PCR-based screening platform for an oilseed rape BAC library

A multidimensional screening platform was developed for a Brassica napus BAC library which encompasses more than 80,000 clones. The 216 microtitre plates that contain all clones of the library were conceptually arranged in a cube consisting of 36 layers, 48 columns and 48 rows. The clones were pooled according to their position in the cube along the six distinct coordinate axes to yield a total of 276 pools. Even screening of all clone pools for a particular locus does not allow for the unambiguous deconvolution of all clone coordinates that may harbour this locus because the BAC library covers between 8 and 10 genome equivalents, therefore additional clone pools were generated.

Gene-specific SNP assays were designed and used for the screening of all clone pools with an Illumina Veracode® system. The results of this experiment demonstrated that the multidimensional screening platform can be used successfully for PCR assays in a highly multiplexed fashion. The design of gene-specific SNP assays for the allopolyploid Brassica napus genome is hampered by high sequence identities between homoeologous genes but the high frequency of SNPs between homoeologous oilseed rape genes („intergenomic SNPs“) may represent an interesting alternative source for a BAC screening platform of this species. We are currently evaluating whether intergenomic SNPs are suitable for identification and differentiation of BACs containing homoeologous genes.