Variability and Number of Circulating Complementary Sex Determiner (Csd) Alleles in a Breeding Population of Italian Honeybees under Controlled Mating.

In Apis mellifera, csd is the primary gene involved in sex determination: haploid hemizygous eggs develop as drones, while females develop from eggs heterozygous for the csd gene. If diploid eggs are homozygous for the csd gene, diploid drones will develop, but will be eaten by worker bees before they are born. Therefore, high csd allelic diversity is a priority for colony survival and breeding. This study aims to investigate the variability of the hypervariable region (HVR) of the csd gene in bees sampled in an apiary under a selection scheme. To this end, an existing dataset of 100 whole-genome sequences was analyzed with a validated pipeline based on de novo assembly of sequences within the HVR region. In total, 102 allelic sequences were reconstructed and translated into amino acid sequences. Among these, 47 different alleles were identified, 44 of which had previously been observed, while 3 are novel alleles. The results show a high variability in the csd region in this breeding population of honeybees.

Analysis of Complementary Sex-Determiner (csd) Allele Diversity in Different Honeybee Subspecies from Italy Based on NGS Data.

Sexual regulation in Apis mellifera is controlled by the complementary sex-determiner (csd) gene: females (queens and workers) are heterozygous at this locus and males (drones) are hemizygous. When homozygous diploid drones develop, they are eaten by worker bees. High csd allelic diversity in honeybee populations is a priority for colony survival. The focus of this study is to investigate csd variability in the genomic sequence of the hypervariable region (HVR) of the csd gene in honeybee subspecies sampled in Italy. During the summer of 2017 and 2018, worker bees belonging to 125 colonies were sampled. The honeybees belonged to seven different A. mellifera subspecies: A. m. ligustica, A. m. sicula, A. m cecropia, A. m. carnica, A. m. mellifera, Buckfast and hybrid Carnica. Illumina genomic resequencing of all samples was performed and used for the characterization of global variability among colonies. In this work, a pipeline using existing resequencing data to explore the csd gene allelic variants present in the subspecies collection, based on de novo assembly of sequences falling within the HVR region, is described. On the whole, 138 allelic sequences were successfully reconstructed. Among these, 88 different alleles were identified, 68 of which match with csd alleles present in the NCBI GenBank database.

Automated image analysis to assess hygienic behaviour of honeybees.

Focus of this study is to design an automated image processing pipeline for handling uncontrolled acquisition conditions of images acquired in the field. The pipeline has been tested on the automated identification and count of uncapped brood cells in honeybee (Apis Mellifera) comb images to reduce the workload of beekeepers during the study of the hygienic behavior of honeybee colonies. The images used to develop and test the model were acquired by beekeepers on different days and hours in summer 2020 and under uncontrolled conditions. This resulted in images differing for background noise, illumination, color, comb tilts, scaling, and comb sizes. All the available 127 images were manually cropped to approximately include the comb area. To obtain an unbiased evaluation, the cropped images were randomly split into a training image set (50 images), which was used to develop and tune the proposed model, and a test image set (77 images), which was solely used to test the model. To reduce the effects of varied illuminations or exposures, three image enhancement algorithms were tested and compared followed by the Hough Transform, which allowed identifying individual cells to be automatically counted. All the algorithm parameters were automatically chosen on the training set by grid search. When applied to the 77 test images the model obtained a correlation of 0.819 between the automated counts and the experts' counts. To provide an assessment of our model with publicly available images acquired by a different equipment and under different acquisition conditions, we randomly extracted 100 images from a comb image dataset made available by a recent literature work. Though it has been acquired under controlled exposure, the images in this new set have varied illuminations; anyhow, our pipeline obtains a correlation between automatic and manual counts equal to 0.997. In conclusion, our tests on the automatic count of uncapped honey bee comb cells acquired in the field and on images extracted from a publicly available dataset suggest that the hereby generated pipeline successfully handles varied noise artifacts, illumination, and exposure conditions, therefore allowing to generalize our method to different acquisition settings. Results further improve when the acquisition conditions are controlled.

