Genetic characterization of a reciprocal translocation present in a widely grown barley variety.

Artificially induced translocation stocks have been used to physically map the barley genome; however, natural translocations are extremely uncommon in cultivated genotypes. Albacete is a barley variety widely grown in recent decades in Spain and carrying a reciprocal translocation which obviously does not affect its agronomical fitness. This translocation has been characterized by a combination of cytological and molecular genetic approaches. Firstly, recombination frequencies between markers on chromosomes 1H and 3H were estimated to determine the boundaries of the reciprocal interchange. Secondly, 1H-3H wheat barley telosome addition lines were used to assign selected markers to chromosome arms. Thirdly, fluorescence in situ hybridization (FISH) with rDNA probes (5S and 18S-5.8S-26S) and microsatellite probes [(ACT)(5), (AAG)(5) and (CAG)(5)] was used to determine the locations of the translocation breakpoints more precisely. Fourthly, fine-mapping of the regions around the translocation breakpoints was used to increase the marker density for comparative genomics. The results obtained in this study indicate that the translocation is quite large with breakpoints located on the long arms of chromosomes 1H and 3H, between the pericentromeric (AAG)(5) bands and above the (ACT)(5) interstitial distal bands, resulting in the reciprocal translocation 1HS.1HL-3HL and 3HS.3HL-1HL. The gene content around the translocation breakpoints could be inferred from syntenic relationships observed among different species from the grass family Poaceae (rice, Sorghum and Brachypodium) and was estimated at approximately 1,100 and 710 gene models for 1H and 3H, respectively. Duplicated segments between chromosomes Os01 and Os05 in rice derived from ancestral duplications within the grass family overlap with the translocation breakpoints on chromosomes 1H and 3H in the barley variety Albacete.

Comparative mapping of the Oregon Wolfe Barley using doubled haploid lines derived from female and male gametes.

The Oregon Wolfe Barley mapping population is a resource for genetics research and instruction. Prior reports are based on a population of doubled haploid (DH) lines developed by the Hordeum bulbosum (H.b.) method, which samples female gametes. We developed new DH lines from the same cross using anther culture (A.C.), which samples male gametes. Linkage maps were generated in each of the two subpopulations using the same 1,328 single nucleotide polymorphism markers. The linkage maps based on DH lines derived from the products of megasporogeneis and microsporogenesis revealed minor differences in terms of estimated recombination rates. There were no differences in locus ordering. There was greater segregation distortion in the A.C.-derived subpopulation than in the H.b.-derived subpopulation, but in the region showing the greatest distortion, the cause was more likely allelic variation at the ZEO1 plant height locus rather than to DH production method. The effects of segregation distortion and pleiotropy had greater impacts on estimates of quantitative trait locus effect than population size for reproductive fitness traits assayed under greenhouse conditions. The Oregon Wolfe Barley (OWB) population and data are community resources. Seed is available from three distribution centers located in North America, Europe, and Asia. Details on ordering seed sets, as well as complete genotype and phenotype data files, are available at http://wheat.pw.usda.gov/ggpages/maps/OWB/ .

Mixed model association scans of multi-environmental trial data reveal major loci controlling yield and yield related traits in Hordeum vulgare in Mediterranean environments.

An association panel consisting of 185 accessions representative of the barley germplasm cultivated in the Mediterranean basin was used to localise quantitative trait loci (QTL) controlling grain yield and yield related traits. The germplasm set was genotyped with 1,536 SNP markers and tested for associations with phenotypic data gathered over 2 years for a total of 24 year × location combinations under a broad range of environmental conditions. Analysis of multi-environmental trial (MET) data by fitting a mixed model with kinship estimates detected from two to seven QTL for the major components of yield including 1000 kernel weight, grains per spike and spikes per m(2), as well as heading date, harvest index and plant height. Several of the associations involved SNPs tightly linked to known major genes determining spike morphology in barley (vrs1 and int-c). Similarly, the largest QTL for heading date co-locates with SNPs linked with eam6, a major locus for heading date in barley for autumn sown conditions. Co-localization of several QTL related to yield components traits suggest that major developmental loci may be linked to most of the associations. This study highlights the potential of association genetics to identify genetic variants controlling complex traits.

Linkage map construction involving a reciprocal translocation.

