An eco-epidemiological modeling approach to investigate dilution effect in two different tick-borne pathosystems.

Disease (re)emergence appears to be driven by biodiversity decline and environmental change. As a result, it is increasingly important to study host-pathogen interactions within the context of their ecology and evolution. The dilution effect is the concept that higher biodiversity decreases pathogen transmission. It has been observed especially in zoonotic vector-borne pathosystems, yet evidence against it has been found. In particular, it is still debated how the community (dis)assembly assumptions and the degree of generalism of vectors and pathogens affect the direction of the biodiversity-pathogen transmission relationship. The aim of this study was to use empirical data and mechanistic models to investigate dilution mechanisms in two rodent-tick-pathogen systems differing in their vector degree of generalism. A community was assembled to include ecological interactions that expand from purely additive to purely substitutive. Such systems are excellent candidates to analyze the link between vector ecology, community (dis)assembly dynamics, and pathogen transmission. To base our mechanistic models on empirical data, rodent live-trapping, including tick sampling, was conducted in Wales across two seasons for three consecutive years. We have developed a deterministic single-vector, multi-host compartmental model that includes ecological relationships with non-host species, uniquely integrating theoretical and observational approaches. To describe pathogen transmission across a gradient of community diversity, the model was populated with parameters describing five different scenarios differing in ecological complexity; each based around one of the pathosystems: Ixodes ricinus (generalist tick)-Borrelia burgdorferi and I. trianguliceps (small mammals specialist tick)-Babesia microti. The results suggested that community composition and interspecific dynamics affected pathogen transmission with different dilution outcomes depending on the vector degree of generalism. The model provides evidence that dilution and amplification effects are not mutually exclusive in the same community but depend on vector ecology and the epidemiological output considered (i.e., the "risk" of interest). In our scenarios, more functionally diverse communities resulted in fewer infectious rodents, supporting the dilution effect. In the pathosystem with generalist vector we identified a hump shaped relationship between diversity and infections in hosts, while for that characterized by specialist tick, this relationship was more complex and more dependent upon specific parameter values.

Upgrading short-read animal genome assemblies to chromosome level using comparative genomics and a universal probe set.

Most recent initiatives to sequence and assemble new species' genomes de novo fail to achieve the ultimate endpoint to produce contigs, each representing one whole chromosome. Even the best-assembled genomes (using contemporary technologies) consist of subchromosomal-sized scaffolds. To circumvent this problem, we developed a novel approach that combines computational algorithms to merge scaffolds into chromosomal fragments, PCR-based scaffold verification, and physical mapping to chromosomes. Multigenome-alignment-guided probe selection led to the development of a set of universal avian BAC clones that permit rapid anchoring of multiple scaffolds to chromosomes on all avian genomes. As proof of principle, we assembled genomes of the pigeon (Columbia livia) and peregrine falcon (Falco peregrinus) to chromosome levels comparable, in continuity, to avian reference genomes. Both species are of interest for breeding, cultural, food, and/or environmental reasons. Pigeon has a typical avian karyotype (2n = 80), while falcon (2n = 50) is highly rearranged compared to the avian ancestor. By using chromosome breakpoint data, we established that avian interchromosomal breakpoints appear in the regions of low density of conserved noncoding elements (CNEs) and that the chromosomal fission sites are further limited to long CNE "deserts." This corresponds with fission being the rarest type of rearrangement in avian genome evolution. High-throughput multiple hybridization and rapid capture strategies using the current BAC set provide the basis for assembling numerous avian (and possibly other reptilian) species, while the overall strategy for scaffold assembly and mapping provides the basis for an approach that (provided metaphases can be generated) could be applied to any animal genome.

Genetic and Methylome Variation in Turkish Brachypodium Distachyon Accessions Differentiate Two Geographically Distinct Subpopulations.

