环境DNA宏条形码在生物多样性研究与监测中的应用

环境DNA宏条形码(eDNA metabarcoding)技术通过提取水体、土壤、空气中的环境DNA,使用引物PCR扩增与高通量测序,进行物种鉴定与生物多样性评估.作为一种新的监测技术,相比于传统监测技术更加快捷、准确以及对自然环境的破坏小,因此在一定程度上改变了我们调查地球生物多样性的方式.本文综述了环境DNA宏条形...     

线粒体基因组测序策略和方法

本文综述了线粒体基因组测序策略和方法,在传统测序方法中介绍了基于物理分离线粒体DNA的克隆文库测序方法和基于PCR扩增产物的直接测序方法,后者重点介绍了基于长PCR扩增产物的引物步移法和基于总DNA的引物步移法;应用新一代高通量测序方法有基于总DNA样品的方法,包括需要预扩增mtDNA的多物种平行高通量和无需预扩增mtDNA的高通量方法,基于总RNA样品的转录组测序方法等。在实际工作中,选择哪种方法取决于研究规模、样品大小和保存状态、经费情况等。总的来说,基于长PCR扩增产物的引物步移法尤其适合小规模昆虫线粒体基因组研究,而对于大规模线粒体基因组研究来说,NGS技术无疑是省时省力的最佳选择。     

植物罹病组织中南方、爪哇、花生根结线虫的LAMP快速检测

【目的】建立一种基于环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术,从植物罹病组织中直接检测3种常见的根结线虫,为根结线虫的监测和防治提供技术支持。【方法】分别采用3种根结线虫的种类特异性引物对所选择的根结线虫的DNA片段进行PCR扩增,扩增产物纯化、回收并测序。根据3种根结线虫的测序结果,针对种类特异区段,采用PrimerExplorerV4软件,分别设计3种根结线虫的LAMP引物。设计的引物组人工合成后,以提取的纯化种群线虫DNA为模板,分别进行引物组的特异性测试,筛选出分别针对3种根结线虫的最佳引物组。【结果】研究设计的3种根结线虫的LAMP特异性引物能够直接从植物根结中检测出南方、花生、爪哇3种常见根结线虫,LAMP快速检测体系为:dNTPS浓度为1 mmol·L~(-1),Mg~(2+)的浓度为5 mmol·L~(-1),不添加甜菜碱,反应时间为45 min。【结论】本实验建立的南方、花生、爪哇根结线虫LAMP快速分子检测方法,具有特异性强、灵敏度高、简单、快速、经济等特征,能够从罹病植物组织中快速准确地检测出南方、花生和爪哇根结线虫,具有极高的实践应用价值。     

基于新一代测序技术的mtDNA突变检测方法学的建立

目的:大量研究证实线粒体DNA(mtDNA)突变与肿瘤发生及进展密切相关,但使用传统测序方法难以高通量、高精确度的检测mtDNA突变,为此本研究建立了基于新一代测序技术的mtDNA突变检测方法.方法:提取肝癌患者癌、癌旁组织以及外周血细胞总DNA,利用PCR技术对线粒体基因组进行富集并对PCR产物进行平末端、粘性末端连接或对PCR引物进行氨基修饰,构建mtDNA测序文库.经Illumina HiSeq 2000平台测序后利用生物信息学方法与人类mtDNA参考序列进行比对,并进行测序数据分析.结果:通过对不同质量基因组DNA进行评估后,发现三对引物法适用于大部分DNA样本的mtDNA富集.进一步我们发现PCR引物的氨基修饰可显著提高测序数据覆盖均一性,降低测序成本.结论:本研究利用新一代测序技术通过对线粒体DNA富集方法以及测序覆盖度均一性进行优化,建立了一套灵敏、特异、高通量的mtDNA突变检测策略,为mtDNA突变与疾病研究提供了新方法.     

