Characterization of an Integrated Active Glu-1Ay Allele in Common Wheat from Wild Emmer and Its Potential Role in Flour Improvement

Glu-1Ay, one of six genes encoding a high molecular weight glutenin subunit (HMW-GS), is frequently silenced in hexaploid common wheat. Here, an active allele of Glu-1Ay was integrated from wild emmer wheat (Triticum turgidum ssp. dicoccoides) accession D97 into the common wheat (Triticum aestivum) cultivar Chuannong 16 via the repeated self-fertilization of the pentaploid interspecific hybrid, culminating in the selection of a line TaAy7-40 shown to express the wild emmer Glu-1Ay allele. The open reading frame of this allele was a 1830 bp long sequence, demonstrated by its heterologous expression in Escherichia coli to encode a 608-residue polypeptide. Its nucleotide sequence was 99.2% identical to that of the sequence within the wild emmer parent. The TaAy7-40 introgression line containing the active Glu-1Ay allele showed higher protein content, higher sodium dodecyl sulfate (SDS) sedimentation value, higher content of wet gluten in the flour, higher grain weight, and bigger grain size than Chuannong 16. The end-use quality parameters of the TaAy7-40 were superior to those of the medium gluten common wheat cultivars Mianmai 37 and Neimai 9. Thus, the active Glu-1Ay allele might be of potential value in breeding programs designed to improve wheat flour quality.


Introduction
High molecular weight glutenin subunits (HMW-GSs) account for 10% of the total protein deposited in the wheat endosperm and have a major influence over dough quality as a result of their ability to form the aggregate referred to as gluten [1][2][3][4]. The genes encoding the HMW-GS are located at three loci, Glu-1A, Glu-1B, and Glu-1D on the long arms of the homologous chromosomes 1A, 1B, and 1D, respectively [4,5]. It has been confirmed that the two tightly linked genes, Glu1-1 and Glu1-2, at each Glu locus, encode the x-and y-type subunits, respectively. The x-type subunit typically has a higher molecular weight than that of the y-type subunit [6,7]. In spite of the presence of six Glu-1 genes in common wheat (Triticum aestivum, AABBDD, 2n = 6x = 42) (1Ax, 1Dx, 1Bx, 1By, 1Dy, and 1Ay), the number of HMW-GS expressed varies from three to five, because the 1Ax and 1By subunits are not usually expressed and 1Ay is always silenced [8][9][10][11] except in two Swedish bread wheat lines [12,13]. The silencing of 1Ay occurs due to either the presence of a premature stop codon [9] or due to the insertion of a transposon-like element within its coding region [14].

Phenotype and Karyotype Characteristics
The TaAy7-40 resembled CN16 with respect to plant height, spike, and spikelet number, but all were significantly different from those of wild emmer D97 ( Figure 1A, Table 1). Interestingly, the TaAy7-40 had an earlier flowering time than both its parents ( Table 1). The grain traits, including kernel length, kernel width, kernel thickness, 1000-kernel weight, and grain weight per plant, showed significant differences between TaAy7-40 and D97, while slight differences occurred between TaAy7-40 and CN16 ( Figure 1B, Table 2). Cytological observations confirmed that the chromosome number of TaAy7-40 in root-tip cells was 2n = 42 ( Figure 1C). Therefore, our results demonstrated that the introgression line TaAy7-40 reached the genetic background of common wheat (AABBDD).  Note: The data are presented as the mean ± SD. The capital letters A and B indicate the significant differences at 0.01 level with Turkey's two-way test between each of two comparison samples. * Sample size, n = 30 per replicate, and three biological replicates per variety. Note: The data are presented as the mean ± SD. The capital letters A, B, and C indicate the significant differences at 0.01 level with Turkey's two-way test between each of two comparison samples. * Sample size n = 30; § Sample size n = 300.

