This is the accepted version of the article: Bâlteanu, Valentin Adrian; Cardoso, Tainã Figueiredo; Amills i Eras, Marcel; [et al.]. «The footprint of recent and strong demographic decline in the genomes of Mangalitza pigs». Animal, Vol. 13, issue 11 (Nov. 2019) , p. 2440-2446. DOI 10.1017/S1751731119000582 This version is avaible at https://ddd.uab.cat/record/214411 under the terms of the license This is the accepted version of the article: Bâlteanu, Valentin Adrian; Cardoso, Tainã Figueiredo; Amills i Eras, Marcel; [et al.]. «The footprint of recent and strong demographic decline in the genomes of Mangalitza pigs». Animal, Vol. 13, issue 11 (Nov. 2019) , p. 2440-2446. DOI 10.1017/S1751731119000582 This version is avaible at https://ddd.uab.cat/record/214411 under the terms of the license 1 The footprint of recent and strong demographic decline in the genomes of 2 Mangalitza pigs 3 4 V.A. Bâlteanu1*, T.F. Cardoso2,3*, M. Amills2,4, I. Egerszegi5, I. Anton6, A. Beja- 5 Pereira7, A. Zsolnai6 6 1 7 Institute of Life Sciences, University of Agricultural Sciences and Veterinary 8 Medicine, Cluj-Napoca, Romania. 2 9 Department of Animal Genetics, Centre for Research in Agricultural Genomics 10 (CRAG), CSIC-IRTA-UAB-UB, Campus de la Universitat Autònoma de Barcelona, 11 Bellaterra, Spain. 3 12 CAPES Foundation, Ministry of Education of Brazil, Brasilia D. F., Brazil. 4 13 Departament de Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona, 14 Bellaterra, Spain. 5 15 Szent István University, Gödöllő, Hungary 6 16 NAIK-Research Institute for Animal Breeding, Nutrition and Food Science, 17 Herceghalom, Hungary. 7 18 Centro de Investigacao em Biodiversidade e Recursos Geneticos, Universidade do 19 Porto (CIBIO-UP), Vairao, Portugal 20 21 *V. A. Bâlteanu and T. F. Cardoso share the first authorship 22 Corresponding author: A. Zsolnai. E-mail: attila.zsolnai@gmail.com 23 1 24 SHORT TITLE: Inbreeeding in Mangalitza pigs 25 26 Abstract 27 28 The Mangalitza pig breed has suffered strong population reductions due to 29 competition with more productive cosmopolitan breeds. In the current work, we 30 aimed to investigate the effects of this sustained demographic recession on the 31 genomic diversity of Mangalitza pigs. By using the Porcine Single Nucleotid 32 Polymorphism BeadChip, we have characterized the genome-wide diversity of 350 33 individuals including 45 Red Mangalitza (number of samples; N = 20 from Hungary 34 and N = 25 from Romania), 37 Blond Mangalitza, 26 Swallow-belly Mangalitza, 48 35 Blond Mangalitza x Duroc crossbreds, 5 Bazna swine, 143 pigs from the Hampshire, 36 Duroc, Landrace, Large White and Pietrain breeds and 46 wild boars from Romania 37 (N=18) and Hungary (N=28). Performance of a multidimensional scaling plot showed 38 that Landrace, Large White and Pietrain pigs clustered independently from 39 Mangalitza pigs and Romanian and Hungarian wild boars. The number and total 40 length of ROH (runs of homozygosity), as well as FROH coefficients (proportion of the 41 autosomal genome covered ROH) did not show major differences between 42 Mangalitza pigs and other wild and domestic pig populations. However, Romanian 43 and Hungarian Red Mangalitza pigs displayed an increased frequency of very long 44 ROH (> 30 Mb) when compared to other porcine breeds. These results indicate that 45 Red Mangalitza pigs underwent recent and strong inbreeding probably as a 46 consequence of severe reductions in census size. 47 48 Keywords: polymorphisms, population, structure, admixture, homozygosity 2 49 Implications: The excellent meat quality of the Mangalitza pig breed has favoured its 50 expansion in Hungary and several other countries. Here, we have analysed the 51 population structure and genome-wide diversity of Mangalitza pigs. When compared 52 to cosmopolitan breeds, Red Mangalitza pigs show a high genomic coverage of very 53 long (> 30 Mb) ROH (runs of homozygosity). These results indicate the occurrence of 54 recent and strong inbreeding. In order to avoid the detrimental consequences of 55 inbreeding depression, Mangalitza breeder associations should implement 56 reproductive strategies aiming to minimize inbreeding. 57 58 Introduction 59 60 Many European local pig breeds have experienced a sustained demographic 61 recession due to indiscriminate crossbreeding, competition with more productive 62 breeds, decline of traditional production systems, progressive abandonment of rural 63 activities and loss of grazing land (FAO, 2015). In Europe and the Caucasus, at least 64 90 breeds have disappeared and many others are endangered or face extinction 65 (FAO, 2015). The progressive loss of local porcine breeds may have a negative 66 impact on the production of highly specialized traditional fresh and cured meat 67 products. 