Development of microsatellite markers for identifying Brazilian Coffea arabica varieties

Microsatellite markers, also known as SSRs (Simple Sequence Repeats), have proved to be excellent tools for identifying variety and determining genetic relationships. A set of 127 SSR markers was used to analyze genetic similarity in twenty five Coffea arabica varieties. These were composed of nineteen commercially important Brazilians and six interspecific hybrids of Coffea arabica, Coffea canephora and Coffealiberica. The set used comprised 52 newly developed SSR markers derived from microsatellite enriched libraries, 56 designed on the basis of coffee SSR sequences available from public databases, 6 already published, and 13 universal chloroplast microsatellite markers. Only 22 were polymorphic, these detecting 2-7 alleles per marker, an average of 2.5. Based on the banding patterns generated by polymorphic SSR loci, the set of twenty-five coffee varieties were clustered into two main groups, one composed of only Brazilian varieties, and the other of interspecific hybrids, with a few Brazilians. Color mutants could not be separated. Clustering was in accordance with material genealogy thereby revealing high similarity

Allelic diversity of S‑RNase alleles in diploid potato species

S-ribonucleases (S-RNases) control the pistil specificity of the self-incompatibility (SI) response in the genus Solanum and several other members of the Solanaceae. The nucleotide sequences of S-RNases corresponding to a large number of S-alleles or S-haplotypes have been characterised. However, surprisingly few S-RNase sequences are available for potato species. The identification of new S-alleles in diploid potato species is desirable as these stocks are important sources of traits such as biotic and abiotic resistance. S-RNase sequences are reported here from three distinct diploid types of potato: cultivated Solanum tuberosum Group Phureja, S. tuberosum Group Stenotomum, and the wild species Solanum okadae. Partial S-RNase sequences were obtained from pistil RNA by RT-PCR or 3’RACE (Rapid Amplification of cDNA Ends) using a degenerate primer. Full length sequences were obtained for two alleles by 5’RACE. Database searches with these sequences, identified sixteen S-RNases in total, all of which are novel. The sequence analysis revealed all the expected features of functional S-RNases. Phylogenetic analysis with selected published S-RNase and S-like-RNase sequences from the Solanaceae revealed extensive trans-generic evolution of the S-RNases and a clear distinction from S-like-RNases. Pollination tests were used to confirm the self-incompatibility status and cross-compatibility relationships of the S. okadae accessions. All the S. okadae accessions were found to be self-incompatible as expected with crosses amongst them exhibiting both cross-compatibility and semi-compatibility consistent with the S-genotypes determined from the S-RNase sequence data. The progeny analysis of four semi-compatible crosses examined by allele-specific PCR provided further confirmation that these are functional S-RNases

Ex vivo immunosuppressive effects of mesenchymal stem cells on Crohn's disease mucosal T cells are largely dependent on indoleamine 2,3-dioxygenase activity and cell-cell contact

Introduction: Crohn's disease (CD) is a disabling chronic enteropathy sustained by a harmful T-cell response toward antigens of the gut microbiota in genetically susceptible subjects. Growing evidence highlights the safety and possible efficacy of mesenchymal stem cells (MSCs) as a new therapeutic tool for this condition. Therefore, we aimed to investigate the effects of bone marrow-derived MSCs on pathogenic T cells with a view to clinical application. Methods: T-cell lines from both inflamed and non-inflamed colonic mucosal specimens of CD patients and from healthy mucosa of control subjects were grown with the antigen muramyl-dipeptide in the absence or presence of donors' MSCs. The MSC effects were evaluated in terms of T-cell viability, apoptotic rate, proliferative response, immunophenotype, and cytokine profile. The role of the indoleamine 2,3-dioxygenase (IDO) was established by adding a specific inhibitor, the 1-methyl-DL-tryptophan, and by using MSCs transfected with the small interfering RNA (siRNA) targeting IDO. The relevance of cell-cell contact was evaluated by applying transwell membranes. Results: A significant reduction in both cell viability and proliferative response to muramyl-dipeptide, with simultaneous increase in the apoptotic rate, was found in T cells from both inflamed and non-inflamed CD mucosa when co-cultured with MSCs and was reverted by inhibiting IDO activity and expression. A reduction of the activated CD4(+) CD25(+) subset and increase of the CD3(+) CD69(+) population were also observed when T-cell lines from CD mucosa were co-cultured with MSCs. In parallel, an inhibitory effect was evident on the expression of the pro-inflammatory cytokines tumor necrosis factor-alpha, interferon-gamma, interleukin-17A and -21, whereas that of the transforming growth factor-beta and interleukin- 6 were increased, and production of the tolerogenic molecule soluble HLA-G was high. These latter effects were almost completely eliminated by blocking the IDO, whose activity was upregulated in MSCs co-cultured with CD T cells. The use of a semipermeable membrane partially inhibited the MSC immunosuppressive effects. Finally, hardly any effects of MSCs were observed when T cells obtained from control subjects were used. Conclusion: MSCs exert potent immunomodulant effects on antigen-specific T cells in CD through a complex paracrine and cell-cell contact-mediated action, which may be exploited for widespread therapeutic use