真菌内共生细菌研究进展

自真菌内共生细菌在1970年被首次发现以来,各个时期的学者都采用当时流行的研究方法关注宿主真菌及其内共生细菌之间的关联现象。近年来科技手段日益多样,对二者相互作用的探索逐渐成为新的研究热点,随着研究的不断拓展和深入,越来越多的生物学现象和原理被揭示。本文在真菌内共生细菌的研究方法、定殖位置、形态、分类、宿主类群、共生关联的建立、生物学功能、宿主治愈、分离和重新植入等方面进行综述,并在此基础上进行展望,以期为真菌内共生细菌的广泛深入研究提供借鉴。     

云南文山健康三七种植年限对根际微生物群落的影响

【背景】三七的连作障碍与微生物区系失衡有一定关联,了解种植年限对三七微生物区系的影响,判断根际微生物区系是否失衡,对三七连作障碍的防治具有重要意义。【目的】探究文山不同种植年限健康三七根际土壤细菌和真菌群落结构、多样性差异,以及与种植年限之间的关联,为后续缓解三七连作障碍提供理论依据。【方法】采集一年生、二年生和三年生健康三七根际土壤,采用16S rRNA基因和ITS序列进行高通量测序。对测序结果进行分析,比较一年生、二年生和三年生健康三七根际土壤细菌和真菌的多样性和群落分布规律及与种植年限的关联。【结果】一年生健康三七根际细菌和真菌群落的α指数均显著高于二年生和三年生。通过β多样性分析发现,一年生健康三七根际土壤中真菌群落结构与其他年限的真菌群落结构有较大差异。相关性分析结果表明,真菌的均匀度和香农指数与种植年限呈现显著的负相关性,其余指数不显著相关。三七根际丰富度前三的门和属,除Acidobacteria和Aquicella丰富度随着三七种植年限的增加而显著增加外,其余群落丰度都与种植年限呈负相关。细菌中Bacteroidetes、Proteobacteria和真菌中Ascomycota、Basidiomycota、Chytridiomycota的丰富度随着种植年限增加而降低。在属水平上,细菌中Opitutus和真菌中Mortierella、Clitopilus、Pholiota的丰富度随着三七种植年限的增加而降低。在二年生三七根际土壤中病原真菌Alternaria、Cylindrocarpon、Fusarium和Pestalotiopsis的丰富度低于一年生和三年生,而有益真菌Mucor和Bacillus的丰富度却呈相反趋势。同时,有益细菌Flavobacterium和有益真菌Myxocephala、Aspergillus的丰富度随着种植年限的增加呈下降趋势。【结论】三七种植年限的增加,会引起三七根际微生物区系失衡,增加根际病原真菌属的丰度,同时降低一些有益真菌属的丰度。     

人类环境病原真菌的毒力和进化

侵袭性病原真菌极大威胁了人类公共健康安全。绝大多数的病原真菌来源于环境,它们与人类宿主并无共进化与共生关系,被称为环境病原真菌。该类病原真菌的致病能力可能衍生于其出色的环境适应策略,且可通过有性生殖依赖/非依赖的方式实现基因组快速进化,加速了自然界中高毒菌株和抗性菌株的产生。本文将主要以环境病原真菌的模式菌——新生隐球菌为例,阐述其复杂环境适应策略与致病力和毒力进化之间的关联,并对人类环境病原真菌的相关理论研究和临床应用提供一点思考。     

DNA分子标记技术在真菌系统学研究中的应用及影响

DNA分子标记技术为真菌系统进化研究提供了许多新的方法,真菌分子系统学已成为一门成熟的学科。简述了真菌分子系统学的发展简史和代表性的研究方法以及对真菌系统学的主要贡献,包括将广义的真菌划分为3个类群,粘菌和卵菌不再属于真菌界成员。真菌生命之树项目的研究结果对真菌界高阶分类系统作出重大调整,将先前的4个门(壶菌门、接合菌门、子囊菌门和担子菌门)变为7个门(微孢子虫门、壶菌门、新丽鞭毛菌门、芽枝霉门、球囊菌门、子囊菌门和担子菌门)和4个亚门,并对真菌各类群概念作出修订。此外,DNA分子标记技术对真菌种概念的认识、有性型-无性型关联及分子生态学等研究领域产生了重要影响。     

