有害甲藻Stoeckeria algicida在辽东湾的时空分布

Stoeckeria algicida为甲藻纲胸甲球藻科,有侵噬鱼类细胞杀鱼的能力,可导致鱼类成群死亡,同时也会杀死其他海洋微藻。由于该藻个体微小、形态学鉴定困难,研究较为迟缓,我国海域几乎没有该藻的研究报道。近几年,高通量测序技术的发展极大地推动了微型/微微型浮游植物的鉴定研究,为了解我国辽东湾海域是否存在Stoeckeria algicida及其分布情况,以18S rD NA V4区作为目标基因,结合高通量测序技术,专门设计了微型/微微型浮游植物鉴定引物对V4(F/R),随后对辽东湾2014年四季海水中微型和微微型浮游植物多样性进行了检测。结果发现,Stoeckeria algicida除了春季未检出外,其他季节均有检出,温度是影响该藻繁殖的主要因素。虽然Stoeckeria algicida在整个环境样品中优势度不太明显,但其夏季密度较高(最高达2.753×10~3个/L),高值区主要分布在辽东湾东西两岸,致灾风险较高,应引起有关方面足够重视。Stoeckeria algicida在我国海域首次报道,其危害后果严峻,必须加强监测监管。     

核糖体小亚基V4和V9区引物扩增大亚湾真核浮游生物的比较研究

对大亚湾水体的环境DNA分别进行18S rDNA的V4和V9区的引物扩增,通过高通量测序技术进行测序,并比较分析二者浮游真核生物基因多样性和相对丰度。18S rDNA V4区引物扩增共检测出浮游动物56纲, 101目,浮游植物52纲, 69目; 18S rDNA V9区引物扩增共检测出浮游动物47纲, 81目,浮游植物56纲, 101目。两对引物对浮游真核生物扩增都具有较高覆盖度,在纲级别上二者的结果相近:颚足纲(Maxillopoda)是浮游动物优势类群;甲藻纲(Dinophyceae)、圆筛藻纲(Coscinodiscophyceae)、小豆藻纲(Mamiellophyceae)是浮游植物优势类群,其中甲藻纲多样性与丰度的结果相近,而18S rDNA V9区引物扩增得到的圆筛藻纲丰度高于18S rDNA V4区引物。分析结果表明, 18S rDNAV4区引物扩增的浮游动物多样性比18SrDNAV9区引物高,而18SrDNAV9区引物扩增的浮游植物多样性比18S rDNA V4区引物高。同时,通过高通量测序技术首次确定大亚湾海区大量存在着寄生型甲藻(Syndiniales),小豆藻目...     

基于环境DNA技术的辽东湾真核微藻群落结构特征

以18S rDNA V4区作为目标基因,利用自行设计的真核微藻鉴定引物V4（F/R）,结合高通量测序技术,对辽东湾2014年四季海水中真核微藻多样性进行了检测。结果发现,辽东湾海域注释到种的真核微藻有136种,41%的种类在中国海域未见报道,其中自养型占60%、异养型占10%、混合营养型占30%。研究对丰富中国海域微藻名录和外来海洋微藻背景数据库具有较大意义。     

榕江下游流域夏秋季网采浮游植物群落结构特征

根据2013 年7 月(夏季)和11 月(秋季)的调查数据, 对榕江流域揭阳至汕头段浮游植物物种组成、时间分布及多样性等群落结构特征进行了分析。结果显示: 共鉴定出浮游植物5 门106 种, 其中, 硅藻54 种、绿藻32 种、蓝藻12 种、甲藻和裸藻各4 种, 分别占种类总数的50.94%、30.19%、11.32%和3.77%。浮游植物种类数目秋季(79 种)多于夏季(52 种), 夏季丰度(12571.94×10⁴ 细胞·m^-3)是秋季(342.30×104 细胞·m^-3)的36.73 倍。丰度在空间上表现出夏季呈无规则的变化, 秋季则呈上游站位高于下游站位的变化规律。优势种夏季主要为蓝绿藻类的微小色球藻Chroococcus minutus、细小隐球藻Aphanocapsa elachista、月牙藻Selenastrum bibraianum 和普通小球藻Chlorella vulgaris 等6 种; 秋季主要为颗粒直链藻Aulacoseira granulata 和胶网藻Dictyosphaerium ehrenbergianum 2 种。Shannon-Wiener 多样性指数H′、Pielou 均匀度指数J′和Margalef 物种丰富度指数D 在秋季(2.42, 0.60, 2.79)高于夏季(1.46, 0.36, 1.12)。榕江流域水质状况的生物多样性指数综合评价显示, 该流域水质总体属于中度污染型, 生态环境受到了一定程度的污染破坏。     

