Advancing Eucalyptus genomics: identification and sequencing of lignin biosynthesis genes from deep-coverage BAC libraries

<p>Abstract</p> <p>Background</p> <p><it>Eucalyptus </it>species are among the most planted hardwoods in the world because of their rapid growth, adaptability and valuable wood properties. The development and integration of genomic resources into breeding practice will be increasingly important in the decades to come. Bacterial artificial chromosome (BAC) libraries are key genomic tools that enable positional cloning of important traits, synteny evaluation, and the development of genome framework physical maps for genetic linkage and genome sequencing.</p> <p>Results</p> <p>We describe the construction and characterization of two deep-coverage BAC libraries EG_Ba and EG_Bb obtained from nuclear DNA fragments of <it>E. grandis </it>(clone BRASUZ1) digested with <it>Hind</it>III and <it>BstY</it>I, respectively. Genome coverages of 17 and 15 haploid genome equivalents were estimated for EG_Ba and EG_Bb, respectively. Both libraries contained large inserts, with average sizes ranging from 135 Kb (Eg_Bb) to 157 Kb (Eg_Ba), very low extra-nuclear genome contamination providing a probability of finding a single copy gene ≥ 99.99%. Libraries were screened for the presence of several genes of interest <it>via </it>hybridizations to high-density BAC filters followed by PCR validation. Five selected BAC clones were sequenced and assembled using the Roche GS FLX technology providing the whole sequence of the <it>E. grandis </it>chloroplast genome, and complete genomic sequences of important lignin biosynthesis genes.</p> <p>Conclusions</p> <p>The two <it>E. grandis </it>BAC libraries described in this study represent an important milestone for the advancement of <it>Eucalyptus </it>genomics and forest tree research. These BAC resources have a highly redundant genome coverage (> 15×), contain large average inserts and have a very low percentage of clones with organellar DNA or empty vectors. These publicly available BAC libraries are thus suitable for a broad range of applications in genetic and genomic research in <it>Eucalyptus </it>and possibly in related species of <it>Myrtaceae</it>, including genome sequencing, gene isolation, functional and comparative genomics. Because they have been constructed using the same tree (<it>E. grandis </it>BRASUZ1) whose full genome is being sequenced, they should prove instrumental for assembly and gap filling of the upcoming <it>Eucalyptus </it>reference genome sequence.</p

Evidence for Reductive Genome Evolution and Lateral Acquisition of Virulence Functions in Two Corynebacterium pseudotuberculosis Strains

Ruiz JC, D'Afonseca V, Silva A, et al. Evidence for Reductive Genome Evolution and Lateral Acquisition of Virulence Functions in Two Corynebacterium pseudotuberculosis Strains. PLoS ONE. 2011;6(4): e18551.Background: Corynebacterium pseudotuberculosis, a Gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL). CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economic impact in Oceania, Africa and South America. Although C. pseudotuberculosis causes major health and productivity problems for livestock, little is known about the molecular basis of its pathogenicity. Methodology and Findings: We characterized two C. pseudotuberculosis genomes (Cp1002, isolated from goats; and CpC231, isolated from sheep). Analysis of the predicted genomes showed high similarity in genomic architecture, gene content and genetic order. When C. pseudotuberculosis was compared with other Corynebacterium species, it became evident that this pathogenic species has lost numerous genes, resulting in one of the smallest genomes in the genus. Other differences that could be part of the adaptation to pathogenicity include a lower GC content, of about 52%, and a reduced gene repertoire. The C. pseudotuberculosis genome also includes seven putative pathogenicity islands, which contain several classical virulence factors, including genes for fimbrial subunits, adhesion factors, iron uptake and secreted toxins. Additionally, all of the virulence factors in the islands have characteristics that indicate horizontal transfer. Conclusions: These particular genome characteristics of C. pseudotuberculosis, as well as its acquired virulence factors in pathogenicity islands, provide evidence of its lifestyle and of the pathogenicity pathways used by this pathogen in the infection process. All genomes cited in this study are available in the NCBI Genbank database (http://www.ncbi.nlm.nih.gov/genbank/) under accession numbers CP001809 and CP001829

Causas químicas de resistência de Lycopersicon peruvianum (L.) a Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae)

