Genome and time-of-day transcriptome of Wolffia australiana link morphological minimization with gene loss and less growth control.

Rootless plants in the genus Wolffia are some of the fastest growing known plants on Earth. Wolffia have a reduced body plan, primarily multiplying through a budding type of asexual reproduction. Here, we generated draft reference genomes for Wolffia australiana (Benth.) Hartog & Plas, which has the smallest genome size in the genus at 357 Mb and has a reduced set of predicted protein-coding genes at about 15,000. Comparison between multiple high-quality draft genome sequences from W. australiana clones confirmed loss of several hundred genes that are highly conserved among flowering plants, including genes involved in root developmental and light signaling pathways. Wolffia has also lost most of the conserved nucleotide-binding leucine-rich repeat (NLR) genes that are known to be involved in innate immunity, as well as those involved in terpene biosynthesis, while having a significant overrepresentation of genes in the sphingolipid pathways that may signify an alternative defense system. Diurnal expression analysis revealed that only 13% of Wolffia genes are expressed in a time-of-day (TOD) fashion, which is less than the typical ∼40% found in several model plants under the same condition. In contrast to the model plants Arabidopsis and rice, many of the pathways associated with multicellular and developmental processes are not under TOD control in W. australiana, where genes that cycle the conditions tested predominantly have carbon processing and chloroplast-related functions. The Wolffia genome and TOD expression data set thus provide insight into the interplay between a streamlined plant body plan and optimized growth.

The complex architecture and epigenomic impact of plant T-DNA insertions.

The bacterium Agrobacterium tumefaciens has been the workhorse in plant genome engineering. Customized replacement of native tumor-inducing (Ti) plasmid elements enabled insertion of a sequence of interest called Transfer-DNA (T-DNA) into any plant genome. Although these transfer mechanisms are well understood, detailed understanding of structure and epigenomic status of insertion events was limited by current technologies. Here we applied two single-molecule technologies and analyzed Arabidopsis thaliana lines from three widely used T-DNA insertion collections (SALK, SAIL and WISC). Optical maps for four randomly selected T-DNA lines revealed between one and seven insertions/rearrangements, and the length of individual insertions from 27 to 236 kilobases. De novo nanopore sequencing-based assemblies for two segregating lines partially resolved T-DNA structures and revealed multiple translocations and exchange of chromosome arm ends. For the current TAIR10 reference genome, nanopore contigs corrected 83% of non-centromeric misassemblies. The unprecedented contiguous nucleotide-level resolution enabled an in-depth study of the epigenome at T-DNA insertion sites. SALK_059379 line T-DNA insertions were enriched for 24nt small interfering RNAs (siRNA) and dense cytosine DNA methylation, resulting in transgene silencing via the RNA-directed DNA methylation pathway. In contrast, SAIL_232 line T-DNA insertions are predominantly targeted by 21/22nt siRNAs, with DNA methylation and silencing limited to a reporter, but not the resistance gene. Additionally, we profiled the H3K4me3, H3K27me3 and H2A.Z chromatin environments around T-DNA insertions using ChIP-seq in SALK_059379, SAIL_232 and five additional T-DNA lines. We discovered various effect s ranging from complete loss of chromatin marks to the de novo incorporation of H2A.Z and trimethylation of H3K4 and H3K27 around the T-DNA integration sites. This study provides new insights into the structural impact of inserting foreign fragments into plant genomes and demonstrates the utility of state-of-the-art long-range sequencing technologies to rapidly identify unanticipated genomic changes.

High contiguity Arabidopsis thaliana genome assembly with a single nanopore flow cell.

The handheld Oxford Nanopore MinION sequencer generates ultra-long reads with minimal cost and time requirements, which makes sequencing genomes at the bench feasible. Here, we sequence the gold standard Arabidopsis thaliana genome (KBS-Mac-74 accession) on the bench with the MinION sequencer, and assemble the genome using typical consumer computing hardware (4 Cores, 16 Gb RAM) into chromosome arms (62 contigs with an N50 length of 12.3 Mb). We validate the contiguity and quality of the assembly with two independent single-molecule technologies, Bionano optical genome maps and Pacific Biosciences Sequel sequencing. The new A. thaliana KBS-Mac-74 genome enables resolution of a quantitative trait locus that had previously been recalcitrant to a Sanger-based BAC sequencing approach. In summary, we demonstrate that even when the purpose is to understand complex structural variation at a single region of the genome, complete genome assembly is becoming the simplest way to achieve this goal.

Robust single-cell DNA methylome profiling with snmC-seq2.

Single-cell DNA methylome profiling has enabled the study of epigenomic heterogeneity in complex tissues and during cellular reprogramming. However, broader applications of the method have been impeded by the modest quality of sequencing libraries. Here we report snmC-seq2, which provides improved read mapping, reduced artifactual reads, enhanced throughput, as well as increased library complexity and coverage uniformity compared to snmC-seq. snmC-seq2 is an efficient strategy suited for large-scale single-cell epigenomic studies.

Single-cell methylomes identify neuronal subtypes and regulatory elements in mammalian cortex.

The mammalian brain contains diverse neuronal types, yet we lack single-cell epigenomic assays that are able to identify and characterize them. DNA methylation is a stable epigenetic mark that distinguishes cell types and marks regulatory elements. We generated >6000 methylomes from single neuronal nuclei and used them to identify 16 mouse and 21 human neuronal subpopulations in the frontal cortex. CG and non-CG methylation exhibited cell type-specific distributions, and we identified regulatory elements with differential methylation across neuron types. Methylation signatures identified a layer 6 excitatory neuron subtype and a unique human parvalbumin-expressing inhibitory neuron subtype. We observed stronger cross-species conservation of regulatory elements in inhibitory neurons than in excitatory neurons. Single-nucleus methylomes expand the atlas of brain cell types and identify regulatory elements that drive conserved brain cell diversity.