SPInDel: a multifunctional workbench for species identification using insertion/deletion variants

The majority of the available methods for the molecular identification of species use pairwise sequence divergences between the query and reference sequences (DNA barcoding). The presence of multiple insertions and deletions (indels) in the target genomic regions is generally regarded as a problem, as it introduces ambiguities in sequence alignments. However, we have recently shown that a high level of species discrimination is attainable in all taxa of life simply by considering the length of hypervariable regions defined by indel variants. Each species is tagged with a numeric profile of fragment lengths—a true numeric barcode. In this study, we describe a multifunctional computational workbench (named SPInDel for SPecies Identification by Insertions/Deletions) to assist researchers using variable‐length DNA sequences, and we demonstrate its applicability in molecular ecology. The SPInDel workbench provides a step‐by‐step environment for the alignment of target sequences, selection of informative hypervariable regions, design of PCR primers and the statistical validation of the species‐identification process. In our test data sets, we were able to discriminate all species from two genera of frogs (Ansonia and Leptobrachium) inhabiting lowland rainforests and mountain regions of South‐East Asia and species from the most common genus of coral reef fishes (Apogon). Our method can complement conventional DNA barcoding systems when indels are common (e.g. in rRNA genes) without the required step of DNA sequencing. The executable files, source code, documentation and test data sets are freely available at http://www.portugene.com/SPInDel/SPInDel_webworkbench.html .     

Analysis of microcalorimetric curves for bacterial identification

A numeric method is suggested for the treatment of microcalorimetric curves of bacterial growth to provide a new tool for their automatic identification. In this method the microcalorimetric curves are searched against certain reference profiles (stored in a library) by means of a cross-correlation analysis and a parametric comparison. The matching between the new curve and each reference profile is evaluated by means of a specific identification coefficient which provides an objective criterion for the identification of each species. The reliability of the method is discussed.     

High-Resolution Melting of 12S rRNA and Cytochrome b DNA Sequences for Discrimination of Species within Distinct European Animal Families

The cheap and easy identification of species is necessary within multiple fields of molecular biology. The use of high-resolution melting (HRM) of DNA provides a fast closed-tube method for analysis of the sequence composition of the mitochondrial genes 12S rRNA and cytochrome b. We investigated the potential use of HRM for species identification within eleven different animal groups commonly found in Europe by animal-group-specific DNA amplification followed by DNA melting. Influence factors as DNA amount, additional single base alterations, and the existence of mixed samples were taken into consideration. Visual inspection combined with mathematical evaluation of the curve shapes did resolve nearly all species within an animal group. The assay can therefore not only be used for identification of animal groups and mixture analysis but also for species identification within the respective groups. The use of a universal 12S rRNA system additionally revealed a possible approach for species discrimination, mostly by exclusion. The use of the HRM assay showed to be a reliable, fast, and cheap method for species discrimination within a broad range of different animal species and can be used in a flexible “modular” manner depending on the question to be solved.     

Distinguishing Anuran species by high‐resolution melting analysis of the COI barcode (COI‐HRM)

Taxonomic identification can be difficult when two or more species appear morphologically similar. DNA barcoding based on the sequence of the mitochondrial cytochrome c oxidase 1 gene (COI) is now widely used in identifying animal species. High‐resolution melting analysis (HRM) provides an alternative method for detecting sequence variations among amplicons without having to perform DNA sequencing. The purpose of this study was to determine whether HRM of the COI barcode can be used to distinguish animal species. Using anurans as a model, we found distinct COI melting profiles among three congeners of both Lithobates spp. and Hyla spp. Sequence variations within species shifted the melting temperature of one or more melting domains slightly but do not affect the distinctness of the melting profiles for each species. An NMDS ordination plot comparing melting peak profiles among eight Anuran species showed overlapping profiles for Lithobates sphenocephala and Gastrophryne carolinensis. The COI amplicon for both species contained two melting domains with melting temperatures that were similar between the two species. The two species belong to two different families, highlighting the fact that COI melting profiles do not reveal phylogenetic relationships but simply reflect DNA sequence differences among stretches of DNA within amplicons. This study suggests that high‐resolution melting analysis of COI barcodes (COI‐HRM) may be useful as a simple and rapid method to distinguish animal species that appear morphologically similar.     

