Breeding without Breeding: Is a Complete Pedigree Necessary for Efficient Breeding?

Complete pedigree information is a prerequisite for modern breeding and the ranking of parents and offspring for selection and deployment decisions. DNA fingerprinting and pedigree reconstruction can substitute for artificial matings, by allowing parentage delineation of naturally produced offspring. Here, we report on the efficacy of a breeding concept called “Breeding without Breeding” (BwB) that circumvents artificial matings, focusing instead on a subset of randomly sampled, maternally known but paternally unknown offspring to delineate their paternal parentage. We then generate the information needed to rank those offspring and their paternal parents, using a combination of complete (full-sib: FS) and incomplete (half-sib: HS) analyses of the constructed pedigrees. Using a random sample of wind-pollinated offspring from 15 females (seed donors), growing in a 41-parent western larch population, BwB is evaluated and compared to two commonly used testing methods that rely on either incomplete (maternal half-sib, open-pollinated: OP) or complete (FS) pedigree designs. BwB produced results superior to those from the incomplete design and virtually identical to those from the complete pedigree methods. The combined use of complete and incomplete pedigree information permitted evaluating all parents, both maternal and paternal, as well as all offspring, a result that could not have been accomplished with either the OP or FS methods alone. We also discuss the optimum experimental setting, in terms of the proportion of fingerprinted offspring, the size of the assembled maternal and paternal half-sib families, the role of external gene flow, and selfing, as well as the number of parents that could be realistically tested with BwB

Hv-CBF2A overexpression in barley accelerates COR gene transcript accumulation and acquisition of freezing tolerance during cold acclimation

Abstract C-Repeat Binding Factors (CBFs) are DNAbinding transcriptional activators of gene pathways imparting freezing tolerance. Poaceae contain three CBF subfamilies, two of which, HvCBF3/CBFIII and HvCBF4/CBFIV, are unique to this taxon. To gain mechanistic insight into HvCBF4/CBFIV CBFs we overexpressed Hv-CBF2A in spring barley (Hordeum vulgare) cultivar ‘Golden Promise’. The Hv-CBF2A overexpressing lines exhibited stunted growth, poor yield, and greater freezing tolerance compared to non-transformed ‘Golden Promise’. Differences in freezing tolerance were apparent only upon cold acclimation. During cold acclimation freezing tolerance of the Hv-CBF2A overexpressing lines increased more rapidly than that of ‘Golden Promise’ and paralleled the freezing tolerance of the winter hardy barley ‘Dicktoo’. Transcript levels of candidate CBF target genes, COR14B and DHN5 were increased in the overexpressor lines at warm temperatures, and at cold temperatures they accumulated to much higher levels in the Hv-CBF2A overexpressors than in ‘Golden Promise’. Hv-CBF2A overexpression also increased transcript levels of other CBF genes at FROST RESISTANCE-H2-H2 (FR-H2) possessing CRT/DRE sites in their upstream regions, the most notable of which was CBF12. CBF12 transcript levels exhibited a relatively constant incremental increase above levels in ‘Golden Promise’ both at warm and cold. These data indicate that Hv-CBF2A activates target genes at warm temperatures and that transcript accumulation for some of these targets is greatly enhanced by cold temperatures

The RNA Helicase Rm62 Cooperates with SU(VAR)3-9 to Re-Silence Active Transcription in Drosophila melanogaster

Gene expression is highly dynamic and many genes show a wide range in expression over several orders of magnitude. This regulation is often mediated by sequence specific transcription factors. In addition, the tight packaging of DNA into chromatin can provide an additional layer of control resulting in a dynamic range of gene expression covering several orders of magnitude. During transcriptional activation, chromatin barriers have to be eliminated to allow an efficient progression of the RNA polymerase. This repressive chromatin structure has to be re-established quickly after it has been activated in order to tightly regulate gene activity. We show that the DExD/H box containing RNA helicase Rm62 is targeted to a site of rapid induction of transcription where it is responsible for an increased degree of methylation at H3K9 at the heat shock locus after removal of the heat shock stimulus. The RNA helicase interacts with the well-characterized histone methyltransferase SU(VAR)3-9 via its N-terminus, which provides a potential mechanism for the targeting of H3K9 methylation to highly regulated genes. The recruitment of SU(VAR)3-9 through interaction with a RNA helicase to a site of active transcription might be a general mechanism that allows an efficient silencing of highly regulated genes thereby enabling a cell to fine tune its gene activity over a wide range

