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Summary The number of teats is a reproductive‐related trait of great economic relevance as it affects the mothering ability of the sows and thus the number of properly weaned piglets. Moreover, genetic improvement of this trait is fundamental to parallelly help the selection for increased litter size. We present the results of single‐marker and haplotypes‐based genome‐wide association studies for the number of teats in two large cohorts of heavy pig breeds (Italian Large White and Italian Landrace) including 3990 animals genotyped with the 70K GGP Porcine BeadChip and other 1927 animals genotyped with the Illumina PorcineSNP60 BeadChip. In the Italian Large White population, genome scans identified three genome regions (SSC7, SSC10, and SSC12) that confirmed the involvement of the VRTN gene (as we previously reported) and highlighted additional loci known to affect teat counts, including the FRMD4A and HOXB1 gene regions. A different picture emerged in the Italian Landrace population, with a total of 12 genome regions in eight chromosomes (SSC3, SSC6, SSC8, SSC11, SSC13, SSC14, SSC15, and SSC16) mainly detected via the haplotype‐based genome scan. The most relevant QTL was close to the ARL4C gene on SSC15. Markers in the VRTN gene region were not significant in the Italian Landrace breed. The use of both single‐marker and haplotype‐based genome‐wide association analyses can be helpful to exploit and dissect the genome of the pigs of different populations. Overall, the obtained results supported the polygenic nature of the investigated trait and better elucidated its genetic architecture in Italian heavy pigs.

Single nucleotide polymorphism (SNP) genotyping tools, which can analyse thousands of SNPs covering the whole genome, have opened new opportunities to estimate the inbreeding level of animals directly using genome information. One of the most commonly used genomic inbreeding measures considers the proportion of the autosomal genome covered by runs of homozygosity (ROH), which are defined as continuous and uninterrupted chromosome portions showing homozygosity at all loci. In this study, we analysed the distribution of ROH in three commercial pig breeds (Italian Large White, n = 1968; Italian Duroc, n = 573; and Italian Landrace, n = 46) and four autochthonous breeds (Apulo-Calabrese, n = 90; Casertana, n = 90; Cinta Senese, n = 38; and Nero Siciliano, n = 48) raised in Italy, using SNP data generated from Illumina PorcineSNP60 BeadChip. We calculated ROH-based inbreeding coefficients (FROH) using ROH of different minimum length (1, 2, 4, 8, 16 Mbp) and compared them with several other genomic inbreeding coefficients (including the difference between observed and expected number of homozygous genotypes (FHOM)) and correlated all these genomic-based measures with the pedigree inbreeding coefficient (FPED) calculated for the pigs of some of these breeds. Autochthonous breeds had larger mean size of ROH than all three commercial breeds. FHOM was highly correlated (0.671 to 0.985) with FROH measures in all breeds. Apulo-Calabrese and Casertana had the highest FROH values considering all ROH minimum lengths (ranging from 0.273 to 0.189 and from 0.226 to 0.152, moving from ROH of minimum size of 1 Mbp (FROH1) to 16 Mbp (FROH16)), whereas the lowest FROH values were for Nero Siciliano (from 0.072 to 0.051) and Italian Large White (from 0.117 to 0.042). FROH decreased as the minimum length of ROH increased for all breeds. Italian Duroc had the highest correlations between all FROH measures and FPED (from 0.514 to 0.523) and between FHOM and FPED (0.485). Among all analysed breeds, Cinta Senese had the lowest correlation between FROH and FPED. This might be due to the imperfect measure of FPED, which, mainly in local breeds raised in extensive production systems, cannot consider a higher level of pedigree errors and a potential higher relatedness of the founder population. It appeared that ROH better captured inbreeding information in the analysed breeds and could complement pedigree-based inbreeding coefficients for the management of these genetic resources.

