湖北钉螺多态微卫星DNA位点筛选和特征初步分析

目的分离湖北钉螺的微卫星DNA序列,筛选多态的微卫星DNA位点并分析其特征。方法应用湖北钉螺基因组DNA的酶切片段与生物素标记的(AAT)17、(GA)25、(CCT)17、(CA)25等10个寡核苷酸探针杂交,富集、浓缩、克隆并测序,构建微卫星DNA库。挑选合适的微卫星DNA位点设计并合成引物,扩增钉螺样本经聚丙烯酰胺凝胶电泳筛选多态性。结果获得湖北钉螺微卫星DNA序列205条,GenBank注册登记号GU204044～GU204248,其中完整重复序列74条,占36.10%;非完整重复序列102条,占49.76%;复合重复序列29条,占14.15%。设计合成的20对微卫星DNA位点引物中,经鉴定显示13个位点具有多态性。结论分离建立了湖北钉螺微卫星DNA序列库,为湖北钉螺群体遗传、种群溯源等相关研究提供了分子标志。     

分子标记技术在寄生虫分类鉴定中的应用

寄生虫在生物学上有差异的亚种和株具有不同的致病性, 其分类学对于寄生虫病的病原学、流行病学和防治等方面具有实际意义。DNA分子标记是以生物大分子的多态性为基础的遗传标记, 具备多态性高、无基因多效性、能够明确辨别等位基因等优点。本文综述第1代（限制性片段长度多态性、随机扩增多态性DNA等）、第2代（微卫星锚定PCR、简单重复序列间扩增等）和第3代（单核苷酸多态性）分子标记技术在寄生虫分类鉴定中的应用。     

中华白蛉微卫星DNA序列的分离和多态位点筛选的初步研究

目的 分离中华白蛉的微卫星DNA序列,并筛选其中具有多态性的位点。 方法 应用中华白蛉基因组DNA的酶切片段与生物素标记的寡核苷酸探针（AAT）₁₇、（GA）₂₅、（CCT）₁₇和（TG）₁₈杂交,亲合素富集和超滤离心浓缩目的片段,扩增放大后克隆并测序,构建中华白蛉微卫星DNA库。挑选合适的微卫星位点,建立PCR扩增体系。应用中华白蛉现场标本对不同的微卫星DNA进行扩增,聚丙烯酰胺凝胶电泳筛选具有多态性的位点。 结果 本研究分离中华白蛉微卫星DNA的方法效率高,重组克隆阳性率为78.6%。构建的微卫星DNA库含有118条序列,GenBank登录号为FJ919812～FJ919932（登录号为FJ919833、FJ919836和FJ919869除外）,其中典型微卫星DNA序列72条（占61.0%）,非典型序列46条。在构建的中华白蛉微卫星DNA库中选择22个位点进行多态性筛选,电泳结果显示14个为多态位点,双核苷酸重复的位点比三核苷酸和多核苷酸重复位点的多态性高。 结论 首次构建了含有118条序列的中华白蛉微卫星DNA库,共获得14个新的多态微卫星位点。     

新疆哈萨克民族血管紧张素原基因GT重复序列多态性与高血压病相关性研究

目的探讨研究血管紧张素原基因3'端GT重复序列多态性与新疆哈萨克族人原发性高血压的关系.方法以人群为基础进行病例-对照研究,应用PCR扩增和毛细管电泳和GeneticProfiler自动分析软件进行微卫星基因组扫描分析技术,检测89例原发性高血压患者与81例正常对照者的AGT的3'端GT重复序列多态性.结果血管紧张素原基因3'端GT重复序列存在13种等位基因按片段由小至大分别命名为A1、A2、A3……(GT的重复次数依次为13、14、15……),扩增长度为160bp-184bp,其频率介于0.003～0.197间.以扩增片段长度为170bp的A6等为基因最为常见,其次为A3和A4,分布符合Hardy-Weinberg遗传平衡定律.该位点杂合度为0.862,多态信息量为0.847.研究表明,哈萨克族血管紧张素原基因3'端GT重复序列在原发性高血压人群和正常人群的分布无显著性差异(x2=1.773,P＞0.05).结论AGT基因3'端GT重复序列多态性与新疆哈萨克族人高血压病不相关.     

