Diversidade genética e tamanho efetivo de duas populações de Myracrodruon urundeuva Fr. All., sob conservação ex situ

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Genotypic characterization of microsatellite markers in broiler and layer selected chicken lines and their reciprocal F1s

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

Genetic diversity between and within populations of Handroanthus heptaphyllus (Vell.) Mattos using microsatellite markers

Handroanthus heptaphyllus (Vell.) Mattos, popularmente conhecida por ipê-roxo, é uma espécie pertencente à família Bignoneaceae, muito apreciada por sua beleza, madeira de excelente qualidade e utilizada em produtos medicinais e programas de reflorestamento de áreas degradadas, paisagismo e restauração. Neste trabalho, objetivou-se estudar a diversidade genética entre e dentro das populações de H. heptaphyllus por meio de marcadores microssatélites. Foram estudadas 192 plântulas, formadas a partir de sementes colhidas de duas populações, em um total de 30 árvores, de fragmentos florestais naturais na região de Botucatu - SP. Foram analisados oito locos microssatélites, com polimorfismo alélico, variando de seis alelos para o loco TAU22 a 14 alelos para os locos TAU12, TAU30 e TAU31, com número efetivo médio de alelos por loco (Âe) igual a 4,9. As médias para a heterozigosidade esperada (Ĥe), para as duas populações foi de 0,785, a heterozigosidade observada (Ĥo)foi de 0,609 e o índice de fixação (^F) variou pouco entre as populações, com média de 0,222. O valor médio da divergência genética entre as duas populações (Ĝst') foi de 0,100. Conclui-se que a maior diversidade genética ocorre dentro das populações; portanto, em programa de coleta de germoplasma, para a região de Botucatu, é recomendado realizar uma maior amostragem de indivíduos dentro das populações, o que possibilitaria uma boa representatividade genética. As populações estudadas possuem diversidade genética para subsidiar programas de melhoramento genético e conservação de germoplasma.

Evaluation of nine microsatellite loci and misidentification paternity frequency in a population of Gyr breed bovines

Erros de identificação de paternidade são prejudiciais por reduzir o ganho genético anual e comprometer um programa eficiente de melhoramento genético. O objetivo principal deste trabalho foi avaliar o potencial de uso de nove microssatélites em testes de paternidade e investigar a freqüência de erro de identificação de famílias de um rebanho de animais da raça Gir. No experimento foram utilizadas amostras de sangue de quarenta famílias (touro/ vaca/ bezerro) de animais da raça Gir, Puros de Origem e registrados na Associação Brasileira dos Criadores de Zebu (ABCZ). A maior parte dos microssatélites avaliados neste trabalho são recomendados, para Testes de Paternidade em bovinos, pela Sociedade Internacional de Genética Animal (ISAG). As regiões microssatélites TGLA122, TGLA126, BM1824, BMS2533, SPS115, ETH3, ETH10, ETH225 e POTCHA foram amplificadas por meio da técnica de PCR. Os produtos da amplificação foram separados por eletroforese em gel de poliacrilamida desnaturante. A partir dos dados obtidos foram calculadas as freqüências alélicas, diversidade gênica, conteúdo de polimorfismo informativo e probabilidade de exclusão para cada microssatélite. Também foram calculadas as freqüências genotípicas, heterozigosidade, probabilidade de exclusão combinada e probabilidade de Paternidade nas famílias consideradas. A probabilidade de exclusão combinada para todos os microssatélites estudados foi de 0,9789. Os resultados dos testes de paternidade acusaram erro de identificação em onze das 40 famílias estudadas, ou seja, 27,5% da amostra. A probabilidade de paternidade variou entre 0,8691 e 0,9999, com valor médio de 0,9512.

Structure and genetic relationships between Brazilian naturalized and exotic purebred goat domestic goat (Capra hircus) breeds based on microsatellites

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

Allelic imbalance studies of chromosome 9 suggest major differences in chromosomal instability among nonmelanoma skin carcinomas

