西亚试剂：The willow genome and divergent evolution from poplar after

Willows (Salix) and poplars (Populus) are known worldwide as woody species with diverse uses1,2. Although these two genera diverged from each other around the early Eocene3, they share numerous traits, including the same chromosome number of 2n = 38 and the common 'Salicoid' genome duplication with a high macrosynteny4,5. However, most willow species flower early in their lives with short, small and sometimes indistinct stems, and thus differ from poplars in their life histories and habits2. In addition, multiple inter- and intrachromosomal rearrangements have been detected involving chromosomal regions present in both lineages6, suggestive of the likely genomic divergence after the common genome duplication.

In order to test this hypothesis, we sequenced the genome of a shrub willow S. suchowensis, which flowers within two years1,2. Genome sequencing was conducted with a combined approach using Roche/454 and Illumina/HiSeq-2000 sequencing technologies (Supplementary information, Table S1A and S1B), and the statistics of the genome assembly were listed in Supplementary Table S1C. The size of the S. suchowensis genome was estimated to be ~425 Mb and ~429 Mb based on 17-mer analysis and flow cytometry, respectively (Supplementary information, Figure S1A and Table S1D), about 60 Mb smaller than that of P. trichocarpa (approximately 485 ± 10 Mb)4, which is largely consistent with the previous genome measurements of other willow species7. The final length of the assembled sequence was amounted to about 71% (303.8/425 Mb) of the estimated genome size. Sequencing depth distribution showed that ~94% of the assemblies had greater than 20-fold coverage, ensuring a high level of single-base accuracy (Supplementary information, Figure S1B). A detailed analysis found that the ~120 Mb of unassembled genomic sequence consisted mainly of repetitive sequences, and the protein-coding regions were assembled to near completeness. The S. suchowensis genome assembly is of comparable quality to other sequenced plant genomes based on next-generation sequencing (Supplementary information, Table S1E). We further evaluated the assembled genome using two independent EST datasets. The first comprises the EST assemblies from various tissues of the sequenced individual, including tender root, young leaves, bark, non-lignified shoot and vegetative buds. The second consists of EST assemblies from flower buds collected from natural willow stands. These two analyses identified 97.6% and 94.2% of the two EST assemblies in our S. suchowensis genome assembly, separately, with > 95% identity and > 50% coverage of query length, confirming the nearly complete coverage of genic regions (Supplementary information, Table S1F). Core eukaryotic genes identified by CEGMA were further mapped to the predicted gene set, and 97.8% of the NCBI euKaryotic clusters of Orthologous Groups (KOGs) are covered by the predicted willow gene set, confirming the nearly completeness of the genic regions by the present genome assembly (Supplementary information, Table S1G). Assessment of the quality of gene prediction revealed that the overall gene length distributions were similar between S. suchowensis and other plant species with genomes sequenced (Supplementary information, Figure S1C).

In total, we identified 26 599 putative protein-coding genes (Supplementary information, Table S1H and Data S1), 20 261 of which constitute homolgous gene pairs with the P. trichocarpa reference gene set4. Synteny and collinearity analyses indicate that the chromosomal structures are highly similar between the willow and poplar genomes (Figure 1A and 1B), supporting the concept that these two genera share an additional common (Salicoid) whole genome duplication (WGD) event in their evolutionary history4. Estimation with four-fold synonymous third-codon transversion (4DTv) values of the orthologous pairs suggests that the divergence between Salix and Populus took place around 52 million years ago, approximately 6 million years after the additional WGD. The shared WGD was indicated by sharp peaks in 4DTv values of 0.2842 and 0.2052 for S. suchowensis and P. trichocarpa, respectively (Figure 1C). These different 4DTv values might be caused by different evolutionary rates of nucleotide substitution in these two lineages. Using 1 232 single-copy orthologous genes (Supplementary information, Table S1I), we estimated mean substitution rates of 1.09 × 10−9 and 0.67 × 10−9/site/year for S. suchowensis and P. trichocarpa, respectively. This estimated substitution rate is largely consistent with a previous study of other Salix species based on fewer genes6. Our comparisons suggest that S. suchowensis has a significantly higher substitution rate than P. trichocarpa (P < 2.2e-16). However, substitution rates of both species were substantially lower than those of Arabidopsis thaliana and Oryza sativa (Figure 1D). The great differences in evolutionary rates between these two species are highly correlated with their flowering habits: the early-flowering species has faster substitution rates than the long-generation one. This finding is consistent with previous expectations8,9.

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