Identification and Evaluation of Reference Genes for Accurate Transcription Normalization in Safflower under Different Experimental Conditions

Abstract

Safflower (Carthamus tinctorius L.) has received a significant amount of attention as a medicinal plant and oilseed crop. Gene expression studies provide a theoretical molecular biology foundation for improving new traits and developing new cultivars. Real-time quantitative PCR (RT-qPCR) has become a crucial approach for gene expression analysis. In addition, appropriate reference genes (RGs) are essential for accurate and rapid relative quantification analysis of gene expression. In this study, fifteen candidate RGs involved in multiple metabolic pathways of plants were finally selected and validated under different experimental treatments, at different seed development stages and in different cultivars and tissues for real-time PCR experiments. These genes were ABCS, 60SRPL10, RANBP1, UBCL, MFC, UBCE2, EIF5A, COA, EF1-β, EF1, GAPDH, ATPS, MBF1, GTPB and GST. The suitability evaluation was executed by the geNorm and NormFinder programs. Overall, EF1, UBCE2, EIF5A, ATPS and 60SRPL10 were the most stable genes, and MBF1, as well as MFC, were the most unstable genes by geNorm and NormFinder software in all experimental samples. To verify the validation of RGs selected by the two programs, the expression analysis of 7 CtFAD2 genes in safflower seeds at different developmental stages under cold stress was executed using different RGs in RT-qPCR experiments for normalization. The results showed similar expression patterns when the most stable RGs selected by geNorm or NormFinder software were used. However, the differences were detected using the most unstable reference genes. The most stable combination of genes selected in this study will help to achieve more accurate and reliable results in a wide variety of samples in safflower.

Stability evaluation of reference genes for gene expression analysis by RT-qPCR in soybean under different conditions

“…During the past few years, there have been lots of reports published with the aim of identifying and evaluating suitable reference genes for expression analysis in different plant species under abiotic stress [ 11 , 13 , 16 , 19 , 23 , 24 ], at different developmental stages or tissues [ 1 , 3 , 12 , 21 , 22 ], after infection with fungi, virus or bacteria [ 3 , 13 , 17 , 18 , 20 , 24 , 25 ]. However, there is a lack of validated reference genes under soybean mosaic virus (SMV) infection and nitrogen stress.…”

Selection of reliable reference genes for RT-qPCR analysis during developmental stages and abiotic stress in Setaria viridis

“…Thus, it is necessary to validate the stability of the reference gene under determined experimental condition to ensure proper normalization and a robust RT-qPCR analysis 14 . Several studies have been published with the aim of identifying suitable reference genes for expression analysis under different stages of the plant life cycle 15 16 17 18 or under biotic and abiotic stresses 19 20 21 22 .…”

Identification And Evaluation of Reference Genes For Cynanchum Auriculatum Under Various Stress Conditions

“…Improper application of reference genes often leads to the misunderstanding expression of target gene 41 . qRT-PCR is a tool commonly used in the modern gene research eld 42 .…”