【作者】 袁明龙；

【导师】 王进军；

【作者基本信息】 西南大学 ， 农业昆虫与害虫防治， 2011， 博士

【摘要】 柑橘全爪螨(Panonychus citri)隶属蛛形纲(Arachnida)、蜱螨亚纲(Acari)、叶螨科(’Tetranychidae),是一种世界性分布的柑橘害螨。在我国,该螨发生面积广、种群密度高、为害时间长,是大多数柑橘种植区的关键害虫(螨)之一。该螨能够快速地对多种杀螨剂产生高水平抗性,增加了生产中对其有效防控的难度。目前,对该螨的研究很少涉及种群遗传与进化,这很大程度上源于缺乏有效的分子遗传标记以及单雌DNA提取的困难。深入研究柑橘全爪螨不同地理种群的遗传结构,有利于从分子水平上了解柑橘全爪螨的生态适应机制和成灾规律,为制定有效的控制策略提供理论依据。近年来,叶螨对新型线粒体靶标杀螨剂联苯肼酯等产生了严重抗性,据报道该类抗性源于线粒体cob基因的点突变。因此,测定柑橘全爪螨全线粒体基因组序列,不仅可以丰富蜱螨线粒体基因组数量,推动蜱螨及其它节肢动物动物分子系统学的研究,还可针对突变了的线粒体基因设计引物,进而建立快速、准确的田间抗性诊断技术。本学位论文紧密围绕柑橘全爪螨种群遗传与进化这一重要科学问题,致力于发掘柑橘全爪螨的微卫星位点,重点解析该螨的种群遗传结构,测序获得全线粒体基因组序列,分析叶螨线粒体基因组的进化特点,鉴定适于叶螨分子系统学研究的线粒体分子标记,并构建基于全线粒体基因组序列数据的蜱螨系统发生关系。主要研究结果如下：1.在提取、纯化柑橘全爪螨基因组DNA的基础上,采用磁珠富集法构建了柑橘全爪螨AC、TC和ATG等3个微卫星富集文库。对3个富集文库的部分阳性克隆进行测序,获得了44个可进行引物设计的微卫星位点。序列分析表明,柑橘全爪螨AC富集文库的微卫星位点大部分以多拷贝的形式存在,部分以微卫星DNA家族的形式存在。因此,TC文库和ATG文库中发掘的微卫星位点更适合作为分子标记研究该螨的种群遗传结构。在富集文库的构建过程中,笔者通过将酶切、接头连接一步化,减少了酶切产物的损失,提高了酶切产物的量,有效地提高了微卫星的富集效率。2.联合线粒体cox1基因和核糖体内转录间隔区ITS1序列,分析了15个柑橘全爪螨地理种群的遗传多样性、种群遗传分化及基因交流模式,结果发现：(1)基于线粒体coxl基因的数据显示,15个地理种群中共发现22个单倍型；基于核ITS1序列的数据,共发现134个单倍型。综合两种分子标记的结果推测,15个柑橘全爪螨地理种群具有相对较高的遗传多样性,这可能是该螨在各地能够频繁成灾、对杀螨剂快速产生抗性的遗传基础；(2)系统发育树和单倍型进化网络图显示,来自同一种群或相同柑橘种植区的柑橘全爪螨的cox1单倍型或ITS1单倍型,均未能聚集在一起。AMOVA分析表明,柑橘全爪螨在本论文所涉及的长江中上游(UMYR)、云贵高原(YGP)和华南(SC)等3个柑橘种植区间无显著的种群遗传结构。如果分析时去除云南玉溪和湖北丹江口种群时,仅基于线粒体cox1基因数据的AMOVA分析发现,柑橘全爪螨在3个柑橘种植区间存在显著的种群遗传结构。仅基于核ITS1序列数据的AMOVA分析表明,当来自长江中上游柑橘种植区(UMYR)和云贵高原柑橘种植区(YGP)的种群作为一个组群时,柑橘全爪螨存在显著的种群遗传结构。此外,基于核ITS1序列数据的AMOVA分析还发现,寄主植物对柑橘全爪螨种群遗传结构具有显著影响。总体上看,柑橘全爪螨大多数种群间遗传分化不显著,这可能是因为受到部分种群之间存在高水平的基因流、保留有较大的祖先多态性以及自然选择(如寄主植物、杀螨剂等)等多个因素的共同影响；(3)尽管大多数种群间遗传分化不显著,特别是一些地理上相距很远的种群间还存在高水平的基因流(如眉山和广东种群间),但基于两种分子标记的Mantel检验结果均表明,地理隔离仍然是柑橘全爪螨种群遗传分化的重要原因之一。柑橘全爪螨体小、无翅,主动迁移能力非常有限。因此,柑橘全爪螨的长距离迁移和种群间的基因交流,很大程度上依赖于柑橘种苗的运输等人类活动;(4)基于两种分子标记的中性检验、误配分析及星状分布的单倍型进化网络图均支持,柑橘全爪螨在历史上经历了种群扩张事件；(5)基于上述研究结果及柑橘全爪螨快速发展的抗药性问题,在制定该螨的控制策略时,对于抗性基因在不同种群间的扩散及进化应予高度重视。3.采用Long-PCR和Sub-PCR技术,克服了叶螨线粒体基因序列高A+T含量及基因高度重排等因素造成的PCR扩增、测序困难等问题,成功测定了柑橘全爪螨全线粒体基因组序列。柑橘全爪螨全线粒体基因组具有以下特点：(1)柑橘全爪螨线粒体基因组在目前已测序的节肢动物中最小,但并不缺失经典后生动物线粒体基因组所包含的37个基因；(2)与节肢动物线粒体基因的模式排序(鲎的线粒体基因排序)相比,柑橘全爪螨线粒体基因组发生了一系列的重排事件,其中2个蛋白质编码基因区块的倒位最为显著,此外,24个RNA基因也高度重排；(3)柑橘全爪螨线粒体最大的非编码区长度仅为57 bp,完全由碱基A和T组成,且可形成稳定的茎环二级结构,推测该非编码区是线粒体控制区；(4)柑橘全爪螨线粒体基因组具有极高的A+T含量,在已测序的蜱螨线粒体基因中仅略低于苹果全爪螨。这种高的A+T含量,也反应在蛋白质编码基因的密码子使用上,即以A或T结尾的密码子,占绝对优势。在62种无脊椎动物线粒体密码子中,柑橘全爪螨线粒体基因组仅使用了其中的57个密码子,而未使用5个富含GC的密码子。此外,与大多数后生动物相反,柑橘全爪螨线粒体基因组J-链的GC-偏斜为正值；(5)柑橘全爪螨线粒体基因组中仅3个tRNA基因(trnN、trnL2和trnK)能形成经典的三叶草结构,而其余19个tRNA基因均缺少D-臂或T-臂；trnI的反密码子环为非典型的8个核苷酸,trnM和trnSl在反密码子茎上各有1个碱基错配,13个tRNA基因在氨基酸接受臂上存在1至3个不等的碱基错配；(6)柑橘全爪螨线粒体基因组2个rRNA基因发生倒位而由J-链编码, rrnL和rrnS长度的显著减小导致其丢失了多个螺旋结构,但与苍白纤恙螨(Leptotrombidium pallidum)和户尘螨(Dermatophagoides pteronyssinus)具有相似的茎环二级结构。4.以全爪螨属的柑橘全爪螨(包括2个地理种群)、苹果全爪螨和叶螨属的二斑叶螨、神泽叶螨为对象,采用比较基因组学和生物信息学的研究手段,从科内、属内和种内等三个层次,系统分析了叶螨线粒体基因组的分子进化特征及其遗传分化水平。结果表明：(1)4种叶螨线粒体基因组在已测节肢动物中最小,但均包含经典后生动物线粒体基因组所具有的37个基因。与其它蜱螨和节肢动物相比,叶螨线粒体基因组的相对减小,主要体现在蛋白质编码基因、rrnL口控制区的长度都显著缩短；(2)4种叶螨线粒体基因排序完全一致,具有极高的A+T含量,蛋白质编码基因存在强烈的密码子使用偏向性,但全爪螨属物种未使用的富含GC的密码子数量略高于叶螨属物种；(3)全爪螨属物种J-链的GC-偏斜为正值,A+T富集区的二级结构具有2个茎环结构,而叶螨属J-链的GC-偏斜为负值,A+T富集区的二级结构仅有1个茎环结构；(4)4种叶螨线粒体基因组中的tRNA基因极不典型,19个tRNA基因均缺少D-臂或T-臂,有的tRNA基因甚至同时缺少D-臂和T-臂。此外,部分tRNA基因与邻近基因高度重叠,部分tRNA基因在氨基酸接受臂或反密码子茎上存在碱基错配,而trnl的反密码子环均为非典型的8个核苷酸。