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Gymnosperms are a group of land plants comprising the extant taxa, cycads, Ginkgo, gnetophytes and conifers. Gymnosperms first appeared more than 300 million years ago (Myr ago)1, well before the angiosperm lineage separated from the stem group of extant gymnosperms2. The major radiation of conifer families occurred 250–65 Myr ago3, and during their evolution the morphology of conifers has changed relatively little. There are approximately 630 conifer species, representing about 70 currently recognized genera, which dominate many terrestrial ecosystems, especially in the Northern Hemisphere. Conifers also dominated both before and after the major mass extinction events at the end of the Permian and Cretaceous periods, around 250 and 65 Myr ago, respectively. Conifers are of immense ecological and economic value; coniferous forests cover enormous areas in the Northern Hemisphere, and conifers are keystone species in many other ecosystems. Conifers contribute a large fraction of terrestrial photosynthesis and biomass, and the cultural and economic values of conifers are also paramount; early civilizations used conifers for firewood, tools and artefacts and today several national economies depend on commodities produced from conifers. However, despite their abundance and importance, our understanding of conifer genomes is limited. Most conifers have 12 (2n = 24) chromosomes, probably reflecting the ancestral karyotype4, which are typically of similar size, each being roughly comparable to the size of the human genome, and containing high proportions of repetitive elements5,6. The gene space of conifer genomes has not been well characterized, although several reports have suggested that gene families in conifers may be larger than their angiosperm counterparts7 and that conifer genomes contain numerous pseudogenes8.

Because their genomes are among the largest—typically 20–30 gigabases pairs (Gb)—of all organisms, genome-wide analyses of conifers are particularly challenging. Thus, no full genome sequence of a gymnosperm species is available at present, whereas 30 angiosperm and more basal plant genomes have been sequenced. However, size is not the only challenge to sequencing presented by conifer genomes. Conifers are typically outbreeding, produce wind-dispersed pollen, have very large effective population sizes, and their genomes are highly heterozygous, although their nucleotide substitution rates are lower than those of most angiosperms8,9, perhaps owing to long lifespan (decades to centuries). Furthermore, inbreeding depression negates the production of inbred lines that could facilitate genome assembly.

The availability of conifer genome sequences would enable comparative analyses of genome architecture and the evolution of key traits for seed plants, including flower or fruit development and life history (perennial versus annual), and help to determine how and why conifer genomes became so large. To address these issues and aid forest tree breeding, biodiversity and conservation studies by, for example, enabling the genome-wide design of genetic markers, we used data from massively parallel DNA sequencing to assemble a draft of the 20-Gb nuclear genome of Norway spruce (Picea abies (L.) Karst), one of the most widespread, ecologically and economically important plants in Europe. We analysed the protein-coding and non-coding fractions of the genome and compared them to the low-coverage draft genome assemblies of five other gymnosperms—Scots pine (P. sylvestris), Siberian fir (A. sibirica), juniper (J. communis), yew (Taxus baccata) and Gnetum gnemon—to gain insight into conifer genome evolution.

Sequencing and assembly

We sequenced a 43-year-old root-grafted copy of the P. abies clone Z4006, which originated from a tree in Ragunda, central Sweden, collected in 1959. Many copies of this clone are available in clone archives and seed orchards, and it has been extensively used in Swedish breeding programs. We estimated its genome size to be 19.6 Gb (C = 20.02 ± 0.95 pg (mean ± s.d.); Supplementary Information 1.1), in accordance with previous reports10.

De novo sequencing and assembly of large, repeat-containing, heterozygous genomes remains challenging. To assemble the P. abies genome, we developed a hierarchical strategy combining fosmid pools11 with both haploid and diploid whole genome shotgun (WGS) data, and RNA sequencing (RNA-Seq) data12,13,14 (Supplementary Information 1.2–1.3). The resulting assembly (P.abies 1.0) included 4.3 Gb in >10-kilobase (kb) scaffolds (Table 1), and we estimated that approximately 63% of protein-coding genes15 were fully covered (>90% of their length), and 96% partially covered (>30% of their length) within single scaffolds (Supplementary Information 1.4). By mapping diploid reads to the P.abies 1.0 assembly, the single nucleotide polymorphism (SNP) frequency was estimated to be 0.77% and the short insertion/deletion (indel) frequency to be 0.05% (Supplementary Information 1.5).

