Genome analysis of the domestic dog (Korean Jindo) by massively parallel sequencing

Although pioneering sequencing projects have shed light on the boxer and poodle genomes, a number of challenges need to be met before the sequencing and annotation of the dog genome can be considered complete. Here, we present the DNA sequence of the Jindo dog genome, sequenced to 45-fold average coverage using Illumina massively parallel sequencing technology. A comparison of the sequence to the reference boxer genome led to the identification of 4 675 437 single nucleotide polymorphisms (SNPs, including 3 346 058 novel SNPs), 71 642 indels and 8131 structural variations. Of these, 339 non-synonymous SNPs and 3 indels are located within coding sequences (CDS). In particular, 3 non-synonymous SNPs and a 26-bp deletion occur in the TCOF1 locus, implying that the difference observed in cranial facial morphology between Jindo and boxer dogs might be influenced by those variations. Through the annotation of the Jindo olfactory receptor gene family, we found 2 unique olfactory receptor genes and 236 olfactory receptor genes harbouring non-synonymous homozygous SNPs that are likely to affect smelling capability. In addition, we determined the DNA sequence of the Jindo dog mitochondrial genome and identified Jindo dog-specific mtDNA genotypes. This Jindo genome data upgrade our understanding of dog genomic architecture and will be a very valuable resource for investigating not only dog genetics and genomics but also human and dog disease genetics and comparative genomics.     

Molecular variation in pigmentation genes contributing to coat colour in native Korean Hanwoo cattle

Pigmentation genes such as TYR (tyrosinase), TYRP1 (tyrosinase-related protein 1), DCT (previously TYRP2, or tyrosinase-related protein 2), ASIP (agouti) and MC1R (melanocortin receptor 1) play a major role in cattle coat colour. To understand the genotypic profile underlying coat colour in native Korean Hanwoo cattle and Angus black cattle, portions of the above-mentioned genes were amplified. Sequence analysis revealed variation in the TYRP1 (exon 5) and MC1R genes. Restriction enzyme analysis of these two genes could distinguish between different colours of Hanwoo cattle. Quantitative estimates of melanin and eumelanin in hair from three different-coloured Hanwoo phenotypes and Angus black showed significant differences at the breed and phenotypic levels. Finally, sequence variants in MC1R were associated with total melanin and eumelanin in breeds as well as in Hanwoo phenotypes.     

Two SNPs in the SILV gene are associated with silver coat colour in ponies

In horses, a pigment dilution acting only on black eumelanin is the so-called silver coat colour, which is characterized by a chocolate-to-reddish body with a white mane and tail. Using information from other species, we focused our study on SILV as a possible candidate gene for the equine silver phenotype. A 1559-bp genomic fragment was sequenced in 24 horses, and five SNPs were detected. Two of the five SNPs (DQ665301:g.697A>T and DQ665301:g.1457C>T) were genotyped in 112 horses representing eight colour phenotypes. Both mutations were completely associated with the silver phenotype: all eumelanin-producing horses (blacks and bays) with atypical white mane and tail were carriers of the [g.697T; g.1457T] haplotype. We identified this haplotype as well as the silver phenotype only in Shetland ponies and Icelandic horses. Horses without eumelanin (chestnuts) were carriers of the [g.697T; g.1457T] haplotype, but they showed no phenotypic effect. The white or flaxen mane often detected in chestnuts is presumably based on another SILV mutation or on polymorphisms in other genes.     

Comparative linkage mapping of the Grey coat colour gene in horses

Grey horses are born coloured, turn progressively grey and often develop melanomas late in life. Grey shows an autosomal dominant inheritance and the locus has previously been mapped to horse chromosome 25 (ECA25), around the TXN gene. We have now developed eight new single nucleotide polymorphisms (SNPs) associated with genes on ECA25 using information on the linear order of genes on human chromosome 9q, as well as the human and mouse coding sequences. These SNPs were mapped in relation to the Grey locus using more than 300 progeny from matings between two Swedish Warmblood grey stallions and non-grey mares. Grey was firmly assigned to an interval with flanking markers NANS and ABCA1. This corresponds to a region of approximately 6.9 Mb on human chromosome 9q. Furthermore, no recombination was observed between Grey, TGFBR1 and TMEFF1, the last two being 1.4 Mb apart in human. There are no obvious candidate genes in this region and none of the genes has been associated with pigmentation disorders or melanoma development, suggesting that the grey phenotype is caused by a mutation in a novel gene.     

