Mapping centromeres of microchromosomes in the zebra finch (Taeniopygia guttata) using half-tetrad analysis.

Centromeres usually consist of hundreds of kilobases of repetitive sequence which renders them difficult to assemble. As a consequence, centromeres are often missing from assembled genomes and their locations on physical chromosome maps have to be inferred from flanking sequences via fluorescence in situ hybridization (FISH). Alternatively, centromere positions can be mapped using linkage analyses in accidentally triploid individuals formed by half-tetrads (resulting from the inheritance of two chromatids from a single meiosis). The current genome assembly of the zebra finch (Taeniopygia guttata) comprises 32 chromosomes, but only for the ten largest chromosomes centromere positions have been mapped using FISH. We here map the positions of most of the remaining centromeres using half-tetrad analyses. For this purpose, we genotyped 37 zebra finches that were triploid or tetraploid due to inheritance errors (and mostly died as embryos) together with their parents at 64 microsatellite markers (at least two per chromosome). Using the information on centromere positions on the ten largest chromosomes, we were able to identify 12 cases of non-disjunction in maternal meiosis I and 10 cases of non-disjunction in maternal meiosis II. These 22 informative cases allowed us to infer centromere positions on additional 19 microchromosomes in reference to the current genome assembly. This knowledge will be valuable for studies of chromosome evolution, meiotic drive and species divergence in the avian lineage.

A raça Gir Mocha na região Nordeste do Brasil: estrutura genética populacional via análise de pedigree / The Polled Gir Polled in Northeastern Brazil: population genetic structure via analysis of pedigree

Foram utilizados dados de pedigree de 2.558 bovinos da raça Gir Mocha nascidos no período de 1954 a 2005. As análises foram realizadas utilizando-se o programa Endog. Do total de animais estudados, 61,9%; 10,6% e 0,1% possuíam pedigree na primeira, segunda e terceira gerações, respectivamente.O número efetivo de rebanhos que forneceram machos reprodutores foi de 10,25 para pais e 3,87 para avôs, confirmando a baixa integralidade do pedigree. O número de animais fundadores foi de 975,5, e o número efetivo de fundadores de 141,34. O número de ancestrais na população referência foi de 924 animas, dos quais apenas 39 explicaram 50% da variabilidade genética da população.O coeficiente médio de relação foi estimado em 0,75%, sendo o maior coeficiente individual de 25%. O coeficiente de endogamia foi igual a zero de 1954 a 1984. Vale salientar que, neste período, estão incluídos os animais sem ascendência conhecida. A endogamia e o coeficiente médio de relação da população foram baixos, contudo podem estar subestimados em razão da pequena integralidade do pedigree.

In this study we used data from the 2558 pedigree cattle polled Gir born from 1954 to 2005. Analyses were performed using the Endog program. Of all animals studied, 61.86%, 10.56% and 0.10% had a pedigree in the first, second and third generation, respectively. The effective number of herds that provide breeding males was 10.25 for parents and 3.87 for grandparents, corroborating the low completeness of the pedigree. The number of founder animals was 975.5 and the effective number of founders were 141.34. The number of ancestors in the reference population was 924 animals from which only 39 accounted for 50% of the genetic variability of the population. The average relationship coefficient was estimated at 0.75%, the largest individual coefficient was 25%. The inbreeding coefficient was zero from 1954 to 1894. It is noteworthy that during this period included the population was low, but may be underestimated because of the small pedigree integrity.

The sheep genome reference sequence: a work in progress.

Until recently, the construction of a reference genome was performed using Sanger sequencing alone. The emergence of next-generation sequencing platforms now means reference genomes may incorporate sequence data generated from a range of sequencing platforms, each of which have different read length, systematic biases and mate-pair characteristics. The objective of this review is to inform the mammalian genomics community about the experimental strategy being pursued by the International Sheep Genomics Consortium (ISGC) to construct the draft reference genome of sheep (Ovis aries). Component activities such as data generation, sequence assembly and annotation are described, along with information concerning the key researchers performing the work. This aims to foster future participation from across the research community through the coordinated activities of the consortium. The review also serves as a 'marker paper' by providing information concerning the pre-publication release of the reference genome. This ensures the ISGC adheres to the framework for data sharing established at the recent Toronto International Data Release Workshop and provides guidelines for data users.