Table 1 Characteristics of the P. abies genome
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The chloroplast genome (124 kb) revealed considerable structural variation within the genus Picea (Supplementary Information 1.6). The draft mitochondrial genome (>4 Mb) was among the largest reported for plants and was rich in short open-reading frames (ORFs), which appeared to be gene remnants derived from repeat-driven mitochondrial rearrangements16 (Supplementary Information 1.7).

Presence of long introns and gene-like fragments

We generated >1 billion RNA-Seq reads and used transcript assemblies of these in combination with public expressed sequence tags (ESTs) and transcripts to perform ab initio prediction of protein-coding genes, which identified a high confidence set of 28,354 loci with >70% coverage by supporting evidence from the total set of 70,968 predicted loci. A notable characteristic of the predicted gene structures was the presence of numerous long introns (Fig. 1b), with mean intron length being higher than in most available plant genomes, although similar to the repeat-rich genomes of Vitis vinifera and Zea mays17,18. The longest intron in the high-confidence genes was 68 kb (Supplementary Table 2.6), and 2,384 high-confidence genes contained 2,880 longer than 5-kb introns (20 of which we confirmed by PCR amplification; Supplementary Information 2.14), 2,679 of which contained a repeat, suggesting that repeat insertions account for intron expansion. By contrast, exon size was consistent among the species considered (Supplementary Information 2.6.3). Numerous genes (∼30%) remained split across scaffolds owing to assembly fragmentation, and as such, the longest introns were not represented in the P.abies 1.0 assembly. Long introns (either individual or cumulative intron length) did not influence expression levels (Fig. 1c) and introns containing repeats have not contracted despite a lack of recent repeat activity (see below).

Figure 1: The gene-space and transcribed fraction of the P.abies 1.0 assembly.
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a, Gene family loss and gain in eight sequenced plant genomes (Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera, Oryza sativa, Zea mays, Picea abies, Selaginella moellendorffii and Physcomitella patens). Gene families were identified using TribeMCL (inflation value 4), and the DOLLOP program from the PHYLIP package was used to determine the minimum gene set for ancestral nodes of the phylogenetic tree. We used plant genome annotations filtered to remove transposable elements. ‘Orphans’ refers to gene families containing only a single gene. Blue numbers indicate the number of gene families. b, Boxplot representation of length distribution for the 10% longest introns in the same eight genomes. c, Scatter plots of cumulative intron length against log10 expression calculated as fragments per kilobase per million mapped reads (FPKM) for high-confidence gene loci (top, coloured orange) and green for lncRNA loci (middle, shaded green). The bottom panel shows a histogram of cumulative intron size in the two sets of loci. d, Distribution of small (18–24-nucleotide (nt)) RNAs and their co-alignment-based colocation to genomic features (repeats, high-confidence genes and their promoter/UTRs). CDS, coding sequence.

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Analysis of gene families in the high-confidence gene set and seven sequenced plant genomes (five angiosperms: Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera, Oryza sativa and Zea mays, and two basal plants: Selaginella moellendorffii and Physcomitrella patens) identified 1,021 P. abies-specific gene families (Fig. 1a and Supplementary Information 2.8). P. abies-specific families included over-representation of Gene Ontology categories involved in DNA repair and methylation of DNA and chromatin (Supplementary Information 2.8). As for most draft genomes, these results probably overestimate gene numbers19 and will be refined as we further improve the genome assembly.

A common mechanism leading to genome size expansion is the occurrence of a whole genome duplication (WGD) event. We calculated the number of synonymous substitutions per synonymous site (Ks) of paralogues within the high-confidence genes but found no evidence for any recent WGD; there was a clear, exponential decay in the number of retained paralogues with increasing Ks values (Supplementary Information 2.9 and Supplementary Fig. 2.6). However, a population dynamics model that takes into account both small- and large-scale modes of gene duplication20 suggested the presence of a small peak (around Ks of 1.1), which, considering the slow substitution rate of conifers, might represent the ancient WGD predating the divergence of angiosperms and gymnosperms (350 Myr ago21).

Previous examinations of small genomic subsets indicated that conifer genomes contain numerous pseudogenes5,6,22,23. The gene-like fraction of the P.abies 1.0 assembly was identified by alignment of RNA-Seq reads and de novo assembled transcripts (Supplementary Information 2.10). Within this subset of the genome, loci with valid spliced alignments of de novo assembled transcripts or the presence of a high-confidence gene were also identified. The high-confidence gene set represented 27 Mb of protein-coding sequence, whereas 72 Mb of regions were identified with a valid spliced alignment or a high-confidence gene. In stark contrast, 524 Mb of gene-like regions were identified by less stringent alignments. The presence of such a large gene-like fraction lacking predicted gene structures supports the presence of numerous pseudogenes.

