Optimization of PCR-based TYLCV molecular markers by response surface methodology
| Autor: | Claudia Villicaña, Sanjuana Elizabeth Alemán-Castillo, Mayra Janeth Esparza-Araiza, Josefina León-Félix, Humberto Martínez-Montoya, Richecarde Lafrance, José Benigno Valdez-Torres, Octelina Castillo-Ruiz |
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| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Genetic Markers Central composite design Polymerase Chain Reaction 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Solanum lycopersicum Genetics Tomato yellow leaf curl virus Response surface methodology Gene Disease Resistance Plant Diseases Detection limit biology food and beverages General Medicine biology.organism_classification 030104 developmental biology chemistry 030220 oncology & carcinogenesis Begomovirus Primer (molecular biology) Solanum DNA |
| Zdroj: | Gene. 785 |
| ISSN: | 1879-0038 |
| Popis: | Tomato (Solanum lycopersicum L.) is one of the most economically important vegetables worldwide. However, its production is affected by the tomato yellow leaf curl virus (TYLCV), causing the greatest devastation in the crop. One strategy to cope with TYLCV implies the use of resistant varieties, whose development can be accelerated by molecular markers. The aim of this study was to optimize endpoint PCR protocols for the detection of the molecular markers TG178, TG105A and P6-25, linked to Ty-1, Ty-2 and Ty-3 resistance genes, respectively, through a response surface methodology (RSM) using a central composite design (CCD) for four factors (temperature of annealing (Ta), DNA amount, MgCl2 and primer concentrations). Applicability, the limit of detection and dynamic range were also analyzed. The optimized PCR conditions were: for TG178: Ta = 60 °C, 90 ng DNA, 3.36 mM MgCl2 and 0.13 µM primers; for TG105A: Ta = 54.4 °C, 10 ng DNA, 1.5 mM MgCl2 and 0.9 µM primers; for P6-25, Ta = 52.5 °C, 50 ng DNA, 2.5 mM MgCl2 and 0.5 µM primers. Dynamic ranges varied from 0.42 to 103.3 ng of DNA, while the limit of detection was 3.82, 0.42 and 11.47 ng of DNA for the TG178, TG105A and P6-25 molecular makers respectively and was 100% positive in replicates. CCD allowed the optimization of PCR protocols for molecular markers, which may further apply in identifying TYLCV resistant tomato lines. |
| Databáze: | OpenAIRE |
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