Cenostigma pyramidale is a native legume of the Brazilian semiarid region which performs symbiotic association with arbuscular mycorrhizal fungi (AMF), being an excellent model for studying genes associated with tolerance against abiotic and biotic stresses. In RT-qPCR approach, the use of reference genes is mandatory to avoid incorrect interpretation of the relative expression. This study evaluated the stability of ten candidate reference genes (CRGs) from C. pyramidale root tissues under salt stress (three collection times) and associated with AMF (three different times of salinity). The de novo transcriptome was obtained via RNA-Seq sequencing. Three algorithms were used to calculate the stability of CRGs under different conditions: (i) global (Salt, Salt+AMF, AMF and Control, and collection times), (ii) only non-inoculated plants, and (iii) AMF (only inoculated plants). HAG2, SAC1, aRP3 were the most stable CRGs for global and AMF assays, whereas HAG2, SAC1, RHS1 were the best for salt stress assay. This CRGs were used to validate the relative expression of two up-regulated transcripts in Salt2h (RAP2-3 and PIN8). Our study provides the first set of reference genes for C. pyramidale under salinity and AMF, supporting future researches on gene expression with this species.

中文翻译：

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盐胁迫和菌根关联下 Cenostigma pyramidale 根定量实时 PCR 归一化的参考基因。

Cenostigma pyramidale 是巴西半干旱地区的原生豆科植物，与丛枝菌根真菌 (AMF) 共生，是研究与非生物和生物胁迫耐受性相关的基因的绝佳模型。在 RT-qPCR 方法中，必须使用参考基因​​，以避免对相对表达的错误解释。本研究评估了盐胁迫下（三个收集时间）和与 AMF（三个不同盐度时间）相关的来自锥体锥体根组织的十个候选参考基因 (CRG) 的稳定性。从头转录组是通过 RNA-Seq 测序获得的。三种算法用于计算不同条件下 CRG 的稳定性：（i）全局（盐、盐+AMF、AMF 和对照，以及收集时间），（ii）仅未接种植物，(iii) AMF（仅接种植物）。HAG2、SAC1、aRP3 是全局和 AMF 测定中最稳定的 CRG，而 HAG2、SAC1、RHS1 是盐胁迫测定中最好的。该 CRG 用于验证 Salt2h 中两个上调转录本（RAP2-3 和 PIN8）的相对表达。我们的研究为盐度和 AMF 下的锥体锥虫提供了第一组参考基因，支持未来对该物种基因表达的研究。