S. Sartore

HotSHOT Plus ThermalSHOCK, a new and efficient technique for preparation of PCR-quality mite genomic DNA

The present study adapted the HotSHOT method, a technique which has been successfully applied on different kinds of tissues, to studies of Sarcoptes. Some modifications of this technique were made which allowed the quick preparation of PCR-quality Sarcoptes genomic DNA (gDNA), namely applying sodium hydroxide as a substrate for three cycles of thermal shock, followed by a short incubation and pH adjustment with a Tris solution (HotSHOT Plus ThermalSHOCK). The performance of this technique was tested by amplifying a ~450-bp rDNA fragment of the second internal transcribed spacer (ITS-2) and by multi-locus genotyping using ten microsatellites on 520 individual Sarcoptes samples. No difference in performance was observed between gDNA samples prepared using the HotSHOT Plus ThermalSHOCK technique and those prepared using a commercial kit utilizing proteinase K digestion. The results demonstrated that the HotSHOT Plus ThermalSHOCK technique is time-saving, economic, and easily automatable for the preparation of PCR-quality mite gDNA, which has implications for studying the molecular biology of mites with human and animal health significance. Although tested in the present study using Sarcoptes mites as a model, this technique may find broad applicability in extraction of gDNA from other parasites with small sizes and hard bodies.

Microsatellites as markers for comparison among different populations of Sarcoptes scabiei

Abstract The aim of the present investigation was to analyse genetic variation and relationships of epizootic mange mites from sympatric Alpine chamois and red fox populations. The results of multi-locus genotyping using microsatellite marker loci support the hypothesis that gene flow between mite varieties on sympatric Alpine chamois and red fox is absent or extremely rare. Although the number of samples analysed until now is very small, the transmission of parasites seem to be more frequent when phylogenetically related host species are involved.

Genetic variability of two Italian indigenous chicken breeds inferred from microsatellite marker analysis.

Abstract The objective of this study was to determine the genetic structure and variability of Bionda Piemontese and Bianca di Saluzzo (Piedmont, Northwest Italy) using an international set of microsatellite loci (AVIANDIV-FAO). Differences compared with commercial lines and other Italian breeds were verified to justify the implementation of conservation programmes. Flock contribution to genetic variability was assessed following the approach implemented in the MolKin software. Comparison was performed using the fixation index and the Reynolds genetic distance. The most likely number of different populations was estimated using the clustering procedure implemented in STRUCTURE. The molecular information suggests that management practices could have prevented random mating and produced inbreeding and heterogeneity across flocks. In this respect, Bionda and Bianca show substructuring and are more similar to British breeds than other continental European breeds. Bionda and Bianca fit into the European breeds provided with the highest number of alleles and expected heterozygosity. There is a clear distinction between the Piedmont breeds and the other populations. The Piedmont poultry differ from both commercial lines and other Italian breeds and retain a high level of genetic variability. As for other indigenous breeds, Bionda and Bianca could make an original contribution to the industry in the future. A collective planned approach to restoration is essential, because the flocks are managed with poor regulation. Enhancing connection between breeders with an efficient replacement interchange and mating plan is the right way of controlling inbreeding, preventing substructuring and increasing variability within the flocks.

MUC1 gene polymorphism in three Nelore lines selected for growth and its association with growth and carcass traits

The objective of this study was to describe the VNTR polymorphism of the mucin 1 gene (MUC1) in three Nelore lines selected for yearling weight to determine whether allele and genotype frequencies of this polymorphism were affected by selection for growth. In addition, the effects of the polymorphism on growth and carcass traits were evaluated. Birth, weaning and yearling weights, rump height, Longissimus muscle area, backfat thickness, and rump fat thickness, were analyzed. A total of 295 Nelore heifers from the Beef Cattle Research Center, Instituto de Zootecnia de Sertãozinho, were used, including 41 of the control line, 102 of the selection line and 152 of the traditional. The selection and traditional lines comprise animals selected for higher yearling weight, whereas control line animals are selected for yearling weight close to the average. Five alleles were identified, with allele 1 being the most frequent in the three lines, especially in the lines selected for higher means for yearling weight. Heterozygosity was significantly higher in the control line. Association analyses showed significant effects of allele 1 on birth weight and weaning weight while the allele 3 exert significant effects on yearling weight and back fat thickness. Despite these findings, application of this marker to marker-assisted selection requires more consistent results based on the genotyping of a larger number of animals in order to increase the accuracy of the statistical analyses.

Analysis of genetic variation in Agerolese cattle breed

The Agerolese is a local Italian cattle breed reared in the province of Naples, in the proximity of the town of Agerola. It is a dual-purpose breed, which originated during the nineteenth century from an autochthonous nucleus of Podolian cows crossed with Swiss Brown, Dutch Friesian, and Jersey bulls. After 1940, the breed was severely threatened by the substitution with purebred Italian Brown and Holstein Friesian cattle (Felius, 1995). In 2002 it numbered only 13 males in natural service and 100 breeding females (EAAP, http://www. tiho-hannover.de/einricht/zucht/eaap/). Therefore, according to the FAO criteria for breed categorization, based on the number of breeding subjects as well as on the trend in population size (http://dad.fao.org), the status of the Agerolese should be classified as critical. Moreover, its effective genetic size, Ne, is approximately 46, slightly below the critical value of 50 that corresponds to a rate of inbreeding of 1% per generation, which is commonly accepted as the maximum tolerable level. The reasons for conservation of the breed rely on its integration into agricultural production systems of low to medium input. Agerolese is well adapted to mountainous country and can be fed on products of pruning and undergrowth. The milk is used to produce the Provolone del Monaco, a cheese of remote origins protected by the Slow Food Foundation for Biodiversity. Recently, a specification has been developed to apply for the Protected Designation of Origin (Peretti, personal

