Colonic Microbial Abundances Predict Adenoma Formers.

OBJECTIVE: We aimed to examine associations between the oral, fecal, and mucosal microbiome communities and adenoma formation. SUMMARY BACKGROUND DATA: Data are limited regarding the relationships between microbiota and preneoplastic colorectal lesions. METHODS: Individuals undergoing screening colonoscopy were prospectively enrolled and divided into adenoma and nonadenoma formers. Oral, fecal, nonadenoma and adenoma-adjacent mucosa were collected along with clinical and dietary information. 16S rRNA gene libraries were generated using V4 primers. DADA2 processed sequence reads and custom R-scripts quantified microbial diversity. Linear regression identified differential taxonomy and diversity in microbial communities and machine learning identified adenoma former microbial signatures. RESULTS: One hundred four subjects were included, 46% with adenomas. Mucosal and fecal samples were dominated by Firmicutes and Bacteroidetes whereas Firmicutes and Proteobacteria were most abundant in oral communities. Mucosal communities harbored significant microbial diversity that was not observed in fecal or oral communities. Random forest classifiers predicted adenoma formation using fecal, oral, and mucosal amplicon sequence variant (ASV) abundances. The mucosal classifier reliably diagnosed adenoma formation with an area under the curve (AUC) = 0.993 and an out-of-bag (OOB) error of 3.2%. Mucosal classifier accuracy was strongly influenced by five taxa associated with the family Lachnospiraceae, genera Bacteroides and Marvinbryantia, and Blautia obeum. In contrast, classifiers built using fecal and oral samples manifested high OOB error rates (47.3% and 51.1%, respectively) and poor diagnostic abilities (fecal and oral AUC = 0.53). CONCLUSION: Normal mucosa microbial abundances of adenoma formers manifest unique patterns of microbial diversity that may be predictive of adenoma formation.

Early detection of Pseudocapillaria tomentosa by qPCR in four lines of zebrafish, Danio rerio (Hamilton 1882).

The intestinal nematode Pseudocapillaria tomentosa in zebrafish (Danio rerio) causes profound intestinal lesions, emaciation and death and is a promoter of a common intestinal cancer in zebrafish. This nematode has been detected in zebrafish from about 15% of the laboratories. Adult worms are readily detected about 3 weeks after exposure by either histology or wet mount preparations of the intestine, and larval worms are inconsistently observed in fish before this time. A quantitative PCR (qPCR) test was recently developed to detect the worm in fish and water, and here we determined that the test on zebrafish intestines was effective for earlier detection. Four lines of zebrafish (AB, TU, 5D and Casper) were experimentally infected and evaluated by wet mounts and qPCR at 8, 15-, 22-, 31- and 44-day post-exposure (dpe). At the first two time points, only 8% of the wet mounts from exposed fish were identified as infected, while the same intestines screened by qPCR showed 78% positivity, with low and consistent cycle threshold (Ct) values at these times. Wet mounts at later time points showed a high prevalence of infection, but this was still surpassed by qPCR.

Distinct argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline.

Endogenous small RNAs (endo-siRNAs) interact with Argonaute (AGO) proteins to mediate sequence-specific regulation of diverse biological processes. Here, we combine deep-sequencing and genetic approaches to explore the biogenesis and function of endo-siRNAs in C. elegans. We describe conditional alleles of the Dicer-related helicase, drh-3, that abrogate both RNA interference and the biogenesis of endo-siRNAs, called 22G-RNAs. DRH-3 is a core component of RNA-dependent RNA polymerase (RdRP) complexes essential for several distinct 22G-RNA systems. We show that, in the germline, one system is dependent on worm-specific AGOs, including WAGO-1, which localizes to germline nuage structures called P granules. WAGO-1 silences certain genes, transposons, pseudogenes, and cryptic loci. Finally, we demonstrate that components of the nonsense-mediated decay pathway function in at least one WAGO-mediated surveillance pathway. These findings broaden our understanding of the biogenesis and diversity of 22G-RNAs and suggest additional regulatory functions for small RNAs.

Multimegabase silencing in nucleolar dominance involves siRNA-directed DNA methylation and specific methylcytosine-binding proteins.

