Research Article

The Genome of Black Cottonwood, Populus trichocarpa (Torr. & Gray)

See allHide authors and affiliations

Science  15 Sep 2006:
Vol. 313, Issue 5793, pp. 1596-1604
DOI: 10.1126/science.1128691

Abstract

We report the draft genome of the black cottonwood tree, Populus trichocarpa. Integration of shotgun sequence assembly with genetic mapping enabled chromosome-scale reconstruction of the genome. More than 45,000 putative protein-coding genes were identified. Analysis of the assembled genome revealed a whole-genome duplication event; about 8000 pairs of duplicated genes from that event survived in the Populus genome. A second, older duplication event is indistinguishably coincident with the divergence of the Populus and Arabidopsis lineages. Nucleotide substitution, tandem gene duplication, and gross chromosomal rearrangement appear to proceed substantially more slowly in Populus than in Arabidopsis. Populus has more protein-coding genes than Arabidopsis, ranging on average from 1.4 to 1.6 putative Populus homologs for each Arabidopsis gene. However, the relative frequency of protein domains in the two genomes is similar. Overrepresented exceptions in Populus include genes associated with lignocellulosic wall biosynthesis, meristem development, disease resistance, and metabolite transport.

Forests cover 30% (about 3.8 billion ha) of Earth's terrestrial surface, harbor substantial biodiversity, and provide humanity with benefits such as clean air and water, lumber, fiber, and fuels. Worldwide, one-quarter of all industrial feedstocks have their origins in forest-based resources (1). Large and long-lived forest trees grow in extensive wild populations across continents, and they have evolved under selective pressures unlike those of annual herbaceous plants. Their growth and development involves extensive secondary growth, coordinated signaling and distribution of water and nutrients over great distances, and strategic storage and redistribution of metabolites in concordance with interannual climatic cycles. Their need to survive and thrive in fixed locations over centuries under continually changing physical and biotic stresses also sets them apart from short-lived plants. Many of the features that distinguish trees from other organisms, especially their large sizes and long-generation times, present challenges to the study of the cellular and molecular mechanisms that underlie their unique biology. To enable and facilitate such investigations in a relatively well-studied model tree, we describe here the draft genome of black cottonwood, Populus trichocarpa (Torr. & Gray), and compare it to other sequenced plant genomes.

P. trichocarpa was selected as the model forest species for genome sequencing not only because of its modest genome size but also because of its rapid growth, relative ease of experimental manipulation, and range of available genetic tools (2, 3). The genus is phenotypically diverse, and interspecific hybrids facilitate the genetic mapping of economically important traits related to growth rate, stature, wood properties, and paper quality. Dozens of quantitative trait loci have already been mapped (4), and methods of genetic transformation have been developed (5). Under appropriate conditions, Populus can reach reproductive maturity in as few as 4 to 6 years, permitting selective breeding for large-scale sustainable plantation forestry. Finally, rapid growth of trees coupled with thermochemical or biochemical conversion of the lignocellulosic portion of the plant has the potential to provide a renewable energy resource with a concomitant reduction of greenhouse gases (68).

Sequencing and Assembly

A single female genotype, “Nisqually 1,” was selected and used in a whole-genome shotgun sequence and assembly strategy (9). Roughly 7.6 million end-reads representing 4.2 billion high-quality (i.e., Q20 or higher) base pairs were assembled into 2447 major scaffolds containing an estimated 410 megabases (Mb) of genomic DNA (tables S1 and S2). On the basis of the depth of coverage of major scaffolds (∼7.5 depth) and the total amount of nonorganellar shotgun sequence that was generated, the Populus genome size was estimated to be 485 ± 10 Mb (±SD), in rough agreement with previous cytogenetic estimates of about 550 Mb (10). The near completeness of the shotgun assembly in protein-coding regions is supported by the identification of more than 95% of known Populus cDNA in the assembly.

The ∼75 Mb of unassembled genomic sequence is consistent with cytogenetic evidence that ∼30% of the genome is heterochromatic (9). The amount of euchromatin contained within the Populus genome was estimated in parallel by subtraction on the basis of direct measurements of 4′,6′-diamidino-2-phenylindole–stained prophase and metaphase chromosomes (fig. S4). On average, 69.5 ± 0.3% of the genome consisted of euchromatin, with a significantly lower proportion of euchromatin in linkage group I (LGI) (66.4 ± 1.1%) compared with the other 18 chromosomes (69.7 ± 0.03%, P ≤ 0.05). In contrast, Arabidopsis chromosomes contain roughly 93% euchromatin (11). The unassembled shotgun sequences were derived from variants of organellar DNA, including recent nuclear translocations; highly repetitive genomic DNA; haplotypic segments that were redundant with short subsegments of the major scaffolds (separated as a result of extensive sequence polymorphism and allelic variants); and contaminants of the template DNA, such as endophytic microbes inhabiting the leaf and root tissues used for template preparation (12) (fig. S1 and table S3). The end-reads corresponding to chloroplast (fig. S5) and mitochondrial genomes were assembled into circular genomes of 157 and 803 kb, respectively (9).

