Patterns of genetic and eco-geographical diversity in Spanish barleys.

The pool of Western Mediterranean landraces has been under-utilised for barley breeding so far. The objectives of this study were to assess genetic diversity in a core collection of inbred lines derived from Spanish barley landraces to establish its relationship to barleys from other origins, and to correlate the distribution of diversity with geographical and climatic factors. To this end, 64 SSR were used to evaluate the polymorphism among 225 barley (Hordeum vulgare ssp. vulgare) genotypes, comprising two-row and six-row types. These included 159 landraces from the Spanish barley core collection (SBCC) plus 66 cultivars, mainly from European countries, as a reference set. Out of the 669 alleles generated, a large proportion of them were unique to the six-row Spanish barleys. An analysis of molecular variance revealed a clear genetic divergence between the six-row Spanish barleys and the reference cultivars, whereas this was not evident for the two-row barleys. A model-based clustering analysis identified an underlying population structure, consisting of four main populations for the whole genotype set, and suggested further possible subdivision within two of these populations. Most of the six-row Spanish landraces clustered into two groups that corresponded to geographic regions with contrasting environmental conditions. The existence of wide genetic diversity in Spanish germplasm, possibly related to adaptation to a broad range of environmental conditions, and its divergence from current European cultivars confirm its potential as a new resource for barley breeders, and make the SBCC a valuable tool for the study of adaptation in barley.

Chloroplast DNA microsatellite analysis supports a polyphyletic origin for barley.

Five barley chloroplast DNA microsatellites (cpSSRs) were used to study genetic relationships among a set of 186 barley accessions-34 Hordeum vulgare ssp. spontaneum (HS accessions) from Morocco, Ethiopia, Cyprus, Crete, Libya, Iraq, Iran, Turkey, Afghanistan and Israel, 122 H. vulgare ssp. vulgare landraces (HV landraces) from Spain, Bolivia (old Spanish introductions), Morocco, Libya and Ethiopia and 20 modern European spring barleys (HV cultivars). All loci were polymorphic in the material studied, with the number of alleles per locus ranging from two to three. Fifteen multi-locus haplotypes were observed, 11 in HS accessions and seven in HV landraces and cultivars. Of the seven haplotypes found in the HV lines, three were shared with the HS accessions, and four were unique. Cluster analysis revealed two main groups, one consisting of HS accessions from Ethiopia and the HV landraces from Spain, Bolivia (old Spanish), Morocco and Ethiopia, whereas the other larger group contained all of the other accessions studied. Based on these grouping and the existence of haplotypes found in the HV landraces and cultivars but not in the HS wild barley, a polyphyletic origin is proposed for barley, with further centres of origin in Ethiopia and the Western Mediterranean.

Use of new EST markers to elucidate the genetic differences in grain protein content between European and North American two-rowed malting barleys.

A population comprising 102 doubled haploid lines were produced from a cross between Beka, a barley cultivar widely grown in Spain, and Logan, a north American cultivar with inherently low protein content, a character considered to derive from the cultivar Karl. The intentions were to determine whether low-nitrogen malting barleys could be developed in Spain, and if genetic factors that influenced protein content were similarly expressed in widely diverse environments, i.e. northeastern Spain and eastern Scotland. An extensive map comprising 187 molecular markers was developed. Expressed sequence-tagged-derived markers were used in addition to anonymous simple sequence repeats to determine the potential for identifying candidate genes for quantitative trait loci (QTLs), and 22 such markers were mapped for the first time. There was transgressive segregation for both yield and protein content, and the gene for low protein from Logan was not expressed in the Scottish environment. In 2002, high yield was associated with earlier heading date in Spain, while late heading at the Scottish site was associated with greater lodging and lower thousand-kernel weight. These appeared to be possible pleiotropic effects of a factor detected on chromosome 2H. Using information from a consensus map, it was shown that this locus on 2H was in the region of the photoperiod response gene Eam6. A QTL explaining 18% of the variation in grain protein content was detected on chromosome 5H in a region in which a gene for nitrate reductase was previously observed. No effect on grain protein was associated with chromosome 6H, which has been suggested as the location of the low protein gene from Karl. However, it is likely that Karl contained more than one genetic factor reducing protein, and we postulate that the gene on 6H may have been lost during the breeding of Logan.

Genetic control of dormancy in a Triumph/Morex cross in barley.