Recent ENCODE publications24,25 characterized numerous long non-coding RNA (lncRNA) loci in the human genome, but this class of RNA remains largely uncharacterized in plants. Using short-read de novo transcript assemblies, 13,031 spruce-specific and 9,686 conserved intergenic lncRNAs were identified (Supplementary Information 2.4.3). In common with the ENCODE results, P. abies lncRNA loci contained fewer exons, were shorter (Fig. 1c), and had more tissue-specific expression than protein-coding loci (Supplementary Fig. 2.8).

There has been conflicting evidence about the presence of 24-nucleotide short RNAs (sRNAs) in gymnosperms26,27,28,29, a class of sRNA that silence transposable elements by the establishment of DNA methylation30. Across 22 samples, we identified numerous 24-nucleotide sRNAs, but these were highly specific to reproductive tissues, largely associated with repeats but present at substantially lower levels than in angiosperms (Fig. 1d and Supplementary Fig. 2.10). By contrast, 21-nucleotide sRNAs were associated with genes, repeats and promoters/untranslated regions (UTRs) (Fig. 1d). De novo microRNA (miRNA) prediction identified 2,719 loci, including 20 known miRNA families, with target sites predicted within the high-confidence gene set for 1,378 of these (Supplementary Information 2.13). Furthermore, 55 known miRNA families had >5 aligned sRNA reads and mature miRNAs, representing 49 known families aligned to the genome (Supplementary Information 2.13).

Conifer genomes grew by insertion of LTR-RT elements

We constructed a manually curated library of 1,773 repetitive sequences, approximately half of which could be assigned to known transposable element repeat families (Supplementary Information 3.1–3.3). Long terminal repeat-retrotransposons (LTR-RTs) comprised the most abundant fraction of transposable elements, with the Ty3/Gypsy superfamily being more abundant than the Ty1/Copia superfamily (Fig. 2a and Table 1). We also identified and characterized transposable elements using 454 reads from randomly sheared genomic DNA in five other gymnosperms (P. sylvestris, A. sibirica, J. communis, T. baccata and G. gnemon) and, in all six species, LTR-RTs were the most abundant class (Fig. 2a, Supplementary Information 4.1 and Supplementary Table 3.1).

Figure 2: Conifer genomes contain expansions of a diverse set of LTR-RTs.
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a, Distribution of different classes of transposable elements from six gymnosperm species. The figure is based on the total fraction of transposable elements (TE) identified and grouped into different classes from the different species. Genome sizes of the six species are given in circles and their phylogenetic relationship is shown, with tentative dating of divergence times (x-axis) based on 64 chloroplast genes over 39 species and five fossil calibration points. b, c, Heuristic neighbour-joining trees constructed from 5,922 sequences similar to the Ty3/Gypsy (b) and 3,052 sequences similar to the Ty1/Copia (c) reverse transcriptase domain from nine plant species. The trees to the right have only sequences from P. abies and Z. mays coloured, whereas the grey dots are the uncoloured versions of the other species represented on the left. d, Distributions of insertion times calculated for LTR-RTs in Picea abies, Picea glauca and Oryza glaberrima/O. sativa, using mutation rates (per base per year) of 2.2 × 10−9 for the Picea spp. and 1.8 × 10−8 for O. glaberrima50.

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To trace the history of transposable elements in vascular plants we inferred phylogenies of a domain of the reverse transcriptase genes of both Ty1/Copia and Ty3/Gypsy elements. The phylogenies revealed several diverse and ancient transposable element subfamilies, present in almost all of the examined conifer genera, whereas only a few subfamilies were expanded in the angiosperm genomes (Fig. 2b, c and Supplementary Information 3.11). Most internal clades with significant bootstrap support were consistently species-specific, indicating that most expansions of extant transposable element families occurred after divergence. Two species-specific amplification bursts were evident: a Ty1/Copia family in J. communis and a Ty3/Gypsy family in T. baccata. We used complete LTR-RTs from P. abies and P. glauca to investigate further the timing of conifer transposable element insertions31 (Supplementary Information 3.4–3.8). In contrast to a similar set of elements identified in Oryza sativa and O. glaberrima (Fig. 2d), we detected no evidence of recent activity (that is, less than 5 Myr ago) in P. abies. Instead, insertions seem to have occurred over several tens of millions of years (older insertions are more likely to escape detection). Analysis of 68 orthologous transposable element insertions in P. abies and P. glauca further supported this: 63 insertions apparently predated divergence, and only five occurred after the lineages separated 13–20 Myr ago (Supplementary Information 3.9).