Organization and genomic complexity of bovine lambda-light chain gene locus.

Complete characterization and physical mapping of bovine lambda (lambda) light chain locus, spanning 412kbp, on chromosome 17, has revealed twenty-five V(lambda) genes, seventeen being functional, organized in three sub-clusters 23.7kbp 5' of the J(lambda)-C(lambda) units. Three V(lambda) sub-clusters are separated by two large introns of 126.8 and 138.3kbp. The predominantly expressed V(lambda)1 genes are present in the two 5' sub-clusters, while J(lambda)-proximal V(lambda) sub-cluster comprises rarely expressed V(lambda)2 and V(lambda)3 genes. The preferential expression of V(lambda)1 genes in the bovine immunoglobulin repertoire is influenced by the composition of recombination signal sequences (RSS). Of the J(lambda)-C(lambda) cluster, it is mainly J(lambda)3-C(lambda)3 unit that is expressed in reading frame 2, though J(lambda)2 and J(lambda)3 have identical RSS. The predominant expression of J(lambda)3-C(lambda)3 genes over J(lambda)2-C(lambda)2 is likely due to endogenous counter selection for J(lambda)2 encoded CDR3 and framework 4 regions. Differences in the genomic complexity of V(lambda) genes in Hereford and Holstein cattle are due to polymorphism at the lambda-light chain gene locus. Despite more potential germline encoded combinatorial diversity, restricted V(lambda)1-J(lambda)3-C(lambda)3 recombinations encode the most lambda-light chain repertoire in cattle.

Fine mapping quantitative trait loci affecting milk production traits on bovine chromosome 6 in a Chinese Holstein population.

To fine map the previously detected quantitative trait loci (QTLs) affecting milk production traits on bovine chromosome 6 (BTA6), 15 microsatellite markers situated within an interval of 14.3 cM spanning from BMS690 to BM4528 were selected and 918 daughters of 8 sires were genotyped. Two mapping approaches, haplotype sharing based LD mapping and single marker regression mapping, were used to analyze the data. Both approaches revealed a quantitative trait locus (QTL) with significant effects on milk yield, fat yield and protein yield located in the segment flanked by markers BMS483 and MNB209, which spans a genetic distance of 0.6 cM and a physical distance of 1.5 Mb. In addition, the single marker regression mapping also revealed a QTL affecting fat percentage and protein percentage at marker DIK2291. Our fine mapping work will facilitate the cloning of candidate genes underlying the QTLs for milk production traits.

A 4,103 marker integrated physical and comparative map of the horse genome.

A comprehensive second-generation whole genome radiation hybrid (RH II), cytogenetic and comparative map of the horse genome (2n = 64) has been developed using the 5000rad horse x hamster radiation hybrid panel and fluorescence in situ hybridization (FISH). The map contains 4,103 markers (3,816 RH; 1,144 FISH) assigned to all 31 pairs of autosomes and the X chromosome. The RH maps of individual chromosomes are anchored and oriented using 857 cytogenetic markers. The overall resolution of the map is one marker per 775 kilobase pairs (kb), which represents a more than five-fold improvement over the first-generation map. The RH II incorporates 920 markers shared jointly with the two recently reported meiotic maps. Consequently the two maps were aligned with the RH II maps of individual autosomes and the X chromosome. Additionally, a comparative map of the horse genome was generated by connecting 1,904 loci on the horse map with genome sequences available for eight diverse vertebrates to highlight regions of evolutionarily conserved syntenies, linkages, and chromosomal breakpoints. The integrated map thus obtained presents the most comprehensive information on the physical and comparative organization of the equine genome and will assist future assemblies of whole genome BAC fingerprint maps and the genome sequence. It will also serve as a tool to identify genes governing health, disease and performance traits in horses and assist us in understanding the evolution of the equine genome in relation to other species.

Fluorescent in situ hybridization mapping of the epidermal growth factor receptor gene in donkey.

