Comparison of mtDNA control region among descendant breeds of the extinct Zaupel sheep revealed haplogroup C and D in Central Europe

Abstract The consideration of the descendance is indispensable in the preservation of endangered animal breeds. The authors compared mitochondrial DNA (mtDNA) control region sequence in three descendant breeds of the extinct Zaupel sheep, firstly. Their investigation was carried out in order to prove the common origin of Waldschaf (Austria), Bovec sheep (Slovenia) and Cikta (Hungary). A total of 118 biological samples were taken from non‐related representatives of the three breeds between 2015 and 2017. A newly designed primer pair was also used to amplify the segment (1180 bp) to be tested. The total number of haplotypes in the whole study population was 49. The majority of which fell into haplogroup B. The significant negative value of the Fu's Fs statistic (Fs statistic = −3.296, p = 0.013) based on haplotype frequencies demonstrated a moderate foreign gene flow. As a novel observation haplogroups C and D appeared in Cikta and Bovec sheep, respectively. The Tajima D‐test value in the entire study population was −0.914 (p > 0.10), meaning that the separation of the three descendant breeds did not cause genetic drift, these are collectively in genetic equilibrium. The genetic information confirmed the common origin of the breeds known from the breed history.

arrival of larger, better muscled, yet finer-wooled foreign sheep, the fate of the Zaupel sheep was sealed from the middle of the 15th century (Adlung, 1912). The breed was permanently lost by the middle of the 20th century.
Of the extinct but historically important Zaupel sheep, four independent, still existing gene reserve breeds originated in Central Europe (Seibold, 1990). Today, as transboundary breeds, there are the Bavarian Waldschaf in Germany (its versions are Waldschaf in Austria and the Sumavska in the Czech Republic), the Alpine Steinschaf in Austria (versions of this are Steinschaf in Bavaria, and Bovec sheep or Bovska in Slovenia) and Mountain Sheep (Bergschaf; also with brown colour variations living as Engadiner Fuchsschaf in Switzerland, and as Braunes Bergschaf in Germany and Austria). The fourth Zaupel sheep descendant who came to Hungary with Swabian immigrants of the 18th century is the Cikta (Koppány, 2000).
It is likely that each descendant breed of Zaupel sheep would also have become extinct decades ago if the interest in conserving native farm animals had not been strengthened.
Mitochondrial DNA (mtDNA) is an extra nuclear, non-recombining material of inheritance in the mitochondria. This circular genome is mainly used in evolutionary research, in the phylogenetic study of mammalian species and populations by maternal origin (e.g., Tapio et al., 2006), but also in forensic genetics (e.g., Zenke et al., 2017). mtDNA, which, like in humans, is about 16,500 base pairs in sheep, is also used to study the history of domestication, and in this respect we can get an idea of the relative history of the breeds. According to the sheep mtDNA control region (CR), there are several major lineages (haplogroups A, B, C, D, E, F and G; later two were extinct), of which group B is found mainly in European breeds (Dymova et al., 2017).
Research conducted in the neighbouring countries of the Balkan Peninsula has dealt with the classification of domestic sheep breeds into haplogroups. For example, Cinkulov et al. (2008) found that in the West Balkan Pramenka sheep, the B haplogroup is predominant, but in addition, haplogroup A occurred in traces. The investigation of Ferencakovic et al. (2013)  Within Europe haplogroup C has been found, so far, only on the Iberian Peninsula (in Portugal: Pereira et al., 2006 andin Spain: Pedrosa et al., 2007) and in the southern countries of the Balkan Peninsula (in Albania and Greece: Pariset et al., 2011). The haplotype C, also identified in Egyptian breeds (Othman et al., 2015) is also in agreement with the assumption of early spread in sheep. The Iberian Peninsula was a springboard for the appearance of Asia Minor sheep on the European stage since it was connected directly to the North Africa and Asia Minor through the Mediterranean Sea.
The European spread of sheep could have occurred on a number of highly probable routes, partly along the Eastern European plain and along the Danube Valley, through the Carpathian Basin (Schmölcke et al., 2018).
Haplogroups D and E are the least frequent and have only been identified in samples from Turkey and the Caucasus (Meadows et al., 2007;Tapio et al., 2006). In the study of Liu et al. (2016)

on 15 indigenous
Tibetan sheep populations, the frequency of the rarest haplogroup D was less than 0.2%. Fossils in Anatolia showed a 3% presence of haplogroup E in the Bronze Age (Demirci et al., 2013). Dymova et al. (2017) carried out archaeological mitochondrial DNA CR analysis based on about 4000-1000 years old sheep bone remains in Altai and found all the five recent haplogroups including lineages D and E. That richness of diversity led them to conclude that the Altai region had been a migratory area for many sheep and peoples in the past.
Haplogroup E was detected in Iran in 10 Iranian native breeds (Rafia & Tarang, 2016). Kirikci et al. (2018) stated the Karayaka breed from Northern Anatolia cannot be categorized as a genetically homogeneous population, but even has four different haplogroups (A, B, C and E).
Our hypothesis was that, with modern genetic knowledge, we would be able to show common roots and connections in breeds considered to be of common origin. If this is true, then statistical processing of the data does not justify a significant genetic difference between descendant breeds of Zaupel. In this sense, we surveyed the diversity of maternal genetic background in the following three breeds: Waldschaf, Bovec sheep and Cikta based on the sequence order of the mutable CR of the mitochondrion. With our results we wanted to prove genetically the common origin of the related varieties known from their history, and on the other hand to create a basis for the further maintenance of the genetic characteristics of the representatives in the Zaupel breed group.