Investigating Genetic and Phenotypic Variability of Queen Bees: Morphological and Reproductive Traits.

The quality of the honeybee queen has an important effect on a colony's development, productivity, and survival. Queen failure or loss is considered a leading cause for colonies' mortality worldwide. The queen's quality, resulting from her genetic background, developmental conditions, mating success, and environment, can be assessed by some morphological measures. The study aims to investigate variability for traits that could assess the quality of the queen. Related animals were enrolled in this study. Variance components were estimated fitting a mixed animal model to collected data. Heritabilities of body and tagmata weights ranged from 0.46 to 0.54, whereas lower estimates were found for the tagmata width and wing length. Heritabilities estimated for the spermatheca diameter and volume, number of ovarioles, and number of sperms were 0.17, 0.88, 0.70, and 0.57, respectively. Many phenotypic correlations related to size were high and positive, while weak correlations were found between morphology and reproductive traits. Introducing a queen's traits in a selection program could improve colonies' survivability. Further research should focus on better defining the correlations between the individual qualities of a queen and her colony's performance.

Whole-Genome Sequence Analysis of Italian Honeybees (Apis mellifera).

At the end of the last glaciation, Apis mellifera was established in northern Europe. In Italy, Apis melliferaligustica adapted to the mild climate and to the rich floristic biodiversity. Today, with the spread of Varroa destructor and with the increasing use of pesticides in agriculture, the Ligustica subspecies is increasingly dependent on human action for its survival. In addition, the effects of globalization of bee keeping favored the spread in Italy of other honeybee stocks of A. mellifera, in particular the Buckfast bee. The purpose of this study was to characterize the Italian honeybee's population by sequencing the whole genome of 124 honeybees. Whole genome sequencing was performed by Illumina technology, obtaining a total coverage of 3720.89X, with a mean sample coverage of 29.77X. A total of 4,380,004 SNP variants, mapping on Amel_HAv3.1 chromosomes, were detected. Results of the analysis of the patterns of genetic variation allowed us to identify and subgroup bees according to their type. The investigation revealed the genetic originality of the Sicula, and in A.m. ligustica limited genetic introgression from the other breeds. Morphometric analysis of 5800 worker bees was in agreement with genomic data.

The genetic heritage of Alpine local cattle breeds using genomic SNP data.

BACKGROUND: Assessment of genetic diversity and population structure provides important control metrics to avoid genetic erosion, inbreeding depression and crossbreeding between exotic and locally-adapted cattle breeds since these events can have deleterious consequences and eventually lead to extinction. Historically, the Alpine Arc represents an important pocket of cattle biodiversity with a large number of autochthonous breeds that provide a fundamental source of income for the entire regional economy. By using genotype data from medium-density single nucleotide polymorphism (SNP) arrays, we performed a genome-wide comparative study of 23 cattle populations from the Alpine Arc and three cosmopolitan breeds. RESULTS: After filtering, we obtained a final genotyping dataset consisting of 30,176 SNPs for 711 individuals. The local breeds showed high or intermediate values of genetic diversity compared to the highly selected cosmopolitan breeds. Patterns of genetic differentiation, multidimensional scaling, admixture analysis and the constructed phylogenetic tree showed convergence, which indicates the presence of gene flow among the breeds according to both geographic origin and historical background. Among the most differentiated breeds, we identified the modern Brown cattle. In spite of admixture events, several local breeds have preserved distinctive characteristics, which is probably due to differences in genetic origin and geographic location. CONCLUSIONS: This study represents one of the most comprehensive genome-wide analysis of the Alpine cattle breeds to date. Using such a large dataset that includes the majority of the local breeds found in this region, allowed us to expand knowledge on the evaluation and status of Alpine cattle biodiversity. Our results indicate that although many of the analyzed local breeds are listed as endangered, they still harbor a large amount of genetic diversity, even when compared to some cosmopolitan breeds. This finding, together with the reconstruction of the phylogeny and the relationships between these Alpine Arc cattle breeds, provide crucial insights not only into the improvement of genetic stocks but also into the implementation of future conservation strategies.

Functional SNP panel for parentage assessment and assignment in worldwide goat breeds.

