Mitochondrial DNA sequencing illuminates genetic diversity and origin of Hunagrian Nonius horse breed and his relatives – Danubian horse and Serbian Nonius

Georgi Yordanov; Nadezhda Palova; Ivan Mehandjyiski; Peter Hristov

doi:10.1080/10495398.2023.2237533

. 2023 Jul 25;34(8):3897–3907. doi: 10.1080/10495398.2023.2237533

Mitochondrial DNA sequencing illuminates genetic diversity and origin of Hunagrian Nonius horse breed and his relatives – Danubian horse and Serbian Nonius

Georgi Yordanov ^a, Nadezhda Palova ^b, Ivan Mehandjyiski ^c, Peter Hristov ^d,^✉

PMCID: PMC13353399 PMID: 37489100

Abstract

From a historical perspective, horse breeding in Bulgaria has been very well developed since the time of the Thracians (early Bronze Age c. 3000 BCE). Archaeological discoveries from this era present us with an extremely rich type diversity, including wild and local primitive horses, the prototype of heavy draft horses, and fine riding horses.

The objective of this study was to investigate the genetic structure of unexamined populations of three closely related horse breeds – the Danubian Nonius Hungarian Nonius and Serbian Nonius horses. A 608 bp long fragment of the mtDNA D-loop region was amplified and sequenced. The obtained results showed completely different genetic profiles between the investigated breeds. We identified nine of the 17 haplogroups described in modern horses. Most of the obtained sequences fell into M, L, G, and O’P lineages, which reflects the genetic profiles of the ancestral mares that were probably used at the initial stages of formation of the breeds. The population of the Danubian horse was characterized by a high prevalence of Central Asian specific haplogroup G (45%), followed by Western Eurasian specific haplogroups L and M (both about 21%). In contrast to the Danubian horse, in the Nonius breed the highest frequency of Western Eurasian haplogroup M (43.5%) was found, followed by Middle Eastern haplogroups O’P (26.1%) Central Asian specific E (13.0%) and G (13.1%). The Serbian Nonius horse showed a completely different genetic profile with a prevalence of the rare for Europe haplogroup D (66.7%), followed by Central Asian specific G (16.7%). The high mitochondrial haplotype diversity (Hd = 0.886) found in the investigated samples is evidence for multiple maternal origins in all populations.

In conclusion, the obtained results demonstrated a high percentage of haplogroup sharing especially in the Danubian and Hungarian Nonius horse breeds, which reflects the possible common origins of the two breeds. In contrast to these breeds, the Serbian Nonius, despite the small number of investigated animals, showed a specific genetic profile, which could be explained by different and independent origins.

Keywords: D-loop region, Nonius horse, horse mitochondrial haplogroups, genetic diversity

Introduction

The horse has played a significant role in the history and development of human civilizations. The history of the use of horses can be traced in the rise and fall of empires, the conquest of entire continents, great battles, the development of transport systems, agriculture, and forest management.^1–3 Until the middle of the 19th century, draft breeds of horses were developed for agricultural and forestry works and as power for pulling carts.⁴ The mechanization of transport and agriculture increased the attention of many horse breeds on development for sport and leisure activities.^5–7 However, they are still used in farmed agricultural areas, especially in Eastern Europe, Asia, Africa, Central and South America.⁸^,⁹ Nowadays, the changing social and economic conditions have led to the use of many breeds of horses for competitive events, as sports horses and for equestrian tourism.^10–12

Historically, the development of horse breeding in Bulgaria has always been a priority for the country. After 1885, the newly formed Bulgarian army and agricultural enterprises needed the creation of various breeds of horses which led to the creation of stud farms for rearing and breeding them.¹³ As a predominantly agrarian country, in the early 20th century the question arose of creating more massive, strong and durable horses for the needs of agriculture. To this end, the Hungarian Nonius breed provided a favorable opportunity in breeding and improvement of native horse breeds.¹³ Nonius, or Noniusz, is one of the most famous Hungarian horse breeds. The main goal in the formation of this breed was to create a light draft and utility horse for Hungary’s military. The beginning of the creation of the breed was undertaken in 1816, when a stallion by the name of Nonius Senior (foundation sire), was captured by the Rosieres-aux-Salines, a stud farm in France, and brought to the Mezohegyes stud farm.¹⁴ After that, the founder stallion gave very good foals out of Spanish mares, the advantageous traits of which could be fixed by inbreeding, and its female descendants were crossed with Thoroughbred and Arabian stallions to correct the balance and elegance of the breed.¹⁵ Also in the creation of the breed, mares of Arabian, Lipizzan, Thoroughbred, Spanish and Norman origins were involved. Independent of the breeding programme at the Mezohegyes stud farm, in 1948 another stud – Mata stud farm (about 3 km north of Hortobágy village) located in Debrecen (Hungary) begun to form the breed in its own way.¹⁶ This kind was heavier than most of the Nonius of Mezohegyes and was recognized by the name of Hortobagy Nonius or Hortobagy Landrace.¹⁷ In 1961, the two subtypes of horses were united into one breed. At present, the Nonius horse population consists of about 450 mares and 80 stallions, with the largest number of animals in the Mezohegyes stud farm.¹⁴ Currently, the breed is mainly used for riding, competitive driving sports, equestrian tourism and less in agriculture.¹⁸

