Pyramiding QTLs to improve malting quality in barley: gains in phenotype and genetic diversity

The ability of barley (Hordeum vulgare L.) breeders to deliver germplasm that combine elite malt quality characteristics, disease resistances, and important agronomic traits has been greatly enhanced by the use of molecular marker technologies. These technologies facilitate the rapid transfer of desirable traits from diverse, elite, germplasm into locally adapted varieties. This present study sought to obtain an additive genetic effect by combining favourable alleles associated with the malting quality of two elite donor parents (Harrington and Morex) such that the resultant progeny would possess quality superior to either parent. Analysis of genetic diversity, based on whole-genome profiling with 700 DArT markers, showed clear separation of the BC₆F₁-derived doubled haploid lines from existing malting barley germplasm, indicating they represent a distinctly different source population for genetic improvement. Micro-malting quality results of the BC-derived lines showed substantial quality improvements, compared with the recurrent parent. Malt extract levels were increased by 1.5–2.0%, while diastase levels increased from approximately 320 WKE to 400–460 WKE. Similarly, α-amylase levels were increased from 160 units to between 218 and 251 units, and wort viscosities lowered from 1.90 cps to 1.72–1.82 cps. Other quality improvements include increases in β-glucanase levels from 375 to between 447 and 512 units, and reductions in wort β-glucan levels by 30–60%. Whilst the genetic gains compared to the recurrent parent were impressive, quality of the derived lines were largely equivalent to the levels now available in the recently released varieties, Buloke and Flagship. In a few cases, the backcross-derived lines also showed similarities to the original donors, Harrington and Morex, but in none of the cases did quality of these lines exceed those of either Harrington or Morex.     

Localization of quantitative trait loci (QTL) for agronomic important characters by the use of a RFLP map in barley (Hordeum vulgare L.)

Two hundred and fifty doubled haploid lines were studied from a cross between two 2-row winter barley varieties. The lines were evaluated for several characters in a field experiment for 3 years on two locations with two replications. From a total of 431 RFLP probes 50 were found to be polymorphic and subsequently used to construct a linkage map. Quantitative trait loci (QTLs) were determined and localized for resistance against Rhynchosporium secalis and Erysiphe graminis, for lodging, stalk breaking and ear breaking tendency, for the physical state before harvest, plant height, heading date, several kernel parameters and kernel yield. The heritability of the traits ranged from 0.56 to 0.89. For each trait except for kernel thickness, QTLs have been localized that explain 5–52% of the genetic variance. Transgressive segregation occurred for all of the traits studied.     

AB-QTL analysis in spring barley: III. Identification of exotic alleles for the improvement of malting quality in spring barley (<Emphasis Type="Italic">H. vulgare</Emphasis> ssp. <Emphasis Type="Italic">spontaneum</Emphasis>)

Malting quality is genetically determined by the complex interaction of numerous traits which are expressed prior to and, in particular, during the malting process. Here, we applied the advanced backcross quantitative trait locus (AB-QTL) strategy (Tanksley and Nelson, Theor Appl Genet 92:191–203, 1996), to detect QTLs for malting quality traits and, in addition, to identify favourable exotic alleles for the improvement of malting quality. For this, the BC₂DH population S42 was generated from a cross between the spring barley cultivar Scarlett and the wild barley accession ISR42-8 (Hordeum vulgare ssp. spontaneum). A QTL analysis in S42 for seven malting parameters measured in two different environments yielded 48 QTLs. The exotic genotype improved the trait performance at 18 (37.5%) of 48 QTLs. These favourable exotic alleles were detected, in particular, on the chromosome arms 3HL, 4HS, 4HL and 6HL. The exotic allele on 4HL, for example, improved α-amylase activity by 16.3%, fermentability by 0.8% and reduced raw protein by 2.4%. On chromosome 6HL, the exotic allele increased α-amylase by 16.0%, fermentability by 1.3%, friability by 7.3% and reduced viscosity by 2.9%. Favourable transgressive segregation, i.e. S42 lines exhibiting significantly better performance than the recurrent parent Scarlett, was recorded for four traits. For α-amylase, fermentability, fine-grind extract and VZ45 20, 16, 2 and 26 S42 lines, respectively, surpassed the recurrent parent Scarlett. The present study hence demonstrates that wild barley does harbour valuable alleles, which can enrich the genetic basis of cultivated barley and improve malting quality traits.     

