Performance Comparison of Different Approaches in Genotyping MHC-DRB: The Contrast between Single-Locus and Multi-Locus Species.

Major histocompatibility complex (MHC) genes are widely recognised as valuable markers for wildlife genetic studies given their extreme polymorphism and functional importance in fitness-related traits. Newly developed genotyping methods, which rely on the use of next-generation sequencing (NGS), are gradually replacing traditional cloning and Sanger sequencing methods in MHC genotyping studies. Allele calling in NGS methods remains challenging due to extreme polymorphism and locus multiplication in the MHC coupled with allele amplification bias and the generation of artificial sequences. In this study, we compared the performance of molecular cloning with Illumina and Ion Torrent NGS sequencing in MHC-DRB genotyping of single-locus species (roe deer) and species with multiple DRB loci (red deer) in an attempt to adopt a reliable and straightforward method that does not require complex bioinformatic analyses. Our results show that all methods work similarly well in roe deer, but we demonstrate non-consistency in results across methods in red deer. With Illumina sequencing, we detected a maximum number of alleles in 10 red deer individuals (42), while other methods were somewhat less accurate as they scored 69-81% of alleles detected with Illumina sequencing.

Neutral and Selective Processes Shape MHC Diversity in Roe Deer in Slovenia.

Disease control and containment in free-ranging populations is one of the greatest challenges in wildlife management. Despite the importance of major histocompatibility complex (MHC) genes for immune response, an assessment of the diversity and occurrence of these genes is still rare in European roe deer, the most abundant and widespread large mammal in Europe. Therefore, we examined immunogenetic variation in roe deer in Slovenia to identify species adaptation by comparing the genetic diversity of the MHC genes with the data on neutral microsatellites. We found ten MHC DRB alleles, three of which are novel. Evidence for historical positive selection on the MHC was found using the maximum likelihood codon method. Patterns of MHC allelic distribution were not congruent with neutral population genetic findings. The lack of population genetic differentiation in MHC genes compared to existing structure in neutral markers suggests that MHC polymorphism was influenced primarily by balancing selection and, to a lesser extent, by neutral processes such as genetic drift, with no clear evidence of local adaptation. Selection analyses indicated that approx. 10% of amino acids encoded under episodic positive selection. This study represents one of the first steps towards establishing an immunogenetic map of roe deer populations across Europe, aiming to better support science-based management of this important game species.

Population genetic structure of European wildcats inhabiting the area between the Dinaric Alps and the Scardo-Pindic mountains.

Habitat fragmentation and loss have contributed significantly to the demographic decline of European wildcat populations and hybridization with domestic cats poses a threat to the loss of genetic purity of the species. In this study we used microsatellite markers to analyse genetic variation and structure of the wildcat populations from the area between the Dinaric Alps and the Scardo-Pindic mountains in Slovenia, Croatia, Serbia and North Macedonia. We also investigated hybridisation between populations of wildcats and domestic cats in the area. One hundred and thirteen samples from free-leaving European wildcats and thirty-two samples from domestic cats were analysed. Allelic richness across populations ranged from 3.61 to 3.98. The observed Ho values ranged between 0.57 and 0.71. The global FST value for the four populations was 0.080 (95% CI 0.056-0.109) and differed significantly from zero (P < 0.001). The highest FST value was observed between the populations North Macedonia and Slovenia and the lowest between Slovenia and Croatia. We also found a signal for the existence of isolation by distance between populations. Our results showed that wildcats are divided in two genetic clusters largely consistent with a geographic division into a genetically diverse northern group (Slovenia, Croatia) and genetically eroded south-eastern group (Serbia, N. Macedonia). Hybridisation rate between wildcats and domestic cats varied between 13% and 52% across the regions.