In genetic hybrids, the silencing of nucleolar rRNA genes inherited from one progenitor is the epigenetic phenomenon known as nucleolar dominance. An RNAi knockdown screen identified the Arabidopsis de novo cytosine methyltransferase, DRM2, and the methylcytosine binding domain proteins, MBD6 and MBD10, as activities required for nucleolar dominance. MBD10 localizes throughout the nucleus, but MBD6 preferentially associates with silenced rRNA genes and does so in a DRM2-dependent manner. DRM2 methylation is thought to be guided by siRNAs whose biogenesis requires RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). Consistent with this hypothesis, knockdown of DCL3 or RDR2 disrupts nucleolar dominance. Collectively, these results indicate that in addition to directing the silencing of retrotransposons and noncoding repeats, siRNAs specify de novo cytosine methylation patterns that are recognized by MBD6 and MBD10 in the large-scale silencing of rRNA gene loci.

PRG-1 and 21U-RNAs interact to form the piRNA complex required for fertility in C. elegans.

In metazoans, Piwi-related Argonaute proteins have been linked to germline maintenance, and to a class of germline-enriched small RNAs termed piRNAs. Here we show that an abundant class of 21 nucleotide small RNAs (21U-RNAs) are expressed in the C. elegans germline, interact with the C. elegans Piwi family member PRG-1, and depend on PRG-1 activity for their accumulation. The PRG-1 protein is expressed throughout development and localizes to nuage-like structures called P granules. Although 21U-RNA loci share a conserved upstream sequence motif, the mature 21U-RNAs are not conserved and, with few exceptions, fail to exhibit complementarity or evidence for direct regulation of other expressed sequences. Our findings demonstrate that 21U-RNAs are the piRNAs of C. elegans and link this class of small RNAs and their associated Piwi Argonaute to the maintenance of temperature-dependent fertility.

The zebrafish gut microbiome influences benzo[a]pyrene developmental neurobehavioral toxicity.

Early-life exposure to environmental toxicants like Benzo[a]pyrene (BaP) is associated with several health consequences in vertebrates (i.e., impaired or altered neurophysiological and behavioral development). Although toxicant impacts were initially studied relative to host physiology, recent studies suggest that the gut microbiome is a possible target and/or mediator of behavioral responses to chemical exposure in organisms, via the gut-brain axis. However, the connection between BaP exposure, gut microbiota, and developmental neurotoxicity remains understudied. Using a zebrafish model, we determined whether the gut microbiome influences BaP impacts on behavior development. Embryonic zebrafish were treated with increasing concentrations of BaP and allowed to grow to the larval life stage, during which they underwent behavioral testing and intestinal dissection for gut microbiome profiling via high-throughput sequencing. We found that exposure affected larval zebrafish microbiome diversity and composition in a manner tied to behavioral development: increasing concentrations of BaP were associated with increased taxonomic diversity, exposure was associated with unweighted UniFrac distance, and microbiome diversity and exposure predicted larval behavior. Further, a gnotobiotic zebrafish experiment clarified whether microbiome presence was associated with BaP exposure response and behavioral changes. We found that gut microbiome state altered the relationship between BaP exposure concentration and behavioral response. These results support the idea that the zebrafish gut microbiome is a determinant of the developmental neurotoxicity that results from chemical exposure.

Virus-derived gene expression and RNA interference vector for grapevine.

The improvement of the agricultural and wine-making qualities of the grapevine (Vitis vinifera) is hampered by adherence to traditional varieties, the recalcitrance of this plant to genetic modifications, and public resistance to genetically modified organism (GMO) technologies. To address these challenges, we developed an RNA virus-based vector for the introduction of desired traits into grapevine without heritable modifications to the genome. This vector expresses recombinant proteins in the phloem tissue that is involved in sugar transport throughout the plant, from leaves to roots to berries. Furthermore, the vector provides a powerful RNA interference (RNAi) capability of regulating the expression of endogenous genes via virus-induced gene-silencing (VIGS) technology. Additional advantages of this vector include superb genetic capacity and stability, as well as the swiftness of technology implementation. The most significant applications of the viral vector include functional genomics of the grapevine and disease control via RNAi-enabled vaccination against pathogens or invertebrate pests.

MicroRNA gene evolution in Arabidopsis lyrata and Arabidopsis thaliana.