BACKGROUND: International standard panels of single nucleotide polymorphisms (SNPs) have replaced microsatellites in several species for parentage assessment and assignment (PA) purposes. However, such a resource is still lacking in goats. The application of a cheap tool for PA would help the management of goat populations by improving the reliability of pedigree registration and, consequently, allow a better implementation of breeding schemes or conservation programs. RESULTS: Using data from the current GoatSNP50 chip, starting from a worldwide dataset of more than 4000 animals belonging to more than 140 breeds and populations from the AdaptMap initiative, we selected a panel of 195 SNPs. The assignment rate of this panel was up to 100% on an additional dataset that included 2000 Alpine and Saanen animals and highly related candidate sires. CONCLUSIONS: In this study, we defined a highly informative SNP panel, which will be publicly available to worldwide breeders and laboratories. Its development on such a large number of breeds and populations, together with validation on a second set of cosmopolitan breeds, makes it a promising and important genomic tool for the goat species.

Conservation status and historical relatedness of Italian cattle breeds.

BACKGROUND: In the last 50 years, the diversity of cattle breeds has experienced a severe contraction. However, in spite of the growing diffusion of cosmopolite specialized breeds, several local cattle breeds are still farmed in Italy. Genetic characterization of breeds represents an essential step to guide decisions in the management of farm animal genetic resources. The aim of this work was to provide a high-resolution representation of the genome-wide diversity and population structure of Italian local cattle breeds using a medium-density single nucleotide polymorphism (SNP) array. RESULTS: After quality control filtering, the dataset included 31,013 SNPs for 800 samples from 32 breeds. Our results on the genetic diversity of these breeds agree largely with their recorded history. We observed a low level of genetic diversity, which together with the small size of the effective populations, confirmed that several breeds are threatened with extinction. According to the analysis of runs of homozygosity, evidence of recent inbreeding was strong in some local breeds, such as Garfagnina, Mucca Pisana and Pontremolese. Patterns of genetic differentiation, shared ancestry, admixture events, and the phylogenetic tree, all suggest the presence of gene flow, in particular among breeds that originate from the same geographical area, such as the Sicilian breeds. In spite of the complex admixture events that most Italian cattle breeds have experienced, they have preserved distinctive characteristics and can be clearly discriminated, which is probably due to differences in genetic origin, environment, genetic isolation and inbreeding. CONCLUSIONS: This study is the first exhaustive genome-wide analysis of the diversity of Italian cattle breeds. The results are of significant importance because they will help design and implement conservation strategies. Indeed, efforts to maintain genetic diversity in these breeds are needed. Improvement of systems to record and monitor inbreeding in these breeds may contribute to their in situ conservation and, in view of this, the availability of genomic data is a fundamental resource.

Distribution of ncRNAs expression across hypothalamic-pituitary-gonadal axis in Capra hircus.

BACKGROUND: Molecular regulation of the hypothalamic-pituitary-gonadal (HPG) axis plays an essential role in the fine tuning of seasonal estrus in Capra hircus. Noncoding RNAs (ncRNAs) are emerging as key regulators in sexual development and mammalian reproduction. In order to identify ncRNAs and to assess their expression patterns, along the HPG axis, we sequenced ncRNA libraries from hypothalamus, pituitary and ovary of three goats. RESULTS: Among the medium length noncoding RNAs (mncRNAs) identified, small nucleolar RNAs (snoRNAs) and transfer RNAs (tRNAs) were found to be more abundant in ovary and hypothalamus, respectively. The observed GC content was representative for different classes of ncRNAs, allowing the identification of a tRNA-derived RNA fragments (tRFs) subclass, which had a peak distribution around 32-38% GC content in the hypothalamus. Differences observed among organs confirmed the specificity of microRNA (miRNA) profiles for each organ system. CONCLUSIONS: Data on ncRNAs in organs constituting the HPG axis will contribute to understanding their role in the physiological regulation of reproduction in goats.

Studies of modern Italian dog populations reveal multiple patterns for domestic breed evolution.

