TY - JOUR
T1 - Assessment of gene copy number variation of Scots pine thaumatin-like protein gene using real-time PCR based methods
AU - Šķipars, Vilnis
AU - Rauda, Elza
AU - Snepste, Ilze
AU - Krivmane, Baiba
AU - Rungis, Dainis
N1 - Funding Information:
Acknowledgments This study was funded by the Latvian Council of Science project BInvestigation of molecular defense mechanisms in Scots pine (Pinus sylvestris L.)^ (No. 284/2012). We would like to thank Carl Gunnar Fossdal and Adam Vivian-Smith from Norwegian Institute of Bioeconomy Research for digital PCR.
Publisher Copyright:
© 2017, Springer-Verlag GmbH Germany, part of Springer Nature.
PY - 2017/12/1
Y1 - 2017/12/1
N2 - 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 (R2 TLP3′ = 0.88; R2 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 (R2 = 0.90).
AB - 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 (R2 TLP3′ = 0.88; R2 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 (R2 = 0.90).
KW - Comparative high resolution melting curve analysis
KW - Copy number variation
KW - Heterobasidion annosum
KW - Pinus sylvestris L
KW - qPCR
KW - Thaumatin-like protein
UR - http://www.scopus.com/inward/record.url?scp=85035128702&partnerID=8YFLogxK
U2 - 10.1007/s11295-017-1209-x
DO - 10.1007/s11295-017-1209-x
M3 - Article
AN - SCOPUS:85035128702
SN - 1614-2942
VL - 13
JO - Tree Genetics and Genomes
JF - Tree Genetics and Genomes
IS - 6
M1 - 127
ER -