DnaSP Version 5. DNA Sequence Polymorphism

DnaSP, DNA Sequence Polymorphism, is a software package for the analysis of nucleotide polymorphism from aligned DNA sequence data. DnaSP can estimate several measures of DNA sequence variation within and between populations (in noncoding, synonymous or nonsynonymous sites, or in various sorts of codon positions), as well as linkage disequilibrium, recombination, gene flow and gene conversion parameters. DnaSP can also carry out several tests of neutrality: Hudson, Kreitman and Aguadé (1987), Tajima (1989), McDonald and Kreitman (1991), Fu and Li (1993), and Fu (1997) tests. Additionally, DnaSP can estimate the confidence intervals of some test-statistics by the coalescent. The results of the analyses are displayed on tabular and graphic form.

Major Histocompatibility Complex (MHC) Class II sequence polymorphism in long-finned pilot whale (Globicephala melas) from the North Atlantic

Funding Silvia S. Monteiro and Marisa Ferreira were supported by a Ph.D. grant from Fundação para a Ciência e Tecnologia (ref SFRH/BD/38735/2007 and SFRH/BD/30240/2006, respectively). Alfredo López was supported by a postdoctoral grant from Fundação para a Ciência e Tecnologia (ref SFRH/BPD/82407/2011). Catarina Eira is supported by CESAM (UID/AMB/50017), from FCT/MEC through national funds and FEDER (PT2020, Compete 2020). The work related with strandings and tissue collection in Portugal was partially supported by the SafeSea Project EEAGrants PT 0039 (supported by Iceland, Liechtenstein and Norway through the EEA Financial Mechanism), by the Project MarPro–Life09 NAT/PT/000038 (funded by the European Union–Program Life+) and by the project CetSenti FCT RECI/AAG-GLO/0470/2012; FCOMP-01-0124-FEDER-027472 (Funded by the Program COMPETE and Fundação para a Ciência e Tecnologia).

Phylogenetic relationships in Solanaceae and related species based on cpDNA sequence from plastid trnE-trnT region

Intergenic spacers of chloroplast DNA (cpDNA) are very useful in phylogenetic and population genetic studies of plant species, to study their potential integration in phylogenetic analysis. The non-coding trnE-trnT intergenic spacer of cpDNA was analyzed to assess the nucleotide sequence polymorphism of 16 Solanaceae species and to estimate its ability to contribute to the resolution of phylogenetic studies of this group. Multiple alignments of DNA sequences of trnE-trnT intergenic spacer made the identification of nucleotide variability in this region possible and the phylogeny was estimated by maximum parsimony and rooted with Convolvulaceae Ipomoea batalas, the most closely related family. Besides, this intergenic spacer was tested for the phylogenetic ability to differentiate taxonomic levels. For this purpose, species from four other families were analyzed and compared with Solanaceae species. Results confirmed polymorphism in the trnE-trnT region at different taxonomic levels.

Differentiation of Acidithiobacillus ferrooxidans and A-thiooxidans strains based on 16S-23S rDNA spacer polymorphism analysis

Restriction fragment length polymorphism (RFLP) and sequence analyses of the PCR-amplified 16S-23S rDNA intergenic spacer (ITS) were used for differentiating Acidithiobacillus thiooxidans strains from other related acidithiobacilli, including A. ferrooxidans and A. caldus. RFLP fingerprints obtained with AluI, DdeI, HaeIII, HinfI and MspI enabled the differentiation of all Acidithiobacillus reference strains into species groups. The A. thiooxidans strains investigated (metal mine isolates) yielded identical RFLP patterns to the A. thiooxidans type strain (ATCC 19377(T)), except for strain DAMS, which had a distinct pattern for all enzymes tested. Fourteen A. ferrooxidans mine strains were assigned to 3 RFLP groups, the majority of which were grouped with A. ferrooxidans ATCC 23270(T). The spacer region of one representative strain from each of the RFLP groups obtained was subjected to sequence analysis, in addition to eleven additional A. thiooxidans strains isolated from sediment and water samples, and A. caldus DSM 8584(T). The tRNA(IIe) and tRNA(Ala) genes, present in all strains analyzed, showed high sequence similarity. Phylogenetic analysis of the ITS sequences differentiated all three Acidithiobacillus species. Inter- and infraspecific genetic variations detected were mainly due to the size and sequence polymorphism of the ITS3 region. Mantel tests showed no significant correlation between ITS sequence similarity and the geographical origin of strains. The results showed that the 16S-23S rDNA spacer region is a useful target for the development of molecular-based methods aimed at the detection, rapid differentiation and identification of acidithiobacilli. (C) 2004 Elsevier SAS. All rights reserved.

