Circulating microRNAs in the identification of biological fluids: A new approach to standardization of expression-based diagnostics

Molecular profiling of normal tissues is a regular and necessary step when developing systems for expression analyses in biological samples, including diagnostic panels for various diseases and conditions. Yet there are still no rigorous criteria to allow precise typing of normal tissues. A main problem is that the methods employed in diagnostic expression testing are difficult to standardize. While various technologies, instruments, and reagents are available, universal protocols of handling biological material are lacking, thus impairing the reproducibility of data from independent studies. The review describes a new approach to standardizing circulating microRNA studies in forensic biology, which has relatively recently (7–8 years ago) come to employ RNA markers in molecular typing of tissues and biological fluids. Forensic biology is now one of the few disciplines where several panels of tissue mRNA markers have been developed within a short period of time and a number of specific microRNA markers have been established and validated for several biological fluids. To allow their successful use, new protocols have been combined with the available, rigidly standardized system of genetic personal identification. Although a ready diagnostic product has still not been obtained with this well-working approach, the apparent efficiency of the standardization methods clearly demonstrates that the problem is possible to solve in other biomedical fields, including those where RNA-based diagnostic protocols are still under development.     

Genetic identification of missing persons: DNA analysis of human remains and compromised samples

Human identification has made great strides over the past 2 decades due to the advent of DNA typing. Forensic DNA typing provides genetic data from a variety of materials and individuals, and is applied to many important issues that confront society. Part of the success of DNA typing is the generation of DNA databases to help identify missing persons and to develop investigative leads to assist law enforcement. DNA databases house DNA profiles from convicted felons (and in some jurisdictions arrestees), forensic evidence, human remains, and direct and family reference samples of missing persons. These databases are essential tools, which are becoming quite large (for example the US Database contains 10 million profiles). The scientific, governmental and private communities continue to work together to standardize genetic markers for more effective worldwide data sharing, to develop and validate robust DNA typing kits that contain the reagents necessary to type core identity genetic markers, to develop technologies that facilitate a number of analytical processes and to develop policies to make human identity testing more effective. Indeed, DNA typing is integral to resolving a number of serious criminal and civil concerns, such as solving missing person cases and identifying victims of mass disasters and children who may have been victims of human trafficking, and provides information for historical studies. As more refined capabilities are still required, novel approaches are being sought, such as genetic testing by next-generation sequencing, mass spectrometry, chip arrays and pyrosequencing. Single nucleotide polymorphisms offer the potential to analyze severely compromised biological samples, to determine the facial phenotype of decomposed human remains and to predict the bioancestry of individuals, a new focus in analyzing this type of markers.     

Comparison of Biochemical Polymorphisms and Short Tandem Repeat (STR) DNA Markers for Paternity Testing in Rhesus Monkeys (Macaca mulatta)

Genetic markers are indispensable for molecular and statistical genetic research involving nonhuman primates. Genetic markers must be used to ascertain parentage and to confirm the accuracy of pedigrees based solely on housing or demographic records; otherwise, the results of pedigree, linkage, or quantitative genetic analyses may be unreliable. Until recently, most genetic markers used in nonhuman primates were plasma proteins or isozyme polymorphisms, which were required in large numbers, because levels of genetic variation revealed by these markers were rather low. We compared the newer, PCR-amplified short tandem repeat markers (STRs) with a panel of classical biochemical polymorphic markers, for paternity determination among captive-bred rhesus monkeys. The STR markers exhibited an average genetic diversity of 64% and an expected paternity exclusion probability of 0.443. Both of these were greater than the average 54.5% genetic diversity and 0.298 exclusion probability exhibited by the biochemical markers. The STRs were much more efficient than the biochemical markers for parentage determination, since they required only half the amount of genetic typing data to resolve an average paternity case. Thus, the results of applying these two classes of genetic markers in paternity tests were somewhat different than expected on the basis of theoretical exclusion probabilities. These differences were probably due to inbreeding and other genetic differences among breeding colonies. Because they are more informative and provide rapid and efficient genetic data, STRs are now the method of choice for parentage determination and pedigree corroboration among nonhuman primates.     

