Étude du polymorphisme intra- et inter-spécifique du gène β-tubuline chez des espèces de champignons mycorhiziens à arbuscules en vue de développer des marqueurs moléculaires

Les champignons mycorhiziens à arbuscules (CMA), classés dans le phylum Glomeromycota, ne peuvent pas être facilement identifiés par la morphologie de leurs spores et leurs mycélia à l'intérieur ou à l'extérieur des racines de leurs hôtes. Ce problème fondamental d'identification rend l'étude de leur diversité, en particulier dans leur habitat naturel (sol et racine) extrêmement difficile. Les gènes ribosomaux ont été largement utilisés pour développer des amorces spécifiques et en inférer des arbres phylogénétiques. Cependant, ces gènes sont très polymorphes et existent en plusieurs copies dans le génome des CMA, ce qui complique l’interprétation des résultats. Dans notre étude, nous avons étudié le polymorphisme intra- et inter-spécifique du gène β-tubuline, présent en faible nombre de copies dans le génome des CMA, afin d’obtenir de nouvelles séquences nucléotidiques pour développer des marqueurs moléculaires. Les gènes β-tubuline amplifiés à partir de l'ADN génomique de cinq espèces du genre Glomus ont été clonés et séquencés. L’analyse des séquences indique un polymorphisme intraspécifique chez trois espèces de CMA. Deux séquences paralogues très variables ont été nouvellement identifiées chez les G. aggregatum, G. fasciculatum et G. cerebriforme. Aucun polymorphisme n’a été détecté chez les G. clarum et G. etunicatum. Toutes les séquences montrent la présence de deux introns hautement variables. La majorité des substitutions ont été localisées dans les exons et sont synonymes à 90%. La conservation des acides aminés suggère un niveau élevé de sélection négative sur le gène β-tubuline et nous permet de confirmer que les CMA représentent un ancien groupe fongique (400 million d’années). L’analyse phylogénétique, réalisée avec vingt et une séquences nucléotidiques du gène β-tubuline, a révélé que les séquences des Glomaceae forment un groupe monophylétique bien supporté, avec les Acaulosporaceae et Gigasporaceae comme groupe frère. Les séquences paralogues nouvellement identifiées chez les G. aggregatum et G. fasciculatum n'ont pas été monophylétiques au sein de chaque espèce. Les oligonucléotides ont été choisis sur la base des régions variables et conservées du gène β-tubuline. Le test PCR des amorces β-Tub.cerb.F/ β-Tub.cerb.R a révélé des bandes spécifiques de 401 pb pour les séquences paralogues du G. cerebriforme. Deux paires d’amorces ont été développées afin d’identifier les séquences du groupe nommé Tub.1. Les tests PCR nous ont permis d’identifier certaines séquences du groupe Tub.1. Une paire d’amorce β-Tub.2.F/ β-Tub.2.R nous a permis d’identifier certaines séquences paralogues du groupe nommé Tub.2. L’analyse d’autres gènes combinée à celle du gène β-tubuline permettra le développement de marqueurs moléculaires plus spécifiques pour l’identification de CMA.

Propidium monoazide (PMA) and ethidium bromide monoazide(EMA) improve DNA array and high-throughput sequencing ofporcine reproductive and respiratory syndrome virus identification

Pan-viral DNA array (PVDA) and high-throughput sequencing (HTS) are useful tools to identify novel viruses of emerging diseases. However, both techniques have difficulties to identify viruses in clinical samples because of the host genomic nucleic acid content (hg/cont). Both propidium monoazide (PMA) and ethidium bromide monoazide (EMA) have the capacity to bind free DNA/RNA, but are cell membrane-impermeable. Thus, both are unable to bind protected nucleic acid such as viral genomes within intact virions. However, EMA/PMA modified genetic material cannot be amplified by enzymes. In order to assess the potential of EMA/PMA to lower the presence of amplifiable hg/cont in samples and improve virus detection, serum and lung tissue homogenates were spiked with porcine reproductive and respiratory virus (PRRSV) and were processed with EMA/PMA. In addition, PRRSV RT-qPCR positive clinical samples were also tested. EMA/PMA treatments significantly decreased amplifiable hg/cont and significantly increased the number of PVDA positive probes and their signal intensity compared to untreated spiked lung samples. EMA/PMA treatments also increased the sensitivity of HTS by increasing the number of specific PRRSV reads and the PRRSV percentage of coverage. Interestingly, EMA/PMA treatments significantly increased the sensitivity of PVDA and HTS in two out of three clinical tissue samples. Thus, EMA/PMA treatments offer a new approach to lower the amplifiable hg/cont in clinical samples and increase the success of PVDA and HTS to identify viruses.

