Efficient targeting of plant disease resistance loci using NBS profiling

Abstract

The conserved sequences in the nucleotide-binding sites of the nucleotide-binding site-leucine-rich repeat (NBS-LRR) class of disease resistance (R) genes have been used for PCR-based R-gene isolation and subsequent development of molecular markers. Here we present a PCR-based approach (NBS profiling) that efficiently targets R genes and R-gene analogs (RGAs) and, at the same time, produces polymorphic markers in these genes. In NBS profiling, genomic DNA is digested with a restriction enzyme, and an NBS-specific (degenerate) primer is used in a PCR reaction towards an adapter linked to the resulting DNA fragments. The NBS profiling protocol generates a reproducible polymorphic multilocus marker profile on a sequencing gel that is highly enriched for R genes and RGAs. NBS profiling was successfully used in potato with several restriction enzymes, and several primers targeted to different conserved motifs in the NBS. Across primers and enzymes, the NBS profiles contained 50-90% fragments that were significantly similar to known R-gene and RGA sequences. The protocol was similarly successful in other crops (including tomato, barley, and lettuce) without modifications. NBS profiling can thus be used to produce markers tightly linked to R genes and R-gene clusters for genomic mapping and positional cloning and to mine for new alleles and new sources of disease resistance in available germplasm.

Genetic mapping and transcription analyses of resistance gene loci in potato using NBS profiling

“…Transcription levels of the target genes are therefore expected to be low, which could lead to problems related to PCR kinetics and sensitivity (Vos et al 1998). However, when using the standard NBS proWling protocol as developed by van der Linden et al (2004), between 20 and 35 fragments were ampliWed, which is approximately half the number of bands typically ampliWed with NBS proWling on genomic DNA, suggesting that NBS proWling is suitable to analyse the expression of relatively low expressed genes, although further exploration of the limits of detection of NBS proWling, e.g. through QRT-PCR analysis, is required to support this conclusion.…”

“…NBS proWling using genomic DNA NBS proWling on genomic DNA was carried out as described by van der Linden et al (2004). The restriction enzymes MseI, RsaI or HaeIII were used for digestion of genomic DNA.…”

“…Conservation of several structural motifs within the NBS domain encoded by plant R genes has prompted the development of homology-based approaches aimed at identiWcation of structurally related sequences, termed R gene analogues (RGAs) (Kanazin et al 1996;Yu et al 1996;Leister et al 1996;Aarts et al 1998;Shen et al 1998;Pan et al 2000;van der Linden et al 2004). Cosegregation of speciWc RGAs and R loci and/or quantitative trait loci (QTL) involved in disease resistance has been reported (Hunger et al 2002;Kuhn et al 2003), suggesting that NBS proWling can be a powerful tool for the development of markers linked to resistance loci.…”

“…Motif directed Wngerprinting techniques which combine the advantage of a neutral marker system with a bias towards candidate genes are a better option. The use of degenerate primers that target NBS speciWc motifs in combination with adapter based ampliWcation techniques generates complex Wngerprinting patterns containing several RGA derived fragments (Hayes et al 2000;van der Linden et al 2004). By applying NBS based proWling techniques on individuals of an F1 mapping population, the genetic variation at RGA loci is sampled, resulting in the direct mapping of these fragments relative to other genetic markers or R loci that segregate in the mapping population (Calenge et al 2005).…”

Resistance gene analogues identified through the NBS-profiling method map close to major genes and QTL for disease resistance in apple

“…Most often, the cloned RGAs are mapped by restriction fragment length polymorphism (RFLP), which is often time-consuming. Hayes and Saghai-Maroof (2000), and more recently, Van der Linden et al (2004), proposed new strategies simultaneously to generate polymorphism and specifically amplify highly conserved motifs. Both methods are based on the simultaneous use of an adapter primer matching a restriction enzyme site and of a degenerate primer targeting the NBS-encoding region in PCR reactions.…”