Brachypodium distachyon (Brachypodium) is a non-domesticated model grass species that can be used to test if variation in genetic sequence or methylation are linked to environmental differences. To assess this, we collected seeds from 12 sites within five climatically distinct regions of Turkey. Seeds from each region were grown under standardized growth conditions in the UK to preserve methylated sequence variation. At six weeks following germination, leaves were sampled and assessed for genomic and DNA methylation variation. In a follow-up experiment, phenomic approaches were used to describe plant growth and drought responses. Genome sequencing and population structure analysis suggested three ancestral clusters across the Mediterranean, two of which were geographically separated in Turkey into coastal and central subpopulations. Phenotypic analyses showed that the coastal subpopulation tended to exhibit relatively delayed flowering and the central, increased drought tolerance as indicated by reduced yellowing. Genome-wide methylation analyses in GpC, CHG and CHH contexts also showed variation which aligned with the separation into coastal and central subpopulations. The climate niche modelling of both subpopulations showed a significant influence from the "Precipitation in the Driest Quarter" on the central subpopulation and "Temperature of the Coldest Month" on the coastal subpopulation. Our work demonstrates genetic diversity and variation in DNA methylation in Turkish accessions of Brachypodium that may be associated with climate variables and the molecular basis of which will feature in ongoing analyses.

Mendel's pea crosses: varieties, traits and statistics.

A controversy arose over Mendel's pea crossing experiments after the statistician R.A. Fisher proposed how these may have been performed and criticised Mendel's interpretation of his data. Here we re-examine Mendel's experiments and investigate Fisher's statistical criticisms of bias. We describe pea varieties available in Mendel's time and show that these could readily provide all the material Mendel needed for his experiments; the characters he chose to follow were clearly described in catalogues at the time. The combination of character states available in these varieties, together with Eichling's report of crosses Mendel performed, suggest that two of his F3 progeny test experiments may have involved the same F2 population, and therefore that these data should not be treated as independent variables in statistical analysis of Mendel's data. A comprehensive re-examination of Mendel's segregation ratios does not support previous suggestions that they differ remarkably from expectation. The χ2 values for his segregation ratios sum to a value close to the expectation and there is no deficiency of extreme segregation ratios. Overall the χ values for Mendel's segregation ratios deviate slightly from the standard normal distribution; this is probably because of the variance associated with phenotypic rather than genotypic ratios and because Mendel excluded some data sets with small numbers of progeny, where he noted the ratios "deviate not insignificantly" from expectation.

Transcriptional and Metabolomic Analyses Indicate that Cell Wall Properties are Associated with Drought Tolerance in Brachypodium distachyon.

Brachypodium distachyon is an established model for drought tolerance. We previously identified accessions exhibiting high tolerance, susceptibility and intermediate tolerance to drought; respectively, ABR8, KOZ1 and ABR4. Transcriptomics and metabolomic approaches were used to define tolerance mechanisms. Transcriptional analyses suggested relatively few drought responsive genes in ABR8 compared to KOZ1. Linking these to gene ontology (GO) terms indicated enrichment for "regulated stress response", "plant cell wall" and "oxidative stress" associated genes. Further, tolerance correlated with pre-existing differences in cell wall-associated gene expression including glycoside hydrolases, pectin methylesterases, expansins and a pectin acetylesterase. Metabolomic assessments of the same samples also indicated few significant changes in ABR8 with drought. Instead, pre-existing differences in the cell wall-associated metabolites correlated with drought tolerance. Although other features, e.g., jasmonate signaling were suggested in our study, cell wall-focused events appeared to be predominant. Our data suggests two different modes through which the cell wall could confer drought tolerance: (i) An active response mode linked to stress induced changes in cell wall features, and (ii) an intrinsic mode where innate differences in cell wall composition and architecture are important. Both modes seem to contribute to ABR8 drought tolerance. Identification of the exact mechanisms through which the cell wall confers drought tolerance will be important in order to inform development of drought tolerant crops.

Transcriptomic analysis of crustacean neuropeptide signaling during the moult cycle in the green shore crab, Carcinus maenas.

