Transposon signatures: species-specific molecular markers that utilize a class of multiple-copy nuclear DNA

Transposable elements are mobile sequences found in nuclear genomes and can potentially serve as molecular markers in various phylogenetic and population genetic investigations. A PCR-based method that utilizes restriction site variation of element copies within a genome is developed. These patterns of site variation, referred to as transposon signatures, are useful in differentiating between closely related groups. Signature data using the magellan retrotransposon, for example, is useful in examining relationships within the genus Zea and Tripsacum. This method allows transposable elements, or even other multiple-copy nuclear DNA sequences, to be generally utilized as molecular markers in discriminating between other closely related species and subspecies.     

A mobile element based phylogeny of Old World monkeys

SINEs (Short INterspersed Elements) are a class of non-autonomous mobile elements that are <500 bp in length and have no open reading frames. Individual SINE elements are essentially homoplasy free with known ancestral states, making them useful genetic systems for phylogenetic studies. Alu elements are the most successful SINE in primate genomes and have been utilized for resolving primate phylogenetic relationships and human population genetics. However, no Alu based phylogenetic analysis has yet been performed to resolve relationships among Old World monkeys. Using both a computational approach and polymerase chain reaction display methodology, we identified 285 new Alu insertions from sixteen Old World monkey taxa that were informative at various levels of catarrhine phylogeny. We have utilized these elements along with 12 previously reported loci to construct a phylogenetic tree of the selected taxa. Relationships among all major clades are in general agreement with other molecular and morphological data sets but have stronger statistical support.     

Development, characterisation, and across-taxa utility of oil palm (Elaeis guineensis Jacq.) microsatellite markers.

The results of the development of oil palm (Elaeis guineensis Jacq.) microsatellite markers are given step by step, from the screening of libraries enriched in (GA)n, (GT)n, and (CCG)n simple-sequence repeats (SSRs) to the final characterisation of 21 SSR loci. Also published are primer sequences, estimates of allele size range, and expected heterozygosity in E. guineensis and in the closely related species E. oleifera, in which an optimal utility of the SSR markers was observed. Multivariate data analyses showed the ability of SSR markers to efficiently reveal the genetic-diversity structure of the genus Elaeis in accordance with known geographical origins and with measured genetic relationships based on previous molecular studies. High levels of allelic variability indicated that E. guineensis SSRs will be a powerful tool for genetic studies of the genus Elaeis, including variety identification and intra- or inter-specific genetic mapping. PCR amplification tests on a subset of 16 other palm species and allele-sequence data showed that E. guineensis SSRs are putative transferable markers across palm taxa. In addition, phenetic information based on SSR flanking region sequences makes E. guineensis SSR markers a potentially useful molecular resource for any researcher studying the phylogeny of palm taxa.     

Horny sponges and their affairs: on the phylogenetic relationships of keratose sponges

The demosponge orders Dictyoceratida and Dendroceratida are historically assigned to the keratose (or "horny") sponges, which are mostly devoid of primary skeletal elements, but possess an elaborate skeleton of organic fibres instead. This paucity of complex mineral skeletal elements makes their unambiguous classification and phylogenetic reconstruction based on morphological features difficult. Here we present the most comprehensive molecular phylogeny to date for the Dendroceratida, Dictyoceratida, and also other sponge orders that largely lack a mineral skeleton or skeletal elements at all (i.e. Verongida, Halisarcida, Chondrosida), based on independent mitochondrial and nuclear markers. We used molecular data to validate the coherence of all recognised orders, families and subfamilies that are currently defined using morphological characteristics. We discussed the significance of morphological and chemotaxonomic characters for keratose sponges, and suggested adapted definitions for the classification of dendroceratid, dictyoceratid, and verongid higher taxa. Also, we found that chondrosid sponges are non-monophyletic with respect to Halisarcida. Verongida and Dendroceratida were monophyletic, however most of their classically recognised families were not recovered. This indicated that the current distinction between dendritic and mesh-like fibre skeletons is not significant at this level of classification. Dysideidae were found to be the sister-group to the remaining Dictyoceratida. Irciniidae formed a distinct clade, however Thorectidae and Spongiidae could not be separated with the molecular markers used. Finally, we are establishing the name Verongimorpha for the clade combining verongid, chondrosid and halisarcid taxa and readjust the content of its sister-clade Keratosa.     

