biologia plantarum

Biologia plantarum 46:27-32, 2003 | DOI: 10.1023/A:1027368528704

Isolation of Resistance Gene Analogs in Pepper Using Modified AFLPs

C. Egea-Gilabert¹, M.J. Dickinson², G. Bilotti³, M.E. Candela³

¹ Department of Agrarian Production, E.T.S.I.A., Polytechnic University of Cartagena, Cartagena, Spain

² Faculty of Sciences, School of Biological Sciences, University of Nottingham, Nottingham, U.K

³ Department of Plant Biology, Faculty of Biology, University of Murcia, Espinardo, Murcia, Spain

An efficient technique for isolation of resistant gene analogs (RGAs) in pepper from silver stained denaturing polyacrylamide gel was developed using a modified amplified fragment length polymorphism (AFLP) strategy. Pepper DNA was digested, ligated and pre-amplified as in a normal AFLP method. The selective amplification was made by using combinations with oligonucleotide primers based on conserved motifs in and around nucleotide binding site (NBS) of known NBS-leucine-rich repeats resistance proteins from known resistant genes. The amplified products were separated by using denaturing polyacrylamide gels and silver staining instead of radioactive labelling. We isolated specific polymorphic AFLP bands directly from the gels with one round of polymerase chain reaction amplification, in order to confirm, after sequencing, that these bands have homologies with products of resistance genes described so far. Two bands (R2: 250 bp and R6: 150 bp) are particularly highlighted because they could be considered as RGAs related to resistance to Phytophthora capsici in pepper, because their sequences have a very high homology with other resistant gene analogs that have already been described. Besides, they were only detected in the resistant parent and in the bulked resistant segregants but not in the susceptible parent or susceptible F₂ segregants. We can conclude that the technique used is clean, quick and efficient for the isolation of RGAs in pepper.

Keywords: amplified fragment length polymorphism; Capsicum annuum; Phytophthora capsici

Subjects: amplified fragment length polymorphism (AFLP); Capsicum annuum; pepper, resistant gene analogues (RGA) isolation; Phytophthora capsici; polymerase chain reaction (PCR); resistant gene analogues isolation (RGA), amplified fragment length polymorphism (AFLP); root rot of pepper

Published: July 1, 2003  Show citation