Mariasilvia D’Andrea

Expression profiles of Toll-like receptors 1, 2 and 5 in selected organs of commercial and indigenous chickens

Toll-like receptors (TLRs) are members of the cellular receptors that constitute a major component of the evolutionary conserved pattern recognition system (PRR). TLRs are expressed in a wide variety of tissues and cell types. In this study we compared the expression profiles of the chicken TLR1, TLR2 and TLR5 genes in a range of organs (lung, ovary, liver, thymus, duodenum, spleen and large intestine) in commercial Hy-Line (HL) and indigenous Green-legged Partridgelike (GP) chickens. The level of mRNA was determined with RT-qPCR using the TaqMan probes for target and reference (ACTB) genes. We determined that the tissue profiles differed with respect to each TLR and they were ranked as follows: spleen, lungs, large intestine (TLR1), large intestine, lungs, thymus/ovary (TLR2) and lungs, thymus, liver (TLR5). A differential expression between HL and GP chickens was determined for TLR1 and TLR5 genes in large intestine and thymus of HL (P < 0.05) and GP (P < 0.05) chickens. We conclude that the commercial chickens expressed higher levels of TLR1 mRNA in large intestine and TLR5 mRNA in thymus than indigenous chickens.

Merino and Merino-derived sheep breeds: a genome-wide intercontinental study

BackgroundMerino and Merino-derived sheep breeds have been widely distributed across the world, both as purebred and admixed populations. They represent an economically and historically important genetic resource which over time has been used as the basis for the development of new breeds. In order to examine the genetic influence of Merino in the context of a global collection of domestic sheep breeds, we analyzed genotype data that were obtained with the OvineSNP50 BeadChip (Illumina) for 671 individuals from 37 populations, including a subset of breeds from the Sheep HapMap dataset.ResultsBased on a multi-dimensional scaling analysis, we highlighted four main clusters in this dataset, which corresponded to wild sheep, mouflon, primitive North European breeds and modern sheep (including Merino), respectively. The neighbor-network analysis further differentiated North-European and Mediterranean domestic breeds, with subclusters of Merino and Merino-derived breeds, other Spanish breeds and other Italian breeds. Model-based clustering, migration analysis and haplotype sharing indicated that genetic exchange occurred between archaic populations and also that a more recent Merino-mediated gene flow to several Merino-derived populations around the world took place. The close relationship between Spanish Merino and other Spanish breeds was consistent with an Iberian origin for the Merino breed, with possible earlier contributions from other Mediterranean stocks. The Merino populations from Australia, New Zealand and China were clearly separated from their European ancestors. We observed a genetic substructuring in the Spanish Merino population, which reflects recent herd management practices.ConclusionsOur data suggest that intensive gene flow, founder effects and geographic isolation are the main factors that determined the genetic makeup of current Merino and Merino-derived breeds. To explain how the current Merino and Merino-derived breeds were obtained, we propose a scenario that includes several consecutive migrations of sheep populations that may serve as working hypotheses for subsequent studies.

Promoter polymorphisms in genes involved in porcine myogenesis influence their transcriptional activity.

BackgroundSuccess of meat production and selection for improvement of meat quality is among the primary aims in animal production. Meat quality traits are economically important in swine; however, the underlying genetic nature is very complex. Therefore, an improved pork production strongly depends on identifying and studying how genetic variations contribute to modulate gene expression. Promoters are key regions in gene modulation as they harbour several binding motifs to transcription regulatory factors. Therefore, polymorphisms in these regions are likely to deeply affect RNA levels and consequently protein synthesis. In this study, we report the identification of single nucleotide polymorphisms (SNPs) in promoter regions of candidate genes involved in development, cellular differentiation and muscle growth in Sus scrofa. We identified SNPs in the promoter regions of genes belonging to the Myogenic Regulatory Factors (MRF) gene family (the Myogenic Differentiation gene, MYOD1) and to Growth and Differentiation Factors (GDF) gene family (Myostatin gene, MSTN, GDF8), in Casertana and Large White breeds. The purpose of this study was to investigate if polymorphisms in the promoters could affect the transcriptional activity of these genes. With this aim, we evaluated in vitro the functional activity of the luciferase reporter gene luc2 activity, driven by two constructs carrying different promoter haplotypes.ResultsWe tested the effects of the G302A (U12574) transition on the promoter efficiency in MYOD1 gene. We ascertained a difference in transcription efficiency for the two variants. A stronger activity of the A-carrying construct is more evident in C2C12. The luciferase expression driven by the MYOD1-A allelic variant displayed a 3.8-fold increased transcriptional activity. We investigated the activity of two haplotype variants (AY527152) in the promoter of GDF8 gene. The haploptype-1 (A435-A447-A879) up-regulated the expression of the reporter gene by a two-fold increase, and hence presumably of the GDF8 gene, in both CHO and C2C12 cultured cells.ConclusionsIn vitro the MYOD1-A allelic variant could up-regulate the expression of MYOD1 gene. Additionally, we could assess a different response of in vitro gene expression according to cell type used to transfect constructs, suggesting that MyoD activation is regulated by mechanisms that are specific of myoblasts.

