Application of molecular markers to investigate genetic diversity in Sicilian livestock

La diversità genetica delle specie e razze di interesse zootecnico, rappresenta un’importante risorsa in tutti i sistemi di allevamento . Per lo studio della diversità genetica, nel corso dei decenni sono stati sviluppati diversi metodi che si basano su informazioni del pedigree o su dati molecolari (microsatelliti e SNPs, Single Nucleotide Polymorphisms). Con l’aumento della disponibilità di marcatori molecolari per la maggior parte delle specie di interesse zootecnico, e con lo sviluppo di sofisticate tecniche analitiche, sta crescendo la capacità di caratterizzare la variabilità genetica delle razze. Inoltre, ad oggi, poche sono le informazioni sulla diversità genetica delle razze e delle popolazioni autoctone siciliane. L’obiettivo di questa tesi è stato quello di studiare la diversità e la struttura genetica nelle razze e popolazioni zootecniche autoctone siciliane mediante l’uso di marcatori molecolari. Nel capitolo 2 è stata sequenziata la regione promoter del gene della -lattoglobulina (BLG) ovina al fine di individuare i polimorfismi, calcolare e analizzare gli aplotipi e studiare le relazione filogenetiche tra la razza Valle del Belice e le altre razze considerate sue progenitrici. L’allineamento e l’analisi delle sequenze ottenute hanno evidenziato la presenza di 36 SNPs e una delezione, sottolineando un’elevata variabilità all’interno delle razze. Sono stati individuati quattro siti di legame per Milk Protein Binding Factors (MPBFs) e cinque siti di legame Nuclear Factor-I (NF-I). Utilizzando i siti polimorfici identificati, sono stati calcolati 22 aplotipi, usati per la ricostruzione di un albero filogenetico tramite l’algoritmo Neighbor-Joining. L’analisi degli indici di diversità genetica ha mostrato valori più bassi di diversità nucleotidica per la razza Sarda, mentre la razza Comisana ha presentato i valori più alti. Confrontando la diversità nucleotidica tra le razze, il valore più alto è stato ottenuto tra le razze Valle del Belice e Pinzirita, mentre quello più basso è stato ottenuto tra la razza Valle del Belice e la razza Sarda. Considerando che i polimorfismi nella regione promoter del gene della BLG potrebbero avere un ruolo funzionale associato alla composizione del latte, il valore più basso della diversità nucleotidica tra le razze Valle del Belice e Sarda, potrebbe essere correlato ad una maggiore somiglianza nella produzione qualitativa del latte di queste due razze rispetto alle altre. Nel capitolo 3, un pannello di 20 marcatori microsatelliti è stato utilizzato per studiare e caratterizzare la struttura genetica delle quattro razze ovine autoctone siciliane (Barbaresca, Comisana, Pinzirita e Valle del Belice) e della razza Sarda, e per determinare le relazioni filogenetiche che intercorrono tra esse. Il numero di alleli trovati (259) ed il contenuto di informazione polimorfica (PIC) indicano che il pannello utilizzato è altamente informativo (0,76). I valori di Fst, Fis e Fit hanno mostrato una bassa differenziazione genetica e bassi livelli di consanguineità all’interno delle razze e tra esse. Il dendrogramma Neighbor-Joining, ottenuto sulla base delle distanze genetiche di Reynold, e l’analisi delle corrispondenti fattoriali, hanno evidenziato una marcata separazione tra la razza Barbaresca e le altre razze ovine. Il test di assegnazione bayesiano ha mostrato una struttura genetica più omogenea per le razze Barbaresca e Sarda, mentre il più basso valore di assegnazione è stato trovato nella razza Pinzirita. I nostri risultati indicano la presenza di una elevata variabilità, bassa consanguineità e bassa differenziazione genetica, fatta eccezione per la razza Barbaresca, in accordo con la posizione geografica, i possibili flussi genici e le pratiche di allevamento. La bassa robustezza del test di assegnazione rende inutilizzabile l’uso dei marcatori microsatelliti ai fini della tracciabilità delle produzioni lattiero casearie, a causa dell’elevata promiscuità e del flusso genico, in particolare per le razze ovine da latte autoctone siciliane. Nel capitolo 4 vengono riportate per la prima volta, le stime riguardanti la struttura