Use of the Multivariate Discriminant Analysis for Genome-Wide Association Studies in Cattle.

Genome-wide association studies (GWAS) are traditionally carried out by using the single marker regression model that, if a small number of individuals is involved, often lead to very few associations. The Bayesian methods, such as BayesR, have obtained encouraging results when they are applied to the GWAS. However, these approaches, require that an a priori posterior inclusion probability threshold be fixed, thus arbitrarily affecting the obtained associations. To partially overcome these problems, a multivariate statistical algorithm was proposed. The basic idea was that animals with different phenotypic values of a specific trait share different allelic combinations for genes involved in its determinism. Three multivariate techniques were used to highlight the differences between the individuals assembled in high and low phenotype groups: the canonical discriminant analysis, the discriminant analysis and the stepwise discriminant analysis. The multivariate method was tested both on simulated and on real data. The results from the simulation study highlighted that the multivariate GWAS detected a greater number of true associated single nucleotide polymorphisms (SNPs) and Quantitative trait loci (QTLs) than the single marker model and the Bayesian approach. For example, with 3000 animals, the traditional GWAS highlighted only 29 significantly associated markers and 13 QTLs, whereas the multivariate method found 127 associated SNPs and 65 QTLs. The gap between the two approaches slowly decreased as the number of animals increased. The Bayesian method gave worse results than the other two. On average, with the real data, the multivariate GWAS found 108 associated markers for each trait under study and among them, around 63% SNPs were also found in the single marker approach. Among the top 118 associated markers, 76 SNPs harbored putative candidate genes.

Use of canonical discriminant analysis to study signatures of selection in cattle.

BACKGROUND: Cattle include a large number of breeds that are characterized by marked phenotypic differences and thus constitute a valuable model to study genome evolution in response to processes such as selection and domestication. Detection of "signatures of selection" is a useful approach to study the evolutionary pressures experienced throughout history. In the present study, signatures of selection were investigated in five cattle breeds farmed in Italy using a multivariate approach. METHODS: A total of 4094 bulls from five breeds with different production aptitudes (two dairy breeds: Italian Holstein and Italian Brown Swiss; two beef breeds: Piemontese and Marchigiana; and one dual purpose breed: Italian Simmental) were genotyped using the Illumina BovineSNP50 v.1 beadchip. Canonical discriminant analysis was carried out on the matrix of single nucleotide polymorphisms (SNP) genotyping data, separately for each chromosome. Scores for each canonical variable were calculated and then plotted in the canonical space to quantify the distance between breeds. SNPs for which the correlation with the canonical variable was in the 99th percentile for a specific chromosome were considered to be significantly associated with that variable. Results were compared with those obtained using an FST-based approach. RESULTS: Based on the results of the canonical discriminant analysis, a large number of signatures of selection were detected, among which several had strong signals in genomic regions that harbour genes known to have an impact on production and morphological bovine traits, including MSTN, LCT, GHR, SCD, NCAPG, KIT, and ASIP. Moreover, new putative candidate genes were identified, such as GCK, B3GALNT1, MGAT1, GALNTL1, PRNP, and PRND. Similar results were obtained with the FST-based approach. CONCLUSIONS: The use of canonical discriminant analysis on 50 K SNP genotypes allowed the extraction of new variables that maximize the separation between breeds. This approach is quite straightforward, it can compare more than two groups simultaneously, and relative distances between breeds can be visualized. The genes that were highlighted in the canonical discriminant analysis were in concordance with those obtained using the FST index.

Detection of selection signatures in Piemontese and Marchigiana cattle, two breeds with similar production aptitudes but different selection histories.

BACKGROUND: Domestication and selection are processes that alter the pattern of within- and between-population genetic variability. They can be investigated at the genomic level by tracing the so-called selection signatures. Recently, sequence polymorphisms at the genome-wide level have been investigated in a wide range of animals. A common approach to detect selection signatures is to compare breeds that have been selected for different breeding goals (i.e. dairy and beef cattle). However, genetic variations in different breeds with similar production aptitudes and similar phenotypes can be related to differences in their selection history. METHODS: In this study, we investigated selection signatures between two Italian beef cattle breeds, Piemontese and Marchigiana, using genotyping data that was obtained with the Illumina BovineSNP50 BeadChip. The comparison was based on the fixation index (Fst), combined with a locally weighted scatterplot smoothing (LOWESS) regression and a control chart approach. In addition, analyses of Fst were carried out to confirm candidate genes. In particular, data were processed using the varLD method, which compares the regional variation of linkage disequilibrium between populations. RESULTS: Genome scans confirmed the presence of selective sweeps in the genomic regions that harbour candidate genes that are known to affect productive traits in cattle such as DGAT1, ABCG2, CAPN3, MSTN and FTO. In addition, several new putative candidate genes (for example ALAS1, ABCB8, ACADS and SOD1) were detected. CONCLUSIONS: This study provided evidence on the different selection histories of two cattle breeds and the usefulness of genomic scans to detect selective sweeps even in cattle breeds that are bred for similar production aptitudes.

Analysis of runs of homozygosity and their relationship with inbreeding in five cattle breeds farmed in Italy.

