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Animal Biotechnology

ISSN: 1049-5398 (Print) 1532-2378 (Online) Journal homepage: http://www.tandfonline.com/loi/labt20

Genetic diversity of autochthonous pig breeds analyzed by microsatellite markers and mitochondrial DNA D-loop sequence polymorphism Kristina Gvozdanović, Vladimir Margeta, Polona Margeta, Ivona Djurkin Kušec, Dalida Galović, Peter Dovč & Goran Kušec To cite this article: Kristina Gvozdanović, Vladimir Margeta, Polona Margeta, Ivona Djurkin Kušec, Dalida Galović, Peter Dovč & Goran Kušec (2018): Genetic diversity of autochthonous pig breeds analyzed by microsatellite markers and mitochondrial DNA D-loop sequence polymorphism, Animal Biotechnology, DOI: 10.1080/10495398.2018.1478847 To link to this article: https://doi.org/10.1080/10495398.2018.1478847

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ANIMAL BIOTECHNOLOGY https://doi.org/10.1080/10495398.2018.1478847

ORIGINAL ARTICLE

Genetic diversity of autochthonous pig breeds analyzed by microsatellite markers and mitochondrial DNA D-loop sequence polymorphism Kristina Gvozdanovica, Vladimir Margetaa, Polona Margetaa, Ivona Djurkin Kuseca, Dalida Galovica, Peter Dovcb and Goran Kuseca a Department of Applied Zootechnics, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia; bDepartment of Animal Science, University of Ljubljana, Domzale, Slovenia

KEYWORDS

ABSTRACT

The evaluation of the genetic structure of autochthonous pig breeds is very important for conservation of local pig breeds and preservation of diversity. In this study, 18 microsatellite loci were used to detect genetic relationship between autochthonous pig breeds [Black Slavonian (BS), Turopolje pig (TP), and Croatian wild boar] and to determine phylogenetic relationship among Croatian autochthonous pig breeds and certain Asian and European pigs using the mitochondrial DNA (mtDNA) D-loop sequence polymorphism. Relatively high degree of genetic variation was found between the observed populations. The analysis of mtDNA showed that haplotypes of the studied pig populations are different from the other European and Chinese haplotypes. BS pigs showed some similarities with Mangalitsa and Duroc breeds. The genetic distances of TP can be explained by high degree of inbreeding during the past century. Despite the European origin of Croatian pig breeds with some impact of Chinese breeds in the past, the results of present study show that genetic diversity is still pronounced within investigated breeds. Furthermore, the genetic diversity is even more pronounced between Croatian breeds and other European and Chinese pig breeds. Thus, conservation of Croatian pig breeds will contribute to overall genetic diversity preservation of pig breeds.

Introduction Both Black Slavonian (BS) pig and Turopolje pig (TP) are two critically endangered autochthonous pig breeds in Croatia. BS pig is an old breed characterized by good productivity traits, excellent meat quality, and high intramuscular fat content.1 For this reason, it is very appreciated in the production of traditional dry-cured pork products. TP is thought to be the oldest pig breed in Croatia, and thus can be considered as the one of the oldest pig breeds in Europe. Characteristic carcass and meat quality traits of this breed were defined by specific historical conditions, breeding, and selection.2 It is worth mentioning that meat of TPs is particularly suitable for the production of high quality pork products. In the context of breed preservation and determination of phylogenetic relationships among particular breeds, the application of molecular genetic methods has proven to be very successful; comprehensive review is provided by Yang et al.3 One of the most frequently used markers in the studies of genetic diversity in domestic animals,

Black Slavonian pig; molecular markers; pig; phylogeny; Turopolje pig

particularly in breed characterization, are simple sequences repeats (SSRs) or short tandem repeats, also known as microsatellites. Due to their abundance, even distribution in the genome, high polymorphism, and ease of genotyping, microsatellites are considered to be very efficient markers for evaluation of genetic uniqueness and breed diversity in pigs.4 For example, Li et al.5 used 20 microsatellites that were proven to be useful for studying the genetic diversity of 10 local Chinese breeds. More recently, Oh et al.6 were able to differentiate two phenotypically similar pig breeds using 13 microsatellite markers by estimating the genetic diversity and population structure of the investigated breeds. The authors found that 10 microsatellite markers were sufficient to genetically discriminate studied breeds. The genetic diversity of black pigs from Greece was investigated by Michailidou et al.7 using 11 microsatellites. The results of the study showed that, despite low population size, Greek black pig has a high degree of genetic variability allowing a maintenance of the breed by breeding programs based on microsatellite analysis. In addition,

CONTACT Ivona Djurkin Kusec [email protected] Josip Juraj Strossmayer University of Osijek, Faculty of Agriculture in Osijek, Vladimira Preloga 1, 31000 Osijek, Croatia. Supplemental data for this article can be accessed on the publisher's website. ß 2018 Taylor & Francis Group, LLC.