Recent Advances in the Identification of Replication Origins Based on the Z-curve Method

Precise DNA replication is critical for the maintenance of genetic integrity in all organisms. In all three domains of life, DNA replication starts at a specialized locus, termed as the replication origin, oriC or ORI, and its identification is vital to understanding the complex replication process. In bacteria and eukaryotes, replication initiates from single and multiple origins, respectively, while archaea can adopt either of the two modes. The Z-curve method has been successfully used to identify replication origins in genomes of various species, including multiple oriCs in some archaea. Based on the Z-curve method and comparative genomics analysis, we have developed a web-based system, Ori-Finder, for finding oriCs in bacterial genomes with high accuracy. Predicted oriC regions in bacterial genomes are organized into an online database, DoriC. Recently, archaeal oriC regions identified by both in vivo and in silico methods have also been included in the database. Here, we summarize the recent advances of in silico prediction of oriCs in bacterial and archaeal genomes using the Z-curve based method.     

Genome trees from conservation profiles

The concept of the genome tree depends on the potential evolutionary significance in the clustering of species according to similarities in the gene content of their genomes. In this respect, genome trees have often been identified with species trees. With the rapid expansion of genome sequence data it becomes of increasing importance to develop accurate methods for grasping global trends for the phylogenetic signals that mutually link the various genomes. We therefore derive here the methodological concept of genome trees based on protein conservation profiles in multiple species. The basic idea in this derivation is that the multi-component "presence-absence" protein conservation profiles permit tracking of common evolutionary histories of genes across multiple genomes. We show that a significant reduction in informational redundancy is achieved by considering only the subset of distinct conservation profiles. Beyond these basic ideas, we point out various pitfalls and limitations associated with the data handling, paving the way for further improvements. As an illustration for the methods, we analyze a genome tree based on the above principles, along with a series of other trees derived from the same data and based on pair-wise comparisons (ancestral duplication-conservation and shared orthologs). In all trees we observe a sharp discrimination between the three primary domains of life: Bacteria, Archaea, and Eukarya. The new genome tree, based on conservation profiles, displays a significant correspondence with classically recognized taxonomical groupings, along with a series of departures from such conventional clusterings.     

Fluorescence energy transfer in two dimensions. A numeric solution for random and nonrandom distributions.

A method of Monte Carlo calculations has been applied to the problem of fluorescence energy transfer in two dimensions in order to provide a quantitative measure of the effects of nonideal mixing of lipid and protein molecules on the quenching profiles of membrane systems. These numerical techniques permit the formulation of a detailed set of equations that describes in a precise manner the quenching and depolarization properties of planar donor-acceptor distributions as a function of specific spectroscopic and organizational parameters. Because of the exact nature of the present numeric method, these results are used to evaluate critically the validity of previous approximate treatments existing in the literature. This method is also used to examine the effects of excluded volume interactions and distinct lattice structures on the expected transfer efficiencies. As a specific application, representative quenching profiles for protein-lipid mixtures, in which donor groups are covalently linked to the protein molecules and acceptor species are randomly distributed within lipid domains, have been obtained. It is found that the existence of phase-separated protein domains gives rise to a shielding effect that significantly decreases the transfer efficiencies with respect to those expected for an ideal distribution of protein molecules. The results from the present numerical study indicate that the experimental application of fluorescence energy transfer measurements in multicomponent membrane systems can be used to obtain organizational parameters that accurately reflect the lateral distribution of protein and lipid molecules within the bilayer membrane.     

