A). FBN2 gene has a non-synonymous SNP in the first exon. This C > G non-synonymous SNP leads to alanine as an alternative allele with proline as reference allele in the Donggyeong dog (Fig. 1B). In OR5K4 gene, only the Donggyeong dog breed had an alternative allele at three positions in the first exon. Other dog breeds and related species had the same reference allele (Fig. 1C).
Donggyeong dog tail length-specific genes. Donggyeong dog-specific genes were further classified into three gene sets based on the uniqueness of non-synonymous SNPs in these three Donggyeong dog subpopulations (Materials and Methods). A total of 39 genes contained unique non-synonymous SNPs only in the subpopulation with tails (long tail or short tail) while 20 genes contained unique non-synonymous SNPs only in the non-tail subpopulation. If a gene contained non-synonymous SNPs both in the tail and non-tail subpopulations, it was classified as a common gene (a total of 98 genes, Supplementary Table 4). To identify related functions for these gene sets, Gene Ontology enrichment analysis was conducted. In terms of biological processes, 6, 1 and 10 enriched functions were found in tail-specific, non-tail-specific, and common gene sets, respectively. Among them, regulation of nucleobase-containing compound metabolic process (GO:0019219), organelle organization (GO:0006996), and nitrogen compound metabolic process (GO:0006807) were observed only in the tail-specific gene set while translation (GO:0006412) was uniquely observed in the non-tail-specific gene set. In common gene set, several sensory-related functions were found as enriched functions. Details of enriched functions in biological processes, molecular functions, and cellular processes are shown in Supplementary Table 5.

Protein interactions of Donggyeong dog tail length-specific genes. Donggyeong dog tail
length-specific gene sets were used to find corresponding protein sets. They were then extended by including additional proteins having direct or indirect interactions with original protein sets (Materials and Methods). Supplementary Fig. 2 shows the Venn diagram of the top 5% proteins of the three extended protein sets based on interaction score (690, 669, and 798 proteins of tail-specific, non-tail-specific, and common protein sets, respectively, Materials and Methods, Supplementary Table 6). A total of 155 proteins were shared by these three protein sets while 35% and 36% of common proteins were also observed in tail-specific and non-tail-specific protein set, respectively. The tail-specific and non-tail-specific protein sets contained 209 common proteins. There was no overlap among seed protein sets used for extension. By using the top 5% of these extended protein sets, protein-protein interaction networks were constructed based on interaction data in STRING database and orthologous protein information in orthoDB (Fig. 2, Materials and Methods). In the constructed network ( Fig. 2A, Supplementary Table 7), significantly more numbers of interactions were observed within each of tail-specific and non-tail-specific protein set (Fisher's exact test, p-value < 2.2e-16). Subnetworks for tail-specific and non-tail-specific protein sets by only considering interactions absent in other related species (cat, horse, pig, and cow) are shown in Fig. 2B. A total of 84 tail-specific proteins and 186 non-tail-specific proteins had 2,407 and 2,331 interactions, respectively. Clustering pattern of interactions with the same type of protein set was more obvious (Supplementary Table 8).
