CBF gene copy number variation at Frost Resistance-2 is associated with levels of freezing tolerance in temperate-climate cereals

Frost Resistance-1 (FR-1) and FR-2 are two loci affecting freezing tolerance and winterhardiness of the temperate-climate cereals. FR-1 is hypothesized to be due to thepleiotropic effects of VRN-1. FR-2 spans a cluster of C-Repeat Binding Factor (CBF)genes. These loci are genetically and functionally linked. Recent studies indicate CBFtranscripts are down-regulated by the VRN-1 encoded MADS-box protein or a factor inthe VRN-1 pathway. Here we report that barley genotypes ‘Dicktoo’ and ‘Nure’ carryinga vrn-H1 winter allele at VRN-H1 harbor increased copy numbers of CBF codingsequences relative to Vrn-H1 spring allele genotypes ‘Morex’ and ‘Tremois’. Sequencingbacteriophage lambda genomic clones from these four genotypes alongside DNA blothybridizations indicate approximately half of the eleven CBF orthologs at FR-H2 areduplicated in individual genomes. One of these duplications discriminates vrn-H1genotypes from Vrn-H1 genotypes. The vrn-H1 winter allele genotypes harbor tandemsegmental duplications through the CBF2A–CBF4B genomic region and maintain twodistinct CBF2 paralogs, while the Vrn-H1 spring allele genotypes harbor single copies ofCBF2 and CBF4. An additional CBF gene, CBF13, is a pseudogene interrupted bymultiple nonsense codons in ‘Tremois’ whereas CBF13 is a complete uninterruptedcoding sequence in ‘Dicktoo’ and ‘Nure’. DNA blot hybridization with wheat DNAsreveals greater copy numbers of CBF14 also occurs in winter wheats than in springwheats. These data indicate that variation in CBF gene copy numbers is widespread in theTriticeae and suggest selection for winter hardiness co-selects winter alleles at both VRN-1 and FR-2.