Letizia Nicoloso

Assessing SNP markers for assigning individuals to cattle populations

The effectiveness of single nucleotide polymorphisms (SNPs) for the assignment of cattle to their source breeds was investigated by analysing a panel of 90 SNPs assayed on 24 European breeds. Breed assignment was performed by comparing the Bayesian and frequentist methods implemented in the STRUCTURE 2.2 and GENECLASS 2 software programs. The use of SNPs for the reallocation of known individuals to their breeds of origin and the assignment of unknown individuals was tested. In the reallocation tests, the methods implemented in STRUCTURE 2.2 performed better than those in GENECLASS 2, with 96% vs. 85% correct assignments respectively. In contrast, the methods implemented in GENECLASS 2 showed a greater correct assignment rate in allocating animals treated as unknowns to a reference dataset (62% vs. 51% and 80% vs. 65% in field tests 1 and 2 respectively). These results demonstrate that SNPs are suitable for the assignment of individuals to reference breeds. The results also indicate that STRUCTURE 2.2 and GENECLASS 2 can be complementary tools to assess breed integrity and assignment. Our findings also stress the importance of a high-quality reference dataset in allocation studies.

Stearoyl CoA desaturase (SCD) gene polymorphisms in Italian cattle breeds.

Stearoyl CoA desaturase (SCD) is the key enzyme involved in the endogenous synthesis of conjugated linoleic acid (CLA) in ruminants. Changes in the enzymatic activity as a result of SCD gene polymorphism and regulation have been hypothesized to cause diet-independent variations of CLA content in milk. Evidences for the direct influence of SCD polymorphism on fatty acid composition of milk and beef have also been reported. To evaluate genetic differences because of breed and/or selection goal, we investigated the polymorphism of three previously reported single nucleotide polymorphisms located in exon 5 of the SCD gene in 11 cattle breeds raised in Italy and selected for different production goals. Results obtained: (i) evidenced a high variability in the allele frequencies across breeds; (ii) detected three novel haplotypes, one of which is private to indigenous beef breeds, and (iii) showed a significant association between haplotypes and selective goal.

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.

Recent Advance in DNA-based Traceability and Authentication of Livestock Meat PDO and PGI Products

This review updates the available molecular techniques and technologies and discusses how they can be used for traceability, food control and enforcement activities. The review also provides examples on how molecular techniques succeeded to trace back unknowns to their breeds of origin, to fingerprint single individuals and to generate evidence in court cases. The examples demonstrate the potential of the DNA based traceability techniques and explore possibilities for translating the next generation genomics tools into a food and feed control and enforcement framework.

Assessing the spatial dependence of adaptive loci in 43 European and Western Asian goat breeds using AFLP markers

Background During the past decades, neutral DNA markers have been extensively employed to study demography, population genetics and structure in livestock, but less interest has been devoted to the evaluation of livestock adaptive potential through the identification of genomic regions likely to be under natural selection. Methodology/Principal findings Landscape genomics can greatly benefit the entire livestock system through the identification of genotypes better adapted to specific or extreme environmental conditions. Therefore we analyzed 101 AFLP markers in 43 European and Western Asian goat breeds both with Matsam software, based on a correlative approach (SAM), and with Mcheza and Bayescan, two FST based software able to detect markers carrying signatures of natural selection. Matsam identified four loci possibly under natural selection – also confirmed by FST-outlier methods – and significantly associated with environmental variables such as diurnal temperature range, frequency of precipitation, relative humidity and solar radiation. Conclusions/Significance These results show that landscape genomics can provide useful information on the environmental factors affecting the adaptive potential of livestock living in specific climatic conditions. Besides adding conservation value to livestock genetic resources, this knowledge may lead to the development of novel molecular tools useful to preserve the adaptive potential of local breeds during genetic improvement programs, and to increase the adaptability of industrial breeds to changing environments.