微卫星DNA在寄生虫学研究中的应用

微卫星DNA(Microsatellite DNA)，又称为短小串联重复(Short Tandem Repeats，STR)或简单重复序列(simple repeat sequence，SRS或SSR)，是近年来飞速发展起来的一种新型的DNA高度多态性遗传标记系统，具有种类多分布广泛、高度多态性、杂合性高、重组率低的特点，在群体中变异范围大，构成了丰富的长度多态性，有高度的个体特异性，并遵循     

Na-K-2Cl转运蛋白基因5′端GT重复序列多态性与新疆哈萨克民族原发性高血压的相关性

目的:探讨研究Na-K-2Cl转运蛋白(NKCC2,SLC12A1,BSC-1)基因GT重复序列多态性与新疆哈萨克族人原发性高血压(EH)的关系。方法:以人群为基础进行病例-对照研究,随机选取新疆牧区30~55岁的哈萨克族牧民303例(EH患者152例,正常对照151例),采用经典的饱和酚/氯仿抽提法提取白细胞基因组DNA,运用荧光标记多聚酶链式反应(PCR)技术、毛细管电泳和Genetic Profiler自动分析软件进行微卫星基因组扫描分析技术,根据片断大小分离等位基因。检测EH患者与正常对照者的NKCC2基因GT重复序列多态性。结果:在所有研究人群中NKCC2基因重复序列存在14种等位基因(A1~A14),扩增长度为210~238 bp,其频率介于0.002~0.281,以扩增长度为224 bp的A9等位基因最为常见,其次为A5和A8,分布符合Hardy-Weinberg遗传平衡定律,GT重复多态杂合率为0.812,多态信息量为0.803。EH人群NKCC2基因GT重复多态分布与正常人群差异无统计学意义(χ2=16.885,P>0.05)。结论:NKCC2基因5′端GT重复多态性标记与新疆哈萨克民族EH可能不相关。     

Na-K-2C1转运蛋白基因5′端GT重复序列多态性与新疆哈萨克民族原发性高血压的相关性

目的:探讨研究Na-K-2Cl转运蛋白(NKCC2,SLC12A1,BSC-1)基因GT重复序列多态性与新疆哈萨克族人原发性高血压(EH)的关系.方法:以人群为基础进行病例-对照研究,随机选取新疆牧区30～55岁的哈萨克族牧民303例(EH患者152例,正常对照151例),采用经典的饱和酚/氯仿抽提法提取白细胞基因组DNA,运用荧光标记多聚酶链式反应(PCR)技术、毛细管电泳和Genetic Profiler自动分析软件进行微卫星基因组扫描分析技术,根据片断大小分离等位基因.检测EH患者与正常对照者的NKCC2基因GT重复序列多态性.结果:在所有研究人群中NKCC2基因重复序列存在14种等位基因(A1～A14),扩增长度为210～238 bp,其频率介于0.002～0.281,以扩增长度为224 bp的A9等位基因最为常见,其次为A5和A8,分布符合Hardy-Weinberg遗传平衡定律,GT重复多态杂合率为0.812,多态信息量为0.803.EH人群NKCC2基因GT重复多态分布与正常人群差异无统计学意义(x2=16.885,P＞0.05).结论:NKCC2基因5′端GT重复多态性标记与新疆哈萨克民族EH可能不相关.     

