核基因序列在昆虫分子系统学上的应用

核基因中含有更加丰富的生物学信息,运用核基因序列或将核基因序列与线粒体基因序列相结合研究昆虫的系统发育正成为分子系统学领域的一种发展趋势.核糖体基因中18S rDNA、28S rDNA、ITS已在昆虫分子系统学中得到了广泛的应用.与核糖体基因相比,虽然编码蛋白的核基因应用于昆虫分子系统学的种类不少,但大部分都是应用于双翅目和鳞翅目昆虫的分子系统学研究中,能够成功地普遍用于多个目昆虫的系统学研究的核基因并不多.本文简要介绍了应用于昆虫分子系统学的核中核糖体基因和编码蛋白的核基因,并分析了核基因序列在分子系统学应用上的局限性和应用前景.     

核基因在两栖爬行动物分子系统学中的研究进展

从DNA水平探索生物进化的理论、生物类群的演化历史具有重要的意义,应用DNA序列研究生物的系统发育和进化规律成为当前分子系统学研究的热点,与线粒体DNA相比,核基因由于包含有更加丰富的生物学信息,运用核基因序列或将核基因序列与线粒体基因序列相结合研究两栖爬行动物的系统发育,正成为分子系统学领域的新的发展趋势.Rag-1、Rag-2、tyrosinase、rhodopsin、C-mos等核基因已在两栖爬行动物分子系统学中得到了广泛的应用.由于目前的技术手段等诸多因素,限制了更多的核基因用于两栖爬行动物分子系统学研究.为此简要介绍了目前核基因在两栖爬行动物分子系统学方面的研究进展,并分析了核基因序列在分子系统学应用上面临的问题和应用前景.     

直翅目昆虫分子系统学研究新进展

对1994年以来国内外在直翅目昆虫种群遗传变异及进化、种及种下阶元的分类鉴定、种上阶元的系统发育分析及分子进化等分子系统学方面的研究进展进行了综述。近年来,蝗亚目昆虫分子系统学方面的研究成果较为丰富,而有关螽亚目的分子系统学研究较少。线粒体基因和核基因序列联合分析、整个基因组全序列分析以及分子数据与形态学的密切结合将是分子系统学未来发展的主要研究手段。     

ITS假基因对栎属系统学研究的影响及其对分子系统学研究的启示

核糖体rDNA ITS是被子植物系统发育研究中应用最广泛的分子标记之一。以前人们认为同一物种中的ITS序列因致同进化而使不同拷贝高度一致,在分子系统学研究中常以ITS1-5.8S-ITS2序列作为构建系统进化树的基础。近年来,在对一些被子植物的研究中发现这段序列在同一物种中具有多态性,有些拷贝中的5．8S区不具编码功能,人们把含有不具编码功能5．8S区的ITS1-5．8S-ITS2序列定义为ITS假基因序列,它对同源基因致同进化的假设形成了新的挑战。在诸多应用ITS序列重建系统进化关系的研究中,栎属系统学研究因ITS假基因的发现而倍受关注。本文以栎属为例回顾了ITS假基因的发现过程,分析了其对该属系统学研究的影响,为分子生物学在植物系统进化研究中的应用提供一些新的参考。     

DNA序列在悬钩子属植物分子系统学研究中的应用进展

悬钩子属植物种类繁多,类群复杂,而且多为多倍体和杂种。该文就近年来国内外有关DNA序列在悬钩子属植物分子系统学研究中的应用现状和进展进行了综述,并对中国悬钩子属植物系统发育研究进行了展望。研究认为:叶绿体DNA序列多应用非编码区,且多与ITS序列联合分析;核基因组中ITS序列应用最为广泛,主要用于研究悬钩子属空心莓组与木莓组的进化关系、栽培品种间亲缘关系及部分杂种和多倍体的起源等;在该属植物中发现了ITS个体内多态性,但未进行ITS假基因检测,其系统学应用价值需重新评价;低拷贝核基因只有GBSSI和LEAFY有相关应用。同时认为,悬钩子属植物系统学研究中应用的DNA序列及研究类群均较少,缺乏对整个悬钩子属全面而系统的研究。指出应进一步选择具有代表性的样本、筛选合适的DNA片段,并结合形态学、孢粉学和细胞学等手段对中国悬钩子属植物系统关系进行深入研究。     