SDS-PAGE Analysis of HMW-GSs
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the female CN16 had five HMW-GSs, including 1Ax1 at the Glu-A1 locus, 1Bx20 + 1By20 at the Glu-B1 locus, and 1Dx5 + 1Dy10 at the Glu-D1 locus. 1Ay was not detected in CN16. The male D97 had three HMW-GSs, including 1Ax2.2 [36] + 1Ay at the Glu-A1 locus and 1By8.1 at the Glu-B1 locus. However, the resulting introgression line TaAy7-40 possessed six HMW-GSs, including 1Ax1 and 1Ay at the Glu-A1 locus, 1Bx20 and 1By8.1 at the Glu-B1 locus, and 1Dx5 and 1Dy10 at the Glu-D1 locus, compared with the HMW-GSs composition of D97, XY6, and CN16 ( Figure 2A). Further analysis by eight randomly sampled grains confirmed that the six HMW-GSs were highly stable in TaAy7-40 ( Figure 2B) and that its 1Ay had electrophoretic mobility similar to the fast subunit 1Ay in the D97 profile (Figure 2A,B).

Molecular Characterization of 1Ay Genes
The amplicons consisting of several products were generated from a genomic DNA template of TaAy7-40, CN16, and D97 using the primer pair PF1/PR1 ( Figure 3A). Previous studies have demonstrated that the most active Glu-1Ay genes show approximately 1800 bp in length [19,20]. Therefore, in the current study, the PCR bands with such specific fragment sizes were first selected for further cloning and three open reading frames (ORFs) of HMW-GSs sequences were obtained. The 1Ay ORFs of TaAy7-40 and D97 were each of length 1830 bp and encode for 608 amino acid residues. The 1Ay ORFs of CN16 were shorter (1791 bp) and a premature stop codon at 1000 to 1002 bp was identified ( Figure 4). The authenticity of the 1Ay alleles isolated from TaAy7-40 and D97 was further confirmed by successful expression in Escherichia coli ( Figure 3B). The sequences of the three 1Ay alleles were deposited in GenBank with the accession number KC545952 for TaAy7-40, KC545955 for CN16, and KC545956 for D97.

Molecular Characterization of 1Ay Genes
The amplicons consisting of several products were generated from a genomic DNA template of TaAy7-40, CN16, and D97 using the primer pair PF1/PR1 ( Figure 3A). Previous studies have demonstrated that the most active Glu-1Ay genes show approximately 1800 bp in length [19,20]. Therefore, in the current study, the PCR bands with such specific fragment sizes were first selected for further cloning and three open reading frames (ORFs) of HMW-GSs sequences were obtained. The 1Ay ORFs of TaAy7-40 and D97 were each of length 1830 bp and encode for 608 amino acid residues. The 1Ay ORFs of CN16 were shorter (1791 bp) and a premature stop codon at 1000 to 1002 bp was identified ( Figure 4). The authenticity of the 1Ay alleles isolated from TaAy7-40 and D97 was further confirmed by successful expression in Escherichia coli ( Figure 3B). The sequences of the three 1Ay alleles were deposited in GenBank with the accession number KC545952 for TaAy7-40, KC545955 for CN16, and KC545956 for D97.

Molecular Characterization of 1Ay Genes
The amplicons consisting of several products were generated from a genomic DNA template of TaAy7-40, CN16, and D97 using the primer pair PF1/PR1 ( Figure 3A). Previous studies have demonstrated that the most active Glu-1Ay genes show approximately 1800 bp in length [19,20]. Therefore, in the current study, the PCR bands with such specific fragment sizes were first selected for further cloning and three open reading frames (ORFs) of HMW-GSs sequences were obtained. The 1Ay ORFs of TaAy7-40 and D97 were each of length 1830 bp and encode for 608 amino acid residues. The 1Ay ORFs of CN16 were shorter (1791 bp) and a premature stop codon at 1000 to 1002 bp was identified ( Figure 4). The authenticity of the 1Ay alleles isolated from TaAy7-40 and D97 was further confirmed by successful expression in Escherichia coli ( Figure 3B). The sequences of the three 1Ay alleles were deposited in GenBank with the accession number KC545952 for TaAy7-40, KC545955 for CN16, and KC545956 for D97.  IPTG, the protein from E. coli cultured with isopropyl β-∆-thiogalactopyranoside (IPTG) using the general extraction method. IPTG+AS, the IPTG induced 1Ay protein extracted using acetone sedimentation (AS). CK indicates the protein from E. coli cultured without IPTG using the general traction method. Arrowheads point to the expressed 1Ay subunit protein from E. coli culture; triangles point to the 1Ay protein extracted from endosperms of D97 and TaAy7-40 (F 9 to F 11 progenies).

