P. Ajmone Marsan

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.

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.

Inbreeding and purging at the genomic Level: the Chillingham cattle reveal extensive, non-random SNP heterozygosity

Local breeds of livestock are of conservation significance as components of global biodiversity and as reservoirs of genetic variation relevant to the future sustainability of agriculture. One such rare historic breed, the Chillingham cattle of northern England, has a 350-year history of isolation and inbreeding yet shows no diminution of viability or fertility. The Chillingham cattle have not been subjected to selective breeding. It has been suggested previously that the herd has minimal genetic variation. In this study, high-density SNP genotyping with the 777K SNP chip showed that 9.1% of loci on the chip are polymorphic in the herd, compared with 62-90% seen in commercial cattle breeds. Instead of being homogeneously distributed along the genome, these loci are clustered at specific chromosomal locations. A high proportion of the Chillingham individuals examined were heterozygous at many of these polymorphic loci, suggesting that some loci are under balancing selection. Some of these frequently heterozygous loci have been implicated as sites of recessive lethal mutations in cattle. Linkage disequilibrium equal or close to 100% was found to span up to 1350 kb, and LD was above r(2) = 0.25 up to more than 5000 kb. This strong LD is consistent with the lack of polymorphic loci in the herd. The heterozygous regions in the Chillingham cattle may be the locations of genes relevant to fitness or survival, which may help elucidate the biology of local adaptation in traditional breeds and facilitate selection for such traits in commercial cattle.

Pattern of ancient goat migration revealed by AFLP molecular markers

Riassunto I marcatori AFLP ricostruiscono gli spostamenti di Capra hircus al seguito delle antiche migrazioni umane. Capra hircus è adattabile a condizioni ambientali molto differenti e possiede un areale di distribuzione estremamente ampio. Studi recenti suggeriscono che la capra abbia giocato un ruolo chiave nel sostentamento delle popolazioni umane durante le migrazioni demiche verso ovest, successive alla rivoluzione agricola del Neolitico. Lo studio della variabilità genetica di questa specie può quindi essere un utile strumento sia per ricostruire le antiche migrazioni umane, sia per preservare la diversità biologica di razze storiche e localmente adattate. L’analisi statistica dei marcatori AFLP effettuata su 44 razze autoctone (campionate in Europa, Medio Oriente, Anatolia) e 2 cosmopolite, affiancata da metodi di interpolazione geografica, ha permesso di evidenziare: - la peculiare composizione genetica delle razze Orobica e Tauernshecken (entrambe di origine incerta) rispetto ai popolamenti europeo e medio-orientale; - un significativo gradiente di distribuzione delle distanze genetiche tra razze che da sud-est procede verso nord-ovest, sovrapponibile al modello di variazione genetica umana, probabile traccia di almeno due differenti eventi di migrazioni umane lungo la via di espansione neolitica dell’agricoltura.

Nanotechnologies Applied to the Analysis of the Animal Genome

Ajmone Marsan, P., Tramontana, S. and Mazza, R., 2007. Nanotechnologies applied to the analysis of the animal genome. Veterinary Research Communications, 31(Suppl. 1), 153–159ABSTRACTWhole genome sequence information and high throughput technologies are speeding up the investigation of cellular processes leading to the phenotypic expression of genetic information. Nanotechnologies provide innovative tools to accomplish this task, increasing throughput and sensitivity and decreasing cost and time of analyses. Goals as ambitious as the sequencing of a mammalian-sized genome in a matter of hours, and of detecting gene expression from a single cell, are just around the corner. Animal breeding will benefit from these advances in the understanding of the genetic basis of complex traits and in the application of molecular information for marker and gene assisted selection.

Assessment of AFLP® marker behaviour in enriching STS radiation hybrid maps

Radiation hybrid (RH) mapping provides a powerful tool to build high-resolution maps of genomes. Here, we demonstrate the use of the AFLP technique for high-throughput typing of RH cell lines. Cattle were used as the model species because an RH panel was available to investigate the behaviour of AFLP markers within the microsatellite- and STS-based maps of this species. A total of 747 AFLP markers were typed on the TM112 RH radiation panel and 651 of these were assigned by two-point analysis to the 29 bovine autosomes and sex chromosomes. AFLP markers were added to the 1222 microsatellite and STS markers that were included in earlier RH maps. Multipoint maps were constructed for seven example chromosomes, which retained 248 microsatellite and STS markers, and added 123 AFLP markers at LOD 4. The addition of the AFLP markers increased the number of markers by 42.1% and the map length by 10.4%. The AFLP markers showed lower retention frequency (RF) values than the STS markers. The comparison of RF values in AFLP markers and their corresponding AFLP-derived STSs demonstrated that the lower RF values were due to the lower detection sensitivity of the AFLP technique. Despite these differences, AFLP and AFLP-derived STS markers mapped to identical or similar positions. These results demonstrate that it is possible to merge AFLP and microsatellite markers in the same map. The application of AFLP technology could permit the rapid construction of RH maps in species for which extensive genome information and large numbers of SNP and microsatellite markers are not available.

Potential neoplastic evolution of Vero cells: in vivo and in vitro characterization

Vero cell lines are extensively employed in viral vaccine manufacturing. Similarly to all established cells, mutations can occur during Vero cells in vitro amplification which can result in adverse features compromising their biological safety. To evaluate the potential neoplastic evolution of these cells, in vitro transformation test, gene expression analysis and karyotyping were compared among low- (127 and 139 passages) and high-passage (passage 194) cell lines, as well as transformed colonies (TCs). In vivo tumorigenicity was also tested to confirm preliminary in vitro data obtained for low passage lines and TCs. Moreover, Vero cells cultivated in foetal bovine serum-free medium and derived from TCs were analysed to investigate the influence of cultivation methods on tumorigenic evolution. Low-passage Vero developed TCs in soft agar, without showing any tumorigenic evolution when inoculated in the animal model. Karyotyping showed a hypo-diploid modal chromosome number and rearrangements with no difference among Vero cell line passages and TCs. These abnormalities were reported also in serum-free cultivated Vero. Gene expression revealed that high-passage Vero cells had several under-expressed and a few over-expressed genes compared to low-passage ones. Gene ontology revealed no significant enrichment of pathways related to oncogenic risk. These findings suggest that in vitro high passage, and not culture conditions, induces Vero transformation correlated to karyotype and gene expression alterations. These data, together with previous investigations reporting tumour induction in high-passage Vero cells, suggest the use of low-passage Vero cells or cell lines other than Vero to increase the safety of vaccine manufacturing.

New approaches to investigate the genetic basis of animal adaptation to different environments

The understanding of the genetic mechanisms of livestock adaptation to environmental challenges is becoming an important research topic in this time of rapid climate change. New tools and approaches are now available to investigate this complex phenomenon. Low-cost high-throughput technologies have opened the genomic era to agricultural species. All major farm animals have been completely sequenced and HapMap projects are completed or in progress. Low, medium and high-density SNP panels are available or under construction. Molecular information on many thousand markers has initiated the era of population genomics, which is the application of genomic approaches to population genetics. The comparison of patterns of diversity along the genome in animals originating in different environments and new GIScience-based models able to associate molecular markers to environmental variables promise to discover genomic regions associated to traits important for adaptation and to pave the way to the identification of causative genes. Local breeds adapted to a sustainable production in extreme and harsh environments will play a fundamental role in this process.