Esta pesquisa foi conduzida em casa de vegetação na Universidade Federal de Viçosa e objetivou estudar a resistência dos acessos CNPH 101, 374 e 402 de Lycopersicon peruvianum a Tuta absoluta e as causas químicas dessa resistência. Os tratamentos foram as espécies de tomateiro Lycopersicon esculentum (cvs. Santa e IPA-5: padrões de suscetibilidade) e os acessos de L. peruvianum. Avaliaram-se o número de ovos / folha, de minas pequenas (comprimento 0,5 cm) e a percentagem de folíolos minados por T. absoluta. Realizou-se extração hexânica nas folhas, e os extratos obtidos foram submetidos a cromatografia gasosa associada a espectrômetro de massa. Nos acessos de L. peruvianum contataram-se menores taxas de oviposição, percentagens de folíolos minados, números de minas pequenas e grandes de T. absoluta do que nas cultivares IPA-5 e Santa Clara. Foram detectados 69 picos nos cromatogramas do extrato hexânico das folhas com concentração relativa > a 10⁶ íons x seg. Verificou-se efeito significativo (p 0.5 cm) and the percentage of mined leaflets by T. absoluta were evaluated. All hexanic extracts were analyzed with a gas chromatography-mass spectrometer (GC-MS). Accessions of L. peruvianum showed lower number of eggs per leaf, percentage of mined leaflets and number of leaf mines (small and big) than the susceptible cultivars (IPA-5 and Santa Clara). The total number of compounds in the chromatograms was 69, with relative concentrations > 10⁶ ions x second. The effect between T. absoluta attack and relative concentrations of the compounds showed by the five peaks (28, 42, 46, 55 and 58) was significant (p<0.05). The main substances related with L. peruvianum were: cyclobutanol; hexadecanoic acid; 9-octadecenoic acid and hexadecane (peaks 28, 42, 46 and 55, respectively). Tetracosane (peak 58) was related with L. esculentum. All substances in the leaf extracts were associated with the susceptibility of tomatoes to T. absoluta

Hipersensibilidade e necrose sistêmica em Nicotiana benthamiana transformada com o gene de resistência Sw- 5 de tomateiro

O gene Sw-5 do tomateiro confere resistência a várias espécies de tospovírus e codifica uma proteína contendo domínios de ligação a nucleotídeos e repetições ricas em leucina. Tomateiros com Sw-5 exibem reações necróticas nas folhas inoculadas com tospovírus. Estas reações e a estrutura da proteína Sw-5 indicam que a resistência ocorre por meio do reconhecimento do patógeno e desencadeamento da resposta de hipersensibilidade. A capacidade de Sw-5 de conferir resistência a tospovírus em tabaco selvagem (Nicotiana benthamiana Domin.) foi avaliada em plantas transgênicas. Uma construção com a seqüência aberta de leitura de Sw-5 e sua região 3 não-traduzida sob controle do promotor 35S do CaMV foi utilizada para transformação de N. benthamiana via Agrobacterium tumefaciens. Plantas de progênies R1 foram inoculadas com um isolado de tospovírus e avaliadas quanto à ocorrência de reação de hipersensibilidade e resistência à infecção sistêmica. Em uma progênie com segregação 3:1 (resistente:suscetível), foi selecionada uma planta homozigota e sua progênie avaliada quanto ao espectro da resistência a tospovírus. Plantas com o transgene exibiram resposta de hipersensibilidade 48 h após a inoculação, sendo resistentes à infecção sistêmica. O fenótipo da resistência foi dependente do isolado viral e um isolado de Tomato chlorotic spot virus (TCSV) causou necrose sistêmica em todas as plantas inoculadas, enquanto que isolados de Groundnut ringspot virus (GRSV) e um isolado relacionado a Chrysanthemum stem necrosis virus (CSNV) ficaram restritos ao sítio de infecção. Comparações do espectro da resistência obtido neste trabalho com aquele observado em outros membros da família Solanaceae indicam que as vias de transdução de sinais e as respostas de defesa ativadas por Sw-5 são conservadas dentro desta família e polimorfismos genéticos nas vias de transdução de sinais ou em componentes das respostas de defesa podem resultar em diferentes níveis de resistência.The tomato gene Sw-5 confers resistance against tospovirus species and codes for a nucleotide binding site and leucine rich protein. Tomatoes with the Sw-5 gene develop a necrotic reaction when mechanically inoculated with tospoviruses. The necrotic lesions on inoculated leaves of the resistant plants and the structure of the protein codified by Sw-5 suggests that the resistance depends on recognition of the pathogen and activation of hypersensitive response (HR). The capacity of the Sw-5 to confer resistance in wild tobacco (Nicotiana benthamiana) was evaluated in transgenic plants transformed by Agrobacterium tumefaciens. The Sw-5 ORF and its own 3' UTR region were placed under 35S promoter control. Plants of R1 progenies were inoculated with tospovirus and evaluated for local and systemic symptoms. In one progeny with 3:1 (resistant:susceptible) segregation ratio a homozygous plant was selected and the resistance spectrum of its progeny was evaluated. Transgenic plants showed hypersensitive response 48 h after inoculations and were resistant to tospovirus infection. The resistance was isolate-specific and a Tomato chlorotic spot virus (TCSV) isolate caused systemic necrosis in the transformed plants, while Groundnut ringspot virus (GRSV) isolates and one Chrysanthemum stem necrosis virus (CSNV) related isolate was restricted to the inoculation site. Comparisons of the resistance spectrum with that observed in other members of the Solanaceae suggest that the signal transduction pathways and resistance responses triggered by Sw-5 are conserved in Solanaceae, and the genetic polymorphism in the signal transduction pathways or defense response components may result in different resistance levels

