Study of simple sequence repeat markers from coffee expressed sequences associated to leaf miner resistance

O objetivo deste trabalho foi identificar marcadores do tipo microssatélite (SSR) associados à resistência ao bicho-mineiro. A identificação dos marcadores foi feita por meio de buscas dirigidas no banco de seqüências expressas (EST) de café. Foram selecionadas 32 sequências microssatélites expressas em folhas e relacionadas a mecanismos de defesa. Os locos SSR avaliados apresentaram, em sua maioria, repetições mais complexas, com: 65% das repetições de tetranucleotídeos, 21% de trinucleotídeos e 14% de dinucleotídeos. Os locos avaliados estão freqüentemente localizados na região 5'-UTR do gene e envolvidos com mecanismos de defesa. Polimorfismos foram avaliados em progênies avançadas de cruzamentos entre Coffea racemosa x Coffea arabica, segregantes para resistência ao bicho-mineiro. Destes 32 oligonucleotídeos, 29 amplificaram os locos SSR esperados, e o número médio de alelos por loco foi de 2,1. Não foram identificados alelos polimórficos associados à resistência ao bicho-mineiro. Esses resultados sugerem que a seleção assistida por marcadores, em café, deve ser realizada em cruzamentos iniciais, nos quais a variabilidade genética é maior.The objective of this work was to identify expressed simple sequence repeats (SSR) markers associated to leaf miner resistance in coffee progenies. Identification of SSR markers was accomplished by directed searches on the Brazilian Coffee Expressed Sequence Tags (EST) database. Sequence analysis of 32 selected SSR loci showed that 65% repeats are of tetra-, 21% of tri- and 14% of dinucleotides. Also, expressed SSR are localized frequently in the 5'–UTR of gene transcript. Moreover, most of the genes containing SSR are associated with defense mechanisms. Polymorphisms were analyzed in progenies segregating for resistance to the leaf miner and corresponding to advanced generations of a Coffea arabica x Coffea racemosa hybrid. Frequency of SSR alleles was 2.1 per locus. However, no polymorphism associated with leaf miner resistance was identified. These results suggest that marker-assisted selection in coffee breeding should be performed on the initial cross, in which genetic variability is still significant

Caracterização da diversidade genética de linhagens comerciais de Coffea arabica através de marcadores moleculares do tipo RAPD, AFLP e SSR

A identificação de linhagens de Coffea arabica a partir de descritores botânicos e agronômicos é um problema para o desenvolvimento de cultivares. Basicamente, a limitada variação fenotípica observada em cultivares é o resultado de uma estreita variabilidade genética em C. arabica associada com uma origem genealógica próxima. Recentemente, os uso de marcadores moleculares tem contribuído para a caracterização e identificação de várias espécies de interesse comercial. O objetivo deste trabalho foi comparar a confiabilidade de três tipos de marcadores moleculares, RAPD, AFLP e SSR, para a caracterização da variabilidade genética e uma possível identificação de linhagens comerciais de Coffea desenvolvidas pelo IAC. Os métodos avaliados permitiram identificar polimorfismos entre cultivares. A variabilidade genética detectada por eles é muito semelhante, ainda que reduzida. Marcadores do tipo RAPD e SSR foram mais eficientes em análises de parentesco, e o agrupamento das linhagens correspondeu à sua origem genealógica. No entanto, nenhum dos métodos testados permitiu a identificação individual de linhagens. Neste caso, a utilização conjunta de descritores botânicos, agronômicos e marcadores moleculares é recomendada para a identificação precisa de linhagens, visando processos de proteção legal de cultivares de Coffea.One of the greatest problems in Coffea arabica breeding is identifying precisely any inbred line, based only on botanical and agronomical descriptors, because of the reduced genetic variability of the species, close pedigree origin, which results in small phenotypic variation. Recently, molecular markers have been used for plant germplasm characterization and identification in several commercial species. This work evaluates the reliability of three marker systems: RAPD, AFLP and SSR, to characterize the genetic variability of commercially-used Coffea inbred lines developed by the Instituto Agronômico (IAC), and their potential for cultivar identification. All methods identified polymorphisms among the cultivars. The genetic diversity recognized by the methods is very similar, although is very narrow. RAPD and SSR marker systems grouped more efficiently the evaluated cultivars according to parental origin. None of the methods allowed inbred line identification. Therefore for varietal protection, it would be necessary using a combination of botanical, agronomical and molecular markers descriptors for precise cultivar identification

