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.     

Diversity and geographical distribution of seed lipoxygenase‐1 thermostability types in barley

To understand the diversity in the thermostability of the seed lipoxygenase‐1 (LOX‐1), 1040 cultivars of worldwide barley (Hordeum vulgare ssp. vulgare) genetic resources were investigated. The relative thermostability of LOX‐1 (LOX‐RTS) in these lines showed a bimodal frequency distribution and these lines were categorized into the high and low thermostability types (H‐type and L‐type, respectively). The H‐type lines predominated in the wild progenitor, ssp. spontaneum. The geographical distribution of these types in the cultivars was surveyed. The frequencies of the H‐ and L‐types were almost equal to one another in southwestern Asia. The occurrence of the H‐type predominated in eastern Asia and Africa, whereas in Europe and Turkey, the L‐type did. The predominance of the L‐type in Europe and Turkey can be understood through the hypothesis that the Fertile Crescent domestication contributed the majority of diversity in Europe. The uneven geographical distribution of the LOX‐1 thermostability types in the cultivars may reflect a polyphyletic origin of barley.     

The Effect of Cultivar and Environment on β-amylase Activity is Associated with the Change of Protein Content in Barley Grains

Eight malt barley cultivars were grown in seven locations with wide ecological differences in China for two successive years to study the cultivar and environmental variation of β‐amylase activity and its association with the change of protein content in grains. The results showed that the variation in β‐amylase activity was mainly attributable to the environment (location and year), although the effect of cultivar was also highly significant. The response of β‐amylase activity to the environment differed markedly among cultivars, reflected by large difference in coefficients of variation for the cultivars grown across diverse locations. The effect of cultivar and environment on protein content showed a pattern similar to that on β‐amylase, but the variations caused by cultivar and, in particular, environment were relatively smaller for protein content than for enzymatic activity. Regression analysis showed that there was a highly significant correlation between protein content and β‐amylase activity for all cultivars but ZAU 3. However, an obvious difference existed in the constants and regressive coefficients of the equation among cultivars, suggesting genetic differences in the effect of the changed protein content on β‐amylase activity.     

Identification of novel alleles from wild barley for the improvement of alpha‐amylase and related malt quality traits

Germplasms consisting of 64 wild barley accessions (Hordeum vulgare ssp. spontaneum) were examined for polymorphisms in α‐amylase using both isoelectric focusing (IEF) and thermostability assays. Wide variation was found for the high pI α‐amylase with 20 IEF band patterns identified. Enzyme activity and thermostability assays showed large differences among α‐amylase isoenzymes. Two wild accessions Tel‐Shoket CPI 77146‐32 and Afiq CPI 77128‐41 showed superior enzyme activity and thermostability compared with commercial varieties such as ‘Baudin’, ‘Flagship’ or ‘Navigator’. The functionality of the Tel‐Shoket allele was validated in backcross lines with ‘Flagship’ as the recurrent parent. The Tel‐Shoket allele at the amy1 locus increased α‐amylase thermostability at 75°C by 8.4% and α‐amylase activity in kilned malt by 18.7%. The introgression of the wild allele also led to significant improvements in fermentability, hot water extract and viscosity. Gene sequencing showed that there are three single nucleotide polymorphisms in the Tel‐Shoket amy1 sequence, which can be used as diagnostic markers for the selection of this allele in breeding programmes.     

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.     

Genetic analysis of resistance in barley to barley yellow dwarf virus

The inheritance of resistance to barley yellow dwarf virus (BYDV) was studied in the selected 24 spring and winter barley cultivars that showed a high or intermediate resistance level in 1994‐97 field infection tests. The polymerase chain reaction diagnostic markers YLM and Ylp were used to identify the resistance gene Yd2. The presence of the Yd2 gene was detected with both markers in all the resistant spring barley cultivars and lines from the CIMMYT/ICARDA BYDV nurseries. The results of field tests and genetic analyses in winter barley corresponded with marker analyses only when the Ylp marker was used. Genes non‐allelic with Yd2 were detected by genetic analyses and the Ylp marker in moderately resistant spring barley cultivars ‘Malvaz’, ‘Atribut’ and ‘Madras’, and in the winter barley cultivars ‘Perry’ and ‘Sigra’. Significant levels of resistance to BYDV were obtained by combining the resistance gene Yd2 with genes detected in moderately resistant cultivars. The utilization of analysed resistance sources in barley breeding is discussed.     

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.     