Mast Cells Express 11 beta-hydroxysteroid Dehydrogenase Type 1: A Role in Restraining Mast Cell Degranulation:a role in restraining mast cell degranulation

Mast cells are key initiators of allergic, anaphylactic and inflammatory reactions, producing mediators that affect vascular permeability, angiogenesis and fibrosis. Glucocorticoid pharmacotherapy reduces mast cell number, maturation and activation but effects at physiological levels are unknown. Within cells, glucocorticoid concentration is modulated by the 11β-hydroxysteroid dehydrogenases (11β-HSDs). Here we show expression and activity of 11β-HSD1, but not 11β-HSD2, in mouse mast cells with 11β-HSD activity only in the keto-reductase direction, regenerating active glucocorticoids (cortisol, corticosterone) from inert substrates (cortisone, 11-dehydrocorticosterone). Mast cells from 11β-HSD1-deficient mice show ultrastructural evidence of increased activation, including piecemeal degranulation and have a reduced threshold for IgG immune complex-induced mast cell degranulation. Consistent with reduced intracellular glucocorticoid action in mast cells, levels of carboxypeptidase A3 mRNA, a glucocorticoid-inducible mast cell-specific transcript, are lower in peritoneal cells from 11β-HSD1-deficient than control mice. These findings suggest that 11β-HSD1-generated glucocorticoids may tonically restrain mast cell degranulation, potentially influencing allergic, anaphylactic and inflammatory responses

Tissue stiffening coordinates morphogenesis by triggering collective cell migration in vivo.

Collective cell migration is essential for morphogenesis, tissue remodelling and cancer invasion. In vivo, groups of cells move in an orchestrated way through tissues. This movement involves mechanical as well as molecular interactions between cells and their environment. While the role of molecular signals in collective cell migration is comparatively well understood, how tissue mechanics influence collective cell migration in vivo remains unknown. Here we investigated the importance of mechanical cues in the collective migration of the Xenopus laevis neural crest cells, an embryonic cell population whose migratory behaviour has been likened to cancer invasion. We found that, during morphogenesis, the head mesoderm underlying the cephalic neural crest stiffens. This stiffening initiates an epithelial-to-mesenchymal transition in neural crest cells and triggers their collective migration. To detect changes in their mechanical environment, neural crest cells use mechanosensation mediated by the integrin-vinculin-talin complex. By performing mechanical and molecular manipulations, we show that mesoderm stiffening is necessary and sufficient to trigger neural crest migration. Finally, we demonstrate that convergent extension of the mesoderm, which starts during gastrulation, leads to increased mesoderm stiffness by increasing the cell density underneath the neural crest. These results show that convergent extension of the mesoderm has a role as a mechanical coordinator of morphogenesis, and reveal a link between two apparently unconnected processes-gastrulation and neural crest migration-via changes in tissue mechanics. Overall, we demonstrate that changes in substrate stiffness can trigger collective cell migration by promoting epithelial-to-mesenchymal transition in vivo. More broadly, our results raise the idea that tissue mechanics combines with molecular effectors to coordinate morphogenesis

A Switch in Hepatic Cortisol Metabolism across the Spectrum of Non Alcoholic Fatty Liver Disease

