A study of maternal variability of Russian local sheep breeds based on analysis of cytochrome B gene polymorphism

Analysis of mitochondrial DNA (mtDNA) polymorphism is one of the most effective modern approaches to assess the genetic diversity of livestock species. The mtDNA sequencing is the most efficient approach for identifying mtDNA haplogroups in sheep ( Ovis aries ). Although this approach is widely used abroad, a systematic and comprehensive study of Russian sheep breeds with its aid has not yet been conducted. In this work, we analyzed the polymorphism of the complete sequence of the cytochrome b ( CytB ) gene in Russian sheep breeds of various origins. For the first time, we established the belonging of sheep from 25 Russian breeds to haplogroups and showed haplotype relationships between coarse wool, fine wool and semi-fine wool sheep breeds based on the analysis of polymorphism of the mitochondrial cytochrome b gene. The maternal variability of a wide range of local sheep breeds in comparison with transboundary breeds was assessed. In this research, we aimed to evaluate genetic diversity and to determine the haplotype variability and haplogroup belonging of Russian local sheep breeds based on the CytB gene sequences. The study was performed on 106 samples from 25 Russian sheep breeds in 2020-2021. Tissue samples (ear notches) were retrieved from the biological collection “Bank of genetic material of domestic and wild animal species and poultry” (registered by the Ministry of Education and Science of the Russian Federation No. 498808), which is established and maintained at the Ernst Federal Research Center for Animal Husbandry. The final study sample included nine fine-wool breeds, including Baikal ( n = 3), Dagestan Mountain ( n = 4), Groznensk ( n = 5), Kulundin ( n = 5), Manych Merino ( n = 5), Salsky ( n = 5), Soviet Merino ( n = 3), Stavropol ( n = 5) and Volgograd ( n = 5); five semi-fine wool breeds, including Altai mountain( n = 5), Kuibyshev ( n = 1), North Caucasian meat-wool ( n = 5), Russian long-haired ( n = 3) and Tsigai ( n = 2); eleven coarse-wool breeds, including Romanov ( n = 3), Andean black ( n = 5), Buubei ( n = 5), Karakul ( n = 3), Karachaev ( n = 5), Kuchugur (n = 3), Lezgin (n = 5), Tushin ( n = 5), Tuva short-fat-tailed ( n = 4), Edilbai ( n = 5) and Kalmyk ( n = 5). The complete sequences of the CytB gene of the studied sheep breeds were determined using the next generation sequencing (NGS) technology. To achieve this goal, three overlapping mtDNA fragments (overlapping region of more than 290 bp) with lengths of 6500, 5700, and 6700 bp were amplified. The obtained polymerase chain reaction (PCR) products were used to prepare libraries, which were then sequenced by the method of paired terminal reads of 300 bp each with a MiSeq System Sequencer (Illumina, Inc., USA). The CytB gene sequence was recovered from the complete mtDNA sequence after alignment, which was performed using the MUSCLE algorithm in the MEGA 7.0.26 software. All studied breeds had high haplotype (HD = 0.400-1,000) and nucleotide diversity (p = 0.00058-0.00760). In total, we identified 82 haplotypes. Tuva short-fat-tailed sheep breed was represented by only one haplotype. The AMOVA results showed that genetic diversity was mainly determined by intrabreed differences (90.55 %). Four haplogroups including A, B, C and D were identified in the study sample. Such a haplogroup diversity might be explained by a wide geographical range of habitats of the studied animals. The most frequent haplogroups in Russian local sheep breeds were B ( n = 64) and A ( n = 34), which are typical for sheep of European and Asian origin respectively. Seven animals were assigned to haplogroup C, and haplogroup D was represented by one animal. The results contribute to a deeper understanding of the processes of migration and settlement of domestic sheep in Eurasia.