Prophage Carriage and Diversity within Clinically Relevant Strains of Clostridium difficile

Prophages are encoded in most genomes of sequenced Clostridium difficile strains. They are key components of the mobile genetic elements and, as such, are likely to influence the biology of their host strains. The majority of these phages are not amenable to propagation, and therefore the development of a molecular marker is a useful tool with which to establish the extent and diversity of C. difficile prophage carriage within clinical strains. To design markers, several candidate genes were analyzed including structural and holin genes. The holin gene is the only gene present in all sequenced phage genomes, conserved at both terminals, with a variable mid-section. This allowed us to design two sets of degenerate PCR primers specific to C. difficile myoviruses and siphoviruses. Subsequent PCR analysis of 16 clinical C. difficile ribotypes showed that 15 of them are myovirus positive, and 2 of them are also siphovirus positive. Antibiotic induction and transmission electron microscope analysis confirmed the molecular prediction of myoviruses and/or siphovirus presence. Phylogenetic analysis of the holin sequences identified three groups of C. difficile phages, two within the myoviruses and a divergent siphovirus group. The marker also produced tight groups within temperate phages that infect other taxa, including Clostridium perfringens, Clostridium botulinum, and Bacillus spp., which suggests the potential application of the holin gene to study prophage carriage in other bacteria. This study reveals the high incidence of prophage carriage in clinically relevant strains of C. difficile and correlates the molecular data to the morphological observation.     

A Molecular Phylogeny of the Tiger Beetles (Cicindelidae): Congruence of Mitochondrial and Nuclear rDNA Data Sets

As a basis for comparative studies in the family Cicindelidae (tiger beetles), a phylogenetic hypothesis for major groups was established based on molecular data, focusing on the taxa of the New World. Two markers, mitochondrial 16S rRNA and nuclear 18S rRNA, were sequenced for a total of 912 nucleotides and 30 taxa. All Cicindelidae were inferred to be a monophyletic group by the presence of a segment in the 5′ region of the 18S rDNA which is predicted to fold into a single stem-loop structure not present in the outgroup. The total number of variable and potentially informative positions was smaller in the 18S data set, but their higher internal consistency resulted in a strong phylogenetic signal. Inferred relationships were stable over a wide range of alignment parameters, but the inclusion of alignment-sensitive positions and presumptive gap characters added substantially to the resolution and overall support of the tree. Both data sets were in general congruence. The combined evidence suggests that relationships in Cicindelidae as currently recognized have to be reconsidered. No convincing evidence could be found for the traditional subdivision into two subfamilies, Cicindelinae and Collyrinae. The latter is embedded within the Cicindelinae and clearly not the sister to all other cicindelids. Most of the current tribes and subtribes, particularly in the Megacephalini, are paraphyletic groups, whereas monophyly of the large tribe Cicindelini is well supported by the molecular data.     

Mobile element-based forensic genomics

Mobile elements are commonly referred to as selfish repetitive DNA sequences. However, mobile elements represent a unique and underutilized group of molecular markers. Several of their characteristics make them ideally suited for use as tools in forensic genomic applications. These include their nature as essentially homoplasy-free characters, they are identical by descent, the ancestral state of any insertion is known to be the absence of the element, and many mobile element insertions are lineage specific. In this review, we provide an overview of mobile element biology and describe the application of certain mobile elements, especially the SINEs and other retrotransposons, to forensic genomics. These tools include quantitative species-specific DNA detection, analysis of complex biomaterials, and the inference of geographic origin of human DNA samples.     

反转录转座子标记及在作物遗传育种中的应用

反转录转座子通过RNA中间体进行反转录而转座,广泛分布于各种植物基因组中,拷贝数多,异质性高,在种内和种间表现出较高的序列差异性和丰富的插入多态性。针对这些特点,开发出了几种基于反转录转座子的分子标记,如SSAP、RIVPI、RAP、REMAP和RBIP等。由于反转录转座子标记能揭示出丰富的多态性,因而在遗传多样性和系谱研究、遗传连锁图谱构建及性状基因定位等方面得到了应用。随着分离技术的不断改进,获取序列信息更加容易,反转录转座子作为分子标记用于作物遗传育种将具有广阔前景。     

Inferring genome-scale rearrangement phylogeny and ancestral gene order: a <Emphasis Type="Italic">Drosophila</Emphasis>case study

A simple, fast, and biologically inspired computational approach for inferring genome-scale rearrangement phylogeny and ancestral gene order has been developed. This has been applied to eight Drosophila genomes. Existing techniques are either limited to a few hundred markers or a small number of taxa. This analysis uses over 14,000 genomic loci and employs discrete elements consisting of pairs of homologous genetic elements. The results provide insight into evolutionary chromosomal dynamics and synteny analysis, and inform speciation studies.     