We clustered LTRs of complete elements to identify transposable element families32. More than 86% of the elements remained as singletons, indicating that LTR-RTs are quite divergent and that there are several low-abundance families. We searched three LTR-RT families for signatures of unequal intra-element recombination events in scaffolds >50 kb and 20 complete fosmids33. For families ALISEI, 3K05 and 4D08_5 we identified 21, 22 and 39 complete elements, and four, five and no solo LTRs, respectively (Supplementary Information 3.10). Although this data set is limited, the analysis suggested that LTR-RT-related sequences might be removed less frequently by unequal recombination than in other plant genomes. The ratio of solo-LTRs to complete elements in P. abies is ∼1:9, whereas in A. thaliana, rice and barley it is 1:1 (ref. 33), 0.6:1 (ref. 34) and 16:1 (for the abundant BARE 1 element35), respectively. Taken together, these findings indicated that the extant set of transposable elements in P. abies accumulated slowly over tens or hundreds of millions years, mainly by the insertion of LTR-RT elements with limited transposable element removal.

An analysis of introns across taxa provided further insight into the genome of the last common ancestor to the conifers. We identified orthologues of normal sized (50–300 bp) and long (1–20 kb) introns in spruce within draft genome assemblies of P. sylvestris and G. gnemon (Supplementary Information 4.2). Introns identified as orthologous to a long intron in P. abies were also atypically long (Fig. 3a, b), suggesting that intron expansions started early in the history of conifers.

Figure 3: Intron sizes are conserved among gymnosperms.
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a, b, Intron size comparisons between P. abies, P. sylvestris (a) and G. gnemon (b), respectively. Orthologues of introns that were categorised as short (50–300 bp) or long (1–20 kb) in P. abies were identified in P. sylvestris and G. gnemon, and the corresponding intron size was scored.

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The evolution of important conifer traits

Two major differences between angiosperms and gymnosperms are their contrasting reproductive development and the development of water-conducting xylem cells. We therefore manually identified P. abies loci homologous to genes known to be centrally involved in these processes in angiosperms.

In angiosperms, homologues of the A. thaliana phosphatidylethanolamine-binding protein (PEBP) FLOWERING LOCUS T (FT) are key activators of flowering. It has been suggested that gymnosperms lack orthologues of FT genes, instead containing a group of FT/TFL1-like genes that probably act as flowering repressors36,37. We identified four putative FT/TFL1-like genes in the P.abies 1.0 genome that have not been previously described and confirmed that the genome does indeed lack FT-like genes (Supplementary Information 5.1).

MADS-box genes are involved in controlling most aspects of angiosperm development38. A total of 278 sequences with clear homology to MADS boxes were identified in the P.abies 1.0 assembly (Supplementary Information 5.2), 41 of which had transcript support. Type I and II MADS-box genes are distinguished in plants. Only 5% of the identified MADS boxes were of type I (Supplementary Fig. 5.2.), the lowest percentage of potential type I genes recorded in any plant genome. Type II MADS-box genes are subdivided into about a dozen ancient clades. We observed remarkable expansions in the TM3-like (or SOC1-like), STMADS11-like and TM8-like gene clades in P. abies. Because members of these gene clades are involved in vegetative development and phase changes such as the floral transition in angiosperms39, we propose that the expansion of these gene clades has contributed to the evolution of developmental phase changes in gymnosperms.

The xylem tissue of most gymnosperms comprises a single water-transporting cell type, tracheids. By contrast, the xylem tissue of angiosperm species contains fibres, originating from tracheids that have to a large extent lost the capacity to conduct mass water flows, and vessels that have taken over the water-transport function in the stem. Formation of vessels is controlled by the VASCULAR NAC DOMAIN (VND) gene family, which has seven members in A. thaliana40. We detected two VND genes in P. abies (Supplementary Information 5.3), suggesting that co-option and expansion of the VND gene family in vessel formation might have been important for angiosperm evolution.

