Novel pGreen/pSoup dual-binary vector system in multiple T-DNA co-cultivation as a method of producing marker-free (clean gene) transgenic rice (Oriza sativa L) plant

The possibility of producing marker-free transgenic rice plants using a novel dual binary pGreen/pSoup vectors, in multiple T-DNA co-cultivation, was investigated and demonstrated to be feasible. The T-DNA in pSoup (pRT47) vector was engineered to contain the selection marker hygromycin phosphotransferase (aphIV) gene (plus intron in 5′ UTR), and the green fluorescent protein (gfp) as a reporter gene both driven by the CaMV35S promoter and the nopaline synthase terminator. T-DNA in the pGreen (pRT18) vector harboured the phosphinothricin acetyl transferase (bar), as selection marker gene, and the b- glucuronidase (gusA) plus intron as a reporter gene, both driven by the maize 5′ ubiquitin region and the nopaline synthase terminator. Both the pGreen and pSoup plasmids were transformed into E. coli strain DH5α using the PEG-transformation technique and into Agrobacterium strains AGL1using a freeze-thaw method. AGL1 was then used to transform embryogenic nodular units (ENU), derived from mature seeds of the model rice genotype Nipponbare. Selection on herbicide (PPT) or antibiotic (hygromycin) of co-cultured ENUs led to the production of numerous independently transformed callus clones containing both T-DNAs from the selected and unselected vector. While co- ransformation frequencies were 71% and 80% for the hygromycin only and herbicide (PPT) only selection, respectively, data showed that co-expression frequency is most useful for the production of marker free transgenic rice. About half (50%) of the independent transgenic plant lines contained at least one unlinked T-DNA integration. In this work, we showed for the first time, that the novel dual-binary pGreen/pSoup can efficiently produce marker-free transgenic rice.African Journal of Biotechnology Vol. 4 (6), pp. 531-540, 200

Chromosomal location of genes encoding for resistance to septoria tritici blotch (Mycosphaerella graminicola) in substitution lines of wheat

Chromosomal location of resistance to Mycosphaerella graminicola was studied in substitution lines of resistant Triticum genotypes into the (susceptible) cultivar Chinese Spring (T. aestivum). (Moderately) resistant genotypes for which substitution lines were available were tested in a first screening. We selected a synthetic hexaploid wheat (Synthetic 6x; T. dicoccoides × T. tauschii), T. spelta and the wheat (T. aestivum) cultivars Cheyenne and Cappelle-Desprez. In a second screening the most suitable Argentinian isolates were identified. We decided to use the isolate IPO 92067 (all sets of substitution lines), IPO 93014 (substitution lines of Synthetic 6x, Cappelle-Desprez and T. spelta) and IPO 92064 (substitution lines of Cheyenne). In the final experiments, substitution lines of the selected genotypes into Chinese Spring were grown in two different environments and inoculated with the selected isolates at the seedling stage (lines of all four selected genotypes) or the adult stage (lines of Synthetic 6x and Cheyenne). Resistance was expressed as (reduction in) necrosis percentage or pycnidial coverage percentage; the two measures were highly (linearly) correlated. When tested in the seedling stage, all chromosomes seemed to carry genes effective against M. graminicola. Many genes were effective against only one isolate or in only one environment or their effects only showed in one resistance parameter. Often these effects were minor. Only chromosome 7D of Synthetic 6x was found with a major effect against both isolates tested. When tested in the adult stage, all lines but the one carrying chromosome 4B from the resistant parent seemed to show genes effective against M. graminicola. The line carrying chromosome 7D from Synthetic 6x showed a level of resistance similar to the resistant parent for isolate IPO 92067, but not for isolate IPO 93014. Major genes, effective against both isolates, were also found on chromosomes 5A and 5D from Synthetic 6x. Lines carrying chromosome 1B, 5D or 6D from Cheyenne showed major effects against isolate IPO 92064. For both necrosis percentage and pycnidial coverage percentage, highly significant linear correlations were found between resistance in the seedling stage and resistance in the adult stage. However, the variance accounted for was only small (20-24%; n = 184).Facultad de Ciencias Agrarias y Forestale

Chromosome manipulation and its exploitation in the genetics and breeding of wheat

