Bioinformatic analysis of ESTs collected by Sanger and pyrosequencing methods for a keystone forest tree species: oak

<p>Abstract</p> <p>Background</p> <p>The Fagaceae family comprises about 1,000 woody species worldwide. About half belong to the <it>Quercus </it>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.</p> <p>Results</p> <p>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 <url>http://genotoul-contigbrowser.toulouse.inra.fr:9092/Quercus_robur/index.html</url>.</p> <p>Conclusions</p> <p>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.</p

Génomique et diversité du débourrement chez les chênes

La productivité et la distribution des espèces longévives telles que les arbres forestiers, sont intimement liées à la phénologie, et en particulier à la date de débourrement. Sous l’effet du réchauffement climatique, le processus de débourrement pourrait être fortement affecté, et la question de l’adaptation des essences forestières à ces changements se pose clairement. Leur capacité d’adaptation dépend, pour une part, de la diversité génétique présente dans les populations naturelles. Cette thèse a pour objectif de déterminer les régions génomiques contrôlant le débourrement chez les chênes afin de mettre au point des outils d’évaluation de la diversité adaptative de ce caractère. Sur le plan des méthodes, nous avons procédé en trois étapes, associant des approches génétiques et moléculaires. Nous avons tout d’abord suivi une approche transcriptomique pour identifier des gènes candidats (GC) pertinents au regard de leur profil d’expression au cours du débourrement chez le chêne sessile (Quercus petraea). Cette étape a été menée à l’aide de banques SSH, d’expériences de macroarrays et de RT-PCR quantitative. Dans un deuxième temps, ce lot de gènes candidats et certains GC identifiés dans la littérature, ont été cartographiés chez Quercus robur (chêne pédonculé) et Castanea sativa (châtaignier européen), et leur position comparée à celle des QTL impliqués dans le débourrement et conservés chez les deux espèces. Une étude de diversité au niveau nucléotidique a été menée sur sept de ces GC, dont deux ont fait l’objet d’une étude d’association entre les variants nucléotidiques et la date de débourrement. L’étude a permis la mise en évidence des processus fonctionnels régulés au cours du débourrement, ainsi que l’identification d’un certain nombre de transcrits sans équivalent dans les bases de données. Le caractère de débourrement apparaît remarquablement conservé entre le chêne et le châtaignier et les EST se sont révélés un formidable outil pour les études de cartographie comparée. Le niveau de diversité moyen est élevé au sein des locus analysés, dont certains présentent une structure compatible (niveaux de différenciation, écart à la neutralité) avec l’action de pressions de sélection liées au débourrement. Cependant, aucune association significative n’a été détectée entre variants nucléotidiques et note de débourrement. Ce travail a permis d’aboutir à un certain nombre de résultats intéressants et encourageants concernant le déterminisme génétique du caractère de débourrement et ouvrent des perspectives pour l’étude de la diversité adaptative dans les populations naturelles.Productivity and distribution of long-lived species, such as forest trees, are closely related to phenological events, especially bud burst. Forest trees may be greatly affected by global warming in the near future and their adaptation is challenged by the rapid climatic changes. Their capacity to respond will depend on the levels of genetic diversity in natural populations. The main objective of this thesis is to identify genes contributing to the variability of bud burst in oaks in order to develop tools for assessing diversity of these genes in natural populations. We used a three steps strategy, by using genetic and molecular approaches to identify genes of interest. First, we followed a transcriptomic approach in sessile oak (Quercus petraea) to identify relevant expressional candidate genes (CG), by using SSH libraries, macroarrays experiments and quantitative RTPCR. These candidate genes, complemented by genes already published, were mapped for Quercus robur (pedunculate oak) and Castanea sativa (european chestnut) on existing genetic maps and their locations compared to those of QTLs of bud burst. Then, nucleotide diversity was assessed for seven CG, and we studied associations between nucleotide diversity and the date of bud burst for two of them. During this study, an inventory was made of transcripts that are differentially expressed during bud burst, some of these transcripts were reported for the first time. We also used EST as markers to align linkage groups between oak and chestnut, and we showed that QTLs of bud burst are remarkably conserved between the two species. When compared to other species and genes, average nucleotide diversity is high in oaks across the seven studied loci. Some genes exhibited diversity patterns (differentiation levels, departure from neutrality) suggesting that selection might be involved as an evolutionary force. However, no significant association between nucleotide variants and bud burst score has been found. This work has produced several interesting and promising results that can be implemented for future research on adaptive diversity in oaks

