Regulatory mutations in TBX3 disrupt asymmetric hair pigmentation that underlies Dun camouflage color in horses.

Dun is a wild-type coat color in horses characterized by pigment dilution with a striking pattern of dark areas termed primitive markings. Here we show that pigment dilution in Dun horses is due to radially asymmetric deposition of pigment in the growing hair caused by localized expression of the T-box 3 (TBX3) transcription factor in hair follicles, which in turn determines the distribution of hair follicle melanocytes. Most domestic horses are non-dun, a more intensely pigmented phenotype caused by regulatory mutations impairing TBX3 expression in the hair follicle, resulting in a more circumferential distribution of melanocytes and pigment granules in individual hairs. We identified two different alleles (non-dun1 and non-dun2) causing non-dun color. non-dun2 is a recently derived allele, whereas the Dun and non-dun1 alleles are found in ancient horse DNA, demonstrating that this polymorphism predates horse domestication. These findings uncover a new developmental role for T-box genes and new aspects of hair follicle biology and pigmentation.

Evolutionary developmental transcriptomics reveals a gene network module regulating interspecific diversity in plant leaf shape.

Despite a long-standing interest in the genetic basis of morphological diversity, the molecular mechanisms that give rise to developmental variation are incompletely understood. Here, we use comparative transcriptomics coupled with the construction of gene coexpression networks to predict a gene regulatory network (GRN) for leaf development in tomato and two related wild species with strikingly different leaf morphologies. The core network in the leaf developmental GRN contains regulators of leaf morphology that function in global cell proliferation with peripheral gene network modules (GNMs). The BLADE-ON-PETIOLE (BOP) transcription factor in one GNM controls the core network by altering effective concentration of the KNOTTED-like HOMEOBOX gene product. Comparative network analysis and experimental perturbations of BOP levels suggest that variation in BOP expression could explain the diversity in leaf complexity among these species through dynamic rewiring of interactions in the GRN. The peripheral location of the BOP-containing GNM in the leaf developmental GRN and the phenotypic mimics of evolutionary diversity caused by alteration in BOP levels identify a key role for this GNM in canalizing the leaf morphospace by modifying the maturation schedule of leaves to create morphological diversity.

Dog leukocyte antigen class II-associated genetic risk testing for immune disorders of dogs: simplified approaches using Pug dog necrotizing meningoencephalitis as a model.

A significantly increased risk for a number of autoimmune and infectious diseases in purebred and mixed-breed dogs has been associated with certain alleles or allele combinations of the dog leukocyte antigen (DLA) class II complex containing the DRB1, DQA1, and DQB1 genes. The exact level of risk depends on the specific disease, the alleles in question, and whether alleles exist in a homozygous or heterozygous state. The gold standard for identifying high-risk alleles and their zygosity has involved direct sequencing of the exon 2 regions of each of the 3 genes. However, sequencing and identification of specific alleles at each of the 3 loci are relatively expensive and sequencing techniques are not ideal for additional parentage or identity determination. However, it is often possible to get the same information from sequencing only 1 gene given the small number of possible alleles at each locus in purebred dogs, extensive homozygosity, and tendency for disease-causing alleles at each of the 3 loci to be strongly linked to each other into haplotypes. Therefore, genetic testing in purebred dogs with immune diseases can be often simplified by sequencing alleles at 1 rather than 3 loci. Further simplification of genetic tests for canine immune diseases can be achieved by the use of alternative genetic markers in the DLA class II region that are also strongly linked with the disease genotype. These markers consist of either simple tandem repeats or single nucleotide polymorphisms that are also in strong linkage with specific DLA class II genotypes and/or haplotypes. The current study uses necrotizing meningoencephalitis of Pug dogs as a paradigm to assess simple alternative genetic tests for disease risk. It was possible to attain identical necrotizing meningoencephalitis risk assessments to 3-locus DLA class II sequencing by sequencing only the DQB1 gene, using 3 DLA class II-linked simple tandem repeat markers, or with a small single nucleotide polymorphism array designed to identify breed-specific DQB1 alleles.

A comprehensive linkage map of the dog genome.

We have leveraged the reference sequence of a boxer to construct the first complete linkage map for the domestic dog. The new map improves access to the dog's unique biology, from human disease counterparts to fascinating evolutionary adaptations. The map was constructed with approximately 3000 microsatellite markers developed from the reference sequence. Familial resources afforded 450 mostly phase-known meioses for map assembly. The genotype data supported a framework map with approximately 1500 loci. An additional approximately 1500 markers served as map validators, contributing modestly to estimates of recombination rate but supporting the framework content. Data from approximately 22,000 SNPs informing on a subset of meioses supported map integrity. The sex-averaged map extended 21 M and revealed marked region- and sex-specific differences in recombination rate. The map will enable empiric coverage estimates and multipoint linkage analysis. Knowledge of the variation in recombination rate will also inform on genomewide patterns of linkage disequilibrium (LD), and thus benefit association, selective sweep, and phylogenetic mapping approaches. The computational and wet-bench strategies can be applied to the reference genome of any nonmodel organism to assemble a de novo linkage map.