Effect of sequence variation in Plasmodium falciparum Histidine-Rich protein 2 on binding of specific monoclonal antibodies: Implications for rapid diagnostic tests for malaria

This Article Right arrow Full Text Right arrow Full Text (PDF) Right arrow Supplemental material Right arrow Alert me when this article is cited Right arrow Alert me if a correction is posted Services Right arrow Similar articles in this journal Right arrow Similar articles in PubMed Right arrow Alert me to new issues of the journal Right arrow Download to citation manager Right arrow Reprints and Permissions Right arrow Copyright Information Right arrow Books from ASM Press Right arrow MicrobeWorld Citing Articles Right arrow Citing Articles via HighWire Right arrow Citing Articles via Google Scholar Google Scholar Right arrow Articles by Lee, N. Right arrow Articles by McCarthy, J. Right arrow Search for Related Content PubMed Right arrow PubMed Citation Right arrow Articles by Lee, N. Right arrow Articles by McCarthy, J. Right arrow Pubmed/NCBI databases * Substance via MeSH Previous Article | Next Article Journal of Clinical Microbiology, August 2006, p. 2773-2778, Vol. 44, No. 8 0095-1137/06/$08.00+0 doi:10.1128/JCM.02557-05 Copyright © 2006, American Society for Microbiology. All Rights Reserved. Effect of Sequence Variation in Plasmodium falciparum Histidine- Rich Protein 2 on Binding of Specific Monoclonal Antibodies: Implications for Rapid Diagnostic Tests for Malaria{dagger} Nelson Lee,1,2 Joanne Baker,2 Kathy T. Andrews,1 Michelle L. Gatton,1,3 David Bell,4 Qin Cheng,2,3 and James McCarthy1* Australian Centre for International and Tropical Health and Nutrition, Queensland Institute of Medical Research and School of Population Health, University of Queensland, Queensland, Australia,1 Department of Drug Resistance and Diagnostics, Australian Army Malaria Institute, Brisbane, Australia,2 Malaria Drug Resistance and Chemotherapy, Queensland Institute of Medical Research, Queensland, Australia,3 World Health Organization, Regional Office for the Western Pacific, Manila, Philippines4 Received 8 December 2005/ Returned for modification 23 February 2006/ Accepted 26 May 2006 The ability to accurately diagnose malaria infections, particularly in settings where laboratory facilities are not well developed, is of key importance in the control of this disease. Rapid diagnostic tests (RDTs) offer great potential to address this need. Reports of significant variation in the field performance of RDTs based on the detection of Plasmodium falciparum histidine-rich protein 2 (HRP2) (PfHRP2) and of significant sequence polymorphism in PfHRP2 led us to evaluate the binding of four HRP2-specific monoclonal antibodies (MABs) to parasite proteins from geographically distinct P. falciparum isolates, define the epitopes recognized by these MABs, and relate the copy number of the epitopes to MAB reactivity. We observed a significant difference in the reactivity of the same MAB to different isolates and between different MABs tested with single isolates. When the target epitopes of three of the MABs were determined and mapped onto the peptide sequences of the field isolates, significant variability in the frequency of these epitopes was observed. These findings support the role of sequence variation as an explanation for variations in the performance of HRP2-based RDTs and point toward possible approaches to improve their diagnostic sensitivities

