Search

An association panel consisting of 185 accessions representative of the barley germplasm cultivated in the Mediterranean basin was used to localise quantitative trait loci (QTL) controlling grain yield and yield related traits. The germplasm set was genotyped with 1,536 SNP markers and tested for associations with phenotypic data gathered over 2 years for a total of 24 year × location combinations under a broad range of environmental conditions. Analysis of multi-environmental trial (MET) data by fitting a mixed model with kinship estimates detected from two to seven QTL for the major components of yield including 1000 kernel weight, grains per spike and spikes per m(2), as well as heading date, harvest index and plant height. Several of the associations involved SNPs tightly linked to known major genes determining spike morphology in barley (vrs1 and int-c). Similarly, the largest QTL for heading date co-locates with SNPs linked with eam6, a major locus for heading date in barley for autumn sown conditions. Co-localization of several QTL related to yield components traits suggest that major developmental loci may be linked to most of the associations. This study highlights the potential of association genetics to identify genetic variants controlling complex traits.

Frost Resistance-1 (FR-1) and FR-2 are two loci affecting freezing tolerance and winter hardiness of the temperate-climate cereals. FR-1 is hypothesized to be due to the pleiotropic effects of VRN-1. FR-2 spans a cluster of C-Repeat Binding Factor (CBF) genes. These loci are genetically and functionally linked. Recent studies indicate CBF transcripts are downregulated by the VRN-1 encoded MADS-box protein or a factor in the VRN-1 pathway. Here, we report that barley genotypes 'Dicktoo' and 'Nure' carrying a vrn-H1 winter allele at VRN-H1 harbor increased copy numbers of CBF coding sequences relative to Vrn-H1 spring allele genotypes 'Morex' and 'Tremois'. Sequencing bacteriophage lambda genomic clones from these four genotypes alongside DNA blot hybridizations indicate approximately half of the eleven CBF orthologs at FR-H2 are duplicated in individual genomes. One of these duplications discriminates vrn-H1 genotypes from Vrn-H1 genotypes. The vrn-H1 winter allele genotypes harbor tandem segmental duplications through the CBF2A-CBF4B genomic region and maintain two distinct CBF2 paralogs, while the Vrn-H1 spring allele genotypes harbor single copies of CBF2 and CBF4. An additional CBF gene, CBF13, is a pseudogene interrupted by multiple non-sense codons in 'Tremois' whereas CBF13 is a complete uninterrupted coding sequence in 'Dicktoo' and 'Nure'. DNA blot hybridization with wheat DNAs reveals greater copy numbers of CBF14 also occurs in winter wheats than in spring wheats. These data indicate that variation in CBF gene copy numbers is widespread in the Triticeae and suggest selection for winter hardiness co-selects winter alleles at both VRN-1 and FR-2.

Population structure and genome-wide linkage disequilibrium (LD) were investigated in 192 Hordeum vulgare accessions providing a comprehensive coverage of past and present barley breeding in the Mediterranean basin, using 50 nuclear microsatellite and 1,130 DArT((R)) markers. Both clustering and principal coordinate analyses clearly sub-divided the sample into five distinct groups centred on key ancestors and regions of origin of the germplasm. For given genetic distances, large variation in LD values was observed, ranging from closely linked markers completely at equilibrium to marker pairs at 50 cM separation still showing significant LD. Mean LD values across the whole population sample decayed below r (2) of 0.15 after 3.2 cM. By assaying 1,130 genome-wide DArT((R)) markers, we demonstrated that, after accounting for population substructure, current genome coverage of 1 marker per 1.5 cM except for chromosome 4H with 1 marker per 3.62 cM is sufficient for whole genome association scans. We show, by identifying associations with powdery mildew that map in genomic regions known to have resistance loci, that associations can be detected in strongly stratified samples provided population structure is effectively controlled in the analysis. The population we describe is, therefore, shown to be a valuable resource, which can be used in basic and applied research in barley.

Barley is an economically important model for the Triticeae tribe. We recently developed a new resource: the 'Nure' x 'Tremois' mapping population. Two low temperature QTLs were found to segregate on the long arm of chromosome 5H (Fr-H1, distal; Fr-H2, proximal). With the final aim of positional cloning of the genetic determinants of Fr-H1 and Fr-H2, a large segregating population of 1,849 F(2) plants between parents 'Nure' and 'Tremois' was prepared. These two QT loci were first validated by using a set of F(3) families, marker-selected to harbor pairs of reciprocal haplotypes, with one QTL fixed at homozygosity and the alternate one in heterozygous phase. The study was then focused towards the isolation of the determinant of Fr-H2. Subsequent recombinant screens and phenotypic evaluation of F(4) segregants allowed us to estimate (P < or = 0.01) a refined genomic interval of Fr-H2 (4.6 cM). Several barley genes with the CBF transcription factor signature had been already roughly mapped in cluster at Fr-H2, and they represent likely candidate genes underlying this QTL. Using the large segregating population (3,698 gametes) a high-resolution genetic map of the HvCBF gene cluster was then constructed, and after fine mapping, six recombinations between the HvCBFs were observed. It was therefore possible to genetically divide seven HvCBF subclusters in barley, in a region spanning 0.81 cM, with distances among them varying from 0.03 to 0.32 cM. The few recombinants between the different HvCBF subclusters are being marker-selected and taken to homozygosity, to phenotypically separate the effects of the single HvCBF genes.

