Extensive allele mining discovers novel genetic diversity in the loci controlling frost tolerance in barley.

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.

Transcriptome profiling of short-term response to chilling stress in tolerant and sensitive Oryza sativa ssp. Japonica seedlings.

Low temperature is a major factor limiting rice growth and yield, and seedling is one of the developmental stages at which sensitivity to chilling stress is higher. Tolerance to chilling is a complex quantitative trait, so one of the most effective approaches to identify genes and pathways involved is to compare the stress-induced expression changes between tolerant and sensitive genotypes. Phenotypic responses to chilling of 13 Japonica cultivars were evaluated, and Thaibonnet and Volano were selected as sensitive and tolerant genotypes, respectively. To thoroughly profile the short-term response of the two cultivars to chilling, RNA-Seq was performed on Thaibonnet and Volano seedlings after 0 (not stressed), 2, and 10 h at 10 °C. Differential expression analysis revealed that the ICE-DREB1/CBF pathway plays a primary role in chilling tolerance, mainly due to some important transcription factors involved (some of which had never been reported before). Moreover, the expression trends of some genes that were radically different between Thaibonnet and Volano (i.e., calcium-dependent protein kinases OsCDPK21 and OsCDPK23, cytochrome P450 monooxygenase CYP76M8, etc.) suggest their involvement in low temperature tolerance too. Density of differentially expressed genes along rice genome was determined and linked to the position of known QTLs: remarkable co-locations were reported, delivering an overview of genomic regions determinant for low temperature response at seedling stage. Our study contributes to a better understanding of the molecular mechanisms underlying rice response to chilling and provides a solid background for development of low temperature-tolerant germplasm.

Copy number variation at the HvCBF4-HvCBF2 genomic segment is a major component of frost resistance in barley.

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.