Characterization and molecular mapping of genes determining semidwarfism in barley.

The semidwarf trait is desired in cereal breeding programs for increased lodging resistance. We characterized 27 brachytic (brh) semidwarf mutants in barley (Hordeum vulgare L.) and located the genes on barley chromosome linkage maps. All brachytic genes were transferred into the two-rowed cultivar Bowman by backcrossing four to seven times and selecting for semidwarf plants. The brachytic lines were evaluated for 10 phenotypic traits: plant height, awn, peduncle, and rachis internode length, leaf length and width, lodging, grain yield, number of kernels per spike, and kernel weight. We intercrossed the lines to determine which mutants were at independent loci and which were alleles at the same locus. F2 populations from 18 brh semidwarfs were constructed for genetic mapping using simple sequence repeat (SSR) markers. The brachytic semidwarf near-isogenic lines were significantly shorter than their normal counterparts and most had lower yields (16/27); shorter awns (26/27), peduncles (26/27), and rachis internodes (24/27); and reduced kernel weight (22/27). Twelve of the lines had shorter penultimate leaves and 15 had reduced lodging. Four lines had increased kernels per spike, while one had fewer kernels per spike. Allelism tests and mapping comparisons indicated that the 27 semidwarfs comprise 18 independent genetic loci. SSR mapping placed these loci in five of the seven barley chromosomes. Knowledge of the effects and locations of these brachytic semidwarf genes will help barley breeders select appropriate lines for barley improvement.

Identification of QTLs associated with Fusarium head blight resistance in Zhedar 2 barley.

Fusarium head blight (FHB) in barley and wheat, caused by Fusarium graminearum, is a continual problem worldwide. Primarily, FHB reduces yield and quality, and results in the production of the toxin deoxynivalenol (DON), which can affect food safety. Identification of QTLs for FHB severity, DON level and related traits heading-date (HD) and plant-height (HT) with consistent effects across a set of environments, would provide the basis for marker-assisted selection (MAS) and potentially increase the efficiency of selection for resistance. A segregating population of 75 double-haploid lines, developed from the three-way cross Zhedar 2/ND9712//Foster, was used for genome mapping and FHB severity evaluation. A linkage map of 214 RFLP, SSR and AFLP markers was constructed. Phenotypic data were collected in replicated field trials from five environments in two growing seasons. The data were analyzed using MQTL software to detect quantitative trait locus (QTL) x environment (E) interactions. Because of the presence of QTL x E, the MQM procedure in MAPQTL was applied to identify QTLs in single environments. We identified nine QTLs for FHB severity and five for low DON. Many of the disease-related QTLs identified were coincident with FHB QTLs identified in previous studies. Only two of the QTLs identified in this study were consistent across all five environments, and both were Zhedar 2 specific. Five of the FHB QTLs were associated with HD, and two were associated with HT. Regions that appear to be promising candidates for MAS and further genetic analysis include the two FHB QTLs on chromosome 2H and one on 6H, which were also associated with low DON and later heading-date in multiple environments. This study provides a starting point for manipulating Zhedar 2-derived resistance by MAS in barley to develop cultivars that will show effective resistance under disease pressure.

Enhancement of plant regeneration from embryogenic callus of commercial barley cultivars.

Genotypic restrictions on plant regeneration from cultured cells have hindered the genetic transformation of most barley cultivars. Optimizing culturing protocols for specific cultivars of commercial interest may facilitate their genetic transformation. Plant regeneration from embryogenic callus of `Harrington', `Morex', and `Hector' as affected by certain protocol modifications was examined in replicated experiments. Regeneration was improved for all cultivars by separately autoclaving certain components of the culture media and by reducing the amount of embryogenic callus cultured per petri dish. Regeneration improvements in response to various concentrations of copper and 2,4-dichlorophenoxyacetic acid were more genotype specific. This study suggests that the development and use of genotype-specific protocols can enhance plant regeneration. Enhancements in plant regeneration are expected to facilitate the transformation of commercial barley germplasm.

Use of a subset of doubled-haploid lines for RAPD interval mapping in barley.

