18 research outputs found
Genetic diversity of Ralstonia solanacearum isolates and molecular characterization as to phylotypes and sequevars
O objetivo deste trabalho foi identificar isolados brasileiros de Ralstonia solanacearum quanto a filotipos e sequevares, determinar sua diversidade genética, realizar a associação da estrutura genética do patógeno com sua classificação e origem geográfica e identificar um marcador molecular para a diagnose do moko-da-bananeira. Um grupo de 33 isolados de R. solanacearum, da coleção da Embrapa Tabuleiros Costeiros, coletado de diversos hospedeiros, foi caracterizado por meio de PCR em sequência palindrômica extragênica repetitiva (rep‑PCR) e RAPD. Deste grupo, 19 perteciam à raça 2 do patógeno e 14 à raça 1, e 15 isolados eram associados à cultura da bananeira. Os filotipos e sequevares foram caracterizados e determinados por PCR Multiplex. Verificou‑se que 82% dos isolados pertencem ao filotipo II, e 12% ao filotipo III. Todos os isolados da bananeira pertencem ao filotipo II. A técnica de RAPD foi eficiente em agrupar os isolados de acordo com sua origem geográfica; entretanto, ela requer um número elevado de marcas moleculares. Foi possível relacionar os isolados pela análise rep‑PCR. O iniciador com sequências repetitivas enterobacterianas intergênicas de consenso (ERIC) possibilitou a separação dos isolados de acordo com a raça, e o iniciador REP permitiu a discriminação entre os filotipos. Estas duas análises foram as mais informativas.The objective of this work was to identify Brazilian isolates of Ralstonia solanacearum according to phylotypes and sequevars, to determine their genetic diversity, to associate the pathogen genetic structure with its taxonomy and geographical origin, and to identify a specific molecular marker to diagnose banana moko disease. A group of 33 isolates of R. solanacearum, from the collection of Embrapa Tabuleiros Costeiros, collected from different plant hosts, was characterized using the repetitive extragenic palindromic sequence‑based PCR (rep‑PCR) and RAPD. From this group, 19 belonged to the pathogen race 2 and 14 to the race 1, and 15 isolates were associated with banana crop. Phylotypes and sequevars were characterized and determined by Multiplex PCR. It was verified that the isolates belonged to phylotypes II (82%) and III (12%). All isolates from banana plants belonged to phylotype II. The RAPD technique was efficient in grouping these isolates according to their geographical origin; however, it requires a large number of molecular markers. It was possible to establish the relationships among the isolates by rep‑PCR. The enterobacterial repetitive intergenic consensus primer (ERIC) made it possible to separate the isolates according to the race, and the REP primer allowed for the discrimination among phylotypes. These were the two most informative analyses
Assessment of genetic diversity of a native population of Eplingiella fruticosa: a plant with therapeutic potential
Eplingiella fruticosa (Salzm. ex Benth.) Harley & J.F.B. Pastore, ex Hyptis fruticosa Salzm. ex Benth. is an aromatic and medicinal plant of the family Lamiaceae, found mainly in regions with intense anthropic activity. Information on the genetic diversity of this species is scarce. However, it can be assessed using molecular markers that identify the level of diversity among phenotypically identical individuals. The present study aimed to characterize the genetic diversity of a native population of E. fruticosa from the State of Sergipe using ISSR molecular markers. Samples of 100 plants were collected in 11 municipalities of the State of Sergipe and analyzed using eight ISSR primers, resulting in 72 informative bands. The cluster analysis obtained using the neighbor joining method resulted in three groups: Group I consisted of 50 plants, mainly from the municipalities of Areia Branca, Estância, Japaratuba, Moita Bonita, Pirambu, and Salgado; Group II was formed by 21 plants, with nine representatives from the municipality of Itaporanga d’Ajuda and 13 representatives from other municipalities; Group III was composed by 29 plants, being represented mainly by the municipalities of Malhada dos Bois and São Cristóvão. The smallest genetic distance occurred between plants EPF94 and EPF96 (0.250), and the greatest distance occurred between plants EPF50 and EPF96 (0.9778). The Shannon index had a mean value of 0.42, and diversity was considered moderate. Heterozygosity had a mean value of 0.267 and was considered low. Polymorphic information content (0.253) was considered moderately informative. Genetic diversity of E. fruticosa plants was intermediate, and the results of the present study can assist in the conservation and use of the genetic resources of this species
Physical maping of the chromosomal region close to Meloidogyne exigua resistance gene (Mex-1) in Coffea arabica L.
