Analysis of Mitogenome Variability in Indigenous Sheep Breeds of the Republic of Croatia

Ovčarstvo je predstavlja važan tip ekstenzivnog stočarstva u Hrvatskoj. Proučavanje genetske varijabilnosti ovaca postaje sve važnije, posebno uzimajući u obzir male efektivne veličine populacija naših autohtonih pasmine ovaca, koje mogu rezultirati smanjenom genetskom raznolikošću, što može povećati rizik od negativnih genetskih anomalija i smanjiti otpornost stada na bolesti ili druge ekološke stresore. U ovom radu prvi put je analizirana varijabilnost rodovo na molekularnoj razini proučavanjem cijelog mitogenoma 23 jedinke koje predstavljaju osam autohtonih pasmina ovaca (creska ovca, dalmatinska pramenka, dubrovačka ruda, istarska ovca, krčka ovca, lička ovca, paška ovca i rapska ovca ) i dva muflona iz Hrvatske. Pomoću bioinformatičkih alata iz cijelog genoma ovaca i muflona 12x pokrivenosti izdvojeni su mitogenomi s prosječnom dubinom pokrivenosti 1626, a genoma 30x pokrivenosti izdvojeni su mitogenomi s prosječnom dubinom pokrivenosti 1851. Fasta set od 25 mitogenoma duljine 16616 bp sastojao se od 247 singleton i 112 parsim informativnih mjesta čineći 24 haplotipa s raznolikošću haplotipova 0.997 pružajući korisne informacije o velikoj genetskoj raznolikosti i između naših pasmina ovaca i muflona. U svrhu podrobnije filogenetske i filogeografske analize mitogenoma, navedenih 25 mitogenoma analizirano je s dodatnih 348 mitogenoma različitih svjetskih pasmina ovaca iz GenBank repozitorija. Median joining mreža pokazala je da svi hapotipovi naših autohtonih pasmine ovaca i muflona iz Hrvatske pripadaju haplogrupi B te zanimljivo jedan haplotip krčke ovce A i jedan haplotip dubrovačke rude C haplogrupi. Beast filogenetska analiza temeljena na Bayesian metodi također je potvrdila pripadnost navednih uzoraka A, B i C haplogrupi uz posteriori vjerojatnost 1 grananja navedenih haplogrupa. Filogeografska distribucija haplogrupa potvrdila je visoku učestalost haplogrupe A u pasminama iz srednje i istočne Azije, te visoku učestalost haplogrupe B u Europi i Anatoliji. Rijetke haplogrupe C pojavljuju se na području Pirenejskog poluotoka, Bliskog istoka, regije Kaspijskog mora, Kine, Indije i Nepala, dok se haplogrupe D i E mogu pronaći na Bliskom istoku. Haplotipovi naših muflona svrstani su u haplogrupu B zajedno s ostalim europskim muflonima. Prisutnost dva haplotipa pripisana haplogrupama A i C sugerira migracijske kontakte između Balkana i Bliskog istoka koje je potrebno dodatno istražiti.Sheep farming represents an important type of extensive livestock farming in Croatia. Studying the genetic variability of sheep is becoming increasingly crucial, especially considering the small effective population sizes of our indigenous sheep breeds. These small populations can result in reduced genetic diversity, which may increase the risk of negative genetic anomalies and reduce the herd's resistance to diseases or other environmental stressors. This study, for the first time, analyzed gender variability at the molecular level by examining the entire mitogenome of 23 individuals representing eight indigenous sheep breeds (Cres Island sheep, Dalmatian Pramenka sheep, Dubrovnik Ruda sheep, Istrian sheep, Krk sheep, Lika sheep, Pag sheep, and Rab sheep) and two mouflons from Croatia. Using bioinformatics tools, mitogenomes with an average coverage depth of 1626 were extracted from the whole genomes of sheep and mouflons at 12x coverage, and mitogenomes with an average coverage depth of 1851 were extracted from genomes at 30x coverage. The fasta set of 25 mitogenomes, with a length of 16616 bp, consisted of 247 singletons and 112 parsimony-informative sites, comprising 24 haplotypes with a haplotype diversity of 0.997. This provided valuable information about high genetic diversity both within and between our sheep breeds and mouflons. For a more detailed phylogenetic and phylogeographic analysis of the mitogenomes, these 25 mitogenomes were analyzed alongside an additional 348 mitogenomes from various sheep breeds worldwide from the GenBank repository. The median-joining network revealed that all haplotypes of our indigenous sheep breeds and Croatian mouflons belong to haplogroup B. Interestingly, one Krk sheep haplotype belonged to haplogroup A, and one Dubrovnik Ruda sheep haplotype belonged to haplogroup C. Bayesian-based Beast phylogenetic analysis also confirmed the membership of these haplotypes in haplogroups A, B, and C, with posterior probabilities of 1 for the branching of these haplogroups. The phylogeographic distribution of haplogroups confirmed a high frequency of haplogroup A in breeds from Central and Eastern Asia, and a high frequency of haplogroup B in Europe and Anatolia. Rare haplogroups C were found in the Pyrenees, the Middle East, the Caspian Sea region, China, India, and Nepal, while haplogroups D and E were present in the Middle East. The haplotypes of our mouflons were grouped into haplogroup B, along with other European mouflons. The presence of two haplotypes attributed to haplogroups A and C suggests migratory contacts between the Balkans and the Middle East, which require further investigation