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.

Assessment of the existing PCR-gel electrophoresis-based methods for detection of Rx1 and Rx2, the genes that independently control extreme resistance (ER) to Potato virus X (PVX), indicated that the 5Rx1F/5Rx1R primer pair led to reliable detection of Rx1, whereas the 106Rx2F/106Rx2R primer pair detected Rx2 despite some nonspecific reactions in potato clones/cultivars without Rx2. However, the methodology is time consuming and does not differentiate the absence of Rx1/Rx2 from a failed PCR reaction. A newly designed primer pair that targets Rx1 and Rx2 as well as rx1 and rx2 produced an amplicon for all alleles. When the primer pair was combined with 5Rx1F/5Rx1R, respective amplicons were produced, although they were not distinguishable by regular agarose gel electrophoresis. When subjected to a high-resolution DNA melting (HRM) assay, two distinct melting profiles for Rx1 and rx1, respectively, were detected. Triplex PCR-gel electrophoresis and -HRM assay for detection of Rx1, Rx2, and rx1/rx2 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 Rx1 or Rx2. Duplex PCR-HRM assays of over 600 progeny from 12 crosses involving various parents correctly detected the presence or absence of Rx1 in each progeny, allowing accurate prediction of the phenotype. Progeny that tested positive for Rx1 by HRM exhibited ER to PVX whereas progeny that tested negative for Rx1 were susceptible to PVX infection. The genotype of each parent and the possible presence of Nx in two Rx1-possessing parents are also discussed.

Deletion of AcMNPV ac146 eliminates the production of budded virus.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac146 is a highly conserved gene in the Alpha- and Betabaculovirus genera that has an unknown function. Northern blot analysis and transcript mapping showed that ac146 is transcribed at late times post infection as a 1.2 kb mRNA. To determine the role of ac146 in the baculovirus life cycle ac146 knock out viruses were constructed. Transfection and plaque assays showed that all the ac146 deletions produced a single cell phenotype indicating that no infectious budded virus (BV) was produced, however occlusion bodies were formed. The lack of BV production was confirmed by viral titration utilizing both qPCR and TCID50. Analysis of BV and occlusion derived virus (ODV) revealed that AC146 is associated with both forms of the virus and is modified specifically in ODV. This study therefore demonstrates that AC146 is a late virion associated protein and is essential for the viral life cycle.