Endangered Pinzgauer cattle subtype Jochberger Hummel are genetically distinct.

Autochthonous cattle breeds are an important part of cultural heritage and reservoir of genetic diversity. Usually, such breeds have been selected over centuries and reflect adaptation to a specific local environment and human demands. However, owing to low effective population size in conjunction with increased inbreeding and genetic drift, many of these lineages are threatened with extinction. The Jochberger Hummel ('Jochberger Bumblebee') is such an endangered subtype of the Pinzgauer cattle originating from a single polled female calf. To evaluate the suspected uniqueness of this subtype and to assess whether it should be kept separate or managed together with the Pinzgauer cattle as one population, I examined the genetic diversity in a set of 844 cattle (Angus, Charolais, Holstein, Jochberger Hummel, Pinzgauer, Uckermaerker and Tux-Zillertaler) using principal component and biogeographical ancestry analysis. The analysis showed that the Jochberger Hummel is still a distinct subtype of Pinzgauer cattle with less than 5% admixture and a low inbreeding coefficient.

Suitability of ultrasound-guided fine-needle aspiration biopsy for transcriptome sequencing of the canine prostate.

Ultrasound-guided fine-needle aspiration (US-FNA) biopsy is a widely used minimally invasive sampling procedure for cytological diagnosis. This study investigates the feasibility of using US-FNA samples for both cytological diagnosis and whole transcriptome RNA-sequencing analysis (RNA-Seq), with the ultimate aim of improving canine prostate cancer management. The feasibility of the US-FNA procedure was evaluated intra vitam on 43 dogs. Additionally, aspirates from 31 euthanised dogs were collected for standardising the procedure. Each aspirate was separated into two subsamples: for cytology and RNA extraction. Additional prostate tissue samples served as control for RNA quantity and quality evaluation, and differential expression analysis. The US-FNA sampling procedure was feasible in 95% of dogs. RNA isolation of US-FNA samples was successfully performed using phenol-chloroform extraction. The extracted RNA of 56% of a subset of US-FNA samples met the quality requirements for RNA-Seq. Expression analysis revealed that only 153 genes were exclusively differentially expressed between non-malignant US-FNAs and tissues. Moreover, only 36 differentially expressed genes were associated with the US-FNA sampling technique and unrelated to the diagnosis. Furthermore, the gene expression profiles clearly distinguished between non-malignant and malignant samples. This proves US-FNA to be useful for molecular profiling.

Species identification and quantification in meat and meat products using droplet digital PCR (ddPCR).

Species fraud and product mislabelling in processed food, albeit not being a direct health issue, often results in consumer distrust. Therefore methods for quantification of undeclared species are needed. Targeting mitochondrial DNA, e.g. CYTB gene, for species quantification is unsuitable, due to a fivefold inter-tissue variation in mtDNA content per cell resulting in either an under- (-70%) or overestimation (+160%) of species DNA contents. Here, we describe a reliable two-step droplet digital PCR (ddPCR) assay targeting the nuclear F2 gene for precise quantification of cattle, horse, and pig in processed meat products. The ddPCR assay is advantageous over qPCR showing a limit of quantification (LOQ) and detection (LOD) in different meat products of 0.01% and 0.001%, respectively. The specificity was verified in 14 different species. Hence, determining F2 in food by ddPCR can be recommended for quality assurance and control in production systems.

A study based on records taken at time of hoof trimming reveals a strong association between the IQ motif-containing GTPase-activating protein 1 (IQGAP1) gene and sole hemorrhage in Holstein cattle.

