Non-sequenced Species Research Articles

Tulostoma subreticulatum (Agaricomycetes, Agaricales): a new species of stalked puffball from Mexico

Tulostoma subreticulatum is a new species to science. This species is characterized by its small to medium-size basidiomes, tubular stoma, verrucose exoperidium with flat and angulose verrucae up to 0.8 mm, reddish-brown endoperidium, and subreticulated 3.8–5 μm basidiospores. This new species was collected in the tropical forest of Tabasco, Mexico. Description of the specimens, macroscopic images of the basidiomes, light microscopy, and scanning electron micrographs are provided. DNA extraction was carried out following a CTAB protocol. GeneBank and newly generated ITS, LSU, and Tef1α sequences were used in Maximum Likelihood and Bayesian Inference analyses. Phylogenetically, T. subreticulatum is related to T. deltaconcavum and forms a sister group from Clade 11 of the monophyletic genus Tulostoma. Comparisons with related sequenced and non-sequenced species are discussed. With this species, a total of 51 species of Tulostoma are known in Mexico.

Redescription of the last instar larva of Stenophylax fissus (McLachlan 1875) and the DNA barcode of Stenophylax crossotus McLachlan 1884 (Trichoptera: Limnephilidae) with an updated diagnostic matrix of the known larvae of Stenophylax species of the Iberian Peninsula.

The larva of Stenophylax fissus (McLachlan 1875) is redescribed, illustrated, and compared with morphologically similar Limnephilidae larvae from the Iberian Peninsula. In addition, the sequence of the mtCOI gene in the barcode region of one individual is reported. Likewise, the barcode and the conspecificity between two larvae and one female of the non-sequenced species S. crossotus are given. In addition, the high genetic diversity among S. sequax populations is highlighted, suggesting a complex of cryptic species. Finally, a diagnostic matrix of the known larvae of Stenophylax species from the Iberian Peninsula is provided.

Mass Spectrometry-Based Sequencing and SRM-Based Quantitation of Two Novel Vitellogenin Isoforms in the Leatherback Sea Turtle (Dermochelys coriacea)

No biomarker has yet been discovered to identify the reproductive status of the endangered leatherback sea turtle (Dermochelys coriacea). Although vitellogenin (VTG) could be used for this, its sequence is not known in D. coriacea and no quantitative assay has been carried out in this species to date. Using de novo sequencing-based proteomics, we unambiguously characterized sequences of two different VTG isoforms that we named Dc-VTG1 and Dc-VTG2. To our knowledge, this is the first clear evidence of different VTG isoforms and the structural characterization of derived yolk proteins in reptiles. This work illustrates how massive de novo sequencing can characterize novel sequences when working on "exotic" nonmodel species in which even nucleotide sequences are not available. We developed assays for absolute quantitation of these two isoforms using selected reaction monitoring (SRM) mass spectrometry, thus providing the first SRM assays developed specifically for a nonsequenced species. Plasma levels of Dc-VTG1 and Dc-VTG2 decreased as the nesting season proceeded, and were closely related to the increased levels of reproductive effort. The SRM assays developed here therefore provide an original and efficient approach for the reliable monitoring of reproduction cycles not only in D. coriacea, but potentially in other turtle species.

Wheat Zapper: a flexible online tool for colinearity studies in grass genomes

In the course of evolution, the genomes of grasses have maintained an observable degree of gene order conservation. The information available for already sequenced genomes can be used to predict the gene order of nonsequenced species by means of comparative colinearity studies. The "Wheat Zapper" application presented here performs on-demand colinearity analysis between wheat, rice, Sorghum, and Brachypodium in a simple, time efficient, and flexible manner. This application was specifically designed to provide plant scientists with a set of tools, comprising not only synteny inference, but also automated primer design, intron/exon boundaries prediction, visual representation using the graphic tool Circos 0.53, and the possibility of downloading FASTA sequences for downstream applications. Quality of the "Wheat Zapper" prediction was confirmed against the genome of maize, with good correlation (r > 0.83) observed between the gene order predicted on the basis of synteny and their actual position on the genome. Further, the accuracy of "Wheat Zapper" was calculated at 0.65 considering the "Genome Zipper" application as the "gold" standard. The differences between these two tools are amply discussed, making the point that "Wheat Zapper" is an accurate and reliable on-demand tool that is sure to benefit the cereal scientific community. The Wheat Zapper is available at http://wge.ndsu.nodak.edu/wheatzapper/ .

