Plant Meiosis pp 237-258 | Cite as

Isolating Male Meiocytes from Maize and Wheat for “-Omics” Analyses

  • Stefanie Dukowic-SchulzeEmail author
  • Nelson Garcia
  • Arun S. K. Shunmugam
  • Sateesh Kagale
  • Changbin Chen
Part of the Methods in Molecular Biology book series (MIMB, volume 2061)


Genome-wide gene expression studies have become a routine approach due to the advances in sequencing technologies, their ease of use, and increasing affordability. Simultaneous investigation of small RNA expression adds further valuable information but is not adopted as widely yet. Both RNA-seq and small RNA-seq benefit from the use of specific cell types. Here, we describe a protocol for the isolation of male meiotic cells from maize or wheat plants, along with the application of downstream RNA sequencing, extendable to other -omics approaches.

Key words

Meiocytes Meiosis Maize Wheat Plants Omics Sequencing 



This work was supported by the National Science Foundation (IOS: 1025881, IOS-1546792), a grant-in-aid fund from the University of Minnesota, and Genome Canada funded Canadian Triticum Applied Genomics2 (CTAG2) initiative.


  1. 1.
    Caryl AP, Jones GH, Franklin FCH (2003) Dissecting plant meiosis using Arabidopsis thaliana mutants. J Exp Bot 54:25–38CrossRefGoogle Scholar
  2. 2.
    Dukowic-Schulze S, Harris A, Li J, Sundararajan A, Mudge J, Retzel E, Pawlowski W, Chen C (2014) Comparative transcriptomics of early meiosis in Arabidopsis and maize. J Genet Genomics 41:139–152CrossRefGoogle Scholar
  3. 3.
    Dukowic-Schulze S, Sundararajan A, Ramaraj T, Kianian S, Pawlowski WP, Mudge J, Chen C (2016) Novel meiotic miRNAs and indications for a role of phasiRNAs in meiosis. Front Plant Sci 7:762CrossRefGoogle Scholar
  4. 4.
    Sánchez-Morán E, Mercier R, Higgins JD, Armstrong SJ, Jones GH, Franklin FCH (2005) A strategy to investigate the plant meiotic proteome. Cytogenet Genome Res 109:181–189CrossRefGoogle Scholar
  5. 5.
    Li J, Yuan J, Ji K, Zhang Y, Wang J, Zhang S, Liu S, Qiu P, Li N, Li M (2016) Small-scale isolation of meiocytes from Arabidopsis thaliana anthers for cytological analyses. Pak J Bot 48:1989–1992Google Scholar
  6. 6.
    Flórez-Zapata NMV, Reyes-Valdés MH, Hernandez-Godínez F, Martinez O (2014) Transcriptomic landscape of prophase I sunflower male meiocytes. Front Plant Sci 5:277PubMedPubMedCentralGoogle Scholar
  7. 7.
    Collado-Romero M, Alós E, Prieto P (2014) Unravelling the proteomic profile of rice meiocytes during early meiosis. Front Plant Sci 5:356CrossRefGoogle Scholar
  8. 8.
    Chen C, Retzel EF (2013) Analyzing the meiotic transcriptome using isolated meiocytes of Arabidopsis thaliana. In: Pawlowski WP, Grelon M, Armstrong S (eds) Plant meiosis. Humana Press, New York, pp 203–213CrossRefGoogle Scholar
  9. 9.
    Dukowic-Schulze S, Sundararajan A, Ramaraj T, Mudge J, Chen C (2014) Sequencing-based large-scale genomics approaches with small numbers of isolated maize meiocytes. Front Plant Sci 5:57PubMedPubMedCentralGoogle Scholar
  10. 10.
    Shunmugam ASK, Bollina V, Dukowic-Schulze S, Bhowmik PK, Ambrose C, Higgins JD, Pozniak C, Sharpe AG, Rozwadowski K, Kagale S (2018) MeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheat. BMC Plant Biol 18:293CrossRefGoogle Scholar
  11. 11.
    Chen C, Farmer AD, Langley RJ, Mudge J, Crow JA, May GD, Huntley J, Smith AG, Retzel EF (2010) Meiosis-specific gene discovery in plants: RNA-Seq applied to isolated Arabidopsis male meiocytes. BMC Plant Biol 10:280CrossRefGoogle Scholar
  12. 12.
    Chang P, Tseng Y-F, Chen P-Y, Wang C-JR (2018) Using flow cytometry to isolate maize meiocytes for next generation sequencing: a time and labor efficient method. Curr Protoc Plant Biol 3:e20068CrossRefGoogle Scholar
  13. 13.
    Barra L, Aiese-Cigliano R, Cremona G, De Luca P, Zoppoli P, Bressan RA, Consiglio FM, Conicella C (2012) Transcription profiling of laser microdissected microsporocytes in an Arabidopsis mutant (Atmcc1) with enhanced histone acetylation. J Plant Biol 55:281–289CrossRefGoogle Scholar
  14. 14.
    Sims J, Chen C, Schlögelhofer P, Kurzbauer M-T Targeted analysis of chromatin events (TACE). Plant meiosis: methods and protocolsGoogle Scholar
  15. 15.
    Hsu SY, Huang YC, Peterson PA (1988) Development pattern of microspores in Zea mays L.—the maturation of upper and lower florets of spikelets among an assortment of genotypes. Maydica 33:77–98Google Scholar
