Centro de Investigación en Alimentación y Desarrollo (CIAD), A.C., Culiacán, Sinaloa, Mexico; Unidad Académica Multidisciplinaria Reynosa-Aztlán, Universidad Autónoma de Tamaulipas, Reynosa, Tamaulipas, Mexico.
CONACYT-Centro de Investigación en Alimentación y Desarrollo, A.C., Culiacán, Sinaloa, Mexico.
Gene. 2021 Jun 15;785:145606. doi: 10.1016/j.gene.2021.145606. Epub 2021 Mar 23.
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, MgCl 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 MgCl and 0.13 µM primers; for TG105A: Ta = 54.4 °C, 10 ng DNA, 1.5 mM MgCl and 0.9 µM primers; for P6-25, Ta = 52.5 °C, 50 ng DNA, 2.5 mM MgCl 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.
番茄（Solanum lycopersicum L.）是世界上最重要的经济蔬菜之一。然而，其生产受到番茄黄曲叶病毒（TYLCV）的影响，对作物造成最大的破坏。应对 TYLCV 的策略之一是使用抗性品种，通过分子标记可以加速其开发。本研究旨在通过中心复合设计（CCD）使用响应面法（RSM）优化端点 PCR 协议，以检测分别与 Ty-1、Ty-2 和 Ty-3 抗性基因相关的分子标记 TG178、TG105A 和 P6-25。还分析了适用性、检测限和动态范围。优化的 PCR 条件为：TG178：退火温度（Ta）= 60°C，90ng DNA，3.36mM MgCl 和 0.13µM 引物；TG105A：Ta = 54.4°C，10ng DNA，1.5mM MgCl 和 0.9µM 引物；P6-25，Ta = 52.5°C，50ng DNA，2.5mM MgCl 和 0.5µM 引物。动态范围从 0.42 到 103.3ng DNA 不等，而 TG178、TG105A 和 P6-25 分子标记的检测限分别为 3.82、0.42 和 11.47ng DNA，重复检测均为 100%阳性。CCD 允许优化用于分子标记的 PCR 协议，这可能进一步应用于鉴定 TYLCV 抗性番茄品系。
