Abstract
BackgroundWith the availability of genome sequences, gene expression analysis of jute has drawn considerable attention for understanding the regulatory mechanisms of fiber development and improving fiber quality. Gene expression profiles of a target gene can provide valuable clues towards the understanding of its biological function. Reverse transcription quantitative real-time PCR (qRT-PCR) is the best method for targeted gene expression analysis due to its sensitivity and reproducibility. However, calculating relative expression requires reference genes, which must be stable across various biological conditions. For this purposes, 11 prospective genes namely, 28S RNA, ACT7, CYP, EF1A, EF2, ETIF3E, GAPDH, PP2Ac, PTB, UBC2 and UBI1 were evaluated for their potential use as reference genes in jute.ResultsThe expression stabilities of eleven prospective genes were analyzed in various jute plant tissues, such as the root, stick, bark, leaf, flower, seed and fiber, as well as under abiotic (waterlogged, drought and salinity) and biotic stress (infestation with Macrophomina phaseolina) conditions with different time points. All 11 genes were variably expressed in different tissues and stress conditions. To find suitable reference genes in different sample sets, a comprehensive approach based on four statistical algorithms such as GeNorm, BestKeeper, NormFinder the ΔCt was used. The PP2Ac and EF2 genes were the most stably expressed across the different tissues. ACT7 and UBC2 were suitable reference genes under drought stress, and CYP and PP2Ac were the most appropriate after inoculation with Macrophomina phaseolina. Under salinity stress, PP2Ac and UBC2 were the best genes, and ACT7 and PP2Ac were the most suitable under waterlogged conditions.ConclusionExpression stability of reference genes from jute varied in different tissues and selected experimental conditions. Our results provide a valuable resource for the accurate normalization of gene expression experiments in fiber research for important bast fiber crops.
Highlights
With the availability of genome sequences, gene expression analysis of jute has drawn considerable attention for understanding the regulatory mechanisms of fiber development and improving fiber quality
The results of the present study describes a systematic approach to identify suitable reference genes for the accurate normalization of gene expression analysis using quantitative real-time PCR (qRT-PCR) across different tissues and under various stress conditions in C. olitorius
The expression pattern of target gene ethylene-responsive transcription factor (ERF7A) and hypoxia-responsive/ethyleneresponsive transcription factor (ERF7B) was determined under waterlogged condition to further verify the reliability of the identified stable reference genes
Summary
With the availability of genome sequences, gene expression analysis of jute has drawn considerable attention for understanding the regulatory mechanisms of fiber development and improving fiber quality. In relative gene expression studies, the use of an internal gene reference to normalize test conditions, which must be stable across various biological and ontogenetic conditions, is a prerequisite [15]. [17], to identify suitable reference genes (RGs) under different stress conditions and among only three different tissue such as roots, leaves and stems from 15 day old seedlings. These studies were conducted before the availability of genomic data of jute. We report the validation of reference genes to identify the most suitable internal control for normalization of qRTPCR data from different types of tissue obtained from various organs of jute (C. olitorius) in various environmental conditions
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