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Table 1. List of microsatellite markers used in the study Name SSC Size range (bp) Annealing temp.,  C SO155 SW240 SO226 SO002 SO227 SO005 IGF1 SW122 SW632 SO225 SO178 SW911 SW951 SO090 SO215 SW857 SW24 SO218

1 2 2 3 4 5 5 6 7 8 8 9 10 12 13 14 17 X

150–166 96–115 181–205 190–216 231–256 205–248 197–209 110–122 159–180 170–196 110–124 153–177 125–133 244–251 135–169 144–160 96–121 164–184

57 53 57 57 56 56 57 53 53 56 57 53 53 57 55 56 54 55

Forward primer

Reverse primer

TGTTCTCTGTTTCTCCTCTGTTTG AGAAATTAGTGCCTCAAATTGG GCACTTTTAACTTTCATGATACTCC GAAGCCCAAAGAGACAACTGC GATCCATTTATAATTTTAGCACAAAGT TCCTTCCCTCCTGGTAACTA GCTTGGATGGACCATGTTG TTGTCTTTTTATTTTGCTTTTGG TGGGTTGAAAGATTTCCCAA GCTAATGCCAGAGAAATGCAGA TAGCCTGGGAACCTCCACACGCTG CTCAGTTCTTTGGGACTGAACC TTTCACAACTCTGGCACCAG CCAAGACTGCCTTGTAGGTGAATA TAGGCTCAGACCCTGCTGCAT TGAGAGGTCAGTTACAGAAGACC CTTTGGGTGGAGTGTGTGC GTGTAGGCTGGCGGTGGTTGT

AAAGTGGAAAGAGTCAATGGCTAT AAACCATTAAGTCCCTAGCAAA GGTTAAACTTTTNCCCCAATACA GTTCTTTACCCACTGAGCCA GCATGGTGTGATGCTATGTCAAGC GCACTTCCTGATTCTGGGTA CATATTTTTCTGCATAACTTGAACCT CAAAAAAGGCAAAAGATTGACA GGAGTCAGTACTTTGGCTTGA CAGGTGGAAAGAATGGAATGA GGCACCAGGAATCTGCAATCCAGT CATCTGTGGAAAAAAAAAGCC GATCGTGCCCAAATGGAC GCTATCAAGTATTGTACCATTAGG TGGGAGGCTGAAGGATTGGGT GATCCTCCTCCAAATCCCAT ATCCAAATGCTGCAAGCG CCCTGAAACCTAAAGCAAAG

microsatellite analysis can be applied for a product traceability as shown by Scali et al.8 who used a set of eight microsatellite markers as a unique fingerprint, which was later used as a tracer in the processed products. Apart from nuclear DNA analyses, animal mitochondrial DNA (mtDNA) has shown to have properties very useful for analyses in the studies of genetic diversity, population structure, and evolution of animal populations. Gupta et al.9 defined mtDNA as very abundant within the cells, small genome size, haploid, almost exclusively maternally inherited, with high mutation rate, and without genetic recombination. These characteristics make it a very good biomarker for studying genetic origin of animal breeds and populations including determination of genetic relationships. The control region of mtDNA (‘displacement loop’ or D-loop) is particularly interesting for studying intraspecific and interspecific relationships, maternal contributions, and tracing the origin of animals. For example, Molnar et al.10 provided the first DNAbased genetic evidence that Mangalitsa (MNG) is related to pigs that lived in the Carpathian basin. By studying the D-loop region of indigenous pigs and commercial swine on a global level Zhang et al.11 identified 334 haplotypes and 136 polymorphic sites. Lower level of diversity was found in the indigenous breeds when compared to the commercial type pigs. Authors concluded that introgression of commercial breeds into indigenous ones decreased their genetic diversity. In this study, a set of 18 microsatellite markers was used to evaluate the genetic structure of two Croatian autochthonous breeds assuming that genetic diversity within the breeds is sufficiently pronounced for their characterization. Furthermore, our intention was to investigate the genetic relationship among these breeds, as well as among other European and Asian breeds by mtDNA D-loop region analysis. Finally, this study should contribute to other comparative studies showing the importance of local breeds’ conservation as it is essential for the preservation of overall diversity in domestic animals.

Material and methods Samples and microsatellite genotyping The study was carried out on 20 BS pigs, 20 TP, and 20 Croatian wild boars (WCR). Hair samples have been collected from each animal and total genomic DNA was extracted using the phenol-chloroform extraction method.12 All animals were genotyped on 18 microsatellite loci chosen in accordance with their normativity, allele size, and location in the porcine genome and representing 15 porcine chromosomes including chromosome X (Table 1). Polymerase chain reactions (PCR) were performed in a 20.0 ll reaction volume containing 50 ng of genomic DNA, 100 lM of dNTPs, 1.5 mM MgCl2, 5 pMol of each primer, 0.5 Units of DNA Taq polymerase (Thermofisher Scientific), and 1  PCR buffer (10 mM Tris, pH 8.3, 50 mM KCl; Thermofisher Scientific). PCR was performed on PTC-100 thermal cycler (MJ Research, USA) with following conditions: initial denaturation at 95  C (5 minutes), followed by 35 cycles of denaturation at 95  C (45 seconds), annealing step (30 seconds) at annealing temperature characteristic for each primer (Table 1), and extension at 72  C (20 seconds), followed by final extension at 72  C (6 minutes). Each obtained PCR product (0.5–1.6 ll) was mixed with 12.0 ll formamide and 1.0 ll of Genescan-350 ROX standard and genescan analysis was performed on ABI 3730 DNA analyzer (Applied Biosystems, Foster City, CA, USA).

