Genetic characterization of Fagaceae by molecular and cytogenetic approaches
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2015
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A família Fagaceae inclui arbustos e árvores de folha perene e caduca, estando distribuída
por todo o Hemisfério Norte. O Sudeste Asiático é aceite como centro de origem das fagáceas,
que depois se dispersaram para a Europa e América do Norte.
Uma vez que dados moleculares têm vindo a ser utilizados como um recurso importante
para inferências filogenéticas, efetuou-se a caracterização genética de algumas fagáceas com
elevada importância ecológica e/ou económica usando abordagens moleculares e citogenéticas
com vista a obter ferramentas úteis para discriminação de espécies e DNA fingerprinting.
Neste trabalho, foram estudados trinta fagáceas: vinte e quatro espécies do género
Quercus pertencentes a quatro grupos infragenéricos; três espécies do género Castanea; e duas
espécies e uma cultivar do género Fagus.
As abordagens moleculares utilizadas basearam-se em sistemas marcadores dominantes
(ISSR e iPBS) e codominantes (IGS rDNA PCR-RFLP e ITS rDNA PCR-RFLP).
Avaliou-se o potencial dos marcadores ISSR, IPBS e ITS PCR-RFLP para DNA
fingerprinting, tendo em vista avaliar a diversidade inter- e/ou intra-específica, a estrutura
genética, a discriminação taxonómica, e a filogenia entre indivíduos dos três géneros estudados.
Os ISSRs agruparam os indivíduos por género, espécie e grupos infragenéricos de acordo
com a classificação taxonómica e origens ecológicas, provando ser adequados para DNA
fingerprinting e inferências taxonómicas.
Visto que os elementos transponíveis influenciam o tamanho do genoma e contribuem
para a especiação das espécies, o sistema de marcadores iPBS foi aqui usado, pela primeira vez
em Fagáceas tendo-se revelado adequado para DNA fingerprinting e para estimar a estrutura
genética e filogenias.
Os marcadores ITS PCR-RFLP também provaram a sua capacidade de discriminação
taxonómica, tendo agrupado todos os indivíduos por género, e a maior parte dos carvalhos por
grupo infragenérico ou distribuição ecológica.
Globalmente, os sistemas de marcadores forneceram dados moleculares robustos e fiáveis
que corroboraram a taxonomia adotada neste trabalho.
Espécies do género Quercus foram caracterizadas molecular e citogeneticamente ao nível
do DNA ribossomal (rDNA) usando: os marcadores IGS PCR-RFLP para a análise de
polimorfismos e deteção de padrões discriminativos; a técnica de coloração por nitrato de prata foi usada para a inferir sobre a atividade nucleolar; e a técnica FISH foi realizada para localizar
fisicamente os loci 5S e 35S rDNA.
Relativamente à atividade nucleolar, os 22 carvalhos estudados revelaram um número
máximo de quatro Ag-NOR por célula metafásica, e um máximo de dois nucléolos por núcleo
interfásico.
A análise citogenética também revelou um elevado nível de conservação no número e
localização dos loci de rDNA. Detetaram-se dois loci 5S e quatro loci 35S por célula mitótica em
todas as espécies. O locus 5S foi sempre localizado no segundo maior par de cromossomas
metacêntricos. Os quatro loci 35S foram observados em dois pares distintos de cromossomas: um
subtelocêntrico com o major locus 35S em posição subterminal; e um par metacêntrico com o
minor locus 35S numa posição pericentromérica. Adicionalmente, realizou-se a primeira
localização física por FISH de dois loci 5S e quatro loci 35S de rDNA em Quercus castaneifolia,
Quercus x crenata, Quercus frainetto, Quercus kelloggii, Quercus libani, Quercus mongolica,
Quercus phellos, Quercus phillyraeoides e Quercus velutina. Foi ainda atribuído, pela primeira
vez, um complemento cromossómico de 2n=2x=24 ao Q. phellos.
Os dados citogenéticos não revelaram a existência de polimorfismo, quer entre as 22
espécies de Quercus, quer entre os seus grupos infragenéricos, não permitindo a sua
discriminação. No entanto, o facto dos espaçadores do rDNA serem usualmente mais variáveis do
que os genes ribossomais e estarem com eles intercalados, levou-nos a avaliar o polimorfismo do
espaçador IGS da região 35S utilizando a técnica de PCR-RFLP. Estes marcadores agruparam a
maioria dos carvalhos estudados por grupo infragenérico, constituindo assim uma ferramenta
complementar para a discriminação de espécies, e avaliação de polimorfismo do rDNA.