O nematóide de galhas, Meloidogyne exigua, parasita o cafeeiro (Coffea sp.) e sua erradicação das regiões infestadas é impraticável. Estudos anteriores mostraram que a resistência a M. exigua é controlada por um gene de herança simples, designado Mex-1, presente em C. canephora. Linhagens de café arábica apresentando resistência têm sido desenvolvidas em programas de melhoramento genético por meio da introgressão deste gene. Uma biblioteca BAC de café foi avaliada inicialmente utilizando-se três marcadores (Exi-2, Exi-3 e Exi-4) ligados a Mex-1, obtidos a partir de marcadores AFLP previamente clonados. Os clones positivos associados à região Mex-1 foram identificados e isolados. O DNA destes clones foi então analisado por hibridização e amplificação com marcadores SCAR (Exi-2 e Exi- 3) e 9 combinações de primers das extremidades BAC. Os clones BAC positivos, relativos aos marcadores Exi-2, Exi-3 e Exi-4, foram reunidos em 5 contigs. O primeiro contig apareceu composto de dois clones BAC identificados com o marcador Exi-4. Dos cinco clones BAC identificados com o marcador Exi-2, dois contigs designados Exi-2 I e Exi-2 II foram distintos. Os clones BAC Exi-3 foram organizados em dois contigs distintos chamados Exi-3 I e Exi-3 II. No total, nove sítios de restrição ou de PCR, correspondendo a sete marcadores físicos, foram localizados na região Exi-3. As três regiões cromossômicas correspondentes ao contig Exi-4, aos dois contigs Exi-2 e aos dois contigs Exi-3 não apresentaram sobreposição. A presença de clones BAC contendo análogos de gene de resistência (RGA) na região Mex-1 foi investigado por meio de amplificação com primers degenerados e hibridização com sondas RGA de café. Alguns clones BAC contendo RGAs foram identificados. Para confirmar a introgressão do gene Mex-1, vinte e uma linhagens de Icatu foram comparadas a três testemunhas resistentes, Iapar 59, Híbrido de Timor e Sarchimor e a uma testemunha susceptível, Catuaí. Foi avaliada, por meio de marcadores AFLP, a presença do fragmento introgredido associado ao locus Mex-1. Nove marcadores AFLP foram amplificados. Destes, os marcadores Exi-1, Exi-2, Exi-3, Exi-11 e Exi-13 confirmaram a presença do fragmento associado à resistência a M. exigua, mostrando que esta marca pode ser utilizada em programas de seleção assistida. Uma biblioteca de cDNA de Caturra foi utilizada para se isolar fragmentos de genes associados à resistência, através da hibridização com sondas RGA. Após a utilização das nove famílias RGA de café, 32 colônias foram isoladas e seqüenciadas. Destas, 25 seqüências não apresentaram similaridade com outras seqüências disponíveis na base de dados do NCBI ou não puderam ser seqüenciadas. Seis amostras continham seqüências muito pequenas e não confiáveis, e apenas uma amostra resultou em uma seqüência de 428 nucleotídeos (142 aminoácidos). Esta seqüência apresentou alta homologia a catalases de outras plantas como Nicotiana plumbaginifolia, Glycine max, Phaseolus vulgaris, entre outras. A esta catalase descoberta em café foi dado o nome de Cat-C. A catalase de ligação ao ácido salicílico (SR1) e a seqüência parcial de aminoácidos da proteína de ligação ao ácido salicílico (SABP), ambos de N. tabacum, apresentaram 82% e 77% de identidade, respectivamente, quando comparadas a Cat- C. Na região C-terminal desta catalase foi detectada a presença de uma seqüência peroxissomal. No entanto, ao invés de uma seqüência SRL (Serina-Arginina- Leucina), foi encontrada uma seqüência TRL (Treonina-Arginina-Leucina). Esta diferença é devido à troca de uma timina por uma adenina. Estes dados sugerem que Cat-C pode estar associado ao mecanismo de resistência em café.The root-knot nematode Meloidogyne exigua parasites the arabica coffee tree (Coffea sp.) and its eradication of the infested areas is impracticable. Previous studies showed that the resistance to M. exigua is controlled by a simple inheritance gene, designated Mex-1, present in C. canephora. Lineages of