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

Thind, A.K., Wicker, T., Šimková, H., Fossati, D., Moullet, O., Brabant, C., Vrána, J., Doležel, J., Krattinger, S.G.
NATURE BIOTECHNOLOGY 35: 793-796, 2017

Abstract: 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 species1–4. 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.
DOI: 10.1038/nbt.3877
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IEB authors: Jaroslav Doležel, Hana Šimková, Jan Vrána