All methods were carried out in accordance with relevant guidelines and regulations for laboratory work as well as ARRIVE guidelines. The local Ethics Committee of the Institute of Cytology and Genetics approved the study protocols.
black crystal (Cr/ + 10 individuals and Cr/Cr 7 individuals), shadow silverblue (Sh/ +p/p 1 individuals), black cross (S/ + 1 individual), violet (a/a m/m p/p 1 individual), Royal paste (b/b 1 individual), Hedlund white (h/h1 individual), moyle (m/m1 individual, silverblue (p/p3 individuals) and standard dark brown (25 individuals) farm-bred American minks were maintained in the Experimental Fur Farm of the Institute of Cytology and Genetics (Novosibirsk, Russia). Collected tissues were rapidly dissected and frozen in liquid nitrogen and then stored at − 70 °C until DNA extraction.
Genomic DNA from mink tissues was extracted using QIAGEN Mini Spin Columns following the manufacturer’s protocol (QIAGEN, Germany). Library preparation from the DNA of completely white animal (mink_4-131), which were expected to be homozygous for the Black crystal mutation (Cr/Cr), was performed with the TruSeq PCR Free Kit (Illumina, USA) following the manufacturer’s protocol. Library validation was performed with an Agilent 2100 Bioanalyzer with a DNA High Sensitivity chip (Agilent, USA) and quantified with qPCR using a KAPA Library Quantification Illumina Kit protocol (KAPA Biosystems, USA). The paired-end library was sequenced in 2 x 76 and 2 x 101 cycles with the Illumina RapidRun SBS v2 kit (Illumina, USA), and in 2 x 101 cycles with the Illumina TruSeq SBS v3 kit (Illumina, USA) on a HiSeq 2000/2500 sequencer (Illumina, USA) at the Vavilov Institute of General Genetics RAS (Moscow, Russia).
Additionally, we used whole-genome sequencing data from 3 standard dark brown, 3 silverblue, 1 moyle and 1 violet mink from our previous studies (Table 1)8,9.
The resulting reads were mapped to the mink genome (NNQGG.v01) using a BWA-MEM algorithm (bwa v.0.7.13-r112)21. Duplicate reads were detected with the MarkDuplicates algorithm from picard-tools v.1.133 (http://broadinstitute.github.io/picard) and excluded from further analysis.
Genetic variants in sequenced mink genomes were predicted using the Genome Analysis Toolkit (GATK) HaplotypeCaller package version 4.022.
To detect the genetic factor underlying the Black crystal phenotype, we selected homozygous variants with a depth of coverage greater than 2 in the mink_4-131 genome that were not homozygous or heterozygous in all other color phenotypes (standard dark brown, silverblue, moyle and violet ). Sample mink_4-131 has completely white fur and normal hearing and was expected to be homozygous for the Black crystal mutation (Cr/Cr).
Annotation and effect prediction of selected variants were performed in SnpEff23 using mink genome annotation.
We performed Sanger sequencing to validate the selected mutation. Primers for PCR amplification were designed in Primer3 software (Table 3), and PCR was performed with GenPack PCR Core (Isogen, Russia). Resultant amplicons were cleaned with a Cleanup Standard Kit (Evrogen, Russia) and processed with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) following the manufacturer’s protocol. Probes were purified using a DyeEx 2.0 Spin Kit (QIAGEN, Germany) and sequenced in a 3730xl DNA Analyzer (Applied Biosystems, USA).