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

Abstract

(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-α 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-α 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-α as reference genes. (4) Conclusions: our study of reference genes selection in hazelnut shows that the two reference genes, ChaActin and ChaEF1-α, 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.

Stigmatic Transcriptome Analysis of Self-Incompatible and Compatible Pollination in Corylus heterophylla Fisch. × Corylus avellana L.

“…Thermal cycler parameters were set at 95°C for 3 min, 95°C for 30 s, then 40 cycles of 95°C for 5 s and 50–60°C for 5 s. The qRT-PCR analysis with three biological replicates was conducted with the Bio-Rad CFX Manager version 3.0 software (Bio-Rad, Hercules, CA, United States). The relative expression level was calculated using the 2 –ΔΔCt method ( Livak and Schmittgen, 2001 ) with ChaActin and ChaEF1- α serving as the reference genes ( Hou et al, 2021 ).…”

Molecular cloning and yeast two‐hybrid provide new evidence for unique sporophytic self‐incompatibility system of Corylus

“…ChaActin and ChaEF1‐α were used as reference genes to normalize expression levels of target genes (Hou et al . 2021). All primers were designed based on the full‐length cDNA of five homologous genes (Table S3).…”

“…Total RNA (1.0 lg) for each sample was reverse-transcribed into cDNA with the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) for qRT-PCR analysis. ChaActin and ChaEF1a were used as reference genes to normalize expression levels of target genes (Hou et al 2021). All primers were designed based on the full-length cDNA of five homologous genes (Table S3).…”

Selection of Suitable Reference Genes for Gene Expression Normalization Studies in Dendrobium huoshanense

“…Reference genes frequently come from common housekeeping genes, such as glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) [ 27 ], actin ( ACT ) [ 28 ], α-tubulin ( α-TUB ) [ 29 ], ribosomal RNA ( 18S rRNA ) [ 30 ], and elongation factor 1α ( EF-1α ) [ 31 ]. They are usually used to calibrate qPCR results, so a stable reference gene is needed for qPCR.…”