Selective mode excitation in dye-doped DNA polyvinyl alcohol thin film

A solid-state laser based on a dye-doped deoxyribonucleic acid (DNA) matrix is described. A thin solid film of DNA has been fabricated by treating with polyvinyl alcohol (PVA) and used as a host for the laser dye Rhodamine 6G. The edge emitted spectrum clearly indicated the existence of laser modes and amplified spontaneous emission. Lasing was obtained by pumping with a frequency-doubled Nd:YAG laser at 532 nm. For a pump energy of 10 mJ/pulse, an intense line with FWHM ≈0.2 nm was observed at 566 nm due to selective mode excitation.

Evaluation of Somaclonal Variation in Callus Cultures of Jatropha curcas Maintained on Different Hormonal Combinations Using RAPD Markers

Random genetic changes generated during in vitro culture are not desirable for plant micropropagation and genetic transformation. RAPD markers were used to detect the variation in leaf disc callus cultures of Jatropha curcas, maintained in Murashige and Skoog (MS) medium with different auxin and cytokinin combinations. In total 41 scorable bands were produced with 11 primers. Out of 41 bands, 37 were polymorphic (91.12%). The average number of polymorphic bands was 3.36 per primer. The highest similarity (0.82) with mother plant was seen in callus maintained on MS with hormonal combination Indole butyric acid - 0.4mg/l+ N6-benzyladenine purine - 4.0 mg/l. The callus grown on MS with hormonal combinations IBA- 0.4mg/l+ BAP- 2.0mg/l, IBA- 0.4mg/l+ BAP- 2.5mg/l and IBA- 0.6 mg/l+ BAP- 2.0 mg/l also showed similarity with the mother plant. Callus maintained on MS with hormonal combination IBA- 0.2mg/l+ BAP- 2.0 mg/l was found to show least similarity (0.53) with mother plant

Polimorfismos del gen Butirilcolinesterasa responsables de reacciones adversas en pacientes consumidores de “cocaína”

La butirilcolinesterasa humana (BChE; EC 3.1.1.8) es una enzima polimórfica sintetizada en el hígado y en el tejido adiposo, ampliamente distribuida en el organismo y encargada de hidrolizar algunos ésteres de colina como la procaína, ésteres alifáticos como el ácido acetilsalicílico, fármacos como la metilprednisolona, el mivacurium y la succinilcolina y drogas de uso y/o abuso como la heroína y la cocaína. Es codificada por el gen BCHE (OMIM 177400), habiéndose identificado más de 100 variantes, algunas no estudiadas plenamente, además de la forma más frecuente, llamada usual o silvestre. Diferentes polimorfismos del gen BCHE se han relacionado con la síntesis de enzimas con niveles variados de actividad catalítica. Las bases moleculares de algunas de esas variantes genéticas han sido reportadas, entre las que se encuentra las variantes Atípica (A), fluoruro-resistente del tipo 1 y 2 (F-1 y F-2), silente (S), Kalow (K), James (J) y Hammersmith (H). En este estudio, en un grupo de pacientes se aplicó el instrumento validado Lifetime Severity Index for Cocaine Use Disorder (LSI-C) para evaluar la gravedad del consumo de “cocaína” a lo largo de la vida. Además, se determinaron Polimorfismos de Nucleótido Simple (SNPs) en el gen BCHE conocidos como responsables de reacciones adversas en pacientes consumidores de “cocaína” mediante secuenciación del gen y se predijo el efecto delos SNPs sobre la función y la estructura de la proteína, mediante el uso de herramientas bio-informáticas. El instrumento LSI-C ofreció resultados en cuatro dimensiones: consumo a lo largo de la vida, consumo reciente, dependencia psicológica e intento de abandono del consumo. Los estudios de análisis molecular permitieron observar dos SNPs codificantes (cSNPs) no sinónimos en el 27.3% de la muestra, c.293A>G (p.Asp98Gly) y c.1699G>A (p.Ala567Thr), localizados en los exones 2 y 4, que corresponden, desde el punto de vista funcional, a la variante Atípica (A) [dbSNP: rs1799807] y a la variante Kalow (K) [dbSNP: rs1803274] de la enzima BChE, respectivamente. Los estudios de predicción In silico establecieron para el SNP p.Asp98Gly un carácter patogénico, mientras que para el SNP p.Ala567Thr, mostraron un comportamiento neutro. El análisis de los resultados permite proponer la existencia de una relación entre polimorfismos o variantes genéticas responsables de una baja actividad catalítica y/o baja concentración plasmática de la enzima BChE y algunas de las reacciones adversas ocurridas en pacientes consumidores de cocaína.