The Hungarian Nonius horse breed was the basis of the creation of the Danubian horse in Bulgaria, officially recognized as a breed in 1951.¹³ The Danube horse was created in the stud farm ‘Clementina‘ in the village of Pobeda (Pleven region). The first import was made by Hungary in 1893, when the following breeds were imported – Hungarian Nonius, Lipizzan, Trotters, Anglo-Arabian, Shagya Arabian, etc., as unsystematic crossbreeding had been carried out for 25 years. The basis of the herd nucleus were stallions and mares of the Hungarian Nonius breed, imported from Hungary (imported from Mezohegyes stud farm) and the former Republic of Yugoslavia in 1956.¹⁹ From the outset, purebred breeding had been carried out with the imported stallions and males of the Hungarian Nonius breed.¹³ At the same time as purebred breeding was taking place, crossbreeding with Hungarian Nonius stallions and partial reproductive crossbreeding, the so called ‘grading-up,’ i.e., sires of the Hungarian Nonius breed were continually backcrossed with females of the previous generation of local and improved mares.¹³ In the application of crossbreeding and in partially reproducible crossbreeding, mares of different origin were used – Anglo-Arabian, half-bred English and native mares.¹⁹ These three breeding methods, along with the strict selection have contributed to the creation of a peculiar kind of Bulgarian Nonius, which differs from Hungarian Nonius breeding in Hungary, Romania and the former Republic of Yugoslavia. According to the data published by National Association of Horse Breeding, in 2019 the number of horses controlled by the association registered in the Stud book and the Register of the breed was 250 in total.¹³ The small population size of the breed threatens it with decay due to inbreeding. This fact imposes the following prerequisites for the development of the breed: (1) The use of Thoroughbred stallions for the production of horses suitable for equestrian sports; (2) Lightening the type of the horses and production of horses for driving championship through the use of stallions of the Nonius breed.¹³

The creation of the Serbian Nonius was started at the Karadjordjevo stud farm in 1885, using mainly horses of the Nonius breed from the Mezohegyes stud farm in Hungary.²⁰ In this breeding programme mainly Thoroughbred horses were used. Depending on the size of the impact of the Thoroughbred horse, two types of Nonius were created: a lighter type (higher impact), with finer skeletons and lively temperament, and a heavier type (less influence), which was much larger and stronger, being intended primarily for agricultural work. Currently, the largest number of animals from the breed is concentrated in the territory of Vojvodina, which is mostly represented by the Karadjordjevo horse farm (50 horses), while the rest of the horses are owned by a relatively small number of individual breeders.²⁰ The breeding strategy is to preserve strong harness Nonius horses, with distinctive breed specific phenotype, correct movement for use in harness and in riding, which is suitable for both traditional agricultural work and for equestrian tourism, therapeutic riding, recreation etc.

Despite the widespread popularity of the Hungarian Nonius horse, so far there is no data on the genetic structure of the breed. There is also lack of information about the genetic profile of the breeds derived from the Hungarian Nonius horse, those being the, Danubian and the Serbian Nonius horse. This study was designed and implemented to investigate these matters.

Materials and methods

Animal welfare/ethical statement

All experimental procedures were reviewed and approved by the Animal Research Ethics Committee of the Bulgarian Food Safety Agency (BFSA), (Ar. 154 from of the Law on Veterinary Activity) in accordance with the European Union Directive 86/609.

Sample collection

Hair samples from mane and/or tail were collected from 93 animals belonging to three horse populations: 23 Hungarian Nonius horses from Mata stud farm (Debrecen, Hungary), 58 Danubian horses mainly from private tribal horse farms in Bulgaria stud and former Klementina stud farm in the village of Pobeda (Pleven region, Bulgaria) and 12 Serbian Nonius horses from Stud Farm Karadjordjevo (Karadjordjevo, Autonomous Province of Vojvodina). The samples were collected ensuring that each extracted hair contained a follicle and sufficient length for the shaft samples.

For all three breeds, Danubian horse, Hungarian Nonius and Serbian Nonius, analyzed in this study, genealogical data was recorded in Studbooks. Genealogical information was considered in order to select unrelated animals, while some samples from the Danubian horse were randomly selected from private tribal horse farms.

DNA extractions, PCR amplification and sequencing

Total DNA was extracted from the hair follicle and hair shaft samples using a GeneMATRIX Tissue DNA purification kit (Cat. no. E3550, EURx Ltd., Gdansk, Poland) as per the manufacturer’s instruction. In brief, 1–3 hair follicles or 3 pieces of hair shaft approximately 0.5 cm in length were cut and placed in an Eppendorf tube. Afterwards they were mixed with 350 μl of buffer Lyse T, 20 μl of 1 M DTT and 20 μl of Proteinase K and incubated overnight at 56 °C with shaking. The quality and quantity of the isolated DNA was checked by 1% agarose gel electrophoresis and then visualized under UV trans-illuminator gel documentation systems after staining with SimpliSafe™ (cat. no. E4600; EURx Ltd., Gdansk, Poland). The isolated DNA was stored at − 20 °C before analysis.

A fragment of 665 bp (from 15,434 to 16,098 bp, horse mtDNA reference sequence X79547)²¹ of the mtDNA D-loop region (hypervariable region I, HVR1) was amplified using the following primers: 15453 5′-CACCCAAAGCTGAAATTCTAC-3′ and R16078 5′-ATAACACCTTATGGTTGCTG-3′.²²

All PCR mixtures contained 25 µL of Color Taq PCR Master Mix (2× (Cat. No. E2525, EURx Ltd., Gdansk, Poland), 1 µM of each virus specific primer (FOR/REV), and 5 µL of template cDNA in a total volume of 50 µL. All PCR amplifications were carried out using a LifeExpress Classic Thermal Cycler (BIOER Technology Co., Ltd, Kampenhout, Belgium) under the following conditions: initial denaturation at 94 °C for 5 min; 35 cycles (denaturation at 94 °C for 30 s; primer annealing at 50 °C for 30 s; extension at 72 °C for 30 s) and final extension at 72 °C for 10 min. The PCR products were visualized on 1% agarose gel stained with SimplySafe™ (Cat. No. E4600, EURx Ltd., Gdansk, Poland). The fragment size was determined using MassRuler Low Range DNA Ladder (Cat. No. SM0383, Thermo Fisher Scientific Inc., Massachusetts, USA). The successfully amplified products were purified by a PCR purification kit (PCR/DNA Clean-Up Purification Kit, Cat. No. E3520, EURx Ltd., Gdansk, Poland) and sequenced in both directions by a PlateSeq kit (Eurofins Genomics Ebersberg, Germany).

