Analysis of Animal Genetic Resources for the Identification of Polymorphisms Associated with Phenotypic Features and Evolutionary Aspects

Coat colour has been one of the main target of both natural and artificial selection and in particular one of the first of domestication process. This Thesis has been focused on the detection of genetic variability of new variants in coat colour genes in different species, especially those associated with particular coat colour phenotypes and introgression of domesticated alleles in wild populations. The aim of this Thesis was to identify and analyse polymorphisms associated with phenotypic traits that have been selected during the domestication processes and may differentiate breeds or populations in several livestock species, i.e. rabbit, donkey and pig, and evaluate the potential evolutionary effects on the wild counterparts, considering the specific example of the introgression between domestic pigs and wild boar populations. In particular, two main genes involved in melanin production (TYR and TYRP1) have been characterized in donkeys and rabbits respectively and variants in these genes have been significantly associated with albinism, suggesting that rabbits and donkeys could be considered as animal models for human albinism because these variants have never been detected in humans. Another aim of this Thesis, was the possibilty to use coat colour genes as introgression markers and the results suggest that accurate monitoring and management of rural stock populations, in order to avoid backcrossing events, are needed both for the conservation of the ecologic natural equilibrium of wild environments and to make wild species less invasive for human activities. Obtained results have been also evaluated for applications that consider allele and genotype frequency differences for authentication purposes, in order to differentiate meat products originating from wild boars, considered an upper market niche, against pig meat products with a less economic value

Honey Environmental DNA Can Be Used to Detect and Monitor Honey Bee Pests: Development of Methods Useful to Identify Aethina tumida and Galleria mellonella Infestations

Environmental DNA (eDNA) contained in honey derives from the organisms that directly and indirectly have been involved in the production process of this matrix and that have played a role in the hive ecosystems where the honey has been produced. In this study we set up PCR-based assays to detect the presence of DNA traces left in the honey by two damaging honey bee pests: the small hive beetle (Aethina tumida) and the greater wax moth (Galleria mellonella). DNA was extracted from 82 honey samples produced in Italy and amplified using two specific primer pairs that target the mitochondrial gene cytochrome oxidase I (COI) of A. tumida and two specific primer pairs that target the same gene in G. mellonella. The limit of detection was tested using sequential dilutions of the pest DNA. Only one honey sample produced in Calabria was positive for A. tumida whereas about 66% of all samples were positively amplified for G. mellonella. The use of honey eDNA could be important to establish early and effective measures to contain at the local (e.g., apiary) or regional scales these two damaging pests and, particularly for the small hive beetle, to prevent its widespread diffusion

A genotyping by sequencing approach can disclose Apis mellifera population genomic information contained in honey environmental DNA

Awareness has been raised over the last years on the genetic integrity of autochthonous honey bee subspecies. Genomic tools available in Apis mellifera can make it possible to measure this information by targeting individual honey bee DNA. Honey contains DNA traces from all organisms that contributed or were involved in its production steps, including the honey bees of the colony. In this study, we designed and tested a genotyping by sequencing (GBS) assay to analyse single nucleotide polymorphisms (SNPs) of A. mellifera nuclear genome using environmental DNA extracted from honey. A total of 121 SNPs (97 SNPs informative for honey bee subspecies identification and 24 SNPs associated with relevant traits of the colonies) were used in the assay to genotype honey DNA, which derives from thousands of honey bees. Results were integrated with information derived from previous studies and whole genome resequencing datasets. This GBS method is highly reliable in estimating honey bee SNP allele frequencies of the whole colony from which the honey derived. This assay can be used to identify the honey bee subspecies of the colony that produced the honey and, in turn, to authenticate the entomological origin of the honey

Distribution of the Main Apis mellifera Mitochondrial DNA Lineages in Italy Assessed Using an Environmental DNA Approach

Simple Summary The conservation of the genetic diversity of the native honey bee subspecies is a hot topic in many European countries. Mitochondrial DNA (mtDNA) analyses can provide some information that is useful to monitor the genetic integrity of Apis mellifera populations. A preliminary distribution of the main honey bee mitotypes in Italy was obtained more than 20 years ago. In this study we obtained an updated and more detailed distribution map of the main groups of honey bee mitotypes using an unconventional method that exploits the information derived from the environmental DNA contained in the honey. The results were quite different from the picture taken two decades ago. The African mtDNA lineage was spread all over Italy and not only in Sicily, where it is mainly attached to the A. m. siciliana subspecies, and where it was identified in the previous investigation. A reduction in the frequency of the M lineage was also evident, and, on the other hand, a counterbalanced increase in the C mitotypes was observed in regions all over. The obtained results provided an updated distribution map of the A, C and M groups of mitotypes in Italy, which could be a starting point to design appropriate conservation programs for native honey bee subspecies. Growing interest has been emerging on the need to monitor the genetic integrity of the European Apis mellifera subspecies that could be threatened by the human-mediated dispersion of non-native populations and lines. Mitochondrial DNA (mtDNA) lineages can provide useful information for this purpose. In this study, we took advantage of the environmental DNA (eDNA) contained in the honey, which can be analyzed to detect the main groups of mitotypes of the honey bees that produced it. In this study, we applied this eDNA to produce a distribution map all over the Italian peninsula and the two major islands (Sicily and Sardinia) of the following three honey bee mtDNA lineages: A, C and M. A total of 607 georeferenced honey samples, produced in all Italian regions, was analyzed to detect these lineages. The A lineage was widespread in Sicily, as expected, considering that A. m. siciliana carries the African lineage. Surprisingly, this lineage was also reported in about 14% of all other samples produced in almost all continental regions, and in Sardinia. The applied method obtained an updated distribution map of honey bee mtDNA lineages that could be useful to design policies for the conservation of Italian honey bee genetic resources

