A Preliminary Approach in Mendelizing Barley FR-H1 and FR-H2 QTLs

Freezing temperatures is one of the major limitations among the abiotic stresses in Triticeae crops, and two major Quantitative Trait Loci of frost resistance (namely, FR-H1 and FR-H2) have been mapped in barley on the long arm of chromosome 5. FR-H1 co-segregated with the vernalization requirement gene VRN-H1, while FR-H2 encompasses a cluster of at least 13 different C-repeat Binding Factor (CBF) genes that activate cold acclimation. To disentangle the effect of FR-H1 and FR-H2, four reciprocals Near Isogenic Lines (NILs) were developed. The donor of the winter/resistance alleles was cv. Nure, while the donor of the spring/susceptible alleles was cv. Tremois. Marker-assisted backcross scheme was used to separate FR-H1 and FR-H2 in alternative backgrounds. PCR-based molecular markers for HvCBF3 (FR-H2) and HvBM5 (FR-H1) were used for selection, and after at least four backcrossing generations, selected plants underwent multiple selfing. The genotypic status of the lines was initially tested using 19 SSR markers scattered throughout the genome, then with via 9k iSelect array genotyping. After preliminary experiments in controlled conditions , two locations were selected to assess the winter survival rate (WSR) and agronomical traits: Reggio Emilia, Po valley area, and Wooster, Ohio, USA. In Wooster the winter was cold enough to assess the WSR and the winter allele of FR-H2 increased the WSR in the susceptible (Trèmois) background; conversely, the winter allele of FR-H1 did not significantly increase the resistance in the susceptible (Trèmois) background. For the lines with the Nure genomic background, allele substitution at either FR-H1 or FR-H2 with spring alleles did not reduce WSR, indicating an important role by the other factors in winter (Nure) genomic background in regulating the frost resistance. In Reggio Emilia WSR was the same for QTL-NILs, Nure and Tremois due to mild winter. These results indicate that winter FR-H2 allele is sufficient to enhance freezing tolerance in susceptible genotypes, but that the winter FR-H1 allele might require other factors for winter survival. In addition, the QTL-NILs, Nure and Tremois were tested for a preliminary experiment aimed at evaluating the immediate and early changes in CBF’s expression in the first two days of cold acclimation. Plants at third-leaf stage were sampled in the morning, afternoon, and night for three consecutive days, grown under control (20/15°C, 8/16 h day/night) and acclimation (3/1°C, 8/16 h day/night) conditions. RT-qPCR analysis showed different expression patterns in the CBF gene cluster. Phenotypic characterization for agronomic traits is currently underway, and, combined with gene expression results, will be preparatory for more in-depth (RT-ddPCR and RNAseq) studies.