Increased inbreeding is an inevitable consequence of selection in livestock populations. The analysis of high-density single nucleotide polymorphisms (SNPs) facilitates the identification of long and uninterrupted runs of homozygosity (ROH) that can be used to identify chromosomal regions that are identical by descent. In this work, the distribution of ROH of different lengths in five Italian cattle breeds is described. A total of 4095 bulls from five cattle breeds (2093 Italian Holstein, 749 Italian Brown, 364 Piedmontese, 410 Marchigiana and 479 Italian Simmental) were genotyped at 54K SNP loci. ROH were identified and used to estimate molecular inbreeding coefficients (FROH ), which were compared with inbreeding coefficients estimated from pedigree information (FPED ) and using the genomic relationship matrix (FGRM ). The average number of ROH per animal ranged from 54 ± 7.2 in Piedmontese to 94.6 ± 11.6 in Italian Brown. The highest number of short ROH (related to ancient consanguinity) was found in Piedmontese, followed by Simmental. The Italian Brown and Holstein had a higher proportion of longer ROH distributed across the whole genome, revealing recent inbreeding. The FPED were moderately correlated with FROH > 1 Mb (0.662, 0.700 and 0.669 in Italian Brown, Italian Holstein and Italian Simmental respectively) but poorly correlated with FGRM (0.134, 0.128 and 0.448 for Italian Brown, Italian Holstein and Italian Simmental respectively). The inclusion of ROH > 8 Mb in the inbreeding calculation improved the correlation of FROH with FPED and FGRM . ROH are a direct measure of autozygosity at the DNA level and can overcome approximations and errors resulting from incomplete pedigree data. In populations with high linkage disequilibrium (LD) and recent inbreeding (e.g. Italian Holstein and Italian Brown), a medium-density marker panel, such as the one used here, may provide a good estimate of inbreeding. However, in populations with low LD and ancient inbreeding, marker density would have to be increased to identify short ROH that are identical by descent more precisely.

Use of locally weighted scatterplot smoothing (LOWESS) regression to study selection signatures in Piedmontese and Italian Brown cattle breeds.

Selection is the major force affecting local levels of genetic variation in species. The availability of dense marker maps offers new opportunities for a detailed understanding of genetic diversity distribution across the animal genome. Over the last 50 years, cattle breeds have been subjected to intense artificial selection. Consequently, regions controlling traits of economic importance are expected to exhibit selection signatures. The fixation index (Fst ) is an estimate of population differentiation, based on genetic polymorphism data, and it is calculated using the relationship between inbreeding and heterozygosity. In the present study, locally weighted scatterplot smoothing (LOWESS) regression and a control chart approach were used to investigate selection signatures in two cattle breeds with different production aptitudes (dairy and beef). Fst was calculated for 42 514 SNP marker loci distributed across the genome in 749 Italian Brown and 364 Piedmontese bulls. The statistical significance of Fst values was assessed using a control chart. The LOWESS technique was efficient in removing noise from the raw data and was able to highlight selection signatures in chromosomes known to harbour genes affecting dairy and beef traits. Examples include the peaks detected for BTA2 in the region where the myostatin gene is located and for BTA6 in the region harbouring the ABCG2 locus. Moreover, several loci not previously reported in cattle studies were detected.

CD34 glycoprotein identifies putative stem cells located in the isthmic region of canine hair follicles.

It is widely documented that a pool of multipotent stem cells located in humans and mice hair follicle outer root sheath (bulge region) is involved in the restoration of the whole follicular unit during each anagen phase. To the authors' knowledge, data regarding the location and characterization of hair follicle stem compartment in dogs have not been reported in the recent relevant literature. In this study, we investigated the haematopoietic stem and progenitor cell antigen CD34 as a marker of putative stem cells located in a bulge-like region of canine hair follicles. The presence of CD34 mRNA and glycoprotein was assessed on formalin-fixed, paraffin-embedded canine skin samples by in situ hybridization technique and by standard immunohistochemistry, respectively. A strong expression of CD34 mRNA and glycoprotein was observed in a well-defined area of the hair follicle isthmic region and appeared uniformly concentrated at the level of the basal layer of the outer root sheath. These findings provide compelling support to the hypothesis that in dogs, a subpopulation of basal keratinocytes located in the hair follicle isthmic region and characterized by the selective expression of CD34 is potentially associated with the stem cell compartment of this skin appendage.

Molecular characterization and expression of a divergent alpha-tubulin in planarian Schmidtea polychroa.

We report the cloning and sequencing of a cDNA from planarian Schmidtea polychroa (Platyhelminthes, Turbellaria, Tricladida) encoding for an unusual tubulin isoform (SpTub-1) which is specifically expressed in testis. Sequence comparison of SpTub-1 with other known tubulins reveals that it has the highest homology with alpha-tubulins, even though the analysis of the molecular features shows that this isoform is significantly divergent. Hybridization of SpTub-1 to restriction-digested genomic DNA to Southern blotting produced a multiple banding pattern indicating that in planarian, a tubulin multigene family exists. Using in situ hybridization, we showed that the transcript is specifically detectable in planarian testis, suggesting that it may play a role in spermatogenesis.