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/ .

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.

Structural and functional characterization of a winter malting barley.

The development of winter malting barley (Hordeum vulgare L.) varieties is emerging as a worldwide priority due to the numerous advantages of these varieties over spring types. However, the complexity of both malting quality and winter hardiness phenotypes makes simultaneous improvement a challenge. To obtain an understanding of the relationship between loci controlling winter hardiness and malt quality and to assess the potential for breeding winter malting barley varieties, we structurally and functionally characterized the six-row accession "88Ab536", a cold-tolerant line with superior malting quality characteristics that derives from the cross of NE76129/Morex//Morex. We used 4,596 SNPs to construct the haplotype structure of 88Ab536 on which malting quality and winter hardiness loci reported in the literature were aligned. The genomic regions determining malting quality and winter hardiness traits have been defined in this founder germplasm, which will assist breeders in targeting regions for marker-assisted selection. The Barley1 GeneChip array was used to functionally characterize 88Ab536 during malting. Its gene expression profile was similar to that of the archetypical malting variety Morex, which is consistent with their similar malting quality characteristics. The characterization of 88Ab536 has increased our understanding of the genetic relationships of malting quality and winter hardiness, and will provide a genetic foundation for further development of more cold-tolerant varieties that have malt quality characteristics that meet or exceed current benchmarks.

Enhanced induction of microspore embryogenesis after n-butanol treatment in wheat (Triticum aestivum L.) anther culture.

The aim of this study was the improvement of embryo production in wheat anther culture. Three butanol alcohols, n-butanol, sec-butanol and tert-butanol, were evaluated for their effect on microspore embryogenesis in two spring cultivars of wheat, Pavon and Caramba. Application of n-butanol, at 0.1 and 0.2% (v/v) in the induction media for 5 h, highly improved embryo production in both cultivars. Sec- and tert-butanol performed similarly to control plates. Regeneration ability was unaffected by any butyl-alcohol treatment. As a consequence of the higher embryo production after n-butanol treatment, the number of green regenerated plants increased up to five times in cultivar Pavon and up to three times in cultivar Caramba. The percentage of green plants was improved or unaffected by the treatment. Doubled haploid plant production was between 2 and 4 times higher after n-butanol treatment than in control plates. Therefore, n-butanol was successfully applied in the production of wheat doubled haploids. This primary alcohol is known as an activator of phospholipase D and has been previously reported to disrupt cortical microtubules and detach them from the plasma membrane in plants. Its effects on androgenetic induction could confirm the importance of microtubule regulation in plant cell fate, specifically in microspore development. A possible implication of phospholipase D is discussed.

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

The objective of this work was to produce doubled haploid plants from durum wheat through the induction of androgenesis. A microspore culture technique was developed and used to produce fertile doubled haploid plants of agronomic interest. Five cultivars, one selected line, plus a collection of 20 F(1) crosses between different genotypes of high breeding value were used. Studies on several factors such as pre-treatments and media components were carried out in order to develop a protocol to regenerate green haploid plantlets. Anthers were pre-treated in 0.7 M mannitol. Microspores, from anther maceration, were plated on a C(17) induction culture medium with ovary co-culture. The optimum regeneration medium J25-8 was used. From 35 microspore isolations, 407 green plantlets were obtained. With this technique mature embryos were obtained. Green plants were regenerated from all genotypes used and approximately 67% of them were spontaneously doubled haploids. Some haploids and a very few polyploids plants were obtained. From the 407 plants, 275 were completely fertile and gave enough seeds to be assayed in the field. This protocol could be used complementary to or instead of the intergeneric crossing with maize as an economically feasible method to obtain doubled haploids from most durum wheat genotypes.

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.

Low responsiveness of six-rowed genotypes to androgenesis in barley does not have a pleiotropic basis.

A heterozygous mutant for the two- and six-rowed character was isolated in the barley cultivar Igri through application of sodium azide to isolated microspore cultures and posterior regeneration. Six-rowed and two-rowed homozygotic plants were subsequently identified in the self-pollinated M2 progenies of the original heterozygous M1. Detailed molecular markers confirmed the isogenic nature of this recovered mutant and the original cultivar Igri. A comparative study of the anther culture response of this six-rowed induced mutant vs. diploid 'Igri' was performed to assess whether the two- or six-rowed gene influences anther culture response in barley through a pleiotropic effect or via linkage disequilibrium. No significant differences for any of the recorded variables throughout the in vitro regeneration process were detected between the 'Igri' six-rowed mutant and any of their two-rowed isogenic lines. This suggests that row-type association with anther culture response in barley cultivars is due to the effect of a tight linkage with other genes directly responsible for androgenic response.

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.

Iron deficiency decreases the Fe(III)-chelate reducing activity of leaf protoplasts.

The ferric-chelate reductase (FC-R) activity of mesophyll protoplasts isolated from Fe-sufficient (control) and Fe-deficient sugar beet (Beta vulgaris L.) leaves has been characterized. Measurements were made in an ionic environment similar to that in the apoplastic space of the sugar beet mesophyll cells. The FC-R activity of Fe-sufficient and Fe-deficient protoplasts was dependent on light. Fe deficiency decreased markedly the FC-R activity per protoplast surface unit. The optimal pH for the activity of the FC-R in mesophyll protoplasts was in the range 5.5 to 6.0, typical of the apoplastic space. Beyond pH 6.0, the activity of the FC-R in mesophyll protoplasts decreased markedly in both Fe-sufficient and Fe-deficient protoplasts. These data suggest that both the intrinsic decrease in FC-R activity per protoplast surface and a possible shift in the pH of the apoplastic space could lead to the accumulation of physiologically inactive Fe pools in chlorotic leaves.

Somatic embryogenesis and plant regeneration from barley cultivars grown in Spain.

Thirty-two barley cultivars grown in Spain, 18 of the two-row type and 14 of the six-row type, were screened for plant regeneration from cultured immature embryos. Although there was much variation in regeneration capacity among the cultivars, plants were obtained from all cultivars except Almunia. No statistical differences were found in the percentage of regeneration between two- and six-row types. The influence of the auxins 2,4-dichlorophenoxyacetic acid, dicamba, and picloram on the induction and maintenance of embryogenesis and regeneration capacity after 3-4 months in culture, were evaluated for cultivars Cobra, Hop and Reinette. Hop had the highest rates of maintenance of embryogenic capacity and plant regeneration. The medium containing dicamba gave the best embryogenic callus induction, maintenance and regeneration. Five regeneration media, differing in growth regulators and micronutrient composition, as well as partial desiccation of the calli before regeneration, were tested. The regeneration medium containing 10 µM copper sulfate gave the best results. Regeneration frequencies after 3-4 months in culture of cultivar Hop were raised from 59.5 to 93.7% in this medium. Silver nitrate and partial desiccation of the calli also enhanced plant regeneration, but the medium containing 10 µM of silver nitrate reduced root formation.

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.

Production of gynogenic haploids of Hordeum vulgare L.

Haploid plants were regenerated from cultured unfertilized ovaries of Hordeum vulgare L. (barley). Optimal response was obtained by the addition of 0.6 µM 4-chloro-2-methylphenoxyacetic acid (MCPA), 2.8 µM indole-3-acetic acid (IAA) and 4.4 µM 6-benzyladenine (BA) in the N6 medium. Further increase in the rate of callus formation and the number of green plants produced was possible with the addition of 90 g/l sucrose and 100 g/l coconut water. The stage of development of the ovaries at the time of culture was critical; the largest number of plants being produced by ovaries from flowers at the trinucleate stage of pollen.