Insights into the evolution of the nucleolus by an analysis of its protein domain repertoire

Recently, the first investigation of nucleoli using mass spectrometry led to the identification of 271 proteins. This represents a rich resource for a comprehensive investigation of nucleolus evolution. We applied a protocol for the identification of known and novel conserved protein domains of the nucleolus, resulting in the identification of 115 known and 91 novel domain profiles. The phyletic distribution of nucleolar protein domains in a collection of complete proteomes of selected organisms from all domains of life confirms the archaebacterial origin of the core machinery for ribosome maturation and assembly, but also reveals substantial eubacterial and eukaryotic contributions to nucleolus evolution. We predict that, in different phases of nucleolus evolution, protein domains with different biochemical functions were recruited to the nucleolus. We suggest a model for the late and continuous evolution of the nucleolus in early eukaryotes and argue against an endosymbiotic origin of the nucleolus and the nucleus. Supplementary material for this article can be found on the BioEssays website at http://www.interscience.wiley.com/jpages/0265-9247/suppmat/index.html.     

Ernst Mayr,the tree of life,and philosophy of biology

Ernst Mayr’s influence on philosophy of biology has given the field a particular perspective on evolution, phylogeny and life in general. Using debates about the tree of life as a guide, I show how Mayrian evolutionary biology excludes numerous forms of life and many important evolutionary processes. Hybridization and lateral gene transfer are two of these processes, and they occur frequently, with important outcomes in all domains of life. Eukaryotes appear to have a more tree-like history because successful lateral events tend to occur among more closely related species, or at a lower frequency, than in prokaryotes, but this is a difference of degree rather than kind. Although the tree of life is especially problematic as a representation of the evolutionary history of prokaryotes, it can function more generally as an illustration of the limitations of a standard evolutionary perspective. Moreover, for philosophers, questions about the tree of life can be applied to the Mayrian inheritance in philosophy of biology. These questions make clear that the dichotomy of life Mayr suggested is based on too narrow a perspective. An alternative to this dichotomy is a multidimensional continuum in which different strategies of genetic exchange bestow greater adaptiveness and evolvability on prokaryotes and eukaryotes.     

Identification of domains and domain interface residues in multidomain proteins from graph spectral method

We present a novel method for the identification of structural domains and domain interface residues in proteins by graph spectral method. This method converts the three-dimensional structure of the protein into a graph by using atomic coordinates from the PDB file. Domain definitions are obtained by constructing either a protein backbone graph or a protein side-chain graph. The graph is constructed based on the interactions between amino acid residues in the three-dimensional structure of the proteins. The spectral parameters of such a graph contain information regarding the domains and subdomains in the protein structure. This is based on the fact that the interactions among amino acids are higher within a domain than across domains. This is evident in the spectra of the protein backbone and the side-chain graphs, thus differentiating the structural domains from one another. Further, residues that occur at the interface of two domains can also be easily identified from the spectra. This method is simple, elegant, and robust. Moreover, a single numeric computation yields both the domain definitions and the interface residues.     

The Conservation and Management of Tunas and Their Relatives: Setting Life History Research Priorities

Scombrids (tunas, bonitos, Spanish mackerels and mackerels) support important fisheries in tropical, subtropical and temperate waters around the world, being one of the most economically- and socially-important marine species globally. Their sustainable exploitation, management and conservation depend on accurate life history information for the development of quantitative fisheries stock assessments, and in the fishery data-poor situations for the identification of vulnerable species. Here, we assemble life history traits (maximum size, growth, longevity, maturity, fecundity, spawning duration and spawning interval) for the 51 species of scombrids globally. We identify major biological gaps in knowledge and prioritize life history research needs in scombrids based on their biological gaps in knowledge, the importance of their fisheries and their current conservation status according to the International Union for Conservation of Nature Red List. We find that the growth and reproductive biology of tunas and mackerel species have been more extensively studied than for Spanish mackerels and bonitos, although there are notable exceptions in all groups. We also reveal that reproductive biology of species, particular fecundity, is the least studied biological aspect in scombrids. We identify two priority groups, including 32 species of scombrids, and several populations of principal market tunas, for which life history research should be prioritized following the species-specific life history gaps identified in this study in the coming decades. By highlighting the important gaps in biological knowledge and providing a priority setting for life history research in scombrid species this study provides guidance for management and conservation and serves as a guide for biologists and resource managers interested in the biology, ecology, and management of scombrid species.     