应用微卫星DNA技术研究蚊虫群体遗传的现状

微卫星DNA是目前较为理想的群体遗传研究的分子标志,该文阐述了微卫星DNA的特点和获取途径,综述了已分离的多态性微卫星DNA的蚊种及对其进行群体遗传研究的现状。     

应用微卫星DNA技术研究蚊虫群体遗传的现状

微卫星DNA是目前较为理想的群体遗传研究的分子标志，该文阐述了微卫星DNA的特点和获取途径，综述了已分离的多态性微卫星DNA的蚊种及对其进行群体遗传研究的现状。     

用微卫星分析我国不同地区间日疟原虫的种群结构

目的 了解我国不同疟疾流行区间日疟原虫种群结构和遗传多样性特征,积累我国间日疟原虫遗传相关数据.方法 收集云南、海南、河南流行区间日疟患者血样,血涂片鉴定间日疟原虫阳性者抽提血液基因组,采用巢式/半巢氏PCR方法扩增特异性2.21微卫星片段,对扩增阳性产物进行基因扫描检测,根据检测微卫星的重复序列进行STR分型,并应用GENALEX软件计算等位基因频率、等位基因数目以及期望杂合度(expected heterozygosity,He).结果 间日疟原虫2.21微卫星在不同地区间日疟原虫样本中呈现高度的多态性,其等位基因数目变化范围为4～9,期望杂合度为0.613～0.853,比较不同地区,云南地区间日疟原虫等位基因数目为9,期望杂合度为0.853,变异度最高.结论我国不同地区间日疟原虫基因组具有较高的遗传多样性,其种群结构具有地区特征,这可能与各种群传疟媒介种类不同及地理环境差异等相关.     

Genetic analysis of traditional and evolved Basmati and non-Basmati rice varieties by using fluorescence-based ISSR-PCR and SSR markers

The objective of the present study was to make use of efficient molecular marker systems to reveal genetic relationships in traditional and evolved Basmati (EB) and semidwarf non-Basmati (NB) rice varieties. A subset of three rice groups was analyzed by using 19 simple sequence repeat (SSR) loci and 12 inter-SSR-PCR primers. A total of 70 SSR alleles and 481 inter-SSR-PCR markers were revealed in 24 varieties from the three groups. The lowest genetic diversity was observed among the traditional Basmati varieties, whereas the EB varieties showed the highest genetic diversity by both the marker assays. The results indicated that the subset of aromatic rice varieties analyzed in the present study is probably derived from a single land race. The traditional Basmati (TB) and semidwarf NB rice varieties used in the present study were clearly delineated by both marker assays. A number of markers, which could unambiguously distinguish the TB varieties used in the present study from the evolved and NB rice varieties, were identified. The potential use of these markers in Basmati rice-breeding programs and authentication of TB varieties used in the present study are envisaged.     

Plasmodium falciparum populations from northeastern Myanmar display high levels of genetic diversity at multiple antigenic loci

Levels of genetic diversity of the malaria parasites and multiclonal infections are correlated with transmission intensity. In order to monitor the effect of strengthened malaria control efforts in recent years at the China–Myanmar border area, we followed the temporal dynamics of genetic diversity of three polymorphic antigenic markers msp1, msp2, and glurp in the Plasmodium falciparum populations. Despite reduced malaria prevalence in the region, parasite populations exhibited high levels of genetic diversity. Genotyping 258 clinical samples collected in four years detected a total of 22 PCR size alleles. Multiclonal infections were detected in 45.7% of the patient samples, giving a minimum multiplicity of infection of 1.41. The majority of alleles experienced significant temporal fluctuations through the years. Haplotype diversity based on the three-locus genotypes ranged from the lowest in 2009 at 0.33 to the highest in 2010 at 0.80. Sequencing of msp1 fragments from 36 random samples of five allele size groups detected 13 different sequences, revealing an additional layer of genetic complexity. This study suggests that despite reduced prevalence of malaria infections in this region, the parasite population size and transmission intensity remained high enough to allow effective genetic recombination of the parasites and continued maintenance of genetic diversity.     

