in common wheat.
Additional file 6: The FISH pattern of DS1E(1D) and DS6E(6D). Chromosomes 1E and 6E from Th. elongatum were indicated by arrows respectively. Additional file 7: A-PAGE patterns of gliadin in CS and its derived substitution lines.