藻类快速鉴定研究进展

综述了传统形态学方法和生物化学法、分子生物学法在藻类鉴定中的应用及其原理,系统阐述了以上方法的研究进展。在藻类鉴定中,生物化学法和分子生物学法是新兴技术,其中的分子鉴定技术,是根据物种的基因差异来定性分类物种,可以快速稳定地鉴定藻类。分子生物学法在水华藻类快速鉴定方面具有很大的发展潜力,有望得到广泛应用。     

DNA barcoding of stoneflies (Plecoptera) in a general genetics course

DNA Barcoding is a powerful molecular biology tool for the identification of species, analysis of gene flow from within and between populations and evaluating species concepts. The process can be incorporated into a college genetics or bioinformatics core curriculum. Here we demonstrate how, by simply using Plecoptera, we actively engaged students in DNA extraction, amplification and DNA Barcoding. As a result, 38 new DNA sequences were accepted by an international DNA database, with potentially three of them being new species to the database. More importantly, through multiple assessment measurements, students showed a high degree of learning took place and that all student learning objectives were met.     

论中药分子鉴定的方法和原则

中药的准确鉴定涉及到人民生命安全和切身利益。传统的鉴定方法,即感官评价、显微鉴定和理化鉴定均存在不同程度的局限性,而分子鉴定则为中药的快速和准确鉴定带来了新的契机。为了使中药的分子鉴定得到更加广泛和有效的应用,应该高度重视相关标准、规范的制订。本文提出了中药分子鉴定的一些主要方法：1)真伪品鉴定：特异聚合酶链式反应法;2)正品和替代品鉴定：DNA条形码鉴定法;3)多基源鉴定：群体遗传学分析法;4)产地鉴别：分子谱系地理学分析法。经上述方法仍无法鉴别的贵重药材可进一步应用人类亲子鉴定的方法,开发特异微卫星标记进行进一步的鉴定。     

On the Methods and Principles of MolecularIdentification of Chinese Herbs

Accurate identification of herbal medicinal materials is relevant to the safety of human life and economic interests. Traditional identification methods, including sensory evaluation, microscopic identification, physical and chemical identification, all have their limitations. Molecular identification brings a new opportunity for accurate identification of herbal medicinal materials. However, prior to its wide adoption, the methods and principles of molecular identification should be fully discussed. In this paper, we proposed a set of new methods for molecular authentication of herbal medicinal materials: 1) Identification between authenticity and adulteration by using specific polymerase chain reaction; 2) Identification between official herb and substitute by using the method of DNA barcoding; 3) Identification among multiple species of one official herbs by constructing genealogy among closely related species based on population genetics; 4) Identification among herbs of different geographical origins by phylogeography based analysis. For those that can not be identified by above four methods, more rapidly evolved markers such as microsatellite should be employed and individual based analysis could be adopted.     

A tree of life based on protein domain organizations

It is desirable to estimate a tree of life, a species tree including all available species in the 3 superkingdoms, Archaea, Bacteria, and Eukaryota, using not a limited number of genes but full-scale genome information. Here, we report a new method for constructing a tree of life based on protein domain organizations, that is, sequential order of domains in a protein, of all proteins detected in a genome of an organism. The new method is free from the identification of orthologous gene sets and therefore does not require the burdensome and error-prone computation. By pairwise comparisons of the repertoires of protein domain organizations of 17 archaeal, 136 bacterial, and 14 eukaryotic organisms, we computed evolutionary distances among them and constructed a tree of life. Our tree shows monophyly in Archaea, Bacteria, and Eukaryota and then monophyly in each of eukaryotic kingdoms and in most bacterial phyla. In addition, the branching pattern of the bacterial phyla in our tree is consistent with the widely accepted bacterial taxonomy and is very close to other genome-based trees. A couple of inconsistent aspects between the traditional trees and the genome-based trees including ours, however, would perhaps urge to revise the conventional view, particularly on the phylogenetic positions of hyperthermophiles.     

