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.     

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.     

Molecular and chemical analysis of β-amylase-less mutant barley in Tibet

The molecular and biochemical aspects of Tibetan β‐amylase‐less mutant barley were investigated. An enzyme assay showed that there were eight β‐amylase‐less mutant lines in the Tibetan landrace barley. Following Southern analysis and polymerase chain reaction, it is suggested that these mutants result from the same insert mutation in the latter part of the β‐amylase structural gene. The α‐glucosidase activity level of the mutant line was high in the initial period of germination, and the glucose ratio in the sugar conformation was remarkably high in the germinated seed of the mutant line. These results suggest that this β‐amylase‐less mutant line could be a new breeding material for beer production.     

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.     

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.     

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.     

Genetic analysis of heading date and other agronomic characters in barley (Hordeum vulgare L.)

Genetic analyses of heading date, tiller number, plant height, grain yield, kernel weight, and plump and thin kernels were made in three six-rowed barley crosses (Hordeum vulgare L.) involving four cultivars. Six populations, P₁ , P₂ , F₁ , F₂ , BC₁ , and BC₂ , from each cross were grown and evaluated at Fargo and Prosper, North Dakota, 1982. Parental means within crosses generally were different except for tiller number. Comparison of generation means suggested that late heading was dominant to early, high kernel weight was dominant to low, and kernel plumpness was influenced by additive gene action. The relationship between yield and heading date was not consistent among crosses and positive r values were quite low. It should be possible to select early maturing, high yielding segregates with plump kernels. Heterosis over the mid-parent was quite similar among crosses for heading date, but there was no heterosis over the high parent. Inbreeding depression was fairly constant for heading date, but was less consistent for yield. The lack of uniformity for estimates of inbreeding depression can be related to environmental variation and to its influence on type of gene action. The ratio of additive to dominance variance was inconsistent among crosses for heading date and yield. These data suggest selection for these characters should be delayed past the F 2 generation. Broad sense heritabilities for heading date ranged from 42 to 86%. Values obtained for grain yield were more consistent among broad sense than narrow sense estimates. Genetic advance estimates were low due to lack of additive variance. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic analysis of some chlorophyll fluorescence and productivity parameters in barley (Hordeum vulgare)

Reciprocal crosses were made between five barley genotypes of diverse origin. Parents and 20 F₁S were grown in a greenhouse in a randomized block design with three replications. Each replication consisted of one pot containing three plants. Chlorophyll fluorescence activity of the penultimate leaf (below the flag leaf) of the main stem in each plant was measured at two stages of development (six-leaf stage and anthesis). At anthesis, and after measuring fluorescence parameters, a drought stress was applied. Yield components were measured at complete maturity. Statistical analysis for chlorophyll fluorescence parameters showed high genetic variability for the traits studied at both the six-leaf stage and anthesis, but most fluorescence parameters had decreased by anthesis. General and specific combining abilities of a diallel analysis were significant for most of the characters studied. General combining ability (GCA) values were usually more important than those of specific combining ability (SCA), showing the importance of additive genetic control for the fluorescence traits. Reciprocal effects were not significant for the fluorescence traits studied. A high genetic variability was also observed for yield components. Additive gene effects for plant weight, spike number, spike weight, number of grains per plant and 1000-grain weight were demonstrated. Potential photosynthetic activity (Rfd) at anthesis was positively correlated with plant weight, spike number per plant and 1000-grain weight.     

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.     

Genetic diversity in landrace populations of Hordeum vulgare L. from Sardinia, Italy, as revealed by RAPDs, isozymes and morphophenological traits

Barley is the second most widely cultivated cereal on the island of Sardinia. It is grown for green fodder, grain and straw, all of which are used for animal feet. The cultivation of modem varieties is limited and many farmers grow local populations of the six-row landrace ‘S'orgiu sardu’. Twelve local populations (20 strains per population) were evaluated for 13 random amplified polymorphic DNA (RAPD) markers, sisisozyme loci and five morphophenological traits. Results showed that Sardinian barley is characterized by a lax six-row spike and is. in general, a spring type. Isozyme polymorphism was delected for only two esterase loci (Est and Est 4). The genetic variation for all types of murkers, was found located mostly within population (about 84%, for isozymes aud morphaphenological traits and 89%, for RAPDs). This finding appears to confirm the hypothesis that the populations studied belong to the same land race called ‘S'orgiu sardu’ by farmers. A geographical differential ion between southern and northern populations was foand in relation to distinct environmental characteristics. Taking into account all types of marker 209 (90.5%) different genotypes were identified out of 231 individuals. The overall results confirm the high variability that can be found within landrace populations, underlining the value of landraccs as a source of genetic variation.     

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.     