The physical localization of the epidermal growth factor receptor (EGFR) gene was performed on donkey chromosomes. Bacterial artificial chromosome DNA containing the equine EGFR gene was used to map this gene by fluorescent in situ hybridization on donkey metaphase chromosomes. The gene was mapped on donkey 1q21.1 region.

Quantitative trait loci affecting fertility and calving traits in Swedish dairy cattle.

Impaired fertility is the main reason for involuntary culling of dairy cows in Sweden. The objective of this study was to map quantitative trait loci (QTL) influencing fertility and calving traits in the Swedish dairy cattle population. The traits analyzed were number of inseminations, 56-d nonreturn rate, interval from calving to first insemination, fertility treatments, heat intensity score, stillbirth, and calving performance. A genome scan covering 20 bovine chromosomes was performed using 145 microsatellite markers. The mapping population consisted of 10 sires and their 417 sons in a granddaughter design. Nine of the sires were of the Swedish Red Breed, and one was a Swedish Holstein. Least squares regression was used to map loci affecting the analyzed traits, and permutation tests were used to set significance thresholds. Cofactors were used in the analyses of individual chromosomes to adjust for QTL found on other chromosomes. The use of cofactors increased both the number of QTL found and the significance level. In the initial analysis, we found 13 suggestive QTL that were mapped to chromosomes 6, 7, 9, 11, 13, 15, 20, and 29. When cofactors were included, 30 QTL were detected on chromosomes 1, 3, 4, 18, 19, 22, and 25, in addition to the 8 previously mentioned chromosomes. Some of the results from the cofactor analysis may be false positives and require further validation. In conclusion, we were able to map several QTL affecting fertility and calving traits in Swedish dairy cattle.

Characterization of bovine FAS-associated death domain gene.

The FAS-associated death domain (FADD) protein is an adapter/signaling molecule that has been shown to function in human cells to promote apoptosis and to inhibit NF-kappaB activation. Because of the critical role that apoptosis and NF-kappaB play in a variety of disease states, we mapped the bovine FADD gene, sequenced bovine FADD cDNA, and characterized its expression in endothelial cells (EC). Sequencing of bovine FADD revealed approximately 65 and 58% amino acid sequence identity to its human and murine homologues, respectively. Bovine FADD was mapped to chromosome 29 by radiation hybrid mapping. In addition, the functionality of bovine FADD was studied. Expression of a bovine FADD dominant-negative construct blocked bacterial lipopolysaccharide (LPS)- and TNF-alpha-induced apoptosis in bovine EC consistent with previous studies of human FADD. In contrast to human FADD, elevated expression of bovine FADD had no effect on LPS- or TNF-alpha-induced upregulation of NF-kappaB-dependent gene products as assayed by E-selectin expression. Thus, while the role of FADD in mediating apoptosis is conserved across species, its role in regulating NF-kappaB-dependent gene expression is not.

The telomeric region of BTA18 containing a potential QTL region for health in cattle exhibits high similarity to the HSA19q region in humans.

We have applied a targeted physical mapping approach, based on the isolation of bovine region-specific large-insert clones using homologous human sequences and chromosome microdissection, to enhance the physical gene map of the telomeric region of BTA18 and to prove its evolutionary conservation. The latter is a prerequisite to exploit the dense human gene map for future positional cloning approaches. Partial sequencing and homology search were used to characterize 20 BACs targeted to the BTA18q2.4-q2.6 region. We used fluorescence in situ hybridization (FISH) to create physical maps of 11 BACs containing 15 gene loci; these BACs served as anchor loci. Using these approaches, 12 new gene loci (CKM, STK13, PSCD2, IRF3, VASP, ACTN4, ITPKC, CYP2B6, FOSB, DMPK, MIA, SIX5) were assigned on BTA18 in the bovine cytogenetic map. A resolved physical map of BTA18q2.4-q2.6 was developed, which encompasses 28 marker loci and a comparative cytogenetic map that contains 15 genes. The mapping results demonstrate the high evolutionary conservation between the telomeric region of BTA18q and HSA19q.

Mapping of 195 genes in cattle and updated comparative map with man, mouse, rat and pig.

