Abstract Understanding genome organization and gene regulation requires insight into RNA transcription, processing and modification. We adapted nanopore direct RNA sequencing to examine RNA from a wild-type accession of the model plant Arabidopsis thaliana and a mutant defective in mRNA methylation (m 6 A). Here we show that m 6 A can be mapped in full-length mRNAs transcriptome-wide and reveal the combinatorial diversity of cap-associated transcription start sites, splicing events, poly(A) site choice and poly(A) tail length. Loss of m 6 A from 3’ untranslated regions is associated with decreased relative transcript abundance and defective RNA 3′ end formation. A functional consequence of disrupted m 6 A is a lengthening of the circadian period. We conclude that nanopore direct RNA sequencing can reveal the complexity of mRNA processing and modification in full-length single molecule reads. These findings can refine Arabidopsis genome annotation. Further, applying this approach to less well-studied species could transform our understanding of what their genomes encode.
Abstract Sequence exchange between homologous chromosomes through crossing over and gene conversion is highly conserved among eukaryotes, contributing to genome stability and genetic diversity. Lack of recombination limits breeding efforts in crops, therefore increasing recombination rates can reduce linkage-drag and generate new genetic combinations. We use computational analysis of 13 recombinant inbred mapping populations to assess crossover and gene conversion frequency in the hexaploid genome of wheat ( Triticum aestivum ). We observe that high frequency crossover sites are shared between populations and that closely related parental founders lead to populations with more similar crossover patterns. We demonstrate that gene conversion is more prevalent and covers more of the genome in wheat than in other plants, making it a critical process in the generation of new haplotypes, particularly in centromeric regions where crossovers are rare. We have identified QTL for altered gene conversion and crossover frequency and confirm functionality for a novel RecQ helicase gene that belongs to an ancient clade that is missing in some plant lineages including Arabidopsis . This is the first gene to be demonstrated to be involved in gene conversion in wheat. Harnessing the RecQ helicase has the potential to break linkage-drag utilizing widespread gene conversions.
Abstract In response to exogenous rhythms of light and temperature, most organisms exhibit endogenous circadian rhythms (i.e. cycles of behavior and gene expression with a periodicity of approximately 24 h). One of the defining characteristics of the circadian clock is its ability to synchronize (entrain) to an environmental rhythm. Entrainment is arguably the most salient feature of the clock in evolutionary terms. Previous quantitative trait studies of circadian characteristics in Arabidopsis (Arabidopsis thaliana) considered leaf movement under constant (free-running) conditions. This study, however, addressed the important circadian parameter of phase, which reflects the entrained relationship between the clock and the external cycle. Here it is shown that, when exposed to the same photoperiod, Arabidopsis accessions differ dramatically in phase. Variation in the timing of circadian LUCIFERASE expression was used to map loci affecting the entrained phase of the clock in a recombinant population derived from two geographically distant accessions, Landsberg erecta and Cape Verde Islands. Four quantitative trait loci (QTL) were found with major effects on circadian phase. A QTL on chromosome 5 contained SIGNALING IN RED LIGHT REDUCED 1 and PSEUDORESPONSE REGULATOR 3, both genes known to affect the circadian clock. Previously unknown polymorphisms were found in both genes, making them candidates for the effect on phase. Fine mapping of two other QTL highlighted genomic regions not previously identified in any circadian screens, indicating their effects are likely due to genes not hitherto considered part of the circadian system.
Abstract RNA-Seq is a fundamental technique in genomics, yet reference bias, where transcripts derived from non-reference alleles are quantified less accurately, can undermine the accuracy of RNA-Seq quantification and thus the conclusions made downstream. Reference bias in RNA-Seq analysis has yet to be explored in complex polyploid genomes despite evidence that they are often a complex mosaic of wild relative introgressions, which introduce blocks of highly divergent genes. Here we use hexaploid wheat as a model complex polyploid, using both simulated and experimental data to show that RNA-Seq alignment in wheat suffers from widespread reference bias which is largely driven by divergent introgressed genes. This leads to underestimation of gene expression and incorrect assessment of homoeologue expression balance. By incorporating gene models from ten wheat genome assemblies into a pantranscriptome reference, we present a novel method to reduce reference bias, which can be readily scaled to capture more variation as new genome and transcriptome data becomes available.
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