Genetic basis of fr-h2 in barley (Hordeum vulgare L.): significant improvements in the understanding of the “CBF number game”

Freezing temperatures can severely compromise barley yield potential. Freezing tolerance depends on a period of cold acclimation mediated by CBFs (C-repeat binding factors), a class of transcriptional factors that modulate the expression of multiple effector genes. In order to investigate the relationship between frost tolerance effect due to the major QTL Fr-H2 and copy number variation (CNV) for a specific cluster of CBF genes, a qPCR approach was used on a contrasting set of barley cultivars. The panel, composed of 41 genotypes with different growth habits, was accurately phenotyped by field survival and Fv/Fm measurements. Data showed that there was a positive correlation between frost tolerance and CNV for the HvCBF2-HvCBF4 genomic region in the proximal cluster and a lower yet significant negative correlation between CNV for HvCBF3 in the distal cluster and frost tolerance. These results strongly support the hypothesis that Fr-H2 effect is due to a sort of“HvCBF-number game” rather than to a CNV at a single HvCBF gene. In addition to this, a protocol to discriminate the HvCBF2 forms showed an increased copy number of HvCBF2A form in highly resistant facultative and winter genotypes. Resequencing by third generation sequencing technology (PacBio) of Fr-H2 in the frost tolerant cv. ‘Nure’ is in progress. A comparison of the region with the structure of the locus in both the susceptible reference genotype ‘Morex’ and the wheat genomes allowed a detailed characterization of the genic and intergenic space of the proximal subcluster harbouring HvCBF4 and HvCBF2. The obtained results are being validated by detailed expression analysis aimed to define how each gene involved in CNV contribute in terms of CBF transcripts accumulation. These results represent a significant step forward the understanding of the genetic bases of frost tolerance in barley.