保护性耕作增强了真菌群落生态网络稳定性北大核心

【目的】解析不同耕作措施下的土壤真菌群落组成以及病原真菌生态网络特征,为了解真菌在维持和改善农业生态系统稳定性方面提供科学依据。【方法】通过高通量测序技术,对传统犁耕(moldboard plow,MP)和保护性耕作[少耕(reduced tillage,RT)、免耕(no-tillage,NT)]下根际和非根际土壤中真菌群落的组成、多样性和真菌病原菌的关联网络特征进行了综合分析。【结果】与NT对比,MP和RT显著增加了土壤全碳(total carbon,TC)、全氮(total nitrogen,TN)和速效钾(available potassium,AK)的含量。根际效应对真菌群落的影响显著高于耕作措施。根际土壤中,RT处理的Shannon指数显著高于MP和NT。NT和RT处理显著降低了根际土壤中镰孢菌(Fusarium)和链格孢菌(Alternaria)的相对丰度。此外,RT降低了病原真菌的网络复杂性、减少了病原菌与其他真菌间的交互作用,增强了网络稳定性。【结论】保护性耕作RT是具有增加土壤养分、提升真菌网络稳定性且具有减轻病原菌有效传播作用的最佳耕作模式,为东北地区的耕作应用提供了新的视角。     

大兴安岭棕色针叶林土壤的真菌多样性

为探究大兴安岭棕色针叶林土壤的真菌群落分布特征及其与土壤环境因子间的关系,采用高通量测序技术对大兴安岭呼中自然保护区兴安落叶松林的表浅棕色针叶林土、生草棕色针叶林土和典型棕色针叶林土的土壤真菌群落结构和多样性进行分析,运用R语言、SPSS 17.0等软件对土壤真菌群落与土壤环境因子间进行冗余分析和相关性分析.结果表明: 3个亚类的棕色针叶林土壤的理化性质间存在显著性差异.测序共获得6门23纲46目74科88属111种的土壤真菌,其中,子囊菌门、担子菌门为优势菌门;门分类水平上各样地菌群的相对丰度存在显著性差异.土壤真菌Ace和Chao1多样性指数在各亚类土壤间无显著性差异, 浅棕色针叶林土壤样地的Shannon和Simpson指数与其他样地存在显著差异.典型关联分析和相关性分析发现,土壤真菌的α和β多样性与土壤含水率、pH值、有机质、全氮、全钾等显著相关.说明土壤环境因子是影响大兴安岭兴安落叶松棕色针叶林土壤真菌多样性的重要因素.     

百里杜鹃保护区马缨杜鹃土壤真菌群落特征

近年来,贵州百里杜鹃保护区戛木管理区发现有部分马缨杜鹃感染根腐病,造成其大量死亡,致病机制尚不明确。为了研究马缨杜鹃根腐病发病区土壤真菌群落结构组成,探讨真菌微生物种类组成变化与根腐病发病的关联,以感病马缨杜鹃根部土壤和未感病马缨杜鹃根部土壤的土壤为对象,利用高通量测序技术进行测序,发现2份感病杜鹃根部土壤中分别有846和911个操作分类单元(operating taxonomic unit, OTUs),而未感病杜鹃根部土壤仅有439个OTUs。通过分析土壤真菌物种丰度和分类、α-多样性、β-多样性、主成分(principal component analysis, PCA)分析和聚类分析。结果表明:感病马缨杜鹃和未感病马缨杜鹃根部土壤真菌多样性和群落结构存在较大差异,未感病杜鹃根部土壤的真菌微生物种类明显少于感病杜鹃根部土壤。     

Illumina高通量测序揭示艾比湖湿地6种盐生植物根际土壤真菌群落组成及多样性

【目的】通过研究新疆艾比湖湿地不同盐生植物根际土壤真菌的多样性和群落结构,为艾比湖湿地退化恢复工作和真菌深入研究提供理论支持。【方法】利用高通量测序技术对真菌扩增子ITS1区进行测定,从而分析艾比湖湿地6种盐生植物根际土壤真菌群落多样性,并结合相关土壤理化因子分析环境与真菌群落多样性和丰富度的关联。【结果】艾比湖湿地6种盐生植物根际土壤真菌群落多样性及丰富度存在差异,碱蓬根际土壤真菌多样性最高,芦苇根际土壤真菌群落丰富度最高。真菌群落组成分析表明,土壤样品中真菌菌落主要隶属于子囊菌门(Ascomycota)和担子菌门(Basidiomycota),其中子囊菌门为主要优势菌门;链格孢霉属(Alternaria)是6种植物共有的优势菌属,但是其在不同植物之间的丰度存在差异,在戟叶鹅绒藤中的丰度最高,在准噶尔大戟中的丰度最低。pH与真菌多样性呈显著负相关,全磷(TP)与真菌群落丰富度呈显著正相关,pH、电导率(EC)和有机质(OM)对优势菌属的影响最大。【结论】艾比湖湿地6种盐生植物根际土壤真菌群落组成及多样性具有显著差异,碱蓬和芦苇根际土壤真菌的多样性和丰度高于其他植物,子囊菌门和链格孢霉...     

