Abstract
The desert pioneer plant Stipagrostis pennata plays an important role in sand fixation, wind prevention, and desert ecosystem recovery. An absence of reference genes greatly limits investigations into the regulatory mechanism by which S. pennata adapts to adverse desert environments at the molecular and genetic levels. In this study, eight candidate reference genes were identified from rhizosheath development transcriptome data from S. pennata, and their expression stability in the rhizosheaths at different development stages, in a variety of plant tissues, and under drought stress was evaluated using four procedures, including geNorm, NormFinder, BestKeeper, and RefFinder. The results showed that GAPDH and elF were the most stable reference genes under drought stress and in rhizosheath development, and ARP6 and ALDH were relatively stable in all plant tissues. In addition, elF was the most suitable reference gene for all treatments. Analysis of the consistency between the reverse transcription-quantitative PCR (RT-qPCR) and RNA sequencing data showed that the identified elF and GAPDH reference genes were stable during rhizosheath development. These results provide reliable reference genes for assuring the accuracy of RT-qPCR and offer a foundation for further investigations into the genetic responses of S. pennata to abiotic stress.
Highlights
Abbreviations ACT Actin aldehyde dehydrogenase (ALDH) Aldehyde dehydrogenase ARP Actin related protein Cq Quantification cycle coefficient of variation (CV) Coefficient of variation CYP Cyclophilin days post-germination (DPG) Day post germination elongation factor-1α (EF-1α) Elongation factor-1α eukaryotic translation initiation factor (elF) Eukaryotic translation initiation factor FPKM Fragments per Kilobase of exon model per Million mapped reads glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Glyceraldehyde-3-phosphate dehydrogenase HIS-3 Histone H3 PEG Polyethylene glycol phosphoglycerate kinase gene (PGK) Phosphoglycerate kinase gene phosphatase 2A (PP2A) Protein phosphotase 2A RNA-Seq RNA sequencing Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) Reverse transcription quantitative polymerase chain reaction SAMSC S-Adenosylmethionine synthase standard deviation (SD) Standard deviation
The conditions of |log2FoldChange| < 1, q-value ≥ 0.05, and FPKM (Fragments per Kilobase of exon model per Million mapped reads) ≥ 6 were used to screen the RNA-Seq data, and the relatively low coefficient of variation (CV) of FPKM was set as a high standard at all sampling points, generating a total of eight candidate reference genes (Table 1)
The FPKM-based heatmap of the eight genes during different stages of rhizosheath development is provided in Fig. 1 and indicates that GAPDH, α-TUB, TIP41, Histone H3 (HIS-3), elF, and ARP6 exhibited stable expression in different stages of rhizosheath development, whereas ALDH and protein phosphatase 2A (PP2A) showed relative unsteady expression
Summary
Abbreviations ACT Actin ALDH Aldehyde dehydrogenase ARP Actin related protein Cq Quantification cycle CV Coefficient of variation CYP Cyclophilin DPG Day post germination EF-1α Elongation factor-1α elF Eukaryotic translation initiation factor FPKM Fragments per Kilobase of exon model per Million mapped reads GAPDH Glyceraldehyde-3-phosphate dehydrogenase HIS-3 Histone H3 PEG Polyethylene glycol PGK Phosphoglycerate kinase gene PP2A Protein phosphotase 2A RNA-Seq RNA sequencing RT-qPCR Reverse transcription quantitative polymerase chain reaction SAMSC S-Adenosylmethionine synthase SD Standard deviation. The region is subject to land and wind erosion and is characterized by shifting sand dunes, which seriously hampers normal agricultural production and human development Plants growing in this area have adapted to the extreme environment. Stipagrostis pennata is a desert plant distributed in the Gurbantünggüt Desert in Xinjiang that mainly grows on mobile and semi-mobile sand dunes, having important roles in wind prevention, sand fixation, and the protection of the desert e cosystem[1] This plant has a typical rhizosheath structure around the roots, which allows the roots to resist the unfavorable external conditions of the arid desert environment and endows the plant with sand fixation a bility[2]. Based on a comprehensive evaluation of their ranking, the genes of GAPDH and eukaryotic translation initiation factor (elF) under drought stress, actin related protein (ARP6) and aldehyde dehydrogenase (ALDH) in the plant tissues, elF and GAPDH in rhizosheath development, as well as ARP6 and elF in all samples, were identified as the most stable reference genes. Our results provide reliable reference genes for RT-qPCR and further genetic function studies in S. pennata
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