Selection of reference genes for qPCR normalization in buffalobur (Solanum rostratum Dunal)

Dandan Zhao,Chaoxian Zhang,Shouhui Wei,Hongjuan Huang,Cuilan Jiang,Jingchao Chen,Xu Wang,Zhaofeng Huang,Heqiang Huo

doi:10.1038/s41598-019-43438-6

Abstract

Buffalobur (Solanum rostratum Dunal), which belongs to the Solanaceae family, is a worldwide noxious invasive weed and is listed as one of the top 10 alien invasive species in China. It is harmful to humans and livestock because the entire plant is covered with spines containing toxins. Many studies have analysed the gene expression in this weed species under different stress conditions using quantitative real-time PCR (qPCR). However, until now, there has been no report on suitable reference genes in buffalobur. Herein, 14 candidate reference genes were selected and evaluated for their expression stability in buffalobur in different tissues, at different developmental stages, and in response to several stress conditions using the geNorm, NormFinder, BestKeeper and RefFinder statistical algorithms. The results showed that EF1α, ACT and SAND are suitable reference genes across all samples tested. We recommend the normalization of target gene expression under different experimental conditions using these three genes together. Validation of selected reference genes was achieved by assessing the relative expression levels of P5CS and GI. This work identified the appropriate reference genes for transcript normalization in buffalobur, which will be helpful in future genetic studies of this invasive weed.

Highlights

Due to its advantages of high sensitivity and specificity, quantitative real-time Polymerase chain reaction (PCR) (qPCR) has been widely used to quantify gene expression to discover the genetic basis of physiological patterns during the plant life cycle[4]
According to the Cq values, we can make a preliminary judgement that TIP41 and RUBP might not be suitable candidate reference genes
Three software packages and one web tool were used to test the statistical reliability of candidate reference genes

Summary

Introduction

Due to its advantages of high sensitivity and specificity, qPCR has been widely used to quantify gene expression to discover the genetic basis of physiological patterns during the plant life cycle[4]. The expression level of the appropriate internal control gene should remain relatively constant and should not change significantly across experimental conditions, types of tissues, developmental stages or stress treatments[6,7]; in practice, no gene exhibits fully stable expression throughout all growth stages and experimental conditions. It has been suggested that multiple reference genes can achieve accurate normalization[8]. There is general agreement that the expression stability of candidate genes should be validated prior to initiating normalization studies using qPCR in a particular species. The expression stabilities of these genes were tested with respect to different developmental periods, tissues, abiotic stresses, and hormone stimuli and with glyphosate treatment using geNorm[8], NormFinder[14], BestKeeper[15] and RefFinder[16] to identify the most stable gene for qPCR normalization in buffalobur. Tissuesb geNorm Gene eIF SAND UBQ EF1α ACT PP2Acs GR CYP GAPDH RPL8 TUB TIP41 RUBP DNAJ eIF SAND CYP EF1α RPL8 GAPDH PP2Acs UBQ ACT GR TUB TIP41 DNAJ RUBP

Methods

Results

Conclusion