Diversidade genetica entre linhagens de milho tropical : estudo com base em marcadores moleculares

Orientadores: Anete Pereira de Souza, Antonio Augusto Franco GarciaDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Conhecer a variabilidade genética de plantas de interesse econômico permite manipular recursos naturais de forma eficiente, visando o melhoramento vegetal. A milho é uma das mais importantes plantas cultivadas e a diversidade que o germoplasma tropical apresenta ainda não está bem estabelecida. Atualmente, os marcadores moleculares são a principal maneira de analisar essa diversidade, e muitas possibilidades estatísticas foram desenvolvidas para organizar informações de dados moleculares. Este trabalho usou os marcadores AFLP e SSR para avaliar a diversidade genética em linhagens de milho tropical pertencentes ao Banco de Germoplasma do Instituto Agronômico de Campinas (IAC), e revelou grande nível de variabilidade. Os três tipos de coeficiente usados (Jaccard, MRD e coeficiente de parentesco molecular) exibiram baixos valores de similaridade entre as linhagens e, dependendo do marcador usado, diferentes agrupamentos foram obtidos. Entretanto, altas correlações entre as matrizes geradas por Jaccard e pelo coeficiente de parentesco molecular com AFLP, SSR e AFLP/SSR foram observadas. As correlações entre AFLP e SSR foram baixas, independentemente do coeficiente de similaridade aplicado. Devido à habilidade dos SSRs em revelar vários aspectos da variação genômica, essa técnica foi considerada mais apropriada para análises de diversidade. Notou-se que a grande diversidade existente no material tropical levou a resultados distintos se marcadores diferentes foram usados, e que o uso simultâneo de AFLPs e SSRs pode não ser a maneira mais eficiente de avaliar a variabilidade em materiais altamente divergentesAbstract: Knowledge about genetic variability of agronomically important crops allows efficient manipulation of resources in order to achieve plant improvement. Maize is one of the most important crops, but the extent of its diversity in the tropical germplasm has still not been well established. Nowadays, molecular markers are the principal way of analyzing genetic diversity and many statistical possibilities have been developed to compile data from molecular assays. This work used AFLP and SSR markers to access genetic diversity in tropical maize inbred lines from the Maize Genebank of the Agronomic Institute of Campinas (IAC), and revealed a great level of variability, with high rates of polymorphism. All three types of coefficients applied (Jaccard, MRD and molecular coefficient of coancestry) exhibited very low genetic similarities among the inbreds, and depending on the marker used, different clusters were obtained. Nevertheless, high correlations between matrices generated by Jaccard and by the molecular coefficient of coancestry with AFLP, SSR and AFLP/SSR were seen. AFLP and SSR were poorly correlated with both similarity coefficients. Because of SSR's ability of uncovering many aspects of genomic variation, it was considered more appropriate for diversity analysis. It was observed that the great amount of diversity found in tropical maize led to distinct results with different markers, and that using both AFLP and SSR together may not be the most efficient manner of accessing variability in highly diverse materialsMestradoGenetica Vegetal e MelhoramentoMestre em Genética e Biologia Molecula

Genome Sequence Of Bacillus Safensis Cfa06, Isolated From Biodegraded Petroleum In Brazil.

Bacillus safensis is a microorganism recognized for its biotechnological and industrial potential due to its interesting enzymatic portfolio. Here, as a means of gathering information about the importance of this species in oil biodegradation, we report a draft genome sequence of a strain isolated from petroleum.

Evaluating Genetic Relationships Between Tropical Maize Inbred Lines By Means Of Aflp Profiling.

