Patterns of genetic diversity and linkage disequilibrium in a highly structured Hordeum vulgare association-mapping population for the Mediterranean basin

Population structure and genome-wide linkage disequilibrium (LD) were investigated in 192 Hordeum vulgare accessions providing a comprehensive coverage of past and present barley breeding in the Mediterranean basin, using 50 nuclear microsatellite and 1,130 DArT^® markers. Both clustering and principal coordinate analyses clearly sub-divided the sample into five distinct groups centred on key ancestors and regions of origin of the germplasm. For given genetic distances, large variation in LD values was observed, ranging from closely linked markers completely at equilibrium to marker pairs at 50 cM separation still showing significant LD. Mean LD values across the whole population sample decayed below r ² of 0.15 after 3.2 cM. By assaying 1,130 genome-wide DArT^® markers, we demonstrated that, after accounting for population substructure, current genome coverage of 1 marker per 1.5 cM except for chromosome 4H with 1 marker per 3.62 cM is sufficient for whole genome association scans. We show, by identifying associations with powdery mildew that map in genomic regions known to have resistance loci, that associations can be detected in strongly stratified samples provided population structure is effectively controlled in the analysis. The population we describe is, therefore, shown to be a valuable resource, which can be used in basic and applied research in barley.     

Acrotrisomic analysis in linkage mapping in barley (Hordeum vulgare L.)

Summary Three acrotrisomic lines, Triplo IL^1S, 3L^3S, and 4L^4S, each carrying an extra acrocentric chromosome, were used for cytogenetic linkage mapping of barley chromosomes. The cytological structures of the acrocentric chromosome of the three acrotrisomic lines were studied with an improved Giemsa N-banding technique. The long (1L) and short arm (1S) of chromosome 1 had deficiencies of approximately 38% and 65%, respectively. The percentages of deficiencies were 0 and 77.8% for 3L and 3S, and 31.7 and 59.3% for 4L and 4S, respectively. All three genes tested (br, f _c , gs3) in 1S and all three genes tested, f8, n and 1k2 in 1L showed a disomic ratio indicating that they are located in the deficient segments. Two genes (a _c , yst2) located in the middle segment of 3S in linkage map showed a trisomic ratio, and two others a _n , x _s showed a disomic ratio. The only gene(f9) tested in 4L showed a trisomic ratio. Two genes (1g4, g1) located in the proximal segment of 4S in the linkage map showed a trisomic ratio, whereas two genes (br2, g13) located distally in 4S showed a disomic ratio, indicating that the breakage occurred between g1 and br2. This experiment demonstrates a new method for physical localization of genes on chromosome segments in material such as barley in which pachytene analysis can not be effectively used for accurate determination of break points in structural changes. Problems associated with this new technique are discussed.Contribution from the Department of Agronomy and published with the approval of the Director of Colorado State University Experiment Station as Scientific Series Paper No. 2823. Supported by USDA/SEA Competitive Research Grant Nos. 5901-0410-9-0334-0 and 82-CRCR-1-1020 and USDA-CSU Cooperative Research Grant 58-9AHZ-2-265     

Associative overdominance: Evaluating the effects of inbreeding and linkage disequilibrium