Genetics of seedling and adult plant resistance to net blotch (Pyrenophora teres f. teres) and spot blotch (Cochliobolus sativus) in barley

Net blotch (caused by Pyrenophora teres f. teres) and spot blotch (Cochliobolus sativus) are important foliar diseases of barley in the midwestern region of the USA. To determine the number and chromosomal location of Mendelian and quantitative trait loci (QTL) controlling resistance to these diseases, a doubled haploid population (Steptoe/Morex) was evaluated to the pathogens at the seedling stage in the greenhouse and at the adult plant stage in the field. Alleles at two or three unlinked loci were found to confer resistance to the net blotch pathogen at the seedling stage depending on how progeny exhibiting an intermediate infection response were classified. This result was corroborated in the quantitative analysis of the raw infection response data as 2 major QTL were identified on chromosomes 4 and 6M. A third QTL was also identified on chromosome 6P. Seven QTL were identified for net blotch resistance at the adult plant stage and mapped to chromosomes 1P, 2P, 3P, 3M, 4, 6P, and 7P. The 7 QTL collectively accounted for 67.6% of the phenotypic variance under a multiple QTL model. Resistance to the spot blotch pathogen was conferred by a single gene at the seedling stage. This gene was mapped to the distal region of chromosome 1P on the basis of both qualitative and quantitative data analyses. Two QTL were identified for spot blotch resistance at the adult plant stage: the largest QTL effect mapped to chromosome 5P and the other mapped to chromosome 1P near the seedling resistance locus. Together, the 2 QTL explained 70.1% of the phenotypic variance under a multiple QTL model. On the basis of the chromosomal locations of resistance alleles detected in this study, it should be feasible to combine high levels of resistance to both P. teres f. teres and C. sativus in barley cultivars.     

Genetic mapping of a quantitative trait locus (QTL) that enhances the shoot differentiation rate in Hordeum vulgare L.

A quantitative trait locus (QTL) controlling shoot differentiation from immature embryo callus was identified by linkage analysis with morphological and isozyme markers in barley, Hordeum vulgare L. Immature embryos were isolated from cvs Azumamugi (difficult to differentiate), Kanto Nakate Gold (easy to differentiate), their hybrids (F₁) and a backcross population derived from a cross Azumamugi x F₁. The embryos were cultured in vitro for callus initiation and subsequent shoot differentiation. The shoot differentiation rate was closely associated with ear type (v locus), isocitrate dehydrogenase isozyme (Idh-2), and esterase isozyme (Est-11). These markers were found to reside in a chromosome segment of approximately 30cM on chromosome 2. Recombination frequency was 9.9% between v and a proposed QTL named Shd1 (shoot differentiation), 11.5% between Idh-2 and Shd1, and 21.3% between Est-11 and Shd1. All data showed the Idh-2, v, Shdl and Est-11 loci to be arranged in this order from proximal to distal on the long arm of chromosome 2.     

Effects of chromosome reconstruction of barley genome on tissue culture response using two different regeneration systems

Mature embryos and seedlings from mature embryos of one standard and five reconstructed karyotypes of barley (Hordeum vulgare L.) were cultured in vitro to study the influence of repositioning of particular chromosome segments of barley genome on the regeneration response. A comparative analysis of the regeneration response of a reconstructed karyotype having complete and well characterized rearrangement of the chromosome complement, and its four parental lines were used as experimental material. Depending on the source of explants two systems of in vitro culture were applied. The regeneration ability was found to be significantly influenced by both chromosome reconstruction and protocol applied. Possible reasons underlying the effects of chromosomal reconstruction on the regeneration response of karyotypes are briefly discussed.     