Phylogeographic analysis of the chloroplast DNA variation in wild common bean (Phaseolus vulgaris L.) in the Americas

The wild common bean (Phaseolus vulgaris) is widely but discontinuously distributed from northern Mexico to northern Argentina on both sides of the Isthmus of Panama. Little is known on how the species has reached its current disjunct distribution. In this research, chloroplast DNA polymorphisms in seven non-coding regions were used to study the history of migration of wild P. vulgaris between Mesoamerica and South America. A penalized likelihood analysis was applied to previously published Leguminosae ITS data to estimate divergence times between P. vulgaris and its sister taxa from Mesoamerica, and divergence times of populations within P. vulgaris. Fourteen chloroplast haplotypes were identified by PCR-RFLP and their geographical associations were studied by means of a Nested Clade Analysis and Mantel Tests. The results suggest that the haplotypes are not randomly distributed but occupy discrete parts of the geographic range of the species. The current distribution of haplotypes may be explained by isolation by distance and by at least two migration events between Mesoamerica and South America: one from Mesoamerica to South America and another one from northern South America to Mesoamerica. Age estimates place the divergence of P. vulgaris from its sister taxa from Mesoamerica at or before 1.3 Ma, and divergence of populations from Ecuador-northern Peru at or before 0.6 Ma. As these ages are taken as minimum divergence times, the influence of past events, such as the closure of the Isthmus of Panama and the final uplift of the Andes, on the migration history and population structure of this species cannot be disregarded.

Novel high-resolution characterization of ancient DNA reveals C > U-type base modification events as the sole cause of post mortem miscoding lesions

Ancient DNA (aDNA) research has long depended on the power of PCR to amplify trace amounts of surviving genetic material from preserved specimens. While PCR permits specific loci to be targeted and amplified, in many ways it can be intrinsically unsuited to damaged and degraded aDNA templates. PCR amplification of aDNA can produce highly-skewed distributions with significant contributions from miscoding lesion damage and non-authentic sequence artefacts. As traditional PCR-based approaches have been unable to fully resolve the molecular nature of aDNA damage over many years, we have developed a novel single primer extension (SPEX)-based approach to generate more accurate sequence information. SPEX targets selected template strands at defined loci and can generate a quantifiable redundancy of coverage; providing new insights into the molecular nature of aDNA damage and fragmentation. SPEX sequence data reveals inherent limitations in both traditional and metagenomic PCR-based approaches to aDNA, which can make current damage analyses and correct genotyping of ancient specimens problematic. In contrast to previous aDNA studies, SPEX provides strong quantitative evidence that C U-type base modifications are the sole cause of authentic endogenous damage-derived miscoding lesions. This new approach could allow ancient specimens to be genotyped with unprecedented accuracy.

Amplified fragment length polymorphism genetic fingerprinting challenges the taxonomic status of the near-endemic species Chara curta Nolte ex Kutz. (Characeae)

Amplified fragment length polymorphism (AFLP) genetic fingerprinting of 14 accessions of Chara curta and Chara aspera Willd., sampled across a range of habitats and morphologies in Britain, suggests that these taxa are part of the variation within a single species complex. Two primer combinations generating 397 fragments (97% of which were polymorphic), analysed by Jaccard's similarity coefficient and principal co-ordinate analysis, did not recover groups which reflect the current taxonomy. By contrast with the genetic study, a Gower general similarity coefficient and principal co-ordinate analysis of 52 morphological characters recovered the currently recognized species groups. A Mantel test showed no significant correlation between the genetic data and the morphological data, supporting the hypothesis that phenotypic variability in Chara L. is either to some extent environmentally induced or represents developmental stages. Implications for the conservation status of C. curta in Britain are discussed. (c) 2007 The Linnean Society of London, Botanical Journal of the Linnean Society, 2007, 155, 467-476.

