A study on the minimum number of loci required for genetic evaluation using a finite locus model

For a finite locus model, Markov chain Monte Carlo (MCMC) methods can be used to estimate the conditional mean of genotypic values given phenotypes, which is also known as the best predictor (BP). When computationally feasible, this type of genetic prediction provides an elegant solution to the problem of genetic evaluation under non-additive inheritance, especially for crossbred data. Successful application of MCMC methods for genetic evaluation using finite locus models depends, among other factors, on the number of loci assumed in the model. The effect of the assumed number of loci on evaluations obtained by BP was investigated using data simulated with about 100 loci. For several small pedigrees, genetic evaluations obtained by best linear prediction (BLP) were compared to genetic evaluations obtained by BP. For BLP evaluation, used here as the standard of comparison, only the first and second moments of the joint distribution of the genotypic and phenotypic values must be known. These moments were calculated from the gene frequencies and genotypic effects used in the simulation model. BP evaluation requires the complete distribution to be known. For each model used for BP evaluation, the gene frequencies and genotypic effects, which completely specify the required distribution, were derived such that the genotypic mean, the additive variance, and the dominance variance were the same as in the simulation model. For lowly heritable traits, evaluations obtained by BP under models with up to three loci closely matched the evaluations obtained by BLP for both purebred and crossbred data. For highly heritable traits, models with up to six loci were needed to match the evaluations obtained by BLP.     

Epigenetic silencing of LncRNA ANRIL enhances liver fibrosis and HSC activation through activating AMPK pathway

Long non‐coding RNAs (LncRNAs) and DNA methylation are important epigenetic mark play a key role in liver fibrosis. Currently, how DNA methylation and LncRNAs control the hepatic stellate cell (HSC) activation and fibrosis has not yet been fully characterized. Here, we explored the role of antisense non‐coding RNA in the INK4 locus (ANRIL) and DNA methylation in HSC activation and fibrosis. The expression levels of DNA methyltransferases 3A (DNMT3A), ANRIL, α‐Smooth muscle actin (α‐SMA), Type I collagen (Col1A1), adenosine monophosphate‐activated protein kinase (AMPK) and p‐AMPK in rat and human liver fibrosis were detected by immunohistochemistry, qRT‐PCR and Western blotting. Liver tissue histomorphology was examined by haematoxylin and eosin (H&E), Sirius red and Masson staining. HSC was transfected with DNMT3A‐siRNA, over‐expressing ANRIL and down‐regulating ANRIL. Moreover, cell proliferation ability was examined by CCK‐8, MTT and cell cycle assay. Here, our study demonstrated that ANRIL was significantly decreased in activated HSC and liver fibrosis tissues, while Col1A1, α‐SMA and DNMT3A were significantly increased in activated HSC and liver fibrosis tissues. Further, we found that down‐regulating DNMT3A expression leads to inhibition of HSC activation. Reduction in DNMT3A elevated ANRIL expression in activated HSC. Furthermore, we performed the over expression ANRIL suppresses HSC activation and AMPK signalling pathways. In sum, our study found that epigenetic DNMT3A silencing of ANRIL enhances liver fibrosis and HSC activation through activating AMPK pathway. Targeting epigenetic modulators DNMT3A and ANRIL, and offer a novel approach for liver fibrosis therapy.     

Development of a novel,high resolution melting analysis based genotyping method for Cryptosporidium parvum

This study employed the post-real-time PCR application, high resolution melting (HRM) analysis, in order to differentiate between characterised clinical and reference Cryptosporidium parvum samples obtained from Cork University Hospital (Cork, Ireland) and the Cryptosporidium Reference Unit (Swansea, Wales). A sample set composed of 18 distinct C. parvum gp60-subtypes of the IIa gp60-subtype family (an allele family accounting for over 80% of all cryptosporidiosis cases in Ireland) was employed. HRM analysis-based interrogation of the gp60, MM5 and MS9-Mallon tandem repeat loci was found to completely differentiate between 10 of the 18 studied gp60-subtypes. The remaining eight gp60-subtypes were differentiated into three distinct groupings, with the designations within these groupings resolved to two to three potential gp60-subtypes.The current study aimed to develop a novel, reproducible, real-time PCR based multi-locus genotyping method to distinguish between C. parvum gp60-subtypes. These preliminary results support the further expansion of the multi-locus panel in order to increase the discriminatory capabilities of this novel method.     

Tolerance to oxidative stress is associated with both oxidative stress response and inherent growth in a fungal wheat pathogen

Reactive oxygen species are toxic byproducts of aerobic respiration that are also important in mediating a diversity of cellular functions. Reactive oxygen species form an important component of plant defenses to inhibit microbial pathogens during pathogen–plant interactions. Tolerance to oxidative stress is likely to make a significant contribution to the viability and pathogenicity of plant pathogens, but the complex network of oxidative stress responses hinders identification of the genes contributing to this trait. Here, we employed a forward genetic approach to investigate the genetic architecture of oxidative stress tolerance in the fungal wheat pathogen Zymoseptoria tritici. We used quantitative trait locus (QTL) mapping of growth and melanization under axenic conditions in two cross-populations to identify genomic regions associated with tolerance to oxidative stress. We found that QTLs associated with growth under oxidative stress as well as inherent growth can affect oxidative stress tolerance, and we identified two uncharacterized genes in a major QTL associated with this trait. Our data suggest that melanization does not affect tolerance to oxidative stress, which differs from what was found for animal pathogens. This study provides a whole-genome perspective on the genetic basis of oxidative stress tolerance in a plant pathogen.     