植物分子系统学近五年的研究进展概况

本文综述了与分子系统学发展密切相关的4个因素：1.分子生物学方法的不断改进;2.基因组的全序列测定;3.用于分子系统学研究的基因种类不断增加,对这些基因进化规律的认识不断深入;4.化石DNA的研究。本文还阐述了核基因及叶绿体基因在系统学研究中的应用,例举了rbcL、matk、18s rDNA和ITS序列分析在植物系统发育研究中取得的重要成果,同时提出了分子系统学研究中应注意的一些问题。     

ITS 假基因对栎属系统学研究的影响及其对分子系统学研究的启示*

核糖体rDNA ITS 是被子植物系统发育研究中应用最广泛的分子标记之一。以前人们认为同一物种
中的ITS 序列因致同进化而使不同拷贝高度一致, 在分子系统学研究中常以ITS1- 518S- ITS2 序列作为构建
系统进化树的基础。近年来, 在对一些被子植物的研究中发现这段序列在同一物种中具有多态性, 有些拷
贝中的518S 区不具编码功能, 人们把含有不具编码功能518S 区的ITS1-51 8S- ITS2 序列定义为ITS 假基因序
列, 它对同源基因致同进化的假设形成了新的挑战。在诸多应用ITS 序列重建系统进化关系的研究中, 栎
属系统学研究因ITS 假基因的发现而倍受关注。本文以栎属为例回顾了ITS 假基因的发现过程, 分析了其
对该属系统学研究的影响, 为分子生物学在植物系统进化研究中的应用提供一些新的参考。     

叶蝉科昆虫分子系统发育的研究进展

从基因组DNA的提取、研究的基因片段、PCR引物选用、扩增条件以及叶蝉科不同阶元的分子系统发育分析等方面,综述叶蝉科(半翅目:叶蝉科)昆虫分子系统发育的研究进展。目前角顶叶蝉类的研究成果相对较多,大叶蝉亚科次之,其余类群的研究较少或无。线粒体基因与核基因序列联合分析以及线粒体全序列分析以及基因序列与形态数据相结合分析,分子鉴定叶蝉与共生菌之间的协同进化的研究,将是叶蝉分子系统学未来发展的主要研究手段。     

蚕类昆虫线粒体DNA研究及其在起源与进化研究中的应用

线粒体DNA(mtDNA)属母系遗传,进化速率较核基因快且基因组结构相对简单,已作为理想的分子标记广泛应用于昆虫群体遗传学及分子系统学等研究。本文对蚕类昆虫线粒体DNA在分子水平上的最新研究进展进行了较详细的阐述,重点介绍了蚕类昆虫线粒体基因组的组成及特征、mtDNA克隆与多态性及在蚕类昆虫分子系统学研究中的应用等。     

串珠藻目分子系统学研究进展

串珠藻目(Batrachospermales)是淡水红藻中最主要的类群。近年来, 应用DNA序列分析探讨串珠藻目的系统发育, 并与传统的形态学和生态学特征相结合, 为串珠藻目系统学研究拓展了新的思路。本文回顾了串珠藻目的建立及其所含类群的研究历史, 归纳了目前在串珠藻目系统发育与进化研究中常用的分子标记方法, 其中包括核基因组的18S rDNA、26S rDNA和ITS序列, 叶绿体基因组的rbcL序列, 线粒体基因组的cox2-3序列, 以及新兴的ISSR技术, 并对各种分子标记的特点及适用范围做了评述。结果表明, ITS序列多适用于种群分化及相近种间遗传分析, ISSR标记适用于种下分类群间及同一种群不同个体间基因多态性分析, cox2-3序列在一定程度上也可用于同一种群不同个体间的基因多态性分析, 而18S rDNA 与rbcL序列既可用于种间关系分析, 又可用于更高水平分析的构建系统树。这些分子标记已被证明在研究串珠藻目系统地理、物种起源和散布机制方面有着广泛的应用前景。同时, 本文对串珠藻目分子系统学研究的最新进展也进行了概述,并对今后的研究方向做了展望。     

