Molecular genetic analysis of phosphomannomutase genes in Triticum monococcum

Chunmei Yu*,Xinyan Liu,Qian Zhang,Xinyu He,Wan Huai,Baohua Wang,Yunying Cao,Rong Zhou

School of Life Science,Nantong University,Nantong,Jiangsu 226019,China

1.Introduction

In the Tribe Triticeae,the diploid A genome wheat species Triticum monococcum(AmAm)and Triticum urartu(AuAu)evolved from a common ancestor about 0.5–3.0 million years ago(MYA) [1,2].T.urartu is a wild species,and the donor of the A genome in tetraploid wheat (Triticum turgidum,AABB,2n =4x = 28) and hexaploid common wheat (Triticum aestivum,AABBDD,2n = 6x = 42)[1,3].T.monococcum is mainly cultivated,although one of its subspecies(T.monococcum ssp.aegilopoides)is wild [4].T.monococcum holds large genetic diversity in multiple traits,and has been found to be suitable for mining important genes useful for improving wheat and related Triticeae crops[5,6].For example,a number of T.monococcum accessions exhibit strong resistance to powdery mildew,leaf rust and cereal cyst nematode[7–10].Some disease resistance genes in T.monococcum were transferred to common wheat through marker-assisted selection[7,8].Puroiudoline and high-molecular-weight glutenin subunit genes in T.monococcum were also introgressed into common wheat to improve grain milling and processing qualities[11–14].Furthermore,the comparatively smaller diploid genome of T.monococcum has made it an efficient experimental model for map-based cloning of genes controlling important traits in Triticeae crops,such as the powdery mildew resistance gene Pm3,leaf rust resistance gene Lr10,the VRN1 and VRN2 genes regulating vernalization response,and the Q gene controlling spike morphology [15–19].Characterization of T.monococcum alleles of Pm3,Lr10,Q,VRN1 and VRN2 not only expanded our understanding of the evolutionary and functional diversification patterns of important genes in Triticeae plants,but also helped to overcome the narrow genetic diversity of common wheat due to the bottleneck of polyploidization[20,21].

In eukaryotic cells,phosphomannomutase (PMM) is a conserved enzyme catalyzing the interconversion between mannose-6-phosphate(M6P)and mannose-1-phosphate(M1P),with M1P being the major substrate for synthesizing the key cellular metabolite GDP-mannose.In higher plants,GDPmannose is essential for biosynthesis of the potent antioxidant ascorbic acid (AsA) through the Smirnoff–Wheeler pathway[22–24].Overexpression of the PMM gene in plants increased AsA content and tolerance to oxidative stress [22,23].On the other hand,complete functional deficiency of PMM is lethal to organisms,and mild mutations of PMM were observed to decrease cellular tolerance to high temperature stress in both fungi and higher plants [24,25].In humans the genetically inherited disease CDG-Ia,associated with inadequate glycosylation of macromolecules,is caused by point mutations in PMM2[26–29].Dysfunction of PMM may also result in accumulation of M6P,the PMM substrate,and may cause a blockage of glycolysis in plant cells [30],and destruction of lipid-linked oligosaccharide in mammalian cells[31,32].Major agricultural crops such as rice,maize and wheat are mannose-sensitive plants,and cannot tolerate high M6P content in the cells[33–35].Therefore,it is very important to maintain an appropriate level of PMM enzyme activity in the cells of eukaryotes.So far,no report has shown that a eukaryotic organism can survive without PMM activity[23–25,36].

Because of its fundamental importance,many in-depth studies on PMM have been reported,mostly in mammals and model organisms.By contrast,less is known about PMM genes and their functions in polyploid plants.We previously reported a detailed characterization of the PMM gene family in common wheat [37].We identified six TaPMM genes,with TaPMM-A1,B1 and D1 located on group 2 chromosomes and TaPMM-A2,B2 and D2 on group 4 chromosomes.TaPMM-A2 is a pseudogene,while the protein encoded by TaPMM-B2 lacks PMM activity.Although TaPMM-D2 exhibits PMM activity,its transcript level is relatively low.By contrast,all three TaPMM-1 members are more highly transcribed and their proteins show potent PMM activity.Clearly,TaPMM-A1,B1 and D1 are the dominant PMM genes,whereas TaPMM-A2,B2 and D2 are either debilitated or functionally diverged.To enrich our understanding of PMM genes in Triticeae species,the main objective of this work was to characterize PMM gene members in T.monococcum.Owing to close similarity between the A genome of common wheat and the Amgenome of T.monococcum,it was anticipated that the outcome of the study should expand our knowledge on the existence and potential functional differences of PMM members in common wheat and related species,and possibly provide useful clues for further functional and applied studies of PMM in crop plants.

