【作者】 张建珍；

【导师】 马恩波； Kun Yan Zhu；

【作者基本信息】 山西大学 ， 环境科学， 2006， 博士

【摘要】 稻蝗属(Oxya)昆虫属于直翅目(Orthoptera)斑腿蝗科(Catantopidae),世界范围内广泛分布于非洲区、古北区东南部、东洋区、澳大利亚等地区。本属昆虫栖息的环境多为稻田及湿度较大的河边、水库周围,喜食各种禾本科植物,是水稻、甘蔗、谷类等农作物的主要害虫。有关稻蝗属的分类研究由来已久,自1831年Audient-Serville建立本属一个半世纪以来,世界范围内许多学者曾先后进行过较为系统的分类研究工作。据Hollis 1971年对世界范围内稻蝗属全面的分类学订正工作以及我国蝗虫分类学工作者的研究结果,目前世界上已知稻蝗有22种,中国有15种。其中,国内分布较广的主要有中华稻蝗(Oxya chinensis)、无齿稻蝗(O.adentata)日本稻蝗(Oxya japonica)、山稻蝗(Oxya agavisa)和小稻蝗(Oxya intricata)等物种。 稻蝗属种类分布广泛,形态学差异在有些种间不十分显著,因此给常规分类带来一定的困难,部分物种的鉴定在分类学界尚存在争议。由于稻蝗属昆虫是农业生产上的重要害虫,对它的系统分类学研究不仅具有理论意义,同时在稻蝗防治的农业植保实践中也具有较为重要的应用价值。以往的分类学研究工作除应用常规形态分类特征予以鉴别外,也曾采用染色体核型和带型指标以及同工酶生化指标进行过较为深入的研究。但我们在工作中发现,对该属的种间、种群间更深入的系统分类学关系的探讨还需要采用分子水平更加适合的遗传标记来补充和完善。 RAPD(随机扩增多态性DNA)技术是1990年由Williams和Welsh同时提出的一项DNA分子水平上的多态性检测技术,由于它具有简捷、快速、成本低、信息含量丰富等特点,被广泛应用于生物类群种属分类鉴定、遗传图谱构建、物种亲缘关系和种群遗传学等领域的研究。并很快被昆虫学家所认可,广泛用于分类学及系统发育研究。本实验室多年来一直开展稻蝗属细胞及分子水平系统进化研究,本项研究在依据外部形态分类鉴定及前人工作的基础上,采用RAPD技术对稻蝗属部分种类基因组DNA的RAPD带型进行比较,在分子水平上探讨这些物种的分类地位和亲缘关系,为更好地开展稻蝗属系统分类研究和稻蝗防治提供分更多还原

【Abstract】 The grasshoppers of the genus Oxya (Orthoptera: Acridoidea) distribute from Pakistan region, China, southeastern Russia, and the Australia. The morphologically monotypic speices of Oxya are commonly found in fields of graminoid plants with relatively abundant water supply. They are agricultural pests damaging rice, sugar cane and other crops. This genus was established by J. G. Audinet-Serville in 1831 and had been revised several times, especially by D. Hollis in his revision publications in 1971 and 1975. Hollis described the genus of Oxya as a unnatural grouping of species which was retained as a taxonomic unit for purely practical purpose, and recognized the difficulty to determine its phylogenetic relationships. He re-documented 18 species based on 8 morphological characters of the phallic complex. In Hollis’s classification, 8 species are found in China: O. chinensis, O. agavisa, O. intricata, O. japonica, O. ningpoensis, O. tinkhami, O. adentata, and O. velox. Since then, serveral new species in Oxya from China have been described, e.g., O. hainanensis, O. yunnana, and O. anagavisa;O. brachyptera and O. flavefemura, O. termacingula and O. bicingula. The taxonomic status of these Oxya species were assessed using morphological and cytological characteristics. At present, there are 15 described species in China, among which O. chinensis, O. adentata, O. japonica, O. agavisa and O. intricate are widely distributed and usually abundant in numbers.The grasshoppers of the genus Oxya almost range throughout China. Two factors may have contributed to the possible population differentiation among the speices. First, the survival and reproduction of Oxya depends on abundant water in their habitats. Second, their dispersal capacity is relatively limited in term of flight ability. So it seems that the genetic differentiation is likely to occur among the different populations and species of Oxya. The controversial taxonomic status of several Oxya species based solely on morphological characteristics calls for more information from other biological aspects, e.g. cytological and genetic studies. Attempts have been made usingchromosomal characteristics including C-band types and allozyme markers to study taxonomic relationships of Oxya. While having provided valuable information from cytological and allozyme investigations, diffculties still exist in delineating populations at local scale. Therefore, more genetic markers are needed for exploring the phylogenetic relationships of grasshoppers.RAPD (Random Amplification Polymorphic DNA) method using for the identification of polymorphism and analyzing phylogenetic relationships among and within closely related species was first described by Williams and Welsh in 1990. RAPD markers are based on the amplification of unknown DNA sequences using single, short, random oligonucleotide primers. The RAPD method is advantageous over RFLPs in technical ease, quickness, low cost and less amount of samples required. It has been widely applied to the organisms including bacteria, plants, and vertebrates for taxonomy and species identification, as well as construction of genetic mapping, genetic relationships among species and population, and locating pathological important genes. The RAPD method has also been soon adopted by entomologists and widely used to study taxonomy and phylogenetic relationships of insects.Our laboratory has attempted to study phylogenetic relationships of Oxya using chromosome, allozyme and DNA markers. So far, data on effective population sizes and relative genetic variability are insufficient, although