Cinzia Marchitelli

Double muscling in Marchigiana beef breed is caused by a stop codon in the third exon of myostatin gene

Double muscling is a partially recessive trait present in some beef breeds. It shows a high frequency in some breeds, while in others the frequency is low, and double-muscled individuals are rare. The double muscling is caused by an allelic series of mutations that cause a loss of function of the myostatin gene (GDF8). We describe here a new mutation in the myostatin gene in Marchigiana breed, a typical beef breed of Central Italy, in which rare double-muscling individuals have been described. A PCR product of the third exon was sequenced in subjects phenotypically showing double muscling, and a G > T transversion was discovered that introduces a premature stop codon. The variant found adds to the large series of mutations present in cattle, and particularly to the only two causative of double muscling in the third exon. A PCR-RFLP test is described for the rapid and effective identification of both heterozygous and homozygous subjects. It was applied to a larger survey carried on the same and also in two other beef breeds, Chianina and Romagnola. Further individuals carrying the new variant were found in Marchigiana, but none in the other breeds. The results may be important for a better comprehension of the role of myostatin in muscular development, for commercial use and for the inference of phylogeny of this gene.

Massive screening of copy number population-scale variation in Bos taurus genome

BackgroundCopy number variations (CNVs) represent a significant source of genomic structural variation. Their length ranges from approximately one hundred to millions of base pair. Genome-wide screenings have clarified that CNVs are a ubiquitous phenomenon affecting essentially the whole genome. Although Bos taurus is one of the most important domestic animal species worldwide and one of the most studied ruminant models for metabolism, reproduction, and disease, relatively few studies have investigated CNVs in cattle and little is known about how CNVs contribute to normal phenotypic variation and to disease susceptibility in this species, compared to humans and other model organisms.ResultsHere we characterize and compare CNV profiles in 2654 animals from five dairy and beef Bos taurus breeds, using the Illumina BovineSNP50 genotyping array (54001 SNP probes). In this study we applied the two most commonly used algorithms for CNV discovery (QuantiSNP and PennCNV) and identified 4830 unique candidate CNVs belonging to 326 regions. These regions overlap with 5789 known genes, 76.7% of which are significantly co-localized with segmental duplications (SD).ConclusionsThis large scale screening significantly contributes to the enrichment of the Bos taurus CNV map, demonstrates the ubiquity, great diversity and complexity of this type of genomic variation and sets the basis for testing the influence of CNVs on Bos taurus complex functional and production traits.

A second generation radiation hybrid map to aid the assembly of the bovine genome sequence

BackgroundSeveral approaches can be used to determine the order of loci on chromosomes and hence develop maps of the genome. However, all mapping approaches are prone to errors either arising from technical deficiencies or lack of statistical support to distinguish between alternative orders of loci. The accuracy of the genome maps could be improved, in principle, if information from different sources was combined to produce integrated maps. The publicly available bovine genomic sequence assembly with 6× coverage (Btau_2.0) is based on whole genome shotgun sequence data and limited mapping data however, it is recognised that this assembly is a draft that contains errors. Correcting the sequence assembly requires extensive additional mapping information to improve the reliability of the ordering of sequence scaffolds on chromosomes. The radiation hybrid (RH) map described here has been contributed to the international sequencing project to aid this process.ResultsAn RH map for the 30 bovine chromosomes is presented. The map was built using the Roslin 3000-rad RH panel (BovGen RH map) and contains 3966 markers including 2473 new loci in addition to 262 amplified fragment-length polymorphisms (AFLP) and 1231 markers previously published with the first generation RH map. Sequences of the mapped loci were aligned with published bovine genome maps to identify inconsistencies. In addition to differences in the order of loci, several cases were observed where the chromosomal assignment of loci differed between maps. All the chromosome maps were aligned with the current 6× bovine assembly (Btau_2.0) and 2898 loci were unambiguously located in the bovine sequence. The order of loci on the RH map for BTA 5, 7, 16, 22, 25 and 29 differed substantially from the assembled bovine sequence. From the 2898 loci unambiguously identified in the bovine sequence assembly, 131 mapped to different chromosomes in the BovGen RH map.ConclusionAlignment of the BovGen RH map with other published RH and genetic maps showed higher consistency in marker order and chromosome assignment than with the current 6× sequence assembly. This suggests that the bovine sequence assembly could be significantly improved by incorporating additional independent mapping information.