Table 1-Indel and SNP polymorphisms of TmPMM genes in T.monococcum accessions in common wheat[37].We identified six TaPMM genes,with.

2.Materials and methods

2.1.Plant materials,growth conditions and oligonucleotide primers

T.monococcum accessions used for cloning TmPMM genes are listed in Table 1.Growth of T.monococcum in the greenhouse was accomplished as described previously [37].The oligonucleotide primers used in this work are listed in Table S1.The general molecular methods for handling nucleic acid and protein samples,PCR amplification,DNA cloning,and protein expression in bacterial cells were described previously [38].High fidelity Taq DNA polymerases were used to minimize errors in PCR amplification.

2.2.Cloning of PMM cDNA and genomic sequences in T.monococcum

Total RNA samples were prepared from seedling leaves using a RNeasy Plant Mini Kit (Qiagen,Düsseldorf,Germany),and were converted into cDNAs using M-MLV reverse transcriptase (Promega,Madison,USA).PMM cDNAs of T.monococcum were amplified by RT-PCR,cloned and sequenced.Genomic DNA samples were extracted from leaf materials as detailed previously [39],and were used for isolating DNA sequences of PMM genes of T.monococcum by genomic PCR.The final nucleotide sequence for each cDNA or genomic DNA coding region was constructed from sequencing information of at least three independent clones.The newly isolated PMM sequences in this work were submitted to GenBank and the accession numbers are listed in Table S2.

2.3.Phylogenetic analysis

Phylogenetic analysis was conducted using the Mega 5.02 program [40].Briefly,the genomic coding region of TmPMM-1 and TmPMM-2b (isolated from the T.monococcum accession TA2034,Table 1)was aligned to that of 16 Triticeae PMM genes reported previously [37] using ClustalX software,and was subsequently used for constructing phylogenetic trees by the methods of neighbor-joining and maximum parsimony.The PMM gene of Brachypodium distachyon,BdPMM[37],was used as an out-group control in the phylogenetic analysis.

2.4.Bacterial expression and activity assay of recombinant PMM protein

The cDNA coding region of TmPMM-2a was cloned into the bacterial expression vector pET-30a (Novagen,Darmstadt,Germany) with the aim to express a recombinant protein containing the histidine tag at the C-terminal end.Cloning was facilitated by PCR amplification using primers listed in Table S1,and the accuracy of the resultant bacterial expression construct was confirmed by DNA sequencing.The induction of TmPMM-2a expression and its subsequent purification by nickel affinity chromatography were conducted following previously described protocols [37].The PMM activity of recombinant TmPMM-2a was tested via a coupled assay,where the conversion of M1P to M6P was coupled to the reduction of NADP+to NADPH [23,24,37].The reaction was monitored through increased absorbance at 340 nm as a result of increased NADPH content.Recombinant TaPMM-D1,which was found to exhibit high PMM activity previously[37],was used as a positive control.The assay was repeated three times for both TmPMM-2a and TaPMM-D1,with highly reproducible results obtained for both proteins.

2.5.Quantitative RT-PCR

T.monococcum accessions were grown in the glasshouse for four weeks.For each accession,leaf samples were collected from five individual plants and pooled together for isolating total RNA samples as described above.After cDNA synthesis(see above),quantitative RT-PCR was performed in an ABI 7500 Real-time PCR system with 7500 Software v2.0.4.The PCR mixture contained 2 μL of diluted cDNA,10 μL of 2× SYBR Premix Ex Taq II (TaKaRa,Dalian,China),0.4 μL of 50 × ROX Reference Dye II,and 400 nmol L-1of the appropriate primer set(Table S1)in a final volume of 20 μL.Thermo-amplification was performed in a 96-well plate (Applied Biosystems,Carlsbad,USA) with the following parameters: 30 s at 95 °C,followed by 40 cycles of 5 s at 95 °C and 34 s at 60 °C.The specificity of the amplicon was verified by melting curve analysis (60 to 95 °C) after 40 cycles.Each assay included at least three technical replicates.The amplification of a wheat 26S rRNA gene served as the internal control for the assay[41].Two independent assays were conducted for each gene (or primer set),with nearly identical results being obtained.