CONTEXTO: Vários estudos de perda de heterozigozidade na região 9p21-p22, que abriga os genes supressores tumorais CDKN2a/p16INK4a, p19ARF e p15INK4b, têm sido realizados em uma ampla série de tumores humanos, incluindo os melanomas familiares. Perdas e ganhos em outras regiões do cromossomo 9 também têm sido observados com freqüência e podem indicar mecanismos adicionais no processo de tumorigênese dos carcinomas basocelulares da pele. OBJETIVO: Investigar o equilíbrio alélico existente na região 9p21-p22 em carcinomas basocelulares. TIPO DE ESTUDO: Análise molecular de marcadores de microssatélites em tumores e controles. LOCAL: Dois serviços de dermatologia de atendimento terciário em universidades públicas de São Paulo e o Laboratório de Genética Molecular do Câncer da Universidade Estadual de Campinas (UNICAMP), Brasil. PARTICIPANTES: Examinamos 13 casos benignos, incluindo 4 queratoses solares, 3 queratoacantomas, 3 nevos melanocíticos, 2 doenças de Bowen e 1 neurofibroma cutâneo, além de 58 tumores malignos da pele: 14 de células escamosas, 40 carcinomas basocelulares e 4 melanomas; em pacientes consecutivamente encaminhados à clínica de Dermatologia da Unicamp e que concordaram em participar do estudo. VARIÁVEIS ESTUDADAS: O tumor principal e uma porção normal de pele não-adjacente foram removidos cirurgicamente de pacientes que consecutivamente procuraram os ambulatórios de dermatologia da Universidade Estadual de Campinas (UNICAMP) e da Universidade Estadual de São Paulo (Unesp), São Paulo, por causa de lesões cutâneas. Extraímos DNA tanto de tecido tumoral como do correspondente tecido normal de cada paciente. Para amplificar regiões de repetição polimórfica de microssatélites do cromossomo 9, foram utilizados quatro pares de primers, sendo dois deles destinados à região 9p21-p22. RESULTADOS: Identificamos oito casos (20%) de desequilíbrio alélico entre os carcinomas basocelulares, sendo dois casos de perda de heterozigozidade e seis casos de instabilidade de microssatélite na região 9p21-p22. Outros marcadores também mostravam anormalidades em três destes tumores, enquanto nenhuma alteração foi detectada entre os casos benignos e nos outros tumores malignos. CONCLUSÃO: Esta dependência fenotípica sugere que existem diferenças importantes no comportamento das formas mais comuns de tumores cutâneos não-melanocíticos em relação à sua tendência para instabilidade de microssatélite no cromossomo 9. Considerando-se que os genes CDKN2a/p16INK4a, p19ARF e p15INK4b não parecem responsáveis pelas anormalidades observadas, outros genes em 9p21-p22 podem estar envolvidos na etiopatogenia e na progressão dos carcinomas basocelulares.

Comparative genomic hybridization analysis detects frequent over-representation of DNA sequences at 3q, 7p, and 8q in head and neck carcinomas

Comparative genomic hybridization (CGH) was used to identify chromosomal imbalances in 19 samples of squamous cell carcinoma of the head and neck (HNSCC). The chromosome arms most often or er-represented were 3q (48%), 8q (42%), and 7p (32%); in many cases, these changes were observed at high copy number. Other commonly over-represented sites were 1q, 2q, 6p, 6q, and 18q. The most frequently under-represented segments were 3p and 22q. Loss of heterozygosity of two polymorphic microsatellite loci from chromosome 22 was observed in two tongue tumors, in agreement with the CGH analysis. Gains of 1q and 2q material were detected in patients exhibiting a clinical history of recurrence and/or metastasis followed by terminal disease. This association suggests that gain of 1q and 2q map be a new marker of head and neck tumors with a refractory clinical response. (C) 2000 Elsevier B.V. All rights reserved.

Quantitative real-time PCR identifies a critical region of deletion on 22q13 related to prognosis in oral cancer

Quantitative real time PCR was performed on genomic DNA from 40 primary oral carcinomas and the normal adjacent tissues. The target genes ECGFB, DIA1, BIK, and PDGFB and the microsatellite markers D22S274 and D22S277, mapped on 22q13, were selected according to our previous loss of heterozygosity findings in head and neck tumors. Quantitative PCR relies on the comparison of the amount of product generated from a target gene and that generated from a disomic reference gene (GAPDH-housekeeping gene). Reactions have been performed with normal control in triplicates, using the 7700 Sequence Detection System (PE Applied Biosystems). Losses in the sequences D22S274 (22q13.31) and in the DIA1 (22q13.2-13.31) gene were detected in 10 out of 40 cases (25%) each. Statistically significant correlations were observed for patients with relative copy number loss of the marker D22S274 and stages T3-T4 of disease (P=0.025), family history of cancer (P = 0.001), and death (P = 0.021). Relative copy number loss involving the DIA1 gene was correlated to family history of cancer (P<0.001), death (P=0.002), and consumption of alcohol (P=0.026). Log-rank test revealed a significant decrease in survival (P=0.0018) for patients with DIA1 gene loss. Relative copy number losses detected in these sequences may be related to disease progression and a worse prognosis in patients with oral cancer.

Isolation and characterization of microsatellite loci in the woolly mouse opossum, Micoureus paraguayanus (Marsupialia : Didelphimorphia)

Five microsatellite loci were isolated and characterized within the woolly mouse opossum (Micoureus paraguayanus), a Neotropical marsupial, using an enrichment cloning procedure. Between four and seven alleles were detected per locus, with expected heterozygosity ranging from 0.358 to 0.560. These microsatellites should provide useful markers in a variety of genetic analyses to examine parentage, inbreeding, population structure and population dynamics in fragmented forest habitats.

