Haidi Arbanasić

First evidence of hybridization between golden jackal (Canis aureus) and domestic dog (Canis familiaris) as revealed by genetic markers

Interspecific hybridization is relatively frequent in nature and numerous cases of hybridization between wild canids and domestic dogs have been recorded. However, hybrids between golden jackals (Canis aureus) and other canids have not been described before. In this study, we combined the use of biparental (15 autosomal microsatellites and three major histocompatibility complex (MHC) loci) and uniparental (mtDNA control region and a Y-linked Zfy intron) genetic markers to assess the admixed origin of three wild-living canids showing anomalous phenotypic traits. Results indicated that these canids were hybrids between golden jackals and domestic dogs. One of them was a backcross to jackal and another one was a backcross to dog, confirming that golden jackal–domestic dog hybrids are fertile. The uniparental markers showed that the direction of hybridization, namely females of the wild species hybridizing with male domestic dogs, was common to most cases of canid hybridization. A melanistic 3bp-deletion at the K locus (β-defensin CDB103 gene), that was absent in reference golden jackal samples, but was found in a backcross to jackal with anomalous black coat, suggested its introgression from dogs via hybridization. Moreover, we demonstrated that MHC sequences, although rarely used as markers of hybridization, can be also suitable for the identification of hybrids, as long as haplotypes are exclusive for the parental species.

Extensive polymorphism and evidence of selection pressure on major histocompatibility complex DLA-DRB1, DQA1 and DQB1 class II genes in Croatian grey wolves

The genes of the major histocompatibility complex (MHC) are a key component of the mammalian immune system and have become important molecular markers for measuring fitness-related genetic variation in wildlife populations. Because of human persecution and habitat fragmentation, the grey wolf has become extinct from a large part of Western and Central Europe, and remaining populations have become isolated. In Croatia, the grey wolf population, part of the Dinaric-Balkan population, shrank nearly to extinction during the 20th century, and is now legally protected. Using the cloning-sequencing method, we investigated the genetic diversity and evolutionary history of exon 2 of MHC class II DLA-DRB1, DQA1 and DQB1 genes in 77 individuals. We identified 13 DRB1, 7 DQA1 and 11 DQB1 highly divergent alleles, and 13 DLA-DRB1/DQA1/DQB1 haplotypes. Selection analysis comparing the relative rates of non-synonymous to synonymous mutations (d(N)/d(S)) showed evidence of positive selection pressure acting on all three loci. Trans-species polymorphism was found, suggesting the existence of balancing selection. Evolutionary codon models detected considerable difference between alpha and beta chain gene selection patterns: DRB1 and DQB1 appeared to be under stronger selection pressure, while DQA1 showed signs of moderate selection. Our results suggest that, despite the recent contraction of the Croatian wolf population, genetic variability in selectively maintained immune genes has been preserved.

Extensive polymorphism of the major histocompatibility complex DRA gene in Balkan donkeys: perspectives on selection and genealogy.

The major histocompatibility complex (MHC) contains genes important for immune response in mammals, and these genes exhibit high polymorphism and diversity. The DRA gene, a member of the MHC class II family, is highly conserved across a large number of mammalian species, but it displays exceptionally rich sequence variations in Equidae members. We analyzed allelic polymorphism of the DRA locus in 248 donkeys sampled across the Balkan Peninsula (Albania, Bulgaria, Croatia, Macedonia, Greece and Montenegro). Five known alleles and two new alleles were identified. The new allele Eqas-DRA*0601 was found to carry a synonymous mutation, and new allele Eqas-DRA*0701, a non-synonymous mutation. We further analyzed the historical selection and allele genealogy at the DRA locus in equids. Signals of positive selection obtained by various tests were ambiguous. A conservative conclusion is that DRA polymorphism occurred relatively recently and that positive selection has been acting on the DRA locus for a relatively brief period.

Diversity of equine major histocompatiblity complex class II DRA locus in Posavina and Croatian Coldblood horse: a new polymorphism detected

Abstract Domestic equidae display polymorphism within ELA-DRA locus which is not characteristic for other species. We characterised sequence polymorphism present at ELA-DRA locus exon 2 and estimated allele frequencies in two autochthonous breeds, Posavina and Croatian Coldblood. In 88 horses, four different alleles were found, one of them not reported before in horses. The new allele shows non-synonymous mutation at position 65 (T→A) causing amino acid change (Phe→Tyr) in antigen binding site and synonymous mutation at position 105 (C→T). Our findings emphasize the importance of DRA polymorphism among equids and some specific DRA frequency pattern potentially specific in draught horses.