浮游植物群落对海南小水电建设的响应

为探讨浮游植物群落对海南省小水电建设的响应,分别在海南省主要河流的上游支流已建小水电的蓄水水域与河道、规划(未建)小水电河段采集浮游植物样品进行比较分析.共鉴定出浮游植物种类62个属178种,曲壳藻(Achnanthaceae)、异极藻(Gomphonema)、菱形藻(Nitzschia)、颗粒直链藻(Melosira granulat)、席藻(Phormidium)、颤藻(Oscillatoria)、小球藻(Chlorella vulgaris)、平裂藻(Merismopedia)、舟形藻(Navicula)为主要的优势藻类,浮游植物丰度在5.1-163.6×104个/L之间,浮游植物Shannon-Wiener多样性指数在2.73-4.53之间.研究结果表明,小水电建设对浮游植物的种类组成、优势种、丰度及多样性均有较大的影响.就浮游植物优势种而言,规划小水电河道以蓝藻及部分硅藻为主要优势种,已建小水电河道曲壳藻、异极藻、菱形藻等大型硅藻为主要优势种.在浮游植物组成及生物多样性上,未建小水电河道浮游植物Shannon-Wiener多样性指数略高,且种属分布更加均衡,而已建设水电站均趋向某一类藻占主导优势种.就浮游植物丰度而言,规划小水电河道浮游植物丰度均保持在20-30×104个/L内,已建小水电河段浮游植物保持在5-160×104个/L内且浮游植物丰度差异性较规划小水电大,小水电建设促进了浮游植物丰度的提升,但降低了浮游植物群落结构的稳定性、均衡性.虽然存在水电站阻隔,同一河流水系浮游植物种属来源仍可表现一定的趋同性,梯级水电特别是相邻水电间浮游植物群落组成存在较大的相似性.     

生物防治稻田与普通稻田水体中浮游植物的生态特征研究

通过对大沙镇生物防治稻田及普通稻田水体中浮游植物的调查研究,共检出藻类112种,其中生物防治稻田中82种,普通稻田中89种.稻田水体中的浮游植物以硅藻、裸藻和绿藻占优势.在普通稻田中,硅藻种类数超过生物防治稻田,其优势度最高的5种藻类中除双对栅藻外,其余4种均为硅藻;而在生物防治稻田中,裸藻种类数高于普通稻田,且其优势度最高的5种藻类中有两种为裸藻.通过比较发现,生物防治稻田水体中浮游生物的密度明显高于普通稻田.对水体浮游植物多样性及均匀度的分析表明,稻田水体中的浮游植物自秧苗插上至干田期间,多样性指数略有上升(主要是由于种类数的增加引起的),而均匀度呈下降趋势.     

生物防治稻田与普通稻田水体中浮游植物的生态特征研究

通过对大沙镇生物防治稻田及普能稻田水体中浮游植物的调查研究,共检出藻类112种,其中生物防治稻田中82种,普通稻田中89种,稻田水体中的浮游植物以硅藻、裸藻和绿藻占优势,在普通稻田中,硅藻种类数超过生物防治稻田,其优势度最高的5种藻类中除双对栅藻外,其余4种均为硅藻;而在生物防治稻田中,裸藻种类数高于普通稻田,且其优势度最高的5种藻类中有两种为裸藻,通过比较发现,生物防治稻田不体中浮游生物的密度明显高于普通稻田,对水体浮游植物多样性及均匀度的分析表明,稻田水体中的浮游植物自秧苗插上至干田期间,多样性指数略有上升（上要是由于种类数的增加引起的）,而均匀度呈下降趋势。     