Assignment of the appaloosa coat colour gene (LP) to equine chromosome 1

A single autosomal dominant locus, leopard complex (LP) controls the presence of appaloosa pigmentation patterns in the horse. The causative gene for LP is unknown. This study was undertaken to map LP in the horse. Two paternal half sib families segregating for the LP locus and including a total of 47 offspring were used to perform a genome scan which localized LP to horse chromosome 1 (ECA1). LP was linked to ASB08 (LOD = 9.99 at Theta = 0.02) and AHT21 (LOD = 5.03 at Theta = 0.14). To refine the map position of LP, eight microsatellite markers on ECA1 (UM041, LEX77, 1CA41, TKY374, COR046, 1CA32, 1CA43, and TKY002) were analysed in the two half sib families. Results from this linkage analysis showed LP was located in the interval between ASB08 and 1CA43. Tight junction protein (TJP1), which lies within the LP interval on ECA1, was used to determine the homologous chromosomes in humans (HSA15) and mice (mouse chromosome 7). We propose that the pink eyed dilution (p) gene and transient receptor potential cation channel subfamily M, member 1 (TRPM1) are positional candidate genes for LP.     

Linkage of the grey coat colour locus to microsatellites on horse chromosome 25

The progressive loss of colour in the hair of grey horses is controlled by a dominantly inherited allele at the Grey locus (GG). In this study, two paternal Quarter Horse (QH) families segregating for the GG allele were genotyped with a set of 101 microsatellite markers spanning the 31 autosomes and the X chromosome. This genome scan demonstrated linkage of Grey to COR018 (RF=0.02, LOD=12.04) on horse chromosome 25 (ECA25). Further chromosome-specific analysis of seven total QH families confirmed the linkage of Grey to a group of ECA25 markers and the map order of NVHEQ43-(0.24)-UCDEQ405-(0.09)-COR080-(0.05)-GREY-(0.14)-UCDEQ464 was produced. Although G was found to be linked to TXN and COR018 in the chromosome-specific analysis, the data were not sufficiently informative to place either marker on our ECA25 map with significant LODs. Our results excluded the equine tyrosinase related protein 1 (TYRP1) and melanocyte protein 17 (Pmel17) genes as possible candidates for the grey phenotype in horses.     

Assignment of the horse grey coat colour gene to ECA25 using whole genome scanning

The dominant grey coat colour gene of horses has been mapped using a whole genome scanning approach. Samples from a large half-sibling pedigree of Thoroughbred horses were utilized in order to map the grey coat colour locus, G. Multiplex groups of microsatellite markers were developed and used to efficiently screen the horse genome at a resolution of approximately 22 cM, based on an estimated map length for the horse genome of 2720 cM. The grey gene was assigned to chromosome 25 (ECA25), one of the smaller acrocentric horse chromosomes. Based on the current state of knowledge of conserved synteny and coat colour genetics in other mammalian species, there are no obvious candidate genes for the grey gene in the region.     

Characterization of the porcine KIT ligand gene: expression analysis, genomic structure, polymorphism detection and association with coat colour traits

Kit ligand (KITLG) is the ligand for the type III receptor tyrosine kinase KIT. Studies of the KIT/KITLG pathway in a number of mammalian species have shown that it is important for the development of stem cell populations in haematopoietic tissues, germ cells in reproductive organs and the embryonic migrating melanoblasts that give rise to melanocytes. Consequently, mutations in the pathway may result in a range of defects including anaemia, sterility and de-pigmentation. The cDNA sequence of the porcine KITLG gene has been reported previously, and is an attractive candidate locus for moderating coat colour in pigs. In this paper we report the gene structure and physical mapping of the porcine gene. We also report the identification of polymorphisms in the gene, one of which was used to confirm linkage to chromosome 5. Preliminary RNA expression studies using a panel of tissues have shown that in addition to the known variant lacking exon 6, there is alternative splicing of exon 4. However, little evidence was found for the KITLG gene being linked to variation in colour in a Meishan x Large White cross.     

