Application of High Throughput Sequencing in Map-Based Cloning of An Eastern Filbert Blight Resistance Gene in Hazelnut

Eastern filbert blight (EFB), caused by the pyrenomycete Anisogramma anomala (Peck) E. Müller, is a devastating disease of European hazelnut (Corylus avellana L.) in the Pacific Northwest where it is a serious threat to the industry's existence.  A dominant allele at a single locus from the obsolete pollenizer 'Gasaway' confers complete resistance, for which several linked random amplified polymorphic DNA (RAPD) markers have been identified.  Seedlings in a mapping population were scored for RAPD markers UBC152₈₀₀ and UBC268₅₈₀, which flank the resistance locus, and recombinants used for fine-mapping.  Our map-based cloning efforts use a BAC library for 'Jefferson', which is heterozygous for resistance from 'Gasaway'.  Amplification with primers designed from RAPD marker and BAC end sequences identified 56 BACs in the EFB resistance region which were then sequenced using an Illumina IIG genome analyzer.  Multiplexing with barcoded adapters reduced the cost, and paired-end reads facilitated de novo sequence assembly.  With the help of high-information content fingerprinting (HICF), the BACs were aligned and a 275 kb contig was constructed.  New primers designed from BAC sequences were used to amplify DNA of the resistant parent, susceptible parent and 'Jefferson'.  The resulting PCR products were sequenced and compared, and single-nucleotide polymorphisms (SNPs) were identified.  Sequence differences were used to assign the 'Jefferson' contigs to the resistant or susceptible parent.  High-resolution melting (HRM) analysis and newly-developed SNP markers are being used for fine-mapping of the region.