*,Kunpu ZhngLingli Dong,Zhenying Dong,Yiwen Li,Arr Hussin,c,Huijie Zhi

aThe State Key Laboratory of Plant Cell and Chromosome Engineering,Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,Beijing 100101,China

bCollege of Agronomy,State Key Laboratory of Wheat and Maize Crop Science,Henan Agricultural University,Zhengzhou 450002,Henan,China

cDepartment of Biosciences,COMSATS Institute of Information Technology,Sahiwal Campus,Pakistan

1.Introduction

Wheat grain quality (WGQ) is largely determined bymilling yield,end-use,flour color and nutritional properties.Genetically,WGQ is mainly the outcome of independent and interactive actions of a number of traits,including(but not limited to)grain hardness,gluten protein quality,flour color,starch quality,and contents of health-promoting substances.These traits are all controlled by polygenes and affected by agro-environmental factors.Consequently,WGQ varies from cultivar to cultivar,year to year and across ecological environments.Nevertheless,genetic factors are the main determinants[1].A better understanding of the genetic,genomic and molecular bases of WGQ traits will enable more effective improvement of WGQ.

Although basic investigations of certain WGQ traits(e.g.,gluten protein quality)have a long history,molecular genetic studies of these traits started in the 1990s when recombinant DNA technologies gained applications in plant biology and genetic research.Genomic research of WGQ traits began much later compared to similar studies in model plants such as Arabidopsis thaliana and rice.This is because the most widely cultivated crop,common wheat(Triticum aestivum,also called bread wheat),has a large and complex hexaploid genome(AABBDD,2n=6x=42,～17 G)[2,3].A draft reference genome sequence of common wheat was not available until 2014[3].In contrast,a high-quality reference genome sequence for rice was published in 2005[4].Difficulty in genetic transformation of common wheat also hampered molecular and genomic studies of agronomic traits including traits associated with WGQ.Reproducible and highly efficient Agrobacterium-mediatedplant transformation became well established for rice in the 1990s[5],but only became available for some wheat genotypes in 2015[6].Despite these difficulties,substantial worldwide effort has been devoted to molecular genetic and genomic studies of WGQ traits[1,7].In the following sections,we review recent progress made in China in molecular genetic and genomic analysis of grain milling and end-use traits of wheat.For each trait,a brief summary of current understanding is provided,and the progress made by Chinese researchers is outlined.Owing to limited space,achievements made in regard to wheat flour color,starch quality and nutritional traits are not included,but information relating to these aspects can be found in several recent articles[1,7–10].

2.Grain hardness and milling traits

The molecular genetic basis of wheat grain hardness(also referred as grain texture)was reviewed recently[11–16].Although this trait is conditioned by multiple genes,Puroindoline a(Pina)and Puroindoline b(Pinb),located at the Hardness(Ha)locus on chromosome 5DS,are the major determinants of grain texture in common wheat.In general,the wild type(WT)alleles of Pina and Pinb,which encode two small and similar cysteine-rich proteins(～13 kDa,68%similarity),confer soft kernel texture,but mutation of one or both genes leads to an increase in grain hardness.Both Pina and Pinb have multiple alleles in common wheat landraces and modern cultivars.The mutations are caused by either large deletions in the Ha locus,which results in the loss of Pina and/or Pinb genes,or SNPs in the coding regions,which create amino acid substitutions or truncations in the Pina or Pinb proteins.Detailed molecular and functional studies of these alleles are essential for not only systematically understanding the evolution and action of Pina and Pinb but also fine-tuning the kernel texture and milling quality of commercial varieties according to the end-use purpose.

A number of studies have been conducted in China to analyze allelic variations of Pina and Pinb[17–26].These efforts have largely clarified the role of Pina and Pinb alleles and their combinations present in Chinese wheat landraces and commercial cultivars.The allelic diversities of Pina and Pinb are relatively high in Chinese wheat compared to those detected in the wheat populations from other countries(regions).For example,13 allelic combinations of Pina and Pinb were found in 204 Chinese wheat landraces,whereas only three such combinations were detected in 104 wheat lines gathered from the International Maize and Wheat Improvement Center[26].A number of Pina and Pinb alleles were first discovered in Chinese wheat lines,and some of them may have originated in China[26].Pinb-D1b is the main allele detected in hard wheat varieties from the main wheat cultivation zone of China(i.e.,the Yellow and Huai River Valley),followed by several other alleles,e.g.,Pinb-D1p,Pina-D1b,and Pina-D1r[17–19,22,24–26].Chen et al.[26]provided a comprehensive summary of the Pina and Pinb alleles detected so far in common wheat,along with the molecular changes in variant alleles,and their effects on grain hardness.

Chinese researchers have also investigated Pina and Pinb orthologs in related Triticeae grasses.For example,two new alleles of Sina and Sinb,the orthologs of Pina and Pinb in rye(Secale cereale,RR,2n=2x=14),were identified[27].Dina and Dinb,the orthologs of Pina and Pinb in Haynaldia villosa(VV,2n=2x=14),were described[28].The latter study is particularly interesting because the chromosomal fragment carrying Dina and Dinb was transferred to the genome of a soft kernel common wheat cultivar, leading to a further reduction of kernel softness. This is valuable for developing environmentally stable supersoft wheat lines with improved flour yield and novel applications in food processing[28].

Apart from the studies described above,Chinese scientists have reported the effects of transgenically expressed Pina on grain hardness and flour yield,suggesting that grain hardness and flour yield can be effectively modulated by adjusting the expression level of Pina with transgenic technology[29,30].More recently,a series of investigations were reported on Pinb-like genes(Pinb-2)locatedon group7chromosomes[31–33].However,further research is needed to ascertain the functions of these genes and the mechanisms underlying their actions[34].Collectively,the molecular genetic studies of Pina and Pinb in common wheat and their orthologs in related Triticeae species in China have contributed to understanding the global allelic diversities of these important genes,thus providing new resources and avenues for modifying grain texture and milling quality in the future.

3.Gluten protein quality and end-use traits

Gluten proteins are the dominant grain storage proteins in common wheat and related species[35–37].They constitute 80%–85%of total wheat flour proteins,and are major determinants of wheat end-use traits[37].The main components of wheat gluten proteins include high-molecular-weight glutenin subunits(HMW-GSs),low-molecular-weight glutenin subunits(LMW-GSs)and gliadins,which account for approximately7.5%,32.5%,and 40.0%of total flour proteins,respectively[37,38].During flour processing,a gluten complex,largely composed of HMW-GSs,LMW-GSs and gliadins,with minor quantities of other seed proteins,is formed in the dough,whose elasticity and extensibility properties determine the suitability of the flour for making a particular type of food product[1,35–37].Consequently,both the amount and composition of HMW-GSs,LMW-GSs and gliadins have important effects on gluten and dough functionalities and flour end-use properties.

Current understanding of the functional mechanisms of gluten proteins in controlling dough functionality and end-use quality include the following points.First,HMW-GSs and LMW-GSs form glutenin macropolymers(GMPs)through intermolecular disulphide bonds in the gluten complex in the dough[35–39].Both the amount and molecular weight distribution of GMP complexes have direct and strong effects on dough elasticity and extensibility;the GMP complexes with a molec-ular mass larger than 250 kD are strongly correlated with dough functionality and end-use quality.Second,most gliadins interact with GMPs non-covalently,and impart viscosity to the dough.However,a few of them can interact with GMPs through disulphide bonding,and act as GMP chain terminators to weaken gluten strength[40–45](see also below).Third,the glutenin/gliadin(Glu/Gli)ratio is an important factor influencing dough functionality and end-use quality.For bread making and noodle processing,the higher the Glu/Gli ratio,the better the performance[1,46].Finally,apart from HMW-GSs,LMW-GSs and gliadins,there are additional and minor protein and lipid components in the gluten complex.These additional proteins include certain species of farinins,serpins and triticins,and some of the mcan interact covalently with GMPs,and have thus been found to affect gluten strength and dough functionality[1,45](see also below).Biochemically,the GMP complexes can be fractionated into two components using0.5% SDS:unextractable polymeric protein(UPP)containing larger GMP complexes and extractable polymeric protein(EPP)composed of smaller GMP complexes[45–47].The amount of UPP has been found positively correlated with gluten and dough strength and bread making quality.The glutenins in the flour can also be separated into soluble and insoluble fractions using different concentrations of 1-propanol,with the level of insoluble glutenin(IG)positively correlated with gluten and dough strength and bread making parameters[48–51].

Besides their roles in end-use quality control, gluten proteins are key contributors to wheat food sensitivities suffered by susceptible human individuals[52,53].Manygliadins,especially the α-gliadins encoded by the Gli-D2 locus,carry epitopes causing celiac disease(CD);various gliadins,HMW-GSs and LMW-GSs have been found to possess the epitopes involved in wheat-dependent excised-induced anaphylaxis[52–56].An s-type LMW-GS has recently been identified as a major wheat allergen because it reacted with IgE antibodies of about 80%of the patients suffering from wheat food allergy[57].

In China,molecular genetic studies of gluten protein genes in common wheat and related species started in the 1990s,with initial efforts directed to isolation and characterization of gluten proteins(genes)and their alleles[58].This was increasingly followed by more functional and application studies of important genes and alleles.Genomic research on gluten gene expression and function began after 2010,and expanded rapidly with availability of functional genomics tools and high-quality reference genome sequences.

