Identification of reliable reference genes for qRT-PCR in the ephemeral plant Arabidopsis pumila based on full-length transcriptome data

Arabidopsis pumila, an annual ephemeral plant, plays important roles in preventing wind and sand erosion, water and soil conservation, and microhabitat improvement in the North of Xinjiang, China. Studies of adaptive mechanisms in harsh desert environments at the genetic and genomic levels can be used to more effectively develop and protect this species. The quantitative real-time polymerase chain reaction (qRT-PCR) method is one of the essential means to achieve these goals, and the selection of an appropriate reference gene is the prerequisite for qRT-PCR. In this study, 10 candidate reference genes were identified from the full-length transcriptome data of A. pumila, and their expression stabilities under four abiotic stresses (drought, heat, cold and salt) and in seven different tissues (roots, hypocotyl, cotyledon, leaves, stems, flowers and siliques) were evaluated with four programmes geNorm, NormFinder, Bestkeeper and RefFinder. Although the most stable reference genes were variable under different treatments using different software, comprehensive ranking revealed that UEP and HAF1 under drought stress, UBQ9 and GAPDH under heat stress, UBC35 and GAPDH under cold stress, GAPDH and ACT1 under salt stress, and ACT1 and GAPDH in different tissues were the most stable reference genes. Moreover, GAPDH and UBQ9 were the most suitable reference gene combinations for all samples. The expression pattern of the K+ uptake permease gene KUP9 further validated that the selected reference genes were suitable for normalization of gene expression. The identification of reliable reference genes guarantees more accurate qRT-PCR quantification for A. pumila and facilitates functional genomics studies of ephemeral plants.