Expressions are obtained for the expected phenotypic values of homozygous and heterozygous genotypes for a neutral marker locus linked to a locus segregating for a recessive deleterious gene. The phenotypic values are functions of the allele frequencies at the marker locus, the inbreeding coefficient and the degree of association of the deleterious gene with the marker alleles. The analysis is extended to more than two alleles at the marker locus. Either linkage disequilibrium or inbreeding alone can produce an apparent superiority of heterozygotes for the marker locus (unless specified otherwise, the terms ‘homozygote’ and ‘heterozygote’ will refer to the marker locus). The effect of linkage disequilibrium on the difference between the heterozygote and homozygote values can be positive (associative overdominance) or negative (associative underdominance), depending on the frequencies of the marker alleles and the degree of their association with the deleterious gene. Inbreeding has always a positive effect. In general, the expected value of a homozygote is a positive function of its allele frequency. When the various homozygous genotypes are combined into one class and the various heterozygous genotypes into another, the phenotypic difference of the two classes is a function of the evenness of the allelic frequency distribution. Inbreeding is a more likely explanation of associative overdominance if the frequency of the deleterious gene is low, but its effect on the character high. Conversely, linkage disequilibrium is more likely if the frequency is high and the effect low. The degrees of association between marker alleles and the deleterious gene can, in principle, be estimated from the observed phenotypic scores and used to calculate expected multi-locus genotype scores. This could provide the basis for statistical tests of the associative overdominance hypothesis as an explanation of observed correlations between multi-locus heterozygosity and phenotypic traits.     

Prediction of multi-locus inbreeding coefficients and relation to linkage disequilibrium in random mating populations

An algorithm to predict the level of identity by descent simultaneously at multiple loci is presented, which can in principle be extended to any number of loci. The model assumes a random mating population, with random association of haplotypes. The relationship is shown between coefficients of multi-locus identity or non-identity by descent and moments of multi-locus linkage disequilibrium. Thus, these moments can be computed from the multilocus identity or, using algorithms derived previously to predict the disequilibria moments, vice-versa. The results can be applied to predict multi-locus identity in, for example, gene mapping.     

Characterising seedling and adult plant resistance to Puccinia hordei in Hordeum vulgare

A set of 113 genotypes of barley (Hordeum vulgare subsp. vulgare), along with the susceptible control genotype Gus, was tested for response to the barley leaf rust pathogen Puccinia hordei in the greenhouse (as seedlings) and field (as adult plants). The tests revealed that 68 lines carried adult plant resistance (APR), 23 lines carried uncharacterised seedling resistance (USR) and that three lines carried the seedling resistance gene Rph3. Nineteen lines lacked detectable seedling resistance and were also susceptible in the field at adult plant growth stages. The presence of marker bPb‐0837, linked to the APR gene Rph20, in 35 of the 68 lines carrying APR, suggested they carry this gene. The remaining 33 lines, which lacked the Rph20 linked marker, are likely sources of new uncharacterised APR. Pedigree analysis of the 68 lines found to carry APR revealed that 32 were related to cv. Gull and to Hordeum laevigatum; two were related to cv. Bavaria and one related to cvv. Manchuria and Taganrog, suggesting that these genotypes may be the ancestral sources of the APR carried by each.     

Analysis of linkage disequilibrium in an island model

Linkage disequilibria for two loci in a finite island model were parameterized. The total linkage disequilibrium was decomposed into three components, gametic, demic, and population, for which corresponding unbiased estimators were established. Other statistics encountered provided measures of differentiation corresponding to the hierarchical structure of the ecological model. Under the assumption of linkage equilibrium, the variances and covariances of these estimators and statistics were formulated in terms of descent measures, functions of gene frequencies, and the numbers of individuals, demes, and populations sampled. The functions of gene frequencies fall into two classes, one representing the differentiation of genes at each locus, and the other representing the association of genes between the loci. For a neutral model with extinction, migration, and linkage, transition equations were derived for the descent measures which also take into account deme size and numbers of dems within the population. With the addition of unequal mutation rates for a finite number of alleles at each locus, the transition equations were solved for the descent measures in the equilibrium state. This permitted the exact numerical evaluation of the effects of the sampling and ecological dimensions and of extinction, migration, and mutation rates in any parameter range. Some numerical results were presented for the effects of linkage, extinction, migration, and sampling on the variances of various measures of linkage disequilibrium and genetic differentiation. Also, some results were compared with the approximate numerical results of Ohta which agreed fairly well in the parameter ranges she considered, but not so well in other ranges.     