genetica, i livelli di inbreeding (F) e coancestry (f) e il linkage disequilibrium(LD) nelle razze bovine autoctone siciliane Cinisara e Modicana, utilizzando l’Illumina Bovine SNP50K v2 BeadChip. L’Analisi delle Componenti Principali (PCA) e l’algoritmo di assegnazione basato sulla statistica bayesiana, hanno mostrato che gli animali delle due razze formano due clusters distinti. I coefficienti F e f erano moderatamente elevati, mentre le attuali stime sulla effettiva dimensione della popolazione (Ne) erano basse in entrambe le razze, sottolineando una bassa variabilità genetica. Il valore medio del LD calcolato con r2 è risultato notevolmente inferiore rispetto ai valori medi riportati in letteratura per le altre razze bovine. I più alti valori di r2 sono stati trovati nel cromosoma 14, all’interno del quale sono state descritte diverse mutazioni che influenzano la produzione quanti-qualitativa del latte. Il basso valore di LD suggerisce che il presente chip non è ottimale, e che un pannello a più alta densità è necessario per acquisire le informazioni riguardanti i livelli di LD, mentre i parametri riguardanti la consanguineità e Ne, indicano la necessità di avviare programmi di conservazione per il recupero di queste razze bovine autoctone. Il capitolo 5 riporta i risultati relativi al calcolo del LD, alla stima del numero di blocchi di aplotipi per cromosoma e alla diversità genetica nelle razze ovine autoctone siciliane allevate per la produzione di latte, utilizzando gli Ovine Beadchip SNP50K v1 BeadChip. I valori del LD diminuivano in funzione della distanza e la media del coefficiente r2 era notevolmente inferiore al valore osservato nelle altre razze ovine. Sono stati trovati un ridotto numero di blocchi di aplotipi e un ridotto numero di SNPs per blocco, in particolare nelle razze Comisana e Pinzirita. La PCA ha mostrato che, mentre la Valle del Belice e la Pinzirita formano due gruppi omogenei e distinti per razza, la Comisana evidenzia la presenza di sottostrutture. La PCA condotta con un subset di SNPs ha mostrato una scarsa capacità di discriminare le razze tra loro. La razza Pinzirita ha evidenziato i più alti livelli di diversità genetica, mentre i valori più bassi sono stati riscontrati nella razza Valle del Belice. Le informazioni generate da questo studio potrebbero essere utilizzate per gli studi di associazione, nonché per lo sviluppo di programmi di selezione e miglioramento genetico.The genetic diversity of farm animal species and breeds is an important resource in all livestock systems. To evaluate genetic diversity in livestock populations several methods have been developed based on pedigree information or on molecular data. The increasing availability of molecular markers for most farm animal species and the development of techniques to analyze molecular variation are widening the capacity to characterize breeds genetic variation. Moreover, little is known about the genetic diversity in Sicilian livestock breeds and populations. The aim of this thesis was to explore the genetic diversity of the Sicilian autochthonous breeds and populations using molecular markers. In chapter 2, the promoter region of ovine -lactoglobulin (BLG) gene was sequenced in order to identify polymorphisms, infer and analyze haplotypes, and phylogenetic relationship among the Valle del Belice sheep breed and other three breeds considered as ancestors. Sequencing analysis and alignment of the obtained sequences showed the presence of 36 single nucleotide polymorphisms (SNPs) and one deletion. We found four binding sites for milk protein binding factors (MPBFs) and five binding sites for nuclear factor-I (NF-I). The number of identified polymorphisms showed high variability within breeds. A total of 22 haplotypes found in best reconstruction were inferred considering all 37 polymorphic sites. Haplotypes were used for the reconstruction of a phylogenetic tree using the Neighbor-Joining algorithm. Analysis of genetic diversity indexes showed that the Sarda sheep breed presented the lowest nucleotide diversity, whereas the Comisana sheep breed presented the highest one. Comparing the nucleotide diversity among breeds, the highest value was obtained between