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.

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.

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.

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.

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.

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.

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.

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.

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.

FISH-mapping of LEP and SLC26A2 genes in sheep, goat and cattle R-banded chromosomes: comparison between bovine, ovine and caprine chromosome 4 (BTA4/OAR4/CHI4) and human chromosome 7 (HSA7).

Sheep (OAR), goat (CHI) and cattle (BTA) R-banded chromosome preparations, obtained from synchronized cell cultures, were used to FISH-map leptin (LEP) and solute carrier family 26 member 2 (SLC26A2) genes on single chromosome bands. LEP maps on OAR4q32 and CHI4q32, being the first assignment of this gene to these two species. SLC26A2 maps on BTA7q24, OAR5q24 and CHI7q24. This gene, too, was assigned for the fist time to both sheep and goat chromosomes, while it was more precisely localized on a single chromosome band in cattle. Improved cytogenetic maps of BTA4/OAR4/CHI4 were constructed and compared with HSA7 revealing five main conserved segments and complex chromosome rearrangements, including a centromere repositioning, differentiating HSA7 and BTA4/OAR4/CHI4.

Molecular cloning of the porcine beta-1,2-N-acetylglucosaminyltransferase II gene and assignment to chromosome 1q23-q27.

Glycosyltransferases play an important role in the synthesis of glycoproteins. Here we report the isolation of a brain cDNA coding for 89% of the porcine UDP-N-acetylglucosamine:alpha-6-D-mannoside-beta-1,2-N-acetylglucosaminy ltransferase II (EC 2.4.1.143) (GnTII). The cDNA was used for screening a genomic liver DNA library and isolation of a recombinant lambda FIX II phage containing the complete porcine GnTII gene and upstream and downstream sequences. The beta-1,2-N-acetylglucosaminyltransferase II gene harbours a single exon with an open reading frame of 1338 bp coding for a 446 amino acid protein with a calculated molecular mass of 51.1 kDa. The promoter of the GnTII gene is lacking a TATA-box and shows variable transcription start sites. In the 3'-untranslated region a polymorphic polyadenosine stretch was detected. The porcine GnTII gene contains four polyadenylation sites. PCR analysis of a porcine-rodent hybrid cell panel revealed the chromosomal location of the GnTII gene on SSC 1q23-q27. The mapping data of the cell panel were confirmed by fluorescence in situ hybridization (FISH) on metaphase chromosomes.

X- and Y-chromosome specific variants of the amelogenin gene allow sex determination in sheep (Ovis aries) and European red deer (Cervus elaphus).

BACKGROUND: Simple and precise methods for sex determination in animals are a pre-requisite for a number of applications in animal production and forensics. However, some of the existing methods depend only on the detection of Y-chromosome specific sequences. Therefore, the abscence of a signal does not necessarily mean that the sample is of female origin, because experimental errors can also lead to negative results. Thus, the detection of Y- and X-chromosome specific sequences is advantageous. RESULTS: A novel method for sex identification in mammals (sheep, Ovis aries and European red deer, Cervus elaphus) is described, using a polymerase chain reaction (PCR) and sequencing of a part of the amelogenin gene. A partial sequence of the amelogenin gene of sheep and red deer was obtained, which exists on both X and Y chromosomes with a deletion region on the Y chromosome. With a specific pair of primers a DNA fragment of different length between the male and female mammal was amplified. CONCLUSION: PCR amplification using the amelogenin gene primers is useful in sex identification of samples from sheep and red deer and can be applied to DNA analysis of micro samples with small amounts of DNA such as hair roots as well as bones or embryo biopsies.

Molecular phylogeny of selected predaceous leeches with reference to the evolution of body size and terrestrialism.

The phylogenetic relationships of erpobdellid leeches collected throughout Europe were investigated using newly obtained mitochondrial cytochrome c oxidase subunit I (CO-I) gene sequence data from 10 taxa. Monophyly of the five European Erpobdella species (sub-family Erpobdellinae) was supported, but a newly discovered leech, E. wuttkei Kutschera, 2004 (the smallest member of its genus, discovered in an aquarium) was only distantly related to this clade. Three members of the semiaquatic Trochetinae were included in this study. The largest European leech species discovered so far, Trocheta haskonis Grosser, 2000, was found to be a terrestrial predator that feeds on earthworms. The rare species T. haskonis is the sister taxon of T. bykowskii Gedroyc, 1913, a well-known amphibious leech. Based on a comparison of body sizes and a phylogenetic tree the evolution of terrestrialism in the family Erpobdellidae is discussed.

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.