mtDNA analysis Two pairs of primers (Table 2) amplified a 487-bp fragment (Mit1.F and Mit1.R) and a 734-bp fragment (Mit2.F and Mi2.R) of the mitochondrial control region, covering 1168-bp between sites 16569 and 1124 of the Sus scrofa mitochondrion complete genome sequence (NCBI Reference Sequence: NC_000845.1).

ANIMAL BIOTECHNOLOGY

Table 2. Set of oligonucleotides used for amplification of two mtDNA fragments Primer name Mit1.F Mit1.R Mit2.F Mit2.R

Sequence 50 CGCCATCAGCACCCAAAGCT30 50 TGGGCGATTTTAGGTGAGATGGT30 50 CCGTGGGGGTTTCTATTGA30 50 ATTTTGGGAGGTTATTGTGTTGTA30

PCR reaction was performed in a 20.0 ll reaction volume as described in previous section. Reaction profiles included 2 minutes denaturation step at 94  C followed by 35 cycles of denaturation (50 seconds at 94  C), annealing (50 seconds at 60  C), extension (30 seconds at 72  C); with final 6-minutes extension step at 72  C. PCR products were purified using GeneJET Gel Extraction Kit (ThermoFisher Scientific) following agarose gel electrophoresis and sequenced on ABI 3730 DNA analyzer (Applied Biosystems, Foster City, CA, USA). To determine variable positions in the mtDNA D-loop regions of investigated pig breeds, aligned sequences were compared with mtDNA sequence GeneBank AJ002189.9 The GeneBank accession numbers of sequences, used together with the investigated pig breeds sequences in this study were EU117375, AY232892, AY232887, AY463076, AY463079, D17739, AF276929, AF276930, D42180, D42179, AB015094, AF276936, and AF304202.

Data analysis Number of alleles per locus, allelic frequencies, observed, expected heterozygosity and inbreeding coefficient (Fis) were calculated using Genetix version 4.05.2.13 All microsatellite loci were tested for deviation from the Hardy-Weinberg equilibrium (HWE) using a Markov Chain Monte Carlo simulation with 10,000 iteration steps and followed by 100 batches of 5000 iterations per batch14 in the GENEPOP version 3.4.15 Population structure was calculated using a factorial correspondence analysis (FCA) performed with the Genetix version 4.05.2.13 The STRUCTURE software16 was run to evaluate genetic structure in the analyzed dataset. The most likely K-value was identified according to Evanno et al.17 Program was run within 10 simulations with different starting points for each K-value. Program parameters were burn-in period of 100,000 iterations and 1,000.000 iterations for data collection periods. Visualization of STRUCTURE results was conducted in Clumpak software.18 Mitochondrial D-loop DNA sequences of Croatian pig breeds and 14 other pig breeds (from Europe, China, and New Zealand) were aligned in ClustalX1.83.19 Previously published sequences (GenBank accession numbers EU117375.1, AY232892.1, AY232887.1, AY463076.1, AY463079.1, D17739.1, AF276929.1, AF276930.1, D42180.1, D42179.1, AB015094.1, AF276936.1, and AF304202.1) were used for the alignment. The aligned