2.6.Assay of PMM activity in the leaf tissues

For in planta PMM activity assay,leaf tissues were collected from appropriate T.monococcum accessions as described above.After grinding in liquid nitrogen,total leaf protein was extracted with buffer containing 50 mmol L-1Hepes (pH 7.1),10 mmol L-1MgCl2,5 mmol L-1dithiothreitol,1 mmol L-1EDTA,1 mmol L-1ethylene glycol-bis (β-aminoethyl ether) N,N,N′,N′-tetraacetic acid,1 mmol L-1benzamidine hydrochloride,and 0.5 mmol L-1phenylmethylsulfonyl fluoride.Cell debris was removed by centrifugation,and the supernatant was desalted using Thermo Scientific Dextran Desalting Columns (Thermo Fisher Scientific Inc.,Shanghai,China) [24].Protein concentration in each supernatant was measured using a Bio-Rad protein assay kit(Bio-Rad,Hercules,USA).An in planta PMM activity assay was set up and executed as described previously[24,37],except that the recombinant PMM protein was replaced by 40 μg total leaf protein.For each T.monococcum accession,the activity assay was repeated twice with each containing three technical replicates.The data were statistically analyzed using Tukey's multiple comparison tests in the software SPSS10 in order to compare the relative PMM activity levels in the leaf tissues of four T.monococcum accessions.

2.7.Protein blot

Total leaf proteins(extracted as outlined above)were separated in 10% SDS-PAGE,followed by transference to polyvinylidene fluoride membranes.Protein blot assays were made with a monoclonal antibody(PMMAb3,recognizing PMM protein from both monocot and dicot plants)as described in a previous study[23].Equal amounts of total leaf proteins(20 μg)were loaded in SDS-PAGE in order to compare the PMM protein levels in four T.monococcum accessions.The protein blot assay was repeated twice with highly similar results.

3.Results

3.1.Cloning and analysis of PMM genes in T.monococcum

Using a homologous cloning approach,PMM cDNA and genomic DNA sequences were isolated from a range of T.monococcum accessions(Table 1).By comparing these sequences to previously reported PMM genes in grass species [37],we deduced that T.monococcum carried two distinct PMM genes,and their exon and intron patterns were identical to those of other Triticeae PMM genes (i.e.,HvPMM,TuPMM,TtPMM,AetPMM and TaPMM)[37].According to the phylogenetic tree constructed with PMM gDNA sequences of Triticeae species(Fig.1),PMM genes isolated in this study belonged to the PMM-1 and PMM-2 clades,and were thus named TmPMM-1 and 2,respectively.

The cDNA sequences of TmPMM-1 from four accessions carried an intact coding region (750–756 bp from start to stop codons) (Table 1).One indel and seven SNPs were present in the coding region of TmPMM-1; the indel caused variation in the length of the short polyalanine tract in the N-terminus while the SNPs at positions 25 and 73 resulted in amino acid residue changes (Fig.S1-A).TmPMM-2 cDNA sequence was cloned from nine accessions,with a total of eight SNPs detected in its coding region (Table 1).The SNP at position 68 led to an amino acid substitution,whereas the one at position 569 converted a “tryptophan” codon(TGG) into a premature stop codon (TAG) in seven accessions (Table 1,Fig.S1-B).Thus,the TmPMM-2 coding sequence was intact (750 bp from start to stop codons) in only two of the nine accessions (TA2026 and TA2726,Table 1)examined.To facilitate further analysis,we divided TmPMM-2 alleles into two types,one carrying an intact coding region (named as TmPMM-2a) and the other being a pseudogene (TmPMM-2b).

Fig.1-Phylogenetic analysis of PMM genes among Triticeae species.The tree was constructed based on an alignment of genomic DNA sequences of PMM genes by the neighbor joining method(with P distance and complete deletion options).Two distinct clades (indicated by PMM-1 and PMM-2) were resolved.Bootstrap values were estimated using 1000 replications.The PMM gene of B.distachyon (BdPMM,GenBank accession GQ412275) was used as an outgroup control.TmPMM-1 and TmPMM-2b (shown in bold,GenBank accessions JX559845 and JX559841,respectively) were isolated from T.monococcum accession TA2034 (Table 1).The other PMM sequences were reported previously[37].Their GenBank accession numbers are GQ412259 to GQ412264(for TaPMM-A1,A2,B1,B2,D1 and D2),GQ412265 to GQ412268(for TtPMM-A1,A2,B1 and B2),GQ412269 and GQ412270 (for TuPMM-1 and 2),GQ412271 and GQ412272 (for AetPMM-1 and 2),and GQ412273 and GQ412274(for HvPMM-1 and 2).The topology of the tree built using an alternative program(i.e.,maximum parsimony)was identical to that shown here.