长江下游3省湖北钉螺不同地理种群的群体遗传结构研究

目的 目的 对长江下游江西、 安徽和江苏3省湖北钉螺的不同地理种群进行群体遗传结构分析, 以探讨rDNA?ITS分 子标记在钉螺扩散路径监测中的应用价值。 方法 方法 采集3省9个种群的钉螺样本, 提取基因组DNA, PCR特异性扩增rD? NA?ITS基因并克隆测序, 统计分析群体间的遗传分化系数 （Fst ）、 遗传距离和种群遗传多样性等参数, 利用单倍型构建家系 网络图。结果 结果 9个种群共获得有效序列93条, 检测到78个单倍型, 平均单倍型多样性、 核苷酸多样性分别为0.988和 0.012 88, 表明钉螺种群的遗传多样性水平较高; 其遗传变异主要来自群体内, 种群间遗传距离为0.001 6～0.002 3, 显示钉 螺种群间遗传分化程度较高; 家系网络图显示所有单倍型虽可形成3个主要的家系分支, 但各地理种群在家系网络图中无 明显分化。结论 结论 长江下游江西、 安徽和江苏3省沿长江分布的湖北钉螺群体遗传多样性主要存在个体间, 群体间未形成 明显的遗传分化。rDNA?ITS分子标记在钉螺扩散路径监测中的应用价值值得进一步探讨。     

利用ssr标记浅析云南登革热重点地区埃及伊蚊种群遗传特征

目的 通过研究云南省不同地理来源的埃及伊蚊自然种群遗传结构,探究不同群体间的遗传特征与联系.方法 捕获景洪、勐腊、勐海、耿马、瑞丽的埃及伊蚊,提取DNA并采用12对荧光标记的微卫星引物进行PCR扩增,用毛细管电泳检测扩增片段,分析5个埃及伊蚊自然种群的遗传相关指标.结果 在5个采集点共采集234个有伊蚊幼虫的容器,采集...     

Population structure and transmission dynamics of Plasmodium vivax in rural Amazonia

Understanding the genetic structure of malaria parasites is essential to predict how fast some phenotypes of interest originate and spread in populations. In the present study, we used highly polymorphic microsatellite markers to analyze 74 Plasmodium vivax isolates, which we collected in cross-sectional and longitudinal surveys performed in an area of low malaria endemicity in Brazilian Amazonia, and to explore the transmission dynamics of genetically diverse haplotypes or strains. P. vivax populations are more diverse and more frequently comprise multiple-clone infections than do sympatric Plasmodium falciparum isolates, but these features paradoxically coexist with high levels of inbreeding, leading to significant multilocus linkage disequilibrium. Moreover, the high rates of microsatellite haplotype replacement that we found during 15 months of follow-up most likely do not result from strong diversifying selection. We conclude that the small-area genetic diversity in P. vivax populations under low-level transmission is not severely constrained by the low rates of effective meiotic recombination, with clear public health implications.     

Reduced genetic variation and the success of an invasive species

Despite the severe ecological and economic damage caused by introduced species, factors that allow invaders to become successful often remain elusive. Of invasive taxa, ants are among the most widespread and harmful. Highly invasive ants are often unicolonial, forming supercolonies in which workers and queens mix freely among physically separate nests. By reducing costs associated with territoriality, unicolonial species can attain high worker densities, allowing them to achieve interspecific dominance. Here we examine the behavior and population genetics of the invasive Argentine ant (Linepithema humile) in its native and introduced ranges, and we provide a mechanism to explain its success as an invader. Using microsatellite markers, we show that a population bottleneck has reduced the genetic diversity of introduced populations. This loss is associated with reduced intraspecific aggression among spatially separate nests, and leads to the formation of interspecifically dominant supercolonies. In contrast, native populations are more genetically variable and exhibit pronounced intraspecific aggression. Although reductions in genetic diversity are generally considered detrimental, these findings provide an example of how a genetic bottleneck can lead to widespread ecological success. In addition, these results provide insights into the origin and evolution of unicoloniality, which is often considered a challenge to kin selection theory.     

Genetic variability in geographical populations of Culex quinquefasciatus Say (Diptera: Culicidae) from India based on random amplified polymorphic DNA analysis

Genetic variability and environmental factors may influence the refractiveness, propagation of pathogen and transmission of disease. Random amplified polymorphic DNA (RAPD) is one of the widely used molecular markers for population genetic diversity studies. In present study, RAPD is used to ascertain the genetic variability in Culex quinquefasciatus populations collected from various Indian geographical locations. Out of 50 RAPD primers screened, 14 primers exhibited clear, concrete and distinct banding pattern showing up to 100% polymorphism. Primer OPBD3 was tested with DNA of 14 geographical populations from India (including one laboratory population) showed 21 loci representing 14 populations with 100% polymorphism. The genetic diversity among the populations indicated the Shannon index (I) and gene diversity index (H_ST), 0.48 and 0.31, respectively among the population, displaying rich genetic variation among the Cx. quinquefasciatus populations. Consensus tree showed two clusters indicating the genetic variation among the various geographical populations. The findings of this study may be useful to understand the population variation under different ecological conditions and development of effective vector management strategies.     

