Letter | Published:

Rapid cloning of genes in hexaploid wheat using cultivar-specific long-range chromosome assembly

Nature Biotechnology volume 35, pages 793796 (2017) | Download Citation


Cereal crops such as wheat and maize have large repeat-rich genomes that make cloning of individual genes challenging. Moreover, gene order and gene sequences often differ substantially between cultivars of the same crop species1,2,3,4. A major bottleneck for gene cloning in cereals is the generation of high-quality sequence information from a cultivar of interest. In order to accelerate gene cloning from any cropping line, we report 'targeted chromosome-based cloning via long-range assembly' (TACCA). TACCA combines lossless genome-complexity reduction via chromosome flow sorting with Chicago long-range linkage5 to assemble complex genomes. We applied TACCA to produce a high-quality (N50 of 9.76 Mb) de novo chromosome assembly of the wheat line CH Campala Lr22a in only 4 months. Using this assembly we cloned the broad-spectrum Lr22a leaf-rust resistance gene, using molecular marker information and ethyl methanesulfonate (EMS) mutants, and found that Lr22a encodes an intracellular immune receptor homologous to the Arabidopsis thaliana RPM1 protein.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


Primary accessions

NCBI Reference Sequence


  1. 1.

    et al. Major haplotype divergence including multiple germin-like protein genes, at the wheat Sr2 adult plant stem rust resistance locus. BMC Plant Biol. 14, 379 (2014).

  2. 2.

    et al. A haplotype map of allohexaploid wheat reveals distinct patterns of selection on homoeologous genomes. Genome Biol. 16, 48 (2015).

  3. 3.

    et al. Maize HapMap2 identifies extant variation from a genome in flux. Nat. Genet. 44, 803–807 (2012).

  4. 4.

    et al. 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 (2016).

  5. 5.

    et al. Chromosome-scale shotgun assembly using an in vitro method for long-range linkage. Genome Res. 26, 342–350 (2016).

  6. 6.

    FAO. The State of the World's Land and Water Resources for Food and Agriculture (SOLAW)—Managing Systems at Risk (Food and Agriculture Organization of the United Nations, Rome and Earthscan, London, 2011).

  7. 7.

    , & in Genetics and Genomics of the Triticeae (eds. Feuillet, C. & Muehlbauer, G.J.) 337–357 (Springer, New York, 2009).

  8. 8.

    , , , & Subgenome chromosome walking in wheat: a 450-kb physical contig in Triticum monococcum L. spans the Lr10 resistance locus in hexaploid wheat (Triticum aestivum L.). Proc. Natl. Acad. Sci. USA 97, 13436–13441 (2000).

  9. 9.

    & Analysis methods for studying the 3D architecture of the genome. Genome Biol. 16, 183 (2015).

  10. 10.

    et al. Chromosome-scale scaffolding of de novo genome assemblies based on chromatin interactions. Nat. Biotechnol. 31, 1119–1125 (2013).

  11. 11.

    Leaf rust of wheat: pathogen biology, variation and host resistance. Forests 4, 70–84 (2013).

  12. 12.

    & Inheritance in hexaploid wheat of adult-plant leaf rust resistance derived from Aegilops squarrosa. Can. J. Genet. Cytol. 12, 175–180 (1970).

  13. 13.

    , , , & Microsatellite mapping of adult-plant leaf rust resistance gene Lr22a in wheat. Theor. Appl. Genet. 115, 877–884 (2007).

  14. 14.

    , & Characterization of adult-plant resistance to leaf rust of wheat conferred by the gene Lr22a. Plant Dis. 71, 542–545 (1987).

  15. 15.

    Virulence in Puccinia recondita f. sp. tritici isolates from Canada to genes for adult-plant resistance to wheat leaf rust. Plant Dis. 81, 267–271 (1997).

  16. 16.

    , & Physiologic specialization of Puccinia triticina, the causal agent of wheat leaf rust, in Canada in 2009. Can. J. Plant Pathol. 35, 338–345 (2013).

  17. 17.

    & Maintaining the efficiency of MAS method in cereals while reducing the costs. J. Plant Breed. Genet. 2, 97–100 (2014).

  18. 18.

    et al. Chromosomes in the flow to simplify genome analysis. Funct. Integr. Genomics 12, 397–416 (2012).

  19. 19.

    et al. Development of chromosome-specific BAC resources for genomics of bread wheat. Cytogenet. Genome Res. 129, 211–223 (2010).

  20. 20.

    et al. A 4-gigabase physical map unlocks the structure and evolution of the complex genome of Aegilops tauschii, the wheat D-genome progenitor. Proc. Natl. Acad. Sci. USA 110, 7940–7945 (2013).

  21. 21.

    et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).

  22. 22.

    , , , & Arabidopsis RIN4 negatively regulates disease resistance mediated by RPS2 and RPM1 downstream or independent of the NDR1 signal modulator and is not required for the virulence functions of bacterial type III effectors AvrRpt2 or AvrRpm1. Plant Cell 16, 2822–2835 (2004).

  23. 23.

    , , & RIN4 interacts with Pseudomonas syringae type III effector molecules and is required for RPM1-mediated resistance in Arabidopsis. Cell 108, 743–754 (2002).

  24. 24.

    et al. Rapid cloning of disease-resistance genes in plants using mutagenesis and sequence capture. Nat. Biotechnol. 34, 652–655 (2016).

