Standardization of a SNP panel for parentage verification and identification in the domestic cat (Felis silvestris catus).

The domestic cat (Felis silvestris catus) is a valued companion animal throughout the world. Over 60 different cat breeds are accepted for competition by the cat fancy registries in different countries. Genetic markers, including short tandem repeats and SNPs, are available to evaluate and manage levels of inbreeding and genetic diversity, population and breed structure relationships, and individual identification for forensic and registration purposes. The International Society of Animal Genetics (ISAG) hosts the Applied Genetics in Companion Animals Workshop, which supports the standardization of genetic marker panels and genotyping for the identification of cats via comparison testing. SNP panels have been in development for many species, including the domestic cat. An ISAG approved core panel of SNPs for use in cat identification and parentage analyses is presented. SNPs (n = 121) were evaluated by different university-based and commercial laboratories using 20 DNA samples as part of the ISAG comparison testing procedures. Different SNP genotyping technologies were examined, including DNA arrays, genotyping-by-sequencing and mass spectroscopy, to select a robust and efficient panel of 101 SNPs as the ISAG core panel for cats. The SNPs are distributed across all chromosomes including two on the X chromosome and an XY pseudo-autosomal sexing marker (zinc-finger XY; ZFXY). A population study demonstrated that the markers have an average polymorphic information content of 0.354 and a power of exclusion greater than 0.9999. The SNP panel should keep testing affordable while also allowing for the development of additional panels to monitor health, phenotypic traits, hybrid cats and highly inbred cats.

Targeted genotyping by sequencing: a new way to genome profile the cat.

Targeted GBS is a recent approach for obtaining an effective characterization for hundreds to thousands of markers. The high throughput of next-generation sequencing technologies, moreover, allows sample multiplexing. The aims of this study were to (i) define a panel of single nucleotide polymorphisms (SNPs) in the cat, (ii) use GBS for profiling 16 cats, and (iii) evaluate the performance with respect to the inference using standard approaches at different coverage thresholds, thereby providing useful information for designing similar experiments. Probes for sequencing 230 variants were designed based on the Felis_catus_8.0. 8.0 genome. The regions comprised anonymous and non-anonymous SNPs. Sixteen cat samples were analysed, some of which had already been genotyped in a large group of loci and one having been whole-genome sequenced in the 99_Lives Cat Genome Sequencing Project. The accuracy of the method was assessed by comparing the GBS results with the genotypes already available. Overall, GBS achieved good performance, with 92-96% correct assignments, depending on the coverage threshold used to define the set of trustable genotypes. Analyses confirmed that (i) the reliability of the inference of each genotype depends on the coverage at that locus and (ii) the fraction of target loci whose genotype can be inferred correctly is a function of the total coverage. GBS proves to be a valid alternative to other methods. Data suggested a depth of less than 11× is required for greater than 95% accuracy. However, sequencing depth must be adapted to the total size of the targets to ensure proper genotype inference.

First genome-wide CNV mapping in FELIS CATUS using next generation sequencing data.

BACKGROUND: Copy Number Variations (CNVs) have becoming very significant variants, representing a major source of genomic variation. CNVs involvement in phenotypic expression and different diseases has been widely demonstrated in humans as well as in many domestic animals. However, genome wide investigation on these structural variations is still missing in Felis catus. The present work is the first CNV mapping from a large data set of Next Generation Sequencing (NGS) data in the domestic cat, performed within the 99 Lives Consortium. RESULTS: Reads have been mapped on the reference assembly_6.2 by Maverix Biomics. CNV detection with cn.MOPS and CNVnator detected 592 CNVs. These CNVs were used to obtain 154 CNV Regions (CNVRs) with BedTools, including 62 singletons. CNVRs covered 0.26% of the total cat genome with 129 losses, 19 gains and 6 complexes. Cluster Analysis and Principal Component Analysis of the detected CNVRs showed that breeds tend to cluster together as well as cats sharing the same geographical origins. The 46 genes identified within the CNVRs were annotated. CONCLUSION: This study has improved the genomic characterization of 14 cat breeds and has provided CNVs information that can be used for studies of traits in cats. It can be considered a sound starting point for genomic CNVs identification in this species.

