GENOME-WIDE DETECTION OF QTL AND CNVS IN DAIRY CATTLE POPULATION

The QTL involved in susceptibility/resistance of infectious diseases and in the productive traits variations, are characterized by genetic heterogeneity and multifactorial inheritance, involving gene polymorphisms from different alternative pathways. With the availavility of single nucleotide polymorphism (SNP) genotyping arrays, the genome-wide association studies (GWAS) have been frequently used to determine the genetic component of complex trait. The Copy Number Variations (CNVs) are another genomic marker that can be possibly used in GWAS and that can be identified from SNP chips themselves. The aims and related discussions for each of the studies presented in this thesis were grouped into three different chapters. \u2022 Chapter 1 described the QTL mapping analysis to identify the existence of genetic variability associated to the CLA, VA and D9D contents in milk of the Italian Brown Swiss dairy cattle breed. For this study a selective DNA pooling in a daughter design was adopted, using the Illumina Bovine SNP50 Bead Chip to genotype the pools. Milk samples from 60 animals with higher values (after correction for environmental factors) and 60 animals with lower values for each of these traits from each of five half-sib families were pooled separately. Allele frequencies were compared between pools of high and low value at the sire and marker level for each SNPs for which the sires were heterozygous. An R procedure was implemented to perform data analysis. A correction for multiple tests was applied using the proportion of false positives approach. BTA 19 showed the largest number of markers in association with CLA. Associations between SNPs and the VA and D9-desaturase traits were found on several chromosomes. A bioinformatics survey identified genes with an important role in pathways for milk fat and fatty acids metabolism within 1 Mb distance from SNP markers associated with fatty acids contents. This is the first available mapping for fatty acid content in the Brown Swiss population. \u2022 Chapter 2 described a genome-wide association study for somatic cell score (SCS) in the Valdostana Red Pied cattle, with a selective DNA pooling analysis, using the Illumina BovineHD BeadChip. The phenotypes of 275 sires for SCS were expressed as Deregressed Proofs (DP-EBVs) for SCS. The sires were ranked according to DP-EBVs for SCS and the 20% high and 20% low sires included in the pools. The multiple marker test was performed in R software. On BTAs 1, 2, 3, 4, 9, 13, 15, 17, 21 and 22 the largest number of markers in association to the trait was found identifying novel genomic regions related to mastitis (1-Mb SNP windows) and confirming others already mapped. The largest number of significant SNPs exceeding the threshold for genome-wide significant signal was found on BTA 15, located at 50.43-51.63 Mb. The genomic regions identified in this study contribute to a better understanding of the genetic control of the mastitis immune response in cattle and may allow the inclusion of more detailed QTL information in selection programs. \u2022 Chapter 3 described a genome wide CNVs discovery in 651 bulls of the Italian Brown Swiss breed using the Illumina Bovine SNP50 BeadChip data. Hidden Markov Model (HMM) of PennCNV and SVS7 software (Golden Helix) were used for the identification of the CNVs and Copy Number Variation Regions (CNVRs). A total of 5,099 and 1,289 CNVs were identified using PennCNV and SVS7 software, respectively. These were grouped at the population level into 1,101 (220 losses, 774 gains, 107 complex) and 277 (185 losses, 56 gains and 36 complex) CNVRs, covering 682 Mb (27.14%) and 33.7 Mb (1.35%) of the autosome, respectively. Ten of the selected CNVRs were experimentally validated with qPCR and the proportions of confirmed positive samples for each region varied from 50% to 100%. The GO and pathway analyses identified genes (false discovery rate corrected) in the CNVRs related to biological processes, cellular component, molecular function and metabolic pathways. Although there is variability in the CNVRs detection across methods, platforms, this study allowed the identification CNVRs in Italian Brown Swiss, overlapping those already detected in other breeds and finding additional ones

Characterization and functional roles of paternal RNAs in 2–4 cell bovine embryos