An investigation into the genetic background of coat colour dilution in a Charolais x German Holstein F2 resource population

The molecular background of many loci affecting coat colour inheritance in cattle is still incompletely characterized, although it is known that coat colour results from the joint effects of several loci, e.g. agouti, extension and dilution. Dilution alleles are responsible for a dilution effect on the original coat colour of an individual, which is determined by the agouti and extension loci. Different loci affecting dilution of pigment are suggested in Charolais (Dc) and Simmental (Ds). To enable chromosomal mapping of the Dc mutation, 133 animals from an F2 full-sib resource population generated from a cross of Charolais and German Holstein were scored for the coat colour dilution phenotype. Linkage analysis covering all autosomes revealed a significant linkage of the dilution phenotype with microsatellite markers on bovine chromosome 5. No recombination was observed between marker ETH10 and the Dc locus. Positional and functional information identified the bovine silver homolog (SILV) gene as a candidate for the Dc mutation. Results from comparative sequencing of the SILV gene in individuals with different dilution coat colour phenotypes confirmed the presence of a c.64G>A non-synonymous mutation, which had previously been identified in the Charolais breed. The alleles at this locus were associated with coat colour dilution in this study. However, further investigation of colour inheritance within the F2 resource population indicated that a single diallelic mutation in the SILV gene cannot explain the total observed variation of coat colour dilution.     

Plant tumor reversal associated with the loss of marker chromosomes in tobacco cells

Summary The determination of partial genotype, B/b and D/d, for coat colour in dogs, from phenotypic observations, is discussed. It is shown that the probability of a given genotype can be reliably determined where multiple observations on the mating of a given dog are available.     

Differences in the expression of the ASIP gene are involved in the recessive black coat colour pattern in sheep: evidence from the rare Xalda sheep breed

Here we have tested the hypothesis of association between different levels of agouti signalling peptide (ASIP) mRNA and the recessive black coat colour in the rare Xalda breed of sheep. To deal with this task, we first tested the possible action of both the dominant black extension allele (E(D)) and a 5-bp deletion (X99692:c.100_104del; A(del)) in the ovine ASIP coding sequence on the black coat colour pattern in 188 Xalda individuals. The E(D) allele was not present in the sample and only 11 individuals were homozygous for the A(del)ASIP allele. All Xalda individuals carrying the A(del)/A(del) genotype were phenotypically black. However, most black-coated individuals (109 out of 120) were not homozygous for the 5-bp deletion, thus rejecting the A(del)/A(del) genotype as the sole cause of recessive black coat colour in sheep. Differences in expression of ASIP mRNA were assessed via RT-PCR in 14 black-coated and 10 white-coated Xalda individuals showing different ASIP genotypes (A(wt)/A(wt), A(wt)/A(del) and A(del)/A(del)). Levels of expression in black animals were significantly (P < 0.0001) lower than those assessed for white-coated individuals. However, the ASIP genotype did not influence the ASIP mRNA level of expression. The consistency of these findings with those recently reported in humans is discussed, and the need to isolate the promoter region of ovine ASIP to obtain further evidence for a role of ASIP in recessive black ovine pigmentation is pointed out.     

Transgenic tobaccos transformed with a gene encoding a truncated form of the coat protein of tomato black ring nepovirus are resistant to viral infection

 Tomato black ring virus (TBRV) belongs to the nepoviruses, an important genus of phytoviruses characterized by isometric particles and by their transmission by longidorid nematodes. As for all other nepoviruses, the coat protein (CP) of TBRV is encoded by the 3′ terminal part of the viral RNA2 (positions 2801–4334). A hybrid gene driving the expression of a truncated form of the TBRV CP was constructed. It contains a frameshift deletion at position T₄₀₆₅ so that in the encoded protein the last 90 amino acids of the wild-type CP are replaced by 52 amino acids encoded by a different reading frame of the viral RNA. This hybrid gene was introduced into the genome of Nicotiana tabacum cv 'Xanthi' plants. When compared to control plants, progeny of some transformants expressing the mutated CP gene (CPm+ plants) showed resistance against TBRV infection. This resistance is characterized by a delay in the appearance of symptoms, a reduction in the number of infected plants and a reduction in virus accumulation. Received: 28 February 1997 / Revision received: 1 August 1997 / Accepted: 24 March 1999     

