Uterine infusion of conceptus fragments changes the protein profile from cyclic mares

This experiment aimed to compare at day seven after ovulation, the protein profile of uterine fluid in cyclic mares with mares infused two days before with Day 13 conceptus fragments. Experimental animals were ten healthy cyclic mares, examined daily to detect ovulation (Day 0) as soon as estrus was confirmed. On day seven, after ovulation, uterine fluid was collected, constituting the Cyclic group (n = 10). The same mares were examined in the second cycle until ovulation was detected. On day five, after ovulation, fragments from a previously collected concepti were infused into each mare's uterus. Two days after infusion, uterine fluid was collected, constituting the Fragment group (n = 10). Two-dimensional electrophoresis technique processed uterine fluid samples. A total of 373 spots were detected. MALDI-TOF/TOF and NanoUHPLC-QTOF mass spectrometry identified twenty spots with differences in abundance between the Cyclic and Fragment group. Thirteen proteins were identified, with different abundance between groups. Identified proteins may be related to embryo-maternal communication, which involves adhesion, nutrition, endothelial cell proliferation, transport, and immunological tolerance. In conclusion, conceptus fragments signalized changes in the protein profile of uterine fluid seven days after ovulation in comparison to the observed at Day 7 in the same cyclic mares

Phylogeographic study and comparative analysis of classification models of Porcine circovirus-2 (PCV-2)