The number of teats is a morphological trait of high economic relevance for the pig industry. Here, to dissect the genomic architecture of this trait in the Italian Duroc pig population, we present the results of genome-wide association studies in this Italian heavy pig breed. A total of 1,162 pigs, for which the number of teats was recorded, was genotyped with two high-throughput single nucleotide polymorphism (SNP) genotyping platforms (60 K and 70 K). Genome-wide association analyses were based on a single-marker approach and on a haplotype-based approach. Two quantitative trait loci (QTL) affecting the number of teats were identified. The most significant QTL, identified by the single-marker analysis and confirmed by the haplotype-based method, was located on Sus scrofa chromosome (SSC) 7, in the region of the vertnin (VRTN) gene. Suggestively associated markers (SNPs and haplotypes) were located on SSC10, in the region of the FERM domain containing 4A (FRMD4A) gene, the second identified QTL. These findings confirm previous results obtained in a few other Duroc populations. Overall, this study further supported the important role of variability in the VRTN gene region in affecting the number of teats in pigs. Moreover, the results also indicated that this trait in the Italian Duroc breed, as in many other pig breeds, is affected by few QTL, with the contribution of many other genetic factors with small effects, following the classical theory of quantitative traits.

Teat number (TN) is an important parameter for the mothering ability of the sows. Being an economically relevant reproductive trait, TN is the objective of genetic selection which generally impose a minimum threshold value in breeding programmes. Several studies investigated TN in different pig populations, reporting high variability (from 8 to 21 teats), a medium/high heritability (from 0.10 to 0.70) and a polygenic control of the trait. We previously investigated TN in Italian Large White (ILW) pigs and associated its variability to polymorphisms in the VRTN gene. Here, we refined those results in a population of about 4,000 ILW pigs genotyped with the 70K GGP Porcine BeadChip. Moreover, about 2,000 Italian Landrace (ILA) pigs were for the first time investigated. Single-SNP and haplotype-based genome scans were performed via linear mixed model analyses (GEMMA tool). TN for the ILW pigs was in the range 12-20, whereas ILA pigs had 10-18 teats. The two populations had about 53.4 and 43.5% of animals with more than 14 teats, respectively. Genomic heritability of TN was 0.30 (ILW) and 0.38 (ILA). We identified a total of 22 quantitative trait loci (QTL) on 12 porcine chromosomes associated with TN variability in these two populations. Obtained results confirmed the association of the VRTN gene variants in the ILW population and pointed out other genome regions harbouring QTLs for reproductive traits (e.g. litter size, number of vertebra as reported in other populations). ILA pigs showed a completely different set of genome variants affecting TN. Overall, these results provided information that could be used to design breeding programmes to improve TN in Italian pig breeds.

The level of inbreeding in a population should be limited to avoid the inbreeding depression and maintain variability needed to assure long term genetic progress. Inbreeding coefficient (FPED) is usually calculated using pedigree information. Single nucleotide polymorphism (SNP) genotyping tools give the possibility to calculate genomic inbreeding parameters. The proportion of the autosomal genome covered by runs of homozygosity (ROH) can be used to estimate the level of genomic inbreeding (FROH). The breeding program of Italian pig breeds started about 25 years ago. In this work, we retrospectively analysed FROH over this period in Italian Large White (ILW) and Italian Duroc (ID) heavy pig breeds and compared this parameter with FPED. A total of 1,420 ILW and 572 ID pigs born over the last 25 years have been genotyped with the Illumina PorcineSNP60 BeadChip. ROH were identified with PLINK version 1.9, considering a minimum of 15 SNPs, and a minimum length of 1 Mbp. Then FROH was computed for all animals and averaged over all animals born by year. Averaged FROH over all considered years was higher in ID than in ILW and was constant over all analysed time window. FROH and FPED were parallel for both breeds over the same period. The results indicated that both FROH and FPED can be used to manage inbreeding levels in Italian heavy pig breeds.

