Using online tools at the Bovine Genome Database to manually annotate genes in the new reference genome.

With the availability of a new highly contiguous Bos taurus reference genome assembly (ARS-UCD1.2), it is the opportune time to upgrade the bovine gene set by seeking input from researchers. Furthermore, advances in graphical genome annotation tools now make it possible for researchers to leverage sequence data generated with the latest technologies to collaboratively curate genes. For many years the Bovine Genome Database (BGD) has provided tools such as the Apollo genome annotation editor to support manual bovine gene curation. The goal of this paper is to explain the reasoning behind the decisions made in the manual gene curation process while providing examples using the existing BGD tools. We will describe the sources of gene annotation evidence provided at the BGD, including RNA-seq and Iso-Seq data. We will also explain how to interpret various data visualizations when curating gene models, and will demonstrate the value of manual gene annotation. The process described here can be applied to manual gene curation for other species with similar tools. With a better understanding of manual gene annotation, researchers will be encouraged to edit gene models and contribute to the enhancement of livestock gene sets.

Origins of cattle on Chirikof Island, Alaska, elucidated from genome-wide SNP genotypes.

Feral livestock may harbor genetic variation of commercial, scientific, historical or esthetic value. The origins and uniqueness of feral cattle on Chirikof Island, Alaska, are uncertain. The island is now part of the Alaska Maritime Wildlife Refuge and Federal wildlife managers want grazing to cease, presumably leading to demise of the cattle. Here we characterize the cattle of Chirikof Island relative to extant breeds and discern their origins. Our analyses support the inference that Yakut cattle from Russia arrived first on Chirikof Island, then ~120 years ago the first European taurine cattle were introduced to the island, and finally a large wave of Hereford cattle were introduced on average 40 years ago. In addition, this mixture of European and East-Asian cattle is unique compared with other North American breeds and we find evidence that natural selection in the relatively harsh environment of Chirikof Island has further impacted their genetic architecture. These results provide an objective basis for decisions regarding conservation of the Chirikof Island cattle.

Genome scan for meat quality traits in Nelore beef cattle.

Meat quality traits are economically important because they affect consumers' acceptance, which, in turn, influences the demand for beef. However, selection to improve meat quality is limited by the small numbers of animals on which meat tenderness can be evaluated due to the cost of performing shear force analysis and the resultant damage to the carcass. Genome wide-association studies for Warner-Bratzler shear force measured at different times of meat aging, backfat thickness, ribeye muscle area, scanning parameters [lightness, redness (a*), and yellowness] to ascertain color characteristics of meat and fat, water-holding capacity, cooking loss (CL), and muscle pH were conducted using genotype data from the Illumina BovineHD BeadChip array to identify quantitative trait loci (QTL) in all phenotyped Nelore cattle. Phenotype count for these animals ranged from 430 to 536 across traits. Meat quality traits in Nelore are controlled by numerous QTL of small effect, except for a small number of large-effect QTL identified for a*fat, CL, and pH. Genomic regions harboring these QTL and the pathways in which the genes from these regions act appear to differ from those identified in taurine cattle for meat quality traits. These results will guide future QTL mapping studies and the development of models for the prediction of genetic merit to implement genomic selection for meat quality in Nelore cattle.

Genome-wide association analysis for quantitative trait loci influencing Warner-Bratzler shear force in five taurine cattle breeds.

We performed a genome-wide association study for Warner-Bratzler shear force (WBSF), a measure of meat tenderness, by genotyping 3360 animals from five breeds with 54 790 BovineSNP50 and 96 putative single-nucleotide polymorphisms (SNPs) within µ-calpain [HUGO nomenclature calpain 1, (mu/I) large subunit; CAPN1] and calpastatin (CAST). Within- and across-breed analyses estimated SNP allele substitution effects (ASEs) by genomic best linear unbiased prediction (GBLUP) and variance components by restricted maximum likelihood under an animal model incorporating a genomic relationship matrix. GBLUP estimates of ASEs from the across-breed analysis were moderately correlated (0.31-0.66) with those from the individual within-breed analyses, indicating that prediction equations for molecular estimates of breeding value developed from across-breed analyses should be effective for genomic selection within breeds. We identified 79 genomic regions associated with WBSF in at least three breeds, but only eight were detected in all five breeds, suggesting that the within-breed analyses were underpowered, that different quantitative trait loci (QTL) underlie variation between breeds or that the BovineSNP50 SNP density is insufficient to detect common QTL among breeds. In the across-breed analysis, CAPN1 was followed by CAST as the most strongly associated WBSF QTL genome-wide, and associations with both were detected in all five breeds. We show that none of the four commercialized CAST and CAPN1 SNP diagnostics are causal for associations with WBSF, and we putatively fine-map the CAPN1 causal mutation to a 4581-bp region. We estimate that variation in CAST and CAPN1 explains 1.02 and 1.85% of the phenotypic variation in WBSF respectively.