Sequences Comparison of 1Ay Alleles
In the current study, the sequences isolated from TaAy7-40 (KC545952) and D97 (KC545956) shared a higher level of similarity (from 83.9 to 99.5%) to published 1Ay ORFs than to either 1By or 1Dy ORFs (from 60.7 to 70.7%); their sequences were also quite distinct from any of the genes encoding x-type HMW-GSs, sharing a level of similarity uniformly below 47.0%. The CN16 amplicon sequence (KC545955) was similar (from 77.8 to 98.5%) to that of the 1Ay ORFs (Table S1). The 1Ay of D97 had a similarity of 99.2% to the TaAy7-40, whereas both of them shared a relatively lower level of similarity (96.2%, 96.2%) to the 1Ay sequence of CN16 (Table S1).
Comparison of the three cloned 1Ay ORFs revealed that a total of three insertions and deletions (InDels) at position 805-822, 1198-1200, and 1468-1485 were detected ( Figure 4). Simultaneously, 38 single nucleotide polymorphisms (SNPs), including 32 transitions and six transversions, were identified. In total, 14 SNPs, including 13 transitions and one transversion, were found between D97and TaAy7-40. In contrast, 30 SNPs, including 25 transitions and five transversions, were identified between CN16 and TaAy7-40. extraction method. IPTG+AS, the IPTG induced 1Ay protein extracted using acetone sedimentation (AS). CK indicates the protein from E. coli cultured without IPTG using the general traction method. Arrowheads point to the expressed 1Ay subunit protein from E. coli culture; triangles point to the 1Ay protein extracted from endosperms of D97 and TaAy7-40 (F9 to F11 progenies).

Sequences Comparison of 1Ay Alleles
In the current study, the sequences isolated from TaAy7-40 (KC545952) and D97 (KC545956) shared a higher level of similarity (from 83.9 to 99.5%) to published 1Ay ORFs than to either 1By or 1Dy ORFs (from 60.7 to 70.7%); their sequences were also quite distinct from any of the genes encoding x-type HMW-GSs, sharing a level of similarity uniformly below 47.0%. The CN16 amplicon sequence (KC545955) was similar (from 77.8 to 98.5%) to that of the 1Ay ORFs (Table S1). The 1Ay of D97 had a similarity of 99.2% to the TaAy7-40, whereas both of them shared a relatively lower level of similarity (96.2%, 96.2%) to the 1Ay sequence of CN16 (Table S1).
Comparison of the three cloned 1Ay ORFs revealed that a total of three insertions and deletions (InDels) at position 805-822, 1198-1200, and 1468-1485 were detected ( Figure 4). Simultaneously, 38 single nucleotide polymorphisms (SNPs), including 32 transitions and six transversions, were identified. In total, 14 SNPs, including 13 transitions and one transversion, were found between D97and TaAy7-40. In contrast, 30 SNPs, including 25 transitions and five transversions, were identified between CN16 and TaAy7-40. A similar comparison was made for the predicted polypeptide sequences. All of the three amino acid sequences shared the typical characteristic structure of HMW-GS ( Figure 5), which contains a signal peptide, N-terminal, C-terminal, and central repetitive region [2]. Three deletion polymorphisms at position 269-274, 400, and 490-495 were detected in CN16, five single amino acid polymorphisms (SAPs) were identified between TaAy7-40 and D97, and 17 SAPs were found between TaAy7-40 and CN16 ( Figure 5). TaAy7-40 and D97 possessed complete 1Ay polypeptide structures, while CN16 encoded a truncated polypeptide as a result of the premature termination codon TAG at position 1000 to 1002 of 1Ay ORFs. A similar comparison was made for the predicted polypeptide sequences. All of the three amino acid sequences shared the typical characteristic structure of HMW-GS ( Figure 5), which contains a signal peptide, N-terminal, C-terminal, and central repetitive region [2]. Three deletion polymorphisms at position 269-274, 400, and 490-495 were detected in CN16, five single amino acid polymorphisms (SAPs) were identified between TaAy7-40 and D97, and 17 SAPs were found between TaAy7-40 and CN16 ( Figure 5). TaAy7-40 and D97 possessed complete 1Ay polypeptide structures, while CN16 encoded a truncated polypeptide as a result of the premature termination codon TAG at position 1000 to 1002 of 1Ay ORFs.