Tissue culture and plant regeneration from immature embryo explants of Barley,Hordeum vulgare

Summary Immature embryo explants taken 8 days after anthesis were used to establish callus cultures of spring barley. Two types of calli were observed. A soft, watery callus produced a limited number of shoots and a harder, more compact, yellowish callus gave rise to numerous green primordia and shoots. Gamborg's B5 basal medium supplemented with either 2,4-D (2,4-dichlorophenoxyacetic acid) or Cl₃ POP (2,4,5-trichlorophenoxypropionic acid) was found to give good callus growth and shoot initiation. Media containing 2,4-D at 1.0 mg L^–1 or Cl₃ POP at 5.0 mg L^–1 produced numerous cultures resulting in regeneration of plants. Plantlets developed roots on basal medium with Cl₃ POP at 1.0 mg L^–1 or on auxin-free medium. Twenty genetically diverse genotypes were screened to determine if these techniques were suitable for a wide range of spring barley cultivars. Regeneration of plantlets was obtained for 19 of the 20 genotypes approximately 4 months after culture initiation. Lines differed in the ability to develop vigorously growing calli and in the ability of calli to develop large numbers of shoots and regenerated plantlets.Contribution from Department of Crop Science, Oregon State University, Corvallis, OR 97331. Oregon Agric. Exp. Stn. No. 7582     

The influence of linkage and inbreeding on patterns of nucleotide sequence diversity at duplicate alcohol dehydrogenase loci in wild barley (Hordeum vulgare ssp. spontaneum)

Patterns of nucleotide sequence diversity are analyzed for three duplicate alcohol dehydrogenase loci (adh1-adh3) within a species-wide sample of 25 accessions of wild barley (Hordeum vulgare ssp. spontaneum). The adh1 and adh2 loci are tightly linked (recombination fraction <0.01) while the adh3 locus is inherited independently. Wild barley is predominantly self-fertilizing (approximately 98%), and as a consequence, effective recombination is restricted by the extreme reduction in heterozygosity. Large reductions in effective recombination, in turn, widen the conditions for linkage to influence nucleotide sequence diversity through the action of selective sweeps or background selection. These considerations would appear to predict (1) homogeneity in patterns of nucleotide sequence diversity, especially between closely linked loci, and (2) extensive linkage disequilibrium relative to random-mating species. In contrast to these expectations, the wild barley data reveal heterogeneity in patterns of nucleotide sequence diversity and levels of linkage disequilibrium that are indistinguishable from those observed at adh1 in maize, an outbreeding grass species.     

Molecular-genetic polymorphism of barley varieties (Hordeum vulgare L.) detected by the AFLP-method

Analysis of molecular-genetic polymorphism of barley varieties was performed using the AFLP-method. A system for identification and differentiation of barley varieties based on AFLP-markers was developed. Results of testing of 19 varieties indicate a high differential ability of the developed system. Identification of varieties can be conducted using one of two offered discriminatory sets of AFLP-markers. Based on the calculated genetic distances, a dendrogram of phylogenetic relations between varieties was constructed. The dendrogram revealed a separated origin of varieties of brewer and feed directions.     

Shed Pollen Culture in Hordeum vulgare

Anthers of Hordeum vulgare cv. Sabarlis at the mid-unicellularpollen stage, pretreated in the excised spike for 14 d at 7°C, dehisce within 24 h of being floated on the surfaceof liquid medium. About half the pollen (1500 grains per anther)is liberated into the medium. If the anthers are then removedand the cultures re-incubated, calluses develop from the shedpollen in high yields. At low anther densities, 10p–20(1–3 x 10⁴ grains) per ml, medium preconditioned by anthersand supplemented with m-inositol (1000 mg 1^–1) is required,but at high densities, 120 anthers (2 x 10⁵ grains) per ml,preconditioning is less important, the cultured anthers themselveshaving a sufficient conditioning influence. Large-scale dissectionof anthers can be avoided by use of drops of medium, the volumebeing increased gradually as culture proceeds. Pollen remainingin the anthers after 3 d gives rise to calluses if isolatedmechanically and cultured in the inositol medium. The use ofshed pollen is seen as particularly valuable for culture inspecies whose anthers are small, tedious to dissect out anddifficult to process without severe damage.     

