In this study we assessed the level of genetic introgression between red foxes bred on fur farms in Poland and the native wild population. We also evaluated the impact of a geographic barrier and isolation by distance on gene flow between two isolated subpopulations of the native red fox and their genetic differentiation. Nuclear and mitochondrial DNA was collected from a total of 308 individuals (200 farm and 108 wild red foxes) to study non-native allele flow from farm into wild red fox populations. Genetic structure analyses performed using 24 autosomal microsatellites showed two genetic clusters as being the most probable number of distinct populations. No strong admixture signals between farm and wild red foxes were detected, and significant genetic differentiation was identified between the two groups. This was also apparent from the mtDNA analysis. None of the concatenated haplotypes detected in farm foxes was found in wild animals. The consequence of this was that the haplotype network displayed two genetically distinct groups: farm foxes were completely separated from native ones. Neither the River Vistula nor isolation by distance had a significant impact on gene flow between the separated wild red fox subpopulations. The results of our research indicate a low probability of genetic introgression between farm and native red foxes, and no threat to the genetic integrity of this species.

The tissue samples were taken post mortem. The samples from the farm foxes were collected after they had been killed at the end of the farm season (during pelting), while those from wild foxes were collected after they had been killed by hunters or in road accidents.

Genomic DNA was extracted using specific reagents and protocols [Boodram 2004] used in the ARK Genomics Lab (currently Edinburgh Genomics). The tissue samples from all the foxes (n=310) were genotyped at 24 autosomal microsatellite loci using previously published primers [Breen M. et al. 2001, Ladon D. et al. 1998, Guyon R. et al. 2003, Neff M.W. et al. 1999, Holmes N.G. et al. 1995, Jouquand, S. et al. 2000]. The following microsatellites were used: AHT137, FH2613, FH2097, FH2980, FH3970, FH3241, FH3713, FH2295, FH3775, FH3824, FH3771, FH3287, FH4001, REN135K06, REN210I14, REN307J23, REN88H03, REN258F18, REN248F14, REN64E19, REN252E18, REN75M10, ZUBECA6 and UOR4101. The polymerase chain reaction (PCR) was performed using Qiagen Multiplex PCR and the PCR protocol was as follow: 95°C for 15 minutes, then 30 cycles of 94°C for 30 sec, 55°C for 90 sec, 72°C for 60 sec; final extention 60°C for 30 minutes. The PCR products were electrophoresed along with the Genescan 500 LIZ internal size standard (Applied Biosystems) on an ABI 3130XL Genetic Analyzer (Applied Biosystems). The data set was analysed using GeneMapper v4.0 (Applied Biosystems). 

DNA for mitochondrial genes analysis was extracted using the Sherlock AX kit (A&A Biotechnology). Eighty nine samples (48 from farm foxes and 41 from wild foxes) were used in this analysis and two regions of mtDNA were amplified: a 878-bp segment of the cytochrome b gene and a 443-bp segment of the D-loop. The PCR mixtures were prepared using a DreamTaq Master Mix kit (Thermo Scientific) according to the manufacturer’s protocol. A new set of primers for both sequences, designed and described by Zatoń-Dobrowolska et al. 2019, were used.  The thermal cycle conditions for both markers were as follows: 94°C for 5 minutes, then 40 cycles of 94°C for 45 sec, 55°C for 30 sec and 72°C for 1 min 10 sec, followed by 10 min at 72°C. The PCR products were purified and sequenced using an ABI BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems) and ABI 3730 capillary sequencer (Applied Biosystems) for cytochrome b (sequenced from primer VVGluF3) and D-loop (sequenced from primer VVCRR2). The sequences were analysis using Bioedit and Mega6 tools. The BLAST method was used to identify haplotypes.