但是,叶螨的这些不典型tRNA基因不可能是假基因,因为22个tRNA基因序列在4种叶螨中高度保守,特别是反密码子臂；氨基酸接受臂上或反密码子茎上的碱基错配可通过转录后的RNA编辑而得以校正；在线虫中已证实,缺少T-臂的tRNA基因具有正常的生物学功能；不典型的tRNA基因在真螨总目中非常普遍。然而,由于对同时缺少D-臂和T-臂的tRNA基因是否具有正常的生物学功能还未知,因此有必要进一步开展功能性验证实验；(5)4种叶螨线粒体基因组的rRNA基因具有相似的二级结构,但柑橘全爪螨的rrnL有其余3种叶螨均缺少的H3螺旋结构。与5′-端相比,rrnL的3′-端在4种叶螨线粒体基因组中更为保守,特别是螺旋结构G16-G20,而rrnS二级结构中的19、21、32、33、49和50等6个螺旋结构最为保守；(6)基于全线粒体基因组序列、13个蛋白质编码基因序列、24个RNA基因序列的分析表明,4种叶螨属间的遗传分化程度远大于属内和种内。二斑叶螨和神泽叶螨之间的P-距离小于苹果全爪螨和柑橘全爪螨间的P-距离,但远高于柑橘全爪螨两个品系间的P-距离；(7)叶螨线粒体基因组的13个蛋白质编码基因中,仅3个基因(cob、cox3和nad1)的密码子数在4种叶螨中完全一致,其余10个基因的密码子数均不相同。序列进化分析表明,cox1、cox2和cob最为保守,表明适于叶螨较高分类阶元的系统发生关系研究；而atp8、nad2、nad6和nad4L等4个基因的遗传分化较高,适于作为分子标记来研究叶螨种内的遗传多样性或界定近缘物种间的系统亲缘关系。5.基于已测序的28种蜱螨线粒体全基因组序列数据,在系统评价碱基组成异质性的基础上,采用最大似然法(ML)和贝叶斯推断法(BI)构建了蜱螨亚纲内各类群的系统发生关系。尽管28种蜱螨线粒体基因组序列存在显著的碱基组成异质性,但基于PCG123、PCG12和PCG2等3个数据集构建的ML树和BI树,以及基于PCG-RNA和PCG1等2个数据集构建的ML树,均很好地反应出目前普遍接受的蜱螨亚纲内各类群的系统发育关系,支持蜱螨亚纲中寄螨总目和真螨总目均为单系群的观点。寄螨总目的蜱目和中气门目均为单系群,蜱目由硬蜱和软蜱两个互为姊妹群关系的单系群构成,而中气门目中的植绥螨总科与皮刺螨总科互为姊妹群,两者再与胭螨总科构成姊妹群关系。真螨总目由绒螨目和疥螨目两个互为姊妹群的单系群组成；绒螨目的游殖螨亚目和寄殖螨亚目为两个单系群。更多还原

【Abstract】 The citrus red mite, Panonychus citri (McGregor) (Arachnida, Acari: Tetranychidae), has a worldwide distribution and is regarded as one of the most important citrus pests in many countries. In China, the distribution range of this mite covers all citrus planting regions. This mite is often difficult to manage because their ability to rapidly develop resistance to various acaricides. Currently, few studies on P. citri focus on the population genetics and evolution, largely because of the lack of efficient genetic markers and the difficulty in DNA extraction from a single mite. Reliable estimates of genetic diversity and population genetic structure of P. citri from different geographical regions are crucial to gain insight into the role of different evolutionary forces and environmental factors in determining population dynamics, and to make a decision on effective pest management strategies. Recently, it has been reported that the resistance of spider mites to the acaricide bifenazate is highly correlated with the remarkable mutations in the mitochondrial cob gene. Therefore, sequencing the complete mitochondrial (mt) genome of P. citri not only increases the amount of Acari mt genomes, prompts the molecular systematics of Acari and other arthropods, but also allows the identification of specific changes of mitochondrial cob and the subsequent development of robust diagnostics, which are essential in resistance management.In this study, we constructed the P. citri microsatellite-enriched libraries, analyzed the genetic structure of the P. citri populations using the mitochondrial coxl gene and ribosomal internal transcribed spacer 1 (ITS1), sequenced the complete mt genome of P. citri and provided a comparison to other Acari. Additionally, we identified several mitochondrial genes as potential markers for population genetics/phylogenetics studies for spider mites, and assessed the utility of complete mt genome sequences as molecular markers for phylogenetic analyses of Acari. The main results are as follows:1. Three microsatellite-enriched libraries of AC-repeat, TC-repeat, and ATG-repeat were constructed for P. citri using microsatellite-enrichment method. A total of 44 unique