MicroRNAs (miRNAs) are short regulatory RNAs processed from partially self-complementary foldbacks within longer MIRNA primary transcripts. Several MIRNA families are conserved deeply through land plants, but many are present only in closely related species or are species specific. The finding of numerous evolutionarily young MIRNA, many with low expression and few if any targets, supports a rapid birth-death model for MIRNA evolution. A systematic analysis of MIRNA genes and families in the close relatives, Arabidopsis thaliana and Arabidopsis lyrata, was conducted using both whole-genome comparisons and high-throughput sequencing of small RNAs. Orthologs of 143 A. thaliana MIRNA genes were identified in A. lyrata, with nine having significant sequence or processing changes that likely alter function. In addition, at least 13% of MIRNA genes in each species are unique, despite their relatively recent speciation (approximately 10 million years ago). Alignment of MIRNA foldbacks to the Arabidopsis genomes revealed evidence for recent origins of 32 families by inverted or direct duplication of mostly protein-coding gene sequences, but less than half of these yield miRNA that are predicted to target transcripts from the originating gene family. miRNA nucleotide divergence between A. lyrata and A. thaliana orthologs was higher for young MIRNA genes, consistent with reduced purifying selection compared with deeply conserved MIRNA genes. Additionally, target sites of younger miRNA were lost more frequently than for deeply conserved families. In summary, our systematic analyses emphasize the dynamic nature of the MIRNA complement of plant genomes.

Disentangling the link between zebrafish diet, gut microbiome succession, and Mycobacterium chelonae infection.

BACKGROUND: Despite the long-established importance of zebrafish (Danio rerio) as a model organism and their increasing use in microbiome-targeted studies, relatively little is known about how husbandry practices involving diet impact the zebrafish gut microbiome. Given the microbiome's important role in mediating host physiology and the potential for diet to drive variation in microbiome composition, we sought to clarify how three different dietary formulations that are commonly used in zebrafish facilities impact the gut microbiome. We compared the composition of gut microbiomes in approximately 60 AB line adult (129- and 214-day-old) zebrafish fed each diet throughout their lifespan. RESULTS: Our analysis finds that diet has a substantial impact on the composition of the gut microbiome in adult fish, and that diet also impacts the developmental variation in the gut microbiome. We further evaluated how 214-day-old fish microbiome compositions respond to exposure of a common laboratory pathogen, Mycobacterium chelonae, and whether these responses differ as a function of diet. Our analysis finds that diet determines the manner in which the zebrafish gut microbiome responds to M. chelonae exposure, especially for moderate and low abundance taxa. Moreover, histopathological analysis finds that male fish fed different diets are differentially infected by M. chelonae. CONCLUSIONS: Overall, our results indicate that diet drives the successional development of the gut microbiome as well as its sensitivity to exogenous exposure. Consequently, investigators should carefully consider the role of diet in their microbiome zebrafish investigations, especially when integrating results across studies that vary by diet.

Best practice for wildlife gut microbiome research: A comprehensive review of methodology for 16S rRNA gene investigations.

Extensive research in well-studied animal models underscores the importance of commensal gastrointestinal (gut) microbes to animal physiology. Gut microbes have been shown to impact dietary digestion, mediate infection, and even modify behavior and cognition. Given the large physiological and pathophysiological contribution microbes provide their host, it is reasonable to assume that the vertebrate gut microbiome may also impact the fitness, health and ecology of wildlife. In accordance with this expectation, an increasing number of investigations have considered the role of the gut microbiome in wildlife ecology, health, and conservation. To help promote the development of this nascent field, we need to dissolve the technical barriers prohibitive to performing wildlife microbiome research. The present review discusses the 16S rRNA gene microbiome research landscape, clarifying best practices in microbiome data generation and analysis, with particular emphasis on unique situations that arise during wildlife investigations. Special consideration is given to topics relevant for microbiome wildlife research from sample collection to molecular techniques for data generation, to data analysis strategies. Our hope is that this article not only calls for greater integration of microbiome analyses into wildlife ecology and health studies but provides researchers with the technical framework needed to successfully conduct such investigations.

Behavioral and Cognitive Performance Following Exposure to Second-Hand Smoke (SHS) from Tobacco Products Associated with Oxidative-Stress-Induced DNA Damage and Repair and Disruption of the Gut Microbiome.

