Background: Quantitative PCR (qPCR) is a widely used technique for gene expression analysis. A common
normalization method for accurate qPCR data analysis involves stable reference genes to determine relative gene
expression. Despite extensive research in the forest tree species Populus, there is not a resource for reference genes
that meet the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) standards for
qPCR techniques and analysis. Since Populus is a woody perennial species, studies of seasonal changes in gene
expression are important towards advancing knowledge of this important developmental and physiological trait.
The objective of this study was to evaluate reference gene expression stability in various tissues and growth
conditions in two important Populus genotypes (P. trichocarpa “Nisqually 1” and P. tremula x P. alba 717 1-B4)
following MIQE guidelines.
Results: We evaluated gene expression stability in shoot tips, young leaves, mature leaves and bark tissues from
P. trichocarpa and P. tremula. x P. alba grown under long-day (LD), short-day (SD) or SD plus low-temperatures
conditions. Gene expression data were analyzed for stable reference genes among 18S rRNA, ACT2, CDC2, CYC063,
TIP4-like, UBQ7, PT1 and ANT using two software packages, geNorm
PLUS
and BestKeeper. GeNorm
PLUS
ranked TIP4-like
and PT1 among the most stable genes in most genotype/tissue combinations while BestKeeper ranked CDC2 and
ACT2 among the most stable genes.
Conclusions: This is the first comprehensive evaluation of reference genes in two important Populus genotypes
and the only study in Populus that meets MIQE standards. Both analysis programs identified stable reference genes
in both genotypes and all tissues grown under different photoperiods. This set of reference genes was found to
be suitable for either genotype considered here and may potentially be suitable for other Populus species and
genotypes. These results provide a valuable resource for the Populus research community.https://doi.org/10.1186/1756-0500-5-36