Increased taxon sampling provides new insights into the phylogeny and evolution of the subclass Calcaronea (Porifera,Calcarea)

Calcaronean sponges are acknowledged to be taxonomically difficult, and generally, molecular data does not support the current morphology-based classification. In addition, molecular markers that have been successfully employed in other sponge taxa (e.g., COI mtDNA) have proven challenging to amplify due to the characteristics of calcarean mitochondrial genomes. A short fragment of the 28S rRNA gene (C-region) was recently proposed as the most phylogenetically informative marker to be used as a DNA barcode for calcareous sponges. In this study, the C-region and a fragment of the 18S rRNA gene were sequenced for a wide range of calcareous taxa, mainly from the subclass Calcaronea. The resulting dataset includes the most comprehensive taxon sampling of Calcaronea to date, and the inclusion of multiple specimens per species allowed us to evaluate the performance of both markers, as barcoding markers. 18S proved to be highly conserved within Calcaronea and does not have sufficient signal to resolve phylogenetic relationships within the subclass. Although the C-region does not exhibit a “proper” barcoding gap, it provides good phylogenetic resolution for calcaronean sponges. The resulting phylogeny supports previous findings that the current classification of the subclass Calcaronea is highly artificial, and with high levels of homoplasy. Furthermore, the close relationship between the order Baerida and the genus Achramorpha suggest that the order Baerida should be abandoned. Although the C-region currently represents the best available marker for phylogenetic and barcoding studies in Calcaronea, it is necessary to develop additional molecular markers to improve the classification within this subclass.     

Development of an efficient retrotransposon-based fingerprinting method for rapid pea variety identification

Fast and efficient DNA fingerprinting of crop cultivars and individuals is frequently used in both theoretical population genetics and in practical breeding. Numerous DNA marker technologies exist and the ratio of speed, cost and accuracy are of importance. Therefore even in species where highly accurate and polymorphic marker systems are available, such as microsatellite SSR (simple sequence repeats), also alternative methods may be of interest. Thanks to their high abundance and ubiquity, temporary mobile retrotransposable elements come into recent focus. Their properties, such as genome wide distribution and well-defined origin of individual insertions by descent, predetermine them for use as molecular markers. In this study, several Ty3-gypsy type retrotransposons have been developed and adopted for the inter-retrotransposon amplified polymorphism (IRAP) method, which is suitable for fast and efficient pea cultivar fingerprinting. The method can easily distinguish even between genetically closely related pea cultivars and provide high polymorphic information content (PIC) in a single PCR analysis.     

PATTERNS OF VARIATION IN LEVELS OF HOMOPLASY

Patterns of variation in levels of homoplasy were explored through statistical analyses of standardized consistency indexes. Data were obtained from 60 recent cladistic analyses of a wide variety of organisms based on several different kinds of characters. Consistency index is highly correlated with the number of taxa included in an analysis, with homoplasy increasing as the number of taxa increases. This observation is compatible with a simple model of character evolution in which 1) the probability of character-state change increases with the total number of branches in a tree and 2) the number of possible states of a character is limited. Consistency index does not show a significant relationship to the number of characters utilized in an analysis or to the taxonomic rank of the terminal taxa. When the relationship between consistency index and number of taxa is taken into account, there is no significant difference between plant and animal data sets in the amount of homoplasy. Likewise, the level of homoplasy in morphological and molecular data sets does not appear to differ significantly, although there are still too few molecular studies to be confident of this result. Future comparisons of consistency indexes, including studies along the lines established here, must take into account the influence of the number of taxa on homoplasy.     

Conserved flanking microsatellite sequences (ReFS) differentiate between Lepidoptera species,and provide insight into microsatellite evolution

High rates of mutation and homoplasy mean that microsatellites generally are not considered to be useful molecular markers for inferring systematic relationships between species. However, an earlier pilot study suggested that conserved flanking microsatellite sequences, also known as repetitive flanking sequences (ReFS), may form a basis for a dominant marker that can differentiate between species of Lepidoptera. We present data that demonstrate that ReFS are quick and easy to use, and generate highly repeatable banding patterns from a range of Lepidoptera species. Sequence data from a subset of ReFS‐amplified bands revealed microsatellite families with flanking sequences that are more conserved within than among species: this is probably attributable to recombination‐mediated events, transposition of mobile elements or a combination of the two. Our data support the use of ReFS as dominant interspecific molecular markers, and add to the growing literature on the evolution of microsatellites in Lepidoptera.     

