Mapping a factor controlling the thermostability of seed lipoxygenase-1 in barley

Barley lipoxygenase (LOX)‐1 is believed to affect the stability of flavour and the foam of beer. The purpose of this study was to investigate the genetic variation of the LOX‐1 thermostability, and to analyse the mode of inheritance of this trait. A simple method was established to evaluate the LOX‐1 relative thermostability (LOX‐RTS). With this method, 153 barley cultivars were screened for LOX‐RTS. The frequency of the LOX‐RTS values was distributed in a bimodal manner. Based on these values, the barley lines were categorized into two groups: an H‐type with relatively thermostable LOX‐1 and an L‐type with relatively thermolabile LOX‐1. Using a ‘Steptoe’/‘Morex’ doubled haploid population, a major quantitative trait locus (QTL) associated with LOX‐RTS was identified on chromosome 4H of barley, explaining 82% of the variance. Mapping of a CAPS marker specific for the LoxA locus revealed co‐segregation with this QTL. In this study, the existence of the thermostability types of barley seed LOX‐1 and the locus controlling the thermostability were made clearer.     

Ribosomal DNA spacer-length polymorphisms in three samples of wild and cultivated barleys and their relevance to the origin of cultivated barley

This study was conducted to provide additional data for evaluating two important issues surrounding the origin of cultivated barley: (i) the level of genetic diversity of the two‐rowed wild barley from Tibet, and (ii) the distribution of rDNA allele 104 in wild and cultivated barleys in the Occidental region. A total of 198 accessions consisting of three distinct samples were used: 82 entries of two‐rowed wild barley from Tibet, 57 accessions of two‐rowed wild barley from 8 countries with a broad range of representation of two‐rowed wild barley in the world, and 59 landrace accessions from four countries representing a part of the barley‐growing areas in the Middle East. These were assayed for rDNA spacer‐length variants (slvs). In all, 27 rDNA space length pheno types were detected, from which 10 slvs were identified as alleles at the two rDNA loci. The two‐rowed wild barley samples from Tibet had the lowest level of genetic variation as evaluated by rDNA polymorphism. Together with results of previous studies, the two wild forms (two‐rowed and six‐rowed) from Tibet could not account for the large genetic diversity observed in the cultivated barley of this region, suggesting that Tibet is unlikely a centre of origin for cultivated barley. In samples from the Occidental region, allele 104 of Rm2 was very rare in wild barley, but occurred at the highest frequency in cultivated barley, while the reverse is the case for allele 107, which is consistent with previous results. The implications of such a contrasting distribution of these rDNA alleles between wild and cultivated barleys in the origin and evolution of cultivated barley were discussed.     

Genetic diversity and population structure of wild and cultivated barley from West Asia and North Africa

Population structure and relationships within and among 185 accessions of wild (Hordeum vulgare ssp. spontaneum) and cultivated barley (H. v. ssp. vulgare) from five countries in the West Asia and North Africa (WANA) region were studied using 36 simple sequence repeat (SSR) markers. The accessions were divided into subspecies/origin (S/O)‐groups and marker results were analysed in relation to genetic diversity and genetic structure. Wild barley from WANA was found to be highly diverse. The landraces from different countries of the Near East showed genetic diversity that was nearly as high as the wild barley from the same country. Further analyses showed that wild barley from Palestine/Israel represented the group with the highest diversity and the most complex structure. However, this group was distantly related to the cultivated barley in WANA, while the wild barley from the rest of the WANA region was closely related to the cultivated WANA barley. The high diversity and the close relationship to the wild barley make the WANA landraces an interesting genetic resource for both conservation and exploitation.     

Screening for resistance in the primary and secondary gene pool of barley against the root-lesion nematode Pratylenchus neglectus

Root-lesion nematodes of the genus Pratylenchus are significant pests in crop cultivation throughout many parts of the world. A study was initiated to determine the resistance of Hordeum vulgare and H. vulgare ssp. spontaneum (wild barley) against one major representative of the genus Pratylenchus , P. neglectus . A glasshouse test was first established. Barley seedlings were grown in 20 cm³ tubes filled with sand. Each plant was inoculated with 400 P. neglectus juveniles. After 12 weeks of cultivation nematodes were isolated from roots and sand using a misting chamber. The nematodes were counted under a microscope. A representative collection of 565 barley and wild barley accessions was tested in this way. The average number of nematodes per accession ranged from 350 to 12 000. In a verification experiment, 35 accessions with low and high infection rates were tested. This experiment identified a number of accessions with low infection rates. The perspectives for future breeding of barley cultivars resistant to root-lesion nematodes are discussed.     

