Linkage and Association Mapping for Two Major Traits Used in the Maritime Pine Breeding Program: Height Growth and Stem Straightness

BackgroundIncreasing our understanding of the genetic architecture of complex traits, through analyses of genotype-phenotype associations and of the genes/polymorphisms accounting for trait variation, is crucial, to improve the integration of molecular markers into forest tree breeding. In this study, two full-sib families and one breeding population of maritime pine were used to identify quantitative trait loci (QTLs) for height growth and stem straightness, through linkage analysis (LA) and linkage disequilibrium (LD) mapping approaches.ResultsThe populations used for LA consisted of two unrelated three-generation full-sib families (n = 197 and n = 477). These populations were assessed for height growth or stem straightness and genotyped for 248 and 217 markers, respectively. The population used for LD mapping consisted of 661 founders of the first and second generations of the breeding program. This population was phenotyped for the same traits and genotyped for 2,498 single-nucleotide polymorphism (SNP) markers corresponding to 1,652 gene loci. The gene-based reference genetic map of maritime pine was used to localize and compare the QTLs detected by the two approaches, for both traits. LA identified three QTLs for stem straightness and two QTLs for height growth. The LD study yielded seven significant associations (P ≤ 0.001): four for stem straightness and three for height growth. No colocalisation was found between QTLs identified by LA and SNPs detected by LD mapping for the same trait.ConclusionsThis study provides the first comparison of LA and LD mapping approaches in maritime pine, highlighting the complementary nature of these two approaches for deciphering the genetic architecture of two mandatory traits of the breeding program.     

Antimicrobial peptides from the skin of the Japanese mountain brown frog Rana ornativentris: evidence for polymorphism among preprotemporin mRNAs

A previous study led to the isolation of antimicrobial peptides belonging to the temporin and brevinin-2 families from a pooled extract of the skin of adult specimens of the Japanese mountain brown frog Rana ornativentris Werner 1903. In order to ascertain whether individual frogs expressed the full complement of temporin genes, we individually cloned cDNAs encoding the temporin precursors from total RNA extracted from the skins of 12 frogs by RT-PCR using a set of preprotemporin-specific primers. All the specimens examined contained mRNAs directing the synthesis of the novel, but inactive, temporin-1Oe (ILPLLGNLLNGLL x NH2). Nucleotide sequence analysis revealed marked polymorphism among individual frogs. Twenty-seven distinct preprotemporin-1Oe mRNAs were identified that contained synonymous substitutions in the antimicrobial peptide region and both synonymous and non-synonymous substitutions in the signal peptide and intervening sequence regions. Up to eight preprotemporin-1Oe mRNA variants were found within a single frog. In addition, several cDNAs encoding preprotemporin-1Oa and -1Ob and a single cDNA encoding preprotemporin-1Oc were characterized. Peptidomic analysis of norepinephrine-stimulated skin secretions revealed the presence of temporin-1Oe, temporin-1Of (SLILKGLASIAKLF x NH2), temporin-1Og (FLSSLLSKVVSLFT x NH2), four members of the ranatuerin-2 family and one member of the palustrin-2 family in addition to previously characterized temporin and brevinin-2 peptides.     

Effector and memory CD8+ T cells can be generated in response to alloantigen independently of CD4+ T cell help

There is now considerable evidence suggesting that CD8(+) T cells are able to generate effector but not functional memory T cells following pathogenic infections in the absence of CD4(+) T cells. We show that following transplantation of allogeneic skin, in the absence of CD4(+) T cells, CD8(+) T cells become activated, proliferate, and expand exclusively in the draining lymph nodes and are able to infiltrate and reject skin allografts. CD44(+)CD8(+) T cells isolated 100 days after transplantation rapidly produce IFN-gamma following restimulation with alloantigen in vitro. In vivo CD44(+)CD8(+) T cells rejected donor-type skin allografts more rapidly than naive CD8(+) T cells demonstrating the ability of these putative memory T cells to mount an effective recall response in vivo. These data form the first direct demonstration that CD8(+) T cells are able to generate memory as well as effector cells in response to alloantigen during rejection in the complete absence of CD4(+) T cells. These data have important implications for the design of therapies to combat rejection and serve to reinforce the view that CD8(+) T cell responses to allografts require manipulation in addition to CD4(+) T cell responses to completely prevent the rejection of foreign organ transplants.     