Characterisation of insect and plant origins using DNA extracted from small volumes of bee honey

A DNA-based tool was validated that potentially enables the characterisation of both plant and insect of origin of small (approximately 1 ml) samples of bee honey. Using this method, mitochondrial, nuclear and chloroplast DNA (mtDNA, nuDNA, cpDNA) markers were successfully extracted, PCR amplified, and sequenced from a range of honeys, and the relative amount of plant nuDNA and cpDNA, and bee mtDNA in the samples was quantified using quantitative real-time PCR. Short, but taxonomically informative lengths of insect and plant organelle DNA could be routinely recovered from all honey samples tested, and longer organelle, and nuclear DNA sequences can be recovered from many. The data also enabled preliminary characterisation of the quality of these different DNA sources in honey. Although the absolute quantity of the different genetic markers varied considerably between sample, a general trend was observed of insect mtDNA dominating over plant organelle DNA, and with plant nuclear DNA at the lowest levels. Furthermore there was a clear correlation between the plant DNA content and the success of the PCR assays. To maximise successful characterisation of samples, future studies are recommended to focus on the use of organelle markers, and limit the size of PCR amplicons targeted, although with appropriate sample selection and assay optimisation, other approaches may be possible.     

On the value of Elongation factor-1α for reconstructing pterygote insect phylogeny

Pterygota are traditionally divided in two lineages, the “Palaeoptera” and Neoptera. Despite several efforts neither morphology nor molecular systematics have resolved the phylogeny of the pterygote insects. Too few markers have yet been identified for adequately tracking mesozoic-aged divergences. We tested the Elongation factor-1α for its phylogenetic value in pterygote insect systematics. This highly conserved nuclear protein-coding gene has previously been reported to be useful in other groups for phylogenetic analyses at the intraordinal level as well as at the interordinal level. The analyses suggest that EF-1α DNA sequences as well as intron positions provide informative markers for pterygote phylogenetics.     

Nuclear DNA analyses in genetic studies of populations: practice,problems and prospects

Population-genetic studies have been remarkably productive and successful in the last decade following the invention of PCR technology and the introduction of mitochondrial and microsatellite DNA markers. While mitochondrial DNA has proven powerful for genealogical and evolutionary studies of animal populations, and microsatellite sequences are the most revealing DNA markers available so far for inferring population structure and dynamics, they both have important and unavoidable limitations. To obtain a fuller picture of the history and evolutionary potential of populations, genealogical data from nuclear loci are essential, and the inclusion of other nuclear markers, i.e. single copy nuclear polymorphic (scnp) sequences, is clearly needed. Four major uncertainties for nuclear DNA analyses of populations have been facing us, i.e. the availability of scnp markers for carrying out such analysis, technical laboratory hurdles for resolving haplotypes, difficulty in data analysis because of recombination, low divergence levels and intraspecific multifurcation evolution, and the utility of scnp markers for addressing population-genetic questions. In this review, we discuss the availability of highly polymorphic single copy DNA in the nuclear genome, describe patterns and rate of evolution of nuclear sequences, summarize past empirical and theoretical efforts to recover and analyse data from scnp markers, and examine the difficulties, challenges and opportunities faced in such studies. We show that although challenges still exist, the above-mentioned obstacles are now being removed. Recent advances in technology and increases in statistical power provide the prospect of nuclear DNA analyses becoming routine practice, allowing allele-discriminating characterization of scnp loci and microsatellite loci. This certainly will increase our ability to address more complex questions, and thereby the sophistication of genetic analyses of populations.     