Exposure to second-hand Smoke (SHS) remains prevalent. The underlying mechanisms of how SHS affects the brain require elucidation. We tested the hypothesis that SHS inhalation drives changes in the gut microbiome, impacting behavioral and cognitive performance as well as neuropathology in two-month-old wild-type (WT) mice and mice expressing wild-type human tau, a genetic model pertinent to Alzheimer's disease mice, following chronic SHS exposure (10 months to ~30 mg/m3). SHS exposure impacted the composition of the gut microbiome as well as the biodiversity and evenness of the gut microbiome in a sex-dependent fashion. This variation in the composition and biodiversity of the gut microbiome is also associated with several measures of cognitive performance. These results support the hypothesis that the gut microbiome contributes to the effect of SHS exposure on cognition. The percentage of 8-OHdG-labeled cells in the CA1 region of the hippocampus was also associated with performance in the novel object recognition test, consistent with urine and serum levels of 8-OHdG serving as a biomarker of cognitive performance in humans. We also assessed the effects of SHS on the percentage of p21-labeled cells, an early cellular marker of senescence that is upregulated in bronchial cells after exposure to cigarette smoke. Nuclear staining of p21-labeled cells was more prominent in larger cells of the prefrontal cortex and CA1 hippocampal neurons of SHS-exposed mice than in sham-exposed mice, and there was a significantly greater percentage of labelled cells in the prefrontal cortex and CA1 region of the hippocampus of SHS than air-exposed mice, suggesting that exposure to SHS may result in accelerated brain aging through oxidative-stress-induced injury.

Formation of complexes at plasmodesmata for potyvirus intercellular movement is mediated by the viral protein P3N-PIPO.

Intercellular transport of viruses through cytoplasmic connections, termed plasmodesmata (PD), is essential for systemic infection in plants by viruses. Previous genetic and ultrastructural data revealed that the potyvirus cyclindrical inclusion (CI) protein is directly involved in cell-to-cell movement, likely through the formation of conical structures anchored to and extended through PD. In this study, we demonstrate that plasmodesmatal localization of CI in N. benthamiana leaf cells is modulated by the recently discovered potyviral protein, P3N-PIPO, in a CI:P3N-PIPO ratio-dependent manner. We show that P3N-PIPO is a PD-located protein that physically interacts with CI in planta. The early secretory pathway, rather than the actomyosin motility system, is required for the delivery of P3N-PIPO and CI to PD. Moreover, CI mutations that disrupt virus cell-to-cell movement compromise PD-localization capacity. These data suggest that the CI and P3N-PIPO complex coordinates the formation of PD-associated structures that facilitate the intercellular movement of potyviruses in infected plants.

Small RNAs serve as a genetic buffer against genomic shock in Arabidopsis interspecific hybrids and allopolyploids.

Small RNAs, including microRNAs (miRNAs), small interfering RNAs (siRNAs), and trans-acting siRNAs (tasiRNAs), control gene expression and epigenetic regulation. Although the roles of miRNAs and siRNAs have been extensively studied, their expression diversity and evolution in closely related species and interspecific hybrids are poorly understood. Here, we show comprehensive analyses of miRNA expression and siRNA distributions in two closely related species Arabidopsis thaliana and Arabidopsis arenosa, a natural allotetraploid Arabidopsis suecica, and two resynthesized allotetraploid lines (F(1) and F(7)) derived from A. thaliana and A. arenosa. We found that repeat- and transposon-associated siRNAs were highly divergent between A. thaliana and A. arenosa. A. thaliana siRNA populations underwent rapid changes in F(1) but were stably maintained in F(7) and A. suecica. The correlation between siRNAs and nonadditive gene expression in allopolyploids is insignificant. In contrast, miRNA and tasiRNA sequences were conserved between species, but their expression patterns were highly variable between the allotetraploids and their progenitors. Many miRNAs tested were nonadditively expressed (deviating from the mid-parent value, MPV) in the allotetraploids and triggered unequal degradation of A. thaliana or A. arenosa targets. The data suggest that small RNAs produced during interspecific hybridization or polyploidization serve as a buffer against the genomic shock in interspecific hybrids and allopolyploids: Stable inheritance of repeat-associated siRNAs maintains chromatin and genome stability, whereas expression variation of miRNAs leads to changes in gene expression, growth vigor, and adaptation.

Experimental methods modestly impact interpretation of the effect of environmental exposures on the larval zebrafish gut microbiome.