We anchored the 410 Mb of assembled scaffolds to a sequence-tagged genetic map (fig. S3). In total, 356 microsatellite markers were used to assign 155 scaffolds (335 Mb of sequence) to the 19 P. trichocarpa chromosome-scale linkage groups (13). The vast majority (91%) of the mapped microsatellite markers were colinear with the sequence assembly. At the extremes, the smallest chromosome, LGIX [79 centimorgans (cM)], is covered by two scaffolds containing 12.5 Mb of assembled sequence, whereas the largest chromosome, LGI (265 cM), contains 21 scaffolds representing 35.5 Mb (fig. S3). We also generated a physical map based on bacterial artificial chromosome (BAC) fingerprint contigs using a Nisqually-1 BAC library representing an estimated 9.5-fold genome coverage (fig. S2). Paired BAC- end sequences from most of the physical map were linked to the large-scale assembly, permitting 2460 of the physical map contigs to be positioned on the genome assembly. Combining the genetic and physical map, nearly 385 Mb of the 410 Mb of assembled sequence are placed on a linkage group.

Unlike Arabidopsis, where predominantly self-fertilizing ecotypes maintain low levels of allelic polymorphism, Populus species are predominantly dioecious, which results in obligate outcrossing. This compulsory outcrossing, along with wind pollination and wind-dispersed plumose seeds, results in high levels of gene flow and high levels of heterozygosity (that is, within individual genetic polymorphisms). Within the heterozygous Nisqually-1 genome, we identified 1,241,251 single-nucleotide polymorphisms (SNPs) or small insertion/deletion polymorphisms (indels) for an overall rate of approximately 2.6 polymorphisms per kilobase. Of these polymorphisms, the overwhelming majority (83%) occurred in noncoding portions of the genome (Table 1). Short indels and SNPs within exons resulted in frameshifts and nonsense stop codons within predicted exons, respectively, suggesting that null alleles of these genes exist in one of the haplotypes. Some of the polymorphisms may be artifacts from the assembly process, although these errors were minimized by using stringent criteria for SNP identification (9).

Table 1.

Characterization of polymorphisms according to their positions relative to predicted coding sequences, introns, and untranslated regions (UTRs). Rate shows the percentage of potential sites of each class that were polymorphic. Most indels within exons resulted in frame shifts, but we could not quantify this due to difficulties with assembly and sequencing of regions containing indels. Nonsense mutations created stop codons within predicted exons.

SourceNumber of lociRate (%)
Noncoding 1,027,322 0.32
INTRON 141,199 0.25
3′UTR 6,731 0.25
5′UTR 3,306 0.24
Exon 62,656 0.14
Within exons:
    Indels 2,722 0.01
    Nonsense 926 0.02
    Nonsynonymous 32,207 0.10

Gene Annotation

We tentatively identified a first-draft reference set of 45,555 protein-coding gene loci in the Populus nuclear genome (www.jgi.doe.gov/poplar) using a variety of ab initio, homology-based, and expressed sequence tag (EST)–based methods (1417) (table S5). Similarly, 101 and 52 genes were annotated in the chloroplast and mitochondrial genomes, respectively (9). To aid the annotation process, 4664 full-length sequences, from full-length enriched cDNA libraries from Nisqually 1, were generated and used in training the gene-calling algorithms. Before gene prediction, repetitive sequences were characterized (fig. S15 and table S14) and masked; additional putative transposable elements were identified and subsequently removed from the reference gene set (9). Given the current draft nature of the genome, we expect that the gene set in Populus will continue to be refined.

About 89% of the predicted gene models had homology [expectation (E) value ≤ 1 10–8] to the nonredundant (NR) set of proteins from the National Center for Biotechnology Information, including 60% with extensive homology that spans 75% of both model and NR protein lengths. Nearly 12% (5248) of the predicted Populus genes had no detectable similarity to Arabidopsis genes (E value ≤ 1 × 10–3); conversely, in the more refined Arabidopsis set, only 9% (2321) of the predicted genes had no similarity to the Populus reference set. Of the 5248 Populus genes without Arabidopsis similarity, 1883 have expression evidence from the manually curated Populus EST data set, and of these, 274 have no hits (E value ≥ 1 × 10–3) to the NR database (9). Whole-genome oligonucleotide microarray analysis provided evidence of tissue-based expression for 53% of the reference gene models (Fig. 1). In addition, a signal was detected from 20% of genes that were initially annotated and excluded from the reference set, suggesting that as many as 4000 additional genes (or gene fragments) may be present. Within the reference gene set, we identified 13,019 pairs of orthologs between genes in Populus and Arabidopsis using the best bidirectional Basic Local Alignment Search Tool (BLAST) hits, with average mutual coverage of these alignments equal to 93%; 11,654 pairs of orthologs had greater than 90% alignment of gene lengths, whereas only 156 genes had less than 50% coverage. As of 1 June 2006, ∼10% (4378) gene models have been manually validated and curated.

Fig. 1.

Whole-genome oligonucleotide microarray expression data for all predicted gene models in P. trichocarpa. Values represent the proportion of genes expressed above negative controls at a 5% false discovery rate. The x axis represents the subsets of predicted genes that were analyzed for the annotated and promoted P. trichocarpa gene set (42,373 genes), chloroplast gene set (49 genes), mitochondria gene set (49 genes), annotated, nonpromoted gene set (10,875 genes), and microRNAs (48 miRNAs).

Genome Organization

Genome duplication in the Salicaceae. Populus and Arabidopsis lineages diverged about 100 to 120 million years ago (Ma). Analysis of the Populus genome provided evidence of a more recent duplication event that affected roughly 92% of the Populus genome. Nearly 8000 pairs of paralogous genes of similar age (excluding tandem or local duplications) were identified (Fig. 2). The relative age of the duplicate genes was estimated by the accumulated nucleotide divergence at fourfold synonymous third-codon transversion position (4DTV) values. A sharp peak in 4DTV values, corrected for multiple substitutions, representing a burst of gene duplication, is evident at 0.0916 ± 0.0004 (Fig. 3A). Comparison of 1825 Populus and Salix orthologous genes derived from Salix EST suggests that both genera share this whole-genome duplication event (Fig. 3B). Moreover, the parallel karyotypes and collinear genetic maps (18) of Salix and Populus also support the conclusion that both lineages share the same large-scale genome history.

