Extensive microsatellite diversity in the human malaria parasite Plasmodium vivax

The population structure of Plasmodium vivax remains elusive. The markers of choice for large-scale population genetic studies of eukaryotes, short tandem repeats known as microsatellites, have been recently reported to be less polymorphic in R vivax. Here we investigate the microsatellite diversity and geographic structure in P vivax, at both local and global levels, using 14 new markers consisting of tri- or tetranucleotide repeats. The local-level analysis, which involved 50 field isolates from Sri Lanka, revealed unexpectedly high diversity (average virtual heterozygosity [H-E], 0.807) and significant multilocus linkage disequilibrium in this region of low malaria endemicity. Multiple-clone infections occurred in 60% of isolates sampled in 2005. The global-level analysis of field isolates or monkey-adapted strains identified 150 unique haplotypes among 164 parasites from four continents. Individual P. vivax isolates could not be unambiguously assigned to geographic populations. For example, we found relatively low divergence among parasites from Central America, Africa, Southeast Asia and Oceania, but substantial differentiation between parasites from the same continent (South Asia and Southeast Asia) or even from the same country (Brazil). Parasite relapses, which may extend the duration of P. vivax carriage in humans, are suggested to facilitate the spread of strains across continents, breaking down any pre-existing geographic structure. (C) 2008 Elsevier B.V. All rights reserved.

Trypanosoma cruzi maxicircle heterogeneity in Chagas disease patients from Brazil

The majority of individuals in the chronic phase of Chagas disease are asymptomatic (indeterminate form, IF). Each year, similar to 3% of them develop lesions in the heart or gastrointestinal tract. Cardiomyopathy (CCHD) is the most severe manifestation of Chagas disease. The factors that determine the outcome of the infection are unknown, but certainly depend on complex interactions amongst the genetic make-up of the parasite, the host immunogenetic background and environment. In a previous study we verified that the maxicircle gene NADH dehydrogenase (mitochondrial complex 1) subunit 7 (ND7) from IF isolates had a 455 bp deletion compared with the wild type (WT) ND7 gene from CCHD strains. We proposed that ND7 could constitute a valuable target for PCR assays in the differential diagnosis of the infective strain. In the present study we evaluated this hypothesis by examination of ND7 structure in parasites from 75 patients with defined pathologies, from Southeast Brazil. We also analysed the structure of additional mitochondrial genes (ND4/CR4, COIII and COII) since the maxicircle is used for clustering Trypanosoma cruzi strains into three clades/haplogroups. We conclude that maxicircle genes do not discriminate parasite populations which induce IF or CCHD forms. Interestingly, the great majority of the analysed isolates belong to T cruzi 11 (discrete typing unit, (DTU) IIb) genotype. This scenario is at variance with the prevalence of hybrid (DTU IId) human isolates in Bolivia, Chile and Argentina. The distribution of WT and deleted ND7 and ND4 genes in T cruzi strains suggests that mutations in the two genes occurred in different ancestrals in the T cruzi 11 cluster, allowing the identification of at least three mitochondrial sub-lineages within this group. The observation that T. cruzi strains accumulate mutations in several genes coding for complex I subunits favours the hypothesis that complex I may have a limited activity in this parasite. (C) 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Estrutura genética populacional de Heliconius erato e Heliconius melpomene (Lepidoptera: Nymphalidae) em fragmentos de Mata Atlântica do Rio Grande do Norte