Phenological and agronomic attributes in arabica cultivars of coffee tree

O objetivo deste trabalho foi avaliar o efeito do ano agrícola sobre os atributos fenológicos e agronômicos em diferentes cultivares de cafeeiro arábica. Os atributos fenológicos determinados foram estádios de frutificação, de gema dormente até fruto seco e duração do ciclo. Como atributos agronômicos, foram avaliados o rendimento, a produtividade, os tipos e o tamanho de grãos. O período entre antese até fase chumbinho não variou entre as cultivares para o ano agrícola 2004/2005, já para o ano agrícola seguinte a cultivar 'Icatu Precoce' apresentou antecipação da fase chumbinho, e a cultivar 'Obatã' atrasou o início do ciclo fenológico em relação às demais cultivares estudadas. A duração do ciclo variou com o ano agrícola, o que possibilitou a confirmação da influência dos efeitos ambientais nessa característica. Os atributos agronômicos (produtividade, rendimento, tipo e tamanho de grãos), independentemente das cultivares, também variaram com o ano agrícola.The aim of this study was to evaluate cultivars of Coffea arabica on the basic of phenology and agronomic attributes a function of the year of production. The phenological attributes were evaluated regarding phenology and percentage of cherry fruits at harvesting time. Agronomic traits evaluated included productivity and outturn, type of seeds and grain size. The period between the blooms till the fruits at the beginning of growth did not vary among the coffee trees in the year of production 2004/2005. While in the following year the Icatu Precoce cultivar presented anticipation of the fruits at the beginning of growth and the 'Obatã' cultivar delayed the beginning of the phenological cycle due to the other cultivars studied. The duration of each cycle varied with the year of production and then all the coffee trees in this study could be differentiated on the timing of maturation (early, middle and late). The agronomic attributes (productivity, outturn, type and size of grains), regardless the different cultivars of coffee also varied with the year of production

Gene expression profile during coffee fruit development and identification of candidate markers for phenological stages

The objective of this work was to identify genes that could be used as suitable markers for molecular recognition of phenological stages during coffee (Coffea arabica) fruit development. Four cultivars were evaluated as to their differential expression of genes associated to fruit development and maturation processes. Gene expression was characterized by both semi‑quantitative and quantitative RT‑PCR, in fruit harvested at seven different developmental stages, during three different seasons. No size polymorphisms or differential expression were observed among the cultivars for the evaluated genes; however, distinct expression profiles along fruit development were determined for each gene. Four out of the 28 evaluated genes exhibited a regular expression profile in all cultivars and harvest seasons, and, therefore, they were validated as candidate phenological markers of coffee fruit. The gene α‑galactosidase can be used as a marker of green stage, caffeine synthase as a marker of transition to green and yellowish‑green stages, and isocitrate lyase and ethylene receptor 3 as markers of late maturation.O objetivo deste trabalho foi identificar genes que possam ser utilizados como marcadores moleculares para reconhecimento de fases fenológicas, durante o desenvolvimento de frutos do cafeeiro (Coffea arabica). Quatro cultivares foram avaliadas quanto à expressão diferencial de genes associados a processos de desenvolvimento e maturação de frutos. A caracterização da expressão gênica foi realizada pelas técnicas de RT‑PCR semi‑quantitativa e quantitativa, em frutos coletados em sete estádios de desenvolvimento, durante três safras. Não foi observado nenhum polimorfismo de tamanho ou expressão diferencial entre as cultivares, para os genes avaliados; porém, perfis de expressão distintos durante o desenvolvimento dos frutos foram determinados para cada gene. Quatro entre os 28 genes avaliados apresentaram perfil de expressão constante, em todas as cultivares e safras e, portanto, foram validados como genes candidatos a marcadores fenológicos de frutos de cafeeiro. O gene α‑galactosidase pode ser usado como marcador do estágio de fruto verde, o gene de cafeína sintase como marcador do estádio de transição entre fruto verde e fruto verde‑cana, e o isocitrato liase e o etileno‑receptor 3 como marcadores das fases finais de maturação

Perfil da expressão gênica durante o desenvolvimento de frutos de café \ud e identificação de genes marcadores para fases fenológicas