68 One appropriate model to investigate the consequences of strong 69 demographic contractions on genetic diversity is the Mangalitza breed, which is 70 distributed in Hungary and, with a much lower population census, in Romania, 71 Germany, Austria and Switzerland (Egerszegi et al., 2003). In 1927-1930, there were 72 1 000 - 1 920 Mangalitza pigs in Hungary, and this number peaked to 17 691 73 individuals in 1955. However, this breed experienced a very serious demographic 3 74 decline during the two subsequent decades, mainly due to competition with more 75 productive breeds. Noteworthy, only 34 breeding sows were registered in the herd- 76 book in 1975 (Egerszegi et al., 2003). Fortunately, the establishment of conservation 77 genetic plans allowed the demographic recovery of this breed (Egerszegi et al., 78 2003). In Romania, the Red Mangalitza breed accounted for 500 individuals in 1983, 79 but only 34 pigs remained in 1996, and nowadays this population faces extinction 80 (Egerszegi et al., 2003) and it has a low diversity (Manunza et al., 2016). In principle, 81 this sustained demographic decline is expected to reduce genetic variation and to 82 increase the levels of inbreeding of Mangalitza pigs, two features that might threaten 83 the genetic conservation of this ancient traditional breed. 84 In the current work, we aimed to characterize the genome-wide diversity of the 85 Hungarian and Romanian Mangalitza breeds, other porcine and wild boar 86 populations in order to assess if the strong population reduction that the Mangalitza 87 breed has experienced during the second half of the 20th century has resulted in a 88 substantial increase of whole-genome homozygosity. Such results would be relevant 89 to ascertain if Mangalitza breeder associations need to implement reproductive and 90 genetic strategies aiming to minimize inbreeding. 91 92 Materials and Methods 93 94 Sample collection, DNA extraction and genotyping 95 Blood samples from Blond (number of samples; N = 37) and Swallow-belly (N 96 = 26) Mangalitza pigs as well as from Red Mangalitza pigs from Hungary (N = 20) 97 and Romania (N = 25) were collected in EDTA coated vacutainer tubes. For 98 comparative purposes, several reference populations were included in this study: 4 99 Blond Mangalitza x Duroc crossed pigs (N = 48), Bazna (N = 5), Duroc (N = 56), 100 Hampshire (N = 11), Landrace (N = 29), Large White (N = 27), Pietrain (N = 20) and 101 wild boar from Hungary (N = 28) and Romania (N = 18, reported in Manunza et al., 102 2016). Bazna is a local breed native to Romania with a black coat and white belt 103 reminiscent of Hampshire pigs (Draganescu et al., 2008). The Bazna breed is 104 famous because of its hardiness and high quality marbled meat. 105 Total DNA was isolated from the samples using a simple protocol (Zsolnai et 106 al., 2003). Single nucleotide polymorphisms (SNPs) were genotyped with the Porcine 107 SNP60 BeadChip (Illumina, San Diego, CA) or GeneSeek Genomic Profiler 108 50K Porcine SNP chip (Neogen, Scotland, UK). A series of quality control procedures 109 were conducted on the raw data using the SNP & Variation Suite software v.8.8.1 110 (Golden Helix, Bozeman, MT, USA). Linkage disequilibrium pruning (genotype 111 correlation coefficient; r2 > 0.5) was applied to the whole dataset. Linkage 112 disequilibrium between adjacent SNPs was measured with r2 calculated with the SNP 113 & Variation Suite software v.8.8.1. In addition, monomorphic markers and unmapped 114 SNPs, as well as those with a call rate < 95%, were eliminated from the dataset. In 115 addition, we removed SNPs with a minor allele frequency lower than 0.05. Duplicated 116 samples (identical by descent value > 0.95) and individuals with a genotype call rate 117 < 95% were removed. After filtering steps, the final dataset included 350 animals and 118 30 121 SNPs (Table 1). 119 120 Population genetics analyses 121 The proportion of mixed ancestry and population structure were evaluated with 122 the ADMIXTURE software v.1.3 (Alexander et al., 2009), by using default 5 123 parameters, and with FastStructure (Raj et al., 2014) by performing five test sets for 124 cross-validation and with Structure software (Evanno et al., 2005), considering burnin 125 periods of 10 000 steps followed by 100 000 additional Markov Chain Monte Carlo 126 iterations. ADMIXTURE calculates maximum likelihood estimates of individual 127 ancestries based on data provided by multiple loci (Alexander et al., 2009). 128 FastStructure is an algorithm for inferring population structure from SNP genotype 129 data based on a variational Bayesian framework for posterior inference (Raj et al., 130 2014). We evaluated different numbers of clusters (K-value, from 1 to 30) by 131 considering the mixed ancestry model in each software. The optimal K-value was 132 determined by taking into account (a) the estimates of the cross-validation errors 133 (Alexander and Lange, 2011) for ADMIXTURE outputs, (b) the highest probability for 134 FastStructure (Raj et al., 2014) and (c) the highest deltaK for Structure results 135 (Evanno et al., 2005) estimated with the STRUCTURE HARVESTER software 136 v0.6.94 (Dent and VonHoldt 2011) as implemented in the Web 137 server http://taylor0.biology.ucla.edu/structureHarvester. Generation of phylogenetic 138 trees and bootstrap analysis were performed with the TreeMix software (Pickrell and 139 Pritchard, 2012). A neighbour-joining tree including all analysed individuals was built 140 with MEGA7 (Kumar et al., 2016). The PLINK software v1.9 (Purcell et al., 2007) was 141 used to calculate observed (Ho) and expected (He) heterozygosities as well as to 142 build a multidimensional scaling (MDS) plot by using a genome-wide identity-by-state 143 pairwise distances matrix (--mds-plot 2 and --cluster options). The -het command of 144 the PLINK software v1.9 (Purcell et al., 2007) was used to compute the method-of- 145 moments relatedness F coefficient. The polymorphism information content (PIC) 146 within each population was computed with the Botstein’s (1980) method. The PIC 147 parameter for any given SNP i is defined as: 6 148 𝑃𝐼𝐶 = 1 − (𝑎 + (1 − 𝑎 ) ) − 2 ∗ 𝑎 ∗ (1 − 𝑎 ) , 149 where ai represents the minor allele frequency of SNP i. 150 The detection of ROH was carried out with the PLINK v.1.9 software (Purcell 151 et al., 2007). The minimum length of a ROH was set to 1 Mb in order to minimize the 152 detection of spurious ROH. To make sure that ROH length is not affected by low 153 SNP density, the minimum number of SNPs that constituted a ROH (𝑙) was set to 50, 154 considering the calculation method proposed by Lencz et al. (2007): 𝑙𝑜𝑔 𝑙= 𝑙𝑜𝑔 (1 − ℎ𝑒𝑡) 155 where ns is the number of SNPs per individual, ni is the number of individuals, 156 α is the percentage of false positive ROH (set to 0.05 in the present study) 157 and het is the mean SNP heterozygosity across all SNPs. The density of SNPs was 158 set to one SNP for each 100 Kb and a maximum distance of 1 000 Kb was allowed 159 between two consecutive SNPs. The scanning window contained 50 SNPs, and the 160 maximum number of missing SNPs per window was set to five with allowance for one 161 heterozygous SNP. 162 Each ROH was classified based on its physical length into four size 163 categories: 1 to ≤5 Mb, 5 to ≤15 Mb, 15 to ≤30 Mb, and >30 Mb. For each ROH 164 category, the mean sum of ROH per breed was calculated by summing the lengths of 165 all ROH in a given individual for each one of the categories under consideration. The 166 inbreeding coefficient derived from ROH genomic coverage (FROH) was calculated by 167 dividing total ROH length per individual by total genome length across all 18 168 autosomes (2 444 Mb) for each individual. The effective population number in the 169 recent past was calculated with the SNeP software (Barbato et al., 2015). 7 170 Results 171 172 Autosomal diversity and population structure 173 To investigate the genetic relationships between Red, Swallow-belly and 174 Blond Mangalitza swine and other pig and wild boar populations, we built an MDS 175 plot based on genome-wide identity-by-state pairwise distances calculated with 176 PLINK (Figure 1). The first principal component separated Duroc pigs from the 177 remaining breeds (Figure 1). As expected, Blond Mangalitza x Duroc crossed pigs 178 occupied an intermediate position between both parental populations. In the second 179 component, the cosmopolitan breeds Landrace, Large White and Pietrain clustered 180 independently from Mangalitza pigs and Romanian and Hungarian wild boars. The 181 Hampshire and Bazna pigs occupied an intermediate position between these two 182 clusters. We detected a close relationship between Mangalitza pigs and Hungarian 183 wild boars. By making a three-dimensional rotation of the MDS plot, we noted that 184 Romanian wild boar is clearly separated from Mangalitza pigs and, on the other 185 hand, it shows a close relationship with the Bazna breed (Supplementary Figure 186 S1). Descriptive statistics of genetic diversity are shown in Table 1. In purebred 187 populations, both observed and expected heterozygosities ranged between 0.28- 188 0.38, whilst Blond Mangalitza x Duroc swine showed high levels of heterozygosity 189 because of their hybrid origin (Ho = 0.42, He = 0.35). 190 The Admixture, fastStructure and Structure analyses showed that the most 191 probable K-value is K = 13 (Figure 2). Indeed, this was the K-value with the lowest 192 cross-validation error (Admixture), the highest probability (fastStructure) and also the 193 highest DeltaK value (Structure). Population structure analyses provided evidence of 194 genetic admixture for Blond Mangalitza x Duroc crossbreds, a result that could be 8 195 anticipated due to their hybrid origin, and also for Bazna pigs. Indeed, the Bazna 196 breed has a composite origin i.e. it was created in 1872 by crossing Mangalitza and 197 Berkshire pigs and, subsequently, it was introgressed with blood from Angler 198 Sattelschwein, Large White and Berkshire pigs (Ciobanu et al., 2001). We also 199 observed that Romanian Red Mangalitza, Hungarian Red Mangalitza and Blond and 200 Swallow-belly Mangalitza pigs have distinct genetic backgrounds. At K≥ 6, they split 201 up into three groups with different genetic background representing the Red, 202 Swallow-belly and Blond Mangalitza varieties (Supplementary Figure S2). 