Extensive studies using molecular markers on butterflies have shown how a highly fragmented landscape may result in the reduction of gene flow among patches of habitat and, consequently, increase genetic differentiation among populations. However, little is known about Heliconius geographical structure and the effects of fragmentation on the connectivity of populations. Furthermore, findings on the effects of the population structure on the dynamics of mimicry evolution in Heliconius butterflies need to be tested in H. erato and H. melpomene specimens found in other locations other than Central and northern South Americas. For the present study, we had two motivations: (1) compare the population structure of H. erato and H. melpomene given the highly fragmented Brazil s Atlantic Forest habitat; and (2) studying population structure of co-mimics could give us insights into the dynamics of mimicry evolution. For this, we analysed the spatial structure and connectivity of eight populations of Heliconius butterflies, in a total of 137 H. erato specimens and 145 H. melpomene specimens, using nine microsatellites loci, 1144 AFLPs markers and 282 mitochondrial DNA sequences. In general, both species exhibited evidence of population subdivision but no isolation by distance indicating some extent of genetic differentiation among populations. Contrary to Kronforst & Gilbert s (2008) Costa Rican Heliconius, H. melpomene exhibited more genetic differentiation than H. erato based on nuclear markers. However, for mitochondrial DNA, H. erato populations showed more genetic differentiation than H. melpomene. Our results corroborate to other studies on Heliconius butterflies concerning the pronounced population subdivision and local genetic drift found in this genus. Nevertheless, the pattern of this differentiation varies significantly from the pattern found in studies conducted in Central America, where H. erato is generally more differentiated and structured than H. melpomene, based on nuclear markers. This different pattern may reflect different evolutionary histories of Heliconius species in Northeastern Brazil s Atlantic Forest

Genetic variability among the wild boars (Sus scrofa scrofa), crossbred animals and pigs using microsatellite markers (STRs)

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

Development and characterization of microsatellite loci in the marsh deer (Blastocerus dichotomus Cervidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Diversidade genética em populações-núcleo de Eucalyptus grandis

No melhoramento genético de espécies florestais, uma população base ou indivíduos superiores pré-selecionados tem importância fundamental para a manutenção do programa. Indivíduos de melhores procedências e de ampla base genética propiciam a obtenção de ganhos de forma contínua. O objetivo deste trabalho foi avaliar a diversidade genética em duas populações-núcleo de Eucalyptus grandis. Foram avaliados 39 indivíduos, sendo 19 pertencentes à população 1 e 20, à população 2, utilizando-se 14 primers microssatélite. Os fragmentos foram identificados e analisados a partir dos programas GeneScan e Genotyper, utilizando-se um sequenciador automático ABI Prism 3100. O número de alelos encontrados para cada primer variou de cinco a 15 para a população 1 e, de 8 a 18 para a população 2. A heterozigosidade estimada foi maior na população 2, 0,869, contra 0,843 na população 1. A média da distância genética entre os indivíduos da população 1 foi 0,6220 e na população 2 foi 0,6112. Com a caracterização molecular dos indivíduos destas populações foi construído um banco de dados que permitirá, a partir dos parâmetros de genética de populações, monitorar esses programas de melhoramento em diferentes ciclos de seleção.

Avaliação do transplante de medula óssea alogênico por meio do estudo de regiões de repetições seqüenciais no genoma humano (VNTRs e STRs)

Os objetivos deste estudo foram estabelecer um protocolo para a análise de minissatélites ou VNTRs e microssatélites ou STRs em pacientes que se submeteram ao TMO alogênico; verificar a validade da metodologia e dos loci estudados e avaliar o tipo de recuperação do paciente. Foram analisados o DNA do paciente anterior e posterior ao transplante de 14 indivíduos e dos respectivos doadores. Amplificações por PCR de seis loci: D1S80, SE33, HumTH01, 33.6, HumARA e HumTPO foram realizadas. Os produtos amplificados foram separados por eletro­forese vertical em gel de poliacrilamida, e os fragmentos visualizados por coloração pela prata. Esse procedimento mostrou ser válido na verificação da recuperação alogênica, autóloga e provavelmente na quimérica. da somatória dos loci estudados, 63,1% apresentaram resultados possíveis de serem avaliados e, desses, 19,0% mostraram resultado informativo, 13,1% parcialmente informativo e 31,0% não informativo. Os 36,9% restantes não foram possíveis de avaliação. Dos loci avaliados, o que demostrou maior índice de resultado informativo foi o SE33, parcialmente informativo o HumTPO e não informativo o HumTH01, sendo o locus 33.6 o que mais apresentou resultados não possíveis de serem avaliados. Por outro lado, determinou-se a recuperação do paciente posterior ao transplante em 71,4% dos indivíduos, sendo que, desses, 90% apresentaram recuperação alogênica e 10% recuperação autóloga.