Génomique et diversité du débourrement chez les chênes

La productivité et la distribution des espèces longévives telles que les arbres forestiers, sont intimement liées à la phénologie, et en particulier à la date de débourrement. Sous l'effet du réchauffement climatique, le processus de débourrement pourrait être fortement affecté, et la question de l'adaptation des essences forestières à ces changements se pose clairement. Leur capacité d'adaptation dépend, pour une part, de la diversité génétique présente dans les populations naturelles. Cette thèse a pour objectif de déterminer les régions génomiques contrôlant le débourrement chez les chênes afin de mettre au point des outils d'évaluation de la diversité adaptative de ce caractère. Sur le plan des méthodes, nous avons procédé en trois étapes, associant des approches génétiques et moléculaires. Nous avons tout d'abord suivi une approche transcriptomique pour identifier des gènes candidats (GC) pertinents au regard de leur profil d'expression au cours du débourrement chez le chêne sessile (Quercus petraea). Cette étape a été menée à l'aide de banques SSH, d'expériences de macroarrays et de RT-PCR quantitative. Dans un deuxième temps, ce lot de gènes candidats et certains GC identifiés dans la littérature, ont été cartographiés chez Quercus robur (chêne pédonculé) et Castanea sativa (châtaignier européen), et leur position comparée à celle des QTL impliqués dans le débourrement et conservés chez les deux espèces. Une étude de diversité au niveau nucléotidique a été menée sur sept de ces GC, dont deux ont fait l'objet d'une étude d'association entre les variants nucléotidiques et la date de débourrement. L'étude a permis la mise en évidence des processus fonctionnels régulés au cours du débourrement, ainsi que l'identification d'un certain nombre de transcrits sans équivalent dans les bases de données. Le caractère de débourrement apparaît remarquablement conservé entre le chêne et le châtaignier et les EST se sont révélés un formidable outil pour les études de cartographie comparée. Le niveau de diversité moyen est élevé au sein des locus analysés, dont certains présentent une structure compatible (niveaux de différenciation, écart à la neutralité) avec l'action de pressions de sélection liées au débourrement. Cependant, aucune association significative n'a été détectée entre variants nucléotidiques et note de débourrement. Ce travail a permis d'aboutir à un certain nombre de résultats intéressants et encourageants concernant le déterminisme génétique du caractère de débourrement et ouvrent des perspectives pour l'étude de la diversité adaptative dans les populations naturelles.BORDEAUX1-BU Sciences-Talence (335222101) / SudocSudocFranceF

Imprints of natural selection along environmental gradients in phenology-related genes of Quercus petraea

We explored single nucleotide polymorphism (SNP) variation in candidate genes for bud burst from Quercus petraea populations sampled along gradients of latitude and altitude in Western Europe. SNP diversity was monitored for 106 candidate genes, in 758 individuals from 32 natural populations. We investigated whether SNP variation reflected the clinal pattern of bud burst observed in common garden experiments. We used different methods to detect imprints of natural selection (FST outlier, clinal variation at allelic frequencies, association tests) and compared the results obtained for the two gradients. FST outlier SNPs were found in 15 genes, 5 of which were common to both gradients. The type of selection differed between the two gradients (directional or balancing) for 3 of these 5. Clinal variations were observed for six SNPs, and one cline was conserved across both gradients. Association tests between the phenotypic or breeding values of trees and SNP genotypes identified 14 significant associations, involving 12 genes. The results of outlier detection on the basis of population differentiation or clinal variation were not very consistent with the results of association tests. The discrepancies between these approaches may reflect the different hierarchical levels of selection considered (inter- and intrapopulation selection). Finally, we obtained evidence for convergent selection (similar for gradients) and clinal variation for a few genes, suggesting that comparisons between parallel gradients could be used to screen for major candidate genes responding to natural selection in trees

