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Open AccessArticle

Identification of Reference Genes for Quantitative Gene Expression Studies in Pinus massoniana and Its Introgression Hybrid

by 1,2,3, 1,2,3,*, 1,2,3, 1,2,3, 1,2,3 and 1,2,3
1
Key Laboratory of Forest Genetics & Biotechnology of Ministry of Education, Nanjing Forestry University, Nanjing 210037, China
2
Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China
3
College of Forestry, Nanjing Forestry University, Nanjing 210037, China
*
Author to whom correspondence should be addressed.
Forests 2019, 10(9), 787; https://doi.org/10.3390/f10090787
Received: 25 July 2019 / Revised: 5 September 2019 / Accepted: 8 September 2019 / Published: 11 September 2019
(This article belongs to the Special Issue Forest Genetic and Tree Improvement)
qRT-PCR is a powerful molecular research tool to study the regulation of gene expression. However, to accurately calculate gene expression levels, an experiment should include proper reference genes that show no changes in their expression level. Pinus massoniana, P. hwangshanensis, and their introgression hybrid in Mountain Lushan, China, are an ideal model for studying introgression and speciation. Although some research on reference gene selection for P. massoniana has been reported before, no studies on this subject have been performed where P. massoniana and its introgression hybrid were evaluated simultaneously. Here, we investigated ten genes (upLOC, SDH, ACT, EF, TOC75, DMWD, FBOX, PGK1, UBQ, and CL2417C7) identified from transcriptome data of these two taxa for reference gene potential. These ten genes were then screened across multiple tissues such as cone, young and mature stems, and young needles according to qRT-PCR thermal cycling and dissociation. Correlation coefficient, amplification efficiency, and cycle threshold value (Ct) range were applied to evaluate the reliability of each gene. The stability of candidate reference gene expression was calculated using three algorithms: geNorm, NormFinder, and BestKeeper. Base on the reliability and stability, we then offered a list of genes of recommended and not recommended for seven different tissue type and species. Our results demonstrated that different sample lines require different genes as reference to evaluate. View Full-Text
Keywords: Pinus massoniana; reference gene; qRT-PCR; introgression; cone Pinus massoniana; reference gene; qRT-PCR; introgression; cone
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Mo, J.; Xu, J.; Jin, W.; Yang, L.; Yin, T.; Shi, J. Identification of Reference Genes for Quantitative Gene Expression Studies in Pinus massoniana and Its Introgression Hybrid. Forests 2019, 10, 787.

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