The genus Liriodendron belongs to the family Magnoliaceae, which resides within the magnoliids, an early diverging lineage of the Mesangiospermae. However, the phylogenetic relationship of magnoliids with eudicots and monocots has not been conclusively resolved and thus remains to be determined1–6. Liriodendron is a relict lineage from the Tertiary with two distinct species—one East Asian (L. chinense (Hemsley) Sargent) and one eastern North American (L. tulipifera Linn)—identified as a vicariad species pair. However, the genetic divergence and evolutionary trajectories of these species remain to be elucidated at the whole-genome level7. Here, we report the first de novo genome assembly of a plant in the Magnoliaceae, L. chinense. Phylogenetic analyses suggest that magnoliids are sister to the clade consisting of eudicots and monocots, with rapid diversification occurring in the common ancestor of these three lineages. Analyses of population genetic structure indicate that L. chinense has diverged into two lineages—the eastern and western groups—in China. While L. tulipifera in North America is genetically positioned between the two L. chinense groups, it is closer to the eastern group. This result is consistent with phenotypic observations that suggest that the eastern and western groups of China may have diverged long ago, possibly before the intercontinental differentiation between L. chinense and L. tulipifera. Genetic diversity analyses show that L. chinense has tenfold higher genetic diversity than L. tulipifera, suggesting that the complicated regions comprising east–west-orientated mountains and the Yangtze river basin (especially near 30° N latitude) in East Asia offered more successful refugia than the south–north-orientated mountain valleys in eastern North America during the Quaternary glacial period.

CRISPR (clustered regularly interspaced short palindromic repeats), first discovered as an immune system of prokaryotes, has become a powerful tool for genome editing in eukaryotes (Gaj et al., 2013). Co-expression of the CRISPR-associated endonuclease (Cas9) with a chimeric guide-RNA (gRNA) targeting a GN 19 NGG motif results in a double-strand DNA break near NGG, the protospacer adjacent motif (PAM) (Jinek et al., 2012). Processing by the endogenous DNA repair machinery generates small indels that, when located within a coding sequence, can disrupt the reading frame and render the gene nonfunctional. The CRISPR/ Cas9 system has been successfully applied in several herbaceous systems (Belhaj et al., 2013;Harrison et al., 2014). Here we report its application in the woody perennial Populus using the 4-coumarate:CoA ligase (4CL) gene family as a case study. We achieved 100% mutational efficiency for two 4CL genes targeted, with every transformant examined carrying biallelic modifications. The CRISPR/Cas9 system is highly sensitive to single nucleotide polymorphisms (SNPs), as cleavage for a third 4CL gene was abolished due to SNPs in the target sequence. For outcrossing species with a highly heterozygous genome, gRNA design must take into account the frequent occurrence of SNPs to achieve efficient genome editing.Two 4CL genes, 4CL1 and 4CL2, associated with lignin and flavonoid biosynthesis, respectively (Hu et al., 1998;Harding et al., 2002), were targeted for CRISPR/Cas9 editing. The Populus tremula 9 alba clone 717-1B4 (717) routinely used for transformation is divergent from the genome-sequenced Populus trichocarpa (Hamzeh & Dayanandan, 2004). Therefore, the 4CL1 and 4CL2 gRNAs designed from the reference genome were interrogated with in-house 717 RNA-Seq data to ensure the absence of SNPs which could limit Cas9 efficiency (Supporting Information Fig. S1). A third gRNA designed for 4CL5, a genome duplicate of 4CL1, was also included. The corresponding 717 sequence harbors one SNP in each allele near/within the PAM, both of which are expected to abolish targeting by the 4CL5-gRNA (Fig. S1). All three gRNA target sites are located within the first exon (Fig. S1a).

Small RNAs (sRNAs) are common and effective modulators of gene expression in eukaryotic organisms. To characterize the sRNAs expressed during rice seed development, massively parallel signature sequencing (MPSS) was performed, resulting in the obtainment of 797 399 22-nt sequence signatures, of which 111 161 are distinct ones. Analysis on the distributions of sRNAs on chromosomes showed that most sRNAs originate from interspersed repeats that mainly consist of transposable elements, suggesting the major function of sRNAs in rice seeds is transposon silencing. Through integrative analysis, 26 novel miRNAs and 12 miRNA candidates were identified. Further analysis on the expression profiles of the known and novel miRNAs through hybridizing the generated chips revealed that most miRNAs were expressed preferentially in one or two rice tissues. Detailed comparison of the expression patterns of miRNAs and corresponding target genes revealed the negative correlation between them, while few of them are positively correlated. In addition, differential accumulations of miRNAs and corresponding miRNA*s suggest the functions of miRNA*s other than being passenger strands of mature miRNAs, and in regulating the miRNA functions.