Characterization of the COMMD1 (MURR1) mutation causing copper toxicosis in Bedlington terriers

Copper toxicosis is an autosomal recessive disorder affecting Bedlington terriers, characterized by elevated liver copper levels and early death of affected dogs. Genetic linkage mapping studies initially identified linkage between the disease and the microsatellite marker C04107. Subsequently, the deletion of exon 2 of the copper metabolism domain containing 1 (COMMD1) gene (formerly MURR1) was shown to be the major cause of copper toxicosis, although the deletion breakpoints were not defined. In this investigation, polymerase chain reaction (PCR)-based techniques and sequencing were used to isolate the deletion breakpoints, utilizing the newly available dog genome sequence. The breakpoints were positioned at 65.3091 and 65.3489 Mb of dog chromosome 10, in intron 1 and intron 2 of COMMD1 respectively, a deletion of 39.7 kb. The two breakpoints share sequence homology suggesting that homologous recombination may have been responsible for the deletion. Using this information, a genomic diagnostic test for the COMMD1 deletion was developed and compared with microsatellite C04107 genotypes of 40 Bedlington terriers. Results from the 40 samples showed allele 2 of C04107 to be in linkage disequilibrium with the COMMD1 deletion.     

Evaluation of the present breeding programme against copper toxicosis in Danish Bedlington terriers

A breeding programme to eradicate copper toxicosis in Danish Bedlington terriers has been established based on a DNA marker test. Genotyping of both parents is compulsory and after 1 January 2000, only homozygous non-carriers are used for breeding. In this study, two groups of Bedlington terriers were genotyped at 18 microsatellite loci. One group represented the original population of Bedlington terriers before introducing the breeding programme (n = 23); the other represented a group of homozygous non-carriers (n = 24) available for breeding after year 2000. Allele numbers, allele frequencies, observed heterozygosities (Ho), expected heterozygosities (He), locus-specific coefficients of inbreeding (Fl) and Nei's genetic distance (D) was calculated. Individual coefficients of inbreeding (Fi) were calculated from the pedigrees and an assignment test was performed. Four rare alleles were lost in the group of homozygous non-carriers. No significant differences were observed between the mean values of allele numbers, Ho, He, Fl and Fi of the two populations of dogs. Nei's genetic distance between the two populations was 0.06 and 88% of the homozygous non-carriers were assigned correctly in the assignment test. The overall diversity of the breed was low (Ho = 0.41) and the breeders were advised to include the heterozygous carriers again.     

Inherited copper toxicosis with emphasis on copper toxicosis in Bedlington terriers

Canine copper toxicosis is an important inherited disease in Bedlington terriers, because of its high prevalence rate and similarity to human copper storage disease. It can lead to chronic liver disease and occasional haemolytic anaemia due to impaired copper excretion. The responsible gene for copper toxicosis in Bedlington terriers has been recently identified and was found not to be related to human Wilson’s disease gene ATP7B. Although our understanding of copper metabolism in mammals has improved through genetic molecular technology, the diversity of gene mutation related to copper metabolism in animals will help identify the responsible genes for non-Wilsonian copper toxicoses in human. This review paper discusses our knowledge of normal copper metabolism and the pathogenesis, molecular genetics and current research into copper toxicosis in Bedlington terriers, other animals and humans.     

微卫星DNA在分子遗传标记研究中的应用

随着种群遗传学的发展 ,分子遗传标记特别是微卫星标记已经成为研究种群遗传的有力工具。本文就微卫星遗传标记的研究背景、技术应用以及优势与不足等方面进行了综述。     

Development of microsatellite DNA markers of silver carp (Hypophthalmichthys molitrix) and their cross‐species application in bighead carp (Aristichthys nobilis)

Forty‐four microsatellite DNA markers were developed for silver carp, and used to investigate polymorphisms of 41 wild silver carps and seven wild bighead carps collected from Jingzhou fragment of Yangtze River. In silver carp, 40 markers were polymorphic. A total of 297 alleles were detected at 40 polymorphic loci. The number of alleles per locus varied from two to 16 with an average of 7.4 and the expected heterozygosities of each locus ranged from 0.07 to 0.91 with an average of 0.69. All markers amplified both silver carp and bighead carp DNAs.     

