Mireille Dardevet

Genetic diversity of wheat storage proteins and bread wheat quality

To understand the genetic and biochemical basis of the bread makingquality of wheat varieties, a large experiment was carried out with a set of162 hexaploid bread wheat varieties registered in the French or EuropeanWheat Catalogue. This material was used to analyse their allelic compositionat the twelve main storage protein loci. A large genetic and biochemicaldiversity of the gluten proteins was found. Several gliadin encoding lociexhibited the highest allelic diversity whereas the lowest diversity was foundfor Glu-A1 and Glu-D3 loci encoding some high molecularweight glutenin subunits (HMW-GS) and LMW-GS respectively. Thevarieties were grown in three experimental locations in France. Qualityevaluation was carried out from material harvested in each location usingseven technological tests: grain protein content (Prot), grain hardness(GH), Zeleny sedimentation test (Zel), Pelshenke test (Pel), water solublepentosans (relative viscosity: Vr ), mixograph test (giving 11 parameters)and the alveograph test (dough strength W, tenacity P , extensibility L,swelling G, ratio P/L and the elasticity index Ie). Genetic and locationeffects as well as broad-sense heritability of each of the 22 technologicalparameters were calculated. GH, corresponding to the major Ha gene, Pel,and MtxW (mixograph parameter) had the highest heritability coefficients,alveograph parameters like W, P, the relative viscosity Vr and severalmixograph parameters had medium heritability coefficients whereas Protand L had the lowest. Variance analysis (using GLM procedure) allowed theeffect of the allelic diversity of the storage proteins, on the geneticvariations of each quality parameters, to be estimated. Glu-1 and Glu-3 loci had significant additive effects in the genetic variations of manyparameters. Gliadin alleles encoded at Gli-1 and Gli-2 were alsofound to play significant effect on several quality parameters. The majorpart of the phenotypic variation of the different quality parameters like Zel,Pel, W or mixograph peak time MPT was explained with the GH and allelesencoded at Glu-1 and Glu-3. Allelic variants encoded at Glu3and Gli-2 had similar contribution to the phenotypic variations ofquality parameters and accounted for 4% up to 21% each.

Allelic diversity of HMW and LMW glutenin subunits and omega-gliadins in French bread wheat (Triticum aestivum L.)

Wheat endosperm storage proteins, namely gliadins and glutenins, are the major components of gluten. They play an important role in dough properties and in bread making quality in various wheat varieties. In the present study, the different alleles encoded at the 6 glutenin loci and at 3 ω-gliadin loci were identified from a set of 200 hexaploid wheat cultivars grown primarily in France using SDS PAGE. At Glu-A1, Glu-B1 and Glu-D1, encoding high molecular weight glutenin subunits (HMW-GS), 3, 8 and 5 alleles were observed respectively. Low molecular weight glutenin subunits (LMW-GS) displayed similar polymorphism, as 5 and 11 alleles were identified at loci Glu-A3 and Glu-B3 respectively. Four alleles were observed at Glu-D3 loci. Omega-gliadin diversity was also very high, as 7, 13 and 9 alleles were found at Gli-A1, Gli-B1 and Gli-D1, respectively. A total of 147 (or 149) patterns resulted from the genetic combination of the alleles encoding at the six glutenin loci (or Glu-1 and Gli-1 loci). Although Glu-1 and Glu-3 loci were located on different chromosome arms and were theoretically independent, some associations were revealed due to pedigree relatedness between some French wheat cultivars. The usefulness of allelic identification of LMW-GS together with HMW-GS and gliadins for future genetic and technological wheat improvement is discussed.

Identification of Glu-B1-1 as a candidate gene for the quantity of high-molecular-weight glutenin in bread wheat (Triticum aestivum L.) by means of an association study

A previous study in wheat (Triticum aestivum L.) identified two candidate genes controlling a quantitative trait locus (QTL) for high-molecular-weight glutenin subunit (HMW-GS) GluBx. These candidates were Glu-B1-1, the structural gene coding for Glu1Bx, and the B homoeologous gene coding for SPA (spa-B), a seed storage protein activator. The goal of this study was to identify the best candidate gene for this QTL. Single nucleotide polymorphisms (SNPs) are an abundant source of DNA polymorphisms that have been successfully used to identify loci associated with particular phenotypes. As no linkage disequilibrium was detected between Glu-B1-1 and spa-B, we performed an association study to identify the individual gene responsible for the QTL. Six SNPs, three located in Glu-B1-1 and three in spa-B, were genotyped by mass spectrometry in a collection of 113 bread wheat lines. These lines were also evaluated for protein content as well as the total quantity of HMW-GSs and of each HMW-GS in seed samples from two harvest years. Significant associations were detected only between Glu-B1-1 polymorphism and most of the traits evaluated. Spa-B was unambiguously discarded as a candidate. To our knowledge, this is the first report on an association study that was successfully used to discriminate between two candidate genes.