Transgenic pigs carrying cDNA copies encoding the murine Mx1 protein which confers resistance to influenza virus infection.

An important aspect of gene transfer into farm animals is the improvement of disease resistance. The mouse Mx1 protein is known to be sufficient to confer resistance to influenza viruses. Gene constructs containing the mouse Mx1 cDNA controlled by the human metallothionein IIA promoter (hMTIIA::Mx), the SV40 early enhancer/promoter region (SV40::Mx) and the mouse Mx1 promoter (mMx::Mx) were transferred into pigs. The results of the gene transfer experiments with the hMTIIA::Mx and the SV40::Mx constructs indicate that the permanent high-level synthesis of Mx1 might be deleterious to the organism: the gene transfer efficiency was surprisingly low, and all transgenic piglets born had rearrangements in their transgene copies that abolished protein synthesis. The use of the interferon (IFN)- and virus-inducible mMx::Mx construct resulted in normal gene transfer efficiency. Two transgenic pig lines could be established which expressed IFN-inducible mouse Mx1 mRNA. Extensive protein analysis did not detect mouse Mx1 in IFN-treated transgenic animals.

Genomic organization and analysis of the 5' end of the porcine ryanodine receptor gene (ryr1).

In this study we describe the isolation of genomic clones of the 5' region of the porcine ryanodine receptor gene, a candidate for malignant hyperthermia in pigs and humans. The recombinants were isolated from a porcine liver, genomic DNA library in phage EMBL3A after screening with PCR amplified DNA fragments. The exon/intron structure of the ryanodine receptor gene was determined by DNA sequencing. Based on the sequence data it was possible to develop a simple test for the detection of malignant hyperthermia susceptible and normal pigs.

cDNA cloning and sequencing of the human ryanodine receptor type 3 (RYR3) reveals a novel alternative splice site in the RYR3 gene.

The human ryanodine receptor type 3 (RYR3) was cloned from a fetal brain cDNA library and its complete sequence was determined (EMBL accession number AJ001515). The sequenced cDNA spanned 15,564 bp and contained an open reading frame of 14,613 bp. The corresponding protein consisted of 4870 amino acids with a calculated molecular mass of 552 kDa. Amino acid sequence identities to the RYR3 proteins from rabbit, mink, and chicken were 96%, 95%, and 83% respectively. A previously unidentified alternative splice site was detected generating a transcript that lacked bases 11,569-11,650 and encoded a truncated protein.

Molecular characterization of porcine hyaluronidase genes 1, 2, and 3 clustered on SSC13q21.

Hyaluronidase genes (HYAL) encode hyaluronidase enzymes required for hyaluronan degradation. Both in humans and in mouse, clustered hyaluronidase genes have been identified. Here, the porcine hyaluronidase cluster consisting of genes HYAL1, HYAL2 and HYAL3 was characterized. The porcine cDNA sequences and proteins share homologies to human orthologs of 85 and 81% for HYAL1, 87 and 89% for HYAL2 and 86 and 83% for HYAL3, respectively. The porcine hyaluronidase proteins approximately share a 40% homology with each other. Furthermore, genes FUS1 and FUS2 were found within this cluster, which was assigned to SSC13q21. A total of seven SNPs were detected in the genes (four in HYAL1, two in HYAL2 and one in HYAL3). Three of the four SNPs in HYAL1 led to amino acid exchanges (C622G --> Asp24 to Glu; C633T --> Pro28 to Leu, and G1298T --> Ala250 to Ser). The amino acid replacements induce putative changes in the extended strand at Asp24, in the extended strand and the random coil at Pro28, and finally in the random coil and the alpha helix at Ala250. Frequency estimations for four SNPs located in genes HYAL1 and HYAL3 using animals (n = 295) of nine European and six Chinese pig breeds indicated several significant deviations. For example, there were no significant differences in allele frequencies between pigs representing breeds Hampshire and Jiangquhai at SNP C633T (HYAL1), but between Hampshire respectively Jiangquhai animals and Rongchang pigs. Analysis of the same breeds at SNP C588T (HYAL3) indicates significant differences between Hampshire and Jiangquhai respectively Rongchang, but not between Jiangquhai and Rongchang. The breed Göttingen Minipig displayed significant differences concerning two SNPs with respect to the other European pig breeds tested. For all three hyaluronidase genes, N-glycosylation sites are typical. For HYAL2 the lysosomal character was proven. The catalytic site responsible for HAase activity is conserved in the three enzymes. Expression of hyaluronidases was determined by RT-PCR and quantitative PCR. Broad gene expression was observed in different tissues for the three genes, respectively.