Development and characterization of 14 microsatellite loci from an enriched genomic library of Eucalyptus camaldulensis Dehnh

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

Genética e biologia reprodutiva de vriesea minarum (Bromeliaceae): em busca de estratégias de conservação no quadrilátero ferrífero, minas gerais

Vriesea minarum is a rupiculous bromeliad species, with naturally fragmented populations, restricted to the Iron Quadrangle, Minas Gerais, Brazil. It is a threatened species, which is suffering from habitat loss due to the growth of cities and mining activities. The knowledge of genetic variability in plant populations is one of the main branches of conservation genetics, linking genetic data to conservation strategies while the knowledge about plant reproductive biology can aid in understanding key aspects of their life story, as well as in the comprehension of their distribution and survival strategies. Thus, the study of diversity, richness, and genetic structure, as well as the reproductive biology of populations of V. minarum can contribute to the development of conservation actions. Chapter 1 presents the transferability of 14 microsatellite loci for V. minarum. Among the results of this chapter, we highlight the successful transferability of 10 microsatellite loci described for other species of Bromeliaceae, all of which are polymorphic. In Chapter 2, we present the genetic analyses of 12 populations of V. minarum that are distributed throughout the Iron Quadrangle. We used the 10 microsatellite loci tested in Chapter 1. The results show a low population structuring (Fst = 0.088), but with different values of genetic richness (mean = 2.566) and gene diversity (mean = 0.635) for all populations; and a high inbreeding coefficient (Gis = 0.376). These may be the result of pollinators action and/or efficient seed dispersal, thus allowing a high connectivity among populations of naturally fragmented outcrops. The reproductive biology and floral morphology of a population of V. minarum, located in the Parque Estadual da Serra do Rola-Moça, are studied in Chapter 3. This reserve is the only public environmental protection area where the species occurs. As a result of field experiments and observations, we found that the species has its flowering period from January to March, with flowers that last for two days and that it has a mixed pollination syndrome. It is primarily alogamous, but also has the capacity to be self-ferilized. It is expected that data obtained in chapters 1, 2 and 3 serve as basis for other studies with species from the ferruginous rocky fields, since until now, to our knowledge, there are no other survey of endemic species from the Iron Quadrangle, seeking to merge the genetic knowledge, with the data of the reproductive biology, with the ultimate aim of biodiversity conservation. Considering the great habitat loss for the species by mining, it becomes crucial to analyze the creation of new protected areas for its conservation

Genetic diversity analysis in the section Caulorrhizae (genus Arachis) using microsatellite markers

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

Characterization and transferability of microsatellite markers of the cultivated peanut (Arachis hypogaea)

Background: the genus Arachis includes Arachis hypogaea (cultivated peanut) and wild species that are used in peanut breeding or as forage. Molecular markers have been employed in several studies of this genus, but microsatellite markers have only been used in few investigations. Microsatellites are very informative and are useful to assess genetic variability, analyze mating systems and in genetic mapping. The objectives of this study were to develop A. hypogaea microsatellite loci and to evaluate the transferability of these markers to other Arachis species.Results: Thirteen loci were isolated and characterized using 16 accessions of A. hypogaea. The level of variation found in A. hypogaea using microsatellites was higher than with other markers. Cross-transferability of the markers was also high. Sequencing of the fragments amplified using the primer pair AhII from 17 wild Arachis species showed that almost all wild species had similar repeated sequence to the one observed in A. hypogaea. Sequence data suggested that there is no correlation between taxonomic relationship of a wild species to A. hypogaea and the number of repeats found in its microsatellite loci.Conclusion: These results show that microsatellite primer pairs from A. hypogaea have multiple uses. A higher level of variation among A. hypogaea accessions can be detected using microsatellite markers in comparison to other markers, such as RFLP, RAPD and AFLP. The microsatellite primers of A. hypogaea showed a very high rate of transferability to other species of the genus. These primer pairs provide important tools to evaluate the genetic variability and to assess the mating system in Arachis species.

