Pyruvate kinase deficiency mutant gene carriage in stray cats and rescued cats from animal hoarding in Hokkaido, Japan.

The mutant allele frequency of the Pyruvate kinase (PK) gene has been investigated mostly in pure breed cats. We investigated the PK mutant gene in stray and animal hoarding mongrel cats in Hokkaido, Japan. We also investigated the kinship of individuals carrying the mutant gene. Genotyping was conducted using the previously reported real-time PCR method. Fourteen microsatellite markers were used to identify the parents and offspring of cats carrying the PK mutant gene, and some kinship such as parent-offspring and siblings was observed. Some stray and animal hoarding cats carried the PK mutation gene and that consanguinity was confirmed among these cats indicated that the PK mutation gene was spread by unregulated interbreeding.

Molecular Epidemiological Survey for Degenerative Myelopathy in German Shepherd Dogs in Japan: Allele Frequency and Clinical Progression Rate.

Canine degenerative myelopathy (DM) is an adult-onset, chronic, progressive neurodegenerative disease reported in multiple canine breeds, including the German Shepherd Dog (GSD). Clinical signs include progressive motor neuron paralysis, which begins in the pelvic limbs and eventually leads to respiratory distress, which may necessitate euthanasia. A common DM-associated mutation is a single nucleotide substitution that causes an amino acid substitution (c.118G>A, p.E40K) in the canine SOD1 gene. This SOD1 mutation and the clinical progression rate of A/A risk genotype in the Japanese GSD population have not been analyzed before. Therefore, the aim of this study was to determine the frequency of the mutated allele and analyze the clinical progression rate in the Japanese GSD population. We studied 541 GSDs registered with the Japanese German Shepherd Dog Registration Society between 2000 and 2019. Genotyping was performed using real-time PCR with DNA extracted from the hair roots of each dog. The study revealed 330 G/G dogs (61%), 184 G/A dogs (34%), and 27 A/A dogs (5%), indicating a frequency of the mutant allele of 0.220, which are in Hardy−Weinberg equilibrium. We analyzed the clinical signs in A/A dogs with an age limit of 10 years based on information obtained from the dogs' owners. Of the seven A/A dogs older than 10 years, owners reported DM-related clinical signs, indicating a clinical progression rate of 100%. These results, further genotyping, and thorough clinical examinations of SOD1 A/A risk genotype will help control and prevent DM in the Japanese GSD population.

An inter-laboratory study of DNA-based identity, parentage and species testing in animal forensic genetics.

The probative value of animal forensic genetic evidence relies on laboratory accuracy and reliability. Inter-laboratory comparisons allow laboratories to evaluate their performance on specific tests and analyses and to continue to monitor their output. The International Society for Animal Genetics (ISAG) administered animal forensic comparison tests (AFCTs) in 2016 and 2018 to assess the limitations and capabilities of laboratories offering forensic identification, parentage and species determination services. The AFCTs revealed that analyses of low DNA template concentrations (≤300 pg/µL) constitute a significant challenge that has prevented many laboratories from reporting correct identification and parentage results. Moreover, a lack of familiarity with species testing protocols, interpretation guidelines and representative databases prevented over a quarter of the participating laboratories from submitting correct species determination results. Several laboratories showed improvement in their genotyping accuracy over time. However, the use of forensically validated standards, such as a standard forensic short tandem repeat (STR) kit, preferably with an allelic ladder, and stricter guidelines for STR typing, may have prevented some common issues from occurring, such as genotyping inaccuracies, missing data, elevated stutter products and loading errors. The AFCTs underscore the importance of conducting routine forensic comparison tests to allow laboratories to compare results from each other. Laboratories should keep improving their scientific and technical capabilities and continuously evaluate their personnel's proficiency in critical techniques such as low copy number (LCN) analysis and species testing. Although this is the first time that the ISAG has conducted comparison tests for forensic testing, findings from these AFCTs may serve as the foundation for continuous improvements of the overall quality of animal forensic genetic testing.

Genetic association between GHSR1a 5'UTR-microsatellite and nt-7(C>A) loci and growth and carcass traits in Japanese Black cattle.