Cereal crop yield is greatly affected in many growing areas by abiotic stresses, mainly low temperature and drought. In order to find candidates for the tolerance genes for these stresses, 13 genes encoding for transcription factors and upstream regulators were screened by amplification and SSCP on six parental genotypes of three barley mapping populations ('Nure' x 'Tremois', 'Proctor' x 'Nudinka', and 'Steptoe' x 'Morex'), and mapped as newly developed STS, SNP, and SSCP markers. A new consensus function map was then drawn using the three maps above, including 16 regulatory candidate genes (CGs). The positions of barley cold and drought tolerance quantitative trait loci (QTLs) presently described in the literature were added to the consensus map to find positional candidates from among the mapped genes. A cluster of six HvCBF genes co-mapped with the Fr-H2 cold tolerance QTL, while no QTLs for the same trait were positioned on chromosome 7H, where two putative barley regulators of CBF expression, ICE1 and FRY1, found by homology search, were mapped in this work. These observations suggest that CBF gene(s) themselves, rather than their two regulators, are at present the best candidates for cold tolerance. Four out of 12 drought tolerance QTLs of the consensus map are associated with regulatory CGs, on chromosomes 2H, 5H, and 7H, and two QTLs with effector genes, on chromosomes 5H and 6H. The results obtained could be used to guide MAS applications, allowing introduction into an ideal genotype of favourable alleles of tolerance QTLs.

Barley ( Hordeum vulgare subsp. vulgare) is an economically important diploid model for the Triticeae; and a better understanding of low-temperature tolerance mechanisms could significantly improve the yield of fall-sown cereals. We developed a new resource for genetic analysis of winter hardiness-related traits, the 'Nure' x 'Tremois' linkage map, based on a doubled-haploid population that is segregating for low-temperature tolerance and vernalization requirement. Three measures of low-temperature tolerance and one measure of vernalization requirement were used and, for all traits, QTLs were mapped on chromosome 5H. The vernalization response QTL coincides with previous reports at the Vrn-1/Fr1 region of the Triticeae. We also found coincident QTLs at this position for all measures of low-temperature tolerance. Using Composite Interval Mapping, a second proximal set, of coincident QTLs for low-temperature tolerance, and the accumulation of two different COR proteins (COR14b and TMC-Ap3) was identified. The HvCBF4 locus, or another member of the CBF loci clustered in this region, is the candidate gene underlying this QTL. There is a CRT/DRE recognition site in the promoter of cor14b with which a CBF protein could interact. These results support the hypothesis that highly conserved regulatory factors, such as members of the CBF gene family, may regulate the stress responses of a wide range of plant species.

Leaf stripe caused by the fungus Pyrenophora graminea represents a serious threat to grain yield in organically grown barley and in conventional Nordic and Mediterranean districts, for which resistant cultivars are necessary. A medium-density, molecular marker map derived from a 'Steptoe' (partially resistant) x 'Morex' (susceptible) spring barley cross and its derived doubled-haploid mapping population inoculated with the fungus made it possible to identify QTLs of resistance to leaf stripe. In order to investigate isolate-specificity of partial resistance, the 'Steptoe' x 'Morex' segregating population was inoculated with two highly virulent P. graminea isolates, Dg2 and Dg5. The present study demonstrates that partial resistance to leaf stripe of cv 'Steptoe' is governed in part by shared loci and in part by isolate-specific ones. One QTL is common to the resistance for the two isolates, on the long arm of chromosome 2 (2H), two QTLs are linked on chromosome 3 (3H), and the remaining two are isolate-specific, respectively for isolate Dg2 on chromosome 2 (2H) and for isolate Dg5 on chromosome 7 (5H). The QTL in common is that with the major effect on the resistance for each isolate, explaining 18.3% and 30.9% R(2) respectively for Dg2 and Dg5. The isolate-specific QTLs mapped in the 'Steptoe' x 'Morex' barley reference map support the assumption of Parlevliet and Zadoks (1977) that partial resistance may be due to minor gene-for-minor-gene interactions. Map comparisons of the QTLs with the known qualitative resistance genes to leaf stripe, Rdg1 (2H) and Rdg2 (7H), as well as with other QTLs of partial resistance in barley, show that the QTL for resistance to both isolates mapped on the long arm of chromosome 2 (2H) does not coincide with the qualitative Rdg1 gene but is linked to it at about 30 cM. One isolate-specific QTL of resistance to P. graminea, mapped on the short arm of chromosome 2 (2H), is coincident with a QTL for resistance to Pyrenophora teres previously mapped in the 'Steptoe' x 'Morex' cross.

A major gene underlying quantitative resistance of barley against Pyrenophora graminea, a seedborne pathogen causing leaf stripe, was mapped with molecular markers in a barley doubled haploid (DH) population derived from the cross 'Proctor' x 'Nudinka'. This quantitative trait locus (QTL) accounts for r (2)= 58.5% and was mapped on barley chromosome 1, tightly linked to the "naked" gene. A second resistance QTL accounting for 29.3% of the variation in the trait was identified on the P arm of barley chromosome 2. Another two minor QTLs were detected by further analysis. None of the QTLs was found in the barley chromosome 2 "Vada" region studied by Giese et al. (1993).

Barley middle-repeat sequences were screened for their ability to discriminate 51 barley commercial varieties. Two hordein clones, a clone encoding a leaf-specific thionin, a desiccation induced cDNA clone, a clone coding for 5S-rRNA and one corresponding to ubiquitin genes were tested. A very sensitive RFLP technique including four cutter restriction enzymes and denaturing 4% polyacrylamide gels were used to evidence the highest level of polymorphism.The RFLP data were analyzed by computer. Some probe/enzyme combinations were able to differentiate a large number of the cultivars tested, whereas three probe/enzyme combinations succeeded in identifying all the varieties. The use of this RFLP method can thus be suggested for cultivar identification in barley.