Design and evaluation of a panel os single-nucleotide polymorphisms in microRNA genomic regions for association studies in human disease

MicroRNAs (miRNA) are recognized posttranscriptional gene repressors involved in the control of almost every biological process. Allelic variants in these regions may be an important source of phenotypic diversity and contribute to disease susceptibility. We analyzed the genomic organization of 325 human miRNAs (release 7.1, miRBase) to construct a panel of 768 single-nucleotide polymorphisms (SNPs) covering approximately 1 Mb of genomic DNA, including 131 isolated miRNAs (40%) and 194 miRNAs arranged in 48 miRNA clusters, as well as their 5-kb flanking regions. Of these miRNAs, 37% were inside known protein-coding genes, which were significantly associated with biological functions regarding neurological, psychological or nutritional disorders. SNP coverage analysis revealed a lower SNP density in miRNAs compared with the average of the genome, with only 24 SNPs located in the 325 miRNAs studied. Further genotyping of 340 unrelated Spanish individuals showed that more than half of the SNPs in miRNAs were either rare or monomorphic, in agreement with the reported selective constraint on human miRNAs. A comparison of the minor allele frequencies between Spanish and HapMap population samples confirmed the applicability of this SNP panel to the study of complex disorders among the Spanish population, and revealed two miRNA regions, hsa-mir-26a-2 in the CTDSP2 gene and hsa-mir-128-1 in the R3HDM1 gene, showing geographical allelic frequency variation among the four HapMap populations, probably because of differences in natural selection. The designed miRNA SNP panel could help to identify still hidden links between miRNAs and human disease.

Phylogenetic relationships in the subfamily Psychodinae (Diptera, Psychodidae)

Thanks to recent advances in molecular systematics, our knowledge of phylogenetic relationships within the order Diptera has dramatically improved. However, relationships at lower taxonomic levels remain poorly investigated in several neglected groups, such as the highly diversified moth-fly subfamily Psychodinae (Lower Diptera), which occurs in numerous terrestrial ecosystems. In this study, we aimed to understand the phylogenetic relationships among 52 Palearctic taxa from all currently known Palearctic tribes and subtribes of this subfamily, based on mitochondrial DNA. Our results demonstrate that in light of the classical systematics of Psychodinae, none of the tribes sensu Je?ek or sensu Vaillant is monophyletic, whereas at least five of the 12 sampled genera were not monophyletic. The results presented in this study provide a valuable backbone for future work aiming at identifying morphological synapomorphies to propose a new tribal classification.

The dermatophyte species Arthroderma benhamiae: intraspecies variability and mating behaviour.

Arthroderma benhamiae is a zoophilic dermatophyte belonging to the Trichophyton mentagrophytes species complex. Here, a population of A. benhamiae wild strains from the same geographical area (Switzerland) was studied by comparing their morphology, assessing their molecular variability using internal transcribed spacer (ITS) and 28S rRNA gene sequencing, and evaluating their interfertility. Sequencing of the ITS region and of part of the 28S rRNA gene revealed the existence of two infraspecific groups with markedly different colony phenotypes: white (group I) and yellow (group II), respectively. For all strains, the results of mating type identification by PCR, using HMG (high-mobility group) and α-box genes in the mating type locus as targets, were in total accordance with the results of mating type identification by strain confrontation experiments. White-phenotype strains were of mating type + (mt+) or mating type - (mt-), whilst yellow-phenotype strains were all mt-. White and yellow strains were found to produce fertile cleistothecia after mating with A. benhamiae reference tester strains, which belonged to a third group intermediate between groups I and II. However, no interfertility was observed between yellow strains and white strains of mt+. A significant result was that white strains of mt- were able to mate and produce fertile cleistothecia with the white A. benhamiae strain CBS 112371 (mt+), the genome of which has recently been sequenced and annotated. This finding should offer new tools for investigating the biology and genetics of dermatophytes using wild-type strains.