Globalmente, as abordagens citogenéticas revelaram elevada conservação do rDNA entre
as espécies de carvalhos analisadas. Por outro lado, ambos os sistemas de marcadores moleculares
dominantes e codominantes utilizados para análise das espécies de fagáceas comprovaram: i) a
existência de elevada diversidade genética; ii) o potencial para serem usados na avaliação das
relações filogenéticas; e iii) a aptidão para amplificar marcadores moleculares específicos para os
taxon estudados, que poderão corresponder a regiões codificantes para características
interessantes, potencialmente úteis para gene-tagging e seleção assistida por marcadores.
The family Fagaceae includes evergreen and deciduous shrubs and trees, and is worldwide distributed throughout the Northern Hemisphere. The Southeast Asia is accepted as the centre of origin of this family, which then migrated to Europe and North America. Since molecular data have been widely used as an important resource for phylogenetic inferences, herein, we performed the genetic characterization of some Fagaceae species with high ecological and/or economic importance, using both molecular and cytogenetic approaches in order to achieve useful tools for species discrimination and DNA fingerprinting. In this work, were studied thirty different Fagaceae: twenty four oak species belonging to four infrageneric group; three species from genus Castanea; two species and one cultivar from genus Fagus. The molecular approaches used in this work relied on dominant (ISSR and iPBS) and codominant (IGS rDNA PCR-RFLP and ITS rDNA PCR-RFLP) marker systems. The potential of the ISSR, iPBS and ITS PCR-RFLP markers for fingerprinting, assessment of inter- and/or intraspecific genetic diversity and structure, taxonomic discrimination, and estimation of phylogenies among individuals from the three genera, was evaluated. The ubiquitous ISSRs clustered the individuals per genus, species and infrageneric groups in agreement with the taxonomic classification and geographic distributions, being suitable for DNA fingerprinting, and for taxonomic inferences. Because transposable elements influence genome size and contribute for species speciation, the iPBS marker system is used for the first time in Fagaceae, which proved to be appropriate for DNA fingerprinting and estimation of genetic structure and phylogenies. The ITS PCR-RFLP markers also prove their ability for taxonomic discrimination, clustering all the individuals per genus, and most of the Quercus per infrageneric group or geographic distribution. Overall, the marker systems provided robust and reliable molecular data that corroborated the adopted taxonomy in this work. Species from genus Quercus were molecularly and cytogenetically characterized at the rDNA level using: IGS PCR-RFLP markers for detection of polymorphic and discriminative patterns; silver nitrate staining for inference about nucleolar activity; and FISH to localize physically the 5S and 35S rDNA loci. Concerning the nucleolar activity, all oaks revealed a maximum number of four Ag-NORs per metaphase cell, and a maximum of two nucleoli per interphase nucleus. The cytogenetic analysis also revealed a high degree of conservation regarding the number and position of the rDNA loci among the 22 studied oaks. Four 35S rDNA and two 5S rDNA loci per mitotic cell were detected in each species. The 5S rDNA locus was always located in the second largest submetacentric chromosome pair. The four 35S rDNA loci were positioned in two distinct pairs: a medium-sized subtelocentric pair bearing a subterminal 35S major locus, and a medium-sized metacentric pair harbouring the pericentromeric 35S minor locus. Additionally, this study performs the first physical localization by FISH of two 5S and four 35S rDNA loci in Quercus castaneifolia, Quercus x crenata, Quercus frainetto, Quercus kelloggii, Quercus libani, Quercus mongolica, Quercus phellos, Quercus phillyraeoides and Quercus velutina. Also, a chromosome complement of 2n=2x=24 was ascribed for the first time to Q. phellos. The cytogenetic data did not reveal polymorphism among the 22 Quercus species or their infrageneric groups, preventing their discrimination. However, since the rDNA spacers tend to be more variable and are interspersed with the ribosomal RNA (rRNA) genes, the polymorphism of the IGS spacer of the 35S rDNA was evaluated using the PCR-RFLP technique. These markers clustered most of the studied oaks per infrageneric group, revealing that the IGS PCR-RFLP markers may constitute a complementary tool for species discrimination, and for the assessment of rDNA polymorphism. Globally, the cytogenetic approaches revealed high degree of conservation at the rDNA level among the analysed Quercus species. On the other hand, both dominant and codominant molecular markers systems revealed in the studied Fagaceae species: i) high genetic diversity; ii) potential to be used for estimation of the phylogenetic relationships; and iii) suitability for the identification of molecular markers specifically amplified per taxon that could correspond to coding regions for interesting traits, and might be further useful for gene-tagging or markers assisted selection.