Fig.2-Purification of recombinant TmPMM-2a and its PMM activity.(A)Inducible expression and affinity purification of a histidine tagged TmPMM-2a protein in the bacterial cells examined by 10%SDS-PAGE.Compared to bacterial protein extracts not induced by isopropyl-β-D-1-thiogalactopyranoside(IPTG)(lanes 1 and 2),those treated with IPTG over-accumulated TmPMM-2a(indicated by arrowhead,lanes 3 and 4).The histidine-tagged TmPMM-2a (lanes 5 and 6,arrowed) was partially purified using nickel affinity chromatography.Protein size(kD)markers(lane M)are shown on the left side of the graph.(B)Relative PMM activity of recombinant TmPMM-2a is compared to that of TaPMM-D1(arbitrarily set as 1 to facilitate the comparison).The values shown were means ± SD calculated from three technical repeats.The dataset was typical of three separate tests.

Fig.3-Comparative analysis of the relative transcript levels of TmPMM genes in leaf tissues of four T.monococcum accessions.Values are means ± SD of three technical repeats.The dataset was representative of two different experiments.(A)Combined transcript level of TmPMM-1 and TmPMM-2.(B)Transcript level of TmPMM-1.(C)Transcript level of TmPMM-2.

3.2.Test of PMM activity of recombinant TmPMM-2a protein

Following the findings above,it became necessary to test if TmPMM-2a might encode a protein with PMM activity.Towards this end,TmPMM-2a was expressed in the bacterial cells,and the resultant recombinant protein was purified(Fig.2-A).Subsequently,the recombinant TmPMM-2a was subjected to biochemical assay with TaPMM-D1 as a positive control.As shown in Fig.2-B,TmPMM-2a displayed a PMM activity level similar to that of TaPMM-D1.

3.3.Analysis of PMM transcript levels in T.monococcum

Based on the results presented above,two types of T.monococcum accessions were selected for analyzing PMM transcript levels in the leaf tissues.Accessions TA2026 and TA2726(Table 1)carried TmPMM-1 and TmPMM-2a,both with intact coding regions.Accessions TA2024 and TA2722 (Table 1) hosted TmPMM-1 and TmPMM-2b,with TmPMM-2b lacking coding capacity.The total TmPMM (TmPMM-1 + TmPMM-2) transcript levels in TA2026 and TA2726 leaf tissues were substantially higher than those in TA2024 and TA2722(Fig.3-A).The transcript level of TmPMM-1 did not differ among TA2722,TA2026 and TA2726,but was decreased in TA2024 (Fig.3-B).Finally,compared to TmPMM-2a,the transcript level of TmPMM-2b was drastically reduced(Fig.3-C).

3.4.Investigation of total PMM protein and activity levels in T.monococcum

The total PMM (TmPMM-1 + TmPMM-2) protein levels in leaf tissues of TA2024,TA2026,TA2722 and TA2726 were investigated using a monoclonal antibody that recognizes higher plant PMM proteins [23].From Fig.4-A,it is clear that PMM protein levels did not vary considerably among the four accessions.The in planta PMM activity levels of TA2722,TA2026 and TA2726 were approximately similar,whereas that of TA2024 was significantly lower(Fig.4-B).

4.Discussion

4.1.New information on PMM genes in T.monococcum

In this study,we identified two TmPMM genes (TmPMM-1 and 2) in a range of T.monococcum accessions.The main characteristics of TmPMM genes are summarized as follows:1) TmPMM-1 has a functional coding region,and its deduced protein sequence is highly conserved among different T.monococcum accessions despite the some indel and SNP variations; 2) the TmPMM-2 coding region is intact in only a few T.monococcum accessions,and in many accessions is disrupted by a premature stop codon.For the TmPMM-2a allele with an intact coding sequence,the recombinant protein was biochemically active and possessed high PMM activity.Consequently,some T.monococcum accessions(about 22% of genotypes examined in this study) carry two highly active PMM members (TmPMM-1 and TmPMM-2a),whereas the majority of T.monococcum germplasm has only one active PMM gene (TmPMM-1).3) TmPMM-1 and 2 are orthologous to the PMM-1 and 2 genes of Triticeae plants,respectively.Compared to PMM genes of common wheat and barley characterized previously [37],TmPMM-1 is likely the ortholog of TaPMM-A1,TaPMM-B1,TaPMM-D1 and HvPMM-1,whereas TmPMM-2 may be orthologous to TaPMM-A2,TaPMM-B2,TaPMM-D2 and HvPMM-2.Conservation of a functional TmPMM-1 coding region in T.monococcum germplasm reinforces our previous suggestion that,in Triticeae plants,the functionality of PMM-1 genes is selectively maintained compared to that of PMM-2 members [37].On the other hand,the occurrence of non-functional alleles is probably common to Triticeae PMM-2 genes.For the PMM-2 alleles with an intact coding region,their protein may either retain PMM activity (as represented by TaPMM-D2 and TmPMM-2) or have lost such activity (such as TaPMM-B2)[37].

