Genetic diversity in an indigenous horse breed: implications for mating strategies and the control of future inbreeding

The Franches-Montagnes is an indigenous Swiss horse breed, with approximately 2500 foalings per year. The stud book is closed, and no introgression from other horse breeds was conducted since 1998. Since 2006, breeding values for 43 different traits (conformation, performance and coat colour) are estimated with a best linear unbiased prediction (BLUP) multiple trait animal model. In this study, we evaluated the genetic diversity for the breeding population, considering the years from 2003 to 2008. Only horses with at least one progeny during that time span were included. Results were obtained based on pedigree information as well as from molecular markers. A series of software packages were screened to combine best the best linear unbiased prediction (BLUP) methodology with optimal genetic contribution theory. We looked for stallions with highest breeding values and lowest average relationship to the dam population. Breeding with such stallions is expected to lead to a selection gain, while lowering the future increase in inbreeding within the breed.

Mapping genetic variants associated with beta-adrenergic responses in inbred mice

β-blockers and β-agonists are primarily used to treat cardiovascular diseases. Inter-individual variability in response to both drug classes is well recognized, yet the identity and relative contribution of the genetic players involved are poorly understood. This work is the first genome-wide association study (GWAS) addressing the values and susceptibility of cardiovascular-related traits to a selective β(1)-blocker, Atenolol (ate), and a β-agonist, Isoproterenol (iso). The phenotypic dataset consisted of 27 highly heritable traits, each measured across 22 inbred mouse strains and four pharmacological conditions. The genotypic panel comprised 79922 informative SNPs of the mouse HapMap resource. Associations were mapped by Efficient Mixed Model Association (EMMA), a method that corrects for the population structure and genetic relatedness of the various strains. A total of 205 separate genome-wide scans were analyzed. The most significant hits include three candidate loci related to cardiac and body weight, three loci for electrocardiographic (ECG) values, two loci for the susceptibility of atrial weight index to iso, four loci for the susceptibility of systolic blood pressure (SBP) to perturbations of the β-adrenergic system, and one locus for the responsiveness of QTc (p<10(-8)). An additional 60 loci were suggestive for one or the other of the 27 traits, while 46 others were suggestive for one or the other drug effects (p<10(-6)). Most hits tagged unexpected regions, yet at least two loci for the susceptibility of SBP to β-adrenergic drugs pointed at members of the hypothalamic-pituitary-thyroid axis. Loci for cardiac-related traits were preferentially enriched in genes expressed in the heart, while 23% of the testable loci were replicated with datasets of the Mouse Phenome Database (MPD). Altogether these data and validation tests indicate that the mapped loci are relevant to the traits and responses studied.

Comparison of genetic gains per year for carcass traits among breeding programs in the Japanese Brown and the Japanese Black cattle

The breeding program for beef cattle in Japan has changed dramatically over 4 decades. Visual judging was done initially, but progeny testing in test stations began in 1968. In the 1980s, the genetic evaluation program using field records, so-called on-farm progeny testing, was first adopted in Oita, Hyogo, and Kumamoto prefectures. In this study, genetic trends for carcass traits in these 3 Wagyu populations were estimated, and genetic gains per year were compared among the 3 different beef cattle breeding programs. The field carcass records used were collected between 1988 and 2003. The traits analyzed were carcass weight, LM area, rib thickness, s.c. fat thickness, and beef marbling standard number. The average breeding values of reproducing dams born the same year were used to estimate the genetic trends for the carcass traits. For comparison of the 3 breeding programs, birth years of the dams were divided into 3 periods reflecting each program. Positive genetic trends for beef marbling standard number were clearly shown in all populations. The genetic gains per year for all carcass traits were significantly enhanced by adopting the on-farm progeny testing program. These results indicate that the on-farm progeny testing program with BLUP is a very powerful approach for genetic improvement of carcass traits in Japanese Wagyu beef cattle.

EmsB, a tandem repeated multi-loci microsatellite, new tool to investigate the genetic diversity of Echinococcus multilocularis

In order to explore the genetic diversity within Echinococcus multilocularis (E. multilocularis), the cestode responsible for the alveolar echinococcosis (AE) in humans, a microsatellite, composed of (CA) and (GA) repeats and designated EmsB, was isolated and characterized in view of its nature and potential field application. PCR-amplification with specific primers exhibited a high degree of size polymorphism between E. multilocularis and Echinococcus granulosus sheep (G1) and camel (G6) strains. Fluorescent-PCR was subsequently performed on a panel of E. multilocularis isolates to assess intra-species polymorphism level. EmsB provided a multi-peak profile, characterized by tandemly repeated microsatellite sequences in the E. multilocularis genome. This "repetition of repeats" feature provided to EmsB a high discriminatory power in that eight clusters, supported by bootstrap p-values larger than 95%, could be defined among the tested E. multilocularis samples. We were able to differentiate not only the Alaskan from the European samples, but also to detect different European isolate clusters. In total, 25 genotypes were defined within 37 E. multilocularis samples. Despite its complexity, this tandem repeated multi-loci microsatellite possesses the three important features for a molecular marker, i.e. sensitivity, repetitiveness and discriminatory power. It will permit assessing the genetic polymorphism of E. multilocularis and to investigate its spatial distribution in detail.

