Quantitative trait loci with sex-specific effects for internal organs weights and hematocrit value in a broiler-layer cross

Rapid growth in broilers is associated with susceptibility to metabolic disorders such as pulmonary hypertension syndrome (ascites) and sudden death. This study describes a genome search for QTL associated with relative weight of cardio respiratory and metabolically important organs (heart, lungs, liver and gizzard), and hematocrit value in a Brazilian broiler-layer cross. QTL with similar or different effects across sexes were investigated. At 42 days of age after fasted for 6 h, the F2 chickens were weighed and slaughtered. Weights and percentages of the weight relative to BW42 of gizzard, heart, lungs, liver and hematocrit were used in the QTL search. Parental, F1 and F2 individuals were genotyped with 128 genetic markers (127 microsatellites and 1 SNP) covering 22 linkage groups. QTL mapping analyses were carried out using mixed models. A total of 11 genome-wide significant QTL and five suggestive linkages were mapped. Thus, genome-wide significant QTL with similar effects across sexes were mapped to GGA2, 4 and 14 for heart weight, and to GGA2, 8 and 12 for gizzard %. Additionally, five genome-wide significant QTL with different effects across sexes were mapped to GGA 8, 19 and 26 for heart weight; GGA26 for heart % and GGA3 for hematocrit value. Five QTL were detected in chromosomal regions where QTL for similar traits were previously mapped in other F2 chicken populations. Seven novel genome-wide significant QTL are reported here, and 21 positional candidate genes in QTL regions were identified.

History and fate of a small isolated population of Weddell seals at White Island, Antarctica

Weddell seals (Leptonychotes weddellii Lesson) at White Island, Antarctica form a small, completely enclosed, natural population hypothesized to be of recent origin, likely founded by individuals from nearby Erebus Bay. This population constitutes an ideal model to document a founder event and ensuing genetic drift, with implications for conservation. Here we combined historical accounts, census and tagging data since the late 1960s, and genetic data (41 microsatellite loci and mitochondrial DNA sequences) from 84 individuals representing nearly all individuals present between 1990 and 2000 to investigate the history of the founding of the White Island population, document its population dynamics and evaluate possible future threats. We fully resolved parental relationships over three overlapping generations. Cytonuclear disequilibrium among the first generation suggested that it comprised the direct descendants of a founding group. We estimated that the White Island population was founded by a small group of individuals that accessed the island during a brief break in the surrounding sea ice in the mid-1950s, consistent with historical accounts. Direct and indirect methods of calculating effective population size were highly congruent and suggested a minimum founding group consisting of three females and two males. The White Island population showed altered reproductive dynamics compared to Erebus Bay, including highly skewed sex ratio, documented inbred mating events, and the oldest known reproducing Weddell seals. A comparison with the putative source population showed that the White Island population has an effective inbreeding coefficient (Fe) of 0.29. Based on a pedigree analysis including the hypothesized founding group, 86% of the individuals for whom parents were known had inbreeding coefficients ranging 0.09–0.31. This high level of inbreeding was correlated with reduced pup survival. Seals at White Island therefore face the combined effects of low genetic variability, lack of immigration, and inbreeding depression. Ultimately, this study provides evidence of the effects of natural isolation on a large, long-lived vertebrate and can provide clues to the potential effects of anthropogenic- caused isolation of similar taxa.

Genetic characterization of fig tree mutants with molecular markers

The fig (Ficus carica L.) is a fruit tree of great world importance and, therefore, the genetic improvement becomes an important field of research for better crops, being necessary to gather information on this species, mainly regarding its genetic variability so that appropriate propagation projects and management are made. The improvement programs of fig trees using conventional procedures in order to obtain new cultivars are rare in many countries, such as Brazil, especially due to the little genetic variability and to the difficulties in obtaining plants from gamete fusion once the wasp Blastophaga psenes, responsible for the natural pollinating, is not found in Brazil. In this way, the mutagenic genetic improvement becomes a solution of it. For this reason, in an experiment conducted earlier, fig plants formed by cuttings treated with gamma ray were selected based on their agronomic characteristics of interest. We determined the genetic variability in these fig tree selections, using RAPD and AFLP molecular markers, comparing them to each other and to the Roxo-de-Valinhos, used as the standard. For the reactions of DNA amplification, 140 RAPD primers and 12 primer combinations for AFLP analysis were used. The selections did not differ genetically between themselves and between them and the Roxo-de-Valinhos cultivar. Techniques that can detect polymorphism between treatments, such as DNA sequencing, must be tested. The phenotypic variation of plants may be due to epigenetic variation, necessitating the use of techniques with methylation-sensitive restriction enzymes.