Detection of QTL for milk protein percentage in Italian Friesian cattle by AFLP markers and selective genotyping

We targeted quantitative trait loci (QTL) for milk protein percentage (P%) in two Italian Holstein granddaughter design families using selective genotyping in combination with high throughput amplified fragment length polymorphism (AFLP) markers. A total of 64 extreme high and low sires in respect to estimated breeding value (EBV) for P% (EBVP%) were genotyped with 25 AFLP primer combinations that revealed 305 and 291 polymorphisms in the two families. Association between markers and EBVP% was investigated by a linear model only on bands having paternal origin (105 and 96 AFLP bands in family D and S, respectively). Although no marker was significantly associated with the target trait after correction for multiple comparisons, 17 AFLP markers, significant without correction for multiple tests, were considered suggestive of the presence of a QTL. Eleven of these were successfully located on six Bos taurus (BTA) chromosomes by radiation hybrid or in-silico mapping. Ten of these mapped in the immediate neighbourhood (less than 10 cM) of already described QTL for P%. Suggestive association was verified in four regions by microsatellites analysis: one on BTA 10; one on BTA 28; and two on BTA 18. Microsatellites identified significant effects by single marker and interval mapping analyses on BTA 10 and BTA 28, while they were only suggestive of the presence of QTL on BTA 18. In summary, our results firstly indicate that AFLP markers may be used to seek QTL exploiting a selective genotyping approach in GDD, a wide used experimental design in cattle; secondly, propose two approaches for AFLP mapping, namely in-silico mapping exploiting most updated release from the bovine whole genome sequencing project, and physical mapping exploiting a panel of Bovine/Hamster Radiation Hybrids; and thirdly, provide new information on QTLs for an economic important trait in a never investigated Holstein cattle population. AFLP in combination with selective genotyping can be a useful strategy for QTL searching in minor livestock species, sometimes having large economic impact in marginal areas, where more informative markers are still poorly developed.

A method for single nucleotide polymorphism selection for parentage assessment in goats

Accurate pedigrees are essential to optimize genetic improvement and conservation of animal genetic resources. In goats, the use of mating groups and kidding management procedures hamper the identification of parentage. Small panels of single nucleotide polymorphisms (SNP) have been proposed in other species to substitute microsatellites for parentage assessment. Using data from the current GoatSNP50 chip, we developed a new 3-step procedure to identify a low-density SNP panel for highly accurate parentage assessment. Methodologies for SNP selection used in other species are less suitable in the goat because of uncertainties in the genome assembly. The procedure developed in this study is based on parent-offspring identification and on estimation of Mendelian errors, followed by canonical discriminant analysis identification and stepwise regression reduction. Starting from a reference sample of 109 Alpine goats with known pedigree relationships, we first identified a panel of 200 SNP that was further reduced to 2 final panels of 130 and 114 SNP with random coincidental match inclusion of 1.51×10(-57) and 2.94×10(-34), respectively. In our reference data set, all panels correctly identified all parent-offspring combinations, revealing a 40% pedigree error rate in the information provided by breeders. All reference trios were confirmed by official tests based on microsatellites. Panels were also tested on Saanen and Teramana breeds. Although the testing on a larger set of breeds in the reference population is still needed to validate these results, our findings suggest that our procedure could identify SNP panels for accurate parentage assessment in goats or in other species with unreliable marker positioning.

On the way to functional agro biodiversity: coat colour gene variability in goats

Functional agro biodiversity defines the exploitation of biodiversity to provide ecosystem services, support sustainable agricultural production and benefit the regional and global environment and the public at large (ELN-FAB, 2009; www.eln_fab.eu). Tracking of animal products back to the breed of origin based on their genetic make-up undoubtedly falls in this category. The aim of this paper was to identify and validate a set of single nucleotide polymorphisms (SNPs) in goat coat colour genes, most of which have not been investigated before, to trace five goat populations of the Italian Alps and their product. Several regions of 28 genes influencing coat colour pathways were amplified in eight animals (two per breed). Sequence comparison revealed 48 SNPs and three INDEL (INsertion DELetion). No breed-specific alleles were detected; however, several SNPs showed an uneven frequency distribution between breeds. In BIO, the genotype frequency distribution of a non-synonymous SNP suggested a possible role of TYRP1 in brown eumelanic goat coat colour. A total of 29 independent SNPs in 20 genes were selected and used to allocate 159 minimally related goat samples using STRUCTURE 2.2 and GeneClass 2 software. STRUCTURE 2.2 assigns 99% of individuals to the correct breed considering the prior information on putative breed of origin for each sample and 81% using only the genotypic data. The three algorithms available in GeneClass 2 performed with nearly equal efficiency, with 86% and 87% correct allocations. All the methods yielded an average probability of assignment >0.92 and a specificity index >0.86. Despite their coat colour variability, individuals belonging to ORO were fully assigned, showing that, in the absence of a breed-specific allele tied to coat colour, the best assignment resulted for the most genetically distinct breed. The lowest rate of correct assignment was observed in Verzaschese (73%), not ascertained in the breed panel used in the SNP discovery phase.