Genetic Distance between Species Predicts Novel Trait Expression in Their Hybrids

Interspecific hybridization can generate transgressive hybrid phenotypes with extreme trait values exceeding the combined range of the parental species. Such variation can enlarge the working surface for natural selection, and may facilitate the evolution of novel adaptations where ecological opportunity exists. The number of quantitative trait loci fixed for different alleles in different species should increase with time since speciation. If transgression is caused by complementary gene action or epistasis, hybrids between more distant species should be more likely to display transgressive phenotypes. To test this prediction we collected data on transgression frequency from the literature, estimated genetic distances between the hybridizing species from gene sequences, and calculated the relationship between the two using phylogenetically controlled methods. We also tested if parental phenotypic divergence affected the occurrence of transgression. We found a highly significant positive correlation between transgression frequency and genetic distance in eudicot plants explaining 43% of the variance in transgression frequency. In total, 36% of the measured traits were transgressive. The predicted effect of time since speciation on transgressive segregation was unconfounded by the potentially conflicting effects of phenotypic differentiation between species. Our analysis demonstrates that the potential impact hybridization may have on phenotypic evolution is predictable from the genetic distance between species.

Phenotypic novelty in experimental hybrids is predicted by the genetic distance between species of cichlid fish

Background: Transgressive segregation describes the occurrence of novel phenotypes in hybrids with extreme trait values not observed in either parental species. A previously experimentally untested prediction is that the amount of transgression increases with the genetic distance between hybridizing species. This follows from QTL studies suggesting that transgression is most commonly due to complementary gene action or epistasis, which become more frequent at larger genetic distances. This is because the number of QTLs fixed for alleles with opposing signs in different species should increase with time since speciation provided that speciation is not driven by disruptive selection. We measured the amount of transgression occurring in hybrids of cichlid fish bred from species pairs with gradually increasing genetic distances and varying phenotypic similarity. Transgression in multi-trait shape phenotypes was quantified using landmark-based geometric morphometric methods. Results: We found that genetic distance explained 52% and 78% of the variation in transgression frequency in F1 and F2 hybrids, respectively. Confirming theoretical predictions, transgression when measured in F2 hybrids, increased linearly with genetic distance between hybridizing species. Phenotypic similarity of species on the other hand was not related to the amount of transgression. Conclusion: The commonness and ease with which novel phenotypes are produced in cichlid hybrids between unrelated species has important implications for the interaction of hybridization with adaptation and speciation. Hybridization may generate new genotypes with adaptive potential that did not reside as standing genetic variation in either parental population, potentially enhancing a population's responsiveness to selection. Our results make it conceivable that hybridization contributed to the rapid rates of phenotypic evolution in the large and rapid adaptive radiations of haplochromine cichlids.

Multiplex strategy for multilocus sequence typing, fla typing, and genetic determination of antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolates collected in Switzerland

We present an optimized multilocus sequence typing (MLST) scheme with universal primer sets for amplifying and sequencing the seven target genes of Campylobacter jejuni and Campylobacter coli. Typing was expanded by sequence determination of the genes flaA and flaB using optimized primer sets. This approach is compatible with the MLST and flaA schemes used in the PubMLST database and results in an additional typing method using the flaB gene sequence. An identification module based on the 16S rRNA and rpoB genes was included, as well as the genetic determination of macrolide and quinolone resistances based on mutations in the 23S rRNA and gyrA genes. Experimental procedures were simplified by multiplex PCR of the 13 target genes. This comprehensive approach was evaluated with C. jejuni and C. coli isolates collected in Switzerland. MLST of 329 strains resulted in 72 sequence types (STs) among the 186 C. jejuni strains and 39 STs for the 143 C. coli isolates. Fourteen (19%) of the C. jejuni and 20 (51%) of the C. coli STs had not been found previously. In total, 35% of the C. coli strains collected in Switzerland contained mutations conferring antibiotic resistance only to quinolone, 15% contained mutations conferring resistance only to macrolides, and 6% contained mutations conferring resistance to both classes of antibiotics. In C. jejuni, these values were 31% and 0% for quinolone and macrolide resistance, respectively. The rpoB sequence allowed phylogenetic differentiation between C. coli and C. jejuni, which was not possible by 16S rRNA gene analysis. An online Integrated Database Network System (SmartGene, Zug, Switzerland)-based platform for MLST data analysis specific to Campylobacter was implemented. This Web-based platform allowed automated allele and ST designation, as well as epidemiological analysis of data, thus streamlining and facilitating the analysis workflow. Data networking facilitates the exchange of information between collaborating centers. The described approach simplifies and improves the genotyping of Campylobacter, allowing cost- and time-efficient routine monitoring.