Technical note: Advantages and limitations of authenticating Palmera goat dairy products by pyrosequencing the melanocortin 1 receptor (MC1R) gene

Inferring the breed of origin of dairy products can be achieved through molecular analysis of genetic markers with a population-specific pattern of segregation. The goal of the current work was to generate such markers in goats by resequencing several pigmentation genes [melanocortin 1 receptor (MC1R), v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT), tyrosinase (TYR), and tyrosinase-related protein 2 (TYRP2)]. This experiment revealed 10 single nucleotide polymorphisms (SNP), including 5 missense mutations and 1 nonsense mutation. These markers were genotyped in 560 goats from 18 breeds originally from Italy, the Iberian Peninsula, the Canary Islands, and North Africa. Although the majority of SNP segregated at moderate frequencies in all populations (including 2 additional markers that were used as a source of information), we identified a c.764G>A SNP in MC1R that displayed highly divergent allelic frequencies in the Palmera breed compared with the Majorera and Tinerfeña breeds from the Canary Islands. Thus, we optimized a pyrosequencing-based technique that allowed us to estimate, very accurately, the allele frequencies of this marker in complex DNA mixtures from different individuals. Once validated, we applied this method to generating breed-specific DNA profiles that made it possible to detect fraudulent cheeses in which Palmero cheese was manufactured with milk from Majorera goats. One limitation of this approach, however, is that it cannot be used to detect illegal manufacturing where Palmero dairy products are produced by mixing milk from Palmera and Majorera goats, because the c.764G>A SNP segregates in both breeds.

Identification of c.483C>T polymorphism in the caprine tyrosinase-related protein 1 (TYRP1) gene

Tyrosinase-related protein 1 (TYRP1) has been shown to play a fundamental role in pigmentation both in human and mouse. In this work, we aimed to characterize the variability of the caprine TYRP1 gene and investigate its segregation in a wide array of goat breeds. By partially sequencing the coding region of the TYRP1 gene in 18 individuals from eight different breeds, we were able to identify a synonymous nucleotide substitution at exon 3 (c.483C>T). An extensive survey of Iberian and Balearic (N=175), Italian (N=99), Swiss (N=54), Asian (N=14), Canarian (N=92) and North African (N=117) goats with different coat colours was carried out. We found that the C-allele has a different distribution in European vs African breeds, being almost fixed in the latter. Moreover, the C-allele showed an increased frequency in white coated breeds (Girgentana, Grigia Molisana, Blanca de Rasquera and Saanen) when compared with those displaying a dark pigmentation (Cilentana Nera, Azpi Gorri and Murciano-Granadina). This could be due to genetic drift, migration and other factors associated with the demographic history of breeds under analysis or to a genetic hitchhiking event (c.483C>T frequencies would be shaped by a neighbouring causal mutation differentially selected in white and black goats). More refined studies will be needed to distinguish between these two alternative explanations.

Differential distribution of Y-chromosome haplotypes in Swiss and Southern European goat breeds

The analysis of Y-chromosome variation has provided valuable clues about the paternal history of domestic animal populations. The main goal of the current work was to characterize Y-chromosome diversity in 31 goat populations from Central Eastern (Switzerland and Romania) and Southern Europe (Spain and Italy) as well as in reference populations from Africa and the Near East. Towards this end, we have genotyped seven single nucleotide polymorphisms (SNPs), mapping to the SRY, ZFY, AMELY and DDX3Y Y-linked loci, in 275 bucks from 31 populations. We have observed a low level of variability in the goat Y-chromosome, with just five haplotypes segregating in the whole set of populations. We have also found that Swiss bucks carry exclusively Y1 haplotypes (Y1A: 24%, Y1B1: 15%, Y1B2: 43% and Y1C: 18%), while in Italian and Spanish bucks Y2A is the most abundant haplotype (77%). Interestingly, in Carpathian goats from Romania the Y2A haplotype is also frequent (42%). The high Y-chromosome differentiation between Swiss and Italian/Spanish breeds might be due to the post-domestication spread of two different Near Eastern genetic stocks through the Danubian and Mediterranean corridors. Historical gene flow between Southern European and Northern African goats might have also contributed to generate such pattern of genetic differentiation.

Breed and adaptive response modulate bovine peripheral blood cells’ transcriptome

BackgroundAdaptive response includes a variety of physiological modifications to face changes in external or internal conditions and adapt to a new situation. The acute phase proteins (APPs) are reactants synthesized against environmental stimuli like stress, infection, inflammation.MethodsTo delineate the differences in molecular constituents of adaptive response to the environment we performed the whole-blood transcriptome analysis in Italian Holstein (IH) and Italian Simmental (IS) breeds. For this, 663 IH and IS cows from six commercial farms were clustered according to the blood level of APPs. Ten extreme individuals (five APP+ and APP- variants) from each farm were selected for the RNA-seq using the Illumina sequencing technology. Differentially expressed (DE) genes were analyzed using dynamic impact approach (DIA) and DAVID annotation clustering. Milk production data were statistically elaborated to assess the association of APP+ and APP- gene expression patterns with variations in milk parameters.ResultsThe overall de novo assembly of cDNA sequence data generated 13,665 genes expressed in bovine blood cells. Comparative genomic analysis revealed 1,152 DE genes in the comparison of all APP+ vs. all APP- variants; 531 and 217 DE genes specific for IH and IS comparison respectively. In all comparisons overexpressed genes were more represented than underexpressed ones. DAVID analysis revealed 369 DE genes across breeds, 173 and 73 DE genes in IH and IS comparison respectively. Among the most impacted pathways for both breeds were vitamin B6 metabolism, folate biosynthesis, nitrogen metabolism and linoleic acid metabolism.ConclusionsBoth DIA and DAVID approaches produced a high number of significantly impacted genes and pathways with a narrow connection to adaptive response in cows with high level of blood APPs. A similar variation in gene expression and impacted pathways between APP+ and APP- variants was found between two studied breeds. Such similarity was also confirmed by annotation clustering of the DE genes. However, IH breed showed higher and more differentiated impacts compared to IS breed and such particular features in the IH adaptive response could be explained by its higher metabolic activity. Variations of milk production data were significantly associated with APP+ and APP- gene expression patterns.