盐湖微微型浮游植物多样性研究进展

盐湖在地球表层分布较广,中国是世界上盐湖分布稠密的国家。尽管盐湖生态环境极端恶劣,但它们依然是陆地特别是高原生态系统中十分重要的组成部分。微微型浮游植物(Picophytoplankton)通常是指粒径在0.2—3μm之间的光合自养型浮游生物,在这一生态类群中既有原核生物也有真核生物。微微型浮游植物不仅是海洋生态系统中生物量和生产力的最重要贡献者,也是盐湖生态系统最重要的组成部分。最近的调查数据显示,在东非Bogoria和Nakuru苏打盐湖中微微型浮游植物的生产力占整个浮游植物生产力的53%—68%。迄今为止,对盐湖微微型浮游植物多样性的研究多集中于盐田(Solar salterns)、盐池(Saline ponds)以及碱湖(Soda lakes)等超盐水体中;超盐盐湖中最常见的微微型浮游植物主要是蓝细菌(Cyanobacteria)类群,包括席蓝细菌属(Phormidium)、节旋藻属(Arthrospira)和隐杆藻属(Aphanothece)。分子生物学的研究显示,低碱浅水盐湖中微微型浮游植物具有丰富的多样性,主要类群是微微型原核浮游植物,还有部分种类与真核藻类质体序列高度相似。盐湖生态系统中微微型浮游植物群落结构演替变化的研究已经逐渐引起更多研究者的关注。已有的研究资料显示,水体矿化度是影响微微型浮游植物平面分布及群落结构组成的重要因子;光照、营养成分和温度等也会影响盐湖水体中微微型浮游植物平面分布及群落结构组成。综述了国内外对不同类型盐湖生态系统中微微型浮游植物多样性研究的概况,探讨了不同地理区域各种盐湖中微微型浮游植物群落结构的演替变化,并就我国未来加强盐湖中微微型浮游植物多样性构成、分布与变动及驱动变化的因子等研究提出了建议。     

淮南矿区小型煤矿塌陷湖泊浮游植物群落结构特征

在淮南潘谢矿区内设置3个水文生态环境条件差异较大的小型煤矿塌陷湖泊研究站点, 即潘谢潘集站(PXPJ)、潘谢顾桥站 (PXGQ)和潘谢谢桥站(PXXQ), 于20132014年4个季度分别对塌陷湖泊的浮游植物结构组成特征及其水生态环境因子的关系进行了分析。3个小型塌陷湖泊共鉴定出浮游植物7门9纲18目34科70属131种, 浮游植物种类主要由蓝藻、绿藻和硅藻组成。其中绿藻门种类最多, 共59种, 占浮游植物总种数45.0%; 其次是蓝藻, 总共24种, 占浮游植物总种数18.3%; 硅藻22种, 占浮游植物总种数16.8%。从各门类藻细胞密度的百分比看, PXPJ站点以绿藻、硅藻和隐藻为主, 范围77.5%90.5%; PXGQ站点蓝藻在夏秋季数量上均占据绝对优势, 分别占藻类总细胞密度的61.5%和46.2%; PXXQ站点隐藻在春季为绝对优势类群, 在总细胞密度中占的比率为94.6%, 夏秋以蓝藻为主, 分别为74.7%和81.8%。3个湖泊由于水文生态环境条件的不同, 浮游植物丰度、多样性和均匀度体现出了一定的差异。典范对应分析(CCA)表明, 光照、水温和营养盐含量与比率(TN/TP)是影响塌陷湖泊浮游植物群落结构的重要环境因子。       

中国东部陆架边缘海网采浮游植物种类组成和季节变化

为揭示中国东部陆架边缘海浮游植物群落季节变化规律,根据2006年6–7月、2007年1–2月、2007年11月和2009年4–5月在中国东部陆架边缘海域(25.00°–39.00°N,118.00°–129.00°E)进行的综合采样调查,对调查海域网采浮游植物(网孔直径77μm)的物种多样性和分布特征进行了研究。4个航次共鉴定出浮游植物4门70属257种(不包括未定名种),其中硅藻是主要功能群,其次是甲藻,主要的优势种为骨条藻(Skeletonema spp.)、细弱海链藻(Thalassiosira subtilis)、囊状海链藻(T.scrotiformis)、伏氏海线藻(Thalassionema frauenfeldii)、菱形海线藻(T.nitzschioides)、具槽帕拉藻(Paralia sulcata)、洛氏角毛藻(Chaetoceros lorenzianus)、旋链角毛藻(C.curvisetus)、尖刺伪菱形藻(Pseudo-nitzschia pungens)和夜光藻(Noctiluca scintillans)。浮游植物细胞丰度为0.02×104–31,350.21×104cells/m3,最低值出现在冬季黄海海域,最高值出现在春季长江口邻近海域。4个季节的浮游植物细胞密度呈现春季夏季秋季冬季的趋势,浮游植物各生物多样性指数的等值线均呈现西北–东南走向。     