We carried out a genetic association study between five nucleotide polymorphisms (5'UTR microsatellite ((TG)(n)), nt-7(C>A), L24V, DelR242 and Intron 1 microsatellite) of the GHSR1a gene and growth and carcass traits in 1285 steers sired by 117 Japanese Black bulls in a progeny testing program. We report herein, a significant association between the 5'UTR microsatellite and nt-7(C>A) loci and growth and carcass traits. We also propose a translational hypothesis that the association is due to differences in the secondary structure of GHSR1b mRNA (the non-spliced type with the 5'UTR microsatellite) among the GHSR1a gene haplotypes. Furthermore, we predicted the potential increase in profitability due to increased carcass weight in cow-calf fattening enterprises through planned matings based on DNA testing of the 5'UTR microsatellite. Statistical analysis revealed that the 5'UTR microsatellite locus had a significant additive effect on carcass weight (CW) and average daily gain (ADG), but not on beef marbling score (BMS). One of the four major microsatellite alleles (19-TG allele) with an allele frequency of 0.145, had a significantly (P < 0.0007) desirable effect on CW and ADG. We concluded that the 19-TG allele could potentially be economically useful nucleotide markers for growth and carcass traits in Japanese Black cattle.

Nucleotide polymorphisms and the 5'-UTR transcriptional analysis of the bovine growth hormone secretagogue receptor 1a (GHSR1a) gene.

Growth hormone secretagogue receptor 1a (GHSR1a) mediates the different actions of its endogenous ligand, ghrelin. Ghrelin-GHSR is involved in many important functions that include growth hormone secretion and food intake. We evaluated the haplotype variety and characterized the microsatellite ((TG)(n) , 5'-UTR) and nucleotide polymorphisms of the bovine GHSR1a gene. The nucleotide sequencing of this gene (∼6 kb) revealed 47 single nucleotide polymorphisms (SNPs), four indels and the microsatellite ((GTTT)(n) , Intron 1). The 19 haplotypes were constructed from all nucleotide viability patterns and were divided into three major groups. Four SNPs (L24V, nt456(G>A), D191N and nt667(C>T)) and DelR242 in Exon 1 and a haplotype block of approximately 2.2 kb (nt667(C>T) ∼ nt2884 (A>G)) were found in Bos taurus breeds. Breed differences in allele frequencies of the two microsatellites, nt-7(C>A), L24V, and DelR242 loci were found (P < 0.005). A DelR242 was found in the Japanese Shorthorn (frequency: ∼ 0.44), Japanese Brown, five European cattle breeds, the Philippine native cattle, but none detected in the Japanese Black or the Mishima island cattle. Additionally, 5'-rapid amplification of cDNA ends and RT-PCR analyses revealed that there were two different kinds of transcripts: spliced, without a microsatellite within 5'-UTR (GHSR1a); and non-spliced, with the microsatellite (GHSR1b).

Electrochemical detection of E. coli 16S rDNA sequence using air-plasma-activated fullerene-impregnated screen printed electrodes.

A new DNA modified electrode for the electrochemical detection of 16S rDNA extracted from Escherichia coli (JCM1649) is proposed. The electrodes were fabricated by screen printing a fullerene-impregnated carbon ink onto a poly(methylmethacrylate) substrate and immobilizing a probe DNA on the surface after activating the electrode with air plasma. The results indicated a dramatic improvement in the surface coverage of the immobilized probe DNA, and of the reduction peak of the redox indicator (Co(phen)(3)(3+)) due to the incorporation of fullerene. By immobilizing the probe onto the fullerene-impregnated screen-printed electrodes, the PCR product of the 16S rDNA extracted from E. coli was directly detected without any pretreatment. A well defined signal difference was observed between the perfectly matching oligonucleotide and the mismatching one, and it was possible to detect the target at the modified electrode. This method enabled us to clearly detect the two base mismatches in the ca. 1500-bases long 16S rDNA sequence.

Assignment of the gene for porcine insulin-like growth factor binding protein 1 to chromosome 18 and detection of polymorphisms in intron 2 by PCR-RFLP.

We have obtained a partial cDNA and three BAC clones for the porcine insulin-like growth factor binding protein 1 gene (IGFBP-1). Results of fluorescence in situ and radiation hybrid (RH) mapping assigned this gene to porcine chromosome (SSC) 18q24-qter. We found two types of polymerase chain reaction-restriction-fragment-length polymorphisms (PCR-RFLP) in intron 2 by using FokI and AluI.

A comprehensive radiation hybrid map of the bovine genome comprising 5593 loci.