  16. 16.
    Bennett MD, Smith JB (1972) The effects of polyploidy on meiotic duration and pollen development in cereal anthers. Proc R Soc Lond B 181:81–107CrossRefGoogle Scholar
  17. 17.
    Nan G-L, Zhai J, Arikit S, Morrow D, Fernandes J, Mai L, Nguyen N, Meyers BC, Walbot V (2017) MS23, a master basic helix-loop-helix factor, regulates the specification and development of the tapetum in maize. Development 144:163–172CrossRefGoogle Scholar
  18. 18.
    Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, Gassmann M, Lightfoot S, Menzel W, Granzow M, Ragg T (2006) The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol 7:3CrossRefGoogle Scholar
  19. 19.
    Mills JD, Kawahara Y, Janitz M (2013) Strand-specific RNA-seq provides greater resolution of transcriptome profiling. Curr Genomics 14:173–181CrossRefGoogle Scholar
  20. 20.
    Afgan E, Baker D, van den Beek M et al (2016) The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update. Nucleic Acids Res 44:W3–W10CrossRefGoogle Scholar
  21. 21.
    R Development Core Team (2008) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, AustriaGoogle Scholar
  22. 22.
    RStudio Team (2015) RStudio: integrated development for R. RStudio, Inc., Boston, MAGoogle Scholar
  23. 23.
    Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15:550CrossRefGoogle Scholar
  24. 24.
    Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP (2011) Integrative genomics viewer. Nat Biotechnol 29:24–26CrossRefGoogle Scholar
  25. 25.
    Du Z, Zhou X, Ling Y, Zhang Z, Su Z (2010) agriGO: a GO analysis toolkit for the agricultural community. Nucleic Acids Res 38:W64–W70CrossRefGoogle Scholar
  26. 26.
    Axtell MJ (2013) ShortStack: comprehensive annotation and quantification of small RNA genes. RNA 19:740–751CrossRefGoogle Scholar
  27. 27.
    Pertea M, Pertea GM, Antonescu CM, Chang T-C, Mendell JT, Salzberg SL (2015) StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol 33:290–295CrossRefGoogle Scholar
  28. 28.
    Bray NL, Pimentel H, Melsted P, Pachter L (2016) Near-optimal probabilistic RNA-seq quantification. Nat Biotechnol 34:525–527CrossRefGoogle Scholar
  29. 29.
    Grabherr MG, Haas BJ, Yassour M et al (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 29:644–652CrossRefGoogle Scholar
  30. 30.
    Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26:139–140CrossRefGoogle Scholar
  31. 31.
    Anders S, Huber W (2012) Differential expression of RNA-Seq data at the gene level – the DESeq package. EMBL, Heidelberg, GermanyGoogle Scholar
  32. 32.
    Lin Y, Golovnina K, Chen Z-X, Lee HN, Negron YLS, Sultana H, Oliver B, Harbison ST (2016) Comparison of normalization and differential expression analyses using RNA-Seq data from 726 individual Drosophila melanogaster. BMC Genomics 17:28CrossRefGoogle Scholar
  33. 33.
    Aanes H, Winata C, Moen LF, Østrup O, Mathavan S, Collas P, Rognes T, Aleström P (2014) Normalization of RNA-sequencing data from samples with varying mRNA levels. PLoS One 9:e89158CrossRefGoogle Scholar
  34. 34.
    Sahraeian SME, Mohiyuddin M, Sebra R et al (2017) Gaining comprehensive biological insight into the transcriptome by performing a broad-spectrum RNA-seq analysis. Nat Commun 8:59CrossRefGoogle Scholar
  35. 35.
    Axtell MJ (2014) Butter: high-precision genomic alignment of small RNA-seq data. bioRxiv 007427Google Scholar
  36. 36.
    Kozomara A, Griffiths-Jones S (2014) miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res 42:D68–D73CrossRefGoogle Scholar
  37. 37.
    Smedley D, Haider S, Ballester B, Holland R, London D, Thorisson G, Kasprzyk A (2009) BioMart – biological queries made easy. BMC Genomics 10:22CrossRefGoogle Scholar
  38. 38.
    Begcy K, Dresselhaus T (2017) Tracking maize pollen development by the Leaf Collar Method. Plant Reprod 30:171–178CrossRefGoogle Scholar
  39. 39.
    Mackenzie A, Heslop-Harrison J, Dickinson HG (1967) Elimination of ribosomes during meiotic prophase. Nature 215:997–999CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Stefanie Dukowic-Schulze
    • 1
    Email author
  • Nelson Garcia
    • 1
  • Arun S. K. Shunmugam
    • 2
  • Sateesh Kagale
    • 2
  • Changbin Chen
    • 1
  1. 1.Department of Horticultural ScienceUniversity of MinnesotaSaint PaulUSA
  2. 2.National Research Council CanadaSaskatoonCanada

Personalised recommendations