Clinical, genetic, and functional characterization of adrenocorticotropin receptor mutations using a novel receptor assay.

The ACTH receptor (MC2R) is expressed predominantly in the adrenal cortex, but is one of five G protein-coupled, seven-transmembrane melanocortin receptors (MCRs), all of which bind ACTH to some degree. Testing of MC2R activity is difficult because most cells express endogenous MCRs; hence, ACTH will elicit background activation of assayable reporter systems. Inactivating mutations of MC2R lead to hereditary unresponsiveness to ACTH, also known as familial glucocorticoid deficiency (FGD). These patients are usually seen in early childhood with very low cortisol concentrations, normal mineralocorticoids, hyperpigmentation, and increased bodily growth. Several MC2R mutations have been reported in FGD, but assays of the activities of these mutants are cumbersome. We saw two patients with typical clinical findings of FGD. Genetic analysis showed that patient 1 was homozygous for the mutation R137W, and patient 2 was a compound heterozygote for S74I and Y254C. We tested the activity of these mutations in OS-3 cells, which are unresponsive to ACTH but have intact downstream cAMP signal transduction. OS-3 cells transfected with a cAMP-responsive luciferase reporter plasmid (pCREluc) were unresponsive to ACTH, but cotransfection with a vector expressing human MC2R increased luciferase activity more than 40-fold. Addition of ACTH to cells cotransfected with the pCREluc reporter and wild-type MC2R activated luciferase expression with a 50% effective concentration of 5.5 x 10(-9) M ACTH, which is similar to previously reported values. By contrast, the MC2R mutant R137W had low activity, and the S74I or Y254C mutants elicited no measurable response. This assay provides excellent sensitivity in an easily assayed transient transfection system, providing a more rapid and efficient measurement of ACTH receptor activity.

Determination of genetic transferrin variants in human serum by high-resolution capillary zone electrophoresis(†)

High-resolution capillary zone electrophoresis in the routine arena with stringent quality assurance is employed for the determination of carbohydrate-deficient transferrin in human serum. The assay comprises mixing of human serum with a Fe(III) -containing solution prior to analysis of the iron-saturated mixture in a dynamically double-coated capillary using a commercial buffer at alkaline pH. In contrast to other assays, it provides sufficient resolution for proper recognition of genetic transferrin variants. Analysis of 7290 patient sera revealed 166 isoform patterns that could be assigned to genetic variants, namely, 109 BC, 53 CD, one BD and three CC variants. Several subtypes of transferrin D can be distinguished as they have large enough differences in pI values. Subtypes of transferrin C and B cannot be resolved. However, analysis of the detection time ratios of tetrasialo isoforms of transferrin BC and transferrin CD variants revealed multimodal frequency histograms, indicating the presence of subtypes of transferrin C, B and D. The data gathered over 11 years demonstrate the robustness of the high-resolution capillary zone electrophoresis assay. This is the first account of a capillary zone electrophoresis based carbohydrate-deficient transferrin assay with a broad overview on transferrin isoform patterns associated with genetic transferrin variants.

Genetic analysis of white facial and leg markings in the Swiss Franches-Montagnes Horse Breed

White markings and spotting patterns in animal species are thought to be a result of the domestication process. They often serve for the identification of individuals but sometimes are accompanied by complex pathological syndromes. In the Swiss Franches-Montagnes horse population, white markings increased vastly in size and occurrence during the past 30 years, although the breeding goal demands a horse with as little depigmented areas as possible. In order to improve selection and avoid more excessive depigmentation on the population level, we estimated population parameters and breeding values for white head and anterior and posterior leg markings. Heritabilities and genetic correlations for the traits were high (h(2) > 0.5). A strong positive correlation was found between the chestnut allele at the melanocortin-1-receptor gene locus and the extent of white markings. Segregation analysis revealed that our data fit best to a model including a polygenic effect and a biallelic locus with a dominant-recessive mode of inheritance. The recessive allele was found to be the white trait-increasing allele. Multilocus linkage disequilibrium analysis allowed the mapping of the putative major locus to a chromosomal region on ECA3q harboring the KIT gene.