Molecular ordering and structure of quasi-spherical solutes by liquid crystal NMR and Monte Carlo simulations: the case of norbornadiene.

Norbornadiene (a C2v symmetry bicyclic rigid hydrocarbon) dissolved in three different nematic mesophases has been studied by liquid crystal NMR, to contribute to a better understanding of the influence of solvents on the solute's ordering and structure. The main results achieved by this work can be summarized as follows: (i) the order parameters obtained by the analysis of the 1H NMR spectra (at different temperatures) were successfully reproduced by a recently proposed model of solute/liquid crystal interactions, by using Monte Carlo numerical simulations; (ii) the theoretical (B3LYP/6-31++G**) "equilibrium" geometry of norbornadiene, vibrationally corrected by using the force field calculated at the same level, is compatible (within, at most, a 5% error) with experimental LXNMR data. This leads to the conclusion that the structure is not significantly distorted by the tested solvents.

An investigation of the structure and bond rotational potential of some fluorinated ethanes by NMR spectroscopy of solutions in nematic liquid crystalline solvents.

NMR spectra of 1,2-dibromo-1,1-difluoroethane and 1-bromo-2-iodo-tetrafluoroethane dissolved in nematic liquid crystalline solvents have been analysed to yield the magnitudes and signs of the scalar couplings, J(ij), and total anisotropic couplings, T(ij), between all the (1)H, (19)F, and (13)C nuclei, except for those between two (13)C nuclei. The values obtained for T(ij) in principle contain a contribution from J(ij)(aniso), the component along the static applied magnetic field of the anisotropic part of the electron-mediated spin-spin coupling. Neglecting this contribution allows partially averaged dipolar couplings, D(ij), to be extracted from the T(ij), and these were used to determine the structure, orientational order, and the conformational distribution generated by rotation about the C-C bond. The values obtained are compared with the results of calculations by ab initio and density functional methods. The differences found are no greater than those obtained for similar compounds which do not contain fluorine, so that there is no definitive evidence for significant contributions from J(CF)(aniso) or J(FF)(aniso) in the two compounds studied.

Short communication: quantitative trait loci affecting the somatic cell score on chromosomes 4 and 26 in Italian Holstein cattle.

This work aimed to confirm previously reported quantitative trait loci (QTL) affecting the somatic cell score (SCS) in dairy cattle on Bos taurus autosomes (BTA) 4 and 26. A granddaughter design with selective genotyping was implemented that included half-sib families from 12 male lines of Italian Holstein cattle. The animals were genotyped for 5 microsatellite markers each on regions of BTA 4 (average marker spacing 9.42 cM) and BTA 26 (average marker spacing 5.26 cM), previously reported by other authors as carrying QTL for somatic cell count. Quantitative trait loci analyses were performed using interval mapping by regressing sire breeding values for SCS onto genotype probabilities at 1-cM intervals along the 2 chromosome regions. Breeding values for SCS were estimated for the whole population using a test-day repeatability animal model. Results were not significant on a chromosome basis, but a possible QTL was found at BM4505 on BTA 26, confirming this region for further studies of QTL affecting SCS in the Italian Holstein population.

Changes in serum and urine SAA concentrations and qualitative and quantitative proteinuria in Abyssinian cats with familial amyloidosis: a five-year longitudinal study (2009-2014).

