Long-read sequencing can overcome the weaknesses of short reads in the assembly of eukaryotic genomes; however, at present additional scaffolding is needed to achieve chromosome-level assemblies. We generated Pacific Biosciences (PacBio) long-read data of the genomes of three relatives of the model plant Arabidopsis thaliana and assembled all three genomes into only a few hundred contigs. To improve the contiguities of these assemblies, we generated BioNano Genomics optical mapping and Dovetail Genomics chromosome conformation capture data for genome scaffolding. Despite their technical differences, optical mapping and chromosome conformation capture performed similarly and doubled N50 values. After improving both integration methods, assembly contiguity reached chromosome-arm-levels. We rigorously assessed the quality of contigs and scaffolds using Illumina mate-pair libraries and genetic map information. This showed that PacBio assemblies have high sequence accuracy but can contain several misassemblies, which join unlinked regions of the genome. Most, but not all, of these misjoints were removed during the integration of the optical mapping and chromosome conformation capture data. Even though none of the centromeres were fully assembled, the scaffolds revealed large parts of some centromeric regions, even including some of the heterochromatic regions, which are not present in gold standard reference sequences.

Background: Global warming severely affects flowering time and reproductive success of plants. Alternative splicing of pre-messenger RNA (mRNA) is an important mechanism underlying ambient temperature-controlled responses in plants, yet its regulation is poorly understood. An increase in temperature promotes changes in plant morphology as well as the transition from the vegetative to the reproductive phase in Arabidopsis thaliana via changes in splicing of key regulatory genes. Here we investigate whether a particular histone modification affects ambient temperature-induced alternative splicing and flowering time.Results: We use a genome-wide approach and perform RNA-sequencing (RNA-seq) analyses and histone H3 lysine 36 tri-methylation (H3K36me3) chromatin immunoprecipitation sequencing (ChIP-seq) in plants exposed to different ambient temperatures. Analysis and comparison of these datasets reveal that temperature-induced differentially spliced genes are enriched in H3K36me3. Moreover, we find that reduction of H3K36me3 deposition causes alteration in temperature-induced alternative splicing. We also show that plants with mutations in H3K36me3 writers, eraser, or readers have altered high ambient temperature-induced flowering. Conclusions: Our results show a key role for the histone mark H3K36me3 in splicing regulation and plant plasticity to fluctuating ambient temperature. Our findings open new perspectives for the breeding of crops that can better cope with environmental changes due to climate change.

Spliceosomal introns are noncoding sequences that separate exons in eukaryotic genes and are removed from pre-messenger RNAs by the splicing machinery. Their origin has remained a mystery in biology since their discovery because intron gains seem to be infrequent in many eukaryotic lineages. Although a few recent intron gains have been reported, none of the proposed gain mechanisms can convincingly explain the high number of introns in present-day eukaryotic genomes. Here we report on particular spliceosomal introns that share high sequence similarity and are reminiscent of introner elements. These elements multiplied in unrelated genes of six fungal genomes and account for the vast majority of intron gains in these fungal species. Such introner-like elements (ILEs) contain all typical characteristics of regular spliceosomal introns (RSIs) but are longer and predicted to harbor more stable secondary structures. However, dating of multiplication events showed that they degenerate in sequence and length within 100,000 years to eventually become indistinguishable from RSIs. We suggest that ILEs not only account for intron gains in six fungi but also in ancestral eukaryotes to give rise to most RSIs by a yet unknown multiplication mechanism.

The reproductive strategies of plants are highly variable. Short-lived annuals flower abundantly soon after germination, whereas longer-lived perennials postpone and spatially restrict flowering. We used CRISPR/Cas9 and interspecies gene transfer to understand divergence in reproductive patterns between annual and perennial crucifers. We show that in perennial Arabis alpina, flowering in response to winter cold depends on the floral integrator SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 15 (SPL15), whose activity is limited to older shoots and branches during cold exposure. In annuals, this regulatory system is conserved, but cold-induced flowering occurs in young shoots, without requirement for SPL15, through the photoperiodic pathway when plants return to warm. By reconstructing the annual response in perennials, we conclude that characteristic patterns of reproduction in annuals and perennials are conferred through variation in dependency on distinct flowering pathways acting in parallel.