茶园茶黄蓟马及其近似种的DNA条形码鉴定

基于 mtDNA COI 基因的 DNA 条形码技术是一种新的物种鉴定手段,本文尝试应用该技术对6个不同地理种群的茶黄蓟马与茶园中的其他4种常见蓟马进行了鉴别。测定各种类 mtDNA COI 基因序列并在 BOLD 系统进行比对,采用 MEGA5构建系统进化树,结果表明基于 mtDNACOI 基因的DNA 条形码技术可实现对茶园中茶黄蓟马的快速鉴定。     

基于DNA条形码技术的湖南省柑橘主产区实蝇幼虫分子鉴定

为明确湖南省柑橘主产区的实蝇入侵为害现状,从该省7个市(自治州)22个地点收集柑橘蛆果中的幼虫,利用DNA条形码技术对其进行分子鉴定,并以DNA条形码序列作为分子标记探究橘大实蝇Bactrocera minax的20个中国地理种群(湖南省6个组群共16个种群、其它3省市4个种群)以及1个印度地理种群间的亲缘地理关系,分析橘大实蝇在我国的遗传进化关系。结果表明,仅采集自邵阳市城步苗族自治县柑橘蛆果中的5头幼虫被鉴定为蜜柑大实蝇B. tsuneonis,其余21个地点采集的595头幼虫均被鉴定为橘大实蝇。21个橘大实蝇地理种群的平均单倍型多样性为0.75,核苷酸多样性为0.0032,核苷酸差异数为2.13,中国所有地理种群均具有较高的遗传多样性;单倍型网络进化图显示湖南、重庆、贵州种群共享的单倍型H3为原始单倍型,表明其为比较原始的种群;AMOVA分析结果显示种群内个体间遗传变异占总体变异的59.04%,是遗传变异的主要来源;遗传分化结果表明湖南省6个组群间均出现了中度至高度的遗传分化,FST在0.0521～0.7795之间。表明DNA条形码技术可用于蜜柑大实蝇和橘大实蝇幼虫的分子鉴定及其种群遗传进化分析。     

DNA条形码技术在小花蝽属昆虫分类鉴定中的应用

本研究利用DNA条形码技术对13种小花蝽属Orius Wolff昆虫进行了鉴定,进行了59条COI基因序列碱基组成及种内、种间遗传距离的分析,采用邻接法、最大简约法、贝叶斯推论法构建了系统发育树。结果表明,小花蝽属昆虫COI基因序列碱基组成与典型的昆虫线粒体DNA一致,A+T平均含量(66.4%)明显高于G+C含量,密码子的第3位A+T含量高达90.7%,碱基替换多为同义替换;13种小花蝽种内平均遗传距离为0.008,种间平均遗传距离为0.128,种内、种间遗传距离没有重叠区域。3种方法构建的系统发育树的聚类分析与形态学鉴定结果基本一致,除微小花蝽Orius minutus(Linnaeus)可能存在隐存种现象外,其他同一种群的不同个体单独聚为一支。利用DNA条形码技术对小花蝽属昆虫进行物种快速分子鉴定具有可行性。     

西花蓟马分类鉴定方法的研究进展

西花蓟马Frankliniella occidentalis(Pergande)是一种危险性外来入侵害虫,虫体小,鉴定困难。本文总结了国内外西花蓟马分类鉴定方法的研究进展,包括形态学鉴别、分子生物学方法以及基于表皮碳氢化合物分析的生化分类方法,并讨论了各种分类方法的优势和弊端,最后对西花蓟马的分类研究前景进行了展望。     

基于DNA条形码的向日葵田弯管列当种群遗传多样性研究

弯管列当是危害我国向日葵最严重的寄生杂草。为了明确我国向日葵田弯管列当种群的遗传多样性,本试验利用rbcL、matK、ITS2条形码序列对采自我国向日葵主产区的58份弯管列当样品进行PCR扩增及测序,采用Vector NTI软件对测序结果进行剪切比对,利用MEGA 6.0软件计算种内遗传距离并构建系统发育树。利用扫描电镜观察其种子的显微形态特征。结果表明,3个DNA条形码序列中仅ITS2片段扩增测序结果理想并表现出较好的聚类结果。各样品ITS2序列剪切比对后长度为453 bp,种内遗传距离为0.002～0.007,通过比较各样品ITS2序列的碱基组成和差异位点,能将不同弯管列当种群区分开。ITS2聚类结果表明58份弯管列当样品聚为3类,分别为Ⅰ型、Ⅱ型和Ⅲ型。形态分类结果表明,不同类型弯管列当在植株形态、种子形状及微观形态结构等方面存在差别。由于不同弯管列当种群的生境、寄主(向日葵栽培品种)不同,其种群遗传进化差异显著。基于ITS2条形码和扫描电镜形态学观察相结合的方法可用于弯管列当种群遗传多样性研究。     