Characterization of a continuous feline mammary epithelial cell line susceptible to feline epitheliotropic viruses.

Mucosal epithelial cells are the primary targets for many common viral pathogens of cats. Viral infection of epithelia can damage or disrupt the epithelial barrier that protects underlying tissues. In vitro cell culture systems are an effective means to study how viruses infect and disrupt epithelial barriers, however no true continuous or immortalized feline epithelial cell culture lines are available. A continuous cell culture of feline mammary epithelial cells (FMEC UCD-04-2) that forms tight junctions with high transepithelial electrical resistance (>2000Omegacm(-1)) 3-4 days after reaching confluence was characterized. In addition, it was shown that FMECs are susceptible to infection with feline calicivirus (FCV), feline herpesvirus (FHV-1), feline coronavirus (FeCoV), and feline panleukopenia virus (FPV). These cells will be useful for studies of feline viral disease and for in vitro studies of feline epithelia.

A high-resolution radiation hybrid map of rhesus macaque chromosome 5 identifies rearrangements in the genome assembly.

A 10,000-rad radiation hybrid (RH) cell panel of the rhesus macaque was generated to construct a comprehensive RH map of chromosome 5. The map represents 218 markers typed in 185 RH clones. The 4846-cR map has an average marker spacing of 798 kb. Alignments of the RH map to macaque and human genome sequences confirm a large inversion and reveal a previously unreported telomeric inversion. The macaque genome sequence indicates small translocations from the ancestral homolog of macaque chromosome 5 to macaque chromosomes 1 and 6. The RH map suggests that these are probably assembly artifacts. Unlike the genome sequence, the RH mapping data indicate the conservation of synteny between macaque chromosome 5 and human chromosome 4. This study shows that the 10,000-rad panel is appropriate for the generation of a high-resolution whole-genome RH map suitable for the verification of the rhesus genome assembly.

Cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) mutations associated with the domestic cat AB blood group.

BACKGROUND: The cat has one common blood group with two major serotypes, blood type A that is dominant to type B. A rare type AB may also be allelic and is suspected to be recessive to A and dominant to B. Cat blood type antigens are defined, N-glycolylneuraminic acid (NeuGc) is associated with type A and N-acetylneuraminic acid (NeuAc) with type B. The enzyme cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) determines the sugar bound to the red cell by converting NeuAc to NeuGc. Thus, mutations in CMAH may cause the A and B blood types. RESULTS: Genomic sequence of CMAH from eight cats and the cDNA of four cats representing all blood types were analyzed to identify causative mutations. DNA variants consistent with the blood types were genotyped in over 200 cats. Five SNPs and an indel formed haplotypes that were consistent with each blood type. CONCLUSION: Mutations in type B cats likely disrupt the gene function of CMAH, leading to a predominance of NeuAc. Type AB concordant variants were not identified, however, cDNA species suggest an alternative allele that activates a downstream start site, leading to a CMAH protein that would be altered at the 5' region. The cat AB blood group system is proposed to be designated by three alleles, A > aab > b. The A and b CMAH alleles described herein can distinguish type A and type B cats without blood sample collections. CMAH represents the first blood group gene identified outside of non-human primates and humans.

Identification of positional candidate genes for body weight and adiposity in subcongenic mice.

We previously constructed a congenic mouse, B6.S-D2Mit194-D2Mit311 (B6.S-2) with 27 Mb of SPRET/Ei donor DNA on distal chromosome 2 in a C57BL/6J background that captured an obesity quantitative trait locus (QTL). Mice homozygous for SPRET/Ei alleles at the donor region had decreased body weight and obesity-related phenotypes (Diament AL, Farahani P, Chiu S, Fisler J, Warden CH. Mamm Genome 15: 452-459, 2004). In this study, we constructed five overlapping subcongenics with smaller SPRET/Ei donor regions to fine map the underlying gene(s). One of the five subcongenic lines derived from the B6.S-2 founding congenic, B6.S-2A, captured the body weight and adiposity phenotypes in a donor region with a maximum size of 7.4 Mb. Homozygous SPRET/Ei donor alleles in both the founding congenic and the derived B6.S-2A subcongenic exhibited significant decreases in body weight, multiple fat pad weights, and adiposity index (total fat pad weight divided by body weight). Interval-specific microarray analysis in four tissues for donor region genes from the founding B6.S-2 congenic identified several differentially expressed genes mapping to the B6.S-2A subcongenic donor region, including prohormone convertase 2 (PC2; gene name: Pcsk2). Quantitative real-time PCR confirmed a modest decrease of PC2 expression in brains of mice homozygous for SPRET/Ei donor alleles. Analysis of the relative levels of mRNA for B6 and SPRET/Ei in heterozygous congenic mice showed differentially higher expression of the C57BL/6J allele over the SPRET/Ei allele, indicating a cis regulation of differential expression. Using subcongenic mapping, we successfully narrowed a body weight and obesity QTL interval and identified PC2 as a positional candidate gene.