Toward developing a genome-wide microsatellite marker set for linkage analysis in the rhesus macaque (Macaca mulatta): identification of 76 polymorphic markers

Linkage analysis can be problematic in humans because of the lack of large, multigenerational pedigrees and the difficulties in obtaining phenotypic data on all family members. In contrast, large, captive colonies of rhesus macaque are a potentially valuable resource for linkage studies because detailed phenotypic and genealogical data are kept, inbreeding is avoided, and DNA samples can usually be obtained. Microsatellite marker sets for genome-wide screening are available in a number of species, but not for the rhesus macaque. We tested primers to 400 human microsatellite markers from a genome-wide mapping set using DNA from nine unrelated female rhesus macaques. We found that 76 (19%) of the primers amplified a polymorphic product using the standard protocols for human DNA. The average heterozygosity of the markers in humans was 0.80, compared to 0.65 in the rhesus macaques. This study provides preliminary data, which could be used toward the development of a linkage mapping set in this species. There would be a need, however, to confirm the Mendelian inheritance of the markers.     

Isolation and characterization of novel tetranucleotide microsatellite DNA markers for the spotted salamander,Ambystoma maculatum

Fifteen tetranucleotide microsatellite loci were identified and characterized for spotted salamanders (Ambystoma maculatum) collected from three vernal pools in the south‐eastern USA. These markers revealed a high degree of genetic diversity (7–32 alleles per locus), heterozygosity (31.6–86.3%) and allelic heterogeneity (91% of comparisons were statistically significant). Considerable differentiation among populations was observed as genetic distances (chord) ranged between 0.50 and 0.65, and all F_ST values (0.08–0.14) were statistically significant. Moreover, genotypic assignment tests correctly classified all individuals to their respective collection. These markers should prove useful for investigating fine‐scale population structure and mating system.     

Allozyme and microsatellite DNA markers of toothfish population structure in the Southern Ocean

Toothfish represent a major finfish resource in the Southern Ocean, where there has been a rapid increase in exploitation of the Patagonian toothfish Dissostichus eleginoides. Molecular genetic methods have been applied to address questions relating to stock discrimination in D. eleginoides. Eight microsatellite loci were tested in population samples from the Atlantic, Indian, and Pacific Ocean sectors of the Southern Ocean. Some loci were characterised by a large number of alleles, but no alleles were restricted to specific ocean basins. However there was a significant allelic heterogeneity in the total data indicative of population differentiation, and a high level of genetic sub-division was measured with F _ST and R _ST. Seven polymorphic allozyme loci revealed no significant heterogeneity among Pacific and Indian Ocean sector samples. The microsatellite DNA heterogeneity suggests that there is restricted gene flow through the Southern Ocean, and that the different fishing grounds may support independent stocks.     

Variation of 15 autosomal microsatellite DNA loci in the Russian population

The allele frequencies of 15 autosomal STR loci (D3S1358, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317, D7S820, D16S539, D2S1338, D8S1179, D21S11, D18S51, and D19S433) used in forensic medicine were determined for the Russian population of European Russia (N = 176). The power of discrimination (PD) and power of exclusion (PE) of the system of the 15 STR loci were 0.999 999 999 999 999 986 and 0.999 999 331 310 171 000, respectively. The allele and genotype frequency distributions in the Russian population corresponded to the Hardy-Weinberg equilibrium. The D2S1338, D18S51, D21S11, and FGA loci were identified as the most informative markers for the Russian population and proposed as a reference for forensic studies in the Russian Federation.     

Cloning and characterization of novel microsatellite DNA markers for the grass rockfish,Sebastes rastrelliger,and cross‐species amplification in 10 related Sebastes spp

Here we report development and characterization of seven polymorphic loci derived from grass rockfish (Sebastes rastrelliger) genomic DNA phagemid libraries enriched for microsatellite motifs. Within grass rockfish, average allelic diversity was 11.3 alleles per locus and average heterozygosity was 0.73. The seven loci also were surveyed in 10 related species of Sebastes, where allelic diversity ranged from highly polymorphic to monomorphic. Deviations from Hardy–Weinberg were nonsignificant in all but one species/locus combination suggesting low occurrence of null alleles. Significant linkage disequilibrium was detected among three loci, but these events were limited to a single species in each case.     

Isolation and characterization of a female-specific DNA marker in the giant freshwater prawn Macrobrachium rosenbergii

In this study, a female-specific DNA marker in the freshwater prawn Macrobrachium rosenbergii was identified through amplified fragment length polymorphism (AFLP). The AFLP-derived sequence-characterized amplified region (SCAR) marker was tested in over 200 individuals, giving reproducible sex identification. Further molecular characterization of the sex-marker's genomic region (～ 3 kb long) revealed the presence of tandem and inverted repeats. The ～ 3-kb sequence was identified both in male and female prawns, but with subtle differences: a deletion of 3 bp (present in female prawn but absent in male prawn) identified upstream of the SCAR marker sequence and two female-specific single-nucleotide polymorphisms, both indicating that male prawns are homozygous, whereas female prawns are heterozygous in this locus. Fluorescent in situ hybridization showed the ～ 3-kb sequence to be unique: to the best of our knowledge, this is the first report of a unique sex-specific sequence observed in situ in crustaceans. The sex-specific marker identified in M. rosenbergii may have considerable applied merit for crustacean culture in that it will enable the determination of genetic sex at early developmental stages when phenotypic differences are not identifiable.     