A set of primers for plastid indels and nuclear microsatellites in the invasive plant Heracleum mantegazzianum (Apiaceae) and their transferability to Heracleum sphondylium

This study reports the isolation and polymorphism characterization of four plastid indels and six nuclear microsatellite loci in the invasive plant Heracleum mantegazzianum. These markers were tested in 27 individuals from two distant H. mantegazzianum populations. Plastid indels revealed the presence of five chlorotypes while five nuclear microsatellite loci rendered polymorphism. Applications of these markers include population genetics and phylogeography of H. mantegazzianum. A very good transferability of markers to Heracleum sphondylium was demonstrated.

Phylogenetic structures of the Holarctic Sorex araneus group and its relationships with S-samniticus, as inferred from mtDNA sequences

The shrews of the Sorex araneus group, characterized by the sexual chromosome complex XY1, Y2 have been intensively studied by morphological, karyotypical, and biochemical analyses. Nevertheless, the phylogenetic relationships among the species belonging to the araneus complex are still under debate, as different approaches gave often contradictory results. In this paper, partial nucleotide sequences of the mitochondrial DNA cytochrome b gene (1011 bp) were determined for 6 species of the araneus group from Eurasia and North America. We also included in the data set the sequences of Sorex samniticus, whose relationships with the araneus group remain controversial. Three other species representing two major karyological groups were also examined. Both parsimony and distance trees strongly support the monophyly of the araneus group. Sorex sumniticus is significantly more closely related to the araneus complex than to the other species included in the analysis. Based on the branching pattern within the araneus group, an attempt has been made to reconstruct the colonization history of the Holarctic region.

Evidence for the evolution of multiple genomes in arbuscular mycorrhizal fungi.

Ancient asexuals directly contradict the evolutionary theories that explain why organisms should evolve a sexual life history. The mutualistic, arbuscular mycorrhizal fungi are thought to have been asexual for approximately 400 million years. In the absence of sex, highly divergent descendants of formerly allelic nucleotide sequences are thought to evolve in a genome. In mycorrhizal fungi, where individual offspring receive hundreds of nuclei from the parent, it has been hypothesized that a population of genetically different nuclei should evolve within one individual. Here we use DNA-DNA fluorescent in situ hybridization to show that genetically different nuclei co-exist in individual arbuscular mycorrhizal fungi. We also show that the population genetics techniques used in other organisms are unsuitable for detecting recombination because the assumptions and underlying processes do not fit the fungal genomic structure shown here. Instead we used a phylogenetic approach to show that the within-individual genetic variation that occurs in arbuscular mycorrhizal fungi probably evolved through accumulation of mutations in an essentially clonal genome, with some infrequent recombination events. We conclude that mycorrhizal fungi have evolved to be multi-genomic.

Simple method for detection of MYH11 DNA rearrangements in patients with inv(16)(p13q22) and acute myeloid leukemia.

The pericentric inversion on chromosome 16 [inv(16)(p13q22)] and related t(16;16)(p13;q22) are recurrent aberrations associated with acute myeloid leukemia (AML) M4 Eo. Both abberations result in a fusion of the core binding factor beta (CBFB) and smooth muscle myosin heavy chain gene (MYH11). A selected genomic 6.9-kb BamHl probe detects MYH11 DNA rearrangements in 18 of 19 inv(16)/t(16;16) patients tested using HindIII digested DNA. The rearranged fragments were not detectable after remission in two cases tested, while they were present after relapse in one of these two cases tested.

Phylogeography of Mabuya maculilabris (Reptilia) from São Tomé Island (Gulf of Guinea) inferred from mtDNA sequences

The pattern of genetic variation of the lizard Mabuya maculilabris from São Tomé Island (Gulf of Guinea) was investigated using a combination of three mitochondrial DNA gene fragments. Forty-eight haplotypes were recovered among 66 individuals covering the whole island. The genealogy inferred from the most parsimonious network of haplotypes allows us to detect two main and long branches departing from the putative group of oldest haplotypes. The tips of these branches exhibit star-like phylogenies, which may indicate of recently expanded populations, most probably from a small number of founders. A nested clade analysis suggests a complex pattern of past events that gave rise to the extant geographical pattern found in the haplotype distribution: past and allopatric fragmentation, range expansion, restricted gene Xow and long-distance dispersal. These results are consistent with the complex geological history of the island where important volcanic activity with extensive lava Xows has occurred during several periods. Mismatch- distribution analysis and AMOVA also support these conclusions. Substantial genetic structuring among these lizards was detected as well as high levels of diVerentiation between the southern edge populations (particularly those from the Rolas Islet) and the remaining ones. However, variation is low relative to the geological age of the island. Our results indicate that patterns of variation observed in reptiles in other oceanic islands are not indicative of those observed in the islands of the Gulf of Guinea.

