A rapid and simple method for the separation of pure lymphocytes from horse blood

A method for the separation of pure and viable lymphocytes and granulocytes from the same blood sample in horses was reported. By centrifuging equine heparinized blood at 100 xg for 10 min at room temperature (r.t.), the resulting supernatant plasma was an almost pure (97.71 +/- 0.30%; n = 15) suspension of highly viable (98.72 +/- 0.28%) lymphocytes. When sodium citrate was used as an anticoagulant, lymphocyte suspensions collected in the same manner showed lower purity (87.89 +/- 1.59%; n = 9) and higher yields (56.56 +/- 3.89%, n = 9 versus 36.11 +/- 2.23%, n = 15). Where needed, a further centrifugation at 250 xg for 3 min (r.t.) of heparinized lymphocyte preparations removed an average of 87.39% (n = 15) contaminating platelets. A suspension of 85.96 +/- 2.20% pure granulocytes (93.23 +/- 1.74% neutrophils; n = 14) with minimal contamination by erythrocytes and high viability (93.11 +/- 1.26%) was obtained by performing a flash red blood cell lysis on the white-greyish layer resulting from the centrifugation of the heparinized blood samples. Among the several methods available, the procedure described herein is easy, rapid, cheap and reproducible.     

Synthesis and in-vitro antifungal activity of 6-trifluoromethyipyrazolo[3,4-6d]pyrimidin-4(5H)-thiones

Twelve 6-trifluoromethylpyrazolo[3,4-d]pyrimidin-4(5H)-thiones were prepared by the reaction between 4-thiocarbamoyl-5-aminopyrazoles and trifluoroacetic anhydride. They were tested in vitro for antifungal activity against a series of phytopathogenic fungi of different taxonomic classes. The EC50 and MIC values of four compounds were comparable or inferior to those of reference commercial fungicides in controlling Sclerotinia minor, Corticium solani and Phytium ultimum.     

Novel Prunus rootstock somaclonal variants with divergent ability to tolerate waterlogging

Plants require access to free water for nutrient uptake, but excess water surrounding the roots can be injurious or even lethal because it blocks the transfer of free oxygen between the soil and the atmosphere. Genetic improvement efforts in this study were focused on the increased tolerance in roots to waterlogging. Among a pool of clones generated in vitro from leaf explants of rootstock Mr.S.2/5 of Prunus cerasifera L., the S.4 clone was flood tolerant whereas the S.1 clone was sensitive. The S.4 clone formed adventitious roots on exposure to flooding. Moreover, the chlorophyll content and mitochondrial activity in the leaf and root, soluble sugar content, alcohol dehydrogenase activity and ethylene content were different between the clones. The sorbitol transporter gene (SOT1) was up-regulated during hypoxia, the alcohol dehydrogenase genes (ADH1 and ADH3) were up-regulated in the leaves and down-regulated in the roots of the S.4 clone during hypoxia, and the 1-aminocyclopropane-1-oxidase gene (ACO1) was up-regulated in the leaves and roots of the S.4 clone during hypoxia and down-regulated in the wild-type roots. In addition, in the S.4 root, hypoxia induced significant down-regulation of a glycosyltransferase-like gene (GTL), which has a yet-undefined role. Although the relevant variation in the S.4 genome has yet to be determined, genetic alteration clearly conferred a flooding-tolerant phenotype. The isolation of novel somaclonals with the same genomic background but with divergent tolerance to flooding may offer new insights in the elucidation of the genetic machinery of resistance to flooding and aid in the selection of new Prunus rootstocks to be used in various adverse environments.     

Molecular markers for improving control of soil-borne pathogen <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> in sugar beet

Fusarium spp. cause severe damage in many agricultural crops, including sugar beet, with Fusarium oxysporum historically being considered as the most damaging of all species. Sugar beet needs to be protected from this class of soil-borne pathogens in order to ensure an optimal sugar yield in the field. Genetic control of the disease is crucial in managing these pathogens. Identification of single nucleotide polymorphism (SNP) markers linked to resistance can be a powerful tool for the introgression of valuable genes needed to develop Fusarium-resistant varieties. A candidate gene approach was carried out to identify SNP markers linked to putative Fusarium resistance sources in sugar beet. Five resistant analogue genes (RGAs) were screened by means of high resolution melting (HRM) analysis in a set of sugar beet lines, considered as resistant and susceptible to Fusarium oxysporum. HRM polymorphisms were observed in 80% of amplicons. Two HRM polymorphisms were significantly associated with Fusarium resistance (P < 0.05). The amplicons that showed association were sequenced and two SNPs were identified. The association was further validated on 96 susceptible and 96 resistant plants using competitive allele-specific PCR (KASPar) technology. The selected SNPs could be used for marker-assisted breeding of Fusarium resistance in sugar beet.     

Genetic variability following selection for scrapie resistance in six autochthonous sheep breeds in the province of Bolzano (northern Italy)

Scrapie is an ovine transmissible spongiform encephalopathy, and its susceptibility is associated with polymorphisms in the prion protein gene (PRNP). Genetic selection is currently the most effective mean for eradication of the susceptible VRQ allele in favour of resistant ARR allele. Maintenance of genetic diversity should be one of the major objectives in breeding programmes, especially in endangered breeds, and genetic information are an excellent alternative to pedigree data where these information are missing. The aim of our study was to determine changes of genetic variability in six native sheep breeds from autonomous province of Bolzano, northern Italy, following simulation of scrapie selection scenarios. A total of 684 rams were investigated for PRNP polymorphisms and for 10 microsatellite loci to estimate genetic variability. Across all loci, a total of 163 alleles were detected with a mean of 10.4 alleles per locus. Average observed (Ho) and unbiased expected (uHe) heterozygosity overall loci were 0.74 and 0.78, respectively, showing a statistically significant deviation from Hardy–Weinberg equilibrium (HWE) in all breeds. This heterozygosity deficit was confirmed by a positive fixation index (Fis), determining a moderate inbreeding in each breed. Simulating a soft selection, where only rams having at least a VRQ allele should be excluded from reproduction, Ho, uHe and Fis values remained almost unchanged, indicating that genetic variability should not be affected by the removal of these individuals. With a mild selection scenario, considering only rams with at least one ARR allele, we observed a decrease in the mean alleles per breed (8.9) and the maintenance of heterozygosity deficiency, except for two breeds, where it was any longer significant. These results showed that selection strategies allowing use of heterozygous as well homozygous ARR rams might be the right compromise to improve resistance to scrapie and to do not dramatically affect genetic variability of these breeds.