Genetic diversity, provenances extrapolation and cytogenetics analysis of Portuguese Pinus nigra Arn. populations
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2017-07-04
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Durante o século XX instalaram-se povoamentos de Pinus nigra em Portugal.
Actualmente, o número de populações Portuguesas de P. nigra alóctones é restrito estando
distribuídas por uma área reduzida. A sua origem e taxonomia são desconhecidas. A
caracterização botânica destes povoamentos em 1980 revelou a presença das subespécies
nigra, salzmannii e laricio.
Neste estudo pretendeu-se: i) caracterizar molecularmente estas populações alóctones;
ii) extrapolar as suas proveniências; iii) determinar a sua taxonomia; iv) caracterizar amostras
estrangeiras de diferentes taxa infraespecíficos e proveniências para comparar com as
Portuguesas, utilizando marcadores ISSR e SCoT. Portugal constitui o limite mais ocidental
de distribuição de P.nigra e Pinus sylvestris L.. Neste estudo objectivou-se ainda a
caracterização citogenética de uma população de P.nigra (Campeã) e sua comparação com
pinheiro-silvestre (população autóctone da Biduiça) usando as técnicas citogenéticas de FISH
com uma sonda de rDNA, de ND-FISH com sondas SSR e de coloração com nitrato de prata.
O estudo de diversidade genética requereu a prévia extração de DNA de P.nigra.
Devido ao elevado conteúdo de polissacáridos e polifenóis, o protocolo de extração foi
optimizado para DNA de agulhas congeladas e desidratadas, sendo extensível a outros tecidos
e espécies.
A caracterização molecular e extrapolação das proveniências das populações de P.nigra
(Manteigas, Caminha, Vila Pouca de Aguiar, Vale do Zêzere, Paredes de Coura e Campeã)
foram efectuadas em 127 indivíduos com oito primers para amplificação de ISSRs e oito
primers para produção de SCoTs. Foram detetadas uma elevada diversidade genética intrapopulacional
(expectável para uma espécie Pinus) e uma estrutura genética definida
(agrupamento dos indivíduos por população).
Os perfis genéticos das amostras estrangeiras foram determinados em bulks
correspondentes a 120 indivíduos de 12 proveniências, representativos das subespécies (nigra,
salzmannii e laricio) e das variedades (corsicana, calabrica e austriaca) usando ISSRs e SCoTs.
A subespécie salzmannii revelou-se geneticamente distante das amostras Portuguesas
e restantes estrangeiras. Uma elevada identidade genética foi obtida entre amostras
Portuguesas e estrangeiras da subespécie laricio. Os ISSRs evidenciaram maior similaridade
genética entre os indivíduos Portugueses e amostras da subsp. laricio var. calabrica enquanto
os SCoTs detetaram maior similaridade com a subespécie laricio var. corsicana. Segundo a
caracterização botânica de 1980, esta subespécie foi apontada como a melhor adaptada e
predominante em Portugal. Como a amplificação de SCoTs se baseia na ligação do primer a
uma curta sequência conservada nos genes, consideraram-se estes resultados mais fidedignos.
Globalmente, os dois sistemas marcadores demonstraram especificidade para determinar
estrutura genética, extrapolar proveniências e discriminar taxa infraespecíficos em P.nigra.
Após ND-FISH observaram-se diferenças na hibridação de SSRs entre P.nigra e
P.sylvestris. A sonda rDNA detetou 14 e 16 loci em P.sylvestris e P.nigra, respetivamente. O
número de nucléolos por núcleo (1 a 12) em ambas as espécies revela que nem todos os loci
rDNA estão transcripcionalmente activos. A presença de micronucléolos associada à
ocorrência de irregularidades cromossómicas (e.g. cromossomas em anel, policêntricos e
fragmentados) sugere que estas populações periféricas ainda estão em adaptação.
Os resultados deste trabalho contribuirão para o delineamento de estratégias de
plantação/reflorestação de P. nigra em Portugal.
