Specific mapping of disease resistance genes in tetraploid cut roses

Control of fungal diseases is a major constraint of cut-rose cultivation in greenhouses and in transportation around the world. Therefore, development of resistant cultivars is a promising way to reduce the use of chemicals required for controlling the diseases. Genetic analyses and breeding for resistance, however, are hampered by the high degree of heterozygosity and the polyploid nature of cultivated roses. Nucleotide-binding site (NBS) profiling of Van der Linden et al. (2004) was used as a tool enabling a more directed way of studying the genetics of resistance to pathogens responsible for diseases such as powdery mildew. NBS profiling is a multiplex screening technique, producing amplified resistance gene (R-gene) and resistance gene analogue (RGA) fragments by using degenerated primers based on the conserved motifs present in the NBS domain of resistance genes. Since RGAs are abundantly distributed and highly polymorphic within the plant genome, NBS profiling generates multiple markers of putative resistance genes. Twelve NBS degenerated primer/ restriction enzyme combinations were used to genotype the whole rose tetraploid K5 population (Yan, 2005) and its parents. To generate RGA profiles, the restriction enzymes: AluI, HaeIII, Mse and RsaI were used in combination with degenerated primers NBS1, NBS3, and NBS5a6. The profiles were dominantly scored resulting in 106 polymorphic RGA markers which segregated in a 1:1 or 3:1 ratio. Uni-and bi-parental simplex markers will be mapped on the two available AFLP/SSR K5 maps (Yan, 2005) with Joinmap 4.0. The resulting parental tetraploid maps will be used to dissect the genetic variation for resistance to powdery mildew resistance. Additional Rosaceae SSRs mentioned in the literature are currently tested on the K5 population to obtain allelic bridges between the tetraploid and diploid genetic maps in rose and related species in order to align them. These bridges will improve cross-ploidy comparisons in roses in order to strengthen cut rose breedin

Screening Genes for Association with Loci for Nitrogen-Use Efficiency in Perennial Ryegrass by Pyrosequencing\u3csup\u3eTM\u3c/sup\u3e

The application of marker-assisted selection to improve quantitative traits in perennial ryegrass (Lolium perenne) is cumbersome. It requires a priori knowledge on the association of markers and genes. The knowledge on the chromosomal location of major genes for quantitative traits as well as on gene sequences is rapidly growing. However, determination of the genetic constitution of parents prior to their use in breeding still is impractical. More realistic is to collect association data along with the testing activities needed for breeding new varieties. This study uses changes in allele frequency due to selection as a criterion for gene-trait association. Selection-dependent changes are detected with single nucleotide polymorphisms (SNPs) of candidate genes using DNA-pools of F2 plants differing in nitrogen-use efficiency (NUE). The procedure and its feasibility are outlined for one locus

Voluntary suppression of associated activity decreases force steadiness in the active hand

Unilateral muscle contractions are often accompanied by the activation of the ipsilateral hemisphere, producing associated activity (AA) in the contralateral homologous muscles. However, the functional role of AA is not fully understood. We determined the effects of voluntary suppression of AA in the first dorsal interosseous (FDI), on force steadiness during a constant force isometric contraction of the contralateral FDI. Participants (n = 17, 25.5 years) performed two trials of isometric FDI contractions as steadily as possible. In Trial 1, they did not receive feedback or explicit instructions for suppressing the AA in the contralateral homologous FDI. In Trial 2, participants received feedback and were asked to voluntarily suppress the AA in the contralateral nontarget FDI. During both trials, corticospinal excitability and motor cortical inhibition were measured. The results show that participants effectively suppressed the AA in the nontarget contralateral FDI (-71%), which correlated with reductions in corticospinal excitability (-57%), and the suppression was also accompanied by increases in inhibition (27%) in the ipsilateral motor cortex. The suppression of AA impaired force steadiness, but the decrease in force steadiness did not correlate with the magnitude of suppression. The results show that voluntary suppression of AA decreases force steadiness in the active hand. However, due to the lack of association between suppression and decreased steadiness, we interpret these data to mean that specific elements of the ipsilateral brain activation producing AA in younger adults are neither contributing nor detrimental to unilateral motor control during a steady isometric contraction

DESC9115 Lab Report 1

The implementation of the Vibrato and Flanger effect by Oscar GonzalezArchitecture & Allied Art

A Polymorphism in the Splice Donor Site of ZNF419 Results in the Novel Renal Cell Carcinoma-Associated Minor Histocompatibility Antigen ZAPHIR