Feet and leg problems have a major effect on the well-being and lifespan of the dairy cow and thus are economically important to the dairy farmer. Apart from approaches using genetic selection for classical traits from conformation scoring, attempts for genetic improvement can be based either on records of individual disease cases or on records of disorder status at time of hoof trimming. In this study, 1,962 first-lactation cows were subjected to hoof trimming with an assessment of disorder status for sole hemorrhage as a binary trait. Cows were from 7 large commercial herds in Mecklenburg-Western Pomerania (northeastern Germany) that had similar housing with cubicles, slatted flooring, little use of straw for bedding, and total mixed ration feeding. Cows were trimmed and assessed once, focusing on cows in the first half of the lactation. Herds were visited at intervals to enable recording of cohorts at a similar stage of lactation. Each cohort or herd-visit included between 31 and 165 cows. Additional measurements included body weight, back fat thickness, and body condition at time of trimming. Further data on dairy production, conformation scores, and reproductive performance were merged after collection of records had finished. The DNA extracted from blood of 1,183 cows was used for analysis with a custom-made array of 384 single nucleotide polymorphisms (SNP). The SNP were selected according to results from the literature for effects in classical conformation traits, from biochemical pathway analysis, and from comparative analysis of putative candidate genes in cattle, pigs, and sheep. Selection of cohorts of cows for SNP chip analysis was such that cohorts with extreme frequencies of disorders and cohorts with slightly deviating housing systems were excluded in this first step. The results from a mixed threshold model analysis with genotype included as a fixed effect and accounting for relationships among animals revealed that the intronic SNP rs29017173 (A/G) within the IQ motif-containing GTPase-activating protein 1 (IQGAP1, Bos taurus autosome 21) was significantly associated with disorder status. Back-transformed means of disorder status for the 3 genotypes were 0.37 (AA), 0.52 (AG), and 0.56 (GG). Using the full data set of 1,962 cows, including the less-suitable cohorts, gave back-transformed means of 0.51 (AA), 0.58 (AG), and 0.62 (GG). As SNP rs29017173 is included on the Illumina BovineSNP50 DNA Analysis BeadChip (Illumina Inc., San Diego, CA), a sample of 2,394 artificial insemination sires from the German calibration sample for genomic selection from birth years 1998 to 2003 was studied for possible correlated effects. The A/G polymorphism of SNP rs29017173 studied here was also associated with substantial effects for feet and leg traits from the classical conformation score system. Selection using this polymorphism will be facilitated by the fact that the same allele is favored for all traits with substantial effects.

Molecular genetics of coat colour variations in White Galloway and White Park cattle.

White Galloway cattle exhibit three different white coat colour phenotypes, that is, well marked, strongly marked and mismarked. However, mating of individuals with the preferred well or strongly marked phenotype also results in offspring with the undesired mismarked and/or even fully black coat colour. To elucidate the genetic background of the coat colour variations in White Galloway cattle, we analysed four coat colour relevant genes: mast/stem cell growth factor receptor (KIT), KIT ligand (KITLG), melanocortin 1 receptor (MC1R) and tyrosinase (TYR). Here, we show that the coat colour variations in White Galloway cattle and White Park cattle are caused by a KIT gene (chromosome 6) duplication and aberrant insertion on chromosome 29 (Cs29 ) as recently described for colour-sided Belgian Blue. Homozygous (Cs29 /Cs29 ) White Galloway cattle and White Park cattle exhibit the mismarked phenotype, whereas heterozygous (Cs29 /wt29 ) individuals are either well or strongly marked. In contrast, fully black individuals are characterised by the wild-type chromosome 29. As known for other cattle breeds, mutations in the MC1R gene determine the red colouring. Our data suggest that the white coat colour variations in White Galloway cattle and White Park cattle are caused by a dose-dependent effect based on the ploidy of aberrant insertions and inheritance of the KIT gene on chromosome 29.

Polymorphism and gene organization of water buffalo MHC-DQB genes show homology to the BoLA DQB region.

In cattle (Bos taurus), there is evidence of more than 50 alleles of BoLA-DQB (bovine lymphocyte antigen DQB) that are distributed across at least five DQB loci, making this region one of the most complex in the BoLA gene family. In this study, DQB alleles were analysed for the water buffalo (Bubalus bubalis), another economically important bovine species. Twelve alleles for Bubu-DQB (Bubalis bubalis DQB) were determined by nucleotide sequence analysis. A phylogenetic analysis revealed numerous trans-species polymorphisms, with alleles from water buffalo assigned to at least three different loci (BoLA-DQB1, BoLA-DQB3 and BoLA-DQB4) that are also found in cattle. These presumptive loci were analysed for patterns of synonymous (d(S)) and non-synonymous (d(N)) substitution. Like BoLA-DQB1, Bubu-DQB1 was observed to be under strong positive selection for polymorphism. We conclude that water buffalo and cattle share the current arrangement of their DQB region because of their common ancestry.

Further resolution of porcine phylogeny in Southeast Asia by Thai mtDNA haplotypes.

