Copy Number Variation of CBF Genes Rules the Effect of FR-H2 QTL for Frost Tolerance in Barley

Freezing temperatures represent a major limitation to barley yield potential. Freezing Tolerance (FT) depends on a period of cold acclimation mediated by C-repeat binding factors (CBFs) that modulate the expression of multiple effector genes. Two QTLs on chromosome 5H, Fr-H1 and Fr-H2, explain a large part of the phenotypic variation in FT. Fr-H1 coincides with Vrn-H1, while Fr-H2 segregates with a cluster of more than 13 HvCBFs. In order to study the Fr-H2 QTL effect, a qPCR approach was used to identify resistance-associated copy number variation (CNV) at specific subclusters of HvCBFs in a diversity panel with different growth habits. Simultaneously an accurate phenotyping that combines F_v/F_m and field survival was carried out. A direct involvement of CNV at the HvCBF4-HvCBF2 genomic segment in the proximal cluster of HvCBF element was demonstrated, in combination with a negative role of HvCBF3 CNV in the distal cluster. A protocol to discriminate the HvCBF2 forms (2A, 2B, 2A/B) was developed and showed an increased copy number of HvCBF2A in highly resistant facultative and winter genotypes. Resequencing of Fr-H2 in the frost tolerant cv. ‘Nure’ is in progress and allowed the comparison of the region with the structure of the locus in both the susceptible reference genotype ‘Morex’ and the wheat genomes. In particular, a detailed characterization of the genic and intergenic space of the proximal subcluster harbouring HvCBF4 and HvCBF2 was obtained. These results represent a significant step forward the understanding of the genetic bases of the frost tolerant phenotype in barley.