Application of Microsatellite Primers Developed for Polistes in the Independent-Founding Wasp Polists satan Bequaert (Hymenoptera: Vespidae)

Microsatellite primers developed for a given species are sometimes useful for another in the same genus and in other genera within the same family, making possible to search for pre-existing suitable primers in the databanks such as GenBank. We examined whether existing primers developed for Polistes could be used for Polistes satan Bequaert. We tested 50 microsatellite primers from three Polistes species and found that six microsatellite loci show polymorphism in size in P. satan. These six loci were highly polymorphic, having four to 15 alleles in P. satan with an expected heterozygosity of 0.525-0.832. These loci can be used to study parameters concerning genetic relatedness such as social interactions in colonies and genetic conflicts of interest among nestmate individuals.

Genetic diversity of polysporic isolates of Moniliophthora perniciosa (Tricholomataceae)

The causal agent of witches' broom disease, Moniliophthora perniciosa is a hemibiotrophic and endemic fungus of the Amazon basin and the most important cocoa disease in Brazil. The purpose of this study was to analyze the genetic diversity of polysporic isolates of M. perniciosa to evaluate the adaptation of the pathogen from different Brazilian regions and its association with different hosts. Polysporic isolates obtained previously in potato dextrose agar cultures of M. perniciosa from different Brazilian states and different hosts (Theobroma cacao, Solanum cernuum, S. paniculatum, S. lycocarpum, Solanum sp, and others) were analyzed by somatic compatibility grouping where the mycelium interactions were distinguished after 4-8 weeks of confrontation between the different isolates of M. perniciosa based on the precipitation line in the transition zone and by protein electrophoresis through SDS-PAGE. The diversity of polysporic isolates of M. perniciosa was grouped according to geographic proximity and respective hosts. The great genetic diversity of M. perniciosa strains from different Brazilian states and hosts favored adaptation in unusual environments and dissemination at long distances generating new biotypes.

Low polymorphism revealed in new microsatellite markers for Bemisia tabaci (Hemiptera: Aleyrodidae)

The silverleaf whitefly (Bemisia tabaci) is actually a complex of morphologically indistinct species that vary in their capacity to transmit plant viruses and to induce physiological disorders in plants of economic importance. The worldwide impact of this whitefly has increased greatly, as it is a vector of various types of phytovirus, especially geminiviruses, in plants of economic importance. The adaptability of B. tabaci to many regions of the world has fostered the appearance of various biotypes that attack a broad spectrum of host plants. We developed microsatellite markers to study genetic variability and population structure of this whitefly in Brazil. Thirteen polymorphic microsatellite markers were isolated and characterized in 20 individuals from a natural population that were collected in soybean in Campinas (SP). The number of alleles per locus ranged from one to two, and the expected heterozygosity ranged from 0.000 to 0.505. These microsatellite markers will be useful for studies and management of B. tabaci. The low polymorphism found in these molecular markers is probably associated with homology of genes expressed in these markers.

MICROSATELLITE MARKERS FOR PLATHYMENIA RETICULATA (LEGUMINOSAE)

Premise of the study: Microsatellite markers were developed and characterized to investigate genetic diversity and gene flow and to help in conservation efforts for the endangered timber species Plathymenia reticulata. Methods and Results: Eleven microsatellite loci were characterized using 60 adult trees of two populations of P. reticulata from the Atlantic Forest of southern Bahia, Brazil. Of these, nine loci were polymorphic, with an average of 4.39 alleles per locus. The average expected heterozygosity per population ranged from 0.47 to 0.55. The combined exclusion probability was 0.99996. Conclusions: Our results reveal that the microsatellite markers developed in this study are an effective tool for paternity and genetic structure analysis that may be useful for conservation strategies.

