紫芝基因组rDNA序列特征及ITS2二级结构比较

紫芝栽培品种‘紫芝S2’(武芝2号)的ITS序列与NCBI数据库中5个紫芝菌株/分离株相似度高达99.79%-100%,在系统进化树上相聚成一类。本研究预测‘紫芝S2’基因组与参考基因组中的rRNA基因簇,分析rDNA结构及各构件序列间的多态性。从高质量‘紫芝S2’基因组中挖掘得到完整rDNA,序列全长40.377 kb,由4组串联重复的(18S、5.8S、28S、5S) rRNA基因簇组成,并含有完整的基因内间隔区(ITS1、ITS2)和基因间间隔区(IGS1、IGS2)。在紫芝S2的rDNA中,高度保守的28S rRNA基因间出现3个SNP和2个插入(1 bp,10 bp)位点;虽然第4条ITS2中有1个SNP位点,但紫芝S2的4条ITS2在二级结构上的分子形态高度一致,与ITS2数据库中其他紫芝菌株仅存在螺旋区间夹角的微小差异。由‘紫芝S2’基因组rDNA的ITS2生成的DNA条形码与二维码,可以作为该栽培品种鉴定与同源物种其他菌株鉴别的分子标记。     

我国代表地区须癣毛癣菌复合体的分子鉴定与分型研究

目的对我国代表地区的须癣毛癣菌菌株进行分子再鉴定和分型研究。方法选取我国南北方8个省市地区经表型鉴定的须癣毛癣菌菌株47株,通过再培养形态观察、生理试验;PCR扩增核糖体DNA(rDNA)的内转录间隔区(ITS)和核糖体大亚基(LSU)D1-D2区,测序后利用数据库进行序列比对,对须癣毛癣菌复合体进行再鉴定;PCR扩增rDNA非转录间隔区(NTS)的三个串联重复亚单位S0、S1和S2区,进行种内分型,并比较不同部位来源菌株型别的差异性。结果我国南北方8个省市地区47株须癣毛癣菌中3株鉴定为断发毛癣菌,6株鉴定为无性型苯海姆节皮菌,其余均鉴定为万博节皮菌中的亲人型趾间毛癣菌;三对不同引物扩增38株趾间型毛癣菌和2株苯海姆节皮菌NTS区,共产生28种特征性带型。带型和菌株来源及发生部位无相关性。结论我国分离自人类须癣毛癣菌复合体的主要组成菌种为趾间毛癣菌;ITS区结合LSU D1-D2区测序有助于鉴定须癣毛癣菌复合体至种水平;NTS区的三个串联重复亚单位所产生的特征性指纹图提供了一种快速、稳定的分子生物学种内分型方法,可应用于趾间毛癣菌感染的流行病学研究。     

rRNA基因序列分析在阿萨希毛孢子菌鉴定中的应用

作者对近年有关毛孢子菌属rRNA分析的研究进行了综述,目前的研究结果表明。RNA的26S亚基的D1／D2区、ITS和IGS区的序列分析对毛孢子菌属的鉴定均有一定价值,其中ITS转录间区与18S、5．8S以及26S相比具有较高的特异性;IGS区序列分析优于ITS区序列分析;IGS1区优于IGS2区。     

人工栽培蛹虫草退化现象的分子分析*

利用PCR-RFLP和RAPD方法对野生驯化蛹虫草及其退化菌种进行了基因水平的分析。PCR-RFLP实验,采用真菌通用引物ITS1和ITS4,扩增出5.8S和其两端的两个转录间隔区(ITS),选用5种识别四个碱基的内切酶(HaeIII、AfaI、TagI、AluI和XspI),其中只XspI酶切结果在两个菌种中存在差异,进一步测序表明,整个片段全长534bp,共有13个位点发生碱基突变,且都为c转换为t。RAPD实验,选用4组计80个随机引物进行PCR扩增,共筛选出9个对所有供试个体均存在明显差异的引物。结果表明了退化菌株较野生驯化菌株在DNA水平上发生了频率较高的突变,这些突变可能直接与导致菌种退化的基因相关联。     