Os agentes infecciosos de suínos têm recebido grande atenção desde a última década, quando muitos países produtores acumularam perdas econômicas significativas devido a patógenos como o Porcine circovirus-2 (PCV-2). O PCV-2 é um vírus classificado na família Circoviridae e está associado a um conjunto de diferentes síndromes em suínos denominado Porcine Circovirus Associated Diseases (PCVAD). Desde a sua identificação e caracterização, o PCV-2 alcançou uma distribuição mundial e a PCVAD tornou-se uma síndrome endêmica na maioria dos países produtores, sendo considerada a principal causa por perdas nas granjas. Neste trabalho, as nomenclaturas propostas para a classificação das linhagens virais foram comparadas e as vias de dispersão do PCV-2 entre os países suinocultores foram analisadas. Uma pesquisa por sequências genômicas do PCV-2 foi realizada no GenBank e 350 sequências de isolados virais foram aleatoriamente selecionadas. Os modelos de classificação do PCV-2 em genótipos, recomendado pelo Consórcio Europeu que estuda as Porcine circovirus diseases (PCVD) e definido por análises de mismatch distibution, e o modelo de classificação em subgrupos filogenéticos, modelo alternativo e definido por hipóteses filogenéticas, foram aplicados ao conjunto de sequências selecionado. Tanto nas análises de mismatch distribution quanto nas análises filogenéticas, os agrupamentos formados e as variações observadas foram muito semelhantes. Nessas análises, observou-se a sobreposição entre os agrupamentos em genótipos e em subgrupos filogenéticos. O genótipo PCV-2a foi correspondente ao Grupo 2 e reuniu os subgrupos 2A, 2B, 2C, 2D e 2E. O genótipo PCV-2b foi correspondente ao Grupo 1 e reuniu os subgrupos 1A, 1B e 1C. O genótipo PCV-2c reuniu apenas a sequência do isolado viral que o define. Esses resultados mostram que as subdivisões existentes nos genótipos PCV-2a e PCV-2b não devem ser desconsideradas para que a diversidade genética existente entre os isolados virais do PCV-2 não seja subestimada. No estudo filogeográfico, as vias de dispersão do PCV-2 foram preditas por meio de abordagens filogenéticas e filogeográficas. Nas análises filogenéticas, as árvores foram calculadas por inferência bayesiana e os isolados virais foram agrupados nos genótipos PCV-2a, PCV-2b e PCV-2c. Nas análises filogeográficas, uma rede de haplótipos foi calculada pelo algoritmo Median Joining, para o estabelecimento da genealogia entre os isolados virais, e as vias de dispersão entre os países foram preditas. Para dar suporte a essas análises, dois bancos de dados foram organizados. O primeiro banco de dados reuniu todas as informações disponíveis sobre os isolados do PCV-2 no GenBank e nas publicações relacionadas ao isolamento viral. O segundo banco de dados reuniu as estatísticas do comércio internacional de suínos vivos disponíveis no United Nations Commodity Trade Statistics Database DESA/UNSD e foi organizado para estabelecer um contexto econômico às vias preditas na rede de haplótipos. A partir dessas análises e desses bancos de dados, as seguintes vias de dispersão do PCV-2 entre os países produtores de suínos foram preditas: América do Norte → África, América do Norte → América do Sul, América do Norte → Caribe, Europa → América do Norte, Europa → América do Sul, Europa → Ásia, Oceania → Ásia e Oceania → América do Norte. Nessas vias de dispersão, os principais países de origem das vias são o Canadá, os Estados Unidos, a Dinamarca, a França e a Holanda, que são os principais exportadores de animais no mercado internacional de suínos vivos. As correlações observadas entre as vias de dispersão do PCV-2 preditas nas análises filogeográficas e as estatísticas do comércio internacional de suínos vivos mostram a importância do movimento de animais no rebanho mundial para a emergência de novos patógenos e a necessidade da criação de barreiras sanitárias cada vez mais eficazes no comércio de animais.The pig infectious agents have received great attention since the last decade, when many pig producing countries accumulated economic losses due to pathogens such as Porcine circovirus-2 (PCV-2). The PCV-2 is a virus classified in Circoviridae family and is associated with a number of different syndromes in pigs called Porcine Circovirus Associated Diseases (PCVAD). Since its identification and characterization, PCV-2 has reached a worldwide distribution and the PCVAD became a syndrome endemic in most producer countries and currently considered the main cause for losses on pig farms. In this study, the nomenclatures proposed for the classification of viral isolates were compared, and the spread routes of PCV-2 among pig producing countries were analyzed. A search for genomic sequences of PCV-2 was performed in GenBank and 350 sequences of viral isolates were randomly selected. The classification model of PCV-2 in genotypes, recommended by the European consortium that studies the Porcine circovirus diseases (PCVD) and defined by analysis of mismatch distribution, and the classification model in phylogenetic subgroups, an alternative model defined by phylogenetic hypotheses, were applied to the set of selected sequences. Both in the mismatch distribution analysis and in the phylogenetic analysis, the groups formed and the changes observed were very similar. In these analyses, there was an overlap between genotypes and phylogenetic subgroups. The genotype PCV-2a was equivalent to Group 2 and grouped the subgroups 2A, 2B, 2C, 2D and 2E. The genotype PCV-2b was equivalent to Group 1 and grouped the subgroups 1A, 1B and 1C. The genotype PCV- 2c only grouped the sequence of the viral isolate that defines it. These results show that the existing subdivisions in the genotypes of PCV-2a and PCV-2b should not be neglected for don ́t underestimate the genetic diversity among isolates of PCV-2. In the phylogeographic study, the spread routes of PCV-2 were predicted through phylogenetic and phylogeographic approaches. In phylogenetic analysis, the phylogenetic trees were calculated by Bayesian inference and the isolates were grouped in the genotypes PCV-2a, PCV-2b and PCV-2c. In phylogeographical analysis, a haplotype network was calculated by Median Joining algorithm for the establishment of genealogy among the viral isolates, and the spread routes of PCV-2 among the countries were predicted. To support these analyses, two databases were organized. The first database grouped all available information on the isolates of PCV-2 in GenBank and in publications related to viral isolation. The second data set grouped the statistics of live pigs trade available at United Nations Commodity Trade Statistics Database DESA/UNSD and was organized to establish an economic context to establish an economic context in the phylogeographic analysis. From these analyses, the following spread routes of PCV-2 were predicted: North America → Africa, North America → South America, North America → Caribbean, Europe → North America, Europe → South America, Europe → Asia, Oceania → Asia and Oceania → North America. In these spread routes, the main countries of origin of the routes are Canada, United States, Denmark, France and the Netherlands, that are the major exporters of animals in the international trade of live pigs. In this study, were identified correlations between the means of dispersal of PCV-2 in pigs predicted by the phylogeographic context and the statistics on international trade in live pigs. This correlation shows the importance of the movement of animals in the world to the emergence of new pathogens and the need to establish sanitary barriers that are increasingly effective for trade in animals.Conselho Nacional de Desenvolvimento Científico e Tecnológic

Characterization of nuclear, chloroplastidial and mitochondrial genomes of sugarcane using next-generation sequencing data