Componentes de resistência em cebola a Colletotrichum gloeosporioides

A antracnose foliar causada por Colletotrichum gloeosporioides é importante nas regiões cebolicultoras da América Latina, África e Ásia, mas há poucos estudos relacionados à resistência ao patógeno. Assim, neste trabalho, conduzido em condições de casa de vegetação, avaliaram-se componentes de resistência de oito cultivares e dois acessos de cebola (Allium cepa) a quatro isolados do patógeno, inoculados por atomização de suspensão de inóculo ou deposição de discos de micélio em folhas. Detectaram-se diferenças significativas entre cultivares/acessos, na inoculação por atomização, quanto à freqüência de infecção inicial e à taxa de progresso monocíclico da doença (rg) e, na inoculação com disco de micélio, quanto ao período de incubação e área da lesão. Determinaram-se os coeficientes de correlação (r) dos componentes de resistência estimados na inoculação por atomização. Os valores de r foram 0,98 entre severidade estimada visualmente aos nove dias da inoculação (SEV9) e área abaixo da curva de progresso da doença (AACPD); 0,80 entre SEV9 e severidade estimada por medidor de área foliar (SEV); 0,72 entre SEV9 e rg; 0,64 entre SEV9 e freqüência de infecção aos nove dias da inoculação; 0,81 entre SEV e AACPD e 0,64 entre SEV e rg. Em vista dos valores significativos de r associados a SEV9 e da não necessidade de escala para estimar esse componente, SEV9 é potencialmente útil na avaliação de germoplasma de cebola frente a C. gloeosporioides. Na inoculação por atomização, mais rápida e simples de execução, obtiveram-se maior eficiência de infecção e menor variabilidade de respostas, e deverá ser adotada em estudos futuros.Despite the importance of onion (Allium cepa) leaf anthracnose caused by Colletotrichum gloeosporioides in Latin America, Africa, and Asia, few studies have focused on host resistance to the pathogen. Therefore, in this study, resistance components of two cultivars and eight accessions of onion to four isolates of C. gloeosporioides were evaluated under greenhouse conditions. Inoculations were performed either by spraying inoculum suspension or by placing a mycelial disc on the leaf. The cultivars and accessions differed significantly regarding initial infection frequency and monocyclic progress rate (rg) with the spray-inoculation, and regarding incubation period and lesion area with the mycelial-disc inoculation. Correlation coefficient (r) values were estimated between the components with the mycelial disk inoculations. Values of r were 0.98 between disease severity visually assessed nine days after inoculation (SEV9) and area under the disease progress curve (AUDPC), 0.80 between SEV9 and disease severity assessed with the leaf area meter (SEV), 0.72 between SEV9 and rg, 0.64 between SEV9 and infection frequency nine days after inoculation, 0.81 between SEV and AUDPC, and 0.64 between SEV and rg. Considering both the significant r values associated with SEV9 and that to estimate SEV9 there is no need of rating diagrams, this component is potentially useful to evaluate onion germplasm against C. gloeosporiodes. The spray inoculation procedure was faster, simpler, and provided higher infection efficiency and lower variability than the mycelial disk inoculation technique. Therefore, this should be the preferred inoculation procedure when assessing onion germplasm

