Fr-H2 is one of the two major loci affecting freezing tolerance and winter hardiness of barley. A cluster of more than 14 genes encoding CBF transcription factors maps coincident with Fr-H2 suggesting these as candidates; however, the molecular basis of the QTL is still to be demonstrated. The resistant phenotype could either result from a single CBF, or a copy number variation of CBF genes, or an effect of other sequences independent from the candidates. A large segregating population (2,849 F2) was developed from the ‘Nure’ (frost tolerant) x ‘Tremois’ (frost susceptible) cross (NxT) for fine mapping and locus dissection. These experiments indicated that the locus spans 0.81 cM and identified recombinants between seven of the 14 CBFs. Phenotyping F4/F5 NxT lines harbouring alternative allelic states of the CBF genes was performed through five independent experiments, and highlighted significant differences in frost resistance. Reciprocal QTL-NILs (Near-Isogenic Lines) are also in preparation (now BC4F1 and BC5F1). 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 barcoded BAC pools is being performed for the structural characterization of the genomic region of barley chromosome 5H, including the Fr-H2 locus. Altogether, the results will hopefully increase knowledge about the role of different CBFs in barley, and will be further efforts to sequence barley chromosome 5H.
Structural characterisation of a chromosome 5H genomic region including the Fr-H2 frost resistance locus / Francia, Enrico; Pasquariello, Marianna; Milc, Justyna Anna; Tondelli, A; Barabaschi, D; Schulte, D; Ariyadasa, R; Himmelbach, A; Knox, Ak; Dhillon, T; Stanca, Am; Stockinger, Ej; Stein, N; Pecchioni, Nicola. - STAMPA. - -:(2012), pp. ---. (Intervento presentato al convegno 11th International Barley Genetics Symposium tenutosi a Hangzhou nel April 15-20, 2012).
Structural characterisation of a chromosome 5H genomic region including the Fr-H2 frost resistance locus
FRANCIA, Enrico;PASQUARIELLO, Marianna;MILC, Justyna Anna;PECCHIONI, Nicola
2012
Abstract
Fr-H2 is one of the two major loci affecting freezing tolerance and winter hardiness of barley. A cluster of more than 14 genes encoding CBF transcription factors maps coincident with Fr-H2 suggesting these as candidates; however, the molecular basis of the QTL is still to be demonstrated. The resistant phenotype could either result from a single CBF, or a copy number variation of CBF genes, or an effect of other sequences independent from the candidates. A large segregating population (2,849 F2) was developed from the ‘Nure’ (frost tolerant) x ‘Tremois’ (frost susceptible) cross (NxT) for fine mapping and locus dissection. These experiments indicated that the locus spans 0.81 cM and identified recombinants between seven of the 14 CBFs. Phenotyping F4/F5 NxT lines harbouring alternative allelic states of the CBF genes was performed through five independent experiments, and highlighted significant differences in frost resistance. Reciprocal QTL-NILs (Near-Isogenic Lines) are also in preparation (now BC4F1 and BC5F1). 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 barcoded BAC pools is being performed for the structural characterization of the genomic region of barley chromosome 5H, including the Fr-H2 locus. Altogether, the results will hopefully increase knowledge about the role of different CBFs in barley, and will be further efforts to sequence barley chromosome 5H.
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