This paper is concerned with a novel statistical-genetic approach for the construction of linkage maps in populations obtained from reciprocal translocation heterozygotes of barley (Hordeum vulgare L.). Using standard linkage analysis, translocations usually lead to 'pseudo-linkage': the mixing up of markers from the chromosomes involved in the translocation into a single linkage group. Close to the translocation breakpoints recombination is severely suppressed and, as a consequence, ordering markers in those regions is not feasible. The novel strategy presented in this paper is based on (1) disentangling the "pseudo-linkage" using principal coordinate analysis, (2) separating individuals into translocated types and normal types and (3) separating markers into those close to and those more distant from the translocation breakpoints. The methods make use of a consensus map of the species involved. The final product consists of integrated linkage maps of the distal parts of the chromosomes involved in the translocation.

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((R)) 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((R)) 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.

Improvements in the production of doubled haploids in durum wheat (Triticum turgidum L.) through isolated microspore culture.

The objective of this study was to produce durum wheat doubled haploid (DH) plants through the induction of microspore embryogenesis. The microspore culture technique was improved to maximize production of green plants per spike using three commercial cultivars. Studies on factors such as induction media composition, induction media support and the stage and growth of donor plants were carried out in order to develop an efficient protocol to regenerate green and fertile DH plants. Microspores were plated on a C(17) induction culture medium with ovary co-culture and a supplement of glutathione plus glutamine; 300 g/l Ficoll Type-400 was incorporated to the induction medium support. Donor plants were fertilized with a combination of macro and microelements. With the cultivars 'Ciccio' and 'Claudio' an average of 36.5 and 148.5 fertile plants were produced, respectively, from 1,000 anthers inoculated. This technique was then used to produce fertile DH plants of potential agronomic interest from a collection of ten F(1) crosses involving cultivars of high breeding value. From these crosses 849 green plants were obtained and seed was harvested from 702 plants indicating that 83% of green plants were fertile and therefore were spontaneously DHs. No aneuploid plant was obtained. The 702 plants yielded enough seeds to be field tested. One of the DH lines obtained by microspore embryogenesis, named 'Lanuza', has been sent to the Spanish Plant Variety Office for Registration by the Batlle Seed Company. This protocol can be used instead of the labor-intensive inter-generic crossing with maize as an economically feasible method to obtain DHs for most crosses involving the durum wheat cultivars grown in Spain.

Use of new EST markers to elucidate the genetic differences in grain protein content between European and North American two-rowed malting barleys.

A population comprising 102 doubled haploid lines were produced from a cross between Beka, a barley cultivar widely grown in Spain, and Logan, a north American cultivar with inherently low protein content, a character considered to derive from the cultivar Karl. The intentions were to determine whether low-nitrogen malting barleys could be developed in Spain, and if genetic factors that influenced protein content were similarly expressed in widely diverse environments, i.e. northeastern Spain and eastern Scotland. An extensive map comprising 187 molecular markers was developed. Expressed sequence-tagged-derived markers were used in addition to anonymous simple sequence repeats to determine the potential for identifying candidate genes for quantitative trait loci (QTLs), and 22 such markers were mapped for the first time. There was transgressive segregation for both yield and protein content, and the gene for low protein from Logan was not expressed in the Scottish environment. In 2002, high yield was associated with earlier heading date in Spain, while late heading at the Scottish site was associated with greater lodging and lower thousand-kernel weight. These appeared to be possible pleiotropic effects of a factor detected on chromosome 2H. Using information from a consensus map, it was shown that this locus on 2H was in the region of the photoperiod response gene Eam6. A QTL explaining 18% of the variation in grain protein content was detected on chromosome 5H in a region in which a gene for nitrate reductase was previously observed. No effect on grain protein was associated with chromosome 6H, which has been suggested as the location of the low protein gene from Karl. However, it is likely that Karl contained more than one genetic factor reducing protein, and we postulate that the gene on 6H may have been lost during the breeding of Logan.

Genetic control of dormancy in a Triumph/Morex cross in barley.