Fig. 2.

Chromosome-level reorganization of the most recent genome-wide duplication event in Populus. Common colors refer to homologous genome blocks, presumed to have arisen from the salicoid-specific genome duplication 65 Ma, shared by two chromosomes. Chromosomes are indicated by their linkage group number (I to XIX). The diagram to the left uses the same color coding and further illustrates the chimeric nature of most linkage groups.

Fig. 3.

(A) The 4DTV metrics for paralogous gene pairs in Populus-Populus and Populus-Arabidopsis. Three separate genome-wide duplications events are detectable, with the most recent event contained within the Salicaceae and the middle event apparently shared among the Eurosids. (B) Percent identity distributions for mutual best EST hit to Populus trichocarpa CDS.

If we naively calibrated the molecular clock using synonymous rates observed in the Brassicaceae (19) or derived from the Arabidopsis-Oryza divergence (20), we would conclude that the genome duplication in Populus is very recent [8 to 13 Ma, as reported by Sterk (21)]. Yet the fossil record shows that the Populus and Salix lineages diverged 60 to 65 Ma (2225). Thus, the molecular clock in Populus must be ticking at only one-sixth the estimated rate for Arabidopsis (that is, 8 to 13 Ma divided by 60 to 65 Ma). Qualitatively similar slowing of the molecular clock is found in the Populus chloroplast and mitochondrial genomes (9). Because Populus is a long-lived vegetatively propagated species, it has the potential to successfully contribute gametes to multiple generations. A single Populus genotype can persist as a clone on the landscape for millennia (26), and we propose that recurrent contributions of “ancient gametes” from very old individuals could account for the markedly reduced rate of sequence evolution. As a result of the slowing of the molecular clock, the Populus genome most likely resembles the ancestral eurosid genome.

To test whether the burst of gene creation 60 to 65 Ma was due to a single whole-genome event or to independent but near-synchronous gene duplication events, we used a variant of the algorithm of Hokamp et al. (27) to identify segments of conserved synteny within the Populus genome. The longest conserved syntenic block from the 4DTV ∼0.09 epoch spanned 765 pairs of paralogous genes. In total, 32,577 genes were contained within syntenic blocks from the salicoid epoch; half of these genes were contained in segments longer than 142 paralogous pairs. The same algorithm, when applied to randomly shuffled genes, typically yields duplicate blocks with fewer than 8 to 9 genes, indicating that the Populus gene duplications occurred as a single genome-wide event. We refer to this duplication event as the “salicoid” duplication event.

Nearly every mapped segment of the Populus genome had a parallel “paralogous” segment elsewhere in the genome as a result of the salicoid event (Fig. 2). The pinwheel patterns can be understood as a whole-genome duplication followed by a series of reciprocal tandem terminal fusions between two separate sets of four chromosomes each—the first involving LGII, V, VII, and XIV and the second involving LGI, XI, IV, and IX. In addition, several chromosomes appear to have experienced minor reorganizational exchanges. Furthermore, LGI appears to be the result of multiple rearrangements involving three major tandem fusions. These results suggest that the progenitor of Populus had a base chromosome number of 10. After the whole-genome duplication event, this base chromosome number experienced a genome-wide reorganization and diploidization of the duplicated chromosomes into four pairs of complete paralogous chromosomes (LGVI, VIII, X, XII, XIII, XV, XVI, XVIII, and XIX); two sets of four chromosomes, each containing a terminal translocation (LGI, II, IV, V, VII, IX, and XI); and one chromosome containing three terminally joined chromosomes (LGIII with I or XVII with VII). The colinearity of genetic maps among multiple Populus species suggests that the genome reorganization occurred before the evolution of the modern taxa of Populus.

Genome duplication in a common ancestor of Populus and Arabidopsis. The distribution of 4DTV values for paralogous pairs of genes also shows that a large fraction of the Populus genome falls in a set of duplicated segments anchored by gene pairs with 4DTV at 0.364 T 0.001, representing the residue of a more ancient, large-scale, apparently synchronous duplication event (Fig. 3A). This relatively older duplication event covers about 59% of the Populus genome with 16% of genes in these segments present in two copies. Because this duplication preceded and is therefore superimposed upon the salicoid event, each genomic region is potentially covered by four such segments. Similarly, the Arabidopsis genome experienced an older “beta” duplication that preceded the Brassicaceae-specific “alpha” event (2832).

We next asked whether the Arabidopsis “beta” (30, 32) and Populus 4DTV ∼0.36 duplication events were (i) independent genome-wide duplications that occurred after the split from the last common eurosid ancestor (H1) or (ii) a single shared duplication event that occurred in an ancestral lineage (i.e., before the divergence of eurosid lineages I and II) (H2). These two hypotheses have very different implications for the interpretation of homology between Populus and Arabidopsis. Under H1, each genomic segment in one species is homologous to four segments in the other; whereas under H2, each segment is homologous to only two segments in the other species. These hypotheses were tested by comparing the relative distances between gene pairs sampled within and between Populus and Arabidopsis. H2 was generally supported (9), but we could not reject H1. We can only conclude that the Populus genome duplication occurred very close to the time of divergence of the eurosid I and II lineages (9), with slight support for a shared duplication. This coincident timing raises the possibility of a causal link between this duplication and rapid diversification early in eurosid (and perhaps core eudicot) history. We refer to this older Populus/Arabidopsis duplication event as the “eurosid” duplication event. We note that the salicoid duplication occurred independently of the eurosid duplication observed in the Arabidopsis genome.

