High Resolution DNA Melting Assays for Detection of Rx1 and Rx2 for High-Throughput Marker-Assisted Selection for Extreme Resistance to Potato virus X in Tetraploid Potato

摘要

Assessment of the existing PCR-gel electrophoresis-based methods for detection of Rxl and Rx2, the genes that independently control extreme resistance (ER) to Potato virus X (PVX), indicated that the 5RxIF/ 5RxlR primer pair led to reliable detectionof Rxl, whereas the 106Rx2F/106Rx2R primer pair detected Rxl despite some nonspecific reactions in potato clones/cultivars without Rxl. However, the methodology is time consuming and does not differentiate the absence of Rxl/Rxl from a failed PCR reaction. A newly designed primer pair that targets Rxl and Rx2 as well as rxl and rxl produced an amplicon for all alleles. When the primer pair was combined with 5Rx 1 F/5Rx 1R, respective amplicons were produced, although they were not distinguishable by regular agarose gel electrophoresis. When subjected to a high-resolution DNAmelting (HRM) assay, two distinct melting profdes for Rxl and rxl, respectively, were detected. Triplex PCR-gel electrophoresis and -HRM assay for detection of Rxl, Rxl, and rxllrxl were also performed. The efficacy of the HRM assays were validated in potato cultivars/clones with known phenotypes, indicating its potential for high-throughput selection of potato clones/cultivars carrying Rxl or Rxl. Duplex PCR-HRM assays of over 600 progeny from 12 crosses involving various parents correctly detected thepresence or absence of Rxl in each progeny, allowing accurate prediction of the phenotype. Progeny that tested positive for Rxl by HRM exhibited ER to PVX whereas progeny that tested negative for Rxl were susceptible to PVX infection. The genotype of each parent and the possible presence of Nx in two /fx/-possessing parents are also discussed.