Statistical analysis

All 93 obtained DNA sequences were manually edited and aligned with the MEGA7 program,²³ using the horse reference mtDNA sequence X79547.²¹ The obtained sequences (about 640 bp covered tRNA-Pro genes and the beginning of the D-loop region, HVR1) were deposited in the National Center for Biotechnology Information (NCBI) GenBank database under accession numbers MG420898–MG420955 (Danubian Horse), MG420956–MG420978 (Hungarian Nonius) and MG420979–MG420990 (Serbian Nonius). Sequences were analyzed by polymorphic single-nucleotide polymorphism position, and haplogroups were determined according to Achilli et al.²⁴ The phylogenetic relationships among haplotypes were visualized through the construction of two median-joining trees using Network 10.0 (www.fluxus-engineering.com).

In order to graphically display (and summarize) the mitochondrial relationships among the analyzed samples, the principal component analysis (PCA) was applied, using Excel software implemented by XLSTAT.²⁵ The number of polymorphic sites (S), haplotypes (H), haplotype diversity (Hd), nucleotide diversity (π), and average number of pair-wise nucleotide differences within a population (K) were estimated using DnaSP6.0.²⁶ Values of non-synonymous (dN) andsynonymous (dS) substitutions were estimated and compared using the Z test (p < 0.05 was considered significant) based on the methodology²⁷ with Jukes and Cantor correction. Tajima’s D value²⁸ and Fu and Li’s D and F values²⁹ were analyzed using DnaSP6.0 to evaluate the neutral theory of evolution.²⁶

Results

mtDNA sequence polymorphism and tests of neutrality

The sequence analysis of 608 bp from the mitochondrial D-loop region showed a high average haplotype diversity between studied horse breeds (Hd = 0.886). In the Hungarian Nonius and Serbian Nonius we observed 12 haplotypes (Hap), while in the Danubian horse 13 haplotypes was estimated (Table 1). Overall, we identified from one to six haplotypes in the investigated breeds from a total of 49 distinct haplotypes (Table S1). Only one haplotype was common between Hungarian Nonius and Serbian Nonius as well as between Danubian horse and Serbian Nonius. The haplotype with the highest frequency among our samples was Hap34, which was found in six sequences out of 93 (6.45%), followed by Hap5 and Hap28 (found in five sequences, 5.38%) (Table S1). The number of polymorphic sites (S, excluding gaps and ambiguous sites) was detected to be highest in the Hungarian Nonius horse (53) and lowest in Danubian horse (16). These results confirmed a different genetic structure in all of the investigated horse breeds.

Table 1.

mtDNA sequence polymorphism and tests of neutrality in Hungarian Nonius, Serbian Nonius and Danubian horse.

Breed	S	Eta	K	H	Hd ± SD	π ± SD	Fu and Li’s D test	Fu and Li’s F test	Tajima’s D
Hungarian Nonius	55	58	13,557	11	0.909 ± 0.036	0.02282 ± 0.00302	−1.14089	−1.11830	−0.54020
Hungarian Nonius	55	58	13,557	11	0.909 ± 0.036	0.02282 ± 0.00302	(p > 0.10)^a	(p > 0.10)^a	(p > 0.10)^a
Serbian Nonius	45	49	19,121	9	1.000 ± 0.034	0.03119 ± 0.00298	0.80420	0.92111	0.81844
Serbian Nonius	45	49	19,121	9	1.000 ± 0.034	0.03119 ± 0.00298	(p > 0.10)^a	(p > 0.10)^a	(p > 0.10)^a
Danubian horse	16	16	2,445	29	0.748 ± 0.047	0.01519 ± 0.00179	−1.59001 (p	−1.59565 (p	−0.88460
Danubian horse	16	16	2,445	29	0.748 ± 0.047	0.01519 ± 0.00179	> 0.10)^a	> 0.05)^a	(p > 0.10)^a

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S: number of polymorphic sites; Eta: total number of mutations; K: average number of nucleotide differences; H: number of haplotypes; Hd: haplotype diversity; π: observed average pairwise nucleotide diversity.

^aNot significant.

Highest nucleotide differences were found in the Serbian Nonius (K = 19,121), whereas the lowest were found in the Danubian horse (K = 2,445). Haplotype diversity was higher for Serbian Nonius (1.000 ± 0.034) than for Hungarian Nonius (0.909 ± 0.036) or Danubian horse (0.692 ± 0.020) (Table 1). The π value was highest in Serbian Nonius (0.03119 ± 0.00298), suggesting that most nucleotide diversities were concentrated in this horse breed. The estimated Tajima’s D value and Fu and Li’s D and L tests were negative in Hungarian Nonius and Danubian horse, suggesting population size expansion (e.g., after a bottleneck or a selective sweep) and/or purifying selection, i.e., a rare or previously non-existing allele increased the fitness of the carrier (relative to other members of the population) and increased rapidly in frequency due to natural selection (genetic hitchhiking). In contrast to the Hungarian Nonius and Danubian horse, in the Serbian Nonius positive Tajima’s D value and Fu and Li’s D and L tests were observed, which is an indicator for a decrease in population size and/or balancing selection, since balancing selection maintains mutations at intermediate frequencies.