3.1.HMW-GS proteins

HMW-GSs account for only7%–15%of the total gluten proteins in common wheat,but explain 45%–70%of the variation in gluten,dough and end-use parameters[35,59,60].Therefore,HMW-GSs are key determinants of end-use quality.Genetically,HMW-GSs are specified by three homoeologous loci(Glu-A1,Glu-B1,and Glu-D1)located on the long arms of group 1 chromosomes[35,61](Fig.1-A).Within each locus,there exist two HMW-GS genes,encoding one x-and one y-type subunits,respectively[62](Fig.1-B).Each gene has multiple alleles(including a null variant)[1,35].The 1Ay gene is silenced in common wheat,although it is frequently expressed in diploid and tetraploid wheat species(see below).Common wheat cultivars usually express three to five HMW-GSs,but the composition of HMW-GSs often differs among different varieties.For both x-and y-type subunits,their primary structure is composed of a signal peptide(removed from mature protein),a N-terminal domain(ND),a central repetitive domain(RD),and aC-terminal domain(CD);the x-type subunits generally have four conserved cysteine(cys)residues(three in ND and one in CD),whereas the y-type subunits usually have seven conserved cys residues(five in ND,one in RD and one in CD)(Fig.1-C).These cys residues are involved in intermolecular disulphide bonding in the formation of GMPs,and play an important role in the function of HMW-GSs[35–39].The size of the RD often varies among different HMW-GSs and allelic variants(Fig.1-C),and a longer RD has been suggested to enhance the functionality of HMW-GSs in controlling end-use quality related parameters[35–39].

Fig.1–Schematic representations of prolamin gene loci on wheat homoeologous group 1 chromosomes(1A,1B,and 1D)(a),organization of x-and y-type HMW-GS genes in Glu-A1(1Ax and 1Ay),Glu-B1(1Bx and 1By),and Glu-D1(1Dx and 1Dy)(b),and primary structure of representative HMW-GSs(c).For convenience,the Gli-1 and Glu-3 loci are separated in the diagram(a),but in reality they are closely adjacent to each other(see Fig.2).In(b),the size of the genomic DNA sequence for Glu-A1,Glu-B1,and Glu-D1 and the large physical distances between the x-and y-type HMW-GS genes in the three Glu-1 loci are illustrated based on the work by Gu et al.[62].In(c),the capital letters S,N,R and C mark the signal peptide,N-terminal domain,repetitive domain and C-terminal domain,respectively.Conserved and non-conserved cys residues are represented by red-and green lowercase letters,respectively.1Bx14,1Fx3.7,Gy7*,and 1Ay-PI428339 are listed in Table 1.GenBank accession numbers for 1Dx2,1Dx5,1Dx2.2*,and 1Dy12 are X03346,X12928,AY893508,and X03041,respectively.

A large number of HMW-GS genes have been isolated and characterized by Chinese scientists.Table 1 lists those with novel structural variations[63–77],some of which(i.e.,1Bx14,1Fx3.7,Gy7*,and 1Ay-PI428339)are also illustrated in Fig.1-C.For example,the x-type HMW-GS of Aegilops umbellulata and the x-and y-type HMW-GSs of Ae.bicornis,Ae.Longissimi,and Ae.sharonensis carry much longer RDs[63,71,72];1Bx14 lacks two of the three cys residues conserved in the ND of x-type HMW-GSs[64](Fig.1-C).Many actively expressed 1Ay alleles have been cloned from diploid and tetraploid wheat species[68,76,77].For some of the active 1Ay alleles,their deduced proteins display novel changes compared to canonical HMW-GSs,e.g.,carrying one or more extra cys residues in non-conserved positions or having a much-extended RD(Table 1).The Glu-D1d allele(encoding 1Dx5 and 1Dy10 subunits)is widely used in international wheat quality breeding programs.However,genetic diversity in 1Dx5 is very limited in modern wheat cultivars[78].Interestingly,Glu-D1d was not detected in a large population of Ae.tauschii,the D genome donor of common wheat,indicating that it may have evolved during or after the development of hexaploid wheat[78].As an evidence for this proposition,Glu-D1d was present in a number of accessions of spelt wheat[78],a more primitive form of cultivated common wheat[79].The wide variety of HMW-GS alleles listed in Table 1 represent potentially useful resources for enhancing the function of HMW-GSs,providing that their beneficial effects are verified by appropriate means.In this context,it is worth noting that the positive contributions of 1Bx14 to dough functionality(especially to dough extensibility)and breadmaking quality have been validated using knockout mutants[60](see also below).The functional superiority of 1Bx13 and 1By16,both specified by Glu-B1f,were verified by analyzing a large set of isogenic lines differing in gluten gene composition[80].Positive effects of Ae.longissima HMW-GSs on breadmaking quality were revealed by analyzing a wheat substitution linewhose 1B chromosome was replaced by a 1Slchromosome from Ae.longissima[72].

Table 1–Representative HMW-GS genes with novel structural variations.

During the course of these studies,three interesting observations were made.First,there are natural “hybrid”HMW-GS proteins between x-and y-types of subunits.Sun et al.[67]characterized the first hybrid HMW-GS,1Ssx49077,in Ae.searsii(SsSs,2n=2x=14)accession IG49077.This subunit resembled a typical x-type subunit in its N-terminal domain,but its last 159 residues were identical to those of the y-type HMW-GS isolated from the same accession.The amino acid sequence of 1Ssx49077 was genuine as it was validated by mass spectrometry analysis of the corresponding protein in the grain.Therefore,it was a true hybrid with an x-type N-terminal domain and a y-type C-terminal domain.Unequal crossing-over was proposed for generating the coding sequence of 1Ssx49077.Since then,additional hybrid HMW-GSs have been found in different wheat species and genotypes(Table 1).Apart from the mechanism discussed for generating 1Ssx49077,homoeologous recombination between HMW-GS genes located in different subgenomes of polyploid Triticeae species has also been proposed for generating a hybrid subunit ChAy/Bx detected ina wild emmer wheat accession[73].Hybrid HMW-GSs seem to be widespread in decaploid Thinopyrum(Agropyron)elongatum(2n=10x=70)and hexaploid Thinopyrum(Agropyron)intermedium(2n=2x=42)[70,75].In these species,the hybrid subunits may have evolved through multiple mechanisms,including unequal crossing-over between the x and y HMW-GS genes located at the same locus and/or homoeologous recombination between the HMW-GS genes located in different subgenomes.Second,wide hybridization between wheat and related species seems to be particularly effective for inducing molecular variation in HMW-GSs,which include abundant single nucleotide polymorphisms(SNPs)and indels of various sizes[65,81,82].Finally,the HMW-GSs in some Triticeae species differ strongly in primary structure from those of wheat and closely related species.For example,the HMW-GSs in four diploid and tetraploid Pseudoroegneria species were unusually small in size due to a much-reduced repetitive domain[69].TheGlu-1St1subunit isolated from Pd. stipifolia had a y-type N-terminal domain but an x-type C-terminal domain,and both domains shared conserved sequence elements with barley D-hordein proteins[69].The x-type subunit(1Fx3.7)cloned from Eremopyrum bonaepartis is the largest HMW-GS characterized so far because it carries a dramatically enlarged repetitive domain[74](Fig.1-C).Moreover,the number and position of cys residues in the N-terminal domain of 1Fx3.7 resembled that of a typical y-type subunit,and three cys residues were present in the repetitive domain(2)and C-terminal domain(1)of 1Fx3.7 in non-conserved positions[74].Clearly,the extent of HMW-GS variation in natural Triticeae populations is likely wider than currently recognized,and wide hybridization can beemployed to further enhance the molecular diversity of HMW-GSs in wheat.Whether naturally evolved or artificially created,the availability of a large reptoire of molecularly characterized HMW-GSs should be beneficial for more systematic basic and applied studies of these important proteins in the future.

3.2.LMW-GS proteins

The genes and proteins of the LMW-GSs are more complex than those of HMW-GSs.The main loci hosting LMW-GS genes,i.e.,Glu-A3,Glu-B3,and Glu-D3,are located on the short arms of homoeologous group 1 chromosomes,and physically linked with the Gli-A1,Gli-B1,and Gli-D1 loci that carry the genes encoding γ-,δ-,and ω-gliadins[35](Figs.1-A,2).A joint Sino-USA study,which sequenced and analyzed the Gli-D1/Glu-D3 region of Ae.tauschii,showed that the Glu-D3 locus(～1600 kb)was immediately downstream of that of Gli-D1(～667 kb),and harbored five different LMW-GS genes and a number of other genes[83](Fig.2).In the latest version of the Chinese Spring(CS)genomic sequence the cumulative length of the scaffolds containing the LMW-GS genes was 182.9 kb for Glu-A3,435.1 kb for Glu-B3,and 1127.3 kb for Glu-D3,and the numbers of intact LMW-GS genes annotated for the three loci were two,three and six,respectively[84].These data may not reflect the actual size and gene content of the homoeologous Glu-3 loci,but they illustrate the complexity of these loci in common wheat.In addition to this complexity there is wide allelic variation in LMW-GS alleles among different accessions[85].Together,these factors have made it challenging to understand the genomic organization,expression,accumulation and function of LMW-GSs,especially in common wheat.

A comprehensive understanding of the LMW-GS genes in the elite common wheat variety Xiaoyan 54 was achieved through BAC sequencing,gene cloning,genetic mapping and analysis of recombinant inbred lines,which demonstrated convincingly that the contribution of LMW-GSs to gluten functionality depends on the number of actively expressed LMW-GS genes;the more actively expressed LMW-GS members,the greater the contribution to wheat gluten quality[86].Subsequently,a molecular marker system based on length polymorphisms of PCR amplicons of LMW-GSgenes was established[87].Although this system cannot differentiate active LMW-GS genes from their inactive homologs,it is useful for estimating the total number of LMW-GS genes in common wheat cultivars.When this system was employed to investigate the diversity of LMW-GS genes in the micro-core collection of Chinese wheat germplasm,15–19 different LMW-GS genes were detected among 262 bread wheat cultivars,with four to six members in Glu-A3,three to five members in Glu-B3,and eight members in Glu-D3;the magnitude of allelic variation was highest in Glu-A3,intermediate in Glu-B3,and relatively low in Glu-D3[88].This marker systemhas also proven useful for investigating LMW-GS genes in the diploid wheat T.urartu(AA,2n=2x=14)[89].