Seed dormancy in barley ( Hordeum vulgare L.) is one of the most important parameters affecting malting. Seed dormancy is quantitatively inherited and variously influenced by the environment. The objectives of the present study were to determine the genome location and effects of quantitative trait loci (QTLs) involved in the expression of seed dormancy in a barley cross between two varieties derived from different germplasm pools. Using a doubled-haploid population of 107 lines of the cross between the malting types Triumph (two-row, dormant) and Morex (six-row, non-dormant), seed dormancy phenotypic data sets from five environments and a 147-marker linkage map were developed in order to perform QTL analyses with simple interval mapping and simplified composite interval mapping procedures. Two different types of variables were considered for seed dormancy characterization: (1) level of dormancy induced during seed development, which was indirectly measured as germination percentage at 3 days and 7 days, GP3 and GP7 respectively; (2) rate of dormancy release in the course of a period after seed harvest (after-ripening). Different mechanisms of genetic control were detected for these two types of dormancy-related traits. A major and consistent dormancy QTL near the centromere on chromosome 7(5H) was associated with the establishment of dormancy during seed development and accounted for 52% and 33% of the variability for GP3 and GP7, respectively. Two other QTLs located in the vicinity of the vrs1 locus on chromosome 2(2H) and near the long arm telomere on chromosome 7(5H) explained 9% and 19% of variation, respectively, for the rate of dormancy release during after-ripening. Likewise, seed dormancy was assessed in an F(2) population derived from the cross between two dormant types of distinct germplasm groups, Triumph (European, two-row, malt) and Steptoe (North American, six-row, feed), which showed similar but not identical genetic control for dormancy. Interestingly, there is remarkable dormancy QTL conservation in both regions on chromosome 7(5H) identified in this study and among other barley mapping populations. These widely conserved QTLs show potential as targets for selection of a moderate level of seed dormancy in breeding programs.

Mildew-resistant mutants induced in North American two- and six-rowed malting barley cultivars.

Mildew-resistant mutants were induced with sodium azide in three North American malting barley cultivars, two in the six-rowed Ursula (URS1 and URS2), one in the six-rowed Gertrud (GER1), and one in the two-rowed Prudentia (PRU1). Two of the mutants, URS1 and PRU1, showed complete resistance and were shown to have two new alleles at the mlo locus; these were designated, respectively, mlo31 and mlo32. Mutant URS2, showing partial resistance, was inherited as a dominant gene, but was not an allele at the Mla locus. The mean yield of each mutant was higher than that of its parental line, but yield levels varied across environments, although this was independent of the severity of the mildew attack. Other reasons, for example, the severity of the necrotic lesions in the mutants, may account for yield variations. The malting quality of the GER1 mutant proved similar to that of Gertrud, but both URS1 and URS2 showed lower malt extract than Ursula. This lower extract might be due to the smaller grain size of the mutants that could, in turn, result from necrotic lesions in the leaves, as implied by the effects on grain yield.

Dormancy, ABA content and sensitivity of a barley mutant to ABA application during seed development and after ripening.

Assessment of dormancy inception, maintenance and release was studied for artificially dried immature seeds harvested throughout seed development in the barley cv. Triumph and its mutant line TL43. Each was grown in Spain and Scotland under low and high dormancy inducing conditions, respectively. Both TL43 and Triumph followed a similar pattern of release from dormancy across the seasons, although seeds of TL43 were able to germinate at an earlier seed development stage. Abscisic acid (ABA) content was also studied in immature grains throughout the seed development period. Total amount of ABA in seeds of Triumph and TL43 was higher in plants grown in Scotland than in Spain. However, no clear genotypic differences in ABA pattern in the course of grain development could be detected whilst significant genotypic differences were observed for germination percentage (GP). Endogenous ABA content alone throughout grain development did not explain genetic differences in GP within environments. Environmental and genetic differences in dormancy were also observed on mature seeds throughout the after-ripening period. The initial germination (GP(0)) played a key role in the sensitivity to ABA of post-harvest mature seeds. For the same after-ripening stage, TL43 was more insensitive to exogenous ABA than Triumph. However, ABA responses in seeds of the two genotypes with similar GP(0) at different after-ripening stages were comparable. Therefore, differences in exogenous ABA sensitivity of post-harvest mature grain of these two genotypes seemed to be determined by, or coincident with, the initial germination percentage.

Morphological and agronomical diversity patterns in the Spanish barley core collection.