Gene Content

Although Populus has substantially more protein-coding genes than Arabidopsis, the relative frequency of domains represented in protein databases (Prints, Prosite, Pfam, ProDom, and SMART) in the two genomes is similar (9). However, the most common domains occur in Populus compared with Arabidopsis in a ratio ranging from 1.4:1 to 1.8:1. Noteworthy outliers in Populus include genes and gene domains associated with disease and insect resistance (such as, in Populus versus Arabidopsis, respectively: leucine-rich repeats, 1271 versus 527; NB-ARC domain, 302 versus 141; and thaumatin, 55 versus 24), meristem development (such as NAC transcription factors, 157 versus 100, respectively), and metabolite and nutrient transport [such as oligopeptide transporter of the proton-dependent oligopeptide transporter (POT) and oligopeptide transporter (OPT) families, 129 versus 61, and potassium transporter, 30 versus 13, respectively].

Some domains were underrepresented in Populus compared with Arabidopsis. For example, the F-box domain was twice as prevalent in Arabidopsis as in Populus (624 versus 303, respectively). The F-box domain is involved in diverse and complex interactions involving protein degradation through the ubiquitin-26S proteosome pathway (33). Many of the ubiquitin-associated domains are underrepresented in Populus compared with Arabidopsis (for example, the Ulp1 protease family and the C-terminal catalytic domain, 10 versus 63, respectively). Moreover, the RING-finger domains are nearly equally present in both genomes (503 versus 407, respectively), suggesting that protein degradation pathways in the two organisms are metabolically divergent.

The common eurosid gene set. The Populus and Arabidopsis gene sets were compared to infer the conserved gene complement of their common eurosid ancestor, integrating information from nucleotide divergence, synteny, and mutual best BLAST-hit analysis (9). The ancestral eurosid genome contained at least 11,666 protein-coding genes, along with an undetermined number that were either lost in one or both of the lineages or whose homology could not be detected. These ancestral genes were the progenitors of gene families of typically one to four descendents in each of the complete plant genomes and account for 28,257 Populus and 17,521 Arabidopsis genes. Gene family lists are accessible at www.phytozome.net. The gene predictions in these two genomes that could not be accounted for in the eurosid clusters were often fragmentary or difficult to categorize, and we could not confidently assign orthology to them. They may include previously unidentified or rapidly evolving genes in the Populus and/or Arabidopsis lineages, as well as poorly predicted genes.

Noncoding RNAs. Based on a series of publicly available RNA detection algorithms (34), including tRNAScan-SE, INFERNAL, and snoScan, we identified 817 putative tRNAs; 22 U1, 26 U2, 6 U4, 23 U5, and 11 U6 spliceosomal small nuclear RNAs (snRNAs); 339 putative C/D small nucleolar RNAs (snoRNAs); and 88 predicted H/ACA snoRNAs in the Populus assembly. All 57 possible anticodon tRNAs were found. One selenocysteine tRNA was detected and two possible suppressor tRNAs (anticodons that bind stop codons) were also identified. Populus has nearly 1.3 times as many tRNA genes as Arabidopsis. In contrast to Arabidopsis (fig. S7A), the copy number of tRNA in Populus was significantly and positively correlated with amino acid occurrence in predicted gene models (fig. S7B). The ratio of the number of snRNAs in Populus compared with the number in Arabidopsis is 1.3 to 1.0, yet U1, U2, and U5 are overrepresented in Populus, whereas U4 is underrepresented. Furthermore, U14 was not detected in Arabidopsis. The snRNAs and snoRNAs have not been experimentally verified in Populus.

There are 169 identified microRNA (miRNA) genes representing 21 families in Populus (table S7). In Arabidopsis, these 21 families contain 91 miRNA genes, representing a 1.9X expansion in Populus, primarily in miR169 and miR159/319. All 21 miRNA families have regulatory targets that appear to be conserved among Arabidopsis and Populus (table S8). Similar to the miRNA genes themselves, the number of predicted targets for these miRNA is expanded in Populus (147) compared with Arabidopsis (89). Similarly, the genes that mediate RNA interference (RNAi) are also overrepresented in Populus (21) compared to Arabidopsis (11) [e.g., AGO1 class, 7 versus 3; RNA helicase 2 versus 1; HEN, 2 versus 1; HYL1-like (double-stranded RNA binding proteins), 9 versus 5, respectively].

Tandem duplications. In Populus there were 1518 tandemly duplicated arrays of two or more genes based on a Smith-Waterman alignment E value ≤ 10–25 and a 100-kb window. The total number of genes in such arrays was 4839 and the total length of tandemly duplicated segments in Populus was 47.9 Mb, or 15.6% of the genome (fig. S8). By the same criteria, there are 1366 tandemly duplicated segments in Arabidopsis, covering 32.4 Mb, or 27% of the genome. By far the most common number of genes within a single array was two, with 958 such arrays in Populus and 805 in Arabidopsis. Arabidopsis had a larger number of arrays containing six or more genes than did Populus. Tandem duplications thus appear to be relatively more common in Arabidopsis than in Populus. This may in part be due to difficulties in assembling tandem repeats from a whole-genome shotgun sequencing approach, particularly when tandemly duplicated genes are highly conserved. Alternatively, the Populus genome may be undergoing rearrangements at a slower rate than the Arabidopsis genome, which is consistent with our observations of reduced chromosomal rearrangements and slower nucleotide substitution rates in Populus.

