Natural variation in Brachypodium distachyon responses to combined abiotic stresses.

The demand for agricultural production is becoming more challenging as climate change increases global temperature and the frequency of extreme weather events. This study examines the phenotypic variation of 149 accessions of Brachypodium distachyon under drought, heat, and the combination of stresses. Heat alone causes the largest amounts of tissue damage while the combination of stresses causes the largest decrease in biomass compared to other treatments. Notably, Bd21-0, the reference line for B. distachyon, did not have robust growth under stress conditions, especially the heat and combined drought and heat treatments. The climate of origin was significantly associated with B. distachyon responses to the assessed stress conditions. Additionally, a GWAS found loci associated with changes in plant height and the amount of damaged tissue under stress. Some of these SNPs were closely located to genes known to be involved in responses to abiotic stresses and point to potential causative loci in plant stress response. However, SNPs found to be significantly associated with a response to heat or drought individually are not also significantly associated with the combination of stresses. This, with the phenotypic data, suggests that the effects of these abiotic stresses are not simply additive, and the responses to the combined stresses differ from drought and heat alone.

Integrated annotations and analyses of small RNA-producing loci from 47 diverse plants.

Plant endogenous small RNAs (sRNAs) are important regulators of gene expression. There are two broad categories of plant sRNAs: microRNAs (miRNAs) and endogenous short interfering RNAs (siRNAs). MicroRNA loci are relatively well-annotated but compose only a small minority of the total sRNA pool; siRNA locus annotations have lagged far behind. Here, we used a large data set of published and newly generated sRNA sequencing data (1333 sRNA-seq libraries containing more than 20 billion reads) and a uniform bioinformatic pipeline to produce comprehensive sRNA locus annotations of 47 diverse plants, yielding more than 2.7 million sRNA loci. The two most numerous classes of siRNA loci produced mainly 24- and 21-nucleotide (nt) siRNAs, respectively. Most often, 24-nt-dominated siRNA loci occurred in intergenic regions, especially at the 5'-flanking regions of protein-coding genes. In contrast, 21-nt-dominated siRNA loci were most often derived from double-stranded RNA precursors copied from spliced mRNAs. Genic 21-nt-dominated loci were especially common from disease resistance genes, including from a large number of monocots. Individual siRNA sequences of all types showed very little conservation across species, whereas mature miRNAs were more likely to be conserved. We developed a web server where our data and several search and analysis tools are freely accessible.

Large structural variations in the haplotype-resolved African cassava genome.

Cassava (Manihot esculenta Crantz, 2n = 36) is a global food security crop. It has a highly heterozygous genome, high genetic load, and genotype-dependent asynchronous flowering. It is typically propagated by stem cuttings and any genetic variation between haplotypes, including large structural variations, is preserved by such clonal propagation. Traditional genome assembly approaches generate a collapsed haplotype representation of the genome. In highly heterozygous plants, this results in artifacts and an oversimplification of heterozygous regions. We used a combination of Pacific Biosciences (PacBio), Illumina, and Hi-C to resolve each haplotype of the genome of a farmer-preferred cassava line, TME7 (Oko-iyawo). PacBio reads were assembled using the FALCON suite. Phase switch errors were corrected using FALCON-Phase and Hi-C read data. The ultralong-range information from Hi-C sequencing was also used for scaffolding. Comparison of the two phases revealed >5000 large haplotype-specific structural variants affecting over 8 Mb, including insertions and deletions spanning thousands of base pairs. The potential of these variants to affect allele-specific expression was further explored. RNA-sequencing data from 11 different tissue types were mapped against the scaffolded haploid assembly and gene expression data are incorporated into our existing easy-to-use web-based interface to facilitate use by the broader plant science community. These two assemblies provide an excellent means to study the effects of heterozygosity, haplotype-specific structural variation, gene hemizygosity, and allele-specific gene expression contributing to important agricultural traits and further our understanding of the genetics and domestication of cassava.

Analysis of RDR1/RDR2/RDR6-independent small RNAs in Arabidopsis thaliana improves MIRNA annotations and reveals unexplained types of short interfering RNA loci.

Plant small RNAs (sRNAs) modulate key physiological mechanisms through post-transcriptional and transcriptional silencing of gene expression. Small RNAs fall into two major categories: those are reliant on RNA-dependent RNA polymerases (RDRs) for biogenesis and those that are not. Known RDR1/2/6-dependent sRNAs include phased and repeat-associated short interfering RNAs, while known RDR1/2/6-independent sRNAs are primarily microRNAs (miRNA) and other hairpin-derived sRNAs. In this study we produced and analyzed sRNA-seq libraries from rdr1/rdr2/rdr6 triple mutant plants. We found 58 previously annotated miRNA loci that were reliant on RDR1, -2, or -6 function, casting doubt on their classification. We also found 38 RDR1/2/6-independent sRNA loci that are not MIRNAs or otherwise hairpin-derived, and did not fit into other known paradigms for sRNA biogenesis. These 38 sRNA-producing loci have as-yet-undescribed biogenesis mechanisms, and are frequently located in the vicinity of protein-coding genes. Altogether, our analysis suggests that these 38 loci represent one or more undescribed types of sRNA in Arabidopsis thaliana.

Several phased siRNA annotation methods can frequently misidentify 24 nucleotide siRNA-dominated PHAS loci.

Small RNAs regulate key physiological functions in land plants. Small RNAs can be divided into two categories: microRNAs (miRNAs) and short interfering RNAs (siRNAs); siRNAs are further subdivided into transposon/repetitive region-localized heterochromatic siRNAs and phased siRNAs (phasiRNAs). PhasiRNAs are produced from the miRNA-mediated cleavage of a Pol II RNA transcript; the miRNA cleavage site provides a defined starting point from which phasiRNAs are produced in a distinctly phased pattern. 21-22 nucleotide (nt)-dominated phasiRNA-producing loci (PHAS) are well represented in all land plants to date. In contrast, 24 nt-dominated PHAS loci are known to be encoded only in monocots and are generally restricted to male reproductive tissues. Currently, only one miRNA (miR2275) is known to trigger the production of these 24 nt-dominated PHAS loci. In this study, we use stringent methodologies in order to examine whether or not 24 nt-dominated PHAS loci also exist in Arabidopsis thaliana. We find that highly expressed heterochromatic siRNAs were consistently misidentified as 24 nt-dominated PHAS loci using multiple PHAS-detecting algorithms. We also find that MIR2275 is not found in A. thaliana, and it seems to have been lost in the last common ancestor of Brassicales. Altogether, our research highlights the potential issues with widely used PHAS-detecting algorithms which may lead to false positives when trying to annotate new PHAS, especially 24 nt-dominated loci.

More than meets the eye? Factors that affect target selection by plant miRNAs and heterochromatic siRNAs.

MicroRNAs, which target mRNAs for post-transcriptional regulation, and heterochromatic siRNAs, which target chromatin causing DNA methylation, make up the majority of the endogenous regulatory small RNA pool in most plant specimens. They both function to guide Argonaute proteins to targeted nucleic acids on the basis of complementarity. Recent work on plant miRNA-target interactions has clarified the general ''rules' of complementarity, while also providing several intriguing exceptions to these rules. In addition, emerging evidence suggests that several factors besides miRNA-target complementarity affect plant miRNA function. For heterochromatic siRNAs, recent work has made progress towards comprehensively identifying potential target regions, but numerous fundamental questions remain to be answered.