The mtDNA control region of 72 Thai native pigs and 11 Thai wild boars indigenous to Northern Thailand was comparatively sequenced. In total, 36 nucleotide variations that accounted for 24 haplotypes have been described (TNH01 to TNH20 and TWH01 to TWH04). These haplotypes and further publicly available mtDNA haplotypes were used to assess phylogenetic relationships. Twenty-three of the 24 haplotypes became integrated into the Asian clade of the phylogenetic tree and eight of them recapitulated another major cluster of haplotypes within this clade (Thai haplogroup, THG). Only haplotype TNH01 fit in with the European clade of the phylogenetic tree. An additional analysis using 510 bp of the mtDNA incorporated the THG haplotypes in to clade MTSEA (mountainous and Southeast Asian distribution) to form haplogroup MTSEA-THG. Recently, MTSEA was renamed in MC3. MC3 contains only signatures of pigs scattered across the Indo-Burma Biodiversity Hotspot (IBBH), a region including Thailand to the Kra Isthmus. Here we propose a putative independent porcine domestication event in South-east Asia (SEA). All haplotypes of haplogroup MTSEA-THG have revealed unique and previously unknown nucleotide signatures at positions 24 (nucleotide A) and at positions 183 (nucleotide C) that differentiate them from all other porcine mtDNA haplotypes.

Shotgun metagenomics of biological stains using ultra-deep DNA sequencing.

A detailed molecular analysis of blood or other biological stains at a crime scene is often hampered by the low quantity and quality of the extractable DNA. However, the determination of the origin and composition of a stain is in most cases a prerequisite for the final elucidation of a criminal case. Standard methodologies, e.g. amplification of DNA followed by microsatellite typing or mitochondrial DNA sequencing, are often not sensitive enough to result in sufficient and conclusive data. We have applied ultra-deep DNA sequencing using the 454 pyrosequencing technology on a whole genome amplified (WGA) environmental biological stain, which was analysed unsuccessfully with standard methodologies following WGA. With the combination of WGA and 454 pyrosequencing, however, we were able to generate 7242 single sequences with an average length of 195bp. A total of 1,441,971bp DNA sequences were generated and compared with public DNA sequence databases. Using RepeatMasker and basic logical alignment search tool (BLAST) searches against known microbial and mammalian genomes it was possible to determine the metagenomic composition of the stain, i.e. 4.2% bacterial DNA, 0.3% viral DNA, 2.7% fungal DNA, 10.3% mammalian repetitive DNA, 0.9% porcine DNA, 0.13% human DNA and 81.5% DNA of unknown origin. Our data demonstrate that 454 pyrosequencing has the potential to become a powerful tool not only in basic research but also in the metagenomic analysis of biological trace materials for forensic genetics.

Molecular analysis of carbohydrate N-acetylgalactosamine 4-O sulfotransferase 8 (CHST8) as a candidate gene for bovine spongiform encephalopathy susceptibility.

Endogenous prion proteins (PrP) play the central role in the pathogenesis of transmissible spongiform encephalopathies. The carbohydrate N-acetylgalactosamine 4-O sulfotransferase 8 (CHST8) promotes the conversion of the cellular PrP(C) into the pathogenic PrP(d). Six sequence variants within the CHST8 gene were identified by comparative sequencing and genotyped for a sample of 623 animals comprising bovine spongiform encephalopathy (BSE)-affected and healthy control cows representing German Fleckvieh (German Simmental), German Holstein (Holstein-Friesian) and Brown Swiss. Significant differences in the allele, genotype and haplotype frequencies between BSE-affected and healthy cows indicate an association of sequence variant g.37254017G>T with the development of the disease in Brown Swiss cattle.

Detection of classical and atypical/Nor98 scrapie by the paraffin-embedded tissue blot method.

The paraffin-embedded tissue (PET) blot method was used to investigate sections of the central nervous system and lymphatic tissues from 24 cases of classical scrapie and 25 cases of atypical/Nor98 scrapie in sheep and four healthy control sheep. The PET blot detected deposits of PrP(Sc) in the brain tissue of all 49 sheep with scrapie but no PrP(Sc) labelling could be detected in the control sheep. By contrast, not all the atypical/Nor98 scrapie cases were detectable by immunohistochemistry. The high sensitivity of the PET blot method made it possible to observe that in some atypical/Nor98 cases, deposits of PrP(Sc) may be restricted to supratentorial brain structures and that the diagnosis may be missed when only testing the obex area, where deposits are common in classical scrapie, and the cerebellar structures, where deposits are considered to be common in atypical/Nor98 cases.