Short report: Molecular evaluation of the efficacy of chloroquine treatment of uncomplicated Plasmodium falciparum malaria in East Timor

The efficacy of chloroquine treatment of uncomplicated Plasmodium falciparum malaria in East Timor was investigated via molecular tools. Genotyping of the polymorphic markers msp1 and msp2 was performed to investigate the number and type of parasite alleles in pre- and posttreatment blood samples collected from 48 patients. Patients were infected with a minimum of 8 msp1 and 14 msp2 allelic types of parasite, and 43% of the patients had more than one allelic type before treatment. The genotyping also revealed that 66.7% of the patients were infected with at least one identical allelic type of parasite before and after treatment and therefore were likely to have experienced recrudescence. All parasites in pre- and posttreatment blood samples carried the K76T mutation in pfcrt, regardless of the clinical response to chloroquine. The sequence polymorphism patterns in pfcrt in the majority of parasites examined were identical to those observed in Bougainville, Papua New Guinea.

Molecular evolutionary patterns in the Glomeromycota;

Abstract Arbuscular mycorrhizal fungi (AMF) form symbiosis with roots of approximately 80% of known land plants. These fungi play a key role in the ecology and adaptation of plants to various ecosystems.by increasing the plant resources for various nutrients. Despite their important ecological role, we still have poor understanding of their genetic structure and their molecular evolution. The work presented in this thesis aims to isolate and analyse AMF genes with various molecular techniques, in order to obtain new insights about their genetics, phylogeny and molecular evolution. Some AMF genes were shown through phylogenetic analyses to be more related with plants or mycoparasites than with other fungal organisms. These results led to the prediction that lateral gene transfers (LGT) occurred between AMF and plants during their long-term co-évolution. By phylogenetic and molecular analyses, in the chapter 2 I demonstrate that the hypothesis of LGT is most likely a consequence of analyses carried out on contaminant non AMF-DNA. In addition, various features characteristic of AMF genes have been determined, allowing researchers to scan their own sequence databases for potential non-AMF contaminants. Phylogenetic relationships of AMF with other fungi has been mostly analysed using molecular markers of ribosomal origin. In chapter 2 I successfully isolated gene encoding α- and ß-tubulins from several AMF genera. Consequently, phylogenetic analyses showed that AMF possess an unexpected relationship with ancestral aquatic fungi (chytrids). These results are consistent with the prediction stating that AMF may have played an important role in the colonisation of land by green plants through the establishment of a symbiosis and after the divergence of AMF from aquatic ancestors. In Chapter 4 I tried to isolate the entire AMF gene family encoding P-Type II ATPases, in order to determine their molecular evolution with the fungal kingdom. These genes were further analysed to detect the level of sequence polymorphism that is present within an AMF population. The results obtained show that mutational events previously thought as occurring only among divergent evolutionary lineages (gene duplications, indel mutations in coding regions) can occur within a single population of AMF. These results have far reaching consequences for our understanding of the genetics and ecology of AMF. Résumé Les champignons endomycorrhiziens arbusculaires (CEA) forment une symbiose racinaire avec environ 80% des plantes vasculaires connues. Ces champignons possèdent un rôle important dans l'écologie et l'adaptation des plantes au sein de différents écosystèmes en .augmentant leurs ressources en nutriments. Le travail présenté dans cette thèse se propose d'isoler et d'analyser certains gènes de CEA avec différentes techniques moléculaires à fin d'obtenir de pÌus amples informations concernant l'évolution moléculaire, la phylogénie et leur diversité génétique à diverses échelles taxonomiques. Certaines analyses phylogénétiques des CEA ont conduit à l'hypothèse que des transferts horizontaux de gènes (THG) ont pu avoir lieu durant leur longue co-évolution avec les plantes vasculaires. Dans le chapitre 2 de cette thèse nous démontrons par analyses moléculaire et phylogénétique que l'hypothèse de THG est une conséquence de contaminations à partir d'ADN de plante ou d'autres micro-organismes. De plus, de nombreuses caractéristiques moléculaires de CEA ont pu être déterminées, permettant la mise en place d'un plan à suivre lors de l'analyse de gènes de CEA dans les études futures. Les relations évolutives des. CEA avec d'autres champignons ont été analysées majoritairement à l'aide de marqueurs moléculaires d'origine ribosomiale. Dans les chapitres 2 et 3 j'ai isolé des gènes codant pour l'a- et la ß-tubuline chez différents genres, de CEA. Les analyses phylogénétiques ont démontré une parenté entre les CEA et des champignons aquatiques ancestraux (chytrides). Ces résultats sont en accord avec l'hypothèse selon laquelle les CEA ont probablement joué un rôle primordial dans l'établissement des plantes sur terre à travers une symbiose et suite à leur évolution à partir d'ancêtres vivant dans des milieux aquatiques: Dans le chapitre 4 j'ai isolé une entière famille de gènes chez les CEA codant des ATPases de la membrane plasmique, et étudié leur évolution moléculaire dans le règne des champignons. Ces mêmes gènes ont été analysés ultérieurement à fin de déterminer le degré de polymorphisme de séquence qui peut être présent au sein d'une population de CEA. Les résultats obtenus montrent que des évènements mutationnels considérés comme apparaissant exclusivement dans des lignées évolutives très divergentes (duplication de gènes, insertions/délétions dans des régions transcrites du génome) ont lieu sein d'une même population de CEA. Cette découverte a un impact important sur nos connaissances concernant la génétique des populations des CEA et leur écologie.