BACKGROUND: Ecdysis is an innate behaviour programme by which all arthropods moult their exoskeletons. The complex suite of interacting neuropeptides that orchestrate ecdysis is well studied in insects, but details of the crustacean ecdysis cassette are fragmented and our understanding of this process is comparatively crude, preventing a meaningful evolutionary comparison. To begin to address this issue we identified transcripts coding for neuropeptides and their putative receptors in the central nervous system (CNS) and Y-organs (YO) within the crab, Carcinus maenas, and mapped their expression profiles across accurately defined stages of the moult cycle using RNA-sequencing. We also studied gene expression within the epidermally-derived YO, the only defined role for which is the synthesis of ecdysteroid moulting hormones, to elucidate peptides and G protein-coupled receptors (GPCRs) that might have a function in ecdysis. RESULTS: Transcriptome mining of the CNS transcriptome yielded neuropeptide transcripts representing 47 neuropeptide families and 66 putative GPCRs. Neuropeptide transcripts that were differentially expressed across the moult cycle included carcikinin, crustacean hyperglycemic hormone-2, and crustacean cardioactive peptide, whilst a single putative neuropeptide receptor, proctolin R1, was differentially expressed. Carcikinin mRNA in particular exhibited dramatic increases in expression pre-moult, suggesting a role in ecdysis regulation. Crustacean hyperglycemic hormone-2 mRNA expression was elevated post- and pre-moult whilst that for crustacean cardioactive peptide, which regulates insect ecdysis and plays a role in stereotyped motor activity during crustacean ecdysis, was elevated in pre-moult. In the YO, several putative neuropeptide receptor transcripts were differentially expressed across the moult cycle, as was the mRNA for the neuropeptide, neuroparsin-1. Whilst differential gene expression of putative neuropeptide receptors was expected, the discovery and differential expression of neuropeptide transcripts was surprising. Analysis of GPCR transcript expression between YO and epidermis revealed 11 to be upregulated in the YO and thus are now candidates for peptide control of ecdysis. CONCLUSIONS: The data presented represent a comprehensive survey of the deduced C. maenas neuropeptidome and putative GPCRs. Importantly, we have described the differential expression profiles of these transcripts across accurately staged moult cycles in tissues key to the ecdysis programme. This study provides important avenues for the future exploration of functionality of receptor-ligand pairs in crustaceans.

Identification of Stipules reduced, a leaf morphology gene in pea (Pisum sativum).

Pea (Pisum sativum) is one of relatively few genetically amenable plant species with compound leaves. Pea leaves have a variety of specialized organs: leaflets, tendrils, pulvini and stipules, which enable the identification of mutations that transform or affect distinct parts of the leaf. Characterization of these mutations offers insights into the development and evolution of novel leaf traits. The previously characterized morphological gene Cochleata, conferring stipule identity, was known to interact with Stipules reduced (St), which conditions stipule size in pea, but the St gene remained unknown. Here we analysed Fast Neutron irradiated pea mutants by restriction site associated DNA sequencing. We identified St as a gene encoding a C2H2 zinc finger transcription factor that is regulated by Cochleata. St regulates both cell division and cell expansion in the stipule. Our approach shows how systematic genome-wide screens can be used successfully for the analysis of traits in species for which whole genome sequences are not available.

MetaPred2CS: a sequence-based meta-predictor for protein-protein interactions of prokaryotic two-component system proteins.

MOTIVATION: Two-component systems (TCS) are the main signalling pathways of prokaryotes, and control a wide range of biological phenomena. Their functioning depends on interactions between TCS proteins, the specificity of which is poorly understood. RESULTS: The MetaPred2CS web-server interfaces a sequence-based meta-predictor specifically designed to predict pairing of the histidine kinase and response-regulator proteins forming TCSs. MetaPred2CS integrates six sequence-based methods using a support vector machine classifier and has been intensively tested under different benchmarking conditions: (i) species specific gene sets; (ii) neighbouring versus orphan pairs; and (iii) k-fold cross validation on experimentally validated datasets. AVAILABILITY AND IMPLEMENTATION: Web server at: http://metapred2cs.ibers.aber.ac.uk/, Source code: https://github.com/martinjvickers/MetaPred2CS or implemented as Virtual Machine at: http://metapred2cs.ibers.aber.ac.uk/download CONTACT: naf4@aber.ac.ukSupplementary information: Supplementary data are available at Bioinformatics online.

