Centro de Investigación en Alimentación y Desarrollo (CIAD), A.C., Culiacán 80110, Sinaloa, Mexico.
CONACYT-Centro de Investigación en Alimentación y Desarrollo, A.C., Culiacán 80110, Sinaloa, Mexico.
Genes (Basel). 2023 Jan 28;14(2):337. doi: 10.3390/genes14020337.
Molecular markers linked to disease resistance genes which affect economically important crops are of great interest. In the case of tomato, a major focus on resistance breeding to multiple fungal and viral pathogens such as (TYLCV), (TSWV) and f. sp. (), have led to the introgression of several resistance genes; therefore, molecular markers have become important in molecular-assisted selection (MAS) of tomato varieties resistant to those pathogens. However, assays that allow simultaneous evaluation of resistant genotypes, such as multiplex PCR, need to be optimized and evaluated to demonstrate their analytical performance, as many factors can affect them. This work aimed to generate multiplex PCR protocols for the joint detection of the molecular markers associated with pathogen resistance genes in tomato plants that are sensitive, specific and repeatable. For the optimization a central composite design of a response surface methodology (RSM-CCD) was used. For analytical performance evaluation, specificity/selectivity and sensibility (limit of detection and dynamic range) were analyzed. Two protocols were optimized: the first one with a desirability of 1.00, contained two markers (At-2 and P7-43) linked to and -resistant genes. The second one with a desirability of 0.99, contained markers (SSR-67, SW5 and P6-25) linked to , and -resistant genes. For protocol 1, all the commercial hybrids (7/7) were resistant to and for protocol 2, two hybrids were resistant to , one to TSWV and one to TYLCV with good analytical performance. In both protocols, the varieties considered susceptible to the pathogens, no-amplicon or susceptible amplicons, were observed. The optimized multiplex PCR protocols showed dynamic ranges from 5.97 up to 161.3 ng DNA. The limit of detection was 17.92 ng and 53.76 ng DNA for protocols 1 and 2, respectively, giving 100% positive results in the test replicates. This method allowed to develop optimized multiplex PCR protocols with few assays which translates into less time and resources, without sacrificing method performance.
与影响经济重要作物的抗病基因相关的分子标记备受关注。以番茄为例，针对多种真菌和病毒病原体（如 TYLCV、TSWV 和 f. sp. ( )）的抗性育种重点，已经导致了几种抗性基因的导入；因此，分子标记在番茄品种对这些病原体的分子辅助选择（MAS）中变得非常重要。然而，需要对允许同时评估抗性基因型的检测方法（如多重 PCR）进行优化和评估，以证明其分析性能，因为许多因素会影响它们。这项工作旨在为番茄植株中与病原体抗性基因相关的分子标记的联合检测生成多重 PCR 方案，这些方案应具有敏感性、特异性和可重复性。为了优化，使用响应面法（RSM-CCD）的中心复合设计进行了优化。为了评估分析性能，分析了特异性/选择性和敏感性（检测限和动态范围）。优化了两个方案：第一个方案的理想值为 1.00，包含与 和 -抗性基因相关的两个标记（At-2 和 P7-43）。第二个方案的理想值为 0.99，包含与 、 和 -抗性基因相关的标记（SSR-67、SW5 和 P6-25）。对于方案 1，所有商业杂种（7/7）均对 具有抗性，而对于方案 2，有两种杂种对 具有抗性，一种对 TSWV 具有抗性，一种对 TYLCV 具有抗性，且具有良好的分析性能。在两个方案中，观察到对病原体敏感的品种没有扩增子或敏感扩增子。优化的多重 PCR 方案显示出从 5.97 到 161.3 ng DNA 的动态范围。方案 1 和 2 的检测限分别为 17.92 ng 和 53.76 ng DNA，在测试重复中均给出了 100%的阳性结果。该方法允许开发具有较少检测的优化多重 PCR 方案，这转化为更少的时间和资源，而不会牺牲方法性能。
