The full mitochondrial genomes of Mangalica pig breeds and their possible origin

Abstract The mitogenomes of one animal of each of the three Mangalica breeds, Blonde, Red, and Swallow-belly were assembled from reads obtained by Next Generation Sequencing of the three genomes. Features of the mitogenomes were identical in the three breeds, apart from a second tRNA-Val gene on the L strand in Swallow-belly. Phylogenetic comparison of the three mitogenomes with 112 full mtDNA sequences clearly put Mangalicas into the European clade. Comparing the mitogenome of eight Mangalica animals revealed particular differences between them. The mitogenome of some Mangalicas was closely related to the Croatian Turopolje breed and this indicates either the common origin of their maternal lineages or admixture of some populations of the breeds. However, the origin of the mitogenome of certain purebred Mangalicas kept in the Hungarian Mangalica Gene Reserve still remains unknown.


Introduction
The three fatty-type Mangalica pig breeds, Blonde, Red, and Swallow-belly, are farmed in growing number in Hungary and also kept in several other European and overseas countries ) for up-market pork production. They have a quite complex history (B€ ok€ onyi 1974;Egerszegi et al. 2003), which goes back to the late 1700s, early 1800s, when their spontaneous and conscious breeding started involving the old Serbian pig breeds Sumadia (Sumadinka) and Syrmian (sometimes also called Black Mangalica) and three, already extinct, Carpathian Basin pig races, Bakonyi, Szalontai, and Alf€ oldi, which might be originated from old domesticated pigs brought to the Carpathian Basin by ancient Romans.
Previously, we have studied the relationship between maternal lineages of Mangalica and other pig breeds . The results indicated that 197 out of the 203 examined Mangalica individuals carried the ancient ANC-Aside D-loop signature (Larson et al. 2007). One hundred and twenty-two Mangalicas belonged to three Mangalica-specific D-loop haplotypes, while 64 animals belonged to two major European haplotypes .
In this study, we present the mitogenome of the three Mangalica breeds assembled form next-generation-sequencing data and investigate the molecular phylogenetics of full mtDNA sequences of different pig breeds in order to determine a presumptive relatedness of maternal lineages of Mangalica pigs.

Materials and methods
To assemble the mitogenome of the three Mangalica and a Duroc animal, 100 bp Illumina reads obtained from genome sequencing of one male animal of each bread  were separately aligned to the AJ002189 (Ursing and Arnason 1998) pig mtDNA reference sequence using the Burrows-Wheeler Alignment Tool . The mitochondrial sequences were assembled from the aligned reads by using SAMtools ).
Full mitochondrial nucleotide sequences of domestic pig (Sus scrofa) breeds were downloaded from the National Center for Biotechnology Information website. The mitochondrial genome of bearded pig (Sus barbatus) was also downloaded to use as outgroup. The H strand sequences of the mitogenomes were aligned using ClustalW2 (Larkin et al. 2007) and manually adjusted in a few instances. Phylogenetic analyses were performed by a Bayesian approach using BEAST version 1.8.2 (Drummond et al. 2012). Maximum likelihood analysis was carried out with the bootstrap test, with 1000 replicates, using complete deletion for gaps/missing data, with the general time reversible model. Bayesian Markov chain Monte Carlo (MCMC) sampling was performed for 10 000 000 iterations using estimated model parameters as starting values. The phylogenetic tree was drawn from the 50% majority rule consensus of trees sampled every 1000 generations. Sequence alignments were performed using the software Geneious v.9.1.7 (geneious.com).

