Dataset Information

Validation of reference genes for qRT-PCR data normalisation in lentil (Lens culinaris) under leaf developmental stages and abiotic stresses.

ABSTRACT: Lentil (Lens culinaris) is one of the most important staple food crops of developing countries. Transcriptome based global gene expression profiling followed by validation of expression of important genes through quantitative real time-PCR (qRT-PCR) has achieved significance in recent years. However, there is a severe scarcity of information regarding stable reference genes in lentil, which is mandatory for qRT-PCR data normalisation. Hence, the present study was under-taken to identify the most stable reference gene(s) in lentil. Expression stability of eight candidate genes viz. ribulose 1,5-bisphosphate carboxylase large subunit (Rbcl), ribosomal protein L2 (RPL2), 18S rRNA, tubulin (Tub), elongation factor 1? (EF1?), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), heat shock protein (HSP70), and Maturase (mat K) was evaluated in five varieties of lentil at three different stages of leaf development and abiotic stress conditions using qRT-PCR. The results were analysed using four types of statistical software viz., geNorm, BestKeeper, NormFinder and RefFinder; all softwares identified RPL2 as most stable under abiotic stress conditions and developmental stages followed by Tub and Rbcl; while, HSP70 was identified as least stable. Relative expression of the target genes, defensin and PR4, was evaluated under abiotic stress conditions and data normalisation was done using two stable reference genes, RPL2 and Tub, either alone or in combination and with two least stable genes, HSP70 and 18S. The present work provides a list of potential reference genes in lentil, which will help in selection of appropriate reference gene for qRT-PCR data normalization depending upon the experiment.

SUBMITTER: Sinha R

PROVIDER: S-EPMC6352542 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Validation of reference genes for qRT-PCR data normalisation in lentil (<i>Lens culinaris</i>) under leaf developmental stages and abiotic stresses.

Sinha Ragini R Sharma T R TR Singh Anil Kumar AK

Physiology and molecular biology of plants : an international journal of functional plant biology 20180928 1

Lentil (<i>Lens culinaris</i>) is one of the most important staple food crops of developing countries. Transcriptome based global gene expression profiling followed by validation of expression of important genes through quantitative real time-PCR (qRT-PCR) has achieved significance in recent years. However, there is a severe scarcity of information regarding stable reference genes in lentil, which is mandatory for qRT-PCR data normalisation. Hence, the present study was under-taken to identify t ...[more]

PMID: 30804635

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Project description:Frost is one of the main abiotic stresses limiting plant distribution and crop production. To cope with the stress, plants evolved adaptations known as cold acclimation or chilling tolerance to maximize frost tolerance. Cold acclimation is a progressive acquisition of freezing tolerance by plants subjected to low non-freezing temperatures which subsequently allows them to survive exposure to frost. Lentil is a cool season grain legume that is challenged by winter frost in some areas of its cultivation.To better understand the genetic base of frost tolerance differential gene expression in response to cold acclimation was investigated. Recombinant inbred lines (RILs) from the cross Precoz x WA8649041 were first classified as cold tolerant or cold susceptible according to their response to temperatures between -3 to -15 °C. Then, RILs from both extremes of the response curve were cold acclimated and the leaf transcriptomes of two bulks each of eight frost tolerant and seven cold susceptible RILs were investigated by Deep Super-SAGE transcriptome profiling. Thus, four RNA bulks were analysed: the acclimated susceptible, the acclimated tolerant and the respective controls (non-acclimated susceptible and non-acclimated tolerant). Approximately 16.5 million 26 nucleotide long Super-SAGE tags were sequenced in the four sets (between ~3 and 5.4 millions). In total, 133,077 different unitags, each representing a particular transcript isoform, were identified in these four sets. Tags which showed a significantly different abundance in any of the bulks (fold change ?4.0 and a significant p-value <0.001) were selected and used to identify the corresponding lentil gene sequence. Three hundred of such lentil sequences were identified. Most of their known homologs coded for glycine-rich, cold and drought-regulated proteins, dormancy-associated proteins, proline-rich proteins (PRPs) and other membrane proteins. These were generally but not exclusively over-expressed in the acclimated tolerant lines.This set of candidate genes implicated in the response to frost in lentil represents an useful base for deeper and more detailed investigations into this important agronomic trait in future.

Dataset Information

Validation of reference genes for qRT-PCR data normalisation in lentil (Lens culinaris) under leaf developmental stages and abiotic stresses.

Publications

Validation of reference genes for qRT-PCR data normalisation in lentil (<i>Lens culinaris</i>) under leaf developmental stages and abiotic stresses.

Similar Datasets

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