Phylogenetic and taxonomic incongruence involving nuclear and mitochondrial markers in Korean populations of the freshwater snail genus Semisulcospira (Cerithioidea: Pleuroceridae)

We investigated the population genetic structure and phylogenetic relationships of four morphospecies of Semisulcospira sampled from multiple South Korean drainages. One, S. extensa, displayed modest levels of genetic diversity and formed a species-specific clade for both mitochondrial (mt) and nuclear markers. In contrast, the other three, S. coreana, S. gottschei, and S. libertina, were polyphyletic for both mt and nuclear markers. They formed, together with other nominal congeners (S. forticosta, S. multicincta, S. nodiperda, and S. tegulata), a taxonomically heterogeneous species complex containing population-level admixtures of genotypes from numerically predominant modal clades in addition to rare, phylogenetically divergent, mt and nuclear genotypes. The modal mt haplotypes exhibited far more geographic than taxonomic congruence and typically co-clustered into drainage-specific tip clades, irrespective of nominal taxonomic status. The evolutionary origins of the observed non-extensa phylogenetic heterogeneity are unclear at present although the available data do not support paralogous mt marker phenomena or the presence of cryptic species. We cannot distinguish among retention of ancestral polymorphisms or recticulate evolutionary origins as explanatory mechanisms and it may require the genetic characterization of Semisulcospira lineages throughout much of their collective east Asian range to address this issue. Based on the available data, we consider it best to view all of our non-extensa Korean study taxa as a single polymorphic species complex of the type species S. libertina. There is considerable evidence that similarly complex genetic structuring, at least for mt, may be typical of many other freshwater cerithioidean snail taxa. In light of our Semisulcospira results, we suggest that meaningful molecular phylogenetic characterization of freshwater cerithioidean lineages may require the use of both mt and nuclear markers together with population level sampling of all nominal taxa within regional drainages.     

Nonanchored Inter Simple Sequence Repeat (ISSR) markers: Reproducible and specific tools for genome fingerprinting

Many molecular marker techniques are available today. PCR-based approaches are in demand because of their simplicity and requirement for only small quantities of sample DNA. Nonanchored inter simple sequence repeats (ISSRs) are arbitrary multiloci markers produced by PCR amplification with a microsatellite primer. They are advantageous because no prior genomic information is required for their use. We found the technique stable across a wide range of PCR parameters. Polymorphisms were abundant among 7 dicot species tested with 2 tri-nucleotide and 2 tetra-nucleotide primers. Thus, nonanchored ISSR markers are a good choice for DNA fingerprinting.     

The division "Synergistes"

The "Synergistes" group of organisms are a phylogenetic cluster of Gram-negative anaerobes related to Synergistes jonesii, sufficiently distinct from all other phyla to be considered a distinct phylum or Division. They are widely distributed in nature although normally only a minor constituent of the bacterial community in each habitat. They have evolved to adapt to each habitat, and therefore exhibit a wide range of physiological and biochemical characteristics, although all cultivable taxa so far studied have the ability to degrade amino acids. They are found in the human mouth where they appear to be more numerous in tooth and gum disease than health. They have also been found in the human gut and soft tissue infections. Their role in human disease has yet to be established but improved knowledge of the characteristics that enable their identification should increase the likelihood of their recognition when present at diseased sites.     

Drosophila genomes and the development of affordable molecular markers for species genotyping

The recent completion of genome sequencing of 12 species of Drosophila has provided a powerful resource for hypothesis testing, as well as the development of technical tools. Here we take advantage of genome sequence data from two closely related species of Drosophila, Drosophila simulans and Drosophila sechellia, to quickly identify candidate molecular markers for genotyping based on expected insertion or deletion (indel) differences between species. Out of 64 candidate molecular markers selected along the second and third chromosome of Drosophila, 51 molecular markers were validated using PCR and gel electrophoresis. We found that the 20% error rate was due to sequencing errors in the genome data, although we cannot rule out possible indel polymorphisms. The approach has the advantage of being affordable and quick, as it only requires the use of bioinformatics tools for predictions and a PCR and agarose gel based assay for validation. Moreover, the approach could be easily extended to a wide variety of taxa with the only limitation being the availability of complete or partial genome sequence data.     

