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ORIGINAL RESEARCH article

Front. For. Glob. Change
Sec. Pests, Pathogens and Invasions
Volume 7 - 2024 | doi: 10.3389/ffgc.2024.1458554
This article is part of the Research Topic "Forentomics": forest pest and pathogen biology, ecology, and management using omics View all 9 articles

Robust Reference Gene Selection in Norway Spruce: Essential for Real-Time Quantitative PCR across Different Tissue, Stress and Developmental Conditions

Provisionally accepted
Vivek V. Singh Vivek V. Singh 1*Aisha Naseer Aisha Naseer 1Gothandapani Sellamuthu Gothandapani Sellamuthu 1Kanakachari Mogilicherla Kanakachari Mogilicherla 1,2Roman Gebauer Roman Gebauer 3Amit Roy Amit Roy 1Rastislav Jakuš Rastislav Jakuš 4,5
  • 1 Czech University of Life Sciences Prague, Prague, Czechia
  • 2 Indian Institute of Rice Research (ICAR), Hyderabad, Andhra Pradesh, India
  • 3 Institute of Forest Botany, Dendrology and Geobiocoenology, Faculty of Forestry and Wood Technology, Mendel University in Brno, Brno, South Moravia, Czechia
  • 4 Institute of Forest Ecology, Slovak Academy of Sciences (SAS), Zvolen, Slovakia
  • 5 Global Change Research Institute, Czech Academy of Sciences, Brno, South Moravia, Czechia

The final, formatted version of the article will be published soon.

    Accurate gene expression analysis in Norway spruce (Picea abies) under diverse stress conditions requires the identification of stable reference genes for normalization. Notably, the literature lacks reports on suitable reference genes in Norway spruce. Here, we aimed to address this gap by identifying suitable reference genes for quantitative real-time PCR in Norway spruce across various stress conditions (drought, heat, pathogen infection) in seedlings, tissues (needle, phloem, root), and developmental stages (seedlings, mature trees). We evaluated the stability of 15 candidate reference genes and assessed their expression stability using five statistical algorithms (ΔCt, geNorm, NormFinder, BestKeeper, and RefFinder). Our results highlight ubiquitin-protein ligase (SP1), conserved oligomeric Golgi complex (COG7), and tubby-like Fbox protein (TULP6) as the most stable reference genes, while succinate dehydrogenase (SDH5) and heat shock protein 90 (HSP90) were the least stable under various experimental conditions. COG7 and TULP6 are novel candidate reference genes reported for the first time. The expression stability of the identified reference genes was further validated using dehydrin-like protein 5 (PaDhn5) under drought conditions in Norway spruce. Pairwise variation analysis suggested that two reference genes were sufficient to normalize gene expression across all sample sets. This study provides a comprehensive analysis of reference gene stability under different experimental conditions and a catalog of genes for each condition, facilitating future functional genomic research in Norway spruce and related conifers.

    Keywords: RT-qPCR, housekeeping genes/reference genes, Gene Expression, Developmental stages and tissues, abiotic and biotic stress

    Received: 02 Jul 2024; Accepted: 17 Oct 2024.

    Copyright: © 2024 Singh, Naseer, Sellamuthu, Mogilicherla, Gebauer, Roy and Jakuš. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence: Vivek V. Singh, Czech University of Life Sciences Prague, Prague, Czechia

    Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.