A novel barley scald resistance gene: genetic mapping of the Rrs15 scald resistance gene derived from wild barley, Hordeum vulgare ssp. spontaneum

Most genes for resistance to barley leaf scald map either to the Rrs1 locus on the long arm of chromosome 3H, or the Rrs2 locus on the short arm of chromosome 7H. Other loci containing scald resistance genes have previously been identified using lines derived from wild barley, Hordeum vulgare ssp. spontaneum. A single dominant gene conditioning resistance to scald was identified in a third backcross (BC3F3) line derived from an Israeli accession of wild barley. The resistance gene is linked to three microsatellite markers that map to the long arm of chromosome 7H; the closest of these loci, HVM49, maps 11.5 cM from the resistance gene. As no other scald resistance genes have been mapped to this chromosome arm, it is considered to be a novel scald resistance locus. As the Acp2 isozyme locus is linked to this scald resistance locus, at 17.7 cM, Acp2 is assigned to chromosome 7H. Molecular markers linked to the novel scald resistance gene, designated Rrs15, can be used in breeding for scald resistance.     

Genetic variation of β-amylase thermostability among varieties of barley, Hordeum vulgare L., and relation to malting quality

To investigate variation in the thermostability of β-amylase among varieties of barley, Hordeum vulgare L., crude enzyme was extracted from the seeds, and the relative remaining activity was calculated after heat treatment. Our results indicated that the varieties tested were divided into three groups (types A, B and C). All the latest Japanese malting varieties showed high themostability (type A), while European, North American and Australian varieties showed intermediate (type B) or low thermostability (type C). Isoelectric focusing (IEF) of β-amylase was also investigated. Type B varieties divided into two subtypes (types B1 and B2) based on two different IEF patterns (types I and II), whereas those of types A and C showed only one IEF pattern (type II). We also examined their thermostability in the varieties based on the pedigrees of the Japanese malting barley. Our results indicated that the thermostability of β-amylase had a close relationship to fermentability for the production of beer. This fact suggests that the thermostability of β-amylase has a significant influence on the malting quality of barley.     

Geographical variation of β-amylase thermostability among varieties of barley (Hordeum vulgare) and β-amylase deficiency

Investigations onto the thermostability of β-amylase in 274 varieties of barley (Hordeum vulgare L.) indicated that all varieties except one were distributed into three types of high (type A), intermediate (type B), and low (type C) thermostability, respectively. One variety (TB29) from China showed no β-amylase activity. Geographical variation was observed in the thermostability of β-amylase. Type C varieties were not observed in East Asia (Japan, the Korean Peninsula, China and Nepal), although 36 out of 37 varieties in Ethiopia were type C. Most of the varieties were Type A in Japan, the Korean Peninsula and China, whereas the frequency of type A and type B were nearly equal in Nepal. Varieties in the other five areas (North America, North Africa, Southwest Asia, Turkey and Europe) consisted of types A, B and C. These results support the fact that East Asian cultivars are genetically different from those of the western regions, as previously reported.     

Worldwide distribution of β-amylase thermostability in barley

The thermostability of β-amylase in 6752 lines of worldwide barley genetic resources were investigated. Most of the lines were classified into high (type A), medium (type B) , and low (type C) thermostability. Subsequently the geographical distribution of these types was clarified. About 90% of the East Asian (Japan, the Korean Peninsula, China) lines were type A. More than 95% of Ethiopian barley was type C. The thermostability types of varieties in the western areas (north Africa, southwest Asia, Turkey, Europe) consisted of types A, B and C. These results suggest that there is a clear geographical differentiation in β-amylase thermostability, especially in East Asia and Ethiopia. The phenotype characteristics of each thermostability type line reflected the geographical differentiation. Besides types A, B and C, we found new thermostability types, including such useful mutants as a β-amylase-less mutant and highly-thermostable mutants than type A in both China and Nepal. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mapping of Malt Endopeptidase, NADH Diaphorase and Esterase Loci on Barley Chromosome 3L