Postulation of leaf-rust resistance genes in Czech and Slovak barley cultivars and breeding lines

Leaf‐rust resistance (Rph) genes in 61 Czech and Slovak barley cultivars and 32 breeding lines from registration trials of the Czech Republic were postulated based on their reaction to 12 isolates of Puccinia hordei with different combinations of virulence genes. Five known Rph genes (Rph2, Rph3, Rph4, Rph7, and Rph12) and one unknown Rph gene were postulated to be present in this germplasm. To corroborate this result, the pedigree of the barley accessions was analysed. Gene Rph2, as well as Rph4, originated from old European cultivars. The donor of Rph3, which has been mainly used by Czech and Slovak breeders, is ‘Ribari’ (‘Baladi 16’). Rph12 originates from barley cultivars developed in the former East Germany. Rph7 in the registered cultivar ‘Heris’ originates from ‘Forrajera’. A combination of two genes was found in 10 cultivars. Nine heterogeneous cultivars were identified; they were composed of one component with an identified Rph gene and a second component without any resistance gene. No gene for leaf rust resistance was found in 17 of the accessions tested. This study demonstrates the utility of using selected pathotypes of P. hordei for postulating Rph genes in barley.     

Possibility of exploiting the Yd2 resistance to BYDV in spring barley breeding

The effects of the Yd2 gene on tolerance to barley yellow dwarf virus (BYDV) and other agronomically important characters in spring barley were evaluated in a set of randomly selected doubled haploid (DH) lines of an‘Igri’/‘Atlas 68’ cross and three crosses between CIMMYT Yd2 materials and the Czech malting barley ‘Akcent’. The cleaved amplified polymorphic site (CAPS) diagnostic marker Yd2 was used for identification of the Yd2 gene and this analysis showed high agreement with the results of field infection tests. Yd2 lines exhibited significantly lower symptom scores and lower reductions of some grain yield characters, but their resistance level was not consistent over the years. The presence of secondary stresses (high temperature/drought) in 2000 led to relatively higher sensitivity to BYDV infection, strengthened by the long life cycle of genotypes. In cases where secondary stresses were mild (in 2002), the longer life cycle significantly increased sensitivity to BYDV infection only in the absence of the Yd2 gene (in susceptible genotypes). The examination of different vegetative, grain yield and malting quality characters separately for groups of Yd2 and non‐ Yd2 lines did not show any evidence of adverse effect of the Yd2 gene on any character.     

Use of barley seed vigour to discriminate drought and cold tolerance in crop years with high seed vigour and low trait variation

Seed vigour is a precondition for early and homogenous field emergence of barley, in addition to effective malting. This study aimed to assess the selection of barley varieties by using seed vigour as the indicator. Seed vigour of barley (quantified as the germination percentage) was evaluated under drought (?0.2 MPa) and temperature stress (10°C). At two locations over a 3‐year period, 1 population of 133 Derkado × B83‐12/21/5 doubled haploid (DH) lines (and parents) was evaluated for seed vigour, of which 108 DH lines were assessed for three malting parameters. The relatively high values of vigour during the 3‐year period (overall average 94–95%) probably impeded high variations in genetic potential. A total of 27 DH lines of the 133 evaluated showed transgression for vigour (up to 98%) in comparison with the parents (Derkado: 96%; B83: 92%). In conclusion, caution should be applied when selecting for seed vigour, even in good crop years with high levels of seed vigour and low trait variations. Such selection might improve vigour, particularly in crop years with unsuitable weather conditions.     

Powdery mildew resistance in Czech and Slovak barley cultivars

Fifteen powdery mildew resistance genes and the gene MlaN81 derived from ‘Nepal 81’were found in 76 Czech and Slovak spring and winter barley cultivars when tested for reaction to a set of powdery mildew isolates. Nine cultivars (‘Donum’, ‘Expres’, ‘Jubilant’, ‘Orbit’, ‘Primus’, ‘Progres’, ‘Stabil’, ‘Vladan’ and ‘Zlatan’) are composed of lines with different resistance genes. The Mlat gene is present in nine cultivars and was transferred from the Anatolian landrace ‘A‐516′. The resistances derived from ‘KM‐1192’and ‘CI 7672’were identical and designated Ml(Kr). Five winter barley cultivars possess the Ml(Bw) resistance. The winter barley line ‘KM‐2099’carries the mlo gene. The parental cultivar ‘Palestine 10’was also tested in which the genes Mlk1, MlLa were identified. The German cultivar ‘Salome’, a parent of seven cultivars tested, probably carries the gene MlLa in addition to mlo and Mla7. The gene mlo6 may be present in the cultivar ‘Heris’. Most of the results were confirmed by the pedigrees of the cultivars.     

