Molecular mapping of genomic regions underlying barley yellow dwarf tolerance in cultivated oat (Avena sativa L.).

Barley yellow dwarf (BYD) is one of the most important viral diseases in small grains, including oat (Avena sativa L.). Breeding for BYD tolerance is an effective and efficient means to control the disease. Characterization of major sources of tolerance, and identification of marker and the trait associations, will directly benefit breeding for BYD tolerance. Genomic regions underlying BYD tolerance were mapped and characterized in an oat population consisting of 152 recombinant inbred lines from the cross of 'Ogle' (tolerant)/MAM17-5 (sensitive). Tolerance was evaluated in replicated field trials across 2 years under artificial inoculation with viruliferous aphids harboring BYD virus isolate PAV-IL. Composite interval mapping was used for quantitative trait loci (QTLs) analysis with a framework map consisting of 272 molecular markers. Four QTLs, BYDq1, BYDq2, BYDq3 and BYDq4, for BYD tolerance were identified on linkage groups OM1, 5, 7 and 24, respectively. All but BYDq2 were consistently detected across both years. Significant epistasis was found between some QTLs. The final model including the epistatic effect explained 50.3 to 58.2% of the total phenotypic variation for BYD tolerance. Some QTLs for BYD tolerance were closely linked to QTLs for plant height and days to heading. Potential problems with QTL mapping for BYD tolerance have been discussed. The identified association of markers and tolerance should be useful to pyramid favorable alleles for BYD tolerance into individual oat lines.

A genetic linkage map for hexaploid, cultivated oat (Avena sativa L.) based on an intraspecific cross 'Ogle/MAM17-5'.

Genetic research and breeding of oat ( Avena sativa L.) would be aided by development of a genetic linkage map for a breeding population. Such a map could be used for localization of qualitative and quantitative trait loci, marker-assisted selection and other genetic analysis in an adapted, agronomically useful background. The objectives of this research were to develop a genetic linkage map of hexaploid cultivated oat, to identify homoeologous relationships of linkage groups, and to compare homologous linkage groups between this map and the previously published hexaploid oat map from the cross 'Kanota/Ogle' (KO). A total of 510 markers, including 172 restriction fragment length polymorphisms (RFLP), 324 amplified fragment length polymorphisms (AFLP) and 14 simple sequence repeats (SSR), were assessed on a recombinant inbred population of 152 F(5:6) lines derived from the cross, 'Ogle/MAM17-5' (OM). Twenty eight linkage groups of 5 cM or longer were formed using 476 of the markers, while 34 markers remained either unlinked or in small fragments less than 5 cM. The 28 linkage groups contained from 3 to 33 markers, and varied in size from 5.2 to 123.0 cM, representing a total map length of 1,396.7 cM. Three putative homoeologous groups (OM7, OM8 and OM18; OM2 and OM23; OM13 and OM16) were identified. Comparison with the published KO map indicated that nine OM linkage groups could be determined to be homologous to linkage groups in the KO map. Further comparison of the homologous linkage groups revealed that residual differences in genomic rearrangements existed between the two hexaploid oat populations. Some linkage groups were significantly extended compared with the KO map. Since the OM mapping population is segregating for a number of agronomically important traits, this genetic map will provide a useful tool for identification of qualitative and quantitative loci for these traits.

Epigenetic aspects of somaclonal variation in plants.

Somaclonal variation is manifested as cytological abnormalities, frequent qualitative and quantitative phenotypic mutation, sequence change, and gene activation and silencing. Activation of quiescent transposable elements and retrotransposons indicate that epigenetic changes occur through the culture process. Epigenetic activation of DNA elements further suggests that epigenetic changes may also be involved in cytogenetic instability through modification of heterochromatin, and as a basis of phenotypic variation through the modulation of gene function. The observation that DNA methylation patterns are highly variable among regenerated plants and their progeny provides evidence that DNA modifications are less stable in culture than in seed-grown plants. Future research will determine the relative importance of epigenetic versus sequence or chromosome variation in conditioning somaclonal variation in plants.

Transgenic oat plants via visual selection of cells expressing green fluorescent protein.

New selectable markers and selection systems are needed to increase the efficiency and flexibility of plant transformation. The objective of this research was to determine if the green fluorescent protein (gfp) gene could be utilized as a visual selectable marker for transformation of oat (Avena sativa L.). A modified gfp gene was delivered into oat cells by microprojectile bombardment. Cell clusters expressing gfp were visually identified using fluorescence microscopy and physically isolated at each subculture. Eleven independent transgenic cell lines were obtained, and fertile plants regenerated from all lines. Transgene integration and expression were confirmed in transgenic plants and progeny. Transgene expression segregated in a 3 : 1 ratio in progeny of the majority of the transgenic lines.

Do calcium channel blockers and HMG-CoA reductase inhibitors attenuate allograft arteriopathy?

Recent epidemiologic studies of the risk factors for early and late phases of transplant coronary artery disease (TxCAD) have identified metabolic abnormalities such as hyperlipidemia and insulin resistance as important risk factors, independent of rejection. In randomized trials, calcium channel blockers and hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors were shown to decrease coronary artery intimal thickening and stenosis. Furthermore, HMG-CoA reductase inhibitors significantly decreased allograft loss during the first year after transplantation, resulting in a survival benefit, independent of TxCAD and cholesterol lowering. Prevention of acute allograft failure is consistent with known immunomodulatory actions of HMG-CoA reductase inhibitors, and the effects of calcium blockers in preventing TxCAD might have an immunologic basis by virtue of alterations of cyclosporine pharmacodynamics. Hence these two strategies for targeting antigen-independent mechanisms should lead to a significant reduction in the incidence of TxCAD, a goal that has until this time defied all the advances in immunosuppression during the past three decades of heart transplantation.