海南濒危树种保育圃白木香与降香黄檀幼苗根部真菌谱系与生态型多样性比较

【背景】根部真菌是影响植物幼苗存活、定植和生长的重要因子之一,但是苗圃培育的幼苗根部真菌物种组成与生态学特性尚不清楚。【目的】研究苗圃培育的白木香(Aquilaria sinensis)与降香黄檀(Dalbergia odorifera)幼苗根部真菌群落谱系与生态型多样性,以及宿主植物对根部真菌群落结构的影响。【方法】采集幼苗根尖样品提取基因组DNA,用真菌通用引物与丛枝菌根真菌(AMF)特异性引物扩增真菌r DNA-ITS区,经克隆、测序、序列分析鉴定真菌。通过基于核酸与Metadata数据关联分析的FUNGuild软件,划分根部真菌的营养型和共位群。采用非公制多维尺度分析法(NMDS)研究幼苗根部真菌群落物种组成差异与宿主植物物种及形态指标的关系。【结果】白木香与降香黄檀幼苗根部真菌物种丰富,达51个OTU;谱系多样性较高,涉及毛霉菌门(Mucoromycota,51%)、子囊菌门(Ascomycota,43%)以及担子菌门(Basidiomycota,6%)。这些根部真菌涉及不同的营养型与共位群,包括共生型真菌29种,频度较高的如Glomeromycetes sp.2、Rhizophagus irregularis等,二者均属于AMF共位群;腐生营养型真菌5种,如Talaromyces pinophilus、Rhizopycnis vagum等;病原型真菌2种,是Mycoleptodiscus sp.和Fusarium phaseoli;还有15种其生态类型不确定。NMDS分析结果表明,宿主植物物种、株高、地径、叶面积对根部真菌群落物种组成的影响不显著。然而,株高对AMF群落的物种组成有较弱的影响。【结论】本苗圃条件下,土壤中本土性根部真菌繁殖体较为充足,白木香与降香黄檀幼苗根部真菌群落谱系多样性较高,多种营养型与共位群的根部真菌共存;此外,采用真菌通用引物对ITS1F/ITS4研究根部真菌群落物种多样性时,AMF多样性可能会被极度低估。     

高寒草甸植物系统发育与AM真菌侵染的关系

测定了高寒草甸生态系统中17种常见植物根内AM真菌的侵染率,并将AM侵染率作为植物的一个功能特征,分析了其系统发育保守性。结果显示AM侵染率均无系统发育信号,其植物进化树中AM真菌侵染率的差异更多被最新分支节点所解释,而不是古老分支节点,说明高寒草甸生态系统中亲缘关系较近的植物,其AM侵染水平并不相同,不存在进化的保守性。系统报道了高寒草甸生态系统中植物系统进化发育与AM侵染间的关系,表明了植物系统进化与AM侵染间无显著关联。     

葛根资源遗传多样性和性状关联分析

通过研究葛根资源的遗传多样性和葛根表型性状与分子标记的关联分析,为葛根的分子育种和指纹图谱构建提供理论依据。鉴定分析了127份不同来源葛根资源的10个表型性状,并用ISSR标记研究127份葛根资源的遗传多样性,对ISSR标记与表型性状进行关联分析。表型性状鉴定结果显示葛根资源的10个性状变异大、多样性较好。利用ISSR标记获得多态性条带109条,平均每个引物扩增5.73条、平均Nei's基因多样性0.2085、平均Shannon's指数0.3378,最远遗传距离为0.46。ISSR分子标记聚类分析将127份资源聚为两大类,群体结构分析和PCo A分析结果类似将127份资源分为2个亚群。GLM分析发现3个与茸毛性状关联标记,MLM分析未发现与表型性状关联的标记。本研究收集的资源遗传多样性较好,葛根分子标记聚类结果与地域关系不大,综合GLM和MLM关联分析结果,本试验未发现与表型关联位点。     