Leaf Photosynthesis During Grain Filling Under Mediterranean Environments: Are Barley or Traditional Wheat More Efficient Than Modern Wheats?

Barley is one of the most popular crops in dryland agricultural systems of Mediterranean areas, where it is assumed that barley, or traditional wheat cultivars, performs better than modern wheat under low‐yielding conditions. It was tested whether variations in net leaf photosynthetic rate (P_N) during grain filling provide any basis for the potential better performance of barley and traditional wheat compared to modern wheats in Mediterranean areas. Two groups of field experiments were conducted in Agramunt (NE Spain) during 2005/06 (06) and 2006/07 (07) growing seasons combining low and high nitrogen (N) availabilities under rain‐fed and irrigated conditions. Cultivars used in the first group of experiments were a traditional (Anza) and a modern (Soissons) wheat, whilst in a second group of experiments, a wheat (Soissons) and a barley (Sunrise) modern cultivars were used. Both wheat cultivars showed a similar P_N during grain filling but higher than that of the modern barley cultivar. Differences between species in P_N were maximized under high‐yielding conditions. There were no differences between cultivars in instantaneous water‐use efficiency. The barley cultivar showed a higher specific leaf area, but lower N content per unit of leaf area, than wheat. Photosynthetic nitrogen‐use efficiency was similar between the traditional and the modern cultivar but lower than barley. Decreases in P_N after anthesis were not exactly observable in SPAD measurements. In conclusion, we found no consistent differences between cultivars in terms of post‐anthesis photosynthetic activity to support the assumption of better performance under Mediterranean farm conditions of traditional wheat or barley against modern wheat.     

Genes for resistance to powdery mildew in European barley cultivars registered in the Czech Republic from 2011 to 2015

Barley (Hordeum vulgare) is cultivated on 49.1 million hectares worldwide with 50.2% of the area located in Europe. Powdery mildew, caused by Blumeria graminis f. sp. hordei (Bgh), occurs wherever barley is grown. Cultivar resistance plays an important role in global barley production, especially in parts of Europe where high concentrations of both spring and winter types are grown. The aim of this report was to postulate specific resistance genes in barleys from nine European countries registered in the Czech Republic from 2011 to 2015. Thirty‐five spring cultivars and 27 winter barleys were tested with 56 diverse Bgh isolates. Twenty‐five known resistance genes were postulated, and unknown genes were detected in Sandra, Saturn and Zeppelin. Unidentified specific resistance genes were also present in winter hybrids Hobbit and Wootan. Spring cultivars Arthur and Francin consisted of three and two genotypes, respectively. Resistance gene mlo was present in 26 spring cultivars, and the proportion of cultivars with this gene increased from 62.9% in 2006–2010 to 75.7% in 2011–2015. The gene Mlp1 was identified for the first time in German winter cultivar Saturn. Five spring cultivars registered in Slovakia were included in the tests. All the cultivars that were tested contained one or more specific resistance genes to powdery mildew. Adaptability of the pathogen and possibilities for breeding winter barleys are discussed.     

Molecular assessment of genetic diversity in winter barley and its use in breeding

Summary During the last decades extensive progress has been achieved in winter barley breeding with respect to both, yield and resistance to fungal and viral diseases. This progress is mainly due to the efficient use of the genetic diversity present within high yielding adapted cultivars and – with respect to resistance – to the extensive evaluation of genetic resources followed by genetic analyses and introgression of respective genes by sexual recombination. Detailed knowledge on genetic diversity present on the molecular level regarding specific traits as well as on the whole genome level may enhance barley breeding today by facilitating efficient selection of parental lines and marker assisted selection procedures. In the present paper the state of the art with respect to virus diseases, i.e. Barley mild mosaic virus, Barley yellow mosaic virus, and Barley yellow dwarf virus is briefly reviewed and first results on a project aiming on a genome wide estimation of genetic diversity which in combination with data on yield and additional agronomic traits may facilitate the detection of marker trait associations and a more efficient selection of parental genotypes are presented. By field tests of 49 two-rowed and 64 six-rowed winter barley cultivars the genetic gain in yield for the period 1970–2003 was estimated at 54.6 kg ha⁻¹ year⁻¹ (r² = 0.567) for the six-rowed cultivars and at 37.5 kg ha⁻¹ year⁻¹ (r² = 0.621) for the two-rowed cultivars. Analysis of 30 SSRs revealed a non-homogenous allele distribution between two and six-rowed cultivars and changes of allele frequencies in relation to the time of release. By PCoA a separation between two and six-rowed cultivars was observed but no clear cut differentiation in relation to the time of release. In the two-rowed cultivars an increase in genetic diversity (DI) from older to newly released cultivars was detected.     

