Improved precision of QTL mapping using a nonlinear Bayesian method in a multi-breed population leads to greater accuracy of across-breed genomic predictions

Background

Genomic selection is increasingly widely practised, particularly in dairy cattle. However, the accuracy of current predictions using GBLUP (genomic best linear unbiased prediction) decays rapidly across generations, and also as selection candidates become less related to the reference population. This is likely caused by the effects of causative mutations being dispersed across many SNPs (single nucleotide polymorphisms) that span large genomic intervals. In this paper, we hypothesise that the use of a nonlinear method (BayesR), combined with a multi-breed (Holstein/Jersey) reference population will map causative mutations with more precision than GBLUP and this, in turn, will increase the accuracy of genomic predictions for selection candidates that are less related to the reference animals.

Results

BayesR improved the across-breed prediction accuracy for Australian Red dairy cattle for five milk yield and composition traits by an average of 7% over the GBLUP approach (Australian Red animals were not included in the reference population). Using the multi-breed reference population with BayesR improved accuracy of prediction in Australian Red cattle by 2 – 5% compared to using BayesR with a single breed reference population. Inclusion of 8478 Holstein and 3917 Jersey cows in the reference population improved accuracy of predictions for these breeds by 4 and 5%. However, predictions for Holstein and Jersey cattle were similar using within-breed and multi-breed reference populations. We propose that the improvement in across-breed prediction achieved by BayesR with the multi-breed reference population is due to more precise mapping of quantitative trait loci (QTL), which was demonstrated for several regions. New candidate genes with functional links to milk synthesis were identified using differential gene expression in the mammary gland.

Conclusions

QTL detection and genomic prediction are usually considered independently but persistence of genomic prediction accuracies across breeds requires accurate estimation of QTL effects. We show that accuracy of across-breed genomic predictions was higher with BayesR than with GBLUP and that BayesR mapped QTL more precisely. Further improvements of across-breed accuracy of genomic predictions and QTL mapping could be achieved by increasing the size of the reference population, including more breeds, and possibly by exploiting pleiotropic effects to improve mapping efficiency for QTL with small effects.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-014-0074-4) contains supplementary material, which is available to authorized users.     

Phylogeny and historical biogeography of the cocosoid palms (Arecaceae,Arecoideae, Cocoseae) inferred from sequences of six WRKY gene family loci

Arecaceae tribe Cocoseae is the most economically important tribe of palms, including both coconut and African oil palm. It is mostly represented in the Neotropics, with one and two genera endemic to South Africa and Madagascar, respectively. Using primers for six single copy WRKY gene family loci, we amplified DNA from 96 samples representing all genera of the palm tribe Cocoseae as well as outgroup tribes Reinhardtieae and Roystoneae. We compared parsimony (MP), maximum likelihood (ML), and Bayesian (B) analysis of the supermatrix with three species‐tree estimation approaches. Subtribe Elaeidinae is sister to the Bactridinae in all analyses. Within subtribe Attaleinae, Lytocaryum, previously nested in Syagrus, is now positioned by MP and ML as sister to the former, with high support; B maintains Lytocaryum embedded within Syagrus. Both MP and ML resolve Cocos as sister to Syagrus; B positions Cocos as sister to Attalea. Bactridineae is composed of two sister clades, Bactris and Desmoncus in one, for which there is morphological support, and a second comprising Acrocomia, Astrocaryum, and Aiphanes. Two B and one ML gene tree‐species estimation approaches are incongruent with the supermatrix in a few critical intergeneric clades, but resolve the same infrageneric relationships. The biogeographic history of the Cocoseae is dominated by dispersal events. The tribe originated in the late Cretaceous in South America. Evaluated together, the supermatrix and species tree analyses presented in this paper provide the most accurate picture of the evolutionary history of the tribe to date, with more congruence than incongruence among the various methodologies.     