不同扩增引物对高通量测序分析盐角草内生真菌多样性的影响

【背景】高通量测序分析作为深入了解环境微生物群落组成的重要方法,已成为植物内生真菌多样性研究的有效手段,然而由于引物的扩增差异,采用不同引物可对实验结果分析造成影响。同时,盐角草作为世界上最耐盐的植物之一,存在着多种功能性的内生真菌,而较为全面介绍其内生真菌组成和多样性的报道鲜见。【目的】为了揭示盐角草内生真菌的多样性,解析不同扩增引物对内生菌多样性分析的影响。【方法】分别采用真菌高通量测序常用引物对ITS1-5F、ITS1-1F、ITS2对采自乌鲁木齐达坂城盐湖的盐角草内生真菌进行扩增,开展其内生真菌OTU的分析。【结果】通过不同引物对扩增并测序共获得102个盐角草内生真菌OTU,涉及真菌界8个门和未分类菌群,其中子囊菌门(Ascomycota)占绝对优势,其次为担子菌门(Basidiomycota);在属层次上,盐角草内生真菌共涉及64个属及20个未分类属,其中Alternaria、Cladosporium、Podospora等3个属为盐角草内生真菌优势菌群。对不同引物对扩增测序结果分析表明,不同引物对扩增对分析内生真菌OTU数量和种类具有明显的影响,在全部所得的102个OTU中,ITS1-5F引物对获得44个OTU、ITS1-1F引物对获得55个OTU、ITS2引物对获得25个OTU,但以上3对引物扩增均检测到的OTU数仅为5个。物种组成和多样性分析表明,内生真菌多样性分析中采用以ITS1-1F为主,ITS1-5F为辅的分析策略,可较为全面地展现内生真菌的多样性。【结论】盐角草存在较为丰富的内生真菌资源,不同扩增引物对高通量分析盐角草内生真菌组成和分布具有明显的影响。     

New barcoded primers for efficient retrieval of cercozoan sequences in high‐throughput environmental diversity surveys,with emphasis on worldwide biological soil crusts

We describe the performance of a new metabarcoding approach to investigate the environmental diversity of a prominent group of widespread unicellular organisms, the Cercozoa. Cercozoa is an immensely large group of protists, and although it may dominate in soil and aquatic ecosystems, its environmental diversity remains undersampled. We designed PCR primers targeting the hypervariable region V4 of the small subunit ribosomal RNA (SSU or 18S) gene, which is the recommended barcode marker for Cercozoa. The length of the amplified fragment (c. 350 bp) is suitable for Illumina MiSeq, the most cost‐effective platform for molecular environmental surveys. We provide barcoded primers, an economical alternative to multiple libraries for multiplex sequencing of over a hundred samples. In silico, our primers matched 68% of the cercozoan sequences of the reference database and performed better than previously proposed new‐generation sequencing primers. In mountain grassland soils and in biological soil crusts from a variety of climatic regions, we were able to detect cercozoan sequences encompassing nearly the whole range of the phylum. We obtained 901 operational taxonomic units (OTUs) at 97% similarity threshold from 26 samples, with c. 50,000 sequences per site, and only 8% of noncercozoan sequences. We could report a further increase in the diversity of Cercozoa, as only 43% of the OTUs were 97%–100% similar to any known sequence. Our study thus provides an advanced tool for cercozoan metabarcoding and to investigate their diversity and distribution in the environment.     