Nonmyeloablative allogeneic stem cell transplantation (SCT) can induce remission in patients with renal cell carcinoma (RCC), but this graft-versus-tumor (GVT) effect is often accompanied by graft-versus-host disease (GVHD). Here, we evaluated minor histocompatibility antigen (MiHA)-specific T cell responses in two patients with metastatic RCC who were treated with reduced-intensity conditioning SCT followed by donor lymphocyte infusion (DLI). One patient had stable disease and emergence of SMCY.A2-specific CD8+ T cells was observed after DLI with the potential of targeting SMCY-expressing RCC tumor cells. The second patient experienced partial regression of lung metastases from whom we isolated a MiHA-specific CTL clone with the capability of targeting RCC cell lines. Whole genome association scanning revealed that this CTL recognizes a novel HLA-B7-restricted MiHA, designated ZAPHIR, resulting from a polymorphism in the splice donor site of the ZNF419 gene. Tetramer analysis showed that emergence of ZAPHIR-specific CD8+ T cells in peripheral blood occurred in the absence of GVHD. Furthermore, the expression of ZAPHIR in solid tumor cell lines indicates the involvement of ZAPHIR-specific CD8+ T cell responses in selective GVT immunity. These findings illustrate that the ZNF419-encoded MiHA ZAPHIR is an attractive target for specific immunotherapy after allogeneic SCT

A Human Minor Histocompatibility Antigen Specific for B Cell Acute Lymphoblastic Leukemia

Human minor histocompatibility antigens (mHags) play an important role in the induction of cytotoxic T lymphocyte (CTL) reactivity against leukemia after human histocompatibility leukocyte antigen (HLA)-identical allogeneic bone marrow transplantation (BMT). As most mHags are not leukemia specific but are also expressed by normal tissues, antileukemia reactivity is often associated with life-threatening graft-versus-host disease (GVHD). Here, we describe a novel mHag, HB-1, that elicits donor-derived CTL reactivity in a B cell acute lymphoblastic leukemia (B-ALL) patient treated by HLA-matched BMT. We identified the gene encoding the antigenic peptide recognized by HB-1–specific CTLs. Interestingly, expression of the HB-1 gene was only observed in B-ALL cells and Epstein-Barr virus–transformed B cells. The HB-1 gene–encoded peptide EEKRGSLHVW is recognized by the CTL in association with HLA-B44. Further analysis reveals that a polymorphism in the HB-1 gene generates a single amino acid exchange from His to Tyr at position 8 within this peptide. This amino acid substitution is critical for recognition by HB-1–specific CTLs. The restricted expression of the polymorphic HB-1 Ag by B-ALL cells and the ability to generate HB-1–specific CTLs in vitro using peptide-loaded dendritic cells offer novel opportunities to specifically target the immune system against B-ALL without the risk of evoking GVHD

A Uniform Genomic Minor Histocompatibility Antigen Typing Methodology and Database Designed to Facilitate Clinical Applications

BACKGROUND: Minor Histocompatibility (H) antigen mismatches significantly influence the outcome of HLA-matched allogeneic stem cell transplantation. The molecular identification of human H antigens is increasing rapidly. In parallel, clinical application of minor H antigen typing has gained interest. So far, relevant and simple tools to analyze the minor H antigens in a quick and reliable way are lacking. METHODOLOGY AND FINDINGS: We developed a uniform PCR with sequence-specific primers (PCR-SSP) for 10 different autosomal minor H antigens and H-Y. This genomic minor H antigen typing methodology allows easy incorporation in the routine HLA typing procedures. DNA from previously typed EBV-LCL was used to validate the methodology. To facilitate easy interpretation for clinical purposes, a minor H database named dbMinor (http://www.lumc.nl/dbminor) was developed. Input of the minor H antigen typing results subsequently provides all relevant information for a given patient/donor pair and additional information on the putative graft-versus-host, graft-versus-tumor and host-versus-graft reactivities. SIGNIFICANCE: A simple, uniform and rapid methodology was developed enabling determination of minor H antigen genotypes of all currently identified minor H antigens. A dbMinor database was developed to interpret the genomic typing for its potential clinical relevance. The combination of the minor H antigen genomic typing methodology with the online dbMinor database and applications facilitates the clinical application of minor H antigens anti-tumor targets after stem cell transplantation