Fig.4-Comparison of total PMM proteins and activity levels in leaf tissues of four T.monococcum accessions.M:markers;1:TA2024;2:TA2722;3:TA2026;4:TA2726.Total leaf proteins were extracted from accessions TA2024,TA2722,TA2026 and TA2726 and used in this series of experiments.(A)Separation of total leaf protein extracts of the four accessions in 10%SDS-PAGE(top panel)and detection of their total PMM protein levels by protein blot assay with a monoclonal antibody recognizing higher plant PMM(lower panel).Based on the size(kD)of the protein markers used,the molecular mass of TmPMM in the leaf cells was approximately 24 kD,which is close to that deduced from the cloned cDNA sequence.The dataset shown was representative of two separate experiments.(B)Relative in planta PMM activity levels in leaf tissues of the four accessions with the PMM activity of TA2722 arbitrarily set as 1 to facilitate comparison.The values displayed are means ± SD of three technical replicates.Different letters above the histograms indicate significant differences(P ≤0.05)between the means.The dataset displayed was typical of two independent experiments.

4.2.Complex mechanisms regulating the expression and activity of PMM genes in T.monococcum

In this work,we revealed considerable differences in total PMM transcript level,as well as those of TmPMM-1 and TmPMM-2,among different T.monococcum accessions (Fig.3).The transcript level of TmPMM-2a (with an intact coding sequence) was substantially higher than that of TmPMM-2b(a pseudogene) (Fig.3-C).Nonsense-mediated mRNA decay(NMD) might have reduced the transcript level of TmPMM-2b,because NMD has been implicated in decreased transcription of diverse pseudogenes in plant cells [42–46].On the other hand,TmPMM-2a may have contributed to the total PMM transcript level in accessions containing it,because such accessions tended to exhibit a much higher total PMM transcript level than those carrying TmPMM-2b (Fig.3-A).Surprisingly,the transcript level of TmPMM-1 was very low in one (TA2024) of the four T.monococcum accessions examined(Fig.3-B),suggesting the existence of a specific mechanism for maintaining a low TmPMM-1 transcript level in this genotype.However,the total PMM protein and activity levels in T.monococcum germplasm did not deviate as widely.This suggests the operation of complex mechanisms controlling the expression and activity of PMM genes in T.monococcum.Considering the key importance of PMM in regulating the contents of several vital cellular metabolites (e.g.,M6P,GDP-mannose,and AsA) (see Introduction),it is understandable that there are multiple mechanisms controlling the expression and activity of TmPMM genes.

4.3.Implications for further research

The new findings on TmPMM genes and the features of their expression and activity by this work have not only expanded our understanding of PMM genes in common wheat and related species,but also provided new clues for further study of PMM genes and their functions in Triticeae plants.Firstly,from the existence of both functional and non-functional alleles of TmPMM-2,it will be interesting to investigate if TaPMM-A2 encoded by the A genome of common wheat has a functional allele,and whether the TuPMM-2 gene of T.urartu is similar to TmPMM-2 in allelic differentiation.This type of analysis will further improve knowledge on the allelic diversity of PMM genes in Triticeae species,thus aiding the study of the mechanisms involved in molecular evolution of these genes.Secondly,from the data presented here,it is clear that T.monococcum accessions vary in number of functional PMM genes.Therefore,the next challenge will be to understand the potential consequences of this variation on growth and development of T.monococcum and its response to environmental factors.Finally,from the complex mechanisms regulating the expression and activity of TmPMM genes,it will be essential in the future to study dynamic changes in cellular contents of M6P,GDP-mannose and AsA in relation to variation in PMM protein and activity levels.This should yield a more complete elucidation of the biological function(s) of PMM in Triticeae species and may lead to appropriate strategies for using PMM in the improvement of crop plants.

This work was supported by the Knowledge Innovation Program of Nantong(BK2012062),the National Basic Research Program of China (2009CB118302),and the National Natural Science Foundation of China (30771306).We thank wheat Genetic and Genomic Resourced Center,Kansas State University (Manhattan,KS,USA) for providing the T.monococcum accessions used in this study.