Embryos utilize oocyte-donated RNAs until they become capable of producing RNAs through embryonic genome activation (EGA). The sperm\u2019s influence over pre-EGA RNA content of embryos remains unknown. Recent studies have revealed that sperm donate non-genomic components upon fertilization. Thus, sperm may also contribute to RNA presence in pre-EGA embryos. The first objective of this study was to investigate whether male fertility status is associated with the RNAs present in the bovine embryo prior to EGA. A total of 65 RNAs were found to be differentially expressed between 2\u20134 cell bovine embryos derived from high and low fertility sires. Expression patterns were confirmed for protein phosphatase 1 regulatory subunit 36 (PPP1R36) and ataxin 2 like (ATXN2L) in three new biological replicates. The knockdown of ATXN2L led to a 22.9% increase in blastocyst development. The second objective of this study was to characterize the parental origin of RNAs present in pre-EGA embryos. Results revealed 472 sperm-derived RNAs, 2575 oocyte-derived RNAs, 2675 RNAs derived from both sperm and oocytes, and 663 embryo-exclusive RNAs. This study uncovers an association of male fertility with developmentally impactful RNAs in 2\u20134 cell embryos. This study also provides an initial characterization of paternally-contributed RNAs to pre-EGA embryos. Furthermore, a subset of 2\u20134 cell embryo-specific RNAs was identified

The German Shorthair Pointer Dog Breed (Canis lupus familiaris) : Genomic Inbreeding and Variability

The German Shorthaired Pointer (GSHP) is a breed worldwide known for its hunting versatility. Dogs of this breed are appreciated as valuable companions, effective trackers, field trailers and obedience athletes. The aim of the present work is to describe the genomic architecture of the GSHP breed and to analyze inbreeding levels under a genomic and a genealogic perspective. A total of 34 samples were collected (24 Italian, 10 USA), and the genomic and pedigree coefficients of inbreeding have been calculated. A total of 3183 runs of homozygosity (ROH) across all 34 dogs have been identified. The minimum and maximum number of Single Nucleotide Polymorphisms (SNPs) defining all ROH are 40 and 3060. The mean number of ROH for the sample was 93.6. ROH were found on all chromosomes. A total of 854 SNPs (TOP_SNPs) defined 11 ROH island regions (TOP_ROH), in which some gene already associated with behavioral and morphological canine traits was annotated. The proportion of averaged observed homozygotes estimated on total number of SNPs was 0.70. The genomic inbreeding coefficient based on ROH was 0.17. The mean inbreeding based on genealogical information resulted 0.023. The results describe a low inbred population with quite a good level of genetic variability

Genetic variability in a Holstein population using SNP markers and their use for monitoring mating strategies

As genotyping costs continue to decrease, the demand for genotyping has increased among farmers. In most livestock herds, an important issue is controlling the increase in inbreeding coefficient. While this remains a large motive to genotype, producers are often unaware of the other benefits that genotyping could bring. The aim of this study was to demonstrate that SNP chips could be used as an effective herd management tool by utilizing a population of Italian Holstein-Friesian cattle. After filtering, the total number of animals and SNPs retained for analyses were 44 and 27,365, respectively. The principal component analyses (PCA) were able to identify a sire and origin-of-sire effect within the herd, while determining that sires do not influence individual genomic selection index values. The inbreeding coefficients calculated from genotypes (FIS) provided a glimpse into the herd\u2019s heterozygosity and determined that the genetic variability is being well maintained. On the other hand, inbreeding coefficients on the genomic level were deduced from runs of homozygosity (FROH) and were compared to the inbreeding coefficients based on pedigree (FPED). Furthermore, 1,950 runs of homozygosity (ROH) were identified with the average length of ROH being 4.66 Mb. Genes and QTL within the genomic regions most commonly associated (top 1% and top 5% of SNP) with ROH were characterized. These results indicate that genotyping small herds, albeit at low-density, provides insights to the genetic variability within the herd and thus allows producers the ability to manage their stock from a genetic standpoint

Quantitative Trait Loci affecting the somatic cell score on chromosome 4 and 26 in Italian Holstein cattle