Valle del Belice and Pinzirita sheep breeds, whereas the lowest one was between Valle del Belice and Sarda sheep breeds. Considering that polymorphisms in the promoter region of BLG gene could have a functional role associated with milk composition, the lowest value of nucleotide diversity between Valle del Belice and Sarda sheep breeds may be related to a higher similarity of milk composition of these two breeds compared to the others. In Chapter 3, microsatellite markers were used to explore the genetic structure of the four Sicilian autochthonous and Sarda sheep breeds, and to determine their genetic relationship. A total of 259 alleles were observed with average polymorphic information content (PIC) equal to 0.76, showing that the used microsatellites panel was highly informative. The low value of genetic differentiation among breeds (Fst=0.049) may indicate that these breeds have a low differentiation level probably due to common history and breeding practices. The low Fis and Fit values indicated low level of inbreeding within and among breeds. The Unrooted Neighbor-Joining dendrogram obtained from the Reynold’s genetic distances, and factorial correspondence analysis revealed a separation between Barbaresca and the other sheep breeds. The Bayesian assignment test showed that Barbaresca and Sarda sheep breeds had more defined genetic structure, whereas the lowest assignment value was found in the Pinzirita sheep breed. Our results indicated high genetic variability, low inbreeding and low genetic differentiation, except for Barbaresca sheep breed, and were in accordance with geographical location, history, and breeding practices. The low robustness of the assignment test makes it unfeasible for traceability purposes, due to the high level of admixture, in particular for Sicilian dairy sheep breeds. In Chapter 4 were reported for the first time the estimates of population structure, the levels of inbreeding (F) and coancestry (f), and the linkage disequilibrium (LD) in two Sicilian autochthonous cattle breeds, the Cinisara and the Modicana, using the Illumina Bovine SNP50K v2 BeadChip. Principal Components Analysis and Bayesian clustering algorithm showed that animals from the two Sicilian breeds formed non-overlapping clusters and are clearly separated populations, even from the Holstein control population. The average molecular F and f coefficients were moderately high, and the current estimates of Ne were low in both breeds. These values indicated a low genetic variability. The average r2 was notably lower than the values observed in other cattle breeds. The highest r2 values were found in chromosome 14, where causative mutations affecting variation in milk production traits have been reported. The low value of LD indicated that the present chip is not an optimum, and that a denser SNP array is needed to capture more LD information. The levels of inbreeding and Ne reported in this study point out the need to establish a conservation program for these autochthonous cattle breeds. In Chapter 5 genome wide levels of LD, the number of haplotype blocks for each chromosome in each breed and the genetic diversity was assessed in the Sicilian dairy sheep breeds, using the Illumina Ovine SNP50K v1 BeadChip. The LD declined as a function of distance and average r2 was notably lower than the value observed in other sheep breeds. Few and small haplotype blocks were observed in Comisana and Pinzirita sheep breeds, which contained just two SNPs. The number of haplotype blocks reported in our study for the Sicilian dairy sheep breeds were extremely lower than those reported in other livestock species. PCA showed that while Valle del Belice and Pinzirita sheep breeds formed a unique cluster, the Comisana sheep breed showed the presence of substructure. PCA using a subset of SNPs showed lack of ability to discriminate among the breeds. The Pinzirita sheep breed displayed the highest genetic diversity, whereas the lowest value was found in the Valle del Belice sheep breed. The information generated from this study has important implications for the design and applications of association studies as well as for the development of selection breeding programs