they are of particular importance in management and control of pest populations. This dissertation present a study of RAPD polymorphism within and among populations and species of Oxya. This research is an expansion of our previous study to examine the genetic composition at many loci for geographically distant populations to provide more detailed information about the molecular diversity among species and populations of Oxya. At the same time, we also explored the feasibility of applying RAPD technique in the genus of Oxya and among its closed genera. The main contents andconclusions are as follows:1. The molecular phylogenetic relationships of 7 species of Oxya and 1 specie of Pseudoxya was studied by the approach of RAPD. Among 78 arbitrary primers tested, 28 primers rendered good results for Oxya. Ten primers of them which generated clear and reproducible fragments were used to amplify genome DNA of 305 individuals of these 8 species. In addition, eleven primers were used to amplify genome DNA of 24 individuals of 4 species in Arcypteridae.2. In this study, genome DNA of grasshoppers which was frozen under -20°C, preserved in 100% ethanol, 70% ethanol or kept as a dry sample was extracted using saturated NaCl. Arbitrary regions of the genome DNA were amplified by polymerase chain reaction (PCR). The results indicated that DNA obtained from 70% ethanol fixed samples and some dried samples were visible by ethidium bromide staining of agarose gels. The DNA was of a low molecular size and most likely degraded, which caused either false information or no amplified bands at all. However, the well-preserved dry samples, the frozen samples and 100% ethanol fixed samples were quite good for DNA extraction and RAPD analysis, for which they were considered as the optimal preserving conditions of DNA extraction for studying molecular phylogenetic relationships of grasshoppers.3. The genetic divergence among 4 populations of Oxya chinensis, 3 populations of O.adentata and 1 population of O.brachyptera were analyzed using RAPD markers. Nei’s genetic distance based on the RAPD patterns of 92 individuals showed that genetic distance of O. chinensis was closer to O. adentata more than to O. brachyptera. A molecular phylogenetic tree was constructed by between-groups linkage method, showing O. chinensis was closely related to O. adentata, but O. brachyptera had far phylogenetic relationship with them.4. The genetic relationships of six populations (collected from HebeiPingshan, Jiangsu Xuzhou, Zhejiang Hanzhou, Guangdong Guangzhou, Guangxi Nanning, Yunan Tengchongqushimaluchong) of O. japonica were studied with RAPD. The genomic DNA of 57 individuals using 10 primers were amplified. Genetic diversity within each population and partitioning of the genetic diversity among populations were estimated by Shannon’s and Nei’s diversity index. A molecular phylogenetic tree was then constructed by between-groups linkage, UPGMA and NJ. The results suggested that the proportion of polymorphic loci of Yunnan population was the highest. Shannon’s index showed higher genetic diversity within Yunnan Tengchongqushimaluchong population. A tendency of clustering following a North-South gradient could be observed in the dendrogram, the results also implied that genetic distance of six populations of O. japonica correlated with geographical distance to some extent.Ojaponica is distributed in most area of South of Qingling Mountains, for example, the South of Gansu province, the Midsouth of Shannxi province, Zhejiang province, Guangxi province, Shandong province. No Ojaponica was reported in the North of China, such as Hebei province. We found O japonica and O. chinensis lived in same area when we collected samples in Hebei province, it is difficult to distinguish these two species from shape. Nine O. japonica individuals were identified from samples collected from Pingshan, Hebei province by microscope and molecular markers. The results demonstrated O. japonica dispersed in North of China.5. RAPD was used to analyze genetic relationships of six populations (collected from Chongqin Beibei, Hunan Shimen;Hubei Wufeng;Hubei Shengnongjia;Hunan Tongdao;and Hunan Jiangyong) of O. agavisa. Based on the band presence/absence, Shannon’s index, Nei’s index and a genetic distance matrix were constructed and analyzed. The results showed that higher genetic diversity within Hunan Tongdao population. Gene differentiation coefficient estimated by Shannon’s index (44.5%)was almost identical with Nei’s index (41.3%) and indicated that most of the variation occurred within population. Nei’s genetic distance