Structural and functional evolution of genes in conifers

Le développement de nouvelles techniques a accéléré l'exploration structurale et fonctionnelle des génomes des conifères et contribué à l’étude de leur physiologie et leur adaptation aux conditions environnementales. Cette thèse s’intéresse à l’évolution des gènes chez les conifères et (i) fait le point sur les facteurs génomiques qui ont influencé la structure des gènes et (ii) analyse une grande famille de gènes impliqués dans la tolérance à la sécheresse, les déhydrines. Notre étude de la structure génique s’est fait à partir de diverses séquences de l’épinette blanche (Picea glauca [Moench] Voss) provenant de clones BAC, de l'assemblage du génome et de l’espace génique obtenu à partir de la technologie de «sequence capture». Par le biais d’analyses comparatives, nous avons observé que les conifères présentent plus de séquences introniques par gène que la plupart des plantes à fleurs (angiospermes) et que la longueur moyenne des introns n'était pas directement corrélée à la taille du génome. Nous avons constaté que les éléments répétitifs qui sont responsables de la très grande taille des génomes des conifères affectent également l'évolution des exons et des introns. Dans la deuxième partie de la thèse, nous avons entrepris la première analyse exhaustive de la famille des gènes des déhydrines chez les conifères. Les analyses phylogénétiques ont indiqué l'apparition d'une série de duplications de gènes dont une duplication qui a provoqué l'expansion de la famille génique spécifiquement au sein du genre Picea. L’analyse démontre que les déhydrines ont une structure modulaire et présentent chez les conifères des agencements variés de différents motifs d'acides aminés. Ces structures sont particulièrement diverses chez l'épinette et sont associées à différents patrons d'expression en réponse à la sècheresse. Dans l’ensemble, nos résultats suggèrent que l'évolution de la structure génique est dynamique chez les conifères alors que l'évolution des chromosomes est largement reconnue comme étant lente chez ceux-ci. Ils indiquent aussi que l'expansion et la diversification des familles de gènes liés à l'adaptation, comme les déhydrines, pourraient conférer de la plasticité phénotypique permettant de répondre aux changements environnementaux au cours du long cycle de vie qui est typique de plusieurs conifères.Technical advances have accelerated the structural and functional exploration of conifer genomes and opened up new approaches to study their physiology and adaptation to environmental conditions. This thesis focuses on the evolution of conifer genes and explores (i) the genomic factors that have impacted the evolution of gene structure and (ii) the evolution of a large gene family involved in drought tolerance, the dehydrins. The analysis of gene structure was based on white spruce (Picea glauca [Moench] Voss) sequence data from BAC clones, the genome assembly and the gene space obtained from sequence capture. Through comparative analyses, we found that conifers presented more intronic sequence per gene than most flowering plants (angiosperms) and that the average intron length was not directly correlated to genome size. We found that repetitive elements, which are responsible for the very large size of conifer genomes, also affect the evolution of exons and introns. In the second part of the thesis, we undertook the first exhaustive analysis of the dehydrin gene family in conifers. The phylogenetic analyses indicated the occurrence of a series of gene duplications in conifers and a major lineage duplication, which caused the expansion of the dehydrin family in the genus Picea. Conifer dehydrins have an array of modular amino acid structures, and in spruce, these structures are particularly diverse and are associated with different expression patterns in response to dehydration stress. Taken together, our findings suggest that the evolution of gene structure is dynamic in conifers, which contrast with a widely accepted slow rate of chromosome evolution. They further indicate that the expansion and diversification of adaptation-related genes, like the dehydrins in spruce, may confer the phenotypic plasticity to respond to the environmental changes during their long life span

Exploring the loblolly pine (Pinus taeda L.) genome by BAC sequencing and Cot analysis.

