Barley Cbf3 gene identification, expression pattern, and map location

Abstract

Although cold and drought adaptation in cereals and other plants involve the induction of a large number of genes, inheritance studies in Triticeae (wheat [Triticum aestivum], barley [Hordeum vulgare], and rye [Secale cereale]) have revealed only a few major loci for frost or drought tolerance that are consistent across multiple genetic backgrounds and environments. One might imagine that these loci could encode highly conserved regulatory factors that have global effects on gene expression; therefore, genes encoding central regulators identified in other plants might be orthologs of these Triticeae stress tolerance genes. The CBF/DREB1 regulators, identified originally in Arabidopsis as key components of cold and drought regulation, merit this consideration. We constructed barley cDNA libraries, screened these libraries and a barley bacterial artificial chromosome library using rice (Oryza sativa) and barley Cbf probes, found orthologs of Arabidopsis CBF/DREB1 genes, and examined the expression and genetic map location of the barley Cbf3 gene, HvCbf3. HvCbf3 was induced by a chilling treatment. HvCbf3 is located on barley chromosome 5H between markers WG364b and saflp58 on the barley cv Dicktoo x barley cv Morex genetic linkage map. This position is some 40 to 50 cM proximal to the winter hardiness quantitative trait locus that includes the Vrn-1H gene, but may coincide with the wheat 5A Rcg1 locus, which governs the threshold temperature at which cor genes are induced. From this, it remains possible that HvCbf3 is the basis of a minor quantitative trait locus in some genetic backgrounds, though that possibility remains to be thoroughly explored.

Figure 1

Comparison of CBF amino acid sequences from Arabidopsis, rice, and barley. Amino acids are designated in single-letter code. An asterisk indicates identical, a colon indicates closely related, and a period indicates somewhat related amino acid. Dashes indicate where a sequence has been expanded to optimize alignment. The NLS is in boldface and the AP2-DNA binding domain is boxed. Alignments were performed using Clustal W. A, Arabidopsis CBF1/DREB1B and rice OsCBF1. B, Arabidopsis CBF3/DREB1A versus barley HvCBF3. C, Restriction map of a fragment of bacterial artificial chromosome (BAC) clone 790P15 carrying HvCbf3. The white box indicates the location of the HvCbf3 gene. Restriction enzyme sites are: B, BamHI; H, HindIII; S, SalI; and Sc, SacI.

Figure 2

Expression of HvCbf3 in barley seedlings. Plant materials were 6-d-old seedlings growing with a 16-h photoperiod at 20°C or 2°C, and greenhouse-grown 6-d-old seedlings sprayed with 100 μm ABA. Total RNA was used for standard reverse transcription PCR (A, semiquantitative) and real-time reverse transcription PCR (B, quantitative). Gene-specific primers for HvCbf3, the cold-inducible gene Dhn8, the dehydration- and ABA-inducible gene Dhn4, and 28S rRNA were used (see “Materials and Methods”). A, PCR products were electrophoresed in 1.8% (w/v) agarose gels. B, The RNA amount is expressed relative to the highest value found for each gene and the values are normalized against the expression values of 28S rRNA used as an internal control (see “Materials and Methods”). Insets show the expression data on a logarithmic scale during the first 2 h. Bars are the sd of the mean of four amplifications. ●, HvCbf3; ▪, Dhn8; □, Dhn4.

Figure 3

Map location of the HvCbf3 gene. A, PCR reactions were performed on genomic DNA from wheat cv Chinese Spring (CS), barley cv Betzes (BB), and six wheat-barley disomic addition lines (carrying 2H, 3H, 4H, 5H, 6H, or 7H) using HvCbf3 gene-specific primers (see “Materials and Methods”). Amplified DNAs were electrophoresed in a 1.2% (w/v) agarose gel. B, The genotypes of each HvCbf3 gene in the barley cv Dicktoo × barley cv Morex mapping population were compared with mapping data (available at http://wheat/pw.usda.gov/ggpages/DxM/dmsor.txt). The map location of HvCbf3 genes is boxed. The dark bar on chromosome 5H indicates a quantitative trait locus (QTL) for vernalization requirement and freezing tolerance (Pan et al., 1994). The same region of wheat chromosome 5A (Galiba et al., 1995; Vagujfalvi et al., 2000) is compared with barley chromosome 5H. Units are in cM.