A high density genetic map of tobacco (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L.) obtained from large scale microsatellite marker development

Tobacco (Nicotiana tabacum L.) is a species in the large family of the Solanaceae and is important as an agronomic crop and as a model system in plant biotechnology. Despite its importance, only limited molecular marker resources are available that can be used for genome analysis, genetic mapping and breeding. We report here on the development and characterization of 5,119 new and functional microsatellite markers and on the generation of a high-resolution genetic map for the tetraploid tobacco genome. The genetic map was generated using an F2 mapping population derived from the intervarietal cross of Hicks Broadleaf × Red Russian and merges the polymorphic markers from this new set with those from a smaller set previously used to produce a lower density map. The genetic map described here contains 2,317 microsatellite markers and 2,363 loci, resulting in an average distance between mapped microsatellite markers which is less than 2 million base pairs or 1.5 cM. With this new and expanded marker resource, a sufficient number of markers are now available for multiple applications ranging from tobacco breeding to comparative genome analysis. The genetic map of tobacco is now comparable in marker density and resolution with the best characterized genomes of the Solanaceae: tomato and potato.     

The Polynesian gene pool: an early contribution by Amerindians to Easter Island

It is now generally accepted that Polynesia was first settled by peoples from southeast Asia. An alternative that eastern parts of Polynesia were first inhabited by Amerindians has found little support. There are, however, many indications of a 'prehistoric' (i.e. before Polynesia was discovered by Europeans) contact between Polynesia and the Americas, but genetic evidence of a prehistoric Amerindian contribution to the Polynesian gene pool has been lacking. We recently carried out genomic HLA (human leucocyte antigen) typing as well as typing for mitochondrial DNA (mtDNA) and Y chromosome markers of blood samples collected in 1971 and 2008 from reputedly non-admixed Easter Islanders. All individuals carried HLA alleles and mtDNA types previously found in Polynesia, and most of the males carried Y chromosome markers of Polynesian origin (a few had European Y chromosome markers), further supporting an initial Polynesian population on Easter Island. The HLA investigations revealed, however, that some individuals also carried HLA alleles which have previously almost only been found in Amerindians. We could trace the introduction of these Amerindian alleles to before the Peruvian slave trades, i.e. before the 1860s, and provide suggestive evidence that they were introduced already in prehistoric time. Our results demonstrate an early Amerindian contribution to the Polynesian gene pool on Easter Island, and illustrate the usefulness of typing for immunogenetic markers such as HLA to complement mtDNA and Y chromosome analyses in anthropological investigations.     

Genetic transformation in Aspergilli: tools of the trade

DNA-mediated transformation is a powerful tool that allows the introduction of specific genetic changes in an organism. Transformation of Aspergilli, acclaimed for their wide use in the industry, has been possible for about two decades now. Several basic and applied problems related to fungal biology have been addressed using this technique. Nonetheless, new markers and strategies for transformation are still being developed for these filamentous fungi. Different methods and markers that are currently available for the transformation of Aspergilli are summarized here. The review also brings out the importance of these transformation systems in analyzing fungal gene function. Aspects of Aspergillus niger transformation are selectively emphasized.     

Unexpected genetic diversity of Mycoplasma agalactiae caprine isolates from an endemic geographically restricted area of Spain

ABSTRACT: BACKGROUND: The genetic diversity of Mycoplasma agalactiae (MA) isolates collected in Spain from goats in an area endemic with contagious agalactia (CA) was assessed using a set of validated and new molecular typing methods. Validated methods included pulsed field gel electrophoresis (PFGE), variable number of tandem repeats (VNTR) typing, and Southern blot hybridization using a set of MA DNA probes, including those for typing the vpma genes repertoire. New approaches were based on PCR and targeted genomic regions that diverged between strains as defined by in silico genomic comparisons of sequenced MA genomes. RESULTS: Overall, the data showed that all typing tools yielded consistent results, with the VNTR analyses being the most rapid method to differentiate the MA isolates with a discriminatory ability comparable to that of PFGE and of a set of new PCR assays. All molecular typing approaches indicated that the Spanish isolates from the endemic area in Murcia were very diverse, with different clonal isolates probably restricted to separate, but geographically close, local areas. CONCLUSIONS: The important genetic diversity of MA observed in infected goats from Spain contrasts with the overall homogeneity of the genomic background encountered in MA from sheep with CA in Southern France or Italy, suggesting that assessment of the disease status in endemic areas may require different approaches in sheep and in goats. A number of congruent sub-typing tools are now available for the differentiation of caprine isolates with comparable discriminatory powers.     