High‐throughput single nucleotide polymorphism genotyping in wheat (Triticum spp.)

Over the past few years, considerable progress has been made in high-throughput single nucleotide polymorphism (SNP) genotyping technologies, largely through the investment of the human genetics community. These technologies are well adapted to diploid species. For plant breeding purposes, it is important to determine whether these genotyping methods are adapted to polyploidy, as most major crops are former or recent polyploids. To address this problem, we tested the capacity of the multiplex technology SNPlex with a set of 47 wheat SNPs to genotype DNAs of 1314 lines that were organized in four 384-well plates. These lines represented different taxa of tetra- and hexaploid Triticum species and their wild diploid relatives. We observed 40 markers which gave less than 20% missing data. Different methods, based on either Sanger sequencing or the MassARRAY genotyping technology, were then used to validate the genotypes obtained by SNPlex for 11 markers. The concordance of the genotypes obtained by SNPlex with the results obtained by the different validation methods was 96%, except for one discarded marker. Furthermore, a mapping study on six markers showed the expected genetic positions previously described. To conclude, this study showed that high-throughput genotyping technologies developed for diploid species can be used successfully in polyploids, although there is a need for manual reading. For the first time in wheat species, a core of 39 SNPs is available that can serve as the basis for the development of a complete SNPlex set of 48 markers.

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

Storage protein activator (SPA) is a key regulator of the transcription of wheat (Triticum aestivum) grain storage protein genes and belongs to the Opaque2 transcription factor subfamily. We analyzed the sequence polymorphism of the three homoeologous Spa genes in hexaploid wheat. The level of polymorphism in these genes was high particularly in the promoter. The deduced protein sequences of each homoeolog and haplotype show greater than 93% identity. Two major haplotypes were studied for each Spa gene. The three Spa homoeologs have similar patterns of expression during grain development, with a peak in expression around 300 degree days after anthesis. On average, Spa-B is 10 and seven times more strongly expressed than Spa-A and Spa-D, respectively. The haplotypes are associated with significant quantitative differences in Spa expression, especially for Spa-A and Spa-D. Significant differences were found in the quantity of total grain nitrogen allocated to the gliadin protein fractions for the Spa-A haplotypes, whereas the synthesis of glutenins is not modified. Genetic association analysis between Spa and dough viscoelasticity revealed that Spa polymorphisms are associated with dough tenacity, extensibility, and strength. Except for Spa-A, these associations can be explained by differences in grain hardness. No association was found between Spa markers and the average single grain dry mass or grain protein concentration. These results demonstrate that in planta Spa is involved in the regulation of grain storage protein synthesis. The associations between Spa and dough viscoelasticity and grain hardness strongly suggest that Spa has complex pleiotropic functions during grain development.

High level of conservation between genes coding for the GAMYB transcription factor in barley (Hordeum vulgare L.) and bread wheat (Triticum aestivum L.) collections

The transcription factor GAMYB is involved in gibberellin signalling in cereal aleurone cells and in plant developmental processes. Nucleotide diversity of HvGAMYB and TaGAMYB was investigated in 155 barley (Hordeum vulgare) and 42 wheat (Triticum aestivum) accessions, respectively. Polymorphisms defined 18 haplotypes in the barley collection and 1, 7 and 3 haplotypes for the A, B, and D genomes of wheat, respectively. We found that (1) Hv- and TaGAMYB genes have identical structures. (2) Both genes show a high level of nucleotide identity (>95%) in the coding sequences and the distribution of polymorphisms is similar in both collections. At the protein level the functional domain is identical in both species. (3) GAMYB genes map to a syntenic position on chromosome 3. GAMYB genes are different in both collections with respect to the Tajima D statistic and linkage disequilibrium (LD). A moderate level of LD was observed in the barley collection. In wheat, LD is absolute between polymorphic sites, mostly located in the first intron, while it decays within the gene. Differences in Tajima D values might be due to a lower selection pressure on HvGAMYB, compared to its wheat orthologue. Altogether our results provide evidence that there have been only few evolutionary changes in Hv- and TaGAMYB. This confirms the close relationship between these species and also highlights the functional importance of this transcription factor.

wDBTF: an integrated database resource for studying wheat transcription factor families.