Molecular markers have been used in barley to locate genes and quantitative trait loci. Only a few RAPD markers have been located on barley marker maps. The objectives of this study were (i) to place RAPD markers in specific intervals on the barley linkage map developed from the cross Steptoe (S) x Morex (M), (ii) to examine the distribution of RAPD markers, and (iii) to compare markers amplified by Taq DNA polymerase with those amplified by the Stoffel fragment of Taq DNA polymerase. Screening of DNA from S and M with 362 decamer primers identified 85 that amplified 127 reliable RAPDs. A subset of 15 doubled-haploid (DH) lines from the 150 DH line mapping population was used to place these RAPD markers in intervals on the SM map. This subset can be used for rapid placement of any new markers on the SM linkage map. Most of the RAPD markers were dominant but four codominant RAPDs were identified. The RAPDs were not evenly distributed, with many clustered around the centromeric region of each chromosome. Two of these clusters were located in intervals larger than 15 cM. Testing of 38 to 42 additional DH lines provided more precise placement of eight of the markers in these clusters. Reliable RAPDs were detected with 44% of the primers tested with the Stoffel fragment, but with only 17% of the primers tested with Taq DNA polymerase. These RAPDs provide additional markers for use in barley improvement.

Molecular marker analysis of hypoploid regenerants from cultures of barley x Canada wild rye.

Canada wild rye (CWR, Elymus canadensis L., 2n = 4x = 28) is a potential source of genes for disease resistance and environmental tolerance in barley (Hordeum vulgare L., 2n = 2x = 14). Tissue cultures were initiated from immature inflorescences of CWR x 'Betzes' barley hybrids to promote CWR introgression into barley through possible tissue culture induced chromosome breakage and exchange. Among the plants regenerated, some were missing one (2n = 20) or part of one (2n = 20 + telo) chromosome. The objective of this study was to identify the missing chromosome or chromosome arm in these regenerants through the analysis of molecular (RFLP) markers that previously had been mapped in barley. Forty-six hypoploid regenerants that traced to 30 separate explants obtained from 10 interspecific hybrid plants were evaluated. DNA was digested with the restriction enzyme HindIII, Southern blotted, and probed with 39 genomic and cDNA barley clones that identified sequences polymorphic between barley and CWR. Eight of these probes identified band loss patterns that separated the regenerants into two groups. One group, all with barley cytoplasm, were missing a CWR chromosome homoeologous to barley chromosome 3; a second group, all with CWR cytoplasm, were missing a CWR chromosome homoelogous to barley chromosome 7. These results indicated that chromosome elimination in culture was not random. The two cytoplasm groups were further differentiated by probes that identified band shifts. These band shifts were caused by differences in DNA methylation. Key words : Hordeum vulgare, aneuploidy, Elymus canadensis, tissue culture.

Meiotic and isozymic characterization of plants regenerated from euploid and selfed monosomic tall fescue embryos.

Tissue culture of tall fescue (Festuca arundinacea Schreb., 2n=6x=42) would be enhanced by improving the callus induction and plant regeneration efficiency, and evaluating the meiotic and isozymic variation induced by culture. Mature embryos were cultured from four lines of Kenhy tall fescue and from the progeny of three selfed monosomics. Evaluation of six media-auxin combinations showed callus initiation was greatest on SH medium with 2.5 mg/l 2,4,5-T or 7.4 mg/l pCPA, while plant regeneration was greatest on SH medium with 0.5 mg/l 2,4-D. Cytological analyses of 27 plants derived from euploid parents showed a high frequency of aneuploidy (15/27). Chromosome numbers of aneuploids ranged from 36 to 41, with one plant having 80 chromosomes and two plants being asynaptic. Two of ten monosomic-derived plants were euploid, five were monosomic, one was monosomic with a fragment and two were double monosomic. Zymograms of the parents and regenerants were obtained for the enzymes ACPH, ADH, GOT, 6-PGD and PGI. Isozyme variation was observed for two groups of plants derived from the same Kenhy embryos. One group of four monosomic-derived plants differed for the enzymes GOT and ACPH, and all four plants had a PGI pattern. different from that of the parental monosomic plant. This indicated loss of a PGI allele, probably as a result of callus culture.