Chromosome manipulation in bread wheat uses aneuploid methods to transfer whole chromosomes or segments of chromosomes from one variety into another, or from related species into wheat. These methods have been applied extensively so that an incomparable range of aneuploid and whole chromosome substituion lines now exists in wheat. This has given rise to some of the most detailed genetic studies of economic characters carried out on a crop plant and include the analysis of the semi-dwarfism of Mediterranean wheat and the location of genes affecting grain protein amounts. The applications of this knowledge to wheat breeding is considered as well as the potential that these methods have for promoting the understanding of plant processes and of the molecular organisation of the wheat genome.C. N. LAW, J. W. SNAPE and A. J. WORLAND, Plant Breeding Institute, Maris Lane, Trumpington, Cambridge CB2 2LQ, U. K

Chromosomal location of genes encoding for resistance to septoria tritici blotch (Mycosphaerella graminicola) in substitution lines of wheat

Chromosomal location of resistance to Mycosphaerella graminicola was studied in substitution lines of resistant Triticum genotypes into the (susceptible) cultivar Chinese Spring (T. aestivum). (Moderately) resistant genotypes for which substitution lines were available were tested in a first screening. We selected a synthetic hexaploid wheat (Synthetic 6x; T. dicoccoides × T. tauschii), T. spelta and the wheat (T. aestivum) cultivars Cheyenne and Cappelle-Desprez. In a second screening the most suitable Argentinian isolates were identified. We decided to use the isolate IPO 92067 (all sets of substitution lines), IPO 93014 (substitution lines of Synthetic 6x, Cappelle-Desprez and T. spelta) and IPO 92064 (substitution lines of Cheyenne). In the final experiments, substitution lines of the selected genotypes into Chinese Spring were grown in two different environments and inoculated with the selected isolates at the seedling stage (lines of all four selected genotypes) or the adult stage (lines of Synthetic 6x and Cheyenne). Resistance was expressed as (reduction in) necrosis percentage or pycnidial coverage percentage; the two measures were highly (linearly) correlated. When tested in the seedling stage, all chromosomes seemed to carry genes effective against M. graminicola. Many genes were effective against only one isolate or in only one environment or their effects only showed in one resistance parameter. Often these effects were minor. Only chromosome 7D of Synthetic 6x was found with a major effect against both isolates tested. When tested in the adult stage, all lines but the one carrying chromosome 4B from the resistant parent seemed to show genes effective against M. graminicola. The line carrying chromosome 7D from Synthetic 6x showed a level of resistance similar to the resistant parent for isolate IPO 92067, but not for isolate IPO 93014. Major genes, effective against both isolates, were also found on chromosomes 5A and 5D from Synthetic 6x. Lines carrying chromosome 1B, 5D or 6D from Cheyenne showed major effects against isolate IPO 92064. For both necrosis percentage and pycnidial coverage percentage, highly significant linear correlations were found between resistance in the seedling stage and resistance in the adult stage. However, the variance accounted for was only small (20-24%; n = 184).Facultad de Ciencias Agrarias y Forestale

Full sphere hydrodynamic and dynamo benchmarks

Convection in planetary cores can generate fluid flow and magnetic fields, and a number of sophisticated codes exist to simulate the dynamic behaviour of such systems. We report on the first community activity to compare numerical results of computer codes designed to calculate fluid flow within a whole sphere. The flows are incompressible and rapidly rotating and the forcing of the flow is either due to thermal convection or due to moving boundaries. All problems defined have solutions that allow easy comparison, since they are either steady, slowly drifting or perfectly periodic. The first two benchmarks are defined based on uniform internal heating within the sphere under the Boussinesq approximation with boundary conditions that are uniform in temperature and stress-free for the flow. Benchmark 1 is purely hydrodynamic, and has a drifting solution. Benchmark 2 is a magnetohydrodynamic benchmark that can generate oscillatory, purely periodic, flows and magnetic fields. In contrast, Benchmark 3 is a hydrodynamic rotating bubble benchmark using no slip boundary conditions that has a stationary solution. Results from a variety of types of code are reported, including codes that are fully spectral (based on spherical harmonic expansions in angular coordinates and polynomial expansions in radius), mixed spectral and finite difference, finite volume, finite element and also a mixed Fourier–finite element code. There is good agreement between codes. It is found that in Benchmarks 1 and 2, the approximation of a whole sphere problem by a domain that is a spherical shell (a sphere possessing an inner core) does not represent an adequate approximation to the system, since the results differ from whole sphere results