microsatellite loci, which can be used to design PCR primers, were obtained by sequencing partial positive clones. Sequence analyses showed that a much larger proportion of microsatellite loci of AC library shared the same flanking regions and some were present as multi-copy microsatellite DNA families. Therefore, the microsatellite loci from TC and ATG libraries will be more suitable for the study of population genetic structure of P. citri. The DNA digestion and ligation were performed simultaneously, which enhanced the concentration of enzyme-digested products and the efficiency of microsatellite enrichment.2. The genetic diversity, population differentiation, and gene flow among 15 P. citri populations were investigated using the mitochondrial coxl gene and ITS1 sequence. The main results are:(1) There were 22 haplotypes among 15 geographical populations based on the coxl gene, whereas total 134 haplotypes were found in these populations based on the ITS1 sequence. Comprehensively analyses of these results, we proposed that the P. citri populations may have relatively high genetic diversity, which probably is one of the most important reasons that this mite has the ability to severely infest citrus and rapidly develop resistance to various acaricides. (2) The phylogenetic tree and haplotype network showed that the coxl or ITS1 haplotypes from the same citrus planting regions or populations did not cluster together. AMOVA analyses showed that there was no significant population genetic structure among the P. citri populations examined. However, an AMOVA without Yuxi and Danjiangkou populations found a weak, but significant geographic structuring(coxl). Also, when all populations from citrus belt of upper and middle reaches of Yangtze River (UMYR) and citrus base of Yungui Plateau (YGP) were considered as a large group, a significant population structure was detected at this large scale (ITS1). Additionally, the influence of host plants on the genetic structure of P. citri populations was detected by the AMOVA analysis for five host-related groups based on the ITS1 sequences. On the whole, there was no significant genetic differentiation among most populations, which can be contribute to several factors together, i.e. ongoing gene flow, the retention of ancestral polymorphisms, and natural selection (e.g., host plants, acaricides). (3) Although no significant genetic differentiation among most populations, even there were high levels of gene flow among some geographically far populations (e.g., Meishan and Guangdong populations), the Mantel tests showed that the isolation by distance was a factor responsible for the genetic differentiation. Due to its small body size and wingless, the dispersal ability of P. citri is very limited. Thus, long-distance dispersal and gene flow of P. citri among populations may largely rely on passive dispersal by the movement of plants between populations and other human activities. (4) The results of neutrality tests, mismatch analyses, and star-like network strongly supported that P. citri in China have undergone population expansion in the past. (5) Considering that Chinese P. citri populations have developed resistance to