Table_1_Reference genes identification for qRT-PCR normalization of gene expression analysis in Cucumis sativus under Meloidogyne incognita infection and Pseudomonas treatment.docx

qRT-PCR is a common and key technical means to study gene expression in biological research. However, reliability and accuracy of quantification by qRT-PCR is entirely dependent on the identification of appropriate reference genes. Cucumber as an economical vegetable is widely cultivated worldwide and is subject to serious nematode infection, especially from M. incognita. Plant could employ beneficial soil bacteria in the rhizosphere to enhance plant adaptability to various stresses. In this study, the optimal reference genes in cucumber under M. incognita stress and Pseudomonas treatment were calculated and confirmed. A total of thirteen candidate reference genes were identified across three different treatments. Of these, geNorm, NormFinder and BestKeeper programs combined RefFinder software identified EF1 and UBI are the most suitable reference gene in the root knot and whole root of cucumber infected M. incognita, respectively, and CACS is the most suitable reference gene in the whole root of cucumber treated by Pseudomonas. The work first validated the most suitable reference genes for the normalization gene expression in cucumber by nematode infected or Pseudomonas inoculated, and these results would facilitate the further research on M. incognita or Pseudomonas soil rhizosphere microbe interaction with cucumber.

实时荧光定量PCR（quantitative real-time polymerase chain reaction，qRT-PCR）是生物研究中探究基因表达的常用关键技术手段。然而，qRT-PCR定量的可靠性与准确性完全依赖于合适内参基因的筛选。黄瓜作为一种经济蔬菜在全球范围内广泛栽培，却极易遭受线虫侵染，尤其是南方根结线虫（Meloidogyne incognita）。植物可利用根际有益土壤细菌，提升自身对多种胁迫的适应能力。本研究筛选并验证了南方根结线虫胁迫与假单胞菌（Pseudomonas）处理下黄瓜的最优内参基因。本研究共筛选得到13个候选内参基因，覆盖3种不同处理条件。其中，结合"geNorm"、"NormFinder"、"BestKeeper"三款分析程序与"RefFinder"软件的综合分析结果显示：在被南方根结线虫侵染的黄瓜根结组织与整根组织中，EF1与UBI分别为最适内参基因；而在假单胞菌处理的黄瓜整根组织中，CACS为最适内参基因。本研究首次验证了南方根结线虫侵染或假单胞菌接种条件下黄瓜基因表达标准化分析所需的最优内参基因，上述结果将为后续探究南方根结线虫、根际假单胞菌与黄瓜的互作机制提供重要支撑。