Genetic diversity and population structure of Sicilian sheep breeds using microsatellite markers

Genetic diversity studies in domestic animals aim at evaluating genetic variation within and across breeds mainly for conservation purposes. In Sicily, dairy sheep production represents an important resource for hilly and mountain areas economy. Their milk is used for the production of traditional raw milk cheeses, sometimes protected designation of origin (PDO) cheeses. In some cases, the quality of these products is linked to a specific breed, i.e. mono-breed labelled cheeses and it is therefore important to be able to distinguish the milk of a breed from that of others, in order to guarantee both the consumer and the breed itself. In order to investigate the genetic structure and to perform an assignment test, a total of 331 individuals (Barbaresca, BAR n=57, Comisana, COM n=65, Pinzirita, PIN n=75, Sarda, SAR n=64, and Valle del Belice, VDB n=70) were analysed using a panel of 20 microsatellite markers. A total of 259 alleles were observed with average polymorphic information content equal to 0.76, showing that the microsatellites panel used was highly informative. Estimates of observed heterozygosity ranged from 0.65 in the BAR breed to 0.75 in the COM breed. The low value of genetic differentiation among breeds (Fst=0.049) may indicate that these breeds are little differentiated probably due to common history and breeding practices. The low Fis and Fit values indicated low level of inbreeding within and among breeds. The unrooted neighbor-joining dendrogram obtained from the Reynold's genetic distances, and factorial correspondence analysis revealed a separation between BAR and the other sheep breeds. Recent migration rates were estimated, showing that four out of the five breeds have not received a significant proportion of migrants. Only for the PIN breed a recent introgression rate from the VDB breed (7.2%) was observed. The Bayesian assignment test showed that BAR and SAR breeds had a more definite genetic structure (proportion of assignment of 92% and 86.6%, respectively), whereas the lowest assignment value was found in the PIN breed (67.1%). Our results indicated high genetic variability, low inbreeding and low genetic differentiation, except for BAR breed, and were in accordance with geographical location, history, and breeding practices. The low robustness of the assignment test makes it unfeasible for traceability purposes, due to the high level of admixture, in particular for COM, PIN and VDB

Caratterizzazione genetica mediante microsatelliti di una popolazione caprina siciliana

I microsatelliti sono ad oggi i marcatori molecolari maggiormente utilizzati per la caratterizzazione genetica nei caprini. Lo scopo del presente lavoro è stato quello di caratterizzare la struttura genetica della capra Mascaruna per verificare se può essere definita come una popolazione. L’analisi è stata condotta utilizzando un pannello di 18 microsatelliti. Il DNA è stato estratto da 60 individui di cui 20 Mascaruna (MAS), 20 Girgentana (GIR) e 20 animali derivanti da diversi incroci (MIX). Un totale di 148 alleli sono stati osservati di cui 106 in GIR, 107 in MAS e 129 in MIX; il valore del PIC è di 0,69 e tutti i marcatori hanno mostrato un numero di alleli superiori a 4. Valori più alti di MNA, Ho e He sono stati trovati in MIX (MNA=7,17, Ho=0,700 e He=0,731), seguito da MAS (MNA=5,94, Ho=0,697 e He=0,703) e GIR (MNA= 5,89, Ho=0,590 e He=0,666). Il più alto valore del Fis, che misura il livello di inbreeding all’interno di ciascuna popolazione, è stato trovato in GIR, mentre MAS ha mostrato il più basso valore. Alleli privati, in particolare in MAS, sono stati trovati con frequenza relativamente alta (13% e 20%). L’analisi delle distanze genetiche di Nei e Reynolds indicano una maggiore distanza tra MAS e GIR. Anche l’ACF mostra una chiara separazione di MAS rispetto agli altri due gruppi. Il test di assegnazione effettuato con STRUCTURE mostra per MAS un cluster meno definito rispetto a quello di GIR, ma più definito rispetto a MIX. Questo studio riporta i primi risultati sulla caratterizzazione genetica della capra MAS. I dati ottenuti mostrano una uniformità genetica confrontabile con quella riportata per altre razze o popolazioni ufficialmente riconosciute. Ulteriori studi verranno condotti al fine di confermare i risultati ottenuti e definire le origini della capra MAS

Study of polymorphisms in the promoter region of ovine β-lactoglobulin gene and phylogenetic analysis among the Valle del Belice breed and other sheep breeds considered as ancestors

The aim of this work was to sequence the promoter region of b-lactoglobulin (BLG) gene in four sheep breeds, in order to identify polymorphisms, infer and analyze haplotypes, and phylogenetic relationship among the Valle del Belice breed and the other three breeds considered as ancestors. Sequencing analysis and alignment of the obtained sequences showed the presence of 36 single nucleotide polymorphisms (SNPs) and one deletion. A total of 22 haplotypes found in ‘‘best’’ reconstruction were inferred considering the 37 polymorphic sites identified. Haplotypes were used for the reconstruction of a phylogenetic tree using the Neighbor-Joining algorithm. The number of polymorphisms identified showed high variability within breeds. Analysis of genetic diversity indexes showed that the Sarda breed presented the lowest nucleotide diversity, whereas the Comisana breed presented the highest one. Comparing the nucleotide diversity among breeds, the highest value was obtained between Valle del Belice and Pinzirita breeds, whereas the lowest one was between Valle del Belice and Sarda breeds. Considering that polymorphisms in the promoter region of BLG gene could have a functional role associated with milk composition, the lowest value of nucleotide diversity between Valle del Belice and Sarda breeds may be related to a higher similarity of milk composition of these two breeds compared to the others. Further analyses will be conducted in order to evaluate the possible correlation between the genetic diversity indexes and the BLG content in milk of our breeds