Our on-going goal is to improve and update the comparative genome organization between cattle and man but also among the most detailed mammalian species genomes i.e. cattle, mouse, rat and pig. In this work, we localized 195 genes in cattle and checked all human/bovine non-concordant localizations found in the literature. Next, we compiled all the genes mapped in cattle, goat, sheep and pig (2,166) for which the human ortholog with its chromosomal position is known, added corresponding data in mouse and rat, and ordered the genes relatively to the human genome sequence. We estimate that our compilation provides bovine mapping information for about 89% of the human autosomes. Thus, a near complete, overall and detailed picture of the number, distribution and extent of bovine conserved syntenies (regardless of gene order) on human R-banded autosomes is proposed as well as a comparison with mouse, rat and pig genomes.

A comparative map of bovine chromosome 19 based on a combination of mapping on a bacterial artificial chromosome scaffold map, a whole genome radiation hybrid panel and the human draft sequence.

We have constructed a medium density physical map of bovine chromosome 19 using a combination of mapping loci on both a bovine bacterial artificial chromosome (BAC) scaffold map and a whole genome radiation hybrid (WGRH) panel. The resulting map contains 70 loci spanning the length of bovine chromosome 19. Three contiguous groups of BACs were identified on the basis of multiple loci mapping to individual BAC clones. Bovine chromosome 19 was found in this study to be comprised almost entirely from regions of human chromosome 17, with a small region putatively assigned to human chromosome 10. Fourteen breakpoints between the bovine and human chromosomes were detected, with a possibility of five more based on ordering of the WGRH map.

Genomic organization of the DGAT2/MOGAT gene family in cattle (Bos taurus) and other mammals.

We report the cloning and initial characterization of the genes encoding DGAT2 (diacylglycerol transferase 2), MOGAT1 and MOGAT2 (monoacylglycerol transferases 1 and 2) in domestic cattle (Bos taurus). The three closely related genes belong to a gene family with at least eight members in mammals and are candidate genes for quantitative traits related to dietary fat uptake, lipid synthesis and storage. MOGAT2 and DGAT2 form a tandem and were mapped to bovine chromosome (BTA) 15q25-->q26 by fluorescence in situ hybridization. MOGAT1 was localized to BTA 2q43-->q44. The three genes were investigated for polymorphisms that might be associated with breeding values for milk fat percentage in the dairy breeds German Holstein, German Simmental and German Brown. All the detected polymorphisms were located outside exons or, with one exception, were silent. In MOGAT1, a missense mutation in exon 4 was found that causes a non-conservative substitution of cysteine170 (uncharged, hydrophobic) by lysine (positively charged, hydrophilic). However, allele frequency estimates from pooled DNA samples revealed no significant association of the observed polymorphisms with breeding values for milk fat percentage. A comparative analysis of chromosomal locations and exon-intron structure of the known members of the DGAT2/MOGAT gene family in humans, rodents and cattle indicates an ancient tandem duplication of the ancestor gene combined with an intron gain (or loss) in one copy. Further members of the family may have arisen by duplications of this gene tandem via two rounds of interchromosomal or genome duplications as well as further local (single) gene duplication and loss events.

Bovine Y chromosome microsatellite polymorphisms.

Thirty-eight bovine Y chromosome (BTAY) microsatellites (MS) were assessed for polymorphisms in DNA samples obtained from 17 unrelated bulls. Thirty-three of these microsatellites are new and were used for the construction of a first generation radiation hybrid map for BTAY (Liu et al., 2002). Five MS had been previously reported and were used as positive controls. Fourteen out of 38 MS were found to be polymorphic; the remaining 24 were uninformative among the animals tested. The number of hemizygous loci per MS within individual ranged from two to over 20. Seven MS presented smear- or ladder-like bands, a unique feature for Y chromosome multi-copy hemizygous MS loci. The locus length variance, within individual, ranged from 2 to 42 bp corresponding to the MS with the minimum and maximum number of loci observed, respectively. Within the 14 polymorphic MS, the five pseudoautosomal MS, on average, were more polymorphic (35.3%) than the nine Y-specific MS (19.6%). Haplotypes resulting from combinations of these polymorphic loci will provide a powerful tool for future studies on the origin of domestic cattle and the evolution of bovid species.