203 Population structure analyses also evidenced that Hungarian wild boar share part of 204 their genetic background with Hungarian Red, Swallow-belly and Blond Mangalitza 205 pigs, but not with the four cosmopolitan breeds (Duroc, Large White, Landrace, and 206 Pietrain). As previously published by Manunza et al. (2016), we identified two 207 Romanian wild boars that had been clearly introgressed with cosmopolitan breeds 208 (probably Large White). At K = 4, we observed four genetic backgrounds in domestic 209 pigs: (a) Duroc, (b) Large White, Landrace, and Pietrain, (c) Red Mangalitza from 210 Hungary and Romania, and (d) Blond and Swallow-belly Mangalitza, while Bazna 211 and Blond Mangalitza x Duroc crossed pigs showed evidence of admixture b 212 (Supplementary Figure S2). These results were consistently found at K = 4-9 and 213 reflect the common ancestry of Red Mangalitza pigs as well as the fact that 214 Hungarian Red Mangalitza swine were imported into Romania to “refresh the blood” 215 (i.e. reduce endogamy) of the local Red Mangalitza populations recently. The 216 analysis of Supplementary Figure S2 also shows that at K ≥ 10, Hungarian and 217 Romanian Red Mangalitza pigs display signs of genetic differentiation. The 218 phylogenetic tree depicted in Supplementary Figure S3 support the population 219 structure results reported above. Red Romanian and Hungarian populations are 9 220 genetically divergent. Moreover, the Hungarian wild boar population shows a close 221 relationship with Mangalitza pigs, while Romanian wild boar is genetically 222 differentiated from Mangalitza pigs and Hungarian wild boar. The coefficients of 223 genetic differentiation (FST) (Supplementary Figure S4) also evidence that 224 cosmopolitan breeds form a tightly related group and that Hungarian wild boar is 225 closely related to Mangalitza pigs. Moreover, a relevant genetic affinity between 226 Romanian and Hungarian wild boar can be also appreciated. 227 In the neighbour-joining tree shown in Supplementary Figure S5, Romanian 228 wild boars form a distinctive clade, with the only exception of two individuals 229 (identified as hybrid animals) which group with the cosmopolitan porcine breeds. 230 Duroc and Duroc x Blond F1 individuals are also distributed in independent clusters. 231 Romanian and Hungarian Red Mangalitza pigs display a close relationship though 232 there is some level of genetic divergence (they are located in different branches of 233 the tree). Hungarian wild boars are distributed in the three clades corresponding to 234 Blond, Swallow-belly and Red Hungarian Mangalitza pigs, highlighting the close 235 relatedness between these domestic and wild populations. 236 Estimation of the effective size showed that Red Romanian and Blond 237 Mangalitza pigs have the lowest and the highest values of effective population size 238 (Ne), respectively (Supplementary Figure S6), while Ne values of Swallow-belly and 239 Red Hungarian are in between. Such results are quite consistent with the current 240 census of Blond (N = 6 267), Swallow-belly (N = 1 298), Red Hungarian (N = 2 106) 241 and Red Romanian (N = 120) Mangalitza pigs. 242 243 244 10 245 Analysis of runs of homozygosity 246 We have characterized the length, distribution, and frequency of ROH in the 13 247 pig and wild boar populations under analysis. The number and coverage of ROH 248 (Figure 3) as well as FROH coefficients (Table 1) of Romanian Red and Blond 249 Mangalitza pigs were lower than those recorded in Duroc, Hampshire and Bazna 250 pigs, but similar to those measured in Large White, Landrace and Piétrain pigs. 251 Indeed, the analysis of Figure 3 does not show major departures between 252 Mangalitza pigs and cosmopolitan breeds with regard to the distribution of individuals 253 according to ROH number and genomic coverage. On the other hand, we have 254 observed that the majority of ROH were short (< 5Mb) and medium ROH (5-15 Mb), 255 i.e. 3 902 and 4 651 ROH measured 0-5 Mb and 5-15 Mb, respectively, in contrast 256 only 13,62% of the ROH displayed larger sizes i.e. 1,007 ROH from 15 to 30 Mb and 257 342 ROH larger than 30 Mb. Importantly, a comparative analysis of ROH classes 258 evidenced that Red Mangalitza pigs from Romania and Hungary (together with 259 Romanian wild boars) have a higher average proportion of their autosomal genomes, 260 155.7 Mb (Romanian Red) and 129.4 Mb (Hungarian Red), covered by very long 261 ROH (> 30 Mb). In strong contrast, in the remaining populations only 33 (Blond 262 Mangalitza x Duroc) to 102.4 Mb (Bazna) were covered by very long ROH (Figure 263 4). Finally, the levels of homozygosity in Blond Mangalitza x Duroc crossbred pigs 264 were negligible because of their hybrid nature. 265 266 Discussion 267 268 One of the main goals of our study was to investigate whether the strong 269 population recession experienced by Mangalitza pigs has resulted in a severe 11 270 contraction of their genetic diversity. In general, the observation of Figure 3 and 271 Table 1 does not show a convincing pattern of significantly increased homozygosity 272 in Mangalitza pigs. Indeed, ROH number and coverage and FROH coefficients 273 recorded in Mangalitza pigs are comparable to those measured in the majority of 274 cosmopolitan breeds (with the exception of Duroc). In a previous study, Yang et al. 275 (2017) showed that Mangalitza pigs display higher average FROH values than a broad 276 array of European and Chinese breeds, however the dispersion of the FROH data in 277 Mangalitza pigs was also very high making difficult to predict if such difference would 278 be statistically