François Balfourier

Structural and functional partitioning of bread wheat chromosome 3B

We produced a reference sequence of the 1-gigabase chromosome 3B of hexaploid bread wheat. By sequencing 8452 bacterial artificial chromosomes in pools, we assembled a sequence of 774 megabases carrying 5326 protein-coding genes, 1938 pseudogenes, and 85% of transposable elements. The distribution of structural and functional features along the chromosome revealed partitioning correlated with meiotic recombination. Comparative analyses indicated high wheat-specific inter- and intrachromosomal gene duplication activities that are potential sources of variability for adaption. In addition to providing a better understanding of the organization, function, and evolution of a large and polyploid genome, the availability of a high-quality sequence anchored to genetic maps will accelerate the identification of genes underlying important agronomic traits.

Molecular diversity in French bread wheat accessions related to temporal trends and breeding programmes

A set of 41 wheat microsatellite markers (WMS), giving 42 polymorphic loci (two loci on each chromosome), was used to describe genetic diversity in a sample of 559 French bread wheat accessions (landraces and registered varieties) cultivated between 1800 and 2000. A total of 609 alleles were detected. Allele number per locus ranged from 3 to 28, with a mean allele number of 14.5. On the average, about 72% of the total number of alleles were observed with a frequency of less than 5% and were considered to be rare alleles. WMS markers used showed different levels of gene diversity: the highest PIC value occurred in the B genome (0.686) compared to 0.641 and 0.659 for the A and D genomes, respectively. When comparing landraces with registered varieties gathered in seven temporal groups, a cluster analysis based on an Fst matrix provided a clear separation of landraces from the seven variety groups, while a shift was observed between varieties registered before and after 1970. There was a decrease of about 25% in allelic richness between landraces and varieties. In contrast, when considering only registered varieties, changes in diversity related to temporal trends appeared more qualitative than quantitative, except at the end of the 1960s, when a bottleneck might have occurred. New varieties appear to be increasingly similar to each other in relation to allelic composition, while differences between landraces are more and more pronounced over time. Finally, considering a sub-sample of 193 varieties representative of breeding material selected during the twentieth century by the six most important plant breeding companies, few differences in diversity were observed between the different breeding programmes. The observed structure of diversity in French bread wheat collections is discussed in terms of consequences, both for plant breeders and for managers of crop genetic resources.

A high-density molecular map for ryegrass (Lolium perenne) using AFLP markers

Abstract AFLP markers have been successfully employed for the development of a high-density linkage map of ryegrass (Loliumperenne L.) using a progeny set of 95 plants from a testcross involving a doubled-haploid tester. This genetic map covered 930 cM in seven linkage groups and was based on 463 amplified fragment length polymorphism (AFLP) markers using 17 primer pairs, three isozymes and five EST markers. The average density of markers was approximately 1 per 2.0 cM. However, strong clustering of AFLP markers was observed at putative centromeric regions. Around these regions, 272 markers covered about 137 cM whereas the remaining 199 markers covered approximately 793 cM. Most genetic distances between consecutive pairs of markers were smaller than 20 cM except for five gaps on groups A, C, D, F and G. A skeletal map with a uniform distribution of markers can be extracted from this high-density map, and can be applied to detect and map QTLs. We report here the application of AFLP markers to genome mapping, in Lolium as a prelude to quantitative trait locus (QTL) identification for diverse agronomic traits in ryegrass and for marker-assisted plant breeding.

SSR allelic diversity changes in 480 European bread wheat varieties released from 1840 to 2000

A sample of 480 bread wheat varieties originating from 15 European geographical areas and released from 1840 to 2000 were analysed with a set of 39 microsatellite markers. The total number of alleles ranged from 4 to 40, with an average of 16.4 alleles per locus. When seven successive periods of release were considered, the total number of alleles was quite stable until the 1960s, from which time it regularly decreased. Clustering analysis on Nei’s distance matrix between these seven temporal groups showed a clear separation between groups of varieties registered before and after 1970. Analysis of qualitative variation over time in allelic composition of the accessions indicated that, on average, the more recent the European varieties, the more similar they were to each other. However, European accessions appear to be more differentiated as a function of their geographical origin than of their registration period. On average, western European countries (France, The Netherlands, Great Britain, Belgium) displayed a lower number of alleles than southeastern European countries (former Yugoslavia, Greece, Bulgaria, Romania, Hungary) and than the Mediterranean area (Italy, Spain and Portugal), which had a higher number. A hierarchical tree on Nei’s distance matrix between the 15 geographical groups of accessions exhibited clear opposition between the geographical areas north and south of the arc formed by the Alps and the Carpathian mountains. These results suggest that diversity in European wheat accessions is not randomly distributed but can be explained both by temporal and geographical variation trends linked to breeding practices and agriculture policies in different countries.

