Identification and Validation of Reference Genes for RT-qPCR Analysis in Switchgrass under Heavy Metal Stresses

Abstract

Switchgrass (Panicum Virgatum L.) has been recognized as the new energy plant, which makes it ideal for the development of phytoremediation on heavy metal contamination in soils with great potential. This study aimed to screen the best internal reference genes for the real-time quantitative PCR (RT-qPCR) in leaves and roots of switchgrass for investigating its response to various heavy metals, such as cadmium (Cd), lead (Pb), mercury (Hg), chromium (Cr), and arsenic (As). The stability of fourteen candidate reference genes was evaluated by BestKeeper, GeNorm, NormFinder, and RefFinder software. Our results identified U2AF as the best reference gene in Cd, Hg, Cr, and As treated leaves as well as in Hg, Pb, As, and Cr stressed root tissues. In Pb treated leaf tissues, 18S rRNA was demonstrated to be the best reference gene. CYP5 was determined to be the optimal reference gene in Cd treated root tissues. The least stable reference gene was identified to be CYP2 in all tested samples except for root tissues stressed by Pb. To further validate the initial screening results, we used the different sets of combinatory internal reference genes to analyze the expression of two metal transport associated genes (PvZIP4 and PvPDB8) in young leaves and roots of switchgrass. Our results demonstrated that the relative expression of the target genes consistently changed during the treatment when CYP5/UBQ1, U2AF/ACT12, eEF1a/U2AF, or 18S rRNA/ACT12 were combined as the internal reference genes. However, the time-dependent change pattern of the target genes was significantly altered when CYP2 was used as the internal reference gene. Therefore, the selection of the internal reference genes appropriate for specific experimental conditions is critical to ensure the accuracy and reliability of RT-qPCR. Our findings established a solid foundation to further study the gene regulatory network of switchgrass in response to heavy metal stress.

Validation of Reference Genes for Studying Different Abiotic Stresses in Oat (Avena sativa L.) by RT-qPCR

“…ACT1 showed high stability in leaves of both investigated oat cultivars. In agreement with this, other members of the Actin gene family showed high expression stabilities under Cd exposure, ACT12 in switchgrass (Panicum virgatum L.) [31] and ACT3 in soybean (Glycine max L.) [35]. PGD was a stable reference gene in the roots of both oat genotypes, however, in soybean its expression was greatly influenced by Cd stress [35].…”

Validation of Reference Genes for Studying Different Abiotic Stresses by RT-qPCR in Oat (Avena Sativa L.)

“…That was especially true for TBPII in drought stressed roots of Mv Hópehely, and for PGD in cold stressed leaves of both cultivars, respectively. The difference between the ranking order of Bestkeeper and the rest of the softwares is also mentioned by other studies [22][23][24]. The stability of the reference genes were in uenced by all the experimental conditions, such as tissue type, genotype and treatment type, but the applied stress treatments had the most pronounced effect on stability in general.…”

Transcriptional, metabolic, physiological and developmental responses of switchgrass to phosphorus limitation

“…Selected transcriptional changes observed by RNA Seq analysis were validated by real‐time quantitative PCR (RT‐qPCR) analysis. Three housekeeping genes (Pavir.9NG844500, UBQ1 ; Pavir.9NG259500, UBQ6 ; Pavir.8NG010400, CYP5 ) were used as references (Gimeno, Eattock, Van Deynze, & Blumwald, 2014; Zhao, Zhou, & Meng, 2020). Primers utilized are listed in Table S2d.…”

“…Pavir.9NG259500, UBQ6; Pavir.8NG010400, CYP5) were used as references (Gimeno, Eattock, Van Deynze, & Blumwald, 2014;Zhao, Zhou, & Meng, 2020). Primers utilized are listed in Table S2d.…”