Obtenção de marcador molecular potencialmente envolvido com a resistência do cafeeiro à ferrugem
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Data
2007
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Resumo
O Projeto Brasileiro do Genoma Café (PBGC) gerou um banco de dados contendo mais de 200.000 ESTs (Expressed Sequence Tags). Em trabalho preliminar, o banco foi minerado por meio de análise in silico e identificaram-se várias seqüências potencialmente associadas à resistência do cafeeiro a patógenos. Visando verificar o envolvimento destas seqüências com a resistência do cafeeiro à ferrugem foram desenhados oligonucleotídeos iniciadores (primers) que amplificaram as seqüências mineradas. Noventa pares de oligonucleotídeos iniciadores específicos foram desenhados utilizando o programa computacional Primer3. A estabilidade dos oligonucleotídeos foi verificada por meio do programa PrimerSelect®. Diferentes concentrações dos componentes da reação de PCR foram analisadas. Para a amplificação no termociclador, foram avaliadas diferentes combinações de tempo e temperatura, incluindo touchdown PCR. Utilizando as condições de reação e amplificação otimizadas, 40 iniciadores foram testados em 12 genótipos resistentes e 12 susceptíveis a H. vastatrix. Destes, 29 resultaram em bandas únicas e bem definidas, sendo um polimórfico. Os demais 50 estão sendo testados por PCR. Este trabalho permitiu obter, até o momento, um marcador molecular polimórfico entre os dois grupos de indivíduos resistentes e susceptíveis. Para confirmar a possível ligação e seu potencial, o marcador está sendo testado em diferentes populações do Programa de Melhoramento da UFV/EPAMIG.
The Projeto Brasileiro do Genoma Café (PBGC) generated a database containing more than 200.000 ESTs (Expressed Sequence Tags). In a former work, the bank was mined by in silico analyses and several sequences potentially associated with the coffee tree resistance to pathogens were identified. Aiming to verify the involvement of these sequences with the coffee tree resistance to leaf rust, primers were designed to amplify the mined sequences. Ninety specific primers were synthesized using the computational program Primer3. The primers stability was tested by the program PrimerSelect®. Different PCR conditions were tested: concentrations of components reaction, combinations of time and temperature, including touchdown PCR. Using optimized reaction and amplification conditions, 40 primers were tested in 12 resistant and 12 susceptible genotypes to H. vastatrix. Twenty nine of those resulted in unique and sharp bands, and only one of these was polymorphic. The remaining 50 synthesized primers are being tested. The 40 specific primers permitted to find one molecular marker polymorphic between the resistant and susceptible genotypes. To confirm the possible linkage and its potential, the marker is being tested in different populations of the UFV/EPAMIG Breeding Program.
The Projeto Brasileiro do Genoma Café (PBGC) generated a database containing more than 200.000 ESTs (Expressed Sequence Tags). In a former work, the bank was mined by in silico analyses and several sequences potentially associated with the coffee tree resistance to pathogens were identified. Aiming to verify the involvement of these sequences with the coffee tree resistance to leaf rust, primers were designed to amplify the mined sequences. Ninety specific primers were synthesized using the computational program Primer3. The primers stability was tested by the program PrimerSelect®. Different PCR conditions were tested: concentrations of components reaction, combinations of time and temperature, including touchdown PCR. Using optimized reaction and amplification conditions, 40 primers were tested in 12 resistant and 12 susceptible genotypes to H. vastatrix. Twenty nine of those resulted in unique and sharp bands, and only one of these was polymorphic. The remaining 50 synthesized primers are being tested. The 40 specific primers permitted to find one molecular marker polymorphic between the resistant and susceptible genotypes. To confirm the possible linkage and its potential, the marker is being tested in different populations of the UFV/EPAMIG Breeding Program.
Descrição
Trabalho apresentado no Simpósio de Pesquisa dos Cafés do Brasil (5. : 2007 : Águas de Lindóia, SP). Anais. Brasília, D.F. : Embrapa Café, 2007.
Palavras-chave
Hemileia vastatrix Coffea ESTs Genoma café, Hemileia vastatrix Coffea ESTs Coffee genome
Citação
Maciel, Bárbara Hufnagel; Caixeta, Eveline Teixeira; Andrade, Flávia Thiebaut; Alvarenga, Samuel Mazzinghy; Zambolim, Eunize Maciel; Sakiyama, Ney Sussumu. Obtenção de marcador molecular potencialmente envolvido com a resistência do cafeeiro à ferrugem. In: Simpósio de Pesquisa dos Cafés do Brasil (5. : Águas de Lindóia, SP : 2007). Anais. Brasília, D.F. : Embrapa - Café, 2007. (1 CD-ROM), 4p.