QTL analysis of resistance to the rice blast pathogen in barley (Hordeum vulgare)

Barley is compatible with the rice blast pathogen (Pyricularia oryzae Cav.). Fiftyfour barley cultivars of diverse geographic origin and pedigree were inoculated with three isolates of the rice blast pathogen. All barley genotypes showed blast disease symptoms when inoculated at the seedling stage with each of the three isolates. However, one genotype showed quantitative resistance to all three isolates and three genotypes showed quantitative resistance to one or two of the isolates. By inoculating a set of doubled-haploid lines derived from the cross ’Harrington’ (susceptible) and ’TR306’ (resistant) with isolate Ken 54–20, we mapped quantitative trait loci (QTLs) determining seedling stage blast resistance. At all QTLs, TR306 contributed the resistance alleles. The four QTLs, when considered jointly, explained 43.6% of the phenotypic variation in blast symptom expression. A comparison of the blast resistance QTLs with other disease resistance QTLs reported in this population revealed a region on chromosome 4 (4H) with multiple disease resistance loci. It will be useful to capitalize on the syntenic relationship of rice and barley and to integrate information on species-specific resistance genes with information on the reaction of the two species to the same pathogen. Received: 7 January 2000 / Accepted: 22 September 2000     

Improvement of malting quality of barley by complementing the malt enzyme spectrum

The processing quality of cereals can be modified by altering the structural grain constituents or the enzyme activities that mobilize storage reserves of the seeds. In order to complement the malt enzyme spectrum, a gene encoding for a thermotolerant fungal endo-(1,4)--glucanase was introduced into two barley cultivars, Kymppi and Golden Promise. The gene was expressed in the seeds during germination, thus providing a thermotolerant enzyme that is active under mashing conditions. The amount of thermotolerant -glucanase produced by the seeds (ca. 0.025% soluble seed protein) has been shown to be sufficient to reduce wort viscosity by decreasing the soluble -glucan content. For the safe commercial cultivation of transgenic plants risk assessment of their cultivation is needed. In our study experimental estimates of the transgene flow from transgenic barley by pollen dispersal were produced. Field trials were conducted during the summers of 1996 and 1997. A transgenic barley line homozygous for the gene encoding for neomycin phosphotransferase was used as a source of pollen and male-sterile barley lines as recipients. In order to be able to transform the cross-fertilization frequencies to corresponding values of normal male-fertile barley, plots of normal barley were also included in the experimental plan. On the basis of our study, cross-fertilization in male-sterile recipient barley is possible with very low frequency up to 50 meters from the donor area. However, the frequency dramatically decreases with distance and due to self-pollination the possibility of cross-fertilization remains very low in normal cultivated barley.     

Characterization of a gibberellin sensitive dwarf mutant of barley (Hordeum vulgare L.,Mut. Dornburg 576)

The radiation (³²P) induced dwarf mutant of spring barley,Hordeum vulgare L., Mut. Dornburg 576, was genetically studied by crosses with the mother variety and characterized by seedling assays and investigations on development and yield formation. In comparison to the normal mother variety (Hordeum vulgare L., cv. Saale) mutant plants exhibit drastically reduced culm length, intensive tillering, a prolonged life cycle and a smaller biomass and grain yield formation. These characters are controlled by one gene in a pleiotropic way. The mutant responds with normal growth and development to the application of gibberellins.     

Detection of Fusarium head blight resistance QTLs using five populations of top-cross progeny derived from two-row × two-row crosses in barley

Fusarium head blight (FHB) resistance was evaluated in five recombinant inbred (RI) populations. The RI populations consisted of top-cross progeny derived from a diallel set of crosses. Each of five two-row barley lines differing in response to FHB were crossed with ‘Harbin 2-row’. FHB severity was scored on an 11-point scale, where resistant = 0 and susceptible = 10, based on the ‘cut-spike test’. Disease data were obtained for each population for 2 or 3 years. Linkage maps comprised of expressed sequence tag (EST) markers were developed for each population and used for quantitative trait locus (QTL) detection. Thirty two QTLs were detected using all data sets (individual populations and years). Thirteen QTLs were detected using averages across years; 10 of these were consistent across the individual year and average data sets. These QTLs clustered at 14 regions, with clusters on all chromosomes. At 11 of these clusters, Harbin 2-row contributed FHB resistance alleles. No QTLs were detected near the row type (vrs1) locus in any of the five RI populations, suggesting that the FHB resistance QTL in this region reported in two-row × six-row crosses may be pleiotropic effect of vrs1. QTL were coincident with the flowering type locus (cly1/Cly2) on chromosome 2H in every population. Some QTL × QTL interactions were significant, but these were smaller than QTL main effects. Considering the pleiotropic effect of spike morphology on FHB resistance, future FHB resistance mapping efforts in barley should focus on cross combinations in which alleles at vrs1 are not segregating. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of population size on the estimation of QTL: a test using resistance to barley stripe rust

