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1.  Winemaking and Bioprocesses Strongly Shaped the Genetic Diversity of the Ubiquitous Yeast Torulaspora delbrueckii 
PLoS ONE  2014;9(4):e94246.
The yeast Torulaspora delbrueckii is associated with several human activities including oenology, bakery, distillery, dairy industry, etc. In addition to its biotechnological applications, T. delbrueckii is frequently isolated in natural environments (plant, soil, insect). T. delbrueckii is thus a remarkable ubiquitous yeast species with both wild and anthropic habitats, and appears to be a perfect yeast model to search for evidence of human domestication. For that purpose, we developed eight microsatellite markers that were used for the genotyping of 110 strains from various substrates and geographical origins. Microsatellite analysis showed four genetic clusters: two groups contained most nature strains from Old World and Americas respectively, and two clusters were associated with winemaking and other bioprocesses. Analysis of molecular variance (AMOVA) confirmed that human activities significantly shaped the genetic variability of T. delbrueckii species. Natural isolates are differentiated on the basis of geographical localisation, as expected for wild population. The domestication of T. delbrueckii probably dates back to the Roman Empire for winemaking (∼1900 years ago), and to the Neolithic era for bioprocesses (∼4000 years ago). Microsatellite analysis also provided valuable data regarding the life-cycle of the species, suggesting a mostly diploid homothallic life. In addition to population genetics and ecological studies, the microsatellite tool will be particularly useful for further biotechnological development of T. delbrueckii strains for winemaking and other bioprocesses.
PMCID: PMC3981792  PMID: 24718638
2.  High-density linkage mapping in a pine tree reveals a genomic region associated with inbreeding depression and provides clues to the extent and distribution of meiotic recombination 
BMC Biology  2013;11:50.
The availability of a large expressed sequence tags (EST) resource and recent advances in high-throughput genotyping technology have made it possible to develop highly multiplexed SNP arrays for multi-objective genetic applications, including the construction of meiotic maps. Such approaches are particularly useful in species with a large genome size, precluding the use of whole-genome shotgun assembly with current technologies.
In this study, a 12 k-SNP genotyping array was developed for maritime pine from an extensive EST resource assembled into a unigene set. The offspring of three-generation outbred and inbred mapping pedigrees were then genotyped. The inbred pedigree consisted of a classical F2 population resulting from the selfing of a single inter-provenance (Landes x Corsica) hybrid tree, whereas the outbred pedigree (G2) resulted from a controlled cross of two intra-provenance (Landes x Landes) hybrid trees. This resulted in the generation of three linkage maps based on SNP markers: one from the parental genotype of the F2 population (1,131 markers in 1,708 centimorgan (cM)), and one for each parent of the G2 population (1,015 and 1,110 markers in 1,447 and 1,425 cM for the female and male parents, respectively). A comparison of segregation patterns in the progeny obtained from the two types of mating (inbreeding and outbreeding) led to the identification of a chromosomal region carrying an embryo viability locus with a semi-lethal allele. Following selfing and segregation, zygote mortality resulted in a deficit of Corsican homozygous genotypes in the F2 population. This dataset was also used to study the extent and distribution of meiotic recombination along the length of the chromosomes and the effect of sex and/or genetic background on recombination. The genetic background of trees in which meiotic recombination occurred was found to have a significant effect on the frequency of recombination. Furthermore, only a small proportion of the recombination hot- and cold-spots were common to all three genotypes, suggesting that the spatial pattern of recombination was genetically variable.
This study led to the development of classical genomic tools for this ecologically and economically important species. It also identified a chromosomal region bearing a semi-lethal recessive allele and demonstrated the genetic variability of recombination rate over the genome.
PMCID: PMC3660193  PMID: 23597128
Unigene; SNP array; Linkage mapping; Segregation distortion; Recombination; Maritime pine; Pinus pinaster
3.  Bioinformatic analysis of ESTs collected by Sanger and pyrosequencing methods for a keystone forest tree species: oak 
BMC Genomics  2010;11:650.
The Fagaceae family comprises about 1,000 woody species worldwide. About half belong to the Quercus family. These oaks are often a source of raw material for biomass wood and fiber. Pedunculate and sessile oaks, are among the most important deciduous forest tree species in Europe. Despite their ecological and economical importance, very few genomic resources have yet been generated for these species. Here, we describe the development of an EST catalogue that will support ecosystem genomics studies, where geneticists, ecophysiologists, molecular biologists and ecologists join their efforts for understanding, monitoring and predicting functional genetic diversity.
We generated 145,827 sequence reads from 20 cDNA libraries using the Sanger method. Unexploitable chromatograms and quality checking lead us to eliminate 19,941 sequences. Finally a total of 125,925 ESTs were retained from 111,361 cDNA clones. Pyrosequencing was also conducted for 14 libraries, generating 1,948,579 reads, from which 370,566 sequences (19.0%) were eliminated, resulting in 1,578,192 sequences. Following clustering and assembly using TGICL pipeline, 1,704,117 EST sequences collapsed into 69,154 tentative contigs and 153,517 singletons, providing 222,671 non-redundant sequences (including alternative transcripts). We also assembled the sequences using MIRA and PartiGene software and compared the three unigene sets. Gene ontology annotation was then assigned to 29,303 unigene elements. Blast search against the SWISS-PROT database revealed putative homologs for 32,810 (14.7%) unigene elements, but more extensive search with Pfam, Refseq_protein, Refseq_RNA and eight gene indices revealed homology for 67.4% of them. The EST catalogue was examined for putative homologs of candidate genes involved in bud phenology, cuticle formation, phenylpropanoids biosynthesis and cell wall formation. Our results suggest a good coverage of genes involved in these traits. Comparative orthologous sequences (COS) with other plant gene models were identified and allow to unravel the oak paleo-history. Simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were searched, resulting in 52,834 SSRs and 36,411 SNPs. All of these are available through the Oak Contig Browser
This genomic resource provides a unique tool to discover genes of interest, study the oak transcriptome, and develop new markers to investigate functional diversity in natural populations.