Rapidly growing fields, such as microbiome science, often lack standardization of procedures across research groups. This is especially the case for microbiome investigations in the zebrafish (Danio rerio) model system, which is quickly becoming a workhorse system for understanding the exposure-microbiome-physiology axis. To guide future investigations using this model system, we defined how various experimental decisions affect the outcomes of studies on the effects of exogenous exposure on the zebrafish gut microbiome. Using a model toxicant, benzo[a]pyrene (BaP), we assessed how each of two dissection methods (gut dissection vs. whole fish), three DNA extraction kits (Qiagen Blood & Tissue, Macherey-Nagel NucleoSpin, and Qiagen PowerSoil), and inclusion of PCR replicates (single vs. pooled triplicate reactions) affected our interpretation of how exposure influences the diversity and composition of the gut microbiome, as well as our ability to identify microbiome biomarkers of exposure. We found that inclusion of PCR replicates had the smallest effect on our final interpretations, and the effects of dissection method and DNA extraction kit had significant effects in specific contexts, primarily in the cases of identifying microbial biomarkers.

Computational and analytical framework for small RNA profiling by high-throughput sequencing.

The advent of high-throughput sequencing (HTS) methods has enabled direct approaches to quantitatively profile small RNA populations. However, these methods have been limited by several factors, including representational artifacts and lack of established statistical methods of analysis. Furthermore, massive HTS data sets present new problems related to data processing and mapping to a reference genome. Here, we show that cluster-based sequencing-by-synthesis technology is highly reproducible as a quantitative profiling tool for several classes of small RNA from Arabidopsis thaliana. We introduce the use of synthetic RNA oligoribonucleotide standards to facilitate objective normalization between HTS data sets, and adapt microarray-type methods for statistical analysis of multiple samples. These methods were tested successfully using mutants with small RNA biogenesis (miRNA-defective dcl1 mutant and siRNA-defective dcl2 dcl3 dcl4 triple mutant) or effector protein (ago1 mutant) deficiencies. Computational methods were also developed to rapidly and accurately parse, quantify, and map small RNA data.

Genome-wide profiling and analysis of Arabidopsis siRNAs.

Eukaryotes contain a diversified set of small RNA-guided pathways that control genes, repeated sequences, and viruses at the transcriptional and posttranscriptional levels. Genome-wide profiles and analyses of small RNAs, particularly the large class of 24-nucleotide (nt) short interfering RNAs (siRNAs), were done for wild-type Arabidopsis thaliana and silencing pathway mutants with defects in three RNA-dependent RNA polymerase (RDR) and four Dicer-like (DCL) genes. The profiling involved direct analysis using a multiplexed, parallel-sequencing strategy. Small RNA-generating loci, especially those producing predominantly 24-nt siRNAs, were found to be highly correlated with repetitive elements across the genome. These were found to be largely RDR2- and DCL3-dependent, although alternative DCL activities were detected on a widespread level in the absence of DCL3. In contrast, no evidence for RDR2-alternative activities was detected. Analysis of RDR2- and DCL3-dependent small RNA accumulation patterns in and around protein-coding genes revealed that upstream gene regulatory sequences systematically lack siRNA-generating activities. Further, expression profiling suggested that relatively few genes, proximal to abundant 24-nt siRNAs, are regulated directly by RDR2- and DCL3-dependent silencing. We conclude that the widespread accumulation patterns for RDR2- and DCL3-dependent siRNAs throughout the Arabidopsis genome largely reflect mechanisms to silence highly repeated sequences.

Role of transposable elements in heterochromatin and epigenetic control.

Heterochromatin has been defined as deeply staining chromosomal material that remains condensed in interphase, whereas euchromatin undergoes de-condensation. Heterochromatin is found near centromeres and telomeres, but interstitial sites of heterochromatin (knobs) are common in plant genomes and were first described in maize. These regions are repetitive and late-replicating. In Drosophila, heterochromatin influences gene expression, a heterochromatin phenomenon called position effect variegation. Similarities between position effect variegation in Drosophila and gene silencing in maize mediated by "controlling elements" (that is, transposable elements) led in part to the proposal that heterochromatin is composed of transposable elements, and that such elements scattered throughout the genome might regulate development. Using microarray analysis, we show that heterochromatin in Arabidopsis is determined by transposable elements and related tandem repeats, under the control of the chromatin remodelling ATPase DDM1 (Decrease in DNA Methylation 1). Small interfering RNAs (siRNAs) correspond to these sequences, suggesting a role in guiding DDM1. We also show that transposable elements can regulate genes epigenetically, but only when inserted within or very close to them. This probably accounts for the regulation by DDM1 and the DNA methyltransferase MET1 of the euchromatic, imprinted gene FWA, as its promoter is provided by transposable-element-derived tandem repeats that are associated with siRNAs.

