Alma Mater Studiorum –Università Di Bologna GENOMIC CHARACTERIZATION of ITALIAN and EUROPEAN PIG POPULATIONS

Alma Mater Studiorum –Università di Bologna DOTTORATO DI RICERCA IN Scienze e Tecnologie Agrarie Ambientali e Alimentari Ciclo XXXI Settore Concorsuale: 07/G1 SCIENZE E TECNOLOGIE ANIMALI Settore Scientifico Disciplinare: AGR/17 ZOOTECNICA GENERALE E MIGLIORAMENTO GENETICO GENOMIC CHARACTERIZATION OF ITALIAN AND EUROPEAN PIG POPULATIONS Presentata da: CLAUDIA GERACI Coordinatore Dottorato Supervisore Prof. Massimiliano Petracci Prof. Luca Fontanesi Esame finale anno 2019 0 Abstract Over the past ten years, our knowledge about the pig genome has rapidly evolved leading to complete whole genome sequencing, essential for the dissection of molecular basis of pig evolution and various phenotypic traits. As a result of this genomic revolution, we can now take advantage of molecular and bioinformatic tools that allow to study genetic differences or similarities among modern commercial pig breeds, wild boars and local pig populations. This is extremely important in the context of conservation genetics for autochthonous and endangered pig breeds, seen that in many European regions the genetic architecture of local pig populations is still uncharacterized resulting in an unexploited breeding potential. This project aimed to better investigate genomic features of local autochthonous pig breeds focusing analyses on candidate gene markers associated to disease resistance, coat colour and vertebral number and genes potentially involved in feeding preferences. Considering the economic impact of infectious diseases on the pig production, we used a genotyping approach to define the distribution of disease resistance marker alleles in Italian local pig populations, indirectly confirming, with our results, the robustness of local pig breeds. We also performed an association study between investigated disease resistance markers and production traits, with first results suggesting that it could be possible to introduce disease resistance traits in pig breeding programs without affecting productivity. Referring to the relationship between local pig populations and wild boars, in the context of the domestication process, we carried out an analysis monitoring the allelic distribution at two evolutionary important loci, involved in coat colour and vertebral number determination. Results of this study suggested that Sus scrofa genome is currently experiencing bidirectional introgression of wild and domestic alleles, with autochthonous breeds experiencing a sort of “de-domestication” process and wild resources challenged by a “domestication” drift. The last part of this project was dedicated to the study of genetic variability of taste receptor genomic regions across different European pig populations. We performed a SNP discovery study to find out similarities and differences in taste sensing system among local breeds. Considering that taste perception is strongly connected to the diet and the environment, comparing different pig breeds and detecting differences in taste receptor genetic sequences, could be informative about the history of breeds and about the impact of ecology in their biodiversity. Altogether these results can be considered a basis for the use of genetic variability within and among local pig populations and for further studies regarding their full characterization. 1 Table of contents General introduction p. 3 Chapter 1: The domestic pig p. 4 1.1 The origins of pig and the domestication process p. 4 1.2 The molecular perspective in pig domestication p. 6 1.3 Pig genome sequencing p. 9 Chapter 2: Pig biodiversity p. 11 2.1 Breed differentiation and biodiversity challenge p. 11 2.2 Commercial pig lines p. 14 2.3 Autochthonous pig populations p. 15 Chapter 3: The role of genomics in pig breeding p. 23 Aim p. 26 Chapter 4: Genomics and disease resistance in pigs p. 27 4.1 Genetic markers associated with resistance to infectious diseases have no effects p. 29 on production traits and haematological parameters in Italian Large White pigs Chapter 5: Phenotypic changes in the domestication process p. 51 5.1 Signatures of de-domestication in autochthonous pig breeds and of domestication p. 53 in wild boar populations from MC1R and NR6A1 allele distribution Chapter 6: Genomics and nutrient sensing p. 75 6.1 DNA pool-seq approach for the identification of single nucleotide polymorphisms p. 76 in taste receptor genes of European autochthonous pig breeds General conclusions p. 100 2 General introduction The domestic pig (Sus scrofa domesticus) is a member of the Suidae family and it is one of the first animal species in the world to be domesticated. Pig has become over time an important species in animal breeding as it is considered one of the major human nutritional sources of animal-derived proteins (Wang et al., 2017). Pig is also considered an interesting example of domesticated animal whose wild conspecifics have survived and are still living. This is particularly challenging for evolutionary studies together with the great phenotypic varieties represented by existing pig breeds. This variability is the outcome of millions of years of evolution influenced by natural and artificial selections that have impacted morphological, physiological and behavioural traits (Chen et al., 2007). Domestic pig is not only interesting from an evolutionary point of view, but also plays a central role in biomedical studies and translational medicine. Several researches have been, and are still performed, to investigate on pig as animal model for human diseases. This is possible because pigs and humans are very similar in their physiology and, much more importantly, they share the same disease-causing mutations in genes responsible for severe diseases like Parkinson or Alzheimer or multifactorial traits such as obesity and diabetes (Lunney, 2007; Groenen et al., 2012). The possibility to obtain a huge quantity of molecular data on pig genome thanks to the recent development of Next Generation Sequencing and genotyping technologies, has allowed to discover more about the history of domestic pig and to characterize in time the events marking the separation between early domestication and breeding era (Ramirez et al., 2015). At the beginning, breeding was intuition-driven, without scientific basis and structured breeding programs. With their early farming activities, humans have strongly modified ancestor wild boars, with selective events that progressively led to the differentiation of modern breeds (Ibáñez-Escriche et al., 2014). The so called ‘genetic revolution’ in pig history started around 1950, when it became feasible to apply first quantitative genetics approaches and systematic crossbreeding, together with the utilization of estimated breeding values as support to identify animals with the best reproductive qualities for pig industry (Hazel, 1943; Dickerson, 1974; Henderson, 1984; Ibáñez-Escriche et al., 2014). Later, the possibility to employ molecular markers and high-throughput SNP genotyping platforms, led to the discovery of Quantitative Trait Loci (QTLs) and genomic regions, with causal polymorphisms associated with economically important traits for pig industry. In this way, the approach to pig breeding radically changed, opening the path to Genomic Selection (GS) and Marked Assisted Selection (MAS) that are nowadays important tools for pig breeding companies that have implemented breeding schemes and selection strategies (Ibáñez-Escriche et al., 2014). 3 The dissection of complex traits of economic importance to the pig industry through Genome-Wide Association Analysis (GWAS) is currently a hot topic for researchers. For this reason, studies aiming to identify molecular markers (as causal mutations or polymorphisms in linkage with a QTL) are requested for challenging issues like genetic resistance to diseases, animal performances and productivity (Boddicker et al., 2012; Sanchez et al., 2014; Dunkelberger et al., 2017). Together with these topics, a lot of studies are presently aimed to define the genetic differences or similarities among modern commercial pig breeds, wild boars and local pig populations. More in details, genomic tools are widely used for conservation genetics in livestock species and for the identification of peculiar traits characterizing local autochthonous pig breeds for defending biodiversity and enhance local pig breeding and production (Čandek-Potokar et al., 2017) Chapter 1 The domestic pig 1.1 The origins of pig and the domestication process It is generally accepted that among the six species belonging to the Sus genus, Sus scrofa is the only one that underwent a fully domestication process from a wild Sus scrofa ancestor (Larson et al., 2010). Pig is considered a perfect example of animal species whose wild ancestors are still living, so it represents an incredible opportunity for investigating the history of mammalian evolution and for identifying the signatures of selection occurred during domestication process and natural selection (Chen et al., 2007). Several studies investigated on the origins of modern pig domestication process and they all agree that the ancestor Sus scrofa arose from South East Asia around 3.0-3.5 million years ago (Mya) slowly colonizing and spreading in other regions of Asia and then in European area and North Africa (Groenen et al., 2012; Groenen, 2016). This migration of wild boars to new territories, was probably followed by a long period of geographic isolation corresponding to Calabrian stage, that caused the establishment of two differentiated Sus scrofa gene pools, one represented

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