Genomic regions associated with the nitrogen limitation response revealed in a global wheat core collection.

Modern wheat (Triticum aestivum L.) varieties in Western Europe have mainly been bred, and selected in conditions where high levels of nitrogen-rich fertilizer are applied. However, high input crop management has greatly increased the risk of nitrates leaching into groundwater with negative impacts on the environment. To investigate wheat nitrogen tolerance characteristics that could be adapted to low input crop management, we supplied 196 accessions of a wheat core collection of old and modern cultivars with high or moderate amounts of nitrogen fertilizer in an experimental network consisting of three sites and 2 years. The main breeding traits were assessed including grain yield and grain protein content. The response to nitrogen level was estimated for grain yield and grain number per m(2) using both the difference and the ratio between performance at the two input levels and the slope of joint regression. A large variability was observed for all the traits studied and the response to nitrogen level. Whole genome association mapping was carried out using 899 molecular markers taking into account the five ancestral group structure of the collection. We identified 54 main regions involving almost all chromosomes that influence yield and its components, plant height, heading date and grain protein concentration. Twenty-three regions, including several genes, spread over 16 chromosomes were involved in the response to nitrogen level. These chromosomal regions may be good candidates to be used in breeding programs to improve the performance of wheat varieties at moderate nitrogen input levels.

Genome-wide association analysis to identify chromosomal regions determining components of earliness in wheat.

The modification of flowering date is considered an important way to escape the current or future climatic constraints that affect wheat crops. A better understanding of its genetic bases would enable a more efficient and rapid modification through breeding. The objective of this study was to identify chromosomal regions associated with earliness in wheat. A 227-wheat core collection chosen to be highly contrasted for earliness was characterized for heading date. Experiments were conducted in controlled conditions and in the field for 3 years to break down earliness in the component traits: photoperiod sensitivity, vernalization requirement and narrow-sense earliness. Whole-genome association mapping was carried out using 760 molecular markers and taking into account the five ancestral group structure. We identified 62 markers individually associated to earliness components corresponding to 33 chromosomal regions. In addition, we identified 15 other significant markers and seven more regions by testing marker pair interactions. Co-localizations were observed with the Ppd-1, Vrn-1 and Rht-1 candidate genes. Using an independent set of lines to validate the model built for heading date, we were able to explain 34% of the variation using the structure and the significant markers. Results were compared with already published data using bi-parental populations giving an insight into the genetic architecture of flowering time in wheat.

Evaluation of the genetic variability of homoeologous group 3 SSRs in bread wheat.

Thorough characterization of the genetic variability in bread wheat (Triticum aestivum L.) is important for a better improvement of this key crop and to increase cereal yield in the context of sustainable agriculture to face human needs in the next decades. To study the genetic variability of SSRs on wheat homoeologous group 3 chromosomes, we characterized 38 hexaploid and two tetraploid wheat lines using a set of 165 microsatellites that we cytogenetically assigned to the 17 deletion bins for chromosomes group 3. A comparative analysis of the genetic variability through the PIC value study, allele numbers and SSR lengths indicated that there were no statistically significant differences (p > 0.05) between the three chromosomes of this homoeologous group despite the fact that SSRs from chromosome 3B exhibited slightly more alleles per locus compared to chromosomes 3A and 3D as well as slightly higher PIC values compared to chromosome 3D. However, there was a stronger correlation between SSR length and allele number on the short arms compared to the long arms and the correlation increased from the centromeres toward the telomeres. We did not find statistically significant differences in allele numbers and PIC values for SSRs located in more distal bins on 3A and 3B chromosomes. On the contrary, for chromosome 3D, we observed significant differences (p < 0.05) between the PIC values determined for SSRs assigned to deletion bin 3DL3-0.81-1.00 bin that is located distal compare to the more proximal region (C-3DL3-0.81). These results suggest that recombination which is higher in the telomeric regions does not contribute to increase a lot the variability of the SSRs.

[Genetic differentiation of hexaploid wheat inferred from analysis of microsatellite loci].

Landraces of wheat can serve as important potential sources for extending the genetic basis of selection cultivars. Analysis of microsatellites and typing of polymorphism in a representative sample of 347 genotypes, including landraces and selection cultivars, was performed using a set of 38 selected oligonucleotide primer pairs. Classification of genotypes with respect to the level of their similarity was performed using cluster analysis. The data obtained pointed to genetic differentiation of hexaploid wheat. The groups of cultivars, the formation of which was thought to be associated with the main old areas of wheat cultivation in Europe and Asia, were identified. The basis of each of the groups was formed by landraces of common wheat. The differences between the groups identified were associated with multiple changes in the wheat genome and were expressed as quantitative differences in the allele frequencies of microsatellite loci. The results of the study are of interest in terms of understanding the structure of wheat genetic diversity and revealing the pathways of evolution of this culture.