Phylogenetic analyses and haplogroups classification

The analysis of diagnostic mutational motifs showed that all obtained haplotypes belonged to nine haplogroups (D, O’P, G, A, L, C, Q, E and M) (Table 1, Fig. 1). The majority of the samples, 31 out of 93 (33.3%), fell into G haplogroup, and the highest frequency of this group was observed in the Danubian horse (45.0%, 26/58). The second most common haplogroup was M (24.7%, 23/98) with highest values in Hungarian Nonius (43.5%) and Danubian horses (21.8%). This haplogroup was not found in the Serbian Nonius. Haplogroup O’P (9.7%, 9/93) is another represented group which was found with different frequency in Hungarian Nonius (26.1%), Danubian horse (3.5%) and Serbian Nonius (8.3%). In addition to these principal haplogroups, there were ones specific to each breed (Table 1, Fig. 1). For example, in Serbian Nonius a high prevalence of the rare haplogroup D (66.7%, 8/12) was found, which was not observed in the other two breeds. Haplogroup L was specific to the Danubian horse population (21.0%, 12/58), while the other rare haplogroup E was found only in the Hungarian Nonius horse population (13.0%, 3/23).

Figure 1. — A map showing sampling location and haplogroup diversity. Pie charts are proportional to sample size (Supp. Table S1). *Abbreviations*: SN: Serbian Nonius; DH: Danubian horse; EBH: East Bulgarian horse; PH: Pleven horse.

When comparing the mitochondrial profiles of the three equine breeds under investigation with two other Bulgarian plain horse populations (East Bulgarian horse and Pleven horse), significant differences were found with the Hungarian Nonius and Serbian Nonius populations, but also certain similarities with Danubian horse were observed (Fig. 1). The East Bulgarian horse population was characterized with the highest frequency of haplogroup Q (35.90%, 14/39), followed by haplogroup L (33.33% 13/39) and haplogroup A (10.26%, 4/39). Similar to the East Bulgarian horse, in the Pleven horse breed, haplogroup L (45.45% 5/13) was predominant, followed by haplogroup C (27.27% 3/11). In East Bulgarian and Pleven horse breeds the very rare haplogroup N (7.69% and 9.09%, respectively) was observed. The obtained results show that Bulgarian plain horse breeds (Danubian, East Bulgarian and Pleven) share common haplogroup L, which was observed with high frequency in in all three breeds.

Network analysis of the partial D-loop region in three Bulgarian plain horses, Hungarian Nonius and Serbian Nonius showed high haplotype diversity (Fig. 2). Among all 60 identified haplotypes, the most represented belonged to haplogroups G (Hap13, 21.7%), M (Hap10, 16.7%), L (Hap9, 15.0%), and Q (Hap6, 10.0%) (Table S1). We also observed some common haplotypes among the investigated samples. Hap23 was shared with both Danubian and Serbian Nonius horse breeds, Hap32 and Hap39 were detected in Danubian and Pleven horse breeds, Hap36 was found in Danubian and East Bulgarian horses (Table S1). These haplotypes encompassed only Bulgarian horse breeds, which suggested a common maternal ancestry in the formation of modern Bulgarian plains horses.

Figure 2. — Median joining network based on D-loop control-region sequences of three plain Bulgarian horse breeds, Nonius and Serbian Nonius. Median joining network was constructed based on 64 haplotypes in 143 mtDNA sequences. The sequence variations are from Supplementary Table S1. Circle areas are proportional to haplotype frequencies.

Principal component analysis

In order to graphically display and summarize the information concerning haplogroup frequencies, we performed a principal component analysis (PCA). The PCA was carried out by considering only our sample (Fig. 3, Table S2). After reducing the haplogroups to PCs (principal components) we analyzed the coordinates of the observations for the Hungarian Nonius and Serbian Nonius, as well as three Bulgarian plains horse breeds – Danubian horse, East Bulgarian and Pleven horse, in a two-dimensional graph based on haplogroup frequencies from the mitochondrial D-loop region. The outlier position of the Pleven horse is confirmed particularly along the first PC, while the second PC splits the Serbian Nonius from the other breeds. It was observed that the Hungarian Nonius horse was in the same quarter with the Danubian horse due to their sharing the same haplogroups – M, G and O’P as shown by the centroids of the two breeds (Table 1, Fig. 3).

Figure 3. — PCA plot representing the genetic landscape of three modern Bulgarian plain horse breed (Danubian, East Bulgarian and Pleven horse) Hungarian Nonius and Serbian Nonius, based on haplogroup frequencies from mitochondrial D-loop region.

The PCA revealed a specific localization of the East Bulgarian and Pleven horse breeds in contrast to Danubian horse. The PCA analysis suggests that all Bulgarian plain horse breeds have a shared maternal inheritance. The position of the Serbian Nonius on the PCA plot and its clear separation from Hungarian Nonius and Danubian horses is an interesting finding. This suggests a different breed origin and the probable participation of ancestral mares with a specific genetic profile that were used in the initial stages of breeding selections in contrast to Nonius and Danubian horses.