Diversity among tropical maize inbred lines that compose breeding programs, is not well known. The lack of this information has made the arrangement of heterotic groups to be used for breeding purposes difficult. Methods of molecular analysis have been used as efficient alternatives for evaluating genetic diversity, aiming at heterotic group arrangement and acquisition of new hybrids. In this study, AFLP (amplified fragment length polymorphism) was used to investigate the genetic relationships among 96 tropical maize inbred lines from two different origins. The polymorphism level among the genotypes and the possibility of their allocation in heterotic groups were evaluated. Besides, correlations among genetic diversity and flowering time were analyzed. Nine primer combinations were used to obtain AFLP markers, producing 638 bands, 569 of which were polymorphic. Genetic similarities (GS), determined by Jaccard's similarity coefficient, varied from 0.345 to 0.891, with an average of 0.543. The dendrogram based on the GS and on the UPGMA cluster method did not separate the inbred lines in well-defined groups. Aiming at separating the lines into more accurate groups, Tocher's optimization procedure was carried out, 17 groups being identified. Association between flowering time and germplasm pools was detected. AFLP showed itself to be a robust assay, revealing a great power of detection of genetic variability in the tropical germplasm, and also demonstrated to be very useful for guiding breeding programs.14024-3

Transcriptome Profile of Trichoderma harzianum IOC-3844 Induced by Sugarcane Bagasse

Profiling the transcriptome that underlies biomass degradation by the fungus Trichoderma harzianum allows the identification of gene sequences with potential application in enzymatic hydrolysis processing. in the present study, the transcriptome of T. harzianum IOC-3844 was analyzed using RNA-seq technology. the sequencing generated 14.7 Gbp for downstream analyses. de novo assembly resulted in 32,396 contigs, which were submitted for identification and classified according to their identities. This analysis allowed us to define a principal set of T. harzianum genes that are involved in the degradation of cellulose and hemicellulose and the accessory genes that are involved in the depolymerization of biomass. An additional analysis of expression levels identified a set of carbohydrate-active enzymes that are upregulated under different conditions. the present study provides valuable information for future studies on biomass degradation and contributes to a better understanding of the role of the genes that are involved in this process.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Campinas UNICAMP, CBMEG, Campinas, SP, BrazilBrazilian Ctr Res Energy & Mat CNPEM, Brazilian Bioethanol Sci & Technol Lab CTBE, Campinas, SP, BrazilUniv São Paulo, Phys Inst Sao Carlos, Sao Carlos, SP, BrazilFed Univ São Paulo UNIFESP, Inst Sci & Technol, Sao Jose Dos Campos, SP, BrazilUniv Campinas UNICAMP, Dept Plant Biol, Inst Biol, Campinas, SP, BrazilFed Univ São Paulo UNIFESP, Inst Sci & Technol, Sao Jose Dos Campos, SP, BrazilWeb of Scienc

Correlation between the heterosis of maize hybrids and genetic divergence among lines

O objetivo deste trabalho foi avaliar a produtividade de híbridos simples de milho obtidos de cruzamentos dialélicos entre linhagens divergentes, estimar a capacidade de combinação das linhagens e confirmar se a divergência genética entre as linhagens, obtida por marcadores moleculares, é correlacionada com a heterose dos híbridos simples no campo. Trinta e seis híbridos resultantes do dialelo parcial e as 12 linhagens parentais foram avaliadas em Campinas em blocos ao acaso, com três repetições e duas testemunhas. A capacidade de combinação das linhagens foi estimada de acordo com o modelo 4 de Griffing. Estimaram-se a correlação matricial, mediante a estatística de Mantel, entre heterose, produtividade e capacidade específica de combinação com divergência genética por AFLP e SSR. Destacou-se o híbrido PM518 x L111 e as linhagens PM518, IP4035 e L111 apresentaram efeitos positivos da capacidade geral de combinação. As estimativas de heterose variaram de 927 a 6.698 kg ha-1. Houve correlação entre heterose e divergência genética por AFLP e SSR. No entanto, a divergência genética não foi suficiente para determinar a capacidade específica de combinação nem a produtividade dos híbridos.The objective of this work was to evaluate grain yield of maize single cross hybrids obtained from diallel crosses among contrasting lines, to estimate the combining ability of the lines, and finally to confirm if the genetic diversity among those lines assessed by molecular markers is correlated with single cross hybrids heterosis. The 36 single cross hybrids resulting from partial diallel and 12 parental lines were evaluated in Campinas in randomized block design, with three replicates and two control lines checks. General combining ability of the lines was estimated according to Griffing model 4. Correlations among matrices were estimated through Mantel statistics, considering heterosis, yield and specific combining ability with genetic divergence assessed by AFLP and SSR. The hybrids PM518 x L111 exhibited an outstanding yield and the lines PM518, IP4035 and L111 showed positive general combining ability. The estimate heterosis ranged from 927 to 6,698 kg ha-1. A positive and significant correlation was observed in parental lines between heterosis and genetic diversity assessed by AFLP and SSR. The genetic divergence, however, was not enough to determine the specific combining ability and the hybrids yield