Seed dormancy in barley ( Hordeum vulgare L.) is one of the most important parameters affecting malting. Seed dormancy is quantitatively inherited and variously influenced by the environment. The objectives of the present study were to determine the genome location and effects of quantitative trait loci (QTLs) involved in the expression of seed dormancy in a barley cross between two varieties derived from different germplasm pools. Using a doubled-haploid population of 107 lines of the cross between the malting types Triumph (two-row, dormant) and Morex (six-row, non-dormant), seed dormancy phenotypic data sets from five environments and a 147-marker linkage map were developed in order to perform QTL analyses with simple interval mapping and simplified composite interval mapping procedures. Two different types of variables were considered for seed dormancy characterization: (1) level of dormancy induced during seed development, which was indirectly measured as germination percentage at 3 days and 7 days, GP3 and GP7 respectively; (2) rate of dormancy release in the course of a period after seed harvest (after-ripening). Different mechanisms of genetic control were detected for these two types of dormancy-related traits. A major and consistent dormancy QTL near the centromere on chromosome 7(5H) was associated with the establishment of dormancy during seed development and accounted for 52% and 33% of the variability for GP3 and GP7, respectively. Two other QTLs located in the vicinity of the vrs1 locus on chromosome 2(2H) and near the long arm telomere on chromosome 7(5H) explained 9% and 19% of variation, respectively, for the rate of dormancy release during after-ripening. Likewise, seed dormancy was assessed in an F(2) population derived from the cross between two dormant types of distinct germplasm groups, Triumph (European, two-row, malt) and Steptoe (North American, six-row, feed), which showed similar but not identical genetic control for dormancy. Interestingly, there is remarkable dormancy QTL conservation in both regions on chromosome 7(5H) identified in this study and among other barley mapping populations. These widely conserved QTLs show potential as targets for selection of a moderate level of seed dormancy in breeding programs.

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[Heart arrest during fibro-bronchoscopic intubation in a patient with parapharyngeal space neoplasia]. / Parada cardíaca durante intubación con fibrobroncoscopio en paciente con neoplasia del espacio parafaríngeo.

A 37-year-old woman a diagnosis of squamous carcinoma of the tongue and a history of partial/hemiglossectomy with cervical lymphadenectomy and postoperative radiotherapy was scheduled for biopsy under general anesthesia given the suspicion of local recurrence. She underwent routine preparation for intubation with a fiberoptic bronchoscope with topical anesthesia of mucosal tissue without sedation. Syncope and asystole presented suddenly during intubation and external cardiopulmonary resuscitation and difficult ventilation through a face mask were necessary. She was finally intubated using a retrograde technique and recovered heartbeat after 20 minutes. No neurological sequelae were observed over the next few days. The etiologic diagnosis of this rare complication was later based on the appearance of the characteristic clinical profile of glossopharyngeal neuralgia-asystole syndrome in combination with parapharyngeal space lesion syncope syndrome; magnetic resonance findings of extensive neoplastic invasion of the space confirmed the diagnosis. Endotracheal intubation with a fiberoptic bronchoscope plays an important role in the management of the anticipated difficult airway. It is safe in the awake patient and facilitates the identification of structures, particularly when there are anatomical alterations due to neoplastic invasion, surgery, or radiotherapy. In the patient we describe, however, stimulation caused asystole.

Parada cardíaca durante intubación con fibrobroncoscopio en paciente con neoplasia del espacio parafaríngeo / Cardiac arrest during intubation with a fiberoptic bronchoscope in a patient with parapharyngeal neoplasm

Una paciente de 37 años de edad con diagnóstico de carcinoma escamoso de lengua, intervenida un año antes de hemiglosectomía con linfadenectomía cervical y radioterapia posterior fue programada para biopsia bajo anestesia general por sospecha de recidiva local. En quirófano se preparó de forma reglada para intubación con fibrobroncoscopio mediante anestesia tópica de mucosas y sin sedación. En el transcurso de la intubación presentó de forma súbita un cuadro sincopal con asistolia, precisando de maniobras de reanimación cardiopulmonar con masaje cardíaco externo y ventilación con mascarilla facial, que resultó ser dificultosa. Finalmente se intubó a la paciente mediante una técnica retrógrada, recuperando el latido cardíaco transcurridos veinte minutos. No se objetivó ningún tipo de secuela neurológica en los días siguientes. El diagnóstico etiológico de esta rara complicación se estableció con posterioridad basándonos en la aparición de clínica característica del síndrome de neuralgia-asistolia del glosofaríngeo, en combinación con el definido síndrome del síncope de lesión del espacio parafaríngeo, una vez que fueron determinados hallazgos en la resonancia magnética de invasión neoplásica extensa de dicho espacio. La intubación endotraqueal con fibrobroncoscopio desempeña un papel preponderante en el manejo de la vía aérea difícil anticipada. En el paciente despierto aporta seguridad al mismo tiempo que facilita la identificación de estructuras, especialmente en situaciones de distorsión anatómica por invasión neoplásica, cambios postquirúrgicos o postradioterapia. Sin embargo, en esta paciente la estimulación que ocasionó provocó la asistolia (AU)

Dormancy, ABA content and sensitivity of a barley mutant to ABA application during seed development and after ripening.