Chromosome 22q a frequent site of allele loss in head and neck carcinoma

Background. Loss of heterozygosity (LOH) correlates with inactivated tumor suppressor genes. LOH at chromosome arm 22q has been found in a variety of human neoplasms, suggesting that this region contains a tumor suppressor gene(s) other than NF2 important to tumorigenesis. The aim of this study was to evaluate the presence of LOH on chromosome 22q11.2-13 and determine whether there was a relationship between loss in this genomic region and tumor histologic parameters, anatomic site, and survival in patients with squamous cell carcinoma of the head and neck (HNSCC).Methods. Fifty matched blood and HNSCC tumor samples taken at the time of surgical treatment were evaluated for LOH by use of four microsatellite markers mapping to 22q11.2-q13. Clinical information was available for all patients. The frequency and distribution of LOH was correlated with clinical (age, sex, use of tobacco and alcohol, site of primary tumor, clinical stage, adjuvant therapy and overall survival) and histologic parameters (histopathologic stage, tumor differentiation).Results. LOH at 22q was found in 19 of 50 (38%) informative tumors. The respective incidence of allelic loss for the patients was as follows: 28% at D22S421, 10% at D22S277, 8% at D22S44S, and 4% at D22S280. No statistical differences were apparent with a mean follow-up of 30 months. Laryngeal tumors showed a higher incidence of LOH compared with oral tumors.Conclusions. These results suggest that the D22S277 locus may be closely linked to a tumor suppressor gene (TSG) and involved in upper aerodigestive tract carcinogenesis. In particular, laryngeal tumors may harbor another putative TSG on 22q11.2-q12.3 that may play a role in aggressive stage III/IV disease. (C) 2000 John Wiley & Sons, Inc.

A set of polymorphic microsatellite loci for Vriesea gigantea and Alcantarea imperialis (Bromeliaceae) and cross-amplification in other bromeliad species

Fifteen polymorphic microsatellite markers were isolated and characterized in two species of Bromeliaceae: Vriesea gigantea and Alcantarea imperialis. The number of alleles observed for each locus ranged from three to 16. The loci will be used for studies of the genetic structure of natural populations, reproductive biology, and evolutionary relationships among and within these genera. A cross-amplification test in 22 taxa suggests that the markers will be useful for similar applications in numerous other bromeliad species.

Novel polymorphic microsatellite markers in section Caulorrhizae (Arachis, Fabaceae)

This work reports the characterization of 11 polymorphic microsatellite loci in section Caulorrhizae. The primer pairs were designed from Arachis pintoi and showed full transferability to Arachis repens species. These new markers were used to evaluate the genetic diversity in germplasm (accessions and cultivars) of section Caulorrhizae. This new set of markers detected greater gene diversity than morphological and molecular markers such as AFLP (amplified fragment length polymorphism) and RAPD (rapid analysis of polymorphic DNA) previously used in this germplasm.

Isolation and characterization of microsatellite markers for Cascaria sylvestris Sw. (Salicaceae), a neotropical medicinal tree

Casearia sylvestris Sw. is a widespread neotropical tree utilized in popular medicine. Recent research ranked Casearia as one of the most promising genus in the search of drugs against cancer. Despite its wide distribution and pharmacological importance, no microsatellite markers have yet been developed for this genus. In this study, we provide 10 polymorphic microsatellite loci specifically designed for C. sylvestris, used to analyse 90 individuals distributed in two populations from São Paulo state, Brazil. on average, 12.3 alleles per locus were identified, showing the ability of the markers to detect microsatellite polymorphism in this species.