Characterization and PCR multiplexing of polymorphic microsatellite loci in cashew (Anacardium occidentale L.) and their cross-species utilization

Cashew (Anacardium occidentale L.) is the most economically important tropical nut crop in the world, and yet there are no sequence tagged site (STS) markers available for its study. Here we use an automated, high-throughput system to isolate cashew microsatellites from a non-enriched genomic library blotted onto membranes at high density for screening. Sixty-five sequences contained a microsatellite array, of which 21 proved polymorphic among a closely related seed garden population of 49 genotypes. Twelve markers were suitable for multiplex analysis. Of these, 10 amplified in all three related tropical tree species tested: Anacardium microcarpum, Anacardium pumilum and Anacardium nanum.

High throughput, high resolution selection of polymorphic microsatellite loci for multiplex analysis

Background Large-scale genetic profiling, mapping and genetic association studies require access to a series of well-characterised and polymorphic microsatellite markers with distinct and broad allele ranges. Selection of complementary microsatellite markers with non-overlapping allele ranges has historically proved to be a bottleneck in the development of multiplex microsatellite assays. The characterisation process for each microsatellite locus can be laborious and costly given the need for numerous, locus-specific fluorescent primers. Results Here, we describe a simple and inexpensive approach to select useful microsatellite markers. The system is based on the pooling of multiple unlabelled PCR amplicons and their subsequent ligation into a standard cloning vector. A second round of amplification utilising generic labelled primers targeting the vector and unlabelled locus-specific primers targeting the microsatellite flanking region yield allelic profiles that are representative of all individuals contained within the pool. Suitability of various DNA pool sizes was then tested for this purpose. DNA template pools containing between 8 and 96 individuals were assessed for the determination of allele ranges of individual microsatellite markers across a broad population. This helped resolve the balance between using pools that are large enough to allow the detection of many alleles against the risk of including too many individuals in a pool such that rare alleles are over-diluted and so do not appear in the pooled microsatellite profile. Pools of DNA from 12 individuals allowed the reliable detection of all alleles present in the pool. Conclusion The use of generic vector-specific fluorescent primers and unlabelled locus-specific primers provides a high resolution, rapid and inexpensive approach for the selection of highly polymorphic microsatellite loci that possess non-overlapping allele ranges for use in large-scale multiplex assays.

Detection of Chlamydia psittaci DNA in avian clinical samples by polymerase chain reaction

A polymerase chain reaction (PCR) assay was developed to detect Chlamydia psittaci DNA in faeces and tissue samples from avian species. Primers were designed to amplify a 264 bp product derived from part of the 5' non-translated region and part of the coding region of the ompA gene which encodes the major outer membrane protein. Amplified sequences were confirmed by Southern hybridization using an internal probe. The sensitivity of the combined assay was found to be between 60 to 600 fg of chlamydial DNA (approximately 6 to 60 genome copies). The specificity of the assay was confirmed since PCR product was not obtained from samples containing several serotypes of C. trachomatis, strains of C. pneumoniae, the type strain of C. pecorum, nor from samples containing microorganisms commonly found in the avian gut flora. In this study, 404 avian faeces and 141 avian tissue samples received by the Central Veterinary Laboratory over a 6 month period were analysed by PCR, antigen detection ELISA and where possible, cell culture isolation. PCR performed favourably compared with ELISA and cell culture, or with ELISA alone. The PCR assay was especially suited to the detection of C. psittaci DNA in avian faeces samples. The test was also useful when applied to tissue samples from small contact birds associated with a case of human psittacosis where ELISA results were negative and chlamydial isolation was a less favourable method due to the need for rapid diagnosis.

Non-concordance between genetic profiles of olive oil and fruit: a cautionary note to the use of DNA markers for provenance testing.

