Optimization of PCR-based TYLCV molecular markers by response surface methodology

Abstract

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.

Keywords: Endpoint PCR; Protocols; Resistance genes; Solanum lycopersicum L; TYLCD.