A program to test linkage disequilibrium between loci in autotetraploid species

No software currently implements a test of linkage disequilibrium in autotetraploid species. We propose a program, LD4X that performs a Fisher's exact test between pairs of alleles at two loci. All combinations of alleles from two loci are treated in turn. If two alleles of a pair of loci have a nonrandom distribution, the markers are considered as linked. The program was tested on a set of microsatellite markers in synthetic alfalfa populations.     

Cytogenetics of an unstable trisomie in barley (Hordeum vulgare).

The origin, identification, meiotic chromosome behavior, and breeding behavior of an unstable trisomic barley were studied. The extra chromosome originated by breakage and fusion of an acrocentric chromosome 3 in a plant from an F2 population of a cross between acrotrisomic 3L3S (2n = 14 + 1 acro3L3S) and a balanced lethal stock, xc. (xantha) ac (albino). The F2 population segregated only for the albino trait. The genotypic constitution of the trisomic plant was ac ac (for both normal chromosome 3) and Ac (for the unstable metacentric chromosome). The unstable extra metacentric chromosome was designated as metacentric 3B (abbreviated as meta3B). Meiotic chromosome behavior in plants with 2n = 14 + 1 meta3B differed from plant to plant and within spikes. Some plants showed only trisomic cells with a chromosome configuration of 1 III + 6 II and 7 II + 1 I at metaphase I, whereas other plants showed both trisomie and disomic cells (7 II) that resulted from the elimination of the extra meta3B. The frequency of ring trivalents was low (6.8%). An average transmission rate of unstable meta3B ranged from 4.3 to 12.9%. The elimination of meta3B, and hence loss of the dominant Ac allele, resulted in albino seedlings as well as white stripes on plants, leaves, and spikes. Chromosome numbers of albino seedlings in the progeny of 2n = 14 + 1 meta3B were all diploid (2n = 14), while green seedlings contained 2n = 14 + 1 meta3B. However, progenies of some spikes of one trisomic plant showed a low frequency of green diploids and metatrisomics (2n = 14 + 1 meta3B), which was attributed to crossing-over.     

Histological analysis of somatic embryogenesis in Brazilian cultivars of barley, Hordeum vulgare vulgare, Poaceae

 Histological analysis was performed aimed at elucidating the origin and the developmental process of somatic embryos of two Brazilian cultivars of barley (Hordeum vulgare vulgare), 'MN-599' and 'A-05'. The observed site of somatic embryo origin (SSEO) could originate from a superficial callus cell, possibly indicating a unicellular origin, or from epidermal and subepidermal callus cells, representing a multicellular origin. A fold, the somatic embryo scutellum that subsequently develops into a cotyledonary leaf, indicates the somatic embryo differentiation. The somatic embryos also showed a growth increase of the primary root and, occasionally, a delay in root development. A possible alternative pathway for the origin of somatic embryos is suggested, in which a SSEO forms a clump of somatic embryos. Received: 4 June 1998 / Revision received: 28 August 1998 / Accepted: 7 December 1998     

Association mapping of plant height, yield, and yield stability in recombinant chromosome substitution lines (RCSLs) using Hordeum vulgare subsp. spontaneum as a source of donor alleles in a Hordeum vulgare subsp. vulgare background