Seven thousand years of barley cultivation under the environmental hardships typical of the Mediterranean climate have generated genetic singularity of the Spanish barleys, consistently reported in the literature. From the Spanish National Collection of 2289 accessions, a core subset with 159 landraces and 16 old varieties was constituted. Twenty-seven characters were evaluated for the core collection, to define the structure of the diversity. Several evaluation trials were carried out in 1999-2000, whereas yield trials were performed in earlier years. Phenotypic diversity was large for most of the characters studied. Comparisons of genetic diversity between the core and the original collections suggested that the core is a good representation of the existing diversity in the BNG. Comparisons with results of studies on Spanish materials from other collections seem to indicate that the Spanish diversity is not well represented in some world collections. Principal component analyses for quantitative and qualitative characters revealed a clear distinction between two- and six-row cultivars, and also between landraces and commercial varieties. Geographical origins of the landraces were correlated with grain yield, heading date, duration of grain filling period, and growth class. In relation to diseases, altitude played an important role on the resistance to powdery mildew and brown rust. For brown rust, all the resistant landraces came from low altitudes. These geographical gradients seemed consistent with prior knowledge about barley adaptation, and would confirm the agreement between passport data and true adaptive origin of these landraces from a geographical point of view.

Efficient production of androgenic doubled-haploid mutants in barley by the application of sodium azide to anther and microspore cultures.

The aim of this study was to establish a protocol for an efficient production of agronomical and/or physiological mutants from model (cvs. Igri and Cobra) and low-androgenic-responding (cv. Volga) cultivars of barley through the application of a mutagenic agent, sodium azide, to anthers and isolated microspores cultured in vitro. This technology offers the possibilities of screening for recessive mutants in the first generation, selecting for novel genotypes from very large haploid populations, avoiding chimerism and rapidly fixing selected genotypes as fertile true breeding lines. The mutagenic treatment, 10-3-10-5 M sodium azide, was applied during the anther induction pre-treatment or immediately after the microspore isolation procedure. Out of 616 M2 doubled-haploid lines characterised under field conditions, a total of 63 morphological and developmental independent mutant lines were identified. The percentage of M2 doubled-haploid lines carrying mutations per line analysed was 3.8% when 10-4 M sodium azide was applied to anthers from the low-responding cv. Volga; this increased to 8.6% and 15.6% when 10-5 and 10-4 M sodium azide were applied to freshly isolated microspores from model cultivars.

Determination of beta-(1-3),(1-4)-D-glucans in barley by reversed-phase high-performance liquid chromatography.

An HPLC method for the determination of beta-glucan in barley was developed. The beta-glucan was hydrolysed with lichenase [endo-beta-(1-3),(1-4)-D-glucan-4-glucanhydrolase from Bacillus subtilis] to oligosaccharides, which were analysed by reversed-phase HPLC using water as the mobile phase at a flow-rate of 0.7 ml/min. The separation of the oligosaccharides was performed in a C18 stainless-steel column (Spherisorb ODS-2) with 5-microns particles in less than 10 min, with a refractive index detection.

On the origin of Spanish two-rowed barleys.

To investigate the phylogenetic origin of Spanish two-rowed barleys, we studied 44 accessions of old land-races both morphologically and biochemically to ascertain their similarity with 51 entries of old cultivars and land-races of widespread origin across Europe. They were also compared with 20 accessions of Hordeum spontaneum from the Mediterranean basin and other regions of its distribution range, 14 accessions of Moroccan cultivated six-rowed barley land-races, and different six-rowed Spanish and two-and six-rowed European cultivars. CM-(trypsin inhibitors and subunits of the barley tetrameric α-amylase inhibitor) proteins and hordeins, all of which are endosperm proteins, were used as biochemical markers. The appearance of separate clusters of the Spanish barleys in the numerical classifications for both protein systems as a result of the existence of characteristic gene combinations that do not exist in entries from other origins permitted us to postulate the existence of local ancestors for most of the Spanish two-rowed barleys studied, and, therefore, a possible in situ domestication.

Integration of statistical and physiological analyses of adaptation of near-isogenic barley lines.

Seven near-isogenic barley lines, differing for three independent mutant genes, were grown in 15 environments in Spain. Genotype x environment interaction (G x E) for grain yield was examined with the Additive Main Effects and Multiplicative interaction (AMMI) model. The results of this statistical analysis of multilocation yield-data were compared with a morpho-physiological characterization of the lines at two sites (Molina-Cano et al. 1990). The first two principal component axes from the AMMI analysis were strongly associated with the morpho-physiological characters. The independent but parallel discrimination among genotypes reflects genetic differences and highlights the power of the AMMI analysis as a tool to investigate G x E. Characters which appear to be positively associated with yield in the germplasm under study could be identified for some environments.

Quantitative phenotypical expression of three mutant genes in barley and the basis for defining an ideotype for Mediterranean environments.