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sequences have been compared to the mtDNA sequence AJ002189.120 as the reference sequence. To perform phylogenetic analysis, 46 published sequences of mitochondrial control region were aligned with 708-bp part of the control region between positions 3 and 710 of the Sus scrofa mitochondrion complete genome sequence (NCBI Reference Sequence: NC_000845.1) of novel WCR (19 samples), TP (20 samples), and BS pig, BS (20 samples) control region sequences. Published sequences correspond to: MNG (AY232892.1; MNW, MNG white, JN601066.1; MNSB, MNG swallow-belly, JN601069.1), TP (TPGB, JN601073.1), different types of Iberian pig (IB, EU117375; IBHL, Iberian hairless, AY232852.1; BSQ, Basque, AY232891.1; IBR, Iberian red, AY232856.1), Pietrain (PI, AY232887.1; AY232886.1), Landrace (SWL, Swedish landrace, AF034253.1; GL, German landrace, AY230823.1; LAN, Landrace, AY232884.1; AY232885.1), Duroc (DUR, AY337045.1; AY232877.1), Large White (LW, AY574048.1; AY232882.1), Hampshire (HAM, AY574046.1), Westran or Australia feral (WES, AF276921.1), Welsh (WEL, AF276937.2), Large Black (LB, AY463075.1), Berkshire (BERK, AF276936.1) British Saddleback (BSB, DQ379205.2), Tamworth (TAM, AY463073.1), Kune Kune (KUN, AY463076.1), Meishan (MEI, D17739.1), Neinjang (NEI, AF276929.1), Jinghua (JIN, AF276930.1); Jabugo black spotted (JBS, AY232890.1), Erhualian (ERH, AF276922.1), Tongcheng (TON, AF276923.1), Yanxin (YAN, AF276927.1), Wanhua (WAN, AF276932.1), Okinawa native pig (OKN, AB015092.1), Yucatan miniature pig (YMP, AB015093.1), different haplotypes of European wild boar (WEU, AB015094.1; FJ237003.1; FJ236999.1; FJ237002.1; WSP, AY232868.1; WSW, AF304203.1) and different haplotypes of Asian wild boar (WRY; Ryukyu wild boar, AB015087.1); WJP (Japanese wild boar, AB015085.1); WCH (Chinese wild boar, EU333163.1). A warthog sequence (AB046876.1) was used as an outgroup. The tandem repeat motif of the control region was excluded from the analysis due to its high degree of heteroplasmy.21,22 The ClustalW software19 was used for alignment of 104 sequences from the control region. Maximum parsimony (MP) and MP bootstrap (MPB) analyses for ITS (Internal transcribed spacer) and plastid datasets were performed using PAUP 4.0b10.23 The most parsimonious trees were searched for heuristically with 100 replicates of random sequence addition, TBR (Tree bisection and reconnection swapping), and MulTrees off. The swapping was performed on a maximum of 1000 trees (nchuck ¼ 1000). All characters were equally weighted and unordered. The data set was bootstrapped using full heuristics, 1000 replicates, TBR branch swapping, MulTrees option off, and random addition sequence with five replicates. Distantly related warthog sequence, WH (AB046876.1) was used for rooting.

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Bayesian analyses were performed with MrBayes 3.2.124 applying the HKYI þ C substitution model proposed by the Akaike information criterion implemented in MrAIC.pl 1.4.25 Values for all parameters, such as the shape of the gamma distribution, were estimated during MrBayes analyses. The settings for the Metropoliscoupled Markov chain Monte Carlo process included four runs with four chains each (three heated ones using the default heating scheme), run simultaneously for 10,000,000 generations each, sampling trees every 1000th generation using default priors. The posterior probability (PP) of the phylogeny and its branches was determined from the combined set of trees, discarding the first 1001 trees of each run as burn-in. In addition, a NeighborNet of the ingroup (without the outgroup warthog sequence) was produced using SplitsTree4 12.3.26

Results Microsatellite analysis Inter population genetic diversity Allele number (A), observed heterozygosity (Hobs), expected heterozygosity (Hexp), inbreeding coefficient (Fis), and statistical significance of Fis value (Fis sign) for three investigated pig populations are presented in Table 3. For all 18 loci expected heterozygosity (Hexp) ranged from 0.648 for BS pig to 0.310 for TP. The mean value of observed heterozygosity (Hobs) ranged from 0.592 for BS pig to 0.352 for TP. Level of inbreeding coefficient (Fis) ranged from 0.116 for BS pig to 0.105 for TP. The mean number of alleles (A) ranged from 2.722 for TP to 5.722 for the WCRs. Mean number of alleles for BS pig was 5.611. The average Fst of all loci was 0.24. Multilocus Fst values indicated that around 24% of the total genetic

variation was explained by population differences, with the remaining 76% corresponding to differences among individuals within population. Table 4 shows mean FST estimates between observed populations and their significance by Wright.27 Significant values (P < 0.001) were found between all investigated populations.

Structure analysis Although three pig populations were investigated, structure analysis of observed populations, and calculation of DK indicated the existence of the subpopulations in the population of BS pig. Ideal K value was three and was in accordance with value obtained by Evanno et al.17 At K ¼ 2 two pig populations can be observed: BS pig population and TP in first genetic cluster and WCR population in second genetic cluster. At K ¼ 3, a clear clustering of BS pig, TP, and WCR populations can be observed. At K ¼ 4 BS pig population divided into two subpopulations. It can be assumed that the first genetic cluster is represented by crossings of BS pig, and the second genetic cluster is presented by pure animals (Fig. 1). The results of FCA indicate that BS pig was separated from the other investigated breeds by the first and the second axes (Fig. 2). The largest genetic distance was recorded between TP and other two pig populations (BS and WCR). Total variability is accounted with nearly 23.16%.