Supplementary material

Supplementary figure and tables to this article can be found online at http://dx.doi.org/10.1016/j.cj.2014.07.003.

[1] S.Huang,A.Sirikhachornkit,X.Su,J.Faris,B.Gill,R.Haselkorn,P.Gornicki,Genes encoding plastid acetyl-CoA carboxylase and 3-phosphoglycerate kinase of the Triticum/Aegilops complex and the evolutionary history of polyploid wheat,Proc.Natl.Acad.Sci.U.S.A.99(2002)8133–8138.

[2] T.Wicker,N.Yahiaoui,R.Guyot,E.Schlagenhauf,Z.D.Liu,J.Dubcovsky,B.Keller,Rapid genome divergence at orthologous low molecular weight glutenin loci of the A and Amgenomes of wheat,Plant Cell 15(2003)1186–1197.

[3] M.Feldman,F.G.H.Lupton,T.E.Miller,Wheats,2,Longman Scientific,London,1995.

[4] Y.Matsuoka,Evolution of polyploid Triticum wheats under cultivation:the role of domestication,natural hybridization and allopolyploid speciation in their diversification,Plant Cell Physiol.52(2011) 750–764.

[5] H.C.Jing,D.Kornyukhin,K.Kanyuka,S.Orford,A.Zlatska,O.P.Mitrofanova,R.Koebner,K.Hammond-Kosack,Identification of variation in adaptively important traits and genome-wide analysis of trait-marker associations in Triticum monococcum,J.Exp.Bot.58 (2007) 3749–3764.

[6] H.C.Jing,C.Bayon,K.Kanyuka,S.Berry,P.Wenzl,E.Huttner,A.Kilian,K.E.Hammond-Kosack,DArT markers: diversity analyses,genomes comparison,mapping and integration with SSR markers in Triticum monococcum,BMC Genomics 10(2009) 458.

[7] P.Chhuneja,K.Kumar,D.Stirnweis,S.Hurni,B.Keller,H.S.Dhaliwal,K.Singh,Identification and mapping of two powdery mildew resistance genes in Triticum boeoticum L,Theor.Appl.Genet.124 (2012) 1051–1058.

[8] H.Xu,G.Yao,L.Xiong,L.Yang,Y.Jiang,B.Fu,W.Zhao,Z.Zhang,C.Zhang,Z.Ma,Identification and mapping of pm2026:a recessive powdery mildew resistance gene in an einkorn(Triticum monococcum L.) accession,Theor.Appl.Genet.117 (2008) 471–477.

[9] G.Yao,J.Zhang,L.Yang,H.Xu,Y.Jiang,L.Xiong,C.Zhang,Z.Zhang,Z.Ma,M.E.Sorrells,Genetic mapping of two powdery mildew resistance genes in einkorn (Triticum monococcum L.) accessions,Theor.Appl.Genet.114 (2007)351–358.

[10] W.Sodkiewicz,A.Strzembicka,T.Sodkiewicz,M.Majewska,Response to stripe rust (Puccinia striiformis Westend.f.sp.tritici) and its coincidence with leaf rust resistance in hexaploid introgressive triticale lines with Triticum monococcum genes,J.Appl.Genet.50(2009) 205–211.

[11] W.J.Rogers,T.E.Miller,P.I.Payne,J.A.Seekings,E.J.Sayers,L.M.Holt,C.N.Law,Introduction to bread wheat (Triticum aestivum L.) and assessment for bread-making quality of alleles from T.boeoticum Boiss ssp.thaoudar at Glu-A1 encoding two high-molecular-weight subunits of glutenin,Euphytica 93 (1997) 19–29.

[12] D.R.See,M.Giroux,B.S.Gill,Effect of multiple copies of puroindoline genes on grain softness,Crop Sci.44 (2004)1248–1253.

[13] G.Tranquilli,M.Cuniberti,M.C.Gianibelli,L.Bullrich,O.R.Larroque,F.MacRitchie,J.Dubcovsky,Effect of Triticum monococcum glutenin loci on cookie making quality and on predictive tests for bread making quality,J.Cereal Sci.36(2002) 9–18.

[14] G.Tranquilli,J.Heaton,O.Chicaiza,J.Dubcovsky,Substitutions and deletions of genes related to grain hardness in wheat and their effect on grain texture,Crop Sci.42(2002) 1812–1817.

[15] J.D.Faris,J.P.Fellers,S.A.Brooks,B.S.Gill,A bacterial artificial chromosome contig spanning the major domestication locus Q in wheat and identification of a candidate gene,Genetics 164 (2003) 311–321.