A model for conifer genome evolution

We propose the following model of conifer genome evolution. After the lineage that led to angiosperms had branched off and the most recent common ancestor of extant conifers had been established, the 12 ancestral chromosomes expanded at a slow and steady rate due to the activity of a diverse set of Gypsy and Copia LTR transposable elements that are largely shared among extant conifers. The expansion started early and, in contrast to angiosperms genomes in which this has been counteracted by efficient recombination mechanisms33 resulting in only smaller transposable element subsets remaining following recent bursts of activity41,42, these elements have remained in the genome. We propose that mechanisms for transposable element removal (for example by unequal recombination) have been less active in conifers than in most other organisms43, and our data suggest that the insertion of transposable elements into genes gave rise to large introns, and (combined with other mechanisms) abundant pseudogenes. Each chromosome has grown to a similar size—perhaps limited by physical constraints on, for instance, chromosomal replication—with genes separated by large regions of transposable-element-rich, highly polymorphic non-protein-coding regions with low recombination frequencies. The gradual increase in size, the lack of WGDs and a predominately out-crossing mating system have probably also buffered conifer genomes against chromosomal rearrangements (WGD reduces sensitivity to aneuploidy), thereby maintaining synteny over large phylogenetic distances44.

Some angiospems, such as cereals, also have large genomes but it seems as if the “one way ticket towards genome obesity”45 that is barely recognizable in angiosperms prevails in conifers. The underlying mechanism remains unclear, but the low frequency of 24-nucleotide sRNAs, their role in methylation of repeats and their restriction to reproductive tissues may have influenced the process. However, considering the effect of methylation patterns on recombination rates46 and the fact that 24-nucleotide sRNAs trigger methylation, such low recombination frequencies would more likely result from hypermethylation47. A state of ‘genome paralysis’ could potentially have been triggered once an obesity threshold was reached. In the angiosperm lineage, the occurrence of a number of WGDs probably increased diversification potential, allowing morphological innovation (for example, the origin of flowers and fruits) and facilitating speciation46,48,49. By contrast, the conserved genome structure resulting from the paucity of genome rearrangements and lack of WGDs in conifers probably limited the evolution of reproductive barriers (resulting in relatively low rates of speciation), and may explain the high degrees of conservation through time and low morphological diversity. Nevertheless, these processes do not seem to affect fitness as conifers dominate many ecosystems, probably because they contain high degrees of standing genetic variation, allowing them to occupy very wide ecological niches in climatic regions where other plant species are less competitive. The future availability of additional gymnosperm genome sequences will allow further exploration of the unique processes that have driven their evolution and facilitate improvement of this important species.

Methods Summary

We shotgun-sequenced 450 fosmid pools containing around 100–6,000 fosmids per pool (Supplementary Table 1.4). Each fosmid pool was assembled and scaffolded individually. Fosmid pool scaffolds larger than 1 kb (∼6.7 Gb in total) were merged12 (Supplementary Information 1.3) with a 38× haploid WGS assembly (∼9.8 Gb in total, derived from ∼600 ng of DNA extracted from a single megagametophyte). We subsequently performed scaffolding13 using WGS libraries of five different insert sizes (0.3, 0.65, 2.4, 4.4 and 10.4 kb) from diploid tissue. We further increased assembly contiguity of protein-coding regions by scaffolding using a set of ∼38 million unassembled (after digital normalization) RNA-Seq read-pairs generated from 22 samples (Supplementary Information 1.3).

Ab initio prediction of protein-coding genes was performed using ESTs from numerous conifer species, our own short-read de novo transcript assemblies and proteins from other plant species as supporting evidence (Supplementary Information 2.6). Predicted loci were used to perform gene family analysis and to examine the Ks substitution rates of paralogues to identify evidence for a recent WGD event. De novo transcript assemblies were used to identify lncRNA, and sRNA sequencing was performed and used for de novo miRNA prediction.

Repeated sequences were identified de novo using 454 reads longer than 700 bp generated from randomly sheared genomic DNA. Candidates were characterized using similarity searches at the nucleotide and amino acid level against public and custom collections of plant transposable element sequences. Complete LTR-RTs were identified using a combination of de novo searches and manual inspection.

WGS assemblies from shallow sequencing (3.8–12.5×) of P. sylvestris, A. sibirica, J. communis, T. baccata and G. gnemon were produced using the CLC Bio de novo assembler.

For website and accession number information, see Supplementary Information 6.