Discussion

Tracing of the genetic roots of Hungarian Nonius horse

Analysis of genetic diversity in the Nonius horse showed an unusually high frequency of haplogroup M − 43.5% in all 23 investigated samples (Fig. 1, Table 2). Haplogroup M was present at a frequency of about 7% in modern and about 17% in ancient European horse populations.²⁴ Haplogroup M is of typical European origin as it is established during the Mesolithic/Neolithic era (5200–4900 BC) and later during the Neolithic/Bronze age in the Iberian Peninsula.³⁰^,³¹ Native Iberian horses contributed to the ancestry of animals found in the Early Bronze Age in Hungary from the late 3rd millennium BC.³² The native Iberian horses’ contribution to the Iron Age Spanish horse, suggesting genetic influence of these horses continued to persist in Iberia until at least the 7th century BCE in a domestic context.³³ Taking into account the fact that Spanish mares were involved in the creation of Hungarian Nonius horse breed, as well as the direct ancestry of native Spanish horses on Hungarian animals back in the Early Bronze Age, it is not surprising that the haplogroup M is present with such a high frequency in the Hungarian Nonius population. Another haplogroup with high frequency in this breed is O’P (26.1%, 6/23) (Table 1, Fig. 2). In contrast to haplogroup M, haplogroup O’P is a typical Middle Eastern haplogroup with the highest frequency (c. 8%) in Middle Eastern modern horses²⁴ and about 10% in ancient Asian samples.³³ This haplogroup emerged for the first time in prehistoric Anatolia during the late Pleistocene and early Holocene. After that during the Bronze Age (around 2000 BC) the frequency of O’P decreased significantly.³⁴ It is possible for haplogroup O’P to be incorporated into Oriental horse breeds from the Bronze Age Anatolian horses. Perhaps the most famous breed among oriental horses and in the world, created in the Middle East in particular the Arabian Peninsula (over the last 3,000 years) by the nomadic Bedouin people is the Arabian horse.³⁵^,³⁶ For more than 100 years Arabian horses have been used to improve many horse breeds.³⁷ This improvement reached its peak in the 19th and 20th Centuries in the creation of the English Thoroughbred racing horse.³⁸ In addition, the contribution of Arabian mares to Hungarian Nonius horse formation might explain the presence of O’P haplogroup in its population. Another possible explanation for the existence of O’P haplogroup in Nonius breeds is related to the Umayyad Muslims (756–1236 AD) in the Iberian Peninsula.³⁹ The most common assumption was that the term Spanish horse referred primarily to those coming from the region of Andalusia, both for the implications of its Barb ancestry and eastern breeding methods under the Cordoba Caliphate.⁴⁰ The Muslim conquests on the Iberian Peninsula as well as the contribution of Andalusian mares to the formation of the Hungarian Nonius breeds represents another point of view regarding the presence of haplogroup O’P in Hungarian horse. Also, of interest is the presence of haplogroup G (13.1%, 3/23) in the Hungarian Nonius population (Fig. 1, Table 2). This haplogroup is typical of Central Asiatic origin and is present in around 16.0% and 22.0% in modern and ancient horse populations, respectively.²⁴^,⁴¹ Another typical Central Asiatic haplogroup that is observed in the Hungarian Nonius horse is the rare haplogroup E that is also present in modern horse populations, at a frequency of approximately 2%.²⁴ These lineages could have arrived in this region after the appearance of Yamnaya people (with already domesticated horses) in the central and eastern part of the Carpathian Basin in the Early Bronze Age. The western Yamnaya people from the northern Pontic zone began to expand across the steppe in all directions between c. 3100 and 2400 BC.⁴² In this region the domestic horse arrived with the expansion of Yamnaya people from 3000 BC in a wide zone further to the west, in the modern countries of Romania, Bulgaria, Serbia and Hungary.⁴³ Evidence supporting this hypothesis, are the single burials found under kurgans (typical for Yamnaya cultural complex) in prehistoric Europa. Alternatively, the domestic horse might have arrived with the Corded Ware culture (late Neolithic to early Bronze Age) and these wild ancestors of the earliest domestic horses in the Pontic-Caspian steppe in all probability dispersed elsewhere as the domestic horse dispersed.⁴⁴ Another point of view concerning the presence of Central Asiatic haplogroup G and E in the Nonius population is associated with the arrival of ancient Hungarians from the Eastern European steppes to the Carpathian Basin at the end of the ninth century AD.⁴⁵ Haplogroups G and E are prehistoric haplogroups which were present in ancient Central Asian horse populations during the Late Bronze Age (1450–1250 BC).³⁴ According to the general hypothesis, the present-day Hungarians abandoned their original homeland in the Western Ural Mountains and conquered their current homeland in the Carpathian Basin in 895 AD.⁴¹ It is suggested that early Hungarian horses originated from Central Asia – the area of today’s Turkmenistan, Uzbekistan, and south Kazakhstan.⁴⁶ These horses were some of the best in the area at the time and in all probability early Hungarians will have taken into account their valuable qualities, which is why they disseminated them into their new homeland territory – the Carpathian Basin.⁴¹ In support of this is the close genetic relationship between early Hungarian horses and the modern Akhal Teke breeds, with haplogroup G being present in approximately 50% of horses in both populations.⁴¹ Similarly, one can associate the presence of haplogroup E in the Hungarian Nonius breed with the Hungarian conquest of the Carpathian basin. However, both haplogroups are allochthonous for the Caucasus, as they were not established in that territory before 2000 BC, probably being introduced at the end of the third millennium BC from the Pontic-Caspian steppe via the Transcaucasian route.³⁴ Tajima’s D as well as Fu and Li’s D and F statistics was negative in the Hungarian Nonius horse population (Table 1). These values might be the result of population expansion, i.e., rapidly increasing frequency of some favorable neutral alleles due to natural selection (genetic hitchhiking). It could be assumed that haplogroup G was undergoing a selective sweep in the Hungarian Nonius horse population, because ancient Hungarians appreciated the excellent form, speed and stamina of the Turkoman horses.

Table 2.

Haplogroups frequencies in Danubian horse (DH), Hungarian Nonius and Serbian Nonius (SN) populations.

Haplogroup	Hungarian Nonius (n)	%	SN (n)	%	DH (n)	%	Total	%
D	0	0.0	8	66.7	0	0.0	8	8.6
O’P	6	26.1	1	8.3	2	3.5	9	9.7
G	3	13.1	2	16.7	26	45.0	31	33.3
A	0	0.0	1	8.3	1	1.7	2	2.2
L	0	0.0	0	0.0	12	21.0	12	12.9
C	0	0.0	0	0.0	1	1.7	1	1.1
Q	1	4.3	0	0.0	3	5.3	4	4.3
E	3	13.0	0	0.0	0	0.0	3	3.2
M	10	43.5	0	0.0	13	21.8	23	24.7

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Haplogroups affiliation is according to Achilli et al.²⁴