During the 20th century, P.nigra stands were installed in Portugal. Currently, the number of Portuguese P. nigra allochthonous populations is restricted being distributed to a small area. Origin and taxonomy of material used are unknown. The botanic characterization of these stands in 1980 revealed the presence of the subspecies nigra, salzmannii e laricio. In this study, we intend to: i) characterize molecularly these allochthonous populations; ii) extrapolate their provenances; iii) determine their taxonomy; and iv) characterize foreign samples from different infraspecifics taxa and provenances to compare with Portuguese, using ISSR and SCoT markers. Portugal constitutes the limit more occidental of P.nigra and P.sylvestris distribution. In this study, it was also aimed the cytogenetic characterization of a P.nigra population (‘Campeã’) and their comparison with Scots pine (autochthonous population of ‘Biduiça’) using the cytogenetic techniques FISH with a ribosomal rDNA probe, ND-FISH with SSR probes and silver nitrate staining. The genetic diversity study required a previous extraction of P.nigra DNA. Due to the high content of polysaccharides and polyphenols, the extraction protocol was optimized for DNA from frozen and dehydrated needles, and was suitable to other tissues and species. The molecular characterization and provenances extrapolation of P.nigra populations (‘Manteigas’, ‘Caminha’, ‘Vila Pouca de Aguiar’, ‘Vale do Zêzere’, ‘Paredes de Coura’, ‘Campeã’) were performed in 127 individuals with eight primers to ISSRs amplification and eight for SCoTs production. A high genetic diversity within populations (expectable for a Pinus species) and a defined genetic structure (clustering of individuals per population) were detected. The genetic profiles of foreign samples were determined in bulks corresponding to 120 individuals of 12 provenances representatives of subspecies (nigra, salzmannii and laricio) and varieties (corsicana, calabrica and austriaca) using ISSRs e SCoTs. The subspecies salzmannii revealed to be genetically distant from the Portuguese and other foreign samples. A high genetic identity was obtained among Portuguese and foreign samples from laricio subspecies. ISSRs evidenced higher genetic identity among the Portuguese and foreign samples from subsp. laricio var. calabrica whereas SCoTs estimated a higher genetic identity with subsp. laricio var. corsicana. According to botanic characterization of 1980, this subspecies was considered the best adapted and prevalent in Portugal. As the SCoTs amplification is based on the binding of primer to a short conserved sequence in the genes, was considered these results more reliable. Globally, the two marker systems showed specificity for genetic structure determination, provenances extrapolation and infraspecific taxa discrimination of P.nigra. After ND-FISH, differences in SSRs hybridization between P.nigra and P.sylvestris were observed. rDNA probe detected 14 and 16 loci in P.sylvestris and P.nigra, respectively. The number of nucleoli per interphase nucleus (1 to 12) in both species revealed that not all rDNA loci were transcriptionally active. The presence of micronucleoli associated to chromosomic irregularities (e.g. ring, polycentric and fragmented chromosomes) in both species, suggested that these peripheral populations are still in adaptation. The results of this work will contribute to design strategies for planting/reforestation of P.nigra in Portugal.
During the 20th century, P.nigra stands were installed in Portugal. Currently, the number of Portuguese P. nigra allochthonous populations is restricted being distributed to a small area. Origin and taxonomy of material used are unknown. The botanic characterization of these stands in 1980 revealed the presence of the subspecies nigra, salzmannii e laricio. In this study, we intend to: i) characterize molecularly these allochthonous populations; ii) extrapolate their provenances; iii) determine their taxonomy; and iv) characterize foreign samples from different infraspecifics taxa and provenances to compare with Portuguese, using ISSR and SCoT markers. Portugal constitutes the limit more occidental of P.nigra and P.sylvestris distribution. In this study, it was also aimed the cytogenetic characterization of a P.nigra population (‘Campeã’) and their comparison with Scots pine (autochthonous population of ‘Biduiça’) using the cytogenetic techniques FISH with a ribosomal rDNA probe, ND-FISH with SSR probes and silver nitrate staining. The genetic diversity study required a previous extraction of P.nigra DNA. Due to the high content of polysaccharides and polyphenols, the extraction protocol was optimized for DNA from frozen and dehydrated needles, and was suitable to other tissues and species. The molecular characterization and provenances extrapolation of P.nigra populations (‘Manteigas’, ‘Caminha’, ‘Vila Pouca de Aguiar’, ‘Vale do Zêzere’, ‘Paredes de Coura’, ‘Campeã’) were performed in 127 individuals with eight primers to ISSRs amplification and eight for SCoTs production. A high genetic diversity within populations (expectable for a Pinus species) and a defined genetic structure (clustering of individuals per population) were detected. The genetic profiles of foreign samples were determined in bulks corresponding to 120 individuals of 12 provenances representatives of subspecies (nigra, salzmannii and laricio) and varieties (corsicana, calabrica and austriaca) using ISSRs e SCoTs. The subspecies salzmannii revealed to be genetically distant from the Portuguese and other foreign samples. A high genetic identity was obtained among Portuguese and foreign samples from laricio subspecies. ISSRs evidenced higher genetic identity among the Portuguese and foreign samples from subsp. laricio var. calabrica whereas SCoTs estimated a higher genetic identity with subsp. laricio var. corsicana. According to botanic characterization of 1980, this subspecies was considered the best adapted and prevalent in Portugal. As the SCoTs amplification is based on the binding of primer to a short conserved sequence in the genes, was considered these results more reliable. Globally, the two marker systems showed specificity for genetic structure determination, provenances extrapolation and infraspecific taxa discrimination of P.nigra. After ND-FISH, differences in SSRs hybridization between P.nigra and P.sylvestris were observed. rDNA probe detected 14 and 16 loci in P.sylvestris and P.nigra, respectively. The number of nucleoli per interphase nucleus (1 to 12) in both species revealed that not all rDNA loci were transcriptionally active. The presence of micronucleoli associated to chromosomic irregularities (e.g. ring, polycentric and fragmented chromosomes) in both species, suggested that these peripheral populations are still in adaptation. The results of this work will contribute to design strategies for planting/reforestation of P.nigra in Portugal.
Descrição
Dissertação de Mestrado em Genética Molecular Comparativa e Tecnológica
Palavras-chave
Pinus nigra , Citogenética , Portugal , Variação genética , Hibridação in situ fluorescente , Marcadores moleculares