分子标记技术在斑潜蝇中的应用进展

斑潜蝇是农作物、蔬菜和花卉等植物上的重要害虫,尤其是美洲斑潜蝇、南美斑潜蝇和三叶斑潜蝇的入侵,给我国农业生产和对外贸易带来重大损失。采用传统的形态学分类特征对斑潜蝇进行鉴定极易混淆,更难进行地理分布与遗传多样性分析,近年来迅速发展的分子标记技术为解决斑潜蝇遗传及鉴定等问题提供了有效的手段。本文主要概述了DNA分子标记技术在斑潜蝇的分子鉴定、地理分布及传播途径、种群遗传分化及种间杂交等方面的应用现状,并对其应用前景进行了展望。     

运用DNA条形码技术鉴定茶园蜘蛛物种

运用DNA条形码技术,对广东潮州茶园蜘蛛物种进行了分子鉴定。本研究利用基因测序技术获取了凤凰山区域茶园50种蜘蛛的74条线粒体细胞色素C氧化酶亚基I (mitochondrial cytochrome C oxidase subunitⅠ, COⅠ)基因序列。使用邻接法(neighbor joining, NJ)构建系统发育树,运用ABGD(automatic barcode gap discovery)软件对蜘蛛样本进行聚类分析。结果表明:邻接法构建的系统发育树聚类结果与ABGD软件划分结果以及形态分类鉴定结果相一致,运用DNA条形码可以有效地对蜘蛛物种进行分子鉴定。这对茶园蜘蛛疑难物种和新物种的鉴定具有重要意义,在茶园蜘蛛物种多样性的研究中也具有重要价值。由此表明,基于COⅠ基因的DNA条形码对于本研究中所涉及的蜘蛛物种的划分具有较好的区分结果,可以作为一种有效的工具在茶园蜘蛛物种鉴定中进行应用。     

大赖草种群遗传多样性的AFLP分析

采用荧光AFLP标记对大赖草(Leymus racemosus(Lam.)Tzvel)的6个天然种群进行了遗传多样性和遗传结构进行分析,6对选择性扩增引物共扩增出1269条清晰的条带,其中多态性位点1203个(PPL=80.61％).在物种水平上,Nei's遗传多样性指数H=0.188,Shannon多态性信息指数I=0.313;在种群水平上,H =0.177;I=0.288.遗传分化系数Fst为0.1009,表明有10.09％的遗传变异存在于种群之间,基因流Nm为4.4554＞1,说明种群间基因交流可以阻止由于遗传漂变导致的遗传分化,遗传分化呈现均质化的趋势.PCoA聚类结果表明,大赖草种群间的遗传距离与地理距离有一定的相关性,但未达到显著水平.该研究结果显示,遗传多样性水平不仅与物种本身特性和研究方法有关,濒危植物并不一定表现为遗传变异水平的降低而濒危,还与人类活动与气候环境密切相关.     

基于线粒体 COⅠ和 COⅡ基因的5种不同寄主植物 西花蓟马种群遗传多样性研究

西花蓟马Frankliniella occidentalis(Pergande)是一种分布广、危害大的世界性检疫害虫。本研究旨在探讨田间常见5种寄主植物上的西花蓟马种群的寄主专化性和遗传多样性。以采自甘肃、宁夏的辣椒、茄子、蜀葵、月季、美人蕉等5种寄主植物上的西花蓟马为对象,以线粒体COⅠ和COⅡ基因为靶标,应用Arlequin 3.5软件进行种群遗传多样性、遗传分化、基因流水平及分子变异分析,以MEGA 7.0和Network 5.0软件分别构建单倍型系统发育树和中接网状树。结果显示,当以mtDNACOⅠ和COⅡ基因为靶标时,分别检测到13种和12种单倍型,其中单倍型H_1和H_2为优势单倍型;辣椒和茄子上的单倍型数量只有3种(COⅠ基因)或2种(COⅡ基因),而蜀葵、月季和美人蕉上的单倍型数量比较多,为7~9种(COⅠ基因)或8~9种(COⅡ基因);辣椒和茄子上的种群单倍型种类、单倍型多样性、核苷酸多样性和核苷酸平均差异数等均较蜀葵、月季和美人蕉上的种群低。COⅠ和COⅡ基因总寄主种群Fu's Fs检验结果分别为2.36和4.06,种群大小整体保持稳定;总寄主种群固定指数FST和基因流Nm分析表明,西花蓟马各寄主植物种群之间基因交流充分,尚未发生明显的遗传分化;AMOVA分析显示遗传变异主要来自种群内部;基于COⅠ和COⅡ基因序列的单倍型聚类和网状树都明显分为两支,分别对应西花蓟马的温室品系Glasshouse strain和羽扇豆品系Lupin strain;其中温室品系在所有寄主植物种群中均有发生,而羽扇豆品系的单倍型主要来自多年生的蜀葵、月季和美人蕉等植物,在辣椒和茄子上并无羽扇豆品系发生。研究结果对西花蓟马种群扩张机制探讨、遗传动态分析以及有效防控措施的制定具有一定指导意义。     