Intrinsic reactivity of uric acid with dioxygen: Towards the elucidation of the catalytic mechanism of urate oxidase

Urate oxidase catalyzes the transformation of uric acid in 5-hydroxyisourate, an unstable compound which is latter decomposed into allantoïn. Crystallographic data have shown that urate oxidase binds a dianion urate species deprotonated in N3 and N7, while kinetics experiments have highlighted the existence of several intermediates during catalysis. We have employed a quantum mechanical approach to analyze why urate oxidase is selective for one particular dianion and to explore all possible reaction pathways for the oxidation of one uric acid species by molecular dioxygen in presence of water. Our results indicate the urate dianion deprotonated in N3 and N7 is among all urate species that can coexist in solution it is the compound which will lose the most easiestly one electron in presence of molecular dioxygen. In addition, the transformation of this dianion in 5-hydroxyisourate is thermodynamically the most favorable reaction. Finally, several reaction pathways can be drawn, each starting with the spontaneous transfer of one electron from the urate dianion to molecular dioxygen. During that period, the existence of a 5-hydroperoxyisourate intermediate, which has been proposed elsewhere, does not seem mandatory.     

Polymorphism of transferrin and esterase in Alaskan wolves: evidence of close molecular homology with the dog

By the use of isoelectric focusing in polyacrylamide gels serum samples from 146 Alaskan wolves were studied with regard to transferrin (Tf) and esterase (ArE) polymorphism, comparing the phenotypic band patterns with those of selected Norwegian dogs. The study revealed Tf and ArE polymorphisms in the wolf with phenotypic band patterns being indistinguishable from the corresponding ones in dogs. This suggests the occurrence of the same two common Tf alleles in the wolf as in the dog. In the ArE system the results are consistent with the occurrence of three alleles which also occur in dogs whereas a fourth allele, so far not seen in dogs, is seen in Alaskan wolves.     

Single nucleotide polymorphisms in the Melanocortin 1 Receptor gene are linked with lightness of fibre colour in Peruvian Alpaca (Vicugna pacos)

Melanocortin 1 receptor (alpha melanocyte stimulating hormone receptor) (MC1R) is a gene‐controlling melanogenesis in mammals. However, it is not well characterized in alpacas and its association with colour is not known. The aim of this study was to look for polymorphisms in the MC1R gene in Peruvian Huacaya alpacas and to analyse the relationship between MC1R single nucleotide polymorphisms (SNPs) and the variations in the instrumental measurement of colour of alpaca fibre. Sixty alpaca fibre samples from black, brown, cream and white animals (15 for each colour) were used to extract DNA from hair bulbs. Colour was measured with a spectrophotometer to obtain quantitative values (CieL*a*b*). Sixteen samples, four of each colour group, were sequenced. Eighteen SNP mutations, 10 not previously described, were found in these 16 sequences. Three of them were chosen (c.82A>G, c.865C>T, c.901C>T) to analyse genotypes by PCR‐RFLP in the other 44 fibre samples and to determine the association of mutations with instrumental colour. These three polymorphisms showed association with fibre lightness (P < 0.05), although there was no correlation with colour groups.     

Changes in egg colour, egg weight and oviposition rate with the number of eggs laid by wild females of the small heath butterfly, Coenonympha pamphilus

ABSTRACT. 1. The colour of eggs laid by individual Coenonympha pamphilus (L.) (Lep., Satyridae) females changed over their lifespan. The first laid eggs were green, but after having laid about 100 eggs, females laid only yellow eggs.
2. By following females in the field, or by capturing wild females, and noting the colour of their eggs, it was established that younger females showed higher oviposition rates and laid heavier eggs than older females.
3. Hence, wild C.pamphilus females are unable to sustain a constant egg production by extracting amino acids or any other nutrients from nectar. We hypothesize that this is a general phenomenon in nectar feeding butterflies.
4. The egg colour did not seem to match the colour of the substrate on which the female chose to deposit the egg.     