DISMISS: detection of stranded methylation in MeDIP-Seq data.

BACKGROUND: DNA methylation is an important regulator of gene expression and chromatin structure. Methylated DNA immunoprecipitation sequencing (MeDIP-Seq) is commonly used to identify regions of DNA methylation in eukaryotic genomes. Within MeDIP-Seq libraries, methylated cytosines can be found in both double-stranded (symmetric) and single-stranded (asymmetric) genomic contexts. While symmetric CG methylation has been relatively well-studied, asymmetric methylation in any dinucleotide context has received less attention. Importantly, no currently available software for processing MeDIP-Seq reads is able to resolve these strand-specific DNA methylation signals. Here we introduce DISMISS, a new software package that detects strand-associated DNA methylation from existing MeDIP-Seq analyses. RESULTS: Using MeDIP-Seq datasets derived from Apis mellifera (honeybee), an invertebrate species that contains more asymmetric- than symmetric- DNA methylation, we demonstrate that DISMISS can identify strand-specific DNA methylation signals with similar accuracy as bisulfite sequencing (BS-Seq; single nucleotide resolution methodology). Specifically, DISMISS is able to confidently predict where DNA methylation predominates (plus or minus DNA strands - asymmetric DNA methylation; plus and minus DNA stands - symmetric DNA methylation) in MeDIP-Seq datasets derived from A. mellifera samples. When compared to DNA methylation data derived from BS-Seq analysis of A. mellifera worker larva, DISMISS-mediated identification of strand-specific methylated cytosines is 80 % accurate. Furthermore, DISMISS can correctly (p <0.0001) detect the origin (sense vs antisense DNA strands) of DNA methylation at splice site junctions in A. mellifera MeDIP-Seq datasets with a precision close to BS-Seq analysis. Finally, DISMISS-mediated identification of DNA methylation signals associated with upstream, exonic, intronic and downstream genomic loci from A. mellifera MeDIP-Seq datasets outperforms MACS2 (Model-based Analysis of ChIP-Seq2; a commonly used MeDIP-Seq analysis software) and closely approaches the results achieved by BS-Seq. CONCLUSIONS: While asymmetric DNA methylation is increasingly being found in growing numbers of eukaryotic species and is the predominant pattern observed in some invertebrate genomes, it has been difficult to detect in MeDIP-Seq datasets using existing software. DISMISS now enables more sensitive examinations of MeDIP-Seq datasets and will be especially useful for the study of genomes containing either low levels of DNA methylation or for genomes containing relatively high amounts of asymmetric methylation.

De-novo transcriptome assembly for gene identification, analysis, annotation, and molecular marker discovery in Onobrychis viciifolia.

BACKGROUND: Sainfoin (Onobrychis viciifolia) is a highly nutritious tannin-containing forage legume. In the diet of ruminants sainfoin can have anti-parasitic effects and reduce methane emissions under in vitro conditions. Many of these benefits have been attributed to condensed tannins or proanthocyanidins in sainfoin. A combination of increased use of industrially produced nitrogen fertilizer, issues with establishment and productivity in the first year and more reliable alternatives, such as red clover led to a decline in the use of sainfoin since the middle of the last century. In recent years there has been a resurgence of interest in sainfoin due to its potential beneficial nutraceutical and environmental attributes. However, genomic resources are scarce, thus hampering progress in genetic analysis and improvement. To address this we have used next generation RNA sequencing technology to obtain the first transcriptome of sainfoin. We used the library to identify gene-based simple sequence repeats (SSRs) and potential single nucleotide polymorphisms (SNPs). RESULTS: One genotype from each of five sainfoin accessions was sequenced. Paired-end (PE) sequences were generated from cDNA libraries of RNA extracted from 7 day old seedlings. A combined assembly of 92,772 transcripts was produced de novo using the Trinity programme. About 18,000 transcripts were annotated with at least one GO (gene ontology) term. A total of 63 transcripts were annotated as involved in the tannin biosynthesis pathway. We identified 3786 potential SSRs. SNPs were identified by mapping the reads of the individual assemblies against the combined assembly. After stringent filtering a total of 77,000 putative SNPs were identified. A phylogenetic analysis of single copy number genes showed that sainfoin was most closely related to red clover and Medicago truncatula, while Lotus japonicus, bean and soybean are more distant relatives. CONCLUSIONS: This work describes the first transcriptome assembly in sainfoin. The 92 K transcripts provide a rich source of SNP and SSR polymorphisms for future use in genetic studies of this crop. Annotation of genes involved in the condensed tannin biosynthesis pathway has provided the basis for further studies of the genetic control of this important trait in sainfoin.

