Fourth International Bemisia Workshop International Whitefly Genomics Workshop
Progress in Positional Cloning of CMD2 the Gene that Confers High Level of Resistance to the Cassava Mosaic Disease (CMD)
1 Centro Internacional de Agricultura Tropical (CIAT), Cali, Colombia. Correspondence: isa.mo.ca@gmail.com
2 Clemson University Genomics Institute (CUGI), Clemson, South Carolina, USA
3 International Institute for Tropical Agriculture (IITA), Ibadan, Nigeria, Africa
We describe the development of a chromosome walking experiment using different strategies toward cloning a Cassava Mosaic Disease (CMD) resistance gene (CMD2). The first part of our project was the fine mapping of a population made up of 1690 individuals from a cross between TME-3, the source of CMD2 and the improved variety TMS30572. Two SSR markers previously reported NS158 and SSRY 28 were evaluated in the fine-mapping population and 112 recombinant individuals were identified. Two bulks from resistance and susceptible individuals were constructed and evaluated with several molecular marker systems including AFLP (STMPs, AFLP-NBS), ISTRs, RAPDs and SSR in a modified Bulk Segregant Analysis (BSA). Two polymorphic RAPD markers were identified in the gene region. The new linkage map with all markers reported, showed the resistance gene (CMD2) flanked by RME-1 and NS158 markers. A bacterial artificial chromosome (BAC) library with more than 10X coverage for the cassava genome was constructed from cassava variety TME3 for contig mapping. The BAC library was screened with RME-1 and NS158 markers by PCR amplification of BAC pools and 14 RME-1 positives clones and 2 NS158 clones were identified. Restriction enzyme fingerprinting with Hind III was employed to construct two BAC contigs around those markers using the FPC program. Ends of each contig were sequenced to design allele specific and SSCP-SNP primers to map the BAC ends into the CMD2 region. Additionally, new set the molecular markers are currently designed using the BLAST homology between the molecular markers associated with this characteristic and the castor bean genome sequenced. Successive screening with the BAC end markers and new COS markers in the BAC library will be carried out until a contig that contains the CMD2 gene is obtained.