The objective of this work was to identify genes that could be used as suitable markers for molecular recognition of phenological stages during coffee (Coffea arabica) fruit development. Four cultivars were evaluated as to their differential expression of genes associated to fruit development and maturation processes. Gene expression was characterized by both semi-quantitative and quantitative RT-PCR, in fruit harvested at seven different developmental stages, during three different seasons. No size polymorphisms or differential expression were observed among the cultivars for the evaluated genes; however, distinct expression profiles along fruit development were determined for each gene. Four out of the 28 evaluated genes exhibited a regular expression profile in all cultivars and harvest seasons, and, therefore, they were validated as candidate phenological markers of coffee fruit. The gene a-galactosidase can be used as a marker of green stage, caffeine synthase as a marker of transition to green and yellowish-green stages, and isocitrate lyase and ethylene receptor 3 as markers of late maturation.O objetivo deste trabalho foi identificar genes que possam ser utilizados como marcadores \ud moleculares para reconhecimento de fases fenológicas, durante o desenvolvimento de frutos do cafeeiro (Coffea \ud arabica). Quatro cultivares foram avaliadas quanto à expressão diferencial de genes associados a processos de \ud desenvolvimento e maturação de frutos. A caracterização da expressão gênica foi realizada pelas técnicas de \ud RT‑PCR semi‑quantitativa e quantitativa, em frutos coletados em sete estádios de desenvolvimento, durante \ud três safras. Não foi observado nenhum polimorfismo de tamanho ou expressão diferencial entre as cultivares, \ud para os genes avaliados; porém, perfis de expressão distintos durante o desenvolvimento dos frutos foram \ud determinados para cada gene. Quatro entre os 28 genes avaliados apresentaram perfil de expressão constante, \ud em todas as cultivares e safras e, portanto, foram validados como genes candidatos a marcadores fenológicos \ud de frutos de cafeeiro. O gene α‑galactosidase pode ser usado como marcador do estágio de fruto verde, o gene \ud de cafeína sintase como marcador do estádio de transição entre fruto verde e fruto verde‑cana, e o isocitrato \ud liase e o etileno‑receptor 3 como marcadores das fases finais de maturação.Consorcio Brasileiro de Pesquisa e Desenvolvimento do CafeConsorcio Brasileiro de Pesquisa e Desenvolvimento do Caf

Gene expression profile during coffee fruit development and identification of candidate markers for phenological stages

The objective of this work was to identify genes that could be used as suitable markers for molecular recognition of phenological stages during coffee (Coffea arabica) fruit development. Four cultivars were evaluated as to their differential expression of genes associated to fruit development and maturation processes. Gene expression was characterized by both semi-quantitative and quantitative RT-PCR, in fruit harvested at seven different developmental stages, during three different seasons. No size polymorphisms or differential expression were observed among the cultivars for the evaluated genes; however, distinct expression profiles along fruit development were determined for each gene. Four out of the 28 evaluated genes exhibited a regular expression profile in all cultivars and harvest seasons, and, therefore, they were validated as candidate phenological markers of coffee fruit. The gene α-galactosidase can be used as a marker of green stage, caffeine synthase as a marker of transition to green and yellowish-green stages, and isocitrate lyase and ethylene receptor 3 as markers of late maturation

Estudo de um mutante de caratenoide e viviparo causado por transposon em zea mays

Orientador : William Jose da SilvaDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaMestradoGeneticaMestre em Ciências Biológica

Molecular and biochemical characterization of thevp12 mutant of maize (Zea mays L.)

The mutant vp12 is a single locus mutation that conditions lemon endosperm, viviparous embryos and white seedlings. Previous genetic analysis demonstrated that vp12 is not allelic to other viviparous mutants of maize. In this work, the objectives are to characterize vp12 at both molecular and biochemical levels. First, levels of the hormone ABA were measured at several stages during embryo development and also in isolated organs, under drought stress. In mutant embryos, ABA levels were lower than in normal ones, at all stages analyzed. Under drought stress, mutant organs from both 3-day and 9-day old seedlings accumulated less ABA than corresponding normal organs. However, the hormone levels in some stressed mutant organs, such as roots and leaves, are higher than in non-stressed organs. These results suggest that the ability of ABA synthesis is not completely blocked in vp12. Expression of several ABA-responsive genes, such as Em, glb1, glb2 and rab17, was detected in RNA extracts from mutant immature embryos, despite the lower ABA levels observed in those. In addition, transcripts corresponding to vp1, a transcriptional activator for embryo ABA-mediated responses, were also observed in mutant embryos. Therefore, it can be suggested that the ABA signal transduction pathway is not impaired in vp12 embryos.At the carotenoid level, analysis of carotenoid extracts from normal and lemon endosperms showed that no colored precursors are accumulated in vp12 endosperms. Also, HPLC analysis indicated that phytoene, the first non-colored carotenoid precursor, is not present in mutant endosperms. In order to verify the presence of enzymes from the carotenoid biosynthetic pathway, their expression was analyzed in vp12 endosperms. Phytoene synthase accumulation was detected using a corresponding maize polyclonal antibody. Transcripts corresponding to two other enzymes, phytoene desaturase (PDS) and geranylgeranyl pyrophosphate synthase (GGPPS), were detected by RT-PCR. PDS transcripts levels were similar in both normal and vp12 immature endosperm RNA. However, GGPPS transcripts accumulated at lower levels in mutant immature endosperms than normal ones. These results, combined with the profile of carotenoid accumulation, suggest that the block in the carotenoid pathway in vp12 occurs in the GGPP synthesis.U of I OnlyETDs are only available to UIUC Users without author permissio