Genetic diversity of the Danubian horse

There are three modern plain horse breeds in Bulgaria – the Danubian horse, the Pleven horse and the East Bulgarian horse. Mitochondrial DNA analysis of the Danubian horse revealed approximately 45% (26/58) presence of haplogroup G (Fig. 1, Table 2). The presence of this haplogroup in the Danubian horse population seems logical given the contribution of Hungarian Nonius mares in the formation of the breed. The high frequency of Haplogroup G in Danubian horses might be explained by population bottlenecks and/or founder events. It is probable that imported mares with an identical genetic profile came from the Mezohegyes stud farm, which was the regional nucleus for the formation of the Danubian horse. Supporting this breeding programme was a ‘grading-up‘ process involving the use of purebred animals over a series of generations to provide a ‘nearly purebred‘ result, which was used in the formation of the Danubian horse breed¹⁹ as well as calculated negative value of Fu and Li’s D and F tests and Tajima’s D (Table 1). Another haplogroup with relatively high frequency is haplogroup M (21.8%, 13/58) (Fig. 1, Table 2). The origin and distribution of this haplogroup has already been reviewed in the Hungarian Nonius horse and by association it is present in the Danubian horse population. Another haplogroup, which was present in the Danubian horse was haplogroup L (21.0%, 12/58). This haplogroup, along with M, was typical West Eurasian and present in modern and ancient population at approximately38.0 and 21.43%, respectively.²⁴ In contrast to Neolithic/Bronze age Iberian haplogroup M, haplogroup L appeared in the Iberian Peninsula in the Middle Ages either or as an independent domestication event in the Iberian Peninsula or by migration from North Africa.³³ Surprisingly, we did not find evidence of haplogroup L in the Hungarian Nonius horse population, although it was apparent at high frequency in the Iberian horses involved in the creation of the breed (Fig. 1, Table 2). One possible explanation for this is with the different process involved in the formation of the Hungarian Nonius breed at the Mezohegyes and Mata stud farms. The selection process in the Mata stud farm (where our samples come from) was aimed at creating heavier horses for agricultural purposes, whereas at the Mezohegyes stud farm the goal was to create light draft horses for military purposes. Moreover, bay colored horses are more common among the Hungarian Nonius horses from Hortobagy stud farm, which are mostly black. Danubian horse and the other two plain Bulgarian horse breeds – Pleven and East Bulgarian shared common haplogroup L (Fig. 1).⁴⁷ In contrast to the Danubian horse, Pleven and East Bulgarian breeds showed different genetic profiles most likely associated with local Bulgarian breeds (Deliorman and Kamchia, respectively), from which the creation of the breeds began with introgression of local horses along with various exotic horse breeds involved in their formation.

The enigmatic haplogroup D in the Serbian Nonius

The least data on the creation of the breed is available for the Serbian Nonius horse. Despite the small number of animals included in the analysis which was a consequence of the reduced size of the total population, some conclusions could be drawn about its genetic structure. The Sequence analysis of a partial D-loop region showed the presence of the very rare haplogroup D at more than 50% (66.7%, 8/12) (Fig. 1, Table 2). This haplogroup occurred in only about 4.5% of the modern and in about 10.2% of the ancient samples.²⁴ Haplogroup D emerged for the first time in the prehistoric Anatolian hose population during the Bronze Age (around 2000 BC), but it was an allochthonous lineage, and presumably incorporated in Anatolian horses from the Pontic-Caspian steppe via the Transcaucasian route in the Early Bronze Age (the end of the third millennium BC).³⁴ According to this observation, haplogroup D might have disseminated in Europe alongside the expansion of Yamnaya people from the Northern Pontic zone during the Early Bronze age.⁴³ Although we have no data it is possible that it was similar to the creation of Hungarian Nonius and Danubian horses, with the participation of local Serbian breeds such as the Serbian Mountain Pony in the formation of Serbian Nonius horse. It is interesting to note that several studies have considered that haplogoup D was an old clade characteristic of the small ponies distributed on in Northern Europe (Exmoor, Fjord, Icelandic, Scottish Highland etc.) and gathering of wild mares for domestication from geographically different areas.³⁰^,^48–50 These observations represent a convenient possibility for the high frequency of haplogroup D in the Serbian Nonius horse.

Haplogroup G was present in the Serbian Nonius population in 13.1% of horses sampled (2/12) (Fig. 1, Table 2). Like the Danubian horse, the presence of this haplogroup might be explained by the influence of the Hungarian Nonius breed in the creation of the Serbian Nonis. Haplogroup O’P and A were observed with equal frequency of 8.3% in Serbian Nonius horses. These haplogroups are typical representatives of Oriental horses (Arabian, Akhal-Teke, Barb etc.).²⁴^,³⁰^,⁵⁰ The stock of Arab and Barb horses was introduced into England as early as the 3rd century, which marked the beginning of the formation of the Thoroughbred.⁵¹^,⁵² The Thoroughbred horse took active part in the creation of Serbian Nonius horse, which also explains the presence of the haplogroups O’P and A in Serbian Nonius population.

In contrast to Hungarian Nonius and Danubian horses, the Tajima’s D, Fu and Li’s D and F statistics were positive in the Serbian Nonius population (Table 1). These statistics suggest that in this breed balancing selection occurred. It represents a panmictic population – one with the ability of individuals in a population to interbreed without restrictions.

Conclusions

This study is the first to demonstrate the genetic structure of the Nonius horse in Hungary and The closely related Danubian and Serbian Nonius horse populations. The Hungarian Nonius horse population expressed highly diverse genetic structure, which could be traced back to human migration during the Early Bronze Age (around 3000 BCE) concurrent with expansion of Yamnaya pastoralist with horse husbandry westwards into Europe and even much earlier alongside dissemination of the late Neolithic Corded Ware culture.

