Reddy Dumbala Srinivas, Bhatnagar-Mathur Pooja, Reddy Palakolanu Sudhakar, Sri Cindhuri Katamreddy, Sivaji Ganesh Adusumalli, Sharma Kiran Kumar
Genetic Transformation Laboratory, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru-502324, Telangana, India.
PLoS One. 2016 Feb 10;11(2):e0148451. doi: 10.1371/journal.pone.0148451. eCollection 2016.
Quantitative Real-Time PCR (qPCR) is a preferred and reliable method for accurate quantification of gene expression to understand precise gene functions. A total of 25 candidate reference genes including traditional and new generation reference genes were selected and evaluated in a diverse set of chickpea samples. The samples used in this study included nine chickpea genotypes (Cicer spp.) comprising of cultivated and wild species, six abiotic stress treatments (drought, salinity, high vapor pressure deficit, abscisic acid, cold and heat shock), and five diverse tissues (leaf, root, flower, seedlings and seed). The geNorm, NormFinder and RefFinder algorithms used to identify stably expressed genes in four sample sets revealed stable expression of UCP and G6PD genes across genotypes, while TIP41 and CAC were highly stable under abiotic stress conditions. While PP2A and ABCT genes were ranked as best for different tissues, ABCT, UCP and CAC were most stable across all samples. This study demonstrated the usefulness of new generation reference genes for more accurate qPCR based gene expression quantification in cultivated as well as wild chickpea species. Validation of the best reference genes was carried out by studying their impact on normalization of aquaporin genes PIP1;4 and TIP3;1, in three contrasting chickpea genotypes under high vapor pressure deficit (VPD) treatment. The chickpea TIP3;1 gene got significantly up regulated under high VPD conditions with higher relative expression in the drought susceptible genotype, confirming the suitability of the selected reference genes for expression analysis. This is the first comprehensive study on the stability of the new generation reference genes for qPCR studies in chickpea across species, different tissues and abiotic stresses.
定量实时聚合酶链反应（qPCR）是一种用于准确量化基因表达以了解精确基因功能的首选且可靠的方法。总共选择了25个候选参考基因，包括传统和新一代参考基因，并在一组多样的鹰嘴豆样本中进行了评估。本研究中使用的样本包括9种鹰嘴豆基因型（鹰嘴豆属），涵盖栽培种和野生种，6种非生物胁迫处理（干旱、盐度、高蒸汽压亏缺、脱落酸、冷和热激），以及5种不同组织（叶、根、花、幼苗和种子）。用于在四个样本集中鉴定稳定表达基因的geNorm、NormFinder和RefFinder算法显示，UCP和G6PD基因在各基因型中表达稳定，而TIP41和CAC在非生物胁迫条件下高度稳定。虽然PP2A和ABCT基因在不同组织中排名最佳，但ABCT、UCP和CAC在所有样本中最稳定。本研究证明了新一代参考基因对于在栽培和野生鹰嘴豆物种中基于qPCR进行更准确的基因表达定量分析的有用性。通过研究它们对水通道蛋白基因PIP1;4和TIP3;1在高蒸汽压亏缺（VPD）处理下的三种对比鹰嘴豆基因型中的标准化的影响，对最佳参考基因进行了验证。鹰嘴豆TIP3;1基因在高VPD条件下显著上调，在干旱敏感基因型中具有更高的相对表达，证实了所选参考基因适用于表达分析。这是关于鹰嘴豆跨物种、不同组织和非生物胁迫的qPCR研究中新一代参考基因稳定性的首次全面研究。
