Genomic characterization of the Croatian autochthonous sheep breeds

Svakim danom izumire sve veći broj životinja i nepovratno se gubi genetska raznolikost. Selekcijom na određena svojstva (povećanje proizvodnje, kvaliteta vune) životinje gube određenu razinu varijabilnosti. Izvorne pasmine su većinom nisko proizvodne životinje koje su prilagođene specifičnim uvjetima okoliša, te kao takve u pravilu imaju veću genetsku raznolikost. Hrvatske izvorne pasmine ovaca su otporne i izdržljive pasmine, s niskim proizvodnim svojstvima, ali prilagođene na oštre uvjete držanja. Sve izvorne pasmine ovaca spadaju u pramenka tip ili su nastale na osnovi pramenka tipa. U Hrvatskoj je priznato devet izvornih pasmina ovaca, a ovim doktorskim radom je obuhvaćeno njih osam: istarska ovca, creska ovca, krčka ovca, paška ovca, rapska ovca, dalmatinska pramenka, lička pramenka, i dubrovačka ovca. Osim hrvatskih izvornih pasmina ovaca u rad je uključeno 10 muflona iz Kalifronta te 21 javno dostupna europska pasmina s područja Mediterana ili istočne Europe. Ciljevi ovog doktorskog rada bili su utvrditi konzervacijski status hrvatskih izvornih pasmina ovaca, odrediti jedinstvenost genoma hrvatskih izvornih pasmina ovaca i pozicionirati hrvatske izvorne pasmine ovaca u mediteranski kontekst na temelju genotipskih podataka dobivenih s Infinium Ovine HD® SNP čipom koji sadržava 606 000 polimorfnih mjesta. Sve jedinke hrvatskih izvornih pasmina ovaca uspješno su genotipizirane visokorezolutnim SNP čipom te su pokazale visoku genetsku raznolikost. Analizom unutar i između populacijskih odnosa hrvatskih izvornih pasmina utvrđeno je da se pasmine dijele na otočke i kontinentalne obzirom na geografske barijere (more i planine). U otočke pasmine svrstale su se istarska ovca, krčka ovca, creska ovca, rapska ovca i paška ovca, dok su se u kontinentalne pasmine grupirale lička pramenka i dalmatinska pramenka te dubrovačka ovca. Ovakvu podjelu populacija pokazale su analize glavnih komponenti, Structure analiza, analiza Nei genetskih udaljenosti prikazana Neighbour Net mrežom kao i filogenetsko stablo dobiveno SplitsTree softverom. Structure analiza je dodatno pokazala veliku sličnost paške i rapske ovca kao i lička i dalmatinske pramenke. Hrvatske izvorne pasmine pokazale su nisku do srednju razinu genomskog inbridnga (FROH>2Mb = 0,025 - 0,056), jedino je kod dubrovačke ovce utvrđena nešto viša razina inbridinga od 0,070. Kod dalmatinske i ličke pramenke te paške ovce utvrđena je visoka efektivna veličina populacije, dok je najmanja efektivna veličina populacije utvrđena kod krčke ovce. Gledano u širem mediteranskom kontekstu hrvatske izvorne pasmine ovaca grupirale su se zajedno s ostalim ovcama pramenka tipa i smjestile između istočno i zapadnoeuropskih ovaca. Uspoređujući hrvatske izvorne ovce s europskim pasminama utvrđeno je osam genomskih regija specifičnih za hrvatske izvorne pasmine.Every day we witness the reduction of genetic variability of plants and animals. The extinction of animals is a process that is constantly taking place, and the loss of genetic material is inevitable. As we intensify selection for higher production, we reduce the genetic variability of these species. Autochthonous animals are usually lower production animals raised in extensive systems and are better adapted to environmental conditions and therefore have higher genetic variability. Sheep breeding has historically been an important part of agricultural production on Croatian territory. It is believed that sheep came to Croatia with human migrations in the Neolithic period and are an integral part of Croatian culture since. In the 18th century, Dalmatia had the highest number of sheep in Europe. Since then, the number of sheep has steadily decreased and some of the Croatian sheep breeds are threatened with extinction. The Croatian autochthonous sheep breeds are low production animals adapted to harsh environmental conditions and extensive breeding systems. They all belong to the Pramneka sheep type or are descended from the Pramenka sheep type. There are nine recognised autochthonous sheep breeds in Croatia and eight of them are discussed in this doctoral thesis: Istrian sheep, Cres Island sheep, Krk Island sheep, Pag Island sheep, Rab Island sheep, Dalmatian Pramenka, Lika Pramenka and Dubrovnik sheep -ruda. Another 21 European sheep breeds from the Data repository were included in this analysis as well as ten mouflon samples from Kalifront. This PhD aimed to estimate the conservation status of Croatian autochthonous sheep breeds, determine the unique parts of the genome and position Croatian autochthonous sheep breeds in the Mediterranean context. For this purpose, the high-density Infinium Ovine HD SNP chip was used, which contains 606 000 polymorphic positions. All Croatian autochthonous sheep breeds were successfully genotyped. Croatian autochthonous sheep breeds showed high genetic diversity. Based on the analysis within and between populations, it is clear that Croatian autochthonous sheep breeds are divided into two groups based on their geographical characteristics: island breeds and continental breeds. Island breeds include Istrian sheep, Krk Island sheep, Cres Island sheep, Rab Island sheep and Pag Island sheep, while continental breeds include Lika Pramenka, Dalmatian Pramenka and Dubrovnik sheep. This clustering was confirmed with PCA, Structure analysis, Nei distances represented with Neighbour Net network and phylogenetic analysis performed with SplitsTree. Furthermore, the Structure analysis showed that not all breeds are completely separated. The Pag Island sheep and the Rab Island sheep, as well as the Lika Pramenka and the Dalmatian Pramenka, showed similar ancestral breed composition, indicating greater genetic relatedness. The Croatian autochthonous sheep breeds showed a lower degree of genomic inbreeding obtained from runs of homozygosity. The highest inbreeding was observed in Dubrovnik sheep with FROH > 2Mb = 0.07. Higher recent inbreeding (up to 5 generations ago) was observed in all breeds. The lowest effective population size was observed in sheep from the island of Krk (61), but lower Ne was also observed in sheep from the island of Cres (148), Dubrovnik sheep (157) and Istrian sheep (161). The highest effective population size was observed in Dalmatian Pramenka sheep (1039). The Croatian autochthonous sheep breeds observed in the Mediterranean context grouped with other European Pramenka sheep types. In the PCA diagram, they were placed in the middle of the diagram, creating a link between Eastern European and Western European sheep breeds. The Croatian autochthonous sheep breeds had eight specific genomic regions compared to the European breeds. Since some of the Croatian autochthonous sheep breeds have lower effective population size and higher inbreeding, additional caution should be taken when implementing breeding programmes