Genomic synteny between sorghum and sugarcane inferred from a BAC pool sequencing

Orientador: Paulo ArrudaTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O sequenciamento genômico de plantas tem se acelerado nos últimos anos principalmente devido ao avanço das tecnologias de sequenciamento de nova geração, capazes de gerar um grande volume de dados com custo cada vez menor. No entanto, o sequenciamento e a montagem de genomas de plantas ainda continua sendo um grande desafio em função da alta complexidade desses genomas que na sua grande maioria possuem alto grau de ploidia e grande proporção de sequências repetitivas. O sequenciamento de bibliotecas produzidas com DNA genômico de plantas clonados em vetores BACs (bacterial artificial chromosomes) pode ser uma estratégia efetiva para sequenciamento de genomas complexos, por dividir a tarefa de montagem em problemas menores. No geral, bibliotecas de BACs contém fragmentos de DNA de 100 a 200 kilobases, cujo conjunto cobre o genoma clonado várias vezes. Entretanto, mesmo com as novas tecnologias de sequenciamento, o custo de sequenciar bibliotecas de BACs ainda é alto, pois na maioria das vezes o sequenciamento é realizado a partir do DNA isolado de cada BAC individualmente. Uma alternativa seria sequenciar pools contendo centenas de BACs amostrados randomicamente, que dessa forma diminuiria o custo proporcionalmente ao número de BACs do pool. Neste trabalho, desenvolvemos um modelo para sequenciamento e montagem de pools de BACs de uma biblioteca preparada a partir de uma variedade comercial de cana-de-açúcar. Como resultado, um pool com 178 BACs de cana-de-açúcar da variedade SP80-3280 foi sequenciado utilizando-se as tecnologias HighSeq2000 da Illumina e PacBio, e montados utilizando diferentes conjuntos de softwares. Por ser uma amostra de BACs selecionados randomicamente foi possível montar 2.451 scaffolds correspondentes a 88,2% do tamanho estimado total do conjunto de BACs do pool. A completeza da montagem foi verificada de várias maneiras incluindo a análise do número de BACs montados com tamanho esperado, a comparação com BACs depositados no NCBI e pela colinearidade e ordem de genes presentes entre scaffolds de cana e os cromossomos de sorgo. Os scaffolds com tamanho superior a 2 kb foram alinhados contra o genoma de sorgo, e no geral os alinhamentos mostraram uma distribuição uniforme ao longo dos 10 cromossomos do sorgo indicando a aleatoriedade da amostragem. Pela análise sintênica entre os scaffolds de cana e os cromossomos de sorgo, observamos que o genoma monoploide da cana parece ser mais contraído em relação ao genoma do sorgo. No geral o trabalho mostrou que é possível sequenciar pool de BACs de genomas de plantas de alta complexidade como o genoma de cana-de-açúcar com altos níveis de ploidiaAbstract: The genomic sequencing of plants has accelerated in recent years mainly due to the advances of next generation sequencing technologies capable of generating a high volume of data with ever lower cost. However, the sequencing and assembly of plant genomes remains a major challenge due to the high complexity of these genomes that mostly have a high degree of ploidy and large proportion of repetitive sequences. The sequencing of libraries produced with genomic DNA of plants cloned into BAC (bacterial artificial chromosome) vectors can be an effective strategy for sequencing complex genomes, by breaking down the assembly task into smaller problems. Typical BAC libraries contain DNA fragments of 100 to 200 kilobases which together cover the genome cloned several times. However, even with the new sequencing technologies, the cost of sequencing BACs libraries is still high because most of the times the sequencing is individually performed from the isolated DNA of each BAC. An alternative would be the sequencing of pools containing hundreds of randomly sampled BACs, which thereby would decrease the cost in proportion to the number of BACs pooled. In this work we developed a model for sequencing and assembly BAC pools of a library prepared from a commercial sugarcane variety. As a result, a pool of 178 BACs from sugarcane variety SP80-3280 was sequenced using the technologies of the Illumina HighSeq2000 and PacBio and was assembled using different sets of softwares. Being a sample of randomly selected BACs was possible to assemble 2,451 scaffolds corresponding to 88.2% of the estimated total pool size set of BACs. The completeness of the assembly was verified in many ways including the analysis of the number of BACs assembled with expected size, comparison to sugarcane BACs deposited in NCBI and by the collinearity and gene order presented between sugarcane scaffolds and sorghum chromosomes. Scaffolds larger than 2 kb were aligned to the sorghum genome, and in general, alignments showed a uniform distribution over the 10 sorghum chromosomes indicating the randomness of sampling. By syntenic analysis between sugarcane scaffolds and sorghum chromosomes, we found that the monoploid sugarcane genome seems to be more contracted compared to the genome of sorghum. Overall the study showed that it is possible to sequence BAC pools from plant genomes with high complexity like the sugarcane genome with high level of ploidyDoutoradoBioinformaticaDoutor em Genetica e Biologia Molecula

