A 4 Mb High Resolution BAC Contig on Bovine Chromosome 1q12 and Comparative Analysis With Human Chromosome 21q22

The bovine RPCI-42 BAC library was screened to construct a sequence-ready ~4 Mb single contig of 92 BAC clones on BTA 1q12. The contig covers the region between the genes KRTAP8P1 and CLIC6. This genomic segment in cattle is of special interest as it contains the dominant gene responsible for the hornless or polled phenotype in cattle. The construction of the BAC contig was initiated by screening the bovine BAC library with heterologous cDNA probes derived from 12 human genes of the syntenic region on HSA 21q22. Contig building was facilitated by BAC end sequencing and chromosome walking. During the construction of the contig, 165 BAC end sequences and 109 single-copy STS markers were generated. For comparative mapping of 25 HSA 21q22 genes, genomic PCR primers were designed from bovine EST sequences and the gene-associated STSs mapped on the contig. Furthermore, bovine BAC end sequence comparisons against the human genome sequence revealed significant matches to HSA 21q22 and allowed the in silico mapping of two new genes in cattle. In total, 31 orthologues of human genes located on HSA 21q22 were directly mapped within the bovine BAC contig, of which 16 genes have been cloned and mapped for the first time in cattle. In contrast to the existing comparative bovine–human RH maps of this region, these results provide a better alignment and reveal a completely conserved gene order in this 4 Mb segment between cattle, human and mouse. The mapping of known polled linked BTA 1q12 microsatellite markers allowed the integration of the physical contig map with existing linkage maps of this region and also determined the exact order of these markers for the first time. Our physical map and transcript map may be useful for positional cloning of the putative polled gene in cattle. The nucleotide sequence data reported in this paper have been submitted to EMBL and have been assigned Accession Numbers AJ698510–AJ698674

A Frameshift Mutation within LAMC2 Is Responsible for Herlitz Type Junctional Epidermolysis Bullosa (HJEB) in Black Headed Mutton Sheep

Junctional epidermolysis bullosa (JEB) is a hereditary mechanobullous skin disease in humans and animals. A Herlitz type JEB was identified in German Black Headed Mutton (BHM) sheep and affected lambs were reproduced in a breeding trial. Affected lambs showed skin and mucous membranes blistering and all affected lambs died within the first weeks of life. The pedigree data were consistent with a monogenic autosomal recessive inheritance. Immunofluorescence showed a reduced expression of laminin 5 protein which consists of 3 subunits encoded by the genes LAMA3, LAMB3 and LAMC2. We screened these genes for polymorphisms. Linkage and genome-wide association analyses identified LAMC2 as the most likely candidate for HJEB. A two base pair deletion within exon 18 of the LAMC2 gene (FM872310:c.2746delCA) causes a frameshift mutation resulting in a premature stop codon (p.A928*) 13 triplets downstream of this mutation and in addition, introduces an alternative splicing of exon 18 LAMC2. This deletion showed a perfect co-segregation with HJEB in all 740 analysed BHM sheep. Identification of the LAMC2 deletion means an animal model for HJEB is now available to develop therapeutic approaches of relevance to the human form of this disease

A One Base Pair Deletion in the Canine ATP13A2 Gene Causes Exon Skipping and Late-Onset Neuronal Ceroid Lipofuscinosis in the Tibetan Terrier

Neuronal ceroid lipofuscinosis (NCL) is a progressive neurodegenerative disease characterized by brain and retinal atrophy and the intracellular accumulation of autofluorescent lysosomal storage bodies resembling lipofuscin in neurons and other cells. Tibetan terriers show a late-onset lethal form of NCL manifesting first visible signs at 5–7 years of age. Genome-wide association analyses for 12 Tibetan-terrier-NCL-cases and 7 Tibetan-terrier controls using the 127K canine Affymetrix SNP chip and mixed model analysis mapped NCL to dog chromosome (CFA) 2 at 83.71–84.72 Mb. Multipoint linkage and association analyses in 376 Tibetan terriers confirmed this genomic region on CFA2. A mutation analysis for 14 positional candidate genes in two NCL-cases and one control revealed a strongly associated single nucleotide polymorphism (SNP) in the MAPK PM20/PM21 gene and a perfectly with NCL associated single base pair deletion (c.1620delG) within exon 16 of the ATP13A2 gene. The c.1620delG mutation in ATP13A2 causes skipping of exon 16 presumably due to a broken exonic splicing enhancer motif. As a result of this mutation, ATP13A2 lacks 69 amino acids. All known 24 NCL cases were homozygous for this deletion and all obligate 35 NCL-carriers were heterozygous. In a sample of 144 dogs from eleven other breeds, the c.1620delG mutation could not be found. Knowledge of the causative mutation for late-onset NCL in Tibetan terrier allows genetic testing of these dogs to avoid matings of carrier animals. ATP13A2 mutations have been described in familial Parkinson syndrome (PARK9). Tibetan terriers with these mutations provide a valuable model for a PARK9-linked disease and possibly for manganese toxicity in synucleinopathies

