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
| Autor: | Mathuresh Singh, Bihua Nie, Virginia Dickison, David De Koeyer, Xianzhou Nie, Agnes Murphy, Sydney Brooks |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
0301 basic medicine Genetic Markers Plant Science Nucleic Acid Denaturation Real-Time Polymerase Chain Reaction 01 natural sciences law.invention 03 medical and health sciences Nucleic acid thermodynamics law Genotype Antibiosis Selection Genetic Gene Polymerase chain reaction Plant Diseases Plant Proteins Solanum tuberosum Genetics biology Virulence fungi Marker-assisted selection Amplicon Potato virus X biology.organism_classification Molecular biology Potexvirus 030104 developmental biology Agarose gel electrophoresis Agronomy and Crop Science 010606 plant biology & botany |
| Zdroj: | Plant disease. 102(2) |
| ISSN: | 0191-2917 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|