and cluster analysis showed that geographically close populations (from Chongqin beibei, Hunan Shimen, Hubei Wufeng, Hubei Shengnongjia) were grouped into one branch and indicated genetic distance corresponded to geographic distance.6. The genetic relationships of five populations (collected from Henan Xinxiang, Hubei Wufeng, Hubei Shengnongjia, Hunan Changsha, Yunnan Longlingliangjiaoshui) of O. intricata were studied with RAPD markers. The RAPD patterns demonstrated that higher polymorphisms within Yunnan population, no shared bands were observed among populations. Partitioning of the genetic diversity of five populations of O. intricata estimated by Shannon’s index indicated that 54.2% of the variation was from within population and 45.8% between populations, gene differentiation coefficient estimated by Nei’s index was 42.7%, almost identical to Shannon’s index. Nei’s genetic distance and dendrogram showed genetic distance of five populations of O. intricata correlated with geographical distance to some extents.7. DNA polymorphism of Oxya from Longlingliangjiaoshui and Tengchongqushimaluchong, Yunnan was analyzed using random amplified polymorphic DNA (RAPD) markers, the cluster analysis indicated all individuals were failed into three main clusters. According to morphological characteristics, Oxya analyzed were divided into three different species ( O.japonica, O.intricata and a special population with short front wing, might be a new specie that needed to be identified based on the more classification information) correspondence to three main clusters. The results showed that RAPD is very useful as an effective molecular marker to distinguish the closed related species. Compared with other populations of O.japonica collected from Guangzhou, Nanning, Hangzhou, Xuzhou, Pingshan, indicated genetic diversity of O.japonicafrom yunnan is the highest, so is O.intricata from yunnan, reflecting diversity of the insect species and genetic diversity within species or populations of Yunnan.8. The molecular phylogenetic relationships of seven species of Oxya Audient-serville including O. chinensis, O. adentata, O .brachyptera, O, japonica, O. agavisa, O. SR, O. intricata and P. diminuta of Pseudoxya were studied with RAPD. A molecular phylogenetic tree from the RAPD profile of all individual grasshoppers was constructed by between-groups linkage method, the results indicated high within-population similarities. The clustering followed the order of, first the individuals within a population, then the different populations of the same species, later the species of the same genus, and finally the species among genera. Specifically O. chinensis and O. adentata were the most closed to each other, which were latter grouped with O. brachyptera into one cluster, the species of O. japonica then was clustered with them before clustered with O. agavisa, whereas O. intricata and O. SP. had far phylogenetic relationship with them. Finally, P. diminuta clustered with species of Oxya. The results of dendrogram and Nei’s genetic distance were consistent with our previous observations of morphologic classification and cytotaxonomy, and suggested RAPD is very useful as an effective molecular marker to distinguish the divergence in Oxya and its closed related genera. Genetic diversity within population of P. Diminuta, as estimated by Shannon’s and Nei’s diversity index is the lowest. The high uniformity in both RAPD and chromosomes in P. diminuta, along with its narrow distraction range, suggested the species a highly specialized group with close relationship to Oxya.9. Random Amplified Polymorphic DNA (RAPD) markers were applied to analyze genetic relationships among four species of Arcypteridae including Ceracris hoffmanni Uvrov ,Ceracris fasciata fsaciata (Br.-W.), Arcyptera coreana Shiraki and Pararcyptera microptera meridionalis(Ikonn). Genomic DNA of twenty-four individuals was each amplified with eleven oligonucleotide (10 bp) primers selected previously from twenty-four random primers (10 bp), The dendrogram based on Nei’s genetic distance of RAPD markers was constructed using UPGMA and Neighbor-Joining, It is suggested that the species within each genus first grouped together and the 4 species of Acridoidea fell into two branches. One is C. hoffmanni Uvrov and C. fasciata fasciata (Br.-W.) cluster, and another is A. coreana Shiraki and P. microptera meridionalis(lkonn.) cluster. While this result coincides with the results of morphological studies, impling the feasibility of using RAPD analysis for exploring genetic relationships among Oxya and its closed related genera.更多还原