Chinese researchers also played an active role in an international consortium to standardize molecular markers and wheat lines useful for differentiating different Glu-3 alleles.This stimulated subsequent basic and applied investigations of LMW-GSs[90–93].An important outcome of these investigations was an assessment of the relative effectiveness of different LMW-GS alleles on several end-use quality parameters(e.g.,dough strength and extensibility)[91].Based on this assessment,Glu-A3d,Glu-B3b,Glu-B3g,and Glu-B3i were suggested to be elite alleles for bread making quality,whereas Glu-A3e and Glu-B3c were considered to be unfavorable alleles for bread making.

Concurrent to the above studies,a large number of new LMW-GS gene sequences were isolated from wheat and related species[46,58],which may be useful for more systematically studying the molecular evolution of LMW-GSs.Many PCR markers specific for particular LMW-GS genes or Glu-3 alleles were developed,thus facilitating the applications of LMW-GS resources in WGQ improvement[46,58,94].

Fig.2–Types and members of prolamin genes at the Gli-D1 and Glu-D3 loci of Ae.tauschii.The diagram is based on the study of Dong et al.[83].Glu-D3(～1600 kb)is located immediately downstream of Gli-D1(～ 668 kb).

3.3.Gliadin proteins

Loci specifying gliadins are even more complex than those encoding HMW-GSs and LMW-GSs.As indicated above,the Gli-1 loci coding for γ-, δ-,and ω-gliadins are located on 1AS,1BS,and 1DS,respectively(Fig.1-A),whereas the Gli-2 loci(Gli-A2,Gli-B2,and Gli-D2)for α-gliadins are on 6AS,6BS,and 6DS,respectively[35,95–97].As exemplified by the Gli-D2 locus of Ae.tauschii published recently(Fig.3),each Gli locus is large in size(nearly 550 kb),and carries multiple copies of the gliadin genes(12 members)[98].Furthermore,most of the gliadin gene members exhibit rich allelic variation among different accessions[35,95–97].Biochemically, α-, γ-,and δ-gliadins are sulphur-rich,and carry six,eight and eight conserved cys residues,respectively,in their proteins[35,95–98].These cys residues are involved in formation of intra-molecular disulphide bonding,thus excluding typical α-,γ-,and δ-gliadins from interacting with glutenin proteins through intermolecular disulphide bonds.However,some variant α-and γ-gliadins contain extra cys residues in their proteins in addition to the conserved ones,and these become incorporated into the gluten network through intermolecular disulphide bonding,and may act as gluten chain terminators[40–45,95].The ω-gliadins are generally sulphur-poor,and carry no cys residue in their proteins[35,99].This property keeps the majority of ω-gliadins away from interacting with glutenin proteins through intermolecular disulphide bonds.But again some variant ω-gliadins are observed to have one cys residue,and can become incorporated into the gluten network through intermolecular disulphide bonding[99].

The multiplicity and structural heterogeneity of gliadins have made it notoriously difficult to precisely assess the functional importance of different Gli loci and individual gliadin genes in end-use quality.Past studies have suggested that gliadins may act as plasticizers and impart viscosity to dough[36].This viscosity may give rise to the foaming properties of gluten,which enable bread to rise properly during baking[100–102].However,recent molecular genetic studies have shown that decreasing gliadin accumulation in wheat grains is beneficial to gluten and dough functionality[103,104].It is possible that some gliadins do contribute positively to end-use quality,but the overall amount and composition of gliadins may need to be adjusted for different end-use purposes.To test this hypothesis vigorously,systematic molecular genetic and genomic studies involving knockout mutants of different Gli loci and important gliadin genes are necessary.

Fig.3–Organization of α-gliadin genes at the Gli-D2locus of Ae.tauschii.The diagram is prepared according to Huo et al.[98].Gli-D2(～547 kb)is flanked by glutamate-like receptor genes.

A number of Chinese studies have investigated gliadin gene sequences in wheat and related Triticeae species,and some of them have also analyzed the presence of CD epitopes in the gliadins deduced from cloned sequences[reviewed in 46 and 95].However,a more systematic genome-wide study of common wheat gliadins was published only recently.In this study,mutagenic,transcriptomic,proteomic and bioinformatic approaches were combined to analyze the expression and accumulation of four different types of gliadins in the winter wheat cultivar Xiaoyan 81[105].Six deletion mutants(DLGliA1,DLGliB1,DLGliD1,DLGliA2,DLGliB2,and DLGliD2),each lacking one of the six Gli loci,were developed.Full-length transcripts(FLTs)were identified for 52 gliadin genes using transcriptomic data generated from second- and third-generation RNA sequencing.Among the 52 FLTs,42 carried an intact open reading frame(ORF)and were predicted to encode 25 α-,11 γ-,1 δ-,and five ω-gliadins.However,comparative proteomic analysis among Xiaoyan 81 and the six Gli locus deletion mutants identified only 38 gliadins accumulated in the mature grains;these included 21 α-,11 γ-,1 δ-,and 5 ω-gliadins.For the four α-gliadin genes(Gli-α7,Gli-α8,Gli-α18,and Gli-α25)that were transcribed but with no protein products detected,their transcript levels were generally and comparatively lower than those of the α-gliadin genes that had protein products accumulated in the mature grains.Of the 38 accumulated gliadins,28 carried CD epitopes,which included 13 α-,11 γ-,and four ω-gliadins.The remaining 10 members,including eight α-,one δ-and one ω-gliadin proteins did not have CD epitopes.

Based on amino acid sequence variation downstream of the last conserved cys residue,Wang et al.[105]divided α-gliadins into CT and CSTT groups,and interestingly,the CT group α-gliadins all carried CD epitopes,whereas the CSTT group had no or very few CD epitopes(Fig.3).Intriguingly,the nine accumulated α-gliadins encoded by Gli-B2 were all CSTT type,but the six accumulated α-gliadins by Gli-A2 were all CT type,and five of the six accumulated α-gliadins by Gli-D2 were also CT type.The number of transcribed CSTT α-gliadin genes in Gli-A2 and Gli-D2,one copy for each locus,was much lower than that in Gli-B2(10 copies),and the only transcribed CSTT α-gliadin gene in Gli-A2,Gli-α8,did not accumulate a protein product in the mature grain.Similar to previous studies,Wang et al.[105]also found the accumulation in the mature grains of two γ-gliadins(Gli-γ5 and Gli-γ10)each containing an additional cys residue,and two ω-gliadins(Gli-ω2 and Gli-ω5)each with one cys residue,in their proteins.As anticipated,a deletion mutant DLGliD2,lacking the Gli-D2 locus on 6DS,exhibited a significant reduction in the most celiac-toxic α-gliadin and derivative CD epitopes.

The genome-wide study by Wang et al.[105]clearly demonstrated the necessity and effectiveness of a genomics approach in combination with other methods in resolving complex proteins involved in WGQ.The strategy adopted may be useful for genome-wide characterization of gliadins in additional wheat genotypes and other protein families functioning in WGQ.The deletion mutant and FLT resources may be employed for more systematic investigation of the roles of individual Gli loci and gliadin species in WGQ control and the occurrence of CD and other gliadin related illnesses.It will also be interesting to examine the six deletion mutants for changes in gluten,dough and bread making qualities.Since the content of α-gliadins and that of total gliadins were both decreased in DLGliD2,this mutant may have an elevated glutenin/gliadin ratio,and thus improved gluten,dough and bread making qualities.However,this possibility needs to be verified experimentally.

3.4.Other proteins in the gluten complex

Apart from HMW-GSs,LMW-GSs,and gliadins,the gluten complex also contains other types of flour proteins in smaller amounts.These additional gluten proteins include farinins,serpins,triticins,and globulins.Among these proteins,more molecular and functional insights are currently available for farinins[106].Farinins were originally named as avenin-like b proteins by Kan et al.[107]based on their similarity to the oat seed storage protein avenin.They carry 18 or 19 conserved cys residues in the primary structure, and have been detected in the smaller glutenin polymers of common wheat and the glutenin fraction of durum wheat[45,108].More recently,Chen et al.[109]reported that the homoeologous genes encoding farinins,i.e.,TaALP-7A,TaALP-4A,and TaALP-7D,were located on the chromosome arms 7AS,4AL,and 7DS,respectively.A farinin protein with 18 cys residues was incorporated into glutenin macro polymers when transgenically expressed in common wheat,with the transgenic lines exhibiting significantly improved gluten and dough functionalities[110].This is in line with the finding that allelic variation in TaALP-7A affected dough parameters,with the superior allele associated with better processing quality[109].Interestingly,among the three farinins detected in the grains of the common wheat cultivar Butte86,onlyfarininBu-3,having19 cys residues in its deducedprotein,was found in the smaller glutenin polymers[45];farinin Bu-1 might exist as a monomer associated with starch granules based on the analysis of a highly similar protein present in the flour of the common wheat cultivar Scout 66[106].These findings indicate that different farinins may differ in their ability to become incorporated into GMPs,and thus the potential to affect gluten,dough and end-use properties.More systematic genomic studies,coupled with gluten,dough and end-use tests,should help to clarify functional similarities and differences among different farinins.