Bioinformatica de projetos genoma de bacterias

Orientador : João Carlos SetubalDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Matematica, Estatistica e Computação CientificaResumo: Este trabalho apresenta a bioinformática desenvolvida e usada nos projetos genoma XyIelIa fastidiosa e Xanthomonas. O objetivo geral da bioinformática nesses projetos é armazenar, organizar, analisar e disponibilizar os dados biológicos oriundos dos aboratórios de seqüenciamento. Em particular, são apresentados dois sistemas de software usados para a montagem e para a anotação de genomas de bactérias. A dissertação contém também um capítulo detalhando fundamentos de biologia molecular e de técnicas de seqüenciamento de DNAMestradoMestre em Ciência da Computaçã

Bac-pool Sequencing And Assembly Of 19 Mb Of The Complex Sugarcane Genome

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Sequencing plant genomes are often challenging because of their complex architecture and high content of repetitive sequences. Sugarcane has one of the most complex genomes. It is highly polyploid, preserves intact homeologous chromosomes from its parental species and contains 55% repetitive sequences. Although bacterial artificial chromosome (BAC) libraries have emerged as an alternative for accessing the sugarcane genome, sequencing individual clones is laborious and expensive. Here, we present a strategy for sequencing and assembly reads produced from the DNA of pooled BAC clones. A set of 178 BAC clones, randomly sampled from the SP80-3280 sugarcane BAC library, was pooled and sequenced using the Illumina HiSeq2000 and PacBio platforms. A hybrid assembly strategy was used to generate 2,451 scaffolds comprising 19.2 MB of assembled genome sequence. Scaffolds of >= 20 Kb corresponded to 80% of the assembled sequences, and the full sequences of forty BACs were recovered in one or two contigs. Alignment of the BAC scaffolds with the chromosome sequences of sorghum showed a high degree of collinearity and gene order. The alignment of the BAC scaffolds to the 10 sorghum chromosomes suggests that the genome of the SP80-3280 sugarcane variety is similar to 19% contracted in relation to the sorghum genome. In conclusion, our data show that sequencing pools composed of high numbers of BAC clones may help to construct a reference scaffold map of the sugarcane genome.7[FAPESP - 10/50114-4]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Multiplex Amplicon Sequencing For Microbe Identification In Community-based Culture Collections

Microbiome analysis using metagenomic sequencing has revealed a vast microbial diversity associated with plants. Identifying the molecular functions associated with microbiome-plant interaction is a significant challenge concerning the development of microbiome-derived technologies applied to agriculture. An alternative to accelerate the discovery of the microbiome benefits to plants is to construct microbial culture collections concomitant with accessing microbial community structure and abundance. However, traditional methods of isolation, cultivation, and identification of microbes are time-consuming and expensive. Here we describe a method for identification of microbes in culture collections constructed by picking colonies from primary platings that may contain single or multiple microorganisms, which we named community-based culture collections (CBC). A multiplexing 16S rRNA gene amplicon sequencing based on two-step PCR amplifications with tagged primers for plates, rows, and columns allowed the identification of the microbial composition regardless if the well contains single or multiple microorganisms. The multiplexing system enables pooling amplicons into a single tube. The sequencing performed on the PacBio platform led to recovery near-full-length 16S rRNA gene sequences allowing accurate identification of microorganism composition in each plate well. Cross-referencing with plant microbiome structure and abundance allowed the estimation of diversity and abundance representation of microorganism in the CBC.6RepsolRepsol Sinope