Genetic variation within analyzed populations Genetic diversity parameters of 18 microsatellite markers are presented in Table 5. The mean number of alleles was 9.944. Locus SO005 had the highest number of alleles,23 while locus SO227 and SO218 had the lowest number of alleles.4 The genetic differentiation was

Table 3. Number of alleles, heterozygosity, inbreeding coefficient, and statistical significance for observed pig populations BS pig (n ¼ 20) MS locus SW24 SW857 SO225 SO227 SW240 SO215 SO218 SO005 SW122 SO155 SO226 SO090 SO178 SW911 SO002 SW951 SW632 IGF1 Overall

A 8 6 6 2 7 1 2 9 7 8 6 4 8 6 6 4 7 4 5.6111

HExp 0.601 0.722 0.720 0.04 0.795 0.049 0.390 0.828 0.822 0.821 0.826 0.635 0.789 0.715 0.775 0.476 0.748 0.810 0.649

HObs 0.550 0.850 0.500 0.050 0.200 0.050 0.250 0.600 0.800 0.650 0.750 1.000 0.950 0.700 0.800 0.400 0.650 0.900 0.5916

FIS 0.111 –0.152 0.329 –0.000 0.759 –0.000 0.395 0.298 0.053 0.233 0.118 –0.077 –0.244 0.047 –0.007 0.185 0.126 –0.086 0.116

TP (n ¼ 20) FIS sign 0.233 0.990 0.010 – 0.000 – 0.107 0.001 0.082 0.001 0.040 0.917 0.491 0.554 0.174 0.006 0.089 0.020 0.232

A 2 2 2 2 4 2 2 2 2 4 3 5 5 2 3 3 2 2 2.722

HExp 0.499 0.349 0.095 0.489 0.143 0.420 0.000 0.226 0.420 0.611 0.095 0.548 0.303 0.000 0.454 0.141 0.255 0.534 0.310

HObs 0.850 0.450 0.100 0.850 0.100 0.600 0.000 0.250 0.600 0.650 0.000 0.650 0.350 0.000 0.600 0.050 0.200 0.0500 0.3527

WCR (n ¼ 20) FIS –0.691 –0.267 –0.027 –0.727 0.321 –0.407 1.000 –0.080 –0.407 –0.038 1.000 –0.162 –0.132 1.000 –0.299 0.661 0.240 0.911 0.105

FIS sign 0.005 0.528 1.000 0.002 0.077 0.118 – 1.000 0.122 0.535 0.026 0.659 1.000 – 0.392 0.028 0.354 0.000 0.365

A 3 4 4 6 9 3 4 11 6 6 4 6 7 5 8 3 8 6 5.7222

HExp 0.411 0.614 0.556 0.289 0.859 0.379 0.651 0.819 0.781 0.694 0.676 0.791 0.794 0.748 0.741 0.476 0.773 0.761 0.656

A: alleles; FIS: inbreeding coefficient; HExp: expected heterozygosity; HObs: observed heterozygosity; MS locus: microsatellite locus

HObs 0.300 0.600 0.600 0.350 0.500 0.250 0.300 0.900 0.900 0.500 0.350 0.750 0.600 0.650 0.400 0.050 0.900 0.800 0.539

FIS 0.294 0.048 –0.053 –0.187 0.439 0.362 0.557 –0.074 –0.127 0.303 0.502 0.078 0.268 0.156 0.480 –0.000 –0.123 –0.025 0.161

FIS sign 0.106 0.370 0.821 1.000 0.000 0.002 0.000 0.723 0.850 0.081 0.000 0.309 0.012 0.138 0.000 – 0.488 0.453 0.315

ANIMAL BIOTECHNOLOGY

Table 4. Fst between populations and Fst signification values S TP WCR

BS

TP

WCR



0.298 –

0.126 0.3025 –

BS: Black Slavonian pig; TP: Turopolje pig; WCR: Croatian wild boar. P < .001

estimated by F-statistics parameters (inbreeding coefficient, fixation index, overall fixation index). Value of inbreeding coefficient (Fis) ranged from 0.066 for SW240 to 0.115 for the locus SO227. Fixation index (Fit) at 18 loci was in the range from 0.326 at locus SW240 to 0.367 at locus SW122. The highest value of fixation index (Fst) was 0.288 at locus SO155 and the lowest value, 0.266, was recorded at SW24 locus.

Mitochondrial DNA analysis mtDNA D-loop sequences of BS pig, TP, WCR, WEU, BERK, MNG, IB, SWL, LW, PI, DUR, MEI, Neijiang, JIN, Auckland Island Pig (AIP), and Kunekune (KUN) are presented in Table 6. In total 39 polymorphic sites in the 700-bp sequences representing 5.57% of the analyzed sequence were determined. Thirty-five variable positions represented single nucleotide substitutions and the remaining four were insertion/deletion of single base pairs.