A truncated retrotransposon disrupts the GRM1 coding sequence in Coton de Tulear dogs with Bandera's neonatal ataxia.

BACKGROUND: Bandera's neonatal ataxia (BNAt) is an autosomal recessive cerebellar ataxia that affects members of the Coton de Tulear dog breed. OBJECTIVE: To identify the mutation that causes BNAt. ANIMALS: The study involved DNA from 112 Cotons de Tulear (including 15 puppies with signs of BNAt) and 87 DNA samples from dogs of 12 other breeds. METHODS: The BNAt locus was mapped with a genome-wide association study (GWAS). The coding exons of positional candidate gene GRM1, which encodes metabotropic glutamate receptor 1, were polymerase chain reaction (PCR)-amplified and resequenced. A 3-primer PCR assay was used to genotype individual dogs for a truncated retrotransposon inserted into exon 8 of GRM1. RESULTS: The GWAS indicated that the BNAt locus was in a canine chromosome 1 region that contained candidate gene GRM1. Resequencing this gene from BNAt-affected puppies indicated that exon 8 was interrupted by the insertion of a 5'-truncated retrotransposon. All 15 BNAt-affected puppies were homozygous for the insert, whereas all other Cotons de Tulear were heterozygotes (n = 43) or homozygous (n = 54) for the ancestral allele. None of the 87 dogs from 12 other breeds had the insertion allele. CONCLUSIONS AND CLINICAL IMPORTANCE: BNAt is caused by a retrotransposon inserted into exon 8 of GRM1. A DNA test for the GRM1 retrotransposon insert can be used for genetic counseling and to confirm the diagnosis of BNAt.

Genomic tools for characterizing monogenic and polygenic traits in ruminants--using the bovine as an example.

Next generation sequencing platforms have democratized genome sequencing. Large genome centers are no longer required to produce genome sequences costing millions. A few lanes of paired-end sequence on an Illumina Genome Analyzer, costing < $10,000, will produce more sequence than generated only a few years ago to produce the human and cow assemblies. The de novo assembly of large numbers of short reads into a high-quality whole-genome sequence is now technically feasible and will allow the whole genome sequencing and assembly of a broad spectrum of ruminant species. Next-generation sequencing instruments are also proving very useful for transcriptome or resequencing projects in which the entire RNA population produced by a tissue, or the entire genomes of individual animals are sequenced, and the produced reads are aligned to a reference assembly. We have used this strategy to examine gene expression differences in tissues from cattle differing in feed efficiency, to perform genome-wide single nucleotide polymorphism discovery for the construction of ultrahigh-density genotyping assays, and in combination with genome-wide association analysis, for the identification of mutations responsible for Mendelian diseases. The new 800K SNP bovine genotyping assays possess the resolution to map trait associations to the locations of individual genes and the 45 million polymorphisms identified in > 180X genome sequence coverage on over 200 animals can be queried to identify the polymorphisms present within positional candidate genes. These new tools should rapidly allow the identification of genes and mutations underlying variation in cattle production and reproductive traits.

Development of improved electrical-substitution radiometers at the National Research Council of Canada.

We describe the development of a third generation of electrical-substitution radiometers (ESR's) at the National Research Council of Canada. The new ESR's follow the same general design as before, but incorporate improved thermopiles and electrical heating elements. The ESR's have a responsivity between 0.6 and 1.0 VW(-1), a time constant of approximately 2.0 s, a uniformity of 0.1% over a 6-mm-diameter region, and a noise level of approximately 6 nW. Performance characteristics of the new ESR's are discussed. It is shown that calibrations performed with these ESR's agree with those made with the previous generation of ESR's to better than 0.05%.