BACKGROUND: Diagnosis of familial amyloidosis (FA) in Abyssinian cats usually is made on postmortem examination. HYPOTHESIS/OBJECTIVES: Sequential analysis of serum SAA (sSAA), urinary SAA (uSAA), urinary protein:creatinine (UPC) ratio, or sodium-dodecylsulfate agarose gel electrophoresis (SDS-AGE) may facilitate early identification of cats with FA. ANIMALS: Twenty-three Abyssinian cats belonging to cattery A or B (low and high prevalence of FA, respectively). METHODS: Prospective longitudinal study using 109 blood and 100 urine samples collected over 4-year period every 4 months, if possible, or more frequently in case of illness. Cats that died during study were necropsied. Health status of live cats was checked 5 years after enrollment. Serum amyloid A (sSAA) and urinary SAA (uSAA) were measured using ELISA kit. The UPC ratio and SDS-AGE also was performed. RESULTS: Familial amyloidosis was not identified in cattery A, whereas 7/14 cats from cattery B had FA. Serum amyloid A concentrations were not significantly different between cats in catteries A and B or between cats with or without FA, despite frequent peaks in cats from cattery B. Conversely, uSAA was significantly higher in cattery B, especially in the terminal phases of FA. Proteinuria occasionally was found in cats from both catteries, especially in those with FA. Urine protein electrophoresis identified mixed proteinuria only in cats with FA. CONCLUSIONS AND CLINICAL IMPORTANCE: Serum amyloid A and UPC ratio are not helpful for early identification of Abyssinian cats with FA. Conversely, increases in uSAA with or without mixed proteinuria may be found before onset of clinical signs in cats with FA.

Comparative FISH-mapping of the survival of motor neuron gene (SMN) in domestic bovids.

A comparative fluorescence in situ mapping of the SMN gene was performed on R-banded chromosome preparations of cattle (Bos taurus, BTA, 2n = 60), river buffalo (Bubalus bubalis, BBU, 2n = 50), sheep (Ovis aries, OAR, 2n = 54) and goat (Capra hircus, CHI, 2n = 60), as well as on those of a calf from Piedmont breed affected by arthrogryposis. SMN was located on BTA20q13.1, OAR16q13.1, CHI20q13.1 and BBU19q13. These chromosomes and chromosome bands are believed to be homeologous, confirming the high degree of chromosome homeologies among bovids. The position of SMN was refined in cattle, compared to the two previous localizations, while it is a new gene assignment in the other three bovids. A comparative fiber-FISH performed on extended chromatin of both normal cattle and calf affected by arthrogryposis revealed more extended FITC signals in the calf, compared to the normal cattle (control), suggesting a possible duplication of the SMN gene in the calf affected by arthrogryposis. .

The NMR spectra of samples dissolved in liquid-crystalline phases: automatic analysis with the aid of multiple quantum spectra--the case of flexible molecules.

Homonuclear N(S) = 0 and heteronuclear N(S) not equal 0 multiple quantum spectra, involving changes in the magnetic number m(I) by (N(I)-1), (N(I) - 2), and (N(I)-3), with N(I) and N(S) the number of interacting nuclei of magnetogyric ratio gamma(I) and gamma(S), are used for the automatic analysis of (1)H NMR spectra of flexible molecules dissolved in liquid-crystalline phases. The automatic procedure has been applied to study molecules of general formula Ph-CH(2)-X starting from a parameter set having all the spectral parameters set to zero. The results of such an analysis are then used as starting parameters for analysis of the single quantum spectrum. The method was first tested when X = Br and X = H in order to compare strategies differing for the types of parameters used and was then applied to the analysis of 3-phenylprop-1-yne.

Symmetry and phase-selected NMR spectra of liquid crystalline samples.

It is demonstrated that the NMR spectra of liquid crystalline samples can be simplified by using multiple quantum filtering. In a system of N spin-12 nuclei, the N or (N-1)-multiple quantum filtered spectra (NQF or (N-1)QF) contain lines which originate only from transitions among the eigenstates belonging to the highest symmetry class of the spin permutation group. In addition the NQF spectra are divided further into two sets of lines which differ in phase by 180 degrees. A method for simulating and analysing multiple quantum filtered spectra is described, with examples from molecules with up to eight interacting spins.

Myosin-binding protein C DNA variants in domestic cats (A31P, A74T, R820W) and their association with hypertrophic cardiomyopathy.