Nesta tese, os dados do sequenciamento do genoma da variedade de cana-de-açúcar RB867515 e de outros 508 genótipos provenientes de 100 famílias de meios-irmãos são analisados com os seguintes objetivos: (I) montar as sequências dos genomas nuclear, cloroplastidial e mitocondrial da variedade RB867515; (II) predizer e anotar funcionalmente os genes e demais elementos presentes nas sequências obtidas; (III) analisar os polimorfismos de nucleotídeo único presentes nos genomas dos genótipos de cana-de-açúcar; (IV) associar os polimorfismos identificados com as características fenotípicas dos genótipos usando estudos de associação ampla do genoma; (V) criar um banco de dados integrando as sequências, os polimorfismos e as informações funcionais obtidas. As bibliotecas genômicas foram sequenciadas usando as tecnologias de sequenciamento de nova geração da Illumina e as sequências obtidas foram trimadas e selecionadas, produzindo um conjunto de dados contendo 1,39 bilhões de sequências da variedade RB867515 e 2,43 bilhões de sequências dos genótipos. As sequências dos genomas nuclear, cloroplastidial e mitocondrial da variedade RB867515 foram montadas usando algoritmos de montagem De Novo e estratégias baseadas em genomas de referência. Os genes foram identificados usando métodos ab initio e empíricos, e as informações funcionais foram preditas a partir de pesquisas de similaridade. Na genotipagem, as sequências dos genótipos foram mapeadas no genoma da variedade RB867515 e os polimorfismos de nucleotídeo único foram identificados seguindo os métodos recomendados para a descoberta de variantes a partir de dados de sequenciamento de nova geração. No estudo de associação ampla do genoma, a associação dos polimorfismos com as características fenotípicas dos genótipos foi avaliada usando o método enriched compressed mixed linear model (ECMLM). O genoma nuclear da variedade RB867515 foi montado em 484.719 sequências com um tamanho total de 552,97 megabases (Mb), sendo preditos 75.715 genes codificadores de proteínas. Esses genes foram funcionalmente anotados e as suas proteínas foram classificadas em diferentes grupos funcionais. A análise dos polimorfismos identificou 40.663 loci contendo polimorfismos de nucleotídeo único em 7.890 genes e apenas um polimorfismo no gene da enzima isocitrato desidrogenase apresentou associação significativa com a característica fenotípica conteúdo de sacarose na cana em percentagem (PC) no estudo de associação ampla de genoma. O genoma cloroplastidial da RB867515 foi montado em uma única sequência contendo 141,18Kb, sendo identificados 88 genes codificadores de proteínas, 8 genes de rRNA e 39 genes de tRNA. A sequência desse genoma é idêntica ao genoma da variedade de cana-de-açúcar Q155, originária da Austrália. A análise dos polimorfismos mostrou que apenas oito genótipos incluídos na família da variedade RB982639 (g011, g030, g031, g094, g103, g246, g425 e g441) apresentam polimorfismos em suas sequências. Esses polimorfismos estão localizados em regiões intergênicas do genoma e nos genes petB e ndhF. O genoma mitocondrial da variedade RB867515 foi montado em dois segmentos subgenômicos circulares com um tamanho total de 445,52Kb, sendo identificados 34 genes codificadores de proteínas, 6 genes de rRNA e 22 genes de tRNA. A análise dos polimorfismos identificou 6 SNPs e 23 indels localizados em regiões intergênicas do genoma mitocondrial dos genótipos. Todas essas informações geradas nesta tese auxiliarão na aplicação das ciências genômicas nos PMGCA, permitindo a integração entre as sequências dos genes e suas respectivas proteínas, os polimorfismos e as informações funcionais. O banco de dados Sugarcane DB (http://sugarcane.ccb.ufv.br) foi criado para organizar essas informações e torna-las disponíveis aos PMGCA. A missão do Sugarcane DB é ser um repositório público permanente de informações genômicas relacionadas à cana-de-açúcar e se tornar uma ferramenta importante para auxiliar os melhoristas na identificação de novos alvos para o melhoramento genético da cana-de-açúcar.In this thesis, genome sequencing data of the sugarcane cultivar RB867515 and other 508 sugarcane genotypes from 100 half-sib families are analyzed with the following objectives: (I) assemble the sequences of the nuclear, chloroplastidial and mitochondrial genomes of sugarcane cultivar RB867515; (II) predict and functionally annotate the genes and other elements in the assembled sequences; (III) analyze the single nucleotide polymorphisms (SNPs) in the genomes of sugarcane genotypes; (IV) to associate the identified SNPs to the phenotypes of sugarcane genotypes using genome wide association studies (GWAS); (V) create a database integrating sequences, polymorphisms and functional information. Genomic libraries were sequenced using Illumina's next generation sequencing technologies and sequenced reads were trimmed and selected, yielding a dataset containing 1.39 billion reads of the cultivar RB867515 and 2.43 billion reads of the sugarcane genotypes. Nuclear, chloroplastidial and mitochondrial genome sequences of the cultivar RB867515 were assembled using De Novo assembly algorithms and reference-guided approaches. Genes were identified using ab initio and empirical methods, and functional information were predicted by using sequence similarities searches. In genotyping, the sequenced reads of the sugarcane genotypes were mapped in the genome of cultivar RB867515, and SNPs were predicted following guidelines for variant discovery from next-generation sequencing data. In the genome-wide association study, the association of SNPs with the phenotypes of sugarcane genotypes was evaluated using the enriched compressed mixed linear model (ECMLM). The nuclear genome of the cultivar RB867515 was assembled in 484,719 sequences with 552.97 megabases (Mb), containing 75,715 protein-coding genes. These genes were functionally annotated and the encoded proteins were classified into different functional groups. The polymorphisms analysis identified 40,663 loci containing SNPs in 7,890 genes. In GWAS, only one SNP in the isocitrate dehydrogenase enzyme gene showed significant association with sugarcane sucrose content in percentage (PC). The chloroplast genome of the cultivar RB867515 was assembled in a single sequence containing 141.18Kb, containing 88 protein-coding genes, 8 rRNA genes and 39 tRNA genes. Chloroplast genome sequence of cultivar RB867515 is identical to the sequence of Q155 cultivar from Australia. The polymorphisms analysis showed that only eight genotypes of the RB982639 family (g011, g030, g031, g094, g103, g246, g425 and g441) have SNPs in their chloroplast genome sequences. These polymorphisms (7 SNPs and 4 indels) are located in intergenic regions of chloroplast genome and in petB and ndhF genes. The mitochondrial genome of the cultivar RB867515 was assembled in two circular subgenomic segments with a total size of 445.52Kb, containing 34 protein-coding genes, 6 rRNA genes and 22 tRNA genes. The polymorphisms analysis identified 6 SNPs and 23 indels located in intergenic regions of the mitochondrial genome of sugarcane genotypes. All data generated in this thesis will contribute to the application of the genomic sciences in sugarcane breeding, allowing the integration among gene and protein sequences, polymorphisms and functional information. The Sugarcane DB database (http://sugarcane.ccb.ufv.br) was created to organize these data and make it available to the PMGCA. Sugarcane DB aims to be a permanent public repository of sugarcane genomic information and to become an important tool to assist breeders in identifying new targets for the genetic improvement of sugarcane.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Complete chloroplast genome sequence and annotation of the Saccharum hybrid cultivar RB867515