  25. 25.

    et al. Rapid gene isolation in barley and wheat by mutant chromosome sequencing. Genome Biol. 17, 221 (2016).

  26. 26.

    et al. Mapping-by-sequencing in complex polyploid genomes using genic sequence capture: a case study to map yellow rust resistance in hexaploid wheat. Plant J. 87, 403–419 (2016).

  27. 27.

    et al. Structural and functional partitioning of bread wheat chromosome 3B. Science 345, 1249721 (2014).

  28. 28.

    et al. Insular organization of gene space in grass genomes. PLoS One 8, e54101 (2013).

  29. 29.

    , & A new DNA extraction method for high-throughput marker analysis in a large-genome species such as Triticum aestivum. Plant Breed. 120, 354–356 (2001).

  30. 30.

    et al. Characterization of Lr75: a partial, broad-spectrum leaf rust resistance gene in wheat. Theor. Appl. Genet. 130, 1–12 (2017).

  31. 31.

    et al. The gene Sr33, an ortholog of barley Mla genes, encodes resistance to wheat stem rust race Ug99. Science 341, 786–788 (2013).

  32. 32.

    et al. Flow sorting of mitotic chromosomes in common wheat (Triticum aestivum L.). Genetics 156, 2033–2041 (2000).

  33. 33.

    , , , & Flow karyotyping and chromosome sorting in bread wheat (Triticum aestivum L.). Theor. Appl. Genet. 104, 1362–1372 (2002).

  34. 34.

    et al. FISHIS: fluorescence in situ hybridization in suspension and chromosome flow sorting made easy. PLoS One 8, e57994 (2013).

  35. 35.

    et al. Analysis and sorting of rye (Secale cereale L.) chromosomes using flow cytometry. Genome 46, 893–905 (2003).

  36. 36.

    et al. Coupling amplified DNA from flow-sorted chromosomes to high-density SNP mapping in barley. BMC Genomics 9, 294 (2008).

  37. 37.

    , , , & Preparation of HMW DNA from plant nuclei and chromosomes isolated from root tips. Biol. Plant. 46, 369–373 (2003).

  38. 38.

    , & Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).

  39. 39.

    et al. Meraculous: de novo genome assembly with short paired-end reads. PLoS One 6, e23501 (2011).

  40. 40.

    International Brachypodium Initiative. Genome sequencing and analysis of the model grass Brachypodium distachyon. Nature 463, 763–768 (2010).

  41. 41.

    et al. Genotype-specific SNP map based on whole chromosome 3B sequence information from wheat cultivars Arina and Forno. Plant Biotechnol. J. 11, 23–32 (2013).

  42. 42.

    et al. Aegilops tauschii draft genome sequence reveals a gene repertoire for wheat adaptation. Nature 496, 91–95 (2013).

  43. 43.

    et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011).

  44. 44.

    , , & Plant intracellular innate immune receptor Resistance to Pseudomonas syringae pv. maculicola 1 (RPM1) is activated at, and functions on, the plasma membrane. Proc. Natl. Acad. Sci. USA 108, 7619–7624 (2011).

  45. 45.

    et al. LRR conservation mapping to predict functional sites within protein leucine-rich repeat domains. PLoS One 6, e21614 (2011).

  46. 46.

    Phylogenetic analysis using PHYLIP. Methods Mol. Biol. 132, 243–258 (2000).

Download references


We are grateful to the staff at Dovetail Genomics for constructing the CH Campala Lr22a scaffolds. We thank M. Karafiátová for supervising chromosome 2D flow sorting and estimation of purity in flow sorted fractions, and Z. Dubská, R. Šperková and J. Weiserová for technical assistance. We also thank B. Senger and L. Luthi for assistance with field experiments and B. Keller for continuous support. This work was financed by an Ambizione fellowship of the Swiss National Science Foundation. J.V., H.Š., and J.D. were supported by the Ministry of Education, Youth and Sports of the Czech Republic (grant award LO1204 from the National Program of Sustainability I).

Author information

Author notes

    • Anupriya Kaur Thind
    •  & Thomas Wicker

    These authors contributed equally to this work.


  1. Department of Plant and Microbial Biology, University of Zurich, Zurich, Switzerland.

    • Anupriya Kaur Thind
    • , Thomas Wicker
    •  & Simon G Krattinger
  2. Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research, Olomouc, Czech Republic.

    • Hana Šimková
    • , Jan Vrána
    •  & Jaroslav Doležel
  3. Institute for Plant Production Sciences, Agroscope, Switzerland.

    • Dario Fossati
    • , Odile Moullet
    •  & Cécile Brabant


  1. Search for Anupriya Kaur Thind in:

  2. Search for Thomas Wicker in:

  3. Search for Hana Šimková in:

  4. Search for Dario Fossati in:

  5. Search for Odile Moullet in:

  6. Search for Cécile Brabant in:

  7. Search for Jan Vrána in:

  8. Search for Jaroslav Doležel in:

  9. Search for Simon G Krattinger in:


A.K.T., T.W., H.Š., J.D. and S.G.K. designed the experiments and wrote the manuscript, A.K.T., and S.G.K. performed phenotypic and molecular analyses, H.Š., J.V., and J.D. flow-sorted chromosome 2D and prepared high molecular weight (HMW) DNA, O.M., C.B., and D.F. developed the CH Campala Lr22a backcross line.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Simon G Krattinger.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–8 and Supplementary Tables 1–4.

About this article

Publication history






Further reading