Marcadores moleculares derivados de sequências expressas do genoma café potencialmente envolvidas na resistência à ferrugem

Abstract

Resumo -O objetivo deste trabalho foi identificar marcadores moleculares relacionados à resistência do cafeeiro (Coffea arabica) à ferrugem (Hemileia vastatrix). Foram identificadas sequências de DNA potencialmente envolvidas na resistência do cafeeiro a doenças, por meio de análise "in silico", a partir das informações geradas pelo Projeto Brasileiro do Genoma Café. A partir das sequências mineradas, foram desenhados 59 pares de iniciadores para amplificá-las. Os 59 iniciadores foram testados em 12 cafeeiros resistentes e 12 susceptíveis a H. vastatrix. Vinte e sete iniciadores resultaram em bandas únicas e bem definidas, enquanto um deles amplificou fragmento de DNA em todos os cafeeiros resistentes, mas não nos suscetíveis. Esse marcador molecular polimórfico amplificou uma região do DNA que corresponde a uma janela aberta de leitura parcial do genoma de C. arabica que codifica uma proteína de resistência a doenças. O marcador CARF 005 é capaz de diferenciar os cafeeiros analisados em resistentes e susceptíveis a H. vastatrix.Termos para indexação: Coffea arabica, Hemileia vastatrix, bioinformática, EST-PCR, genes de resistência, mineração de dados. Molecular markers from coffee genome expressed sequences potentially involved in resistance to rustAbstract -The objective of this work was to identify molecular markers related to the resistance of coffee (Coffea arabica) to rust (Hemileia vastatrix). DNA sequences potentially involved in coffee disease resistance were identified, using "in silico" analysis, from data obtained by the Brazilian coffee genome project. After data mining, 59 primer pairs were designed to amplify the sequences identified. The 59 primers were tested on 12 resistant and 12 susceptible coffee plants to H. vastatrix. Twenty-seven primers resulted in unique and well-defined bands, while one of these amplified a DNA fragment in all resistant plants, but not in the susceptible ones. This polymorphic molecular marker amplified a region of DNA that corresponds to a partial open reading frame of C. arabica genome that encodes a disease resistance protein. The marker CARF 005 can be used to differentiate between resistant and susceptible coffee plants to H. vastatrix.

In silico guided structural and functional analysis of genes with potential involvement in resistance to coffee leaf rust: A functional marker based approach

“…A Sigma made (Sigma-Aldrich, Belo Horizonte, Brazil) disease RGA primer pair, CARF005, (F: and R: ) [ 26 ] was used to screen the differential host clones. PCR reagents were 1x buffer, 0.2 mM dNTPs, 0.2 μM primers, 1 mM MgCl 2 , 0.8 units of Taq polymerase (Invitrogen, Carlsbad, USA) to which 5 ng gDNA was added to form a reaction volume of 20 μl.…”

Structural and functional analysis of genes with potential involvement in resistance to coffee leaf rust: a functional marker based approach

“…A Sigma made (Sigma-Aldrich, Belo Horizonte, Brazil) disease RGA primer pair, CARF005, (F: 5’-GGACATCAACACCAACCTC-3’ and R: 5’-ATCCCTACCATCCACTTCAAC-3’) [26] was used to screen the differential host clones. PCR reagents were 1x buffer, 0.2 mM dNTPs, 0.2 µM primers, 1 mM MgCl 2 , 0.8 units of Taq polymerase (Invitrogen, Carlsbad, USA) to which 5 ng gDNA was added to form a reaction volume of 20 µl.…”

Marker-Assisted Pyramiding of Multiple Disease Resistance Genes in Coffee Genotypes (Coffea arabica)

“…To obtain durable resistance and identify various rust resistance loci, the progenies were also analyzed using the CARF005 marker. This marker amplifies a DNA fragment that corresponds to the nucleotide binding-leucine rich repeat (CC-NBS-LRR) gene [47], which shares conserved sequences with other S H genes and expresses a characteristic polymorphic allele conferring distinct resistance phenotypes [13]. The PCR for CARF005 was performed in a 20 µL reaction mixture containing 50 ng of DNA, 0.1 µM of each primer, 0.15 mM of each dNTP (Promega), 1.0 mM of MgCl 2 , 1.0 U of Taq DNA polymerase (Invitrogen), and 1× PCR reaction buffer.…”