Catherine Ravel

A worldwide bread wheat core collection arrayed in a 384-well plate.

Bread wheat (Triticum aestivum), one of the world’s major crops, is genetically very diverse. In order to select a representative sample of the worldwide wheat diversity, 3,942 accessions originating from 73 countries were analysed with a set of 38 genomic simple sequence repeat (SSR) markers. The number of alleles at each locus ranged from 7 to 45 with an average of 23.9 alleles per locus. The 908 alleles detected were used together with passport data to select increasingly large sub-samples that maximised both the number of observed alleles at SSR loci and the number of geographical origins. A final core of 372 accessions (372CC) was selected with this M strategy. All the different geographical areas and more than 98% of the allelic diversity at the 38 polymorphic loci were represented in this core. The method used to build the core was validated, by using a second set of independent markers [44 expressed sequence tag (EST)-SSR markers] on a larger sample of 744 accessions: 96.74% of the alleles observed at these loci had already been captured in the 372CC. So maximizing the diversity with a first set of markers also maximised the diversity at a second independent set of locus. To relate the genetic structure of wheat germplasm to its geographical origins, the two sets of markers were used to compute a dissimilarity matrix between geographical groups. Current worldwide wheat diversity is clearly divided according to wheat’s European and Asian origins, whereas the diversity within each geographical group might be the result of the combined effects of adaptation of an initial germplasm to different environmental conditions and specific breeding practices. Seeds from each accession of the 372CC were multiplied and are now available to the scientific community. The genomic DNA of the 372CC, which can be entirely contained in a 384-deep-well storage plate, will be a useful tool for future studies of wheat genetic diversity.

Phylogenetic analysis in the Festuca-Lolium complex using molecular markers and ITS rDNA

Abstract Molecular markers were used to investigate phylogenetic relationships among the eight species of ryegrass (Lolium) and 11 species of fescue (Festuca). RAPD and RFLP analyses were carried out on total bulked DNA from each population. Factorial analysis of a phenetic distance matrix yielded three major groups: (1) fine-leaved fescues, (2) broad-leaved fescues and (3) ryegrasses. Six non-coding regions of chloroplastic DNA were PCR-amplified, then digested by 20 restriction enzymes. Nuclear rDNA sequences, including internal transcribed spacers (ITSs) were used to estimate the average proportion of nucleotide substitutions. The correlation between substitution rate estimated from ITS sequences and that estimated from organelle DNA restriction sites was very high (0.94), and the corresponding UPGMA trees were very similar, with a slightly better resolution of the ITS tree in the Lolium genus. The time-scale inferred from substitution rates indicated that the period since divergence of the broad-leaved fescues from the fine-leaved fescues was four times as long as that since divergence of the genus Lolium from the former. Among the broad-leaved fescues, meadow fescue was closer to the Lolium group, while F. glaucescens and tall fescue were very closely related. North-African fescues were clustered together and giant fescue was the most differentiated species in this group. Our dataset was merged with ITS sequences recovered from the EMBL database, and the neighbor-joining method was used to draw a phylogenetic tree. In this tree, the tribe Poeae was clearly monophyletic, and more closely related to the Aveneae than to the Triticeae or Bromoideae. The genus Festuca appeared somewhat artificial, since Vulpia myuros and Dactylis glomerata were placed between fine-leaved and broad-leaved fescues.

The HEALTHGRAIN Wheat Diversity Screen: Effects of Genotype and Environment on Phytochemicals and Dietary Fiber Components

Analysis of the contents of bioactive components (tocols, sterols, alkylresorcinols, folates, phenolic acids, and fiber components) in 26 wheat cultivars grown in six site x year combinations showed that the extent of variation due to variety and environment differed significantly between components. The total contents of tocols, sterols, and arabinoxylan fiber were highly heritable and hence an appropriate target for plant breeding. However, significant correlations between the contents of bioactive components and environmental factors (precipitation and temperature) during grain development also occurred, with even highly heritable components differing in amount between grain samples grown in different years on different sites.

Anthesis date mainly explained correlations between post-anthesis leaf senescence, grain yield, and grain protein concentration in a winter wheat population segregating for flowering time QTLs

The genetic variability of the duration of leaf senescence during grain filling has been shown to affect both carbon and nitrogen acquisition. In particular, maintaining green leaves during grain filling possibly leads to increased grain yield, but its associated effect on grain protein concentration has not been studied. The aim of this study was to dissect the genetic factors contributing to correlations observed at the phenotypic level between leaf senescence during grain filling, grain protein concentration, and grain yield in winter wheat. With this aim in view, an analysis of quantitative trait locus (QTL) co-locations for these traits was carried out on a doubled haploid mapping population grown in a large multienvironment trial network. Pleiotropic QTLs affecting leaf senescence and grain yield and/or grain protein concentration were identified on chromosomes 2D, 2A, and 7D. These were associated with QTLs for anthesis date, showing that the phenotypic correlations with leaf senescence were mainly explained by flowering time in this wheat population. Study of the allelic effects of these pleiotropic QTLs showed that delaying leaf senescence was associated with increased grain yield or grain protein concentration depending on the environments considered. It is proposed that this differential effect of delaying leaf senescence on grain yield and grain protein concentration might be related to the nitrogen availability during the post-anthesis period. It is concluded that the benefit of using leaf senescence as a selection criterion to improve grain protein concentration in wheat cultivars may be limited and would largely depend on the targeted environments, particularly on their nitrogen availability during the post-anthesis period.