3.5.Transcriptional regulation of gluten gene expression

An important feature of cereal prolamin genes is that their expression is restricted to the developing grains[35].Although substantial insights have been gained on the molecular genetic mechanisms regulating rice,maize and barley prolamin gene expression in the developing grains[111–116],less progress has been made in this field of research in wheat and its ancestral species.The promoter regions of HMW-GS,LMW-GS and gliadin genes are rich in cis-regulatory elements,and for HMW-GS genes,their promoters have been found to carry a number of conserved cis-regulatory modules(CCRM)[117–122].Furthermore,the GCN4-likemotifin CCRM4 was suggested to be functional because it binds the bZIP transcriptional factor SPA known to regulate expression of different types of gluten genes[122].By comparing the genes encoding several allelic 1Bx HMW-GSs,Geng et al.[123]suggested that large indels in the promoter regions influenced the expression of these genes.A miniature inverted-repeat transposable element(185 bp),inserted in the promoter of the 1Bx14 gene[64],was beneficial for its expression;a 43 bp insertion unique to the promoter of 1Bx7OEconferred enhanced expression of this allele.In contrast,a 54 bp deletion in the promoter of 1Bx13 reduced its expression in the endospermic tissues.Recently,Guo et al.[124]identified a regulatory module important for controlling the expression of HMW-GS genes.In this module,a TaGAMyb transcription factor interacted with the histone acetyltransferase TaGCN5 in the promoter region of HMW-GS genes,facilitating establishment of histone H3 acetylation(H3K9 and H3K14)and activation of HMW-GS gene expression in the endospermic tissues.Collectively,the available studies demonstrated the complexities of cis-elements and trans-factors involved in regulating tissue-specific expression of gluten genes.However,the functions of the majority of these elements need to be experimentally verified,and many of the factors are yet to be isolated and functionally characterized.

4.Toward establishing a mutagenetic platform for systematically studying WGQ related chromosomal loci and genes

A major difficulty frequently encountered in the studies of WGQ-related chromosomal loci and genes is the lack of well-defined genetic mutants.This has hampered systematic and in-depth functional and mechanistic investigations.Although the development of genome-wide mutant libraries(e.g.,T-DNA insertion)using Agrobacterium-mediated transformation is difficult in wheat,the efficiencies of chemical and radiation mutagenesis are quite high in both durum and tetraploid wheat species[125–127].To facilitate systematic genetic,molecular and genomic studies of WGQ-related chromosomal loci and genes,efforts have been made in China to develop appropriate mutants for various gluten genes and loci using elite winter wheat cultivars as progenitors.

For HMW-GSs,ethyl methanesulfonate(EMS)mutants were developed for the five subunits(1Ax1,1Bx14,1By15,1Dx2,and1Dy10)expressed inXiaoyan54,withboth knockout and missense mutants identified[60].In a pilot study involving the use of single and double knockout mutants,the contributions of 1Ax1 and 1Bx14 to important gluten,dough and breadmaking parameters were shown to be clearly different[60].While 1Ax1 had stronger effects on gluten strength,dough elasticity and bread loaf volume,1Bx14 exerted a better influence on dough extensibility.This study also revealed the strong beneficial effects of a missense mutant of1Ax1,caused byaG330Esubstitution,on breadmaking quality[60].Concomitantly,a complete series of seven ion beam-induced deletion mutants lacking one,two or all three Glu-1 loci was developed[51].Comparative genetic analysis of these mutants confirmed the different effects of Glu-1 loci(i.e.,Glu-D1＞Glu-B1＞Glu-A1)on breadmaking quality and related parameters,which was originally suggested by Lawrence et al.[128]based on a correlative study of wheat recombinant lines.Simultaneous analysis of EMS and ion beam mutants revealed that the x-type subunit 1Dx2 contributed more to breadmaking quality than the y-type subunit 1Dy12,both of which are encoded by Glu-D1a[51].For either Glu-D1a or 1Dx2,their superior functionality was attributed to enhanced capacities to enable the incorporation of HMW-GSs and LMW-GSs into functional GMPs.

For LMW-GSs and gliadins,ion beam-induced deletion mutants,each lacking an entire target locus,were prepared,because the deletion of one or a few LMW-GS or gliadin genes may not have sufficient effect on gluten,dough and end-use performance.Therefore,six distinct deletion mutants missing Gli-A1/Glu-A3,Gli-B1/Glu-B3,Gli-D1/Glu-D3,Gli-A2,Gli-B2,or Gli-D2 were prepared[105].The use of these mutants aided genomic analysis of transcribed gliadin genes and the gliadins accumulated in mature wheat grains.These mutants also facilitated the identification of transcribed LMW-GS genes and LMW-GS accumulation in mature common wheat grains,and a more comprehensive assessment of the genetic effects of Gli-A1/Glu-A3,Gli-B1/Glu-B3,Gli-D1/Glu-D3,Gli-A2,Gli-B2,and Gli-D2 on gluten,dough and end-use performance(D.Wang,unpublished data).

The value of this mutagenetic platform can be further enhanced in two ways.First,by developing mutants miss two or more different types of gluten loci(genes)through genetic recombination.For example,mutants lacking the major gluten gene loci in each of the three subgenomes can be developed.These lines can be used to unravel the contributions of individual subgenomes to different WGQ traits;they can also be analyzed using functional genomics approaches to reveal molecular and functional interactions among the gluten loci in the three subgenomes.Second,by identifying the mutants lacking genes encoding other types of proteins present in the gluten complex,e.g.,farinins,serpins and triticins.The contributions of these proteins to WGQ traits are still poorly understood(see above),and even less is known about their interactions with the major gluten proteins(i.e.,HMW-GSs,LMW-GSs,and gliadins).With continued effort a rich mutagenetic platform for WGQ-related chromosomal loci and genes can be established.The analysis of these mutants with genomics tools(see below)will very likely shed new light on the genetic basis of WGQ traits.

5.Future perspectives and concluding remarks

The pace of wheat genetic and genomics research has accelerated with the availability of increasing amounts of genomics information for common wheat and related Triticeae species.This has dramatically changed the way by which wheat agronomic traits(including WGQ)are studied and improved.Genome-wide association studies with abundant molecular markers have been successfully used to reveal the genomic basis of complex traits[129,130].Gene expression involved in the formation and variation of important wheat traits can now be effectively uncovered using functional genomics tools,e.g.,transcriptomics and proteomics[131–134].Concomitant with the surge in genomic studies comes genome editing, a revolution in the manipulation of genome expression and function[135].An ability to co-edit all three homoeologs of a common wheat gene provides unprecedented opportunities to dissect the individual and interactive actions of different homoeologs in trait control[136–138].Base editing allows precise improvement of the function of specific members of homeologous gene sets[139].Furthermore,multiplex editing makes it possible to destruct en mass unwanted genes (or genetic elements), or to simultaneously up-regulate the functions of multiple genes controlling the same trait or process[140,141].

Fig.4–Perspectives on future research and improvement of wheat WGQ traits.

Against the background outlined above,genomic analysis and genome editing will be the top choice in future efforts aimed at dissecting and enhancing WGQ traits(Fig.4).Genomic analysis will unravel the genes,and define the molecular interactions,key to WGQ traits,which can be validated,and improved,by genome editing.Molecular breeding tools,including genetic transformation,marker assisted gene pyramiding,whole genome selection and precision genome engineering,will be used to incorporate functionally improved genes into suitable varietal background,resulting in elite cultivars with good adaptability, high yield potential and desirable WGQ traits.Chinese scientists have made significant contributions to sequencing the A genome of T.urartu and D genome of Ae.tauschii[142,143].They are also pioneering wheat genome editing research[136–139].The combination of these achievements with the rich genetic resources accumulated in past studieswill enable China tomake even greater progress inWGQ research in the genomics era.Acknowledgments

Acknowledgments

We thank the Ministry of Science and Technology of China(2016YFD0100500)and Chinese Academy of Sciences(XDA08020302,2017PB0044)for supporting our research.The authors are grateful to Professor Robert McIntosh for improving the English.We apologize for not being able to cite many published works related to the subjects reviewed due to limited space.

R E F E R E N C E S

[1]C.Wrigley,R.Asenstorfer,I.Batey,G.Cornish,L.Day,D.Mares,K.Mrva,The biochemical and molecular basis of wheat quality,in:B.F.Carver(Ed.),Wheat Science and Trade,Iowa,USA 2009,pp.495–520.

[2]R.Brenchley,M.Spannagl,M.Pfeifer,G.L.Barker,R.D'Amore,A.M.Allen,N.McKenzie,M.Kramer,A.Kerhornou,D.Bolser,S.Kay,D.Waite,M.Trick,I.Bancroft,Y.Gu,N.Huo,M.C.Luo,S.Sehgal,B.Gill,S.Kianian,O.Anderson,P.Kersey,J.Dvorak,W.R.McCombie,A.Hall,K.F.Mayer,K.J.Edwards,M.W.Bevan,N.Hall,Analysis of the bread wheat genome using whole-genome shotgun sequencing,Nature 491(2012)705–710.

[3]International Wheat Genome Sequencing Consortium,A chromosome-based draft sequence of the hexaploid bread wheat(Triticum aestivum)genome,Science 345(2014)1251788.

[4]International Rice Genome Sequencing Project,The map-based sequence of the rice genome,Nature 436(2005)793–800.

[5]Y.Hiei,T.Komari,T.Kubo,Transformation of rice mediated by Agrobacterium tumefaciens,Plant Mol.Biol.35(1997)205–218.

[6]Y.Ishida,M.Tsunashima,Y.Hiei,Y.Komari,Wheat(Triticum aestivum L.)transformation using immature embryos,in:K.Wang(Ed.),Agrobacterium Protocols,Vol.1,Methods in Molecular Biology,1223,Springer Science+Business Media,New York 2015,pp.189–198.

[7]A.M.Kiszonas,C.F.Morris,Wheat breeding for quality:a historical review,Cereal Chem.(2017)https://doi.org/10.1094/CCHEM-05-17-0103-FI.

[8]S.Zhai,Z.He,W.Wen,H.Jin,J.Liu,Y.Zhang,Z.Liu,X.Xia,Genome-wide linkage mapping of flour color-related traits and polyphenol oxidase activity in common wheat,Theor.Appl.Genet.129(2016)377–394.

