【作者】 任俐；

【导师】 马恩波；

【作者基本信息】 山西大学 ， 动物学， 2005， 硕士

【摘要】 随机扩增多态DNA技术(简称RAPD)是二十世纪90年代发展起来的一项DNA分子多态检测技术,它建立在PCR技术基础上,以一系列不同随机排列的碱基序列—单链寡核苷酸为引物—对所研究的基因组DNA进行PCR扩增。该技术能够在没有任何遗传背景的情况下,对物种基因组进行DNA多态性分析。它不但能通过众多的引物检测大量的基因位点,且具有高效、快速、样品用量少和对材料要求不高等优点,目前已广泛应用于动植物的遗传多样性研究中。 飞蝗是直翅目中较大的一个类群,它广泛分布于各种生态环境中,由于食性及环境条件的适应,形成了不同的生态型和自然地理种群。长期隔离和演化的结果,同种蝗虫不同种群之间逐渐产生了一定的遗传分化。飞蝗引起的蝗灾是我国农业历史上重大的自然灾害,本文的研究工作为我国飞蝗种群遗传学的研究提供了丰富的资料。 本文采用随机扩增多态性DNA(RAPD)技术检测了飞蝗Locusta migratoria(Linnaeus)9个自然种群的遗传多样性及遗传分化。用筛选的11条随机引物对飞蝗的9个种群113个个体进行扩增,共获得163个清晰稳定的条带,多态位点共计161个,总的多态位点百分率为98.77%。不同引物在不同种群中所检测出的RAPD位点及多态位点百分率不同。Shannon信息指数和Nei’s指数对RAPD数据的分析表明:东亚飞蝗Locusta migratoria manilensis(Meyen)不同种群存在较高的遗传多样性。其中,东亚飞蝗山西临漪种群由Shannon信息指数和Nei’s指数估算的遗传多样性最高分别为0.2767和0.1896,河南中牟种群由这两种指数估算的遗传多样性最低分别为0.1648和0.1109;同时,东亚飞蝗种群间出现一定程度的遗传分化,由这两种指数估算的遗传分化系数分别为42.90%和40.10%。 用NJ法对Nei’s遗传距离作聚类分析,结果显示:东亚飞蝗山西临漪和山西永济种群聚为一支,东亚飞蝗河南中牟种群和江苏沛县种群聚为一支,两支相聚后,辽宁葫芦岛种群和它们相聚;东亚飞蝗河北黄骅种群和河北平山种群聚为一支后与天津北大港种群相聚再与前三支聚为一起,最后新疆亚洲飞蝗Locusta migratoria migratoria(Linnaeus)种群与更多还原

