Identification of mutant gene for black crystal coat and non-allelic gene interactions in Neogale mink
All methods were performed in accordance with applicable 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 (VSr/ + 10 individuals and VSr/VSr 7 individuals), silverblue grayling (Sh/ +p/p 1 individuals), black cross (S/ + 1 individual), violet (y/am/mp/p 1 individual), pastel Royle (b/w 1 individual), Hedlund white (h/h1 individual), moyle (m/m1 individual), silverblue (p/p3 individuals) and standard dark brown (25 individuals) farm-bred American mink were kept in the experimental fur farm of the Institute of Cytology and Genetics (Novosibirsk, Russia). Collected tissues were quickly dissected and frozen in liquid nitrogen, 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 a completely white animal (mink_4-131), which was expected to be homozygous for the black crystal mutation (VSr/VSr), 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 high-sensitivity microarray (Agilent, USA) and quantified by qPCR using a KAPA Library Quantification Illumina Kit protocol (KAPA Biosystems, USA). United). The matched library was sequenced in 2 × 76 and 2 × 101 cycles with the Illumina RapidRun SBS v2 kit (Illumina, USA), and in 2 × 101 cycles with the Illumina TruSeq SBS v3 kit (Illumina, USA) on a HiSeq Sequencer 2000/2500 (Illumina, USA) at the Vavilov Institute of General Genetics RAS (Moscow, Russia).
Additionally, we used whole genome sequence data of 3 standard dark brown, 3 silver blue, 1 moyle, and 1 purple 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 of 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 HaplotypeCaller package version 4.0 of the Genome Analysis Toolkit (GATK)22.
To detect the genetic factor underlying the crystal black phenotype, we selected homozygous variants with 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, silver blue, moyle and purple). ). Sample mink_4-131 has completely white fur and normal hearing and was expected to be homozygous for the black crystal mutation (VSr/VSr).
Annotation and prediction of effects of selected variants was done 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). The resulting amplicons were cleaned with a standard cleaning 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).