Discovery, characterization and validation of single nucleotide polymorphisms within 206 bovine genes that may be considered as candidate genes for beef production and quality

A large number of putative single nucleotide polymorphisms (SNPs) have been identified from the bovine genome-sequencing project. However, few of these have been validated and many will turn out to be sequencing artefacts or have low minor allele frequencies. In addition, there is little information available on SNPs within coding regions, which are likely to be responsible for phenotypic variation. Therefore, additional SNP discovery is necessary to identify and validate polymorphisms both in specific genes and genome-wide. Sequence-tagged sites within 286 genes were resequenced from a panel of animals representing a wide range of European cattle breeds. For 80 genes, no polymorphisms were identified, and 672 putative SNPs were identified within 206 genes. Fifteen European cattle breeds (436 individuals plus available parents) were genotyped with these putative SNPs, and 389 SNPs were confirmed to have minor allele frequencies above 10%. The genes containing SNPs were localized on chromosomes by radiation hybrid mapping and on the bovine genome sequence by Blast. Flanking microsatellite loci were identified, to facilitate the alignment of the genes containing the SNPs in relation to mapped quantitative trait loci. Of the 672 putative SNPs discovered in this work, only 11 were found among the validated SNPs and 100 were found among the approximately 2.3 million putative SNPs currently in dbSNP. The genes studied in this work could be considered as candidates for traits associated with beef production and the SNPs reported will help to assess the role of the genes in the genetic control of muscle development and meat quality. The allele frequency data presented allows the general utility of the SNPs to be assessed.

Milk fatty acid variability: effect of some candidate genes involved in lipid synthesis.

In this work, the genetic variation of milk FA was investigated in three different bovine breeds, the Jersey, the Piedmontese and the Valdostana, and at different lactation stages. All animals were genotyped for 21 Single Nucleotide Polymorphisms located within nine candidate genes involved in lipid synthesis: diacylglycerol acyltransferase 1 and 2 (DGAT1, 2); stearoyl-CoA desaturase (SCD); growth hormone receptor (GHR); fatty acid synthase (FASN); acyl-CoA dehydrogenase (ACAD); fatty acid binding protein (FABP4); lipoprotein lipase (LPL); and leptin gene (LEP). The highest milk-fat Jersey breed also showed the highest content of saturated FA. Throughout lactation, the breeds showed a similar variation in the FA, with a decrease in the short-chain, this was accompanied by a general increase in the long chain FA at the end of lactation. The increase in long chain saturated FA was particularly evident in the case of the Jersey. The effect of SCD gene on the C14 desaturation index was confirmed; the DGAT1 gene was polymorphic only in the Jersey breed, but its effect was confirmed only on milk fat content; three further potential candidate genes were identified: first, the FABP4 gene, which was found to influence medium and long chain FA in all the breeds, but not the desaturation indices; second, the FASN gene, which was found to influence the amount of PUFA in the Piedmontese and the Valdostana, and third, the LPL gene, which was found to affect fat content in the Piedmontese.

Splicing Variants of SERPINA1 Gene in Ovine Milk: Characterization of cDNA and Identification of Polymorphisms

The serine protease inhibitor, clade A, member 1 (SERPINA1) is the gene for a protein called alpha-1-antitrypsin (AAT), which is a member of the serine protease inhibitor (serpin) superfamily of proteins. By conformational change, serpins control several chemical reactions inhibiting the activity of proteases. AAT is the most abundant endogenous serpin in blood circulation and it is present in relatively high concentration in human milk as well as in bovine and porcine colostrum. Here we report for the first time the molecular characterization and sequence variability of the ovine SERPINA1 cDNA and gene. cDNAs from mammary gland and from milk were PCR amplified, and three different transcripts (1437, 1166 and 521bp) of the SERPINA1 gene were identified. We amplified and sequenced different regions of the gene (5’ UTR, from exon 2 to exon 5 and 3’ UTR), and we found that the exon-intron structure of the gene is similar to that of human and bovine. We detected a total of 97 SNPs in cDNAs and gene sequences from 10 sheep of three different breeds. In adult sheep tissues a SERPINA1 gene expression analysis indicated a differential expression of the three different transcripts. The finding reported in this paper will aid further studies on possible involvement of the SERPINA1 gene in different physiological states and its possible association with production traits.

Mutations and sequence variants in GDF9, BMP15, and BMPR1B genes in Maremmana cattle breed with single and twin births

The families of TGF-β (transforming growth factor-β) proteins are the most important growth factors in the ovary, and three related oocyte-derived members, namely GDF9 (growth differentiation factor 9), BMP15 (bone morphogenetic protein 15), and BMPR1B (bone morphogenetic protein receptor 1B), have been shown to be essential for follicular growth and ovulation. Although the essential role of these genes in determining litter size in sheep and mouse and in controlling folliculogenesis in human has been demonstrated, there is limited information on their action in other species, especially in bovine. Bovine is a monotocous specie, as humans, with one or sometimes two newborns per birth. The twinning is a complex trait determined by both genetic and environmental factors. This study aimed at investigating the nucleotide sequences of different fragments of GDF9, BMP15, and BMPR1B genes in Maremmana cows reared in Castelporziano Presidential Estate (Rome). In this herd, in the period between 1996 and 2008, a twinning rate of 12 % (on average) was observed. We identified nine single-nucleotide polymorphisms (SNPs), five in the coding region, and four in the noncoding region: Two polymorphisms caused non-synonymous mutations, g.6045 G>A (V202I) in the BMP15 gene, and g.231 T>C (L66S) in GDF9 gene. The mutation L66S was found only in cows with double birth. In the literature, there are different evidences that mutations in proregion of GDF9 protein could affect its correct function. A relationship between mutations in this region of protein and granulosa cells proliferation and oocyte development was hypothesized.