Genome-wide prediction of prokaryotic two-component system networks using a sequence-based meta-predictor.

BACKGROUND: Two component systems (TCS) are signalling complexes manifested by a histidine kinase (receptor) and a response regulator (effector). They are the most abundant signalling pathways in prokaryotes and control a wide range of biological processes. The pairing of these two components is highly specific, often requiring costly and time-consuming experimental characterisation. Therefore, there is considerable interest in developing accurate prediction tools to lessen the burden of experimental work and cope with the ever-increasing amount of genomic information. RESULTS: We present a novel meta-predictor, MetaPred2CS, which is based on a support vector machine. MetaPred2CS integrates six sequence-based prediction methods: in-silico two-hybrid, mirror-tree, gene fusion, phylogenetic profiling, gene neighbourhood, and gene operon. To benchmark MetaPred2CS, we also compiled a novel high-quality training dataset of experimentally deduced TCS protein pairs for k-fold cross validation, to act as a gold standard for TCS partnership predictions. Combining individual predictions using MetaPred2CS improved performance when compared to the individual methods and in comparison with a current state-of-the-art meta-predictor. CONCLUSION: We have developed MetaPred2CS, a support vector machine-based metapredictor for prokaryotic TCS protein pairings. Central to the success of MetaPred2CS is a strategy of integrating individual predictors that improves the overall prediction accuracy, with the in-silico two-hybrid method contributing most to performance. MetaPred2CS outperformed other available systems in our benchmark tests, and is available online at http://metapred2cs.ibers.aber.ac.uk, along with our gold standard dataset of TCS interaction pairs.

Generation of whole genome sequences of new Cryptosporidium hominis and Cryptosporidium parvum isolates directly from stool samples.

BACKGROUND: Whole genome sequencing (WGS) of Cryptosporidium spp. has previously relied on propagation of the parasite in animals to generate enough oocysts from which to extract DNA of sufficient quantity and purity for analysis. We have developed and validated a method for preparation of genomic Cryptosporidium DNA suitable for WGS directly from human stool samples and used it to generate 10 high-quality whole Cryptosporidium genome assemblies. Our method uses a combination of salt flotation, immunomagnetic separation (IMS), and surface sterilisation of oocysts prior to DNA extraction, with subsequent use of the transposome-based Nextera XT kit to generate libraries for sequencing on Illumina platforms. IMS was found to be superior to caesium chloride density centrifugation for purification of oocysts from small volume stool samples and for reducing levels of contaminant DNA. RESULTS: The IMS-based method was used initially to sequence whole genomes of Cryptosporidium hominis gp60 subtype IbA10G2 and Cryptosporidium parvum gp60 subtype IIaA19G1R2 from small amounts of stool left over from diagnostic testing of clinical cases of cryptosporidiosis. The C. parvum isolate was sequenced to a mean depth of 51.8X with reads covering 100 % of the bases of the C. parvum Iowa II reference genome (Bioproject PRJNA 15586), while the C. hominis isolate was sequenced to a mean depth of 34.7X with reads covering 98 % of the bases of the C. hominis TU502 v1 reference genome (Bioproject PRJNA 15585). The method was then applied to a further 17 stools, successfully generating another eight new whole genome sequences, of which two were C. hominis (gp60 subtypes IbA10G2 and IaA14R3) and six C. parvum (gp60 subtypes IIaA15G2R1 from three samples, and one each of IIaA17G1R1, IIaA18G2R1, and IIdA22G1), demonstrating the utility of this method to sequence Cryptosporidium genomes directly from clinical samples. This development is especially important as it reduces the requirement to propagate Cryptosporidium oocysts in animal models prior to genome sequencing. CONCLUSION: This represents the first report of high-quality whole genome sequencing of Cryptosporidium isolates prepared directly from human stool samples.