Isoelectric focusing (IEF) and immunoblotting were used to detect genetic variants of malt endopeptidase (MEP1), an enzyme related to malting quality in barley and coded by the CepB locus on barley chromosome 3 (= 3H). A variant was found in a Turkish accession of wild barley (Hordeum vulgare ssp. spontaneum). The self progeny of a hybrid between this accession and the barley cultivar ‘Clipper’ were analyzed for recombination between the CepB locus and other isozyme loci. The estimates of recombination linked the CepB locus to an NADH diaphorase locus (Ndh2), which in turn was linked to the seedling esterase complex (Est1, Est2, and Est4) situated near the distal end of the long arm of chromosome 3. The Ndh2 locus was independent of two other NADH dehydrogenase loci (Ndh3, Ndh5) which were mapped on barley chromosome 5 (= 1H) in relation to the three hordein loci (Hor1, Hor2, and Hor3).     

Genetic analysis of β-amylase thermostability to develop a DNA marker for malt fermentability improvement in barley, Hordeum vulgare

β‐Amylase thermostability is one of the major factors affecting fermentability in the brewing process; consequently, it could be used as a selection marker for the trait. In order to clarify what controls its thermostability, the linkage analysis of β‐amylase thermostability and its genotype as restriction fragment length polymorphism patterns was performed in three cross populations. Then, β‐amylase cDNAs cloned from the three varieties which had a different thermostability type were expressed in Escherichia coli. According to the results of the linkage analysis and gene expression test, it was concluded that β‐amylase thermostability resulted from a difference in its structural gene. Furthermore, to construct an STS marker for the gene, the gDNA sequences of β‐amylase were compared among the three varieties, which had different thermostabilities. Although there were many differences in the intron sequence, few nucleotides differed in the exon region. Based on the variation in the intron region, a sequence‐tagged‐site marker was constructed to detect β‐amylase genotypes in breeding material.     

Genetic regulation of growth and nutrient content under phosphorus deficiency in the wild barley introgression library S42IL

Phosphorus (P) is an important macronutrient required for plant growth and yield formation. Since decades, breeders aim to optimize P efficiency in crops. We studied a set of 47 wild barley (Hordeum vulgare ssp. spontaneum, Hsp) introgression lines (ILs) in hydroponic culture to identify quantitative trait loci (QTLs) improving growth and nutrient content under P deficiency. Applying a mixed model analysis, a total of 91 independent QTLs were located among 39 ILs, of which 64 QTLs displayed trait‐improving Hsp effects. For example, an unknown Hsp allele on barley chromosome 4H increased shoot dry weight under P deficiency in three overlapping ILs by 25.9%. Likewise, an Hsp allele on barley chromosome 6H increased root dry weight under P deficiency in two overlapping ILs by 27.6%. In total, 31 QTLs confirmed Hsp effects already identified in previous field and glasshouse experiments with the same ILs. We conclude that wild barley contains numerous trait‐improving QTL alleles, which are active under P deficiency. In future, the underlying genes can be subjected to cloning and, simultaneously, used in elite barley breeding.     

Genetic mapping of the barley Rrs14 scald resistance gene with RFLP, isozyme and seed storage protein markers

The scald susceptible barley cultivar ‘Clipper’ and a third‐backcross (BC₃) line homozygous for the Rrs14 scald resistance gene that originally came from Hordeum vulgare ssp. spontaneum were grown in replicated field trials. The level of resistance that Rrs14 confers against field populations of the pathogen Rhynchosporium secalis, the causal agent of scald disease, was evaluated. The Rrs14 BC₃ line exhibited 80% and 88% less leaf damage than ‘Clipper’ in 1995 and 1996, respectively. Given this effectiveness of Rrs14, research was undertaken to identify a linked marker locus suitable for indirect selection of Rrs14. Based on linkage to a set of previously mapped loci, Rrs14 was positioned to barley chromosome 1H between the seed storage protein (hordein) loci Hor1 and Hor2, approximately 1.8 cM from the latter locus. The Hor2 locus is thus an ideal codominant molecular marker for Rrs14. The tight linkage between Rrs14 and Hor2 and the availability of alternative biochemical and molecular techniques for scoring Hor2 genotypes, permits simple indirect selection of Rrs14 in barley scald resistance breeding programmes.     

