Reference Genes for qPCR Analysis in Resin-Tapped Adult Slash Pine As a Tool to Address the Molecular Basis of Commercial Resinosis

Abstract

Pine oleoresin is a major source of terpenes, consisting of turpentine (mono- and sesquiterpenes) and rosin (diterpenes) fractions. Higher oleoresin yields are of economic interest, since oleoresin derivatives make up a valuable source of materials for chemical industries. Oleoresin can be extracted from living trees, often by the bark streak method, in which bark removal is done periodically, followed by application of stimulant paste containing sulfuric acid and other chemicals on the freshly wounded exposed surface. To better understand the molecular basis of chemically-stimulated and wound induced oleoresin production, we evaluated the stability of 11 putative reference genes for the purpose of normalization in studying Pinus elliottii gene expression during oleoresinosis. Samples for RNA extraction were collected from field-grown adult trees under tapping operations using stimulant pastes with different compositions and at various time points after paste application. Statistical methods established by geNorm, NormFinder, and BestKeeper softwares were consistent in pointing as adequate reference genes HISTO3 and UBI. To confirm expression stability of the candidate reference genes, expression profiles of putative P. elliottii orthologs of resin biosynthesis-related genes encoding Pinus contorta β-pinene synthase [PcTPS-(−)β-pin1], P. contorta levopimaradiene/abietadiene synthase (PcLAS1), Pinus taeda α-pinene synthase [PtTPS-(+)αpin], and P. taeda α-farnesene synthase (PtαFS) were examined following stimulant paste application. Increased oleoresin yields observed in stimulated treatments using phytohormone-based pastes were consistent with higher expression of pinene synthases. Overall, the expression of all genes examined matched the expected profiles of oleoresin-related transcript changes reported for previously examined conifers.

Validation of Reference Genes for Quantitative Real-Time PCR during Bicolor Tepal Development in Asiatic Hybrid Lilies (Lilium spp.)

“…However, when different tissues and total samples were considered, all V n /V n + 1 values were still above 0.15 ( Figures 5C,D ), and thus the cut-off value of 0.15 was somewhat strict in these cases. Many studies have shown that the use of 0.15 as a cut-off value is just a recommendation, and whether 0.15 is actually used is dependent on the data ( Silveira et al, 2009 ; Fernandez et al, 2011 ; Lu et al, 2013 ; De Lima et al, 2016 ). Generally, a reliable result could be obtained by using three reference genes in the majority of experiments ( Toegel et al, 2007 ; Maroufi et al, 2010 ; Wang et al, 2014 ; Wang and Lu, 2015 ; Li M.Y.…”

A transcriptome-based association study of growth, wood quality, and oleoresin traits in a slash pine breeding population

“…The cDNA was amplified according to the Takara rr820a kit (Takara, Shiga, Japan), and the expression levels of the genes were calculated using the 2 -ΔΔCt method [39]. The UBI [40] gene was used to normalize the transcript profiles. ).…”

Assessment of Appropriate Reference Genes for Quantitative Real-Time Polymerase Chain Reaction Normalisation in <i>Magnolia sieboldii</i> K. Koch across Various Experimental Conditions

“…Hence, the threshold of 0.15 was slightly rigorous in these experimental conditions. Numerous studies have indicated that the application of a threshold of 0.15 is merely a suggestion, and whether it is practically applied is determined by the data [52][53][54].…”