MATERIALS AND METHODS
To avoid the random sampling ancestries provided information. Biological samples were taken from non-closely related individuals in Waldschaf (Austria, n = 27) and Bovec sheep (Slovenia, n = 21) breeds using three-generation pedigrees. In Bovec sheep, blood samples were collected from potential breeding rams included in the regular prion genotyping program, determined by order of the Ministry of Agriculture, Forestry and Food of the Republic of Slovenia. All rams were offspring of different ewes. In Cikta sheep (Hungary, n = 70), the so-called 'founder sampling' was used (Maróti-Agóts et al., 2008). In this case, sample collection was preceded by processing of the entire pedigree (Posta et al., 2019). In this breed, we collected samples from the liv- DNA was isolated using the GenElute Blood Genomic DNA Kit (Sigma-Aldrich) according to the manufacturer's instructions. The 25 μL PCR reaction mixture prepared for each sample contained 2.5 μL dNTP (10 mM), 2.5 μL 10× PCR buffer, 1.5 μL MgCl 2 (25 mM), 2 μL primer (10μM), 1 μL BSA (20 mg/mL), 0.4 μL Taq polymerase (5 U/μL) (ThermoFisher Scientific) and 10 ng DNA template and PCR grade water to volume. Two primer pairs were used to amplify the segment to be tested: newly designed primers (Primer Designer 4.0 software) for the beginning of the region, and with a second pair as described by Hiendleder et al. (1998). First, the following amplification protocol was used for the newly designed primers (MtOA_F15400 5′-ACACCCAAAGCTGAAGTTCTAC-3′ and Mutations were evaluated using the test proposed by Fu and Li (1993) first, and then, we used the Tajima D-test, developed by Tajima (1989) as a method for population genetic evaluation, to analyse the detected sequence mutations.
Using DNAsp version 6.0 software (Rozas et al., 2017), the number of polymorphic sites in the entire assay sample was determined and the mean nucleotide difference within and between groups was calculated.
The resulting sequences were arranged with MEGAX (Kumar et al., 2018), aligned, and the related dendrogram was drawn.
The corrected number of base substitutions within the sequences was determined by the method of Jukes and Cantor (1969) and Jukes (1990).

RESULTS
In the CR region of the whole study sample, the number of monomorphic base sites was 1048, while that of the polymorphic base sites The total number of haplotypes in the whole study population was 49.
The haplogroup and haplotype distribution of the studied breeds is shown in Figure 1. The number of haplogroups identified was four. The most populous of the haplogroups was B, followed by A, with 35, and 10 haplotypes, respectively. The dendrogram illustrates convincing the separation of haplogroups D and C from these, which include one and three haplotypes represented by one Bovec sheep and six Cikta individuals, respectively.
The comparison of related breeds is reported in Table 2   The ratio of shared mutations between breeds occurred in inverse proportion to the values of k and π.
According to the Tajima test performed in the entire study population, the average number of pairwise nucleotide differences (k) was 20.880 and the average nucleotide diversity (π) was 17.71 * 10 −3 . The Tajima D-test value was −0.914, not statistically significant The network of the connections between the found CR haplotypes and the reference CR haplogroups calculated by the median-joining method clearly shows that the haplotypes are mainly located around the reference haplogroup B (Figure 2).

DISCUSSION
In the Tsigai, another Hungarian native sheep breed (81 individuals), 98 variable base sites were found in the CR region by Annus et al. (2015), of which 47 singleton and 51 parsimony mutations were detected.
From these, it can be deduced that several related breeds show an overall more diverse genetic composition than a single breed, for example, the Tsigai which is also gene reserve breed.
The average nucleotide diversity of both breeds Gyimesi Racka and Turcana was lower (both 5 * 10 −3 ) than that of all breeds currently studied, and the Tajima Wassmuth et al. (2002) found that all individuals of Steinschaf Among 11 Austrian sheep breeds, the shortest genetic distance was proven by Baumung et al. (2006) between Alpines Steinschaf and Waldschaf based on microsatellites, nuclear information.
The Copper Age sheep, which, according to Olivieri et al. (2012), also belongs to haplogroup B, is the possible link between the peat sheep and its distant ancestor, a certain Asiatic sub-species of Ovis ammon.
The common genetic origin confirming the breed history may be the reason for the similarity of the three studied breeds.
The complete identity between the breeds is supported by the value of the Tajima  The primordial haplogroup C was first separated during phylogenetic development from B and A. The peculiarity of the processing result is that we detected the haplogroup C in the Cikta breed (with six individuals). A 9% incidence of haplogroup C indicates an even more complex maternal background of that breed. This means that ewes have entered Hungary in the past from the closer and farther areas, either from the Asia Minor (Meadows et al., 2007), Caucasus (Tapio et al., 2006), Georgia (Kunelauri et al., 2019) and Caspian See region (Lv et al., 2015) or from Mongolia (Ganbold et al., 2019) and China (Chen et al., 2006;Guo et al., 2005) where, to the best of our knowl- In addition, our work draws attention to the importance of maintaining families and within family selection. The increased emphasis on the maternal side is also justified by the fact that females are present in a higher proportion than males and remain in breeding for a longer period of time, thus, they can be a greater custodian of the implementation and maintenance of genetic diversity.

CONCLUSIONS
Our working hypothesis, which was based on the breed histories, was In addition to the haplogroups B and A typical of Europe, we were also able to detect the presence of haplogroups C and D in Central Europe.  and sequencing with, and Ákos Maróti-Agóts had substantial role in statistical data analysis and created a first draft of the article.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are openly available under MW428078 in GenBank at https://www.ncbi.nlm.nih.gov.