In some cases, genes were highly duplicated in both species, and some tandem duplications predated the Populus-Arabidopsis split (9). The largest number of tandem repeats in Populus in a single array was 24 and contained genes with high homology to S locus–specific glycoproteins. Genes of this class also occur as tandem repeats in Arabidopsis, with the largest segments containing 14 tandem duplicates on chromosome 1. One of the InterPro domains in this protein, IPR008271, a serine/threonine protein kinase active site, was the most frequent domain in tandemly repeated genes in both species (fig. S8). Other common domains in both species were the leucine-rich repeat (IPR007090, primarily from tandem repeats of disease resistance genes), the pentatricopeptide repeat RNA-binding proteins (IPR002885), and the uridine diphosphate (UDP)-glucuronosyl/UDP-glucosyltransferase domain (IPR002213) (table S9).

In contrast, some genes were highly expanded in tandem duplicates in one genome and not in the other (fig. S8). For example, one of the most frequent classes of tandemly duplicated genes in Arabidopsis was F-box genes, with a total of 342 involved in tandem duplications, the largest segment of which contained 24 F-box genes. Populus contains only 37 F-box genes in tandem duplications, with the largest segment containing only 3 genes.

Postduplication Gene Fate

Functional expression divergence. In Populus, 20 of the 66 salicoid-event duplicate gene pairs contained in 19 Populus EST libraries (2.3% of the total) showed differential expression (9) [displaying significant deviation in EST frequencies per library (Fig. 4)]. Out of 18 eurosid-event duplicate gene pairs (2.7% of the total), 11 also displayed significant deviation in EST frequencies per library. Many of the duplicate gene pairs that displayed significant overrepresentation in one or more of the 19 sampled libraries were involved in protein-protein interactions (such as annexin) or protein folding (such as cyclophilins). In the eurosid set, there was a greater divergence in the best BLAST hit among pairwise sets of genes. These results support the premise of functional expression divergence among some duplicated gene pairs in Populus.

Fig. 4.

Kolmogorov-Smirnov (K-S) test for differential expression for 5-methyltetrahydropteroyl-triglutamate-homocysteine S-methyltransferase genes [for descriptions of the EST data set, see Sterky et al. (79)]. Results suggest that the duplicated genes in Populus are differentially expressed in alternate tissues. Tissue types include: cambial zone (1), young leaves (2), flower buds (3), tension wood (4), senescing leaves (5), apical shoot (6), dormant cambium (7), active cambium (8), cold stressed leaves (9), roots (10), bark (11), shoot meristem (12), male catkins (13), dormant buds (14), female catkins (15), petioles (16), wood cell death (17), imbibed seeds (18) and infected leaves (19).

To further test for variation in gene expression among duplicated genes, we examined whole-genome oligonucleotide microarray data containing the 45,555 promoted genes (9). There was significantly lower differential expression in the salicoid duplicated pairs of genes (mean = 5%) relative to eurosid duplications (mean = 11%), again suggesting that differential expression patterns for retained paralogous gene pairs is an ongoing process that has had more time to occur in eurosid pairs (Fig. 5). This difference could also be due to absolute expression level, which may vary systematically between the two duplication events. Moreover, differential expression was more evident in the wood-forming organs. Almost 14 and 13% (2632 pairs of genes) of eurosid duplicated genes in the nodes and internodes, respectively, displayed differential expression, compared with 8% or less in roots and young leaves (Fig. 5).

Fig. 5.

Proportion of eurosid and salicoid duplicated gene sets differentially expressed in stems (nodes and internode), leaves (young and mature), and whole roots. Samples from four biological replicates collected from the reference genotype Nisqually 1 were individually hybridized to whole-genome oligonucleotide microarrays containing three 60-oligomer oligonucleotide probes for each gene. Differential expression between duplicated genes was evaluated in t tests and declared significant at a 5% false discovery rate (9).

Single-nucleotide polymorphisms. Populus is a highly polymorphic taxon and substantial numbers of SNPs are present even within a single individual (Table 1). The ratio of nonsynonymous to synonymous substitution rate (ω = dN/dS) was calculated as an index of selective constraints for alleles of individual genes (9). The overall average dN across all genes was 0.0014, whereas the dS value was 0.0035, for a total ω of 0.40, suggesting that the majority of coding regions in the Populus genome are subject to purifying selection. There was a significant, negative correlation between ω and the 4DTV distance to the most closely related paralog (r = –0.034, P = 0.028), which is consistent with the expectation of higher levels of nonsynonymous polymorphism in recently duplicated genes as a result of functional redundancy (20, 35). Similarly, genes with recent tandem duplicates (4DTV ≤ 0.2) had significantly higher ω than did genes with no recent tandem duplicates (Wilcoxon rank sum Z = 8.65, P ≤ 0.0001) (table S10).

The results for tandemly duplicated genes were consistent with expectations for accelerated evolution of duplicated genes (20). However, this expectation was not upheld for paralogous pairs of genes from the whole-genome duplication events. Relative rates of nonsynonymous substitution were actually lower for genes with paralogs from the salicoid and eurosid whole-genome duplication events than for genes with no paralogs (table S11). One possible explanation for this discrepancy is that the apparent single-copy genes have a corresponding overrepresentation of rapidly evolving pseudogenes. However, this does not appear to be the case, as demonstrated by an analysis of gene size, synonymous substitution rate, and minimum genetic distance to the closest paralog as covariates in an analysis of variance with ω as the response variable (table S11). Therefore, genes with no paralogs from the salicoid and eurosid duplication events seem to be under lower selective constraints, and purifying selection is apparently stronger for genes with paralogs retained from the whole-genome duplications. Chapman et al. (36) have recently proposed the concept of functional buffering to account for similar reduction in detected mutations in paralogs from whole-genome duplications in Arabidopsis and Oryza. The vegetative propagation habit of Populus may also favor the conservation of nucleotide sequences among duplicated genes, in that complementation among duplicate pairs of genes would minimize loss of gene function associated with the accumulation of deleterious somatic mutations.