Loblolly pine (LP; Pinus taeda L.) is an economically and ecologically important tree in the southeastern U.S. To advance understanding of the loblolly pine (LP; Pinus taeda L.) genome, we sequenced and analyzed 100 BAC clones and performed a Cot analysis. The Cot analysis indicates that the genome is composed of 57, 24, and 10% highly-repetitive, moderately-repetitive, and single/low-copy sequences, respectively (the remaining 9% of the genome is a combination of fold back and damaged DNA). Although single/low-copy DNA only accounts for 10% of the LP genome, the amount of single/low-copy DNA in LP is still 14 times the size of the Arabidopsis genome. Since gene numbers in LP are similar to those in Arabidopsis, much of the single/low-copy DNA of LP would appear to be composed of DNA that is both gene- and repeat-poor. Macroarrays prepared from a LP bacterial artificial chromosome (BAC) library were hybridized with probes designed from cell wall synthesis/wood development cDNAs, and 50 of the "targeted" clones were selected for further analysis. An additional 25 clones were selected because they contained few repeats, while 25 more clones were selected at random. The 100 BAC clones were Sanger sequenced and assembled. Of the targeted BACs, 80% contained all or part of the cDNA used to target them. One targeted BAC was found to contain fungal DNA and was eliminated from further analysis. Combinations of similarity-based and ab initio gene prediction approaches were utilized to identify and characterize potential coding regions in the 99 BACs containing LP DNA. From this analysis, we identified 154 gene models (GMs) representing both putative protein-coding genes and likely pseudogenes. Ten of the GMs (all of which were specifically targeted) had enough support to be classified as intact genes. Interestingly, the 154 GMs had statistically indistinguishable (α = 0.05) distributions in the targeted and random BAC clones (15.18 and 12.61 GM/Mb, respectively), whereas the low-repeat BACs contained significantly fewer GMs (7.08 GM/Mb). However, when GM length was considered, the targeted BACs had a significantly greater percentage of their length in GMs (3.26%) when compared to random (1.63%) and low-repeat (0.62%) BACs. The results of our study provide insight into LP evolution and inform ongoing efforts to produce a reference genome sequence for LP, while characterization of genes involved in cell wall production highlights carbon metabolism pathways that can be leveraged for increasing wood production

De novo SNP discovery and genetic linkage mapping in poplar using restriction site associated DNA and whole-genome sequencing technologies

Detailed information on genetic distance and linkage phase between adjacent SNP markers on the genetic linkage map of the female P. deltoides ‘I-69’. The corresponding identical SNPs identified based on the P. trichocarpa reference genome are also included. (XLS 452 kb

Insect herbivory (Choristoneura fumiferana, Tortricidea) underlies tree population structure (Picea glauca, Pinaceae)

Variation in insect herbivory can lead to population structure in plant hosts as indicated by defence traits. In annual herbaceous, defence traits may vary between geographic areas but evidence of such patterns is lacking for long-lived species. This may result from the variety of selection pressures from herbivores, long distance gene flow, genome properties, and lack of research. We investigated the antagonistic interaction between white spruce (Picea glauca) and spruce budworm (SBW, Choristoneura fumiferana) the most devastating forest insect of eastern North America in common garden experiments. White spruces that are able to resist SBW attack were reported to accumulate the acetophenones piceol and pungenol constitutively in their foliage. We show that levels of these acetophenones and transcripts of the gene responsible for their release is highly heritable and that their accumulation is synchronized with the most devastating stage of SBW. Piceol and pungenol concentrations negatively correlate with rate of development in female SBW and follow a non-random geographic variation pattern that is partially explained by historical damage from SBW and temperature. Our results show that accumulation of acetophenones is an efficient resistance mechanism against SBW in white spruce and that insects can affect population structure of a long-lived plant

Utilization of Tissue Ploidy Level Variation in de Novo Transcriptome Assembly of Pinus sylvestris

Compared to angiosperms, gymnosperms lag behind in the availability of assembled and annotated genomes. Most genomic analyses in gymnosperms, especially conifer tree species, rely on the use of de novo assembled transcriptomes. However, the level of allelic redundancy and transcript fragmentation in these assembled transcriptomes, and their effect on downstream applications have not been fully investigated. Here, we assessed three assembly strategies for short-reads data, including the utility of haploid megagametophyte tissue during de novo assembly as single-allele guides, for six individuals and five different tissues in Pinus sylvestris. We then contrasted haploid and diploid tissue genotype calls obtained from the assembled transcriptomes to evaluate the extent of paralog mapping. The use of the haploid tissue during assembly increased its completeness without reducing the number of assembled transcripts. Our results suggest that current strategies that rely on available genomic resources as guidance to minimize allelic redundancy are less effective than the application of strategies that cluster redundant assembled transcripts. The strategy yielding the lowest levels of allelic redundancy among the assembled transcriptomes assessed here was the generation of SuperTranscripts with Lace followed by CD-HIT clustering. However, we still observed some levels of heterozygosity (multiple gene fragments per transcript reflecting allelic redundancy) in this assembled transcriptome on the haploid tissue, indicating that further filtering is required before using these assemblies for downstream applications. We discuss the influence of allelic redundancy when these reference transcriptomes are used to select regions for probe design of exome capture baits and for estimation of population genetic diversity.Peer reviewe