Proteins in tail-specific (blue) and non-tail-specific (red) subnetwork shown in Fig. 2B were further investigated to identify related functions. In the tail-specific subnetwork, several proteins are chemotactic factors and receptors. For examples, IL8 is one of chemotactic factors involved in response to inflammatory stimulus while ACKR3 is an atypical chemokine receptor related to chemokine levels. Among proteins in the non-tail-specific subnetwork, TAC1 encodes four products of the tachykinin peptide hormone family which are thought to function as neurotransmitters known to induce behavioral response. PMCH also produces melanin-concentrating hormone (MCH) that may act as a neurotransmitter or neuromodulater directed toward the regulation of goal-directed-behavior. GH1 is a member of the somatotropin/prolactin family of hormones which play an important role in growth control. In addition, NTS that makes neurotensin with important role in contraction of smooth muscle appeared in the non-tail-specific subnetwork. Other known functions of these proteins are summarized in Supplementary Table 8. SCIeNtIfIC REPORTS | 7: 17303 | DOI:10.1038/s41598-017-17817-w Evolutionary analysis of protein interactions of Donggyeong dog tail length-specific genes. The mode and extent of changes in protein interactions during evolution were investigated by considering four related species (cat, horse, pig, and cow) with mouse as an outgroup species. From 2,218 protein pairs of tail-specific extended protein set, interactions in other species were obtained and used to identify the existence of protein interactions in ancestors by using the maximum parsimony algorithm (Materials and Methods, Fig. 3A). From A1 to A2, 26.20% of protein pairs without interaction in A1 had interactions. In addition, significantly larger number of protein interactions (99.20%) were found during evolution from A2 to dog compared to other descendant species of A2. In contrast, many protein interactions disappeared during evolution from A1 to pig and cow. A similar pattern was also obtained from the analysis of non-tail-specific extended protein set (Fig. 3B). The statistical significance of the fraction of interaction changes was investigated based on a randomization test. p-values close to zero were obtained for all branches (Materials and Methods).

Discussion
In this study, 22 individuals of Donggyeong dog with different tail lengths were investigated to identify the following: (i) breed-specific genes and proteins associated with their unique traits, (ii) their functions, and (iii) evolutionary changes in their interactions by using population and comparative genomics approaches based on whole-genome sequencing data. Donggyeong dogs are very important Korean native dog breeds because of their unusual shape of tail and superior agility 14. This study was made possible due to the availability of whole-genome sequencing data of mammalian species and other 12 dog breeds. They have been successfully used in other studies investigating evidence of selection during domestication 18 and reconstruction of the evolutionary history of dogs 19.
A total of 1,814 Donggyeong dog-specific genes were identified as having non-synonymous SNPs not found in other dog breeds and related species. Majority of them including FBN2 and BCAN were involved in sensory-related functions such as sensory perception (GO:0007600), sensory perception of chemical stimulus (GO:0007606), and neurological system process (GO:0050877) which might affect the agility and appearance of Donggyeong dogs 14. Among these 1,814 genes, the T gene known to contain a single amino acid variant responsible for short tail length 20 was not present. This was because Yingjiang village dog included as one of the other 12 and non-tail-specific (red) subnetworks with interactions absent in other related species (cat, horse, pig, and cow). Node colors correspond to the colors used in Supplementary Fig. 2 (blue: the tail-specific proteins not shared with the non-tail-specific and the common proteins, red: the non-tail-specific proteins not shared with the tail-specific and the common proteins, green: shared proteins by the tail-specific and the non-tail-specific proteins excluding the common proteins, and gray: the common proteins). The definition of the protein sets is found in Materials and Methods. dog breeds had the same non-synonymous SNP at the same position in the T gene compared to Donggyeong dog, indicating that some Yingjiang dogs might have short tails.
In our study, the key information for identifying Donggyeong dog-specific genes was the presence of non-synonymous SNPs only in Donggyeong dogs. The non-synonymous SNPs are known to change the amino acid sequence of protein. However, it is very important to collect and use only non-synonymous SNPs that have non-neutral functional effect on phenotype to draw unbiased conclusions. SnpEff 21, which is a variant annotation tool, generates additional information, called an impact (or deleteriousness) category, having one of three values, high, moderate, and low. The impact is determined based on the amount of disruption of a gene structure, and high or moderate values indicate the potential effect of a variant on the loss of function or the change of protein effectiveness. In our study, we used only non-synonymous SNPs with the high or moderate impact values as in many recent studies [22][23][24][25][26]. There is also a similar measure, called GERP, to check the phenotypic impact of variants [27][28][29]. GERP is based on the sequence level conversation of a position with a variant obtained from sequence alignment of multiple species, and assigns higher phenotypic impact on variants observed at more conserved positions.
It is known that biological functions are performed through a cascade of interactions among multiple proteins, not just by a single protein. This fact was reflected in the analysis of protein interactions. Starting from Donggyeong dog tail length-specific proteins identified based on the uniqueness of non-synonymous SNPs, additional proteins directly or indirectly interacting with seed proteins were extracted from the