基于mtDNA-COⅠ的云南西花蓟马的遗传分析

西花蓟马是我国重要的入侵害虫之一, 为了解其不同地理种群的遗传分化, 测定了云南6个地理种群141个个体的mtDNA-COⅠ基因片段。结果表明：在比对的433 bp基因片段中, 变异位点19个, 共检测出5个共享单倍型, 单倍型间出现了明显的遗传分化, 证实了云南西花蓟马为一个复合种群。地理种群间的遗传距离较低, 遗传距离与地理距离无显著相关性。各地理种群西花蓟马的遗传分化指数 Fst 为-0.021 19, 种群间变异多为负值, 表明云南西花蓟马种群遗传变异主要来自于种群内部, 种群间未发生明显的遗传分化。     

DNA barcoding: a new module in New Zealand's plant biosecurity diagnostic toolbox

Molecular methods for identification of high risk pests and pathogens have been employed for more than a decade to supplement standard diagnostic protocols. However, as the volume of traded goods continues to increase so does the breadth of taxa that diagnosticians need to deal with. Keeping pace by introducing more molecular tests that are typically species-group specific is not an efficient way to progress. Since 2005 classical DNA barcoding using cytochrome oxidase I sequence has been employed routinely in New Zealand for the highest risk insect species (fruit flies and lymantriid moths). Subsequently a broader range of pests have been considered. Case studies are presented here for three important lepidopteran pests, Lymantria mathura (pink gypsy moth), Conogethes punctiferalis (yellow peach moth) and Hyphantria cunea (fall web worm), as well as a trial to identify miscellaneous border interceptions. While the data support the effectiveness of DNA barcoding for border diagnostics, they also raise issues around cryptic species identification and potential species discovery that could impact on operational biosecurity systems.     

基于DNA条形码的广西苦瓜中实蝇幼虫分子鉴定研究*

实蝇是多种热带、亚热带水果和蔬菜的重要害虫,其卵、幼虫、蛹难以通过形态特征进行种类鉴定。DNA条形码是近年来出现的物种分子鉴定技术,能够实现对昆虫非成熟虫态的快速鉴定。本研究利用DNA条形码技术和BOLD系统,选取线粒体DNA细胞色素氧化酶I（COI）基因片段,针对采自于广西南宁地区苦瓜中的实蝇幼虫样品进行了序列测定、比对和分析。结果显示,在所检测的10头实蝇幼虫样品中,有4头为瓜实蝇［Bactrocera cucurbitae (Coquillett)］、6头为南亚果实蝇［Bactrocera tau (Walker)］。     

入侵种西花蓟马与本地近缘种花蓟马的双基因鉴定

快速准确的害虫种类识别是有效筛选天敌昆虫开展靶向性生物防控的关键。入侵种西花蓟马Frankliniella occidentalis与本地种花蓟马F.intonsa常同域同期同种寄主上发生,因体型小、形态相似,难以快速准确鉴定。本研究以mt DNA COI与r DNA ITS2基因为标记对我国10个采样点的西花蓟马和花蓟马开展分子鉴定研究。结果显示,当以COI或ITS2基因为靶标时,西花蓟马与花蓟马间的平均遗传距离分别为0.221和0.113,是种内遗传距离的22.1和18.8倍,且种内、种间遗传距离间无重叠,并在系统发育树上聚为独立的分支,表明2种基因均可准确区分西花蓟马和花蓟马。此外,基于COI基因构建的系统发育树,西花蓟马的各单倍型聚为了2个亚分支,并分别对应温室品系和羽扇豆品系,表明COI基因还可用于西花蓟马品系的识别鉴定。研究结果对近缘种花蓟马的快速鉴定、专食性天敌昆虫的筛选,及其生防效果评价具有重要意义。     

