Parentage testing in alpacas (Vicugna pacos) using semi-automated fluorescent multiplex PCRs with 10 microsatellite markers

The aim of this study was to assess and apply a microsatellite multiplex system for parentage determination in alpacas. An approach for parentage testing based on 10 microsatellites was evaluated in a population of 329 unrelated alpacas from different geographical zones in Peru. All microsatellite markers, which amplified in two multiplex reactions, were highly polymorphic with a mean of 14.5 alleles per locus (six to 28 alleles per locus) and an average expected heterozygosity (H-E) of 0.8185 (range of 0.698-0.946). The total parentage exclusion probability was 0.999456 for excluding a candidate parent from parentage of an arbitrary offspring, given only the genotype of the offspring, and 0.999991 for excluding a candidate parent from parentage of an arbitrary offspring, given the genotype of the offspring and the other parent. In a case test of parentage assignment, the microsatellite panel assigned 38 (from 45 cases) offspring parentage to 10 sires with LOD scores ranging from 2.19 x 10(+13) to 1.34 x 10(+15) and Delta values ranging from 2.80 x 10(+12) to 1.34 x 10(+15) with an estimated pedigree error rate of 15.5%. The performance of this multiplex panel of markers suggests that it will be useful in parentage testing of alpacas.

In vitro Culture of Bovine Embryos in Murine ES Cell Conditioned Media Negatively Affects Expression of Pluripotency-Related Markers OCT4, SOX2 and SSEA1

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Documenting the advantages and limitations of different classes of molecular markers in a well-established phylogeographic context: lessons from the Iberian endemic Golden-striped salamander, Chioglossa lusitanica (Caudata: Salamandridae)

Previous analyses of mitochondrial (mt)DNA and allozymes covering the range of the Iberian endemic golden-striped salamander, Chioglossa lusitanica, suggested a Pleistocene split of the historical species distribution into two population units (north and south of the Mondego river), postglacial expansion into the northernmost extant range, and secondary contact with neutral diffusion of genes close to the Mondego river. We extended analysis of molecular variation over the species range using seven microsatellite loci and the nuclear P-fibrinogen intron 7 (beta-fibint7). Both microsatellites and beta-fibint7 showed moderate to high levels of population structure, concordant with patterns detected with mtDNA and allozymes; and a general pattern of isolation-by-distance, contrasting the marked differentiation of two population groups suggested by mtDNA and allozymes. Bayesian multilocus analyses showed contrasting results as populations north and south of the Douro river were clearly differentiated based on microsatellites, whereas allozymes revealed differentiation north and south of the Mondego river. Additionally, decreased microsatellite variability in the north supported the hypothesis of postglacial colonization of this region. The well-documented evolutionary history of C. lusitanica, provides an excellent framework within which the advantages and limitations of different classes of markers can be evaluated in defining patterns of population substructure and inferring evolutionary processes across distinct spatio-temporal scales. The present study serves as a cautionary note for investigations that rely on a single type of molecular marker, especially when the organism under study exhibits a widespread distribution and complex natural history. (C) 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95, 371-387.

Fecal Sterols in Estuarine Sediments as Markers of Sewage Contamination in the Cubatao Area, São Paulo, Brazil

Sterol biomarkers serve as an alternative method for detecting sewage pollution. Sterols were extracted from samples of surface sediment collected in Cubato (the Vila dos Pescadores and Vila Esperan double dagger a communities) and quantified using GC-MS after Soxhlet extraction, cleanup, and derivatization. Fecal contamination was evaluated based on the concentration of coprostanol and the ratio of the selected sterols. The most abundant sterol was cholestanol, followed by coprostanol. The concentrations of coprostanol in surface sediments ranged from a minimum of 4.21 mu g g(-1) dry sediment (Vila dos Pescadores station) to a maximum of 8.32 mu g g(-1) dry sediment (Vila Esperan double dagger a station). A coprostanol concentration of about 10 mu g g(-1) was found, indicating areas of high sewage contamination. Coprostanol levels at sewage stations were higher than in other Brazilian coastal areas, which may be attributed to the fraction of the population without sanitation services.

Long-Term Outcome and Prognostic Factors of Juvenile Dermatomyositis: A Multinational, Multicenter Study of 490 Patients

Objective. To investigate the long-term outcome and prognostic factors of juvenile dermatomyositis (DM) through a multinational, multicenter study.Methods. Patients consisted of inception cohorts seen between 1980 and 2004 in 27 centers in Europe and Latin America. Predictor variables were sex, continent, ethnicity, onset year, onset age, onset type, onset manifestations, course type, disease duration, and active disease duration. Outcomes were muscle strength/endurance, continued disease activity, cumulative damage, muscle damage, cutaneous damage, calcinosis, lipodystrophy, physical function, and health-related quality of life (HRQOL).Results. A total of 490 patients with a mean disease duration of 7.7 years were included. At the cross-sectional visit, 41.2-52.8% of patients, depending on the instrument used, had reduced muscle strength/endurance, but less than 10% had severe impairment. Persistently active disease was recorded in 41.2-60.5% of the patients, depending on the activity measure used. Sixty-nine percent of the patients had cumulative damage. The frequency of calcinosis and lipodystrophy was 23.6% and 9.7%, respectively. A total of 40.7% of the patients had decreased functional ability, but only 6.5% had major impairment. Only a small fraction had decreased HRQOL. A chronic course, either polycyclic or continuous, consistently predicted a poorer outcome. Mortality rate was 3.1%.Conclusion. This study confirms the marked improvement in functional outcome of juvenile DM when compared with earlier literature. However, many patients had continued disease activity and cumulative damage at followup. A chronic course was the strongest predictor of poor prognosis. These findings highlight the need for treatment strategies that enable a better control of disease activity over time and the reduction of nonreversible damage.

