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Plant Methods

, 9:37

Next Generation Sequencing technologies for plant research

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.

KeywordsMotif-directed profiling Resistance gene Potato Solanum berthaultii AbbreviationsR geneResistance gene

RGAResistance gene analog

CDPCluster-directed profiling

NBSNucleotide binding site

LRRLeucine-rich repeat

NLRNucleotide binding leucine-rich repeat proteins

RLPReceptor-like proteins

RLKReceptor-like kinases

PKProtein kinases

TIRToll-interleukin-1 receptor-R gene domain

CCCoiled-coil

UID tagUnique identifier tag

BRResistant bulk

BSSusceptible bulk.

Electronic supplementary materialThe online version of this article doi:10.1186-1746-4811-9-37 contains supplementary material, which is available to authorized users.

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Autor: Jack H Vossen - Sara Dezhsetan - Danny Esselink - Marjon Arens - Maria J Sanz - Walter Verweij - Estelle Verzaux - C Ger

Fuente: https://link.springer.com/







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