Selection and validation of reference genes of Paeonia lactiflora in growth development and light stress

Abstract

The stem of Paeonia lactiflora will bend when it grows in greenhouse at a low light intensity. It is important to explore causes of morphological changes of peony to improve its quality. Gene expression can be evaluated by quantitative real-time PCR, based on reference gene. However, systematic selection of reference genes under weak lighting for herbaceous peony is lacking. To address this problem, we first selected 10 candidate reference genes based on a coefficient of variation of gene expression from peony stem transcriptome data. Then, geNorm, NormFinder and BestKeeper were applied to assess the stability of the genes, and RankAggreg was used to give a comprehensive ranking. The results show that there are some differences in optimal reference genes among samples from different organs and under the two lighting conditions, and the optimal number of suitable reference genes is distinct. Two selected suitable reference genes were then used to normalize target genes, and the results were compared with transcriptome data. Consistent gene expression trends were obtained, indicating the reliability of the method. To the best of our knowledge, this is the first time reference genes for herbaceous peony were selected in different organs, developmental stages and under two kinds of lighting conditions. The findings can provide a practical method for selecting reference genes for peony under these conditions and demonstrate a useful combination of reference genes. Keywords Herbaceous peony Á Reference gene Á Light stress Á F-box Á GAPDH Abbreviations DFG Paeonia lactiflora 'Da Fugui' CTH Paeonia lactiflora 'Chui Touhong' qPCR Quantitative real-time PCR CV Coefficient of variation F-box F-box protein GAPDH Glyceraldehyde-3-phosphate dehydrogenase UBQ10 Polyubiquitin CYP Cyclophilin ACT Actin/actin-like conserved site-containing protein EF-a Elongation factor-1 alpha 3 AQU Probable aquaporin PIP1-2 ETI Eukaryotic translation initiation factor 5A-2 E3 E3 ubiquitin protein ligase RIE1 pp2A Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A beta isoform-like Tm The melting temperature Cq Quantification cycle Electronic supplementary material The online version of this article (

Selection of Stable Reference Genes for Quantitative Real-Time PCR on Paeonia ostii T. Hong et J. X. Zhang Leaves Exposed to Different Drought Stress Conditions

“…This indicates that it is essential to choose reference genes in different cultivars, even though they pertain to the same species. Some scholars screened the reference genes under different abiotic stresses such as plant hormone stress [55], light stress [23], cold or hightemperature stress [56,57], and salt stress [58], based on the special experimental needs to solve specific scientific problems. Similarly, due to the high industrial values of P. ostii, research on P. ostii drought stress and its mitigation methods is also very significant.…”

“…In herbaceous peony (Paeonia lactiflora Pall.) under light stress, ETI (eukaryotic translation initiation factor 5A-2) was recommended to be the most stable gene in the flowers, and ACT and pp2A (serine/threonine protein phosphatase 2A) showed the least expression variations in stems [23]. Therefore, it is very important to choose relatively stable housekeeping genes according to specific experimental conditions, especially under specific abiotic stresses, before their use in qRT-PCR normalization.…”

Characteristics of Microsatellites Mined from Transcriptome Data and the Development of Novel Markers in Paeonia lactiflora

“…All the SSR sequences used in this study were obtained from transcriptome data from inflorescence stems of P. lactiflora 'Da Fugui' and 'Chui Touhong' at five developmental stages (i.e., stages representing every seven days from stem elongation to flowering). The transcriptome data have been deposited in the Sequence Read Archive (SRA) database as described previously (accession number: PRJNA528693) [26,27] and were assembled by Trinity 3.0.…”

Identification and Validation of Reference Genes for qRT-PCR Analysis of Petal-Color-Related Genes in Rosa praelucens

“…In addition, the most popular reference genes are the “housekeeping genes”, including actin ( ACT ), tubulin ( TUBA and TUBB ), eukaryotic translation elongation factor 1-α ( EEF1A ), ubiquitin ( UBQ ), and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ). However, more and more studies have indicated that many of these genes are inappropriate for qRT-PCR analysis, as their expression could probably be altered by developmental and environmental factors [ 26 , 27 , 28 ]. It is widely accepted that reference genes are specific to experimental situations and even the ploidy levels [ 29 ], and suitable reference genes have to be experimentally detected for each species and for each certain experiment [ 28 , 30 , 31 ].…”