Plant Meiosis pp 319-330 | Cite as

A Cytological Analysis of Wheat Meiosis Targeted by Virus-Induced Gene Silencing (VIGS)

  • Stuart Desjardins
  • Kostya Kanyuka
  • James D. HigginsEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2061)


Virus-induced gene silencing (VIGS) is a rapid and cost-effective reverse genetic technology that can be used to assess gene function in wheat. This chapter contains a detailed description of how to target wheat meiotic genes by VIGS. The timing of this technique is critical and has been optimized to silence meiotic genes at peak expression, evidenced by silencing of Triticum aestivum disrupted meiotic cDNA1 (TaDMC1). We also describe cytological techniques that have been adapted for the preparation and analysis of meiocytes in wheat, including fluorescent in situ hybridization (FISH) with directly labeled, synthetic oligonucleotide probes, and immunolocalization on spread material.

Key words

VIGS Meiosis Cytology Wheat FISH Chromosomes Immunolocalization 



This work has been supported by the UK Research and Innovation (UKRI) Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/N002628/1. Technical assistance was provided by Daisy Ogle, Inna Guterman, and Wing-Sham Lee.


  1. 1.
    Mercier R, Mézard C, Jenczewski E, Macaisne N, Grelon M (2015) The molecular biology of meiosis in plants. Annu Rev Plant Biol 66:297–327CrossRefGoogle Scholar
  2. 2.
    Osman K, Higgins JD, Sanchez-Moran E, Armstrong SJ, Franklin FC (2011) Pathways to meiotic recombination in Arabidopsis thaliana. New Phytol 190:523–544CrossRefGoogle Scholar
  3. 3.
    International Wheat Genome Sequencing Consortium (IWGSC) (2014) A chromosome-based draft sequence of the hexaploid bread wheat (Triticum aestivum) genome. Science 345:1251788CrossRefGoogle Scholar
  4. 4.
    Krasileva KV, Vasquez-Gross HA, Howell T, Bailey P, Paraiso F, Clissold L, Simmonds J, Ramirez-Gonzalez RH, Wang X, Borrill P, Fosker C, Ayling S, Phillips AL, Uauy C, Dubcovsky J (2017) Uncovering hidden variation in polyploid wheat. Proc Natl Acad Sci U S A 114:E913–E921CrossRefGoogle Scholar
  5. 5.
    Bhalla PL, Sharma A, Singh MB (2017) Enabling molecular technologies for trait improvement in wheat. Methods Mol Biol 1679:3–24CrossRefGoogle Scholar
  6. 6.
    Bennypaul HS, Mutti JS, Rustgi S, Kumar N, Okubara PA, Gill KS (2012) Virus-induced gene silencing (VIGS) of genes expressed in root, leaf, and meiotic tissues of wheat. Funct Integr Genomics 12:143–156CrossRefGoogle Scholar
  7. 7.
    Bhullar R, Nagarajan R, Bennypaul H, Sidhu GK, Sidhu G, Rustgi S, von Wettstein D, Gill KS (2014) Silencing of a metaphase I-specific gene results in a phenotype similar to that of the pairing homeologous 1 (Ph1) gene mutations. Proc Natl Acad Sci U S A 111:14187–14192CrossRefGoogle Scholar
  8. 8.
    Lee W-S, Hammond-Kosack KE, Kanyuka K (2012) Barley stripe mosaic virus-mediated tools for investigating gene function in cereal plants and their pathogens: virus-induced gene silencing, host-mediated gene silencing, and virus-mediated overexpression of heterologous protein. Plant Physiol 160:582–590CrossRefGoogle Scholar
  9. 9.
    Lee W-S, Rudd JJ, Kanyuka K (2015) Virus induced gene silencing (VIGS) for functional analysis of wheat genes involved in Zymoseptoria tritici susceptibility and resistance. Fungal Genet Biol 79:84–88CrossRefGoogle Scholar
  10. 10.
    Yuan C, Li C, Yan L, Jackson AO, Liu Z, Han C, Yu J, Li D (2011) A high throughput barley stripe mosaic virus vector for virus-induced gene silencing in monocots and dicots. PLoS One 6:e26468CrossRefGoogle Scholar
  11. 11.
    Tang Z, Yang Z, Fu S (2014) Oligonucleotides replacing the roles of repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 for FISH analysis. J Appl Genetics 55:313–318CrossRefGoogle Scholar
  12. 12.
    Chelysheva L, Grandont L, Vrielynck N, le Guin S, Mercier R, Grelon M (2010) An easy protocol for studying chromatin and recombination protein dynamics during Arabidopsis thaliana meiosis: immunodetection of cohesins, histones and MLH1. Cytogenet Genome Res 129:143–153CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Stuart Desjardins
    • 1
  • Kostya Kanyuka
    • 2
  • James D. Higgins
    • 1
    Email author
  1. 1.Department of Genetics and Genome BiologyUniversity of LeicesterLeicesterUK
  2. 2.Department of Biointeractions and Crop ProtectionRothamsted ResearchHarpendenUK

Personalised recommendations