Genetic structure of Blastocerus dichotomus populations in the Parana River basin (Brazil) based on protein variability

The population structure of 147 marsh deer (Blastocerus dichotomus) from three areas in the Parana River basin, Brazil, was studied by observing protein polymorphism at 17 loci. Six loci were polymorphic and 11 monomorphic. The proportion of polymorphic loci (P) was 35.29% and the average heterozygosity (H) was 6.31%. Wright's F-ST indicated that only 4.9% of the total variation in allelic frequencies was due to genetic differences between the three groups. The high value of F-IS (0.246) indicated inbreeding in the marsh deer. Genetic distance values (D = 0.014-0.051) showed little divergence between the three areas. We suggest that probable mechanisms accounting for the genetic structure are female phylopatry and polygyny and also that inbreeding has resulted from decreasing areas of wetland leading to isolation, overhunting, and diseases transmitted by cattle.

Phylogeography of the Solanaceae-infecting Basidiomycota fungus Rhizoctonia solani AG-3 based on sequence analysis of two nuclear DNA loci

Background: the soil fungus Rhizoctonia solani anastomosis group 3 (AG-3) is an important pathogen of cultivated plants in the family Solanaceae. Isolates of R. solani AG-3 are taxonomically related based on the composition of cellular fatty acids, phylogenetic analysis of nuclear ribosomal DNA (rDNA) and beta-tubulin gene sequences, and somatic hyphal interactions. Despite the close genetic relationship among isolates of R. solani AG-3, field populations from potato and tobacco exhibit comparative differences in their disease biology, dispersal ecology, host specialization, genetic diversity and population structure. However, little information is available on how field populations of R. solani AG-3 on potato and tobacco are shaped by population genetic processes. In this study, two field populations of R. solani AG-3 from potato in North Carolina (NC) and the Northern USA; and two field populations from tobacco in NC and Southern Brazil were examined using sequence analysis of two cloned regions of nuclear DNA (pP42F and pP89).Results: Populations of R. solani AG-3 from potato were genetically diverse with a high frequency of heterozygosity, while limited or no genetic diversity was observed within the highly homozygous tobacco populations from NC and Brazil. Except for one isolate (TBR24), all NC and Brazilian isolates from tobacco shared the same alleles. No alleles were shared between potato and tobacco populations of R. solani AG-3, indicating no gene flow between them. To infer historical events that influenced current geographical patterns observed for populations of R. solani AG-3 from potato, we performed an analysis of molecular variance (AMOVA) and a nested clade analysis (NCA). Population differentiation was detected for locus pP89 (Phi(ST) = 0.257, significant at P < 0.05) but not for locus pP42F (Phi(ST) = 0.034, not significant). Results based on NCA of the pP89 locus suggest that historical restricted gene flow is a plausible explanation for the geographical association of clades. Coalescent-based simulations of genealogical relationships between populations of R. solani AG-3 from potato and tobacco were used to estimate the amount and directionality of historical migration patterns in time, and the ages of mutations of populations. Low rates of historical movement of genes were observed between the potato and tobacco populations of R. solani AG-3.Conclusion: the two sisters populations of the basidiomycete fungus R. solani AG-3 from potato and tobacco represent two genetically distinct and historically divergent lineages that have probably evolved within the range of their particular related Solanaceae hosts as sympatric species.

Isolation and characterization of polymorphic microsatellites for the natural populations of barker frog Physalaemus cuvieri

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

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.

Isolation and characterization of 15 polymorphic microsatellites in the Plethodontid salamander Ensatina eschscholtzii

We developed 15 new polymorphic microsatellites for the plethodontid salamander Ensatina eschscholtzii. Loci were isolated from a genomic library from Ensatina eschscholtzii xanthoptica enriched for (AAAG)(n) repetitive elements. The number of alleles per locus ranged from 4 to 20 (mean 9) in the sampled population. Observed heterozygosity ranged from 0.37 to 1. None of the loci deviated from Hardy-Weinberg equilibrium or showed significant linkage disequilibrium after a Bonferroni correction for multiple comparisons. All loci amplified in the six other subspecies of the Ensatina eschscholtzii complex. These new markers will prove useful in measuring gene flow and population structure as well as patterns of mating and sperm use in Ensatina.

Isolation and characterization of ten microsatellite loci from the Patagonian notothenioid fish Eleginops maclovinus

Eleginops maclovinus, the Patagonian blennie, is a notothenioid (Perciformes) endemic to South American temperate and sub-Antarctic waters. Here, we report ten polymorphic microsatellite loci isolated from a dinucleotide-enriched E. maclovinus genomic library. Among 48 individuals, the number of alleles per locus ranged from eight to 41, and the observed and expected heterozygosity ranged from 0.688 to 0.938 and from 0.695 to 0.968, respectively. No locus significantly deviated from Hardy-Weinberg equilibrium, and no significant linkage disequilibrium between pairs of loci was found. These polymorphic microsatellite loci will be useful for investigating genetic population structure and connectivity among natural populations.