The genetic profile of the Danubian horse resembled that of the Hungarian Nonius horse, due to the tribal nucleus from which the formation of the breed began and was composed of stallions and mares of the Hungarian Nonius breed as well as grading-up reproductive crossbreeding. In contrast to Hungarian Nonius and Danubian horses, the Serbian Nonius showed a different genetic profile, resulting from balancing of selection and the use of predominantly native mares in the creation of the breed.

Supplementary Material

Supplemental Material

LABT_A_2237533_SM0856.xlsx^{(120.3KB, xlsx)}

Supplemental Material

LABT_A_2237533_SM0852.xlsx^{(77KB, xlsx)}

Acknowledgments

The authors wish to thank Maya Marinova (IBPhBME-BAS, Bulgaria) for the English editing.

Funding Statement

The author(s) reported there is no funding associated with the work featured in this article.

Author contributions

Conceptualization, G.Y. and P.H.; methodology, N.P., and I.M.; software, P.H. and G.Y.; formal analysis, N.P. and I.M.; investigation, P.H., G.Y., N.P. and I.M.; data curation, P.H. and G.Y.; writing—original draft preparation, P.H., G.Y. and N.P.; writing—review and editing, P.H., G.Y., N.P., and I.M.; visualization, P.H.; supervision, P.H. All authors have read and agreed to the published version of the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

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Associated Data

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Supplementary Materials

Supplemental Material

LABT_A_2237533_SM0856.xlsx^{(120.3KB, xlsx)}

Supplemental Material

LABT_A_2237533_SM0852.xlsx^{(77KB, xlsx)}

[CIT0001] 1.Kelekna P. The Horse in Human History. Vol. 799. Cambridge: Cambridge University Press; 2009. [Google Scholar]

[CIT0002] 2.Haring H. The changing role of horses in our society. Lohmann Inf. 2012;47:49. [Google Scholar]

[CIT0003] 3.Ryckman KC, Braithwaite JM.. Changing horses in midstream: leadership changes and the civil war peace process. Confl Manag Peace Sci. 2020;37(1):83–105. [Google Scholar]

[CIT0004] 4.Kȩdzierski W, Pluta M.. The welfare of young Polish Konik horses subjected to agricultural workload. J Appl Anim Welf Sci. 2013;16(1):35–46. [DOI] [PubMed] [Google Scholar]

[CIT0005] 5.Berber N, Gaouar S, Leroy G, Kdidi S, Tabet Aouel N, Saïdi Mehtar N.. Molecular characterization and differentiation of five horse breeds raised in Algeria using polymorphic microsatellite markers. J Anim Breed Genet. 2014;131(5):387–394. [DOI] [PubMed] [Google Scholar]

[CIT0006] 6.Benhamadi MEA, Berber N, Benyarou M, Ameur AA, et al. Molecular characterization of eight horse breeds in Algeria using microsatellite markers. Biodiversitas. 2020;21(9):4107–4115. [Google Scholar]

[CIT0007] 7.Rzekęć A, Vial C, Bigot G.. Green assets of equines in the European context of the ecological transition of agriculture. Animals. 2020;10(1):106. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0008] 8.Lawrence PR, Pearson RA.. Use of draught animal power on small mixed farms in Asia. Agric Syst. 2002;71(1–2):99–110. [Google Scholar]

[CIT0009] 9.Gebregziabher S, Mouazen AM, Van Brussel H, Ramon H, et al. Animal drawn tillage, the Ethiopian and plough, Maresha: a review. Soil Tillage Res. 2006;89(2):129–143. [Google Scholar]

[CIT0010] 10.Akhoondnejad A. Loyalty formation process of tourists in sporting event: the case of Turkmen horse races. J Hosp Tou. Manag. 2018;34:48–57. [Google Scholar]

[CIT0011] 11.Cernaianu S, Sobry CA. A diagnose of equestrian tourism: The case of France and Romania. In: Artal-Tur A, Kozak M, Kozak N, eds. Trends in Tourist Behavior. Tourism, Hospitality & Event Management. Cham: Springer. 10.1007/978-3-030-11160-1_1 [DOI] [Google Scholar]

[CIT0012] 12.Nolte W, Thaller G, Kuehn C.. Selection signatures in four German warmblood horse breeds: Tracing breeding history in the modern sport horse. PLOS One. 2019;14(4):e0215913. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0013] 13.Barzev G, Yordanov G, Yuseinov Y.. The Bulgarian primitive horse in the area of Stara Planina mountain. Trakia J Sci. 2005;3:74–76. [Google Scholar]

[CIT0014] 14.Moravčíková N, Kasarda R, Kukučková V, Vostrý L, et al. Genetic diversity of Old Kladruber and Nonius horse populations through microsatellite variation analysis. Acta Agric Slov. 2016;5:45–49. [Google Scholar]

[CIT0015] 15.Mihók S, Posta J, Komlósi I, Bodó I. Genetic variability of Mezıhegyes horse breeds using genealogical and molecular information. In Book of Abstracts of the 60th Annual Meeting of the European Association for Animal Production; August 2009; Barcelona, Spain: 24–27.

[CIT0016] 16.Toth Z, Kaps M, Sölkner J, Bodo I, Curik I.. Quantitative genetic aspects of coat color in horses. J Anim Sci. 2006;84(10):2623–2628. [DOI] [PubMed] [Google Scholar]

[CIT0017] 17.Matiuți M, Crăiniceanu E.. Research regarding native domesticated endangered animal breeds. Univ Ştiinţe Agr Med Vet. 2008;41(1):221–224. [Google Scholar]

[CIT0018] 18.Sandor N, Adam B.. Equestrian tourism and horse breeding in Hungary and Slovenia–Environmental sustainability and conservation of cultural heritage: a strategic approach. Ecocycles. 2019;5(1):67–97. [Google Scholar]

[CIT0019] 19.Karaivanov R, Barzev G, Karadzhov T. Development and status of families in the Danubian horse breed. International Symposium on Half Breed Equine Breeding; 1989; St. Zagora:82–96. [Google Scholar]

[CIT0020] 20.Štrbac L, Trivunović S, Baban M. Phenotypic and genetic variability of some morphological traits of Nonius horse in Serbia. In 26th international Dagene Symposium; 2015; Dobrna, Slovenia:21.