Here, we present the chloroplast genome sequence of the Saccharum hybrid cultivar RB867515, the most planted sugarcane cultivar in Brazil

Complete chloroplast genome sequence and annotation of the Saccharum hybrid cultivar RB867515

Here, we present the chloroplast genome sequence of the Saccharum hybrid cultivar RB867515, the most planted sugarcane cultivar in Brazil

Genetic evaluation of IS900 partial sequence of Mycobacterium avium subsp. paratuberculosis Brazilian isolates from bovine milk

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis. Insertion sequence IS900 is used for the identification of MAP. The objective of this study was to verify the genetic conservation of IS900 sequences in raw milk samples. To evaluate genetic conservation, 206 quarter milk samples and 16 bulk-tank milk samples were collected. DNA extraction and IS900 PCR were performed in all samples. Six samples amplified the expected fragment. To confirm the identity of the amplified fragments, PCR products were cloned and sequenced. The resulting sequences were compared with other MAP sequences from GenBank, and it was possible to identify eight polymorphic regions and to form five distinct haplotypes. The number of mutations in each haplotype was verified. IS900 sequence is a very well-conserved sequence that could be used as tool for the molecular detection of this agent and epidemiological purposes. The results showed the first genetic analysis on Brazilian isolates of MAP

Near-infrared spectroscopy outperforms genomics for predicting sugarcane feedstock quality traits.

The main objectives of this study were to evaluate the prediction performance of genomic and near-infrared spectroscopy (NIR) data and whether the integration of genomic and NIR predictor variables can increase the prediction accuracy of two feedstock quality traits (fiber and sucrose content) in a sugarcane population (Saccharum spp.). The following three modeling strategies were compared: M1 (genome-based prediction), M2 (NIR-based prediction), and M3 (integration of genomics and NIR wavenumbers). Data were collected from a commercial population comprised of three hundred and eighty-five individuals, genotyped for single nucleotide polymorphisms and screened using NIR spectroscopy. We compared partial least squares (PLS) and BayesB regression methods to estimate marker and wavenumber effects. In order to assess model performance, we employed random sub-sampling cross-validation to calculate the mean Pearson correlation coefficient between observed and predicted values. Our results showed that models fitted using BayesB were more predictive than PLS models. We found that NIR (M2) provided the highest prediction accuracy, whereas genomics (M1) presented the lowest predictive ability, regardless of the measured traits and regression methods used. The integration of predictors derived from NIR spectroscopy and genomics into a single model (M3) did not significantly improve the prediction accuracy for the two traits evaluated. These findings suggest that NIR-based prediction can be an effective strategy for predicting the genetic merit of sugarcane clones