Polymorphisms within the Toll-like receptor (TLR)-2, -4, and -6 genes in cattle

Abstract Toll-like receptors (TLR) have an important role in the defence against a variety of infectious diseases. TLR recognize various microbial components and are important in the innate immune response to pathogens and in crosstalk between innate immunity and adaptive immunity. Polymorphisms in TLRs influence their abilities of recognition of pathogen-derived molecules and therefore could be of great relevance in livestock selection once genetic variation in these loci has been associated with resistance. Several single nucleotide polymorphisms (SNPs) within TLR genes have been described in humans and some of them seem to be associated with susceptibility to infection diseases. Recently, all the 10 TLR genes have been mapped in cattle by radiation hybrid panel. In this study, we screened the nucleotide sequences of bovine TLR2, TLR4, and TLR6 genes (located on BTA17, BTA8 and BTA6, respectively) searching for SNPs potentially linked with disease resistance. We could identify 11 SNPs that were used for screening 900 individuals belonging to 16 different bovine European breeds. Eight of the analysed breeds resulted polymorphic at all the analysed loci, and six of the loci were polymorphic in all the breeds. Allelic frequencies, Gene Diversity, Heterozygosity and PIC were calculated. Heterozygosity and PIC ranged respectively from 0.05 to 0.47 and from 0.06 and 0.37. Heterozygosity and Fst were calculated using the Fdist software (http://www.rubic.rdg.ac.uk/~mab/software.html). Population phylogeny datasets were built by bootstrapping 200,000 replications on real data using a coalescent model. None of the SNPs were found to lie outside the 95% confidence limits assumed for conditional joint distribution of Fst vs mean heterozygosity, and therefore the genes do not appear to be under selection in the analysed samples according to the employed model. However, several SNPs were not in HW equilibrium. We hypothesize that some of the polymorphisms were fixated since many generations within breed and the coalescent model could not be powerful enough to reveal selection event so far in the past.

European cattle breed cluster accordingly to their meat quality parameters

Abstract The concept of breed is rather questionable and it’s used more as a tool for “labelling” production systems than as a biological category. Here, production system is intended as a whole set of animal units, techniques, breeding schemes, marketing, etc. However, man has demonstrated to be very quick in capturing and disseminating good characteristics whence they appear in a breed by mutation or by selection. Therefore, it might be expected that breeds, nevertheless of recent origin, could bear distinguished productive characteristics. Due to the quantitative nature of them, more characteristics should be measured in order to obtain a clear and statistically significant distinction. We have measured several meat characteristics in 15 European breeds (30 individuals for each breed), mostly with beef attitude, reared in similar conditions. This was accomplished to better reveal the genetic background of breeds. A canonical discriminant analysis showed a clear distinction among breeds. In particular lipid composition of meat was able to assign individuals to breeds with 57% and 63% of individuals correctly classified respectively for neutral and phospholipids. The classification is generally good for all breeds except for the Spanish ones, indicating probably some crossing in the past for these breeds. Neutral lipids can classify double muscled breeds with high precision (84% and 95% in Asturiana de los Valles and Piedmontese respectively). Tenderness related measures (collagen, µ-calpain, m-calpain, calpastatin, MFI) poorly assign individuals to breeds (average 22%). The good classification of individuals to breeds for lipid composition suggests distinctive genetic features and encourages to look further to genetic determination of fat composition in the meat, as well as to exploit particular breeds to obtain products suitable for categories of consumers needing/searching for special components in their diet.

Effect of some candidate genes on meat characteristics of three cattle breeds.

Abstract With the aim to assess if some molecular markers can help to select animals for meat characteristics, we studied 84 individuals equally representing the Marchigiana, Maremmana, and Holstein Friesian cattle breeds genotyped at 288 SNPs located within candidate genes. Several SNPs were found associated with meat quality parameters but with P which was higher than the Bonferroni threshold. However, several SNPs had a low P at different times during meat maturation, suggesting their involvement in the meat quality variation. Of particular interest for the biological role and potential for selection were: cathepsin G affecting MFI, IGF1R affecting pH and collagen XVIII affecting colour.