Liliana Di Stasio

Molecular genetics in aquaculture.

Abstract Great advances in molecular genetics have deeply changed the way of doing research in aquaculture, as it has already done in other fields. The molecular revolution started in the 1980’s, thanks to the widespread use of restriction enzymes and Polymerase Chain Reaction technology, which makes it possible to easily detect the genetic variability directly at the DNA level. In aquaculture, the molecular data are used for several purposes, which can be clustered into two main groups. The first one, focused on individuals, includes the sex identification and parentage assignment, while the second one, focused on populations, includes the wide area of the genetic characterization, aimed at solving taxonomic uncertainties, preserving genetic biodiversity and detecting genetic tags. For the future, the increase in the number of molecular markers and the construction of high density genetic maps, as well as the implementation of genomic resources (including genome sequencing), are expected to provide tools for the genetic improvement of aquaculture species through Marked Assisted Selection. In this review the characteristics of different types of molecular markers, along with their applications to a variety of aquaculture issues are presented.

Genetic characterization of the Bardigiano horse using microsatellite markers

Abstract The study was aimed at investigating the genetic structure of the Bardigiano horse and its relationships with the Haflinger, Maremmano and Arabian breeds using 11 microsatellite markers. A total of 94 alleles were detected across the breeds, with a mean of 8.5 alleles per locus and a mean observed heterozygosity of 0.69. Compared to the other breeds, the Bardigiano horse showed quite a high genetic variability, as indicated by the mean number of alleles (7.0 vs 6.1÷7.6) and by the observed heterozygosity (0.72 vs 0.66÷0.71). Moreover, the genotype distributions in the Bardigiano groups of different sex and age were not significantly different. The overall Fst value showed that the genetic differences among breeds accounted for 7.8% (P=0.001) of the total variation, and the pairwise Fst values were all significant. The assignment test allocated between 96.8 and 98.9% of the individuals to the population they were collected from, with a mean probability of assignment of about 97% for all breeds, except for the Arabian, where it approached 100%. The results have highlighted that the Bardigiano breed has a high within and between breed variability, which is considerably more than could be expected by looking at its evolution history. This justifies the need for the development of additional breeding strategies to preserve the existing genetic variability.

Genetic variability in tench (Tinca tinca L.) as revealed by PCR-RFLP analysis of mitochondrial DNA

Four mitochondrial DNA segments, ND1, ND6, cyt b and D-loop, were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 14 tench (Tinca tinca L.) populations located in Europe and Asia; data on 5 Italian populations previously analyzed for the same mtDNA segments were also included in the study. All the considered segments were polymorphic and originated a total of 9 composite haplotypes which were clustered into 2 haplogroups, A and B, possibly corresponding to the Western and Eastern phylogroups previously described in tench. Nine out of 19 populations showed polymorphism, with haplotype diversity ranging from 0.246 to 0.643 and nucleotide diversity from 0.009 to 0.078. Seventy-five percent of the pairwise comparisons were significant, indicating a high between-population variability. The Neighbour-Joining tree revealed the presence of 3 clusters, including pure populations, with only a A or B haplogroup, and mixed populations, with both haplogroups. The possibility of identifying populations with different haplotypes has practical implications for both conservation and supportive stocking.

“Tinca Gobba Dorata del Pianalto di Poirino”: genetic characterization by microsatellite markers

The Tinca Gobba Dorata del Pianalto di Poirino (Golden humped tench of Poirino highland, [PO]), the only Italian fish with the Protected Designation of Origin, was characterized by seven microsatellites and compared to three wild populations living in Italian lakes (Valagola [VA]; Trasimeno [TR]; Bolsena [BO]). The PO population showed high variability values (number of effective alleles: 2.70 vs. 1.62 to 2.20; expected heterozygosity: 0.49 vs. 0.29 to 0.40). The analysis of between-population differentiation indicated that PO significantly differed from the others (FST = 0.039 to 0.097, P<0.05), while BO and TR were the most similar, consistently with their geographic proximity. The Neighbour-Joining tree revealed a clear separation between Northern and Central populations, with a bootstrap support of 97%. The population differentiation was reflected by the results of the assignment test, with 64% to 92% of the individuals correctly assigned to the original population, and a probability ranging from 76% to 95%. No individuals belonging to other populations were erroneously assigned to PO. A more detailed analysis of the PO population showed a similar genetic variability within the 15 considered ponds and a low degree of differentiation between ponds, with the exception of one “historical” pond, which significantly differed from most of the others, thus deserving to be preserved. The results indicate that the PO, despite being farmed, has a high level of within-population diversity and is greatly differentiated from the other populations considered. The possibility of applying the assignment test in the framework of the product traceability deserves further investigation.