Co-Inheritance Analysis within the Domains of Life Substantially Improves Network Inference by Phylogenetic Profiling

Phylogenetic profiling, a network inference method based on gene inheritance profiles, has been widely used to construct functional gene networks in microbes. However, its utility for network inference in higher eukaryotes has been limited. An improved algorithm with an in-depth understanding of pathway evolution may overcome this limitation. In this study, we investigated the effects of taxonomic structures on co-inheritance analysis using 2,144 reference species in four query species: Escherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana, and Homo sapiens. We observed three clusters of reference species based on a principal component analysis of the phylogenetic profiles, which correspond to the three domains of life—Archaea, Bacteria, and Eukaryota—suggesting that pathways inherit primarily within specific domains or lower-ranked taxonomic groups during speciation. Hence, the co-inheritance pattern within a taxonomic group may be eroded by confounding inheritance patterns from irrelevant taxonomic groups. We demonstrated that co-inheritance analysis within domains substantially improved network inference not only in microbe species but also in the higher eukaryotes, including humans. Although we observed two sub-domain clusters of reference species within Eukaryota, co-inheritance analysis within these sub-domain taxonomic groups only marginally improved network inference. Therefore, we conclude that co-inheritance analysis within domains is the optimal approach to network inference with the given reference species. The construction of a series of human gene networks with increasing sample sizes of the reference species for each domain revealed that the size of the high-accuracy networks increased as additional reference species genomes were included, suggesting that within-domain co-inheritance analysis will continue to expand human gene networks as genomes of additional species are sequenced. Taken together, we propose that co-inheritance analysis within the domains of life will greatly potentiate the use of the expected onslaught of sequenced genomes in the study of molecular pathways in higher eukaryotes.     

Genome profiling: a realistic solution for genotype-based identification of species

Species identification is the basis of Biology and has been carried out based on phenotype. Although some genes, such as that for 16S rRNA, have been used for species confirmation, identification of species based only on genotype has never been done before, although recent whole genome sequencing studies have demonstrated it to be possible in principle. However, it is evidently unrealistic for routine experiments of species identification. This paper clarifies that a very limited amount of information derived from a genome sequence is sufficient for identifying the species. It also proves that Genome Profiling [Nishigaki, K., Amano, N., and Takasawa, T. (1991) Chem. Lett. 1097-1100], TGGE analysis of random PCR products, can not only fulfill such requirements, but also serve as a universal method to analyze species. Thus, this compact technology can be used in many fields of biology, especially in microbe-related disciplines such as microbial ecology and epidemiology where exact knowledge about all members of a population is essential but previously difficult to obtain. This is the first demonstration that genotype-based identification of species is possible using a simple and uniform protocol for all organisms.     

Integration of metabolomics and proteomics in molecular plant physiology--coping with the complexity by data-dimensionality reduction

In recent years, genomics has been extended to functional genomics. Toward the characterization of organisms or species on the genome level, changes on the metabolite and protein level have been shown to be essential to assign functions to genes and to describe the dynamic molecular phenotype. Gas chromatography (GC) and liquid chromatography coupled to mass spectrometry (GC- and LC-MS) are well suited for the fast and comprehensive analysis of ultracomplex metabolite samples. For the integration of metabolite profiles with quantitative protein profiles, a high throughput (HTP) shotgun proteomics approach using LC-MS and label-free quantification of unique proteins in a complex protein digest is described. Multivariate statistics are applied to examine sample pattern recognition based on data-dimensionality reduction and biomarker identification in plant systems biology. The integration of the data reveal multiple correlative biomarkers providing evidence for an increase of information in such holistic approaches. With computational simulation of metabolic networks and experimental measurements, it can be shown that biochemical regulation is reflected by metabolite network dynamics measured in a metabolomics approach. Examples in molecular plant physiology are presented to substantiate the integrative approach.