various acaricides and current high gene flow exists between some populations, great attention should be paid to the spread of acaricide-resistance alleles to help gain insight into P. citri resistance management.3. Overcoming the difficulty in the PCR amplification and sequencing resulting from the high A+T content and gene rearrangement, the complete mt genome of P. citri was successfully sequenced using Long-PCR and Sub-PCR techniques. This mt genome has several features:(1) This is the smallest mt genome among arthropods sequenced so far, but it does not lack anyone of 37 genes typical of metazoan mt genomes. (2) Compared to Limulus polyphemus, which is considered as the representative ground pattern for arthropod mt genomes, a series of gene rearrangements have occurred in the evolutionary history of P. citri, and the most striking features are the inversions of two segments containing several protein-coding genes. In addition,24 RNA genes are highly rearranged. (3) The largest non-coding region is only 57 bp long, and is completely comprised of adenines and thymines. and can be folded into stable stem-loop structure, indicating that this region possibly functions as a control region. (4) The mt genome of P. citri has high A+T content, making it the second highest within sequenced Acari. The high A+T content also reflected in the codon usage of protein-coding genes, i.e., codons harbouring A or T in the third position are always overused as compared to other synonymous codons. Among 62 amino-acid encoding codons of invertebrate mitochondrial code, the P. citri mt genome uses 57 codons and never utilizes the five G+C rich codons. The P. citri mt genome is characterized by a positive GC-skew, which is reverse to that of most metazoans mt genomes. (5) The tRNA genes found in the P. citri mt genome are extremely truncated:only three tRNA genes (trnN, trnL2, and trnK) can potentially fold into a typical cloverleaf structure, whereas all the remaining 19 tRNA genes appear to lack the sequence to code the D-or T-arm. Thirteen tRNA genes have 1-3 bp mismatches in the amino acceptor stem, and trnl has eight nucleotides in the anticodon loop. In addition, two tRNA genes(trnM and trnSl) have a single mismatch in the anticodon stem. (6) The two genes encoding the large and small rRNA subunits(rrnL and rrnS) are inverted to the J-strand. The substantial reduction of rrnL and rrnS lead to the loss of several stem-loop structures, as found in the other two mites Leptotrombidium pallidum and Dermatophagoides pteronyssinus.4. The genetic divergence and molecular evolution of mt genomes of spider mites were analyzed from three levels (within family, genus, and species) using comparative genomics and bioinformatics. (1) The mt genomes of spider mites are smallest within the sequenced arthropods, but contain 37 genes typically found in most metazoans. The relatively small size is primarily due to the significant size reduction of PCGs, rrnL, and the putative control region in comparison with other arthropods and Acari. (2) The mt genomes of four spider mites have the same gene order, similarly high A+T content, and strong codon usage bias, but Panonychus had more GC-rich codons