Variability of μ-Calpain and Calpastatin genes in cattle

Abstract A preliminary analysis on the variability of μ-Calpain (CAPN1) and Calpastatin (CAST) genes in six cattle breeds was carried out, focusing the attention on CAPN1 g.5709C>G, CAPN1 g.6545C>T, and CAST g.282C>G SNPs, which have been suggested to affect beef tenderness in cattle. The results indicate that the two genes are polymorphic in all the analysed breeds, with significant between-breed differences. On the basis of their variability, only CAPN1 g.6545C>T and CAST g.282C>G SNPs seem appropriate to be considered as potential markers for beef tenderness.

MC1R gene analysis applied to breed traceability of beef

Abstract Since the breed of origin highly affects the beef price, reliable methods are needed to detect incorrect declarations. As most breeds are standardised for coat colour, the Melanocortin 1 Receptor gene (MC1R), involved in the regulation of eu/pheomelanins synthesis, has been suggested as marker for breed traceability of products of animal origin. The aim of this investigation is to characterise the main breeds reared in the Piedmont Region by MC1R locus and to apply the analysis of the locus to breed traceability of beef cuts purchased in different outlets of the Region. A total of 168 DNA samples of four cattle breeds (Piemontese, Blonde d’Aquitaine, Italian Friesian and Aosta Red Pied) were analysed for MC1R locus by PCR-RFLP. In addition, 28 DNA samples from beef with breed indication were tested. Piemontese and Blonde d’Aquitaine were monomorphic for the E+ and e allele, respectively. In the Friesian breed the EdEd genotype was the most frequent, but Ede was also observed (2%). Aosta Red Pied was the most variable breed, with the presence of the three alleles and five genotypes out of six. The comparison of the genotypic distribution in the four breeds clearly indicates that it is possible to distinguish among Piemontese, Blonde d’Aquitaine and Friesian breeds, but the same is not true for Aosta Red Pied, which has genotypes in common with the other breeds. The results on beef samples revealed a high percentage of mislabelling (about 18%), which concerned Friesian breed and crossbreds. These results indicate that MC1R locus is an effective marker in breed traceability of beef, when the involved breeds are characterised by different genotypes. Moreover, compared to other genetic markers, it has the great advantage of not requiring DNA reference samples. This survey, though limited, has revealed a high percentage of incompatibilities. Therefore, the analysis of MC1R locus is recommended in the framework of product certification, at least for random controls within a system aimed at preventing fraud.

Variability in candidate genes revealed associations with meat traits in the Piemontese cattle breed

In the last years an increasing number of associations between single nucleotide polymorphisms (SNPs) in candidate genes and production traits have been reported in beef cattle, but very often the results were not validated and few studies considered breeds homozygous for the allele responsible for the muscular hypertrophy. Therefore, we analysed the variability of 19 previously reported SNPs in 12 genes (GH, GHR, GDF8, GHRL, IGF2, LEP, LEPR, MYF5, NPY, POMC, UCP2, UCP3) in the hypertrophic Piemontese breed and investigated the effects of the observed polymorphisms on growth and conformation. Fourteen SNPs were polymorphic and a significant linkage disequilibrium was observed between SNPs in GHR, LEP and NPY genes, for which both single-SNP and haplotype effects were estimated. Negligible effects on the investigated traits were observed for GHRL, MYF5, NPY, POMC, UCP2 and UCP3 genes. The GHR gene significantly affected daily gain and its effect was further increased when haplotypes were considered. The C allele at LEP-1 and LEP-2 had moderate negative effects on the considered traits, whereas the C allele at LEP-3 mostly had positive effects; haplotypes in the LEP gene showed weaker but favourable associations with all the traits. The C allele at IGF2 and LEPR had favourable effects on daily gain and negative effects on meat conformation traits. The associations observed for GHR and LEP were consistent with those of previous studies, providing additional evidence of their usefulness as markers. Practical aspects of the applications to the breeding programme of the Piemontese breed need to be examined.

