应用微卫星DNA标志研究我国雷氏按蚊群体遗传结构

发布时间：2018-01-23 20:24

本文关键词： 雷氏按蚊分子鉴别群体遗传微卫星DNA　出处：《第二军医大学》2011年硕士论文　论文类型：学位论文

【摘要】：雷氏按蚊Anopheles lesteri Baisas Hu 1936,隶属按蚊属(Genus Anopheles)、赫坎按蚊种团(An. hyrcanus group),分布于菲律宾、中国、日本、韩国和关岛等地,就医学重要性而言,仅在我国中部具有较高的传疟效能。多年的调查显示,雷氏按蚊在我国不同地理分布区,生态习性和传病能力也存在一定差异,故亟待深入研究雷氏按蚊的群体的遗传差异。本研究应用分子特征首先鉴别我国的按蚊样本;在研究雷氏按蚊多态微卫星DNA位点特征的基础上,应用多态的微卫星DNA位点检测我国雷氏按蚊群体遗传结构。依据分子特征鉴别我国的按蚊样本,提高了准确性,在一定程度上补充和更新了各地按蚊的本地资料。雷氏按蚊不同群体的遗传差异程度,以及群体分化等问题的阐明,可望为制定媒介区的疟疾控制策略提供理论依据。课题研究策略如下:首先,对我国不同分布地的按蚊样本依据rDNA的2个标志,即ITS2和D3,进行分子鉴定;之后,对课题组已分离的雷氏按蚊微卫星DNA位点应用现场样本进行GENESCAN扫描,研究各位点的基本特征;应用获得的多态微卫星位点检测我国雷氏按蚊的群体内和群体间的遗传差异。取得如下主要结果: 1.本研究分子鉴定的样本来自云南、湖北、贵州、河南、广东、山东、辽宁、陕西和海南共126个样本,序列分析结果显示包括:中华按蚊(An. sinensis)(n=44)、迷走按蚊(An. vagus)(n=22)、暗灰按蚊(An. pullus) (n=18)、微小按蚊(An. minimus)(n=13)、林氏按蚊(An. lindesayi)(n=10)、克莱按蚊(An. kleini)(n=4)、筠连按蚊(An. junlianensis)(n=4)、腹簇按蚊(An. kochi)(n=4)、比伦按蚊(An. belenrae)(n=3)、贵阳按蚊(An. kweiyangensis)(n=3)和乌头按蚊(An. aconitus)(n=1)。 2.序列分析发现存在4类无法定名的种类,即LL1、LL2、LL3和LL4,除LL4外,其他3类均与赫坎按蚊种团成员种最为近似,应是该种团的新成员种。另外,在多种按蚊中发现个体内的碱基双峰现象,提示这些按蚊物种近期分化活跃,处于隐种形成中(speciation)。 3.应用分子鉴定确认的雷氏按蚊现场57个样本,研究了雷氏按蚊11个多态微卫星DNA的基本特征,为进一步检测雷氏按蚊的群体遗传结构提供了新的分子标志。 4.应用9个多态的微卫星DNA位点检测了采自我国17个,以及韩国1个采集地的雷氏按蚊样本共216个,各行政省份合并后成1个群体,将获得的9个群体进行后续分析。 5.雷氏按蚊群体内遗传差异的分析结果可见,平均期望杂合度和观察杂合度的范围为0.4848(江苏)～0.7134(辽宁)和0.3309(江苏)～0.4704(韩国);在辽宁群体中自有等位基因最多(9),而云南群体无自有等位基因;近交系数范围为0.2101(海南)～0.5037(湖北),平均为0.3508;经检验,总共30个位点偏离哈代-温伯格平衡,辽宁群体中的位点最多(7),在韩国、云南和湖北群体无位点偏离;连锁不平衡的检验结果显示,存在连锁关联的共38对位点(P0.01),位点ANL10与ANL11在所有群体中均出现连锁关联,其他位点无规律。 6.雷氏按蚊群体间遗传差异分析时去除江苏群体(实验室品系),以及位点ANL11。分析结果可见,成对群体间的FST范围为-0.0127(辽宁与韩国)～0.2539(云南与海南);Mantel检验结果显示群体间遗传差异与地理距离为正相关关系,群体遗传结构符合地理隔离模型;分子变异等级分析显示雷氏按蚊的变异主要存在于群体内,占总变异的86.55%,群体间的变异仅占总变异的13.45%;贝叶斯法的分析结果均显示所有群体可分为2个支,分支I包括广东和辽宁群体,分支II包括即韩国、河南、湖北、广东、海南和四川群体。 7.雷氏按蚊群体的稳定性和规模的分析结果可见,河南、湖北、海南和辽宁共4个群体经历了近期群体的扩张;基于连锁不平衡模型,群体规模的范围为1.2(湖北)～33.7(广东),总体的群体规模为44.6,95%置信区间为40.6～49.0。 8.综合分析雷氏按蚊的群体遗传结构,可以推测雷氏按蚊的祖先群体来自云南,其他群体均为扩散而至,向北、东迁移和扩散,在途中定殖,为适应当地生态环境出现一定比例的变异。雷氏按蚊分布广泛,地理屏障虽在一定程度上阻碍基因的交流,但群体间差异并不大,群体遗传结构符合地理隔离模型(Isolation-by-distance Model)。因其在我国中部的医学重要性,故在分布地长期、大量使用杀虫剂,导致雷氏按蚊的群体数量较小,存在大量近交,偏离哈代-温伯格平衡的现象。
[Abstract]:Anopheles LESTERI Baisas Hu 1936 Anopheles LESTERI, membership (Genus Anopheles), Anopheles HYRCANUS group (An. HYRCANUS group), located in Philippines, Chinese, Japan, South Korea and Guam, terms of medical importance, only high in our country in the middle of the malaria transmission efficiency. According to many years of investigation, Anopheles LESTERI in our country, different geographical distribution, ecological habits and disease ability there exist some differences between the population genetic differences need to be in-depth study of Anopheles LESTERI. The research and application of molecular characteristics first identify our sample of Anopheles LESTERI in Anopheles; polymorphic microsatellite DNA loci based on the characteristics and application of polymorphic microsatellite DNA loci the detection of genetic structure of Anopheles LESTERI group in our country. On the basis of the molecular characteristics of identification in China Anopheles samples, improve the accuracy, supplement and update all Anopheles local data to a certain extent. Ray The explanation of the genetic diversity and population differentiation in different groups of Anopheles' Anopheles is expected to provide a theoretical basis for the formulation of malaria control strategies in the media area.
The following research methods: first, the 2 sign of different distribution in China based on rDNA sample of Anopheles, namely ITS2 and D3, were identified; after GENESCAN scanning of the research group has isolated Anopheles LESTERI microsatellite DNA loci using field samples, study on the basic characteristics of each site; the application of polymorphism microsatellite loci detected genetic differences between Chinese Anopheles LESTERI groups within and between groups. The main results are as follows:
