A novel allelic donkey ß-lactoglobulin I protein isoform generated by a non-AUG translation initiation codon is associated with a nonsynonymous SNP.

ß-lactoglobulin I (ß-LG I) is one of the most important whey proteins in donkey milk. However, to our knowledge, there has been no study focusing on the full nucleotide sequences of this gene (BLG I). Current investigation of donkey BLG I gene is very limited with only 2 variants (A and B) characterized so far at the protein level. Recently, a new ß-LG I variant, with a significantly higher mass (+1,915 Da) than known variants has been detected. In this study, we report the whole nucleotide sequence of the BLG I gene from 2 donkeys, whose milk samples are characterized by the ß-LG I SDS-PAGE band with a normal electrophoretic mobility (18,514.25 Da, ß-LG I B1 form) the first, and by the presence of a unique ß-LG I band with a higher electrophoretic mobility (20,428.5 Da, ß-LG I D form) the latter. A high genetic variability was found all over the 2 sequenced BLG I alleles. In particular, 16 polymorphic sites were found in introns, one in the 5' flanking region, 3 SNPs in the 5' untranslated region and one SNP in the coding region (g.1871G > A) located at the 40th nucleotide of exon 2 and responsible for the AA substitutions p.Asp28 > Asn in the mature protein. Two SNPs (g.920-922CAC > TGT and g.1871G/A) were genotyped in 93 donkeys of 2 Italian breeds (60 Ragusana and 33 Amiatina, respectively) and the overall frequencies of g.920-922CAC and g.1871A were 0.3065 and 0.043, respectively. Only the rare allele g.1871A was observed to be associated with the slower migrating ß-LG I. Considering this genetic diversity and those found in the database, it was possible to deduce at least 5 different alleles (BLG I A, B, B1, C, D) responsible for 4 potential ß-LG I translations. Among these alleles, B1 and D are those characterized in the present research, with the D allele of real novel identification. Haplotype data analysis suggests an evolutionary pathway of donkey BLG I gene and a possible phylogenetic map is proposed. Analyses of mRNA secondary structure showed relevant changes in the structures, as consequence of the g.1871G > A polymorphism, that might be responsible for the recognition of an alternative initiation site providing an additional signal peptide. The extension of 19 AA sequence to the mature protein, corresponding to the canonical signal peptide with an additional alanine residue, is sufficient to provide the observed molecular weight of the slower migrating ß-LG I encoded by the BLG I D allele.

Genome-wide analysis in endangered populations: a case study in Barbaresca sheep.

Analysis of genomic data is becoming increasingly common in the livestock industry and the findings have been an invaluable resource for effective management of breeding programs in small and endangered populations. In this paper, with the goal of highlighting the potential of genomic analysis for small and endangered populations, genome-wide levels of linkage disequilibrium, measured as the squared correlation coefficient of allele frequencies at a pair of loci, effective population size, runs of homozygosity (ROH) and genetic diversity parameters, were estimated in Barbaresca sheep using Illumina OvineSNP50K array data. Moreover, the breed's genetic structure and its relationship with other breeds were investigated. Levels of pairwise linkage disequilibrium decreased with increasing distance between single nucleotide polymorphisms. An average correlation coefficient <0.25 was found for markers located up to 50 kb apart. Therefore, these results support the need to use denser single nucleotide polymorphism panels for high power association mapping and genomic selection efficiency in future breeding programs. The estimate of past effective population size ranged from 747 animals 250 generations ago to 28 animals five generations ago, whereas the contemporary effective population size was 25 animals. A total of 637 ROH were identified, most of which were short (67%) and ranged from 1 to 10 Mb. The genetic analyses revealed that the Barbaresca breed tended to display lower variability than other Sicilian breeds. Recent inbreeding was evident, according to the ROH analysis. All the investigated parameters showed a comparatively narrow genetic base and indicated an endangered status for Barbaresca. Multidimensional scaling, model-based clustering, measurement of population differentiation, neighbor networks and haplotype sharing distinguished Barbaresca from other breeds, showed a low level of admixture with the other breeds considered in this study, and indicated clear genetic differences compared with other breeds. Attention should be given to the conservation of Barbaresca due to its critical conservation status. In this context, genomic information may have a crucial role in management of small and endangered populations.

Genome-wide analysis of Italian sheep diversity reveals a strong geographic pattern and cryptic relationships between breeds.