PCR-based Y chromosome marker for discriminating between golden jackal (Canis aureus) and domestic dog (Canis lupus familiaris) paternal ancestry

AbstractNumerous reports of hybridization between wild Canis species and domestic dog suggest that hybridization between golden jackal and dog is likely. Here we present an assay of a Y chromosome marker for discriminating between golden jackal (Canis aureus) and dog (Canis lupus familiaris) paternal ancestry. Taking advantage of an insertion found in a dog Zfy intron haplotype but not in a golden jackal haplotype, we developed a three-primer PCR system in which species are differentiated based on the number of amplicons. Two amplicons are produced from domestic dog DNA template, while one amplicon is produced from golden jackal DNA template. Both amplicons can be analyzed in a single agarose gel electrophoresis run, while the longer amplicon also serves as an internal control. The method was validated using 27 golden jackal and 25 dog samples; all samples showed the expected amplicons. This simple and cost-effective method should prove useful for determining the direction of golden jackal–dog hybridization.

HEMATOLOGICAL AND SERUM BIOCHEMICAL REFERENCE VALUES FOR THE POSAVINA AND CROATIAN COLDBLOOD HORSE BREEDS

Abstract In this study the hematological and biochemical parameters in the blood of indigenous Croatian working horse breeds were investigated. The Posavina and Croatian Coldblood horses are adapted to harsh environmental conditions and their blood parameters might differ from other horse breeds. The study was carried out on 100 mares and 12 stallions of ages from 2 to 19 years. Fifteen hematological and 19 biochemical parameters were analysed. Values of 22 parameters showed considerable overlapping with values obtained for other horse breeds, and substantial resemblance is evident with values reported for Pakistani working horses. Several reference values showed statistically significant effect of sex (Hb, ALB, CREA, TBIL and Ca where significantly higher in stallions; EOS, CK, ALP, UREA and Mg were significantly higher in mares). None of the parameters studied showed any differences associated with age. The adaptation of Posavina and Croatian Coldblood horses to the harsh environment of flooded pastures and the way of breeding might be reflected in their specific reference values. The obtained results enabled the establishment of hematological and biochemical reference values in the blood of Posavina and Croatian Coldblood horses that will in turn support clinical diagnosis and further research into horse physiology. Abstract U studiji je obavljeno ispitivanje hematoloških i biohemijskih parametara krvi kod radnih hrvatskih autohtonih rasa konja. Posavski i hrvatski hladnokrvnjak su dve rase konja koje su adaptirane na oštre uslove spoljašnje sredine pa se pretpostavlja da njihovi parametri krvi mogu da budu različiti u poređenju sa drugim rasama. Ispitivanje je obavljeno na 100 kobila i 12 pastuva starosti od 2 do 19 godina. Obavljena je analiza u odnosu na pedeset hematoloških i 19 biohemijskih parametara. Vrednosti koje su dobijene u odnosu na 22 parametra pokazale su značajna preklapanja u poređenju sa drugim rasama konja, pri čemu je ustanovljena značajna podudarnost sa vrednostima koje su karakteristične za pakistanskog radnog konja. Nekoliko referentnih vrednosti je pokazalo statistički značajnan utacaj pola (Hb, ALB, CREA, TBIL i Ca vrednosti su bile značajno veće kod pastuva; EOS, CK, ALP, UREA i Mg vrednosti su bile značajno veće kod kobila). Nijedan parametar koji je ispitivan nije pokazao bilo kakvu varijaciju u odnosu na starost životinja. Prilagođenost posavskog i hrvatskog hladnokrvnog konja na oštre i teške uslove spoljašnje sredine koji podrazumevaju ispašu na poplavljenim pašnjacima, kao i način uzgoja, može da ima posledice na specifične referentne vrednosti ispitivanih parametara. Dobijeni podaci omogućavaju definisanje hematoloških i biohemijskih referentnih vrednosti krvi posavske i hrvatske rase hladnokrvnih konja, a te vrednosti bi omogućavale kliničku dijagnostiku i buduća ispitivanja fiziologije konja.

Evolution of MHC class II SLA-DRB1 locus in the Croatian wild boar (Sus scrofa) implies duplication and weak signals of positive selection

The wild boar is an ancestor of the domestic pig and an important game species with the widest geographical range of all ungulates. Although a large amount of data are available on major histocompatibility complex (MHC) variability in domestic pigs, only a few studies have been performed on wild boars. Due to their crucial role in appropriate immune responses and extreme polymorphism, MHC genes represent some of the best candidates for studying the processes of adaptive evolution. Here, we present the results on the variability and evolution of the entire MHC class II SLA-DRB1 locus exon 2 in 133 wild boars from Croatia. Using direct sequencing and cloning methods, we identified 20 SLA-DRB1 alleles, including eight new variants, with notable divergence. In some individuals, we documented functional locus duplication, and SLA-DRB1*04:10 was identified as the allele involved in the duplication. The expression of a duplicated locus was confirmed by cloning and sequencing cDNA-derived amplicons. Based on individual genotypes, we were able to assume that alleles SLA-DRB1*04:10 and SLA-DRB1*06:07 are linked as an allelic combination that co-evolves as a two-locus haplotype. Our investigation of evolutionary processes at the SLA-DRB1 locus confirmed the role of intralocus recombination in generating allelic variability, whereas tests of positive selection based on the dN/dS (non-synonymous/synonymous substitution rate ratio) test revealed atypically weak and ambiguous signals.