A bovine whole genome 7000-rad radiation hybrid (RH) panel, SUNbRH(7000-rad), was constructed to build a high-resolution RH map. The Shirakawa-USDA linkage map served as a scaffold to construct a framework map of 3216 microsatellites on which 2377 ESTs were ordered. The resulting RH map provided essentially complete coverage across the genome, with 1 cR7000 corresponding to 114 kb, and a cattle-human comparative map of 1716 bovine genes and sequences annotated in the human genome, which covered 79 and 72% of the bovine and human genomes, respectively. We then integrated the bovine RH and comparative maps with BAC fingerprint information in to construct a detailed, BAC-based physical map covering a reported 40-cM quantitative trait locus region for intramuscular fat or "marbling" on BTA 4. In summary, the new, high-resolution SUNbRH7000-rad, comparative, Shirakawa-USDA linkage, and BAC fingerprint maps provide a set of genomic tools for fine mapping regions of interest in cattle.

A comprehensive genetic map of the cattle genome based on 3802 microsatellites.

A microsatellite-based high-density genetic map facilitates for fine mapping of hereditary traits of interest, characterization of meiosis, and providing a foundation for physical map construction. Here, we developed a comprehensive genetic map on the basis of >880,000 genotypes across the USDA MARC cattle reference families with a potential genetic resolution of 0.8 cM at the 95% confidence level ( approximately 800 kb in the bovine genome). We incorporated 2325 microsatellites into the second-generation genetic map by linkage analysis based on sex-averaged two-point LOD scores (>3.0), of which 2293 were fine-mapped by multipoint linkage analysis. The new 3160-cM map comprised of 29 sex-averaged autosomal linkage groups and a sex-specific X-chromosome linkage group includes 3960 markers with 2389 positions, resulting in an average interval size of 1.4 cM. More than half (51%) of the total length of the map is covered with intervals of 2.0 cM or less, and the largest gap is a 10.2-cM interval on the X-linkage group. The new map should accelerate fine mapping and positional cloning of genes for genetic diseases and economically important traits in cattle, as well as related livestock species, such as sheep and goat.

A transposable element-mediated gene divergence that directly produces a novel type bovine Bcnt protein including the endonuclease domain of RTE-1.

Ruminant Bcnt protein with a molecular mass of 97 kDa (designated p97Bcnt) includes a region derived from the endonuclease domain of a retrotransposable element RTE-1. Human and mouse Bcnt proteins lack the corresponding region but have a highly conserved 82-amino acid region at the C-terminus that is not present in p97Bcnt. By screening a bovine BAC library, we found two more bcnt-related genes: human-type bcnt (h-type bcnt) and its processed pseudogene. Whereas the pseudogene is localized on chromosome 26, both bcntp97 and the h-type bcnt genes are found on bovine chromosome 18, a synteny region of human chromosome 16 on which human BCNT is localized. Complete nucleotide sequencing of the BAC clone reveals that the bcntp97 and h-type bcnt genes are located just 6 kb apart in a tandem manner. The two h-type bcnt and bcntp97genes are active at both the transcriptional level and the protein level. H-type bovine Bcnt is more like human BCNT than p97Bcnt, when compared at their N-terminal regions. However, phylogenetic analysis using the N-terminal region of the bcnt gene family revealed that the duplication of bovine genes occurred within the bovine lineage with significantly accelerated substitution in bcntp97. This acceleration was not ascribed definitely to positive selection. After duplication, one of the bovine bcnt genes recruited the endonuclease domain of an intronic RTE-1 repeat accompanied by the accelerated substitution at the 5'-ORF, resulting in creation of a novel type of Bcnt protein in bovine.

Positional cloning of the gene LIMBIN responsible for bovine chondrodysplastic dwarfism.

Chondrodysplastic dwarfism in Japanese brown cattle is an autosomal recessive disorder characterized by short limbs. Previously, we mapped the locus responsible for the disease on the distal end of bovine chromosome 6. Here, we narrowed the critical region to approximately 2 cM by using linkage analysis, constructed a BAC and YAC contig covering this region, and identified a gene, LIMBIN (LBN), that possessed disease-specific mutations in the affected calves. One mutation was a single nucleotide substitution leading to an activation of a cryptic splicing donor site and the other was a one-base deletion resulting in a frameshift mutation. Strong expression of the Lbn gene was observed in limb buds of developing mouse embryos and in proliferating chondrocytes and bone-forming osteoblasts in long bones. These findings indicate that LBN is responsible for bovine chondrodysplastic dwarfism and has a critical role in a skeletal development.