BACKGROUND: Two mutations in the MYBPC3 gene have been identified in Maine Coon (MCO) and Ragdoll (RD) cats with hypertrophic cardiomyopathy (HCM). OBJECTIVE: This study examined the frequency of these mutations and of the A74T polymorphism to describe their worldwide distribution and correlation with echocardiography. ANIMALS: 1855 cats representing 28 breeds and random-bred cats worldwide, of which 446 underwent echocardiographic examination. METHODS: This is a prospective cross-sectional study. Polymorphisms were genotyped by Illumina VeraCode GoldenGate or by direct sequencing. The disease status was defined by echocardiography according to established guidelines. Odds ratios for the joint probability of having HCM and the alleles were calculated by meta-analysis. Functional analysis was simulated. RESULTS: The MYBPC3 A31P and R820W were restricted to MCO and RD, respectively. Both purebred and random-bred cats had HCM and the incidence increased with age. The A74T polymorphism was not associated with any phenotype. HCM was most prevalent in MCO homozygote for the A31P mutation and the penetrance increased with age. The penetrance of the heterozygote genotype was lower (0.08) compared with the P/P genotype (0.58) in MCO. CONCLUSIONS AND CLINICAL IMPORTANCE: A31P mutation occurs frequently in MCO cats. The high incidence of HCM in homozygotes for the mutation supports the causal nature of the A31P mutation. Penetrance is incomplete for heterozygotes at A31P locus, at least at a young age. The A74T variant does not appear to be correlated with HCM.

The indirect through-space F-F coupling in peri-difluoronaphthalene: is it anisotropic?

The (1)H, (19)F and (13)C spectra have been obtained of a sample of peri-difluoronaphthalene dissolved in the nematic liquid crystalline solvent ZLI 1695. The (13)C satellite spectra from the six, single-(13)C isotopomers at natural abundance in both the (1)H and (19)F spectra were identified and analysed to yield a set of residual total, anisotropic spin-spin couplings, T(ij). This was achieved by first obtaining residual (13)C-(19)F and (13)C-(1)H couplings from a proton-encoded, (13)C detected, local field 2D spectrum. The 45 values of T(HH), T(HF) and T(CH) were used to obtain the structure of the molecule, and then to estimate whether there is a significant contribution from the component along the magnetic field, J, of the anisotropic, electron-mediated, spin-spin coupling tensors for (13)C-(19)F and (19)F-(19)F pairs. It is found that there is strong evidence for a significant contribution of J to T(FF) but not for the (13)C-(19)F pairs.

An international parentage and identification panel for the domestic cat (Felis catus).

Seventeen commercial and research laboratories participated in two comparison tests under the auspices of the International Society for Animal Genetics to develop an internationally tested, microsatellite-based parentage and identification panel for the domestic cat (Felis catus). Genetic marker selection was based on the polymorphism information content and allele ranges from seven random-bred populations (n = 261) from the USA, Europe and Brazil and eight breeds (n = 200) from the USA. Nineteen microsatellite markers were included in the comparison test and genotyped across the samples. Based on robustness and efficiency, nine autosomal microsatellite markers were ultimately selected as a single multiplex 'core' panel for cat identification and parentage testing. Most markers contained dinucleotide repeats. In addition to the autosomal markers, the panel included two gender-specific markers, amelogenin and zinc-finger XY, which produced genotypes for both the X and Y chromosomes. This international cat parentage and identification panel has a power of exclusion comparable to panels used in other species, ranging from 90.08% to 99.79% across breeds and 99.47% to 99.87% in random-bred cat populations.

Obtaining the structure and bond rotational potential of a substituted ethane by NMR spectroscopy of solutions in nematic liquid-crystalline solvents.

Partially averaged dipolar couplings (also referred to as residual dipolar couplings) D(ij) can be obtained from the analysis of the NMR spectra of molecules dissolved in liquid-crystalline solvents. Their values for a nonrigid molecule depend upon the bond lengths and angles, the rotational potentials, and the orientational order of the molecules. The molecule studied, 1-chloro-2-bromoethane, is one of the simplest example of a substituted alkane in which the rotational potential has three minimum-energy positions, trans and gauche+/-conformations, and the present investigation explores the problems inherent in deriving the form of the potential and the molecular geometry from the set of partially averaged couplings between the protons, and between protons and (13)C nuclei. The geometrical parameters and the rotational potential obtained are compared with the results from a density-functional theory method.