Italy counts several sheep breeds, arisen over centuries as a consequence of ancient and recent genetic and demographic events. To finely reconstruct genetic structure and relationships between Italian sheep, 496 subjects from 19 breeds were typed at 50K single nucleotide polymorphism loci. A subset of foreign breeds from the Sheep HapMap dataset was also included in the analyses. Genetic distances (as visualized either in a network or in a multidimensional scaling analysis of identical by state distances) closely reflected geographic proximity between breeds, with a clear north-south gradient, likely because of high levels of past gene flow and admixture all along the peninsula. Sardinian breeds diverged more from other breeds, a probable consequence of the combined effect of ancient sporadic introgression of feral mouflon and long-lasting genetic isolation from continental sheep populations. The study allowed the detection of previously undocumented episodes of recent introgression (Delle Langhe into the endangered Altamurana breed) as well as signatures of known, or claimed, historical introgression (Merino into Sopravissana and Gentile di Puglia; Bergamasca into Fabrianese, Appenninica and, to a lesser extent, Leccese). Arguments that would question, from a genomic point of view, the current breed classification of Bergamasca and Biellese into two separate breeds are presented. Finally, a role for traditional transhumance practices in shaping the genetic makeup of Alpine sheep breeds is proposed. The study represents the first exhaustive analysis of Italian sheep diversity in an European context, and it bridges the gap in the previous HapMap panel between Western Mediterranean and Swiss breeds.

Towards standardization of canine STRs: a proposed nomenclature for six markers from the ISAG comparison-test panel.

Eight short tandem repeat markers included in the International Society for Animal Genetics panel of 24 loci investigated in canine comparison tests were analysed in a sample of pure-breed dogs, with the purpose of defining an allele nomenclature based on the number of repeats. A regression analysis of the raw data produced by the sequencer, coupled with the direct sequencing of selected alleles, allowed us to propose a system of nomenclature for six of the eight loci (four di-nucleotidic: AHT121, AHTh137, REN169018 and REN64E19, and two tetra-nucleotidic: FH2001 and FH2328). The remaining two loci (INU055 and FH2848) showed a pattern of fragments that did not resolve in a simple allele series. This work may be useful to establish a basis for comparing data across different laboratories for a set of validated canine markers, which can be used in population genetics, forensics and other analyses.

Genetic variability of the Gentile di Puglia sheep breed based on microsatellite polymorphism.

The aim of the present study was to estimate the genetic variability of the Gentile di Puglia sheep breed by using microsatellite markers, to provide information useful for conservation. The DNA of 82 animals, belonging to 6 different farms, was analyzed at 19 microsatellite loci. The number of alleles, allele frequencies, deviations from Hardy-Weinberg proportions, linkage disequilibrium among loci, genetic similarity, genetic distances, and molecular coancestry-based parameters were calculated. Seventeen out of 19 microsatellites showed heterozygote deficiency in the whole population, although only 4 (OarAE129, ILSTS28, ILSTS5, MAF33) showed significant deviations from Hardy-Weinberg proportions (P<0.01). The genetic similarity (an index of allele sharing among individuals) within the whole population was low (0.318), and the average number of alleles per locus was quite high (9.68). The results obtained highlighted a population presenting both a deficiency of heterozygotes and great genetic variability. These results suggest that stratification of the breed in subpopulations, probably derived from different farm management in each herd, led to a population characterized by great genetic diversity among different herds and by great similarity within each herd. Moreover, in the past, Gentile di Puglia sheep have undergone a strong numeric decline, involving genetic drift effects and considerable consanguinity within the breed.

The genetic resistance to gastro-intestinal strongylids in Appenninica sheep: preliminary results.

Heritability parameters of resistance to gastro-intestinal strongylids, measured as FEC (Faecal Egg Count), were evaluated in the Appenninica sheep breed. FEC heritability coefficient was 0.11 +/- 0.061 while FEC repeatability coefficients were 0.58 +/- 0.085 and 0.76 +/- 0.223 in adult females and lambs respectively. Subjects were classified, based on FEC, into three different levels of resistance to strongylids. Ewes belonging to the 'resistant class' should be conveniently exploited in mating schemes, in order to provide a method, alternative to drug administration, for a long-term parasite control; this would result particularly helpful under those production systems, such as organic farming, where the use of drugs is not allowed or limited.

Statistical analysis of individual assignment tests among four cattle breeds using fifteen STR loci.