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Both Bayesian and parsimony analyses (Supplementry material Electronic appendix) resulted in unresolved trees, i.e., none of the clades had PP >0.95, except the one consisting of the accessions JBS, KUN, LB, LW1, and LW2 (PP 0.99); the same clade had a bootstrap support 75% in parsimony analysis. However, in NeighborNet of ingroup sequences (Fig. 3) two clusters of accessions were revealed, separated by strongly weighted splits. One cluster included European-type pigs, whereas the other consisted of Asian-type pigs including BERK, one PI haplotype, Tamworth, LW, and Large Black. Some accessions (Chinese wild boar from north-east; CWB), GL, YMP, two Iberian-type pigs (IB and IBHL), WEL, New Zealand feral, and Swedish wild boar were positioned along the main split between both terminal clusters. In the European terminal cluster, four groups can be distinguished. WCRs clustered together with previously published WEU accessions. This cluster contains also two TPs (TP4 and TP20) and two Iberian breeds (IBR and BSQ). Second cluster consists of modern European pigs, Landrace (LAN1 and LAN2, SWL), DUR 1 and 2, and PI2. Croatian autochthonous pig breeds clustered between modern and old European breeds (three types of MNG and TP from Austria). All TPs are in the middle of this cluster, while BS pigs are more spread, some are closer to old European breeds (BS5-7, 12, 13), some are in the middle together with Turopolje breed (BS3, 4, 9-11, 16,

Figure 1. Population assignment proportions based on results from STRUCTURE for K ¼ 2-7. Visualization was conducted by Clumpak software. Bar plots are read from left to right, with bars representing pig populations and the color of the bar representing the proportion of markers that originated from certain population. BS, Black Slavonian pig; TP, Turopolje pig; WCR, Croatian wild boar.

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Figure 2. Graphical representation of FCA analysis.

17, 19 and 20) and some are closer to modern European breeds (BS2, 8, 14, 15).

Discussion Results of the microsatellite analysis showed high diversity of loci. Average number of alleles per locus was 4.68. Barker28 suggested that microsatellite loci used in studies of genetic distance should have no less than four alleles to reduce the standard errors of distance estimates. The information on number of alleles, heterozygosity, inbreeding coefficient, and statistical significance show that microsatellites used in this study were suitable for genetic diversity analysis. Average heterozygosity Table 5. Parameters of genetic diversity for 18 microsatellite markers Locus

Chr

Na

Ho

He

Fis

Fit

Fst

SW24 SW857 SO225 SO227 SW240 SO215 SO218 SO005 SW122 SO155 SO226 SO090 SO178 SW911 SO002 SW951 SW632 IGF1 Average

17 14 8 4 2 13 X 5 6 1 2 12 8 9 3 10 7 5

9 8 12 4 14 5 4 23 9 11 10 8 12 8 11 5 12 14 9.944

0.503 0.633 0.400 0.416 0.266 0.300 0.183 0.583 0.766 0.600 0.366 0.700 0.650 0.450 0.600 0.166 0.583 0.583 0.486

0.566 0.561 0.457 0.275 0.598 0.282 0.350 0.624 0.674 0.708 0.532 0.657 0.628 0.487 0.656 0.222 0.587 0.701 0.531

0.109 0.110 0.095 0.115 0.066 0.102 0.083 0.098 0.114 0.091 0.084 0.108 0.105 0.098 0.097 0.094 0.102 0.090 0.098

0.346 0.359 0.347 0.362 0.326 0.356 0.341 0.346 0.367 0.353 0.333 0.359 0.357 0.351 0.349 0.350 0.346 0.352 0.350

0.266 0.279 0.273 0.279 0.278 0.282 0.281 0.275 0.285 0.288 0.272 0.281 0.281 0.280 0.278 0.282 0.271 0.287 0.279

Chr: chromosome; FIS: inbreeding coefficient; FST: fixation index; FIT: overall fixation index; Ho: observed heterozygosity; He: expected heterozygosity; Na: number of alleles

(expected and observed) of the selected microsatellite markers was between 0.486 and 0.531. Takezaki and Nei29 determined that an average heterozygosity in the population ranging between 0.3 and 0.8 is required for useful assessment of genetic variation. Markers used in this study fulfilled this criterion for genetic diversity analysis. The average observed heterozygosity was lower than expected in the population of BS and WCR. However, the average observed heterozygosity in TP population was higher than expected. Nevertheless, observed heterozygosity in TP population was 0.35 and mean number of alleles was 2.7, clearly showing that Turopolje breed lacks genetic variability. These results are in accordance with the results of Druml et al.,30 who reported heterozygosity 0.38 and mean number of alleles 3.29 for TPs. The authors explained that higher observed heterozygosity was a result of mating strategy in this herd, where small number of boars was used for reproduction. Although the heterozygosity observed in the studied Croatian pig populations was similar to other economically less important European pig breeds,30 they seemed to be somewhat lower than those reported for Chinese pig breeds.5,31 The Fst statistic is an estimate of genetic variation due to differences among populations, which is the reduction in heterozygosity of a subpopulation due to genetic drift. According to Fst values, Li et al.32 reported that distinction of Asian native pig breeds from European breeds was evidently pronounced. Mean Fst value (0.279) for BS and other investigated breeds based on 18 SSR loci in this work is comparable with other European breeds8 and shows that Croatian pig populations are well differentiated. The highest Fst value was obtained between Turopolje breed and Wild boar population with the coefficient 0.303, and between Turopolje and BS breed with

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Table 6. Variable positions in mtDNA D-loop regions of investigated pig breeds Breed Nucleotide position in porcine D-Loop regiona 1 5 4 3 7 A BS1 G BS2 G BS3 G BS4 G TP1 G TP2 G TP3 G WCR1 . WCR2 . WCR3 . WEU . BERK . MNG . IB . SWL . LW . PI . DUR . MEI . NEI . JIN AIP . KUN .