[CIT0021] 21.Xu X, Arnason U.. The complete mitochondrial DNA sequence of the horse, Equus caballus: extensive heteroplasmy of the control region. Gene. 1994;148(2):357–362. [DOI] [PubMed] [Google Scholar]

[CIT0022] 22.Hristov P, Yordanov G, Ivanova A, et al. Mitochondrial diversity in mountain horse population from the South-Eastern Europe. Mitochondrial DNA A DNA Mapp Seq Anal. 2017;28(6):787–792. [DOI] [PubMed] [Google Scholar]

[CIT0023] 23.Kumar S, Stecher G, Tamura K.. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33(7):1870–1874. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0024] 24.Achilli A, Olivieri A, Soares P, et al. Mitochondrial genomes from modern horses reveal the major haplogroups that underwent domestication. Proc Natl Acad Sci USA. 2012;109(7):2449–2454. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0025] 25.Achilli A, Olivieri A, Pala M, et al. Mitochondrial DNA variation of modern Tuscans supports the near eastern origin of Etruscans. Am J Hum Genet. 2007;80(4):759–768. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0026] 26.Rozas J, Ferrer-Mata A, Sánchez-DelBarrio JC, et al. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Mol Biol Evol. 2017;34(12):3299–3302. [DOI] [PubMed] [Google Scholar]

[CIT0027] 27.Nei M, Gojobori T.. Simple methods for estimating the number of synonymous and nonsynonymous nucleotide substitutions. Mol Biol Evol. 1986;3(5):418–426. [DOI] [PubMed] [Google Scholar]

[CIT0028] 28.Tajima F. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics. 1989;123(3):585–595. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0029] 29.Fu YX, Li WH.. Statistical tests of neutrality of mutations. Genetics. 1993;133(3):693–709. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0030] 30.Cieslak M, Pruvost M, Benecke N, et al. Origin and history of mitochondrial DNA lineages in domestic horses. PLOS One. 2010;5(12):e15311. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0031] 31.Lira J, Linderholm A, Olaria C, et al. Ancient DNA reveals traces of Iberian neolithic and bronze age lineages in modern Iberian horses. Mol Ecol. 2010;19(1):64–78. [DOI] [PubMed] [Google Scholar]

[CIT0032] 32.Fages A, Seguin-Orlando A, Germonpré M, Orlando L.. Horse males became over-represented in archaeological assemblages during the Bronze Age. J Archaeolog Sci: Rep. 2020;31:102364. [Google Scholar]

[CIT0033] 33.Fages A, Hanghøj K, Khan N, et al. Tracking five millennia of horse management with extensive ancient genome time series. Cell. 2019;177(6):1419–1435.e31. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0034] 34.Guimaraes S, Arbuckle BS, Peters J, et al. Ancient DNA shows domestic horses were introduced in the southern Caucasus and Anatolia during the Bronze Age. Sci Adv. 2020;6(38):eabb0030. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0035] 35.Almarzook S, Reissmann M, Arends D, Brockmann GA.. Genetic diversity of Syrian Arabian horses. Anim Genet. 2017;48(4):486–489. [DOI] [PubMed] [Google Scholar]

[CIT0036] 36.Sadeghi R, Moradi-Shahrbabak M, Miraei Ashtiani SR, et al. Genetic diversity of Persian Arabian horses and their relationship to other native Iranian horse breeds. J Hered. 2019;110(2):173–182. [DOI] [PubMed] [Google Scholar]

[CIT0037] 37.Knorr F. A history of the Arabian horse and its influence on modern breeds. J Hered. 1912;3(3):174–180. [Google Scholar]

[CIT0038] 38.Bower MA, Campana MG, Whitten M, et al. The cosmopolitan maternal heritage of the thoroughbred racehorse breed shows a significant contribution from British and Irish native mares. Biol Lett. 2011;7(2):316–320. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0039] 39.Funes-Pérez JA, Rodríguez-López J, Manas A.. Medieval Córdoba: from Umayyad recreations to competitive Christian sport. Int J Hist Sport. 2016;33(10):1009–1027. [Google Scholar]

[CIT0040] 40.Renton K. Breeding techniques and court influence: Charting a ‘Decline’ of the Spanish horse in the Early Modern Period. The Court Historian. 2019;24(3):221–234. [Google Scholar]

[CIT0041] 41.Priskin K, Szabó K, Tömöry G, et al. Mitochondrial sequence variation in ancient horses from the Carpathian Basin and possible modern relatives. Genetica. 2010;138(2):211–218. [DOI] [PubMed] [Google Scholar]

[CIT0042] 42.Anthony DW. The Horse, the Wheel, and Language: How Bronze-Age Riders from the Eurasian Steppes Shaped the Modern World. Princeton, New Jersey: Princeton University Press; 2010. [Google Scholar]

[CIT0043] 43.Gerling C, Bánffy E, Dani J, et al. Immigration and transhumance in the early Bronze age Carpathian Basin: the occupants of a Kurgan. Antiquity. 2012;86(334):1097–1111. [Google Scholar]

[CIT0044] 44.Kvist L, Niskanen M.. Modern northern domestic horses carry mitochondrial DNA similar to Przewalski’s horse. J Mammal Evol. 2021;28(2):371–376. [Google Scholar]

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PERMALINK

Mitochondrial DNA sequencing illuminates genetic diversity and origin of Hunagrian Nonius horse breed and his relatives – Danubian horse and Serbian Nonius

Georgi Yordanov

Nadezhda Palova

Ivan Mehandjyiski

Peter Hristov

Abstract

Introduction