Studies Of Non-model Organisms Research Articles

Deciphering cellular heterogeneity in Spodoptera frugiperda midgut cell line through single cell RNA sequencing

Using the 10x Genomics Chromium single-cell RNA sequencing (scRNA-seq) platform, we discovered unexpected heterogeneity in an established cell line developed from the midgut of the Fall armyworm, Spodoptera frugiperda, a major global pest. We analyzed the sequences of 18,794 cells and identified ten unique cellular clusters, including stem cells, enteroblasts, enterocytes and enteroendocrine cells, characterized by the expression of specific marker genes. Additionally, these studies addressed an important knowledge gap by investigating the expression of genes coding for respiratory and midgut membrane insecticide targets classified by the Insecticide Resistance Action Committee. Dual-fluorescence tagging method, fluorescence microscopy and fluorescence-activated cell sorting confirmed the expression of midgut cell type-specific genes. Stem cells were isolated from the heterogeneous population of SfMG-0617 cells. Our results, validated by KEGG and Gene Ontology analyses and supported by Monocle 3.0, advance the fields of midgut cellular biology and establish standards for scRNA-seq studies in non-model organisms.

MHCtools 1.5: Analysis of MHC Sequencing Data in R.

The genes of the major histocompatibility complex (MHC) play a vital role in the vertebrate immune system and have attracted considerable interest in evolutionary biology. While the MHC has been characterized in detail in humans (human leukocyte antigen, HLA) and in model organisms such as the mouse, studies in non-model organisms often lack prior knowledge about structure, genetic variability, and evolutionary properties of this locus. MHC genotyping in non-model species commonly relies on PCR-based amplicon sequencing, and while several published protocols facilitate generation of MHC sequence data, there is a lack of transparent and standardized tools for downstream data analysis.Here, I present the R package MHCtools version 1.5, which contains 15 tools that (i) assist accurate MHC genotyping from high-throughput amplicon sequencing data, and provide standardized methods to analyze (ii) MHC diversity, (iii) MHC supertypes, and (iv) MHC haplotypes.I hope that MHCtools will be helpful in future studies of the MHC in non-model species and that it may help to advance our understanding of the important roles of the MHC in ecology and evolution.

Evaluating restriction enzyme selection for reduced representation sequencing in conservation genomics.

Conservation genomic studies in non-model organisms generally rely on reduced representation sequencing techniques based on restriction enzymes to identify population structure as well as candidate loci for local adaptation. While the expectation is that the reduced representation of the genome is randomly distributed, the proportion of the genome sampled might depend on the GC content of the recognition site of the restriction enzyme used. Here, we evaluated the distribution and functional composition of loci obtained after a reduced representation approach using Genotyping-by-Sequencing (GBS). To do so, we compared experimental data from two endemic fish species (Symphodus ocellatus and Symphodus tinca, EcoT22I enzyme) and two ecosystem engineer sea urchins (Paracentrotus lividus and Arbacia lixula, ApeKI enzyme). In brief, we mapped the sequenced loci to the phylogenetically closest reference genome available (Labrus bergylta in the fish and Strongylocentrotus purpuratus in the sea urchin datasets), classified them as exonic, intronic and intergenic, and studied their function by using Gene Ontology (GO) terms. We also simulated the effect of using both enzymes in the two reference genomes. In both simulated and experimental data, we detected an enrichment towards exonic or intergenic regions depending on the restriction enzyme used and failed to detect differences between total loci and candidate loci for adaptation in the empirical dataset. Most of the functions assigned to the mapped loci were shared between the four species and involved a myriad of general functions. Our results highlight the importance of restriction enzyme selection and the need for high-quality annotated genomes in conservation genomic studies.

Expanding interactome analyses beyond model eukaryotes.

Interactome analyses have traditionally been applied to yeast, human and other model organisms due to the availability of protein-protein interaction data for these species. Recently, these techniques have been applied to more diverse species using computational interaction prediction from genome sequence and other data types. This review describes the various types of computational interactome networks that can be created and how they have been used in diverse eukaryotic species, highlighting some of the key interactome studies in non-model organisms.

Invertebrate methylomes provide insight into mechanisms of environmental tolerance and reveal methodological biases.

