Skip to main content
Springer Nature Link
Log in

Rapid Gene Isolation Using MutChromSeq

  • Protocol
  • First Online:
Wheat Rust Diseases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1659))

Abstract

MutChromSeq is an approach for isolation of genes and DNA sequences controlling gene expression in plants with complex and polyploid genomes. It involves a lossless complexity reduction by flow cytometric chromosome sorting and shotgun sequencing DNA from isolated chromosomes. Comparison of sequences from wild-type parental chromosome with chromosomes from multiple independently derived mutants identifies causative mutations in a single candidate gene or a noncoding sequence. MutChromSeq does not rely on recombination-based genetic mapping and does not exclude any DNA sequence from being targeted.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Piraino SW, Furney SJ (2016) Beyond the exome: the role of non-coding somatic mutations in cancer. Ann Oncol 27:240–248

    Article  CAS  PubMed  Google Scholar 

  2. Spitz F (2016) Gene regulation at a distance: from remote enhancers to 3D regulatory ensembles. Semin Cell Dev Biol 57:57–67

    Article  CAS  PubMed  Google Scholar 

  3. Fekih R, Takagi H, Tamiru M, Abe A, Natsume S, Yaegashi H, Sharma S, Sharma S, Kanzaki H, Matsumura H et al (2013) MutMap+: genetic mapping and mutant identification without crossing in rice. PLoS One 8:e68529

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Thole JM, Strader LC (2015) Next-generation sequencing as a tool to quickly identify causative EMS-generated mutations. Plant Signal Behav 10:e1000167

    Article  CAS  PubMed  Google Scholar 

  5. Feuillet C, Travella S, Stein N, Albar L, Nublat A, Keller B (2003) Map-based isolation of the leaf rust disease resistance gene Lr10 from the hexaploid wheat (Triticum aestivum L.) genome. Proc Natl Acad Sci USA 100:15253–15258

    Google Scholar 

  6. Huang L, Brooks SA, Li W, Fellers JP, Trick HN, Gill BS (2003) Map-based cloning of leaf rust resistance gene Lr21 from the large and polyploid genome of bread wheat. Genetics 164:655–664

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Uauy C, Distelfeld A, Fahima T, Blechl A, Dubcovsky J (2006) A NAC Gene regulating senescence improves grain protein, zinc, and iron content in wheat. Science 314:1298–1301

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Krattinger SG, Lagudah ES, Spielmeyer W, Singh RP, Huerta-Espino J, McFadden H, Bossolini E, Selter LL, Keller B (2009) A putative ABC transporter confers durable resistance to multiple fungal pathogens in wheat. Science 323:1360–1363

    Article  CAS  PubMed  Google Scholar 

  9. Rawat N, Pumphrey MO, Liu S, Zhang X, Tiwari VK, Ando K, Trick HN, Bockus WW, Akhunov E, Anderson JA, Gill BS (2016) Wheat Fhb1 encodes a chimeric lectin with agglutinin domains and a pore-forming toxin-like domain conferring resistance to Fusarium head blight. Nat Genet 48:1576–1580

    Article  CAS  PubMed  Google Scholar 

  10. Rabinowicz PD (2003) Constructing gene-enriched plant genomic libraries using methylation filtration technology. Methods Mol Biol 236:21–36

    CAS  PubMed  Google Scholar 

  11. Peterson DG, Schulze SR, Sciara EB, Lee SA, Bowers JE, Nagel A, Jiang N, Tibbitts DC, Wessler SR, Paterson AH (2002) Integration of cot analysis, DNA cloning, and high-throughput sequencing facilitates genome characterization and gene discovery. Genome Res 12:795–807

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Shagina I, Bogdanova E, Mamedov IZ, Lebedev Y, Lukyanov S, Shagin D (2010) Normalization of genomic DNA using duplex-specific nuclease. BioTechniques 48:455–459

    Article  CAS  PubMed  Google Scholar 

  13. Oono Y, Kobayashi F, Kawahara Y, Yazawa T, Handa H, Itoh T, Matsumoto T (2013) Characterisation of the wheat (Triticum aestivum L.) transcriptome by de novo assembly for the discovery of phosphate starvation-responsive genes: gene expression in pi-stressed wheat. BMC Genomics 214:77

