E. Milanesi

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.

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.

MOLECULAR DETECTION OF CELL LINE CROSS-CONTAMINATIONS USING AMPLIFIED FRAGMENT LENGTH POLYMORPHISM DNA FINGERPRINTING TECHNOLOGY

SummaryWe have tested amplified fragment length polymorphism (AFLP) technology, in comparison with isoenzyme analysis, for the simultaneous detection of inter-and intraspecific cell line cross-contaminations (CCCs) in the cell line collection held at the Istituto Zooprofilattico della Lombardia e dell’Emilia Romagna. Isoenzyme analysis identified four cases of interspecific CCCs. In a single expreiment, AFLP was able to identify the species of origin of all cell lines for which a reference genomic deoxyribonucleic acid was available and to detect five interspecific contaminations. Four CCCs confirmed data on isoenzymes, whereas the fifth CCC was detected in a species for which isoenzyme analysis was noninformative. In addition, AFLP was able to identify the putative source of the contaminations detected. The utility of the technology in the detection of intraspecific cell line contaminations, depends on the number of cell lines that have to be distinguished in a specific species and on the availability of highly informative fingerprinting systems. In mice, a single AFLP primer pair produced 16 polymorphisms and distinguished all the 15 strains of mouse cell lines analyzed. In humans, 18 AFLPs identified 83 different profiles in the 159 cell lines analyzed. Amplified fragment length polymorphism can conveniently be applied for cell line fingerprinting in species for which hypervariable markers are not available. In species for which a highly informative multiplex of microsatellite markers is available, AFLP can still provide a useful and cheap tool for simultaneously testing inter-and intraspecific contaminations.

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.

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.

Identification of milk protein percentage QTLs in Italian Friesian cattle by selective genotyping two GDD families with AFLP and SSR markers

Abstract In this work we evaluated the combined use of AFLP markers and selective genotyping in a Grand Daughter experimental design (GDD), to identify chromosomal regions candidate to contain QTL for milk protein percentage (PP) in Italian Friesian cattle. We verified the approach by microsatellite analysis of 4 candidate chromosomal regions. Twenty-five AFLP primer combinations, assayed on the extreme minus and plus-variant half-sibs and on the grandsires of 2 GDD families, produced 180 polymorphic bands of paternal origin. Association analysis resulted in 16 AFLPs significantly associated to PP-EBVs (P≤0.05), that mapped on 8 chromosomes, in regions where PP-QTLs were identified in previous studies. Microsatellite analysis confirmed association for 2 regions of BTA10 and BTA28.

Ancient DNA from domestic animal species remains: preliminary approaches

Abstract DNA analysis from ancient and old remains offers new tools to answer archaeozoological questions and investigate the origin of the genetic variability in domestic animal species. Molecular genetics techniques contribute to identify the species supporting classical osteological studies and to establish the relationship to modern species and breeds. Mitochondrial DNA (mtDNA) sequences are useful to reconstruct the history of maternal lineages comparing haplotype variations of present and old DNA samples. Mitochondrial data from modern cattle populations show a high diversity in Anatolia and in the Middle East supporting a near-Eastern matrilineal centre of origin. On the contrary in Europe a single family of mitochondrial haplotypes strongly dominates. A number of recent studies reported the successful recovery of ancient and old nuclear DNA (nuDNA) sequences. Such studies represent an important breakthrough, as nuDNA can be used for the characterisation of genetic loci directly involved in phenotypic traits, answering challenging questions. A bright example is offered by the study on the single nuclear exon of melanocortin type 1 receptor gene from a ca. 43,000 years old mammoth bone from Siberia, showing that mammoth populations were polymorphic with regard to hair colour, harbouring both dark and light haired animals. In contrast, these studies on ancient and old DNA sequences need great caution, due to the analytical problems caused by post-mortem damage of DNA, contamination from exogenous sources of mt- and nuDNA, and the consequent reliability of observed polymorphisms. The present research describes the preliminary analytical approach to DNA study of faunal remains (103 animal bones of different domestic species: Bos taurus 51; Ovis aries/Capra hircus 39; Sus scrofa/Sus domesticus 10; Gallus gallus 1; Equus caballus/Equus sp. 2), collected in seven archaeological sites located within the province of Trento, in the Alpine region of Trentino Alto-Adige (N-E Italy). The chosen sites, dating from the Bronze Age to the late Middle Ages, display different settlement typology and include Iron Age retic houses, votive Bronze Age contexts, a 4th century roman villa and several 13th century medieval buildings. Archaeozoological data will be collected on species, skeletal parts, age of slaughter, method of butchery, evidence of bone working and presence of paleopathologies. We describe the analytical procedure used in preparing and collecting samples and in extracting and analysing DNA from a subset of the bones previously described.