DEVELOPMENT OF MICROSATELLITE PRIMERS FOR JATROPHA CURCAS (EUPHORBIACEAE) AND TRANSFERABILITY TO CONGENERS

Premise of the study: Microsatellite primers were developed for Jatropha curcas (Euphorbiaceae), a tree species with large potential for biofuel production, to investigate its natural genetic diversity and mating system to facilitate the establishment of tree improvement and conservation programs. Methods and Results: Using a protocol for genomic library enrichment, 104 clones containing 195 repeat motifs were identified. Primer pairs were developed for 40 microsatellite loci and validated in 41 accessions of J. curcas from six provenances. Nine loci were polymorphic revealing from two to eight alleles per locus, and six primers were able to amplify alleles in the congeners J. podagrica, J. pohliana, and J. gossypifolia, but not in other Euphorbiaceae species, such as Hevea brasiliensis, Manihot esculenta, or Ricinus communis. Conclusions: The primers developed here revealed polymorphic loci that are suitable for genetic diversity and structure, mating system, and gene flow studies in J. curcas, and some congeners.

DEVELOPMENT OF MICROSATELLITE MARKERS FOR QUALEA GRANDIFLORA (VOCHYSIACEAE), A TYPICAL SPECIES OF THE BRAZILIAN CERRADO

Premise of the study: Microsatellite primers were developed to investigate genetic diversity and population structure of Qualea grandiflora, a typical species of the Brazilian cerrado. Methods and Results: Eight microsatellite loci were isolated using an enrichment cloning protocol. These loci were tested on a population of 110 individuals of Q. grandiflora collected from a cerrado fragment in Sao Paulo State, Brazil. The loci polymorphism ranges from seven to 19 alleles and the average heterozygosity value is 0.568, while the average polymorphic information content is 0.799. Conclusions: The developed markers were found to be highly polymorphic, indicating their applicability to studies of population genetic diversity in Q. grandiflora

Comparison of microsatellites and isozymes in genetic diversity studies of Oryza glumaepatula (Poaceae) populations

The study of the genetic structure of wild plant populations is essential for their management and conservation. Several DNA markers have been used in such studies, as well as isozyme markers. In order to provide a better comprehension of the results obtained and a comparison between markers which will help choose tools for future studies in natural populations of Oryza glumaepatula, a predominantly autogamous species, this study used both isozymes and microsatellites to assess the genetic diversity and genetic structure of 13 populations, pointing to similarities and divergences of each marker, and evaluating the relative importance of the results for studies of population genetics and conservation. A bulk sample for each population was obtained, by sampling two to three seeds of each plant, up to a set of 50 seeds. Amplified products of eight SSR loci were electrophoresed on non-denaturing polyacrylamide gels, and the fragments were visualized using silver staining procedure. Isozyme analyses were conducted in polyacrylamide gels, under a discontinuous system, using six enzymatic loci. SSR loci showed higher mean levels of genetic diversity (A=2.83, p=0.71, A(P)=3.17, H-o=0.081, H-e=0.351) than isozyme loci (A=1.20, p=0.20, A(P)=1.38, H-o=0.006, H-e=0.056). Interpopulation genetic differentiation detected by SSR loci (R-ST=0.631, equivalent to F-ST=0.533) was lower than that obtained with isozymes (F-ST=0.772). However, both markers showed high deviation from Hardy-Weinberg expectations (F-IS=0.744 and 0.899, respectively for SSR and isozymes). The mean apparent outcrossing rate for SSR ((t) over bar (a)=0.14) was higher than that obtained using isozymes ((t) over bar (a)=0.043), although both markers detected lower levels of outcrossing in Amazonia compared to the Pantanal. The migrant number estimation was also higher for SSR (Nm=0.219) than isozymes (Nm=0.074), although a small number for both markers was expected due to the mode of reproduction of this species, defined as mixed with predominance of self fertilization. No correlation was obtained between genetic and geographic distances with SSR, but a positive correlation was found between genetic and geographic distances with isozymes. We conclude that these markers are divergent in detecting genetic diversity parameters in O. glumaepatula and that microsatellites are powerful for detecting information at the intra-population level, while isozymes are more powerful for inter-population diversity, since clustering of populations agreed with the expectations based on the geographic distribution of the populations using this marker. Rev. Biol. Trop. 60 (4): 1463-1478. Epub 2012 December 01.

