Genotype of c-KIT in Chinese Bama miniature pigs
As previously described, the genotype of c-KIT in pigs is various
and complex with changes in copy number and splice mutation (Fig. 3a).
Firstly, we described the genotype of c-KIT in Bama pigs, which
regulates the “two-ends” black phenotype (Fig. 3b). To determine the
copy number of c-KIT , we designed primers and probes to amplifyc-KIT and a single copy control sequence (ESR ) using the
droplet-digital PCR (ddPCR) in five wild-type pigs (Fig. 3c). After
amplification, the number of positive and negative droplets was
determined based on the fluorescence signal (Fig. 3d), and thec-KIT and ESR copy numbers in each sample were calculated
by Poisson statistics based on the ratio of positive to total partitions
(Fig. 3e). The comparative copy numbers of c-KIT and ESRin all five samples implied the presence of two c-KIT copies in
the Bama pig diploid genome (Fig. 3f). Therefore, this genotype is theIBe allele of c-KIT in pigs.
Also, we analyzed the whole c-KIT sequence in Bama pigs. Finally,
a total of 26 polymorphisms (23 single nucleotide polymorphisms (SNPs)
and three indels) were identified in the genomic DNA: 12 (nine SNPs and
three indels) in introns and 14 in exons
(Table. S3). Of the 14 SNPs
located in exons, two were in the 3’-untranslated region (UTR) and 12
were synonymous (n=10) or non-synonymous mutations (n=2). The missense
mutations in exon 3 (g.58390G>A, p.R173K; g.58455
G>A, p.V195M) have been previously reported in cDNA or
genomic sequences of c-KIT (Fontanesi et al., 2010; Lai et al.,
2007). The BLAST analysis of the
sequence of the KIT protein
(NP_001037990.1) predicted an amino acid substitution (p.D309G, exon 5)
in the third Ig-like loop in the extracellular domain of Bama pigs.
Moreover, we detected two critical mutations in c-KIT : a splice
mutation in exon 17 and a 4 bp indel in intron 18 (intron 18:
g.29_32delAGTT). As described previously(Marklund et al., 1998), we
obtained a 175-bp fragment with KIT 17 primers and a 160–164 bp
fragment with Intron 18 primers. Sequence analysis showed high
conservation of the GT dinucleotide in the 5’ splice site of exon 17
(Fig. 3f) in Bama pigs, which confirmed the correlation between thec-KIT splice mutation and the dominant white phenotype. Moreover,
the 4-bp deletion mutation in intron 18 was absent in Bama pigs.