Epigenetic regulation of sex ratios may explain natural variation in self-fertilization rates.

Self-fertilization (selfing) favours reproductive success when mate availability is low, but renders populations more vulnerable to environmental change by reducing genetic variability. A mixed-breeding strategy (alternating selfing and outcrossing) may allow species to balance these needs, but requires a system for regulating sexual identity. We explored the role of DNA methylation as a regulatory system for sex-ratio modulation in the mixed-mating fish Kryptolebias marmoratus. We found a significant interaction between sexual identity (male or hermaphrodite), temperature and methylation patterns when two selfing lines were exposed to different temperatures during development. We also identified several genes differentially methylated in males and hermaphrodites that represent candidates for the temperature-mediated sex regulation in K. marmoratus. We conclude that an epigenetic mechanism regulated by temperature modulates sexual identity in this selfing species, providing a potentially widespread mechanism by which environmental change may influence selfing rates. We also suggest that K. marmoratus, with naturally inbred populations, represents a good vertebrate model for epigenetic studies.

Transcriptomic analysis of the lesser spotted catshark (Scyliorhinus canicula) pancreas, liver and brain reveals molecular level conservation of vertebrate pancreas function.

BACKGROUND: Understanding the evolution of the vertebrate pancreas is key to understanding its functions. The chondrichthyes (cartilaginous fish such as sharks and rays) have often been suggested to possess the most ancient example of a distinct pancreas with both hormonal (endocrine) and digestive (exocrine) roles. The lack of genetic, genomic and transcriptomic data for cartilaginous fish has hindered a more thorough understanding of the molecular-level functions of the chondrichthyan pancreas, particularly with respect to their "unusual" energy metabolism (where ketone bodies and amino acids are the main oxidative fuel source) and their paradoxical ability to both maintain stable blood glucose levels and tolerate extensive periods of hypoglycemia. In order to shed light on some of these processes, we carried out the first large-scale comparative transcriptomic survey of multiple cartilaginous fish tissues: the pancreas, brain and liver of the lesser spotted catshark, Scyliorhinus canicula. RESULTS: We generated a mutli-tissue assembly comprising 86,006 contigs, of which 44,794 were assigned to a particular tissue or combination of tissues based on mapping of sequencing reads. We have characterised transcripts encoding genes involved in insulin regulation, glucose sensing, transcriptional regulation, signaling and digestion, as well as many peptide hormone precursors and their receptors for the first time. Comparisons to mammalian pancreas transcriptomes reveals that mechanisms of glucose sensing and insulin regulation used to establish and maintain a stable internal environment are conserved across jawed vertebrates and likely pre-date the vertebrate radiation. Conservation of pancreatic hormones and genes encoding digestive proteins support the single, early evolution of a distinct pancreatic gland with endocrine and exocrine functions in jawed vertebrates. In addition, we demonstrate that chondrichthyes lack pancreatic polypeptide (PP) and that reports of PP in the literature are likely due cross-reaction with PYY and/or NPY in the pancreas. A three hormone islet organ is therefore the ancestral jawed vertebrate condition, later elaborated upon only in the tetrapod lineage. CONCLUSIONS: The cartilaginous fish are a great untapped resource for the reconstruction of patterns and processes of vertebrate evolution and new approaches such as those described in this paper will greatly facilitate their incorporation into the rank of "model organism".

De novo assembly of red clover transcriptome based on RNA-Seq data provides insight into drought response, gene discovery and marker identification.

