Selection and Validation of Reference Genes for Quantitative RT-PCR Analysis in Corylus heterophylla Fisch. x Corylus avellana L.

(1) Background: the species of Corylus have sporophytic type of self-incompatibility. Several genes related to recognition reaction between pollen and stigma have been identified in hazelnuts. To better understand the self-incompatibility (SI) response, we screened the suitable reference genes by using quantitative real-time reverse transcription PCR (qRT-PCR) analysis in hazelnut for the first time. (2) Methods: the major cultivar Dawei was used as material. A total of 12 candidate genes were identified and their expression profiles were compared among different tissues and in response to various treatments (different times after self- and cross-pollination) by RT-qPCR. The expression stability of these 12 candidate reference genes was evaluated using geNorm, NormFinder, BestKeeper, Delta Ct, and RefFinder programs. (3) Results: the comprehensive ranking of RefFinder indicated that ChaActin, VvActin, ChaUBQ14, and ChaEF1-alpha were the most suitable reference genes. According to the stability analysis of 12 candidate reference genes for each sample group based on four software packages, ChaActin and ChaEF1-alpha were most stable in different times after self-pollination and 4 h after self- and cross-pollination, respectively. To further validate the suitability of the reference genes identified in this study, CavPrx, which the expression profiles in Corylus have been reported, was quantified by using ChaActin and ChaEF1-alpha as reference genes. (4) Conclusions: our study of reference genes selection in hazelnut shows that the two reference genes, ChaActin and ChaEF1-alpha, are suitable for the evaluation of gene expression, and can be used for the analysis of pollen-pistil interaction in Corylus. The results supply a reliable foundation for accurate gene quantifications in Corylus species, which will facilitate the studies related to the reproductive biology in Corylus.

Automated summary

By using quantitative real-time reverse transcription PCR analysis, suitable reference genes were identified in hazelnut, with ChaActin and ChaEF1-alpha showing the most stability in self-incompatibility response. These reference genes can be used for accurate gene quantifications in Corylus species and facilitate studies related to reproductive biology in Corylus.