The family Fagaceae includes evergreen and deciduous shrubs and trees, and is worldwide distributed throughout the Northern Hemisphere. The Southeast Asia is accepted as the centre of origin of this family, which then migrated to Europe and North America. Since molecular data have been widely used as an important resource for phylogenetic inferences, herein, we performed the genetic characterization of some Fagaceae species with high ecological and/or economic importance, using both molecular and cytogenetic approaches in order to achieve useful tools for species discrimination and DNA fingerprinting. In this work, were studied thirty different Fagaceae: twenty four oak species belonging to four infrageneric group; three species from genus Castanea; two species and one cultivar from genus Fagus. The molecular approaches used in this work relied on dominant (ISSR and iPBS) and codominant (IGS rDNA PCR-RFLP and ITS rDNA PCR-RFLP) marker systems. The potential of the ISSR, iPBS and ITS PCR-RFLP markers for fingerprinting, assessment of inter- and/or intraspecific genetic diversity and structure, taxonomic discrimination, and estimation of phylogenies among individuals from the three genera, was evaluated. The ubiquitous ISSRs clustered the individuals per genus, species and infrageneric groups in agreement with the taxonomic classification and geographic distributions, being suitable for DNA fingerprinting, and for taxonomic inferences. Because transposable elements influence genome size and contribute for species speciation, the iPBS marker system is used for the first time in Fagaceae, which proved to be appropriate for DNA fingerprinting and estimation of genetic structure and phylogenies. The ITS PCR-RFLP markers also prove their ability for taxonomic discrimination, clustering all the individuals per genus, and most of the Quercus per infrageneric group or geographic distribution. Overall, the marker systems provided robust and reliable molecular data that corroborated the adopted taxonomy in this work. Species from genus Quercus were molecularly and cytogenetically characterized at the rDNA level using: IGS PCR-RFLP markers for detection of polymorphic and discriminative patterns; silver nitrate staining for inference about nucleolar activity; and FISH to localize physically the 5S and 35S rDNA loci. Concerning the nucleolar activity, all oaks revealed a maximum number of four Ag-NORs per metaphase cell, and a maximum of two nucleoli per interphase nucleus. The cytogenetic analysis also revealed a high degree of conservation regarding the number and position of the rDNA loci among the 22 studied oaks. Four 35S rDNA and two 5S rDNA loci per mitotic cell were detected in each species. The 5S rDNA locus was always located in the second largest submetacentric chromosome pair. The four 35S rDNA loci were positioned in two distinct pairs: a medium-sized subtelocentric pair bearing a subterminal 35S major locus, and a medium-sized metacentric pair harbouring the pericentromeric 35S minor locus. Additionally, this study performs the first physical localization by FISH of two 5S and four 35S rDNA loci in Quercus castaneifolia, Quercus x crenata, Quercus frainetto, Quercus kelloggii, Quercus libani, Quercus mongolica, Quercus phellos, Quercus phillyraeoides and Quercus velutina. Also, a chromosome complement of 2n=2x=24 was ascribed for the first time to Q. phellos. The cytogenetic data did not reveal polymorphism among the 22 Quercus species or their infrageneric groups, preventing their discrimination. However, since the rDNA spacers tend to be more variable and are interspersed with the ribosomal RNA (rRNA) genes, the polymorphism of the IGS spacer of the 35S rDNA was evaluated using the PCR-RFLP technique. These markers clustered most of the studied oaks per infrageneric group, revealing that the IGS PCR-RFLP markers may constitute a complementary tool for species discrimination, and for the assessment of rDNA polymorphism. Globally, the cytogenetic approaches revealed high degree of conservation at the rDNA level among the analysed Quercus species. On the other hand, both dominant and codominant molecular markers systems revealed in the studied Fagaceae species: i) high genetic diversity; ii) potential to be used for estimation of the phylogenetic relationships; and iii) suitability for the identification of molecular markers specifically amplified per taxon that could correspond to coding regions for interesting traits, and might be further useful for gene-tagging or markers assisted selection.
Descrição
Tese de Doutoramento em Genética Molecular Comparativa e Tecnológica
Palavras-chave
Fagaceae , Taxonomia , Variação genética , Marcadores genéticos