Recombinant DRB sequences produced by mismatch repair of heteroduplexes during cloning in Escherichia coli.

Recombinant chimeric sequences originating from a mixture of the sequences of two different alleles are frequently found after amplification and cloning in Escherichia coli of exon 2 of the major histocompatibility complex (MHC) DRB genes. Several authors have suggested that the recombinant molecules result from in vitro recombination during PCR; nevertheless, a clear experimental demonstration of this hypothesis is lacking. In order to understand the mechanism producing the chimeric sequences, we set up a simple experiment based on the different restriction patterns of parental and recombinant sequences. Our data demonstrate that in the analysed case most of the recombinant variants were not produced by in vitro recombination during PCR, but were the result of the mismatch repair of heteroduplex molecules during cloning in E. coli. The high mutation rate in the alpha-helix region of DRB expressed genes, both after cloning in E. coli and after the germ-line differentiation process in vertebrates, suggests that the observed mutations are the result of similar gene conversion processes, probably favoured by chi-dependent microrecombination events.

BoLA class I polymorphism and in vitro immune response to M. bovis antigens.

SUMMARY: From a sample of 119 Friesian calves, serologically typed for BoLA class I, 47 subjects were chosen expressing 9 different MHC types (A6, A6.9, A10, A11, A14, A15, A30, W16, M103) with the same age and reared in the same farm conditions. The animals were s.c. injected with a water in oil suspension of killed M. bovis and the treatment was repeated two days later. Before the treatment and 21 days later, calves were bled and on PBM (peripheral blood mononuclear leucocytes) were performed the following tests: 1. Lymphocyte Stimulation with bovine and avian PPDs (Purified protein derivative of Mycobacterium bovis and Mycobacterium avium, respectively). 2. Phagocytic activity towards M. bovis. 3. Class II molecules expression on cell surface. 4. Percentage of leucocyte populations and subpopulations. In the in vitro Lymphocyte Stimulation test, all the animals and classes were responders. Animals bearing A10 BoLA class I presented c.p.m. (counts per minute) and index values higher than the other cattle; these values were significantly positively related both to bovine and avian PPDs (P < .01). By variance analysis A14 BoLA type showed a slight positive significant correlation with more efficient phagocytic activity. BoLA class I type did not seem to significantly affect percentage of class II positive cells and leucocyte percentages on PBM. ZUSAMMENFASSUNG: Der BoLA Klasse I Polymorphismus und in vitro immunologische Antwort gegen die Antigene von M. bovis Aus einer Stichprobe von 119 für BoLA Klasse I serologisch typisierten Friesian Kälber, wurden 47 Subjekte ausgewählt, die 9 verschiedene MHC Typen ausdrückten (A6, A6.9, A10, A11, A14, A15, A30, W16, M103). Alle waren gleich alt und in gleichen Haltungsbedingungen. Die Tiere wurden mit einer Wasser-in-Ol Suspension abgetöteter M. bovis subkutan injiziert und die Behandlung nach zwei Tagen wiederholt. Vor und 21 Tage nach Behandlung wurden die folgenden Tests ausgeführt: 1. Lymphozyten-Stimulationstest mit bovinen und Geflügel PPDs. 2. Phagozyten Aktivität gegen M. bovis. 3. Zeil-Oberflächen, Expression der Klasse II Moleküle. 4. Anteile der Lymphozyten Populationen und Subpopulationen. Im in vitro Lymphozyten-Stimulationstest waren alle Tiere und Klassen responder. Tiere mit A10 BoLA I zeigten höhere c.p.m. und Indexwerte als die anderen; diese Werte waren in signifikant positiver Beziehung mit der PPD von M. bovis und auch mit M. avium (P < .01). BoLA Typ A14 zeigte leicht signifikant positive Korrelation mit wirksamerer Phagozyten Aktivität. BoLA Klasse I Typ scheint nicht den Prozentsatz der positiven Zellen der Klasse II und der Leukozyten der PBM signifikant zu beeinflussen. RESUMEN: Polimorfismo de BoLA clase I y immunidad a los antigenos del M. bovis Se escojeron 47 novillos dentro de un grupo de 119 animales que segun analisis previamente hecha tenian BoLA de clase I. Estos 47 novillos fueron escojidos de manera que tuvieran 9 distintos tipos de MHC (A6, A6.9, A10, A11, A14, A15, A30, W16, M103), la misma edad, las mismas condiciones de cria. Estos animales fueron inoculados subcutaneo con M. bovis matados en una suspension oleosa y la misma inoculacion fue repetida una secunda vez despues de dos dias. Por cada animal se tomaron muestras de sangre antes y 21 dias despues de la inoculacion de arriba. Las muestras de sangre fueron pruebaoas con: 1. Stimulacion Lymhocitaria con PPD bovina y avicola. 2. Actividad phagocitaria a M. bovis. 3. Expresion sobre la superficie celular de moleculas de clase II. 4. Porcentaje de poblaciones y de subpob-laciones de leucocitos. Todos los animales y todos los tipos de MHC dieron respuestas positivas en las pruebas de Stimulacion Lymphocitaria. Los animales que tenian la BoLA A10 presentaron valores de c.p.m. y indices mas altos de los demas animales. Estos valores se encontraron significativamente y positivamente relacionados sea a la PPD bovina que a la PPD avicola. Por medio de la analisis de varianzas se encontro que el tipo BoLA A14 muestraba una correlacion significativa y algo positiva con una mejor actividad fagocitaria. Los tipos de clase BoLA I no parecieron que influenzaran de manera appreciable el porcentaje de positividad por la clase II y el porcentaje de leucocitos en la sangre PBM.