Construction of a high-resolution comparative gene map between swine chromosome region 6q11-->q21 and human chromosome 19 q-arm by RH mapping of 51 genes.

A comprehensive and comparative map was constructed for the porcine chromosome (SSC) 6q11-->q21 region, where the gene(s) responsible for the maldevelopment of embryos are localized using swine populations of the National Institute of Animal Industry, Japan (NIAI). Since the chromosomal region corresponds to a region of human chromosome (HSA) 19q13.1-->q13.3 based on bi-directional chromosome painting, primer pairs were designed from porcine cDNA sequences identified, on a sequence comparison basis, as being transcripts from genes orthologous to those in the HSA region. Fifty-one genes were successfully assigned to a swine radiation hybrid (RH) map with LOD scores greater than 6. ERF and PSMD8 genes were assigned to SSC4 and SSC1, respectively. The remaining 49 genes were assigned to SSC6, demonstrating that the synteny between the SSC6 and HSA19 chromosomal regions is essentially conserved, therefore confirming, the results of bi-directional chromosome painting. However, when examined precisely, rearrangements have apparently occurred within the region of conserved synteny. For the ERF and PSMD8 genes assigned to SSCs other than SSC6, additional mapping using somatic cell hybrid (SCH) panels was performed to confirm the results of RH-mapping.

Improving the comparative map of porcine chromosome 10 with respect to human chromosomes 1, 9 and 10.

ZOO-FISH mapping shows human chromosomes 1, 9 and 10 share regions of homology with pig chromosome 10 (SSC10). A more refined comparative map of SSC10 has been developed to help identify positional candidate genes for QTL on SSC10 from human genome sequence. Genes from relevant chromosomal regions of the public human genome sequence were used to BLAST porcine EST databases. Primers were designed from the matching porcine ESTs to assign them to porcine chromosomes using the INRA somatic cell hybrid panel (INRA-SCHP) and the INRA-University of Minnesota Radiation Hybrid Panel (IMpRH). Twenty-eight genes from HSA1, 9 and 10 were physically mapped: fifteen to SSC10 (ACO1, ATP5C1, BMI1, CYB5R1, DCTN3, DNAJA1, EPHX1, GALT, GDI2, HSPC177, OPRS1, NUDT2, PHYH, RGS2, VIM), eleven to SSC1 (ADFP, ALDHIB1, CLTA, CMG1, HARC, PLAA, STOML2, RRP40, TESK1, VCP and VLDLR) and two to SSC4 (ALDH9A1 and TNRC4). Two anonymous markers were also physically mapped to SSC10 (SWR1849 and S0070) to better connect the physical and linkage maps. These assignments have further refined the comparative map between SSC1, 4 and 10 and HSA1, 9 and 10.

Generation of a 5.5-Mb BAC/PAC contig of pig chromosome 6q1.2 and its integration with existing RH, genetic and comparative maps.

We generated a sequence-ready BAC/PAC contig spanning approximately 5.5 Mb on porcine chromosome 6q1.2, which represents a very gene-rich genome region. STS content mapping was used as the main strategy for the assembly of the contig and a total of 6 microsatellite markers, 53 gene-related STS and 116 STS corresponding to BAC and PAC end sequences were analyzed. The contig comprises 316 BAC and PAC clones covering the region between the genes GPI and LIPE. The correct contig assembly was verified by RH-mapping of STS markers and comparative mapping of BAC/PAC end sequences using BLAST searches. The use of microsatellite primer pairs allowed the integration of the physical maps with the genetic map of this region. Comparative mapping of the porcine BAC/PAC contig with respect to the gene-rich region on the human chromosome 19q13.1 map revealed a completely conserved gene order of this segment, however, physical distances differ somewhat between HSA19q13.1 and SSC6q1.2. Three major differences in DNA content between human and pig are found in two large intergenic regions and in one region of a clustered gene family, respectively. While there is a complete conservation of gene order between pig and human, the comparative analysis with respect to the rodent species mouse and rat shows one breakpoint where a genome segment is inverted.

