Evaluation of qPCR reference genes for taimen (Hucho taimen) under heat stress

Xiaoxing Yang,Guopan Tang,Kai Ma,Ting Yan,Jiasheng Yin,Yongquan Zhang,Huan Xu,Youyi Kuang,Le Dong,Guangxiang Tong

doi:10.1038/s41598-021-03872-x

Abstract

As a powerful and attractive method for detecting gene expression, qRT-PCR has been broadly used in aquaculture research. Understanding the biology of taimen (Hucho taimen) has drawn increasing interest because of its ecological and economic value. Stable reference genes are required for the reliable quantification of gene expression, but such genes have not yet been optimized for taimen. In this study, the stability levels of 10 commonly used candidate reference genes were evaluated using geNorm, NormFinder, BestKeeper, and RefFinder. The expression levels of the 10 genes were detected using 240 samples from 48 experimental groups consisting of 40 individuals treated under four heat-stress conditions (18, 20, 22, and 24 °C) for 24 h and 26 °C for 4, 24, 48, and 72 h. Six tissues (blood, heart, brain, gill, skin, and liver) were collected from each individual. Ribosomal protein S29 (RPS29) and ribosomal protein L19 (RPL19) were the most stable genes among all of the samples, whereas 28S ribosomal RNA (28S rRNA), attachment region binding protein (ARBP), and 18S ribosomal RNA (18S rRNA) were the least stable. These results were verified by an expression analysis of taimen heat-stress genes (heat shock protein 60, hsp60, and heat shock protein 70, hsp70). In conclusion, RPS29 and RPL19 are the optimal reference genes for qRT-PCR analyses of taimen, irrespective of the tissue and experimental conditions. These results allow the reliable study of gene expression in taimen.

Highlights

Taimen (Hucho taimen), belonging to Salmonidae, is a cold freshwater carnivorous fish[1]
The results showed that a single band for each gene was detected, and no dimers or non-specific amplified bands occurred (Fig. 2), which indicated that the designed primers were appropriate for quantitative real-time PCR (qRT-PCR)
The qRT-PCR analysis using the fluorescent SYBR dye showed that the melting curves of all of the amplicons presented single distinct signal peaks, which indicated that the primers for the 10 reference genes were appropriate for quantifying their expression levels

Summary

Introduction

Taimen (Hucho taimen), belonging to Salmonidae, is a cold freshwater carnivorous fish[1]. Many microsatellite[6,7] markers, transcriptomes obtained by mRNA sequencing[8], high resolution linage maps[9], and genome sequences of huchen, a closely related fish (Hucho hucho, GCA_003317085.1 in GenBank) have been developed These are useful tools to characterize candidate genes related to high-temperature tolerance. Olsvik et al.[23] evaluated six reference genes, including 18S, S20 ribosomal protein (S20), β-actin, GAPDH, and two paralog genes encoding elongation factor 1A (EF1AA and EF1AB), in eight tissues (gill, liver, head kidney, spleen, thymus, brain, muscle, and posterior intestine) of Atlantic salmon (Salmo salar) cultured in normal or smoltification conditions, and they found that EF1AB was the best reference gene, whereas Jorgensen et al.[24] reported that the combination of 18S rRNA, EF1A, and RNA polymerase I (RPL1) was the best normalization method for qRT-PCR in immune-related organs despite viral infection. To validate the selected reference genes, the heat shock protein g enes[31] hsp[60] and hsp[70] were selected as targets to assess the performance of the selected reference genes in different conditions

Methods

Results

Conclusion