Soft selective sweep near a gene that increases plant height in wheat.

Strong selection within a given population locally reduces genetic variability not only in the selected gene itself but also in neighbouring loci. This so-called hitch-hiking effect is related to the initial linkage disequilibrium between markers and the selected gene, and depends mainly on the number of copies of the beneficial allele at the start of the selection phase. Contrary to the classical case, in which selection acts on a single, newly arisen beneficial mutation, we considered selection from standing variation (soft selective sweeps) on a gene (Rht-B1) with a major effect on plant height, a selected trait in an experimental wheat population grown for 17 generations, and we documented the evolution of gene diversity and linkage disequilibrium near this gene. As expected, Rht-B1 was found to be under strong selection (s = 0.15) and its variation in frequency accounted for 15% of the total trait evolution. This led to a smaller genetic effective population size at Rht-B1 (N(eg) = 18) compared to the whole genome estimation (N(eg) = 167). When compared with expectations under genetic drift only, no significant decrease in gene diversity was found at the closest loci. We computed expected di-locus frequencies for any linked marker-Rht-B1 pair due to hitch-hiking effects. We found that hitch-hiking was expected to affect the two most closely linked loci, but expected reduction in gene diversity was not greater than that due to genetic drift, which was consistent with the observations. Such limited effect was attributed to the low level of linkage disequilibrium (0.16) estimated after parental intercrosses, together with a relatively high initial frequency of the gene. This situation is favourable to candidate gene approaches where small linkage disequilibrium around selected genes is expected.

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.

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.

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 F(st) 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.

AFLP markers have been successfully employed for the development of a high-density linkage map of ryegrass (Lolium perenne 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.

Hierarchical clustering of perennial ryegrass populations with geographic contiguity constraint.

An algorithm of automatic classification is proposed and applied to a large collection of perennial ryegrass wild populations from France. This method is based on an ascendant hierarchical clustering using the Euclidian distance from the principal components extracted from the variance-covariance matrix between 28 agronomic traits. A contiguity constraint is imposed: only those pairs of populations which are defined as contiguous are grouped together into a cluster. The definition of contiguity is based on a geostatistical parameter: the range of the variogramme, i.e. the largest distance above which the variance between pairs of population no longer increases. This method yields clusters that are generally more compact than those obtained without constraint. In most cases the contours of these clusters fit well with known ecogeographic regions, namely, for macroclimatic homogeneous conditions. This suggests that selective factors exert a major influence in the genetic differentiation of ryegrass populations for quantitatively inherited adaptive traits. It is proposed that such a method could provide useful genetic and ecogeographic bases for sampling a core collection in widespread wild species such as forage grasses.

Isozyme variation and species relationships in the genus Lolium L. (ryegrasses, Graminaceae).

Thirty-two natural populations belonging to the eight species of the genus Lolium (ryegrass) or to Festuca pratensis (meadow fescue) were recorded for allelic frequencies at 13 isozyme loci. Cultivated ryegrass (L. perenne and L. multiflorum), meadow fescue, and the annual L. rigidum, are true outbreeders. The other species are true inbreeders, except for L. canariense, which shows a moderate level of cross fertilisation (20%). Hierarchical clustering from Nei's unbiased distance leads to four groups. The three self-pollinating, weed species, L. temulentum, L. remotum and L. persicum, belong to the first cluster, which is the most differentiated one. The second cluster comprises L. multiflorum, L. subulatum and most populations of L. rigidum. All L. perenne populations belong to the third cluster, as do two of L. rigidum. The average genetic distance within the L. perenne group is very low. Surprisingly, the fourth cluster groups together L. canariense and Festuca pratensis. The data suggest that L. rigidum is the species with the greatest diversity, and could be a common ancestor of the genus. Knowledge of historical processes of domestication could help to calibrate the molecular clock.

Genotype x environment interactions in a core collection of French perennial ryegrass populations.

A representative sample (core collection) of natural populations of perennial ryegrass (Lolium perenne L.) from France was evaluated for agronomic traits at seven locations. This sample exhibits a high level of genotype-environment interaction for most traits. The interactions for summer-growth (a key-factor of adaptation in most French regions) were studied by means of regression using climatic factors of the evaluation sites and the sites of population origin as covariates. This method succeeded in explaining most of the interaction term and also part of the main effects. It appears that populations from either warm or dry sites generally have a positive interaction when evaluated in a site with similar characteristics, as expected as a consequence of natural selection. A population component of regression on environmental covariates, however, was significant and could be exploited through breeding to improve adaptation of perennial ryegrass to either drier or warmer regions.