昆虫隐存种的鉴定方法及其在有害生物防控与物种保护中的应用

昆虫中存在很多外部形态高度相似但具生殖隔离的隐存种,这给物种分类和相关研究带来更多挑战。目前,关于昆虫隐存种的研究日益受到关注。该文基于筛选出的368篇中文文献对昆虫隐存种的鉴定方法及其潜在研究价值和应用领域进行系统总结,发现自2003年DNA条形码技术提出以来,关于昆虫隐存种分类的研究迅速增加,已有鉴定方法可概括为4类,分别为分子分类（298篇,80.98%）、生态分类（191篇,51.90%）、生化分类（4篇,1.09%）和细胞分类（4篇,1.09%）。基于单一分类方法的研究最多（241篇,65.49%）,其中分子分类的使用最多（172篇,46.74%）,其次是生态分类（64篇,17.39%）;基于2种分类方法的研究次之（125篇,33.97%）,其中分子分类与生态分类的联合使用最多（124篇,33.70%）;采取3种及以上分类方法的研究较少（2篇,0.54%）。目前,昆虫隐存种鉴定研究主要应用于农业病虫害防治（262篇,71.20%）、物种多样性评估（74篇,20.11%）、疾病防控（27篇,7.34%）和生境保护（6篇,1.63%）等领域。开展昆虫隐存种的研究有利于促进有害生物防控和物种保护,建议未来将整合分类学评估体系纳入到昆虫隐存种研究中并在分类实践中探讨隐存种演化机制。     

DNA条形码技术在入侵植物刺萼龙葵检验检疫中的应用

刺萼龙葵(Solanum rostratum)是危害严重的外来入侵植物,基于种子形态的传统检疫方法有一定的局限性,不依赖于形态学特征的DNA条形码技术是基于DNA序列差异对物种进行鉴定的新方法,是传统形态学鉴定方法的有力补充。以刺萼龙葵及其8种龙葵亚属近缘植物为材料,从Gen Bank数据库中下载rbc L、mat K和内转录间隔区(ITS)序列,进行DNA条形码分析,结果显示ITS鉴定效果优于其他片段,可把刺萼龙葵从其近缘植物中鉴别出来。     

Research on plant-parasitic nematode biology conducted by the United States Department of Agriculture-Agricultural Research Service

The recent de-registration of several chemical nematicides and the impending loss of methyl bromide from the pest-control market necessitate the development of new methods for controlling nematode-induced crop damage. One approach for developing novel target-specific controls is by exploiting fundamental differences between the biological processes of nematodes and their host plants. Researchers of the Agricultural Research Service (ARS) of the US Department of Agriculture are actively exploring these differences. Research accomplishments include the discovery of heat shock protein genes possibly involved in developmental arrest of the soybean cyst nematode, the identification of neuropeptides and female-specific proteins in the soybean cyst nematode, the disruption of nematode reproduction with inhibitors of nematode sterol metabolism, the development of novel morphological and molecular (heat shock protein genes and the D3 segment of large subunit ribosomal DNA) features useful for nematode identification and classification, and the elucidation of the population genetics of potato cyst nematode pathotypes. In addition, several ARS researchers are investigating biological determinants of nematode response to management strategies utilized in agricultural fields. These collective efforts should lead to new chemical and non-chemical alternatives to conventional nematode control strategies.     

A definitive method for distinguishing cultivated onion from its weedy mimic,Asphodelus fistulosus,at multiple developmental stages

The onion, Allium cepa, is an important vegetable crop around the world. Globally, India ranks second in onion production, but yields are often much lower than in many other onion producing countries. This study reports on one major contributing factor to low onion yields in India: the presence of a weedy mimic that is morphologically similar to onion until the onset of flowering. We used morphological and molecular methods to identify this mimic as Asphodelus fistulosus, an exotic weed not previously reported in India. Our study indicated that molecular markers derived from the rbcL and/or matK chloroplast genes in a DNA barcoding approach allow accurate identification of this weed using any tissue and even at early developmental stages. Our results also showed that visual examination of seed lots coupled with DNA barcoding analysis of samples of 5–10 individual seeds can be used to confirm identification. The ability to easily identify contaminating material before and during cultivation can allow farmers to minimise production losses and prevent further spreading of this weed.     

A high-density random-oligonucleotide genome microarray for universal diagnostics

Microarrays offer virtually unlimited diagnostics capability, and have already been developed into diagnostic chips for many different plant pests. The full capacity of such chips, however, has lagged far behind their full potential. The main reason for this is that current chip design relies on a priori genetic information for target organisms and on a consensus on the genetic sequences to be used in particular organism groups. Such information is often unavailable and laborious to obtain. Thus, broad-application diagnostic microarrays have been limited to narrow organism groups focused on Genera of pests/pathogens or those affecting individual host crops, without applicability for simultaneous detection of diverse pests affecting many crops. This paper describes the development of a diagnostic microarray platform that has universal application based on genomic fingerprinting of any organism without a need for a priori sequence information. Taxon-specific hybridization patterns are obtained by unique hybridisation of genomic DNA to 100s–1000s of short random oligonucleotide probes. Taxon identification is then achieved by comparison of hybridisation patterns from an unknown sample against a reference-pattern database. Using bacteria as a model pathogen group, these methods deliver highly reproducible hybridisation patterns with high resolution power and enable discrimination at the species and subspecies level.