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.

Evidence for phylogeographic structure in Lolium species related to the spread of agriculture in Europe. A cpDNA study

Abstract In order to explain the present distribution area of natural populations of two forage grasses species (Lolium perenne and L. rigidum), we studied genetic variation for maternally inherited chloroplast DNA (cpDNA) in 447 individual plants from 51 natural populations sampled throughout Europe and the Middle East. The detection of polymorphism by restriction analysis of PCR-amplified cpDNA fragments resulted in the identification of 15 haplotypes. Hierarchical analysis of chloroplastic diversity showed a high level of within-population diversity while, for both species, we found that about 40% of the total diversity still remains among populations. The use of previous isozymes data enabled us to estimate the pollen to seed flow ratio: pollen flow appears to be 3.5 times greater than seed flow for L. perenne and 2.2 times higher for L. rigidum. A stepwise weighted genetic distance between pairs of populations was calculated using the haplotypes frequencies of populations. A hierarchical clustering of populations clearly divides the two species, while two main clusters of L. perenne populations show a strong geographical structure. Different scenario are proposed for explaining the distribution area of the two species. Finally, evidence attesting that these geographical structures are related to the spread of agriculture in Europe are presented and 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.

Transferable bread wheat EST-SSRs can be useful for phylogenetic studies among the Triticeae species

The genetic similarity between 150 accessions, representing 14 diploidand polyploid species of the Triticeae tribe, was investigated following the UPGMA clustering method. Seventy-three common wheat EST-derived SSR markers (EST-SSRs) that were demonstrated to be transferable across several wheat-related species were used. When diploid species only are concerned, all the accessions bearing the same genome were clustered together without ambiguity while the separation between the different sub-species of tetraploid as well as hexaploid wheats was less clear. Dendrograms reconstructed based on data of 16 EST-SSRs mapped on the A genome confirmed that Triticum aestivum and Triticum durum had closer relationships with Triticum urartu than with Triticum monococcum and Triticum boeoticum, supporting the evidence that T. urartu is the A-genome ancestor of polyploid wheats. Similarly, another tree reconstructed based on data of ten EST-SSRs mapped on the B genome showed that Aegilops speltoides had the closest relationship with T. aestivum and T. durum, suggesting that it was the main contributor of the B genome of polyploid wheats. All these results were expected and demonstrate thus that EST-SSR markers are powerful enough for phylogenetic analysis among the Triticeae tribe.

Deciphering the genetics of flowering time by an association study on candidate genes in bread wheat (Triticum aestivum L.)

Earliness is very important for the adaptation of wheat to environmental conditions and the achievement of high grain yield. A detailed knowledge of key genetic components of the life cycle would enable an easier control by the breeders. The objective of the study was to investigate the effect of candidate genes on flowering time. Using a collection of hexaploid wheat composed of 235 lines from diverse geographical origins, we conducted an association study for six candidate genes for flowering time and its components (vernalization sensitivity and earliness per se). The effect on the variation of earliness components of polymorphisms within the copies of each gene was tested in ANOVA models accounting for the underlying genetic structure. The collection was structured in five groups that minimized the residual covariance. Vernalization requirement and lateness tend to increase according to the mean latitude of each group. Heading date for an autumnal sowing was mainly determined by the earliness per se. Except for the Constans (CO) gene orthologous of the barley HvCO3, all gene polymorphisms had a significant impact on earliness components. The three traits used to quantify vernalization requirement were primarily associated with polymorphisms at Vrn-1 and then at Vrn-3 and Luminidependens (LD) genes. We found a good correspondence between spring/winter types and genotypes at the three homeologous copies of Vrn-1. Earliness per se was mainly explained by polymorphisms at Vrn-3 and to a lesser extent at Vrn-1, Hd-1 and Gigantea (GI) genes. Vernalization requirement and earliness as a function of geographical origin, as well as the possible role of the breeding practices in the geographical distribution of the alleles and the hypothetical adaptive value of the candidate genes, are discussed.