Evaluation of qPCR reference genes in two genotypes of Populus for use in photoperiod and low-temperature studies

Abstract

BackgroundQuantitative 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.ResultsWe 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, geNormPLUS and BestKeeper. GeNormPLUS 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.ConclusionsThis 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.

Validation of Reference Genes for Gene Expression by Quantitative Real-Time RT-PCR in Stem Segments Spanning Primary to Secondary Growth in Populus tomentosa

“…Here PtrACT6 was selected for assessment of the stable reference genes across the stem segments with different developmental stages, since this gene had comparable expression in bark, phloem, cambium, and xylem. The 18S rRNA transcripts were much more abundant than the others, as indicated by the lowest Cq value with 100 times dilution of cDNA template in this experiment and supported by other reports [ 33 , 34 , 38 ]. Moreover, the rRNA fraction in total RNA may quite differ from sample to sample [ 38 ].…”

“…In given species and tissues under specific experimental, it is crucial to validate the expression stability of reference genes for an accurate analysis of RNA transcription level by qRT-PCR. In Populus plants, identification of reliable reference genes has been carried out for qRT-PCR normalization in adventitious rooting of hardwood cuttings [ 3 ], in different tissues under long-day, short-day, or short-day plus low-temperature conditions and in different abiotic stress treatments [ 33 , 34 ]. Populus is a woody perennial plant, which can undergo considerable secondary growth and form more wood each growing season.…”

Characterisation of the willow phenylalanine ammonia-lyase (PAL) gene family reveals expression differences compared with poplar

“…PCR conditions were: 2 min at 50 °C and 2 min at 95 °C; 40 cycles of 3 s 95 °C, and 30 s 60 °C followed by a melt curve stage: 15 s at 95 °C, 1 min at 60 °C, 0.11 °C/s increase to 95 °C; 15 s 60 °C. Relative transcript levels were calculated by the comparative threshold cycle method ( Schmittgen and Livak, 2008 ) with the willow orthologue ( SvTIP4-like ) of the previously used ( Pettengill et al, 2012 ) poplar TIP4-like gene (Potri.009G093200) used as a reference. Expression levels were calculated as the means of two biological replicates, each with three technical replicates ± SD.…”

Identification and Analysis of microRNAs in the SAM and Leaves of Populus tomentosa

“…The total RNA was reverse transcribed into cDNA using the PrimeScript RT Reagent Kit (TaKaRa, Dalian, China), and then diluted 10 times with sterile water. The primers of the targets are shown in Table S1, and the reference gene, Actin 2 (ACT2), was selected according to a previous study [33]. qRT-PCR reactions contained 8.…”