Pathogenesis of bovine spongiform encephalopathy in sheep

The pathogenesis of bovine spongiform encephalopathy (BSE) in sheep was studied by immunohistochemical detection of scrapie-associated prion protein (PrPSc) in the gastrointestinal, lymphoid and neural tissues following oral inoculation with BSE brain homogenate. First accumulation of PrPSc was detected after 6 months in the tonsil and the ileal Peyer’s patches. At 9 months postinfection, PrPSc accumulation involved all gut-associated lymphoid tissues and lymph nodes as well as the spleen. At this time point, PrPSc accumulation in the peripheral neural tissues was first seen in the enteric nervous system of the caudal jejunum and ileum and in the coeliac-mesenteric ganglion. In the central nervous system, PrPSc was first detected in the dorsal motor nucleus of the nervus Vagus in the medulla oblongata and in the intermediolateral column in the spinal cord segments T7–L1. At subsequent time points, PrPSc was seen to spread within the lymphoid system to also involve all non-gut-associated lymphoid tissues. In the enteric nervous system, further spread of PrPSc involved the neural plexi along the entire gastrointestinal tract and in the CNS the complete neuraxis. These findings indicate a spread of the BSE agent in sheep from the enteric nervous system through parasympathetic and sympathetic nerves to the medulla oblongata and the spinal cord

Spring emergence and canopy development strategies in miscanthus hybrids in Mediterranean, continental and maritime European climates

Due to its versatility and storability, biomass is an important resource for renewable materials and energy. Miscanthus hybrids combine high yield potential, low input demand, tolerance of certain marginal land types and several ecosystem benefits. To date, miscanthus breeding has focussed on increasing yield potential by maximising radiation interception through: 1) selection for early emergence, 2) increasing the growth rate to reach canopy closure as fast as possible, and 3) delayed flowering and senescence. The objective of this study is to compare early season re-growth in miscanthus hybrids cultivated across Europe. Determination of differences in early canopy development on end-of-year yield traits is required to provide information for breeding decisions to improve future crop performance. For this purpose, a trial was planted with four miscanthus hybrids (two novel seed-based hybrids M. sinensis×sinensis (M sin×sin) and M. sacchariflorus×sinensis (M sac×sin), a novel rhizome-based M sac×sin and a standard Miscanthus×giganteus (M×g) clone) in the UK, Germany, Croatia and Italy, and was monitored in the third and fourth growing season. We determined differences between the hybrids in base temperature, frost sensitivity and emergence strategy. M×g and M sac×sin mainly emerged from belowground plant organs, producing fewer but thicker shoots at the beginning of the growing season, but these shoots were susceptible to air frosts (determined by recording 0°C 2 m above ground surface). By contrast, M sin×sin emerged 10 days earlier, avoiding damage by late spring frosts and producing a high number of thinner shoots from aboveground shoots. Therefore, we recommend cultivating M sac×sin at locations with low risk and M sin×sin at locations with higher risk of late spring frosts. Selecting miscanthus hybrids that produce shoots throughout the vegetation period is an effective strategy to limit the risk of late frost damage and avoid reduction in yield from a shortened growing season

The mode of inheritance in tetraploid cut roses

Tetraploid hybrid tea roses (Rosa hybrida) represent most of the commercial cultivars of cut roses and form the basis for breeding programmes. Due to intensive interspecific hybridizations, modern cut roses are complex tetraploids for which the mode of inheritance is not exactly known. The segregation patterns of molecular markers in a tetraploid mapping population of 184 genotypes, an F1 progeny from a cross of two heterozygous parents, were investigated for disomic and tetrasomic inheritance. The possible occurrence of double reduction was studied as well. We can exclude disomic inheritance, but while our observations are more in line with a tetrasomic inheritance, we cannot exclude that there is a mixture of both inheritance modes. Two novel parental tetraploid linkage maps were constructed using markers known from literature, combined with newly generated markers. Comparison with the integrated consensus diploid map (ICM) of Spiller et al. (Theor Appl Genet 122:489–500, 2010) allowed assigning numbers to each of the linkage groups of both maps and including small linkage groups. So far, the possibility of using marker-assisted selection in breeding of tetraploid cut roses and of other species with a tetrasomic or partly tetrasomic inheritance, is still limited due to the difficulties in establishing marker-trait associations. We used these tetraploid linkage maps to determine associations between markers, two morphological traits and powdery mildew resistance. The knowledge on inheritance and marker-trait associations in tetraploid cut roses will be of direct use to cut rose breeding