Molecular basis for the dominant white phenotype in the domestic pig

Abstract

The change of phenotypic traits in domestic animals and crops as a response to selective breeding mimics the much slower evolutionary change in natural populations. Here, we describe that the dominant white phenotype in domestic pigs is caused by two mutations in the KIT gene encoding the mast/stem cell growth factor receptor (MGF), one gene duplication associated with a partially dominant phenotype and a splice mutation in one of the copies leading to the fully dominant allele. The splice mutation is a G to A substitution in the first nucleotide of intron 17 and leads to skipping of exon 17. The duplication is most likely a regulatory mutation affecting KIT expression, whereas the splice mutation is expected to cause a receptor with impaired or absent tyrosine kinase activity. Immunocytochemistry showed that this variant form is expressed in 17- to 19-day-old pig embryos. Hundreds of millions of white pigs around the world are assumed to be heterozygous or homozygous for the two mutations. [The EMBL accession numbers for porcine KIT1*0101, KIT1*0202, KIT2*0202, and KIT2*0101 are AJ223228-AJ223231, respectively.]

Figure 1

Identification of a KIT transcript lacking exon 17 in pigs with the dominant white allele. Agarose gel electrophoresis of RT–PCR products of KIT exon 16–19. Samples 1–3 and 4–6 are Swedish Large White (homozygous or heterozygous for the I allele) and Swedish Hampshire (i/i) pigs, respectively. The size difference between the 424- and 301-bp fragments is due to the lack of exon 17 in the latter fragment. The two upper bands of samples 1–3 were shown to be heteroduplexes (HDs) formed between the normal and splice mutant form. The tendency for different relative amounts of the 424- and 301-bp fragments may be a PCR artifact but may also have a biological cause, e.g., whether an animal is heterozygous or homozygous for the I allele.

Figure 2

DNA sequence comprizing 48 bp of the exon 17/intron 17 border in KIT1 and KIT2 associated with the I, I^p, and i alleles. The position of the exon/intron border is marked with a vertical line, the splice mutation (nt1^G → A) with a vertical arrow, and sequence identity to the master sequence with a dash. KIT1 and KIT2 sequences from the I allele were obtained by sequencing PCR products from genomic λ clones representing the two forms while direct sequencing of PCR products from genomic DNA was carried out to determine KIT sequences associated with I^p and i.

Figure 3

Quantitative PCR–RFLP analysis estimating the ratio of normal to splice mutant KIT in genomic DNA from F₂ animals in a Wild Boar/Large White intercross with different genotypes. (I/I) n = 13; (I/i) n = 14; (I/I^p) n = 12. The genotypes were deduced by family segregation analysis using the dense linkage map around the I locus.

Figure 4

Light microphotographs of the cardiac wall (m = endomyocardium) and blood cells (arrows) in dominant white I/I (a,b) and colored embryos i/i (a′,b′) after incubation with the Ex21 (a,a′) and Ex16/18 (b,b′) antibodies. Note the negative immunolabeling of Ex16/18 in colored embryos (b′, open arrow). ABC-ELITE, magnification 200×.