Backbone and side-chain assignments of an effector membrane localization domain from Vibrio vulnificus MARTX toxin

¹H, ¹³C, and ¹⁵N chemical shift assignments are presented for the isolated four-helical bundle membrane localization domain from the domain of unknown function 5 (DUF5) effector (MLD_VvDUF5) of the MARTX toxin from Vibrio vulnificus in its solution state. We have assigned 97 % of all backbone and side-chain carbon atoms, including 96 % of all backbone residues. Secondary chemical shift analysis using TALOS+ demonstrates four helices that align with those predicted by structure homology modeling using the MLDs of Pasteurella multocida toxin (PMT) and the clostridial TcdB and TcsL toxins as templates. Future studies will be towards solving the structure and determining the dynamics in the solution state.     

Anti-inflammatory effects of inosine in allergic lung inflammation in mice: evidence for the participation of adenosine A2A and A3 receptors

Inosine, a naturally occurring purine formed from the breakdown of adenosine, is associated with immunoregulatory effects. Evidence shows that inosine modulates lung inflammation and regulates cytokine generation. However, its role in controlling allergen-induced lung inflammation has yet to be identified. In this study, we aimed to investigate the role of inosine and adenosine receptors in a murine model of lung allergy induced by ovalbumin (OVA). Intraperitoneal administration of inosine (0.001–10 mg/kg, 30 min before OVA challenge) significantly reduced the number of leukocytes, macrophages, lymphocytes and eosinophils recovered in the bronchoalveolar lavage fluid of sensitized mice compared with controls. Interestingly, our results showed that pre-treatment with the selective A_2A receptor antagonist (ZM241385), but not with the selective A_2B receptor antagonist (alloxazine), reduced the inhibitory effects of inosine against macrophage count, suggesting that A_2A receptors mediate monocyte recruitment into the lungs. In addition, the pre-treatment of mice with selective A₃ antagonist (MRS3777) also prevented inosine effects against macrophages, lymphocytes and eosinophils. Histological analysis confirmed the effects of inosine and A_2A adenosine receptors on cell recruitment and demonstrated that the treatment with ZM241385 and alloxazine reverted inosine effects against mast cell migration into the lungs. Accordingly, the treatment with inosine reduced lung elastance, an effect related to A₂ receptors. Moreover, inosine reduced the levels of Th₂-cytokines, interleukin-4 and interleukin-5, an effect that was not reversed by A_2A or A_2B selective antagonists. Our data show that inosine acting on A_2A or A₃ adenosine receptors can regulate OVA-induced allergic lung inflammation and also implicate inosine as an endogenous modulator of inflammatory processes observed in the lungs of asthmatic patients.     

Global Impact of Salmonella Pathogenicity Island 2-secreted Effectors on the Host Phosphoproteome