Canine COL4A3 and COL4A4: sequencing, mapping and genomic organization.

Canine alpha3 and alpha4 chains of collagen type IV genes (COL4A3 and COL4A4) are expressed in the renal glomerular basement membrane, where they provide a critical structural and functional matrix for other basement membrane components. These genes are candidates for hereditary nephritis (Alport syndrome) in several dog breeds (e.g. English Cocker Spaniel and Bull Terrier). Using RACE and PCR, the cDNA of both genes was cloned and sequenced. Both COL4A3 and COL4A4, as well as canine NPPC (Natriuretic Peptide Precursor C), were mapped to CFA25 using an RH panel. Conservation of the tight linkage of COL4A3 and COL4A4 as seen in human and mouse was verified in the dog. Intron-exon boundaries in both genes were determined by BLAST analysis of the Canis Familiaris Trace Archive. The elucidation of the cDNA sequences, genomic organization and the open reading frames of canine COL4A3 and COL4A4 provide the groundwork for screening these genes for mutations in hereditary nephritis in dogs.

The first-generation whole-genome radiation hybrid map in the horse identifies conserved segments in human and mouse genomes.

A first-generation radiation hybrid (RH) map of the equine (Equus caballus) genome was assembled using 92 horse x hamster hybrid cell lines and 730 equine markers. The map is the first comprehensive framework map of the horse that (1) incorporates type I as well as type II markers, (2) integrates synteny, cytogenetic, and meiotic maps into a consensus map, and (3) provides the most detailed genome-wide information to date on the organization and comparative status of the equine genome. The 730 loci (258 type I and 472 type II) included in the final map are clustered in 101 RH groups distributed over all equine autosomes and the X chromosome. The overall marker retention frequency in the panel is approximately 21%, and the possibility of adding any new marker to the map is approximately 90%. On average, the mapped markers are distributed every 19 cR (4 Mb) of the equine genome--a significant improvement in resolution over previous maps. With 69 new FISH assignments, a total of 253 cytogenetically mapped loci physically anchor the RH map to various chromosomal segments. Synteny assignments of 39 gene loci complemented the RH mapping of 27 genes. The results added 12 new loci to the horse gene map. Lastly, comparison of the assembly of 447 equine genes (256 linearly ordered RH-mapped and additional 191 FISH-mapped) with the location of draft sequences of their human and mouse orthologs provides the most extensive horse-human and horse-mouse comparative map to date. We expect that the foundation established through this map will significantly facilitate rapid targeted expansion of the horse gene map and consequently, mapping and positional cloning of genes governing traits significant to the equine industry.

Characterization of the beta2-microglobulin gene of the horse.

A clone containing beta(2)-microglobulin (beta(2)-m), the light chain of the major histocompatibility complex class I cell surface molecule, was isolated from an equine bacterial artificial chromosome library. This clone was used as a template for polymerase chain reaction (PCR) and unidirectional sequencing to elucidate the genomic sequence and intron/exon boundaries. We obtained 7,000 bases of sequence, extending from 1,100 nucleotides (nt) upstream of the coding region start through 1,698 nt downstream of the stop codon. The sequence contained regulatory elements in the region upstream of the coding sequence similar to those of the beta(2)-m gene of other species. The beta(2)-m gene was localized to horse chromosome ECA1q23-q25 by fluorescent in situ hybridization. This was confirmed by synteny mapping on a (horse x mouse) somatic cell hybrid panel. The sequence and intron/exon boundaries determined were used to design PCR primers to amplify and sequence the coding region of the beta(2)-m gene in other equids, including five breeds of domestic horse, one Przewalski's horse, five domestic donkeys and five zebras. A high degree of conservation was found among equids, illustrated by >98% (349/354) identity at the nucleotide level and 95% (113/118) at the amino acid level, because of non-synonymous nucleotide substitutions. The promoter detected in the region upstream of the coding sequence was subcloned and used in chloramphenicol acetyl transferase (CAT) assays to demonstrate the presence of a functional promoter. This study provides tools for the analysis of regulation of not only the horse beta(2)-m gene, but also for any genes dependent upon beta(2)-m for expression.