Identification of Reference Genes for Quantitative Gene Expression Studies in Pinus massoniana and Its Introgression Hybrid

Abstract

qRT-PCR is a powerful molecular research tool to study the regulation of gene expression. However, to accurately calculate gene expression levels, an experiment should include proper reference genes that show no changes in their expression level. Pinus massoniana, P. hwangshanensis, and their introgression hybrid in Mountain Lushan, China, are an ideal model for studying introgression and speciation. Although some research on reference gene selection for P. massoniana has been reported before, no studies on this subject have been performed where P. massoniana and its introgression hybrid were evaluated simultaneously. Here, we investigated ten genes (upLOC, SDH, ACT, EF, TOC75, DMWD, FBOX, PGK1, UBQ, and CL2417C7) identified from transcriptome data of these two taxa for reference gene potential. These ten genes were then screened across multiple tissues such as cone, young and mature stems, and young needles according to qRT-PCR thermal cycling and dissociation. Correlation coefficient, amplification efficiency, and cycle threshold value (Ct) range were applied to evaluate the reliability of each gene. The stability of candidate reference gene expression was calculated using three algorithms: geNorm, NormFinder, and BestKeeper. Base on the reliability and stability, we then offered a list of genes of recommended and not recommended for seven different tissue type and species. Our results demonstrated that different sample lines require different genes as reference to evaluate.

Diterpene Resin Acids and Olefins in Calabrian Pine (Pinus nigra subsp. laricio (Poiret) Maire) Oleoresin: GC-MS Profiling of Major Diterpenoids in Different Plant Organs, Molecular Identification and Expression Analysis of Diterpene Synthase Genes

“…As for the reference genes for expression analysis, we looked at those showing stable expression in different pine tissues in the presence of stress conditions of different origin [48,49]. The reference genes selected encode the following proteins: Actin 1 (ACT1, NCBI accession no KM496527), Cyclophilin (CYP, KM496534), Tubulin alpha (TUBα, KM496535), Polyubiquitin 4 (UBI4, KM496539), and uncharacterized protein LOC103705956 (upLOC, MN172175).…”

Selection and Validation of Candidate Reference Genes for Gene Expression Analysis by RT-qPCR in Rubus

“…In the study of gene expression analysis, using a single internal reference gene for correction and standardization sometimes cannot meet the experimental requirements and ensure the accuracy of the experimental results. Therefore, to reduce various errors in the experiment, it is necessary to introduce two or more internal reference genes for standardized correction [ 12 ]. Through comprehensive evaluation combined with validation analysis, Ru EEF1A and Ru 18S were identified as relatively suitable reference genes.…”

“…When RT-qPCR is used to calculate the relative expression level of target genes (data standardization processing), it is necessary to combine relatively stable reference genes for correction and homogenization to improve the accuracy of quantitative results [ 8 , 10 ]. The commonly used reference genes are usually stable housekeeping genes, which are expressed in all kinds of cells and code for proteins necessary to maintain the basic biological activities of cells, for example, the 18S gene, the most abundant ribosomal RNA in eukaryotic organisms, EEF1A and EEF1B proteins of the elongation factor 1 ( EEF1 ) gene family in eukaryotes, polyubiquitin ( UBC ), ubiquitin ( UBQ ), F-box family protein ( F-box ) and actin ( ACT ) [ 11 , 12 ]. Ideal reference genes require relatively constant expression under different treatments, in different tissues or organs, and at different developmental stages of cells.…”

The Key Role of Plant Hormone Signaling Transduction and Flavonoid Biosynthesis Pathways in the Response of Chinese Pine (Pinus tabuliformis) to Feeding Stimulation by Pine Caterpillar (Dendrolimus tabulaeformis)

“…The SYBR Green reaction system was used for PCR reactions, with 18sRNA [ 95 ] and actin [ 96 ] used as internal references. The relative gene expression was calculated based on the 2 −∆∆CT method.…”