Selection of stable housekeeping genes for gene expression studies during inflorescence development in black pepper(pepper nigrum L)

Abstract

The project entitled “Selection of stable housekeeping genes for gene expression studies during inflorescence development in black pepper (Piper nigrum L.) using real time PCR” was conducted at the Department of Plant Biotechnology, College of Agriculture, Vellayani during 2017-2019. The objective of the study was to compare the expression profiles of different housekeeping genes such as Actin, β-Tubulin, Elongation factor, Initiation factor, Ubiquitin and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) during inflorescence development and to identify the most stable genes to be used as reference genes for Reverse Transcription quantitative PCR (RT-qPCR) studies in black pepper (Piper nigrum L.). RT-qPCR is an effective and sensitive technique to measure transcript level modulation of genes and provides significant quantitative information on gene expression. Accuracy of the results obtained by this technique is dependent on normalization using stably expressed genes known as reference genes. An ideal reference gene should be stable under any experimental condition. Spike samples of three cultivars of black pepper viz., Panniyur, Karimunda and Thekken were used for the study. Samples were collected at three different developmental stages viz., stage I (1-2cm), stage II (6-8cm) and stage III (9-12cm) from two different plants of each cultivar. Genomic DNA and RNA were extracted by modified Cetyl Trimethyl Ammonium Bromide (CTAB) method and Trizol method respectively. Primers were designed for six selected housekeeping genes viz., Actin, Elongation factor, GAPDH, Initiation factor, Tubulin and Ubiquitin using “Primer Express” software and absence of secondary structure at the primer binding site was confirmed by “mfold” web server. Specificity of the designed gene specific primers was checked by PCR using genomic DNA. Single amplicon was obtained for all the primers indicating specificity of the designed primers. Amplification efficiency of the designed primers was determined by standard curve analysis and “Lin Reg” software. All the primers exhibited cent per cent amplification efficiency. RNA isolated from the spike samples was converted to cDNA and the quality was confirmed by PCR and agarose gel electrophoresis. cDNA was used for RT-qPCR using SYBR Green dye-based assay. Thermal conditions for RT-qPCR were 95°C for 2 min followed by 40 cycles of 95°C for 15 sec, 55°C for 15 sec and 72°C for 45 sec. RT-qPCR for each gene was performed with three technical replicates for each sample. Cq values obtained were used for further analysis. Stability of the housekeeping genes in the samples were analysed using three softwares viz, “Bestkeeper”, “geNorm” and “Normfinder”. Bestkeeper algorithm takes raw Cq values as input whereas “geNorm” and “Normfinder” uses relative expression values for analysis. Relative expression values were generated from Cq values using “qbase plus” software with an amplification factor of two. High standard deviation of Cq values (>1) for all the genes restricted the use of “Bestkeeper” software in the present study. “geNorm” and “Normfinder” softwares identified Actin as the most stable gene when all the cultivars were analysed together. “Normfinder” software identified Actin as the most stable gene in all the three cultivars, when they were analysed separately also. However, in the analysis using “geNorm” software, Actin emerged to be the most stable gene only in Thekken. In Panniyur, Elongation factor was the most stable gene followed by Actin and Initiation factor while in in Karimunda, Initiation factor was the most stable gene followed by Elongation factor and Actin. When analysed using “geNorm” software, the stability value (M value) was high for all the genes in all the cultivars except in Thekken, indicating high variation for all the genes in Panniyur and Karimunda. To conclude, among the genes studied, Actin was identified as the most stable housekeeping gene during inflorescence development in the genotypes of black pepper studied. The study also emphasizes the necessity of identifying more number of reference genes for improving the accuracy of RT-qPCR studies during inflorescence development in different cultivars of black pepper.