Results and discussion
Genome-sequencing of one male individual of each of the Blonde, Red, and Swallow-belly Mangalica breeds (Moln ar et al. 2014) provided 260,270, 98,832 and 104,478 reads that aligned to the pig reference mitochondrial genome (Ursing and Arnason 1998), resulting in 1,571Â, 602Â, and 638Â coverage, respectively. All three mitogenomes contained two ribosomal RNA and thirteen protein genes, the Blonde and Red individuals carried 22 tRNA genes, while the Swallowbelly animal contained 23 tRNA genes due to the presence of a second tRNA-Val gene on the L-strand ( Table 1). Most of the genes were encoded on the H-strand.
Previously we have studied the relationship between Mangalica and other pig breeds by comparing amplified and sequenced partial mitochondrial D-loop sequences Moln ar et al. 2013). Such D-loop sequences, however, often differ in length, because the primer pairs used for D-loop amplification usually vary from study to study. Those partial sequences, therefore, should have been truncated to the same length for comparison , which obviously results in loss of information. To overcome this limitation, in this study we used the full mitogenomes of 115 pig individuals of a number of different breeds (Supplementary  Table S1) to investigate the phylogenetic relationship between Mangalica and other breeds.
There was a clear separation between the European and Asian breeds in the phylogenetic tree ( Supplementary Fig. S1), as expected, although some Berkshire and Yorkshire individuals were placed into the Asian clade, indicating the introgression of maternal lineages of Asian breeds into their bloodline. The Mangalicas 4S, 15F and 13V with assembled mitogenomes, were kept at the Hungarian Mangalica Gene Reserve in the village of Em} od, Hungary, and represent the bloodlines "B ator" ($) and "B acska" (#), "Szank" ($) and "Csat ar" (#), and "Szolnok" ($) and "Mecs er" (#), respectively. The Mangalicas studied in the USA (Cannon et al. 2015) were imported directly from Austria (Terry Brandebourg, personal communication), but their lineages were not revealed. According to another study though, most of the Austrian Mangalicas were imported from Hungary (Druml et al. 2012), thus it is possible that the USA Mangalicas have Hungarian origin and are not from the separated Mangalica population that remained in Austria after the break-up of the Austro-Hungarian Empire following World War I. The origin and breed of the Mangalica sequenced in China is not known.
To compare the Mangalica mitogenomes, their sequences were aligned, which revealed certain differences between them. In addition to the five one-base-pair deletions being present in different animals, the Mangalica of China and the Hungarian Mangalicas have a 50 and a 90 bp deletion, respectively, compared to the USA Mangalica pigs ( Supplementary Fig. S2). Regarding SNPs, the Mangalica of China has the highest number of differences compared to every other individual, and the number of SNPs is higher between the Hungarian and USA Mangalica groups than within each group ( Supplementary Fig. S3).
Previously we have determined the D-loop haplotype of 2713 pig individuals belonging to five cosmopolitan breeds, 38 local breeds from nine and wild boars from 14 European countries, respectively ). In silico haplotyping of the Mangalica individuals in this study revealed that  Table S2). Amongst the haplotypes, which were identified in domesticated pigs , both HAP16 and HAP45 were considered Mangalica-specific since no individuals of other local European and Cosmopolitan breeds carried those, although HAP45 was found in five Austrian wild boar individuals. HAP16 and HAP45 were the fifth and third most abundant haplotypes amongst the Mangalicas included in that study. In contrast, the Blonde and Swallow-belly Mangalicas sequenced in the USA belonged to haplotypes HAP08 and HAP13 (Supplementary Table S2), respectively, which were the first and fourth most abundant haplotypes amongst all individuals ). HAP08 was found in 132 cosmopolitan pigs, and in 314 and 63 local pigs and wild boars representing eight and six countries, respectively, ). HAP13 was found in 35 cosmopolitan animals, and in 70 and 69 local pigs and wild boars, which originated from four and eight countries, respectively, . The Chinese Mangalica also belonged to haplotype HAP08 (Supplementary Table S2). It is worth to note here that the clades of the European part of the phylogenetic tree (Figure 1.) were consistent with the D-loop haplotypes of the individual pigs ( Supplementary Fig. S4). Based on D-loop haplotyping, Mangalicas were classified into two groups . Animals in the first group carried major European haplotypes, while animals in the second group had Mangalica-specific haplotypes. In this respect, the Mangalicas imported to the USA and China, are the representatives of the first group, while Mangalicas 4S, 15F and 13V belong to the second group. We hypothesized that the two groups should have been evolved by different routes .
It was previously shown ) that 37 of the examined 203 Mangalica and each of the 35 Turopolje individuals shared one major European D-loop haplotype (HAP08). In this respect, it is interesting that the two Turopolje pigs, included in this study, have HAP13. It is also important to note that, although HAP08 and HAP13 differ in only one SNP and are very closely related in a median-joint network of haplotypes , the two HAP08 Blonde Mangalicas from the USA (Cannon et al. 2015) are in one clade with other HAP08 breeds and not with HAP13 Mangalicas (Figure 1). This is very likely due to the seven SNP  Supplementary Fig. 4), according to Marincs et al. (2013); HAP13, HAP16, HAP45, HAP149, NEW, HAP07, HAP44, HAP08. Scale bar represents branch length (substitution per site). differences between the USA Blonde and Swallow-belly Mangalicas ( Supplementary Fig. S3), which are outside of the previously examined D-loop sequence used for haplotyping . The phylogenetic position and the sequence differences between the USA Blonde and other Mangalicas indicate the possibility of introgression of other breeds into the progenitors of the USA Blonde Mangalicas.
