Conifers have dominated forests for more than 200 million years and are of huge ecological and economic importance. Here we present the draft assembly of the 20-gigabase genome of Norway spruce (Picea abies), the first available for any gymnosperm. The number of well-supported genes (28,354) is similar to the .100 times smaller genome of Arabidopsis thaliana, and there is no evidence of a recent whole-genome duplication in the gymnosperm lineage. Instead, the large genome size seems to result from the slow and steady accumulation of a diverse set of long-terminal repeat transposable elements, possibly owing to the lack of an efficient elimination mechanism. Comparative sequencing of Pinus sylvestris, Abies sibirica, Juniperus communis, Taxus baccata and Gnetum gnemon reveals that the transposable element diversity is shared among extant conifers. Expression of 24-nucleotide small RNAs, previously implicated in transposable element silencing, is tissue-specific and much lower than in other plants. We further identify numerous long (.10,000 base pairs) introns, gene-like fragments, uncharacterized long non-coding RNAs and short RNAs. This opens up new genomic avenues for conifer forestry and breeding.

The NLRs or NBS-LRRs (nucleotide-binding, leucine-rich-repeat) form the largest resistance gene family in plants, with lineage-specific contingents of TNL, CNL and RNL subfamilies and a central role in resilience to stress. The origin, evolution and distribution of NLR sequences has been unclear owing in part to the variable size and diversity of the RNL subfamily and a lack of data in Gymnosperms. We developed, searched and annotated transcriptomes assemblies of seven conifers and identified a resource of 3816 expressed NLR sequences. Our analyses encompassed sequences data spanning the major groups of land plants and determinations of NLR transcripts levels in response to drought in white spruce. We showed that conifers have among the most diverse and numerous RNLs in tested land plants. We report an evolutionary swap in the formation of RNLs, which emerged from the fusion of an RPW8 domain to a NB-ARC domain of CNL. We uncovered a quantitative relationship between RNLs and TNLs across all land plants investigated, with an average ratio of 1:10. The conifer RNL repertoire harbours four distinct groups, with two that differ from Angiosperms, one of which contained several upregulated sequences in response to drought while the majority of responsive NLRs are downregulated.

BackgroundA positive relationship between genome size and intron length is observed across eukaryotes including Angiosperms plants, indicating a co-evolution of genome size and gene structure. Conifers have very large genomes and longer introns on average than most plants, but impacts of their large genome and longer introns on gene structure has not be described.ResultsGene structure was analyzed for 35 genes of Picea glauca obtained from BAC sequencing and genome assembly, including comparisons with A. thaliana, P. trichocarpa and Z. mays. We aimed to develop an understanding of impact of long introns on the structure of individual genes. The number and length of exons was well conserved among the species compared but on average, P. glauca introns were longer and genes had four times more intronic sequence than Arabidopsis, and 2 times more than poplar and maize. However, pairwise comparisons of individual genes gave variable results and not all contrasts were statistically significant. Genes generally accumulated one or a few longer introns in species with larger genomes but the position of long introns was variable between plant lineages. In P. glauca, highly expressed genes generally had more intronic sequence than tissue preferential genes. Comparisons with the Pinus taeda BACs and genome scaffolds showed a high conservation for position of long introns and for sequence of short introns. A survey of 1836 P. glauca genes obtained by sequence capture mostly containing introns <1 Kbp showed that repeated sequences were 10× more abundant in introns than in exons.ConclusionConifers have large amounts of intronic sequence per gene for seed plants due to the presence of few long introns and repetitive element sequences are ubiquitous in their introns. Results indicate a complex landscape of intron sizes and distribution across taxa and between genes with different expression profiles.

Temperatures are expected to increase over the next century in all terrestrial biomes and particularly in boreal forests, where drought-induced mortality has been predicted to rise. Genomics research is helping to develop hypotheses regarding the molecular basis of drought tolerance and recent work proposed that the osmo-protecting dehydrin proteins have undergone a clade-specific expansion in the Pinaceae, a major group of conifer trees. The objectives of this study were to identify all of the putative members of the gene family, trace their evolutionary origin, examine their structural diversity and test for drought-responsive expression. We identified 41 complete dehydrin coding sequences in Picea glauca, which is four times more than most angiosperms studied to date, and more than in pines. Phylogenetic reconstructions indicated that the family has undergone an expansion in conifers, with parallel evolution implicating the sporadic resurgence of certain amino acid sequence motifs, and a major duplication giving rise to a clade specific to the Pinaceae. A variety of plant dehydrin structures were identified with variable numbers of the A-, E-, S- and K-segments and an N-terminal (N1) amino acid motif including assemblages specific to conifers. The expression of several of the spruce dehydrins was tissue preferential under non-stressful conditions or responded to water stress after 7-18 days without watering, reflecting changes in osmotic potential. We found that dehydrins with N1 K2 and N1 AESK2 sequences were the most responsive to the lack of water. Together, the family expansion, drought-responsive expression and structural diversification involving loss and gain of amino acid motifs suggests that subfunctionalization has driven the diversification seen among dehydrin gene duplicates. Our findings clearly indicate that dehydrins represent a large family of candidate genes for drought tolerance in spruces and in other Pinaceae that may underpin adaptability in spatially and temporally variable environments.