Genetic and non-genetic factors affecting germination and vitality in spring barley seed

Germination of eight spring barley varieties, grown in two years at eight different locations in State varietal trials in the Czech republic, was analysed. All the combinations were tested under optimal conditions for germination as recommended by ISTA and also at low temperatures (5 and 10°C), and underwater stress (- 2 and - 4 bar). Germination under low temperature and water stress was considered as a character of seed vitality. Varieties in nearly all experiments were a significant source of variation and their relative contribution to the total variation was higher for vitality (5.7–38.7%) than for germination (2.0–4.3%). However, the percentage of germination were not related to the percentage of vitality. The location in nearly all the experiments caused the greatest variation and the impact was greater for germination than for vitality. Provenance effects were different for germination and for vitality; grains harvested in the lowland had the highest germination values, but not the highest vitality values. The results should encourage breeding for seed vitality and some reconsideration of provenance effects on seed vitality.     

Evaluation of cultivated barley (Hordeum vulgare) germplasm for the presence of thermostable alleles of β-amylase

A cleaved amplified polymorphic sequence marker was used to detect the alleles Bmy‐Sd2H and Bmy‐Sd3 identifying highly thermostable isoforms of the enzyme b‐amylase, which improves fermentability during brewing. Among the 889 accessions of barley (Hordeum vulgare) investigated, and two accessions of H. spontaneum a total of 166 accessions were identified carrying the superior b‐amylase alleles. These thermostable alleles of b‐amylase were most frequently observed in six‐rowed varieties originating from Asia, especially Japan, with 61.9% of the accessions from Asia carrying the alleles of interest. Additional six‐rowed barleys carrying the relevant alleles were identified among accessions from America, Africa and the Near East. In the European varieties, the percentage of accessions with the alleles of interest was 5.1% with a strong predominance in two‐rowed spring barleys. A pedigree analysis identified the cross ‘Binder’ x ‘Gull’ as the main source of the thermostable b‐amylase alleles in European varieties. The data suggest that an improvement of malting quality in barley could be achieved by introduction of the Bmy1‐Sd2H and Bmy1‐Sd3 alleles into the European breeding programmes.     

Associations of simple sequence repeats with quantitative trait variation including biotic and abiotic stress tolerance in Hordeum spontaneum

A total of 33 simple sequence repeats (SSRs) was analyzed in 52 genotypes of Hordeum spontaneum originally collected from two different soil types (Terra rossa and Basalt) at Tabigha in Israel. Data on the performance of developmental, morphological, and yield‐related traits under well‐watered control and water‐stress conditions were available from previous experimentation, and powdery mildew susceptibility was scored. Regression analyses based on SSR allele class differences were performed. Highly significant associations were detected at the SSR loci Bmac181 (on chromosome 4H) and Bmac316 (6H) for water ‐stress tolerance and powdery mildew resistance, respectively. The study shows that association mapping using SSRs and genetically diverse germplasm provides an effective means of relating genotypes to complex quantitative phenotypes.     

Outcrossing rate of barley genotypes with different floral characteristics in drought‐stressed environments in Jordan

The flowering characteristics of a highly inbreeding species such as barley might influence the extent of outcrossing that can be expected. Increased outcrossing is a prerequisite to exploit heterosis in inbreeding species, which can lead to considerable yield increase as has been demonstrated for barley grown under marginal conditions. Anther extrusion (AE) in barley has been demonstrated to be a genetically controlled, quantitative trait with intermediate to high heritability. Some authors consider AE and open flowering in barley as identical. The present study was conducted to investigate whether barley genotypes with increased AE exhibit increased outcrossing rates under field conditions in comparison with genotypes showing no AE. Field experiments were conducted in three contrasting environments in Jordan in the 2001/02 growing season using six adapted barley genotypes, three with high AE and three with no AE. Outcrossing rates were investigated employing codominant DNA markers (microsatellites). No significant difference in outcrossing rates was detected between anther‐extruding and non‐extruding barley genotypes. Results indicate that high AE is not a sufficient condition to cause increased outcrossing rates and thus cannot be termed open flowering.     