never used than Tetranychus. (3) The Panonychus J-strand has a positive GC-skew, which is contrast to that of Tetranychus. Two stem-loop structures of A+T-rich region were found in Panonychus, but only one in Tetranychus. (4) The tRNA genes found in the Tetranychidae mt genomes are extremely short, and 19 tRNA genes lack the D-or T-arm, even some tRNA genes lack the two arms simultaneously. There are several unusual features, such as gene overlap between adjacent tRNA genes, mismatched base at amino acid acceptor stem or anticodon stem,8 nucleotides at anticodon loop of trnI. However, these genes are not likely to be pseudogenes. First of all, their sequences are highly conserved among spider mites, especially for anticodon arm. Secondly, it has been shown that in the nematode Ascaris suum the tRNA genes that lack either the D-or T-arm are functional. Thirdly, stem mismatches and sequence overlap are common for mitochondrial tRNA genes of Acariform mites, and are probably repaired by a posttranscriptional editing process. However, functional tRNA genes that lack both the D-and T-arms have not been found before. Therefore, further experiments are needed to investigate whether these truly tRNA genes lack both D-and T-arms and if so, whether they are functional. (5) The rrnS and rrnL of four Tetranychidae mt genomes have similar stem-loop structures, but the helix H3 appears to only be present in P. citri. Compared to the 5’-end, the 3’-end of rrnL structure is more conserved among Tetranychidae. especially for the helices G16-G20. The most conserved sequences of rrnS among Tetranychidae are found in the helices 19,21,32,33,49, and 50. (6) The genetic divergence of complete mt genome sequences,13 protein-coding genes and 24 RNA genes are high among genera compared to that within genus or species. The average values of P-distance between two Tetranychus species are lower than those between two Panonychus species, but higher than those between the two P. citri strains. The cytochrome oxidase subunits (cox1, cox2) and cytochrome b (cob) are the slowest evolving genes and proteins, making them useful markers for investigating phylogenetic relationships at higher taxonomic levels. In addition, the atp8, nad2, nad6 and nad4L show high P-distance and Ka, implying that they can be used as potential markers to analyze intraspecific relationships within the Tetranychidae species.5. Although there was strong compositional heterogeneity among 28 Acari mt genome sequences, the maximum likelihood (ML) and Bayesian inference (BI) trees based on three data sets (PCG123, PCG12, and PCG2), and the ML trees based on another two data sets (PCG-RNA and PCG1) strongly supported the monophyly of Parasitiformes and Acariformes. The monophyly of Ixodida, Mesostigmata, Trombidiformes, and Sarcoptiformes were recovered:the former two consisted of Parasitiformes, and the latter two consisted of Acariformes. The sister-group relationship between Parasitengona and Eleutherengona was resolved within Trombidiformes. Also, the sister-group between Ixodidae and Argasidae was supported within Ixodida. The supported relationship within Mesostigmata was "(Phytoseioidea+ Dermanyssoidea)+Rhodacaroidea".更多还原