Sibship reconstruction from genetic data with typing errors

Likelihood methods have been developed to partition individuals in a sample into full-sib and half-sib families using genetic marker data without parental information. They invariably make the critical assumption that marker data are free of genotyping errors and mutations and are thus completely reliable in inferring sibships. Unfortunately, however, this assumption is rarely tenable for virtually all kinds of genetic markers in practical use and, if violated, can severely bias sibship estimates as shown by simulations in this article. I propose a new likelihood method with simple and robust models of typing error incorporated into it. Simulations show that the new method can be used to infer full- and half-sibships accurately from marker data with a high error rate and to identify typing errors at each locus in each reconstructed sib family. The new method also improves previous ones by adopting a fresh iterative procedure for updating allele frequencies with reconstructed sibships taken into account, by allowing for the use of parental information, and by using efficient algorithms for calculating the likelihood function and searching for the maximum-likelihood configuration. It is tested extensively on simulated data with a varying number of marker loci, different rates of typing errors, and various sample sizes and family structures and applied to two empirical data sets to demonstrate its usefulness.     

A novel multilocus sequence typing scheme identifying genetic diversity amongst Leishmania donovani isolates from a genetically homogeneous population in the Indian subcontinent

In the Indian subcontinent, infection with Leishmania donovani can cause fatal visceral leishmaniasis. Genetic variation in L. donovani is believed to occur rapidly from environmental changes and through selective drug pressures, thereby allowing continued disease occurrence in this region. All previous molecular markers that are commonly in use multilocus microsatellite typing and multilocus sequence typing, were monomorphic in L. donovani originating from the Indian subcontinent (with only a few exceptions) and hence are not suitable for this region. An multilocus sequence typing scheme consisting of a new set of seven housekeeping genes was developed in this study, based on recent findings from whole genome sequencing data. This new scheme was used to assess the genetic diversity amongst 22 autochthonous L. donovani isolates from Bangladesh. Nineteen additional isolates of the L. donovani complex (including sequences of L. donovani reference strain BPK282A1) from other countries were included for comparison. By using restriction fragment length polymorphism of the internal transcribed spacer 1 region (ITS1-RFLP) and ITS1 sequencing, all Bangladeshi isolates were confirmed to be L. donovani. Population genetic analyses of 41 isolates using the seven new MLST loci clearly separated L. donovani from Leishmania infantum. With this multilocus sequence typing scheme, seven genotypes were identified amongst Bangladeshi L. donovani isolates, and these isolates were found to be phylogenetically different compared with those from India, Nepal, Iraq and Africa. This novel multilocus sequence typing approach can detect intra- and inter-species variations within the L. donovani complex, but most importantly these molecular markers can be applied to resolve the phylogenetically very homogeneous L. donovani strains from the Indian subcontinent. Four of these markers were found suitable to differentiate strains originating from Bangladesh, with marker A2P being the most discriminative one.     

Development and mapping of SSR markers for maize

Microsatellite or simple sequence repeat (SSR) markers have wide applicability for genetic analysis in crop plant improvement strategies. The objectives of this project were to isolate, characterize, and map a comprehensive set of SSR markers for maize (Zea mays L.). We developed 1051 novel SSR markers for maize from microsatellite-enriched libraries and by identification of microsatellite-containing sequences in public and private databases. Three mapping populations were used to derive map positions for 978 of these markers. The main mapping population was the intermated B73 × Mo17 (IBM) population. In mapping this intermated recombinant inbred line population, we have contributed to development of a new high-resolution map resource for maize. The primer sequences, original sequence sources, data on polymorphisms across 11 inbred lines, and map positions have been integrated with information on other public SSR markers and released through MaizeDB at URL:www.agron.missouri.edu. The maize research community now has the most detailed and comprehensive SSR marker set of any plant species.     