    Article  Google Scholar 

  14. Steuernagel B, Periyannan SK, Hernández-Pinzón I, Witek K, Rouse MN, Yu G, Hatta A, Ayliffe M, Bariana H, Jones JDG et al (2016) Rapid cloning of disease-resistance genes in plants using mutagenesis and sequence capture. Nat Biotechnol 34:652–655

    Article  CAS  PubMed  Google Scholar 

  15. Sanchez-Martin J, Steuernagel B, Ghosh S, Herren G, Hurni S, Adamski N, Vrána J, Kubaláková M, Krattinger SG, Wicker T et al (2016) Rapid gene isolation in barley and wheat by mutant chromosome sequencing. Genome Biol 17:221

    Google Scholar 

  16. Doležel J, Binarová P, Lucretti S (1989) Analysis of nuclear DNA content in plant cells by flow cytometry. Biol Plant 31:113–120

    Google Scholar 

  17. Vrána J, Šimková H, Kubaláková M, Číhalíková J, Doležel J (2012) Flow cytometric chromosome sorting in plants: the next generation. Methods 57:331–337

    Google Scholar 

  18. Giorgi D, Farina A, Grosso V, Gennaro A, Ceoloni C, Lucretti S (2013) FISHIS: fluorescence in situ hybridization in suspension and chromosome flow sorting made easy. PLoS One 8:e57994

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Šimková H, Svensson JT, Condamine P, Hřibová E, Suchánková P, Bhat PR, Bartoš J, Šafář J, Close TJ, Doležel J (2008) Coupling amplified DNA from flow-sorted chromosomes tohigh-density SNP mapping in barley. BMC Genomics 9:294

    Google Scholar 

  20. Wicker T, Matthews DE, Keller B (2002) TREP: a database for Triticeae repetitive elements. Trends Plant Sci 7:561–562

    Article  CAS  Google Scholar 

  21. Li H, Durbin R (2009) Fast and accurate short read alignment with burrows-wheeler transform. Bioinformatics 25:1754–1760

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, GPDPS (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25:2078–2079

    Article  PubMed  PubMed Central  Google Scholar 

  23. Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP (2011) Integrative genomics viewer. Nat Biotechnol 29:24–26

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Vrána J, Cápal P, Šimková H, Karafiátová M, Čízková J, Doležel J (2016) Flow analysis and sorting of plant chromosomes. Curr Protoc Cytom 78:531–534

    Google Scholar 

Download references

Acknowledgements

This work was supported by the JIC Innovation Fund (IF2015BW22) and the Biotechnological and Biological Sciences Research Council (BBSRC)-UK and grant LO1204 from the Czech National Program of Sustainability I. We also thank Ms. Zdeňka Dubská and Ms. Romana Šperková for excellent technical assistance.

Author information

Author notes

  1. Burkhard Steuernagel and Jan Vrána contributed equally to this work.

Authors and Affiliations

  1. John Innes Centre, Norwich Research Park, Norwich, UK

    Burkhard Steuernagel & Brande B. H. Wulff

  2. Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 31, 783 71, Olomouc, Czech Republic

    Jan Vrána, Miroslava Karafiátová & Jaroslav Doležel

Authors
  1. Burkhard Steuernagel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jan Vrána
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Miroslava Karafiátová
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Brande B. H. Wulff
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jaroslav Doležel
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jaroslav Doležel .

Editor information

Editors and Affiliations

  1. Research School of Biology, Australian National University, Canberra, Aust Capital Terr, Australia

    Sambasivam Periyannan

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Steuernagel, B., Vrána, J., Karafiátová, M., Wulff, B.B.H., Doležel, J. (2017). Rapid Gene Isolation Using MutChromSeq. In: Periyannan, S. (eds) Wheat Rust Diseases. Methods in Molecular Biology, vol 1659. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7249-4_20

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7249-4_20

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7248-7

  • Online ISBN: 978-1-4939-7249-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics