三个山羊品种KAP8基因的PCR-SSCP分析

目的：为了筛选绒山羊产绒性状的候选基因。方法：采用PCR-SSCP法对三个山羊品种角蛋白辅助蛋白8（KAP8）进行了多态性研究。结果：三个品种中KAP8.1均未出现多态,而KAP8.2均出现了三种带型：AA、BB和AB。Hardy-Weinberg分析表明辽宁绒山羊和黎城大青羊未达到平衡状态,辽-苛高代杂种山羊达到了平衡。测序结果显示,和Genebank登陆序列相比,AA和BB均发生了碱基变化。其中AA型出现了以下变异：216bp（G-A）,217（A-G）,BB型出现了以下变异：216bp（G-A）,232bp（C-T）。分析表明AA型变异造成了氨基酸序列发生了变化（R-K）,而BB型突变未造成氨基酸发生改变。结论：KAP8.2可能是影响绒山羊产绒性状的基因之一。     

A single base transversion in the flanking region of an equine microsatellite locus affects amplification of one allele

The equine dinucleotide microsatellite HMS7 is part of a microsatellite panel utilized in a parentage verification programme at the Veterinary Genetics Laboratory (Davis, California, USA). Apparent non-Mendelian inheritance was noted when a Quarter Horse mare was excluded as the parent of two offspring based on analysis of the HMS7 locus. The mare's DNA type qualified her as a parent of the offspring at an additional 20 microsatellite loci. The three animals appeared homozygous for HMS7 with each possessing an allele different from that of the other two animals. Polymerase chain reaction primers designed to bind outside the published primer-binding sites amplified an additional shared allele in all three horses, which qualified the mare as the dam of the two offspring. Sequencing of this newly detected allele revealed a C to A transversion in one of the published primer-binding regions. Apparent non-Mendelian inheritance at the HMS7 locus has been encountered in an additional 26 Quarter Horse parentage cases. In all instances, the lack of amplification and resultant 'null' allele was shown to be caused by the same transversion.     

Rapid changes of microsatellite flanking sequence in the allopolyploidization of new synthesized hexaploid wheat

It was suggested that the rapid changes of DNA sequence and gene expression occurred at the early stages of allopolyploid formation. In this study, we revealed the microsatellite (SSR) differences between newly formed allopolyploids and their donor parents by using 21 primer sets specific for D genome of wheat. It was indicated that rapid changes had occurred in the “shock” process of the allopolyploid formation between tetraploid wheat and Aegilops tauschii. The changes of SSR flanking sequence resulted in appearance of novel bands or disappearance of parental bands. The disappearance of the parental bands showed much higher frequencies in comparison with that of appearance of novel bands. Disappearance of the parental bands was not random. The frequency of disappearance in tetraploid wheat was much higher than in Ae. tauschii, i. e. the disappearance frequency in AABB genome was much higher than in D genome. Changes of SSR flanking sequence occurred at the early stage of F₁ hybrid or just after chromosome doubling. From the above results, it can be inferred that SSR flanking sequence region was very active and was amenable to change in the process of polyploidization. This suggested that SSR flanking sequence probably had special biological function at the early stage of ployploidization. The rapid and directional changes at the early stage of polyploidization might contribute to the rapid evolution of the newly formed allopolyploid and allow the divergent genomes to act in harmony.     

Maximum likelihood estimation of the frequency of null alleles at microsatellite loci

We review three methods for estimating the frequency of null alleles at codominant loci (such as microsatellite loci) and present a new maximum likelihood approach. Computer simulations show that the maximum likelihood estimator has a smaller root mean squared error than previous estimators.     

Cross-species amplification of microsatellite primers in passerine birds

Developing species specific microsatellite primers can be avoidedby using existing markers which amplify across species. However,for passerines, such cross-species markers are mostly lackingand few guidelines exist for selecting them from the wide rangeof existing markers. Here cross-species amplification tests of 40microsatellite primers in 13 passerine species show an increasein probability of amplification and polymorphism with decreasingphylogenetic distance. Primers which successfully amplified inmany species had a higher chance to be polymorphic. However,since the amplification success, across a broad range of species,of particular primersets remains difficult to predict it iscrucial to identify such markers empirically. Here we describesuch widely applicable bird (passerines) microsatellite markers.     

DNA typing and microsatellite associated sequence amplification (MASA) in detecting loss or gain of alleles

Constant exposure of genomic DNA to a variety of damaging agents including radiations poses a major challenge for developing an experimental approach for monitoring such damages. No single approach can be used to address this issue. In the present article, we have provided examples of DNA typing and believe that various approaches may enable to uncover not only loss or gain(s) of the alleles but also reveal subtle changes in the genome. Similarly, our genome analysis of the animal systems provides a method to assess the effects of radiation on domestic animals.     

Co-segregation of alleles at a microsatellite locus within the macrophage expressed lysozyme gene and levels of serum lysozyme activity in two half-sib families of Polish Black and White Lowland cattle

Alleles at a microsatellite locus within the macrophage expressed lysozyme gene were shown to co-segregate with lysozyme activity in two half-sib families of Polish Black and White Lowland cattle. The bimodal distribution of lysozyme activities in both progeny groups is concordant with the occurrence of the alternative paternal alleles. The microsatellite is linked to a locus for high lysozyme activity that accounts for 70–95% of the phenotypic variation of both offspring groups considering the lysozyme activities of animals being older than 1 month.     

Evolution of a perfect simple sequence repeat locus in the context of its flanking sequence

Microsatellites, which have rapidly become the preferred markers in population genetics, reliably assign individual chinook salmon to the winter, fall, late-fall, or spring chinook runs in the Sacramento River in California's Central Valley (Banks et al. 2000. Can. J. Fish. Aquat. Sci. 57:915-927). A substantial proportion of this discriminatory power comes from Ots-2, a simple CA repeat, which is expected to evolve rapidly under the stepwise mutation model. We have sequenced a 300-bp region around this locus and typed 668 microsatellite-flanking sequence haplotypes to explore further the basis of this microsatellite divergence. Three sites of nucleotide polymorphism in the Ots-2 flanking sequence define five haplotypes that are shared by the Californian and Canadian populations. The Ots-2 microsatellite alleles are nonrandomly distributed among these five haplotypes in a pattern of gametic disequilibrium that is also shared among populations. Divergence between the winter run and other Central Valley stocks appears to be caused by a combination of surprisingly static evolution at Ots-2 within a context of more rapidly changing haplotype frequencies.     

Isolation and characterization of microsatellite loci in Merluccius australis and cross-species amplification

Eight novel and two heterologous microsatellite pairs of primers are presented for the Austral hake (Merluccius australis), representing the first microsatellite markers available for this species. Loci were characterized for 50 individuals from two populations in South America (Argentinean and Chilean coasts). All loci were polymorphic within M. australis (5 to 30 alleles per locus; observed heterozygosity between 0.320 and 0.840), and therefore useful for population genetic studies within the species. Cross-species transferability was tested for 100 individuals from four additional species within the Merluccius genus (M. albidus, M. bilinearis, M. gayi and M. hubbsi), and results indicate that most of these primers pairs will likely be useful for population genetic studies on Merluccius species.     

PERMANENT GENETIC RESOURCES: Development of microsatellite markers in Rhinanthus angustifolius and cross-species amplification

Fifteen polymorphic microsatellite loci were developed from an enriched genomic library of the annual plant Rhinanthus angustifolius and characterized using 36 individuals. These markers provided high polymorphism ranging from two to 15 alleles per locus. Four loci showed significant departure from Hardy-Weinberg equilibrium, probably because of the occurrence of null alleles. No significant linkage disequilibrium was detected between pairs of loci. Tests of cross-species transferability were performed on four congeners with a success rate of 100% in Rhinanthus minor, 93% in R. mediterraneus and R. glacialis, and 80% in R. alectorolophus. These microsatellite loci will be useful tools to study mating system, gene flow and hybridization in the genus Rhinanthus.     

Mutations in the KIAA0196 gene at the SPG8 locus cause hereditary spastic paraplegia

Hereditary spastic paraplegia (HSP) is a progressive upper-motor neurodegenerative disease. The eighth HSP locus, SPG8, is on chromosome 8p24.13. The three families previously linked to the SPG8 locus present with relatively severe, pure spastic paraplegia. We have identified three mutations in the KIAA0196 gene in six families that map to the SPG8 locus. One mutation, V626F, segregated in three large North American families with European ancestry and in one British family. An L619F mutation was found in a Brazilian family. The third mutation, N471D, was identified in a smaller family of European origin and lies in a spectrin domain. None of these mutations were identified in 500 control individuals. Both the L619 and V626 residues are strictly conserved across species and likely have a notable effect on the structure of the protein product strumpellin. Rescue studies with human mRNA injected in zebrafish treated with morpholino oligonucleotides to knock down the endogenous protein showed that mutations at these two residues impaired the normal function of the KIAA0196 gene. However, the function of the 1,159-aa strumpellin protein is relatively unknown. The identification and characterization of the KIAA0196 gene will enable further insight into the pathogenesis of HSP.     

