Estudo de bactérias do gênero Gluconobacter: isolamento, purificação, identificação fenotípica e molecular

A importância do estudo de bactérias acéticas, em especial as do gênero Gluconobacter, está baseada em suas aplicações industriais, pois estas possuem a capacidade de bioconversão de sorbitol a sorbose, viabilizando o processo de produção de vitamina C. O estudo envolveu coletas de amostras em indústrias de refrigerante, flores, frutos e mel, seguidas de purificação, identificação fenotípica e identificação molecular, com a utilização de iniciador definido a partir de consulta ao Nucleotide Sequence Database. Preservaram-se as linhagens identificadas como membros da família Acetobacteriaceae, gênero Gluconobacter. Foi isolado um total de 110 linhagens dos substratos: Pyrostegia venusta (Cipó de São João), mel, Vitis vinifera (uva), Pyrus communis (pêra), Malus sp. (maçã) e de duas amostras de refrigerantes envasados em embalagens de PET de 2 L. Deste total, 57 linhagens foram recuperadas em meio MYP (manitol, extrato de levedura, peptona), 12 em meio YGM (glicose, manitol, extrato de levedura, etanol, ácido acético), 41 em meio de enriquecimento e, posteriormente, em meio GYC (glicose, extrato de levedura e carbonato de cálcio). Obtiveram-se 68 linhagens identificadas como bastonetes Gram negativos. Destas, 31 foram caracterizadas bioquimicamente como pertencentes à família Acetobacteriaceae por serem catalase positivas, oxidase negativas e produtoras de ácido a partir de glicose. A caracterização dessas linhagens foi complementada com os testes bioquímicos: liquefação da gelatina, redução de nitrato, formação de indol e H2S e oxidação de etanol a ácido acético. Métodos moleculares foram aplicados para identificação do gênero Gluconobacter. Finalmente, oito linhagens foram caracterizadas como pertencentes ao gênero Gluconobacter. As linhagens encontram-se depositadas em coleção de cultura do laboratório de Microbiologia do Departamento de Biologia da UNESP, campus de Assis, estocadas em extrato de malte 20 a -196 ºC.

Association of CSSM066 and ILSTS011 microsatellite markers and thyroglobulin gene SNP with backfat in Canchim cattle

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

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.

Development and characterization of polymorphic microsatellite markers for the sweet orange (Citrus sinensis L. Osbeck)

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

Evaluation of the genetic diversity of Xylella fastidiosa strains from citrus and coffee hosts by single-nucleotide polymorphism markers

The aim of this study was to obtain information about genetic diversity and make some inferences about the relationship of 27 strains of Xylella fastidiosa from different hosts and distinct geographical areas. Single-nucleotide polymorphism (SNP) molecular markers were identified in DNA sequences from 16 distinct regions of the genome of 24 strains of X. fastidiosa from coffee and citrus plants. Among the Brazilian strains, coffee-dependent strains have a greater number of SNPs (10 to 24 SNPs) than the citrus-based strains (2 to 12 SNPs); all the strains were compared with the sequenced strain 9a5c. The identified SNP markers were able to distinguish, for the first time, strains from citrus plants and coffee and showed that strains from coffee present higher genetic diversity than the others. These markers also have proven to be efficient for discriminating strains from the same host obtained from different geographic regions. X. fastidiosa, the causal agent of citrus variegated chlorosis, possesses genetic diversity, and the SNP markers were highly efficient for discriminating genetically close organisms.

DEVELOPMENT of MICROSATELLITE MARKERS FOR PIMENTA PSEUDOCARYOPHYLLUS (MYRTACEAE), A WILD SOUTH AMERICAN SPECIES

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

Identification of a molecular marker linked to apomixis in Brachiaria humidicola (Poaceae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Molecular tools and analytical approaches for the characterization of farm animal genetic diversity

Genetic studies of livestock populations focus on questions of domestication, within- and among-breed diversity, breed history and adaptive variation. In this review, we describe the use of different molecular markers and methods for data analysis used to address these questions. There is a clear trend towards the use of single nucleotide polymorphisms and whole-genome sequence information, the application of Bayesian or Approximate Bayesian analysis and the use of adaptive next to neutral diversity to support decisions on conservation.

Molecular genetic maps of the group 6 chromosomes of hexaploid wheat (Triticum aestivum L. em. Thell.)