Assignment tests based on multilocus genotypes are becoming increasingly important to certify quality and origin of livestock products and assure food safety and authenticity. The purpose of this study was to determine the potential of microsatellites (STR) for determining the breed origin of beef products among cattle breeds present in the market. We typed 19 STR in 269 animals from 4 cattle breeds. Based on Wright's F-statistics, 4 loci were discarded, and the remaining 15 loci (FIT = 0.101, FST = 0.089, and FIS = 0.013) were used to compute the likelihood that each multilocus genotype of the total sample was drawn from its true breed instead of another breed. To avoid occurrence of zero likelihood when one or more alleles were missing from a tested breed, sample allele frequencies were estimated assuming uniform prior distributions. Log-likelihood ratio [log(LR)] distributions of the individual assignments were determined for all possible breed contrasts, and their means and SD were used to infer the true-positive and false-positive rates at several values of the log(LR). The posterior probability that the animals of a presumed breed were actually drawn from that breed instead of any another breed was then calculated. Given an observed value of log(LR) > 0 and assuming equal priors, these probabilities were > 99.5% in 10 of 12 possible breed contrasts. For the 2 most closely related breeds (FST = 0.041), this probability was 96.3%, and the probability of excluding the origin of an animal from an alleged breed when it was actually derived from another breed was similar.

Genomic identification of the breed of an individual or its tissue.

To elaborate a methodological approach for the assignment of a breed to a carcass or other animal tissue, DNA microsatellites were utilized. Four Italian beef breeds were studied; examining these breeds is a challenge because they display similar morphological and productive characteristics. As comparison 54 Holstein-Friesian subjects were used. To measure the genetic similarities between animals or between groups of animals we used our method (Ciampolini et al, 1995, Individual multilocus genotypes using microsatellite polymorphisms to permit the analysis of the genetic variability within and between italian beef cattle breeds. Journal of Animal science,73, 3259-3268.) based on the consideration of a multilocus genotype of each animal, which enabled us to establish breed genomic formulae. Although the results are only preliminary, they show that we can distinguish easily between Holstein-Friesian subjects and beef animals; moreover we can identify, with a high resolution potential, animals from breeds (the four Italian white) with very high genetic similarities. However all the Holstein-Friesian subjects were identified.

Individual multilocus genotypes using microsatellite polymorphisms to permit the analysis of the genetic variability within and between Italian beef cattle breeds.

We investigated the genetic variability within and between cattle breeds. The polymorphisms of 17 microsatellites were studied in 220 unrelated animals belonging to four Italian beef cattle breeds (Chianina, Marchigiana, Romagnola, and Piemontese). Variations of allelic frequencies were examined to characterize the breeds and their relationships. Wahlund coefficients, Polymorphism Information Content values, and Haldane exact test for Hardy-Weinberg proportions were calculated. The results show that the Hardy-Weinberg equilibrium is not always maintained. Moreover, in addition to the classical genetic distances, a new method, based on the consideration of a multilocus genotype of each animal, was set up to measure the genetic similarity between animals or within groups of animals. All the results showed that, whereas Chianina occupies an intermediate position and Piemontese is the most distinct of all four breeds, Marchigiana and Romagnola display the strongest similarity. The new method also provides evidence that average similarities are always higher within breeds than between breeds. By comparing pairwise the multilocus genotypes, it was also possible to discriminate the individuals with higher or lower genetic similarities so that each breed could be subdivided into two groups of animals in relation to their similarity to the average breed multilocus genotype. High similarities between breeds were detected, somewhat surprisingly, when the most homogeneous groups of each breed were compared. The microsatellite multilocus genotype is particularly efficient in evaluating the between- and within-breeds genetic similarities and for subgrouping genetically more homogeneous animals.

A set of 99 cattle microsatellites: characterization, synteny mapping, and polymorphism.

Cattle microsatellite clones (136) were isolated from cosmid (10) and plasmid (126) libraries and sequenced. The dinucleotide repeats were studied in each of these sequences and compared with dinucleotide repeats found in other vertebrate species where information was available. The distribution in cattle was similar to that described for other mammals, such as rat, mouse, pig, or human. A major difference resides in the number of sequences present in the bovine genome, which seemed at best one-third as large as in other species. Oligonucleotide primers (117 pairs) were synthesized, and a PCR product of expected size was obtained for 88 microsatellite sequences (75%). Synteny or chromosome assignment was searched for each locus with PCR amplification on a panel of 36 hamster/bovine somatic cell hybrids. Of our bovine microsatellites, eighty-six could be assigned to synteny groups of chromosomes. In addition, 10 other microsatellites--HEL 5, 6, 9, 11, 12, 13 (Kaukinen and Varvio 1993), HEL 4, 7, 14, 15--as well as the microsatellite found in the kappa-casein gene (Fries et al. 1990) were mapped on the hybrids. Microsatellite polymorphism was checked on at least 30 unrelated animals of different breeds. Almost all the autosomal and X Chr microsatellites displayed polymorphism, with the number of alleles varying between two and 44. We assume that these microsatellites could be very helpful in the construction of a primary public linkage map of the bovine genome, with an aim of finding markers for Economic Trait Loci (ETL) in cattle.