Effect of hairless gene polymorphism on the breeding values of milk production traits in Valle del Belice sheep

The aim of this work was to assess the association between the hairless genotypes and estimated breeding values (EBVs) for milk yield (MY), fat (FAT) and protein (PRT) content in Valle del Belice dairy sheep breed. A data set from 465 randomly chosen unrelated individuals was analyzed. EBV for MY, FAT and PRT contents were estimated by REML analysis of a single trait repeatability animal model. The genotype effect on EBV was assessed by ANOVA and by the Tukey–Kramer multiple comparison test. The PCR-SSCP test showed the presence of CC and CT genotypes in Valle del Belice individuals. Some differences in milk production traits between the genotypes were found. For MY, individuals with CT genotype produced 1.5 times more daily milk than CC homozygotes. Individuals with CC genotype showed eight times less FAT content and 1.7 times less PRT content than the heterozygous. However, these differences were not statistically different, probably due to the low frequency of the CT genotype. Considering our results, polymorphisms of the hr gene do not directly influence production traits, but if further studies confirm our hypothesis, the hr gene could be used in a marker assisted selection program

Genetic polymorphism at the CSN1S1 gene in Girgentana dairy goat breed

The aim of this work was to evaluate the variability of the s1-casein locus in the endangered Girgentana dairy goat breed in order to define genetic improvement and conservation program for this breed. The study was performed on 200 dairy goats by means of different PCR protocols. The most frequent alleles were A (0.590) and F (0.290) followed by B (0.065) and N (0.047). CSN1S1 E allele was identified with a very low frequency (0.008). The most common genotype was AF (0.365) followed by AA (0.340). The high frequency of the strong genotypes is associated with the production of milk with high fat and protein content and with optimal technological properties. In Girgentana goat breed, the CSN1S1 genotype information could be utilized in selection strategies for milk protein content and milk yield, in order to select genetic lines for the production of “drinking milk” using weak and null genotypes, and for niche products using strong genotypes

Genome-wide homozygosity in Maremmana Cattle

The current availability of large numbers of single nucleotide polymorphisms (SNPs) throughout the genome makes these markers particularly suitable for the detection of patterns of genetic diversity and of genome-wide homozygosity in animal populations. The aim of this work was to estimate genetic diversity and homozygosity in the Maremmana cattle breed. We used a sample of 149 animals (males and females) genotyped with the BovineSNP50 v2 (54K) Illumina BeadChip. After editing for call-rate >0.9 and removing SNP unassigned or on the sex chromosomes, 128 animals and 50,814 SNPs were left. We estimated the following genetic parameters: observed and expected heterozygosity (Ho and He), minor allele frequency (MAF), and the FIS statistic. We also scanned the genome for runs of homozygosity (ROH). In the present study, ROH were detected based on 20-SNP-long sliding-windows, and allowing for a maximum of 1 missing and 1 heterozygote genotype, and a maximum gap between consecutive SNP of 105 bp. ROH contained minimum 10 SNPs, and had a minimum length of 1 Mb and a minimum density of 1 SNP every 50 kbps. The average Ho and He were 0.374 ± 0.132 and 0.365 ± 0.120, respectively, and the average MAF was 0.274 ± 0.130. These values are consistent with the range observed in other cattle breeds. We obtained some negative values for FIS (-0.162 to 0.180) which corresponded to animals with lower than average homozygosity. In total, 10,465 ROH were detected (81.75 per animal), with an average length of 2.69 Mb. Most ROH (74%) had length 2 Mb. ROH are contiguous lengths of homozygous genomic segments where the two inherited haplotypes are identical. ROH indicate genomic regions where a reduction in heterozygosity occurred, and offer new opportunities to estimate inbreeding (F). The inbreeding coefficient based on ROH (FROH) was estimated by the ratio between the total ROH length and the size of the genome in each animal. Average FROH was 0.0869 ± 0.032. Unlike inbreeding estimated based on Ho, FROH is not influenced by allele frequencies (sampling) and can distinguish recent from ancient inbreeding. However, FROH requires SNP positions to be known (unlike Ho). Two genomic regions with ROH in over 60% of the animals were found: one on BTA6 (38.6-39.7 Mbps), one on BTA13 (54.3-54.8 Mbps). These may highlight regions where selective pressures have shaped the genome of the Maremmana breed