There is a growing focus on the role of DNA methylation in the ability of marine invertebrates to rapidly respond to changing environmental factors and anthropogenic impacts. However, genome-wide DNA methylation studies in nonmodel organisms are currently hampered by a limited understanding of methodological biases. Here, we compare three methods for quantifying DNA methylation at single base-pair resolution-whole genome bisulfite sequencing (WGBS), reduced representation bisulfite sequencing (RRBS), and methyl-CpG binding domain bisulfite sequencing (MBDBS)-using multiple individuals from two reef-building coral species with contrasting environmental sensitivity. All methods reveal substantially greater methylation in Montipora capitata (11.4%) than the more sensitive Pocillopora acuta (2.9%). The majority of CpG methylation in both species occurs in gene bodies and flanking regions. In both species, MBDBS has the greatest capacity for detecting CpGs in coding regions at our sequencing depth, but MBDBS may be influenced by intrasample methylation heterogeneity. RRBS yields robust information for specific loci albeit without enrichment of any particular genome feature and with significantly reduced genome coverage. Relative genome size strongly influences the number and location of CpGs detected by each method when sequencing depth is limited, illuminating nuances in cross-species comparisons. As genome-wide methylation differences, supported by data across bisulfite sequencing methods, may contribute to environmental sensitivity phenotypes in critical marine invertebrate taxa, these data provide a genomic resource for investigating the functional role of DNA methylation in environmental tolerance.

The Axenic and Gnotobiotic Mosquito: Emerging Models for Microbiome Host Interactions.

The increasing availability of modern research tools has enabled a revolution in studies of non-model organisms. Yet, one aspect that remains difficult or impossible to control in many model and most non-model organisms is the presence and composition of the host-associated microbiota or the microbiome. In this review, we explore the development of axenic (microbe-free) mosquito models and what these systems reveal about the role of the microbiome in mosquito biology. Additionally, the axenic host is a blank template on which a microbiome of known composition can be introduced, also known as a gnotobiotic organism. Finally, we identify a “most wanted” list of common mosquito microbiome members that show the greatest potential to influence host phenotypes. We propose that these are high-value targets to be employed in future gnotobiotic studies. The use of axenic and gnotobiotic organisms will transition the microbiome into another experimental variable that can be manipulated and controlled. Through these efforts, the mosquito will be a true model for examining host microbiome interactions.

Designing and selection of lineage-specific baits for the genomic study of non-model organisms

Designing and selection of lineage-specific baits for the genomic study of non-model organisms

Prodiamesa olivacea: de novo biomarker genes in a potential sentinel organism for ecotoxicity studies in natural scenarios

Along with traditional ecotoxicological approaches in model organisms, toxicological studies in non-model organisms are being taken into consideration in order to complement them and contribute to more robust approaches. This allows us to figure out the complexity of the exposures involved in natural ecosystems. In this context, in the present research we have used the model species Chironomus riparius (Chironomidae, Diptera) and the non-model species Prodiamesa olivacea (Chironomidae, Diptera) to assess the aquatic toxic effects of acute 4-h and 24-h exposures to 1 μgL−1 of three common environmental pollutants: butyl benzyl phthalate (BBP), bisphenol A (BPA), and benzophenone 3 (BP3). Individuals of both species were collected from a contaminated river (Sar) in Galicia (Spain).Regarding Chironomus, there are four OECD standardized tests for the evaluation of water and sediment toxicity, in which different species in this genus can be used to assess classical toxicity parameters such as survival, immobilization, reproduction, and development. In contrast, Prodiamesa is rarely used in toxicity studies, even though it is an interesting toxicological species because it shares habitats with Chironomus but requires less extreme conditions (e.g., contamination) and higher oxygen levels. These different requirements are particularly interesting in assessing the different responses of both species to pollutant exposure.Quantitative real-time PCR was used to evaluate the transcriptional changes caused by xenobiotics in different genes of interest. Since information about P. olivacea in genomic databases is scarce, its transcriptome was obtained using de novo RNAseq. Genes involved in biotransformation pathways and the oxidative stress response (MnSOD, CAT, PHGPx, Cyp4g15, Cyp6a14-like and Cyp6a2-like) were de novo identified in this species. Our results show differential toxic responses depending on the species and the xenobiotic, being P. olivacea the dipteran that showed the most severe effects in most of the studied biomarker genes.This work represents a multi-species approach that allows us to deepen in the toxicity of BBP, BPA, and BP3 at the molecular level. Besides, it provides an assessment of the tolerance/sensitivity of natural populations of model and non-model insect species chronically exposed to complex mixtures of pollutants in natural scenarios. These findings may have important implications for understanding the adverse biological effects of xenobiotics on P. olivacea, providing new sensitive biomarkers of exposure to BBP, BPA, and BP3. It also highlights the suitability of Prodiamesa for ecotoxicological risk assessment, especially in aquatic ecosystems.

EXFI: Exon and splice graph prediction without a reference genome.