Regional distribution of resistances to powdery mildew in winter and spring barley cultivars grown in the northern part of France

The spatio-temporal distribution of race-specific resistances to powdery mildew was analysed in northern France (the east, the north and the west of Paris). Resistances were identified in 26 winter and six spring barley cultivars. Seedling leaf segments were inoculated with 20 powdery mildew isolates, chosen to identify 14 resistance alleles. As opposed to other European countries, the resistance alleles differed between winter and spring cultivars grown in the three regions. Most of the winter cultivars had no resistance allele, or only the widespread resistance alleles Mlra and/or Mlh, plus Mlg in the west. Mla9 and Mla13 were also present in the north, but at a low frequency. Spring cultivars carried the alleles Mla7, Mla9, Mla12, Mlk, Mlg or MlLa in the east, where a diversification of resistances has occurred since 1987, particularly because of the use of ‘Volga’ (Mla7, Mlk, Mlg and MlLa). In the north and the west, Mla12 dominated after a decrease in the frequency of Mla7, Mla13 has recently been introduced in the north and the west with the cultivar ‘Vodka’.     

Genetic variation for dry matter and nitrogen accumulation and translocation in two-rowed spring barley: I. Dry matter translocation

A 4-year field study was carried out to determine dry matter and nitrogen accumulation until anthesis and at grain filling period and dry matter translocation and utilization in grain filling of barley. Twenty two-rowed spring barley (Hordeum vulgare ssp. distichum L.) cultivars originated from different countries (Yugoslavia, Germany, Australia, the Czeck Republic, Netherlands, France and USA) were grown during 1995–1998 on a non-calcareous chernozem soil near Novi Sad (45° 20′N, 15° 51′E, 86 m asl). Dry matter and nitrogen accumulation depended on the cultivar and year. In a year with favorable weather conditions, 58% of dry matter was accumulated during pre-anthesis, while in a year with less favorable weather the amount was 48%. In the favorable year 91% and in unfavorable year 65% of nitrogen was accumulated until anthesis. The results indicated that the greater amount of dry matter and nitrogen accumulated before anthesis. Dry matter translocation efficiency depended on the cultivar and ranged from 3 to 16.4%, while the contribution of pre-anthesis assimilates to kernel varied from 4 to 24.2%. Cultivars that have been developed for the growing conditions of the area where the experimental site was located, i.e. adapted ones, did not use pre-anthesis dry matter for grain filling. High positive correlations (P<0.01) were found between biomass at anthesis and biological yield, dry matter translocation efficiency, contribution of translocated dry matter to grain yield, and total plant nitrogen at maturity. Accumulated nitrogen at anthesis was positively correlated (P<0.01) with growing degree–days until anthesis, dry matter at anthesis and dry matter translocation parameters. Heritability for the investigated characters was rather high, over 0.60.     

Tolerance of barley seed germination to cold- and drought-stress expressed as seed vigour

In barley, high seed vigour is a precondition for rapid and homogenous field emergence and good malting quality. Seed vigour was defined as germination percentage under stress conditions (10°C, drought stress ‐ 2 Bars) in 7‐8 barley varieties grown in 7‐8 locations in the Czech Republic over 7 years. Three of the 7 years were not suitable for high seed quality, probably because of unsuitable weather, as average seed vigour reached only 61, 77 and 86%, respectively. In the remaining 4 years, the average vigour exceeded 94%. The impact of variety on seed vigour was higher in the ‘bad years’ and the impact of location was higher in the other years. Varieties with higher vigour from all locations in the ‘bad years’ were identified. Lower vigour was related to the high occurrence of fungi (indicated by ergosterol assays) and to lower field emergence rates of seed samples. The results support the possibility of selecting for improvement of barley seed vigour, which is related to tolerance to various conditions during emergence and to homogenous malting.