Phylogenetic Analysis
A neighbor-joining (NJ) phylogenetic tree was constructed based on the multiple alignment of the full-length sequence of the three cloned 1Ay alleles, with 29 1Ay alleles and 13 other HMW-GS genes that have been previously reported ( Figure 6). The result showed that all of the 1Ay alleles were clustered together and separated from the clades of 1By and 1Dy as well as all of the x-type genes, including Glu-1A, Glu-1B, and Glu-1D loci ( Figure 6). In the subclade of 1Ay alleles, the TaAy7-40 1Ay (KC545952) and D97 1Ay (KC545956) were clustered in a sole clade and clustered closely together with a previously reported active 1Ay allele in wild emmer (JF519636). In contrast, both of the 1Ay alleles were estranged from the 1Ay of CN16 (KC545955).

Phylogenetic Analysis
A neighbor-joining (NJ) phylogenetic tree was constructed based on the multiple alignment of the full-length sequence of the three cloned 1Ay alleles, with 29 1Ay alleles and 13 other HMW-GS genes that have been previously reported ( Figure 6). The result showed that all of the 1Ay alleles were clustered together and separated from the clades of 1By and 1Dy as well as all of the x-type genes, including Glu-1A, Glu-1B, and Glu-1D loci ( Figure 6). In the subclade of 1Ay alleles, the TaAy7-40 1Ay (KC545952) and D97 1Ay (KC545956) were clustered in a sole clade and clustered closely together with a previously reported active 1Ay allele in wild emmer (JF519636). In contrast, both of the 1Ay alleles were estranged from the 1Ay of CN16 (KC545955).  (1Ay, 1By, 1Bx, 1Dx, 1Dy, 1Ax). The phylogenetic tree was created based on a multiple alignment of DNA sequences. The three new 1Ay genes are marked by boxes and the 14 1Ay active genes are marked in bold. Figure 6. Phylogenetic relationships between the three new Glu-1Ay alleles with previously published HMW-GS genes in wheat (1Ay, 1By, 1Bx, 1Dx, 1Dy, 1Ax). The phylogenetic tree was created based on a multiple alignment of DNA sequences. The three new 1Ay genes are marked by boxes and the 14 1Ay active genes are marked in bold.

Processing Quality Parameters (PQPs)
Flours milled from TaAy7-40, CN16, Mianmai37, and Neimai9 were compared with respect to their three PQPs: grain protein content, SDS sedimentation value, and wet gluten content. All PQPs scored were significantly higher (p < 0.05) for the TaAy7-40 flour than for those of CN16 at two environments except grain protein content at the Chongzhou location (not significant) (Figure 7). Moreover, the grain protein content, SDS sedimentation value, and wet gluten content of TaAy7-40 were also higher than those of the flour milled from the two medium gluten cultivars Mianmai37 and Neimai9. These results indicated that introgression line TaAy7-40 with the traits integrated from wild emmer D97 had better flour processing properties than CN16 and even exceeded the levels of medium gluten cultivars. Flours milled from TaAy7-40, CN16, Mianmai37, and Neimai9 were compared with respect to their three PQPs: grain protein content, SDS sedimentation value, and wet gluten content. All PQPs scored were significantly higher (p < 0.05) for the TaAy7-40 flour than for those of CN16 at two environments except grain protein content at the Chongzhou location (not significant) (Figure 7). Moreover, the grain protein content, SDS sedimentation value, and wet gluten content of TaAy7-40 were also higher than those of the flour milled from the two medium gluten cultivars Mianmai37 and Neimai9. These results indicated that introgression line TaAy7-40 with the traits integrated from wild emmer D97 had better flour processing properties than CN16 and even exceeded the levels of medium gluten cultivars.

Discussion
The HMW-GS 1Ay is rarely expressed in common wheat. The presence of expressed 1Ay subunits in related Triticum species makes it possible to investigate their molecular information and to apply such genes for wheat flour quality improvement. In the present study, we characterized an active 1Ay gene from wild emmer wheat D97 and transferred this gene into the low-gluten common wheat CN16 via interspecies hybridization. We found that the wild emmer 1Ay gene can be stably expressed in the common wheat background, and that introgression line TaAy7-40 with six HMW-GSs, including 1Ax1, 1Ay, 1Bx20, 1By8.1, 1Dx5, and 1Dy10, had agronomic properties of common wheat as well as improved processing quality in relation to female CN16.