This work aimed to confirm previously reported quantitative trait loci (QTL) affecting the somatic cell score (SCS) in dairy cattle on Bos taurus autosomes (BTA) 4 and 26. A granddaughter design with selective genotyping was implemented that included half-sib families from 12 male lines of Italian Holstein cattle. The animals were genotyped for 5 microsatellite markers each on regions of BTA 4 (average marker spacing 9.42 cM) and BTA 26 (average marker spacing 5.26 cM), previously reported by other authors as carrying QTL for somatic cell count. Quantitative trait loci analyses were performed using interval mapping by regressing sire breeding values for SCS onto genotype probabilities at 1-cM intervals along the 2 chromosome regions. Breeding values for SCS were estimated for the whole population using a test-day repeatability animal model. Results were not significant on a chromosome basis, but a possible QTL was found at BM4505 on BTA 26, confirming this region for further studies of QTL affecting SCS in the Italian Holstein population

Histocompatibility genes and somatic cell count (SCC) in Italian Holstein Friesian

Geni di istocompatibilit\ue0 e conta delle cellule somatiche (SCC) nella Frisona Italiana \u2013 Lo studio ha considerato l\u2019effetto del Complesso Maggiore di Istocompatibilit\ue0 (MHC) sulla mastite, clinica e subclinica, utilizzando l\u2019Indice Genetico (I.G.) per la conta delle cellule somatiche: SCC (Somatic Cell Count). Su un totale di 302 tori di razza Frisona Italiana, valutati geneticamente per le cellule somatiche, sono stati analizzati il polimorfismo degli antigeni di istocompatibilit\ue0 di classe I (test di microlinfocitotossicit\ue0 locus BoLA-A) e di classe II (PCR/RFLP del locus DRB3 esone 2). L\u2019effetto degli antigeni di istocompatibilit\ue0 sugli indici genetici \ue8 stato valutato con un modello di sostituzione genica

Genetic variability of Akhal-Teke horses bred in Italy

Background. The Akhal-Teke horse (AKH) is native of the modern Turkmenistan area. It was introduced in Italy from 1991 to 2000 mainly as an endurance horse. This paper characterizes the genetic variability of the whole Italian AKH horse population and evaluates their inbreeding level by analyzing microsatellite markers and mitochondrial D-Loop sequences. Methods. Seventeen microsatellite marker loci were genotyped on 95 DNA samples from almost all the AKH horses bred in Italy in the last 20 years. Standard genetic variability measures (Ho, He, FIS) were compared against the same variables published on other eight AKH populations. In addition, 397 bp of mtDNA D-loop region were sequenced on a sub-group of 22 unrelated AKH out of the 95 sampled ones, and on 11 unrelated Arab horses. The haplotypes identified in the Italian population were aligned to sequences ofAKH(56), Arab (five), Caspian Pony (13), Przewalskii (two) and Barb (15) horses available in GenBank. The Median Joining Network (MJN), Principal Component Analysis (PCA) and Neighbor-joining (NJ) tree were calculated on the total 126 sequences. Results. Nucleic markers showed a high degree of polymorphism (Ho D 0.642; He D 0.649) and a low inbreeding level (FIS D 0.016) in Italian horses, compared to other AKH populations (ranged from 0.103 AKH from Estonia to 0.114 AKH from Czech Republic). High variability was also recorded in the D-Loop region. 11 haplotypes were identified with haplotype diversity (hd), nucleotide diversity () and average number of nucleotide differences (k) of 0.938, 0.021 and 6.448, respectively. When all the 126 D-Loop sequences were compared, 51 haplotypes were found, and four were here found only in the Italian AKH horses. The 51 haplotypes were conformed to eight recognized mtDNA haplogroups (A, C, F, G, L, M, P and Q) and confirmed by MJN analysis, Italian horses being assigned to five haplogroups (A, C, G, L and M). Using a PCA approach to the same data, the total haplotypes were grouped into two clusters including A+C+M+P and G+F haplogroups, while L and Q haplogroups remained ungrouped. Finally, the NJ algorithm effectively discretizes only the L haplogroup. All the above data univocally indicate good genetic variability and accurate management of the Akhal-Teke population in Italy