关联分析在作物种质资源分子评价中的应用

发掘优异基因资源是作物种质资源分子评价的重要部分,对作物育种尤其是分子育种具有非常重要的实践意义。基于连锁不平衡(LD)的关联分析是基因发掘也是等位基因发掘的有效途径。本文系统介绍了关联分析的基本理论、策略、特点及应用现状,并探讨了其在作物种质资源新(等位)基因发掘中的发展趋势及展望。可以预见,与传统QTL作图及功能基因组学相整合的关联分析必将大大加快我国作物种质资源的研究进程,实现我国种质资源优势向基因资源优势的转变。     

Association mapping for yield and grain quality traits in rice (Oryza sativa L.)

Association analysis was applied to a panel of accessions of Embrapa Rice Core Collection (ERiCC) with 86 SSR and field data from two experiments. A clear subdivision between lowland and upland accessions was apparent, thereby indicating the presence of population structure. Thirty-two accessions with admixed ancestry were identified through structure analysis, these being discarded from association analysis, thus leaving 210 accessions subdivided into two panels. The association of yield and grain-quality traits with SSR was undertaken with a mixed linear model, with markers and subpopulation as fixed factors, and kinship matrix as a random factor. Eight markers from the two appraised panels showed significant association with four different traits, although only one (RM190) maintained the marker-trait association across years and cultivation. The significant association detected between amylose content and RM190 was in agreement with previous QTL analyses in the literature. Herein, the feasibility of undertaking association analysis in conjunction with germplasm characterization was demonstrated, even when considering low marker density. The high linkage disequilibrium expected in rice lines and cultivars facilitates the detection of marker-trait associations for implementing marker assisted selection, and the mining of alleles related to important traits in germplasm.     

Region-Based Association Analysis of Human Quantitative Traits in Related Individuals

Regional-based association analysis instead of individual testing of each SNP was introduced in genome-wide association studies to increase the power of gene mapping, especially for rare genetic variants. For regional association tests, the kernel machine-based regression approach was recently proposed as a more powerful alternative to collapsing-based methods. However, the vast majority of existing algorithms and software for the kernel machine-based regression are applicable only to unrelated samples. In this paper, we present a new method for the kernel machine-based regression association analysis of quantitative traits in samples of related individuals. The method is based on the GRAMMAR+ transformation of phenotypes of related individuals, followed by use of existing kernel machine-based regression software for unrelated samples. We compared the performance of kernel-based association analysis on the material of the Genetic Analysis Workshop 17 family sample and real human data by using our transformation, the original untransformed trait, and environmental residuals. We demonstrated that only the GRAMMAR+ transformation produced type I errors close to the nominal value and that this method had the highest empirical power. The new method can be applied to analysis of related samples by using existing software for kernel-based association analysis developed for unrelated samples.     

Comparison of methods for competitive tests of pathway analysis

It has been suggested that pathway analysis can complement single-SNP analysis in exploring genomewide association data. Pathway analysis incorporates the available biological knowledge of genes and SNPs and is expected to improve the chances of revealing the underlying genetic architecture of complex traits. Methods for pathway analysis can be classified as competitive (enrichment) or self-contained (association) according to the hypothesis tested. Although association tests are statistically more powerful than enrichment tests they can be difficult to calibrate because biases in analysis accumulate across multiple SNPs or genes. Furthermore, enrichment tests can be more scientifically relevant than association tests, as they detect pathways with relatively more evidence for association than the remaining genes. Here we show how some well known association tests can be simply adapted to test for enrichment, and compare their performance to some established enrichment tests. We propose versions of the Adaptive Rank Truncated Product (ARTP), Tail Strength Measure and Fisher's combination of p-values for testing the enrichment null hypothesis. We compare the behaviour of these proposed methods with the established Hypergeometric Test and Gene-Set Enrichment Analysis (GSEA). The results of the simulation study show that the modified version of the ARTP method has generally the best performance across the situations considered. The methods were also applied for finding enriched pathways for body mass index (BMI) and platelet function phenotypes. The pathway analysis of BMI identified the Vasoactive Intestinal Peptide pathway as significantly associated with BMI. This pathway has been previously reported as associated with BMI and the risk of obesity. The ARTP method was the method that identified the largest number of enriched pathways across all tested pathway databases and phenotypes. The simulation and data application results are in agreement with previous work on association tests and suggests that the ARTP should be preferred for both enrichment and association testing.     