Genetic characterization of Trypanosoma brucei gambiense and clinical evolution of human African trypanosomiasis in Côte d'Ivoire

Human African trypanosomiasis is a parasitic infection caused by protozoa belonging to Trypanosoma brucei subspecies. The clinical evolution of this disease is complex and might be because of the parasite itself, as genetic diversity has been observed in T. brucei ssp. We investigated the relationship between the genetic diversity of trypanosomes and the diversity of clinical patterns in Côte d'Ivoire. We studied clinical sleeping sickness cases, and genetically analysed the trypanosomes isolated from these patients. An important genetic monomorphism among stocks isolated in Côte d'Ivoire was observed by using various markers: isoenzymes electrophoresis, random amplified polymorphism DNA and PCR of microsatellite sequences. At the same time, the diversity of clinical patterns and evolutions was confirmed by clinical analysis. The existence of an individual susceptibility to disease (human trypanotolerance) should be taken into account even if our genetic conclusions might be distorted because the isolation success rates were particularly poor. In fact, we observed that the isolation success rate varied significantly depending both on the focus of origin (P=0.0002) and on the ethnic group (P=0.0317) of the patient. Further investigations are required in order to study a possible selective impact of the use of the kit for in vitro isolation of trypanosomes as an isolation technique.     

Source and significance of genetic polymorphism of selected parasitic protozoa

The application of biochemical and molecular techniques in parasitological studies has provided increasing evidences of genetic polymorphism among parasite populations. This review presents possible origins of genetic variation within populations of various protozoan species. Since the mode of reproduction has an important influence on genetic polymorphism within parasite populations these considerations refer mainly to some protozoan parasites which have various life cycles, e.g. Giardia, Trypanosoma, Cryptosporidium, Toxoplasma. Also other factors associated with parasites (such as: transmission and passage history in laboratory conditions; occurrence in different hosts or geographic regions; selective pressure of drugs; competitive interactions between populations) that affect parasite genetic diversity are discussed. However, the number of examined isolates of parasites and genetic markers, assortment of methods, probes, primers and reagents used is also of significance. The significance of genetic variability in parasite populations is still the subject of much interest and controversy. A simple interpretation of such variation is impossible because of the complexity of host-parasite interactions. The knowledge of parasite diversity at the nucleic acids level has continually increased, but a corect interpretation of this phenomenon requires at least the same knowledge of genetic variability in host populations. Nevertheless, genetic variability in protozoan parasites has many important implications, e.g. for taxonomy, epidemiology, control and evolution. Genetic differences within parasite populations might also be associated with phenotypic variability, e.g. virulence, antigenicity, infectivity, drug sensitivity, host preference etc.     

Markers for population genetic analysis of human plasmodia species, P. falciparum and P. vivax

Present report deals with the genetic diversity existing among the field isolates of Plasmodium falciparum and P. vivax in India. Isoenzymes and molecular markers were used to analyse field isolates of P. falciparum and P. vivax. High level of length polymorphism was observed in repeat nucleotide sequences of MSP-1, MSP-2 and GLURP in P. falciparum isolates and CSP, GAM-1 and MSP-3 alpha in P. vivax isolates. In study populations a high proportion of isolates (up to 60%) were comprised of more than one genetically distinct parasite type--multiclonal. Presence of identical allelic forms of enzyme and DNA variations in different geographical areas and in different years suggest that isolates belong to a single random mating population of P. vivax and P. falciparum. Observed random combination of alleles in the field isolates suggest the unlinked nature of loci studied. Study supports the feasibility of using molecular markers for the identification of recrudescence in P. falciparum from fresh infection.