Identification of Two Novel Active 1Ay Alleles
In the present study, we isolated three 1Ay sequences from D97 (KC545956), TaAy7-40 (KC545952), and CN16 (KC545955). The KC545955 of CN16 was silenced due to the presence of a premature stop codon in the 1Ay ORFs (Figures 2 and 5). The authenticity of two functional 1Ay alleles from wild emmer D97 (KC545956) and the hexaploid introgression line TaAy7-40 (KC545952) was further confirmed by heterogeneous expression in E. coli ( Figure 3B). The ORFs of KC545956 and KC545952 had identical length (1830 bp) to the majority of 1Ay sequences from diploid (T. urartu FJ404595, EU984503, AM183223, and AY245578) and tetraploid (T. dococcoides JF519636, T. dicoccum EU984511, and T. timopheevii AJ306977) wheat (Table S1). Their deduced proteins shared the typical characteristic structure of HMW-GS, which contains a signal peptide, N-terminal, C-terminal, and central repetitive region ( Figure 5) [2]. They were different from the previously published active 1Ay alleles due to some nucleotide substitutions ( Figure S1) and were clustered in a distinct clade from other 1Ay genes ( Figure 6). Therefore, our results demonstrated that the KC545952 and KC545956 sequences identified in our research were two novel 1Ay alleles.

Discussion
The HMW-GS 1Ay is rarely expressed in common wheat. The presence of expressed 1Ay subunits in related Triticum species makes it possible to investigate their molecular information and to apply such genes for wheat flour quality improvement. In the present study, we characterized an active 1Ay gene from wild emmer wheat D97 and transferred this gene into the low-gluten common wheat CN16 via interspecies hybridization. We found that the wild emmer 1Ay gene can be stably expressed in the common wheat background, and that introgression line TaAy7-40 with six HMW-GSs, including 1Ax1, 1Ay, 1Bx20, 1By8.1, 1Dx5, and 1Dy10, had agronomic properties of common wheat as well as improved processing quality in relation to female CN16.

Identification of Two Novel Active 1Ay Alleles
In the present study, we isolated three 1Ay sequences from D97 (KC545956), TaAy7-40 (KC545952), and CN16 (KC545955). The KC545955 of CN16 was silenced due to the presence of a premature stop codon in the 1Ay ORFs (Figures 2 and 5). The authenticity of two functional 1Ay alleles from wild emmer D97 (KC545956) and the hexaploid introgression line TaAy7-40 (KC545952) was further confirmed by heterogeneous expression in E. coli ( Figure 3B). The ORFs of KC545956 and KC545952 had identical length (1830 bp) to the majority of 1Ay sequences from diploid (T. urartu FJ404595, EU984503, AM183223, and AY245578) and tetraploid (T. dococcoides JF519636, T. dicoccum EU984511, and T. timopheevii AJ306977) wheat (Table S1). Their deduced proteins shared the typical characteristic structure of HMW-GS, which contains a signal peptide, N-terminal, C-terminal, and central repetitive region ( Figure 5) [2]. They were different from the previously published active 1Ay alleles due to some nucleotide substitutions ( Figure S1) and were clustered in a distinct clade from other 1Ay genes ( Figure 6). Therefore, our results demonstrated that the KC545952 and KC545956 sequences identified in our research were two novel 1Ay alleles.

The 1Ay Sequence Variations
In the current study, the sequence and phylogenetic analyses indicated that the 1Ay allele of TaAy7-40 (KC545952) shared a high similarity (99.2%) to that of D97 (KC545956). Fourteen SNPs were identified in 1Ay genes between the TaAy7-40 and D97. A high-fidelity polymerase was used to avoid potential mistakes introduced by PCR amplification, and 1Ay nucleotide sequences were determined by using sequencing results of multiple single clones. Thus, these sequence variations of 1Ay alleles observed between TaAy7-40 and D97 are probably not due to errors from the PCR amplification and sequencing.
Wide hybridization has served as an important tool to combine distinct genomes together in the same nucleus [37]. However, as McClintock [38] speculated, this method may create a major "genome shock" that leads to significant genomic restructuring in the hybrid. It has been demonstrated that wide hybridization can produce rich variations in different cross-parents in addition to the parental type [39]. For instance, Yuan et al. [40] reported that wide hybridization of common wheat with rye induces novel DNA variation. Jiang et al. [36] identified four SNPs at the 1Ax2.2 allele between wild emmer and its wide hybrid. It has been demonstrated that repeat sequences are easily changed due to wide hybridization [41] and the large number of similar repetitive sequences may lead to gene duplication and structural chromosomal changes which promote the rapid evolution of genomes [42]. Therefore, the repetitive domain of HMW-GS could be easily affected by the wide hybridization that induced nucleotide variations in the 1Ay alleles. In addition, the cross-parent genome is asymmetric because strictly speaking, the female common wheat cv. CN16 should have the genome of A 2 A 2 B 2 B 2 DD while wild emmer the genome of A 1 A 1 B 1 B 1 after a very long evolution, domestication, and genetic improvement history. This genomic asymmetry might drive the genetic variation between 1Ay alleles of D97 and TaAy7-40, which can lead to purposeful and useful genetic changes [36,43].

