High-Throughput Genome-Wide Genotyping To Optimize the Use of Natural Genetic Resources in the Grassland Species Perennial Ryegrass (Lolium perenne L.).

The natural genetic diversity of agricultural species is an essential genetic resource for breeding programs aiming to improve their ecosystem and production services. A large natural ecotype diversity is usually available for most grassland species. This could be used to recombine natural climatic adaptations and agronomic value to create improved populations of grassland species adapted to future regional climates. However describing natural genetic resources can be long and costly. Molecular markers may provide useful information to help this task. This opportunity was investigated for Lolium perenne L., using a set of 385 accessions from the natural diversity of this species collected right across Europe and provided by genebanks of several countries. For each of these populations, genotyping provided the allele frequencies of 189,781 SNP markers. GWAS were implemented for over 30 agronomic and/or putatively adaptive traits recorded in three climatically contrasted locations (France, Belgium, Germany). Significant associations were detected for hundreds of markers despite a strong confounding effect of the genetic background; most of them pertained to phenology traits. It is likely that genetic variability in these traits has had an important contribution to environmental adaptation and ecotype differentiation. Genomic prediction models calibrated using natural diversity were found to be highly effective to describe natural populations for almost all traits as well as commercial synthetic populations for some important traits such as disease resistance, spring growth or phenological traits. These results will certainly be valuable information to help the use of natural genetic resources of other species.

Local Adaptation in European Firs Assessed through Extensive Sampling across Altitudinal Gradients in Southern Europe.

BACKGROUND: Local adaptation is a key driver of phenotypic and genetic divergence at loci responsible for adaptive traits variations in forest tree populations. Its experimental assessment requires rigorous sampling strategies such as those involving population pairs replicated across broad spatial scales. METHODS: A hierarchical Bayesian model of selection (HBM) that explicitly considers both the replication of the environmental contrast and the hierarchical genetic structure among replicated study sites is introduced. Its power was assessed through simulations and compared to classical 'within-site' approaches (FDIST, BAYESCAN) and a simplified, within-site, version of the model introduced here (SBM). RESULTS: HBM demonstrates that hierarchical approaches are very powerful to detect replicated patterns of adaptive divergence with low false-discovery (FDR) and false-non-discovery (FNR) rates compared to the analysis of different sites separately through within-site approaches. The hypothesis of local adaptation to altitude was further addressed by analyzing replicated Abies alba population pairs (low and high elevations) across the species' southern distribution range, where the effects of climatic selection are expected to be the strongest. For comparison, a single population pair from the closely related species A. cephalonica was also analyzed. The hierarchical model did not detect any pattern of adaptive divergence to altitude replicated in the different study sites. Instead, idiosyncratic patterns of local adaptation among sites were detected by within-site approaches. CONCLUSION: Hierarchical approaches may miss idiosyncratic patterns of adaptation among sites, and we strongly recommend the use of both hierarchical (multi-site) and classical (within-site) approaches when addressing the question of adaptation across broad spatial scales.

Evidence of divergent selection for drought and cold tolerance at landscape and local scales in Abies alba Mill. in the French Mediterranean Alps.

Understanding local adaptation in forest trees is currently a key research and societal priority. Geographically and ecologically marginal populations provide ideal case studies, because environmental stress along with reduced gene flow can facilitate the establishment of locally adapted populations. We sampled European silver fir (Abies alba Mill.) trees in the French Mediterranean Alps, along the margin of its distribution range, from pairs of high- and low-elevation plots on four different mountains situated along a 170-km east-west transect. The analysis of 267 SNP loci from 175 candidate genes suggested a neutral pattern of east-west isolation by distance among mountain sites. F(ST) outlier tests revealed 16 SNPs that showed patterns of divergent selection. Plot climate was characterized using both in situ measurements and gridded data that revealed marked differences between and within mountains with different trends depending on the season. Association between allelic frequencies and bioclimatic variables revealed eight genes that contained candidate SNPs, of which two were also detected using F(ST) outlier methods. All SNPs were associated with winter drought, and one of them showed strong evidence of selection with respect to elevation. Q(ST)-F(ST) tests for fitness-related traits measured in a common garden suggested adaptive divergence for the date of bud flush and for growth rate. Overall, our results suggest a complex adaptive picture for A. alba in the southern French Alps where, during the east-to-west Holocene recolonization, locally advantageous genetic variants established at both the landscape and local scales.

Annotation and re-sequencing of genes from de novo transcriptome assembly of Abies alba (Pinaceae).

PREMISE OF THE STUDY: We present a protocol for the annotation of transcriptome sequence data and the identification of candidate genes therein using the example of the nonmodel conifer Abies alba. • METHODS AND RESULTS: A normalized cDNA library was built from an A. alba seedling. The sequencing on a 454 platform yielded more than 1.5 million reads that were de novo assembled into 25149 contigs. Two complementary approaches were applied to annotate gene fragments that code for (1) well-known proteins and (2) proteins that are potentially adaptively relevant. Primer development and testing yielded 88 amplicons that could successfully be resequenced from genomic DNA. • CONCLUSIONS: The annotation workflow offers an efficient way to identify potential adaptively relevant genes from the large quantity of transcriptome sequence data. The primer set presented should be prioritized for single-nucleotide polymorphism detection in adaptively relevant genes in A. alba.

Characterization of microsatellite loci and reliable genotyping in a polyploid plant, Mercurialis perennis (Euphorbiaceae).

For many applications in population genetics, codominant simple sequence repeats (SSRs) may have substantial advantages over dominant anonymous markers such as amplified fragment length polymorphisms (AFLPs). In high polyploids, however, allele dosage of SSRs cannot easily be determined and alleles are not easily attributable to potentially diploidized loci. Here, we argue that SSRs may nonetheless be better than AFLPs for polyploid taxa if they are analyzed as effectively dominant markers because they are more reliable and more precise. We describe the transfer of SSRs developed for diploid Mercurialis huetii to the clonal dioecious M. perennis. Primers were tested on a set of 54 male and female plants from natural decaploid populations. Eight of 65 tested loci produced polymorphic fragments. Binary profiles from 4 different scoring routines were used to define multilocus lineages (MLLs). Allowing for fragment differences within 1 MLL, all analyses revealed the same 14 MLLs without conflicting with merigenet, sex, or plot assignment. For semiautomatic scoring, a combination of as few as 2 of the 4 most polymorphic loci resulted in unambiguous discrimination of clones. Our study demonstrates that microsatellite fingerprinting of polyploid plants is a cost efficient and reliable alternative to AFLPs, not least because fewer loci are required than for diploids.