Integração de pAN7-1 no genoma de Magnaporthe grisea mediada por enzima de restrição

Visando explorar a mutagênese insercional em Magnaporthe grisea, foram avaliadas a transformação dos protoplastos obtidos após adequação do protocolo e a eficiência da integração de pAN7-1 no genoma do ascomiceto na presença da enzima de restrição Hind III. Os protoplastos de M. grisea I-22 foram prontamente transformados para a resistência à higromicina. Quando o vetor linearizado com Hind III foi usado para transformar o fungo na presença de Hind III, a eficiência de transformação foi 1,1 a 8,1 vezes superior ao tratamento sem a adição da enzima. No geral, a melhor concentração de Hind III foi 5 unidades/reação de transformação. Tal concentração promoveu a produção média de 332 transformantes/µg de pAN7-1/107 protoplastos. A presença do gene de seleção hph no genoma de 18 indivíduos resistentes à higromicina foi confirmada por PCR.To investigate insertional mutagenesis in Magnaporthe grisea, we tested the transformation of protoplasts produced after protocol optimization, and analyzed the integration efficiency of pAN7-1 into the M. grisea genome mediated by the restriction endonuclease Hind III. The I-22 protoplasts were readily transformed for hygromycin resistance. When pAN7-1 was linearized with Hind III and used to transform fungal protoplasts in the presence of the corresponding enzyme, the transformation efficiency was increased 1.1 to 8.1-fold. The optimal Hind III concentration for enhanced transformation corresponded to 5 unit/transformation mix. This concentration led to average frequency of 332 transformants/µg de pAN7-1/107 protoplasts. The presence of selection gene hph in the 18 transformant genome was confirmed by PCR

PCR amplification and sequence analyses of Reverse Transcriptase-like genes in Crinipellis perniciosa isolates

Reverse transcriptase (RT) sequence analysis is an important technique used to detect the presence of transposable elements in a genome. Putative RT sequences were analyzed in the genome of the pathogenic fungus C. perniciosa, the causal agent of witches' broom disease of cocoa. A 394 bp fragment was amplified from genomic DNA of different isolates of C. perniciosa belonging to C-, L-, and S-biotypes and collected from various geographical areas. The cleavage of PCR products with restriction enzymes and the sequencing of various RT fragments indicated the presence of several sequences showing transition events (G:C to A:T). Southern blot analysis revealed high copy numbers of RT signals, forming different patterns among C-, S-, and L-biotype isolates. Sequence comparisons of the predicted RT peptide indicate a close relationship with the RT protein from the gypsy family of LTR-retrotransposons. The possible role of these retrotransposons in generating genetic variability in the homothallic C. perniciosa is discussed.A análise de sequências de transcriptase reversa (RT) é uma etapa importante para descobrir a presença de elementos transponíveis e investigar o seu papel na geração de variabilidade genética em C. perniciosa. Seqüências putativas de TR foram analisadas no genoma do fitopatógeno C. perniciosa, o agente causal da doença vassoura-de-bruxa no cacau. Um fragmento de 394 pb foi amplificado a partir do DNA genômico de diferentes isolados de C. perniciosa, pertencentes aos biótipos C, L e S e a distintas áreas geográficas. A clivagem dos produtos de PCR com diferentes enzimas de restrição e sequenciamento de vários fragmentos de TR indicou a presença de diferentes seqüências mostrando eventos de transição G:C para A:T. A análise por hibridização revelou alto número de sinais sugerindo a presença de cópias de TR com diferentes perfis entre os isolados dos biótipos C, S e L. As comparações de seqüências dos peptídeos preditos indicam uma relação próxima com a proteína TR de retrotransposons-LTR da família gypsy