Detection of Rhopalosiphum insertum (apple-grass aphid) predation by the predatory mite Anystis baccarum using molecular gut analysis

Abstract 1 A simple, yet sensitive polymerase chain reaction based technique was developed for the detection of the apple‐grass aphid Rhopalosiphum insertum in the gut of Anystis baccarum, a predatory mite. 2 A range of conserved polymerase chain reaction primers for insect mitochondrial and ribosomal DNA were tested in order to amplify R. insertum DNA. The mitochondrial DNA primers LrRNAR2 + N1F1, amplified a region between the ND1 and large subunit RNA genes. 3 DNA sequencing of the R. insertum ND1‐LRNA polymerase chain reaction product allowed aphid‐specific polymerase chain reaction primers to be designed. These amplified a 283‐bp product from individual aphids. No polymerase chain reaction product was amplified from individual A. baccarum. 4 Using the aphid‐specific primers against A. baccarum fed on R. insertum, the diagnostic 283‐bp product was amplified. 5 Two restriction enzymes (RsaI and AluI) produced patterns that allowed unambiguous identification of R. insertum DNA from that of Macrosiphum euphorbiae and Myzus persicae.     

New specific primers for amplification of the Internal Transcribed Spacer region in Clitellata (Annelida)

Nuclear molecular evidence, for example, the rapidly evolving Internal Transcribed Spacer region (ITS), integrated with maternally inherited (mitochondrial) COI barcodes, has provided new insights into the diversity of clitellate annelids. PCR amplification and sequencing of ITS, however, are often hampered by poor specificity of primers used. Therefore, new clitellate‐specific primers for amplifying the whole ITS region (ITS: 29F/1084R) and a part of it (ITS2: 606F/1082R) were developed on the basis of a collection of previously published ITS sequences with flanking rDNA coding regions. The specificity of these and other ITS primers used for clitellates were then tested in silico by evaluating their mismatches with all assembled and annotated sequences (STD, version r127) from EMBL, and the new primers were also tested in vitro for a taxonomically broad sample of clitellate species (71 specimens representing 11 families). The in silico analyses showed that the newly designed primers have a better performance than the universal ones when amplifying clitellate ITS sequences. In vitro PCR and sequencing using the new primers were successful, in particular, for the 606F/1082R pair, which worked well for 65 of the 71 specimens. Thus, using this pair for amplifying the ITS2 will facilitate further molecular systematic investigation of various clitellates. The other pair (29F/1084R), will be a useful complement to existing ITS primers, when amplifying ITS as a whole.     

Assessment of fungal diversity in deep-sea sediments by multiple primer approach

Increasing evidence of the fungal diversity in deep-sea sediments has come from amplification of environmental DNA with fungal specific or eukaryote primer sets. In order to assess the fungal diversity in deep-sea sediments of the Central Indian Basin (CIB) at ~5,000 m depth, we amplified sediment DNA with four different primer sets. These were fungal-specific primer pair ITS1F/ITS4 (internal transcribed spacers), universal 18S rDNA primers NS1/NS2, Euk18S-42F/Euk18S-1492R and Euk18S-555F/Euk18S-1269R. One environmental library was constructed with each of the primer pairs, and 48 clones were sequenced per library. These sequences resulted in 8 fungal Operational Taxonomic Units (OTUs) with ITS and 19 OTUs with 18S rDNA primer sets respectively by taking into account the 2% sequence divergence cut-off for species delineation. These OTUs belonged to 20 distinct fungal genera of the phyla Ascomycota and Basidiomycota. Seven sequences were found to be divergent by 79–97% from the known sequences of the existing database and may be novel. A majority of the sequences clustered with known sequences of the existing taxa. The phylogenetic affiliation of a few fungal sequences with known environmental sequences from marine and hypersaline habitat suggests their autochthonous nature or adaptation to marine habitat. The amplification of sequences belonging to Exobasidiomycetes and Cystobasidiomycetes from deep-sea is being reported for the first time in this study. Amplification of fungal sequences with eukaryotic as well as fungal specific primers indicates that among eukaryotes, fungi appear to be a dominant group in the sampling site of the CIB.     

