Genome analysis identifies the mutant genes for common industrial Silverblue and Hedlund white coat colours in American mink

Abstract

The fur colour of American mink (Neovison vison) involves over 35 traits, but only three of these have been linked to specific genes. Despite being the most popular, coat colours Silverblue and Hedlund white remain uncharacterized genetically. The former is the first genetic mutant of fur colour identified in minks, while the latter is a commercially valuable phenotype that can be dyed easily. Here, we performed the whole genome sequencing for two American mink breeds with Silverblue and Hedlund white coats. We identified mutations in splice donor sites of genes coding melanophilin (MLPH) and microphthalmia-associated transcription factor (MITF) that regulate melanosome transport and neural-crest-derived melanocyte development, respectively. Both mutations cause mRNA splicing impairments that lead to a shift in open reading frames of MLPH and MITF. We conclude that our data should be useful for tracking economically valuable fur traits in mink breeding programs to contribute to global fur production.

Figure 1

American minks of standard dark brown, Silverblue (p/p), and Hedlund white (h/h) phenotypes.

Figure 2

Effects of MLPH^p mutation on MLPH transcripts. (a) Structure of MLPH gene. Red box indicates exon 7. Green triangle indicates primers used for RT-PCR. Equal introns sizes are shown for simplification. (b) Agarose gel electrophoresis of MLPH cDNA exons 6–9. M – 50 bp DNA Ladder (NEB, USA). (c) An electrophoregram of Sanger sequencing for MLPH cDNA exons 6–9. Blue frame is exon 7 deleted in Silverblue (p/p) minks with homozygous MLPH^p mutation. (d) Effects of MLPH^p mutation on MLPH transcripts. Green triangle indicates primers used for RT-PCR.

Figure 3

Effects of MITF^h mutation on transcripts of the MITF M-isoform. (a) Structure of mink MITF gene. Coloured boxes indicated exons 1 A (orange), 1 J (yellow), 1 C (dark blue), 1E (purple), 1 H (black), 1D (grey), 1B1a (brown), 1B1b (blue), 1 M (red) Green boxes indicate exons 2–9, common to all isoforms. Exons 1 A, 1 J, 1 C, 1E, 1 H, 1D, 1B1a and 1 M were predicted in silico. Green triangle indicates primers used for RT-PCR. Equal introns sizes are shown for simplification. (b) Structure of in silico predicted mink MITF isoforms (expression of MITF-M mRNA was confirmed with RT-PCR). Each isoform, except M, has a unique promoter and a first exon followed by 1B1b and 2–9 exons. The M-isoform is specific to melanocytes and melanoma cells, it does not include exon 1B1b. (c) Effect of MITF^h mutation on MITF-M transcript. This mutation potentially retains the first intron in cDNA and introduces a stop codon after position 51 of the intron (indicated as dotted box). The end product is a truncated 29 polypeptide containing only the first 11 amino acids of MITF-M. Green triangle indicates primers used for RT-PCR. (d) Agarose gel electrophoresis of MITF-M cDNA 1M-2 exons and B2M cDNA 1–2 exons. No MITF-M cDNA 1–2 exons were observed in the cortex of Hedlund white (h/h) minks, which were homozygous for this mutation.

Figure 4

Scheme of tripartite complex (RAB27A-MLPH-MYO5A) in melanosome intracellular trafficking in standard dark brown (a) and Silverblue (b) minks. RBD - RAB27-binding domain; MBD - MYO5A-binding domain; ABD - actin-binding domain.