Types and Rates of Sequence Evolution at the High-Molecular-Weight Glutenin Locus in Hexaploid Wheat and Its Ancestral Genomes

The Glu-1 locus, encoding the high-molecular-weight glutenin protein subunits, controls bread-making quality in hexaploid wheat (Triticum aestivum) and represents a recently evolved region unique to Triticeae genomes. To understand the molecular evolution of this locus region, three orthologous Glu-1 regions from the three subgenomes of a single hexaploid wheat species were sequenced, totaling 729 kb of sequence. Comparing each Glu-1 region with its corresponding homologous region from the D genome of diploid wheat, Aegilops tauschii, and the A and B genomes of tetraploid wheat, Triticum turgidum, revealed that, in addition to the conservation of microsynteny in the genic regions, sequences in the intergenic regions, composed of blocks of nested retroelements, are also generally conserved, although a few nonshared retroelements that differentiate the homologous Glu-1 regions were detected in each pair of the A and D genomes. Analysis of the indel frequency and the rate of nucleotide substitution, which represent the most frequent types of sequence changes in the Glu-1 regions, demonstrated that the two A genomes are significantly more divergent than the two B genomes, further supporting the hypothesis that hexaploid wheat may have more than one tetraploid ancestor.

New insights into the origin of the B genome of hexaploid wheat: Evolutionary relationships at the SPA genomic region with the S genome of the diploid relative Aegilops speltoides

BackgroundSeveral studies suggested that the diploid ancestor of the B genome of tetraploid and hexaploid wheat species belongs to the Sitopsis section, having Aegilops speltoides (SS, 2n = 14) as the closest identified relative. However molecular relationships based on genomic sequence comparison, including both coding and non-coding DNA, have never been investigated. In an attempt to clarify these relationships, we compared, in this study, sequences of the Storage Protein Activator (SPA) locus region of the S genome of Ae. speltoides (2n = 14) to that of the A, B and D genomes co-resident in the hexaploid wheat species (Triticum aestivum, AABBDD, 2n = 42).ResultsFour BAC clones, spanning the SPA locus of respectively the A, B, D and S genomes, were isolated and sequenced. Orthologous genomic regions were identified as delimited by shared non-transposable elements and non-coding sequences surrounding the SPA gene and correspond to 35 268, 22 739, 43 397 and 53 919 bp for the A, B, D and S genomes, respectively. Sequence length discrepancies within and outside the SPA orthologous regions are the result of non-shared transposable elements (TE) insertions, all of which inserted after the progenitors of the four genomes divergence.ConclusionOn the basis of conserved sequence length as well as identity of the shared non-TE regions and the SPA coding sequence, Ae speltoides appears to be more evolutionary related to the B genome of T. aestivum than the A and D genomes. However, the differential insertions of TEs, none of which are conserved between the two genomes led to the conclusion that the S genome of Ae. speltoides has diverged very early from the progenitor of the B genome which remains to be identified.

The effect of imperfect transmission on the frequency of mutualistic seed-borne endophytes in natural populations of grasses

We develop a simple mathematical model to explain the lower than expected levels of infection of wild perennial ryegrass populations in France by endophytic Neotyphodium fungi (formerly named Acremonium). Indeed, seed-borne Neotyphodium endophytes are considered as mutualistic symbionts, because they increase survival, growth and flowering rates of their hosts, and should therefore be present at very high frequencies in all host populations. However, recent surveys have shown that 70% of wild populations of perennial ryegrass harbour such endophytes in France. Moreover, most infected populations exhibit a low level of infection. Our simple model, taking into account the life-cycles of the host and the fungus, shows that these patterns can be satisfactorily explained if the vertical transmission of the fungus is imperfect. Such imperfect transmission, though never measured in natural populations, is likely because of the reported mortality of the endophyte in stored seeds. This process, analogous to the mutation-selection balance of classical population genetics theory, may explain the observed patterns even better when we consider random fluctuations of selection coefficients over time and genetic drift.

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.

New Neotyphodium endophyte species from the grass tribes Stipeae and Meliceae

Several species of Achnatherum (grass tribe Stipeae) and Melica (tribe Meliceae) typically are infected by nonpathogenic, seed-transmissible fungi with characteristics of Neotyphodium species (anamorphic Clavicipitaceae). Molecular phylogenetic studies clearly have distinguished the endophytes from Achnatherum inebrians (from Xinjiang Province, China), A. robustum and A. eminens (both from North America) and indicate that the A. inebrians endophyte comprises a unique nonhybrid lineage within the Epichloë and Neotyphodium phylogeny, whereas the endophytes of A. robustum, and A. eminens are hybrids with multiple Epichloë species (holomorphic Clavicipitaceae) as ancestors. Likewise distinct hybrid origins are indicated for Neotyphodium species from the European Melica species, M. ciliata and M. transsilvanica, the South African species M. decumbens and M. racemosa, and the South American species M. stuckertii. Neotyphodium species have been described from A. inebrians from Gansu Province, China, (N. gansuense), A. eminens (N. chisosum), M. stuckertii (N. tembladerae) and the South African Melica species (N. melicicola). However the endophytes from A. robustum and the European Melica species have not been described and the phylogenetic relationships of N. gansuense have not been investigated. Here we report a comprehensive study of morphological features and phylogenetic analyses of β-tubulin and actin gene sequences on an expanded collection of endophytes from the Stipeae and Meliceae. These data provide a firm foundation for the description of two new Neotyphodium species, N. guerinii from M. ciliata and M. transsilvanica, and N. funkii from A. robustum. We also propose the new variety, N. gansuense var. inebrians for endophytes of A. inebrians from Xinjiang Province, which are morphologically and phylogenetically distinct from, yet clearly related to, N. gansuense from Gansu Province.