Genetic Differentiation of the Cabo Verde Archipelago Population Analysed by STR Polymorphisms

Allele frequencies for 17 STR loci were analyzed in a sample of unrelated males from the Cabo Verde Archipelago. The samples were gathered in such a way that the origin of the subjects was perfectly identified, and they could be included in one of the leeward or windward groups of islands. This study reveals that there are significant differences between both groups of islands, and between Cabo Verdeans and other populations from sub-Sahara Africa including the Guineans, the most probable source population for Cabo Verdeans. This study confirms mtDNA data and, together with HLA and Y chromosome data already published, shows that the Cabo Verde population is substructured and atypical, diverging substantially from mainland sub-Saharan populations. Overall these differences are most probably due to admixture between sub-Saharan slaves brought into the islands and other settlers of European origin. In the absence of a clear indication of a different ethnic composition of the first sub-Saharan settlers of Cabo Verde, the differentiation exhibited in both groups of islands can be most probably be attributed to genetic drift.

Genetic structure of populations of Pissodes castaneus (De Geer) (Coleoptera, Curculionidae) using amplified fragment length polymorphism

Genetic structure of populations of Pissodes castaneus (De Geer) (Coleoptera, Curculionidae) using amplified fragment length polymorphism. The objective of this study was to determine the genetic structure of populations of Pissodes castaneus from different areas and on different species of Pinus using the PCR-AFLP technique. Twenty samples were analyzed, representing 19 populations from Brazil and one from Florence, Italy, which is the region of origin of P. castaneus. The four combinations of primers generated a total of 367 fragments of DNA, and 100% of polymorphic loci, indicating high degree of molecular polymorphism. The dendrogram did not reveal trends for grouping the populations in relation to origin. The low genetic similarity (0.11 between the most distant groups) and genetic distances of 0.13 and 0.44 for 10 out of the 20 samples may indicate several founding events or multiple introductions of heterogeneous strains into Brazil. The allelic fixation index (Fst) was 0.3851, considered high, and the number of migrants (Nm) was 0.3991, indicating low gene flow among populations. The highest genetic distances were between the population from Irani, SC and Cambará do Sul, RS and Bituruna, PR, indicating an independent founding event or a particular allelic fixation in the former location. The high genetic diversity among populations points out that the populations are genetically heterogeneous with a diverse gene pool in the surveyed areas, what makes them to respond differently to control measures.

Simultaneous identification of multiple mammalian species from mixed forensic samples based on mtDNA control region length polymorphism

Molecular species identification in mixed or contaminated biological material has always been problematic. We developed a simple and accurate method for mammal DNA identification in mixtures, based on interspecific mitochondrial DNA control region length polymorphism. Contrary to other published methods dealing with species mixtures, our protocol requires a single universal primer pair and amplification step, and is not based on a pre-defined panel of species. This protocol has been routinely employed by our laboratory for species identification in dozens of human and animal forensic caseworks. Six representative forensic caseworks involving the specific identification of mixed animal samples are reported in this paper, in order to demonstrate the applicability and usefulness of the method.

High-level molecular diversity of copper-zinc superoxide dismutase genes among and within species of arbuscular Mycorrhizal fungi.