Impact of wet-lab protocols on quality of whole-genome short-read sequences from foodborne microbial pathogens

For successful elucidation of a food-borne infection chain, the availability of high-quality sequencing data from suspected microbial contaminants is a prerequisite. Commonly, those investigations are a joint effort undertaken by different laboratories and institutes. To analyze the extent of variability introduced by differing wet-lab procedures on the quality of the sequence data we conducted an interlaboratory study, involving four bacterial pathogens, which account for the majority of food-related bacterial infections: Campylobacter spp., Shiga toxin-producing Escherichia coli, Listeria monocytogenes, and Salmonella enterica. The participants, ranging from German federal research institutes, federal state laboratories to universities and companies, were asked to follow their routine in-house protocols for short-read sequencing of 10 cultures and one isolated bacterial DNA per species. Sequence and assembly quality were then analyzed centrally. Variations within isolate samples were detected with SNP and cgMLST calling. Overall, we found that the quality of Illumina raw sequence data was high with little overall variability, with one exception, attributed to a specific library preparation kit. The variability of Ion Torrent data was higher, independent of the investigated species. For cgMLST and SNP analysis results, we found that technological sequencing artefacts could be reduced by the use of filters, and that SNP analysis was more suited than cgMLST to compare data of different contributors. Regarding the four species, a minority of Campylobacter isolate data showed the in comparison highest divergence with regard to sequence type and cgMLST analysis. We additionally compared the assembler SPAdes and SKESA for their performance on the Illumina data sets of the different species and library preparation methods and found overall similar assembly quality metrics and cgMLST statistics

Molecular characterization and chromosomal assignment of the bovine glycinamide ribonucleotide formyltransferase (GART) gene on cattle chromosome 1q12.1-q12.2

The mammalian glycinamide ribonucleotide formyltransferase (GART) genes encode a trifunctional polypeptide involved in the de novo purine biosynthesis. We isolated a bacterial artificial chromosome (BAC) clone containing the bovine GART gene and determined the complete DNA sequence of the BAC clone. Cloning and characterization of the bovine GART gene revealed that the bovine gene consists of 23 exons spanning approximately 27 kb. RT-PCR amplification of bovine GART in different organs showed the expression of two GART transcripts in cattle similar to human and mouse. The GART transcripts encode two proteins of 1010 and 433 amino acids, respectively. Eleven single nucleotide polymorphisms (SNPs) were detected in a mutation scan of 24 unrelated animals of three different cattle breeds, including one SNP that affects the amino acid sequence of GART. The chromosomal localization of the gene was determined by fluorescence in situ hybridization. Comparative genome analysis between cattle, human and mouse indicates that the chromosomal location of the bovine GART gene is in agreement with a previously published mapping report

A Single Nucleotide Polymorphism within the <i>Interferon Gamma Receptor 2</i> Gene Perfectly Coincides with Polledness in Holstein Cattle

<div><p>Polledness is a high impact trait in modern milk and beef production to meet the demands of animal welfare and work safety. Previous studies have mapped the polled-locus to the proximal region of the bovine chromosome 1 (BTA1) and narrowed it down to approximately 1 Mb. Sequencing of the positional candidate genes within the 1 Mb polled region and whole genome sequencing of Holsteins revealed a single nucleotide polymorphism (SNP) <i>AC000158: g.1390292G>A</i> within intron 3 of the <i>interferon gamma</i> receptor <i>2</i> gene (<i>IFNGR2</i>) in perfect co-segregation with polledness in Holsteins. This complete association was validated in 443 animals of the same breed. This SNP allows reliable genotyping of horned, heterozygous and homozygous polled Holsteins, even in animals that could not be resolved using the previously published haplotype for Holstein.</p> </div

Gene structure of <i>IFNGR2</i>.

<p>Rectangles indicate exons. Coding sequence is filled with black, the 3’ UTR is filled with white. The size of exons and introns is specified in number of base pairs (bp). The black line below illustrates the sections analysed by PCR in Holsteins. Each PCR-product is pictured separately with start and end position. Position of start and stop codon is stated in bp. All detected exonic polymorphisms and intronic polymorphisms showing association with polledness in Holsteins are given with name and position above the gene model. Each position is given with accordance to Bos taurus assembly UMD3.1.</p