Abstract
The most studied DNA methylation pathway in plants is the RNA Directed DNA Methylation (RdDM), a conserved mechanism that involves the role of noncoding RNAs to control the expansion of the noncoding genome. Genome-wide DNA methylation levels have been reported to correlate with genome size. However, little is known about the catalog of noncoding RNAs and the impact on DNA methylation in small plant genomes with reduced noncoding regions. Because of the small length of intergenic regions in the compact genome of the carnivorous plant Utricularia gibba, we investigated its repertoire of noncoding RNA and DNA methylation landscape. Here, we report that, compared to other angiosperms, U. gibba has an unusual distribution of small RNAs and reduced global DNA methylation levels. DNA methylation was determined using a novel strategy based on long-read DNA sequencing with the Pacific Bioscience platform and confirmed by whole-genome bisulfite sequencing. Moreover, some key genes involved in the RdDM pathway may not represented by compensatory paralogs or comprise truncated proteins, for example, U. gibba DICER-LIKE 3 (DCL3), encoding a DICER endonuclease that produces 24-nt small-interfering RNAs, has lost key domains required for complete function. Our results unveil that a truncated DCL3 correlates with a decreased proportion of 24-nt small-interfering RNAs, low DNA methylation levels, and developmental abnormalities during female gametogenesis in U. gibba. Alterations in female gametogenesis are reminiscent of RdDM mutant phenotypes in Arabidopsis thaliana. It would be interesting to further study the biological implications of the DCL3 truncation in U. gibba, as it could represent an initial step in the evolution of RdDM pathway in compact genomes.
Highlights
DNA modifications are chemical additions to DNA and/or histones that are associated with changes in gene expression and can be heritable[1]
U. gibba long-noncoding RNAs (lncRNAs) has a reduced number of intergenic lncRNAs. lncRNAs were obtained from sequencing data of 12 RNA-Seq libraries, 10 from vegetative and 2 from trap tissue from plants subjected to the contrasting abiotic stress or hormone treatments (Additional file 1: Dataset S1)
When putative lncRNAs were mapped onto the U. gibba genome, we determined that 37.03% mapped to regions corresponding to exons of protein coding genes in antisense orientation, 10.89% to intronic regions, 25.26% overlapped gene bodies and intergenic regions (IGR), and 26.82% were located only in IGRs (Fig. 1c)
Summary
DNA modifications are chemical additions to DNA and/or histones that are associated with changes in gene expression and can be heritable[1]. In addition to Pol-IV and Pol-V, other key components in RdDM and other sRNA biogenesis pathways key protein families are the ARGONAUTE (AGO), DICER-LIKE (DCL), and RNA DEPENDENT RNA POLYMERASE (RDR). Methylome analysis has revealed variation of methylation patterns in intergenic and gene body regions among different plant species[11,12] and these patterns could be related to genome architectural features such as genome size, rearrangements, duplications, and content/expansion of transposable elements (TEs), among others. Little is known about the relationship between DNA methylation, number, type of ncRNA loci, and genome size, for plant species with small genomes with reduced TEs and other repetitive sequences. We report that U. gibba has, compared to Arabidopsis, a functionally incomplete canonical pathway for siRNAs biogenesis and de novo methylation that correlates with a rather unusual content and proportion of sRNA. We suggest that Pacific Bioscience (PacBio) Single Molecule Real-Time (SMRT) long read sequencing data to determine 5mC methylomes is a useful alternative to BS-seq
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