Reference genes validation in Phenacoccus solenopsis under various biotic and abiotic stress conditions

Abstract

Real-time PCR (RT-qPCR) expression analysis is a powerful analytical technique, but for normalization of data requires the use of stable reference genes. However, suitable reference genes are still not known in the case of Phenacoccus solenopsis under variable experimental treatments. The present study focused on the identification of stable housekeeping genes as a reference for analysis under different abiotic and biotic factors in P. solenopsis. We analyzed the relative expression of six commonly used candidate reference genes in different developmental stages, host-feeding assay, temperature treatments and field distribution conditions. Expression stabilities were analyzed by geNorm, NormFinder, and RefFinder. Under developmental and field distribution conditions, β-Tubulin was found to be most stable reference genes followed by rpl32 and α-Tubulin. In the case host feeding treatment conditions, β-Tubulin and α-tubulin identified to be the most stable reference genes, while in temperature stress, a combination of α-Tubulin and rpl32 found to be suitable for normalizing the RT-qPCR data. Further, the above-identified genes were validated using RT-qPCR based gene expression analysis of four objective genes namely, Myoinhibitory peptides (MIPs), Zinc_metalloprotease (Zn_Mp), fatty acid synthase (fas) and alpha-glucosidase. Identified reference genes will facilitate gene expression studies in future under different stress treatments in P. solenopsis.

Figure 1

Specificity of qRT-PCR amplicons, (a) 1.5% agarose gel electrophoresis showing amplification of a single product of the expected size for each reference gene. ‘M’ represents 100 bp DNA Ladder. (b) Dissociation curves with single peaks generated from all amplicons.

Figure 2

Expression range of Ct values of six candidate reference genes. (a) Field distribution, n = 12 sample points (b) Developmental stages, n = 12 sample points and (c) Abiotic stress-temperature treatments, n = 12 sample points (d) Different Host types, n = 12 sample points. (e) All Samples group, n = 16 sample Points. Black line across each box represents the median. Box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the inter-quartile range from the 25th and 75th percentiles, outliers are represented by dots. Ct values and genes are shown on Y- and X-axis, respectively.

Figure 3

Gene expression stability and rankings of six candidate reference genes using Expression stability values (M). The average expression stability (M) was calculated following stepwise exclusion of the least stable gene across all the samples within an experimental set, the lowest M value indicates the most stable gene, while the highest value represents the most variable gene.

Figure 4

Determination of the optimal number of reference genes for geNorm analysis. The pairwise variation (Vn/Vn + 1) was analyzed for the normalization factors NFn and NFn + 1 by geNorm program to determine (V < 0.15) the optimal number of reference genes.

Figure 5

Variation in Zn_MP, MIP, fatty acid synthase and alpha glucosidase gene-expression data normalized by different reference genes and their combinations. (a) Zn_MP expression levels in developmental stages (b) MIP expression levels in Host-Feeding Assay’s and developmental stages. (c) Fatty acid synthase expression levels in different temperature treatment conditions. (d) Alpha glucosidase expression levels in field treatment conditions. Bar indicate the standard error (±SE) evaluated from three biological replicates.