[16] K.J.Simons,J.P.Fellers,H.N.Trick,Z.Zhang,Y.S.Tai,B.S.Gill,J.D.Faris,Molecular characterization of the major wheat domestication gene Q,Genetics 172 (2006) 547–555.

[17] N.Stein,C.Feuillet,T.Wicker,E.Schlagenhauf,B.Keller,Subgenome chromosome walking in wheat: a 450-kb physical contig in Triticum monococcum L.spans the Lr10 resistance locus in hexaploid wheat (Triticum aestivum L.),Proc.Natl.Acad.Sci.U.S.A.97(2000) 13436–13441.

[18] L.Yan,A.Loukoianov,G.Tranquilli,M.Helguera,T.Fahima,J.Dubcovsky,Positional cloning of the wheat vernalization gene VRN1,Proc.Natl.Acad.Sci.U.S.A.100(2003)6263–6268.

[19] L.Yan,A.Loukoianov,A.Blechl,G.Tranquilli,W.Ramakrishna,P.SanMiguel,J.L.Bennetzen,V.Echenique,J.Dubcovsky,The wheat VRN2 gene is a flowering repressor down-regulated by vernalization,Science 303 (2004)1640–1644.

[20] J.Dubcovsky,J.Dvorak,Genome plasticity a key factor in the success of polyploid wheat under domestication,Science 316(2007) 1862–1866.

[21] A.Haudry,A.Cenci,C.Ravel,T.Bataillon,D.Brunel,C.Poncet,I.Hochu,S.Poirier,S.Santoni,S.Glemin,J.David,Grinding up wheat:a massive loss of nucleotide diversity since domestication,Mol.Biol.Evol.24 (2007) 1506–1517.

[22] A.A.Badejo,H.A.Eltelib,K.Fukunaga,Y.Fujikawa,M.Esaka,Increase in ascorbate content of transgenic tobacco plants overexpressing the acerola (Malpighia glabra)phosphomannomutase gene,Plant Cell Physiol.50(2009)423–428.

[23] W.Qian,C.Yu,H.Qin,X.Liu,A.Zhang,I.E.Johansen,D.Wang,Molecular and functional analysis of phosphomannomutase (PMM) from higher plants and genetic evidence for the involvement of PMM in ascorbic acid biosynthesis in Arabidopsis and Nicotiana benthamiana,Plant J.49(2007) 399–413.

[24] F.A.Hoeberichts,E.Vaeck,G.Kiddle,E.Coppens,B.van de Cotte,A.Adamantidis,S.Ormenese,C.H.Foyer,M.Zabeau,D.Inze,C.Perilleux,F.van Breusegem,M.Vuylsteke,A temperature-sensitive mutation in the Arabidopsis thaliana phosphomannomutase gene disrupts protein glycosylation and triggers cell death,J.Biol.Chem.283 (2008) 5708–5718.

[25] F.Kepes,R.Schekman,The yeast SEC53 gene encodes phosphomannomutase,J.Biol.Chem.263 (1988) 9155–9161.

[26] M.Aebi,T.Hennet,Congenital disorders of glycosylation:genetic model systems lead the way,Trends Cell Biol.11(2001) 136–141.

[27] H.H.Freeze,M.Aebi,Molecular basis of carbohydratedeficient glycoprotein syndromes type I with normal phosphomannomutase activity,Biochim.Biophys.Acta 1455(1999) 167–178.

[28] G.Matthijs,E.Schollen,E.Pardon,M.Veiga-Da-Cunha,J.Jaeken,J.J.Cassiman,E.Van Schaftingen,Mutations in PMM2,a phosphomannomutase gene on chromosome 16p13,in carbohydrate-deficient glycoprotein type I syndrome(Jaeken syndrome),Nat.Genet.16(1997) 88–92.

[29] G.Matthijs,E.Schollen,E.Van Schaftingen,J.J.Cassiman,J.Jaeken,Lack of homozygotes for the most frequent disease allele in carbohydrate-deficient glycoprotein syndrome type 1A,Am.J.Hum.Genet.62(1998) 542–550.

[30] Z.He,Z.Duan,W.Liang,F.Chen,W.Yao,H.Liang,C.Yue,Z.Sun,F.Chen,J.Dai,Mannose selection system used for cucumber transformation,Plant Cell Rep.25(2006) 953–958.

[31] N.Gao,J.Shang,M.A.Lehrman,Analysis of glycosylation in CDG-Ia fibroblasts by fluorophore-assisted carbohydrate electrophoresis: implications for extracellular glucose and intracellular mannose 6-phosphate,J.Biol.Chem.280 (2005)17901–17909.

