Scenarios: Petroleum and Conflict Risk in Tanzania and Uganda Tanzania and Uganda

Os resultados dos múltiplos e complexos padrões de alinhamento de marcadores DArT-seq com os 100 clones BAC de Pinus taeda confirmaram a natureza complexa do genoma de

Pinus taeda, marcado por duplicações e sequências repetitivas dispersas no genoma.

Esforços na manipulação física do genoma de coníferas em geral têm sido limitados em função do tamanho e da complexidade do seu genoma. Várias abordagens estão sendo considerados para o sequenciamento do genoma de coníferas. Inicialmente foram apresentadas propostas baseadas no sequenciamento de alto rendimento de bibliotecas BAC (Hamberger, Hall et al., 2009; Kovach, Wegrzyn et al., 2010; Magbanua, Ozkan et al., 2011). Kovach et al. (2010) observaram atráves do alinhamento de sequências curtas (40- 60pb) obtidos por whole genome shotgun (WGS) contra 10 clones BAC de Pinus taeda que 80% das sequências BAC continham cópias similares em outra parte do genoma, mas apenas 23% tinham cópias idênticas (99% de identidade). Os dados do alinhamento revelaram apenas três marcadores DArT-seq com cópias idênticas aos clones BAC, com a maioria dos marcadores apresentando cópias similares em outra parte do genoma. Mais recentemente o projeto que visa a geração de uma sequência de referência do genoma de Pinus taeda, está adotando uma combinação de diferentes estratégias complementares visando maximizar a probabilidade de obtenção de uma montagem de qualidade (Crepeau, Puiu et al., 2013). Estas estratégias incluem o sequenciamento do DNA de um megagametófito haplóide único que permite uma redução considerável de complexidade embora seja limitado pela pouca quantidade de DNA disponível. Complementando esta estratégia, uma segunda abordagem envolve a geração de sequências em alta densidade de bibliotecas de grupos (pools) de fosmídeos do DNA diploide do parental doador dos megagametófitos. Esta estratégia, embora cara e potencialmente de representação enviesada, fornece sequências contíguas mais longas que facilitam a montagem do genoma.

Independentemente da estratégia de sequenciamento, a disponibilização de um ou mais mapas genéticos densos em marcadores baseados em sequências, como o mapa construído neste trabalho, constitui um recurso experimental útil para os esforços presentes e futuros de montagem de uma sequência de referência para Pinus taeda. Na medida em que maiores quantidades de sequências do genoma de Pinus taeda forem sendo disponibilizadas, novas

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análises de alinhamento dos marcadores deverão permitir aumentar a quantidade de sequências ancoradas ao mapa. Este estudo preliminar demonstrou, entretanto, que mesmo com um mapa denso, a complexidade da tarefa de montar um genoma grande e complexo como o de Pinus taeda é considerável. Isso ficou evidenciado pelo fato de que mesmo com um número muito pequeno de alinhamentos de alta identidade entre alguns poucos marcadores e alguns poucos BAC, já foram observadas diversas ocorrências que sugerem regiões duplicadas e sequências repetitivas dispersas no genoma. Uma solução para mitigar este problema seria a geração de mapas genéticos de marcadores moleculares baseados em sequências mais longas que permitissem um maior rigor no alinhamento. Genotipagem baseada em técnicas de captura (targeted capture) com sondas seguida de sequenciamento de ambas as extremidades da região capturada poderiam fornecer esta solução que já vem sendo otimizada para Pinus taeda com sucesso (Neves, Davis et al., 2011; Neves, Davis et al., 2013).

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