Grain yield and plant height of 80 recombinant chromosome substitution lines (RCSLs) of barley were measured in six environments with contrasting available moisture profiles. Two environments were in OR, USA (Moro and Pendleton) during one growing season (2004), and four in Chile (Cauquenes and Santa Rosa) during two growing seasons (2004/2005 and 2007/2008). From the yield data obtained in the different environments, yield adaptability (Finlay–Wilkinson slope) and stability (deviations from regression) were calculated. Two commercial cultivars (Harrington and Baronesse) were used as checks in all environments. Marker-quantitative trait associations were identified using 47 simple sequence repeats (SSRs) and the general linear model (GLM) implemented in TASSEL. The mean plant height and grain yield of the 80 RCSLs differed greatly across environments, reflecting differences in water availability. In all environments, there were significant differences (P < 0.05) in grain yield among RCSLs. There was also abundant variation in yield adaptability, indicating a differential response of the RCSLs to environmental conditions across environments. Using principal component analysis, it was possible to identify genotypes with better agronomic performance than the recurrent parent cv. Harrington. The association analysis revealed 21 chromosomal regions that were highly correlated with differences in grain yield, plant height and/or yield adaptability (Finlay–Wilkinson slope). In approximately one-fourth of the cases, the H. spontaneum donor contributed favorable alleles. The associations were referenced to the quantitative trait loci (QTL) for the same traits reported in the literature.     

Organophosphate induced chlorophyll mutations in Hordeum vulgare

Summary Ten Organophosphorus (OP) insecticides are tested for their genetic toxicology in the Hordeum vulgare system. Of these, 8 OPs induced chlorophyll mutations of which 6 are new discoveries.     

Testing linkage disequilibrium in sibships

We describe the use of multivariate regression for testing allelic association in the presence of linkage, using marker genotype data from sibships. The test is valid, provided that the correct mean structure is modeled but does not require the correlation structure within families to be specified. The test can be implemented using standard statistical software such as the SAS programming language. In a simulation study, we evaluated this new test in comparison with one from a standard, matched-case-control analysis. First, we noted that the genetic effect needed to be quite extreme before residual familial correlation due to linkage led to false inference using the standard, matched-pair analysis. Second, we showed that under examples of extreme residual familial correlation, the new test had the correct test size. Third, we found that the test was more powerful than the sibship disequilibrium test of Horvath and Laird. Finally, we concluded that although the standard analysis may lead to correct inference for practical purposes, the new test is valid, even under extreme residual familial correlation and with no cost in power at the causal locus.     

Joint linkage and linkage disequilibrium mapping in natural populations

A new strategy for studying the genome structure and organization of natural populations is proposed on the basis of a combined analysis of linkage and linkage disequilibrium using known polymorphic markers. This strategy exploits a random sample drawn from a panmictic natural population and the open-pollinated progeny of the sample. It is established on the principle of gene transmission from the parental to progeny generation during which the linkage between different markers is broken down due to meiotic recombination. The strategy has power to simultaneously capture the information about the linkage of the markers (as measured by recombination fraction) and the degree of their linkage disequilibrium created at a historic time. Simulation studies indicate that the statistical method implemented by the Fisher-scoring algorithm can provide accurate and precise estimates for the allele frequencies, recombination fractions, and linkage disequilibria between different markers. The strategy has great implications for constructing a dense linkage disequilibrium map that can facilitate the identification and positional cloning of the genes underlying both simple and complex traits.     

Protoplast preparation without centrifugation: plant regeneration of barley (Hordeum vulgare L.)

Summary Using a modification of the alginate film culture technique we show that it is possible to prepare and culture tobacco mesophyll and barley cell suspension protoplasts without centrifugation. Comparable division frequencies and colony development were observed from protoplasts embedded with enzyme and protoplasts purified by centrifugation. A 3 × 30 min washing regime was found to be the minimum time necessary to remove the enzyme from the gelled alginate matrix. The procedure provides a more gentle method for isolating protoplasts. It has the additional benefit of recovering all of the cells released from the starting tissue. In particular, the smaller protoplasts that are frequently lost during conventional isolation, are maintained. In barley, we illustrate the use of the system for recovering plants from embryogenic protoplast-derived calli from the cultivars Dissa and Igri. Finally, using small volumes of enzyme (50 l) single cell aggregates were used to isolate and culture protoplasts.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzyl aminopurine - MES 2-(N-morpholino)ethanesulfonic acid - NAA -naphthaleneacetic acid