AFLP-based STS markers closely linked to a fertility restoration locus (Rfm1) for cytoplasmic male sterility in barley

The Rfm1 gene restores the fertility of the msm1 and msm² male‐sterile cytoplasms in barley. Rfm1 is located on the short arm of chromosome 6H. To develop molecular markers tightly linked to Rfm1 for use in sophisticated marker‐assisted selection and map‐based cloning, an amplified fragment‐length polymorphism (AFLP) marker system with isogenic lines and a segregating BC₁F₁ population was used. Nine hundred primer combinations were screened and a linkage map was constructed around the Rfm1 locus by using 25 recombinant plants selected from 214 BC₁F₁ plants. Three AFLP markers were identified, e34m2, e46m19 and e48m17, linked to the locus. The most closely linked markers were e34m2, at 1.0 cM distally and e46m19, at 1.1 cM proximally. The two AFLP markers were converted to dominant STS markers. These markers should accelerate programmes for breeding restorer lines and will be useful for map‐based cloning.     

Genome-wide association studies in a barley (Hordeum vulgare) diversity set reveal a limited number of loci for resistance to spot blotch (Bipolaris sorokiniana)

Spot blotch caused by Bipolaris sorokiniana is an important disease in barley worldwide, causing considerable yield losses and reduced grain quality. In order to identify QTL conferring resistance to spot blotch, a highly diverse worldwide barley set comprising 449 accessions was phenotyped for seedling resistance with three isolates (No 31, SH 15 and SB 61) and for adult plant resistance at two locations (Russia and Australia) in two years. Genotyping with the 50 k iSelect barley SNP genotyping chip yielded 33,818 informative markers. Genome-wide association studies (GWAS) using a compressed mixed linear model, including population structure and kinship, revealed 38 significant marker-trait associations (MTA) for spot blotch resistance. The MTA corresponded to two major QTL on chromosomes 1H and 7H and a putative new minor QTL on chromosome 7H explaining between 2.79% and 13.67% of the phenotypic variance. A total of 10 and 14 high-confidence genes were identified in the respective major QTL regions, seven of which have a predicted involvement in pathogen recognition or defence.     

The influence of photoperiod on the Vrn-H2 locus (4H) which is a major determinant of plant development and reproductive fitness traits in a facultative × winter barley (Hordeum vulgare L.) mapping population

To determine the effect of Vrn‐H2 locus on plant developmental and agronomic traits, detailed controlled environment tests involving a factorial set of vernalization and photoperiod treatments were carried out using doubled haploid lines developed from a facultative (Vrn‐H2^?) × winter (Vrn‐H2⁺) barley cross. The allele phase at the Vrn‐H2 locus influenced heading date as well as the developmental and agronomic traits. The performance of Vrn‐H2⁺ lines was significantly influenced by vernalization: reproductive fitness traits showed significant decreases without vernalization. However, the effects of alleles at the Vrn‐H2 locus extended beyond simple satisfaction of the vernalization requirement. Vrn‐H2⁺ lines showed increased reproductive fitness compared with Vrn‐H2^? lines when vernalization was followed by a long photoperiod. The responses of the two Vrn‐H2 allele classes to photoperiod duration were quite different in terms of heading date, developmental and agronomic traits. These results suggest that alleles at the Vrn‐H2 locus – and/or tightly linked gene(s) – respond primarily to the exogenous signal of vernalization (temperature), but when the vernalization requirement has been fulfilled, they also respond to photoperiod duration.     

Ability of chromosome 4H to compensate for 4D in response to drought stress in a newly developed and identified wheat–barley 4H(4D) disomic substitution line

A spontaneously developed wheat–barley 4H(4D) disomic substitution line was identified cytogenetically using genomic in situ hybridization (GISH), multicolour fluorescent in situ hybridization (FISH) and microsatellite markers. The ability of the barley 4H chromosome to compensate for wheat 4D in response to mild drought stress was also investigated. In the barley cv. 'Betzes' and the 4H(4D) substitution line, mild osmotic stress induced intensive stomatal closure, resulting in reduced water loss through transpiration and unchanged relative water content in the leaves. As the CO₂ assimilation rate remained relatively high, the water use efficiency, which is an important factor associated with drought tolerance, increased extensively under mild osmotic stress in these lines. In the case of the parental wheat genotypes, however, mild drought stress induced less intense stomatal closure and a greater decrease in the CO₂ assimilation rate than in barley or in the substitution line, resulting in unaugmented or reduced water use efficiency. The results demonstrate that genes localised on the 4H chromosome of barley were able to increase the water use efficiency of the wheat substitution line, which is suitable for improving wheat drought tolerance through intergeneric crossing.     

Revival of a well-known, but ignored correction term for χ2 in linkage analysis with small samples

The ‘classical’χ² test is routinely applied in linkage analysis. The values customarily used, however, are only approximations since continuous χ² terms are used instead of discrete multinomial terms. This error becomes increasingly important as the sample becomes small. In this note, a well-known correction term for improvement of this approximation is revived and numerically calculated for some interesting cases from linkage studies.