New softwares for automated microsatellite marker development

Microsatellites are repeated small sequence motifs that are highly polymorphic and abundant in the genomes of eukaryotes. Often they are the molecular markers of choice. To aid the development of microsatellite markers we have developed a module that integrates a program for the detection of microsatellites (TROLL), with the sequence assembly and analysis software, the Staden Package. The module has easily adjustable parameters for microsatellite lengths and base pair quality control. Starting with large datasets of unassembled sequence data in the form of chromatograms and/or text data, it enables the creation of a compact database consisting of the processed and assembled microsatellite containing sequences. For the final phase of primer design, we developed a program that accepts the multi-sequence ‘experiment file’ format as input and produces a list of primer pairs for amplification of microsatellite markers. The program can take into account the quality values of consensus bases, improving success rate of primer pairs in PCR. The software is freely available and simple to install in both Windows and Unix-based operating systems. Here we demonstrate the software by developing primer pairs for 427 new candidate markers for peanut

ESTs from a wild Arachis species for gene discovery and marker development

BACKGROUND: Due to its origin, peanut has a very narrow genetic background. Wild relatives can be a source of genetic variability for cultivated peanut. In this study, the transcriptome of the wild species Arachis stenosperma accession V10309 was analyzed. RESULTS: ESTs were produced from four cDNA libraries of RNAs extracted from leaves and roots of A. stenosperma. Randomly selected cDNA clones were sequenced to generate 8,785 ESTs, of which 6,264 (71.3%) had high quality, with 3,500 clusters: 963 contigs and 2537 singlets. Only 55.9% matched homologous sequences of known genes. ESTs were classified into 23 different categories according to putative protein functions. Numerous sequences related to disease resistance, drought tolerance and human health were identified. Two hundred and six microsatellites were found and markers have been developed for 188 of these. The microsatellite profile was analyzed and compared to other transcribed and genomic sequence data. CONCLUSION: This is, to date, the first report on the analysis of transcriptome of a wild relative of peanut. The ESTs produced in this study are a valuable resource for gene discovery, the characterization of new wild alleles, and for marker development. The ESTs were released in the [GenBank:EH041934 to EH048197]

Resistência extrema a duas estirpes do Potato virus Y (PVY) de batata transgênica, cv. Achat, expressando o gene da capa protéica do PVYO

The coat protein (CP) gene of the potato virus Y strain “o” (PVYO) was introduced into potato, cultivar Achat, via Agrobacterium tumefaciens-mediated transformation. Sixty three putative transgenic lines were challenged against the Brazilian strains PVY-OBR and PVY-NBR. An extremely resistant phenotype, against the two strains, was observed in one line, denominated 1P. No symptoms or positive ELISA results were observed in 16 challenged plants from this line. Another clone, named as 63P, showed a lower level of resistance. Southern blot analysis showed five copies of the CP gene in the extremely resistant line and at least three copies in the other resistant line. The stability of the integrated transgenes in the extreme resistant line was examined during several in vitro multiplications over a period of three years, with no modification in the Southern pattern was observed. The stability of the transgenes, the absence of primary infections and the relatively broad spectrum of resistance suggest that the extremely resistant line obtained in this work can be useful for agricultural purposes.O gene da capa protéica (CP) do Potato virus Y estirpe “o”, foi introduzido em batata cultivar Achat, via Agrobacterium tumefaciens. Sessenta e três linhas possivelmente transgênicas foram desafiadas com as estirpes brasileiras PVY-OBR e PVY-NBR. Uma linha apresentou extrema resistência às duas estirpes inoculadas, e foi denominado clone 1P. Não foram observados sintomas sistêmicos de infecção e as plantas foram negativas em Elisa. Outra linha, denominada clone 63P, mostrou algum nível de resistência. Análises por Southern blot indicaram a presença de pelo menos cinco cópias do gen CP no clone 1P e pelo menos três cópias no clone 63P. A estabilidade do gene introduzido no clone 1P foi avaliada durante três anos, após várias multiplicações in vitro. Não foram observadas mudanças no padrão do Southern blot. A estabilidade do transgene, na ausência de infecções primárias e relativo largo espectro de resistência sugerem que o clone 1P pode ser utilizado para fins comerciais.Fil: Romano, Eduardo. Embrapa Recursos Genéticos; BrasilFil: Ferreira, Adriana T.. Embrapa Hortaliças; BrasilFil: Dusi, André N.. Embrapa Hortaliças; BrasilFil: Proite, Karina. Embrapa Recursos Genéticos; BrasilFil: Buso, Jose A.. Embrapa Hortaliças; BrasilFil: Avila, Antonio C.. Embrapa Hortaliças; BrasilFil: Nishijima, Marta L.. Embrapa Hortaliças; BrasilFil: Nascimento, Adriana S.. Embrapa Hortaliças; BrasilFil: Bravo Almonacid, Fernando Felix. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Mentaberry, Alejandro Nestor. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Monte, Damares. Embrapa Recursos Genéticos; BrasilFil: Campos, Magnolia A.. Embrapa Recursos Genéticos; BrasilFil: Melo, Paulo Eduardo. Embrapa Hortaliças; BrasilFil: Cattony, Monica K.. No especifica;Fil: Torres, Antonio C.. Embrapa Hortaliças; Brasi

Development of molecular markers for resistance gene analogs in wild Arachis spp.