PMCID: PMC3017864  PMID: 21092232
4.  (Not) Keeping the stem straight: a proteomic analysis of maritime pine seedlings undergoing phototropism and gravitropism 
BMC Plant Biology  2010;10:217.
Plants are subjected to continuous stimuli from the environment and have evolved an ability to respond through various growth and development processes. Phototropism and gravitropism responses enable the plant to reorient with regard to light and gravity.
We quantified the speed of maritime pine seedlings to reorient with regard to light and gravity over 22 days. Seedlings were inclined at 15, 30 and 45 degrees with vertical plants as controls. A lateral light source illuminated the plants and stem movement over time was recorded. Depending on the initial angle of stem lean, the apical response to the lateral light source differed. In control and 15° inclined plants, the apex turned directly towards the light source after only 2 h. In plants inclined at 30° and 45°, the apex first reoriented in the vertical plane after 2 h, then turned towards the light source after 24 h. Two-dimensional gel electrophoresis coupled with mass spectrometry was then used to describe the molecular response of stem bending involved in photo- and gravi-tropism after 22 hr and 8 days of treatment. A total of 486 spots were quantitatively analyzed using image analysis software. Significant changes were determined in the protein accumulation of 68 protein spots. Early response gravitropic associated proteins were identified, which are known to function in energy related and primary metabolism. A group of thirty eight proteins were found to be involved in primary metabolism and energy related metabolic pathways. Degradation of Rubisco was implicated in some protein shifts.
Our study demonstrates a rapid gravitropic response in apices of maritime pine seedlings inclined >30°. Little or no response was observed at the stem bases of the same plants. The primary gravitropic response is concomitant with a modification of the proteome, consisting of an over accumulation of energy and metabolism associated proteins, which may allow the stem to reorient rapidly after bending.
PMCID: PMC3017815  PMID: 20925929
5.  Population structure and genetic bottleneck in sweet cherry estimated with SSRs and the gametophytic self-incompatibility locus 
BMC Genetics  2010;11:77.
Domestication and breeding involve the selection of particular phenotypes, limiting the genomic diversity of the population and creating a bottleneck. These effects can be precisely estimated when the location of domestication is established. Few analyses have focused on understanding the genetic consequences of domestication and breeding in fruit trees. In this study, we aimed to analyse genetic structure and changes in the diversity in sweet cherry Prunus avium L.
Three subgroups were detected in sweet cherry, with one group of landraces genetically very close to the analysed wild cherry population. A limited number of SSR markers displayed deviations from the frequencies expected under neutrality. After the removal of these markers from the analysis, a very limited bottleneck was detected between wild cherries and sweet cherry landraces, with a much more pronounced bottleneck between sweet cherry landraces and modern sweet cherry varieties. The loss of diversity between wild cherries and sweet cherry landraces at the S-locus was more significant than that for microsatellites. Particularly high levels of differentiation were observed for some S-alleles.
Several domestication events may have happened in sweet cherry or/and intense gene flow from local wild cherry was probably maintained along the evolutionary history of the species. A marked bottleneck due to breeding was detected, with all markers, in the modern sweet cherry gene pool. The microsatellites did not detect the bottleneck due to domestication in the analysed sample. The vegetative propagation specific to some fruit trees may account for the differences in diversity observed at the S-locus. Our study provides insights into domestication events of cherry, however, requires confirmation on a larger sampling scheme for both sweet cherry landraces and wild cherry.
PMCID: PMC2933703  PMID: 20727153
6.  In Vitro vs In Silico Detected SNPs for the Development of a Genotyping Array: What Can We Learn from a Non-Model Species? 
PLoS ONE  2010;5(6):e11034.
There is considerable interest in the high-throughput discovery and genotyping of single nucleotide polymorphisms (SNPs) to accelerate genetic mapping and enable association studies. This study provides an assessment of EST-derived and resequencing-derived SNP quality in maritime pine (Pinus pinaster Ait.), a conifer characterized by a huge genome size (∼23.8 Gb/C).
Methodology/Principal Findings
A 384-SNPs GoldenGate genotyping array was built from i/ 184 SNPs originally detected in a set of 40 re-sequenced candidate genes (in vitro SNPs), chosen on the basis of functionality scores, presence of neighboring polymorphisms, minor allele frequencies and linkage disequilibrium and ii/ 200 SNPs screened from ESTs (in silico SNPs) selected based on the number of ESTs used for SNP detection, the SNP minor allele frequency and the quality of SNP flanking sequences. The global success rate of the assay was 66.9%, and a conversion rate (considering only polymorphic SNPs) of 51% was achieved. In vitro SNPs showed significantly higher genotyping-success and conversion rates than in silico SNPs (+11.5% and +18.5%, respectively). The reproducibility was 100%, and the genotyping error rate very low (0.54%, dropping down to 0.06% when removing four SNPs showing elevated error rates).
This study demonstrates that ESTs provide a resource for SNP identification in non-model species, which do not require any additional bench work and little bio-informatics analysis. However, the time and cost benefits of in silico SNPs are counterbalanced by a lower conversion rate than in vitro SNPs. This drawback is acceptable for population-based experiments, but could be dramatic in experiments involving samples from narrow genetic backgrounds. In addition, we showed that both the visual inspection of genotyping clusters and the estimation of a per SNP error rate should help identify markers that are not suitable to the GoldenGate technology in species characterized by a large and complex genome.
PMCID: PMC2882948  PMID: 20543950

Results 1-6 (6)