For population genetic studies in nonmodel organisms, it is important to use every single source of genomic information. This paper presents EXFI, a Python pipeline that predicts the splice graph and exon sequences using an assembled transcriptome and raw whole‐genome sequencing reads. The main algorithm uses Bloom filters to remove reads that are not part of the transcriptome, to predict the intron–exon boundaries, to then proceed to call exons from the assembly, and to generate the underlying splice graph. The results are returned in GFA1 format, which encodes both the predicted exon sequences and how they are connected to form transcripts. EXFI is written in Python, tested on Linux platforms, and the source code is available under the MIT License at https://github.com/jlanga/exfi.

Degradome Sequencing in Plants.

Degradome sequencing provides large amounts of data regarding RNA degradation. The degradome library construction described here is modified from the 5'-rapid amplification of cDNA ends (5'-RACE), and each degradome cDNA is sequenced by next-generation sequencing (NGS). Degradome profiles provide information confirming miRNA-mediated cleavage of target genes and allow the identification of novel targets. Furthermore, degradome sequencing provides additional information for the study of RNA processing, such as information regarding RNA-binding proteins. In this chapter, we describe a detailed optimized protocol for constructing a degradome library with high yield and quality, along with NGS and data mining procedures. We hope that the degradome approach will be applied to further studies of non-model organisms.

Allele frequency-free inference of close familial relationships from genotypes or low-depth sequencing data.

Knowledge of how individuals are related is important in many areas of research, and numerous methods for inferring pairwise relatedness from genetic data have been developed. However, the majority of these methods were not developed for situations where data are limited. Specifically, most methods rely on the availability of population allele frequencies, the relative genomic position of variants and accurate genotype data. But in studies of non‐model organisms or ancient samples, such data are not always available. Motivated by this, we present a new method for pairwise relatedness inference, which requires neither allele frequency information nor information on genomic position. Furthermore, it can be applied not only to accurate genotype data but also to low‐depth sequencing data from which genotypes cannot be accurately called. We evaluate it using data from a range of human populations and show that it can be used to infer close familial relationships with a similar accuracy as a widely used method that relies on population allele frequencies. Additionally, we show that our method is robust to SNP ascertainment and applicable to low‐depth sequencing data generated using different strategies, including resequencing and RADseq, which is important for application to a diverse range of populations and species.

Ionomics suggests niche differences between sympatric heathers (Ericaceae)

The co-existence of large number of competing plant species with the same basic needs is a major question in ecology, particularly when this involves closely related species. We investigated the ecology of six heather species (Calluna vulgaris & Erica spp., Ericaceae) able to cohabit in the same heathlands. We characterised the ionome, i.e. the mineral composition of the plants using a sampling strategy specifically designed to control for soil effects. Nine plant communities in Limousin, France, were investigated, representing eight combinations of different species, on serpentine and non-serpentine substrates. Ionome was influenced by both taxonomic and environmental factors with significant interactions between them. We found that species growing in sympatry had distinct ionomic profiles, i.e. they differ in their leaf mineral content, suggesting different nutritional strategies. Different mycorrhizal associations may be hypothesised to explain these different chemical signatures. Differential use of the soil nutrients could explain the co-existence of closely related species. It may also explain the diversity of certain shrubby ecosystems or large shrubby genera such as Erica. Ionomics is therefore a promising tool for ecological studies in non-model organisms.

Theory, practice, and conservation in the age of genomics: The Galápagos giant tortoise as a case study.

High‐throughput DNA sequencing allows efficient discovery of thousands of single nucleotide polymorphisms (SNPs) in nonmodel species. Population genetic theory predicts that this large number of independent markers should provide detailed insights into population structure, even when only a few individuals are sampled. Still, sampling design can have a strong impact on such inferences. Here, we use simulations and empirical SNP data to investigate the impacts of sampling design on estimating genetic differentiation among populations that represent three species of Galápagos giant tortoises (Chelonoidis spp.). Though microsatellite and mitochondrial DNA analyses have supported the distinctiveness of these species, a recent study called into question how well these markers matched with data from genomic SNPs, thereby questioning decades of studies in nonmodel organisms. Using >20,000 genomewide SNPs from 30 individuals from three Galápagos giant tortoise species, we find distinct structure that matches the relationships described by the traditional genetic markers. Furthermore, we confirm that accurate estimates of genetic differentiation in highly structured natural populations can be obtained using thousands of SNPs and 2–5 individuals, or hundreds of SNPs and 10 individuals, but only if the units of analysis are delineated in a way that is consistent with evolutionary history. We show that the lack of structure in the recent SNP‐based study was likely due to unnatural grouping of individuals and erroneous genotype filtering. Our study demonstrates that genomic data enable patterns of genetic differentiation among populations to be elucidated even with few samples per population, and underscores the importance of sampling design. These results have specific implications for studies of population structure in endangered species and subsequent management decisions.