Restriction fragment length polymorphism (RFLP) maps of chromosomes 6A, 6B, and 6D of hexaploid wheat (Triticum aestivum L. em. Thell.) have been produced. They were constructed using a population of F 7-8 recombinant inbred lines derived from a synthetic wheat X bread wheat cross. The maps consist of 74 markers assigned to map positions at a LOD ≥ 3 (29 markers assigned to 6A, 24 to 6B, and 21 to 6D) and 2 markers assigned to 6D ordered at a LOD of 2.7. Another 78 markers were assigned to intervals on the maps. The maps of 6A, 6B, and 6D span 178, 132, and 206 cM, respectively. Twenty-one clones detected orthologous loci in two homoeologues and 3 detected an orthologous locus in each chromosome. Orthologous loci are located at intervals of from 1.5 to 26 cM throughout 70% of the length of the linkage maps. Within this portion of the maps, colinearity (homosequentiality) among the three homoeologues is strongly indicated. The remainder of the linkage maps consists of three segments ranging in length from 47 to 60 cM. Colinearity among these chromosomes and other Triticeae homoeologous group 6 chromosomes is indicated and a consensus RFLP map derived from maps of the homoeologous group 6 chromosomes of hexaploid wheat, tetraploid wheat, Triticum tauschii, and barley is presented.

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.

Avaliação da variabilidade genética em quatro gerações de Eucalyptus urophylla S.T. Blake por meio do marcador molecular RAPD

Molecular markers have gradually replaced morphological markers in population studies. The advantages of molecular markers are the speed and precision of evaluations, mainly for long cycle cultures, where determinate traits can take years to manifest. The principle objectives of this research were to assess variability and genetic distances in four generations of Eucalyptus urophylla and provide data that help with the continued improvement of these materials. The populations can be found at the Experimental Forestry Sciences Station, Anhembi, SP, belonging to the College of Agriculture Luiz de Queiroz of São Paulo University. The initial base population was introduced by seeds collected in indonesia and designated P0 generation. The subsequent segregated generations, derivatives of recombination starting with open pollination, were designated P1, P2, and P3. One hundred and seventy four individual trees representing the four generations were analysed. The RAPD technique allowed the identification of 86 loci that were analysed with the Jaccard Coefficient, generating a genetic similarity matrix, permitting the estimation of genetic distances. The genetic distance of generation PO was 0.3338333, P1 was 0.336824, P2 was 0.40000, and P3 was 0.381093. In percentage terms the genetic distances between individuals grew in relation to base population, being 0.15% for generation P1, 18.93% for P2, and 13.31% for P3. This shows an increase in genetic variability with the advance of the program, despite the selective processes. From this came the belief that the initial base population was resulting from seed collection from isolated trees. These populations, although going through successive selections, had a high cross efficiency through satisfactory pollination, which then permitted genetic variation to increase, the outcome of effective recombination between individuals. Generations P2 and P3 gave a better perspective for the continuance of the improvement program due to the high number of different groups with standard genetic distances of 35%. The selections made between the diverse genetic groups allowed the efficient use of genetic variability evaluation.

Caracterização silvicultural, botânica e avaliação da variabilidade genética por meio do marcador molecular RAPD em um teste de progênies de Eucalyptus urophylla S. T. Blake

Molecular markers have recently been incorporated into genetic improvement programs. They are already considered as powerful tools with several different uses, for instance the monitoring of genetic variability in tree populations. The main objectives of this study were to evaluate genetic variability in Eucalyptus urophylla progenies and together with silvicultural and botanical information, provide assistance to the improvement program. The Eucalypts population is located at the Experimental Forestry Sciences Station, Anhembi, SP, which belongs to the College of Agriculture Luiz de Queiroz. Sixty-nine progenies were analysed representing one individual by family in open pollinated Eucalyptus urophylla trees. The RAPD technique allowed the identification of 72 loci that were analysed using Jaccard's Coefficient generating a genetic similarity matrix to permit estimation of genetic distances. The results obtained showed genetic distance between individuals of 0.40 with 12 groups of genetic variability using a standardised distance of 40%. The progenies showed different bark patterns, allowing the establishment of bark groups. The groups formed based on genetic distances obtained using DNA analysis did not correspond to those based on bark pattern. Genetic selection was simulated in which silvicultural and genetic variability data were linked, thus avoiding excessive variability losses. The simulation of controlled crossings allowed the maximum genetic difference to be obtained linked with height and individual bark roughness.

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.

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.

Genetic polymorphism, molecular characterization and relatedness of Macrobrachium species (Palaemonidae) based on RAPD-PCR.

The prawn genus Macrobrachium belongs to the family Palaemonidae. Its species are widely distributed in lakes, reservoirs, floodplains, and rivers in tropical and subtropical regions of South America. Globally, the genus Macrobrachium includes nearly 210 known species, many of which have economic and ecological importance. We analyzed three species of this genus (M. jelskii, M. amazonicum and M. brasiliense) using RAPD-PCR to assess their genetic variability, genetic structure and the phylogenetic relationship between them and to look for molecular markers that enable separation of M. jelskii and M. amazonicum, which are closely related syntopic species. Ten different random decamer primers were used for DNA amplification, yielding 182 fragments. Three of these fragments were monomorphic and exclusive to M. amazonicum or M. jelskii and can be used as specific molecular markers to identify and separate these two species. Similarity indices and a phylogenetic tree showed that M. amazonicum and M. jelskii are closest to each other, while M. brasiliense was the most differentiated species among them; this may be attributed to the different habitat conditions to which these species have been submitted. This information will be useful for further studies on these important crustacean species.