BACKGROUND: Red clover (Trifolium pratense L.) is a versatile forage crop legume, which can tolerate a variety of soils and is suitable for silage production for winter feed and for grazing. It is one of the most important forage legumes in temperate livestock agriculture. Its beneficial attributes include ability to fix nitrogen, improve soil and provide protein rich animal feed. It is however, a short-lived perennial providing good biomass yield for two or three years. Improved persistency is thus a major breeding target. Better water-stress tolerance is one of the key factors influencing persistency, but little is known about how red clover tolerates water stress. RESULTS: Plants from a full sib mapping family were used in a drought experiment, in which the growth rate and relative water content (RWC) identified two pools of ten plants contrasting in their tolerance to drought. Key metabolites were measured and RNA-Seq analysis was carried out on four bulked samples: the two pools sampled before and after drought. Massively parallel sequencing was used to analyse the bulked RNA samples. A de novo transcriptome reconstruction based on the RNA-Seq data was made, resulting in 45181 contigs, representing 'transcript tags'. These transcript tags were annotated with gene ontology (GO) terms. One of the most striking results from the expression analysis was that the drought sensitive plants were characterised by having approximately twice the number of differentially expressed transcript tags than the tolerant plants after drought. This difference was evident in most of the major GO terms. Before onset of drought the sensitive plants overexpressed a number of genes annotated as senescence-related. Furthermore, the concentration of three metabolites, particularly pinitol, but also proline and malate increased in leaves after drought stress. CONCLUSIONS: This de novo assembly of a red clover transcriptome from leaf material of droughted and non-droughted plants provides a rich source for gene identification, single nucleotide polymorphisms (SNP) and short sequence repeats (SSR). Comparison of gene expression levels between pools and treatments identified candidate genes for further analysis of the genetic basis of drought tolerance in red clover.

Metaphylogenomic and potential functionality of the limpet Patella pellucida's gastrointestinal tract microbiome.

This study investigated the microbial diversity associated with the digestive tract of the seaweed grazing marine limpet Patella pellucida. Using a modified indirect DNA extraction protocol and performing metagenomic profiling based on specific prokaryotic marker genes, the abundance of bacterial groups was identified from the analyzed metagenome. The members of three significantly abundant phyla of Proteobacteria, Firmicutes and Bacteroidetes were characterized through the literature and their predicted functions towards the host, as well as potential applications in the industrial environment assessed.

The RNA cargo of Myxococcus outer membrane vesicles.

The outer membrane vesicles (OMVs) secreted by some Gram-negative bacteria contain RNA cargo, which can be introduced into target cells, affecting their cellular processes. To test whether the antimicrobial OMVs secreted by predatory myxobacteria might contain cargo RNA with a role in prey killing, we purified OMVs and cells from four different strains of Myxococcus spp. for RNA-seq transcriptome sequencing. Myxobacterial OMVs contained distinct sets of RNA molecules. The abundance of major cellular transcripts correlated strongly with their abundance in OMVs, suggesting non-specific packaging into OMVs. However, many major cellular transcripts were absent entirely from OMVs and some transcripts were found exclusively in OMVs, suggesting OMV RNA cargo loading is not simply a consequence of sampling the cellular transcriptome. Despite considerable variation in OMV RNA cargo between biological replicates, a small number of transcripts were found consistently in replicate OMV preparations. These 'core' OMV transcripts were often found in the OMVs from multiple strains, and sometimes enriched relative to their abundance in cellular transcriptomes. In addition to providing the first transcriptomes for myxobacterial OMVs, and the first cellular transcriptomes for three strains of Myxococcus spp., we highlight five transcripts for further study. These transcripts are 'core' for at least two of the three strains of M. xanthus studied, and encode two alkyl hydroperoxidase proteins (AhpC and AhpD), two ribosome-associated inhibitors (RaiA-like) and a DO-family protease. It will be interesting to test whether the transcripts serve a biological function within OMVs, potentially being transported into prey cells for translation into toxic proteins.

Interpreting alignment-free sequence comparison: what makes a score a good score?