宁南山区不同植被恢复方式下土壤线虫群落特征:形态学鉴定与高通量测序法比较

线虫是土壤食物网的重要组分, 也是土壤健康与生态系统恢复的重要指示生物, 因此准确测定线虫群落特征是发挥其生态指示作用的基础。传统线虫学研究多采用形态学鉴定方法, 但高通量测序技术近年来也逐渐受到重视。然而, 关于这两种方法的对比研究目前仍比较缺乏。本研究同时采用形态学鉴定和高通量测序法, 在黄土高原宁夏南部山区, 对不同植被恢复方式下(农田、自然恢复草地、柠条(Caragana korshinskii)人工林地和苜蓿人工草地)土壤线虫的数量、群落格局和生态指数进行了测定和比较。结果表明: (1)高通量测序仅能提供线虫类群的相对多度, 而形态学鉴定法能够测定土壤线虫的绝对多度, 后者测定结果表明3种植被恢复样地, 特别是自然恢复草地和柠条人工林地, 较农田具有更高的土壤线虫多度; (2)高通量测序法检获的线虫类群数(3纲4目26科42属)高于形态学鉴定法的测定结果(2纲3目18科27属), 但两种方法仅检获15个共有线虫属, 前者检测到的植物寄生线虫属数(22属)较后者(7属)显著增加, 而食细菌线虫和杂食-捕食线虫则相反; (3)在两种方法下, 相比农田, 3种植被恢复样地尤其是自然恢复草地和柠条人工林地, 其食微线虫的相对多度均显著下降, 而植物寄生线虫和杂食-捕食线虫的相对多度大幅上升, 这也导致线虫成熟度指数(MI)和植物寄生线虫指数(PPI)的提高及瓦斯乐卡指数(WI)的显著下降; (4)相比形态学鉴定法, 高通量测序法能检测到更丰富多样的植物寄生线虫, 在该方法下土壤线虫群落的组成、结构和生态指数在植被恢复样地与农田之间的差异也更为显著。综上所述, 采用形态学鉴定和高通量测序法测定的不同植被恢复方式下的线虫群落特征具有显著差异。     

DNA提取对微生物多样性测序分析的影响

运用高通量测序技术分析复杂样品中微生物种群的变化情况,已经成为目前微生物研究领域的热点问题之一。而微生物的样品准备,如DNA提取和16S可变区的扩增等,对于测序完成后的数据分析以及微生物原始群落组成的影响是至关重要的。采用国产试剂盒(天根土壤微生物基因组提取试剂盒)和进口试剂盒(MOBIO土壤微生物基因组提取试剂盒)分别对土壤样品和羊瘤胃食糜样品进行DNA提取。然后选取总DNA起始量为25ng,对16S V3可变区进行PCR扩增和文库构建,最后通过数据分析比较不同试剂盒提取的DNA对微生物多样性变化的影响,包括OTU数目、稀释曲线、微生物数量及物种种类等。研究发现,在相同DNA模板量和PCR条件下,进口试剂盒提取的DNA能够获得更多的微生物种类。     

蚜虫基因组DNA提取方法的改进

蚜虫基因组DNA的提取是蚜虫分子生物学研究中的难点。参照动物基因组DNA的提取方法,根据蚜虫体型微小,体表有外骨骼的特点,对SDS法作了改进。改进的方法无需用组织捣碎棒破碎虫体,操作简便。与现在常用的提取方法相比,改进的SDS法能快速、有效地提取单头蚜虫的基因组DNA,适用于RAPD随机引物和测序引物的PCR扩增。     

线粒体基因组的高通量测序策略

综述了高通量测序技术在线粒体全基因组测序中的策略,利用该技术对线粒体全基因组进行序列测定的方法可以归纳为两种,一种是先对目标mt DNA进行富集,包括mt DNA的提取纯化,目标区域PCR扩增法以及特异性探针杂交富集法(可分为基于微阵列和基于PCR探针的杂交富集法),然后对富集出的线粒体DNA进行高通量测序;另一种是先从待测样本的基因组高通量数据中挖掘出线粒体基因组序列信息,之后利用诱饵序列或者近缘物种的线粒体全基因组参考序列,使用软件MITObim对其进行组装。此外,还给出了线粒体高通量测序的优化流程图和介绍了混合样品的线粒体高通量测序策略。     

高通量测序分析冻融期碳源输入方式对土壤线虫群落的影响

植物碳源输入途径变化对土壤生物群落的影响研究是目前学术界关注的热点问题。为探究温带森林生态系统碳源输入方式对土壤线虫群落的影响,通过Illumina MiSeq技术分析了不同碳源输入方式下土壤线虫群落组成和多样性的变化。研究结果显示:所有处理(凋落物和根系同时输入;仅根系输入;仅凋落物输入;无碳源输入)中共发现土壤线虫68属,其中食细菌线虫的相对丰度最大;各碳源输入方式中仅凋落物输入处理对线虫群落组成的影响最为明显,表现为:与凋落物和根系同时输入处理相比,仅凋落物输入处理中食细菌线虫的相对丰度明显增加,食真菌线虫的相对丰度显著降低以及捕食杂食线虫的相对丰度显著增加。从线虫群落的多样性指数角度看,无碳源输入处理中土壤线虫群落的多样性指数(H'')下降,优势度指数(Dom)增高;仅凋落物输入处理中均匀度指数(J)最高。从线虫群落的生态指数角度看,仅凋落物输入处理的线虫群落成熟度指数(MI)最高;各碳源输入处理的分解过程均以细菌分解通道为主。研究表明,土壤线虫群落受到碳源输入变化的调节,仅凋落物输入处理比仅根系输入处理对土壤线虫群落的影响更大。无碳源输入处理的物种多样性明显下降,仅凋落物输入处理的土壤食物网更加稳定。研究结果丰富了森林土壤线虫多样性的研究内容,并为高通量测序技术在土壤线虫方面的研究提供方法和数据的支持。     

PCR-DGGE技术在农田土壤微生物多样性研究中的应用

变性梯度凝胶电泳技术(DGGE)在微生物生态学领域有着广泛的应用。研究采用化学裂解法直接提取出不同农田土壤微生物基因组DNA,并以此基因组DNA为模板,选择特异性引物F357GC和R515对16S rRNA基因的V3区进行扩增,长约230bp的PCR产物经变性梯度凝胶电泳(DGGE)进行分离后,得到不同数目且分离效果较好的电泳条带。结果说明,DGGE能够对土壤样品中的不同微生物的16S rRNA基因的V3区的DNA扩增片断进行分离,为这些DNA片断的定性和鉴定提供了条件。与传统的平板培养方法相比,变性梯度凝胶电泳(DGGE)技术能够更精确的反映出土壤微生物多样性,它是一种有效的微生物多样性研究技术。     

Are we ready to detect nematode diversity by next generation sequencing?

In a Technical Advance article, Porazinska et al. (2009, Molecular Ecology Resources, 9, 1439–1450) assessed next generation sequencing (NGS) as a method for metagenomic analysis of nematode diversity. We agree that NGS has great potential here. However, it is not an easy path to the successful implementation of NGS for environmental DNA analysis of nematodes. Here, we describe the method's limitations and discuss prospective research questions. For instance, only a few direct extraction kits are suitable for nematode DNA extraction from bulk samples without adaptation. They enable the analysis of extracellular nematode DNA. The most crucial and unresolved issue remains the limited availability of suitable primers.     

Barcoding marine nematodes: an improved set of nematode 18S rRNA primers to overcome eukaryotic co-interference

Nematodes form an important component of many benthic marine ecosystems and DNA barcoding approaches could provide an insight into nematode community composition from different environments globally. We have amplified nematode 18S rRNA sequences using standard nematode18S rRNA primers from environmental DNA extracted from intertidal sediment collected from New Jersey coast, USA to test whether the published marine nematode 18S rRNA sequences from GenBank and EMBL databases can effectively assign unknown nematode sequences into genus or species level. Most of the sequenced clones showed some degree of identities with published marine nematode 18S rRNA sequences. However, relatively very few of the sequences could be assigned even to genus level based on sequence assignment rule. In addition, other eukaryotic 18S rRNA sequences were found to be co-amplified with commonly used nematode 18S rRNA primers. We found that the majority of the current nematode 18S rRNA primers will co-amplify other eukaryotes if environmental DNA is the target template. We therefore designed a new set of nematode 18S rRNA primers and evaluated them using environmental DNA in intertidal sediment from the New Jersey coast. In total, 40 clones were screened and subsequently sequenced and all the sequences showed varying degree of identities with published nematode 18S rRNA sequences from GenBank and EMBL databases, and no obvious eukaryotic co-amplicons were detected with new primers. Only 13 out of 40 clones amplified with the new primer set showed 100% identity to published Daptonema and Metachromadora 18S rRNA sequences. The current molecular databases for nematodes are dominated by sequences from NW Europe and need to be more extensively populated with new full length 18S rRNA nematode sequences collected from different biogeographic locations. The new primers developed in this study, in combination with an updated nematode 18S rRNA sequence database, would help us to better investigate and understand the diversity and community composition of free-living marine nematodes based on DNA barcoding approaches during biodiversity or biomonitoring surveys on a global-scale.     

公路源重金属对青藏高原高寒草甸土壤线虫群落的影响

为了解重金属对青藏高原高寒草甸土壤线虫群落的影响,在邦杰塘草原利用公路源重金属含量差异,采用高通量测序技术,分析重金属对高寒草甸土壤线虫群落多样性及群落组成的影响。结果表明：在重金属含量较低样本中,土壤线虫群落Shannon指数、Ace指数、Chao指数比重金属含量较高样本均要小,且整体上Shannon指数、Ace指数、Chao指数与重金属含量较高样本差异显著（P<0.05）。重金属含量升高改变了高寒草甸土壤线虫纲水平和目水平的群落结构,使近自然状况下的色矛纲（Chromadorea）、小杆目（Rhabdtida）优势土壤线虫群落转变为一类未分类线虫纲（unclassified_p_Nematoda）、一类未分类线虫目（unclassified_p_Nematoda）的优势土壤线虫群落。重金属Cu、Pb、Zn、Cd与线虫门（Nematoda）和刺嘴纲（Enoplea）正相关,但与色矛纲（Chromadorea）负相关,且对土壤线虫Chao指数的变异解释比例依次减小。研究表明土壤重金属含量对青藏高原高寒草甸土壤线虫群落多样性及组成产生很大影响,但仍需对高寒草甸区域进行系统的土壤线虫调查,并丰富土壤线虫生物信息数据库。     