Host-pathogen interactions revealed by human genome-wide surveys

Genome-wide association studies (GWAS) have now convincingly shown that the diverse outcomes (such as the resolution of infection, clinical deterioration to severe disease, or progression from acute infection to persistent infection) that occur following microbial infection can be at least partly explained by human genetic variation. Unbiased whole-genome approaches have revealed unprecedentedly robust associations between genetic markers and susceptibility to disease, providing clear insights into our understanding of infectious disease biology by revealing the crucial host-pathogen interaction sites. Further work characterizing both the host causative variations and pathogenic microbial strains with distinct host interactions and disease outcomes is now required to provide potential new intervention strategies.     

Microsatellite analysis of Toxoplasma gondii shows considerable polymorphism structured into two main clonal groups.

Previous studies on Toxoplasma gondii population structure, based essentially on multilocus restriction fragment length polymorphism analysis or on multilocus enzyme electrophoresis, indicated that T. gondii comprises three clonal lineages. These studies showed a weak polymorphism of the markers (2-4 alleles by locus). In this study, we used eight microsatellite markers to type 84 independent isolates from humans and animals. Two microsatellite markers were present in the introns of two genes, one coding for beta-tubulin and the other for myosin A, and six were found in expressed sequence tags. With 3-16 alleles detected, these markers can be considered as the most discriminating multilocus single-copy markers available for typing T. gondii isolates. This high discriminatory power of microsatellites made it possible to detect mixed infections and epidemiologically related isolates. Evolutionary genetic analyses of diversity show that the T. gondii population structure consists of only two clonal lineages that can be equated to discrete typing units, but there is some evidence of occasional genetic exchange that could explain why one of these discrete typing units is less clearly individualised than the other.     

A microarray system for Y chromosomal and mitochondrial single nucleotide polymorphism analysis in chimpanzee populations

Chimpanzee populations are diminishing as a consequence of human activities, and as a result this species is now endangered. In the context of conservation programmes, genetic data can add vital information, for instance on the genetic diversity and structure of threatened populations. Single nucleotide polymorphisms (SNP) are biallelic markers that are widely used in human molecular studies and can be implemented in efficient microarray systems. This technology offers the potential of robust, multiplexed SNP genotyping at low reagent cost in other organisms than humans, but it is not commonly used yet in wild population studies. Here, we describe the characterization of new SNPs in Y-chromosomal intronic regions in chimpanzees and also identify SNPs from mitochondrial genes, with the aim of developing a microarray system that permits the simultaneous study of both paternal and maternal lineages. Our system consists of 42 SNPs for the Y chromosome and 45 SNPs for the mitochondrial genome. We demonstrate the applicability of this microarray in a captive population where genotypes accurately reflected its large pedigree. Two wild-living populations were also analysed and the results show that the microarray will be a useful tool alongside microsatellite markers, since it supplies complementary information about population structure and ecology. SNP genotyping using microarray technology, therefore, is a promising approach and may become an essential tool in conservation genetics to help in the management and study of captive and wild-living populations. Moreover, microarrays that combine SNPs from different genomic regions could replace microsatellite typing in the future.     

A high-resolution consensus linkage map of the rat, integrating radiation hybrid and genetic maps

We have constructed a high-resolution consensus genetic map of the rat in a single large intercross, which integrates 747 framework markers and 687 positions of our whole-genome radiation hybrid (RH) map of the rat. We selected 136 new gene markers from the GenBank database and assigned them either genetically or physically to rat chromosomes to evaluate the accuracy of the integrated linkage-RH maps in the localization of new markers and to enrich existing comparative mapping data. These markers and 631 D-Got- markers, which are physically mapped but still uncharacterized for evidence of polymorphism, were tested for allele variations in a panel of 16 rat strains commonly used in genetic studies. The consensus linkage map constructed in the GK x BN cross now comprises 1620 markers of various origins, defining 840 resolved genetic positions with an average spacing of 2.2 cM between adjacent loci, and includes 407 gene markers. This whole-genome genetic map will contribute to the advancement of genetic studies in the rat by incorporating gene/EST maps, physical mapping information, and sequence data generated in rat and other mammalian species into genetic intervals harboring disease susceptibility loci identified in rat models of human genetic disorders.     