Polymorphism Analysis of Ch1 and Ch2 Genes in the Siberian Cat

Cats are usually spreaders of allergens that are critical for sensitive people; the Siberian cat is a breed supposed to be low level allergenic, according to some breeders’ statements. The sequence of the two genes, namely Ch1 and Ch2, that code for the allergen Fel d 1, the major allergen responsible for outbreaks of allergy symptoms, is not yet known in the Siberian cat, and finding this was the aim of our investigation. Notably, our work is the first survey of the genetic structure of these genes in Siberian cats. The comparison of the sequences of Siberian cats, non-Siberian cats, and sequences present in the National Center for Biotechnology Information database revealed a considerable number of mutations; some of those detected in the Siberian cat, due to their position in exon regions, could affect the Fel d 1 allergenic properties. Therefore, further investigations are recommended to assess if the identified mutations can be responsible for a reduced-allergen synthesis and can be used as markers for selection of low level allergenic cats.

Distinguishing industrial meat from that of indigenous chickens with molecular markers

&NA; The aim of investigation was to evaluate a traceability system to detect industrial chicken meat among indigenous products, considering issues that could affect assignment accuracy. The dataset included 2 Italian indigenous meat breeds, namely Bionda Piemontese (2 ecotypes) and Bianca di Saluzzo, one broiler line, and 3 layer lines. Assignment tests were performed using a standard panel of 28 microsatellite loci. To evaluate effects of inbreeding and substructure on assignment accuracy, a simulated dataset was prepared. Broilers and layers belong to homogeneous populations and never enter the clusters of indigenous breeds. Ambiguity or misallocation are expected between the Bionda ecotypes and between the 2 indigenous breeds, but it is unlikely that niche products provided by Bionda and Bianca will compete with one another. Non‐random mating reduces accuracy, but only populations having weak genetic differentiation are involved, namely those that are less interesting to discriminate. The dataset can be used as a reference population to distinguish commercial meat from indigenous meat with great accuracy. Misallocations increase as number of loci decreases, but only within or between the indigenous breeds. A subpanel of the most resolving 14 loci keeps sufficient informative content to provide accuracy and to correctly allocate additional test samples within the reference population. This analytical tool is economically sustainable as a method to detect fraud or mislabeling. Adoption of a monitoring system should increase the value of typical products because the additional burden of molecular analyses would improve commercial grade and perception of quality.

Variability of Leptin gene promoter in cattle

Abstract A preliminary analysis on the variability of Leptin gene promoter in seven cattle breeds was carried out, focusing the attention on the SNP at nt 1759, which has been suggested to affect some quantitative traits in cattle. In addition, the linkage disequilibrium with the C305T mutation in exon 2 of Leptin gene was tested. The results indicate that the Leptin gene promoter is polymorphic in all the analysed breeds, with significant betweenbreed differences. Pairwise comparison of genotypes at the two considered SNPs revealed a significant linkage disequilibrium, with the presence of the haplotypes 1759C - 305T and 1759G - 305C.

Predicting the impact of selection for scrapie resistance on PRNP genotype frequencies in goats

The European Union has implemented breeding programmes to increase scrapie resistance in sheep. A similar approach can be applied also in goats since the K222 allele provides a level of resistance equivalent to that of ARR in sheep. The European Food Safety Authority stated that breeding for resistance could be offered as an option for Member States to control classical scrapie in goats. We assessed the impact of different breeding strategies on PRNP genotype frequencies using a mathematical model that describes in detail the evolution of K222 in two goat breeds, Chamois Coloured and Saanen. Different patterns of age structure and replacement rate were modelled as factors affecting response to selection. Breeding for scrapie resistance can be implemented in goats, even though the initial K222 frequencies in these breeds are not particularly favourable and the rate at which the resistant animals increase, both breeding and slaughtered for meat production, is slow. If the goal is not to achieve the fixation of resistance allele, it is advisable to carry out selection only until a desired frequency of K222-carriers has been attained. Nucleus selection vs. selection on the overall populations is less expensive but takes longer to reach the desired output. The programme performed on the two goat breeds serves as a model of the response the selection could have in other breeds that show different initial frequencies and population structure. In this respect, the model has a general applicability.

Variability of some genes of the somatotropic axis in the Piemontese cattle breed

Growth hormone (GH) is a major participant in the control of several complex physiological processes, including growth and metabolism. The growth promoting aspects are generally mediated by the insulin-like growth factor 1 (IGF-1), produced primarily in the liver in response to GH, while the metabolic effects are mainly the result of the GH direct binding to its receptors (GHR) on target cells. The production of GH is modulated by growth hormone releasing hormone (GHRH) and somatostatin (SST); the former stimulates both the synthesis and the secretion of GH, the latter inhibits the GH release (Frohman and Jansson, 1986; Bonneau, 1991; Buyse and Decuypere, 1999; Duclos et al., 1999; Sodhi and Rajput, 2001). Due to the several effects of GH, the genes coding for GH and other hormones and factors involved in growth have been suggested.........