Three mutants induced in the two-rowed barley variety Beka and their three binary recombinants have been used in an attempt to define an ideotype suitable for Mediterranean agroclimatic conditions. Physiological methods (classical plant growth analysis) together with the study of genotype x environment interaction for grain yield were used to characterize the genotypes. That characterization brought out the huge phenotypical variation produced by only three mutant genes, suggesting that single Mendelian genes may alone explain the quantitative variation, including grain yield, without the necessity of using the polygenic concept. The genotype best adapted to the environments studied is later in heading and has shorter straw and denser spikes than Beka; it also has higher inverse of leaf area rate and grain: leaf area ratio, a lower rate of leaf senescence, and a shorter grain filling period than the original variety.

The gene for trypsin inhibitor CMe is regulated in trans by the lys 3a locus in the endosperm of barley (Hordeum vulgare L.).

A cDNA encoding trypsin inhibitor CMe from barley endosperm has been cloned and characterized. The longest open reading frame of the cloned cDNA codes for a typical signal peptide of 24 residues followed by a sequence which is identical to the known amino acid sequence of the inhibitor, except for an Ile/Leu substitution at position 59. Southern blot analysis of wheat-barley addition lines has shown that chromosome 3H of barley carries the gene for CMe. This protein is present at less than 2%-3% of the wild-type amount in the mature endosperm of the mutant Risø 1508 with respect to Bomi barley, from which it has been derived, and the corresponding steady state levels of the CMe mRNA are about 1%. One or two copies of the CMe gene (synonym Itc1) per haploid genome have been estimated both in the wild type and in the mutant, and DNA restriction patterns are identical in both stocks, so neither a change in copy number nor a major rearrangement of the structural gene account for the markedly decreased expression. The mutation at the lys 3a locus in Risø 1508 has been previously mapped in chromosome 7 (synonym 5H). A single dose of the wild-type allele at this locus (Lys 3a) restores the expression of gene CMe (allele CMe-1) in chromosome 3H to normal levels.

Fast-germinating low ß-glucan mutants induced in barley with improved malting quality and yield.

Mutation breeding has been used to improve the speed of germination in the high-yielding spring barley variety Troubadour. Five mutants were selected which combined fast germination and good agronomic performance. Two of these mutants yielded significantly more than did Troubadour over eight environments, and showed a clear improvement in their malting quality through an increase in extract yield. The improvement in malting quality appeared to be due to a decrease in the ß-glucan content, which seemed to enhance the germination speed and thus the starch degradation. The improvement in grain yield is postulated to be due to a better early growth caused by the enhanced germination speed. All the described changes could theoretically be explained by a single mutation event in each of the mutant genotypes, affecting the quantity of ß-glucans present in the endosperm.

Morocco as a possible domestication center for barley: biochemical and agromorphological evidence.

The distribution of genetic variants of a group of low molecular weight, chloroform-methanol soluble proteins (CM proteins), among Moroccan and non-Moroccan accessions of Hordeum spontaneum and among selections from several Moroccan landraces of H. vulgare and cultivars of the same species with widespread European origin, suggests that domestication of barley might have taken place in Morocco. An agromorphological characterization of the H. spontaneum accessions further supports this hypothesis. The possible Moroccan origin of the French cultivar 'Hatif de Grignon' and of several Spanish 6-rowed barleys is also presented.

Genetics of CM-proteins (A-hordeins) in barley.

The CM-proteins, which are the main components of the A-hordeins, include four previously described proteins (CMa-1, CMb-1, CMc-1, CMd-1), plus a new one, CMe-1, which has been tentatively included in this group on the basis of its solubility properties and electrophoretic mobility. The variability of the five proteins has been investigated among 38 Hordeum vulgare cultivars and 17 H. spontaneum accessions. Proteins CMa-1, CMc-1 and CMd-1 were invariant within the cultivated species; CMd was also invariant in the wild one. The inheritance of variants CMb-1/CMb-2 and CMe-1/CMe-2,2' was studied in a cross H. spontaneum x H. vulgare. The first two proteins were inherited as codominantly expressed allelic variations of a single mendelian gene. Components CMe-2,2' were jointly inherited and codominantly expressed with respect to CMe-1. Gene CMb and gene(s) CMe were found to be unlinked. The chromosomal locations of genes encoding CM-proteins were investigated using wheat-barley addition lines. Genes CMa and CMc were associated with chromosome 1, and genes CMb and CMd with chromosome 4. These gene locations further support the proposed homoeology of chromosomes 1 and 4 of barley with chromosomes groups 7 and 4 of wheat, respectively. Gene(s) CMe has been assigned to chromosome 3 of barley. The accumulation of protein CMe-1 is totally blocked in the "high lysine" mutant Riso 1508 and partially so in the high lysine barley Hiproly.