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A recent comprehensive genomic analysis based on 50K SNP profiles has shown that the regional Balkan sheep populations have considerable genetic overlap but are distinctly different from surrounding breeds. All eight Croatian sheep breeds were represented by a small number of individuals per breed. Here, we genotyped 220 individuals representing the native Croatian sheep breeds (Istrian Sheep, Krk Island Sheep, Cres Island Sheep, Rab Island Sheep, Lika Pramenka, Pag Island Sheep, Dalmatian Pramenka, Dubrovnik Sheep) and mouflon using the Ovine Infinium® HD SNP BeadChip (606,006 SNPs). In addition, we included publicly available Balkan Pramenka and other Mediterranean sheep breeds. Our analyses revealed the complex population structure of Croatian sheep breeds and their origin and geographic barriers (island versus mainland). Migration patterns confirmed the historical establishment of breeds and the pathways of gene flow. Inbreeding coefficients (FROH>2 Mb) between sheep populations ranged from 0.025 to 0.070, with lower inbreeding coefficients observed in Dalmatian Pramenka and Pag Island Sheep and higher inbreeding in Dubrovnik sheep. The estimated effective population size ranged from 61 to 1039 for Krk Island Sheep and Dalmatian Pramenka, respectively. Higher inbreeding levels and lower effective population size indicate the need for improved conservation management to maintain genetic diversity in some breeds. Our results will contribute to breeding and conservation strategies of native Croatian sheep breeds.

Sheep are one of the most important livestock species in Croatia, found mainly in the Mediterranean coastal and mountainous regions along the East Adriatic coast, well adapted to the environment and mostly kept extensively. Our main objective was therefore to map the positive selection of the X-chromosome (18,983 SNPs that passed quality control), since nothing is known about the adaptation genes on this chromosome for any of the breeds from the Balkan cluster. Analyses were performed on a sample of eight native Croatian breeds (101 females and 100 males) representing the East Adriatic metapopulation and on 10 mouflons (five females and males), all sampled in Croatia. Three classical within-population approaches (extreme Runs of Homozygosity islands, integrated Haplotype Score, and number of Segregating Sites by Length) were applied along with our new approach called Haplotype Richness Drop (HRiD), which uses only the information contained in male haplotypes. We have also shown that phylogenetic analyses, such as the Median-joining network, can provide additional information when performed with the selection signals identified by HRiD. Our new approach identifies positive selection signals by searching for genomic regions that exhibit a sudden decline in haplotype richness. In total, we identified 14 positive selection signals, 11 using the classical approach and three using the HRiD approach, all together containing 34 annotated genes. The most reliable selection signal was mapped by all four approaches in the same region, overlapping between 13.17 and 13.60 Mb, and assigned to the CA5B, ZRSR2, AP1S2, and GRPR genes. High repeatability (86%) of results was observed, as 12 identified selection signals were also confirmed in other studies with sheep. HRiD offers an interesting possibility to be used complementary to other approaches or when only males are genotyped, which is often the case in genomic breeding value estimations. These results highlight the importance of the X-chromosome in the adaptive architecture of domestic ruminants, while our novel HRiD approach opens new possibilities for research.

The majority of the nearly 400 existing local pig breeds are adapted to specific environments and human needs. The demand for large production quantities and the industrialized pig production have caused a rapid decline of many local pig breeds in recent decades. Black Slavonian pig and Turopolje pig, the latter highly threatened, are the two Croatian local indigenous breeds typically grown in extensive or semi-intensive systems. In order to guide a long-term breeding program to prevent the disappearance of these breeds, we analyzed their genetic diversity, inbreeding level and relationship with other local breeds across the world, as well as modern breeds and several wild populations, using high throughput genomic data obtained using the Illumina Infinium PorcineSNP60 v2 BeadChip. Multidimensional scaling analysis positioned Black Slavonian pigs close to the UK/North American breeds, while the Turopolje pig clustered within the Mediterranean breeds. Turopolje pig showed a very high inbreeding level (FROH>4Mb = 0.400 and FROH>8Mb = 0.332) that considerably exceeded the level of full-sib mating, while Black Slavonian pig showed much lower inbreeding (FROH>4Mb = 0.098 and FROH>8Mb = 0.074), indicating a planned mating strategy. In Croatian local breeds we identified several genome regions showing adaptive selection signals that were not present in commercial breeds. The results obtained in this study reflect the current genetic status and breeding management of the two Croatian indigenous local breeds. Given the small populations of both breeds, a controlled management activity has been implemented in Black Slavonian pigs since their commercial value has been recognized. In contrast, the extremely high inbreeding level observed in Turopolje pig argues for an urgent conservation plan with a long-term, diversity-oriented breeding program.