RNA Sequencing Reveals Differential Expression of Mitochondrial and Oxidation Reduction Genes in the Long-Lived Naked Mole-Rat When Compared to Mice

The naked mole-rat (Heterocephalus glaber) is a long-lived, cancer resistant rodent and there is a great interest in identifying the adaptations responsible for these and other of its unique traits. We employed RNA sequencing to compare liver gene expression profiles between naked mole-rats and wild-derived mice. Our results indicate that genes associated with oxidoreduction and mitochondria were expressed at higher relative levels in naked mole-rats. The largest effect is nearly 300-fold higher expression of epithelial cell adhesion molecule (Epcam), a tumour-associated protein. Also of interest are the protease inhibitor, alpha2-macroglobulin (A2m), and the mitochondrial complex II subunit Sdhc, both ageing-related genes found strongly over-expressed in the naked mole-rat. These results hint at possible candidates for specifying species differences in ageing and cancer, and in particular suggest complex alterations in mitochondrial and oxidation reduction pathways in the naked mole-rat. Our differential gene expression analysis obviated the need for a reference naked mole-rat genome by employing a combination of Illumina/Solexa and 454 platforms for transcriptome sequencing and assembling transcriptome contigs of the non-sequenced species. Overall, our work provides new research foci and methods for studying the naked mole-rat's fascinating characteristics.

Screening Target Specificity of siRNAs by Rapid Amplification of cDNA Ends (RACE) for Non-Sequenced Species

RNA interference (RNAi) is the process of sequence-specific posttranslational gene silencing triggered by double-stranded RNAs (dsRNAs). RNAi is a widely used approach for studying gene function. However, studies have shown that using siRNA can lead to off-target effects when the siRNA contains sufficient sequence identity to non-target mRNA sequences. One of the important steps in designing dsRNA is verification that it has sequence identity to only the target mRNA. In this report, we propose an approach for primary screening dsRNAs for potential off-target effects by using rapid amplification of cDNA ends. This method can be especially useful for model systems using species that have limited availability of sequence data.

In vitro homology search array comprehensively reveals highly conserved genes and their functional characteristics in non-sequenced species

BackgroundWith the increase in genomic and transcriptomic data produced by the recent advancements in next generation sequencers and microarrays, it is now easier than ever to conduct large-scale comparative genomic studies for familiar species. However, there are more than ten million species on earth, and the study of all remaining species is not realistic in terms of cost and time. There have been a number of attempts at using microarrays for cross-species hybridization; however, those approaches only utilized the same probes for each species or different probes designed from orthologous genes. To establish easier and cheaper methods for the large-scale comparative genomic study of non-sequenced species, we developed an in vitro homology search array with the aid of a bioinformatic approach to probe design.ResultsTo perform large-scale genomic comparisons of non-sequenced species, we chose squid, one of the most intelligent species among Protostomes, for comparison with human genes. We designed a microarray using human single copy genes and conducted microarray experiments with mRNAs extracted from the squid. Multi-copy genes could not be detected using the microarray in this study because their sequence similarity caused cross-hybridization. A search for squid homologous genes among human genes revealed that 68% of the human probes tested showed the expression of squid homolog genes and 95 genes were confirmed to be expressed highly in squid. Functional classification analysis showed that these highly expressed genes comprise DNA binding proteins, which are under pressure of DNA level mutation and, consequently, show high similarity at the nucleotide level.ConclusionsOur array could detect homologous genes in squids and humans in spite of the distant phylogenic relationships between the species. This experimental method will be useful for identifying homologs in non-sequenced species, for the development of genetic resources and for the collection of information on biodiversity, particularly when using the genome of sibling or closely related species.