Mitochondrial DNA variation along an altitudinal gradient in the greater white-toothed shrew, Crocidura russula.

The distribution of mitochondrial control region-sequence polymorphism was investigated in 15 populations of Crocidura russula along an altitudinal gradient in western Switzerland. High-altitude populations are smaller, sparser and appear to undergo frequent bottlenecks. Accordingly, they showed a loss of rare haplotypes, but unexpectedly, were less differentiated than lowland populations. Furthermore, the major haplotypes segregated significantly with altitude. The results were inconsistent with a simple model of drift and dispersal. They suggested instead a role for historical patterns of colonization, or, alternatively, present-day selective forces acting on one of the mitochondrial genes involved in metabolic pathways.

Population genomics of eusocial insects: the costs of a vertebrate-like effective population size

The evolution of reproductive division of labour and social life in social insects has lead to the emergence of several life-history traits and adaptations typical of larger organisms: social insect colonies can reach masses of several kilograms, they start reproducing only when they are several years old, and can live for decades. These features and the monopolization of reproduction by only one or few individuals in a colony should affect molecular evolution by reducing the effective population size. We tested this prediction by analysing genome-wide patterns of coding sequence polymorphism and divergence in eusocial vs. noneusocial insects based on newly generated RNA-seq data. We report very low amounts of genetic polymorphism and an elevated ratio of nonsynonymous to synonymous changes - a marker of the effective population size - in four distinct species of eusocial insects, which were more similar to vertebrates than to solitary insects regarding molecular evolutionary processes. Moreover, the ratio of nonsynonymous to synonymous substitutions was positively correlated with the level of social complexity across ant species. These results are fully consistent with the hypothesis of a reduced effective population size and an increased genetic load in eusocial insects, indicating that the evolution of social life has important consequences at both the genomic and population levels.