Known loci in the KIT and TYR genes do not explain the depigmented white coat colour of Austro-Hungarian Baroque donkey

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Next generation semiconductor based sequencing of the donkey (Equus asinus) genome provided comparative sequence data against the horse genome and a few millions of single nucleotide polymorphisms

Few studies investigated the donkey (Equus asinus) at the whole genome level so far. Here, we sequenced the genome of two male donkeys using a next generation semiconductor based sequencing platform (the Ion Proton sequencer) and compared obtained sequence information with the available donkey draft genome (and its Illumina reads from which it was originated) and with the EquCab2.0 assembly of the horse genome. Moreover, the Ion Torrent Personal Genome Analyzer was used to sequence reduced representation libraries (RRL) obtained from a DNA pool including donkeys of different breeds (Grigio Siciliano, Ragusano and Martina Franca). The number of next generation sequencing reads aligned with the EquCab2.0 horse genome was larger than those aligned with the draft donkey genome. This was due to the larger N50 for contigs and scaffolds of the horse genome. Nucleotide divergence between E. caballus and E. asinus was estimated to be similar to 0.52-0.57%. Regions with low nucleotide divergence were identified in several autosomal chromosomes and in the whole chromosome X. These regions might be evolutionally important in equids. Comparing Y-chromosome regions we identified variants that could be useful to track donkey paternal lineages. Moreover, about 4.8 million of single nucleotide polymorphisms (SNPs) in the donkey genome were identified and annotated combining sequencing data from Ion Proton (whole genome sequencing) and Ion Torrent (RRL) runs with Illumina reads. A higher density of SNPs was present in regions homologous to horse chromosome 12, in which several studies reported a high frequency of copy number variants. The SNPs we identified constitute a first resource useful to describe variability at the population genomic level in E. asinus and to establish monitoring systems for the conservation of donkey genetic resources

Investigation of genetic markers for resistance to infectious diseases in different pig breeds: antagonism with production and performance traits?

Infectious diseases have devastating economic impacts on the pig breeding industry worldwide. A few genetic markers associated with disease resistance have been recently identified and used in Marker Assisted Selection (MAS) in a few pig populations as part of disease control programs

Shotgun metagenomics of honey DNA: Evaluation of a methodological approach to describe a multi-kingdom honey bee derived environmental DNA signature

Honey bees are considered large-scale monitoring tools due to their environmental exploration and foraging activities. Traces of these activities can be recovered in the honey that also may reflect the hive ecological micro-conditions in which it has been produced. This study applied a next generation sequencing platform (Ion Torrent) for shotgun metagenomic analysis of honey environmental DNA (eDNA). The study tested a methodological framework to interpret DNA sequence information useful to describe the complex ecosystems of the honey bee colony superorganism, its pathosphere and the heterogeneity of the agroecological environments and environmental sources that left DNA marks in the honey. Analysis of two honeys reported sequence reads from five main organism groups (kingdoms or phyla): arthropods (that mainly included reads from Apis mellifera, several other members of the Hymenotpera, in addition to members of the Diptera, Coleoptera and Lepidoptera, as well as aphids and mites), plants (that clearly confirmed the botanical origin of the two honeys, i.e. orange tree blossom and eucalyptus tree blossom honeys), fungi and bacteria (including common hive and honey bee gut microorganisms, honey bee pathogens and plant pathogens), and viruses (which accounted for the largest number of reads in both honeys, mainly assigned to Apis mellifera filamentous virus). The shotgun metagenomic approach that was used in this study can be applied in large scale experiments that might have multiple objectives according to the multi-kingdom derived eDNA that is contained in the honey

Entomological signatures in honey: an environmental DNA metabarcoding approach can disclose information on plant-sucking insects in agricultural and forest landscapes

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

Exploiting phenotype diversity in a local animal genetic resource: identification of a single nucleotide polymorphism associated with the tail shape phenotype in the autochthonous Casertana pig breed

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