【Abstract】 The Random Amplified Polymorphic DNA (RAPD) technique is a method for examining the DNA polymorphism developed in 1990s. Based on PCR technology it uses the single-stranded oligonucleotide of random sequence as primer to amplify the genomic DNA. The method is favored because it can examine a large amount of loci at the genome by use of many primers.It is advantageous for its high efficiency, quickness, small sample quantity and less requirements for sample quality, which resulted in its broad applications in the genetic fields of Botany, Zoology and other Subjects. However, up to date RAPD has been used widely in the studies of Orthoptera, especially Acridoidea insects. Because the RAPD method fits the law of Mendel genetics, which is used complementarily for the population genetics studies in a comprehensive way.The locust of Acridoidea represents a large insect group that can be found in various ecological habitats. The insects have different ecotypes and geographic populations as a result of feeding habits and the adaptations to each environment. The genetic divergence among the populations occurs during the long-time isolation and evolution. This research, by use of RAPD technique, examined the genetic structure and divergence among the natural populations of the locusts (Locusta migratoria manilensis) collected from different locust breeding areas. The oriental migratory locust is a major agricultural pest due to its huge consumption of crops, and more importantly its periodic massive outbreaks. This studies have rendered abundant value for the research in the genetics population of locust in China.Random amplified polymorphic DNA (RAPD) provides a powerful tool for investigation of genetic variation in natural and domesticated populations. We described the genetic variation of 9 natural populations (collected from Shanxi Linyi, Shanxi Yongji, Jiangsu Peixian, Henan Zhongmou, Hebei Pingshan, Hebei Huanghua, Tianjin Beidagang, Liaoning Huludao in China)of Locusta migratoria manilensis (Meyen) and Locusta migratoria migratoria (Linnaeus) from Xinjiang Wulumuqi. Eleven arbitrary 10 base-pair primers produced 163 clear and reproducible amplification bands, 161 bands of which were polymorphic ones. The total percentage of polymorphisms bands was 98.77%. Different primers had different RAPD loci and percentage of amplification bands among different populations. The RAPD patters demonstrated consistent polymorphisms in different populations and subspecies.Based on the presence/absence of bands, Shannon’s index, Nei’s index and a genetic distance matrix were analyzed. The results showed that higher genetic diversity existed within Locusta migratoria manilensis (Meyen) populations. Shanxi-Linyi population had higher genetic diversity, Shannon’s index and Nei’s index was 0.2767 and 0.1896 respectively, while Henan-Zhongmou population had the lowest level, Shannon’s index and Nei’s index was 0.1648 and 0.1109 respectively. Partitioning of the genetic diversity of 8 populations of Locusta migratoria manilensis estimated by Shannon’s index indicated that 57.10% of the variation occurred within populations and 42.90% of the variation existed among populations, gene differentiation coefficient estimated by Nei’s index was 40.10% which is almost identical with Shannon’s index.The dendrogram based on Nei’s genetic distance of RAPD markers was constructed using NJ indicated high similarities within-population. The nine populations of Locusta migratoria were divided into three big branches. Shanxi-linyi population and Shanxi-Yongji population were clustered into one branch, Henan-Zhongmou population and Jiangsu-Peixian population were clustered into one branch, the two branches were clustered together, then Liaoning-Huludao population clustered with them. Heibei-Pingshan population and Hebei-Huanghua population were clustered into one branch, whereas Tianjin-Beidagang population was clustered with Hebei population before clustered with Huludao population. However, Xinjiang-Wulumuqi population was clustered with them finally. These data showed allpopulation was clustered with them finally. These data showed all populations can be distinguished with RAPD markers, Linyi population was closely related to Yongji poulation, Pingshan population was closely related to Huanghua population, nevertheless all the eight populations of Locusta migratoria manilensis (Meyen) had far genetic distance with Locusta migratoria migratoria (Linnaeus).The mean Nei’s genetic distance and genetic identity between them were 0.1551 and 0.8567 respectively, whereas the mean Nei’s genetic distance and genetic identity among Locusta migratoria manilensis (Meyen) populations were 0.2680 and 0.7651 respectively. The mean genetic distance between them and among them by use of mantel-test were 0.2298 and 0.3905 respectively, whereas the mean genetic distance among Locusta migratoria manilensis (Meyen) populations and Locusta migratoria migratoria (Linnaeus) by use of mantel-test were 0.4671.The result showed the similarity among different populations is higher than that among species.The Mantel test is used to determine if a correlation exists between two matrices of genetic distance and geographic distance. Genetic distance of 8 populations of Locusta detected with Mantel test was r<0.7 that showed cophenetic correlation measured very pool fit for genetic analysis. The cophenetic correlation of genetic distance and geographic distance was r = 0.6278, there seems not to be a tendency of correlations between the genetic and geographic distance among 8 different populations. The results also showed that genetic divergence of eight natural populations of Locusta migratoria manilensis (Meyen) did not correspond to geographic distance and genetic distance.The eight oriental migratory locust we studied can been divided into four areas according to the geography and the breeding habitats: HebeiPingshan, JiangsuPeixian and TianjinBeidagang belonged to the lake -shooed areas; ShanxiLinyi and HennanZhongnou belonged to the river flood areas; Hebei Huanghua and LiaoningHuludao belonged to the seacoast areas; ShanxiYongji belonged to the land plain flood areas. From the eight更多还原