Imprints of natural selection along environmental gradients in phenology-related genes of Quercus petraea

We explored single nucleotide polymorphism (SNP) variation in candidate genes for bud burst from Quercus petraea populations sampled along gradients of latitude and altitude in Western Europe. SNP diversity was monitored for 106 candidate genes, in 758 individuals from 32 natural populations. We investigated whether SNP variation reflected the clinal pattern of bud burst observed in common garden experiments. We used different methods to detect imprints of natural selection (FST outlier, clinal variation at allelic frequencies, association tests) and compared the results obtained for the two gradients. FST outlier SNPs were found in 15 genes, 5 of which were common to both gradients. The type of selection differed between the two gradients (directional or balancing) for 3 of these 5. Clinal variations were observed for six SNPs, and one cline was conserved across both gradients. Association tests between the phenotypic or breeding values of trees and SNP genotypes identified 14 significant associations, involving 12 genes. The results of outlier detection on the basis of population differentiation or clinal variation were not very consistent with the results of association tests. The discrepancies between these approaches may reflect the different hierarchical levels of selection considered (inter- and intrapopulation selection). Finally, we obtained evidence for convergent selection (similar for gradients) and clinal variation for a few genes, suggesting that comparisons between parallel gradients could be used to screen for major candidate genes responding to natural selection in trees

Transcriptional changes in two types of pre-mycorrhizal roots and in ectomycorrhizas of oak microcuttings inoculated with Piloderma croceum

The formation of the ectomycorrhizaimplies an alteration in gene expression of both theplant and fungal partners, a process which starts beforethe formation of any symbiotic interface. However, littleis known on the regulation pattern occurring indiVerent parts of the root system. Our experimentalsystem consisting of a micropropagated oak with ahierarchical root system was shown to exhibit symbiosisfunctional traits prior to any mycorrhizal tissuediVerentiation after the inoculation with the basidiomycetePiloderma croceum. Using a cDNA array, theplant gene regulation was analyzed in the pre-mycorrhizalphase. Seventy-Wve transcripts showed diVerentialexpression in pre-mycorrhizal lateral and principalroots, and both root types exhibited diVerent sets ofresponsive genes. For transcripts selected according toa statistical analysis, the alteration in gene expressionwas conWrmed by RT-PCR and quantitative real-timePCR. Genes regulated in pre-mycorrhizal lateral rootsdisplayed an almost identical expression in mycorrhizas.In contrast, genes regulated in pre-mycorrhizalprincipal roots were often regulated diVerently in ectomycorrhizas.Down-regulation aVected most of the regulatedgenes involved in metabolism, whereas most ofthe regulated genes related to cell rescue functions,water regulation and defence response were up-regulated.Regulation of such genes could explain theincrease of global resistance observed in mycorrhizalplants

Comparison of quantitative trait loci for adaptive traits between oak and chestnut based on an expressed sequence tag consensus map

A comparative genetic and QTL mapping was performed between Quercus robur L. and Castanea sativa Mill., two major forest tree species belonging to the Fagaceae family. Oak EST-derived markers (STSs) were used to align the 12 linkage groups of the two species. Fifty-one and 45 STSs were mapped in oak and chestnut, respectively. These STSs, added to SSR markers previously mapped in both species, provided a total number of 55 orthologous molecular markers for comparative mapping within the Fagaceae family. Homeologous genomic regions identified between oak and chestnut allowed us to compare QTL positions for three important adaptive traits. Colocation of the QTL controlling the timing of bud burst was significant between the two species. However, conservation of QTL for height growth was not supported by statistical tests. No QTL for carbon isotope discrimination was conserved between the two species. Putative candidate genes for bud burst can be identified on the basis of colocations between EST-derived markers and QT

Population genomics of phenological clines in sessile oak (Quercus petraea (matt.) Leibl.)

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