A Wild Emmer 1Ay Allele Was Successfully Transferred and Stably Expressed inCommon Wheat
In the comparison between the Glu-B1 and Glu-D1 loci, the active alleles at the Glu-A1 locus in common wheat are of a much lower number because of the silenced versions of 1Ay [1, 11,[44][45][46][47]. The transfer of Glu-D1 and Glu-A1 loci was successfully carried out in a few previous reports [48,49], and the resulting isohomoeoallelic lines were shown to significantly improve dough and mixing characteristics [50]. Although several active alleles of 1Ay have been isolated and characterized from wild relatives of wheat, the utilization of these genes for wheat quality improvement has been less-studied. The active 1Ay subunit in two Swedish bread wheat lines had superior quality to subunit 1 or 2* and even higher correlation with a specific Zeleny volume than with the subunits 5+10 [12,13]. Rogers et al. [22] demonstrated that the introgression of 1Ax and 1Ay from T. boeoticum to the hexaploid common wheat increases gluten strength. However, the grain yield trait of the hybrid hexaploid wheat was not significantly improved. The introgression of the wild emmer 1Ay locus into durum wheat had a positive effect on enhancing gluten properties [35]. A recent study demonstrated that the expression of an active 1Ay gene in two Australian wheat cultivars showed a positive effect on wheat quality [51]. To our best knowledge, there are no reports on the introgression of the wild emmer 1Ay locus into common wheat for the purpose of dough quality improvement.
Wild emmer wheat harbors an abundant HMW-GS composition [19]. Since the Glu-1Ay gene is usually silenced in common wheat, the utilization of alien active 1Ay alleles from wild emmer might be an effective strategy for improving the flour quality of common wheat. However, the genetic improvement of wheat through chromosome engineering done by wide hybridization is more complex than that done via conventional crosses between cultivars of the same species [41]. Introgression of chromatin to wheat from related species often results in deleterious effects on some agronomic traits [52]. It is sometimes possible to recover the original yield by subsequent genetic manipulation, such as backcrossing of lines with alien Glu alleles [53,54]. In our previous study, we revealed that the wild emmer HMW-GSs were detected in F 1 and F 2 offspring of the interspecies hybridization between common wheat and wild emmer [55]. In the current study, our strategy was to continuously select those single plants with desirable agronomic, high yield, and resistance properties for self-crossing. We identified the novel active 1Ay allele in an advanced generation hybrid. Introgression line TaAy7-40 turned out to be superior to its progenitor lines D97 and CN16 in most of the tested morphological and grain traits, implying that many other genes from the D97 are present in introgression line TaAy7-40.
We found that the 1Ay subunit with similar electrophoresis mobility to that of the parent wild emmer D97 was detected in introgression line TaAy7-40 after eight times' self-crossing (Figure 2). Further sequence and phylogenetic analysis revealed that the 1Ay of TaAy7-40 was highly similar to that of D97 ( Figure 6, Table S1). Therefore, our results demonstrated that the active 1Ay gene from wild emmer could be successfully integrated into common wheat and be stably expressed on a common wheat background. It is believable that wild emmer wheat is beneficial to enriching the genetic bases at the Glu-A1 locus.

