In Arabidopsis thaliana, abundant 24 nucleotide small interfering RNAs (24 nt siRNA) guide the cytosine methylation and silencing of transposons and a subset of genes. 24 nt siRNA biogenesis requires nuclear RNA polymerase IV (Pol IV), RNA-dependent RNA polymerase 2 (RDR2) and DICER-like 3 (DCL3). However, siRNA precursors are mostly undefined. We identified Pol IV and RDR2-dependent RNAs (P4R2 RNAs) that accumulate in dcl3 mutants and are diced into 24 nt RNAs by DCL3 in vitro. P4R2 RNAs are mostly 26-45 nt and initiate with a purine adjacent to a pyrimidine, characteristics shared by Pol IV transcripts generated in vitro. RDR2 terminal transferase activity, also demonstrated in vitro, may account for occasional non-templated nucleotides at P4R2 RNA 3’ termini. The 24 nt siRNAs primarily correspond to the 5’ or 3’ ends of P4R2 RNAs, suggesting a model whereby siRNAs are generated from either end of P4R2 duplexes by single dicing events.

Small RNAs (sRNAs) that are 21 to 24 nucleotides (nt) in length are found in most eukaryotic organisms and regulate numerous biological functions, including transposon silencing, development, reproduction, and stress responses, typically via control of the stability and/or translation of target mRNAs. Major classes of sRNAs in plants include microRNAs (miRNAs) and small interfering RNAs (siRNAs); sRNAs are known to travel as a silencing signal from cell to cell, root to shoot, and even between host and pathogen. In mammals, sRNAs are transported inside extracellular vesicles (EVs), which are mobile membrane-bound compartments that participate in intercellular communication. In addition to sRNAs, EVs carry proteins, lipids, metabolites, and potentially other types of nucleic acids. Here we report that Arabidopsis (Arabidopsis thaliana) EVs also contain diverse species of sRNA. We found that specific miRNAs and siRNAs are preferentially loaded into plant EVs. We also report a previously overlooked class of “tiny RNAs” (10 to 17 nt) that are highly enriched in EVs. This RNA category of unknown function has a broad and very diverse genome origin and might correspond to degradation products.

Background: Theobroma cacao L. cultivar Matina 1-6 belongs to the most cultivated cacao type. The availability of its genome sequence and methods for identifying genes responsible for important cacao traits will aid cacao researchers and breeders. Results: We describe the sequencing and assembly of the genome of Theobroma cacao L. cultivar Matina 1-6. The genome of the Matina 1-6 cultivar is 445 Mbp, which is significantly larger than a sequenced Criollo cultivar, and more typical of other cultivars. The chromosome-scale assembly, version 1.1, contains 711 scaffolds covering 346.0 Mbp, with a contig N50 of 84.4 kbp, a scaffold N50 of 34.4 Mbp, and an evidence-based gene set of 29,408 loci. Version 1.1 has 10x the scaffold N50 and 4x the contig N50 as Criollo, and includes 111 Mb more anchored sequence. The version 1.1 assembly has 4.4% gap sequence, while Criollo has 10.9%. Through a combination of haplotype, association mapping and gene expression analyses, we leverage this robust reference genome to identify a promising candidate gene responsible for pod color variation. We demonstrate that green/red pod color in cacao is likely regulated by the R2R3 MYB transcription factor TcMYB113, homologs of which determine pigmentation in Rosaceae, Solanaceae, and Brassicaceae. One SNP within the target site for a highly conserved trans-acting siRNA in dicots, found within TcMYB113, seems to affect transcript levels of this gene and therefore pod color variation. Conclusions: We report a high-quality sequence and annotation of Theobroma cacao L. and demonstrate its utility in identifying candidate genes regulating traits.

Summary: Camelina (Camelina sativa), a Brassicaceae oilseed, has received recent interest as a biofuel crop and production platform for industrial oils. Limiting wider production of camelina for these uses is the need to improve the quality and content of the seed protein‐rich meal and oil, which is enriched in oxidatively unstable polyunsaturated fatty acids that are deleterious for biodiesel. To identify candidate genes for meal and oil quality improvement, a transcriptome reference was built from 2047 Sanger ESTs and more than 2 million 454‐derived sequence reads, representing genes expressed in developing camelina seeds. The transcriptome of approximately 60K transcripts from 22 597 putative genes includes camelina homologues of nearly all known seed‐expressed genes, suggesting a high level of completeness and usefulness of the reference. These sequences included candidates for 12S (cruciferins) and 2S (napins) seed storage proteins (SSPs) and nearly all known lipid genes, which have been compiled into an accessible database. To demonstrate the utility of the transcriptome for seed quality modification, seed‐specific RNAi lines deficient in napins were generated by targeting 2S SSP genes, and high oleic acid oil lines were obtained by targeting FATTY ACID DESATURASE 2 (FAD2) and FATTY ACID ELONGASE 1 (FAE1). The high sequence identity between Arabidopsis thaliana and camelina genes was also exploited to engineer high oleic lines by RNAi with Arabidopsis FAD2 and FAE1 sequences. It is expected that these transcriptomic data will be useful for breeding and engineering of additional camelina seed traits and for translating findings from the model Arabidopsis to an oilseed crop.

The genomes of most, if not all, flowering plants have undergone whole genome duplication events during their evolution. The impact of such polyploidy events is poorly understood, as is the fate of most duplicated genes. We sequenced an approximately 1 million-bp region in soybean (Glycine max) centered on the Rpg1-b disease resistance gene and compared this region with a region duplicated 10 to 14 million years ago. These two regions were also compared with homologous regions in several related legume species (a second soybean genotype, Glycine tomentella, Phaseolus vulgaris, and Medicago truncatula), which enabled us to determine how each of the duplicated regions (homoeologues) in soybean has changed following polyploidy. The biggest change was in retroelement content, with homoeologue 2 having expanded to 3-fold the size of homoeologue 1. Despite this accumulation of retroelements, over 77% of the duplicated low-copy genes have been retained in the same order and appear to be functional. This finding contrasts with recent analyses of the maize (Zea mays) genome, in which only about one-third of duplicated genes appear to have been retained over a similar time period. Fluorescent in situ hybridization revealed that the homoeologue 2 region is located very near a centromere. Thus, pericentromeric localization, per se, does not result in a high rate of gene inactivation, despite greatly accelerated retrotransposon accumulation. In contrast to low-copy genes, nucleotide-binding-leucine-rich repeat disease resistance gene clusters have undergone dramatic species/homoeologue-specific duplications and losses, with some evidence for partitioning of subfamilies between homoeologues.

The full exploration of gene-environment interactions requires model organisms with well-characterized ecological interactions in their natural environment, manipulability in the laboratory and genomic tools. The waterfleaDaphnia magnais an established ecological and toxicological model species, central to the food webs of freshwater lentic habitats and sentinel for water quality. Its tractability and cyclic parthenogenetic life-cycle are ideal to investigate links between genes and the environment. Capitalizing on this unique model system, the STRESSFLEA consortium generated a comprehensive RNA-Seq data set by exposing two inbred genotypes ofD. magnaand a recombinant cross of these genotypes to a range of environmental perturbations. Gene models were constructed from the transcriptome data and mapped onto the draft genome ofD. magnausing EvidentialGene. The transcriptome data generated here, together with the available draft genome sequence ofD. magnaand a high-density genetic map will be a key asset for future investigations in environmental genomics.