The number of teats is a morphological trait that influences the mothering ability of the sows and thus their reproduction performances. In this study, we carried out GWASs for the total number of teats and other 12 related parameters in 821 Italian Large White heavy pigs. All pigs were genotyped with the Illumina PorcineSNP60 BeadChip array. For four investigated parameters (total number of teats, the number of teats of the left line, the number of teats of the right line and the maximum number of teats comparing the two sides), significant markers were identified on SSC7, in the region of the vertnin (VRTN) gene. Significant markers for the numbers of posterior teats and the absolute difference between anterior and posterior teat numbers were consistently identified on SSC6. The most significant SNP for these parameters was an intron variant in the TOX high mobility group box family member 3 (TOX3) gene. For the other four parameters (absolute difference between the two sides; anterior teats; the ratio between the posterior and the anterior number of teats; and the absence or the presence of extra teats) only suggestively significant markers were identified on several other chromosomes. This study further supported the role of the VRTN gene region in affecting the recorded variability of the number of teats in the Italian Large White pig population and identified a genomic region potentially affecting the biological mechanisms controlling the developmental programme of morphological features in pigs.

Most of the Italian pig genetic resources that existed at the beginning of the last century have been substituted by more productive populations. At present, only six original autochthonous pig breeds are officially recognized in Italy and have their own herd books managed by the National Pig Breeders Association. Another section of the breed herd book includes breeds that were completely lost but that were subsequently reconstructed through specific crossbreeding programmes. This is the case of the Nero di Parma breed that was officially recognized as reconstructed breed in 2016. This is a small breed, reared in the province of Parma (North of Italy), that accounts for 103 and 12 registered sow and boars, respectively. In this study, using single nucleotide polymorphisms genotyped with the Illumina PorcineSNP60 BeadChip array, we compared, at the genome level, the reconstructed Nero di Parma pig breed with three Italian commercial (Italian Large White, Italian Duroc and Italian Landrace) and four autochthonous (Apulo-Calabrese, Casertana, Cinta Senese and Nero Siciliano) pig breeds. Nero di Parma showed the highest level of linkage disequilibrium among all analysed breeds. Effective population size estimated over past generations was the lowest in this reconstructed breed. However, genomic inbreeding parameters did not suggest a high level of inbreeding in Nero di Parma. The comparative analysis against several other Italian pig breeds made it possible to evaluate the obtained information that could be helpful to define potential future conservation and breeding strategies for this small reconstructed breed.

Casertana is an autochthonous pig breed reared in the Central-South of Italy, mainly in extensive or semi-extensive production systems. A small number of animals are registered to the breed herd-book. The population of this breed is not completely fixed for several exterior traits, including ear size (small vs large), ear bearing (forward vs floppy), presence or absence of wattles and coat colour (black vs slate-grey) that we recorded in a total of 101 pigs from six different farms. All these pigs were also genotyped with the Illumina PorcineSNP60 BeadChip. Then, with the aim to identify DNA markers associated with these traits, we run four genome-wide association studies (GWAS) comparing pigs having different phenotypes for each of the four external traits. Genome-wide fixation index (FST) analyses were run to confirm the results. GEMMA software was used in the GWAS. Single nucleotide polymorphisms (SNPs) associated to the wattle trait were identified on porcine chromosome (SSC) 1 and SSC11. Markers associated to the coat colour trait were located on SSC6, SSC8, SSC14 and SSC15. These chromosome regions were confirmed by the independent FST analyses. FST did not confirm the signals detected for the two ear conformation traits. Candidate genes were identified according to the function of the annotated genes in the detected genome regions. Despite the small number of pigs that could be investigated, this study showed that autochthonous animal genetic resources might be useful to disclose genetic factors affecting traits that are not fixed in a population.

