Structural Variation at Fr-H2 Locus Is Associated to Frost Tolerance Trait in Barley

Frost resistance-H2 (Fr-H2) is a major QTL affecting freezing tolerance of barley. The locus includes tandemly duplicated C-REPEAT BINDING FACTOR (CBF) transcription factors and regulates the expression of Cold-regulated genes. With the aim of Fr-H2 structural characterization, a high-resolution linkage map was developed from the Nure × Tremois cross to divide a 0.602 cM total genetic distance into ten recombination segments. Then a PCR-based screening of BAC clones in the reference genotype Morex enabled identification of 26 overlapping BACs from the integrated physical-genetic map. After 454 sequencing, the final overall assembly (42 scaffolds, 1.47 Mbp) included more than 90 % of target region: all genes were identified along the locus, and a general survey of Repetitive Elements obtained. Preliminary evidences of structural variation at the Fr-2 locus in Triticeae have accumulated in recent years and entail copy number variation (CNV) of different CBFs. A further CNV study in the Fr-H2 locus was then performed using a qRT-PCR approach in a barley genotype panel, together with the characterization of a set of biparental F_5:6 recombinant lines. A twofold difference in CNV per diploid genome was found for the HvCBF4B–HvCBF2A genomic segment in winter/facultative vs. spring genotypes confirming the preliminary observations. This CNV at the proximal subclusters of HvCBFs was associated with frost tolerance, and could be further investigated by combinations of CNV of other genes at Fr-H2.