[9]C.Guzmán,J.B.Alvarez,Wheat waxy proteins:polymorphism,molecular characterization and effects on starch properties,Theor.Appl.Genet.129(2016)1–16.

[10]P.R.Shewry,S.J.Hey,The contribution of wheat to human diet and health,Food Energy Secur.4(2015)178–202.

[11]M.J.Giroux,C.F.Morris,Wheat grain hardness results from highly conserved mutations in the friabilin components puroindoline a and b,Proc.Natl.Acad.Sci.U.S.A.95(1998)6262–6266.

[12]A.C.Hogg,T.Sripo,B.Beecher,J.M.Martin,M.J.Giroux,Wheat puroindolines interact to form friabilin and control wheat grain hardness,Theor.Appl.Genet.108(2004)1089–1097.

[13]M.Bhave,C.F.Morris,Molecular genetics of puroindolines and related genes:allelic diversity in wheat and other grasses,Plant Mol.Biol.66(2008)205–219.

[14]A.Nadolska-Orczyk,S.Gasparis,W.Orczyk,The determinants of grain texture in cereals,J.Appl.Genet.50(2009)185–197.

[15]I.Pasha,F.M.Anjum,C.F.Morris,Grain hardness:a major determinant of wheat quality,Food Sci.Technol.Int.16(2010)511–522.

[16]S.Shaaf,R.Sharma,F.S.Baloch,E.D.Badaeva,H.Knüpffer,B.Kilian,H.?zkan,The grain Hardness locus characterized in a diverse wheat panel(Triticum aestivum L.)adapted to the central part of the Fertile Crescent:genetic diversity,haplotype structure,and phylogeny,Mol.Gen.Genomics.291(2016)1259–1275.

[17]L.Q.Xia,F.Chen,Z.H.He,X.M.Chen,C.F.Morris,Occurrence of puroindoline alleles in Chinese winter wheats,Cereal Chem.82(2005)38–43.

[18]F.Chen,Z.H.He,X.C.Xia,M.Lillemo,C.F.Morris,A new puroindoline b mutation presented in Chinese winter wheat cultivar Jingdong 11,J.Cereal Sci.42(2005)267–269.

[19]F.Chen,Z.H.He,X.C.Xia,L.Q.Xia,X.Y.Zhang,M.Lillemo,C.F.Morris,Molecular and biochemical characterization of puroindoline a and b alleles in Chinese landraces and historical cultivars,Theor.Appl.Genet.112(2006)400–409.

[20]C.Chang,H.P.Zhang,J.Xu,W.H.Li,G.T.Liu,M.S.You,B.Y.Li,Identification of allelic variations of puroindoline genes controlling grain hardness in wheat using a modified denaturing PAGE,Euphytica 152(2006)225–234.

[21]F.Chen,Y.X.Yu,Z.H.He,X.C.Xia,Prevalence of a novel puroindoline allele in Yunnan endemic wheats(Triticum aestivum ssp.Yunnanense King),Euphytica 156(2007)39–46.

[22]J.Wang,J.Z.Sun,D.C.Liu,W.L.Yang,D.W.Wang,Y.P.Tong,A.M.Zhang,Analysis of Pina and Pinb alleles in the microcore collections of Chinese wheat germplasm by Ecotilling and identification of a novel Pinb allele,J.Cereal Sci.48(2008)836–842.

[23]L.Wang,G.Y.Li,X.C.Xia,Z.H.He,P.Y.Mu,Molecular characterization of Pina and Pinb allelic variations in Xinjiang landraces and commercial wheat cultivars,Euphytica 164(2008)745–752.

[24]G.Y.Li,Z.H.He,M.Lillemo,Q.X.Sun,X.C.Xia,Molecular characterization of allelic variations at Pina and Pinb loci in Shandong wheat landraces,historical and current cultivars,J.Cereal Sci.47(2008)510–517.

[25]F.Chen,F.Y.Zhang,X.C.Xia,Z.D.Dong,D.Q.Cui,Distribution of puroindoline alleles in bread wheat of the Yellow and Huai Valley of China and discovery of a novel puroindoline a allele without PINA protein,Mol.Breed.29(2012)371–378.

[26]F.Chen,H.Li,D.Cui,Discovery,distribution and diversity of Puroindoline-D1 genes in bread wheat from five countries(Triticum aestivum L.),BMC Plant Biol.13(2013)125.

[27]G.Y.Li,Z.H.He,R.J.Pena,X.C.Xia,M.Lillemoe,Q.X.Sun,Identification of novel secaloindoline-a and secaloindolineb alleles in CIMMYT hexaploid triticale lines,J.Cereal Sci.43(2006)378–386.

[28]R.Zhang,X.Wang,P.Chen,Molecular and cytogenetic characterization of a small alien-segment translocation line carrying the softness genes of Haynaldia villosa,Genome 55(2012)639–646.

[29]L.Xia,H.Geng,X.Chen,Z.H.He,M.Lillemo,C.F.Morris,Silencing of puroindoline a alters the kernel texture in transgenic bread wheat,J.Cereal Sci.47(2008)331–338.

[30]Y.Li,X.Mao,Q.Wang,J.Zhang,X.Li,F.Ma,F.Sun,J.Chang,M.Chen,Y.Wang,K.Li,G.Yang,G.He,Overexpression of Puroindoline a gene in transgenic durum wheat(Triticum turgidum ssp.durum)leads to a medium-hard kernel texture,Mol.Breed.33(2014)545–554.

[31]F.Chen,B.Beecher,C.F.Morris,Physical mapping and a new variant of Puroindoline b-2 genes in wheat,Theor.Appl.Genet.120(2010)745–751.

[32]F.Chen,F.Y.Zhang,X.Y.Cheng,C.F.Morris,H.X.Xu,Z.D.Dong,K.H.Zhan,D.Q.Cui,Association of Puroindoline b-B2 variants with grain traits,yield components and flag leaf size in bread wheat(Triticum aestivum L.)varieties of the Yellow and Huai Valleys of China,J.Cereal Sci.52(2010)247–253.

[33]F.Chen,H.X.Xu,F.Y.Zhang,X.C.Xia,Z.H.He,D.W.Wang,Z.D.Dong,K.H.Zhan,X.Y.Cheng,D.Q.Cui,Physical mapping of puroindoline b-2 genes and molecular characterization of a novel variant in durum wheat(Triticum turgidum L.),Mol.Breed.28(2011)153–161.

[34]H.Geng,B.S.Beecher,Z.H.He,A.M.Kiszonas,C.F.Morris,Prevalence of Puroindoline D1 and Puroindoline b-2 variants in U.S.Pacific Northwest wheat breeding germplasm pools,and their association with kernel texture,Theor.Appl.Genet.124(2012)1259–1269.

[35]P.R.Shewry,N.G.Halford,D.Lafiandra,Genetics of wheat gluten proteins,Adv.Genet.49(2003)111–184.

[36]J.A.Delcour,I.J.Joye,B.Pareyt,E.Wilderjans,K.Brijs,B.Lagrain,Wheat gluten functionality as a quality determinant in cereal-based food products,Annu.Rev.Food Sci.Technol.3(2012)469–492.

[37]H.Goesaert,K.Brijs,W.S.Veraverbeke,C.M.Courtin,K.Gebruers,J.A.Delcour,Wheat flour constituents:how they impact bread quality,and how to impact their functionality,Trends Food Sci.Technol.16(2005)12–30.

[38]P.W.Gras,R.S.Anderssen,M.Keentok,F.Békés,R.Appels,Gluten protein functionality in wheat flour processing:a review,Aust.J.Agric.Res.52(2001)1311–1323.

[39]F.MacRitchie,Theories of glutenin/dough systems,J.Cereal Sci.60(2014)4–6.

[40]D.Kasarda,Glutenin structure in relation to wheat quality,in:Y.Pomeranz(Ed.),Wheat Is Unique,American Association of Cereal Chemists,St.Paul,MN,USA 1989,pp.277–302.

[41]O.D.Anderson,F.C.Greene,The α-gliadin gene family:II.DNA and protein sequence variation,subfamily structure,and origins of pseudogenes,Theor.Appl.Genet.95(1997)59–65.

[42]O.D.Anderson,N.Huo,Y.Q.Gu,The gene space in wheat:the complete γ-gliadin gene family from the wheat cultivar Chinese spring,Funct.Integr.Genomics 13(2013)261–273.

[43]P.Ferrante,S.Masci,R.D'Ovidio,D.Lafiandra,C.Volpi,B.Mattei,Proteomic approach to verify in vivo expression of a novel γ-gliadin containing an extra cysteine residue,Proteomics 6(2006)1908–1914.

[44]S.B.Altenbach,K.M.Kothari,Omega gliadin genes expressed in Triticum aestivum cv. Butte 86: effects of post-anthesis fertilizer on transcript accumulation during grain development,J. Cereal Sci. 46 (2007) 169–177.

[45]W.H.Vensel,C.K.Tanaka,S.B.Altenbach, Protein composition of wheat gluten polymer fractions determined by quantitative two-dimensional gel electrophoresis and tandem mass spectrometry, Proteome Sci. 12 (2014) 8.

[46]A.Rasheed,X.Xia,Y.Yan,R.Appels,T.Mahmood,Z.H.He,Wheat seed storage proteins:advances in molecular genetics,diversity and breeding applications,J.Cereal Sci.60(2014)11–24.

[47]R.B.Gupta,K.Khan,F.MacRitchie,Biochemical basis of flour properties in bread wheats.1.Effects of variation in the quantity and size distribution of polymeric protein,J.Cereal Sci.18(1993)23–41.

[48]H.D.Sapirstein,B.X.Fu,Intercultivar variation in the quantity of monomeric proteins,soluble and insoluble glutenin,and residue protein in wheat flour and relationships to breadmaking quality,Cereal Chem.75(1998)500–507.