Reproducibility of read numbers in high-throughput sequencing analysis of nematode community composition and structure

Although nematodes are the most abundant metazoan animals on Earth, their diversity is largely unknown. To overcome limitations of traditional approaches (labour, time, and cost) for assessing biodiversity of nematode species in environmental samples, we have previously examined the suitability of high-throughput sequencing for assessing species level diversity with a set of control experiments employing a mixture of nematodes of known number and with known sequences for target diagnostic loci. Those initial experiments clearly demonstrated the suitability of the approach for identification of nematode taxa but lacked the replicate experiments necessary to evaluate reproducibility of the approach. Here, we analyze reads generated from three different PCR amplifications and three different sequencing reactions to examine the differential PCR amplification, the possibility of emulsion PCR artefacts, and differences between sequencing reactions. Our results suggest that both qualitative and quantitative data are consistent and highly reproducible. Variation associated with in-house PCR amplification or emPCR and sequencing are present but the representation of each nematode is very consistent from experiment to experiment and supports the use of read counts to estimate relative abundance of taxa in a metagenetic sample.     

新疆长期棉花连作对土壤理化性状与线虫群落的影响

土壤线虫群落特征是评价和指示土壤生态系统健康状况的重要依据。本研究选取不同连作年限(5、10、15、20和25年)的棉田为样地,采用高通量测序技术,探究土壤性状和线虫群落对棉田长期连作的响应。结果表明: 棉田连作10～15年后,土壤pH、电导率显著升高,有机碳、全氮、有效磷、有效钾、硝态氮含量和土壤微生物生物量碳(MBC)显著降低。在连作棉田中共鉴定出土壤线虫3纲7目18科25属,其中螺旋属在不同连作年限的棉田土壤中均为优势属;土壤植物寄生类线虫在不同连作年限中均为优势营养类群,呈现先降低后增加的趋势,连作25年较其他连作年限植物寄生类线虫增加9.1%～208.6%,其中螺旋属线虫增加了392%。随着连作年限的增加,矮化属、茎属、Discopersicus、中环属和中轮属等植物寄生类线虫被检出。连作15年的棉田土壤中,土壤线虫丰富度指数和自由生活线虫成熟度指数显著降低,植物寄生线虫成熟度指数/自由生活线虫成熟度指数显著升高,Shannon多样性指数和瓦斯乐斯卡指数最低;有效磷和MBC是影响土壤线虫群落变化的主要环境因子。这说明棉田连作10～15年会发生土壤养分失衡,土壤线虫多样性降低,土壤食物网稳定性变差,棉花致病类植物寄生线虫增加,产生连作障碍。     

土壤细菌网络互作调控线虫肠道细菌群落

动物肠道细菌群落在联系宿主与生态系统功能中发挥着至关重要的作用。【目的】本研究旨在评估绿肥翻压和水稻生长不同时期对土壤细菌和线虫肠道细菌群落组成和结构的影响,并探究土壤细菌和线虫肠道细菌群落间的潜在关联关系。【方法】基于盆栽试验,结合16S rRNA基因高通量测序技术,分析黑麦草翻压和对照处理下水稻生长的前期(返青期)和后期(收获期)土壤细菌和线虫肠道细菌群落,结合网络分析研究土壤细菌网络互作对线虫肠道细菌群落的潜在影响。【结果】黑麦草翻压对土壤细菌和线虫肠道细菌群落组成和结构没有显著影响(P>0.05);水稻生长后期样品比前期样品具有更高的α多样性。基于随机森林机器学习法获得的土壤细菌和线虫肠道细菌生物标志物之间存在广泛的显著相关关系,为土壤细菌群落变化调控线虫肠道细菌群落组成提供了有力的证据。共现网络分析表明土壤细菌之间的正相互作用显著促进了土壤细菌和线虫肠道细菌之间的正相互作用(P<0.01),进而影响了线虫肠道细菌之间的网络互作。结构方程模型进一步表明土壤养分含量的降低主要通过降低土壤细菌之间正相互作用,从而间接影响线虫肠道细菌之间的互作。【结论】土壤细菌互作可能在...     