A genome-wide scan reveals candidate susceptibility loci for pig hernias in an intercross between White Duroc and Erhualian.

Pig scrotal/inguinal and umbilical hernias are the most prevalent congenital disorders in pigs and often cause animal welfare problems and economic loss. To identify susceptibility loci for these traits, a genome-wide scan with 194 microsatellite markers covering the pig genome was performed in a White Duroc x Erhualian resource population with 23 scrotal/inguinal F(2) animals, 50 umbilical F(2) animals, and their unaffected siblings. A sex-average linkage map with a total length of 2,350.3 cM and an average marker interval of 12.84 cM was constructed. Both nonparametric genome-wide linkage (NPL) analysis and transmission disequilibrium test (TDT) were implemented to detect closely linked markers. The NPL analysis revealed 11 chromosomal regions on SSC1, 2, 3, 6, 7, 8, 10, and 11 for umbilical hernia and 5 regions on SSC2, 4, 8, 13, and 16 for scrotal/inguinal hernia, whereas the TDT test identified susceptibility loci for umbilical hernia on SSC1, 2, 4, 7, 10, 13, 14, and 15 and for scrotal/inguinal hernias on SSC2, 8, 10, and 18. The most promising loci were SWR1928 on SSC7 and SW830 on SSC10 for umbilical hernia, and SW933 on SSC8 for scrotal hernia, which were consistently detected by both NPL and TDT. Several previously reported chromosomal regions for scrotal/inguinal hernia were confirmed, and new evidence for linkage with this pig defect was found. Moreover, susceptibility loci for pig umbilical hernia were detected for the first time.

Tissue calcium content in piglets with inguinal or scrotal hernias or cryptorchidism.

Scrotal and inguinal hernias are of great economic importance to the pig industry. These lesions are thought to result from incomplete closure of the inguinal ring and/or a patent processus vaginalis. Impairment of programmed cell death (PCD) may be involved in these abnormalities. As tissue Ca(2+) overload may be used as a measure of cell death, the aim of this study was to determine the tissue Ca(2+) content in samples of hernia sac, peritoneum, cremaster muscle and aqueous fluid from newborn piglets with scrotal or inguinal hernias (n=18) or cryptorchidism (n=18). Control samples from healthy piglets (n=20) were also evaluated. Tissue Ca(2+) content was determined by atomic absorption spectrophotometry. Significantly less Ca(2+) was found in the sacs (0.005 mg/g wt), peritoneal tissue (0.100 mg/g wt) and cremaster muscles (0.008 mg/g wt) of piglets with inguinal or scrotal hernias compared with control tissues (0.184, 0.144 and 0.048 mg/g wt for sacs, peritoneal tissue and cremaster muscles, respectively). These findings suggest that there may be perturbation of the apoptotic pathway in the urogenital tissues of affected piglets.

A linkage map of the porcine genome from a large-scale White Duroc x Erhualian resource population and evaluation of factors affecting recombination rates.

A porcine genome linkage map composed of 194 microsatellite markers was constructed with a large-scale White Duroc x Erhualian resource population. The marker order on this linkage map was consistent with the USDA-MARC reference map except for two markers on SSC3, two markers on SSC13 and two markers on SSCX. The length of the sex-averaged map (2344.9 cM) was nearly the same as that of the USDA-MARC and NIAI map. Highly significant heterogeneity in recombination rates between sexes was observed. Except for SSC1 and SSC13, the female autosomes had higher average recombination rates than the male autosomes. Moreover, recombination rates in the pseudoautosomal region were greater in males than in females. These observations are consistent with those of previous reports. The recombination rates on each paternal and maternal chromosome of F(2) animals were calculated. Recombination rates were not significantly affected by the age (in days) or parity of the F(1) animals. However, recombination rates on paternal chromosomes were affected by the mating season of the F(1) animals. This could represent an effect of environmental temperature on spermatogenesis.

A whole genome scan for quantitative trait loci for leg weakness and its related traits in a large F2 intercross population between White Duroc and Erhualian.