Insights into Genetic Diversity, Runs of Homozygosity and Heterozygosity-Rich Regions in Maremmana Semi-Feral Cattle Using Pedigree and Genomic Data

Semi-feral local livestock populations, like Maremmana cattle, are the object of renewed interest for the conservation of biological diversity and the preservation and exploitation of unique and potentially relevant genetic material. The aim of this study was to estimate genetic diversity parameters in semi-feral Maremmana cattle using both pedigree- and genomic-based approaches (FIS and FROH), and to detect regions of homozygosity (ROH) and heterozygosity (ROHet) in the genome. The average heterozygosity estimates were in the range reported for other cattle breeds (HE=0.261, HO=0.274). Pedigree-based average inbreeding (F) was estimated at 4.9%. The correlation was low between F and genomic-based approaches (r=0.03 with FIS, r=0.21 with FROH), while it was higher between FIS and FROH (r=0.78). The low correlation between F and FROH coefficients may be the result of the limited pedigree depth available for the animals involved in this study. The ROH islands identified in Maremmana cattle included candidate genes associated with climate adaptation, carcass traits or the regulation of body weight, fat and energy metabolism. The ROHet islands contained candidate genes associated with nematode resistance and reproduction traits in livestock. The results of this study confirm that genome-based measures like FROH may be useful estimators of individual autozygosity, and may provide insights on pedigree-based inbreeding estimates in cases when animals’ pedigree data are unavailable, thus providing a more detailed picture of the genetic diversity

Development and validation of RP-HPLC method for the quantitative estimation of as1-genetic variants in goat milk.

A high-performance liquid chromatographic (HPLC) method was developed and validated for separation and quantification of the most common genetic variants of as1-casein in goat’s milk, to evaluate the effect of as1-casein polymorphisms on casein content. Chromatography was carried out by binary gradient technique on a reversed-phase C8 Zorbax column and the detection was made at a wavelength of 214 nm. The procedure was developed using individual raw milk samples of Girgentana goats. For calibration experiments, pure genetic variants were extracted from individual milk samples of animals with known genotypes, considering that commercial standards for goat genetic variants were not available. The data obtained for Girgentana goat breed showed that A, B, F variants were alleles associated with a content of as1-casein in milk of 3.2 ± 0.4, 5.4 ± 0.5 and 0.7 ± 0.1 g/L, respectively, whereas N variant was a ‘null’ allele associated with the absence of as1-casein in milk

Quantitative determination of casein genetic variants in goat milk: Application in Girgentana dairy goat breed

The study was conducted to develop a high-performance liquid chromatographic (HPLC) method to quantify casein genetic variants (s2-, β-, and κ-casein) in milk of homozygous individuals of Girgentana goat breed. For calibration experiments, pure genetic variants were extracted from individual milk samples of animals with known genotypes. The described HPLC approach was precise, accurate and highly suitable for quantification of goat casein genetic variants of homozygous individuals. The amount of each casein per allele was: s2-casein A=2.9 ± 0.8 g/L and F=1.8 ± 0.4 g/L; β-casein C=3.0 ± 0.8 g/L and C1=2.0 ± 0.7 g/L and κ-casein A=1.6 ± 0.3 g/L and B=1.1 ± 0.2 g/L. A good correlation was found between the quantities of s2-casein genetic variants A and F, and β-casein C and C1 with other previously described method. The main important result was obtained for κ-casein because, till now, no data were available on quantification of single genetic variants for this protein