Alignment-free methods are alternatives to alignment-based methods when searching sequence data sets. The output from an alignment-free sequence comparison is a similarity score, the interpretation of which is not straightforward. We propose objective functions to interpret and calibrate outputs from alignment-free searches, noting that different objective functions are necessary for different biological contexts. This leads to advantages: visualising and comparing score distributions, including those from true positives, may be a relatively simple method to gain insight into the performance of different metrics. Using an empirical approach with both DNA and protein sequences, we characterise different similarity score distributions generated under different parameters. In particular, we demonstrate how sequence length can affect the scores. We show that scores of true positive sequence pairs may correlate significantly with their mean length; and even if the correlation is weak, the relative difference in length of the sequence pair may significantly reduce the effectiveness of alignment-free metrics. Importantly, we show how objective functions can be used with test data to accurately estimate the probability of true positives. This can significantly increase the utility of alignment-free approaches. Finally, we have developed a general-purpose software tool called KAST for use in high-throughput workflows on Linux clusters.

In silico characterisation of the complete Ly6 protein family in Fasciola gigantica supported through transcriptomics of the newly-excysted juveniles.

Fasciola gigantica is one of the aetiological trematodes associated with fascioliasis, which heavily impacts food-production systems and human and animal welfare on a global scale. In the absence of a vaccine, fascioliasis control and treatment is restricted to pasture management, such as clean grazing, and a limited array of chemotherapies, to which signs of resistance are beginning to appear. Research into novel control strategies is therefore urgently required and the advent of 'omics technologies presents considerable opportunity for novel drug and vaccine target discovery. Here, interrogation of the first available F. gigantica newly excysted juvenile (NEJ) transcriptome revealed several protein families of current interest to parasitic flatworm vaccine research, including orthologues of mammalian complement regulator CD59 of the Ly6 family. Ly6 proteins have previously been identified on the tegument of Schistosoma mansoni and induced protective immunity in vaccination trials. Incorporating the recently available F. gigantica genome, the current work revealed 20 novel Ly6 family members in F. gigantica and, in parallel, significantly extended the F. hepatica complement from 3 to 18 members. Phylogenetic analysis revealed several distinct clades within the family, some of which are unique to Fasciola spp. trematodes. Analysis of available proteomic databases also revealed three of the newly discovered FhLy6s were present in extracellular vesicles, which have previously been prioritised in studying the host-parasite interface. The presentation of this new transcriptomic resource, in addition to the Ly6 family proteins here identified, represents a wealth of opportunity for future vaccine research.

Genotyping and Whole-Genome Resequencing of Welsh Sheep Breeds Reveal Candidate Genes and Variants for Adaptation to Local Environment and Socioeconomic Traits.

BACKGROUND: Advances in genetic tools applied to livestock breeding has prompted research into the previously neglected breeds adapted to harsh local environments. One such group is the Welsh mountain sheep breeds, which can be farmed at altitudes of 300 m above sea level but are considered to have a low productive value because of their poor wool quality and small carcass size. This is contrary to the lowland breeds which are more suited to wool and meat production qualities, but do not fare well on upland pasture. Herein, medium-density genotyping data from 317 individuals representing 15 Welsh sheep breeds were used alongside the whole-genome resequencing data of 14 breeds from the same set to scan for the signatures of selection and candidate genetic variants using haplotype- and SNP-based approaches. RESULTS: Haplotype-based selection scan performed on the genotyping data pointed to a strong selection in the regions of GBA3, PPARGC1A, APOB, and PPP1R16B genes in the upland breeds, and RNF24, PANK2, and MUC15 in the lowland breeds. SNP-based selection scan performed on the resequencing data pointed to the missense mutations under putative selection relating to a local adaptation in the upland breeds with functions such as angiogenesis (VASH1), anti-oxidation (RWDD1), cell stress (HSPA5), membrane transport (ABCA13 and SLC22A7), and insulin signaling (PTPN1 and GIGFY1). By contrast, genes containing candidate missense mutations in the lowland breeds are related to cell cycle (CDK5RAP2), cell adhesion (CDHR3), and coat color (MC1R). CONCLUSION: We found new variants in genes with potentially functional consequences to the adaptation of local sheep to their environments in Wales. Knowledge of these variations is important for improving the adaptative qualities of UK and world sheep breeds through a marker-assisted selection.