[49]X.Z.Hu,Y.M.Wei,C.Wang,M.I.P.Kovacs,Quantitative assessment of protein fractions of Chinese wheat flours and their contribution to white salted noodle quality,Food Res.Int.40(2007)1–6.

[50]H.Jin,Z.Wang,D.Li,P.Wu,Z.Dong,C.Rong,X.Liu,H.Qin,H.Li,D.Wang,K.Zhang,Genetic analysis of chromosomal loci affecting the content of insoluble glutenin in common wheat,J.Genet.Genomics 42(2015)495–505.

[51]Z.Wang,Y.Li,Y.Yang,X.Liu,H.Qin,Z.Dong,S.Zheng,K.Zhang,D.Wang,New insight into the function of wheat glutenin proteins as investigated with two series of genetic mutants,Sci.Rep.7(2017)3428.

[52]K.A.Scherf,P.Koehler,H.Wieser,Gluten and wheat sensitivities-an overview,J.Cereal Sci.67(2016)2–11.

[53]P.R.Shewry,A.S.Tatham,Improving wheat to remove celiac epitopes but retain functionality,J.Cereal Sci.67(2016)12–21.

[54]L.Shan,?.Molberg,I.Parrot,F.Hausch,F.Filiz,G.M.Gray,L.M.Sollid,C.Khosla,Structural basis for gluten intolerance in celiac spruce,Science 297(2002)2275–2279.

[55]J.A.Tye-Din,J.A.Stewart,J.A.Dromey,T.Beissbarth,D.A.van Heel,A.Tatham,K.Henderson,S.I.Mannering,C.Gianfrani,D.P.Jewell,A.V.Hill,J.McCluskey,J.Rossjohn,R.P.Anderson,Comprehensive,quantitative mapping of T cell epitopes in gluten in celiac disease,Sci.Transl.Med.2(2010)(41ra51).

[56]C.V.Ozuna,J.C.Iehisa,M.J.Giménez,J.B.Alvarez,C.Sousa,F.Barro,Diversification of the celiac disease α-gliadin complex in wheat:a 33-mer peptide with six overlapping epitopes,evolved following polyploidization,Plant J.82(2015)794–805.

[57]A.Baar,S.Pahr,C.Constantin,S.Scheiblhofer,J.Thalhamer,S.Giavi,N.G.Papadopoulos,C.Ebner,A.Mari,S.Vrtala,R.Valenta,Molecular and immunological characterization of Tria 36,a low molecular weight glutenin,as a novel major wheat food allergen,J.Immunol.189(2012)3018–3025.

[58]Z.H.He,X.C.Xia,A.P.A.Bonjean,Wheat improvement in China,in:Z.H.He,A.P.A.Bonjean(Eds.),Cereals in China,CIMMYT,Mexico 2010,pp.51–68.

[59]P.I.Payne,L.M.Holt,A.F.Krattiger,J.M.Carrillo,Relationships between seed quality characteristics and HMW glutenin subunit composition determined using wheats grown in Spain,J.Cereal Sci.7(1988)229–235.

[60]Y.Li,X.An,R.Yang,X.Guo,G.Yue,R.Fan,B.Li,Z.Li,K.Zhang,Z.Dong,L.Zhang,J.Wang,X.Jia,H.Q.Ling,A.Zhang,X.Zhang,D.Wang,Dissecting and enhancing the contributions of high-molecular-weight glutenin subunits to dough functionality and bread quality,Mol.Plant 8(2015)332–334.

[61]P.I.Payne,C.N.Law,E.E.Mudd,Control by homoeologous group 1 chromosomes of the high-molecular-weight subunits of glutenin,a major protein of wheat endosperm,Theor.Appl.Genet.58(1980)113–120.

[62]Y.Q.Gu,J.Salse,D.Coleman-Derr,A.Dupin,C.Crossman,G.R.Lazo,N.Huo,H.Belcram,C.Ravel,G.Charmet,M.Charles,O.D.Anderson,B.Chalhoub,Types and rates of sequence evolution at the high-molecular-weight glutenin locusin hexaploid wheat and its ancestral genomes,Genetics 174(2006)1493–1504.

[63]Z.Liu,Z.Yan,Y.Wan,K.Liu,Y.Zheng,D.Wang,Analysis of HMW glutenin subunits and their coding sequences in two diploid Aegilops species,Theor.Appl.Genet.106(2003)1368–1378.

[64]W.Li,Y.Wan,Z.Liu,K.Liu,X.Liu,B.Li,Z.Li,X.Zhang,Y.Dong,D.Wang,Molecular characterization of HMW glutenin subunit allele 1Bx14:further insights into the evolution of Glu-B1-1 alleles in wheat and related species,Theor.Appl.Genet.109(2004)1093–1104.

[65]D.Feng,G.Xia,S.Zhao,F.Chen,Two quality-associated HMW glutenin subunits in a somatic hybrid line between Triticum aestivum and Agropyron elongatum,Theor.Appl.Genet.110(2004)136–144.

[66]J.R.Wang,Z.H.Yan,Y.M.Wei,Y.L.Zheng,Characterization of high-molecular-weight glutenin subunit genes from Elytrigia elongate,Plant Breed.125(2006)89–95.

[67]X.Sun,S.Hu,X.Liu,W.Qian,S.Hao,A.Zhang,D.Wang,Characterization of the HMW glutenin subunits from Aegilops searsii and identification of a novel variant HMW glutenin subunit,Theor.Appl.Genet.113(2006)631–641.

[68]X.Li,Y.Zhang,L.Gao,A.Wang,K.Ji,Z.He,R.Appels,W.Ma,Y.Yan,Molecular cloning,heterologous expression,and phylogenetic analysis of a novel y-type HMW glutenin subunit gene from the G genome of Triticum timopheevi,Genome 50(2007)1130–1140.

[69]Z.X.Li,X.Q.Zhang,H.G.Zhang,S.H.Cao,D.W.Wang,S.T.Hao,L.H.Li,H.J.Li,X.P.Wang,Isolation and characterization of a novel variant of HMW glutenin subunit gene from the St genome of Pseudoroegneria stipifolia,J.Cereal Sci.47(2008)429–437.

[70]S.Liu,X.Gao,G.Xia,Characterizing HMW-GS alleles of decaploid Agropyron elongatum in relation to evolution and wheat breeding,Theor.Appl.Genet.116(2008)325–334.

[71]Q.T.Jiang,J.Ma,Y.M.Wei,Y.X.Liu,X.J.Lan,S.F.Dai,Z.X.Lu,S.Zhao,Q.Z.Zhao,Y.L.Zheng,Novel variants of HMW glutenin subunits from Aegilops section Sitopsis species in relation to evolution and wheat breeding,BMC Plant Biol.12(2012)73.

[72]S.Wang,Z.Yu,M.Cao,X.Shen,N.Li,X.Li,W.Ma,H.Wei?gerber,F.Zeller,S.Hsam,Y.Yan,Molecular mechanisms of HMW glutenin subunits from 1Slgenome of Aegilops longissima positively affecting wheat breadmaking quality,PLoS One 8(2013),e58947.

[73]X.H.Guo,Z.G.Bi,B.H.Wu,Z.Z.Wang,J.L.Hu,Y.L.Zheng,D.C.Liu,ChAy/Bx,a novel chimeric high-molecular-weight glutenin subunit gene apparently created by homoeologous recombination in Triticum turgidum ssp.dicoccoides,Gene 531(2013)318–325.

[74]Q.T.Jiang,X.W.Zhang,J.Ma,L.Wei,S.Zhao,Q.Z.Zhao,P.F.Qi,Z.X.Lu,Y.L.Zheng,Y.M.Wei,Characterization of high-molecular-weight glutenin subunits from Eremopyrum bonaepartis and identification of a novel variant with unusual high molecular weight and altered cysteine residues,Planta 239(2014)865–875.

[75]S.Cao,Z.Li,C.Gong,H.Xu,R.Yang,S.Hao,X.Wang,D.Wang,X.Zhang,Identification and characterization of highmolecular-weight glutenin subunits from Agropyron intermedium,PLoS One 9(2014),e87477.

[76]Z.Li,H.Li,G.Chen,C.Kou,S.Ning,Z.Yuan,Q.Jiang,Y.Zheng,D.Liu,L.Zhang,Characterization of a novel y-type HMW-GS with eight cysteine residues from Triticum monococcum ssp.monococcum,Gene 573(2015)110–114.

[77]Z.Dong,Y.Yang,K.Zhang,Y.Li,J.Wang,Z.Wang,X.Liu,H.Qin,D.Wang,Development of a new set of molecular markers for examining Glu-A1 variants in common wheat and ancestral species,PLoS One 12(2017),e0180766.

[78]Z.Dong,Y.Yang,Y.Li,K.Zhang,H.Lou,X.An,L.Dong,Y.Q.Gu,O.D.Anderson,X.Liu,H.Qin,D.Wang,Haplotype variation of Glu-D1 locus and the origin of Glu-D1d allele conferring superior end-use qualities in common wheat,PLoS One 8(2013),e74859.

[79]C.Guzmán,L.Caballero,L.M.Martín,J.B.Alvarez,Waxy genes from spelt wheat:new alleles for modern wheat breeding and new phylogenetic inferences about the origin of this species,Ann.Bot.110(2012)1161–1171.

[80]Y.Li,R.Zhou,G.Branlard,J.Jia,Development of introgression lines with 18 alleles of glutenin subunits and evaluation of the effects of various alleles on quality related traits in wheat(Triticum aestivum L.),J.Cereal Sci.51(2010)127–133.

[81]H.Liu,S.Liu,G.Xia,Generation of high frequency of novel alleles of the high molecular weight glutenin in somatic hybridization between bread wheat and tall wheatgrass,Theor.Appl.Genet.118(2009)1193–1198.