significant. Moreover, Yang et al. (2017) only analysed 20 individuals, 279 while in the current work we have sampled 108 Mangalitza pigs. 280 The moderate homozygosity of cosmopolitan breeds, which have been 281 subjected to a strong artificial selection, could be due to the fact that they carry Asian 282 alleles at variable frequencies since they were introgressed with Chinese sows 283 during the 18th-19th centuries. In contrast, the origin of Mangalitza pigs is exclusively 284 European (Manunza et al., 2016). Although Chen et al. (2013) indicated that 285 Hungarian Mangalitza pigs might have been weakly introgressed with Asian blood, a 286 worldwide analysis of porcine genetic diversity showed clear evidence that Chinese 287 introgression into Hungarian Mangalitza, Iberian, Cinta Senese and Nero Siciliano 288 pigs is negligible (Yang et al., 2017). We can conclude that the Mangalitza breed was 289 mostly spared from the introgression of Asian genes into European pig breeds that 290 took place during the 18th-19th centuries (Giuffra et al., 2000). 291 One of the most relevant results of our study is that the genomic coverage of 292 very long ROH (> 30 Mb) is particularly high in Romanian (155.7 Mb, 6.3% 293 autosomal genome) and Hungarian Red (129.4 Mb, 5.3% autosomal genome) 294 Mangalitza pigs. This finding is a clear indication of strong and recent inbreeding. 12 295 The dramatic reduction in population size combined with matings between related 296 individuals might be the main cause of the relatively high genomic coverage of very 297 long ROH in Red Mangalitza swine. In contrast, such pattern was not obvious in 298 Blond Mangalitza, evidencing that the three Mangalitza populations analysed in the 299 current work underwent different demographic histories. Indeed, the MDS plot 300 (Figure 1), the Admixture results (Figure 2) and the neighbor-joining tree 301 (Supplementary Figure S5) indicated the existence of genetic differentiation 302 between Blond, Swallow-belly and Red Mangalitza pigs. As previously explained, the 303 Red Mangalitza breed was produced by crossing, about one century ago, Blond 304 Mangalitza with Szalontai pigs, a high, bulky, robust and red-coated Hungarian 305 aboriginal race devoted to produce meat (Hankó, 1940). 306 One of the potential sources of genetic variability in Mangalitza pigs could be 307 gene flow with wild boars. Indeed, the genome-wide analysis of the diversity of 308 Mangalitza pigs indicated the existence of a close genetic affinity between this breed 309 and wild boars (Figure 1). In the Admixture analysis (Figure 2), Hungarian Red and 310 Blond Mangalitza pigs shared a common genetic background with Hungarian wild 311 boars. Similar results have been obtained when comparing Iberian pigs and Spanish 312 wild boars (Manunza et al., 2016), a finding that might be explained by the 313 occurrence of ancient and recent genetic exchanges between wild and domestic 314 pigs. Indeed, it is well known that Szalontai sows, which intervened in the formation 315 of the Red Mangalitza variety, produce stripped piglets, a strong indication of their 316 ancient and extensive introgression with local wild boars (Egerszegi et al., 2003). 317 Moreover, as many as 25% pigs of the commune of Bârzava (Arad County, 318 Romania) have been introgressed with wild boars (Matiuti et al., 2010). This high 319 level of introgression could be due to the extensive regime of pastoral management 13 320 that has been traditionally performed in certain areas of Romania, where pigs are 321 allowed to roam free providing a window of opportunity for the occurrence of 322 unintentional mattings with wild boars (Matiuti et al., 2010). As reported by Manunza 323 et al., (2016), two Romanian wild boars showed clear evidence of introgression with 324 domestic pigs, being the Large White breed the most likely source (Supplementary 325 Figure S2, K ≥ 4). Recently, Iacolina et al. (2018) reported a relatively high 326 abundance of hybrid wild boars in Austria, Bosnia and Herzegovina, Bulgaria and 327 Serbia. Moreover, a genome-wide analysis of the diversity of North European wild 328 boars revealed that about 10% of the individuals showed some evidence of 329 introgression from domestic pigs (Goedbloed et al., 2013), thus revealing that the 330 hybridization of pigs and wild boars is relatively frequent even in countries where pig 331 management is essentially intensive. 332 We have demonstrated that Red Mangalitza pigs have a relatively high 333 genomic coverage of very long ROH (> 30 Mb), a clear indication that this breed has 334 been subject to a strong and recent inbreeding. It is well known that inbreeding can 335 have negative consequences on both production (Silió et al., 2013) and reproduction 336 (Saura et al., 2015) performance, a phenomenon known as inbreeding depression 337 (Charlesworth and Willis, 2009). In order to avoid the detrimental consequences of 338 inbreeding depression, Mangalitza breeder associations should devise reproduction 339 and genetic strategies aiming to increase breeding values for production goals and at 340 the same time keeping inbreeding to a minimum. 