O maior grupo de genes de resistência de plantas já clonados codifica para proteínas com um sítio de ligação a nucleotídios (NBS) na região N-terminal, e um domínio rico em repetições de leucina (LRR) na região C-terminal. Genes desta classe conferem resistência a diversos patógenos incluindo vírus, bactérias, fungos e nematóides. Para diferentes espécies do gênero Arachis, primers de "polymerase chain reaction" (PCR) degenerados foram construídos para a região NBS, e o produto de tradução putativo indicou similaridade com proteínas de resistência conhecidas sendo denominados análogos a genes de resistência (RGAs). Doze destes RGAs foram utilizados para o desenvolvimento de marcadores moleculares baseados em seus padrões de hibridização com DNA de Arachis spp. digerido com enzimas de restrição. Inicialmente, avaliou-se o polimorfismo de cada RGA como sonda nos parentais de uma população de mapeamento, contrastantes quanto à resistência as manchas foliares e nematóides das galhas, e no híbrido F1. Os RGAs, mesmo isolados de espécies diferentes do gênero Arachis apresentaram homologia com o DNA das espécies testadas, além de apresentarem múltiplas cópias e alto polimorfismo na progênie F2. Todas estas características tornam estes RGAs marcadores moleculares altamente informativos, sendo que alguns apresentaram segregação em "clusters" na F2, indicando que seus locos estão ligados. Estes marcadores serão incluídos em um mapa genético de Arachis spp., o que será de grande utilidade para os programas de melhoramento do amendoim (Arachis hypogaea) cultivado.The majority of cloned plant pathogen resistance genes (R genes) encode a putative nucleotide binding site (NBS) domain and a leucine-rich repeat (NBS-LRR genes). Genes of this NBS-LRR class confer resistance to diverse pathogens such as viruses, bacteria, fungi, nematodes and aphids. The conserved NBS domain was used to generate resistance gene analogues (RGAs) fragments by polymerase chain reaction (PCR) using degenerated primers in different Arachis species. Twelve of these RGAs were used to develop molecular markers based on their patterns of hybridisation to restricted Arachis spp. DNA. An initial step was the evaluation of the polymorphism generated by each RGA in genomic fragments of contrasting parents of a mapping population that segregates for resistance to leaf spot and nematodes, and of the F1 hybrid. The RGAs isolated from different Arachis species showed high homology to the DNA of the parents and hybrid, multiple copies in the genome and high polymorphism in the F2 generation. Therefore, they were considered highly informative markers, with some segregating in clusters in the F2. These RGAs will be included in the Arachis genetic map, which will be of paramount importance for the Arachis spp. breeding programs

Busca de genes envolvidos na resistência de amendoim silvestre ao nematóide das galhas (Meloidogyne arenaria)

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, 2007.O amendoim cultivado (Arachis hypogaea) é uma espécie importante, amplamente cultivada nas regiões tropicais e subtropicais do mundo. É bastante suscetível a diversos estresses bióticos e abióticos, para os quais muitas das espécies silvestres são resistentes. Neste trabalho foram avaliadas as duas espécies silvestres A. duranensis e A. stenosperma e selecionadas quanto à resistência ao nematóide Meloidogyne arenaria raça 1. O estudo histopatológico mostrou que A. stenosperma é uma espécie resistente sendo esta caracterizada por uma reação de hipersensibilidade (HR). A. duranensis é uma espécie suscetível, mas possui ciclo de infecção diferenciado da espécie hospedeira típica, A. hypogaea. Bibliotecas de cDNA de raízes de A. stenosperma foram construídas para a formação de um banco de ESTs. Os ESTs de bibliotecas de raízes de A. stenosperma inoculadas e não inoculadas com o nematóide foram analisados in silico para a expressão diferencial de genes durante a infecção. A estratégia do estudo dos ESTs evidenciou oito genes diferencialmente expressos e foi uma primeira tentativa de procura dos fatores responsáveis pela resistência ao nematóide em A. stenosperma. A análise da expressão de seis ESTs foi testada por Northern blot para a confirmação da análise in silico e identificação do possível envolvimento destes genes com a resistência. Neste trabalho ainda, foram construídas e validadas duas bibliotecas BAC (Bacterial Artificial Chromosome) das espécies silvestres diplóides, A. duranensis e A. ipaënsis que são as possíveis espécies parentais do alotetraplóide A. hypogaea. Estas bibliotecas servirão como importantes ferramentas genéticas que possibilitarão o isolamento de genes de interesse do amendoim silvestre e sua comparação com o genoma de outras leguminosas. _______________________________________________________________________________ ABSTRACTArachis hypogaea, the cultivated peanut, is an important crop, widely grown in tropical and subtropical regions of the world. It is highly susceptible to several biotic and abiotic stresses to which wild species are resistant. In this work, two wild species, A. duranensis and A. stenosperma were evaluated and selected based upon resistance to the root-knot nematode Meloidogyne arenaria race 1. Histopathological studies showed that A. stenosperma is a resistant species. Its resistance is characterized by a hypersensitive response (HR). A. duranensis is susceptible but the evolution of the nematode cycle is different to the host species, A. hypogaea. cDNA libraries of roots were constructed for the development of an EST databank for wild Arachis species. ESTs generated from non inoculated and M. arenaria race 1 inoculated roots of A. stenosperma were analyzed in silico for differential gene expression during infection. This strategy revealed eight clones differentially expressed and represented a first attempt to search for factors that are responsible for resistance in A. stenosperma. Expression analysis of six of these ESTs was conducted by Northern blot to confirm and validate the in silico analysis for the possible involvement of these genes in resistance. In this study two BAC (Bacterial Artificial Chromosome) libraries of the wild progenitors of A. hypogaea, A. duranensis and A. ipaënsis were also constructed and validated. These libraries will function as genetic tools and may help with the isolation of genes of interest in wild Arachis and in comparison with others legume genomes