During the late stages of infection, Salmonella secretes numerous effectors through a type III secretion system that is encoded within Salmonella pathogenicity island 2 (SPI2). Despite the importance of SPI2 as a major virulence factor leading to the systemic spread of the bacteria and diseases, a global view of its effects on host responses is still lacking. Here, we measured global impacts of SPI2 effectors on the host phosphorylation and protein expression levels in RAW264.7 and in HeLa cells, as macrophage and nonphagocytic models of infection. We observe that SPI2 effectors differentially modulate the host phosphoproteome and cellular processes (e.g. protein trafficking, cytoskeletal regulation, and immune signaling) in a host cell-dependent manner. Our unbiased approach reveals the involvement of many previously unrecognized proteins, including E3 ligases (HERC4, RanBP2, and RAD18), kinases (CDK, SIK3, and WNK1), and histones (H2B1F, H4, and H15), in late stages of Salmonella infection. Furthermore, from this phosphoproteome analysis and other quantitative screens, we identified HSP27 as a direct in vitro and in vivo molecular target of the only type III secreted kinase, SteC. Using biochemical and cell biological assays, we demonstrate that SteC phosphorylates multiple sites in HSP27 and induces actin rearrangement through this protein. Together, these results provide a broader landscape of host players contributing to specific processes/pathways mediated by SPI2 effectors than was previously appreciated.Type III secretion systems (T3SSs)¹ are specialized virulence factors in Gram-negative pathogens that play an important role in delivering effector proteins to host cells. Salmonella enterica employs two distinct T3SSs encoded in Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2), with numerous effectors encoded around the genome, including a small number in SPI1 and SPI2 (1). SPI1 T3SS effectors are required for the bacterial internalization by intestinal epithelial cells at early stages of infection after oral ingestion. Although Salmonella is subsequently taken up by intestinal macrophages via phagocytosis, SPI2 T3SS effectors function to promote intracellular replication. Part of the role of SPI2 effectors is to control the maturation of the membrane-enclosed, Salmonella-containing vacuole (SCV) where Salmonella survives and replicates, eventually leading to a systemic infection known as typhoid fever (2, 3).Approximately 30 effectors are known to be translocated by the SPI2 T3SS but the actions and targets of most of these effectors are largely unknown (1, 3, 4). A recent systematic study using a single mutant collection of SPI2 genes showed particular virulence factors (e.g. SpvB, SifA, and SteC) play a dominant role in replication within macrophages (5). It is known that SpvB induces cytotoxicity through its ADP-ribosyltransferase activity (6), and SifA is required for maturation of the SCV and the formation of Salmonella-induced filaments (7). SteC has been identified as the sole serine/threonine protein kinase encoded in the Salmonella genome (8), but the target substrates of this kinase within the host are not fully understood, although it has been demonstrated that SteC partially targets the MAP kinase MEK (9). Interestingly, SteC is capable of promoting assembly of an F-actin meshwork around the SCV; this is dependent on its kinase activity but does not require activation of signaling pathways through Rho-associated protein kinase (8), Cdc42, Rac, N-WASP, Scar/WAVE, and Arp2/3 (10). These host signaling proteins are the main targets of T3SS-secreted effectors from many pathogens, including the SPI1 system in Salmonella (11) and Shigella (12). Therefore, SteC is thought to manipulate actin in a unique way through phosphorylation of host protein target(s).Recent advances in high throughput measurements allow us to characterize host gene expression profiles (13) and host phosphoproteme dynamics (14) dependent on the presence of SPI1 effectors in an unbiased, comprehensive manner. However, although it is clear that SPI2 T3SS is a major virulence factor contributing to systemic infection, our knowledge of its effects on host responses is limited. In this study, we used a mass spectrometry (MS)-based quantitative proteomics approach and measured global host phosphorylation changes as well as proteome abundance altered by SPI2 effectors. Furthermore, we explore a molecular target of SPI2 effector kinase SteC by integrating the phosphoproteomics data and other quantitative proteomics screens.     

Report on the international workshop on alternative methods for Leptospira vaccine potency testing: State of the science and the way forward

Routine potency testing of Leptospira vaccines is mostly conducted using a vaccination–challenge test that involves large numbers of hamsters and unrelieved pain and distress. NICEATM, ICCVAM, and their international partners organized a workshop to review the state of the science of alternative methods that might replace, reduce, and refine the use of animals for veterinary Leptospira vaccine potency testing and to identify ways to advance improved alternative methods. Vaccine manufacturers were encouraged to initiate or continue product-specific validation using in vitro enzyme-linked immunosorbent assays as replacements for potency testing of four common Leptospira serogroups. Participants discussed the potential for eliminating the back-titration procedure in the hamster challenge assay, which could reduce animal use by 50% for each individual potency test. Further animal reduction may also be possible by using cryopreserved Leptospira stock to replace continual passaging through hamsters. Serology assays were identified as a way to further reduce and refine animal use but should be considered only after attempting in vitro assays. Workshop participants encouraged consideration of analgesics and use of earlier humane endpoints when the hamster vaccination–challenge potency assay is used. International harmonization of alternative potency methods was recommended to avoid duplicative potency testing to meet regionally different requirements.     

Characterisation of the major autoxidation products of 3-hydroxykynurenine under physiological conditions