Genetic control over grain damage in a spring barley mapping population

A genetic map was constructed using DNA‐based markers in a barley mapping population derived from the cross ‘Tankard’בLivet’, that was developed to explore the genetic control over grain damage in spring barley cultivars. Quantitative trait loci (QTL) were located for husk skinning, gape between the lemma and palea and splitting of the fused pericarp/testa/aleurone tissues. The QTL accounted for 70% of the genetic variation in Split and 60% of the genetic variation in Gape and Skinning. The QTL were clustered on chromosomes 1H, 4H, 5H, 6H and 7H. QTL analysis indicates the possibility of transgressive segregation for grain splitting and so the breeding of lines with more extreme splitting. This is of concern to the malting industry as, without extensive phenotypic assessment, such lines could be commercialized, as was the case of Landlord, and put malting barley supplies at risk. These findings are discussed in relation to the genetic control over traits including grain length and width.     

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.     

The transfer of a gene conferring resistance to scald (Rhynchosporium secalis) from Hordeum bulbosum into H. vulgare chromosome 4HS

Scald is a serious foliar disease that infects barley (Hordeum vulgare L.) causing reduced yields and adversely affecting quality. A means to combat the disease is to breed cultivars that possess genetic resistance. However, all known resistance alleles have so far originated from within the primary genepool of barley. This reliance on H. vulgare and H. vulgare ssp. spontaneum as resistance sources may encourage virulent forms of the pathogen to become established. To broaden the genetic base of cultivated barley and provide novel resistances to many diseases we have used a species from the secondary genepool of barley, H. bulbosum, in a resistance‐breeding programme. In this study we describe the development and trialling of a scald‐resistant recombinant line derived from a hybrid between H. vulgare and H. bulbosum. The scald resistance is simply inherited and located on the short arm of barley chromosome 4 (4HS).     

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.     

Geographical differentiation and diallel analysis of seed dormancy in barley

Seed dormancy is one of the most important parameters affecting the malting process and pre-harvest sprouting in barley (Hordeum vulgare L.). Variation of seed dormancy in 4365 cultivated and 177 wild barley (ssp. spontaneum) accessions derived from different regions of the world was investigated in Okayama University, Kurashiki, Japan. Seed dormancy of each accession was estimated from their germination percentages at 0, 5, 10 and 15 weeks post-harvest after-ripening periods. All of the wild barley accessions showed less than 10% germination at 0 week after-ripening period. Level of seed dormancy in 4365 cultivated barley accessions showed a clear geographical differentiation. Seventy seven percent of Ethiopian accessions showed high germination percentages, while 86% of Japanese, Turkish and North African accessions showed low germination percentages at 0 week after-ripening period. A half diallel cross using eleven barley accessions with different level of dormancy revealed that seed dormancy was predominately controlled by additive gene effects. These results suggest that large genetic diversity for seed dormancy in barley is explained as different levels of additive accumulation of genetic factors. Barley varieties showing appropriate dormancy could be developed by crossing among barley germplasm accessions used in the present study.     

Genetic diversity among elite Bulgarian barley varieties evaluated by RFLP and RAPD markers

Genetic variation among five elite winter barley cultivars (H. vulgare L.) currently grown in Bulgaria was assessed at the molecular level using restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) markers. The present study sampled RFLPs in four well characterized multigene families in barley: the seed storage protein loci; the 18S, 5.8S and 26S ribosomal DNA loci; the loci coding for 5S ribosomal RNA and the loci coding subunit α of ATP-A complex in the mitochondrial genome. RFLPs were detected in three out of five investigated chromosomal loci in the barley cultivars studied. RAPD assay using arbitrary 10-base primers was applied to generate amplified length polymorphic markers in barley. Overall a total of 15 polymorphic phenotypes were found among the studied barley cultivars by using 11 out of 25 tested primers. All RAPDs were considered as dominant genetic markers except for two, where PCR and Southern blot analysis indicated the presence of codominant amplification products. Five RAPD polymorphisms in F₁ and F₂ progenies of the cross between Alpha and Obzor were inherited in Mendelian fashion. The determined values for the genetic variation proved a high genetic similarity among the tested cultivars. Genetic similarity (GS) calculated from RFLP and RAPD data ranged from 0.888 to 0.997 with a mean GS – 0.933. This revised version was published online in July 2006 with corrections to the Cover Date.