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.     

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.     

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.     

Evaluation of Czech spring malting barleys with respect to the β-amylase allele incidence

Strong enzymatic activities in the germinating barley grain, together with protein and starch content, are crucial for high extraction values in the resulting malt and, therefore, barley malting quality. The efficient characterization of registered barley cultivars and genetic resources with respect to one of the relevant thermostability enzymes (β‐amylase) is an essential requirement. The template‐directed dye‐terminator incorporation (TDI) assay: based on flourescence resonance energy transfer (TDI‐FRET) ( Chen et al. 1995 ) was used to detect single nucleotide polymorphisms (SNPs) in the β‐amylase coding sequence resulting in low (Sd2L), intermediate (Sd1) and high (Sd2H and Sd3) thermostability enzyme across 84 Czech barley cultivars and genetic resources used over a period of time in the Czech Republic. The incidence of different alleles has changed during the last 100 years. Also the new resources with high thermostabile β‐amylase were identified. They can be used effectively to breed for malting quality improvement.     

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.     

Heredity of Resistance to Botrytis cinerea in Sunflower (Helianthus annuus) Measured by Artificial Infections

Genetic Variability for Some Brewing Quality Characters in Barley Malting and brewing quality of six different barley genotypes and their F1 hybrids were investigated in a diallel cross programme. Plants were grown in pots in a randomized block design with four replications. General and specific combining abilities were determined for 1000 grain weight, percentage of proteins of the grain, percentage of water uptake, and, after micromalting, for β-glucanase and α-amylase activities. Significant general and specific combining abilities were observed for all the traits studied. 1000 grain weight, percentage of proteins and water uptake have been found to be essentially controlled by genetic additive effects, while enzymatic activities of malt have been found to be mostly controlled by dominance effects. ‘Karl’ has interesting characteristics for all traits and general combining abilities, except for 1000 grain weight, and has been introduced in a three-way cross programme with ‘Plaisant’ and ‘Clarine’, also showing interesting values for malting quality and productivity.     

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.     

Locating supplementary RFLP markers on barley chromosome 7 and synteny with homoeologous wheat group 5

In order to develop QTL applications, eight new loci were mapped on barley chromosome 7 using 124 doubled haploid lines of the North American Barley Genome Mapping Project (NABGMP) progeny (‘Steptoe’בMorex’)- These loci involve six genomic DNA restriction fragment length polymorphisms (RFLPs) and two cDNA-RFLPs including a puroindoline gene. The distribution of these markers on barley chromosome 7 was compared with that of homoeologous wheat counterparts, i.e. wheat group 5. One locus on chromosome 7 was associated with a QTL for β-glucanase activity measured in green and finished barley malt.     

Vererbung des β-Glucangehaltes der Haferkaryopse

Inheritance process for β-glucan content in oat caryopsis
Due to the fact that there are only few studies concerning β-glucans in oat caryopsis with regard to plant-breeding aspects, three oat crosses were performed (cross 19 = low × high, cross 86 = low × low, cross 98 = high × high). In all of the three oat crosses, lower β-glucan content (% DM) was recorded in F, than in F, indicating that the β-glucan content (% DM) at a higher heterozygoty grade is lower than at a reduced heterozygoty grade. This points to a dominant inheritance of lower β-glucan content (% DM). A tendency towards the female β-glucan content (% DM) could not be proven. Thus, it can be started from a nuclear genetic inheritance of the β-glucan content (% DM). In most cases, the experiments showed a normal distribution of β-glucan content (% DM), indicating for several genes a quantitative inheritance of β-glucan content (% DM). By means of the regression of the F₃-lines to the F₂-plants, the narrow-sense heritability could be calculated for the cross 86 (h²= 0.51***) and the cross 98 (h²= 0.48***). The heritability for the cross 19 was not significant.     

Identification of RAPD markers closely linked to the mlo-locus in barley

Developing resistance to powdery mildew, Erysiphe graminis f.sp. hordei, is a major goal of many barley breeding programmes. Several resistance genes have been tagged or mapped with molecular markers. The mlo gene confers durable resistance towards all known isolates of the pathogen. In this study, RAPD markers and bulked segregant analysis were used to determine PCR-based markers linked to the mlo-locus. Sixty doubled haploid lines from a cross between an isogenic line of ‘Ingrid’ carrying the mlo11 allele and a susceptible cv. ‘Pokko’ were used as plant material. Seven linked RAPD markers were found, the closest lying 1.6 cM away from the resistance gene. When eight barley varieties were assayed for the presence of this band, F4-980, it was found in the resistant varieties but not in the susceptible ones. The linked marker bands could be amplified from DNA-samples prepared by using three different methods, including a quick squash technique. PCR-based markers linked to the resistance gene can be used as tools for selection in breeding programmes.     