Through thousands of years of breeding and strong human selection, the dog (Canis lupus familiaris) exists today within hundreds of closed populations throughout the world, each with defined phenotypes. A singular geographic region with broad diversity in dog breeds presents an interesting opportunity to observe potential mechanisms of breed formation. Italy claims 14 internationally recognized dog breeds, with numerous additional local varieties. To determine the relationship among Italian dog populations, we integrated genetic data from 263 dogs representing 23 closed dog populations from Italy, seven Apennine gray wolves, and an established dataset of 161 globally recognized dog breeds, applying multiple genetic methods to characterize the modes by which breeds are formed within a single geographic region. Our consideration of each of five genetic analyses reveals a series of development events that mirror historical modes of breed formation, but with variations unique to the codevelopment of early dog and human populations. Using 142,840 genome-wide SNPs and a dataset of 1,609 canines, representing 182 breeds and 16 wild canids, we identified breed development routes for the Italian breeds that included divergence from common populations for a specific purpose, admixture of regional stock with that from other regions, and isolated selection of local stock with specific attributes.

Genomic Analysis Suggests KITLG is Responsible for a Roan Pattern in two Pakistani Goat Breeds.

The roan coat color pattern is described as the presence of white hairs intermixed with pigmented hairs. This kind of pigmentation pattern has been observed in many domestic species, including the goat. The molecular mechanisms and inheritance that underlie this pattern are known for some species and the KITLG gene has been shown associated with this phenotype. To date, no research effort has been carried out to find the gene(s) that control(s) roan coat color pattern in goats. In the present study, after genotyping with the GoatSNP50 BeadChip, 35 goats that showed a roan pattern and that belonged to two Pakistan breeds (Group A) were analyzed and then compared to 740 goats of 39 Italian and Pakistan goat breeds that did not have the same coat color pattern (Group B). Runs of homozygosity-based and XP-EHH analyses were used to identify unique genomic regions potentially associated with the roan pattern. A total of 3 regions on chromosomes 5, 6, and 12 were considered unique among the group A versus group B comparisons. The A region > 1.7 Mb on chromosome 5 was the most divergent between the two groups. This region contains six genes, including the KITLG gene. Our findings support the hypothesis that the KITLG gene may be associated with the roan phenotype in goats.

Identification of additional loci associated with antibody response to Mycobacterium avium ssp. Paratuberculosis in cattle by GSEA-SNP analysis.

Mycobacterium avium subsp. paratuberculosis: (MAP) causes a contagious chronic infection results in Johne's disease in a wide range of animal species, including cattle. Several genome-wide association studies (GWAS) have been carried out to identify loci putatively associated with MAP susceptibility by testing each marker separately and identifying SNPs that show a significant association with the phenotype, while SNP with modest effects are usually ignored. The objective of this study was to identify modest-effect genes associated with MAP susceptibility using a pathway-based approach. The Illumina BovineSNP50 BeadChip was used to genotype 966 Holstein cows, 483 positive and 483 negative for antibody response to MAP, data were then analyzed using novel SNP-based Gene Set Enrichment Analysis (GSEA-SNP) and validated with Adaptive Rank Truncated Product methodology. An allele-based test was carried out to estimate the statistical association for each marker with the phenotype, subsequently SNPs were mapped to the closest genes, considering for each gene the single variant with the highest value within a window of 50 kb, then pathway-statistics were tested using the GSEA-SNP method. The GO biological process "embryogenesis and morphogenesis" was most highly associated with antibody response to MAP. Within this pathway, five genes code for proteins which play a role in the immune defense relevant to response to bacterial infection. The immune response genes identified would not have been considered using a standard GWAS, thus demonstrating that the pathway approach can extend the interpretation of genome-wide association analyses and identify additional candidate genes for target traits.

Genome-wide analysis of DNA methylation in hypothalamus and ovary of Capra hircus.

