Department of Phytopathology, Institute of Phytomedicine, Faculty of Agricultural Sciences, University of Hohenheim, Stuttgart, Germany.
PLoS One. 2020 Aug 6;15(8):e0237273. doi: 10.1371/journal.pone.0237273. eCollection 2020.
Uromyces appendiculatus is a major pathogen on common bean. Like other rust fungi, it uses effectors to influence its host plant. Effectors are assumed to possess characteristic expression profiles, reflecting their activity during the infection process. In order to determine expression profiles using RT-qPCR, stably expressed reference genes are necessary for normalization. These reference genes need to be tested. Using samples representing seven different developmental stages of the urediospore-based infection process we employed RT-qPCR to measure the expression of 14 candidate reference genes and determined the most suitable ones based on the range of Cq values and comparative calculations using the geNorm and NormFinder algorithms. Among the tested genes RPS14 had the smallest Cq range, followed by Elf1a and Elf3; geNorm rated Tub and UbcE2 best with CytB as a third and NormFinder found UbcE2, Tub and Elf3 as best reference genes. Combining these findings using equal weight for the rankings UbcE2, Elf3 and Tub can be considered the best reference genes. A combination of either two reference genes, UbcE2 and Tub or three reference genes, UbcE2, Tub, and Elf3 is recommended for normalization. However, differences between most genes were relatively small, so all tested genes can be considered suitable for normalization with the exception of RPS9, SDH, Ubc and PDK.
节节麦多胞锈菌是普通菜豆的主要病原菌。与其他锈菌一样，它使用效应子来影响其宿主植物。效应子被认为具有特征表达谱，反映了它们在感染过程中的活性。为了使用 RT-qPCR 确定表达谱，需要对稳定表达的参考基因进行归一化。这些参考基因需要进行测试。使用代表基于夏孢子的感染过程的七个不同发育阶段的样本，我们通过 RT-qPCR 测量了 14 个候选参考基因的表达，并根据 Cq 值范围和使用 geNorm 和 NormFinder 算法的比较计算，确定了最合适的基因。在测试的基因中，RPS14 的 Cq 值范围最小，其次是 Elf1a 和 Elf3；geNorm 对 Tub 和 UbcE2 的评价最好，CytB 为第三，NormFinder 发现 UbcE2、Tub 和 Elf3 是最好的参考基因。使用排名的等权重组合，UbcE2、Elf3 和 Tub 可以被认为是最佳的参考基因。推荐使用 UbcE2、Elf3 和 Tub 的两种参考基因组合或 UbcE2、Tub 和 Elf3 的三种参考基因组合进行归一化。然而，大多数基因之间的差异相对较小，因此除了 RPS9、SDH、Ubc 和 PDK 之外，所有测试的基因都可以被认为适合归一化。