Gene family evolution. The expansion of several gene families has contributed to the evolution of Populus biology.

Lignocellulosic wall formation. Among the processes unique to tree biology, one of the most obvious is the yearly development of secondary xylem from the vascular cambium. We identified Populus orthologs of the approximately 20 Arabidopsis genes and gene families involved in or associated with cellulose biosynthesis. The Populus genome has 93 cellulose synthesis–related genes compared with 78 in Arabidopsis. The Arabidopsis genome encodes 10 CesA genes belonging to six classes known to participate in cellulose microfibril biosynthesis (37). Populus has 18 CesA genes (38), including duplicate copies of CesA7 and CesA8 homologs. Populus homologs of Arabidopsis CesA4, CesA7, and CesA8 are coexpressed during xylem development and tension wood formation (39). Furthermore, one pair of CesA genes appears unique to Populus, with no homologs found in Arabidopsis (40). Many other types of genes associated with cellulose biosynthesis, such as KOR, SuSY, COBRA, and FRA2, occur in duplicate pairs in Populus relative to single-copy Arabidopsis genes (39). For example, COBRA, a regulator of cellulose biogenesis (41), is a single-copy gene in Arabidopsis, but in Populus there are four copies.

The repertoire of acknowledged hemicellulose biosynthetic genes in Populus is generally similar to that in Arabidopsis. However, Populus has more genes encoding α-l-fucosidases and fewer genes encoding α-l-fucosyltransferases than does Arabidopsis, which is consistent with the lower xyloglucan fucose content (42) in Populus relative to Arabidopsis.

Lignin, the second most abundant cell wall polymer after cellulose, is a complex polymer of monolignols (hydroxycinnamyl alcohols) that encrusts and interacts with the cellulose/hemi-cellulose matrix of the secondary cell wall (43). The full set of 34 Populus phenylpropanoid and lignin biosynthetic genes (table S13) was identified by sequence alignment to the known Arabidopsis phenylpropanoid and lignin genes (44, 45). The size of the Populus gene families that encode these enzymes is generally larger than in Arabidopsis (34 versus 18, respectively). The only exception is cinnamyl alcohol dehydrogenase (CAD), which is encoded by a single gene in Populus and two genes in Arabidopsis (Fig. 6C); CAD is also encoded by only a single gene in Pinus taeda (46, 47). Two lignin-related Populus C4H genes are strongly coexpressed in tissues related to wood formation, whereas the three Populus C3H genes show reciprocally exclusive expression patterns (48) (Fig. 6, A and B).

Fig. 6.

Phylogenetic analysis of gene families in Populus, Arabidopsis, and Oryza encoding selected lignin biosynthetic and related enzymes. (A) Cinnamate-4-hydroxylase (C4H) gene family. (B) 4-coumaroyl-shikimate/quinate-3-hydroxlase (C3H) gene family. (C) Cinnamyl alcohol dehydrogenase (CAD) and related multifunctional alcohol dehydrogenase gene family. Arabidopsis gene names are the same as those in Ehlting et al. (80). Populus and Oryza gene names were arbitrarily assigned; corresponding gene models are listed in table S13. Genes encoding enzymes for which biochemical data are available are highlighted with a green flash. Yellow circles indicate monospecific clusters of gene family members.

Secondary metabolism. Populus trees produce a broad array of nonstructural, carbon-rich secondary metabolites that exhibit wide variation in abundance, stress inducibility, and effects on tree growth and host-pest interactions (4953). Shikimate-phenylpropanoid–derived phenolic esters, phenolic glycosides, and condensed tannins and their flavonoid precursors comprise the largest classes of these metabolites. Phenolic glycosides and condensed tannins alone can constitute up to 35% leaf dry weight and are abundant in buds, bark, and roots of Populus (50, 54, 55).

The flavonoid biosynthetic genes are well annotated in Arabidopsis (56) and almost all (with the exception of flavonol synthase) are encoded by single-copy genes. In contrast, all but three such enzymes (chalcone isomerase, flavonoid 3′-hydroxylase, and flavanone 3-hydroxylase) are encoded by multiple genes in Populus (53). For example, the chalcone synthase, controlling the committed step to flavonoid biosynthesis, has expanded to at least six genes in Populus. In addition, Populus contains two genes each for flavone synthase II (cytochrome accession number CYP98B) and flavonoid 3′,5′-hydroxylase (CYP75A12 and CYP75A13), both of which are absent in Arabidopsis. Furthermore, three Populus genes encode leucoanthocyanidin reductase, required for the synthesis of condensed tannin precursor 2,3-trans-flavan-3-ols, a stereochemical configuration also lacking in Arabidopsis (57). In contrast to the 32 terpenoid synthase (TPS) genes of secondary metabolism identified in the Arabidopsis genome (58), the Populus genome contains at least 47 TPS genes, suggesting a wide-ranging capacity for the formation of terpenoid secondary metabolites.

