How to perform RT-qPCR accurately in plant species? A case study on flower colour gene expression in an azalea (Rhododendron simsii hybrids) mapping population

Abstract

BackgroundFlower colour variation is one of the most crucial selection criteria in the breeding of a flowering pot plant, as is also the case for azalea (Rhododendron simsii hybrids). Flavonoid biosynthesis was studied intensively in several species. In azalea, flower colour can be described by means of a 3-gene model. However, this model does not clarify pink-coloration. The last decade gene expression studies have been implemented widely for studying flower colour. However, the methods used were often only semi-quantitative or quantification was not done according to the MIQE-guidelines. We aimed to develop an accurate protocol for RT-qPCR and to validate the protocol to study flower colour in an azalea mapping population.ResultsAn accurate RT-qPCR protocol had to be established. RNA quality was evaluated in a combined approach by means of different techniques e.g. SPUD-assay and Experion-analysis. We demonstrated the importance of testing noRT-samples for all genes under study to detect contaminating DNA. In spite of the limited sequence information available, we prepared a set of 11 reference genes which was validated in flower petals; a combination of three reference genes was most optimal. Finally we also used plasmids for the construction of standard curves. This allowed us to calculate gene-specific PCR efficiencies for every gene to assure an accurate quantification. The validity of the protocol was demonstrated by means of the study of six genes of the flavonoid biosynthesis pathway. No correlations were found between flower colour and the individual expression profiles. However, the combination of early pathway genes (CHS, F3H, F3'H and FLS) is clearly related to co-pigmentation with flavonols. The late pathway genes DFR and ANS are to a minor extent involved in differentiating between coloured and white flowers. Concerning pink coloration, we could demonstrate that the lower intensity in this type of flowers is correlated to the expression of F3'H.ConclusionsCurrently in plant research, validated and qualitative RT-qPCR protocols are still rare. The protocol in this study can be implemented on all plant species to assure accurate quantification of gene expression. We have been able to correlate flower colour to the combined regulation of structural genes, both in the early and late branch of the pathway. This allowed us to differentiate between flower colours in a broader genetic background as was done so far in flower colour studies. These data will now be used for eQTL mapping to comprehend even more the regulation of this pathway.

The State of RT-Quantitative PCR: Firsthand Observations of Implementation of Minimum Information for the Publication of Quantitative Real-Time PCR Experiments (MIQE)

“…MIQE guidelines were the community's first attempt to map out the methodology and key validation criteria required to perform qPCR experiments. Since their publication, a number of papers have supported the need for a consistent and rigorous methodology to ensure the publication of accurate results [De Keyser et al, 2013;Dooms et al, 2013;Lanoix et al, 2012;Taylor et al, 2010Taylor et al, , 2011.…”

Keeping qRT-PCR rigorous and biologically relevant

“…De Keyser et al (2013) and Saha and Blumwald (2014) proved that qRT-PCR data can be rigorously conducted and reported without inclusion of a MIQE checklist. However, we do advocate that the items on the checklist be addressed during experimental design and execution and recommend that a checklist be made available to referees during the review process.…”

“…A publication that does not provide a MIQE checklist but is nevertheless clearly inspired by the MIQE guidelines is the work of de Keyser et al (2013). It focuses on the flavonoid pigment biosynthetic pathway in azalea (Rhododendron simsii hybrids) and illustrates methods for checking quantity, purity, and integrity of mRNA; primer design; estimating qPCR efficiency; and selecting, validating, and optimizing the number of reference genes.…”

“…For plant species that lack these resources, other approaches can be effective. In the case of azalea, de Keyser et al (2013) found an azalea EST library to be adequate. Saha and Blumwald (2014) found that foxtail millet formed a bioinformatic bridge between pearl millet and rice, enabling homologues of reference genes validated in rice to be found in foxtail millet and then in pearl millet, where validation was confirmed.…”

Hormonal and Genetic Regulation of Axillary Bud Outgrowth in Chrysanthemum Morifolium During Floral Initiation

“…For gene expression analysis, RNA was extracted using a modified CTAB protocol (Luypaert et al, unpublished) and was converted to cDNA using the iScript cDNA Synthesis kit (BioRad). Expression of both branching genes was quantified using RT-qPCR according to De Keyser et al (2013). Qbase + software (Biogazelle) was used for all data analysis.…”