1 5 5 2 1 G . . . . . . . . . . A . . . . . . . . . . . .

1 5 5 4 0 T . . . . . . . . . . . C . . . C C . C C C . C

1 5 5 4 1 G . . . . . . . . . . . . . A . . . . . . . . .

1 5 5 5 5 A T T T T T T T . . . . . T . . . . T . . . T .

1 5 5 5 9 A . . . . . . . . . . G . . . . . . . . . . . .

1 5 5 6 2 G . . . . . . . . . . . A . . . A A . A A A . A

1 5 5 6 8 C . . . _ . . . . . . . _ . . . _ _ . _ _ _ . _

1 5 5 7 4 A . . . . . G . . . . . . . . . . . . . . . . .

1 5 5 7 5 _ A . . . . . . . . . . . . . . . . . . . . . .

1 5 5 7 7 C . . . . . . . . . . . T . . . T T . T T T . T

1 5 5 8 5 C . . . . . . . . . . T T . . . T T . T T T . T

1 5 5 9 0 A . . . . . . . . . . G G . . . G G . G G G . G

1 5 6 1 3 T

1 5 6 6 6 _

1 5 6 7 4 T

1 5 6 8 6 T

. . . . . . . C . . . C . . . . . . . . G . . . G . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . C . . . C . . . C . . . C . C . C . . C C . . . . . . . C . C .

1 5 8 0 6 C _ _ _ _ _ _ _ _ _ _ _ _ _ _ . _ _ _ _ _ _ _ _

1 5 8 1 0 T . . . . . . . . . . C . . . . . . . . . . . .

1 5 8 1 3 T C C C C C C C C C C . . C . . . . C . C . C C

1 5 8 1 4 T . . . . . . . . . . . . . C . . . . . . . . .

1 5 8 2 8 A . . . . . . . . . . G G . . . G G . G G G . G

1 5 8 4 0 C . . . . . . . . . . . T . . . T T . T T T . T

1 5 8 5 7 T C C C C C C C C C C . . C C . . . C . . . C .

1 5 9 2 4 C . . . . . . . . . . . T . . . T T . T T T . T

1 5 9 3 9 T . . . . . . . . . . . . . . . . . . . . . . C

1 5 9 4 7 C . . . . . . . . . . . . . . . . T . . . . . .

1 5 9 7 7 A . . . . . . . . . . G . . . . . . . . G . . .

1 5 9 8 6 C . . . . . . . . . . . . . . . . . . . T . . T

1 6 0 1 5 T . . . . . . . . . . . C . . . . . . . . . . .

1 6 0 3 5 A . . . . . . . . . . . G . . . G G . . . . . .

1 6 0 5 8 A . . . . . . . . . . . . . . . . . . . . G . .

1 6 0 9 4 T . . . . . . . . . . C . . . . C . . . . . . .

1 6 1 0 9 A . . . . . . . . . . G G . . . G G . G G G . G

1 6 1 4 4 T . . . . . . . . . . G . . . . . . . . . . . .

1 6 1 4 9 T . . . . . . . . . . . C . . . . . . . . . . .

1 6 2 0 2 A . . . . . . . . . . G . . . . . . . . . . . .

1 6 2 2 3 T . . . . . . . . . . C . . . . . . . . . . . .

1 6 2 2 6 A . . G G G G G G G G . G . G . G G G G G G . G

a

Nucleotide positions are numbered according to the reference sequence GenBank AJ002189.1 (Ursing and Arnason, 1998). AIP: Auckland island pig; BERK: Berkshire; BS: Black Slavonian pig; DU: Duroc; IB: Iberian pig; JIN: Jinghua; KUN: Kunekune; LW: Large white; MEI: Meishan; MNG: Mangalitsa; NEI: Neijang; PI: Pietrain; SWL: Swedish landrace; TP: Turopolje pig; WCR: Croatian wild boar; WEU: European wild boar.

