A large duplication associated with dominant white color in pigs originated by homologous recombination between LINE elements flanking KIT.

The Dominant White (I/KIT) locus is one of the major coat color loci in the pig. Previous studies showed that the Dominant White (I) and Patch (IP) alleles are both associated with a duplication including the entire KIT coding sequence. We have now constructed a BAC contig spanning the three closely linked tyrosine kinase receptor genes PDGFRA-KIT-KDR. The size of the duplication was estimated at about 450 kb and includes KIT, but not PDGFRA and KDR. Sequence analysis revealed that the duplication arose by unequal homologous recombination between two LINE elements flanking KIT. The same unique duplication breakpoint was identified in animals carrying the I and IP alleles across breeds, implying that Dominant White and Patch alleles are descendants of a single duplication event. An unexpected finding was that Piétrain pigs carry the KIT duplication, since this breed was previously assumed to be wild type at this locus. Comparative sequence analysis indicated that the distinct phenotypic effect of the duplication occurs because the duplicated copy lacks some regulatory elements located more than 150 kb upstream of KIT exon 1 and necessary for normal KIT expression.

Giuffra E ，Törnsten A ，Marklund S ，Bongcam-Rudloff E ，Chardon P ，Kijas JM ，Anderson SI ，Archibald AL ，Andersson L ... -  《MAMMALIAN GENOME》

Chinese white Rongchang pig does not have the dominant white allele of KIT but has the dominant black allele of MC1R.

The mast/stem cell growth factor receptor (KIT) and melanocortin receptor 1 (MC1R) mutations are responsible for coat color phenotypes in domestic pigs. Rongchang is a Chinese indigenous pig breed with a white coat color phenotype. To investigate the genetic variability of the KIT and MC1R genes and their possible association with the coat color phenotype in this breed, a gene duplication and splice mutation of KIT were diagnosed in a sample of 93 unrelated Rongchang animals. The results show that Rongchang pigs have a single copy of KIT without the splice mutation at the first nucleotide of intron 17, indicating that the dominant white I allele of KIT is not responsible for their white phenotype. The KIT mRNA and MC1R coding sequences were also determined in this breed. Three putative amino acid substitutions were found in the KIT gene between Rongchang and Western white pigs, their association with the Rongchang white phenotype remains unknown. For the MC1R gene, Rongchang pigs were demonstrated to have the same dominant black allele (E(D1)) as other Chinese breeds, supporting the previous conclusion that Chinese and Western pigs have independent domestication origin. We also clarified that the Rongchang white phenotype was recessive to nonwhite color phenotypes. Our results provide a good starting point for the identification of the mutations underlying the white coat color in Rongchang pigs.

Lai F ，Ren J ，Ai H ，Ding N ，Ma J ，Zeng D ，Chen C ，Guo Y ，Huang L ... -  《JOURNAL OF HEREDITY》

[Effect study of white locus (I) on coat color inheritance in Chinese native pig breeds].

The classical White (I) locus is one of the important pig coat color hereditable loci,which is homologous to KIT gene. In this study,PCR-RFLP and PCR-SSCP analysis were commanded on the Intron 17 and 18 nucleotide sequences of KIT gene. The tested results showed that the substitution mutation (G-->A) of Intron 17 was found in white pigs, including Wuzhishan pigs (white), Landrace and Large White,and its genotype (AB) frequency was 1.1 and 0.8 respectively. The genotype frequency was uniformly 0 in the other native pig breeds. Similarly,the deleted mutation (AGTT) of Intron 18 was also found in the same white pigs,its genotype (,AA) frequency was 1.1 and 0.93 separately,while in the other native pig breeds was 0. Accordingly it was considered that KIT gene was an important factor regulating the white coat color genotype, and the classical I locus (KIT gene) was epistatic to the other genetic coat color loci. On the other hand,although the native pig breed Rongchang Pig is similar to Landrace and Large White in phenotype (white coat color), the mutation status found in them was absolutely distinct. So it is presumed that the coat color genetic system of Chinese native pig breeds was different from that of imported breeds.

Shi KR ，Wang AG ，Li N ，Deng XM ... -  《-》

A sensitive method for detecting variation in copy numbers of duplicated genes.

Gene duplications are common in the vertebrate genome, and duplicated loci often show a variation in copy number that may have important phenotypic effects. Here we describe a powerful method for quantification of duplicated copies based on pyrosequencing. A reliable quantification was obtained by amplification of the duplication break-point and a corresponding nonduplicated sequence in a competitive PCR assay. A comparison with an independent method for quantification based on the Invader technology revealed an excellent correlation between the two methods. The pyrosequencing-based method was evaluated by analyzing variation in copy number at the duplicated KIT/Dominant white locus in pigs. We were able to distinguish haplotypes at this locus by combining the duplication breakpoint test with a diagnostic test for a functionally important splice mutation in the duplicated gene. An extensive allelic variation, including the presence of a new allele carrying a single KIT copy expected to encode a truncated KIT receptor, was revealed when analyzing white pigs from commercial lines.

Pielberg G ，Day AE ，Plastow GS ，Andersson L ... -  《GENOME RESEARCH》

Pigs with the dominant white coat color phenotype carry a duplication of the KIT gene encoding the mast/stem cell growth factor receptor.

Comparative mapping data suggested that the dominant white coat color in pigs may be due to a mutation in KIT which encodes the mast/stem cell growth factor receptor. We report here that dominant white pigs lack melanocytes in the skin, as would be anticipated for a KIT mutation. We found a complete association between the dominant white mutation and a duplication of the KIT gene, or part of it, in samples of unrelated pigs representing six different breeds. The duplication was revealed by single strand conformation polymorphism (SSCP) analysis and subsequent sequence analysis showing that white pigs transmitted two nonallelic KIT sequences. Quantitative Southern blot and quantitative PCR analysis, as well as fluorescence in situ hybridization (FISH) analysis, confirmed the presence of a gene duplication in white pigs. FISH analyses showed that KIT and the very closely linked gene encoding the platelet-derived growth factor receptor (PDGFRA) are both located on the short arm of Chromosome (Chr) 8 at band 8p12. The result revealed an extremely low rate of recombination in the centromeric region of this chromosome, since the closely linked (0.5 cM) serum albumin (ALB) locus has previously been in situ mapped to the long arm (8q12). Pig Chr 8 shares extensive conserved synteny with human Chr 4, but the gene order is rearranged.

Johansson Moller M ，Chaudhary R ，Hellmén E ，Höyheim B ，Chowdhary B ，Andersson L ... -  《MAMMALIAN GENOME》