A panel of polymorphic bovine, ovine and caprine microsatellite markers.

A panel of 81 new polymorphic bovine microsatellite markers is described, together with further information on a previously reported group of 16 markers. The mean polymorphism information content of the 97 markers determined in 20 cattle was 0.66. Seventy-three of these markers have been assigned to chromosomes by either linkage analysis or use of hybrid cell panels. Thirty-nine of the markers were polymorphic in sheep, and 32 were polymorphic in goat. This study identified a set of 18 robust markers that were polymorphic in all three species and that covered 14 bovine chromosomes. This provides a single group of markers, which would be suited to genetic distance analysis and parentage control in cattle, sheep and goat.

Association of BoLA class II haplotypes with subclinical progression of bovine leukaemia virus infection in Holstein-Friesian cattle.

The influence of bovine lymphocyte antigen (BoLA) complex polymorphism on subclinical progression of bovine leukaemia virus (BLV) infection was investigated in 41 Holstein-Friesian cows from two herds in Italy. All cows were seropositive for BLV and 22 had persistent lymphocytosis (PL). BoLA-A specificities were defined by serology, and class II haplotypes were defined based on restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR)-RFLP analysis of DQ and DR genes. Chi-square analysis revealed a significant and absolute association of haplotype DQA*3A-DQB*3A-DRB2*2A-DRB3.2*11 with resistance to PL (P chi 2 = 0.028, relative risk (RR) = 0.061). Consistent with this observation, multiple regression analysis revealed that animals carrying this haplotype had lower lymphocyte counts (P = 0.0057). By contrast, haplotype DQA*12-DQB*12-DRB2*3A-DRB3.2*8 was associated with susceptibility to PL (P chi 2 = 0.043, RR = 9.625) and increased lymphocyte counts (P = 0.0537). These results confirm the association of haplotype DQA*3A-DQB*3A-DRB2*2A-DRB3.2*11 with resistance to PL, and substantiate earlier findings of haplotype DQA*12-DQB*12-DRB2*3A-DRB3.2*8 as a risk factor for subclinical progression to PL in BLV-infected Holstein-Friesian cattle.