Small- and large-scale heterogeneity in genetic variation across the collard flycatcher genome: implications for estimating genetic diversity in nonmodel organisms.

Population genetic studies in nonmodel organisms are often hampered by a lack of reference genomes that are essential for whole-genome resequencing. In the light of this, genotyping methods have been developed to effectively eliminate the need for a reference genome, such as genotyping by sequencing or restriction site-associated DNA sequencing (RAD-seq). However, what remains relatively poorly studied is how accurately these methods capture both average and variation in genetic diversity across an organism's genome. In this issue of Molecular Ecology Resources, Dutoit etal. (2016) use whole-genome resequencing data from the collard flycatcher to assess what factors drive heterogeneity in nucleotide diversity across the genome. Using these data, they then simulate how well different sequencing designs, including RAD sequencing, could capture most of the variation in genetic diversity. They conclude that for evolutionary and conservation-related studies focused on the estimating genomic diversity, researchers should emphasize the number of loci analysed over the number of individuals sequenced.

Correlation between Hox code and vertebral morphology in the mouse: towards a universal model for Synapsida

BackgroundThe importance of the cervical vertebrae as part of the skull–neck system in facilitating the success and diversity of tetrapods is clear. The reconstruction of its evolution, however, is problematic because of the variation in the number of vertebrae, making it difficult to identify homologous elements. Quantification of the morphological differentiation in the neck of diverse archosaurs established homologous units of vertebrae (i.e. modules) resulting from Hox gene expression patterns within the cervical vertebral column. The present study aims to investigate the modularity of the cervical vertebral column in the mouse and to reveal the genetic patterns and changes underlying the evolution of the neck of modern mammals and their extinct relatives. In contrast to modern mammals, non-mammalian synapsids are characterized by a variable cervical count, the presence of free cervical ribs and the presence of a separate CV1 centrum. How might these evolutionary modifications be associated with changes in the Hox code?ResultsIn combination with up-to-date information on cervical Hox gene expression including description of the vertebral phenotype of Hox knock-out mutants, the 3D landmark-based geometric morphometric approach demonstrates a correlation between Hox code and vertebral morphology in the mouse. There is evidence that the modularity of the neck of the mouse had already been established in the last common ancestor of mammals, but differed from that of non-mammalian synapsids. The differences that likely occurred during the evolution of synapsids include an anterior shift in HoxA-5 expression in relation to the reduction of cervical ribs and an anterior shift in HoxD-4 expression linked to the development of the highly differentiated atlas-axis complex, whereas the remaining Hox genes may have displayed a pattern similar to that in mammals on the basis of the high level of conservatism in the axial skeleton of this lineage.ConclusionThus, the mouse Hox code provides a model for understanding the evolutionary mechanisms responsible for the great morphological adaptability of the cervical vertebral column in Synapsida. However, more studies in non-model organisms are required to further elucidate the evolutionary role of Hox genes in axial patterning of the unique mammalian body plan.

Population genetics analysis of the Nujiang catfish Creteuchiloglanis macropterus through a genome-wide single nucleotide polymorphisms resource generated by RAD-seq

Advances in genome scanning using high-throughput sequencing technologies has led to a revolution in studies of non-model organisms. The glyptosternoid fish Creteuchiloglanis macropterus, is widely distributed in the main stem and tributaries of the Nujiang River basin. Here, we analyzed IIB restriction-site-associated DNA (2b-RAD) sequences and mitochondrial DNA sequences, to assess the genomic signature of adaptation by detecting and estimating the degree of genetic differentiation among ten Creteuchiloglanis macropterus populations from the Nujiang River. The analyses revealed significant population differentiation among the up-tributaries, main stem, mid-tributary and low-tributary. Annotation of contigs containing outlier SNPs revealed that the candidate genes showed significant enrichment in several important biological process terms between up-tributaries and low-tributary, and exhibited prominent enrichment in the term macromolecular metabolic process between all tributaries and the main stem. Population dynamics analyses indicated that the Late Pleistocene glaciations strongly influenced the demographic history of C. macropterus. Our results provide strong evidence for the utility of RAD-seq in population genetics studies, and our generated SNP resource should provide a valuable tool for population genomics studies of C. macropterus in the future.

