Novel applications of motif-directed profiling to identify disease resistance genes in plants

Abstract

BackgroundMolecular profiling of gene families is a versatile tool to study diversity between individual genomes in sexual crosses and germplasm. Nucleotide binding site (NBS) profiling, in particular, targets conserved nucleotide binding site-encoding sequences of resistance gene analogs (RGAs), and is widely used to identify molecular markers for disease resistance (R) genes.ResultsIn this study, we used NBS profiling to identify genome-wide locations of RGA clusters in the genome of potato clone RH. Positions of RGAs in the potato RH and DM genomes that were generated using profiling and genome sequencing, respectively, were compared. Largely overlapping results, but also interesting discrepancies, were found. Due to the clustering of RGAs, several parts of the genome are overexposed while others remain underexposed using NBS profiling. It is shown how the profiling of other gene families, i.e. protein kinases and different protein domain-coding sequences (i.e., TIR), can be used to achieve a better marker distribution. The power of profiling techniques is further illustrated using RGA cluster-directed profiling in a population of Solanum berthaultii. Multiple different paralogous RGAs within the Rpi-ber cluster could be genetically distinguished. Finally, an adaptation of the profiling protocol was made that allowed the parallel sequencing of profiling fragments using next generation sequencing. The types of RGAs that were tagged in this next-generation profiling approach largely overlapped with classical gel-based profiling. As a potential application of next-generation profiling, we showed how the R gene family associated with late blight resistance in the SH*RH population could be identified using a bulked segregant approach.ConclusionsIn this study, we provide a comprehensive overview of previously described and novel profiling primers and their genomic targets in potato through genetic mapping and comparative genomics. Furthermore, it is shown how genome-wide or fine mapping can be pursued by choosing different sets of profiling primers. A protocol for next-generation profiling is provided and will form the basis for novel applications. Using the current overview of genomic targets, a rational choice can be made for profiling primers to be employed.

Characterisation of the late blight resistance in potato differential MaR9 reveals a qualitative resistance gene, R9a, residing in a cluster of Tm-2 2 homologs on chromosome IX

“…In this interval of chromosome IX, two R gene clusters (C42 and C43) are known (Jupe et al 2012 ). These clusters were targeted for R gene cluster-directed profiling (CDP; Vossen et al 2013 ). Using eight Tm - 2 2 primers, population 3154 was screened for linked markers.…”

“…The CDP primers were used in combination with a labelled adapter primer (fluorescent dye IRD700) and labelled R gene-targeted PCR products were separated on a denaturing polyacrylamide gel using a NEN ® IR 2 DNA analyser (LI-COR ® Biosciences, Lincoln, NE, USA). A set of the ten resistant and the ten susceptible BC 1 plants, including parents, was used to obtain CDP markers linked to resistance in mapping population (Vossen et al 2013 ). If linked CDP markers were found, a second round of CDP was applied to all the remaining individuals of a segregating population.…”

Disease Resistance Gene Analogs (RGAs) in Plants

“…A similar profiling method for other R -gene classes, like peroxidase profiling, developed in barley, revealed the resistance of R- genes for rusts and mildew [ 280 ]. RLK and LRR profiling strategies in potato were also developed [ 281 ]. Meanwhile, other non-RGA gene families can also benefit from this idea, like MYB profiling in pot azalea [ 282 ].…”

Identification of expressed resistance gene analog sequences in coconut leaf transcriptome and their evolutionary analysis

“…The receptor-like kinases encode proteins comprising an extracellular receptor domain connected to a cytoplasmic serine-threonine kinase domain and are further divided into several subclasses based on the presence of various extracellular receptor domains. The receptor domains can contain LRRs and lectin-like or additional domains, which can potentially bind pathogen-derived peptides (Vossen et al, 2013). The protein kinase class of R genes encodes only serine-threonine kinases, no transmembrane domains or LRRs (Vossen et al, 2013).…”