Polymorphisms in Lep and Lepr Genes in Infants: Correlation with Serum Leptin Values in the First 6 Months of Life

ABSTRACT Objective: Because several studies indicate that polymorphisms in leptin (Lep) and leptin receptor (Lepr) genes play a central role in determining obesity, we analyzed 2 single nucleotide polymorphisms (SNPs) in the Lep gene (Lep G2548A and A19G) and one in the Lepr gene (Lepr A668G) to verify the effect of the 3 SNPs on leptin concentrations in infancy. Methods: We enrolled 80 healthy Caucasian infants under 6 months of age, who were genotyped for the 3 SNPs with amplification refractory mutation system–mismatch amplification mutation assay (ARMS-MAMA) real-time polymerase chain reaction (PCR). Serum leptin values were measured with a radioimmunoassay method. Statistical significance was set at p < 0.05. Results: There were no significant differences between individually analyzed leptin polymorphisms Lep G2548A and A19G and serum leptin levels (p > 0.05). Because we found that Lep G2548A and A19G are in linkage disequilibrium on chromosome 7, we performed the haplotype analysis for Lep G2548A and Lep A19G. We obtained higher serum leptin levels in infants with the GG/GG haplotype (p < 0.05). Regarding receptor, we found higher leptin levels in GG-genotype infants for Lepr A668G (p < 0.001). Considering the 3 SNPs together, we found higher serum leptin values in GG/GG-GG infants (LepG2548A/A19G-Lepr A668G; p < 0.001). Conclusion: We obtained higher serum leptin levels in infants with the GG genotype for Lepr A668G, with haplotype GG/GG for Lep G2548A/A19G, and with GG/GG-GG (LepG2548A/A19G-Lepr A668G); thus, it seems that the genotype GG could be a protector against obesity development in infancy and adulthood. Moreover, these data confirm that not variations in the Lep gene as well as in the Lepr gene could play a role in weight gain. Further studies are needed to evaluate the role of genetics and the environment in a predisposition toward obesity later in life.

Mismatch Amplification Mutation Assay Real-Time PCR Analysis of the Leptin Gene G2548A and A19G Polymorphisms and Serum Leptin in Infancy: A Preliminary Investigation

Leptin is a hormone that regulates food intake and energy metabolism. Its coding gene (LEP) is one of the most promising candidates for obesity. Although some studies have detected associations of different single nucleotide polymorphisms (SNPs) in the LEP gene with serum leptin levels and obesity-related traits, the results are still conflicting. We investigated two SNPs to find relationships with leptin concentrations. Thirty healthy Caucasian infants younger than 6 months were genotyped for the SNPs G2548A and A19G with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation system-mismatch amplification mutation assay (ARMS- MAMA) real-time PCR, and serum leptin concentrations were measured with a radioimmunoassay method. Considering the significant linkage disequilibrium observed between the two SNPs, we divided the sample according to the number of GG haplotypes and observed that individuals homozygous for the GG haplotype had higher serum leptin levels in early infancy than the others. Although these preliminary results are based on a limited sample, they suggest that the genetic background seems to play a role in modulating leptin levels in infancy, but changes in leptin levels over infancy and their correlation with obesity need to be further explored. We describe an ARMS-MAMA real-time PCR procedure which could be profitably applied in routine genetic screening.

Genetic characterization of the oxytocin-neurophysin I gene (OXT) and its regulatory regions analysis in domestic Old and New World camelids

Oxytocin is a neurohypophysial peptide linked to a wide range of biological functions, including milk ejection, temperament and reproduction. Aims of the present study were a) the characterization of the OXT (Oxytocin-neurophysin I) gene and its regulatory regions in Old and New world camelids; b) the investigation of the genetic diversity and the discovery of markers potentially affecting the gene regulation. On average, the gene extends over 814 bp, ranging between 825 bp in dromedary, 811 bp in Bactrian and 810 bp in llama and alpaca. Such difference in size is due to a duplication event of 21 bp in dromedary. The main regulatory elements, including the composite hormone response elements (CHREs), were identified in the promoter, whereas the presence of mature microRNAs binding sequences in the 3’UTR improves the knowledge on the factors putatively involved in the OXT gene regulation, although their specific biological effect needs to be still elucidated. The sequencing of genomic DNA allowed the identification of 17 intraspecific polymorphisms and 69 nucleotide differences among the four species. One of these (MF464535:g.622C>G) is responsible, in alpaca, for the loss of a consensus sequence for the transcription factor SP1. Furthermore, the same SNP falls within a CpG island and it creates a new methylation site, thus opening future possibilities of investigation to verify the influence of the novel allelic variant in the OXT gene regulation. A PCR-RFLP method was setup for the genotyping and the frequency of the allele C was 0.93 in a population of 71 alpacas. The obtained data clarify the structure of OXT gene in domestic camelids and add knowledge to the genetic variability of a genomic region, which has received little investigation so far. These findings open the opportunity for new investigations, including association studies with productive and reproductive traits.