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.

Keywords: Cytochrome P450 gene; Hylocereus; Identification; Normalization; Reference genes; qRT-PCR.

Fig. 1

Ct values of six candidate reference genes in different fruit developmental stages and tissues of pitayas. Data represent the average values from three biological replicates (± SD)

Fig. 2

Average expression stability values (M) of the six candidate reference genes using geNorm algorithm. Total represents a comparison between all tissues/fruits between the two cultivars

Fig. 3

Pairwise variation (V) analyses of the six candidate reference genes using geNorm algorithm. Total represents a comparison between all tissues/fruits between the two cultivars

Fig. 4

Expression stability values and ranking of the six candidate reference genes using NormFinder algorithm. Total represents a comparison between all tissues/fruits between the two cultivars

Fig. 5

Expression stability values and ranking of the six candidate reference genes using BestKeeper algorithm. Total represents a comparison between all tissues/fruits between the two cultivars

Fig. 6

Relative expression of cyt P450-like1 in Guanhuahong and Guanhuabai pitayas. Actin(1), EF1-α(1) and Actin(1) + EF1-α(1) were used as one or two most stable reference genes, UBC(2) was used as the least stable reference gene. Different letters indicate significant difference of the expression of the target gene based on three biological replications (P < 0.05, t test; n = 3)