Associations of acetyl-coenzyme A carboxylase α, stearoyl-coenzyme A desaturase, and lipoprotein lipase genes with dairy traits in Alpine goats

Milk yield and composition are of great economic importance for the dairy goat industry. The identification of genes associated with phenotypic differences for these traits could allow for the implementation of gene-assisted selection programs in goats. Associations between polymorphisms at 3 candidate genes and milk production traits in Alpine goats farmed in Italy were investigated in the present research. Considered genes were acetyl-coenzyme A carboxylase α (ACACA), the major regulatory enzyme of fatty acid biosynthesis; stearoyl-coenzyme A desaturase (SCD), involved in the biosynthesis of monounsaturated fatty acids in the mammary gland; and lipoprotein lipase (LPL), which plays a central role in plasma triglyceride metabolism. An approach somewhat similar to the granddaughter design for detecting quantitative trait loci in dairy cattle was followed. Effects of genotypes of a sample of 59 Alpine bucks on phenotypes of their 946 daughters raised in 75 flocks were investigated. Data comprised 13,331 daily records for milk yields (L/d), fat and protein yields (kg/d), and fat and protein contents (%) of 2,200 lactations. Population genetics parameters were calculated and associations between milk production traits and 10 single nucleotide polymorphisms (SNP) at the 3 genes were tested. Two markers at the ACACA, 1 for the SCD and 1 at the LPL locus, deviated significantly from the Hardy-Weinberg equilibrium, with an observed heterozygosity lower than expected. Flock, age of the goat, kidding season, and stage of lactation affected all traits considered, except fat percentage. Three SNP were found to be significantly associated with milk production traits. The SNP located on the ACACA gene showed an effect on milk yield, with daughters of TT bucks having an average test-day milk yield of about 0.3 to 0.25 L/d lower than the other 2 genotypes. The marker on the LPL locus was highly associated with milk yield, with the largest values for CC daughters (about 0.50L more than GG). The TGT deletion located on the untranslated region of the SCD gene showed significant effects on average milk and protein yields. The homozygote-deleted genotype had values about 0.5 L/d and 16 g/d lower for milk and protein daily yield, respectively, compared with the TGT/TGT genotype. Differences between genotypes were quite constant across most of the lactation. Associations found in the present study, which should be tested in a larger sample, especially for those markers that show rare genotypes, may offer useful indications for the genetic improvement of dairy traits in goats.

Assessing SNPs in coat colour genes for cattle breed traceability

Abstract Aim of this research was to identify a panel of SNPs in coat colour genes useful for breed traceability in Rendena, an autochthonous cattle breed raised in the province of Trento, and other 4 Italian cattle breeds. First, we sequenced some regions of several coat colour genes in 10 animals belonging to 5 breeds characterised by different coat colour phenotypes (Rendena, Italian Brown, Grey Alpine, Italian Friesian, and Italian Red Pied), and we detected 21 SNPs in 13 genes. These markers and 6 additional SNPs were used to genotype 180 animals of the same 5 breeds obtaining useful genotyping data for a total of 22 SNPs in 13 genes. Five out of the 22 SNP markers in the MC1R, KIT, MLPH, and SILV genes had the highest discriminating power. The panel of 22 SNPs is useful to trace Rendena particularly from Red Italian Pied and Italian Friesian.