A major locus controls a biologically active pheromone component in Heliconius melpomene

Understanding the production, response, and genetics of signals used in mate choice can inform our understanding of the evolution of both intraspecific mate choice and reproductive isolation. Sex pheromones are important for courtship and mate choice in many insects, but we know relatively little of their role in butterflies. The butterfly Heliconius melpomene uses a complex blend of wing androconial compounds during courtship. Electroantennography in H. melpomene and its close relative Heliconius cydno showed that responses to androconial extracts were not species specific. Females of both species responded equally strongly to extracts of both species, suggesting conservation of peripheral nervous system elements across the two species. Individual blend components provoked little to no response, with the exception of octadecanal, a major component of the H. melpomene blend. Supplementing octadecanal on the wings of octadecanal-rich H. melpomene males led to an increase in the time until mating, demonstrating the bioactivity of octadecanal in Heliconius. Using quantitative trait locus (QTL) mapping, we identified a single locus on chromosome 20 responsible for 41% of the parental species’ difference in octadecanal production. This QTL does not overlap with any of the major wing color or mate choice loci, nor does it overlap with known regions of elevated or reduced F_ST. A set of 16 candidate fatty acid biosynthesis genes lies underneath the QTL. Pheromones in Heliconius carry information relevant for mate choice and are under simple genetic control, suggesting they could be important during speciation.     

Recombinase-mediated cassette exchange (RMCE) — A rapidly-expanding toolbox for targeted genomic modifications

Starting in 1991, the advance of Tyr-recombinases Flp and Cre enabled superior strategies for the predictable insertion of transgenes into compatible target sites of mammalian cells. Early approaches suffered from the reversibility of integration routes and the fact that co-introduction of prokaryotic vector parts triggered uncontrolled heterochromatization. Shortcomings of this kind were overcome when Flp-Recombinase Mediated Cassette Exchange entered the field in 1994. RMCE enables enhanced tag-and-exchange strategies by precisely replacing a genomic target cassette by a compatible donor construct. After “gene swapping” the donor cassette is safely locked in, but can nevertheless be re-mobilized in case other compatible donor cassettes are provided (“serial RMCE”). These features considerably expand the options for systematic, stepwise genome modifications. The first decade was dominated by the systematic generation of cell lines for biotechnological purposes. Based on the reproducible expression capacity of the resulting strains, a comprehensive toolbox emerged to serve a multitude of purposes, which constitute the first part of this review. The concept per se did not, however, provide access to high-producer strains able to outcompete industrial multiple-copy cell lines. This fact gave rise to systematic improvements, among these certain accumulative site-specific integration pathways. The exceptional value of RMCE emerged after its entry into the stem cell field, where it started to contribute to the generation of induced pluripotent stem (iPS-) cells and their subsequent differentiation yielding a variety of cell types for diagnostic and therapeutic purposes. This topic firmly relies on the strategies developed in the first decade and can be seen as the major ambition of the present article. In this context an unanticipated, potent property of serial Flp-RMCE setups concerns the potential to re-open loci that have served to establish the iPS status before the site underwent the obligatory silencing process. Other relevant options relate to the introduction of composite Flp-recognition target sites (“heterospecific FRT-doublets”), into the LTRs of lentiviral vectors. These “twin sites” enhance the safety of iPS re-programming and -differentiation as they enable the subsequent quantitative excision of a transgene, leaving behind a single “FRT-twin”. Such a strategy combines the established expression potential of the common retro- and lentiviral systems with options to terminate the process at will. The remaining genomic tag serves to identify and characterize the insertion site with the goal to identify genomic “safe harbors” (GOIs) for re-use. This is enabled by the capacity of “FRT-twins” to accommodate any incoming RMCE-donor cassette with a compatible design.     

CHROMOSOMAL REARRANGEMENTS AND THE GENETICS OF REPRODUCTIVE BARRIERS IN MIMULUS (MONKEY FLOWERS)

Chromosomal rearrangements may directly cause hybrid sterility and can facilitate speciation by preserving local adaptation in the face of gene flow. We used comparative linkage mapping with shared gene‐based markers to identify potential chromosomal rearrangements between the sister monkeyflowers Mimulus lewisii and Mimulus cardinalis, which are textbook examples of ecological speciation. We then remapped quantitative trait loci (QTLs) for floral traits and flowering time (premating isolation) and hybrid sterility (postzygotic isolation). We identified three major regions of recombination suppression in the M. lewisii × M. cardinalis hybrid map compared to a relatively collinear Mimulus parishii × M. lewisii map, consistent with a reciprocal translocation and two inversions specific to M. cardinalis. These inferences were supported by targeted intraspecific mapping, which also implied a M. lewisii‐specific reciprocal translocation causing chromosomal pseudo‐linkage in both hybrid mapping populations. Floral QTLs mapped in this study, along with previously mapped adaptive QTLs, were clustered in putatively rearranged regions. All QTLs for male sterility, including two underdominant loci, mapped to regions of recombination suppression. We argue that chromosomal rearrangements may have played an important role in generating and consolidating barriers to gene flow as natural selection drove the dramatic ecological and morphological divergence of these species.