Genetic diversity of Leishmania amazonensis strains isolated in northeastern Brazil as revealed by DNA sequencing, PCR-based analyses and molecular karyotyping

Abstract Background Leishmania (Leishmania) amazonensis infection in man results in a clinical spectrum of disease manifestations ranging from cutaneous to mucosal or visceral involvement. In the present study, we have investigated the genetic variability of 18 L. amazonensis strains isolated in northeastern Brazil from patients with different clinical manifestations of leishmaniasis. Parasite DNA was analyzed by sequencing of the ITS flanking the 5.8 S subunit of the ribosomal RNA genes, by RAPD and SSR-PCR and by PFGE followed by hybridization with gene-specific probes. Results ITS sequencing and PCR-based methods revealed genetic heterogeneity among the L. amazonensis isolates examined and molecular karyotyping also showed variation in the chromosome size of different isolates. Unrooted genetic trees separated strains into different groups. Conclusion These results indicate that L. amazonensis strains isolated from leishmaniasis patients from northeastern Brazil are genetically diverse, however, no correlation between genetic polymorphism and phenotype were found.

Genetic characterization and molecular identification of the bloodmeal sources of the potential bluetongue vector Culicoides obsoletus in the Canary Islands, Spain

[EN] Background: Culicoides (Diptera: Ceratopogonidae) biting midges are vectors for a diversity of pathogens including bluetongue virus (BTV) that generate important economic losses. BTV has expanded its range in recent decades, probably due to the expansion of its main vector and the presence of other autochthonous competent vectors. Although the Canary Islands are still free of bluetongue disease (BTD), Spain and Europe have had to face up to a spread of bluetongue with disastrous consequences. Therefore, it is essential to identify the distribution of biting midges and understand their feeding patterns in areas susceptible to BTD. To that end, we captured biting midges on two farms in the Canary Islands (i) to identify the midge species in question and characterize their COI barcoding region and (ii) to ascertain the source of their bloodmeals using molecular tools.Methods: Biting midges were captured using CDC traps baited with a 4-W blacklight (UV) bulb on Gran Canaria and on Tenerife. Biting midges were quantified and identified according to their wing patterns. A 688 bp segment of the mitochondrial COI gene of 20 biting midges (11 from Gran Canaria and 9 from Tenerife) were PCR amplified using the primers LCO1490 and HCO2198. Moreover, after selected all available females showing any rest of blood in their abdomen, a nested-PCR approach was used to amplify a fragment of the COI gene from vertebrate DNA contained in bloodmeals. The origin of bloodmeals was identified by comparison with the nucleotide-nucleotide basic alignment search tool (BLAST). Results: The morphological identification of 491 female biting midges revealed the presence of a single morphospecies belonging to the Obsoletus group. When sequencing the barcoding region of the 20 females used to check genetic variability, we identified two haplotypes differing in a single base. Comparison analysis using the nucleotide-nucleotide basic alignment search tool (BLAST) showed that both haplotypes belong to Culicoides obsoletus, a potential BTV vector. As well, using molecular tools we identified the feeding sources of 136 biting midges and were able to confirm that C. obsoletus females feed on goats and sheep on both islands.Conclusions: These results confirm that the feeding pattern of C. obsoletus is a potentially important factor in BTV transmission to susceptible hosts in case of introduction into the archipelago. Consequently, in the Canary Islands it is essential to maintain vigilance of Culicoides-transmitted viruses such as BTV and the novel Schmallenberg virus.