Context: Non alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome. NAFLD represents a spectrum of liver disease ranging from reversible hepatic steatosis, to non alcoholic steato-hepatitis (NASH) and cirrhosis. The potential role of glucocorticoids (GC) in the pathogenesis of NAFLD is highlighted in patients with GC excess, Cushing's syndrome, who develop central adiposity, insulin resistance and in 20% of cases, NAFLD. Although in most cases of NAFLD, circulating cortisol levels are normal, hepatic cortisol availability is controlled by enzymes that regenerate cortisol (F) from inactive cortisone (E) (11β-hydroxysteroid dehydrogenase type 1, 11β-HSD1), or inactivate cortisol through A-ring metabolism (5α- and 5β-reductase, 5αR and 5βR). Objective and Methods: In vitro studies defined 11β-HSD1 expression in normal and NASH liver samples. We then characterised hepatic cortisol metabolism in 16 patients with histologically proven NAFLD compared to 32 obese controls using gas chromatographic analysis of 24 hour urine collection and plasma cortisol generation profile following oral cortisone. Results: In patients with steatosis 5αR activity was increased, with a decrease in hepatic 11β-HSD1 activity. Total cortisol metabolites were increased in this group consistent with increased GC production rate. In contrast, in patients with NASH, 11β-HSD1 activity was increased both in comparison to patients with steatosis, and controls. Endorsing these findings, 11β-HSD1 mRNA and immunostaining was markedly increased in NASH patients in peri septal hepatocytes and within CD68 positive macrophages within inflamed cirrhotic septa. Conclusion: Patients with hepatic steatosis have increased clearance and decreased hepatic regeneration of cortisol and we propose that this may represent a protective mechanism to decrease local GC availability to preserve hepatic metabolic phenotype. With progression to NASH, increased 11β-HSD1 activity and consequent cortisol regeneration may serve to limit hepatic inflammation

The CBF gene family in hexaploid wheat and its relationship to the phylogenetic complexity of cereal CBFs

Most temperate plants tolerate both chilling and freezing temperatures whereas many species from tropical regions suffer chilling injury when exposed to temperatures slightly above freezing. Cold acclimation induces the expression of cold-regulated genes needed to protect plants against freezing stress. This induction is mediated, in part, by the CBF transcription factor family. To understand the evolution and function of this family in cereals, we identified and characterized 15 different CBF genes from hexaploid wheat. Our analyses reveal that wheat species, T. aestivum and T. monococcum, may contain up to 25 different CBF genes, and that Poaceae CBFs can be classified into 10 groups that share a common phylogenetic origin and similar structural characteristics. Six of these groups (IIIc, IIId, IVa, IVb, IVc and IVd) are found only in the Pooideae suggesting they represent the CBF response machinery that evolved recently during colonization of temperate habitats. Expression studies reveal that five of the Pooideae-specific groups display higher constitutive and low temperature inducible expression in the winter cultivar, and a diurnal regulation pattern during growth at warm temperature. The higher constitutive and inducible expression within these CBF groups is an inherited trait that may play a predominant role in the superior low temperature tolerance capacity of winter cultivars and possibly be a basis of genetic variability in freezing tolerance within the Pooideae subfamily

Genetic architecture of gene expression in ovine skeletal muscle

In livestock populations the genetic contribution to muscling is intensively monitored in the progeny of industry sires and used as a tool in selective breeding programs. The genes and pathways conferring this genetic merit are largely undefined. Genetic variation within a population has potential, amongst other mechanisms, to alter gene expression via cis- or trans-acting mechanisms in a manner that impacts the functional activities of specific pathways that contribute to muscling traits. By integrating sire-based genetic merit information for a muscling trait with progeny-based gene expression data we directly tested the hypothesis that there is genetic structure in the gene expression program in ovine skeletal muscle. Results The genetic performance of six sires for a well defined muscling trait, longissimus lumborum muscle depth, was measured using extensive progeny testing and expressed as an Estimated Breeding Value by comparison with contemporary sires. Microarray gene expression data were obtained for longissimus lumborum samples taken from forty progeny of the six sires (4-8 progeny/sire). Initial unsupervised hierarchical clustering analysis revealed strong genetic architecture to the gene expression data, which also discriminated the sire-based Estimated Breeding Value for the trait. An integrated systems biology approach was then used to identify the major functional pathways contributing to the genetics of enhanced muscling by using both Estimated Breeding Value weighted gene co-expression network analysis and a differential gene co-expression network analysis. The modules of genes revealed by these analyses were enriched for a number of functional terms summarised as muscle sarcomere organisation and development, protein catabolism (proteosome), RNA processing, mitochondrial function and transcriptional regulation. Conclusions This study has revealed strong genetic structure in the gene expression program within ovine longissimus lumborum muscle. The balance between muscle protein synthesis, at the levels of both transcription and translation control, and protein catabolism mediated by regulated proteolysis is likely to be the primary determinant of the genetic merit for the muscling trait in this sheep population. There is also evidence that high genetic merit for muscling is associated with a fibre type shift toward fast glycolytic fibres. This study provides insight into mechanisms, presumably subject to strong artificial selection, that underpin enhanced muscling in sheep populations