3-Hydroxykynurenine (3-OHKyn) is a tryptophan metabolite that is readily autoxidised to products that may be involved in protein modification and cytotoxicity. The oxidation of 3-OHKyn has been studied here with a view to characterising the major products as well as determining their relative rates of formation and the role that H₂O₂ and hydroxyl radical (HO^·) may play in modifying the autoxidation process. Oxidation of 3-OHKyn generated several compounds. Xanthommatin (Xan), formed by the oxidative dimerisation of 3-OHKyn, was the major product formed initially. It was, however, found to be unstable, particularly in the presence of H₂O₂, and degraded to other products including the p-quinone, 4,6-dihydroxyquinolinequinonecarboxylic acid (DHQCA). A compound that has a structure consistent with that of hydroxy-xanthommatin (OHXan) was also formed in addition to at least two minor species that we were unable to identify. Hydrogen peroxide was formed rapidly upon oxidation of 3-OHKyn, and significantly influenced the relative abundance of the different autoxidation species. Increasing either pH (from pH 6 to 8) or temperature (from 25°C to 35°C) accelerated the rate of autoxidation but had little impact on the relative abundance of the autoxidation species. Using electron paramagnetic resonance (EPR) spectroscopy, a clear phenoxyl radical signal was observed during 3-OHKyn autoxidation and this was attributed to xanthommatin radical (Xan^·). Hydroxyl radicals were also produced during 3-OHKyn autoxidation. The HO^· EPR signal disappeared and the Xan^· EPR signal increased when catalase was added to the autoxidation mixture. The HO^· did not appear to play a role in the formation of the autoxidation products as evidenced using HO^· traps/scavengers. We propose that the cytotoxicity of 3-OHKyn may be explained by both the generation of H₂O₂ and by the formation of reactive 3-OHKyn autoxidation products such as the Xan^· and DHQCA.     

Polyunsaturated fatty acids in fish tissues more closely resemble algal than terrestrial diet sources

The River Continuum Concept implies that consumers in headwater streams have greater dietary access to terrestrial basal resources, but recent studies have highlighted the dietary importance of high-quality algae. Algae provide consumers with physiologically important omega-3 (n-3) polyunsaturated fatty acids (PUFA), particularly eicosapentaenoic acid (EPA). However, terrestrial plants and most benthic stream algae lack the long-chain (LC) n-3 PUFA docosahexaenoic acid (DHA, 22:6n-3), which is essential for neural development in fish and other vertebrates. We sampled subalpine streams to investigate how the PUFA composition of neural (brain and eyes), muscle, and liver tissues of freshwater fish is related to their potential diets (macroinvertebrates, epilithon, fresh and conditioned terrestrial leaves). The PUFA composition of consumers was more similar to epilithon than to terrestrial leaves. Storage lipids of eyes most closely resembled dietary PUFA (aquatic invertebrates and algae). However, DHA and arachidonic acid (ARA, 20:4n-6) were not directly available in the diet but abundant in organs. This implies that algal PUFA were selectively retained or were produced internally via enzymatic PUFA conversion by aquatic consumers. This field study demonstrates the nutritional importance of algal PUFA for neural organs in aquatic consumers of headwater regions.

     

A high-throughput fluorescence lifetime-based assay to detect binding of myosin-binding protein C to F-actin

Binding properties of actin-binding proteins are typically evaluated by cosedimentation assays. However, this method is time-consuming, involves multiple steps, and has a limited throughput. These shortcomings preclude its use in screening for drugs that modulate actin-binding proteins relevant to human disease. To develop a simple, quantitative, and scalable F-actin–binding assay, we attached fluorescent probes to actin''s Cys-374 and assessed changes in fluorescence lifetime upon binding to the N-terminal region (domains C0–C2) of human cardiac myosin-binding protein C (cMyBP-C). The lifetime of all five probes tested decreased upon incubation with cMyBP-C C0–C2, as measured by time-resolved fluorescence (TR-F), with IAEDANS being the most sensitive probe that yielded the smallest errors. The TR-F assay was compared with cosedimentation to evaluate in vitro changes in binding to actin and actin–tropomyosin arising from cMyBP-C mutations associated with hypertrophic cardiomyopathy (HCM) and tropomyosin binding. Lifetime changes of labeled actin with added C0–C2 were consistent with cosedimentation results. The HCM mutation L352P was confirmed to enhance actin binding, whereas PKA phosphorylation reduced binding. The HCM mutation R282W, predicted to disrupt a PKA recognition sequence, led to deficits in C0–C2 phosphorylation and altered binding. Lastly, C0–C2 binding was found to be enhanced by tropomyosin and binding capacity to be altered by mutations in a tropomyosin-binding region. These findings suggest that the TR-F assay is suitable for rapidly and accurately determining quantitative binding and for screening physiological conditions and compounds that affect cMyBP-C binding to F-actin for therapeutic discovery.