Microsatellite markers in the investigation of Drosophila mediopunctata genome : development and genetic linkage map construction

Orientador: Anete Pereira de SouzaTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Drosophila mediopunctata, mosca não cosmopolita de distribuição neotropical e pertencente ao grupo tripunctata do subgênero Drosophila, é uma espécie utilizada por alguns grupos no Brasil como modelo em estudos com enfoque evolutivo. Abordagens que exploram a biologia da espécie, tais como a detecção de variação genética natural e a influência de inversões cromossômicas na dinâmica populacional são costumeiramente usadas. Todavia, essa espécie não possui disponíveis marcadores moleculares que facilitem a investigação das bases genéticas que norteiam os eventos explorados até agora. Os microssatélites são marcadores moleculares consagrados e a ferramenta de escolha no estudo de diversos organismos pela simplicidade de uso e de análise. Entretanto, para espécies que ainda não têm grande parte de seu genoma seqüenciado, a obtenção de marcadores desse tipo passa pela necessidade de desenvolvimento via bibliotecas genômicas. Esse trabalho objetivou desenvolver, caracterizar e mapear microssatélites em D. mediopunctata para que estudos de variação morfológica, de identificação de regiões genômicas associadas a fenótipos de interesse, de genética de populações entre outros possam ser realizados com o respaldo de dados moleculares. Uma biblioteca enriquecida em motivos repetitivos foi construída e, após o seqüenciamento de aproximadamente 2000 clones, 600 microssatélites foram identificados e 134 locos desenvolvidos. Os marcadores mostraram tamanho reduzido com relação ao número de repetições e preponderância de motivos AC/GT. A aplicação dos microssatélites de D. mediopunctata para amplificações heterólogas em outras trinta espécies foi feita com aproximadamente 50% de sucesso. Em adição, o uso de dados de presença/ausência nas espécies do grupo tripunctata recuperou algumas relações filogenéticas previamente conhecidas. Um mapa de ligação foi construído com 49 marcadores e revelou 450 cM de extensão. Os cinco principais cromossomos da espécie foram identificados por meio de comparações com o genoma de D. melanogaster e com os elementos de Müller. Essas comparações também confirmaram a grande sintenia prevista dentro do gênero Drosophila. Não houve concordância na ordem dos locos microssatélites nas duas espécies. No cromossomo II de D. mediopunctata foi mapeada uma região associada à característica "número de pintas abdominais"Abstract: Drosophila mediopunctata, a non-cosmopolitan fly of Neotropical distribution that belongs to the tripunctata group of the Drosophila subgenus, was chosen by some Brazilian researchers as a model in evolutionary studies. Several approaches, such as the analysis of natural variation and the influence of chromosome inversions in population dynamics, are traditionally used. Nevertheless, molecular markers, which would enhance the investigation of the genetic bases of the already known phenomena, are still not available for the species. Microsatellites are celebrated molecular markers and the chosen tool for the exploration of various organisms due to their ease of use. Nonetheless, their application in species whose genomes have not yet been sequenced requires a prior development phase. This study intended to develop, characterize and map microsatellites for the species D. mediopunctata so that initiatives concerning morphological variation, identification of genomic regions linked to interesting phenotypes, population genetics, etc can be carried out in the light of molecular data. A repetitive DNA-enriched library was constructed and approximately 2000 clones were sequenced. Six hundred microsatellites were identified and 134 loci were developed. The loci are small in length, with reduced number of motif repetitions, and are mainly composed of AC/GT dinucleotides. The use of D. mediopunctata microsatellites for heterologous amplification in other thirty Drosophila species was done with a 50% success ratio. In addition, a clustering analysis carried out with binary data obtained from the tripunctata species recovered already known phylogenetic relationships. A linkage map was constructed with recombination data of 49 markers and is 450 cM in length. The five major species chromosomes were identified on the basis of comparisons with the D. melanogaster genome and the Müller elements. This strategy also confirmed the great synteny predicted for the genus Drosophila. It was not observed agreement in loci order between both species. A genomic region associated to the number of abdominal spots was mapped to the chromosome II of D. mediopunctataDoutoradoGenetica Animal e EvoluçãoDoutor em Genetica e Biologia Molecula