BACKGROUND: DNA methylation is a frequently studied epigenetic modification due to its role in regulating gene expression and hence in biological processes and in determining phenotypic plasticity in organisms. Rudimentary DNA methylation patterns for some livestock species are publically available: among these, goat methylome deserves to be further explored. RESULTS: Genome-wide DNA methylation maps of the hypothalamus and ovary from Saanen goats were generated using Methyl-CpG binding domain protein sequencing (MBD-seq). Analysis of DNA methylation patterns indicate that the majority of methylation peaks found within genes are located gene body regions, for both organs. Analysis of the distribution of methylated sites per chromosome showed that chromosome X had the lowest number of methylation peaks. The X chromosome has one of the highest percentages of methylated CpG islands in both organs, and approximately 50% of the CpG islands in the goat epigenome are methylated in hypothalamus and ovary. Organ-specific Differentially Methylated Genes (DMGs) were correlated with the expression levels. CONCLUSIONS: The comparison between transcriptome and methylome in hypothalamus and ovary showed that a higher level of methylation is not accompanied by a higher gene suppression. The genome-wide DNA methylation map for two goat organs produced here is a valuable starting point for studying the involvement of epigenetic modifications in regulating goat reproduction performance.

The Valdostana goat: a genome-wide investigation of the distinctiveness of its selective sweep regions.

The Valdostana goat is an alpine breed, raised only in the northern Italian region of the Aosta Valley. This breed's main purpose is to produce milk and meat, but is peculiar for its involvement in the "Batailles de Chèvres," a recent tradition of non-cruel fight tournaments. At both the genetic and genomic levels, only a very limited number of studies have been performed with this breed and there are no studies about the genomic signatures left by selection. In this work, 24 unrelated Valdostana animals were screened for runs of homozygosity to identify highly homozygous regions. Then, six different approaches (ROH comparison, Fst single SNPs and windows based, Bayesian, Rsb, and XP-EHH) were applied comparing the Valdostana dataset with 14 other Italian goat breeds to confirm regions that were different among the comparisons. A total of three regions of selection that were also unique among the Valdostana were identified and located on chromosomes 1, 7, and 12 and contained 144 genes. Enrichment analyses detected genes such as cytokines and lymphocyte/leukocyte proliferation genes involved in the regulation of the immune system. A genetic link between an aggressive challenge, cytokines, and immunity has been hypothesized in many studies both in humans and in other species. Possible hypotheses associated with the signals of selection detected could be therefore related to immune-related factors as well as with the peculiar battle competition, or other breed-specific traits, and provided insights for further investigation of these unique regions, for the understanding and safeguard of the Valdostana breed.

Responses of Bovine Innate Immunity to Mycobacterium avium subsp. paratuberculosis Infection Revealed by Changes in Gene Expression and Levels of MicroRNA.

Paratuberculosis in cattle is a chronic granulomatous gastroenteritis caused by Mycobacterium avium subsp. paratubercolosis (MAP) which is endemic worldwide. In dairy herds, it is responsible for huge economic losses. However, current diagnostic methods do not detect subclinical infection making control of the disease difficult. The identification of MAP infected animals during the sub-clinical phase of infection would play a key role in preventing the dissemination of the pathogen and in reducing transmission. Gene expression and circulating microRNA (miRNA) signatures have been proposed as biomarkers of disease both in the human and veterinary medicine. In this paper, gene expression and related miRNA levels were investigated in cows positive for MAP, by ELISA and culture, in order to identify potential biomarkers to improve diagnosis of MAP infection. Three groups, each of 5 animals, were used to compare the results of gene expression from positive, exposed and negative cows. Overall 258 differentially expressed genes were identified between unexposed, exposed, but ELISA negative and positive groups which were involved in biological functions related to inflammatory response, lipid metabolism and small molecule biochemistry. Differentially expressed miRNA was also found among the three groups: 7 miRNAs were at a lower level and 2 at a higher level in positive animals vs unexposed animals, while 5 and 3 miRNAs were respectively reduced and increased in the exposed group compared to the unexposed group. Among the differentially expressed miRNAs 6 have been previously described as immune-response related and two were novel miRNAs. Analysis of the miRNA levels showed correlation with expression of their target genes, known to be involved in the immune process. This study suggests that miRNA expression is affected by MAP infection and play a key role in tuning the host response to infection. The miRNA and gene expression profiles may be biomarkers of infection and potential diagnostic of MAP infection earlier than the current ELISA based diagnostic tests.