Molecular marker systems in insects: current trends and future avenues

Insects comprise the largest species composition in the entire animal kingdom and possess a vast undiscovered genetic diversity and gene pool that can be better explored using molecular marker techniques. Current trends of application of DNA marker techniques in diverse domains of insect ecological studies show that mitochondrial DNA (mtDNA), microsatellites, random amplified polymorphic DNA (RAPD), expressed sequence tags (EST) and amplified fragment length polymorphism (AFLP) markers have contributed significantly for progresses towards understanding genetic basis of insect diversity and for mapping medically and agriculturally important genes and quantitative trait loci in insect pests. Apart from these popular marker systems, other novel approaches including transposon display, sequence-specific amplification polymorphism (S-SAP), repeat-associated polymerase chain reaction (PCR) markers have been identified as alternate marker systems in insect studies. Besides, whole genome microarray and single nucleotide polymorphism (SNP) assays are becoming more popular to screen genome-wide polymorphisms in fast and cost effective manner. However, use of such methodologies has not gained widespread popularity in entomological studies. The current study highlights the recent trends of applications of molecular markers in insect studies and explores the technological advancements in molecular marker tools and modern high throughput genotyping methodologies that may be applied in entomological researches for better understanding of insect ecology at molecular level.     

Introgression of mitochondrial DNA among lineages in a hybridogenetic ant

We report a remarkable pattern of incongruence between nuclear and mitochondrial variations in a social insect, the desert ant Cataglyphis hispanica. This species reproduces by social hybridogenesis. In all populations, two distinct genetic lineages coexist; non-reproductive workers develop from hybrid crosses between the lineages, whereas reproductive offspring (males and new queens) are typically produced asexually by parthenogenesis. Genetic analyses based on nuclear markers revealed that the two lineages remain highly differentiated despite constant hybridization for worker production. Here, we show that, in contrast with nuclear DNA, mitochondrial DNA (mtDNA) does not recover the two lineages as monophyletic. Rather, mitochondrial haplotypes cluster according to their geographical origin. We argue that this cytonuclear incongruence stems from introgression of mtDNA among lineages, and review the mechanisms likely to explain this pattern under social hybridogenesis.     

Sequence capture across large phylogenetic scales by using pooled PCR‐generated baits: A case study of Lepidoptera

Sequence capture across large phylogenetic scales is not easy because hybridization capture is only effective when the genetic distance between the bait and target is small. Here, we propose a simple but effective strategy to tackle this issue: pooling DNA from a number of selected representative species of different clades to prepare PCR‐generated baits to minimize the genetic distance between the bait and target. To demonstrate the utility of this strategy, we newly developed a set of universal nuclear markers (including 94 nuclear protein‐coding genes) for Lepidoptera, a superdiverse insect group. We used a DNA pool from six lepidopteran species (representing six superfamilies) to prepare PCR baits for the 94 markers. These homemade PCR baits were used to capture sequence data from 43 species of 17 lepidopteran families, and 94% of the target loci were recovered. We constructed two data sets from the obtained data (one containing ~90 kb target coding sequences and the other containing ~120 kb target + flanking coding sequences). Both data sets yielded highly similar and well‐resolved trees with 90% of nodes having >95% bootstrap support. Our capture experiment indicated that using DNA mixtures pooled from different clade‐representative species of Lepidoptera to prepare PCR baits can reliably capture a large number of targeted nuclear markers across different Lepidoptera lineages. We hope that this newly developed nuclear marker set will serve as a new phylogenetic tool for Lepidoptera phylogenetics, and the PCR bait preparation strategy can facilitate the application of sequence capture techniques by researchers to accelerate data collection.     

Pleistocene isolation, secondary introgression and restricted contemporary gene flow in the pig-eye shark, Carcharhinus amboinensis across northern Australia

We examine the structure and phylogeography of the pig-eye shark (Carcharhinus amboinensis) common in shallow coastal environments in northern Australia using two types of genetic markers, two mitochondrial (control region and NADH hydrogenase 4) and two nuclear (microsatellite and Rag 1) DNA. Two populations were defined within northern Australia on the basis of mitochondrial DNA evidence, but this result was not supported by nuclear microsatellite or Rag 1 markers. One possibility for this structure might be sex-specific behaviours such as female philopatry, although we argue it is doubtful that sufficient time has elapsed for any potential signatures from this behaviour to be expressed in nuclear markers. It is more likely that the observed pattern represents ancient populations repeatedly isolated and connected during episodic sea level changes during the Pleistocene epoch, until current day with restricted contemporary gene flow maintaining population genetic structure. Our results show the need for an understanding of both the history and ecology of a species in order to interpret patterns in genetic structure.     

