Wolf outside, dog inside? The genomic make-up of the Czechoslovakian Wolfdog

Background Genomic methods can provide extraordinary tools to explore the genetic background of wild species and domestic breeds, optimize breeding practices, monitor and limit the spread of recessive diseases, and discourage illegal crossings. In this study we analysed a panel of 170k Single Nucleotide Polymorphisms with a combination of multivariate, Bayesian and outlier gene approaches to examine the genome-wide diversity and inbreeding levels in a recent wolf x dog cross-breed, the Czechoslovakian Wolfdog, which is becoming increasingly popular across Europe. Results Pairwise FST values, multivariate and assignment procedures indicated that the Czechoslovakian Wolfdog was significantly differentiated from all the other analysed breeds and also well-distinguished from both parental populations (Carpathian wolves and German Shepherds). Coherently with the low number of founders involved in the breed selection, the individual inbreeding levels calculated from homozygosity regions were relatively high and comparable with those derived from the pedigree data. In contrast, the coefficient of relatedness between individuals estimated from the pedigrees often underestimated the identity-by-descent scores determined using genetic profiles. The timing of the admixture and the effective population size trends estimated from the LD patterns reflected the documented history of the breed. Ancestry reconstruction methods identified more than 300 genes with excess of wolf ancestry compared to random expectations, mainly related to key morphological features, and more than 2000 genes with excess of dog ancestry, playing important roles in lipid metabolism, in the regulation of circadian rhythms, in learning and memory processes, and in sociability, such as the COMT gene, which has been described as a candidate gene for the latter trait in dogs. Conclusions In this study we successfully applied genome-wide procedures to reconstruct the history of the Czechoslovakian Wolfdog, assess individual wolf ancestry proportions and, thanks to the availability of a well-annotated reference genome, identify possible candidate genes for wolf-like and dog-like phenotypic traits typical of this breed, including commonly inherited disorders. Moreover, through the identification of ancestry-informative markers, these genomic approaches could provide tools for forensic applications to unmask illegal crossings with wolves and uncontrolled trades of recent and undeclared wolfdog hybrids

Additional file 8: of Wolf outside, dog inside? The genomic make-up of the Czechoslovakian Wolfdog

Figure S7a. BGC alpha parameter outlier SNPs. Values lower than 0 indicate excess of wolf alleles, values higher than 0 indicate excess of dog alleles. BGC significant outliers are indicated by blue crosses (top or bottom 1% of the empirical distribution of values) and by red dots (95% credibility intervals of 10,000 iterations not including 0). Figure S7b. BayeScan outlier SNPs detected comparing differences in allele frequency between Czechoslovakian Wolfdogs and German Shepherds (right) and between Czechoslovakian Wolfdogs and Carpathian wolves (left). The vertical axis indicates mean FST values between populations, and the horizontal axis indicates the logarithm of posterior odds (log(PO)). The vertical line indicates the log(PO) value corresponding to the false discovery rate threshold of 0.05. Loci on the right of this line are putatively under selection. (PDF 312 kb

Additional file 7: of Wolf outside, dog inside? The genomic make-up of the Czechoslovakian Wolfdog

Table S1a. Wolf-excess genes surrounding outlier wolf-like SNPs (OWS) from PCAdmix. Table S1b. Enrichment in gene ontology categories (EGO) of wolf-excess genes surrounding the OWS from PCAdmix. Table S1c. Dog-excess genes surrounding outlier dog-like SNPs (ODS) from PCAdmix. Table S1d. EGO of dog-excess genes surrounding ODS from PCAdmix. Table S2a. Dog-excess genes included in genomic regions within ROHs. Table S2b. EGO of the dog-excess genes included in genomic regions within ROHs. Table S3a. Wolf-excess genes surrounding OWS from the lowest FST between CWDs and Carpathian wolves. Table S3b. EGO of wolf-excess genes surrounding OWS from the lowest FST between CWDs and Carpathian wolves. Table S3c. Dog-excess genes surrounding ODS from the lowest FST between CWDs and German Shepherds. Table S3d. EGO of dog-excess genes surrounding ODS from the lowest FST between CWDs and German Shepherds. Table S4a. Wolf-excess genes surrounding the outlier wolf-like 10-SNP blocks identified from the lowest FST between CWDs and Carpathian wolves. Table S4b. EGO of the wolf-excess genes surrounding the outlier wolf-like 10-SNP blocks identified from the lowest FST between CWDs and Carpathian wolves. Table S4c. Dog-excess genes surrounding outlier dog-like 10-SNP blocks from the lowest FST between CWDs and German Shepherds. Table S4d. EGO of dog-excess genes surrounding outlier dog-like 10-SNP blocks from the lowest FST between CWDs and German Shepherds. Table S5a. Wolf-excess genes surrounding OWS from BGC alpha parameter. Table S5b. EGO of wolf-excess genes surrounding OWS from BGC alpha parameter. Table S5c. Dog-excess genes surrounding ODS from BGC alpha parameter. Table S5d. EGO of dog-excess genes surrounding ODS from BGC alpha parameter. Table S6a. Wolf-excess surrounding OWS from BayeScan. Table S6b. EGO of wolf-excess genes surrounding OWS from BayeScan. Table S6c. Dog-excess genes surrounding ODS from BayeScan. Table S6d. EGO of dog-excess genes surrounding ODS from BayeScan. (XLSX 781 kb

Additional file 3: of Wolf outside, dog inside? The genomic make-up of the Czechoslovakian Wolfdog