Phylogeny and genetic diversity of Italian species of hares (genus Lepus)

In this study we have analysed the genetic variability in ca. 700 samples belonging to six species of genus Lepus using maternal and biparental molecular markers (mitochondrial DNA, microsatellites, Single Nucleotide Polimorphisms). We aimed to reconstruct the phylogenetic relationships of species of hares living in Europe, and assess the occurrence of hybridization between the European hare Lepus europaeus and the Italian hare Lepus corsicanus. Results showed a deep genetic differentiation and absence of hybridization between L. corsicanus and L. europaeus, confirming that they are distinct and distantly related biological species. In contrast, we showed small genetic distances and a close phylogenetic relationship between the Italian hare and Cantabrian hare L. castroviejoi, which suggest a deeper evaluation of their taxonomic status. Populations of L. corsicanus are geographically differentiated. In particular, the peninsular and Sicilian populations of Italian hares are sharply genetically distinct, which calls for avoiding any translocation between Italy and Sicily. Information on genetic variability and population structure is being used to implement the Italian Action Plan for L. corsicanus.

Genetic characterization, population history and evolutionary medicine perspective in two native south american populations: Yanesha and Wichi

Two Amerindian populations from the Peruvian Amazon (Yanesha) and from rural lowlands of the Argentinean Gran Chaco (Wichi) were analyzed. They represent two case study of the South American genetic variability. The Yanesha represent a model of population isolated for long-time in the Amazon rainforest, characterized by environmental and altitudinal stratifications. The Wichi represent a model of population living in an area recently colonized by European populations (the Criollos are the population of the admixed descendents), whose aim is to depict the native ancestral gene pool and the degree of admixture, in relation to the very high prevalence of Chagas disease. The methods used for the genotyping are common, concerning the Y chromosome markers (male lineage) and the mitochondrial markers (maternal lineage). The determination of the phylogeographic diagnostic polymorphisms was carried out by the classical techniques of PCR, restriction enzymes, sequencing and specific mini-sequencing. New method for the detection of the protozoa Trypanosoma cruzi was developed by means of the nested PCR. The main results show patterns of genetic stratification in Yanesha forest communities, referable to different migrations at different times, estimated by Bayesian analyses. In particular Yanesha were considered as a population of transition between the Amazon basin and the Andean Cordillera, evaluating the potential migration routes and the separation of clusters of community in relation to different genetic bio-ancestry. As the Wichi, the gene pool analyzed appears clearly differentiated by the admixed sympatric Criollos, due to strict social practices (deeply analyzed with the support of cultural anthropological tools) that have preserved the native identity at a diachronic level. A pattern of distribution of the seropositivity in relation to the different phylogenetic lineages (the adaptation in evolutionary terms) does not appear, neither Amerindian nor European, but in relation to environmental and living conditions of the two distinct subpopulations.

Endocarditis due to Tropheryma whipplei: rapid detection, limited genetic diversity, and long-term clinical outcome in a local experience

The characteristic features of Whipple's disease include abdominal pain, diarrhoea, wasting, and arthralgias, with the causative agent, Tropheryma whipplei, being detected mainly in intestinal biopsies. PCR technology has led to the identification of T. whipplei in specimens from various other locations, including the central nervous system and the heart. T. whipplei is now recognized as one of the causes of culture-negative endocarditis, and endocarditis can be the only manifestation of the infection with T. whipplei. Although it is considered a rare disease, the true incidence of endocarditis due to T. whipplei is not clearly established. With the increasing use of molecular methods, it is likely that T. whipplei will be more frequently identified. Questions also remain about the genetic variability of T. whipplei strains, optimal diagnostic procedures and therapeutic options. In the present study, we provide clinical data on four new patients with documented endocarditis due to T. whipplei in the context of the available published literature. There was no clinical involvement of the gastrointestinal tract. Genetic analysis of the T. whipplei strains with DNA isolated from the excised heart valves revealed little to no genetic variability. In a selected case, we describe acridine orange staining for early detection of the disease, prompting early adaptation of the antibiotic therapy. We provide long-term follow-up data on the patients. In our hands, an initial 2-week course of intravenous antibiotics followed by cotrimoxazole for at least 1 year was a suitable treatment option for T. whipplei endocarditis.