BackgroundTranscription factors (TFs) regulate gene expression by interacting with promoters of their target genes and are classified into families based on their DNA-binding domains. Genes coding for TFs have been identified in the sequences of model plant genomes. The rice (Oryza sativa spp. japonica) genome contains 2,384 TF gene models, which represent the mRNA transcript of a locus, classed into 63 families.ResultsWe have created an extensive list of wheat (Triticum aestivum L) TF sequences based on sequence homology with rice TFs identified and classified in the Database of Rice Transcription Factors (DRTF). We have identified 7,112 wheat sequences (contigs and singletons) from a dataset of 1,033,960 expressed sequence tag and mRNA (ET) sequences available. This number is about three times the number of TFs in rice so proportionally is very similar if allowance is made for the hexaploidy of wheat. Of these sequences 3,820 encode gene products with a DNA-binding domain and thus were confirmed as potential regulators. These 3,820 sequences were classified into 40 families and 84 subfamilies and some members defined orphan families. The results were compiled in the Database of Wheat Transcription Factor (wDBTF), an inventory available on the web http://wwwappli.nantes.inra.fr:8180/wDBFT/. For each accession, a link to its library source and its Affymetrix identification number is provided. The positions of Pfam (protein family database) motifs were given when known.ConclusionswDBTF collates 3,820 wheat TF sequences validated by the presence of a DNA-binding domain out of 7,112 potential TF sequences identified from publicly available gene expression data. We also incorporated in silico expression data on these TFs into the database. Thus this database provides a major resource for systematic studies of TF families and their expression in wheat as illustrated here in a study of DOF family members expressed during seed development.

Improving the yellow pigment content of bread wheat flour by selecting the three homoeologous copies of Psy1

The yellow pigment content (YPC) of endosperm affects the quality and nutritional value of wheat grain products. Major quantitative trait loci (QTL) for endosperm YPC have been repeatedly mapped on chromosomes 7A and 7B in durum and bread wheats. The genes coding for phytoene synthase (PSY1), which is involved in the biosynthesis of carotenoids, generally co-segregate with these QTL, indicating their role in determining YPC. Here, to study the genetic factors underlying endosperm YPC in bread wheat, the sequence polymorphism of the homoeologous A, B and D copies of genes coding for PSY1, Psy-A1, Psy-B1, and Psy-D1, was studied in a worldwide core collection, which was also phenotyped for flour YPC. Seven novel alleles of Psy-A1 and two novel alleles of Psy-B1 were detected, which confirms the high level of polymorphism of these genes. Two major QTL with respective candidate genes Psy-A1 and Psy-B1 were identified in the distal region of chromosomes 7A and 7B using progeny of a cross between Apache and Ornicar, high and low YPC cultivars, respectively. Association mapping confirms the role of these genes in YPC and shows that the D copy also significantly influences this trait. These results indicate that breeders need to consider all three Psy1 copies when seeking to improve the YPC of wheat endosperm.

Conserved cis-regulatory modules in promoters of genes encoding wheat high-molecular-weight glutenin subunits

The concentration and composition of the gliadin and glutenin seed storage proteins (SSPs) in wheat flour are the most important determinants of its end-use value. In cereals, the synthesis of SSPs is predominantly regulated at the transcriptional level by a complex network involving at least five cis-elements in gene promoters. The high-molecular-weight glutenin subunits (HMW-GS) are encoded by two tightly linked genes located on the long arms of group 1 chromosomes. Here, we sequenced and annotated the HMW-GS gene promoters of 22 electrophoretic wheat alleles to identify putative cis-regulatory motifs. We focused on 24 motifs known to be involved in SSP gene regulation. Most of them were identified in at least one HMW-GS gene promoter sequence. A common regulatory framework was observed in all the HMW-GS gene promoters, as they shared conserved cis-regulatory modules (CCRMs) including all the five motifs known to regulate the transcription of SSP genes. This common regulatory framework comprises a composite box made of the GATA motifs and GCN4-like Motifs (GLMs) and was shown to be functional as the GLMs are able to bind a bZIP transcriptional factor SPA (Storage Protein Activator). In addition to this regulatory framework, each HMW-GS gene promoter had additional motifs organized differently. The promoters of most highly expressed x-type HMW-GS genes contain an additional box predicted to bind R2R3-MYB transcriptional factors. However, the differences in annotation between promoter alleles could not be related to their level of expression. In summary, we identified a common modular organization of HMW-GS gene promoters but the lack of correlation between the cis-motifs of each HMW-GS gene promoter and their level of expression suggests that other cis-elements or other mechanisms regulate HMW-GS gene expression.

Allelic variation of HMW and LMW glutenin subunits, HMW secalin subunits and 75K gamma-secalins of hexaploid triticale

Although the endosperm storage protein of hexaploid triticale have alreadybeen analysed, the allelic diversity of glutenins and secalins remains to bedescribed. Analysis, by SDS-PAGE, of about one thousand F2 seeds fromten triticale crosses allowed the inheritance of these storage proteins to bestudied in order to determine their allelic forms and to assign them toparticular chromosomes. Two new alleles encoding HMW subunits ofglutenin and five new alleles encoding HMW subunits of secalin weredetermined at Glu-B1 and Glu-R1 loci respectively. In additionto the three allelic forms of 75K gamma-secalins encoded at Gli-R2and previously reported, a null form was found. A nomenclature for theseproteins and their corresponding alleles was suggested. The composition ofB-LMW glutenin subunits of hexaploid triticale was described and allelicforms were determined: Five alleles were encoded at Glu-A3 andseven at Glu-B3 including one and two new allelic formsrespectively.