In the ecologically important arbuscular mycorrhizal fungi (AMF), Sod1 encodes a functional polypeptide that confers increased tolerance to oxidative stress and that is upregulated inside the roots during early steps of the symbiosis with host plants. It is still unclear whether its expression is directed at scavenging reactive oxygen species (ROS) produced by the host, if it plays a role in the fungus-host dialogue, or if it is a consequence of oxidative stress from the surrounding environment. All these possibilities are equally likely, and molecular variation at the Sod1 locus can possibly have adaptive implications for one or all of the three mentioned functions. In this paper, we analyzed the diversity of the Sod1 gene in six AMF species, as well as 14 Glomus intraradices isolates from a single natural population. By sequencing this locus, we identified a large amount of nucleotide and amino acid molecular diversity both among AMF species and individuals, suggesting a rapid divergence of its codons. The Sod1 gene was monomorphic within each isolate we analyzed, and quantitative PCR strongly suggest this locus is present as a single copy in G. intraradices. Maximum-likelihood analyses performed using a variety of models for codon evolution indicated that a number of amino acid sites most likely evolved under the regime of positive selection among AMF species. In addition, we found that some isolates of G. intraradices from a natural population harbor very divergent orthologous Sod1 sequences, and our analysis suggested that diversifying selection, rather than recombination, was responsible for the persistence of this molecular diversity within the AMF population.

Cytochrome b haplotype divergences in West European Apodemus

Sequencing of the cytochrome b mitochondrial gene (732 base pairs) in samples of Apodemus sylvaticus from Central Europe (eastern France, Switzerland, southern Germany and western Austria) revealed significant molecular variation not reflected in previous morphological and genetic studies of this species. A comparison with the sequences (150 bp) of 54 specimens available from GenBank (NCBI) showed that two problematic individuals originating from southern Germany have to be assigned to A. fulvipectus, a species not yet known in western Europe. A. sylvaticus specimens (n = 14) sampled north of the Alps exhibited a maximum intraspecific sequence divergence of about 5.1%, whereas the maximum divergence is much Lower in A. flavicollis (1%, n = 5) and in A. alpicola (0.5%, n = 4), although the samples originate from a similar geographic range of about 350 km. We also found a high rate of erroneously assigned specimens in GenBank, which indicates that the discrimination of Apodemus species remains a problem and requires further investigations.

Typing Candida albicans oral isolates from human immunodeficiency virus-infected patients by multilocus enzyme electrophoresis and DNA fingerprinting.

A total of 189 Candida albicans isolates have been typed by multilocus enzyme electrophoresis. The results obtained confirm the clonal mode of reproduction of C. albicans. The C. albicans populations found in the oropharynx of human immunodeficiency virus (HIV)-infected patients, in the oropharynx of healthy carriers, or in association with invasive candidiasis could not be distinguished. No clone or group of clones could be associated with the appearance of clinical disorders or with a reduced in vitro susceptibility to the antifungal agent fluconazole. Multiple and sequential oral isolates from 24 HIV-infected patients were also typed by restriction enzyme analysis with the enzymes EcoRI and HinfI and by use of the Ca3 repetitive probe. The results obtained by the combination of all three typing methods show that all but one patient each carried a unique major C. albicans clone in their oropharynx. The 21 patients with sequential isolates had the same C. albicans clones in their throats during recurrent oropharyngeal candidiasis episodes, independently of clinical status or of changes of in vitro susceptibility to fluconazole. Finally, several isolates of the same C. albicans clone found simultaneously in the oropharynx of a patient may present different levels of susceptibility to fluconazole.

Análisis del ADN mitocondrial de dos muestras del yacimiento paleolítico de El Pirulejo.

En la presente publicación se presentan los resultados del análisis del ADN mitocondrial (ADNmt) de dos individuos del nivel magdaleniense de El Pirulejo (Córdoba) (Asquerino et al., 1991). Las secuencias obtenidas se encuadran dentro de la variabilidad genética del hombre moderno actual, sin presentar similitud alguna con las secuencias de ADN mitocondrial de neandertal publicadas hasta la fecha. La distribución actual de las variantes mitocondriales encontradas en las dos muestras de El Pirulejo es similar a la encontrada en otros dos individuos Paleolíticos (Caramelli et al., 2003), y coherente con el modelo de expansión del hombre anatómicamente moderno desde África.