Toward functional glycomics by localization of tissue lectins: immunohistochemical galectin fingerprinting during diethylstilbestrol-induced kidney tumorigenesis in male Syrian hamster

The current study focused on galectins (-1, -3, -4, -7, and –8) and deliberately performed immunohistochemical fingerprinting to explore their complexity in a context of experimental renal carcinogenesis. The diethylstilbestrol (DES)-induced renal tumors in male Syrian hamster kidney (SHKT) represent a unique animal model for the study of estrogen-dependent renal malignancies. Kidney sections of DES-treated hamsters (3 days to 11 months of DES exposure) were analyzed by immunohistochemistry using a panel of non-crossreactive antibodies raised against galectins-1, -3, -4, -7, and -8. Levels of expression were quantitatively determined by using computer-assisted microscopy on immunostained tissue sections. Except for galectin-4, all above mentioned galectins were expressed in kidney tumors. Small clusters of galectin-1-positive, most likely preneoplastic cells at the corticomedullary junction were already evident 1 week after DES administration. Galectin-1 and -3 expression was apparently associated with the first steps of the neoplastic transformation, because small tumorous buds were found to be positive after 1 month of treatment. In contrast, galectins-7 and -8 were detected in large tumors and medium-sized tumors, respectively, thereby indicating an involvement in later stages of DES-induced SHKT. Galectins-1, -3, -7, and -8 were also detected by immunofluorescence staining in the HKT-1097 cell line established from SHKT, thus illustrating the stability of galectin expression in tumor cells. Our data document the presence and differential regulation of galectins in the course of renal tumorigenesis in the model of DES-induced SHKT.     

Top‐down systems biology integration of conditional prebiotic modulated transgenomic interactions in a humanized microbiome mouse model

Gut microbiome–host metabolic interactions affect human health and can be modified by probiotic and prebiotic supplementation. Here, we have assessed the effects of consumption of a combination of probiotics (Lactobacillus paracasei or L. rhamnosus) and two galactosyl‐oligosaccharide prebiotics on the symbiotic microbiome–mammalian supersystem using integrative metabolic profiling and modeling of multiple compartments in germ‐free mice inoculated with a model of human baby microbiota. We have shown specific impacts of two prebiotics on the microbial populations of HBM mice when co‐administered with two probiotics. We observed an increase in the populations of Bifidobacterium longum and B. breve, and a reduction in Clostridium perfringens, which were more marked when combining prebiotics with L. rhamnosus. In turn, these microbial effects were associated with modulation of a range of host metabolic pathways observed via changes in lipid profiles, gluconeogenesis, and amino‐acid and methylamine metabolism associated to fermentation of carbohydrates by different bacterial strains. These results provide evidence for the potential use of prebiotics for beneficially modifying the gut microbial balance as well as host energy and lipid homeostasis.     

Antimicrobial peptides from the skin of the Tsushima brown frog Rana tsushimensis

The Tsushima brown frog Rana tsushimensis Stejneger, 1907 exists in reproductive isolation on the island of Tsushima, Japan. Six peptides with antimicrobial activity were isolated in pure form from an extract of the skin of this species and their amino acid sequences identified them as members of the brevinin-1 (one peptide), brevinin-2 (one peptide) and temporin (four peptides) families. The C-terminally alpha-amidated brevinin-1 peptide (FLGSIVGALASALPSLISKIRN.NH2) lacks the cyclic heptapeptide domain Cys18-(Xaa)4-Lys-Cys24 at the COOH-terminus of the molecule that characterizes other members of that family. A structurally similar brevinin-1 peptide, also lacking the cyclic domain, was previously isolated from the skin of the Ryukyu brown frog Rana okinavana, indicative of a close phylogenetic relationship between the species. Brevinin-2TSa (GIMSLFKGVLKTAGKHVAGSLVDQLKCKITGGC) showed broad-spectrum growth inhibitory activity against a range of Gram-negative and Gram-positive bacteria (including methicillin-resistant Staphylococcus aureus) (minimum inhibitory concentrations< or =25 microM) and relatively low hemolytic activity against human erythrocytes (LD50=100 microM). The peptide therefore represents a candidate for drug development.     

Genotypic variation in the response of two perennial grass species to elevated carbon dioxide

Shoot and reproductive biomass of genotypes of Bromus erectus and Dactylis glomerata grown in competition at ambient and elevated CO₂ were examined for 2 consecutive years in order to test whether genetic variation in those traits exists and whether it is maintained over time. At the species level, a positive CO₂ response of shoot biomass of both species was only found in the first year of treatment. At the genotype level, no significant CO₂2genotype interaction was found at any single harvest either for vegetative or reproductive biomass of either species. Analysis over time, however, indicated that there is a potential for evolutionary adaptation only for D. glomerata: (1) repeated measures ANOVA detected a marginally significant CO₂2genotype2time interaction for shoot biomass, because the range of the genotypes CO₂ response increased over time; (2) genotypes that displayed the highest response during the first year under elevated CO₂ also showed the highest response the second year. Null (B. erectus) or weak (D. glomerata) selective potentials of elevated CO₂ were detected in this experiment, but short time series could underestimate this potential with perennial species.