Knowledge of genes involved in the variability of performance of racehorses is, in general, limited. In this study, six single nucleotide polymorphisms (SNPs) of five genes were genotyped in 412 Italian Trotter horses (ITHs) and possible associations between five segregating SNPs and harness racing performance (best racing time in career, earnings, placings, starts, and wins) were investigated. These markers were in genes related to exercise physiology (creatine kinase muscle, CKM; and cytochrome c oxidase, subunit 4, isoform 2, COX412), gait patterns (DMRT3), carbohydrates metabolism (GCK) and morphological phenotypes (LCORL). Two SNPs of the GCK gene were used to predict haplotypes. The mutate A allele of the DMRT3 was almost fixed in the ITHs. Single marker association analyses showed that the COX412 SNP was significantly associated with variation of number of starts (P = 0.0003), and wins (P = 0.0045). This COX4I2 polymorphic site seems therefore to be a promising marker for racing longevity and durability. A haplotype of the GCK was instead positively associated with best racing time (P = 0.0118), earnings (P = 0.0370) and wins (P = 0.0061). This is the first study highlighting the effect of GCK on harness racing performance in horses. Confirmation of these results in independent populations would be important to evaluate the inclusion of the SNPs in marker-assisted selection programmes in ITHs.

Runs of homozygosity (ROH) in a diploid organism can be defined as continuous chromosome regions in which all loci have a homozygous genotype. Shared ROH within a livestock population identify chromosome regions in which a reduced haplotype variability produces ROH islands. ROH islands can provide information on hotspot of selection putatively derived from different selection history, genetic events and adaptation to several production systems. In this study we evaluated the distribution of ROH in the genome of a total of 2860 pigs belonging to seven Italian breeds, three commercial breeds (Italian Large White, Italian Duroc and Italian Landrace) and four autochthonous breeds (Apulo-Calabrese, Casertana, Cinta Senese and Nero Siciliano). All animals were genotyped with the Illumina PorcineSNP60 BeadChip array. PLINK software was used to call ROH. The largest number of ROH per animal was observed in the Italian Duroc breed. The mean largest size of ROH was detected in Apulo-Calabrese pigs. Nero Siciliano pigs had the lowest mean number of ROH per animal. Italian Large White pigs had the lowest mean length of ROH. ROH islands were identified in all breeds except in Nero Siciliano. ROH islands spanned from a total of 25.5 (Cinta Senese) to 33.1 Mbp (Italian Landrace) of genomic regions distributed from four to ten autosomes and encompassing from a total of 126 to 262 annotated genes. These selection hotspot regions differed among breeds. Functional inference of the observed ROH islands provided some insights into the mechanisms of adaptation of these pig genetic resources.

Nero Siciliano (or Sicilian Black) is an Italian autochthonous pig breed reared in the Sicily island, mainly under extensive management systems. Nero Siciliano pigs are black (with black skin and black hair), but animals with white face or partially white face ("suino facciolo") can be registered to the breed herd book. Sometimes, other white patterns on extreme portions of legs could appear in this population. This study took advantage from the rare occurrence of pigs with white patterns in the Nero Siciliano population to carry out a genome-wide association study and comparative genome-wide Fixation index (FST) analysis to identify genomic regions that could affect coat colour variability (solid black vs white patterns over black) in this autochthonous pig breed. Analyses have been conducted on 66 Nero Siciliano pigs: 30 completely black and 36 black with white patterns. All samples have been genotyped for the KIT gene duplication and MC1R mutations, two genes well known to affect coat colours in pigs. Only pigs that did not carry any duplication of the KIT gene and were homozygous for the ED2 black dominant MC1R gene allele (n = 26 completely black and n. 22 with white patterns) were genotyped with the Illumina PorcineSNP60 BeadChip. The genome-wide analyzes identified on chromosome 2 a significant marker (rs81329493) associated with the coat colour white patterns in this breed. The homologous chromosome region in felids contains the gene responsible for the blotched tabby and striped coat colour patterns. Further studies, including a larger number of pigs, are needed to confirm this result and identify the causative mutation(s) affecting this coat colour diversity, which might be used to design a conservation programme in this breed aiming to maintain phenotypic homogeneity (i.e. solid black) that is typically associated with Nero Siciliano pigs. This study demonstrated how genetic diversity segregating in an autochthonous genetic resource can be explored to understand the genetic mechanisms affecting phenotypic traits in a livestock species.

