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.

Keywords: Ping’ou hybrid hazelnut (C. heterophylla Fisch. × C. avellana L.); real-time quantitative PCR; reference gene; self-incompatibility; stability of gene expression.

Figure 1

Agarose electrophoresis of 12 candidate reference gene RT-PCR products amplified from different samples. St, blooming styles; Ck, catkins before elongation; Lv, young leaves; Pn, pollen; Ca, cambium of annual branch; Se, green stem; Ro, root tip; Su, sucker.

Figure 2

Melt curve analyses of RT-qPCR products for 12 candidate reference genes.

Figure 3

Cq values of candidate reference genes in 24 samples. Boxes indicate the interquartile range. Lines across the boxes depict the medians. Small white boxes within colored boxes indicate mean values. Whiskers represent the 95th and 5th percentiles. The × symbols denote minimum and maximum values.

Figure 4

Expression stability (M) of twelve reference genes across different sample groups calculated using geNorm. M values of reference genes (A) in 24 sample groups, (B) in different tissues, (C) at different times after pollination, (D) in styles at 4 h after cross/self-pollination, (E) at different stages of flowering, and (F) in male catkins at different stages of elongation.

Figure 5

Determination of the optimal number of reference genes for different sample groups using geNorm. (A–F) represent different sample groups. (A) in 24 sample groups, (B) in different tissues, (C) at different times after pollination, (D) in styles at 4 h aftecross/self-pollination, (E) at different stages of flowering, and (F) in male catkins at different stages of elongation.

Figure 6

Expression analysis of the target gene, CavPrx, using ChaActin and ChaEF1-α as reference genes. * Control samples. The expression level of CavPrx (A) in different tissues, (B) at different times after self-incompatible pollination, (C) at 4 h after different affinity pollination. StR, styles at red-dot stage; St, blooming styles; StE, styles at the end-flower stage; Ck, catkins before elongation; CkO, catkins during elongation; CkA, catkins after elongation; Lv, young leaves; Pn, pollen; Ca, cambium of annual branch; Se, green stem; Ro, root tip; Su, sucker; 0 h, self-pollination 0 h; 0.5 h, self-pollination 0.5 h; 1 h, self-pollination 1 h; 2 h, self-pollination 2 h; 4 h, self-pollination 4 h; 8 h, self-pollination 8 h; 24 h, self-pollination 24 h; CK/4 h, non-pollination 4 h; 254/4 h: incompatible pollination 4 h with 84–254; 84–69/4 h; incompatible pollination 4 h with 84–69; 226/4 h, compatible pollination 4 h with 84–226; 82–11/4 h, compatible pollination 4 h with 82–11.