Identification and analysis of cassava genotype TME3 bacteria artificial chromosome libraries for characterization of the cassava mosaic disease

Abstract

Cassava is an economically important crop in sub-Saharan Africa; however, its yield potential is constrained by cassava mosaic disease (CMD) infection. Classical genetics and biotechnology are being harnessed to overcome the disease and secure yields for farmers. The CMD2 resistance locus flanked by three simple sequence repeats (SSR) markers and one sequence characterized amplified region (SCAR) marker were mapped in West African genotypes and shown to impart qualitative resistant to all species of CMGs. However, gene(s) associated with the CMD2 locus and their mode of actions remains unknown. In an effort to discover gene(s) located in CMD2 locus region, TME3 BAC collections were screened for the presence of CMD2 flanking markers. CMD susceptible and resistant cassava genotypes were found to contain 100% of the markers flanking CMD2 locus. SNPs and nucleotide deletions were identified within the marker sequences but there was no evidence of trait and marker association. All the SSR markers flanking CMD2, and the more recently characterized CMD3 loci were to be located on chromosome 12. Through BAC pools library hybridization with marker probes, 130 BACs were identified, but only 23 BACs contained at least CMD2 specific two markers. Whole BAC sequencing identified five clones that mapped to the marker regions. BAC29 assembled into a 100 kb contig and encoded tandem repeats of three full length R genes (3.5 kb) and two partial repeats. These R genes were conserved and highly expressed in CMD susceptible and CMD resistant cassava genotypes. Promoter sequences derived from R genes showed similar transient expression of GUS as 35S promoter. On cassava genome V6.1 BAC29 sequences were mapped to chromosome 16, eliminating their potential role in CMD resistant.

Keywords: Bacteria artificial chromosome, CMD2, cassava, cassava mosaic disease