Genomic analysis of a cardinalfish with larval homing potential reveals genetic admixture in the Okinawa Islands

Discrepancies between potential and observed dispersal distances of reef fish indicate the need for a better understanding of the influence of larval behaviour on recruitment and dispersal. Population genetic studies can provide insight on the degree to which populations are connected, and the development of restriction site-associated sequencing (RAD-Seq) methods has made such studies of nonmodel organisms more accessible. We applied double-digest RAD-Seq methods to test for population differentiation in the coral reef-dwelling cardinalfish, Siphamia tubifer, which based on behavioural studies, have the potential to use navigational cues to return to natal reefs. Analysis of 11,836 SNPs from fish collected at coral reefs in Okinawa, Japan, from eleven locations over 3years reveals little genetic differentiation between groups of S.tubifer at spatial scales from 2 to 140km and between years at one location: pairwise FST values were between 0.0116 and 0.0214. These results suggest that the Kuroshio Current largely influences larval dispersal in the region, and in contrast to expectations based on studies of other cardinalfishes, there is no evidence of population structure for S.tubifer at the spatial scales examined. However, analyses of outlier loci putatively under selection reveal patterns of temporal differentiation that indicate high population turnover and variable larval supply from divergent source populations between years. These findings highlight the need for more studies of fishes across various geographic regions that also examine temporal patterns of genetic differentiation to better understand the potential connections between early life-history traits and connectivity of reef fish populations.

Discovery and preliminary multi-species evaluation of single nucleotide polymorphism resources for genus Buteo developed from restriction site-associated DNA paired-end data

Just a few years ago, genomic studies of non-model organisms were extremely time and cost prohibitive, but recent advances in next-generation sequencing have made it possible to generate hundreds to thousands of informative single nucleotide polymorphisms (SNPs) for non-model organisms. We generated a SNP panel using restriction site-associated DNA (RAD) sequencing for species of the genus Buteo (Falconiformes: Accipitridae) with the aim of creating genomic resources in the absence of a reference genome. Using blood and feather samples from three different Buteo species, we used HiSeq Illumina sequencing to generate raw reads. We built 29 162 contigs and discovered 89 921 SNPs using paired-end single-digest RAD data. Using Bayesian clustering and principal componenets analysis, this SNP panel successfully assigned individuals of three Buteo (B. swainsoni, B. jamaicensis, B. lagopus) species correctly to species. We then scanned the SNPs for association with variation in coloration within the three species. This de novo reference will have broad application to the study of Buteo genomic diversity, analysis of population structure within species, identification of hybrids, and other applied studies of the avian genome.

Towards integration of population and comparative genomics in forest trees.

Contents 338 I. 338 II. 339 III. 340 IV. 342 343 References 343 SUMMARY: The past decade saw the initiation of an ongoing revolution in sequencing technologies that is transforming all fields of biology. This has been driven by the advent and widespread availability of high-throughput, massively parallel short-read sequencing (MPS) platforms. These technologies have enabled previously unimaginable studies, including draft assemblies of the massive genomes of coniferous species and population-scale resequencing. Transcriptomics studies have likewise been transformed, with RNA-sequencing enabling studies in nonmodel organisms, the discovery of previously unannotated genes (novel transcripts), entirely new classes of RNAs and previously unknown regulatory mechanisms. Here we touch upon current developments in the areas of genome assembly, comparative regulomics and population genetics as they relate to studies of forest tree species.

DOMINO: development of informative molecular markers for phylogenetic and genome-wide population genetic studies in non-model organisms.

The development of molecular markers is one of the most important challenges in phylogenetic and genome wide population genetics studies, especially in studies with non-model organisms. A highly promising approach for obtaining suitable markers is the utilization of genomic partitioning strategies for the simultaneous discovery and genotyping of a large number of markers. Unfortunately, not all markers obtained from these strategies provide enough information for solving multiple evolutionary questions at a reasonable taxonomic resolution. We have developed Development Of Molecular markers In Non-model Organisms (DOMINO), a bioinformatics tool for informative marker development from both next generation sequencing (NGS) data and pre-computed sequence alignments. The application implements popular NGS tools with new utilities in a highly versatile pipeline specifically designed to discover or select personalized markers at different levels of taxonomic resolution. These markers can be directly used to study the taxa surveyed for their design, utilized for further downstream PCR amplification in a broader set taxonomic scope, or exploited as suitable templates to bait design for target DNA enrichment techniques. We conducted an exhaustive evaluation of the performance of DOMINO via computer simulations and illustrate its utility to find informative markers in an empirical dataset. DOMINO is freely available from www.ub.edu/softevol/domino CONTACT: elsanchez@ub.edu or jrozas@ub.eduSupplementary information: Supplementary data are available at Bioinformatics online.