Identification of the c.2430T>A mutation in exon 17 of c-KIT in mutant pigs
To identify the potential causative gene in the G1-016105 mutant strain, linkage analysis and family-based genome-wide association study (GWAS) were performed in 16 mutants and 17 wild-type littermates (including G1-016105, three wild-type sows, and their offspring). Consequently, parametric analysis revealed a significant linkage at the 32 - 48 M region of chromosome 8 (P value < 1×10−5; LOD=7.8) (Fig. 2a, b). The annotation of the pig reference genome suggested the presence of about 141 genes in this candidate linkage disequilibrium interval. c-KIT (chr8: 43550231–43601377) occurs in this region (Fig. 2c) and is associated with the coat color in pigs; hence, it was selected for further analysis. All coding sequences ofc-KIT genomic DNA were sequenced, and a point mutation (T>A) was identified at the 2430thnucleotide (CDS2430 bp) in exon 17 that completely co-segregated with the mutant phenotype (Fig. 2d). This mutation (c-KIT c.2430 A>T) induced the substitution of the 810th aspartic acid with glutamate acid in the second tyrosine kinase domain of the pig c-KIT protein (p.D810E) (Fig. 2d). Intriguingly, this mutation was not found in the SNP database (dbSNP, https://www.ncbi.nlm.nih.gov/snp/) or other pig breeds, indicating that it was novel and created by ENU mutagenesis. Further sequence alignment with BLAST (https://blast.ncbi.nlm.nih.gov/) and ClustalW (https://www.genome.jp/tools-bin/clustalw) revealed that this site Asp810 (Asp812 in mice) in the protein tyrosine kinase domain was conserved among all mammalian c-KIT proteins (Fig. 2e). Moreover, Asp810 belonged to the DFG motif (Asp810, Phe811, Gly812) and localized in the N-terminus of the activation loop.