PHYSICAL MAP AND DE NOVO SEQUENCING OF BARLEY FR-H2 FROST RESISTANCE LOCUS

Fr-H2 is one of the two major loci affecting freezing tolerance of barley. Genetic mapping evidences suggested a cluster of more than 14 genes encoding CBF transcription factors as lilely candidates to explain the trait; however, the molecular basis of the Fr-H2 QTL is still to be demonstrated. A large segregating population (2,849 F2 plants) was developed from the ‘Nure’ (frost tolerant) x ‘Tremois’ (frost susceptible) cross for fine mapping and locus dissection. Recombinants identified between seven of the 14 CBFs indicated that the locus spans a total genetic distance of 0.81 cM. Sequencing bacteriophage lambda genomic clones in ‘Nure’, ‘Dicktoo’, ‘Morex’ and ‘Tremois’ provided the first structural insights into Fr-H2. Subsequently, two ‘Morex’ genomic BAC libraries were screened using 10 CBF-specific markers, and their first BAC clone addresses obtained. A High-Information-Content Fingerprinting of the selected BACs was performed to create anchor points between the genetic and physical maps of the region. The Minimal Tiling Path was PCR-screened for the presence of all available CBFs and the physical order confirmed via placement of BAC-end specific markers. De novo 454 sequencing of 27 barcoded and pooled BACs was performed using the GS FLX platform and the sequences assembled under iterative change of parameters of MIRA software. Though the resolution of complex repetitive DNA structures iwas difficult to solve, more than 70% of the assembled sequence contigs were anchored to the physical map of Fr-H2. Altogether, these results throw light on the structural characterization of the 5H genomic region of the Fr-H2 locus and the role of different CBFs in barley.