Assessment of gene copy number variation of Scots pine thaumatin-like protein gene using real-time PCR based methods

Abstract

The importance and impact of gene copy number variations (CNVs) as a source of polymorphism in the human and other genomes is being increasingly recognized. Less information is available about CNVs in forest tree species, mainly due to the relative lack of genomic resources. In this study, several methods—quantitative polymerase chain reaction, comparative high-resolution melting curve analysis (C-HRM), and digital polymerase chain reaction (dPCR)—were used to investigate CNV of the Scots pine thaumatin-like protein gene (PsTLP). The obtained results were supported by transcriptome analysis of a single Pinus sylvestris individual and publically available pine genome sequences. Although estimations of gene copy number (CN) varied, depending on the region of the PsTLP gene investigated and the endogenous control utilized, our results revealed the existence of copy number variations of the PsTLP gene between Scots pine individuals. Of 23 individuals analyzed, two had an increased calculated relative CN regardless of the analyzed gene region and endogenous control used, while several samples had increased copy numbers of regions of the PsTLP gene. C-HRM results were highly correlated with qPCR data (R 2 TLP3′ = 0.88; R 2 TLPc = 0.92), but interpretation of gene CN from C-HRM results proved to be difficult. The results from selected samples analyzed by digital PCR also were highly correlated with qPCR results (R 2 = 0.90).