From Cuba to most of the neotropic: habenaria bicornis (orchidaceae) is widespread from Mexico to southeastern Brazil

Habenaria bicornis fue descrita por primera vez en 1835, para Cuba, y era conocida apenas para este país y unos pocos registros de Panamá, de 1920. En el presente estudio, demostramos que H. bicornis y H. goyazensis, esta última conocida para Brasil y Guiana, son específicas y la especie se distribuye desde México hasta el sudeste de Brasil. El modelado de nicho y la recolección de datos indican que esta especie posee una preferencia por sabanas húmedas de tierras bajas y es predicho que su distribución incluye gran parte del Neotrópico con hábitats favorables. Los análisis filogenéticos moleculares con secuencias del ADN nuclear (ITS) y plastidial (matK) ubicaron H. bicornis en una posición aislada próximo a la base del clado Neotropical, aunque con bajo soporte. En términos de su morfología, sus relaciones no están aclaradas, ya que no hay similitudes evidentes entre H. bicornis y los subclados básales o cualquier otro subclado Neotropical. Los análisis citogenéticos indican un número cromosómico básico de x=21, similar a las otras especies basales del Neotrópico.Habenaria bicornis was first described in 1835 from Cuba and has only been known from that country and from a few records in Panama from the 1920s. Here we show that H. bicornis and H. goyazensis, known from Brazil and Guyana, are conspecific and that the species is distributed from Mexico to southeastern Brazil. Niche modeling and collection data indicate that this species has a preference for wet lowland savannas and its distribution is predicted to include most of the Neotropics with suitable habitats. The molecular phylogenetic analyses based on DNA sequences from the nuclear internal transcribed spacer (ITS) region and part of the plastid matK gene placed H. bicornis in an isolated position near the base of the Neotropical clade, although with low support. In terms of its morphology, its relationships are likewise not clear as there are no evident similarities between H. bicornis and the basal subclades or any other Neotropical subclade. Cytogenetic analysis indicated a basic chromosome number of x=21, similar to other basal Neotropical species.

New softwares for automated microsatellite marker

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BAC libraries construction from the ancestral diploid genomes of the allotetraploid cultivated peanut.

International audienceCultivated peanut, Arachis hypogaea is an allotetraploid of recent origin, with an AABB genome. In common with many other polyploids, it seems that a severe genetic bottle-neck was imposed at the species origin, via hybridisation of two wild species and spontaneous chromosome duplication. Therefore, the study of the genome of peanut is hampered both by the crop's low genetic diversity and its polyploidy. In contrast to cultivated peanut, most wild Arachis species are diploid with high genetic diversity. The study of diploid Arachis genomes is therefore attractive, both to simplify the construction of genetic and physical maps, and for the isolation and characterization of wild alleles. The most probable wild ancestors of cultivated peanut are A. duranensis and A. ipaënsis with genome types AA and BB respectively. Results: We constructed and characterized two large-insert libraries in Bacterial Artificial Chromosome (BAC) vector, one for each of the diploid ancestral species. The libraries (AA and BB) are respectively c. 7.4 and c. 5.3 genome equivalents with low organelle contamination and average insert sizes of 110 and 100 kb. Both libraries were used for the isolation of clones containing genetically mapped legume anchor markers (single copy genes), and resistance gene analogues.Conclusion. These diploid BAC libraries are important tools for the isolation of wild alleles conferring resistances to biotic stresses, comparisons of orthologous regions of the AA and BB genomes with each other and with other legume species, and will facilitate the construction of a physical map