Evaluating high-throughput sequencing as a method for metagenomic analysis of nematode diversity

Nematodes play an important role in ecosystem processes, yet the relevance of nematode species diversity to ecology is unknown. Because nematode identification of all individuals at the species level using standard techniques is difficult and time-consuming, nematode communities are not resolved down to the species level, leaving ecological analysis ambiguous. We assessed the suitability of massively parallel sequencing for analysis of nematode diversity from metagenomic samples. We set up four artificial metagenomic samples involving 41 diverse reference nematodes in known abundances. Two samples came from pooling polymerase chain reaction products amplified from single nematode species. Two additional metagenomic samples consisted of amplified products of DNA extracted from pooled nematode species. Amplified products involved two rapidly evolving ~400-bp sections coding for the small and large subunit of rRNA. The total number of reads ranged from 4159 to 14771 per metagenomic sample. Of these, 82% were > 199 bp in length. Among the reads > 199 bp, 86% matched the referenced species with less than three nucleotide differences from a reference sequence. Although neither rDNA section recovered all nematode species, the use of both loci improved the detection level of nematode species from 90 to 97%. Overall, results support the suitability of massively parallel sequencing for identification of nematodes. In contrast, the frequency of reads representing individual species did not correlate with the number of individuals in the metagenomic samples, suggesting that further methodological work is necessary before it will be justified for inferring the relative abundances of species within a nematode community.     

Unveiling the Biodiversity of Deep-Sea Nematodes through Metabarcoding: Are We Ready to Bypass the Classical Taxonomy?

Nematodes inhabiting benthic deep-sea ecosystems account for >90% of the total metazoan abundances and they have been hypothesised to be hyper-diverse, but their biodiversity is still largely unknown. Metabarcoding could facilitate the census of biodiversity, especially for those tiny metazoans for which morphological identification is difficult. We compared, for the first time, different DNA extraction procedures based on the use of two commercial kits and a previously published laboratory protocol and tested their suitability for sequencing analyses of 18S rDNA of marine nematodes. We also investigated the reliability of Roche 454 sequencing analyses for assessing the biodiversity of deep-sea nematode assemblages previously morphologically identified. Finally, intra-genomic variation in 18S rRNA gene repeats was investigated by Illumina MiSeq in different deep-sea nematode morphospecies to assess the influence of polymorphisms on nematode biodiversity estimates. Our results indicate that the two commercial kits should be preferred for the molecular analysis of biodiversity of deep-sea nematodes since they consistently provide amplifiable DNA suitable for sequencing. We report that the morphological identification of deep-sea nematodes matches the results obtained by metabarcoding analysis only at the order-family level and that a large portion of Operational Clustered Taxonomic Units (OCTUs) was not assigned. We also show that independently from the cut-off criteria and bioinformatic pipelines used, the number of OCTUs largely exceeds the number of individuals and that 18S rRNA gene of different morpho-species of nematodes displayed intra-genomic polymorphisms. Our results indicate that metabarcoding is an important tool to explore the diversity of deep-sea nematodes, but still fails in identifying most of the species due to limited number of sequences deposited in the public databases, and in providing quantitative data on the species encountered. These aspects should be carefully taken into account before using metabarcoding in quantitative ecological research and monitoring programmes of marine biodiversity.     

Quantification of the slug parasitic nematode Phasmarhabditis hermaphrodita from soil samples using real time qPCR

Phasmarhabditis hermaphrodita is a nematode parasite that infects and kills several species of slugs. The nematode is produced commercially as a biological control agent for slug pests of agriculture and horticulture. Given the difficulties of distinguishing this species from other nematode species in soil samples, very little is known about its natural ecology or its behaviour and persistence following application for biological control. Here we describe a method to quantify P. hermaphrodita in soil samples based on real time PCR. We designed primers and a dual labelled fluorescent probe that can be used to quantify numbers of P. hermaphrodita and which is capable of distinguishing this species from the morphologically identical Phasmarhabditis neopapillosa. We compared different methods whereby the entire nematode community is extracted prior to DNA extraction, and three methods to extract DNA directly from soil samples. Both nematode extraction and DNA extraction from large (10 g) samples of soil gave reliable estimates of nematode numbers, but methods which extracted DNA from small (1 g or less) soil samples substantially underestimated numbers. However, direct extraction of DNA from soils may overestimate numbers of live nematodes as DNA from dead nematodes was found to persist in soil for at least 6 days. The technique could be modified for detection and quantification of all soil borne parasitic nematodes.