Evidence for the Polyphyly of Haworthia (Asphodelaceae Subfamily Alooideae; Asparagales) Inferred from Nucleotide Sequences of rbcL, matK, ITS1 and Genomic Fingerprinting with ISSR-PCR

Abstract: Four molecular markers have been studied to examine the phylogenetic position of the South African plant genus Haworthia Duval within the succulent Asphodelaceae. Sequence data of the chloroplast genes mat K and rbc L were compared to the nuclear markers ITS1 and ISSR (Inter Simple Sequence Repeat) analysis. Both lines of molecular data, chloroplast and nuclear DNA, indicate that Haworthia is polyphyletic, forming two distinct clades. Most taxa previously combined as Haworthia subgenus Haworthia branch off early in the alooid chloroplast trees forming a strongly monophyletic group, whereas subgenus Hexangulares forms a polyphyletic assemblage comprising other alooid genera. The nuclear markers ITS1 and ISSR fingerprinting support the two groups as distinctly different, therefore confirming the division seen in chloroplast DNA. The practical implication is that the generic concept of Haworthia may have to be restricted to H. subgenus Haworthia or alternatively, that the groups of Haworthia be treated as infrageneric taxa within a broadened (Linnaean) concept of Aloe.     

On the distinction of Dactylorhiza baltica and D. pardalina (Orchidaceae) and the systematic affinities of geographically intermediate populations

The eastern European Dactylorhiza baltica (Klinge) N. I. Orlova and the western European D. pardalina (Pugsl.) Aver. (= D. praetermissa var. junialis (Verm.) Sengh) are usually considered to have non-overlapping geographic distributions, for which reason it has rarely been realized that they are morphologically similar. They have not previously been thoroughly compared by molecular methods, and no existing flora or revision has convincingly demonstrated that they can be distinguished by morphological characters. In reality, they might be 'political' rather than natural taxa. Prompted by the recent discovery of geographically intermediate populations (in eastern Denmark), originally identified as D. baltica , we have addressed this problem by analysis of morphometric data as well as molecular data from allozyme markers, plastid haplotypes, nuclear ITS alleles and nuclear microsatellites. Dactylorhiza baltica and D. pardalina turned out to be clearly distinguished genetically, and although they are morphologically similar, a few characters were identified that distinguish with 81–85% certainty between the two taxa. Molecular and morphometric data place the geographically intermediate populations in D. pardalina . Both taxa were confirmed to be allotetraploids combining diploid genomes from the D. incarnata s.l. and D. maculata s.l. lineages, and they should therefore be recognized as infraspecic taxa under D. majalis s.l. Thus, D. baltica should be called D. majalis subsp. baltica ; D. pardalina is identical with D. praetermissa var. junialis , but the nomenclatural consequences for D. praetermissa , if treated as subspecies under D. majalis , are still unresolved.     

An improved method for the identification of areas of endemism using species co-occurrences

Aim To develop a simple method that (1) combines the notions of biotic elements (groups of taxa with ranges significantly more similar to each other than to the ranges of other taxa) and of areas of endemism (AoE, areas of non‐random distributional congruence among taxa), and (2) overcomes the constraints of a previously suggested null model‐based method that cannot deal with disjunctions and is strictly grid‐dependent. Location We used test data sets from southern Africa and Crete. Methods First, we used a null‐model approach to detect pairs of species that have a significant degree of co‐occurrence, in order to determine biotic elements. Subsequently, we used a parsimony analysis of endemicity to delineate candidate AoE, and multivariate analysis to define groups of biotic elements on the basis of species interactions (co‐occurrence, mutual exclusion, neutral) using only the species detected in the previous step. We applied this method to the well known data set for Sciobius in southern Africa, as well as to endemic invertebrates of Crete (Greece), in order to evaluate its performance. Results Our results are very similar to those of previous analyses, and produce meaningful delineation of AoE and biotic elements in both data sets. The method is flexible regarding null models and significance levels, and eliminates noise in the data. Main conclusions We offer a simple method that provides reasonable identification of both biotic elements and AoE, produces good‐fit statistics, reduces uninformative or junk output, and reduces computational time.