Cloning, bioinformatics analysis, and expression of the dust mite allergen Der f 5 of Dermatophagoides farinae

Crude extracts of house dust mites are used clinically for diagnosis and immunotherapy of allergic diseases, including bronchial asthma, perennial rhinitis, and atopic dermatitis. However, crude extracts are complexes with non-allergenic antigens and lack effective concentrations of important allergens, resulting in several side effects. Dermatophagoides farinae (Hughes; Acari: Pyroglyphidae) is one of the predominant sources of dust mite allergens, which has more than 30 groups of allergen. The cDNA coding for the group 5 allergen of D. farinae from China was cloned, sequenced and expressed. According to alignment using the VECTOR NTI 9.0 software, there were eight mismatched nucleotides in five cDNA clones resulting in seven incompatible amino acid residues, suggesting that the Der f 5 allergen might have sequence polymorphism. Bioinformatics analysis revealed that the matured Der f 5 allergen has a molecular mass of 13604.03 Da, a theoretical pI of 5.43 and is probably hydrophobic and cytoplasmic. Similarities in amino acid sequences between Der f 5 and allergens of other domestic mite species, viz. Der p 5, Blo t 5, Sui m 5, and Lep d 5, were 79, 48, 53, and 37%, respectively. Phylogenetic analysis indicated that Der f 5 and Der p 5 clustered together. Blo t 5 and Ale o 5 also clustered together, although Blomia tropicalis and Aleuroglyphus ovatus belong to different mite families, viz. Echimyopodidae and Acaridae, respectively.

Arbuscular mycorrhizal modulation of diazotrophic and denitrifying microbial communities in the (mycor)rhizosphere of Plantago lanceolata

Impacts of divergent arbuscular mycorrhizal (AM) fungi, Glomus intraradices and Gigaspora margarita, on denitrifying and diazotrophic bacterial communities of Plantago lanceolata in nutrient-limited dune soil were assessed. We hypothesized AM species-related modifications that were confirmed in respective bacterial nirK and nifH sequence polymorphism -based community clustering and community variance allocation. The denitrifying community appeared more responsive to AM fungi than the nitrogen-fixing community. Nevertheless, the main explanatory variable, in both cases, was plant age. We conclude that AM fungi can modify N-cycling microbial rhizosphere communities and future work should aim to verify the functional significance and mechanistic basis.

Trypanosoma rangeli isolates of bats from Central Brazil: Genotyping and phylogenetic analysis enable description of a new lineage using spliced-leader gene sequences

Trypanosoma rangeli infects several mammalian orders but has never confidently been described in Chiroptera, which are commonly parasitized by many trypanosome species. Here, we described trypanosomes from bats captured in Central Brazil identified as T rangeli,.T. dionisii, T cruzimarinkellei and T cruzi. Two isolates, Tra643 from Platyrrhinus lineatus and Tra1719 from Artibeus plamirostris were identified as T rangeli by morphological, biological and molecular methods, and confirmed by phylogenetic analyses. Analysis using SSU rDNA sequences clustered these bat trypanosomes together with T rangeli from other hosts, and separated them from other trypanosomes from bats. Genotyping based on length and sequence polymorphism of PCR-amplified intergenic spliced-leader gene sequences assigned Tra1719 to the lineage A whereas Tra643 was shown to be a new genotype and was assigned to the new lineage E. To our knowledge, these two isolates are the earliest T rangeli from bats and the first isolates from Central Brazil molecularly characterized. Rhodnius stali captured for this study was found infected by T rangeli and T cruzi. (c) 2008 Elsevier B.V. All rights reserved.