coefficient 0.298. These results were supported with results of FCA, where the largest genetic distance was recorded between TP and other two pig populations (BS and WCR). The FCA analysis separated BS pig from other pig breeds by the first and second axes; however, the separation from WCR was not significant by the first axes (Fig. 2). Clustering analysis showed the existence of the crossbreds in BS population indicating the introgression of other pig breeds, such as LW, Landrace, or DUR, which can be attributed to its breeding history and/or production system.33,34 The Structure results showed subclustering of BS pig population at higher K values, which confirmed previous findings30 that BS breed is split into three gene pools. These results are supported by previous studies on MC1R coat color gene, which showed that more than half of BS pigs are heterozygous for black coat color,33 probably due to the uncontrolled crossing with commercial white pigs in the near past. Pairwise distance estimation based on the maximumlikelihood method showed that Croatian pigs had mean distance 0.00237 and 0.00113 within the group, Chinese pigs had mean distance 0.00575 and 0.00238 within the group, domestic European pigs 0.01619 and 0.003, rare European breeds (IB and MNG) 0.00725 and 0.00328, while New Zealand pigs 0.023 and 0.006, respectively. The distances between Croatian and other pig breeds differed considerably (0.015, 0.001, 0.003, and 0.005). Low

genetic distances within TP breed can be explained with high degree of inbreeding during past century.35 Interestingly, the genetic distance between MNG and AIP was zero proposing the possibility that mtDNA sequence of AIP used in this study is of MNG mitochondrial origin. Results of Robins et al.36 showed that AIP is closely related to European pig breeds and therefore could be a descendant of MNG. Similarly, Kim et al.37 found 100% sequence identity in mtDNA D-loop between two Westran pigs. The authors argued that these animals were derived from a feral population and very likely originated from a boar and a sow released on Kangeroo Island by a French explorer Nicholas Baudin in 1803. Some sequence similarity between mtDNA of BS pig and DUR breed can be observed from the alignment. This is most probably a result of uncontrolled crossing with modern pig breeds that occurred before the population became officially monitored by professional service. Bayesian and parsimony analyses resulted in unresolved trees. The tree (Supplementry material Information Electronic appendix) divided pigs into two clades—one with European-type pigs and the other containing Asiantype pigs. The same was observed also in NeighborNet of ingroup sequences (Fig. 3), where these two clades were separated by strongly weighted splits. In the European clade, Wild boars form their own group (containing also Wild boars from Croatia). Croatian autochthonous pig breeds are placed in their own clade, between modern and old European domestic

8

 ET AL. K. GVOZDANOVIC

Figure 3. Phylogenetic tree of 46 pig breeds.

pigs. TPs were positioned close together in the center of the clade, and BS pigs were more spread, some closer to old European breeds, some in the middle together with Turopolje breed and some closer to modern European breeds. While TP breed is among the oldest breeds in Europe, BS pig breed was formed at the end of the 19th century by crossing MNG pigs with BERK and Large Black boars. All investigated BS pigs possessed European-type mtDNA, while BERK and Large Black clustered together with Asian-type pigs. This is in line with the mating pattern used in establishment of BS breed, where only BERK and Large Black boars were crossed with locally raised MNG sows. This also explains why one group of BS pigs is closer to old European breeds (i.e., different MNG types). Some BS and TPs clustered together. This can be explained by the fact that MNG was dominant breed, raised in the same

geographical area than Turopolje breed, which often led to uncontrollable crosses with Turopolje breed.38 The third group of BS pigs, which is closer to modern European pig breeds (Landrace, DUR, and PI) is most likely a consequence of uncontrolled crossings with modern pig breeds in the near past before the breed became officially monitored by professional service. Tree groups of BS regarding mtDNA are in line with the results of microsatellite clustering obtained with Structure. In the Asian clade, beside Chinese, Japanese, and Korean domestic pigs and wild boars, also one PI1, Tamworth, BERK, LW, and Large Black pigs were included. Giuffra et al.39 reported that explanation for fairly high frequency of Asian mtDNA haplotypes in these breeds is due to the introgression of Asian pigs in Europe during the 18th and early 19th century. Authors suggested

ANIMAL BIOTECHNOLOGY

that the maternal inheritance of mtDNA implies that Asian sows were used for introgression. Kim et al.37 reported similar results and suggested that the clustering of BERK and LW breeds with Asian (Chinese) pigs clearly demonstrates that Asian pigs were involved in the development of these breeds. The presence of Asian mtDNA haplotypes in other pig breeds such as PI has been reported for the first time from Jones.40 This introgression could be explained by the contribution of BERK and LW pigs to the origin of PI breed. Genetic analyses based on microsatellite markers and on mtDNA showed that both Croatian autochthonous breeds are well defined. The genetic variability of Turopolje breed is low and additional efforts are needed to increase this breed genetic diversity. On the contrary, negative effects of low genetic diversity should be expected in the future. It seems that BS pig is not genetically uniform and that it consists out of few (two to three) genetic pools. We assume that this is, at least partially, consequence of uncontrolled crossings with modern pig breeds in the near past before the breed became officially monitored by professional service. Further investigations using high-throughput technologies and larger number of animals of different breeds are suggested. In general, the information on the genetic diversity obtained in this study can be further used in Croatian autochthonous pig breeding programs, as well as for parentage analysis, identification of individuals and traceability of the investigated breeds in pork products.

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Acknowledgments The authors thank Bozo Frajman from Department of Botany, University of Innsbruck for help with phylogenetic analyses and for valuable discussion.

Funding This work is supported by Croatian Science Foundation under the project number 3396; Hrvatska Zaklada za Znanost.

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