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》

An accurate method for quantifying and analyzing copy number variation in porcine KIT by an oligonucleotide ligation assay.

Aside from single nucleotide polymorphisms, copy number variations (CNVs) are the most important factors in susceptibility to genetic disorders because they affect expression levels of genes. In previous studies, pyrosequencing, mini-sequencing, real-time PCR, invader assays and other techniques have been used to detect CNVs. However, the higher the copy number in a genome, the more difficult it is to resolve the copies, so a more accurate method for measuring CNVs and assigning genotype is needed. PCR followed by a quantitative oligonucleotide ligation assay (qOLA) was developed for quantifying CNVs. The accuracy and precision of the assay were evaluated for porcine KIT, which was selected as a model locus. Overall, the root mean squares of bias and standard deviation of qOLA were 2.09 and 0.45, respectively. These values are less than half of those in the published pyrosequencing assay for analyzing CNV in porcine KIT. Using a combined method of qOLA and another pyrosequencing for quantitative analysis of KIT copies with spliced forms, we confirmed the segregation of KIT alleles in 145 F1 animals with pedigree information and verified the correct assignment of genotypes. In a diagnostic test on 100 randomly sampled commercial pigs, there was perfect agreement between the genotypes obtained by grouping observations on a scatter plot and by clustering using the nearest centroid sorting method implemented in PROC FASTCLUS of the SAS package. In a test on 159 Large White pigs, there were only two discrepancies between genotypes assigned by the two clustering methods (98.7% agreement), confirming that the quantitative ligation assay established here makes genotyping possible through the accurate measurement of high KIT copy numbers (>4 per diploid genome). Moreover, the assay is sensitive enough for use on DNA from hair follicles, indicating that DNA from various sources could be used. We have established a high resolution quantification method using an oligonucleotide ligation assay to measure CNVs, and verified the reliability of genotype assignment for random animal samples using the nearest centroid sorting method. This new method will make it more practical to determine KIT CNV and to genotype the complicated Dominant White/KIT locus in pigs. This procedure could have wide applications for studying gene or segment CNVs in other species.

Seo BY ，Park EW ，Ahn SJ ，Lee SH ，Kim JH ，Im HT ，Lee JH ，Cho IC ，Kong IK ，Jeon JT ... -  《BMC GENETICS》

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》

Unexpectedly high allelic diversity at the KIT locus causing dominant white color in the domestic pig.

Mutations in KIT encoding the mast/stem cell growth factor receptor (MGF) are responsible for coat color variation in domestic pigs. The dominant white phenotype is caused by two mutations, a gene duplication and a splice mutation in one of the copies leading to skipping of exon 17. Here we applied minisequencing and pyrosequencing for quantitative analysis of the number of copies with the splice form. An unexpectedly high genetic diversity was revealed in white pigs. We found four different KIT alleles in a small sample of eight Large White females used as founder animals in a wild boar intercross. A similar number of KIT alleles was found in commercial populations of white Landrace and Large White pigs. We provide evidence for at least two new KIT alleles in pigs, both with a triplication of the gene. The results imply that KIT alleles with the duplication are genetically unstable and new alleles are most likely generated by unequal crossing over. This study provides an improved method for genotyping the complicated Dominant white/KIT locus in pigs. The results also suggest that some alleles may be associated with negative pleiotropic effects on other traits.

Pielberg G ，Olsson C ，Syvänen AC ，Andersson L ... -  《GENETICS》

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》