Resolving the effects cold temperatures from those of gene dosage and the circadian clock on changes in gene expression during cold acclimation in barley using reciprocal near-isogenic lines differing in their allelic states at Frost Resistance-H1 (FR-H1) and FR-H2.

Barley increases in freezing tolerance in response to low, non-freezing temperatures, a phenomenon known as cold acclimation. Two major quantitative trait loci (QTL), Frost Resistance H1 (FR-H1) and FR-H2, play a prominent role in cold acclimation and freezing tolerance. FR-H2 encompasses a cluster of 13 different C-repeat Binding Factor (CBF) genes, which encode transcription factors that activate cold acclimation. CBF are influenced by cold, lights and circadian clock. FR-H1 co-segregated with VRN-1, the vernalization requirement gene. To test the effects of FR-H1 and FR-H2 on cold acclimation and freezing tolerance, we developed four reciprocals Near Isogenic Lines (NILs) in which the winter allele at each locus was introgressed into the spring background, and vice versa. We used a marker-assisted backcross scheme to follow NILs development and validated the percentage of recurrent parent occurring in each NIL using the 50k array. Here we present an experiment aimed at evaluating the immediate and early changes in CBF’s expression pattern considering the circadian influence. Six genotypes – Nure (winter resistant), Tremois (spring susceptible), and four NILs harboring FR-H1 and FR-H2 in alternative backgrounds – were studied. 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 shuffling effect of the two QTLs and different expression patterns in the CBF gene cluster. Phenotypic characterization is underway and, combined with gene expression results, will be preparatory for more in-depth (RT-ddPCR and RNAseq) studies.