Identification of a premature stop codon in the melanocyte-stimulating hormone receptor gene (MC1R) in Labrador and Golden retrievers with yellow coat colour

We have examined whether black/yellow coat colour in Labrador retrievers is controlled by allelic variants at the extension locus. As the gene encoding the melanocyte-stimulating hormone receptor (MC1R) has been shown to correspond to the extension locus in several species, we have determined the genomic MC1R sequence in Labrador retrievers with black and with yellow coat colour. Using primers based on the fox (Vulpes vulpes) MC1R sequence we initially isolated and sequenced the innerpart of the canine MC1R. By means of inverse PCR we succeeded in the characterization of both flanking regions of the MC1R gene (Genbank: AF064455). Comparison of the complete MC1R sequences of a yellow and a black Labrador retriever revealed a single C-->T mutation at nucleotide position 916 in the yellow dog. This transition changed the codon for arginine at position 305 into a stop codon, resulting in the elimination of the evolutionary strongly conserved 10 carboxyterminal amino acid residues. With an allele-specific-oligonucleotide (ASO) test it was shown that the mutation cosegregated with the recessively inherited yellow coat colour in the Labrador retriever. Golden retrievers also appeared to be homozygous for the mutation. Seventeen other breeds were all negative for the mutation. Since the Labrador and Golden retriever are closely related, we suggest a common founder for the yellow coat colour in Labrador and Golden retrievers.     

Analysis of transitive RNA silencing after grafting in transgenic plants with the coat protein gene of <Emphasis Type="Italic">Sweet potato feathery mottle virus</Emphasis>

We have previously reported the graft transmission of target specificity for RNA silencing using transgenic Nicotiana benthamiana plants expressing the coat protein gene (CP, including the 3′ non-translated region) of Sweet potato feathery mottle virus. Transgenic plants carrying the 5′ 200 and 400 bp regions of CP were newly produced. From these plants, two silenced and two non-silenced lines were selected to investigate the manifestation of transitive RNA silencing by graft experiments. Non-silenced scions carrying the entire transgene were grafted onto either 5′ or 3′ silencing inducer rootstocks. When non-silenced scions were grafted onto 5′ silencing inducer rootstocks, RNA silencing was induced in the non-silenced scions and spread toward the 3′ region of the transgene mRNA. Similarly, when non-silenced scions were grafted onto 3′ silencing inducer rootstocks, RNA silencing was induced in the non-silenced scions, but was restricted to the 3′ region of the transgene and did not spread to the 5′ region. In addition, results from crossing experiments, involving non-silenced and 3′ silencing inducer plants, confirmed the above finding. This indicates that RNA silencing spreads in the 5′–3′ direction, not in the 3′–5′ direction, along the transgene mRNA.     

Redox reactivity of the heme Fe<Superscript>3+</Superscript>/Fe<Superscript>2+</Superscript> couple in native myoglobins and mutants with peroxidase-like activity

The reaction enthalpy and entropy for the one-electron reduction of the ferric heme in horse heart and sperm whale aquometmyoglobins (Mb) have been determined exploiting a spectroelectrochemical approach. Also investigated were the T67R, T67K, T67R/S92D and T67R/S92D Mb-H variants (the latter containing a protoheme-l-histidine methyl ester) of sperm whale Mb, which feature peroxidase-like activity. The reduction potential (E°′) in all species consists of an enthalpic term which disfavors Fe³⁺ reduction and a larger entropic contribution which instead selectively stabilizes the reduced form. This behavior differs from that of the heme redox enzymes and electron transport proteins investigated so far. The reduction thermodynamics in the series of sperm whale Mb variants show an almost perfect enthalpy–entropy compensation, indicating that the mutation-induced changes in are dominated by reduction-induced solvent reorganization effects. The modest changes in E°′ originate from the enthalpic effects of the electrostatic interactions of the heme with the engineered charged residues. The small influence that the mutations exert on the reduction potential of myoglobin suggests that the increased peroxidase activity of the variants is not related to changes in the redox reactivity of the heme iron, but are likely related to a more favored substrate orientation within the distal heme cavity.