Selection of reliable reference genes for quantitative real-time PCR gene expression analysis in Jute (Corchorus capsularis) under stress treatments

Abstract

To accurately measure gene expression using quantitative reverse transcription PCR (qRT-PCR), reliable reference gene(s) are required for data normalization. Corchorus capsularis, an annual herbaceous fiber crop with predominant biodegradability and renewability, has not been investigated for the stability of reference genes with qRT-PCR. In this study, 11 candidate reference genes were selected and their expression levels were assessed using qRT-PCR. To account for the influence of experimental approach and tissue type, 22 different jute samples were selected from abiotic and biotic stress conditions as well as three different tissue types. The stability of the candidate reference genes was evaluated using geNorm, NormFinder, and BestKeeper programs, and the comprehensive rankings of gene stability were generated by aggregate analysis. For the biotic stress and NaCl stress subsets, ACT7 and RAN were suitable as stable reference genes for gene expression normalization. For the PEG stress subset, UBC, and DnaJ were sufficient for accurate normalization. For the tissues subset, four reference genes TUBβ, UBI, EF1α, and RAN were sufficient for accurate normalization. The selected genes were further validated by comparing expression profiles of WRKY15 in various samples, and two stable reference genes were recommended for accurate normalization of qRT-PCR data. Our results provide researchers with appropriate reference genes for qRT-PCR in C. capsularis, and will facilitate gene expression study under these conditions.

Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Salix matsudana under different abiotic stresses

“…α-TUB2 encoding a cytoskeleton structure protein62 and DnaJ encoding a cellular chaperone have the ability to repair heat-induced protein machinery damage6364. Our results are in agreement with several previous studies, which showed that α-TUB2 and DnaJ were established as the most stable reference genes in plants under abiotic stresses; for example, in Syntrichia caninervis under drought, salt, and heavy metal stresses65, Corchorus capsularis under drought stress10, Buchloe dactyloides under salt stress66 , and Platycladus orientalis under salt stress67. Normalization with multiple reference genes is an effective way to avoid erroneous data that may result from using a single reference gene68.…”

“…qRT-PCR is a reliable and accurate technique for measuring gene expression levels. Some suitable reference genes under abiotic stresses, such as GAPDH 5960 and DnaJ 10, have been detected in plants; however, the number of reference genes evaluated is limited, especially for woody plants.…”

“…Four reference genes, DnaJ, α-TUB2, MUB , and ACT under drought stress, were needed for accurate normalization. Two reference genes were found to be sufficient to analyze the expression of target genes in sorghum62, jute10, and moss65.…”

“…Plants with short growth cycles, such as Arabidopsis thaliana 7, soybean8, sorghum9, jute10, Sedum alfredii 11, rice12, and tobacco13, have been the focus of studies on the effects of various abiotic stresses, and a few studies have been performed on plants with long growth cycles under different stress conditions. Short growth cycle plants are limited by low biomass, while plants (especially woody plants) with high biomass and long growth cycles are more able to deal with severe abiotic stress conditions.…”

Identification of genic SSRs in jute (Corchorus capsularis, Malvaceae) and development of markers for phenylpropanoid biosynthesis genes and regulatory genes

“…We identified 236 WRKY TFs in jute, indicating multiple duplication events for WRKY TFs during evolution of jute. Some of the WRKY genes have been found to be associated with stress response and fibre development in jute through the involvement of phenylpropanoid biosynthesis genes (Niu et al., ; Samanta, Sadhukhan, & Basu, ). The 44 WRKY‐associated SSRs designed in this study may thus contribute to marker‐assisted selection for phenylpropanoid pathway‐related traits in jute.…”