Assessment of dormancy inception, maintenance and release was studied for artificially dried immature seeds harvested throughout seed development in the barley cv. Triumph and its mutant line TL43. Each was grown in Spain and Scotland under low and high dormancy inducing conditions, respectively. Both TL43 and Triumph followed a similar pattern of release from dormancy across the seasons, although seeds of TL43 were able to germinate at an earlier seed development stage. Abscisic acid (ABA) content was also studied in immature grains throughout the seed development period. Total amount of ABA in seeds of Triumph and TL43 was higher in plants grown in Scotland than in Spain. However, no clear genotypic differences in ABA pattern in the course of grain development could be detected whilst significant genotypic differences were observed for germination percentage (GP). Endogenous ABA content alone throughout grain development did not explain genetic differences in GP within environments. Environmental and genetic differences in dormancy were also observed on mature seeds throughout the after-ripening period. The initial germination (GP(0)) played a key role in the sensitivity to ABA of post-harvest mature seeds. For the same after-ripening stage, TL43 was more insensitive to exogenous ABA than Triumph. However, ABA responses in seeds of the two genotypes with similar GP(0) at different after-ripening stages were comparable. Therefore, differences in exogenous ABA sensitivity of post-harvest mature grain of these two genotypes seemed to be determined by, or coincident with, the initial germination percentage.

Morphological and agronomical diversity patterns in the Spanish barley core collection.

Seven thousand years of barley cultivation under the environmental hardships typical of the Mediterranean climate have generated genetic singularity of the Spanish barleys, consistently reported in the literature. From the Spanish National Collection of 2289 accessions, a core subset with 159 landraces and 16 old varieties was constituted. Twenty-seven characters were evaluated for the core collection, to define the structure of the diversity. Several evaluation trials were carried out in 1999-2000, whereas yield trials were performed in earlier years. Phenotypic diversity was large for most of the characters studied. Comparisons of genetic diversity between the core and the original collections suggested that the core is a good representation of the existing diversity in the BNG. Comparisons with results of studies on Spanish materials from other collections seem to indicate that the Spanish diversity is not well represented in some world collections. Principal component analyses for quantitative and qualitative characters revealed a clear distinction between two- and six-row cultivars, and also between landraces and commercial varieties. Geographical origins of the landraces were correlated with grain yield, heading date, duration of grain filling period, and growth class. In relation to diseases, altitude played an important role on the resistance to powdery mildew and brown rust. For brown rust, all the resistant landraces came from low altitudes. These geographical gradients seemed consistent with prior knowledge about barley adaptation, and would confirm the agreement between passport data and true adaptive origin of these landraces from a geographical point of view.

Efficient production of androgenic doubled-haploid mutants in barley by the application of sodium azide to anther and microspore cultures.

The aim of this study was to establish a protocol for an efficient production of agronomical and/or physiological mutants from model (cvs. Igri and Cobra) and low-androgenic-responding (cv. Volga) cultivars of barley through the application of a mutagenic agent, sodium azide, to anthers and isolated microspores cultured in vitro. This technology offers the possibilities of screening for recessive mutants in the first generation, selecting for novel genotypes from very large haploid populations, avoiding chimerism and rapidly fixing selected genotypes as fertile true breeding lines. The mutagenic treatment, 10-3-10-5 M sodium azide, was applied during the anther induction pre-treatment or immediately after the microspore isolation procedure. Out of 616 M2 doubled-haploid lines characterised under field conditions, a total of 63 morphological and developmental independent mutant lines were identified. The percentage of M2 doubled-haploid lines carrying mutations per line analysed was 3.8% when 10-4 M sodium azide was applied to anthers from the low-responding cv. Volga; this increased to 8.6% and 15.6% when 10-5 and 10-4 M sodium azide were applied to freshly isolated microspores from model cultivars.

