## 2 Material and Methods
### Functional Analyses  
#### 2.4.1 Functional Analyses within Species  
To reveal the functional implications of the detected candidate genes, an enrichment analysis was performed in terms of biological processes (BP), cellular components (CC), and molecular functions (MF) of Gene Ontology terms [84] and metabolic pathways of the Kyoto Encyclopedia of Genes and Genomes (KEGG; [85]) in the DAVID database [86] for each species. The significance of the terms enriched was tested by Bonferroni multiple-test correction, as implemented in the DAVID platform [86]. As a complementary analysis, an independent whole-genome sequence dataset for various cattle breeds [87] was used to detect the exonic polymorphisms (single nucleotide polymorphisms (SNPs)) for all candidate genes identified in cattle. We focused on exonic SNPs because their variations are most likely to have functional implications and they are the most complete datasets available in the referred data source [87]. There is still a lack of studies in the literature comparing behavioral responses across breeds, especially across animals reared under similar management conditions. Therefore, the breeds were categorized into two groups with likely differential temperament characteristics based on the literature (e.g., [88]), breed association and extension web reports, and personal communication with livestock producers and extension specialists. The first group (Group 1) is classified as more temperamental and includes six breeds, namely Brahman, Charolais, Gyr (or Gir), Nellore (or Nelore), Piedmonetse, and Shorthorn Zebu, while the second group (Group 2) consisted of Hereford, Holstein, Jersey, Angus, and Red Angus, with more docile characteristics [87]. The animals and breeds available had an average DNA sequencing coverage of equal to 12.12 (Group 1; from 4.9 to 18.6) and 13.6 (Group 2; from 6.67 to 24.35). Both coding regions and untranslated regions (UTRs) were targeted for SNP detection within the candidate genes among 45 animals in Group 1 (i.e., 9 Brahman, 14 Charolais, 3 Gyr, 4 Nellore, 5 Piedmonetse, and 10 Shorthorn Zebu) and 119 animals in Group 2 (i.e., 21 Hereford, 45 Holstein, 12 Jersey, 25 Angus, and 16 Red Angus) [87]. This grouping approach is partially subjective and could influence the results, but there is enough evidence about the differences in temperamental across breeds in the literature. Future studies should also include additional breeds as well as larger and more balanced datasets. In total, there were 11,035 SNPs tested across autosomal chromosomes. All SNP with minor allele frequency (MAF) lower than 5% in Group 1 were subjected to statistical testing for intergroup differences using [PERSON]'s exact test (false discovery rate adjusted \(p\)-value \(\leq\) 0.05). In order to minimize bias due to _Bos taurus taurus_ and _Bos taurus indicus_ differences, an adjustment for SNPs significantly different between both subspecies breeds ((Brahman, Nelore, and Shorthorn Zebu) versus (Charolais, and Piedmonetse)) were performed. By way of explanation, we have removed all SNP with the MAF lower than 5% in the _Bos taurus taurus_ groups that were statistically different from the MAF in _Bos taurus indicus_ group. Finally, the statistically significant SNPs adjusted to _Bos taurus taurus_ and _Bos taurus indicus_ differences (\(p\)-value \(\leq\) 0.05; null hypothesis: allele frequencies of Group 1 and Group 2 are equal) were classified as potential causal mutations. Candidate genes with a ratio of significantly different SNPs and all SNPs within the gene region higher than 50% suggest strong association with animalbehavior. Statistical power of the [PERSON]'s exact test was evaluated using the _power,fisher.test_ function from the _statmod_ package in R software [81; 89] considering a significance level of 0.05 and 1000 population simulation. The average of the statistical power across loci was 52%.