Labeling index in pituitary adenomas evaluated by means of MIB-1: is there a prognostic role? A critical review

Objective: The present article presents an overview of the literature, and analyses the methods and the primary questions related to assessment of proliferation index using the Ki-67/MIB-1 labeling index in pituitary adenomas. Although atypical adenomas are characterized by their atypical morphological features by an elevated mitotic index, a Ki-67 (MIB-1) labeling index greater than 3% and extensive nuclear staining for p53, use of the proliferation index (LI) of pituitary adenomas in assessing the degree of tumor aggressiveness is a controversial topic in the literature, and there are disparate results involving many studies.Methods: A review of literature was carried out to correlate the role of Ki-67 LI and its correlation with clinical findings, tumor size, invasiveness, recurrence, adenoma subtype, adenoma doubling time, and pituitary carcinomas is addressed. Results: The prognosis cannot be predicted on the basis of the Ki-67 LI alone. Although there is no direct relation between Ki-67 LI and some of these variables and controversial data were found regarding some topics, our review justify the use of Ki-67 in the analysis of pituitary adenomas as an additional information for clinical decision.Conclusion: Although assessment of proliferative may be helpful in predicting subsequent tumor recurrence or invasiveness, there are many other important and as yet unidentified factors pituitary tumors. It is clear that further research is needed to clarify these molecular mechanisms to predict those with a potentially poor clinical outcome.

Evaluation of microsatellite markers in cultivars of sweet orange (Citrus sinensis [L.] Osbeck)

Sweet orange is considered a very important species in the citrus world market and presents wide morphological variability. However, its characterization at the molecular level by random amplified polymorphic DNA (RAPD) and isozyme markers is not appropriate. Microsatellite or simple sequence repeats (SSRs) have been suggested as ideal for studies in cultures of vegetative propagation and as value markers for mapping in several species. However, information on microsatellite polymorphism in citrus species is scarce. In this work, microsatellite markers (AG-repeats) were developed from an enrichment library of genomic DNA of sweet orange cv. Pera (Citrus sinensis [L.] Osbeck), and 31 cultivars of sweet orange were evaluated. Evaluation of 18 microsatellite primers did not permit differentiation of the varieties studied. New microsatellite primers are being evaluated with the aim of detecting polymorphisms among the cultivars and closely related species to be used in genetic mapping programs.

A Bayesian approach for constructing genetic maps when markers are miscoded

The advent of molecular markers has created opportunities for a better understanding of quantitative inheritance and for developing novel strategies for genetic improvement of agricultural species, using information on quantitative trait loci (QTL). A QTL analysis relies on accurate genetic marker maps. At present, most statistical methods used for map construction ignore the fact that molecular data may be read with error. Often, however, there is ambiguity about some marker genotypes. A Bayesian MCMC approach for inferences about a genetic marker map when random miscoding of genotypes occurs is presented, and simulated and real data sets are analyzed. The results suggest that unless there is strong reason to believe that genotypes are ascertained without error, the proposed approach provides more reliable inference on the genetic map.

Kappa-casein gene study with molecular markers in female buffaloes (Bubalus bubalis)

Caseins comprise make up about 80% of the total protein content of milk and present polymorphism with change in the amino acid sequence. Within this abundance of proteins, kappa-casein is noteworthy, since it has been associated with differences in milk yield, composition and processing. The objective of this study was to observe the existence of polymorphism in the kappa-casein gene in female buffaloes. For this purpose, blood samples from 115 female buffaloes, collected with vacutainer by needle punctionure of the jugular vein, were used. for genomic DNA extraction was done from blood samples. The PCR-RFLP and SSCP techniques demonstrated that the studied animals were monomorphic for the kappa-casein gene. Only allele B was observed in these animals, which was present in homozygosis. Therefore, it was not possible to quantify the gene action on milk yield and its constituents. The monomorphism observed in the population studied would allow the development of a method to identify mixtures of cow and buffalo milk in mozzarella cheese production, especially because, in cattle, the kappa-casein gene is polymorphic. Copyright by the Brazilian Society of Genetics.