Molecular mapping of greenbug (Schizaphis graminum) resistance gene Rsg1 in barley

The greenbug, Schizaphis graminum (Rondani) is an extremely damaging aphid pest of barley (Hordeum vulgare L.) particularly in the southern Great Plains of the USA. The simply inherited, dominant resistance gene Rsg1 is in all greenbug‐resistant US barley cultivars. In this study, we conducted molecular mapping of Rsg1 using an F_2:3 population derived from a cross between the greenbug‐resistant Post 90*4/R015 and susceptible CI2260 inbred lines. Segregation of host responses to greenbug biotype E infestation confirmed that a single dominant gene is responsible for greenbug resistance in Post 90*4/R015. Simple sequence repeat (SSR) markers evenly distributed along the seven barley chromosomes were employed for the construction of a framework genetic map. Linkage analysis placed the Rsg1 locus in the long arm of chromosome 3H (3HL) flanked by SSR markers Bmag0877 and GBM1420 that were 35 cM apart. Polymorphic single‐nucleotide polymorphism (SNP) markers in 3HL were identified from an Illumina GoldenGate SNP assay and used for targeted mapping to locate Rsg1 to an 8.4‐cM interval. Comparative analysis identified syntenic genomic regions in Brachypodium distachyon chromosome 2, in which 37 putative genes were annotated including a NB‐LRR‐type resistance gene homologue that may be a potential candidate gene for the Rsg1 locus of barley. Results from this study offer a starting point for fine mapping and cloning of this aphid resistance gene in barley.     

Molecular analysis of the barley cv. 'Valticky' and its X-ray-derived semidwarf-mutant 'Diamant'

The outstanding semidwarf spring barley cultivar ‘Diamant’ was derived from the cv.‘Valticky’ by X‐ray mutagenesis. More than 120 European spring barley varieties trace back to ‘Diamant’. This variety and nine German spring barley cultivars having ‘Diamant’ in their pedigree, as well as four cultivars not related to ‘Diamant’, were analysed by amplified fragment length polymorphisms (AFLPs) and simple sequence repeats (SSRs) in order to identify genomic regions associated with traits derived from ‘Diamant’. The total number of SSRs tested was 122, of which 88 were polymorphic taking all varieties into account, with 51 for ‘Valticky’ and ‘Diamant’, i.e. 42.8%. A varying level of polymorphism was detected on individual chromosomes (11.1–78.6%). In addition, AFLP revealed a high level of polymorphism. Out of the total number of 1591 bands, 670 were polymorphic for all varieties studied with 182 for ‘Valticky’ and ‘Diamant’, i.e. 11.4%. Based on SSR and AFLP data UPGMA cluster analysis was carried out, but no grouping according to the relatedness with ‘Diamant’ or ‘Valticky’, respectively, was found. It is concluded from these results that the mutagenic effect leading to the development of ‘Diamant’ out of ‘Valticky’ affected many genetic loci and is thus larger than expected.     

Multivariate analysis of traits determining adaptation in cultivated barley

Thirty‐nine barley varieties of different origin, representing different growth types, were included in a series of experiments aimed at analysing the variability in vernalization response, photoperiod sensitivity and earliness per se and establishing the types of ecoclimatic adaptability using multivariate analysis. In the case of spring barley varieties there was no correlation between any of the three traits. For winter barleys, a negative correlation was found between photoperiod sensitivity and vernalization response and between photoperiod sensitivity and earliness per se. Vernalization response and earliness per se showed a positive correlation. Among the winter barley varieties large variations were apparent in photoperiod sensitivity, vernalization response and earliness per se, which resulted in a tremendous variation in flowering patterns and frost tolerance. Between the spring barley varieties only wider variations in photoperiod sensitivity were detected. Based on the cluster analysis, the 39 varieties could be separated into seven groups. The spring barley varieties were placed in two groups, and the winter barleys in five groups representing different adaptational types. Among these five groups two represented the two opposing extreme combinations of photoperiod sensitivity and vernalization response. The combination of large photoperiod sensitivity and no vernalization response resulted in better frost tolerance than did the combination of photoperiod insensitivity and large vernalization response.     

Molecular mapping of the leaf rust resistance gene Rph7 in barley

Leaf rust of barley, caused by Puccinia hordei Otth, is an important foliar disease in most temperate regions of the world. Sixteen major leaf rust resistance (Rph) genes have been described from barley, but only a few have been mapped. The leaf rust resistance gene Rph7 was first described from the cultivar ‘Cebada Capa’ and has proven effective in Europe. Previously mapped restriction fragment length polymorphism (RFLP) markers have been used to determine the precise location of this gene in the barley genome. From the genetic analysis of a ‘Bow‐man’/‘Cebada Capa’ cross, Rph7 was mapped to the end of chromosome 3HS, 1.3 recombination units distal to the RFLP marker cMWG691. A codominant cleaved amplified polymorphic site (CAPS) marker was developed by exploiting allele‐specific sequence information of the cMWG691 site and adjacent fragments of genomic DNA. Based on the large amount of polymorphism present in this region, the CAPS marker may be useful for the marker‐assisted selection of Rph7 in most diverse genetic backgrounds.     