Implication of the RDRio Mycobacterium tuberculosis sublineage in multidrug resistant tuberculosis in Portugal

Multidrug and extensively drug resistant Mycobacterium tuberculosis are a threat to tuberculosis control programs. Genotyping methods, such as spoligotyping and MIRU-VNTR typing (Mycobacterial Interspersed Repetitive Units), are useful in monitoring potentially epidemic strains and estimating strain phylogenetic lineages and/or genotypic families. M. tuberculosis Latin American Mediterranean (LAM) family is a major worldwide contributor to tuberculosis (TB). LAM specific molecular markers, Ag85C(103) single nucleotide polymorphism (SNP) and RDRio long-sequence polymorphism (LSP), were used to characterize spoligotype signatures from 859 patient isolates from Portugal. LAM strains were found responsible for 57.7% of all tuberculosis cases. Strains with the RDRio deletion (referred to as RDRio) were estimated to represent 1/3 of all the strains and over 60% of the multidrug resistant (MDR) strains. The major spoligotype signature SIT20 belonging to the LAM1 RDRio sublineage, represented close to 1/5th of all the strains, over 20% of which were MDR. Analysis of published datasets according to stipulated 12 loci MIRU-VNTR RDRio signatures revealed that 96.3% (129/134) of MDR and extensively drug resistant (XDR) clusters were RDRio. This is the first report associating the LAM RDRio sublineage with MDR. These results are an important contribution to the monitoring of these strains with heightened transmission for future endeavors to arrest MDR-TB and XDR-TB. (c) 2012 Elsevier B.V. All rights reserved.

Species identification, distributioin and reproductive interactions of common smoot-hound and blackspotted smooth-hound shark (Genus mustelus) in the Adriatic Sea

Longstanding taxonomic ambiguity and uncertainty exist in the identification of the common (M. mustelus) and blackspotted (M. punctulatus) smooth-hound in the Adriatic Sea. The lack of a clear and accurate method of morphological identification, leading to frequent misidentification, prevents the collation of species-specific landings and survey data for these fishes and hampers the delineation of the distribution ranges and stock boundaries of the species. In this context, adequate species-specific conservation and management strategies can not be applied without risks of population declining and local extinction. In this thesis work I investigated the molecular ecology of the two smooth-hound sharks which are abundant in the demersal trawl surveys carried out in the NC Adriatic Sea to monitor and assess the fishery resources. Ecological and evolutionary relationships were assessed by two molecular tests: a DNA barcoding analysis to improve species identification (and consequently the knowledge of their spatial ecology and taxonomy) and a hybridization assay based on the nuclear codominant marker ITS2 to evaluate reproductive interactions (hybridization or gene introgression). The smooth-hound sharks (N=208) were collected during the MEDITS 2008 and 2010 campaigns along the Italian and Croatian coasts of the Adriatic Sea, in the Sicilian Channel and in the Algerian fisheries. Since the identification based on morphological characters is not strongly reliable, I performed a molecular identification of the specimens producing for each one the cytochrome oxidase subunit 1 (COI) gene sequence (ca. 640 bp long) and compared them with reference sequences from different databases (GenBank and BOLD). From these molecular ID data I inferred the distribution of the two target species in the NC Adriatic Sea. In almost the totality of the MEDITS hauls I found no evidence of species sympatry. The data collected during the MEDITS survey showed an almost different distribution of M. mustelus (confined along the Italian coasts) and M. punctulatus (confined along the Croatian coasts); just one sample (Gulf of Venice, where probably the ranges of the species overlap) was found to have catches of both the species. Despite these data results suggested no interaction occurred between my two target species at least during the summertime (the period in which MEDITS survey is carried out), I still wanted to know if there were inter-species reproductive interactions so I developed a simple molecular genetic method to detect hybridization. This method is based on DNA sequence polymorphism among species in the nuclear ribosomal Internal Transcribed Spacer 2 locus (ITS2). Its application to the 208 specimens collected raised important questions regarding the ecology of this two species in the Adriatic Sea. In fact results showed signs of hybridization and/or gene introgression in two sharks collected during the trawl survey of 2008 and one collected during the 2010 one along the Italian and Croatian coasts. In the case that it will be confirmed the hybrid nature of these individuals, a spatiotemporal overlapping of the mating behaviour and ecology must occur. At the spatial level, the northern part of the Adriatic Sea (an area where the two species occur with high frequency of immature individuals) could likely play the role of a common nursery area for both species.