质体基因工程中选择标记基因研究进展

与细胞核基因工程相比,质体基因工程能更安全、精确和高效地对外源基因进行表达,作为下一代转基因技术已广泛用于基础研究和生物技术应用领域。与细胞核基因工程一样,质体基因工程中也需要合适的选择标记基因用于转化子的筛选和同质化,但基于质体基因组的多拷贝性和母系遗传特点,转化子的同质化需要一个长期的筛选过程,这就决定了质体基因工程中选择标记基因的选择标准将不同于细胞核基因工程中广泛使用的现行标准。目前,质体基因工程的遗传转化操作中使用较多的是抗生素选择标记基因,出于安全性考虑,需要找到可替换、安全的选择标记基因或有效的标记基因删除方法。本文在对质体基因工程研究的相关文献分析基础之上,对主要使用的选择标记基因及其删除体系进行了综述,并对比了其优缺点,同时探讨了质体基因工程中所使用的报告基因,以期为现有选择标记基因及其删除体系的改进和开发提供一定参考,进一步推动质体基因工程,尤其是单子叶植物质体基因工程的发展。     

Lack of genetic isolation by distance,similar genetic structuring but different demographic histories in a fig‐pollinating wasp mutualism

Historical abiotic factors such as climatic oscillations and extreme climatic events as well as biotic factors have shaped the structuring of species' genetic diversity. In obligate species‐specific mutualisms, the biogeographic histories of the interacting species are tightly linked. This could be particularly true for nuclear genes in the Ficus‐pollinating wasp mutualistic association as the insects disperse pollen from their natal tree. In this study, we compare spatial genetic structure of plant and pollinator for the Ficus hirta–Valisia javana association throughout southeast China including Hainan Island, for both nuclear and cytoplasmic markers. We show that dispersal of the insect leads to plant and insect presenting similar signatures of lack of genetic isolation by distance for nuclear genes on the continent over a distance of 1000 km. But we also show that the demographic histories of plant and insect are strikingly different. This is in agreement with extreme climatic events leading to transient regional extinctions of the insects, associated with local survival of the plants. We also observe evidence of genetic differentiation for both wasps and fig‐tree between the continent and Hainan Island, although the Qiongzhou Strait is only on average 30 km wide, suggesting that geographic isolation by itself has not been sufficient to generate this differentiation. Hence, our results suggest that in highly dispersive mutualistic systems, isolation‐by‐dispersal limitation across a geographic barrier could be supplemented by isolation by adaptation, and maybe by coevolution, allowing further genetic divergence. In such systems, species may frequently be composed of a single population.     

Concordance of genetic divergence among sockeye salmon populations at allozyme, nuclear DNA, and mitochondrial DNA markers

Abstract.— We examined genetic variation at 21 polymorphic allozyme loci, 15 nuclear DNA loci, and mitochondrial DNA in four spawning populations of sockeye salmon ( Oncorhynchus nerka ) from Cook Inlet, Alaska, to test for differences in the patterns of divergence among different types of markers. We were specifically interested in testing the suggestion that natural selection at allozyme loci compromises the effectiveness of these markers for describing the amount and patterns of gene flow among populations. We found concordance among markers in the amount of genetic variation within and among populations, with the striking exception of one allozyme locus ( sAH ), which exhibited more than three times the amount of among-population differentiation as other loci. A consideration of reports of discordance between allozymes and other loci indicates that these differences usually result from one or two exceptional loci. We conclude that it is important to examine many loci when estimating genetic differentiation to infer historical amounts of gene flow and patterns of genetic exchange among populations. It is less important whether those loci are allozymes or nuclear DNA markers.