A Comparison of Shiga-Toxin 2 Bacteriophage from Classical Enterohemorrhagic Escherichia coli Serotypes and the German E. coli O104:H4 Outbreak Strain

Escherichia coli O104:H4 was associated with a severe foodborne disease outbreak originating in Germany in May 2011. More than 4000 illnesses and 50 deaths were reported. The outbreak strain was a typical enteroaggregative E. coli (EAEC) that acquired an antibiotic resistance plasmid and a Shiga-toxin 2 (Stx2)-encoding bacteriophage. Based on whole-genome phylogenies, the O104:H4 strain was most closely related to other EAEC strains; however, Stx2-bacteriophage are mobile, and do not necessarily share an evolutionary history with their bacterial host. In this study, we analyzed Stx2-bacteriophage from the E. coli O104:H4 outbreak isolates and compared them to all available Stx2-bacteriophage sequences. We also compared Stx2 production by an E. coli O104:H4 outbreak-associated isolate (ON-2011) to that of E. coli O157:H7 strains EDL933 and Sakai. Among the E. coli Stx2-phage sequences studied, that from O111:H- strain JB1-95 was most closely related phylogenetically to the Stx2-phage from the O104:H4 outbreak isolates. The phylogeny of most other Stx2-phage was largely concordant with their bacterial host genomes. Finally, O104:H4 strain ON-2011 produced less Stx2 than E. coli O157:H7 strains EDL933 and Sakai in culture; however, when mitomycin C was added, ON-2011 produced significantly more toxin than the E. coli O157:H7 strains. The Stx2-phage from the E. coli O104:H4 outbreak strain and the Stx2-phage from O111:H- strain JB1-95 likely share a common ancestor. Incongruence between the phylogenies of the Stx2-phage and their host genomes suggest the recent Stx2-phage acquisition by E. coli O104:H4. The increase in Stx2-production by ON-2011 following mitomycin C treatment may or may not be related to the high rates of hemolytic uremic syndrome associated with the German outbreak strain. Further studies are required to determine whether the elevated Stx2-production levels are due to bacteriophage or E. coli O104:H4 host related factors

Effects of elevated seawater pCO2 on gene expression patterns in the gills of the green crab, Carcinus maenas

Background: The green crab Carcinus maenas is known for its high acclimation potential to varying environmental abiotic conditions. A high ability for ion and acid-base regulation is mainly based on an efficient regulation apparatus located in gill epithelia. However, at present it is neither known which ion transport proteins play a key role in the acid-base compensation response nor how gill epithelia respond to elevated seawater pCO2 as predicted for the future. In order to promote our understanding of the responses of green crab acid-base regulatory epithelia to high pCO2, Baltic Sea green crabs were exposed to a pCO2 of 400 Pa. Gills were screened for differentially expressed gene transcripts using a 4,462-feature microarray and quantitative real-time PCR. Results: Crabs responded mainly through fine scale adjustment of gene expression to elevated pCO2. However, 2% of all investigated transcripts were significantly regulated 1.3 to 2.2-fold upon one-week exposure to CO2 stress. Most of the genes known to code for proteins involved in osmo- and acid-base regulation, as well as cellular stress response, were were not impacted by elevated pCO2. However, after one week of exposure, significant changes were detected in a calcium-activated chloride channel, a hyperpolarization activated nucleotide-gated potassium channel, a tetraspanin, and an integrin. Furthermore, a putative syntaxin-binding protein, a protein of the transmembrane 9 superfamily, and a Cl-/HCO3 - exchanger of the SLC 4 family were differentially regulated. These genes were also affected in a previously published hypoosmotic acclimation response study. Conclusions: The moderate, but specific response of C. maenas gill gene expression indicates that (1) seawater acidification does not act as a strong stressor on the cellular level in gill epithelia; (2) the response to hypercapnia is to some degree comparable to a hypoosmotic acclimation response; (3) the specialization of each of the posterior gill arches might go beyond what has been demonstrated up to date; and (4) a re-configuration of gill epithelia might occur in response to hypercapnia