Development of a SCAR marker and a strain‐specific genomic marker for the detection of the biocontrol agent strain CPA‐8 Bacillus amyloliquefaciens (formerly B. subtilis)

In this work, reliable tools were developed to detect and identify the biocontrol strain CPA‐8 using DNA amplification techniques. As a first approach, the RAPD (random amplified polymorphic DNA) technique was applied to a collection of 77 related Bacillus species. Among the primers tested, the primer pair OPG1/OPG6 amplified a 668 bp specific product to the strain CPA‐8 that was sequenced and used to design SCAR (sequence‐characterised amplified regions) primer pairs. The SCAR‐4 marker amplified a semi‐specific fragment of 665 bp not only for the strain CPA‐8 but also for other 12 strains whose morphology was completely different from CPA‐8. Another approach was developed to obtain a strain‐specific genomic marker related to ecological adaptations of Bacillus amyloliquefaciens species. The primer pair F2/R2 obtained from RBAM 007760, a gene involved in surface adhesion, amplified a 265 bp fragment unique for strain CPA‐8. Our results revealed that these two molecular markers, SCAR‐4 and RBAM 007760 F2/R2 provide suitable monitoring tools to specifically identify the biocontrol CPA‐8 when applied against brown rot caused by Monilinia spp. in stone fruit. Moreover, our findings demonstrate that the strain CPA‐8 is affiliated with B. amyloliquefaciens species that was formerly designated as Bacillus subtilis.     

A multiplex nested PCR assay for simultaneous detection of Corchorus golden mosaic virus and a phytoplasma in white jute (Corchorus capsularis L.)

A multiplex nested PCR assay was developed by optimizing reaction components and reaction cycling parameters for simultaneous detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma (Group 16Sr V‐C) causing little leaf and bunchy top in white jute (Corchorus capsularis). Three sets of specific primers viz. a CoGMV specific (DNA‐A region) primer, a 16S rDNA universal primer pair P1/P7 and nested primer pair R16F2n/R2 for phytoplasmas were used. The concentrations of the PCR components such as primers, MgCl₂, Taq DNA polymerase, dNTPs and PCR conditions including annealing temperature and amplification cycles were examined and optimized. Expected fragments of 1 kb (CoGMV), 674 bp (phytoplasma) and 370 bp (nested R16F2n/R2) were successfully amplified by this multiplex nested PCR system ensuring simultaneous, sensitive and specific detection of the phytoplasma and the virus. The multiplex nested PCR provides a sensitive, rapid and low‐cost method for simultaneous detection of jute little leaf phytoplasma and CoGMV. Based on BLASTn analyses, the phytoplasma was found to belong to the Group 16Sr V‐C.

Significance and Impact of the Study

Incidence of phytoplasma diseases is increasing worldwide and particularly in the tropical and subtropical world. Co‐infection of phytoplasma and virus(s) is also common. Therefore, use of single primer PCR in detecting these pathogens would require more time and effort, whereas multiplex PCR involving several pairs of primers saves time and reduces cost. In this study, we have developed a multiplex nested PCR assay that provides more sensitive and specific detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma in white jute simultaneously. It is the first report of simultaneous detection of CoGMV and a phytoplasma in Corchorus capsularis by multiplex nested PCR.     

BIPES,a cost-effective high-throughput method for assessing microbial diversity

Pyrosequencing of 16S rRNA (16S) variable tags has become the most popular method for assessing microbial diversity, but the method remains costly for the evaluation of large numbers of environmental samples with high sequencing depths. We developed a barcoded Illumina paired-end (PE) sequencing (BIPES) method that sequences each 16S V6 tag from both ends on the Illumina HiSeq 2000, and the PE reads are then overlapped to obtain the V6 tag. The average accuracy of Illumina single-end (SE) reads was only 97.9%, which decreased from ∼99.9% at the start of the read to less than 85% at the end of the read; nevertheless, overlapping of the PE reads significantly increased the sequencing accuracy to 99.65% by verifying the 3′ end of each SE in which the sequencing quality was degraded. After the removal of tags with two or more mismatches within the medial 40–70 bases of the reads and of tags with any primer errors, the overall base sequencing accuracy of the BIPES reads was further increased to 99.93%. The BIPES reads reflected the amounts of the various tags in the initial template, but long tags and high GC tags were underestimated. The BIPES method yields 20–50 times more 16S V6 tags than does pyrosequencing in a single-flow cell run, and each of the BIPES reads costs less than 1/40 of a pyrosequencing read. As a laborsaving and cost-effective method, BIPES can be routinely used to analyze the microbial ecology of both environmental and human microbiomes.     