The limited population sizes used in many quantitative trait locus (QTL) detection experiments can lead to underestimation of QTL number, overestimation of QTL effects, and failure to quantify QTL interactions. We used the barley/barley stripe rust pathosystem to evaluate the effect of population size on the estimation of QTL parameters. We generated a large (n=409) population of doubled haploid lines derived from the cross of two inbred lines, BCD47 and Baronesse. This population was evaluated for barley stripe rust severity in the Toluca Valley, Mexico, and in Washington State, USA, under field conditions. BCD47 was the principal donor of resistance QTL alleles, but the susceptible parent also contributed some resistance alleles. The major QTL, located on the long arm of chromosome 4H, close to the Mlo gene, accounted for up to 34% of the phenotypic variance. Subpopulations of different sizes were generated using three methods—resampling, selective genotyping, and selective phenotyping—to evaluate the effect of population size on the estimation of QTL parameters. In all cases, the number of QTL detected increased with population size. QTL with large effects were detected even in small populations, but QTL with small effects were detected only by increasing population size. Selective genotyping and/or selective phenotyping approaches could be effective strategies for reducing the costs associated with conducting QTL analysis in large populations. The method of choice will depend on the relative costs of genotyping versus phenotyping. Electronic Supplementary Material Supplementary material is available for this article at     

Effect of seed nitrogen on response to applied nitrogen in six spring barley (Hordeum vulgare L.) cultivars in a glasshouse

Summary Variation in the nitrogen content of seed of six barley cultivars was brought about by growing parent plants at four nitrogen levels. Shoot dry weight of plants grown for 23 days from these seeds was generally enhanced by an increase in seed nitrogen content. The most responsive cultivar was a primitive type of barley from Ethiopia. Cultivars with a longer breeding history were less responsive. Risø 1508 apparently had physiological and biochemical limitations in responding to extra seed nitrogen. In the barley cultivars studied extra seed nitrogen seems to supplement, rather than substitute for, nitrogen fertilizer in the seed bed.     

The transfer of a powdery mildew resistance gene from Hordeum bulbosum L to barley (H. vulgare L.) chromosome 2 (2I)

Hordeum bulbosum L. is a source of disease resistance genes that would be worthwhile transferring to barley (H. vulgare L.). To achieve this objective, selfed seed from a tetraploid H. vulgare x H. bulbosum hybrid was irradiated. Subsequently, a powdery mildew-resistant selection of barley phenotype (81882/83) was identified among field-grown progeny. Using molecular analyses, we have established that the H. bulbosum DNA containing the powdery mildew resistance gene had been introgressed into 81882/83 and is located on chromosome 2 (2I). Resistant plants have been backcrossed to barley to remove the adverse effects of a linked factor conditioning triploid seed formation, but there remains an association between powdery mildew resistance and non-pathogenic necrotic leaf blotching. The dominant resistance gene is allelic to a gene transferred from H. bulbosum by co-workers in Germany, but non-allelic to all other known powdery mildew resistance genes in barley. We propose Mlhb as a gene symbol for this resistance.     

Inheritance and expression of NAD(P)H nitrate reductase in barley

Summary NADH-specific and NAD(P)H bispecific nitrate reductases are present in barley (Hordeum vulgare L.). Wild-type leaves have only the NADH-specific enzyme while mutants with defects in the NADH nitrate reductase structural gene (nar1) have the NAD(P)H bispecific enzyme. A mutant deficient in the NAD(P)H nitrate reductase was isolated in a line (nar1a) deficient in the NADH nitrate reductase structural gene. The double mutant (nar1a;nar7w) lacks NAD(P)H nitrate reductase activity and has xanthine dehydrogenase and nitrite reductase activities similar to nar1a. NAD(P)H nitrate reductase activity in this mutant is controlled by a single codominant gene designated nar7. The nar7 locus appears to be the NAD(P)H nitrate reductase structural gene and is not closely linked to nar1. From segregating progeny of a cross between the wild type and nar1a;nar7w, a line was obtained which has the same NADH nitrate reductase activity as the wild type in both the roots and leaves but lacks NADPH nitrate reductase activity in the roots. This line is assumed to have the genotype Nar1Nar1nar7nar7. Roots of wild type seedlings have both nitrate reductases as shown by differential inactivation of the NADH and NAD(P)H nitrate reductases by a monospecific NADH-nitrate reductase antiserum. Thus, nar7 controls the NAD(P)H nitrate reductase in roots and in leaves of barley.Scientific Paper No. 7617, College of Agriculture Research Center and Home Economics, Washington State University, Pullman, WA, USA. Project Nos. 0233 and 0745     

