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

2017; Nature Portfolio; Volume: 35; Issue: 8 Linguagem: Inglês

10.1038/nbt.3877

1546-1696

Anupriya Kaur Thind, Thomas Wicker, Hana Šimková, Dario Fossati, Odile Moullet, Cécile Brabant, Jan Vrána, Jaroslav Doležel, Simon G. Krattinger,

Plant Virus Research Studies

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 species. 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 linkage 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.