Complete rpoB gene sequencing as a suitable supplement to DNA-DNA hybridization for bacterial species and genus delineation

DNA-DNA hybridization (DDH), the gold standard for bacterial species delineation, is a laborious method and the alternative, average nucleotide identity (ANI), a genomic sequence-derived parameter, is not applicable to non-sequenced species. A universal cut-off value to delineate bacterial species does not exist, yet a DDH value <70 % and ANI <95+/-0.5 % have proved useful in selected examples. We herein compare published values for DDH and ANI with sequence similarity of rpoB gene sequences retrieved from GenBank for strains of 230 bacterial species representative of 45 genera. Intraspecific rpoB sequence similarity was 98.2-100 %. We observed that an rpoB gene sequence similarity <or=97.7 % significantly correlated with a DDH value <70 % and an ANI value <94.3 %. An rpoB gene sequence similarity <85.5 % correlated with membership of different genera. When applied to fastidious and as-yet-uncultivated organisms lacking experimental DDH values, these cut-off values suggested that 'Candidatus Blochmannia pennsylvanicus' and 'Candidatus Blochmannia floridarius' may belong to different genera, that the different endosymbiotic Buchnera aphidicola organisms may belong to different genera and that, while the tsetse fly enteric symbiont Sodalis glossinidius may belong to the Enterobacteriaceae, the endocellular obligate symbiont Wigglesworthia glossinidia from the same host may belong to the group of as-yet-uncultivated gammaproteobacteria. rpoB gene sequence similarity provides an efficient supplement to DDH and ANI measurements to delineate bacterial species and genera, including delineation of as-yet-uncultivated, non-sequenced organisms.

Hyperbolic SOM-based clustering of DNA fragment features for taxonomic visualization and classification

Modern high-throughput sequencing technologies enable the simultaneous analysis of organisms in an environment. The analysis of species diversity and the binning of DNA fragments of non-sequenced species for assembly are two major challenges in sequence analysis. To achieve reasonable binnings and classifications, DNA fragment structure has to be represented appropriately, so it can be processed by machine learning algorithms. Hierarchically growing hyperbolic Self-Organizing maps (H(2)SOMs) are trained to cluster small variable-length DNA fragments (0.2-50 kb) of 350 prokaryotic organisms at six taxonomic ranks Superkingdom, Phylum, Class, Order, Genus and Species in the Tree of Life. DNA fragments are mapped to three different types of feature vectors based on the genomic signature: basic features, features considering the importance of oligonucleotide patterns as well as contrast enhanced features. The H (2)SOM classifier achieves high classification rates while at the same time its visualization allows further insights into the projected data and has the potential to support binning of short sequence reads, because DNA fragments can be grouped into phylogenetic groups. An implementation of the H(2)HSOM classifier in Matlab is provided at www.techfak.uni-bielefeld.de/ags/ani/projects/HHSOMSeqData.

A quantitative trait locus for variation in dopamine metabolism mapped in a primate model using reference sequences from related species.

Non-human primates (NHP) provide crucial research models. Their strong similarities to humans make them particularly valuable for understanding complex behavioral traits and brain structure and function. We report here the genetic mapping of an NHP nervous system biologic trait, the cerebrospinal fluid (CSF) concentration of the dopamine metabolite homovanillic acid (HVA), in an extended inbred vervet monkey (Chlorocebus aethiops sabaeus) pedigree. CSF HVA is an index of CNS dopamine activity, which is hypothesized to contribute substantially to behavioral variations in NHP and humans. For quantitative trait locus (QTL) mapping, we carried out a two-stage procedure. We first scanned the genome using a first-generation genetic map of short tandem repeat markers. Subsequently, using >100 SNPs within the most promising region identified by the genome scan, we mapped a QTL for CSF HVA at a genome-wide level of significance (peak logarithm of odds score >4) to a narrow well delineated interval (<10 Mb). The SNP discovery exploited conserved segments between human and rhesus macaque reference genome sequences. Our findings demonstrate the potential of using existing primate reference genome sequences for designing high-resolution genetic analyses applicable across a wide range of NHP species, including the many for which full genome sequences are not yet available. Leveraging genomic information from sequenced to nonsequenced species should enable the utilization of the full range of NHP diversity in behavior and disease susceptibility to determine the genetic basis of specific biological and behavioral traits.