Nevertheless, our study indicates that Turopolje pigs might be the closest relatives of a certain type of Mangalicas at the mitogenome level (Figure 1), which is interesting because the Turopolje breed was not named as a possible contributor to Mangalicas (B€ ok€ onyi 1974; Egerszegi et al. 2003). In the case of Mangalica, it was hypothesized that their ancestors, the Alf€ oldi, Bakonyi and Szalontai pigs, were evolved from a much older pig race with the presumed origin from domesticated Slovenian pigs and wild boars and possibly from pigs brought to and kept in Pannonia province by the Romans (Egerszegi et al. 2003). The already extinct Serbian Sumadia (Sumadinka) and Syrmian breeds were also involved in the development of Mangalica (B€ ok€ onyi 1974; Egerszegi et al. 2003). Turopolje, named after the region where it emerged, is thought to be developed from some old races, such as Siska, Krskopoljski, black Slovenian and white South Austrian/North Croatian pigs (GENETICRESOURCES).
There are different possibilities to explain the relationship between the maternal lineages of Mangalica and Turopolje pigs. The first is a common wild boar ancestor since wild boars were domesticated in several centres around the World (Ramos-Onsins et al. 2014). The Siska pig, which is thought to be one of the direct descendants of wild boar and wild boar itself, was assumed to be amongst the ancestor of Turopolje and Mangalica, respectively (CEPIB; Egerszegi et al. 2003). Controversially, in ten present-day Hungarian wild boar samples the ANC-Cside ancient signature was found exclusively, while Mangalica and Turopolje haplotypes are associated with the ANC-Aside signature .
The second putative common ancestors for Mangalica and Turopolje are the pigs kept by Romans in or brought to the provinces Pannonia (current Transdanubia and the Western Carpathian Basin) and Dalmatia (covering areas of presentday Slovenia, Croatia and Serbia). Regarding the possible Roman origin of the two breeds, the ANC-Aside signature was found in each examined Hungarian archaeological pig samples, which dated back to either Roman times or A.D., but in none of the Croatian archaeological pig samples, which dated back to B.C. (Larson et al. 2007). Though only a few samples were available in both cases and the Croatian samples were found in such geographic regions (Larson et al. 2007), which are not associated with the known geographic origin of the predecessors of the Turopolje and Mangalica breeds. Regarding the possibility of bringing the ancestors of Mangalica and Turopolje into the Carpathian Basin by Romans, it is interesting to note that the ANC-Aside ancient signature, being exclusively present in both Mangalicas and Turopolje, was more frequently found in Italian domesticated pigs, both modern and archaeological samples, than any other ancient D-loop signatures (Larson et al. 2007). The ANC-Aside signature was also more frequent in modern Italian wild boars but was not found amongst the eight archaeological Italian wild boar samples (Larson et al. 2007). This suggests that, if Romans had brought pigs to the Carpathian Basin, those might have been pigs domesticated outside Italy.
The third possible explanation of the relationship between the Mangalica and Turopolje breeds is a later-day admixture during the development of the breeds. Turopolje was spread along the Sava River as far as the Posavina region (the last refuge of the breed) of Slavonia (GENETICRESOURCES), where Turopolje could come into geographical vicinity and mate with the Syrmian and Sumadia pigs, which were described to be involved in the development of Mangalica . Furthermore, the Turopolje breed also spread to the northern Podravina province of Slavonia and then further into south-west Hungary (ANSI), and thus the geographical distribution of Turopolje and Mangalica became overlapped to such a degree that they have had the opportunity to interbreed. Another indication for the admixture is that in a study involved only three breeds, Mangalica (from Austria and Serbia), Turopolje (from Austria and Croatia) and Black Slavonian (from Croatia), Black Slavonian populations were found to be more closely related either to Mangalica or to Turopolje (Druml et al. 2012). Black Slavonian might have been contributed to the development of the ancestors of Mangalica (Egerszegi et al. 2003) and Turopolje (GENETICRESOURCES), which can explain that observation.
The off-white curly hair of Turopolje (ANSI) resembles the curly coat of Mangalica and Sumadia, a contributor of Mangalica, and might be a sign of the relationship between these breeds. Apart from these three breeds, this very rare phenotype was only described in the Brazilian Canastrao breed, which might have common Mediterranean ancestors with Turopolje (Rhoad 1934), and the already extinct Lincolnshire Curly Coat breed (BPA).

Conclusions
Here we presented the mitogenome of one individual of each of the three Hungarian Mangalica breeds from the Hungarian Mangalica Gene Reserve and compared them with that of other Mangalicas, of which mitogenome was sequenced in other countries. It was found that the mitogenomes of the examined Mangalicas, with the exception of two USA Blonde animals, are highly related to that of the Croatian Turopolje breed. In the case of animals with major European D-loop haplotypes, Roman origin, admixture, and common wild boar ancestors are all possible explanations for the relationship. However, it still remains mysterious that what might be the origin of the breed-specific D-loop haplotypes in very large number of Mangalicas.

Disclosure statement
The authors declare no conflict of interest.

Funding
This work was supported by the Hungarian National Development Agency grant TECH_08-A3/2-2008-0405.