Genetic diversity of Italian goat breeds assessed with a medium-density SNP chip.

BACKGROUND: Among the European countries, Italy counts the largest number of local goat breeds. Thanks to the recent availability of a medium-density SNP (single nucleotide polymorphism) chip for goat, the genetic diversity of Italian goat populations was characterized by genotyping samples from 14 Italian goat breeds that originate from different geographical areas with more than 50 000 SNPs evenly distributed on the genome. RESULTS: Analysis of the genotyping data revealed high levels of genetic polymorphism and an underlying North-south geographic pattern of genetic diversity that was highlighted by both the first dimension of the multi-dimensional scaling plot and the Neighbour network reconstruction. We observed a moderate and weak population structure in Northern and Central-Southern breeds, respectively, with pairwise FST values between breeds ranging from 0.013 to 0.164 and 7.49 % of the total variance assigned to the between-breed level. Only 2.11 % of the variance explained the clustering of breeds into geographical groups (Northern, Central and Southern Italy and Islands). CONCLUSIONS: Our results indicate that the present-day genetic diversity of Italian goat populations was shaped by the combined effects of drift, presence or lack of gene flow and, to some extent, by the consequences of traditional management systems and recent demographic history. Our findings may constitute the starting point for the development of marker-assisted approaches, to better address future breeding and management policies in a species that is particularly relevant for the medium- and long-term sustainability of marginal regions.

Variation in salivary and pancreatic alpha-amylase genes in Italian horse breeds.

The dietary demand of the modern horse relies on high-cereal feeding and limited forage compared with natural grazing conditions, predisposing the horse to several important diseases. Salivary and pancreatic alpha-amylases (coded by AMY1 and AMY2 genes, respectively) play a crucial role in carbohydrate digestion in nonruminants, but little is known about these 2 genes in the horse. Aim of this work has been to distinguish genomic sequences of horse AMY1 and AMY2 genes and to analyze any polymorphisms in breeds historically characterized by marked differences in nutritional management. A single nucleotide polymorphism detection was performed and 7 novel single nucleotide polymorphisms were found. Three single nucleotide polymorphisms are in exons and were genotyped in 112 horses belonging to 6 breeds. One single nucleotide polymorphism in AMY1 gene distinguished Haflinger and the Italian native Murgese from the other breeds, whereas both the single nucleotide polymorphisms in AMY2 gene showed different allelic frequencies in Friesian compared with the other breeds. These differences are confirmed by quite high fixation index (Fst) values for these 2 nonsynonymous single nucleotide polymorphisms. These preliminary results highlight marked divergences in allele frequencies of AMY1 and AMY2 genes, involved in starch digestion, between horse breeds characterized by different histories of selection, thus providing first indications of possible relations between genetics and nutritional management.

The hairless (hr) gene is involved in the congenital hypotrichosis of Valle del Belice sheep.

Congenital hypotrichosis in mammalian species consists of partial or complete absence of hair at birth. The hairless gene is often responsible for this disorder in men, mice and rats. Recent experimental data on Valle del Belice sheep reared in Sicily for milk production, support the genetic control of the ovine hypotrichosis as a Mendelian recessive trait. The ovine hairless gene was chosen as the candidate gene involved in this disorder. Blood samples were collected from Valle del Belice sheep with the normal and hypotrichotic phenotypes. Almost the entire hairless gene was successfully amplified using the long PCR technique. Unrelated sheep with differing phenotypes were randomly chosen for sequencing the amplified products. Different mutations related to the hypotrichotic phenotype were found in exon 3. In fact, sequencing revealed an A/T transversion at position 739, a G/A transition at position 823, and a C/T transition at position 1312. From these nucleotide exchanges, three substitutions of the processed mature protein were deduced at the amino acid positions 247 (Thr/Ser), 275 (Ala/Thr), and 438 (Gln/Stop). A PCR-SSCP based test was developed in order to detect the last mutation, which is responsible for the hypotrichotic phenotype.