341 342 Acknowledgments 343 Part of the research presented in this publication was funded by grant AGL2013– 344 48742-C2–1-R awarded by the Spanish Ministry of Economy as well as by projects 14 345 TKISSE and TGENRE supported by the Hungarian Ministry of Agriculture. We also 346 acknowledge the support of the Spanish Ministry of Economy and Competitivity for 347 the Center of Excellence Severo Ochoa 2016-2019 (SEV-2015-0533) grant awarded 348 to the Centre for Research in Agricultural Genomics (CRAG). Tainã Figueiredo 349 Cardoso was funded with a fellowship from the CAPES Foundation-Coordination of 350 Improvement of Higher Education, Ministry of Education of the Federal Government 351 of Brazil. Thanks also to the CERCA Programme of the Generalitat de Catalunya. 352 353 Declaration of interest. 354 The authors declare that they have no competing interests. 355 356 Ethics statement. 357 Sampling was performed by trained veterinarians following standard procedures and 358 relevant national guidelines to ensure appropriate animal care. 359 360 Software and data repository resources 361 None of the data were deposited in an official repository. 362 363 References 364 Alexander DH and Lange K 2011. Enhancements to the ADMIXTURE algorithm for 365 individual ancestry estimation. BMC Bioinformatics 12, 246. 366 Alexander DH, Novembre J and Lange K 2009. Fast model-based estimation of 367 ancestry in unrelated individuals. Genome Research 19, 1655–1664. 368 Barbato M, Orozco-terWengel P, Tapio M, and Bruford MW 2015. SNeP: a tool to 15 369 estimate trends in recent effective population size trajectories using genome-wide 370 SNP data. 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Code Breed (place of collection) N FROH Ho He F PIC BAZNA Bazna (Romania) 5 0.17 0.31 0.26 - 0.196 BLODU Blond Mangalitza x Duroc 48 0.01 0.42 0.35 -0.221 0.267 (Hungary) BLOMA Blond Mangalitza (Hungary) 37 0.10 0.31 0.30 -0.019 0.246 DUROC Duroc (Hungary) 56 0.16 0.31 0.30 -0.004 0.223 HAMPS Hampshire (Hungary) 11 0.14 0.29 0.25 - 0.200 HUNWB Wild boar (Hungary) 28 0.14 0.30 0.32 0.070 0.263 LANDR Landrace (Hungary) 29 0.12 0.36 0.36 -0.008 0.273 LWHITE Large White (Hungary) 27 0.08 0.37 0.36 -0.019 0.276 PIETRAIN Pietrain (Hungary) 20 0.10 0.36 0.36 -0.016 0.272 REDMAH Red Mangalitza (Hungary) 20 0.14 0.32 0.31 -0.034 0.250 REDMAR Red Mangalitza (Romania) 25 0.09 0.38 0.33 -0.120 0.268 ROMWB Wild boar (Romania) 18 0.09 0.28 0.32 - 0.227 SBMA Swallow-belly Mangalitza 26 0.14 0.29 0.28 -0.030 0.256 (Hungary) N= number of sampled individuals FROH= proportion of the autosomal genome covered by runs of homozygosity Ho= observed heterozygosities He= expected heterozygosities F=method-of-moments relatedness F coefficient PIC= polymorphism information content 20 LEGENDS TO FIGURES Figure 1. Multidimensional scaling plot (MDS) depicting the relationships between Mangalitza pigs and other wild boar and pig populations. The following codes are used: C1 = 1st component; C2 = 2nd component; DUROC = Duroc; BLODU = Blond Mangalitza x Duroc; HAMPS = Hampshire; BAZNA = Bazna; LANDR = Landrace; LWHITE = Large White; PIETRAIN = Pietrain; REDMAH = Red Mangalitza (Hungary); REDMAR = Red Mangalitza (Romania); BLOMA = Blond Mangalitza; SBMA = Swallow-belly Mangalitza; HUNWB = Wild boar (Hungary); ROMWB = Wild boar (Romania). Figure 2. Admixture analysis of Mangalitza pigs and additional wild boar and pig populations for the K-value with the lowest cross-validation error (K = 13). Each individual is represented by a single column divided into K colored segments, where K is the number of assumed clusters. Populations are separated by black lines. The following codes are used: DUROC = Duroc; BLODU = Blond Mangalitza x Duroc; HAMPS = Hampshire; BAZNA = Bazna; LANDR = Landrace; LWHITE = Large White; PIETRAIN = Pietrain; REDMAH = Red Mangalitza (Hungary); REDMAR = Red Mangalitza (Romania); BLOMA = Blond Mangalitza; SBMA = Swallow-belly Mangalitza; HUNWB = Wild boar (Hungary); ROMWB = Wild boar (Romania). Figure 3. Number and total length of runs of homozygosity (ROH) in Mangalitza pigs and additional wild boar and pig reference populations. The number of ROH estimated in each individual genome (y-axis) is plotted against total ROH total size (i.e. the number of Mb covered by ROH in each genome, x-axis). The following codes 21 are used: DUROC = Duroc; BLODU = Blond Mangalitza x Duroc; HAMPS = Hampshire; BAZNA = Bazna; LANDR = Landrace; LWHITE = Large White; PIETRAIN = Pietrain; REDMAH = Red Mangalitza (Hungary); REDMAR = Red Mangalitza (Romania); BLOMA = Blond Mangalitza; SBMA = Swallow-belly Mangalitza; HUNWB = Wild boar (Hungary); ROMWB = Wild boar (Romania). Figure 4. Classification of the runs of homozygosity (ROH) identified in Mangalitza pigs and additional wild boar and pig populations based on their size (x-axis) and mean sum of ROH (y-axis, measured in megabases) within each ROH category and averaged per breed. The following codes are used: DUROC = Duroc; BLODU = Blond Mangalitza x Duroc; HAMPS = Hampshire; BAZNA = Bazna; LANDR = Landrace; LWHITE = Large White; PIETRAIN = Pietrain; REDMAH = Red Mangalitza (Hungary); REDMAR = Red Mangalitza (Romania); BLOMA = Blond Mangalitza; SBMA = Swallow-belly Mangalitza; HUNWB = Wild boar (Hungary); ROMWB = Wild boar (Romania). 