Poster: Molecular & Cellular Plant-Microbe Interactions: Plant Defense Responses
Development of HRM markers tightly linked to extreme resistance to Potato virus Y using next-generation sequencing
X. NIE (1), D. De Koeyer (1), A. Murphy (1), V. Dickison (1), M. Singh (2) (1) Fredericton Research and Development Centre, Agriculture and Agri-Food Canada, Canada; (2) Agricultural Certification Services, Canada
Tetraploid potato breeding clone F87084 possesses a resistance gene (Ry) that confers extreme resistance (ER) to Potato virus Y (PVY). However, markers reported to be highly linked to different Ry genes were not detected in F87084. Next generation sequencing of genomic DNA from F87084 (Rrrr), F02010 (rrrr) and the respective bulk DNA of resistant and susceptible progenies of F87084 x F02010 was carried out by Illumina HiSeq for identification of single nucleotide polymorphisms (SNP). Polymorphic positions matching the expected pattern in parents and progeny bulk samples were determined. The genomic regions flanking the target SNP were extracted and used to design primers and probes for high-resolution DNA melting (HRM) assays. Of the 10 primer/probe sets designed and tested, five produced amplicons that exhibit different HRM profiles between the resistant parent/bulk and the susceptible parent/bulk. Further analysis of the HRM primer/probe sets on a segregating population comprised of 300 progenies revealed near-perfect matches between the HRM assays and the phenotyping results, demonstrating high linkage between these newly developed HRM markers and Ry in F87084. The efficacy of the HRM markers for marker-assisted selection of ER to PVY is validated with different populations involving F87084.