A barley RFLP map: alignment of three barley maps and comparisons to Gramineae species

Several gene linkage maps have been produced for cultivated barley. We have produced a new linkage map for barley, based on a cross between Hordeum vulgare subsp. spontaneum and Hordeum vulgare subsp. vulgare (Hvs x Hvv), having a higher level of polymorphism than most of the previous barley crosses used for RFLP mapping. Of 133 markers mapped in the Hvs x Hvv F₂ population, 69 were previously mapped on other barley maps, and 26 were mapped in rice, maize, or wheat. Two known gene clones were mapped as well as two morphological markers. The distributions of previously mapped markers were compared with their respective barley maps to align the different maps into one consensus map. The distributions of common markers among barley, wheat, rice and maize were also compared, indicating colinear linkage groups among these species.To be considered dual first authorsPublished with the approval of the Director of the Colorado State University/Agricultural Experiment Station.     

Use of the additive main effects and multiplicative interaction model in QTL mapping for adaptation in barley

The additive main effects and multiplicative interaction (AMMI) model has emerged as a powerful analytical tool for genotype x environment studies. The objective of the present study was to assess its value in quantitative trait locus (QTL) mapping. This was done through the analysis of a large two-way table of genotype-by-environment data of barley (Hordeum vulgare L.) grain yields, where the genotypes constituted a genetic population suitable for mapping studies. Grain yield data of 150 doubled haploid lines derived from the Steptoe x Morex cross, and the two parental lines, were taken by the North American Barley Genome Mapping Project (NABGMP) at 16 environments throughout the barley production areas of the USA and Canada. Four regions of the genome were responsible for most of the differential genotypic expression across environments. They accounted for approximately 50% of the genotypic main effect and 30% of the genotype x environment interaction (GE) sums of squares. The magnitude and sign of AMMI scores for genotypes and sites facilitate inferences about specific interactions. The parallel use of classification (cluster analysis of environments) and ordination (principal component analysis of GE matrix) techniques allowed most of the variation present in the genotype x environment matrix to be summarized in just a few dimensions, specifically four QTLs showing differential adaptation to four clusters of environments. Thus, AMMI genotypic scores, when the genotypes constituted a population suitable for QTL mapping, could provide an adequate way of resolving the magnitude and nature of QTL x environment interactions.Ignacio Romagosa was on sabbatical leave from the University of Lleida and the Institut de Recerca i Tecnologia Agroalimentàries, Lleida, Spain, when this study was conducted     

水稻株高构成因素的QTL剖析

利用水稻籼粳杂交 (圭 6 30× 0 2 42 8) F1 的花药离体培养建立的一个含 81个 DH家系的作图群体 ,对水稻株高构成因素 (穗长、第 1节间长、……、第 5节间长 )进行基因定位。DH群体中株高构成因素均呈正态分布。相邻的构成因素间呈极显著的正相关 ,而相距较远的构成因素间的相关较弱或不显著。采用 QTL(Quantitative trait lo-cus)分析 ,定位了影响株高构成因素的 6个 QTL:qtl7同时影响穗长和第 1、2、3节间长 ,qtl1 和 qtl2 同时影响第 4和第 5节间长 ,qtl1 0 a和 qtl1 0 b仅影响第 1节间长 ,qtl3 仅影响第 3节间长。采用 QTL 互作分析 ,检测到 19对显著的互作 ,每个构成因素受 2个或 2个以上的 QTL 互作对的影响。并且还发现 ,同一个 QTL 互作对可能影响不同的性状 ,以及一个 QTL 可以分别与不同的 QTL 产生互作而影响同一个性状或影响不同的性状 ,但总的看来 ,加性效应是主要的。这些结果揭示了株高构成因素间相关的遗传基础 ,在水稻育种中运用这些 QTL 将有助于对株高 ,以及对穗长和上部节间长度进行精细的遗传调控。     

cDNA,amino acid and carbohydrate sequence of barley seed-specific peroxidase BP 1