To investigate the contribution of paternal alleles to the DNA content of olive oil, genetic analyses of olive DNA samples from fruits, leaves, and oil derived from the same tree (cv. Leccino) were carried out. DNA extracted from maternal tissues--leaves and flesh--from different fruits showed identical genetic profiles using a set of DNA markers. Additional simple sequence repeat (SSR) alleles, not found in the maternal samples, were amplified in the embryos (stone), and they were also detected in DNA extracted from the paste obtained by crushing whole fruits and from the oil pressed from this material. These results demonstrate that the DNA profile obtained from olive oil is likely to represent a composite profile of the maternal alleles juxtaposed with alleles contributed by various pollen donors. Therefore, care needs to be taken in the interpretation of DNA profiles obtained from DNA extracted from oil for resolving provenance and authenticity issues.

Identification of Hammondia heydorni oocysts by a heminested-PCR (hnPCR-AP10) based on the H-heydorni RAPD fragment AP10

Toxoplasma gondii, Hammondia hammondi, Neospora caninum, Neospora hughesi and Hammondia heydorni are members of the Toxoplasmatinae sub-family. They are closely related coccidians with similarly sized oocysts. Molecular diagnostic techniques, especially those based on polymerase chain reaction (PCR), can be successfully applied for the differentiation of Hammondia-like oocysts. In this paper, we describe a rapid and simple method for the identification of H. heydorni oocysts among other members of the Toxoplasmatinae sub-family, using a heminested-PCR (hnPCR-AP10) based on a H. heydorni RAPD fragment available in molecular database. DNA of oocysts of H. heydorni yielded a specific fragment of 289-290 bp in the heminested-PCR assay. No product was yielded when the primers were used for the amplification of DNA extracted from T. gondii, N. caninum, N. hughesi and H. hammondi, thus allowing the differentiation of H. heydorni among other members of the Toxoplasmatinae sub-family. The hnPCR-AP10 was capable of detecting H. heydorni genetic sequences from suspensions with at least 10 oocysts. In conclusion, the hnPCR-AP10 proved to be a reliable method to be used in the identification of H. heydorni oocysts from feces of dogs. (C) 2010 Elsevier B.V. All rights reserved.

Mitochondrial DNA Diversity of Orchid Bee Euglossa fimbriata (Hymenoptera: Apidae) Populations Assessed by PCR-RFLP

Euglossa fimbriata is a euglossine species widely distributed in Brazil and occurring primarily in Atlantic Forest remnants. In this study, the genetic mitochondrial structure of E. fimbriata from six Atlantic Forest fragments was studied by RFLP analysis of three PCR-amplified mtDNA gene segments (16S, COI-COII, and cyt b). Ten composite haplotypes were identified, six of which were exclusive and represented singleton mitotypes. Low haplotype diversity (0.085-0.289) and nucleotide diversity (0.000-0.002) were detected within samples. AMOVA partitioned 91.13% of the overall genetic variation within samples and 8.87% (I center dot(st) = 0.089; P < 0.05) among samples. Pairwise comparisons indicated high levels of differentiation among some pairs of samples (I center dot(st) = 0.161-0.218; P < 0.05). These high levels indicate that these populations of E. fimbriata, despite their highly fragmented landscape, apparently have not suffered loss of genetic variation, suggesting that this particular population is not currently endangered.

Intraspecific variation in 16S rRNA gene of Mycoplasma synoviae determined by DNA sequencing

Mycoplasma synoviae (MS) is an important avian pathogen may cause both respiratory disease and joint inflammation synovitis in poultry, causing economic losses to the Brazilian poultry industry. The genotypic variation in 16S rRNA gene is unknown. Partial sequences of 16S rRNA gene of 19 strains of M. synoviae were sequenced and analyzed in order to obtain molecular characterization and evaluation of the genetic variability of strains from distinct Brazilian areas of poultry production. Different polymorphic patterns were observed. The number of polymorphic alterations in the studied strains ranged from 0 to 6. The nucleotide variations, including deletion, insertion and substitutions, ranged from 3 to 5. The genotypic diversity observed in this study may be explained by spontaneous mutations that may occur when a lineage remains in the same flock for long periods. The culling and reposition in poultry flocks may be responsible for the entry of new strains in different areas. (C) 2008 Elsevier Ltd. All rights reserved.