Root tip cell cycle synchronization and metaphase-chromosome isolation suitable for flow sorting in common wheat (Triticum aestivum L.).

An efficient procedure for cell-cycle synchronization in meristematic root tips was achieved in common wheat. Treatment parameters for synchronizing the cell cycle of root tip meristem cells, such as time-course and applied concentrations of various chemicals, were systematically tested and optimized by flow cytometric analysis of isolated nuclei. High mitotic indices (69.5% in the root tip meristematic area) were routinely obtained by treating germinating seeds with 1.25 mM hydroxyurea for 16 h, followed by incubation in a hydroxyurea-free solution for 2 h, and treatment with 1 μM trifluralin for 4 h. Uniform seed germination prior to treatment is very important for achieving consistently high metaphase indices in the root tips. Large numbers of metaphase chromosomes, suitable for flow cytometric analysis and sorting, were isolated from synchronized root tip cells. Flow sorted wheat chromosomes, via univariate and bivariate analysis, showed four major chromosome peaks. Each discrete peak may represent wheat chromosome types with similar DNA content. Bivariate flow karyotyping based on AT and GC content did not improve the separation of wheat chromosomes.

Cell synchronization and isolation of metaphase chromosomes from maize (Zea mays L.) root tips for flow cytometric analysis and sorting.

Accumulation of cells containing metaphase chromosomes is an important step in cytological analyses and chromosome sorting procedures. The goal of this research was to optimize treatment parameters to synchronize the cell cycle of maize root tip meristem cells. Levels of hydroxyurea, a DNA synthesis inhibitor, were assessed for their utility in accumulating cells at the G1 phase of the cell cycle. Trifluralin, amiprophos-methyl, and colchicine were used to accumulate cells containing metaphase chromosomes upon release from hydroxyurea inhibition. Optimal mitotic indices were achieved by treating seedlings with 5 mM hydroxyurea for 18 h, incubating for 1 h without chemical treatment to release the hydroxyurea block, and then treating emerging roots with 1 μM trifluralin for 4 h. The mitotic index of synchronized maize root tips was over 70%. Uniformity of synchronization depended upon selection of seeds with emerging radicles that were similar in length at the time of treatment. Suspensions of intact chromosomes were prepared by a simple slicing procedure. The chromosome preparations were found to be suitable for flow cytometric characterization and sorting. Chromosome peaks of the observed flow karyotype resembled the predicted flow karyotype calculated on the basis of maize chromosome size. Key words : flow karyotype, hydroxyurea, plant chromosome sorting, trifluralin.

Silicon carbide fiber-mediated stable transformation of plant cells.

Maize (Zea mays, cv 'Black Mexican Sweet') (BMS) and tobacco (Nicotiana tabacum, cv 'Xanthi') tissue cultures were transformed using silicon carbide fibers to deliver DNA into suspension culture cells. DNA delivery was mediated by vortexing cells in the presence of silicon carbide fibers and plasmid DNA. Maize cells were treated with a plasmid carrying both the BAR gene, whose product confers resistance to the herbicide BASTA, and a gene encoding ß-glucuronidase (GUS). Tobacco cells were treated with two plasmids to co-transfer genes encoding neomycin phosphotransferase (NPTII) and GUS from the respective plasmids. Thirty-four BASTA-resistant BMS colonies and 23 kanamycin-resistant tobacco colonies recovered following selection contained intact copies of the BAR gene and NPTII genes, respectively, as determined by Southern blot analysis. Sixty-five percent of the resistant BMS colonies and 50% of the resistant tobacco colonies also expressed GUS activity. Intact copies of the GUS gene were observed in Southern blots of all resistant BMS and tobacco colonies that expressed GUS activity. These results indicate that a simple, inexpensive DNA delivery procedure employing silicon carbide fibers can be used to reproducibly transform cells of both monocotyledonous and dicotyledonous plant species.Mention of a trademark, vendor, or proprietary product does not constitute a guarantee or warranty of the product by the University of Minnesota or the USDA, and does not imply its approval to the exclusion of other products or vendors that may also be suitable.

Silicon carbide fiber-mediated DNA delivery into plant cells.

Silicon carbide fiber-mediated delivery of DNA into intact plant cells was investigated. Black Mexican Sweet (BMS) maize (Zea mays) and tobacco (Nicotiana tabacum) suspension culture cells were vortexed in the presence of liquid medium, plasmid DNA encoding ß-glucuronidase (GUS), and silicon carbide fibers. Penetration of BMS cells by the silicon carbide fibers was observed by scanning electron microscopy of vortexed cells. Following fiber and DNA treatment, BMS cells transiently expressed GUS activity at a mean frequency of 139.5 units (one unit = one blue cell or one colony of blue cells) per sample. Treated tobacco cells expressed an average of 373 GUS units per sample. Untreated controls did not exhibit GUS activity. These results indicate that the silicon carbide fibers-vortex procedure can be used to rapidly and inexpensively deliver foreign DNA into intact plant cells for investigations of transient gene expression.