A number of phenylpropanoid-like enzymes have been annotated in the Arabidopsis genome (44, 45, 5961). One example is the family encoding CAD. In addition to the single Populus CAD gene involved in lignin biosynthesis, several other clades of CAD-like (CADL) genes are present, most of which fall within larger subfamilies containing enzymes related to multifunctional alcohol dehydrogenases (Fig. 6). This comparative analysis makes it clear that there has been selective expansion and retention of Populus CADL gene families. For example, Populus contains seven CADL genes (Poptr-CADL1 to PoptrCADL7; Fig. 6C) encoding enzymes related to the Arabidopsis BAD1 and BAD2 enzymes with apparent benzyl alcohol dehydrogenase activities (62). BAD1 and BAD2 are known to be pathogen inducible, suggesting that this group of Populus genes, including the Populus SAD gene, previously characterized as encoding a sinapaldehyde-specific CAD enzyme (63), may be involved in chemical defense.

Disease resistance. The likelihood that a perennial plant will encounter a pathogen or herbivore before reproduction is near unity. The long-generation intervals for trees make it difficult for such plants to match the evolutionary rates of a microbial or insect pest. Aside from the formation of thickened cell walls and the synthesis of secondary metabolites that constitute a first line of defense against microbial and insect pests, plants use a variety of disease-resistance (R) genes.

The largest class of characterized R genes encodes intracellular proteins that contain a nucleotide-binding site (NBS) and carboxy-terminal leucine-rich-repeats (LRR) (64). The NBS-coding R gene family is one of the largest in Populus, with 399 members, approximately twice as high as in Arabidopsis. The NBS family can be divided into multiple subfamilies with distinct domain organizations, including 64 TIR-NBS-LRR genes, 10 truncated TIR-NBS that lack an LRR, 233 non–TIR-NBS-LRR genes, and 17 unusual TIR-NBS–containing genes that have not been identified previously in Arabidopsis (TNLT, TNLN, or TCNL domains) (Table 2). Five gene models coding for TNL proteins contained a predicted N-terminal nuclear localization signal (65). The number of non–TIR-NBS-LRR genes in Populus is also much higher than that in Arabidopsis (209 versus 57, respectively). Notably, 40 non–TIR-NBS genes, not found in Arabidopsis, carry an N-terminal BED DNA-binding zinc finger domain that was also found in the Oryza Xa1 gene. These findings suggest that domain cooption occurred in Populus. Most NBS-LRR (about 65%) in Populus occur as singletons or in tandem duplications, and the distribution of pairwise genetic distances among these genes suggests a recent expansion of this family. That is, only 10% of the NBS-LRR genes are associated with the eurosid and salicoid duplication events, compared with 55% of the extracellular LRR receptor-like kinase genes (for example, fig. S10).

Table 2.

Numbers of genes that encode domains similar to plant R proteins in Populus, Arabidopsis (81), and Oryza (82). *, BED finger and/or DUF1544 domain; CC, coiled coil; –, not detected.

Predicted protein domainsLetter code Populus Arabidopsis Oryza
TIR-NBS TN 10 21
TIR-NBS-LRR TNL 64 83
TIR-NBS-LRR-TIR TNLT 13
TIR-NBS-LRR-NBS TNLN 1
NBS-LRR-TIR NLT 1
TIR-CC-NBS-LRR TCNL 2
CC-NBS CN 19 4 7
CC-NBS-LRR CNL 119 51 159
BED/DUF1544*-NBS BN 5
NBS-BED/DUF1544* NB 1
BED/DUF1544*-NBS-LRR BNL 24
NBS-LRR NL 90 6 40
NBS N 49 1 45
Others 41 284
Total NBS genes 398 207 535

Several conserved signaling components such as RAR1, EDS1, PAD4, and NPR1, known to be recruited by R genes, also contain multiple homologs in Populus. For example, two copies of the PAD4 gene, which functions upstream of salicylic acid accumulation, and five copies of the NPR1 gene, an important regulator of responses downstream of salicylic acid, are found in Populus. Nearly all genes known to control disease resistance signaling in Arabidopsis have putative orthologs in Populus. Populus has a larger number of β-1,3-glucanase and chitinase genes than does Arabidopsis (131 versus 73, respectively). In summary, the structural and genetic diversity that exists among R genes and their signaling components in Populus is remarkable and suggests that unlike the rest of the genome, contemporary diversifying selection has played an important role in the evolution of disease resistance genes in Populus. Such diversification suggests that enhanced ability to detect and respond to biotic challenges through R gene–mediated signaling may be critical over a decades-long life span of this genus.

Membrane transporters. Attributes of Populus biology such as massive interannual, seasonal, and diurnal metabolic shifts and redeployment of carbon and nitrogen may require an elaborate array of transporters. Investigation of gene families coding for transporter proteins (http://plantst.genomics.purdue.edu/) in the Populus genome revealed a general expansion relative to Arabidopsis (1722 versus 959, in Populus versus Arabidopsis, respectively) (table S12). Five gene families, coding for adenosine 5′-triphosphate–binding cassette proteins (ABC transporters, 226 gene models), major facilitator superfamily proteins (187 genes), drug/metabolite transporters (108 genes), amino acid/auxin permeases (95 genes), and POT transporters (90 genes), accounted for more than 40% of the total number of transporter gene models (fig. S14). Some large families such as those encoding POT (4.3X relative to Arabidopsis), glutamate-gated ion channels (3.7X), potassium uptake permeases (2.3X), and ABC transporters (1.9X) are expanded in Populus. We identified a subfamily of five putative aquaporins, lacking in the Arabidopsis. Populus also harbors seven transmembrane receptor genes that have previously only been found in fungi, and two genes, identified as mycorrhizal-specific phosphate transporters, that confirm that the mycorrhizal symbiosis may have an impact on the mineral nutrition of this long-lived species. This expanded inventory of transporters could conceivably play a role in adaptation to nutrient-limited forest soils, long-distance transport and storage of water and metabolites, secretion and movement of secondary metabolites, and/or mediation of resistance to pathogen-produced secondary metabolites or other toxic compounds.