[82]Z.Yuan,M.Liu,Y.Ouyang,X.Zeng,M.Hao,L.Zhang,S.Ning,Z.Yan,D.Liu,The detection of a de novo allele of the Glu-1Dx gene in wheat-rye hybrid offspring,Theor.Appl.Genet.127(2014)2173–2182.

[83]L.Dong,N.Huo,Y.Wang,K.Deal,D.Wang,T.Hu,J.Dvorak,O.D.Anderson,M.C.Luo,Y.Q.Gu,Rapid evolutionary dynamics in a 2.8-Mb chromosomal region containing multiple prolamin and resistance gene families in Aegilops tauschii,Plant J.87(2016)495–506.

[84]B.J.Clavijo,L.Venturini,C.Schudoma,G.G.Accinelli,G.Kaithakottil,J.Wright,P.Borrill,G.Kettleborough,D.Heavens,H.Chapman,J.Lipscombe,T.Barker,F.H.Lu,N.McKenzie,D.Raats,R.H.Ramirez-Gonzalez,A.Coince,N.Peel,L.Percival-Alwyn,O.Duncan,J.Tr?sch,G.Yu,D.M.Bolser,G.Namaati,A.Kerhornou,M.Spannagl,H.Gundlach,G.Haberer,R.P.Davey,C.Fosker,F.D.Palma,A.L.Phillips,A.H.Millar,P.J.Kersey,C.Uauy,K.V.Krasileva,D.Swarbreck,M.W.Bevan,M.D.Clark,An improved assembly and annotation of the allohexaploid wheat genome identifies complete families of agronomic genes and provides genomic evidence for chromosomal translocations,Genome Res.27(2017)885–896.

[85]R.D'Ovidio,S.Masci,The low-molecular-weight glutenin subunits of wheat gluten,J.Cereal Sci.39(2004)321–339.

[86]L.Dong,X.Zhang,D.Liu,H.Fan,J.Sun,Z.Zhang,H.Qin,B.Li,S.Hao,Z.Li,D.Wang,A.Zhang,H.Q.Ling,New insights into the organization,recombination,expression and functional mechanism of low molecular weight glutenin subunit genes in bread wheat,PLoS One 5(2010),e13548.

[87]X.Zhang,D.Liu,W.Yang,K.Liu,J.Sun,X.Guo,Y.Li,D.Wang,H.Ling,A.Zhang,Development of a new marker system for identifying the complex members of the lowmolecular-weight glutenin subunit gene family in bread wheat(Triticum aestivum L.),Theor.Appl.Genet.122(2011)1503–1516.

[88]X.Zhang,D.Liu,J.Zhang,W.Jiang,G.Luo,W.Yang,J.Sun,Y.Tong,D.Cui,A.Zhang,Novel insights into the composition,variation,organization,and expression of the lowmolecular-weight glutenin subunit gene family in common wheat,J.Exp.Bot.64(2013)2027–2040.

[89]G.Luo,X.Zhang,Y.Zhang,W.Yang,Y.Li,J.Sun,K.Zhan,A.Zhang,D.Liu,Composition,variation,expression and evolution of low-molecular-weight glutenin subunit genes in Triticum urartu,BMC Plant Biol.15(2015)68.

[90]L.Liu,T.M.Ikeda,G.Branlard,R.J.Pe?a,W.J.Rogers,S.E.Lerner,M.A.Kolman,X.Xia,L.Wang,W.Ma,R.Appels,H.Yoshida,A.Wang,Y.Yan,Z.He,Comparison of low molecular weight glutenin subunits identified by SDS-PAGE,2-DE,MALDI-TOF-MS and PCR in common wheat,BMC Plant Biol.10(2010)124.

[91]X.Zhang,H.Jin,Y.Zhang,D.Liu,G.Li,X.Xia,Z.He,A.Zhang,Composition and functionalanalysis of low-molecular-weight glutenin alleles with Aroona near-isogenic lines of bread wheat,BMC Plant Biol.12(2012)243.

[92]A.Wang,L.Liu,Y.Peng,S.Islam,M.Applebee,R.Appels,Y.Yan,W.Ma,Identification of low molecular weight glutenin alleles by matrix-assisted laser desorption/ionization timeof-flight mass spectrometry(MALDI-TOF-MS)in common wheat(Triticum aestivum L.),PLoS One 10(2015)e0138981.

[93]Y.Wang,S.Zhen,N.Luo,C.Han,X.Lu,X.Li,X.Xia,Z.He,Y.Yan,Low molecular weight glutenin subunit gene Glu-B3h confers superior dough strength and breadmaking quality in wheat(Triticum aestivum L.),Sci.Rep.6(2016)27182.

[94]Y.Liu,Z.He,R.Appels,X.Xia,Functional markers in wheat:current status and future prospects,Theor.Appl.Genet.125(2012)1–10.

[95]P.F.Qi,Y.M.Wei,Y.W.Yue,Z.H.Yan,Y.L.Zheng,Biochemical and molecular characterization of gliadins,Mol.Biol.40(2006)796–807.

[96]O.D.Anderson,L.Dong,N.Huo,Y.Q.Gu,A new class of wheat gliadin genes and proteins,PLoS One 7(2012),e52139.

[97]Y.Wan,P.R.Shewry,M.J.Hawkesford,A novel family of γgliadin genes are highly regulated by nitrogen supply in developing wheat grain,J.Exp.Bot.64(2013)161–168.

[98]N.Huo,L.Dong,S.Zhang,Y.Wang,T.Zhu,T.Mohr,S.Altenbach,Z.Liu,J.Dvorak,O.D.Anderson,M.C.Luo,D.Wang,Y.Q.Gu,New insights into structural organization and gene duplication in a 1.75-Mb genomic region harboring the αgliadin gene family in Aegilops tauschii,the source wheat D genome,Plant J.(2017)https://doi.org/10.1111/tpj.13675.

[99]A.S.Tatham,P.R.Shewry,The S-poor prolamins of wheat,barley and rye:revisited,J.Cereal Sci.55(2012)79–99.

[100]T.Mita,E.Ishida,H.Matsumoto,Physicochemical studies on wheat protein foams.II.Relationship between bubble size and stability of foams prepared with gluten and gluten components,J.Colloid Interface Sci.64(1978)143–153.

[101]H.J.Van Lonkhuijsen,R.J.Hamer,C.Schreuder,Influence of specific gliadins on the breadmaking quality of wheat,Cereal Chem.69(1992)174–177.

[102]B.G.Thewissen,I.Celus,K.Brijs,J.A.Delcour,Foaming properties of wheat gliadin,J.Agric.Food Chem.59(2011)1370–1375.

[103]J.Gil-Humanes,F.Pistón,S.Tollefsen,L.M.Sollid,F.Barro,Effective shutdown in the expression of celiac diseaserelated wheat gliadin T-cell epitopes by RNA interference,Proc.Natl.Acad.Sci.U.S.A.107(2010)17023–17028.

[104]S.B.Altenbach,C.K.Tanaka,B.W.Seabourn,Silencing of omega-5 gliadins in transgenic wheat eliminates a major source of environmental variability and improves dough mixing properties of flour,BMC Plant Biol.14(2014)393.

[105]D.W.Wang,D.Li,J.Wang,Y.Zhao,Z.Wang,G.Yue,X.Liu,H.Qin,K.Zhang,L.Dong,D.Wang,Genome-wide analysis of complex wheat gliadins,the dominant carriers of celiac disease epitopes,Sci.Rep.7(2017)44609.

[106]D.D.Kasarda,E.Adalsteins,E.J.Lew,G.R.Lazo,S.B.Altenbach,Farinin:characterization of a novel wheat endosperm protein belonging to the prolamin superfamily,J.Agric.Food Chem.61(2013)2407–2417.

[107]Y.C.Kan,Y.F.Wan,F.Beaudoin,D.J.Leader,K.Edwards,R.Poole,D.W.Wang,R.A.C.Mitchell,P.R.Shewry,Transcriptome analysis reveals differentially expressed storage protein transcripts in seeds of Aegilops and wheat,J.Cereal Sci.44(2006)75–85.

[108]S.De Caro,P.Ferranti,F.Addeo,G.Mamone,Isolation and characterization of avenin-like protein type B from durum wheat,J.Cereal Sci.52(2010)426–431.

[109]X.Y.Chen,X.Y.Cao,Y.J.Zhang,S.Islam,J.J.Zhang,R.C.Yang,J.J.Liu,G.Y.Li,R.Appels,G.Keeble-Gagnere,W.Q.Ji,Z.H.He,W.J.Ma,Genetic characterization of cysteine-rich type-b avenin-like protein coding genes in common wheat,Sci.Rep.6(2016)30692.

[110]F.Ma,M.Li,T.Li,W.Liu,Y.Liu,Y.Li,W.Hu,Q.Zheng,Y.Wang,K.Li,J.Chang,M.Chen,G.Yang,Y.Wang,G.He,Overexpression of avenin-like b proteins in bread wheat(Triticum aestivum L.)improves dough mixing properties by their incorporation into glutenin polymers,PLoS One 8(2013),e66758.

[111]Y.Onodera,A.Suzuki,C.Y.Wu,H.Washida,F.Takaiwa,A rice functional transcriptional activator,RISBZ1,responsible for endosperm-specific expression of storage protein genes through GCN4 motif,J.Biol.Chem.276(2001)14139–14152.

[112]M.P.Yamamoto,Y.Onodera,S.M.Touno,F.Takaiwa,Synergism between RPBF Dof and RISBZ1 bZIP activators in the regulation of rice seed expression genes,Plant Physiol.141(2006)1694–1707.

[113]Z.Zhang,J.Yang,Y.Wu,Transcriptional regulation of zein gene expression in maize through the additive and synergistic action of opaque 2,prolamine-box binding factor,and O2 heterodimerizing proteins,Plant Cell 27(2015)1162–1172.