Unlocking The Bacterial And Fungal Communities Assemblages Of Sugarcane Microbiome

Plant microbiome and its manipulation herald a new era for plant biotechnology with the potential to benefit sustainable crop production. However, studies evaluating the diversity, structure and impact of the microbiota in economic important crops are still rare. Here we describe a comprehensive inventory of the structure and assemblage of the bacterial and fungal communities associated with sugarcane. Our analysis identified 23,811 bacterial OTUs and an unexpected 11,727 fungal OTUs inhabiting the endophytic and exophytic compartments of roots, shoots, and leaves. These communities originate primarily from native soil around plants and colonize plant organs in distinct patterns. The sample type is the primary driver of fungal community assemblage, and the organ compartment plays a major role in bacterial community assemblage. We identified core bacterial and fungal communities composed of less than 20% of the total microbial richness but accounting for over 90% of the total microbial relative abundance. The roots showed 89 core bacterial families, 19 of which accounted for 44% of the total relative abundance. Stalks are dominated by groups of yeasts that represent over 12% of total relative abundance. The core microbiome described here comprise groups whose biological role underlies important traits in plant growth and fermentative processes.6RepsolRepsol Sinope

Overexpression of UCP1 in tobacco induces mitochondrial biogenesis and amplifies a broad stress response

Background: Uncoupling protein one (UCP1) is a mitochondrial inner membrane protein capable of uncoupling the electrochemical gradient from adenosine-5′-triphosphate (ATP) synthesis, dissipating energy as heat. UCP1 plays a central role in nonshivering thermogenesis in the brown adipose tissue (BAT) of hibernating animals and small rodents. A UCP1 ortholog also occurs in plants, and aside from its role in uncoupling respiration from ATP synthesis, thereby wasting energy, it plays a beneficial role in the plant response to several abiotic stresses, possibly by decreasing the production of reactive oxygen species (ROS) and regulating cellular redox homeostasis. However, the molecular mechanisms by which UCP1 is associated with stress tolerance remain unknown.Results: Here, we report that the overexpression of UCP1 increases mitochondrial biogenesis, increases the uncoupled respiration of isolated mitochondria, and decreases cellular ATP concentration. We observed that the overexpression of UCP1 alters mitochondrial bioenergetics and modulates mitochondrial-nuclear communication, inducing the upregulation of hundreds of nuclear- and mitochondrial-encoded mitochondrial proteins. Electron microscopy analysis showed that these metabolic changes were associated with alterations in mitochondrial number, area and morphology. Surprisingly, UCP1 overexpression also induces the upregulation of hundreds of stress-responsive genes, including some involved in the antioxidant defense system, such as superoxide dismutase (SOD), glutathione peroxidase (GPX) and glutathione-S-transferase (GST). As a consequence of the increased UCP1 activity and increased expression of oxidative stress-responsive genes, the UCP1-overexpressing plants showed reduced ROS accumulation. These beneficial metabolic effects may be responsible for the better performance of UCP1-overexpressing lines in low pH, high salt, high osmolarity, low temperature, and oxidative stress conditions.Conclusions: Overexpression of UCP1 in the mitochondrial inner membrane induced increased uncoupling respiration, decreased ROS accumulation under abiotic stresses, and diminished cellular ATP content. These events may have triggered the expression of mitochondrial and stress-responsive genes in a coordinated manner. Because these metabolic alterations did not impair plant growth and development, UCP1 overexpression can potentially be used to create crops better adapted to abiotic stress conditions141FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP12/00126-1; 12/00235-