RFLP diversity within and between major groups of barley in Europe

Restriction fragment length polymorphism (RFLP) diversity has been determined and analyzed as expressed by 33 single‐ or low‐copy clone/ enzyme combinations at 32 loci distributed over all chromosomes of the barley genome within a sample of 223 European barley accessions comprised of pure line (single‐head progenies) genotypes. The accessions have been selected to include landraces and widely grown cultivars derived from crossbreeding during the 20th century in North‐, West‐ and Central European countries. Genetic diversity obtained from 83 alleles across all accessions is characterized by the diversity index H = 0.385. The diversity indices determined for landraces and cultivars were almost equal, with the difference between spring (H = 0.260) and winter (H = 0.415) barley approaching statistical significance, while comparisons of other groupings only revealed statistically insignificant trends. A more detailed analysis based on differences in allele frequency distributions at each locus (clone/enzyme combinations resp.) revealed very clear differences related to the existence, continuity and dynamics of changes in group‐specific RFLP profiles. With the majority (69%) of RFLP alleles at 23 out of 32 loci on all barley chromosomes involved, contributions from chromosomes 1H, 3H, 4H and 5H seem to be of special importance. Differences in the overall average of abundance indicate higher levels of genetic diversity within both groups of winter barley compared with both groups of spring barley, from which the most frequent alleles at 15 (2‐rowed spring barley) and 17 (6‐rowed spring barley) RFLP loci approach fixation. The results of this study are discussed in relation to the history of barley cultivation and barley breeding in Europe, and possible explanations for group‐specific differences in the RFLP profiles of landraces and cultivars as well as for the high levels of (nearly) fixed alleles of both subsets of spring barley, and with respect to progress in barley breeding that it has been possible to obtain within the rather narrow RFLP profiles.     

Marker-assisted backcross introgression of the Yd2 gene conferring resistance to barley yellow dwarf virus in barley

YLM, a codaominant polymerase chain reaction (PCR) marker linked to Yd2, could substantially improve the precision and efficiency of barley yellow dwarf virus (BYDV) resistance breeding. The aim of this study was to assess the effectiveness of YLM in a marker‐assisted introgression programme and to quantify associations between the presence of Yd2 and other agronomic and quality traits. The Yd2 gene was introgressed into a BYDV‐susceptible background through two cycles of marker‐assisted backcrossing. BC₂ F₂‐derived lines, either carrying or not carrying the YLM allele associated with resistance, were compared in the presence and absence of BYDV. The YLM marker was shown to be effective in the introgression of Yd2. Lines carrying the YLM allele associated with resistance produced significantly fewer leaf symptoms and showed a reduction in yield loss when infected with BYDV. There were no deleterious effects associated with the introgression of Yd2 on grain yield, grain size or malting quality. The implications of marker‐assisted selection for Yd2 on barley improvement are discussed.     

Genetic analysis of tissue culture traits in barley cv. 'Lenins'

The barley cultivar ‘Lenins’ was found to be a genotype showing high shoot regeneration ability in cultures derived from immature embryos. Five cultivars different from ‘Lenins’ in shoot regeneration ability were reciprocally crossed with ‘Lenins’ and the inheritance of tissue culture traits was investigated. F₂ plants showed continuous distributions in callus growth and percentage of shoot regeneration, suggesting that these traits were controlled by polygenes. The F₂ population, derived from a cross between ‘Lenins’ and ‘6721′, showed a monogenic segregation for the number of regenerated shoots, and the segregation ratio fitted 1:2:1. Tissue culture traits of ‘Lenins’ were controlled by several genes, whereas the number of regenerated shoots related to the efficiency of shoot regeneration is controlled by one major gene.     

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.     

PCR-genotyping of barley seedlings using DNA samples from tissue prints

Screening of large numbers of plant samples with polymerase chain reaction (PCR)‐based techniques is a central task in marker‐assisted plant breeding and molecular genetics. While PCR and electrophoresis have been streamlined as a result of high‐throughput analysis methods, the collection of plant material and its processing to make it accessible for reliable amplification reactions remain labour‐intensive, costly and not amenable to automation. As an alternative to traditional extraction protocols, DNA can be bound to specially treated paper (FTA‐paper). For PCR analysis, instead of being added as a liquid suspension, DNA is then released from small paper disks that are immersed into the reaction mixture. In the present study, several parameters were investigated for the successful amplification of various single‐copy sequence characterized amplified region markers from barley tissue prints. The results show that the FTA‐paper approach offers a quick, reliable and low‐cost alternative for PCR screening even in a crop plant with a large genome.