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

Jiaxing Mo,Tongming Yin,Wenjing Jin,Jisen Shi,Jin Xu,Liwei Yang

doi:10.3390/f10090787

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.

Highlights

Masson pine (Pinus massoniana) is an economically important conifer species that is naturally distributed in southern China
Ten candidate reference genes were tested in different P. massoniana and Z pine tissues
Considering and ACTefficiency showing the highest3b), stability (Figure relatively UBQ, high DMWD, amplification (Figure which may cause uncertainty in normalization, Analyzing the combined data from all P. massoniana tissues (Figure 5e), we found that uncharacterized protein LOC103705956 (upLOC)

Summary

Introduction

Masson pine (Pinus massoniana) is an economically important conifer species that is naturally distributed in southern China. Its wood is often used for standing timber and furniture manufacturing. A secretion from its canals, can be used for the maintenance of musical instrument strings, and is an ingredient in several medicines [1,2]. Because of its fast growth and high biomass, it has been planted extensively in south China during the past fifty years for rapid vegetation recovery and additional ecological purposes. Huangshan pine (P. hwangshanensis) is a species native only to China. It grows at a considerably slower rate than P. massoniana, it has ornamental value as a horticultural plant in higher altitude regions

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