The Wild Emmer 1Ay Might be Partly Associated with Better Flour Quality in Common Wheat
Wheat flour quality is mainly determined by the number and composition of HWM-GSs [3]. That an increasing number of HMW-GS in common wheat might give rise to improvements in dough properties has been confirmed by several studies. For example, Rogers et al. [22] showed that the flour quality of wheat expressing the Glu-A1 subunits 39+40 is superior to that of wheat expressing only the Glu-1Ax1 subunit. Recently, Jiang et al. [41] indicated that the novel HMW-GS 1Ax1.2 or 1Ax2.2 at the Glu-A1 locus from wild emmer might be responsible for the better dough quality in common wheat. In the current study, the authenticity of the 1Ay gene from wild emmer was confirmed by heterogeneous expression ( Figure 3B) and it was stably expressed in common wheat introgression line TaAy7-40. We demonstrated that TaAy7-40 was significantly superior to its female parent CN16 for not only the SDS sedimentation value and wet gluten content in Chongzhou but also for the three PQPs in Wenjiang (Figure 7) even though they had the same positive effect HMW-GS combination 1Dx5 + 1Dy10 [56]. These differences might to a certain extent have resulted from the various HWW-GSs at the Glu-1A locus besides those at the Glu-1B locus, because TaAy7-40 had the added novel subunit 1Ay besides the newly substituted subunit 1By8.1 compared with CN16. Previous studies have demonstrated that the expression of active 1Ay genes has the potential to enhance gluten properties in wheat [12,13,22,35]. Further analyses indicated that TaAy7-40 significantly exceeded the levels of medium gluten cultivars MM37 and N9 in the three PQPs. These results suggested that introgression of the HMW-GSs from wild emmer to common wheat could increase the wheat gluten property in relation to the common wheat acceptor line. That in turn can be co-related to some extent with the active 1Ay, which is similar to the result by Roy et al. [51] who discovered the expressed 1Ay in Australian wheat cultivars indicates a positive effect on wheat quality.
All of the materials for testing processing-quality, including TaAy7-40, CN16, MM37, and N9, were arranged in the field using a randomized complete block design with three replicates at the Wenjiang (30 • 43 9" N 103 • 52 17" E) and Chongzhou (30 • 33 34" N 103 • 38 35" E) experimental stations. Individual plants were spaced 10 cm apart within a 2m row, with 30 cm between rows. Each replicate was planted in three rows.

Characterization of Phenotype and Karyotype
In total, 10 morphological traits were studied as listed in Tables 1 and 2. The plant height, the spike number, and the spikelet number traits were measured in the first culm and spike of each plant. The heading date was recorded at which 50% of the heads had fully emerged from the flag leaf sheath. The flowering time was recorded when anthers were visible on 50% of the plants. The kernel length, width, and thickness were recorded for 30 random kernels from each line, and the average values were used for further statistical analysis. The weight of 300 randomly sampled seeds was recorded with an electronic balance to represent the 1000-kernel weight.
Observations of chromosome number in root-tip cells were performed as described by Zhang et al. [59]. At least 30 root-tip cells were counted for each line. The parent lines D97 and CN16 served as controls.

SDS-PAGE Analysis
The HMW-GSs were extracted from mature grain according to two methods. The first method is the general method for whole protein extraction as described by Wan et al. [60]. The second method is the selective precipitation of the HMW gluten reported by Hu et al. [17]. The HMW-GSs were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to Hu et al. [17]. To ensure the experimental accuracy and to investigate a HMW glutenin subunit's stability, at least eight randomly sampled seeds of introgression line TaAy7-40 were analyzed.

DNA Preparation and PCR Amplification
Genomic DNA was extracted from etiolated leaves according to the CTAB method [61] with minor modifications and used as a template to amplify the set of full length 1Ay genes by priming the reactions via the degenerate pair PF1: 5'-ATGGCTAAGCGGC/TTA/GGTCCTCTTTG-3', and PR1: 5'-CTATCACTGGCTAA/GGCCGACAATGCG-3'. Since the target sequences are known to have a high GC content, a high-fidelity ExTaq polymerase (Takara, Dalian, China) was used for PCR amplifications. The PCR amplifications were performed in a reaction volume of 50 µL containing 5 µL of 10 × ExPCR buffer, 0.2 mmol L −1 of dNTPs, 1 µmol L −1 of each primer, 2.5 U of ExTaq polymerase, and ddH 2 O to 50 µL. PCR was carried out using a Mastercycler pro PCR (Eppendorf, Hamburg, Germany). The cycling program consisted of 94 • C for 5 min, followed by 28 cycles of 94 • C for 40 s, 68 • C for 6 min, and a final extension at 72 • C for 10 min [19]. The amplified PCR products were visualized by 1.0% agarose gel electrophoresis followed by staining with ethidium bromide. The PCR amplification was performed in triplicate for each sample.