Genetic divergence in Eucalyptus spp. clones by molecular RAPD markers

The genetic divergence in 20 Eucalyptus spp. clones was evaluated by multivariate techniques based on 167 RAPD markers, of which 155 were polymorphic and 12 monomorphic. The measures of genetic distances were obtained by the arithmetic complement of the coefficients of Jaccard and of Sorenso-Nei and Li and evaluated by the hierarchical methods of Single Linkage clustering and Unweighted Pair Group Method with Arithmetic Mean (UPGMA). Independent of the dissimilarity coefficient, the greatest divergence was found between clones 7 and 17 and the smallest between the clones 11 and 14. Clone clustering was little influenced by the applied procedure so that, adopting the same percentage of divergence, the UPGMA identified two groups less for the coefficient of Sorenso-Nei and Li. The clones evidenced considerable genetic divergence, which is partly associated to the origin of the study material. The clusters formed by the UPGMA clustering algorithm associated to the arithmetic complement of Jaccard were most consistent.

Transferability and use of microsatellite markers for the genetic analysis of the germplasm of some Arachis section species of the genus Arachis

The Arachis section is the most important of the nine sections of the genus Arachis because it includes the cultivated peanut, Arachis hypogaea. The genetic improvement of A. hypogaea using wild relatives is at an early stage of development in spite of their potential as sources of genes, including those for disease and pests resistance, that are not found in the A. hypogaea primary gene pool. Section Arachis species germplasm has been collected and maintained in gene banks and its use and effective conservation depends on our knowledge of the genetic variability contained in this material. Microsatellites are routinely used for the analysis of genetic variability because they are highly polymorphic and codominant. The objective of this study was to evaluate the transferability of microsatellite primers and the assay of genetic variability between and within the germplasm of some species of the Arachis section. Fourteen microsatellite loci developed for three different species of Arachis were analyzed and 11 (78%) were found to be polymorphic. All loci had transferability to all the species analyzed. The polymorphic loci were very informative, with expected heterozygosity per locus ranging from 0.70 to 0.94. In general, the germplasm analyzed showed wide genetic variation. © 2006 Sociedade Brasileira de Genética.

In situ expression of mononuclear cell markers and interleukin-2 receptor in renal allograft biopsies of acute rejection and borderline cases

The correct diagnosis of renal allograft rejection may be difficult using only clinical and/or histopathological criteria. Immunological assays should be considered in order to evaluate the phenotype of inflammatory infiltrate in renal allograft biopsies. Immunohistochemical studies were performed to detect mononuclear cells, CD4 and CD8 T lymphocytes, B lymphocytes, macrophages, null cells, and positive cells for interleukin-2 receptors. A total of 41 allograft biopsies classified into three groups were studied: acute cellular rejection (28 biopsies/22 patients), borderline (7 biopsies/5 patients) and control (6 biopsies/6 patients). In the rejection group (RG), increased cellularity was found mainly at the tubulo-interstitial level. Expression of CD8 positive cells was higher in RG when compared to borderline (BG) and control (CG) groups, respectively (0.9 vs. 0.0 vs. 0.35 cells/mm2; p < 0.001). Expression of macrophages was not statistically significant among the three groups (RG = 0.6 vs. BG = 0.2 vs. CG = 0.0 cells/mm2; p < 0.02). In the BG, CD4 + cells predominated (BG = 0.2 vs. RG = 0.05 vs. CG = 0.0 cells/mm2; p < 0.05). Clinically these patients were treated as cases of acute rejection. The numbers and different types of infiltrating cells did not correlate with patient's clinical outcome. Copyright © Informa Healthcare.

Taenia solium and Taenia saginata: Identification of sequence characterized amplified region (SCAR) markers

Cysticercosis is one of the most important zoonosis, not only because of the effects on animal health and its economic consequences, but also due to the serious danger it poses to humans. The two main parasites involved in the taeniasis-cysticercosis complex in Brazil are Taenia saginata and Taenia solium. Differentiating between these two parasites is important both for disease control and for epidemiological studies. The purpose of this work was to identify genetic markers that could be used to differentiate these parasites. Out of 120 oligonucleotide decamers tested in random amplified polymorphic DNA (RAPD) assays, 107 were shown to discriminate between the two species of Taenia. Twenty-one DNA fragments that were specific for each species of Taenia were chosen for DNA cloning and sequencing. Seven RAPD markers were converted into sequence characterized amplified region (SCAR) markers with two specific for T. saginata and five specific for T. solium as shown by agarose gel electrophoresis. These markers were developed as potential tools to differentiate T. solium from T. saginata in epidemiological studies. © 2007 Elsevier Inc. All rights reserved.

QTL identification for tolerance to fruit set in tomato by FAFLP markers

This study aimed to detect quantitative trait loci (QTL) by fALP (Fluorescent Amplified Fragment Length Polymorphism) markers associated to the trait tomato fruit set at high temperatures. A biparental cross between line Jab-95 (heat-tolerant) and cultivar Caribe (heat-susceptible) was made. A total of 192 plants of the F2 generation were evaluated, generating 172 polymorphic markers through six primer combinations previously identified by the Bulked Segregant Analysis technique. To construct the genetic map, 106 of the 172 markers that segregated in the expected Mendelian segregation proportion (3:1) were used. The map covered 1191.46 cM of the genome. Six trait-linked QTL were identified in the analysis of simple markers and three others by the interval-mapping methodology. These results could be highly useful in improvement programs, since heat-tolerant plants can be selected rapidly, which improves tomato fruit set.