[114]Z.Zhang,X.Zheng,J.Yang,J.Messing,Y.Wu,Maize endosperm-specific transcription factors O2 and PBF network the regulation of protein and starch synthesis,Proc.Natl.Acad.Sci.U.S.A.113(2016)10842–10847.

[115]I.Diaz,J.Vicente-Carbajosa,Z.Abraham,M.Martínez,I.Isabel-La Moneda,P.Carbonero,The GAMYB protein from barley interacts with the DOF transcription factor BPBF and activates endosperm-specific genes during seed development,Plant J.29(2002)453–464.

[116]N.Sreenivasulu,L.Borisjuk,B.H.Junker,H.P.Mock,H.Rolletschek,U.Seiffert,W.Weschke,U.Wobus,Barley grain development toward an integrative view,Int.Rev.Cell Mol.Biol.281(2010)49–89.

[117]M.S.Thomas,R.B.Flavell,Identification of an enhancer element for the endosperm-specific expression of high molecular weight glutenin,Plant Cell 2(1990)1171–1180.

[118]D.Albani,M.C.Hammond-Kosack,C.Smith,S.Conlan,V.Colot,M.Holdsworth,M.W.Bevan,The wheat transcriptional activator SPA:a seed-specific bZIP protein that recognizes the GCN4-like motif in the bifactorial endosperm box of prolamin genes,Plant Cell 9(1997)171–184.

[119]G.Dong,Z.Ni,Y.Yao,X.Nie,Q.Sun,Wheat Dof transcription factor WPBF interacts with TaQM and activates transcription of an alpha-gliadin gene during wheat seed development,Plant Mol.Biol.63(2007)73–84.

[120]C.Ravel,P.Martre,I.Romeuf,M.Dardevet,R.El-Malki,J.Bordes,N.Duchateau,D.Brunel,F.Balfourier,G.Charmet,Nucleotide polymorphism in the wheat transcriptional activator Spa influences its pattern of expression and has pleiotropic effects on grain protein composition,dough viscoelasticity,and grain hardness,Plant Physiol.151(2009)2133–2144.

[121]A.Juhász,S.Makai,E.Sebestyén,L.Tamás,E.Balázs,Role of conserved non-coding regulatory elements in LMW glutenin gene expression,PLoS One 6(2011),e29501.

[122]C.Ravel,S.Fiquet,J.Boudet,M.Dardevet,J.Vincent,M.Merlino,R.Michard,P.Martre,Conserved cis-regulatory modules in promoters of genes encoding wheat high molecular-weight glutenin subunits,Front.Plant Sci.5(2014)621.

[123]Y.Geng,B.Pang,C.Hao,S.Tang,X.Zhang,T.Li,Expression of wheat high molecular weight glutenin subunit 1Bx is affected by large insertions and deletions located in the upstream flanking sequences,PLoS One 9(2014),e105363.

[124]W.Guo,H.Yang,Y.Liu,Y.Gao,Z.Ni,H.Peng,M.Xin,Z.Hu,Q.Sun,Y.Yao,The wheat transcription factor TaGAMyb recruits histone acetyltransferase and activates the expression of a high-molecular-weight glutenin subunit gene,Plant J.84(2015)347–359.

[125]A.J.Slade,S.I.Fuerstenberg,D.Loeffler,M.N.Steine,D.Facciotti,A reverse genetic,nontransgenic approach to wheat crop improvement by TILLING,Nat.Biotechnol.23(2005)75–81.

[126]C.Uauy,F.Paraiso,P.Colasuonno,R.K.Tran,H.Tsai,S.Berardi,L.Comai,J.Dubcovsky,A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat,BMC Plant Biol.9(2009)115.

[127]H.Guo,Z.Yan,X.Li,Y.Xie,H.Xiong,Y.Liu,L.Zhao,J.Gu,S.Zhao,L.Liu,Development of a high-efficient mutation resource with phenotypic variation in hexaploid winter wheat and identification of novel alleles in the TaAGPL-B1 gene,Front.Plant Sci.8(2017)1404.

[128]G.J.Lawrence,F.MacRitchie,C.W.Wrigley,Dough and baking quality of wheat lines deficient in glutenin subunits controlled by the Glu-A1,Glu-B1 and Glu-D1 loci,J.Cereal Sci.7(1988)109–112.

[129]C.Sun,F.Zhang,X.Yan,X.Zhang,Z.Dong,D.Cui,F.Chen,Genome-wide association study for 13 agronomic traits reveals distribution of superior alleles in bread wheat from the Yellow and Huai Valley of China,Plant Biotechnol.J.15(2017)953–969.

[130]Y.Liu,Y.Lin,S.Gao,Z.Li,J.Ma,M.Deng,G.Chen,Y.Wei,Y.Zheng,A genome-wide association study of 23 agronomic traits in Chinese wheat landraces,Plant J.91(2017)861–873.

[131]M.Pfeifer,K.G.Kugler,S.R.Sandve,B.Zhan,H.Rudi,T.R.Hvidsten,International Wheat Genome Sequencing Consortium,K.F.X.Mayer,O.A.Olsen,Genome interplay in the grain transcriptome of hexaploid bread wheat,Science 345(2014)1250091.

[132]L.Dong,H.Liu,J.Zhang,S.Yang,G.Kong,J.S.Chu,N.Chen,D.Wang,Single-molecule real-time transcript sequencing facilitates common wheat genome annotation and grain transcriptome research,BMC Genomics 16(2015)1039.

[133]G.X.Chen,J.W.Zhou,Y.L.Liu,X.B.Lu,C.X.Han,W.Y.Zhang,Y.H.Xu,Y.M.Yan,Biosynthesis and regulation of wheat amylose and amylopectin from proteomic and phosphoproteomic characterization of granule-binding proteins,Sci.Rep.6(2016)33111.

[134]S.Zhen,X.Deng,M.Zhang,G.Zhu,D.Lv,Y.Wang,D.Zhu,Y.Yan,Comparative phosphoproteomic analysis under high-nitrogen fertilizer reveals central phosphoproteins promoting wheat grain starch and protein synthesis,Front.Plant Sci.8(2017)67.

[135]J.A.Doudna,E.Charpentier,The new frontier of genome engineering with CRISPR-Cas9,Science 346(2014)1258096.

[136]Y.Wang,X.Cheng,Q.Shan,Y.Zhang,J.Liu,C.Gao,J.Qiu,Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew,Nat.Biotechnol.32(2014)947–951.

[137]Y.Zhang,Z.Liang,Y.Zong,Y.Wang,J.Liu,K.Chen,J.Qiu,C.Gao,Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA,Nat.Commun.7(2016)12617.

[138]Z.Liang,K.Chen,T.Li,Y.Zhang,Y.Wang,Q.Zhao,J.Liu,H.Zhang,C.Liu,Y.Ran,C.Gao,Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes,Nat.Commun.8(2017)14261.

[139]Y.Zong,Y.Wang,C.Li,R.Zhang,K.Chen,Y.Ran,J.Qiu,D.Wang,C.Gao,Precise base editing in rice,wheat and maize with a Cas9-cytidine deaminase fusion,Nat.Biotechnol.35(2017)438–440.

[140]L.Yang,M.Güell,D.Niu,H.George,E.Lesha,D.Grishin,J.Aach,E.Shrock,W.Xu,J.Poci,R.Cortazio,R.A.Wilkinson,J.A.Fishman,G.Church,Genome-wide inactivation of porcine endogenous retroviruses(PERVs),Science 350(2015)1101–1104.

[141]X.Ma,Q.Zhang,Q.Zhu,W.Liu,Y.Chen,R.Qiu,B.Wang,Z.Yang,H.Li,Y.Lin,Y.Xie,R.Shen,S.Chen,Z.Wang,Y.Chen,J.Guo,L.Chen,X.Zhao,Z.Dong,Y.G.Liu,A robust CRISPR/Cas9 system for convenient,high-efficiency multiplex genome editing in monocot and dicot plants,Mol.Plant 8(2015)1274–1284.

[142]H.Q.Ling,S.Zhao,D.Liu,J.Wang,H.Sun,C.Zhang,H.Fan,D.Li,L.Dong,Y.Tao,C.Gao,H.Wu,Y.Li,Y.Cui,X.Guo,S.Zheng,B.Wang,K.Yu,Q.Liang,W.Yang,X.Lou,J.Chen,M.Feng,J.Jian,X.Zhang,G.Luo,Y.Jiang,J.Liu,Z.Wang,Y.Sha,B.Zhang,H.Wu,D.Tang,Q.Shen,P.Xue,S.Zou,X.Wang,X.Liu,F.Wang,Y.Yang,X.An,Z.Dong,K.Zhang,X.Zhang,M.C.Luo,J.Dvorak,Y.Tong,J.Wang,H.Yang,Z.Li,D.Wang,A.Zhang,J.Wang,Draft genome of the wheat A-genome progenitor Triticum urartu,Nature 496(2013)87–90.

[143]J.Jia,S.Zhao,X.Kong,Y.Li,G.Zhao,W.He,R.Appels,M.Pfeifer,Y.Tao,X.Zhang,R.Jing,C.Zhang,Y.Ma,L.Gao,C.Gao,M.Spannagl,K.F.Mayer,D.Li,S.Pan,F.Zheng,Q.Hu,X.Xia,J.Li,Q.Liang,J.Chen,T.Wicker,C.Gou,H.Kuang,G.He,Y.Luo,B.Keller,Q.Xia,P.Lu,J.Wang,H.Zou,R.Zhang,J.Xu,J.Gao,C.Middleton,Z.Quan,G.Liu,J.Wang,International Wheat Genome Sequencing Consortium,H.Yang,X.Liu,Z.He,L.Mao,J.Wang,Aegilops tauschii draft genome sequence reveals a gene repertoire for wheat adaptation,Nature 496(2013)91–95.