Cloning, Sequencing, and Prokaryotic Expression of the ORFs of 1Ay
The amplified products with expected size were recovered, purified, and further ligated into pMD19-T vector (TaKaRa, Dalian, China), and the ligation mixtures were transformed into Escherichia coli DH10B-competent cells. Three to four sub-clones of these fragments were selected using the nested deletion method [62]. The sequencing was performed by the BGI Company (Shanghai, China) and TaKaRa Biotechnology (TaKaRa). The final nucleotide sequence for each 1Ay ORF was determined from the sequencing results of three independent clones.
The cloned 1Ay sequence was re-amplified using the primer pair 6-Ay-F1 (5'-ACCCATATGGAAGGTGAGACCTCTAAGC-3') and 6-Ay-R1 (5'-TTCCTCGAGCTATCACTGG CTAGCCGAC-3') to remove the signal peptide and add Nde I and Xho I restriction sites. The resulting fragment was inserted into an expression vector pET-30a (Invitrogen, Waltham, USA) and transformed into E. coli BL21 (DE3) plysS cells. The recombinant cells were grown at 37 • C on 2×YT medium containing 25 µg/mL kanamycin and 34 µg/mL chloromycetin until the OD 600 reached 0.6. The expression of 1Ay in E. coli was induced by the addition of 1mM IPTG for 4-6 h, and the protein output of the cells was extracted as described by Wan et al. [60]. The electrophoretic mobility of the protein expressed in the recombinant cells was compared with that of its native form present in D97 and TaAy7-40 (progenies F 9 to F 11 ).

Sequence Alignmentand Phylogenetic Analysis
The ORFs of HMW-GSs were obtained using the ORF Finder program (www.ncbi.nlm.nih.gov/ orffinder). Multiple sequence alignment at both the nucleotide and peptide levels was facilitated by the Clustal X 2.0 software (The Conway Institute, Dublin, Ireland) with manual adjustments where necessary. Sequence alignment of TaAy7-40, D97, and CN16 was performed using MEGA 5.0 [63], and the equivalent analysis at the polypeptide level was done by the GeneDoc2.6 software (genedoc.software.informer.com). The phylogenetic tree was constructed using the neighbor-joining (NJ) algorithm in the MEGA 5.0 software. The bootstrap values in the phylogenetic tree were estimated based on 1000 replications.

Flour Processing Quality Determination
Mature seeds were harvested for determination of the flour processing quality. Seeds of TaAy7-40, CN16, MM37, and N9 were conditioned to 14% moisture [64] and milled using the Chopin CD1 AUTO (Renault, Boulogne-Billancourt, France) [65]. Grain protein content was determined using an Infratec 1241 Grain Analyzer (FOSS A/S, Hillerød, Denmark) following the methods given by Pettersson and Eckersten [66]. SDS sedimentation values were obtained using the procedure as described by Axford et al. [67]. Wet gluten content was determined according to American Association of Cereal Chemists (AACC) method 38-12A [68], and the wet gluten content value in each genotype was recorded at a 14% moisture basis. All of the experiments were repeated three times.

Statistical Analysis
All of the agronomical characters and quality parameters were subjected to analysis of variance, and differences among genotypes were tested using Tukey's two-way test, which were performed using SPSS version 19.0 software (IBM, Armonk, NY, USA).

Conclusions
An active Glu-1Ay allele derived from wild emmer D97 has been successfully integrated and stably expressed in common wheat CN16 through repeated self-fertilization of the pentaploid interspecific hybrid. The hybrid line TaAy7-40 with active Glu-1Ay had agronomic properties of common wheat. It was better than CN16 and the medium gluten common wheat cultivars MM37 and N9 in wheat flour quality. Our findings demonstrated that the expression of active Glu-1Ay allele in common wheat may have a positive effect on wheat quality, flour quality, and the potential of wild emmer wheat to be a valuable source for enriching the genetic bases at the Glu-A1 locus.