[32] N.Gao,J.Shang,D.Huynh,V.L.Manthati,C.Arias,H.P.Harding,R.J.Kaufman,I.Mohr,D.Ron,J.R.Falck,M.A.Lehrman,Mannose-6-phosphate regulates destruction of lipid-linked oligosaccharides,Mol.Biol.Cell 22(2011)2994–3009.

[33] K.Datta,N.Baisakh,N.Oliva,L.Torrizo,E.Abrigo,J.Tan,M.Rai,S.Rehana,S.Al-Babili,P.Beyer,I.Potrykus,S.K.Datta,Bioengineered ‘golden'indica rice cultivars with beta-carotene metabolism in the endosperm with hygromycin and mannose selection systems,Plant Biotechnol.J.1(2003) 81–90.

[34] P.Lucca,X.D.Ye,I.Potrykus,Effective selection and regeneration of transgenic rice plants with mannose as selective agent,Mol.Breed.7(2001) 43–49.

[35] M.Wright,J.Dawson,E.Dunder,J.Suttie,J.Reed,C.Kramer,Y.Chang,R.Novitzky,H.Wang,L.Artim-Moore,Efficient biolistic transformation of maize (Zea mays L.) and wheat(Triticum aestivum L.) using the phosphomannose isomerase gene,pmi,as the selectable marker,Plant Cell Rep.20(2001)429–436.

[36] K.Cromphout,W.Vleugels,L.Heykants,E.Schollen,L.Keldermans,R.Sciot,R.D'Hooge,P.P.De Deyn,K.von Figura,D.Hartmann,C.Korner,G.Matthijs,The normal phenotype of Pmm1-deficient mice suggests that Pmm1 is not essential for normal mouse development,Mol.Cell.Biol.26 (2006)5621–5635.

[37] C.Yu,Y.Li,B.Li,X.Liu,L.Hao,J.Chen,W.Qian,S.Li,G.Wang,S.Bai,H.Ye,H.Qin,Q.Shen,L.Chen,A.Zhang,D.Wang,Molecular analysis of phosphomannomutase (PMM) genes reveals a unique PMM duplication event in diverse Triticeae species and the main PMM isozymes in bread wheat tissues,BMC Plant Biol.10(2010) 214.

[38] J.Sambrook,R.W.Russell,Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press,New York,2001.

[39] M.A.Saghai-Maroof,K.M.Soliman,R.A.Jorgensen,R.W.Allard,Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance,chromosomal location,and population dynamics,Proc.Natl.Acad.Sci.U.S.A.81(1984)8014–8018.

[40] K.Tamura,D.Peterson,N.Peterson,G.Stecher,M.Nei,S.Kumar,MEGA5: molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods,Mol.Biol.Evol.28(2011)2731–2739.

[41] M.A.Ali-Benali,R.Alary,P.Joudrier,M.F.Gautier,Comparative expression of five Lea genes during wheat seed development and in response to abiotic stresses by real-time quantitative RT-PCR,Biochim.Biophys.Acta 1730 (2005)56–65.

[42] M.Isshiki,Y.Yamamoto,H.Satoh,K.Shimamoto,Nonsensemediated decay of mutant waxy mRNA in rice,Plant Physiol.125 (2001) 1388–1395.

[43] E.Botticella,F.Sestili,A.Hernandez-Lopez,A.Phillips,D.Lafiandra,High resolution melting analysis for the detection of EMS induced mutations in wheat SBEIIa genes,BMC Plant Biol.11(2011) 156.

[44] K.Hori,Y.Watanabe,Context analysis of termination codons in mRNA that are recognized by plant NMD,Plant Cell Physiol.48(2007) 1072–1078.

[45] S.Kertesz,Z.Kerenyi,Z.Merai,I.Bartos,T.Palfy,E.Barta,D.Silhavy,Both introns and long 3′-UTRs operate as cis-acting elements to trigger nonsense-mediated decay in plants,Nucleic Acids Res.34(2006) 6147–6157.

[46] M.Kalyna,C.G.Simpson,N.H.Syed,D.Lewandowska,Y.Marquez,B.Kusenda,J.Marshall,J.Fuller,L.Cardle,J.McNicol,H.Q.Dinh,A.Barta,J.W.Brown,Alternative splicing and nonsense-mediated decay modulate expression of important regulatory genes in Arabidopsis,Nucleic Acids Res.40(2012) 2454–2469.