Abstract
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.
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Abbreviations
- FR :
-
Frost resistance loci
- CBF/DREB:
-
C-Repeat binding factor/dehydration responsive element binding factor proteins
- CRT/DRE:
-
C-RepeaT/dehydration responsive elements
- VRN :
-
Vernalization requirement genes and loci
- CNV:
-
Copy number variation
- CNVs:
-
Copy number variants
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Acknowledgments
This work was partially supported by Fondazione Cassa di Risparmio di Modena, Progetto di Ricerca Internazionale “FROSTMAP—Physical mapping of the barley Frost resistance-H2 (Fr-H2) locus”. Funding from “GENOMORE” project of the Fondazione Cassa di Risparmio di Carpi is also acknowledged. The authors are grateful to Dr. Franz W. Badeck (PIK—Potsdam, Germany) for his critical suggestions during statistical analysis and to Mrs. Donata Pagani (CRA-GPG, Fiorenzuola, Italy) for the excellent technical assistance during phenotypic analysis, and to Dr. Ilja Tom Prášil (Crop Research Institute, Prague, Czech Republic) for providing some barley varieties.
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All the authors have participated in the work and none of them have any conflict of interest. In particular, Enrico Francia and Nicola Pecchioni conceived the study. Enrico Francia and Caterina Morcia designed sequence-specific amplicons on the tested genes, carried out RT-qPCR and copy number calculation. Marianna Pasquariello and Valentina Mazzamurro developed a novel CAPS marker and together with Justyna Milc performed CBF2A/B screening. Fulvia Rizza and Valeria Terzi performed the phenotypic characterization for frost resistance of the plant material. Nicola Pecchioni contributed to analysis and interpretation of data and participated in the discussion of results. The manuscript was written Enrico Francia and Justyna Milc.
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E. Francia and C. Morcia have equally contributed to this work.
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11103_2016_505_MOESM2_ESM.tif
Supplementary Fig. 1 Graphical genotyping and frost resistance of ‘Nure’ x ‘Tremois’ F5:6 recombinants in the HvCBF cluster at Fr-H2. a Graphical representation of chromosome 5H region of the phenotyped F5:6 plants. Markers name and order derive from Francia et al. (2007) and Pasquariello et al. (2014). Chromosomal segments with ‘Nure’ and ‘Tremois’ marker alleles are in dark and light gray, respectively. NT-42 and NT-64 are DH lines of the ‘NxT’ population developed by Francia et al. (2004). The number of genotypes considered for each recombinant class is in parenthesis. b Bar graph for Fv/Fm representing the mean ± s.e. for n = 5 independent experiments in controlled conditions (freezing stress ranged from −11°C to −13°C). c Bar graph for winter survival representing the mean ± s.e. for a field trial in 2012/13 (min temp. −7°C). Lowercase letters in (b) and (c) represent least significant differences (LSD0.05) (TIF 1956 KB)
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Francia, E., Morcia, C., Pasquariello, M. et al. Copy number variation at the HvCBF4–HvCBF2 genomic segment is a major component of frost resistance in barley. Plant Mol Biol 92, 161–175 (2016). https://doi.org/10.1007/s11103-016-0505-4
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DOI: https://doi.org/10.1007/s11103-016-0505-4