Towards physical mapping and sequencing the Fr-H2 (Frost resistance-H2) region of barley chromosome 5H

Frost resistance-H2 is one of two major quantitative trait loci, located on chromosome 5H, that affect freezing tolerance and winter hardiness of barley. Coincident with Fr-H2 is a cluster of more than 14 genes encoding CBF transcription factors, that are at present the best candidates in barley to explain the effects of frost tolerance given by the QTL. It is not known whether the effect of Fr-H2 is either the result of a single CBF gene, or the combined effect of a subset/all the CBF genes, or an effect of other sequences independent from the CBF genes. As a first step towards Fr-H2 physical mapping we have generated a large mapping population derived from the freezing tolerant genotype ‘Nure’, and the freezing susceptible ‘Tremois’, in order to both fine map the Fr-H2 interval, and to generate recombinants between the different CBF genes. Screens for recombinant individuals from F2 populations consisting of 2,849 plants, and their subsequent phenotypic evaluation in F4 lines provided an estimated refined genomic interval of 4.6 cM for Fr-H2. Recombinants between seven out of the 14 CBF genes under Fr-H2 have been identified and showed that the CBF gene cluster spans 0.81 cM on barley chromosome 5H. A positional cloning effort of Fr-H2 has been undertaken. A genomic BAC library of barley (cv. ‘Morex’) was screened with a total of six CBF markers mapping in this locus. Using a PCR-based screening strategy the first BAC clone addresses were obtained for all the CBF markers assayed. To create anchor points between the genetic map and a ‘future’ physical map of barley, in this region, a high information content fingerprinting (HICF) of the selected BACs has been performed and then the selected BAC clones have been assembled into contigs.To close the gaps between the assembled clones, additional BACs belonging to the contigs detected, have been screened with further CBF markers and a total of three BACs were sequenced and assembled using 454 sequencing. The construction of a single physical contig encompassing the Fr-H2 region will be our next purpose. This will provide further information on gene content and structural locus organization and thus provide a fundamental resource for detailed comparative analyses of the genomic organization of the locus in other barley cultivars, like ‘Nure’ and ‘Tremois’.