The major peroxidase of barley seed BP 1 was characterized. Previous studies showed a low carbohydrate content, low specific activity and tissue-specific expression, and suggested that this basic peroxidase could be particularly useful in the elucidation of the structure-function relationship and in the study of the biological roles of plant peroxidases (S.K. Rasmussen, K.G. Welinder and J. Hejgaard (1991) Plant Mol Biol 16: 317–327). A cDNA library was prepared from mRNA isolated from seeds 15 days after flowering. Full-length clones were obtained and showed 3 end length variants, a G+C content of 69% in the translated region, a 90% G or C preference in the wobble position of the codons and a typical signal peptide sequence. N-terminal amino acid sequencing and sequence analysis of tryptic peptides verified 98% of the sequence of the mature BP 1 which contains 309 amino acid residues. BP 1 is the first characterized plant peroxidase which is not blocked by pyroglutamate. BP 1 polymorphism was observed. BP 1 is less than 50% identical to other plant peroxidases which, taken together with its developmentally dependent expression in the endosperm 15–20 days after flowering, suggests a unique biological role of this enzyme. The barley peroxidase is processed at the C-terminus and might be targeted to the vacuole. The single site of glycosylation is located near the C-terminus in the N-glycosylation sequon -Asn-Cys-Ser- in which Cys forms part of a disulphide bridge. The major glycan is a typical plant modified-type structure, Man1-6(Xyl1-2)Man1-4GlcNAc1-4(Fuc1-3)GlcNAc. The BP 1 gene was RFLP-mapped on barley chromosome 3, and we propose Prx5 as the name for this new peroxidase locus.     

Flt-2L, a locus in barley controlling flowering time, spike density, and plant height

Flowering time represents an important adaptive trait for temperate cereal crops and may also impact on frost damage in cereal reproductive tissues by enabling escape or by influencing accumulation of genuine tolerance. The Flowering time-2L (Flt-2L) quantitative trait locus (QTL) on the distal end of barley chromosome arm 2HL overlaps with QTL for rachis internode length and reproductive frost damage. Flt-2L was also found to be associated with plant height. By combining marker analysis with phenotyping in progeny families of selected Amagi Nijo × WI2585 F₆ recombinants, we were able to map quantitative flowering time, rachis internode length, and plant height effects on 2HL as discrete Mendelian traits. The three developmental characters showed codominant modes of expression and perfectly cosegregated with one another in a 1.3-cM marker interval, indicating control by the same gene or closely linked genes. Twelve genes were identified in the related intervals in the rice and Brachypodium distachyon genomes. The HvAP2 gene cosegregated with Flt-2L and represents a plausible candidate for Flt-2L, since it is highly similar to the wheat domestication gene Q which has similar developmental effects. These data will contribute to isolation of the Flt-2L gene(s) and help establish the basis of the frost damage QTL. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fertile transgenic barley generated by direct DNA transfer to protoplasts

We report the generation of transgenic barley plants via PEG-mediated direct DNA uptake to protoplasts. Protoplasts isolated from embryogenic cell suspensions of barley (Hordeum vulgare L. cv Igri) were PEG-treated in a solution containing a plasmid which contained the neomycin phosphotransferase (NPT II) gene under the control of the rice actin promoter and the nos terminator. Colonies developing from the treated protoplasts were incubated in liquid medium containing the selective antibiotic G418. Surviving calli were subsequently transferred to solid media containing G418, on which embryogenic calli developed. These calli gave rise to albino and green shoots on antibiotic-free regeneration medium. NPT II ELISA revealed that approximately half of the morphogenic calli expressed the foreign gene. In total, 12 plantlets derived from NPT-positive calli survived transfer to soil. Southern hybridization analysis confirmed the stable transformation of these plants. However, the foreign gene seemed to be inactivated in plants from one transgenic line. Most of the transgenic plants set seed, and the foreign gene was transmitted and expressed in their progenies, which was ascertained by Southern hybridization and NPT II ELISA.