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Key Message: Exome sequencing-based allele mining for frost tolerance suggests HvCBF14 rather than CNV at Fr-H2 locus is the main responsible of frost tolerance in barley. Wild relatives, landraces and old cultivars of barley represent a reservoir of untapped and potentially important genes for crop improvement, and the recent sequencing technologies provide the opportunity to mine the existing genetic diversity and to identify new genes/alleles for the traits of interest. In the present study, we use frost tolerance and vernalization requirement as case studies to demonstrate the power of allele mining carried out on exome sequencing data generated from > 400 barley accessions. New deletions in the first intron of VRN-H1 were identified and linked to a reduced vernalization requirement, while the allelic diversity of HvCBF2a, HvCBF4b and HvCBF14 was investigated by combining the analysis of SNPs and read counts. This approach has proven very effective to identify gene paralogs and copy number variants of HvCBF2 and the HvCBF4b-HvCBF2a segment. A multiple linear regression model which considers allelic variation at these genes suggests a major involvement of HvCBF14, rather than copy number variation of HvCBF4b-HvCBF2a, in controlling frost tolerance in barley. Overall, the present study provides powerful resource and tools to discover novel alleles at relevant genes in barley., (© 2021. The Author(s).)

A family of CBF transcription factors plays a major role in reconfiguring the plant transcriptome in response to low-freezing temperature in temperate cereals. In barley, more than 13 HvCBF genes map coincident with the major QTL FR-H2 suggesting them as candidates to explain the function of the locus. Variation in copy number (CNV) of specific HvCBFs was assayed in a panel of 41 barley genotypes using RT-qPCR. Taking advantage of an accurate phenotyping that combined Fv/Fm and field survival, resistance-associated variants within FR-H2 were identified. Genotypes with an increased copy number of HvCBF4 and HvCBF2 (at least ten and eight copies, respectively) showed greater frost resistance. A CAPS marker able to distinguish the CBF2A, CBF2B and CBF2A/B forms was developed and showed that all the higher-ranking genotypes in term of resistance harbour only CBF2A, while other resistant winter genotypes harbour also CBF2B, although at a lower CNV. In addition to the major involvement of the HvCBF4-HvCBF2 genomic segment in the proximal cluster of CBF elements, a negative role of HvCBF3 in the distal cluster was identified. Multiple linear regression models taking into account allelic variation at FR-H1/VRN-H1 explained 0.434 and 0.550 (both at p < 0.001) of the phenotypic variation for Fv/Fm and field survival respectively, while no interaction effect between CNV at the HvCBFs and FR-H1/VRN-H1 was found. Altogether our data suggest a major involvement of the CBF genes located in the proximal cluster, with no apparent involvement of the central cluster contrary to what was reported for wheat.

The Osmyb4 rice gene, coding for a transcription factor, proved to be efficient against different abiotic stresses as a trans(cis)gene in several plant species, although the effectiveness was dependent on the host genomic background. Eight barley transgenic lines carrying the rice Osmyb4 gene under the control of the Arabidopsis cold inducible promoter cor15a were produced to test the efficiency of this gene in barley. After a preliminary test, the best performing lines were subjected to freezing at -11°C and -12°C. Frost tolerance was assessed measured the F(v)/F(m) parameter widely used to indicate the maximum quantum yield of photosystem II photochemistry in the dark adapted state. Three transgenic lines showed significantly increased tolerance. These selected lines were further studied under a complex stress applying cold and hypoxia at germinating stage. In these conditions the three selected transgenic lines outperformed the wild type barley in terms of germination vigour. The transgenic plants also showed a significant modification of their metabolism under cold/hypoxia conditions as demonstrated through the assessment of the activity of key enzymes involved in anoxic stress response. None of the transgenic lines showed dwarfism, just a slight retarded growth. These results provide evidence that the cold dependent expression of Osmyb4 can efficiently improved frost tolerance and germination vigour at low temperature without deleterious effect on plant growth.

Two quantitative trait loci (Fr-H1 and Fr-H2) for frost tolerance (FT) have been discovered on the long arm of chromosome 5H in barley. Two tightly linked groups of CBF genes, known to play a key role in the FT regulatory network in A. thaliana, have been found to co- segregate with Fr-H2. Here, we investigate the allelic variations of four barley CBF genes (HvCbf3, HvCbf6, HvCbf9 and HvCbf14) in a panel of European cultivars, landraces and H. spontaneum accessions. In the cultivars a reduction of nucleotide and haplotype diversities in CBFs compared with the landraces and the wild ancestor H. spontaneum, was evident. In particular, in cultivars the loss of HvCbf9 genetic variants was higher compared to other sequences. In order to verify if the pattern of CBF genetic variants correlated with the level of FT, an association procedure was adopted. The pairwise analysis of linkage disequilibrium (LD) among the genetic variants in four CBF genes was computed to evaluate the resolution of the association procedure. The pairwise plotting revealed a low level of LD in cultivated varieties, despite the tight physical linkage of CBF genes analysed. A structured association procedure based on a general liner model was implemented, including the variants in CBFs,of Vrn-H1, and of two reference genes not involved in FT (alpha-Amy1 and Gapdh) and considering the phenotypic data for FT. Association analysis recovered two nucleotide variants of HvCbf14 and one nucleotide variant of Vrn-H1 as statistically associated to FT.

Key message: Exome sequencing-based allele mining for frost tolerance suggests HvCBF14 rather than CNV at Fr-H2 locus is the main responsible of frost tolerance in barley. Wild relatives, landraces and old cultivars of barley represent a reservoir of untapped and potentially important genes for crop improvement, and the recent sequencing technologies provide the opportunity to mine the existing genetic diversity and to identify new genes/alleles for the traits of interest. In the present study, we use frost tolerance and vernalization requirement as case studies to demonstrate the power of allele mining carried out on exome sequencing data generated from > 400 barley accessions. New deletions in the first intron of VRN-H1 were identified and linked to a reduced vernalization requirement, while the allelic diversity of HvCBF2a, HvCBF4b and HvCBF14 was investigated by combining the analysis of SNPs and read counts. This approach has proven very effective to identify gene paralogs and copy number variants of HvCBF2 and the HvCBF4b-HvCBF2a segment. A multiple linear regression model which considers allelic variation at these genes suggests a major involvement of HvCBF14, rather than copy number variation of HvCBF4b-HvCBF2a, in controlling frost tolerance in barley. Overall, the present study provides powerful resource and tools to discover novel alleles at relevant genes in barley. [ABSTRACT FROM AUTHOR]