Utility and limitations of hepascore and transient elastography to detect advanced hepatic fibrosis in HFE hemochromatosis

Aspartate aminotransferase-to-platelet ratio index (APRI) and Fibrosis-4 Index (Fib4) have been validated against liver biopsy for detecting advanced hepatic fibrosis in HFE hemochromatosis. We determined the diagnostic utility for advanced hepatic fibrosis of Hepascore and transient elastography compared with APRI and Fib4 in 134 newly diagnosed HFE hemochromatosis subjects with serum ferritin levels \u3e 300 µg/L using area under the receiver operator characteristic curve (AUROC) analysis and APRI- ( \u3e 0.44) or Fib4- ( \u3e 1.1) cut-offs for AHF, or a combination of both. Compared with APRI, Hepascore demonstrated an AUROC for advanced fibrosis of 0.69 (95% CI 0.56–0.83; sensitivity = 69%, specificity = 65%; P = 0.01) at a cut-off of 0.22. Using a combination of APRI and Fib4, the AUROC for Hepascore for advanced fibrosis was 0.70 (95% CI 0.54–0.86, P = 0.02). Hepascore was not diagnostic for detection of advanced fibrosis using the Fib4 cut-off. Elastography was not diagnostic using either APRI or Fib4 cut-offs. Hepascore and elastography detected significantly fewer true positive or true negative cases of advanced fibrosis compared with APRI and Fib4, except in subjects with serum ferritin levels \u3e 1000 µg/L. In comparison with APRI or Fib4, Hepascore or elastography may underdiagnose advanced fibrosis in HFE Hemochromatosis, except in individuals with serum ferritin levels \u3e 1000 µg/L

Copy Number Variation Affecting the Photoperiod-B1 and Vernalization-A1 Genes Is Associated with Altered Flowering Time in Wheat (Triticum aestivum)

The timing of flowering during the year is an important adaptive character affecting reproductive success in plants and is critical to crop yield. Flowering time has been extensively manipulated in crops such as wheat (Triticum aestivum L.) during domestication, and this enables them to grow productively in a wide range of environments. Several major genes controlling flowering time have been identified in wheat with mutant alleles having sequence changes such as insertions, deletions or point mutations. We investigated genetic variants in commercial varieties of wheat that regulate flowering by altering photoperiod response (Ppd-B1 alleles) or vernalization requirement (Vrn-A1 alleles) and for which no candidate mutation was found within the gene sequence. Genetic and genomic approaches showed that in both cases alleles conferring altered flowering time had an increased copy number of the gene and altered gene expression. Alleles with an increased copy number of Ppd-B1 confer an early flowering day neutral phenotype and have arisen independently at least twice. Plants with an increased copy number of Vrn-A1 have an increased requirement for vernalization so that longer periods of cold are required to potentiate flowering. The results suggest that copy number variation (CNV) plays a significant role in wheat adaptation

Human topoisomerase IIα uses a two-metal-ion mechanism for DNA cleavage

The DNA cleavage reaction of human topoisomerase IIα is critical to all of the physiological and pharmacological functions of the protein. While it has long been known that the type II enzyme requires a divalent metal ion in order to cleave DNA, the role of the cation in this process is not known. To resolve this fundamental issue, the present study utilized a series of divalent metal ions with varying thiophilicities in conjunction with DNA cleavage substrates that replaced the 3′-bridging oxygen of the scissile bond with a sulfur atom (i.e. 3′-bridging phosphorothiolates). Rates and levels of DNA scission were greatly enhanced when thiophilic metal ions were included in reactions that utilized sulfur-containing substrates. Based on these results and those of reactions that employed divalent cation mixtures, we propose that topoisomerase IIα mediates DNA cleavage via a two-metal-ion mechanism. In this model, one of the metal ions makes a critical interaction with the 3′-bridging atom of the scissile phosphate. This interaction greatly accelerates rates of enzyme-mediated DNA cleavage, and most likely is needed to stabilize the leaving 3′-oxygen