22 23 24 25 26 LEGENDS TO SUPPLEMENTARY MATERIALS Supplementary Figure S1. Three-dimensional multidimensional scaling (MDS) plot defining the genetic relationships between Mangalitza pigs and other swine and wild boar populations. The following codes are used: DUROC = Duroc; BLODU = Blond Mangalitza x Duroc; HAMPS = Hampshire; BAZNA = Bazna; LANDR = Landrace; LWHITE = Large White; PIETRAIN = Pietrain; REDMAH = Red Mangalitza (Hungary); REDMAR = Red Mangalitza (Romania); BLOMA = Blond Mangalitza; SBMA = Swallow-belly Mangalitza; HUNWB = Wild boar (Hungary); ROMWB = Wild boar (Romania). Supplementary Figure S2. Admixture analysis of Mangalitza pigs and additional wild boar and pig populations for a range of K-values (K = 2 to 13). Each individual is represented by a single column divided into K colored segments, where K is the number of assumed clusters. Populations are separated by black lines. The following codes are used: DUROC = Duroc; BLODU = Blond Mangalitza x Duroc; HAMPS = Hampshire; BAZNA = Bazna; LANDR = Landrace; LWHITE = Large White; PIETRAIN = Pietrain; REDMAH = Red Mangalitza (Hungary); REDMAR = Red Mangalitza (Romania); BLOMA = Blond Mangalitza; SBMA = Swallow-belly Mangalitza; HUNWB = Wild boar (Hungary); ROMWB = Wild boar (Romania). Supplementary Figure S3. Phylogenetic tree of of Mangalitza pigs and additional wild boar and pig populations constructed with the TreeMix program. The following codes are used: DUROC = Duroc; BLODU = Blond Mangalitza x Duroc; HAMPS = 27 Hampshire; BAZNA = Bazna; LANDR = Landrace; LWHITE = Large White; PIETRAIN = Pietrain; REDMAH = Red Mangalitza (Hungary); REDMAR = Red Mangalitza (Romania); BLOMA = Blond Mangalitza; SBMA = Swallow-belly Mangalitza; HUNWB = Wild boar (Hungary); ROMWB = Wild boar (Romania). Supplementary Figure S4. Graphical representation of pairwise coefficients of genetic differentiation (FST) values between Mangalitza pigs and additional wild boar and pig reference populations. The following codes are used: DUROC = Duroc; BLODU = Blond Mangalitza x Duroc; HAMPS = Hampshire; BAZNA = Bazna; LANDR = Landrace; LWHITE = Large White; PIETRAIN = Pietrain; REDMAH = Red Mangalitza (Hungary); REDMAR = Red Mangalitza (Romania); BLOMA = Blond Mangalitza; SBMA = Swallow-belly Mangalitza; HUNWB = Wild boar (Hungary); ROMWB = Wild boar (Romania). Supplementary Figure S5. Neighbor-joining tree of Mangalitza pigs and additional wild boar and pig individuals. The following codes are used: DUROC = Duroc; BLODU = Blond Mangalitza x Duroc; HAMPS = Hampshire; BAZNA = Bazna; LANDR = Landrace; LWHITE = Large White; PIETRAIN = Pietrain; REDMAH = Red Mangalitza (Hungary); REDMAR = Red Mangalitza (Romania); BLOMA = Blond Mangalitza; SBMA = Swallow-belly Mangalitza; HUNWB = Wild boar (Hungary); ROMWB = Wild boar (Romania). Supplementary Figure S6. Evolution of the effective population size (Ne) of Mangalitza pigs and additional wild boar and pig groups in the last 70 generations. The following codes are used: DUROC = Duroc; BLODU = Blond Mangalitza x 28 Duroc; HAMPS = Hampshire; BAZNA = Bazna; LANDR = Landrace; LWHITE = Large White; PIETRAIN = Pietrain; REDMAH = Red Mangalitza (Hungary); REDMAR = Red Mangalitza (Romania); BLOMA = Blond Mangalitza; SBMA = Swallow-belly Mangalitza; HUNWB = Wild boar (Hungary); ROMWB = Wild boar (Romania). 29 SUPPLEMENTARY MATERIALS The footprint of recent and strong demographic decline in the genomes of Mangalitza pigs V.A. Bâlteanu1*, T.F. Cardoso2,3*, M. Amills2,4, I. Egerszegi5, I. Anton6, A. Beja-Pereira7, A. Zsolnai6 Animal journal 1Instituteof Life Sciences, University of Agricultural Sciences and Veterinary Medicine, Cluj- Napoca, Romania. 2Department of Animal Genetics, Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Campus de la Universitat Autònoma de Barcelona, Bellaterra, Spain. 3CAPES Foundation, Ministry of Education of Brazil, Brasilia D. F., Brazil. 4Departament de Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona, Bellaterra, Spain. 5 Szent István University, Gödöllő, Hungary 6NAIK-Research Institute for Animal Breeding, Nutrition and Food Science, Herceghalom, Hungary. 7 Centro de Investigacao em Biodiversidade e Recursos Geneticos, Universidade do Porto (CIBIO-UP), Vairao, Portugal Supplementary Figure S2. Admixture analysis of Mangalitza pigs and additional wild boar and pig populations for a range of K-values (K = 2 to 13). Each individual is represented by a single column divided into K colored segments, where K is the number of assumed clusters. Populations are separated by black lines. The following codes are used: DUROC = Duroc; BLODU = Blond Mangalitza x Duroc; HAMPS = Hampshire; BAZNA = Bazna; LANDR = Landrace; LWHITE = Large White; PIETRAIN = Pietrain; REDMAH = Red Mangalitza (Hungary); REDMAR = Red Mangalitza (Romania); BLOMA = Blond Mangalitza; SBMA = Swallow-belly Mangalitza; HUNWB = Wild boar (Hungary); ROMWB = Wild boar (Romania). Supplementary Figure S3. Phylogenetic tree of of Mangalitza pigs and additional wild boar and pig populations constructed with the TreeMix program. The following codes are used: DUROC = Duroc; BLODU = Blond Mangalitza x Duroc; HAMPS = Hampshire; BAZNA = Bazna; LANDR = Landrace; LWHITE = Large White; PIETRAIN = Pietrain; REDMAH = Red Mangalitza (Hungary); REDMAR = Red Mangalitza (Romania); BLOMA = Blond Mangalitza; SBMA = Swallow-belly Mangalitza; HUNWB = Wild boar (Hungary); ROMWB = Wild boar (Romania). Supplementary Figure S4. Graphical representation of pairwise coefficients of genetic differentiation (FST) values between Mangalitza pigs and additional wild boar and pig reference populations. The following codes are used: DUROC = Duroc; BLODU = Blond Mangalitza x Duroc; HAMPS = Hampshire; BAZNA = Bazna; LANDR = Landrace; LWHITE = Large White; PIETRAIN = Pietrain; REDMAH = Red Mangalitza (Hungary); REDMAR = Red Mangalitza (Romania); BLOMA = Blond Mangalitza; SBMA = Swallow-belly Mangalitza; HUNWB = Wild boar (Hungary); ROMWB = Wild boar (Romania). Tree scale: 0.1 C34 C36 DURO DURO C37 DUROC2 DURO DUROC1 C38 DURO C50 DURO C47 DURO DUROC49 DUROC9 DUROC13 6 DUROC45 C3 DUROC4 C44 1 C1 C39 DURO C12 DUROC48 3 DURO RO C56 DURO DUROC4 DUROC20 DUROC15 DUROC51 O DUROC10 DUROC19 C33 DURO DUROC22 DU DUROC8 DUR RO DUROC6 RO C14 DU DUROC2 DURO C4 C35 DU 1 RO C4 RO DU RO C18 2 DU C4 DURO C40 1 DU DUROC1 RO O C7 DU C5 DUR 5 C16 C2 RO 9 RO C2 RO 6 DU 7 DU C2 RO DU 2 3 RO 4 DU C5 C2 C2 DU RO RO RO 0 DU DU C3 DU 7 C2 RO 1 RO DU C3 DU RO 3 8 C5 C2 4 DU C5 RO RO RO DU DU DU 7 C5 2 C3 RO RO DU DU BLOD ODU33 1 B6 BL 8 W B1 M B9 U34 RO B3 W 0 MW S1 BLOD B5 M B4 M W MP W RO W RO RO HA M 1 O S1 U3 BL R B2 MP M O W HA S1 O DU B1 OM MP R BL 2 W R B7 HA M S6 O RO 0 W S9 B1 OM MP DU 4 MP BL U4 BL BL W R 11 B1 HA HA 13 BLO OD MW OD BL M O D RO B B8 B12 U OD RO BLO W MW W U2 5 M U RO RO ROM D BLO BLODU3 9 4 DU U 6 3 S2 26 DU 8 B1 MP S4 MW HA BLO BL MP BL 42 RO 7 HA BL O B1 O BLOD DU S8 BLO DU45 OD B MW DU LOD BLOODU19 7 MP BL RO HA BLO 3 B U2 LOD BL DU 12 U DU DU U28 36 BL BLLOD BL LO B 27 21 5 B DU 1 O S5 OD U2 OD DU LO BL DU MP HA 3 BL17 O 15 U 2 U 2 U8 23 D PS7 DU 37 U O PS3 HAM O 39 HAM B D BL BL A4 3 A2 BAZN B15 29 BL 0 OD OD 4 BLO BAZN ROMW BL BL O DU U U O O DU DU 16 A1 A3 BAZN BAZN 4 35 U1 B B16 BL LO A5 ROMW 8 OD DU BAZN RED U1 14 AIN16 DU MAR BL 0 PIETR 20 13 OD U4 BL 7 REDM OD REDM AR5 BL U4 AIN17 OD 3 BL PIETR AR8 U4 O 0 BLBLODU AIN18 OD DU41 PIETR REDM BL U4 48 AR2 OD 6 IN19 N20 U1 PIETRA PIETRAI REDM REDM 1 REDM AR21 AR10 AR15 REDM AR24 3 PIETRAIN REDM AR1 PIETRAIN15 REDMAR14 REDM REDMA AR12 R7 LANDR22 REDMAR4 REDMAR 22 PIETRAIN7 REDMAR 11 REDMAR18 REDMAR20 PIETRAIN2 PIETRAIN11 REDMAR25 REDMAR9 PIETRAIN1 REDMAR17 PIETRAIN9 REDMAR16 PIETRAIN8 REDMAR23 REDMAR3 PIETRAIN13 REDMAR6 LANDR23 REDMAR19 10 PIETRAIN10 REDMAH16 REDMAH 13 REDMAH H2 HUNW B14 REDMA PIETRAIN12 B13 HUNW PIETRAIN14 LWHITE7 B17 HUNW B16 HUNW LWHITE6 AH17 H 20 B18 REDM 14 MA B12 HUNW H18 AHED LWHITE B15 HUNW MA DM R 5 HUNW RED RE 2 H1 AH19 15 H7 AH 4 DM A REDM MAH MA REDM 9 AH 6 RE 6 RED A2 B1 RED AH DM LANDR B2 DM LWHIT 24 4 RE M NW RE 3 A SB 2 HU B3 E1 NW BLO 24 5 A BM 0 11 NW A1 AH A1 3 MA A1 BM S 2 1 HU DM MA A2 SB20 S 25 BM MA E M HU R 3 B9 SB M SB 7 MA33 A28 AH SB LWHIT M S MA 1 M S H1 16 A5 3 21 BLO A7 31 MA A22 W BLOM SBBLO E3 BLO MA3 DM A UN A1 A RE AH8 DM M A1 B6 M B25 6 7 6 8 M S SB H M MA MABLO A1 SB MASB BM RE B HUNWB20 HUNWB21 MA BLO M SB MA1 SB BLO BLOMA1 DM NW MA B8 7 SB A19 M HUNWB24 RE LWHIT S8B BLOMBLOMA S 1 11 MA 5 HUNW BLOMA1 SB A9 0 AH NW WB MA E4 A2 B M LWHI 35 A1 7 2 36 HU DM BLOMA20 M HUNWB23 BLO 26 MA HU HUN SB B26 B28 TE8 BLOMA BLOMA SBMAB2 BLOMA34 MA2BLO RE BLOMA17 M SB 4 MA WB BLO 9 A1 HUNW BLOMA BLOMA14 BLO A32 30 HUNW SB BLOMA M BLO 29 SB BLOMA6 5 BLO LWHI A8 2 MA 1 BLOMA2 B27 HUNW BLO TE10 BLOM BLOM M 22 HUNW MA8 BLO SB B4 UN M HUNWB19 24 HUNW A37 NW H BLOMA13 BLO A12 BLOMA15 3 B10 LWHI 5 TE13 B11 HUNW HU A25 LWHI LWHI TE11 TE14 BLO MA4 LWH ITE 9 BLOM LWH LW ITE1 HIT 5 E24 A19 LW HIT LW E12 HIT E16 LW LW HIT LW HIT HIT LW E26 E1 E2 HIT 9 0 E1 7 LW LA LW H ITE ND LA H 23 R2 ND LA ITE 1 ND 27 R1 7 R5 LW H ITE 25 LW LA HIT LW ND E2 H LW R1 ITE H ITE 9 21 18 PIE LW PIE TR LA HIT TR AIN ND E2 AIN R1 2 4 8 6 PIE LA LA N TR D ND AIN R 29 R2 5 6 LA N LA D R ND 27 R1 0 LA N D R 28 LA ND LA N R1 DR 6 15 LA LA ND LA ND LA LA R2 ND ND ND R2 R7 R1 0 R1 4 1 LA LA ND ND R2 R1 5 3 LA ND R6 LA LA N ND LA DR ND R1 LA 4 R3 ND R9 LA ND R8 LA ND R1 2 Supplementary Figure S5 (above). Neighbor-joining tree of Mangalitza pigs and additional wild boar and pig individuals. The following codes are used: DUROC = Duroc; BLODU = Blond Mangalitza x Duroc; HAMPS = Hampshire; BAZNA = Bazna; LANDR = Landrace; LWHITE = Large White; PIETRAIN = Pietrain; REDMAH = Red Mangalitza (Hungary); REDMAR = Red Mangalitza (Romania); BLOMA = Blond Mangalitza; SBMA = Swallow-belly Mangalitza; HUNWB = Wild boar (Hungary); ROMWB = Wild boar (Romania). Supplementary Figure S6. Evolution of the effective population size (Ne) of Mangalitza pigs and additional wild boar and pig groups in the last 70 generations. The following codes are used: DUROC = Duroc; BLODU = Blond Mangalitza x Duroc; HAMPS = Hampshire; BAZNA = Bazna; LANDR = Landrace; LWHITE = Large White; PIETRAIN = Pietrain; REDMAH = Red Mangalitza (Hungary); REDMAR = Red Mangalitza (Romania); BLOMA = Blond Mangalitza; SBMA = Swallow-belly Mangalitza; HUNWB = Wild boar (Hungary); ROMWB = Wild boar (Romania).