Isolation and characterization of microsatellite loci in Babylonia areolata and cross-species amplification in Babylonia formosae habei

Nine novel microsatellite primer pairs were presented for Babylonia areolata, representing the first microsatellite markers available for this genus. Levels of polymorphism were variable with 2 to 11 alleles per locus and expected heterozygosities ranging from 0.073 to 0.907 in 27 individuals of the population from which the loci were isolated. We found significant heterozygote deficit at one locus that might be attributable to null alleles. We were successful at cross-amplifying six loci in the congeneric B. formosae habei. These markers are therefore potentially useful for conservation studies, population structure assessment, ecological analyses and linkage map construction.     

Development of tetranucleotide microsatellite markers for the cushion star, Asterina gibbosa, and cross-species amplification

We describe nine polymorphic tetranucleotide microsatellite loci from the starfish, Asterina gibbosa. Loci were isolated from a partial genomic library that had been enriched for AAAC repeat sequences. Number of alleles per locus ranged from two to 14 in a sample of 85 individuals from three populations (two from Spain and one from the UK). Observed and expected heterozygosities per population ranged from 0.000 to 0.400 and from 0.040 to 0.784, respectively. All loci presented significant heterozygote deficits in one or more populations. Eight of these loci were amplified and variable in A. pancerii and A. phylactica. These loci will be used to study population structure in A. gibbosa.     

Cross-species amplification and optimization of microsatellite markers for use in six Neotropical parrots

Short amplicon primers were redesigned for 17 microsatellite loci developed in St. Vincent's Amazon and six loci developed in blue-and-yellow macaw and tested using six species of Neotropical parrot. Polymorphism was observed at 12 loci in blue-and-yellow macaw, 10 in red-and-green macaw, 11 in scarlet macaw, 10 in chestnut-fronted macaw, 11 in red-bellied macaw and 16 in mealy parrot. Number of alleles per locus ranged from two to 23 and expected heterozygosity ranged from 0.05 to 0.95. The resulting multiplexed loci will be useful in evaluating genetic diversity, genetic structure and mating system in Neotropical parrots.     

Cross‐species amplification at microsatellite loci in Australian quolls including the description of five new markers from the Chuditch (Dasyurus geoffroii)

The quolls are the largest native predators remaining on mainland Australia. We describe the cross‐species testing of all available microsatellite loci for representative across the entire distribution of quolls, with the additional characterization of five new microsatellite loci from the Chuditch. All primers produced clear and polymorphic amplification patterns containing between nine and 17 alleles and with high levels of diagnostic variability. These highly polymorphic primers make them useful additional tools in planning conservation strategies across related conspecifics, many of which are under threat.     

aPCR-SSCP and DNA sequencing detecting two silent SNPs at KAP8.1 gene in the cashmere goat

Keratin-associated proteins 8.1 gene (KAP8.1) is a structural gene responsible for the cashmere. KAP8.1 protein contains high glycine and tyrosine, which concerns regulation and function of the matrix structure fiber. In this study, the polymorphism of KAP8.1 gene was detected by methods of aPCR-SSCP (asymmetric polymerase chain reaction single-strand conformation polymorphism) and DNA sequencing in 791 individuals from two breeds. The results showed that there were two mutations in this gene. The mutations were described as c.63 T>G and c.66 C>G, which would result in two synonymous mutations in KAP8.1 protein. The findings go against previous research, in which there was not polymorphism at KAP8.1 gene. The reasons might be that different cashmere breeds were detected in two studies. Further analysis of results leads us to believe that the polymorphism of KAP8.1 gene might be relevant to fiber diameter.     

五年制高职护理学生乙型肝炎KAP及需求调查

目的：通过了解高职护理学生对乙型肝炎（简称＂乙肝＂）流行病学知识及在医疗护理过程中对乙肝病毒感染防护措施的知晓程度,了解感染乙肝病毒（HBV）后对学生的心理行为和社会功能的影响,为今后有针对性地开展学校传染病管理和宣传工作提供依据。方法：对盐城卫生职业学院2006级552名高职护理学生采用问卷调查的方式进行乙肝相关知识及职业暴露防护相关措施的调查。结果：高职护理学生对乙肝相关知识的了解比较丰富,主要原因是接受过乙肝流行病学的专业学习;但对在医疗护理过程中预防乙肝病毒感染的措施缺乏了解,并且感染乙肝后对其心理、学习、人际交往等方面均有不同程度的影响。结论：高职护理学生乙肝相关知识掌握的较丰富,但就预防乙肝病毒感染和病毒感染后的行为水平较低,学校应加强学生的职业暴露防护及健康心理行为的教育。