First genome-wide CNV mapping in FELIS CATUS using next generation sequencing data

Background: Copy Number Variations (CNVs) have becoming very significant variants, representing a major source of genomic variation. CNVs involvement in phenotypic expression and different diseases has been widely demonstrated in humans as well as in many domestic animals. However, genome wide investigation on these structural variations is still missing in Felis catus. The present work is the first CNV mapping from a large data set of Next Generation Sequencing (NGS) data in the domestic cat, performed within the 99 Lives Consortium. Results: Reads have been mapped on the reference assembly_6.2 by Maverix Biomics. CNV detection with cn.MOPS and CNVnator detected 592 CNVs. These CNVs were used to obtain 154 CNV Regions (CNVRs) with BedTools, including 62 singletons. CNVRs covered 0.26% of the total cat genome with 129 losses, 19 gains and 6 complexes. Cluster Analysis and Principal Component Analysis of the detected CNVRs showed that breeds tend to cluster together as well as cats sharing the same geographical origins. The 46 genes identified within the CNVRs were annotated. Conclusion: This study has improved the genomic characterization of 14 cat breeds and has provided CNVs information that can be used for studies of traits in cats. It can be considered a sound starting point for genomic CNVs identification in this species

Copy number variation mapping and genomic variation of autochthonous and commercial turkey populations

This study aims at investigating genomic diversity of several turkey populations using Copy Number Variants (CNVs). A total of 115 individuals from six Italian breeds (Colle Euganei, Bronzato Comune Italiano, Parma e Piacenza, Brianzolo, Nero d\u2019Italia, and Ermellinato di Rovigo), seven Narragansett, 38 commercial hybrids, and 30 Mexican turkeys, were genotyped with the Affymetrix 600K single nucleotide polymorphism (SNP) turkey array. The CNV calling was performed with the Hidden Markov Model of PennCNV software and with the Copy Number Analysis Module of SVS 8.4 by Golden Helix\uae. CNV were summarized into CNV regions (CNVRs) at population level using BEDTools. Variability among populations has been addressed by hierarchical clustering (pvclust R package) and by principal component analysis (PCA). A total of 2,987 CNVs were identified covering 4.65% of the autosomes of the Turkey_5.0/melGal5 assembly. The CNVRs identified in at least two individuals were 362\u2014189 gains, 116 losses, and 57 complexes. Among these regions the 51% contain annotated genes. This study is the first CNV mapping of turkey population using 600K chip. CNVs clustered the individuals according to population and their geographical origin. CNVs are known to be indicators also of adaptation, as some researches in different species are suggesting

Genome-Wide Association Study in Mexican Holstein Cattle Reveals Novel Quantitative Trait Loci Regions and Confirms Mapped Loci for Resistance to Bovine Tuberculosis

Bovine tuberculosis (bTB) is a disease of cattle that represents a risk to public health and causes severe economic losses to the livestock industry. Recently, genetic studies, like genome-wide association studies (GWAS) have greatly improved the investigation of complex diseases identifying thousands of disease-associated genomic variants. Here, we present evidence of genetic variants associated with resistance to TB in Mexican dairy cattle using a case-control approach with a selective DNA pooling experimental design. A total of 154 QTLRs (quantitative trait loci regions) at 10% PFP (proportion of false positives), 42 at 5% PFP and 5 at 1% PFP have been identified, which harbored 172 annotated genes. On BTA13, five new QTLRs were identified in the MACROD2 and KIF16B genes, supporting their involvement in resistance to bTB. Six QTLRs harbor seven annotated genes that have been previously reported as involved in immune response against Mycobacterium spp: BTA (Bos taurus autosome) 1 (CD80), BTA3 (CTSS), BTA 3 (FCGR1A), BTA 23 (HFE), BTA 25 (IL21R), and BTA 29 (ANO9 and SIGIRR). We identified novel QTLRs harboring genes involved in Mycobacterium spp. immune response. This is a first screening for resistance to TB infection on Mexican dairy cattle based on a dense SNP (Single Nucleotide Polymorphism) chip