Update of ASRP: the Arabidopsis Small RNA Project database.

Development of the Arabidopsis Small RNA Project (ASRP) Database, which provides information and tools for the analysis of microRNA, endogenous siRNA and other small RNA-related features, has been driven by the introduction of high-throughput sequencing technology. To accommodate the demands of increased data, numerous improvements and updates have been made to ASRP, including new ways to access data, more efficient algorithms for handling data, and increased integration with community-wide resources. New search and visualization tools have also been developed to improve access to small RNA classes and their targets. ASRP is publicly available through a web interface at http://asrp.cgrb.oregonstate.edu/db/.

Effects of Six Sequential Charged Particle Beams on Behavioral and Cognitive Performance in B6D2F1 Female and Male Mice.

The radiation environment astronauts are exposed to in deep space includes galactic cosmic radiation (GCR) with different proportions of all naturally occurring ions. To assist NASA with assessment of risk to the brain following exposure to a mixture of ions broadly representative of the GCR, we assessed the behavioral and cognitive performance of female and male C57BL/6J × DBA2/J F1 (B6D2F1) mice two months following rapidly delivered, sequential 6 beam irradiation with protons (1 GeV, LET = 0.24 keV, 50%), 4He ions (250 MeV/n, LET = 1.6 keV/µm, 20%), 16O ions (250 MeV/n, LET = 25 keV/µm 7.5%), 28Si ions (263 MeV/n, LET = 78 keV/µm, 7.5%), 48Ti ions (1 GeV/n, LET = 107 keV/µm, 7.5%), and 56Fe ions (1 GeV/n, LET = 151 keV/µm, 7.5%) at 0, 25, 50, or 200 cGy) at 4-6 months of age. When the activity over 3 days of open field habituation was analyzed in female mice, those irradiated with 50 cGy moved less and spent less time in the center than sham-irradiated mice. Sham-irradiated female mice and those irradiated with 25 cGy showed object recognition. However, female mice exposed to 50 or 200 cGy did not show object recognition. When fear memory was assessed in passive avoidance tests, sham-irradiated mice and mice irradiated with 25 cGy showed memory retention while mice exposed to 50 or 200 cGy did not. The effects of radiation passive avoidance memory retention were not sex-dependent. There was no effect of radiation on depressive-like behavior in the forced swim test. There was a trend toward an effect of radiation on BDNF levels in the cortex of males, but not for females, with higher levels in male mice irradiated with 50 cGy than sham-irradiated. Finally, sequential 6-ion irradiation impacted the composition of the gut microbiome in a sex-dependent fashion. Taxa were uncovered whose relative abundance in the gut was associated with the radiation dose received. Thus, exposure to sequential six-beam irradiation significantly affects behavioral and cognitive performance and the gut microbiome.

Genome-wide profiling of populus small RNAs.

BACKGROUND: Short RNAs, and in particular microRNAs, are important regulators of gene expression both within defined regulatory pathways and at the epigenetic scale. We investigated the short RNA (sRNA) population (18-24 nt) of the transcriptome of green leaves from the sequenced Populus trichocarpa using a concatenation strategy in combination with 454 sequencing. RESULTS: The most abundant size class of sRNAs were 24 nt. Long Terminal Repeats were particularly associated with 24 nt sRNAs. Additionally, some repetitive elements were associated with 22 nt sRNAs. We identified an sRNA hot-spot on chromosome 19, overlapping a region containing both the proposed sex-determining locus and a major cluster of NBS-LRR genes. A number of phased siRNA loci were identified, a subset of which are predicted to target PPR and NBS-LRR disease resistance genes, classes of genes that have been significantly expanded in Populus. Additional loci enriched for sRNA production were identified and characterised. We identified 15 novel predicted microRNAs (miRNAs), including miRNA*sequences, and identified a novel locus that may encode a dual miRNA or a miRNA and short interfering RNAs (siRNAs). CONCLUSIONS: The short RNA population of P. trichocarpa is at least as complex as that of Arabidopsis thaliana. We provide a first genome-wide view of short RNA production for P. trichocarpa and identify new, non-conserved miRNAs.