Cloacal and buccal swabs are a reliable source of DNA for microsatellite genotyping of reptiles

In this study, a minimally invasive method for DNA sampling of reptiles and amphibians using cloacal and buccal swabs is described. High molecular weight DNA was isolated from the swabs, which were collected from tuatara (Sphenodon punctatus), and stored in 70% ethanol at room temperature for approximately 1 week. Amplification of mitochondrial and microsatellite DNA loci was successful from both cloacal and buccal swabs, and in all cases the genotypes matched those obtained from blood samples. These results show that cloacal and/or buccal swabbing is a useful alternative to blood sampling and toe clipping for genetic studies on reptiles. This method is rapid, inexpensive and easy to implement in field situations.     

商城肥鲵二碱基重复和四碱基重复微卫星DNA的结构特征及对筛选效率的影响

两栖类有尾目物种的微卫星分离中的筛选成功率常常较低。为探索微卫星结构对筛选效率的影响,本研究通过AFLP快速分离法(fast isolation by AFLP of sequences containing repeats,FIASCO)对商城肥鲵(Pachyhynobius shangchengensis)二碱基重复类型和四碱基重复类型微卫星进行分离,并对微卫星序列进行了分析。研究中发现二碱基微卫星位点多以微卫星DNA家族形式存在,并因此导致了微卫星位点分离较低的筛选率;在四碱基重复的微卫星位点中未发现微卫星DNA家族的存在。对研究中得到的3个微卫星DNA家族的分析发现,同一家族的上、下游侧翼序列变异程度存在差异;毗邻微卫星重复单元区的侧翼序列碱基变异程度较高,而较远处的区段则相对保守。这些结构特征可能反映出微卫星DNA家族在演化中的复杂性。本文的研究结果提示在两栖动物的一些类群中,微卫星的筛选必须考虑微卫星DNA家族的影响,选取适宜的碱基重复类型将是决定筛选效率的关键。     

Characterization of 35 novel microsatellite DNA markers from the duck (Anas platyrhynchos) genome and cross-amplification in other birds

In order to study duck microsatellites, we constructed a library enriched for (CA)n, (CAG)n, (GCC)n and (TTTC)n. A total of 35 pairs of primers from these microsatellites were developed and used to detect polymorphisms in 31 unrelated Peking ducks. Twenty-eight loci were polymorphic and seven loci were monomorphic. A total of 117 alleles were observed from these polymorphic microsatellite markers, which ranged from 2 to 14 with an average of 4.18 per locus. The frequencies of the 117 alleles ranged from 0.02 to 0.98. The highest heterozygosity (0.97) was observed at the CAUD019 microsatellite locus and the lowest heterozygosity (0.04) at the CAUD008 locus, and 11 loci had heterozygosities greater than 0.50 (46.43%). The polymorphism information content (PIC) of 28 loci ranged from 0.04 to 0.88 with an average of 0.42. All the above markers were used to screen the polymorphism in other bird species. Two markers produced specific monomorphic products with the chicken DNA. Fourteen markers generated specific fragments with the goose DNA: 5 were polymorphic and 9 were monomorphic. But no specific product was detected with the peacock DNA. Based on sequence comparisons of the flanking sequence and repeat, we conclude that 2 chicken loci and 14 goose loci were true homologous loci of the duck loci. The microsatellite markers identified and characterized in the present study will contribute to the genetic map, quantitative traits mapping, and phylogenetic analysis in the duck and goose.     

Isolation of 39 polymorphic microsatellite loci and the development of a fluorescently labelled marker set for the Eurasian badger (Meles meles) (Carnivora: Mustelidae)

We have isolated 78 microsatellite loci from the Eurasian badger (Meles meles). Of the 52 loci characterized, 39 were found to be polymorphic. A fluorescently labelled primer set was developed to enable individual‐specific 17‐locus genotypes to be obtained efficiently.     

Isolation and characterization of microsatellite DNA loci in the toad‐headed lizards,Phrynocephalus przewalskii complex

To assess mechanisms of hybridization and speciation, we isolated and characterized 12 dinucleotide microsatellite DNA loci for the toad‐headed lizards, Phrynocephalus przewalskii complex. A total of 48 specimens were examined and all loci were polymorphic with seven to 25 alleles per locus. The observed and expected heterozygosities ranged from 0.282 to 0.946 and 0.400 to 0.937, respectively. These loci are therefore suitable for a wide range of population level studies within the P. przewalskii complex.