Honeydew produced from the excretion of plant-sucking insects (order Hemiptera) is a carbohydrate-rich material that is foraged by honey bees to integrate their diets. In this study, we used DNA extracted from honey as a source of environmental DNA to disclose its entomological signature determined by honeydew producing Hemiptera that was recovered not only from honeydew honey but also from blossom honey. We designed PCR primers that amplified a fragment of mitochondrial cytochrome c oxidase subunit 1 (COI) gene of Hemiptera species using DNA isolated from unifloral, polyfloral and honeydew honeys. Ion Torrent next generation sequencing metabarcoding data analysis assigned Hemiptera species using a customized bioinformatic pipeline. The forest honeydew honeys reported the presence of high abundance of Cinara pectinatae DNA, confirming their silver fir forest origin. In all other honeys, most of the sequenced reads were from the planthopper Metcalfa pruinosa for which it was possible to evaluate the frequency of different mitotypes. Aphids of other species were identified from honeys of different geographical and botanical origins. This unique entomological signature derived by environmental DNA contained in honey opens new applications for honey authentication and to disclose and monitor the ecology of plant-sucking insects in agricultural and forest landscapes.

Casertana is a local pig breed mainly raised in Central-South regions of Italy. Pigs of this breed are considered the descendants of the ancient Neapolitan population that largely influenced the constitution of the modern commercial pigs. The pigs of this breed are usually curly-tailed, like several other domestic pig populations. However, Casertana population shows some variability for this trait, including animals having straight tail as observed in wild boars. In this study, we run, for the first time, a genome wide association study (GWAS) comparing the curly tailed (no. = 53) and straight tailed (no. = 19) Casertana pigs to identify genomic regions associated with the tail shape phenotype in Sus scrofa. All animals were genotyped with the Illumina PorcineSNP60 BeadChip v.2. GEMMA software was used in the GWAS for which we were able to correct for stratification in the analysed cohort. A single nucleotide polymorphism (rs81439488), located on porcine chromosome 12, was significantly associated with the investigated trait. This marker is close to the SRY-box 9 (SOX9) gene that encodes for a transcription factor that is required during sequential steps of the chondrocyte differentiation pathway, notochord maintenance and skeletogenesis. As the shape of the tail could be important in relation to the problem of tail biting in pigs, the obtained results might open new perspectives for defining selection programs answering indirectly animal welfare issues. This work demonstrated that autochthonous animal genetic resources might be used to disclose genetic factors affecting peculiar traits by exploiting segregating phenotypes and genetic variability.

Autochthonous pig breeds are usually reared in extensive or semi‐extensive production systems that might facilitate contact with wild boars and, thus, reciprocal genetic exchanges. In this study, we analysed variants in the melanocortin 1 receptor (MC1R) gene (which cause different coat colour phenotypes) and in the nuclear receptor subfamily 6 group A member 1 (NR6A1) gene (associated with increased vertebral number) in 712 pigs of 12 local pig breeds raised in Italy (Apulo‐Calabrese, Casertana, Cinta Senese, Mora Romagnola, Nero Siciliano and Sarda) and south‐eastern European countries (Krškopolje from Slovenia, Black Slavonian and Turopolje from Croatia, Mangalitsa and Moravka from Serbia and East Balkan Swine from Bulgaria) and compared the data with the genetic variability at these loci investigated in 229 wild boars from populations spread in the same macro‐geographic areas. None of the autochthonous pig breeds or wild boar populations were fixed for one allele at both loci. Domestic and wild‐type alleles at these two genes were present in both domestic and wild populations. Findings of the distribution of MC1R alleles might be useful for tracing back the complex genetic history of autochthonous breeds. Altogether, these results indirectly demonstrate that bidirectional introgression of wild and domestic alleles is derived and affected by the human and naturally driven evolutionary forces that are shaping the Sus scrofa genome: autochthonous breeds are experiencing a sort of ‘de‐domestication’ process, and wild resources are challenged by a ‘domestication’ drift. Both need to be further investigated and managed.