Karyotype variability in tropical maize sister inbred lines and hybrids compared with KYS standard line

Maize karyotype variability has been extensively investigated. The identification of maize somatic and pachytene chromosomes has improved with the development of fluorescence in situ hybridization (FISH) using tandemly repeated DNA sequences as probes. We identified the somatic chromosomes of sister inbred lines that were derived from a tropical flint maize population (Jac Duro [JD]), and hybrids between them, using FISH probes for the 180-bp knob repeat, centromeric satellite (CentC), centromeric satellite 4 (Cent4), subtelomeric clone 4-12-1, 5S ribosomal DNA and nucleolus organizing region DNA sequences. The observations were integrated with data based on C-banded mitotic metaphases and conventional analysis of pachytene chromosomes. Heterochromatic knobs visible at pachynema were coincident with C-bands and 180-bp FISH signals on somatic chromosomes, and most of them were large. Variation in the presence of some knobs was observed among lines. Small 180-bp knob signals were invariant on the short arms of chromosomes 1, 6, and 9. The subtelomeric 4-12-1 signal was also invariant and useful for identifying some chromosomes. The centromere location of chromosomes 2 and 4 differed from previous reports on standard maize lines. Somatic chromosomes of a JD line and the commonly used KYS line were compared by FISH in a hybrid of these lines. The pairing behavior of chromosomes 2 and 4 at pachytene stage in this hybrid was investigated using FISH with chromosome-specific probes. The homologues were fully synapsed, including the 5S rDNA and CentC sites on chromosome 2, and Cent4 and subtelomeric 4-12-1 sites on chromosome 4. This suggests that homologous chromosomes could pair through differential degrees of chromatin packaging in homologous arms differing in size. The results contribute to current knowledge of maize global diversity and also raise questions concerning the meiotic pairing of homologous chromosomes possibly differing in their amounts of repetitive DNA

Karyotype variability in tropical maize sister inbred lines and hybrids compared with the KYS standard line

Maize karyotype variability has been extensively investigated. The identification of maize somatic and pachytene chromosomes has improved with the development of fluorescence in situ hybridization (FISH) using tandemly repeated DNA sequences as probes. We identified the somatic chromosomes of sister inbred lines that were derived from a tropical flint maize population (Jac Duro [JD]), and hybrids between them, using FISH probes for the 180-bp knob repeat, centromeric satellite (CentC), centromeric satellite 4 (Cent4), subtelomeric clone 4-12-1, 5S ribosomal DNA and nucleolus organizing region DNA sequences. The observations were integrated with data based on C-banded mitotic metaphases and conventional analysis of pachytene chromosomes. Heterochromatic knobs visible at pachynema were coincident with C-bands and 180-bp FISH signals on somatic chromosomes, and most of them were large. Variation in the presence of some knobs was observed among lines. Small 180-bp knob signals were invariant on the short arms of chromosomes 1, 6 and 9. The subtelomeric 4-12-1 signal was also invariant and useful for identifying some chromosomes. The centromere location of chromosomes 2 and 4 differed from previous reports on standard maize lines. Somatic chromosomes of a JD line and the commonly used KYS line were compared by FISH in a hybrid of these lines. The pairing behavior of chromosomes 2 and 4 at pachytene stage in this hybrid was investigated using FISH with chromosome-specific probes. The homologues were fully synapsed, including the 5S rDNA and CentC sites on chromosome 2, and Cent4 and subtelomeric 4-12-1 sites on chromosome 4. This suggests that homologous chromosomes could pair through differential degrees of chromatin packaging in homologous arms differing in size. The results contribute to current knowledge of maize global diversity and also raise questions concerning the meiotic pairing of homologous chromosomes possibly differing in their amounts of repetitive DNA