Screening the genome to detect an association with hypertension

We report tree-based association analysis as applied to the two Framingham cohorts and to the first replication of the simulated data obtained from the Genetic Analysis Workshop 13. For this analysis, familial association is ignored. The two endpoints examined are hypertension status at initial visit and time-to-hypertension, using a censored data approach. Although linkage association has previously been reported with hypertension, we found no association using the tree-based methodology.     

Analyte-receptor binding on surface plasmon resonance biosensors: a fractal analysis of Cre-loxP interactions and the influence of Cl, O, and S on drug-liposome interactions

A fractal analysis of the association and dissociation (whereever applicable) of Cre-loxP interactions and drug-liposome interactions on a sensor chip surface is presented. In both of these cases a dual-fractal analysis is required to adequately describe the association kinetics. The dissociation kinetics for Cre-loxP interactions is adequately described by a single-fractal analysis. The dual-fractal analysis used to describe the association kinetics of Cre-loxP interactions is consistent with the original two-step mechanism presented using a surface plasmon resonance biosensor. Our analysis includes both diffusion and surface effects by introducing the fractal dimension which makes quantitative the degree of heterogeneity on the sensor chip surface. Affinities are provided. Only the association kinetics were analysed for drug-liposome interactions since the initial sections of the dissociation curves were too steep to obtain reasonable drug-liposome complex concentration values on the sensor chip with time. Attempts made to relate the association rate coefficients with the molecular weight of the drug were unsuccessful. On using desipramine and imipramine as "arbitrarily selected standards" or "references" (only C, H, and N atoms present), it was noticed from the data analysed that the inclusion of the O and S atoms in the drug leads to a decrease in the association rate coefficients, ka1 (or k1) and ka2 (or k2) (compared with the arbitrarily selected standards or references). Similarly, the addition of the Cl atom in the drug leads to an increase in the association rate coefficient (compared with the arbitrarily selected standards or references). More data needs to be analysed to determine whether this is true for other drugs also.     

Association mapping for phenological, morphological, and quality traits in canola quality winter rapeseed (Brassica napus L.)

QTL mapping by association analysis has recently gained interest in plant breeding research as an alternative to QTL mapping in segregating populations from biparental crosses. In a first experiment on whole-genome association analysis in rapeseed, 684 mapped AFLP markers were tested for association with 14 traits in a set of 84 canola quality winter rapeseed cultivars. For association analysis a general linear model was used. By testing significance of marker-trait associations against a false discovery rate of 0.2, between 1 and 34 associated markers were found for 10 of the 14 traits. Taking into account linkage disequilibrium between the significant markers, these markers represent between 1 and 22 putative QTL for the respective traits. The minimum phenotypic variance explained by the QTL for the different traits ranged from 15% to 53%. A subset of 27 markers were significantly associated with two or more traits. These markers were predominantly shared between traits that were significantly correlated at the phenotypic level. The results show clearly that in rapeseed, QTL mapping by association analysis is a viable alternative to QTL mapping in segregating populations.     

Methods to test for association between a disease and a multi-allelic marker applied to a candidate region

We report the analysis results of the Genetic Analysis Workshop 14 simulated microsatellite marker dataset, using replicate 50 from the Danacaa population. We applied several methods for association analysis of multi-allelic markers to case-control data to study the association between Kofendrerd Personality Disorder and multi-allelic markers in a candidate region previously identified by the linkage analysis. Evidence for association was found for marker D03S0127 (p < 0.01). The analyses were done without any prior knowledge of the answers.     

关联分析及其在植物遗传学研究中的应用

植物的很多重要经济性状均属于复杂性状。基于连锁分析的QTL作图是研究复杂性状的有效手段, 但其尚存在一定的局限性。随着现代生物学的发展, 一种基于连锁不平衡的新剖分复杂性状方法--关联分析法, 开始应用于植物遗传学研究。与QTL作图法相比, 应用关联分析法具有不需要构建特殊的群体, 可同时对多个等位基因进行分析, 定位QTL精度可达到单基因水平等优势。该文介绍了关联分析方法学的基础和特性, 简述了其在植物遗传学研究中的进展情况, 并对其未来发展和在植物遗传学研究中的应用进行了展望。