1. of the studies on molecular identification of samples from Yunnan, Hubei, Guizhou, Henan, Guangdong, Shandong, Liaoning, Shaanxi and Hainan, a total of 126 samples, the results of sequence analysis showed: Anopheles sinensis (An. sinensis) (n=44), Anopheles vagus (An. vagus) (n=22), dark grey (An. pullus). (n=18) (An. minimus), Anopheles minimus (n=13), Anopheles (An. LINDESAYI) Lin (n=10), Klein (An. KleinI) from Junlian (n=4), Anopheles (An. junlianensis) (n=4), abdominal cluster (An. Kochi) of Anopheles (n=4), (An. belenrae) than Aaron Anopheles (n=3 Guiyang (An.), Anopheles kweiyangensis and Anopheles) (n=3) (An. aconitus) (n=1).
2. sequence analysis shows that there are 4 kinds of species can not be named, namely LL1, LL2, LL3 and LL4, except LL4, the other 3 were with HYRCANUS group members for the most similar, is a new member of this kind should be group. In addition, the phenomenon of Shuangfeng within the individual bases found in a variety of Anopheles. These species of Anopheles cryptic species in recent differentiation of active form (speciation).
3., 57 samples from Anopheles Rex were identified by molecular identification. The basic characteristics of 11 polymorphic microsatellite DNA were studied, providing new molecular markers for further detection of the population genetic structure of Anopheles leani.
4., 9 polymorphic microsatellite DNA loci were used to detect 17 loci collected from China, and 1 samples of Anopheles leani collected from 1 places in South Korea were collected from 216 provinces.
Analysis of 5. groups of Anopheles LESTERI genetic differences within the visible range, the average expected heterozygosity and the observed heterozygosity was 0.4848 (Jiangsu) to 0.7134 (Liaoning) and 0.3309 (Jiangsu) to 0.4704 (South Korea); in the Liaoning group has most alleles (9), and Yunnan population without its own allele inbreeding coefficient; range of 0.2101 (Hainan) to 0.5037 (Hubei), the average was 0.3508; after inspection, a total of 30 loci deviated from Hardy Weinberg equilibrium, the Liaoning population was the largest (7), in South Korea, Yunnan and Hubei groups site deviation; linkage disequilibrium test results showed that a total of 38 para point linkage Association (P0.01), ANL10 and ANL11 loci were associated with a chain in all groups, other sites without the law.
6. groups of genetic differences between Anopheles LESTERI analysis group (Jiangsu removal laboratory strain), and site ANL11. analysis indicated that the paired groups FST range of -0.0127 (Liaoning and Korea) to 0.2539 (Yunnan and Hainan); Mantel test results show that the genetic diversity and geographic distance among populations was positive correlation between population genetic the structure with geographical isolation model; molecular variance analysis showed that the variation of Anopheles LESTERI mainly existed within populations, accounting for 86.55% of the total variation, variation between groups accounted for 13.45% of the total variance; Bayesian analysis method showed that all groups can be divided into 2 branches, including Guangdong and Liaoning branch of I group, II branch including South Korea, Henan, Hubei, Guangdong, Hainan and Sichuan populations.
7. groups of Anopheles LESTERI stability analysis and scale results, Henan, Hubei, Hainan and Liaoning a total of 4 groups experienced a recent population expansion; linkage disequilibrium model based on the range of population size is 1.2 (Hubei) to 33.7 (Guangdong), the overall population size 44.6,95% CI 40.6 ~ 49.0.
A comprehensive analysis of the genetic structure of 8. Anopheles LESTERI groups, presumably Anopheles LESTERI ancestral populations from Yunnan and other groups are spread to North eastward, and the spread of colonization in the way, in order to adapt to the variation of the local ecological environment. A certain percentage of Anopheles LESTERI distributed widely, although the geographical barrier hindering gene exchange to a certain extent, but the difference between groups is not large, population genetic structure with geographical isolation model (Isolation-by-distance Model). Because of its importance in medicine of central China, so in the long-term distribution, extensive use of pesticides, resulting in the Anopheles LESTERI population is small, there are a lot of inbreeding, deviations from Hardy Weinberg equilibrium phenomenon.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R384.1

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