To detect QTL for leg weakness and its related traits in pigs, a total of 1,484 F(2) pigs were recorded for leg (at 76 and 213 d) and gait scores (at 153 and 223 d) in a White Duroc x Erhualian intercross. The length and weight of the biceps brachii muscle were measured after slaughter at 240 d. A genome scan was performed with 183 microsatellite markers in the population. A total of 42 QTL were detected, including 16 at the 1% genome-wide significant level and 6 at the 5% genome-wide significant level. Thirty-eight of the 42 QTL showed significant additive effects, and 14 had significant dominance effects. At least 2 QTL were detected for each trait except for leg score at 76 d, for which no QTL was identified. Some of the QTL for leg and gait scores confirmed previous findings. Eighteen QTL were detected for weight and length of the biceps brachii muscle. To our knowledge, this was the first report about QTL for weight and length of the biceps brachii muscle in pigs. Two chromosome regions each on SSC4 and SSC7 showed significant and multiple associations with both leg weakness and growth of the biceps brachii muscle, which are worthwhile for further investigation.

Implication of complex vertebral malformation and bovine leukocyte adhesion deficiency DNA-based testing on disease frequency in the Holstein population.

Two inherited lethal disorders, bovine leukocyte adhesion deficiency (BLAD) and complex vertebral malformation (CVM), play a major role in breeding of Holstein cattle. Both inherited diseases are based on single nucleotide polymorphisms that have been known for 12 and 7 yr, respectively. A total of 25,753 cattle were genotyped for BLAD (18,200 tests) and CVM (14,493 tests) in our laboratory since the beginning of the genotyping programs for these diseases. Based on founder effects, the CVM mutation is thought to be linked to milk production. The BLAD was genotyped using RFLP until 2001; then a fluorescence resonance energy transfer assay on a LightCycler was used, as for CVM genotyping. By using single nucleotide polymorphism-aided breeding, the allelic frequency of the BLAD and CVM mutations in the active sire population was reduced from 9.4% in 1997 to 0.3% in 2007 (BLAD) and from 8.3% in 2002 to 2.3% in 2007 (CVM), with calculated half-life of the mutant allele of 2.1 yr for BLAD and 3.6 yr for CVM. An observed increase of BLAD frequency in 1999 could be attributed to the massive use of a BLAD-positive sire tested falsely negative in another laboratory. These data show that marker-assisted selection is capable of substantially reducing the frequency of a mutation within a period of not more than 5 yr. The different selection strategies against the lethal recessive allele in CVM and BLAD are reflected in the different reduction rates of the specific allele frequencies.

Sequence analysis of the equine SLC26A2 gene locus on chromosome 14q15-->q21.

The solute carrier family 26, member 2 (SLC26A2) gene belongs to a family of multifunctional anion exchangers. Mutations in the human SLC26A2 gene are associated with autosomal recessively inherited chondrodysplasias. Hence, we postulate that the equine SLC26A2 could be a candidate gene for conformational traits in horses. An equine BAC clone harboring the SLC26A2 gene was isolated. The complete 142,625 bp insert sequence of this clone was determined by transposon sequencing. Together with the SLC26A2 gene the BAC clone contains four genes, i.e. the macrophage colony stimulating factor 1 receptor precursor (CSF1R), KIAA0194 protein gene similar to the SMF protein (KIAA0194), a tigger transposable element derived 14 (TIGD14), the 3'-5'-cyclic GMP phosphodiesterase alpha-chain (EC 3.1.4.35) and one unidentified open reading frame. The equine SLC26A2 gene encompassing 6,152 bp consists of two exons. The complete open reading frame of 2,211 bp encodes a protein of 736 amino acids. A comparison of the amino acid sequence with other mammalian orthologs revealed homologies with identity in a range between 80% and 88%. By contrast, the equine SLC26A2 protein lacks five C-terminal amino acids. Four single nucleotide polymorphisms (SNP) were identified (three synonymous and one non-synonymous variant Ser210Leu) in the coding region by comparative sequencing of 50 DNA samples representing the German Riding horse. Allele frequencies and distribution were further evaluated in a variety of different breeds: Arabians (for all four SNPs), Old Kladrub Horses, Draught Horses (including Westphalian Draught Horses, Rheinish Westphalian Draught Horses, Saxon-Thuringia Coldbloods, Altmarker Coldbloods), American Saddlebreds, Miniature Horses, Australian Riding Ponies, Appaloosa, Morgan Horses, and Lipizzaner for C629T (Ser210Leu) alone. No animal carrying the homozygous genotype TT has been detected. The overall frequency of the newly described variant T is low (between 2% and 6%). Simulation studies on the protein conformation predict structural protein changes mediated by the SNP.