双歧杆菌属特异性测序引物筛选及优化

【背景】目前双歧杆菌的益生功能被普遍认可,越来越多的研究开始关注肠道中双歧杆菌的生物多样性。然而双歧杆菌是肠道中的低丰度物种,现有技术尚难以深入研究其多样性。【目的】基于双歧杆菌16SrRNA基因序列筛选一对适用于分析粪便样品中低丰度双歧杆菌属多样性的特异性引物。【方法】依据已有引物的相对位置及其与双歧杆菌属16S rRNA基因序列的匹配率,将引物重组优化得到扩增片段800 bp的双歧杆菌属特异性引物;通过PCR扩增和琼脂糖凝胶电泳对引物进行实验筛选和特异性验证;以细菌通用引物(27f/1492r)为参照,通过单分子实时(Single-molecule real-time,SMRT)测序技术对不同引物的3份粪便样品中细菌的DNA扩增子进行测序,在种水平上分析比较不同引物的优劣。【结果】对文献中已有的9对双歧杆菌特异性引物进行重组并优化,其中2对引物的理论特异性较好且扩增产物大于800 bp,它们分别为Bif164-f/Pbi R2和Pbi F1/Pbi R2。PCR扩增和琼脂糖凝胶电泳实验发现,Bif164-f/Pbi R2的扩增条带明亮且无拖尾。此外,利用SMRT测序平台对引物27f/1492r和Bif164-f/Pbi R2的3份粪便样品中细菌的DNA扩增子进行测序并分析。27f/1492r扩增子的分析结果显示,3份样品依次分别含1、3、4个双歧杆菌种且双歧杆菌的平均相对含量为0.34%;而Bif164-f/Pbi R2扩增子的分析结果显示,3份样品依次分别含2、6和8个双歧杆菌种且双歧杆菌的平均相对含量为98.72%。上述结果表明,Bif164-f/Pbi R2可在种水平上特异地检出粪便中低丰度的双歧杆菌,进而实现样品中双歧杆菌的多样性分析。【结论】筛选出一对双歧杆菌特异性引物Bif164-f/PbiR2,可在种水平上分析粪便样品中低丰度双歧杆菌的生物多样性,同时也验证了理论结合实验进行引物筛选这种方法的可行性。     

16S rRNAgene-based metagenomic analysis of the gut microbial community associated with the DUI species Unio crassus (Bivalvia: Unionidae)

What factors determine biome richness: genetic or environmental? Sex, phylogeny, and tolerance indicated by other symbionts (e.g., endosymbionts) or simply is it related to local habitat, especially if the gut biome is considered? To answer these questions, we investigated the gut microbial profile of both sexes of three Unio crassus populations, species with unique system of mitochondrial DNA inheritance called doubly uniparental inheritance (DUI), living in different ecological conditions. High-throughput sequencing of the V3–V4 hypervariable regions in the bacterial 16S rRNA gene fragment was performed, which resulted in a total of 1,051,647 reads, with 58,424 reads/65 OTUs (operational taxonomic units) per sample on average. We identified a core microbiome, with all individual mussels sharing 69 OTUs (representing 23% of the total number of OTUs). Proteobacteria was the dominant phylum in all samples, followed by Firmicutes, Actinobacteria, and Bacteroidetes. There were no significant differences in gut microbiome compositions between the two sexes of this species; however, we observed different phyla in geographically isolated populations. A non-metric multidimensional scaling plot and dendrogram showed that the bacterial profile complies with the genetic structure of populations. Although we found differences in microbiomes between populations, their genetic structure suggests that the microbiome is weakly related to habitat, and more strongly to phylogeography (on both F and M mitotypes). We found no significant differences in beta diversity between the individuals of the bacterial communities measured using the Bray–Curtis index. Finally, we also examined whether OTUs were represented by symbiotic bacteria that enable cellulose digestion and by endosymbiotic bacteria that play important functions in the biology of their hosts and also affect microevolutionary processes and population phenomena. With regard to the endosymbionts, however, there was no relation to sex of the studied individuals, which suggests that there are no straightforward relations between DUI and microbiome.