Genetic markers for study of the anopheline vectors of human malaria

Human malaria is truly a disease of global proportions and is one of the most broadly distributed vector-borne infections. Anopheline mosquitoes are the exclusive vectors of human malaria. A handful of species predominate as the most notorious malaria vectors, but the species and forms involved in the transmission of human malaria world-wide are incredibly diverse. Many of the anophelines that vector malaria exist as members of species complexes that often contain vector and non-vector species. Additionally, single anopheline species often exhibit significant heterogeneity across the species' range. This phenotypic and genotypic plasticity exacerbates the difficulties in identification of vector populations and implementation of effective surveillance and control strategies. Polytene chromosome investigations were among the first to provide researchers with tangible genetic markers that could be used to differentiate between what are now recognised as species and chromosomal forms of anopheline mosquitoes. The advent of the polymerase chain reaction gave access to the molecular genetics of genomes and the techniques that followed have facilitated investigation of the genetics of individual specimens or population size samples. The variety and number of genetic markers available for the study of malaria vectors has literally exploded in the last 10 years. Markers have expanded from the 'traditional tools' to include a vast array of molecular markers. Contemporary markers range from what are now referred to as 'classical genetic markers' to methods used to detect and identify single nucleotide polymorphisms and finally to highly polymorphic markers. One of the greatest advantages of this wide variety of genetic markers is that researchers may choose to utilise any combination of markers or techniques to address multifaceted questions relating to malaria transmission. These molecular markers have proven useful in a wide variety of applications including molecular taxonomy, evolutionary systematics, population genetics, genetic mapping, and investigation of defined phenotypes.     

Conserving adaptive genetic diversity in dynamic landscapes

Genetic variation supplies the raw material for adaptation, evolution and survival of populations and has therefore been a key focus of conservation biology ever since its foundation (Soulé 1985). In previous decades, the neutral component of genetic diversity (generated by mutation and shaped by drift) has been the subject of intense scientific research, fuelled by the increasing availability of molecular markers. On the other hand, the adaptive component of genetic diversity, which is shaped by the action of natural selection, has long remained elusive and difficult to assess, especially at small spatial or temporal scales (Ouborg et al. 2010). Fortunately, new technological and methodological developments now make it possible to identify loci in the genome that are influenced by selection, and thus to get a more complete view of genetic diversity. One article featured in this issue of Molecular Ecology is a good example of this recent breakthrough. Richter-Boix et al. (2011) examined a network of moor frog populations breeding in contrasting habitats in order to understand how landscape features influence patterns of genetic variation. They combined information from both neutral markers and loci putatively under selection to quantify the relative roles of selection and isolation in the evolution of fine-scale local adaptations in these populations. This study nicely illustrates how data on polymorphisms of neutral and adaptive loci can now be judiciously synthesized to help identify the best strategies for preserving adaptive variation, and more generally to enlighten conservation and population-management plans.     

High-resolution typing of Toxoplasma gondii using microsatellite loci.

High-resolution typing of Toxoplasma gondii is essential to understand the effect of genetic differences among strains on the variation in disease manifestation and transmission patterns. Current typing methods discern 3 lineages with minimal within-lineage variation. Described here are 6 new variable loci. These loci, including a minisatellite and 5 microsatellites, were more polymorphic than allozymes, restriction fragment length polymorphisms, and sequence variation in introns. Most importantly, these loci revealed, for the first time, substantial within-lineage variation that was over 6-fold higher than that detected by other markers. Genotyping at these loci facilitates classification of isolates beyond the lineage level.