Distal tibia bone graft for arthrodesis of the foot and ankle.

Forty patients since 1988 have had distal tibial bone grafting for 41 arthrodeses of the foot and ankle. Bone graft is obtained through a cortical window made just above the medial metaphyseal distal tibial flare. Average follow-up was 23.3 months. Forty of 41 arthrodesis sites fused; there was only one nonunion. There were no delayed unions. There were no complications at the donor site based on patient examination and radiographs. Ipsilateral ankle motion was not affected by the bone graft procedure. Cited complications from iliac crest bone graft include donor site pain, blood loss, heterotopic bone formation, pelvic instability, iliac hernia, infection, fracture, and deformity. Complications with allografts include disease transmission and immune response. These are avoided by using locally obtained distal tibia autograft for arthrodeses in the foot and ankle.

Spot-size measurement of an intense CO(2) laser beam.

The determination of a high-power CO(2) laser beam spot size is described. The method consists of measuring burn pattern radii in holographic emulsion as a function of laser pulse energy.

Stable operation of a powerful 3-Hz line tunable TEA CO(2) oscillator-amplifiers system.

We communicate results of the stable operation of a TEA CO(2) oscillator-amplifiers system, producing pulses of 10(14) W/cm(2) at best focus, furnished with a simple provision for retropulse isolation.

A surgical approach to the motor branch of the ulnar nerve.

Isolated injury to the motor branch of the ulnar nerve is a relatively rare injury, often initially misdiagnosed. If repair is attempted through the original laceration without complete motor branch exposure, results can be less than satisfactory. A recent case illustrates this injury and provides us with an opportunity to review the surgical anatomy of the motor branch of the ulnar nerve. The surgical approach to the motor branch has been detailed and specifically emphasizes complete motor branch exposure from the main ulnar nerve trunk to the most distal motor branch entry into the adductor pollicis muscle. This approach permits definition of the exact level of the nerve injury, preservation of any intact proximal fine motor branches, and facilitates the mechanics of nerve repair.

The effects of thionapthene-2-carboxylic acid-lysine on the hypercalcemia of malignancy in the rat.

Thionapthene-2-carboxylic acid (TNCA) was previously shown to lower serum calcium concentrations in hypercalcemic rats: however, oral administration of TNCA may cause gastric irritation. We have assessed thionapthene-2-carboxylic acid lysine salt (TNLY) for its effects on serum calcium concentration and survival in rats bearing the hypercalcemic Leydig cell tumor. TNLY (0.6-1.8 mmol/kg/day) produced a marked and prolonged dose-related decrease in serum calcium concentration. At the highest dose of 1.8 mmol/kg/day, hypocalcemia occurred. Effects were sustained for 96 hours or longer. In tumor-bearing rats that were not yet hypercalcemic, pretreatment with TNLY (0.9 mmol/kg/day) did not induce hypocalcemia and the onset of hypercalcemia was prevented. Neither TNLY nor dichloromethylene diphosphonate (CL2MDP), a potent inhibitor of bone resorption, significantly prolonged overall survival. We concluded that TNLY is a potent antihypercalcemic agent that warrants further testing for use in the treatment of hypercalcemic disorders.

Identification of Leishmania spp. by radiorespirometry.

Preliminary investigation of the application of radiorespirometric technic to protozoan parasites of man indicates a potential for rapid identification. This technic, developed for identification of bacteria, was modified for use with culture forms of Leishmania. Five strains of Leishmania were compared: 2 of L. donovani, 2S and K; L. brasilensis, 2936 and B; and 1 of L. tropica, A. Consisent and rapid (approximately 2 hr) identification was obtained by the radiorespirometric procedure. A computer-type analysis of the radiorespirometric profiles of the 5 strains permitted correct identification of each isolate at the strain level 100% of the time. This technic offers several advantages over many current procedures for identification of protozoan parasites: (A) It is simple, rapid and highly reproducible, (B) Since it does not rely on visual or spectrophotometric determination, it may be conducted in the presence of optically complex substances. (C) It requires relatively low numbers of organisms (approximately 2 x 10(5)/14C-labeled substrate). (D) It is based on differential enzymic activity between species and strains of organisms and therefore, ultimately, on inherent genetic determinates of the parasites. (E) Further development of the procedure and accumulation of a data reference "bank" would allow automation of most of the identification process.