RFLP markers associated with major genes controlling heading date evaluated in a barley germ plasm pool

Adjustment of crop phenology to resources and constraints of the production environment is crucial for barley adaptation to dry-land environments. The aims of this study were to characterize (under field and under controlled conditions) the diverse heading responses of barley cultivars grown in Spain, and to identify phenotypic trait-molecular marker associations with restriction fragment length polymorphism (RFLP) probes which are linked to major loci controlling vernalization (Sh and Sh2) and photoperiod (Ppd-H1 and Ppd-H2) sensitivities in barley. Thirty-two cultivars were sown in field trials for 3 years in four locations in northern Spain (10 autumn and nine winter sowings), and for 1 year in southern Spain (autumn sowing), and 2 years in Scotland (spring sowings); they were also studied under four glasshouse treatments, combining presence/absence of vernalization and short/long photoperiod. These cultivars were examined for the presence of RFLP polymorphisms with four probes and three restriction enzymes. Analyses of variance detected a number of associations which were quite consistent with expectations, suggesting that marker-loci associations found in mapping populations are, to some extent, maintained in a germ plasm pool, and may be useful for germ plasm characterization and marker-assisted selection in breeding programmes. Two possible epistatic interactions among the probes were detected, one of them possibly related to a short-day vernalization response.

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.

Verification of barley seed dormancy loci via linked molecular markers.

Seed dormancy is a relatively complex trait in barley (Hordeum vulgare L.). Several dormancy loci were identified previously by quantitative trait locus analysis. Three reciprocal crosses were made in the present study between parents carrying specific dormancy alleles via linked molecular markers to verify individual dormancy locus effects and potential expression. Analyses of F2 progenies revealed that the dormancy allele at the locus flanked by the markers Ale and ABC302 on the long arm of chromosome 7 had a major effect on dormancy, and was at least partly epistatic to the dormancy locus in the ABC309-MWG851 interval near the telomere of the long arm of chromosome 7. In the absence of the dormancy allele in the Ale-ABC302 interval, the allele in the ABC309-MWG851 interval exerted moderate to large effects on dormancy. Cytoplasmic effects on dormancy were also observed. The germination percentages of progeny with relatively high levels of dormancy were more variable than those of non-dormant or less-dormant progeny, apparently due to environmental effects. Removal of the dormancy allele in the Ale-ABC302 interval, or introducing the dormancy allele in the ABC309-MWG851 interval, should suffice for adjusting dormancy levels in breeding programs to suit various production situations and end uses. The verification of dormancy loci via linked molecular markers allows manipulation of these loci in applied breeding programs.

On the origin of Spanish two-rowed barleys.

To investigate the phylogenetic origin of Spanish two-rowed barleys, we studied 44 accessions of old land-races both morphologically and biochemically to ascertain their similarity with 51 entries of old cultivars and land-races of widespread origin across Europe. They were also compared with 20 accessions of Hordeum spontaneum from the Mediterranean basin and other regions of its distribution range, 14 accessions of Moroccan cultivated six-rowed barley land-races, and different six-rowed Spanish and two-and six-rowed European cultivars. CM-(trypsin inhibitors and subunits of the barley tetrameric α-amylase inhibitor) proteins and hordeins, all of which are endosperm proteins, were used as biochemical markers. The appearance of separate clusters of the Spanish barleys in the numerical classifications for both protein systems as a result of the existence of characteristic gene combinations that do not exist in entries from other origins permitted us to postulate the existence of local ancestors for most of the Spanish two-rowed barleys studied, and, therefore, a possible in situ domestication.

Production of large number of doubled haploid plants from barley anthers pretreated with high concentrations of mannitol.

Pretreatment with increasing concentrations of mannitol, from 0.3 to 0.7 M, was used to induce stress in cultured anthers of barley (Hordeum vulgare L.). Three cultivars with varying degrees of androgenetic ability were studied. A positive linear relationship was found between concentration of mannitol in the pretreatment medium and the number of regenerated green doubled haploid plants in all the cultivars. The pretreatment also resulted in an increasing proportion of embryos to dividing microspores, and in green to albino plantlets. The optimum length of the pretreatment seemed to be genotype dependent. When Ficoll was used as an alternative stress agent a differential genotype response was observed.

Integration of statistical and physiological analyses of adaptation of near-isogenic barley lines.

Seven near-isogenic barley lines, differing for three independent mutant genes, were grown in 15 environments in Spain. Genotype x environment interaction (G x E) for grain yield was examined with the Additive Main Effects and Multiplicative interaction (AMMI) model. The results of this statistical analysis of multilocation yield-data were compared with a morpho-physiological characterization of the lines at two sites (Molina-Cano et al. 1990). The first two principal component axes from the AMMI analysis were strongly associated with the morpho-physiological characters. The independent but parallel discrimination among genotypes reflects genetic differences and highlights the power of the AMMI analysis as a tool to investigate G x E. Characters which appear to be positively associated with yield in the germplasm under study could be identified for some environments.