Figure S3. PC1 vs. PC2 results from an exploratory principal component analysis (PCA) computed in SVS on the 126k SNP dataset and including dogs from 30 pure breeds (extrapolated from the available LUPA project dataset; top side of the graph, in grey inside the circle), Carpathian wolves (WCA; black dots to the left), German Shepherds (GSh; light grey dots in the bottom), and Czechoslovakian Wolfdogs (CWD; dark gray dots in the bottom). The two axes are not to scale, in order to better distinguish individuals along PC2. (PDF 202 kb

Additional file 4: of Wolf outside, dog inside? The genomic make-up of the Czechoslovakian Wolfdog

Figure S4. Comparison between the individual frequency of ROHs (FROH), calculated in SVS as the proportion of ROHs on the genome length spanned by the analysed SNPs (on the horizontal axis), and the individual Wright’s inbreeding coefficient (COI), estimated from the pedigrees with the software U-WGI (on the vertical axis). The two inbreeding indexes are significantly (p < 0.01) correlated. (PDF 71 kb

Additional file 6: of Wolf outside, dog inside? The genomic make-up of the Czechoslovakian Wolfdog

Figure S6. Graphical representation, for each chromosome of each analysed Czechoslovakian Wolfdog, of the ancestry components identified by PCAdmix based on the analysis of 10-SNP haplotype blocks. Each horizontal bar represents the two homologous chromosomes of an individual showing in black the genomic regions assigned as wolf-like and in light grey those assigned as dog-like. (PDF 10810 kb

Additional file 1: of Wolf outside, dog inside? The genomic make-up of the Czechoslovakian Wolfdog

Figure S1. FST heat plot matrix of the genetic distances among groups computed from the 126k dataset in SVS. The most distant breed to Carpathian wolves (WCA) is the English Bulldog (EBD) while the closest one is the ancient breed Shar-Pei (ShP). As expected the least differentiated breed from the Czechoslovakian Wolfdog (CWD) is the German Shepherd (GSh). (PDF 200 kb

Additional file 5: of Wolf outside, dog inside? The genomic make-up of the Czechoslovakian Wolfdog

Figure S5. Linkage disequilibrium (LD) decay plot. The vertical axis indicates the mean Estimated R-squared (r2), and the horizontal axis indicates the distance in kb at which LD decays. (PDF 220 kb

Additional file 2: of Wolf outside, dog inside? The genomic make-up of the Czechoslovakian Wolfdog

Figure S2. Genetic variability indexes computed in SVS using the 126k SNP dataset. a Mean values of observed heterozygosity (Ho) within groups. Czechoslovakian Wolfdogs (in dark gray) show higher levels of heterozygosity than parental populations (Carpathian wolves in black and German Shepherds in light grey), as expected from the recent crossings that originated the breed, but lower than most breeds. Bars indicate standard deviations. b Plots of the mean inbreeding coefficient F per breed. Czechoslovakian Wolfdogs show a mean F value intermediate among the other breeds but lower than both parental populations. c: from left to right: individual F values for Carpathian wolf (black histograms), German Shepherd (light grey histograms) and Czechoslovakian Wolfdog (dark gray histograms) groups. Bars indicate standard deviations. (PDF 94 kb

Additional file 7: of Wolf outside, dog inside? The genomic make-up of the Czechoslovakian Wolfdog

Table S1a. Wolf-excess genes surrounding outlier wolf-like SNPs (OWS) from PCAdmix. Table S1b. Enrichment in gene ontology categories (EGO) of wolf-excess genes surrounding the OWS from PCAdmix. Table S1c. Dog-excess genes surrounding outlier dog-like SNPs (ODS) from PCAdmix. Table S1d. EGO of dog-excess genes surrounding ODS from PCAdmix. Table S2a. Dog-excess genes included in genomic regions within ROHs. Table S2b. EGO of the dog-excess genes included in genomic regions within ROHs. Table S3a. Wolf-excess genes surrounding OWS from the lowest FST between CWDs and Carpathian wolves. Table S3b. EGO of wolf-excess genes surrounding OWS from the lowest FST between CWDs and Carpathian wolves. Table S3c. Dog-excess genes surrounding ODS from the lowest FST between CWDs and German Shepherds. Table S3d. EGO of dog-excess genes surrounding ODS from the lowest FST between CWDs and German Shepherds. Table S4a. Wolf-excess genes surrounding the outlier wolf-like 10-SNP blocks identified from the lowest FST between CWDs and Carpathian wolves. Table S4b. EGO of the wolf-excess genes surrounding the outlier wolf-like 10-SNP blocks identified from the lowest FST between CWDs and Carpathian wolves. Table S4c. Dog-excess genes surrounding outlier dog-like 10-SNP blocks from the lowest FST between CWDs and German Shepherds. Table S4d. EGO of dog-excess genes surrounding outlier dog-like 10-SNP blocks from the lowest FST between CWDs and German Shepherds. Table S5a. Wolf-excess genes surrounding OWS from BGC alpha parameter. Table S5b. EGO of wolf-excess genes surrounding OWS from BGC alpha parameter. Table S5c. Dog-excess genes surrounding ODS from BGC alpha parameter. Table S5d. EGO of dog-excess genes surrounding ODS from BGC alpha parameter. Table S6a. Wolf-excess surrounding OWS from BayeScan. Table S6b. EGO of wolf-excess genes surrounding OWS from BayeScan. Table S6c. Dog-excess genes surrounding ODS from BayeScan. Table S6d. EGO of dog-excess genes surrounding ODS from BayeScan. (XLSX 781 kb