Improving the comparative map of porcine chromosome 9 with respect to human chromosomes 1, 7 and 11.

Conserved segments have been identified by ZOO-FISH between pig chromosome 9 (SSC9) and human chromosomes 1, 7 and 11. To assist in the identification of positional candidate genes for QTL on SSC9, the comparative map was further developed. Primers were designed from porcine EST sequence homologous to genes in regions of human chromosomes 1, 7 and 11. Porcine ESTs were then physically assigned using the INRA somatic cell hybrid panel (INRASCHP) and the high-resolution radiation hybrid panel (IMpRH). Seventeen genes (PEPP3, RAB7L1, FNBP2, MAPKAPK2, GNAI1, ABCB1, STEAP, AKAP9, CYP51A1, SGCE, ROBO4, SIAT4C, GLUL, CACNA1E, PTGS2, C1orf16 and ETS1) were mapped to SSC9, while GUSB, CPSF4 and THG-1 were assigned to SSC3.

A refined comparative map between porcine chromosome 13 and human chromosome 3.

We report here the localisation of BAIAP1 (13q24), HTR1F (13q45), PTPRG (13q23) and UBE1C (13q24) by fluorescence in situ hybridisation (FISH), and BAIAP1 (Swr2114; 21 cR; LOD = 11.03), GATA2 (Sw2448; 37 cR; LOD = 8.26), IL5RA (Swr2114; 64 cR; LOD = 3.85), LMCD1 (Sw2450; 61 cR; LOD = 4.73), MME (CP; 50 cR; LOD = 7.75), RYK (Swc22; 12 cR; LOD = 18.62) and SGU003 (Sw1876; 6 cR; LOD = 16.99) by radiation hybrid (RH) mapping to porcine chromosome 13 (SSC13). The mapping of these 10 different loci (all mapped to human chromosome 3; HSA3) not only confirms the extended conservation of synteny between HSA3 and SSC13, but also defines more precisely the regions with conserved linkage. The syntenic region of the centromeric part of SSC13 was determined by isolating porcine bacterial artificial chromosome (BAC) clones (842D4 and 1031H1) using primers amplifying porcine microsatellite markers S0219 and S0076 (mapped to this region). Sequence comparison of the BAC end sequences with the human genome sequence showed that the centromeric part of SSC13 is homologous with HSA3p24.

Study of candidate genes for glycolytic potential of porcine skeletal muscle: identification and analysis of mutations, linkage and physical mapping and association with meat quality traits in pigs.

Several genes (PRKAA2, PRKAB1, PRKAB2, PRKAG3, GAA, GYS1, PYGM, ALDOA, GPI, LDHA, PGAM2 and PKM2), chosen according to their role in the regulation of the energy balance and in the glycogen metabolism and glycolysis of the skeletal muscle, were studied. Eleven single nucleotide polymorphisms (SNPs) were identified in six of these genes (PRKAB1, GAA, PYGM, LDHA, PGAM2 and PKM2). Allele frequencies were analyzed in seven different pig breeds for these loci and for a polymorphism already described for GPI and for three polymorphic sites already reported at the PRKAG3 locus (T30N, G52S and I199V). Linkage mapping assigned PYGM and LDHA to porcine chromosome (SSC) 2, PKM2 to SSC7, GAA to SSC12, PRKAB1 to SSC14 and PGAM2 to SSC18. Physical mapping, obtained by somatic cell hybrid panel analysis, confirmed the linkage assignments of PRKAB1 and GAA and localized ALDOA, PRKAB2 and GYS1 to SSC3, SSC4 and SSC6, respectively. Pigs selected for the association study, for which several meat quality traits were measured, were first genotyped at the PRKAG3 R200Q polymorphic site (RN locus), in order to exclude carriers of the 200Q allele, and then were genotyped for all the mutations considered in this work. Significant associations (P < or = 0.001) were observed for the PRKAG3 T30N and G52S polymorphic sites with meat colour (L* at 24 h post mortem). PGAM2 and PKM2 were significantly associated (P = 0.01) with drip loss percentage and glycogen content at one hour post mortem, respectively.