Phytohormones. Both physiological and molecular studies have indicated the importance of hormonal regulation underlying plant development. Auxin, gibberellin, cytokinin, and ethylene responses are of particular interest in tree biology.

Many auxin responses (6671) are controlled by auxin response factor (ARF) transcription factors, which work together with cognate AUX/IAA repressor proteins to regulate auxin-responsive target genes (72, 73). A phylogenetic analysis using the known and predicted ARF protein sequences showed that Populus and Arabidopsis ARF gene families have expanded independently since they diverged from their common ancestor. Six duplicate ARF genes in Populus encode paralogs of ARF genes that are single-copy Arabidopsis genes, including ARF5 (MONOPTEROS), an important gene required for auxin-mediated signal transduction and xylem development. Furthermore, five Arabidopsis ARF genes have four or more predicted Populus ARF gene paralogs. In contrast to ARF genes, Populus does not contain a notably expanded repertoire of AUX/IAA genes relative to Arabidopsis (35 versus 29, respectively) (74). Interestingly, there is a group of four Arabidopsis AUX/IAA genes with no apparent Populus orthologs, suggesting Arabidopsis-specific functions.

Gibberellins (GAs) are thought to regulate multiple processes during wood and root development, including xylem fiber length (75). Among all gibberellin biosynthesis and signaling genes, the Populus GA20-oxidase gene family is the only family with approximately two times the number of genes relative to Arabidopsis, indicating that most of the duplicated genes that arose from the salicoid duplication event have been lost. GA20-oxidase appears to control flux in the biosynthetic pathway leading to the bioactive gibberellins GA1 and GA4. The higher complement of GA20-oxidase genes may have biological importance in Populus with respect to secondary xylem and fiber cell development.

Cytokinins are thought to control the identity and proliferation of cell types relevant for wood formation as well as general cell division (67). The total number of members in gene families encoding cytokinin homeostasis related isopentenyl transferases (IPT) and cytokinin oxidases is roughly similar between Populus and Arabidopsis, although there appears to be lineage-specific expansion of IPT subfamilies. The cytokinin signal transduction pathway represents a two-component phosphorelay system, in which a two-component hybrid receptor initiates a phosphotransfer by means of histidine-containing phosphotransmitters (HPt) to phosphoaccepting response regulators (RR). One family of genes, encoding the two-component receptors (such as CKI1), is notably expanded in Populus (four versus one in Populus and Arabidopsis, respectively) (76). Gene families coding for recently identified pseudo-HPt and atypical RR are overrepresented in Populus relative to Arabidopsis (2.5- and 4.0-fold increase in Populus, respectively). Both of these gene families have been implicated in the negative regulation of cytokinin signaling (67, 77), which is consistent with the idea of increased complexity in regulation of cytokinin signal transduction in Populus.

Populus and Arabidopsis genomes contain almost identical numbers of genes for the three enzymes of ethylene biosynthesis, whereas the number of genes for proteins involved in ethylene perception and signaling is higher in Populus. For example, Populus has seven predicted genes for ethylene receptor proteins and Arabidopsis has five; the constitutive triple response kinase that acts just downstream of the receptor is encoded by four genes in Populus and only one in Arabidopsis (78). The number of ethylene-responsive element binding factor (ERF) proteins (a subfamily of the AP2/ERF family) is higher in Populus than in Arabidopsis (172 versus 122, respectively). The increased variation in the number of ERF transcription factors may be involved in the ethylene-dependent processes specific to trees, such as tension wood formation (68) and the establishment of dormancy (71).

Conclusions

Our initial analyses provide a flavor of the opportunities for comparative plant genomics made possible by the generation of the Populus genome sequence. A complex history of whole-genome duplications, chromosomal rearrangements and tandem duplications has shaped the genome that we observe today. The differences in gene content between Populus and Arabidopsis have provided some tantalizing insights into the possible molecular bases of their strongly contrasting life histories, although factors unrelated to gene content (such as regulatory elements, miRNAs, posttranslational modification, or epigenetic modifications) may ultimately be of equal or greater importance. With the sequence of Populus, researchers can now go beyond what could be learned from Arabidopsis alone and explore hypotheses to linking genome sequence features to wood development, nutrient and water movement, crown development, and disease resistance in perennial plants. The availability of the Populus genome sequence will enable continuing comparative genomics studies among species that will shed new light on genome reorganization and gene family evolution. Furthermore, the genetics and population biology of Populus make it an immense source of allelic variation. Because Populus is an obligate outcrossing species, recessive alleles tend to be maintained in a heterozygous state. Informatics tools enabled by the sequence, assembly, and annotation of the Populus genome will facilitate the characterization of allelic variation in wild Populus populations adapted to a wide range of environmental conditions and gradients over large portions of the Northern Hemisphere. Such variants represent a rich reservoir of molecular resources useful in biotechnological applications, development of alternative energy sources, and mitigation of anthropogenic environmental problems. Finally, the keystone role of Populus in many ecosystems provides the first opportunity for the application of genomics approaches to questions with ecosystem-scale implications.

Supporting Online Material

www.sciencemag.org/cgi/content/full/313/5793/1596/DC1

Materials and Methods

Figs. S1 to S15

Tables S1 to S14

References

References and Notes

View Abstract

Navigate This Article