Identification of reliable reference genes for quantitative real-time PCR normalization in pitaya

Abstract

Background A suitable reference gene is an important prerequisite for guarantying accurate and reliable results in quantitative real-time PCR (qRT-PCR) analyses. However, there is no absolute universality in reference genes among different species. It’s hard to find an ideal reference gene to fit for different tissues and growth periods. Pitaya ( Hylocereus ) is commercially produced as a new fruit crop at a large scale in tropical and subtropical regions. To date, there is no report on the identification of the most reliable reference genes for qRT-PCR normalization in pitaya. Results In this study, six candidate reference genes i.e. Actin(1) , GAPDH , UBC(1) , UBC(2) EF1 - α(1) and histone(1) were selected from thirty-nine typical candidate reference genes to determine the most stable reference genes for qRT-PCR normalization in different tissues, temperature stresses and fruit developmental stages of pitaya. Among the six candidate reference genes, Actin(1) and EF1 - α(1) were the most stable gene according to calculations of three statistical methods (GeNorm, NormFinder and BestKeeper) while UBC(1) and UBC(2) showed the lowest expression stability. The six candidate reference genes were further validated by comparing expression profiles of key genes related to betalain biosynthesis at flesh coloration stages of Guanhuahong ( Hylocereus monacanthus ) and Guanhuabai ( H. undatus ) pitayas. Actin(1) was recommended the best reference gene for accurate normalization of qRT-PCR data. Conclusions In this study, the stability of the selected reference genes for normalizing the qRT-PCR data were identified from pitaya. Actin(1) was the most stably expressed genes in different tissues and fruit developmental stages in pitaya. The present work provides the first data of reference gene identification for pitaya and will facilitate further studies in molecular biology and gene function on Hylocereus and other closely related species. Electronic supplementary material The online version of this article (10.1186/s13007-019-0455-3) contains supplementary material, which is available to authorized users.

Integrated sRNAome and RNA-Seq analysis reveals miRNA effects on betalain biosynthesis in pitaya

“…All experiments were performed in triplicate. U6 and actin gene were used as reference genes [ 69 , 70 ]. The sequences of miRNAs and target genes were shown in Tables S2 and S10 , respectively.…”

Integrated sRNAome and RNA-Seq Analysis Reveals miRNA Effects on Betalain Biosynthesis in Pitaya

“…All experiments were performed in triplicate. U6 and actin gene were used as reference genes [69,70]. The sequences of miRNAs and target genes were shown in Table S2 and S10, respectively.…”

Selection and validation of reference genes for quantitative real-time PCR analysis of Nitraria tangutorum

“…In general, some housekeeping genes are used as reference genes, such as ACT, EF1-α, and TUB, which are expressed in various types of cells in an organism, and the expression level is less affected by environmental factors. However, the stability of their expression changes greatly in different species, under different abiotic stresses, and in different tissues [34,16,21]. In other words, the selection of suitable reference genes under specific experimental conditions has become an important prerequisite for the qRT-PCR process.…”

“…The quantitative primers of the candidate reference genes were designed by the Primer3Plus online website (www.primer3plus.com/cgi-bin/dev/primer3plus.cgi). The parameters were as follows: the length of the primers and the amplification product size were [18][19][20][21][22][23][24][25][26][27] bp and 100-200 bp, respectively, the range of melting temperature (Tm) was 65°C-75°C, and the GC was 40%-60%. Primers were synthesized by Sangon Biotech.…”

“…it has become important to study their molecular genetic mechanism, and to determine their appropriate reference genes under different experimental conditions. To date, some non-model plants have been selected for internal reference genes, including Reaumuria [16], Caragana intermedia [17], Setaria viridis [18], Miscanthus lutarioriparia [19], Haloxylon ammodendron [20], pitaya [21], and jute [22].…”