resistant Arabica coffee have been developed in some genetic breeding programs through the introgression of this gene. The BAC library of coffee was initially evaluated by three markers (Exi-2, Exi-3 and Exi-4) linked to Mex-1, previously obtained from cloned AFLP markers. The positive clones associated with Mex-1 region were identified and isolated. DNA clones were then analyzed by hybridization and amplification with SCAR (Exi-2 and Exi-3) markers and 9 combinations of primers of the BAC-end. The positive BAC clones, relative to the Exi-2, Exi-3 and Exi-4 markers, were assembled in 5 contigs. The first contig identified with the Exi-4 marker is composed of two positive BAC clones. Of the five BAC clones identified with Exi-2 markers, two contigs designated Exi-2 I and Exi-2 II were different from each other. The BAC clones Exi-3 were organized in two different contigs called Exi-3 I and Exi-3 II. In the total, nine restriction sites or PCR, corresponding to seven physical markers were located in the Exi-3 region. The three chromosomal regions that corresponded to the Exi-4 contig, the two Exi-2 contigs and the two Exi-3 contigs did not present any superposition. The presence of BAC clones containing resistance gene analogs (RGA) in the Mex-1 region was investigated by both PCR amplification with degenerate primers, and hybridization with coffee RGA probes. Some BAC clones containing RGA were identified. To confirm the introgression of Mex-1 gene, twenty-one Icatu lineages xiiiwere compared to three resistant cultivars, Iapar 59, Hibrido de Timor and Sarchimor and to a susceptible cultivar Catuaí, through AFLP markers, evaluating the presence or not of the introgressed fragment associated with the Mex-1 locus. Nine AFLP markers were amplified. Among these, Exi-1, Exi-2, Exi-3, Exi-11 and Exi-13 markers confirmed the presence of the fragment associated to the resistance to Meloidogyne exigua, showing that this marker can be used in selected assistent programs. A cDNA library of Caturra was used to isolate fragments of genes associated with the resistance, through the hybridization using RGA probes. After the use of the nine coffee RGA families, 32 colonies were isolated and sequenciated. Among these, 25 sequences either did not present similarity with other available sequences in the NCBI data base or could not be sequenced. Six samples contained very small and not trustable sequences, and just one sample resulted in a sequence of 428 nucleotides (142 aminoacids). This sequence presented high homology to catalases of other plants such as Nicotiana plumbaginifolia, Glycine max, Phaseolus vulgaris, etc. To this discovered coffee catalase was given the name of Cat-C. The salicylic acid receptor (SR1) and the partial sequence of aminoacids of the salicylic acid binding protein (SABP), both in Nicotinana tabacum, showed 82% and 77% of identities, respectively, when compared with Cat-C. In the C-terminal region of this catalase was detected the presence of a peroxisomal sequence. However, instead of a SRL sequence (Serine-Arginine-Leucine), a TRL sequence was found (Treonine- Arginine-Leucine). This difference is due to the change of a timine by an adenine. These data suggest that Cat-C can be associated with resistance mecanism in coffee.Conselho Nacional de Desenvolvimento Científico e Tecnológic
A new species of Oecleus Stål (Hemiptera: Fulgoroidea: Cixiidae) from coconut in Brazil
A new species of cixiid planthopper (Hemiptera: Fulgoroidea) in the genus Oecleus Stål, Oecleus sergipensis n. sp., is described from Sergipe State, Brazil. This new taxon is associated with coconut (Cocos nucifera L.) and date palm (Phoenix L). The species was detected in Auchenorrhyncha surveys to find potential vectors of lethal yellowing type syndrome. This is the first report of the genus Oecleus in Brazil. Sequence data from the mitochondrial cytochrome c oxidase subunit I (COI) barcoding region was obtained and accessioned into GenBank