Genomic and biological characterization of a new member of the genus Phikmvvirus infecting phytopathogenic Ralstonia bacteria

Bacterial wilt caused by Ralstonia spp., soil-borne Gram-negative bacteria, is considered one of the most important plant diseases in tropical and subtropical regions of the world. A large number of bacteriophages capable of lysing or physiologically reprogramming cells of Ralstonia spp. have been reported in Asia. Despite the potential use of these organisms in the management of bacterial wilt, information on viruses that infect Ralstonia spp. is nonexistent in the Americas. We isolated a virus that infects Ralstonia spp. from a soil sample in the state of Minas Gerais, Brazil. Microscopy and genomic and phylogenetic analysis allowed us to classify the virus as a member of the family Podoviridae, genus Phikmvvirus. In spite of its relationship to Ralstonia virus RSB3, an Asian isolate, genomic and biological characteristics showed that the virus isolated in Brazil, tentatively named “Ralstonia virus phiAP1” (phiAP1), belongs to a new species. phiAP1 has EPS depolymerase activity and contains two putative virion-associated peptidoglycan hydrolases (VAPGHs), which reveals a robust mechanism of pathogenesis. Furthermore, phiAP1 specifically infects Ralstonia solanacearum, R. pseudosolanacearum and R. syzygii, causing cell lysis, but it was not able to infect thirteen other bacteria that were tested. Together, these characteristics highlight the biotechnological potential of this virus for the management of bacterial wilt

Polymorphism analysis of the apxIA gene of Actinobacillus pleuropneumoniae serovar 5 isolated in swine herds from Brazil.

The bacterium Actinobacillus pleuropneumoniae is the etiological agent of Contagious Porcine Pleuropneumonia, a disease responsible for economic losses in the swine industry worldwide. A. pleuropneumoniae is capable of producing proteinaceous exotoxins responsible for inducing hemorrhagic lesions, one of which is ApxI. Few studies have conducted an in-depth evaluation of polymorphisms of the nucleotides that make up the ApxI toxin gene. Here we analyze the polymorphisms of the apxIA gene region of A. pleuropneumoniae serovar 5 isolated from swine in different regions in Brazil and report the results of molecular sequencing and phylogenetic analysis. Analysis of the apxIA gene in 60 isolates revealed the presence of genetic diversity and variability. The polymorphisms in the nucleotide sequences determined the grouping of the Brazilian sequences and five more sequences from the GenBank database into 14 different haplotypes, which formed three main groups and revealed the presence of mutations in the nucleotide sequences. The estimation of selection pressures suggests the occurrence of genetic variations by positive selective pressure on A. pleuropneumoniae in large groups of animals in relatively small spaces. These conditions presumably favor the horizontal dissemination of apxIA gene mutations within bacterial populations with host reservoirs. As a result, the same serovar can demonstrate different antigenic capacities due to mutations in the apxIA gene. These alterations in sequences of the apxIA gene could occur in other areas of countries with intense swine production, which could lead to differences in the pathogenicity and immunogenicity of each serovar and have implications for the clinical status or diagnosis of A. pleuropneumoniae

Evolutionary analysis of porcine circovirus 3 (PCV3) indicates an ancient origin for its current strains and a worldwide dispersion

Porcine circovirus 3 (PCV3) is an emerging virus that was identified in the United States in 2016. Since its first identification, PCV3 has been identified in Brazil, China, United States, Poland, and Republic of Korea. In this study, we used molecular phylogenetic analysis of available sequences to address questions surrounding the emergence of PCV3 in porcine world industry. Our data indicate that PCV3 did not emerge through recombination events among currently known circoviruses and that its speciation is not a recent evolutionary event. The most common recent ancestor analysis suggests that PCV3 lineages have emerged over the past 50 years. PCV3 is not genetically closely related with other Porcine circovirus and it has been evolving undetected for some time in swine and probably in bovine population. We also found groups of genetically related isolates of PCV3 originated from different countries that may be associated with dispersal routes, suggesting that PCV3 has already been circulating in pig-producing countries for some time before its first detection