人工栽培蛹虫草退化现象的分子分析

利用PCR-RFLP和RAPD方法对野生驯化蛹虫草及其退化菌种进行了基因水平的分析。PCR-RFLP实验,采用真菌通用引物ITS1和ITS4,扩增出5．8S和其两端的两个转录间隔区(ITS),选用5种识别四个碱基的内切酶(HaeⅢ、AfaI、TagI、AluI和XspI),其中只XspI酶切结果在两个菌种中存在差异,进一步测序表明,整个片段全长534bp,共有13个位点发生碱基突变,且都为c转换为t。RAPD实验,选用4组计80个随机引物进行PCR扩增,共筛选出9个对所有供试个体均存在明显差异的引物。结果表明了退化菌株较野生驯化菌株在DNA水平上发生了频率较高的突变,这些突变可能：直接与导致菌种退化的基因相关联。     

金针菇rDNA序列结构特征分析

rDNA序列中的ITS作为DNA barcoding广泛应用于真菌的系统发育与物种辅助鉴定,IGS被认为可以用于种内水平不同菌株的鉴别。食用菌中还没有完整的rDNA序列的报道。本研究采用二代和三代测序技术分别对金针菇单核菌株“6-3”进行测序,用二代测序的数据对三代测序组装得到的基因组序列进行修正,得到一个在基因完整性、连续性和准确性均较好的基因组序列,对比Fibroporia vaillantii rDNA序列,获得金针菇完整的rDNA序列。金针菇rDNA序列结构分析表明,它有8个rDNA转录单元,长度均为5 903bp,有9个基因间隔区,其长度有较大差异,3 909-4 566bp。rDNA转录单元中,各元件的序列长度分别为：18S rDNA 1 796bp、ITS1 234bp、5.8S rDNA 173bp、ITS2 291bp、28S rDNA 3 410bp。基因间间隔区中,IGS1 1 351-1 399bp、5S rDNA 124bp、IGS2 2 435-3 092bp。金针菇的5S、5.8S、18S、28S rDNA序列准确性得到转录组数据的验证,也得到系统发育分析结果的支持。多序列比对发现,不同拷贝的基因间间隔区序列（IGS1和IGS2）存在丰富的多态性,多态性来源于SNP、InDel和TRS（串联重复序列）,而TRS来源于重复单元的类型和数量。9个基因间间隔区之间,IGS1只有少量的SNP和InDel,IGS2不仅有SNP和InDel,还有TRS。本研究结果提示,在应用IGS进行种内水平不同菌株之间的鉴别时,需要选取不同拷贝之间的保守IGS序列。     

中国南北方地区临床孢子丝菌菌种鉴定

目的对分离自我国南、北方地区50株临床孢子丝菌进行菌种鉴定。方法分离菌株分别进行25℃恒温培养和玻片小培养,肉眼和镜下观察形态特征;同时提取菌丝相基因组DNA,用PCR分别扩增部分微管蛋白(β-tubulin)基因和核糖体内部转录间隔区(Ribosomal Internal Transcribed Spacer,ITS),扩增产物进行测序,并采用最大似然法(maximum likelihood,ML)和邻接法(neighbor-joining,NJ)构建联合β-tubulin基因和ITS区域的系统发育树。结果结合形态学及系统发育分析,50株菌均鉴定为球形孢子丝菌(Sporothrix globosa,S.globosa)。结论球形孢子丝菌是目前我国南、北方地区孢子丝菌病的主要致病菌种,为进一步明确我国孢子丝菌病致病菌种的分布提供了依据。     

同宗配合蜜环菌与欧美异宗配合蜜环菌狭义种的分子系统学关系*

从亚洲、欧洲和北美收集18个蜜环菌狭义种菌株,PCR扩增其rDNA的IGS和ITS区域,用AluI、HaeIII、HinfI和TagI四种限制性内切酶进行酶切,同时用随机扩增微卫星(RAMS)多态性,对蜜环菌狭义种的进行了遗传多样性和分子系统学分析。结果蜜环菌狭义种的IGS和ITS-RFLP类型比以前报道的更多,亚洲、欧洲和北美菌株都具有比较明显的特征片段,通过ITS和IGS图谱可将三个大陆的菌株区分开,其中IGS-HinfI图谱能完全区分3个大陆的菌株。RFLP数据的系统学分析表明,该种存在明显的大陆遗传分化,亚、欧、北美的群体分属三个相互独立的进化系,北美群体IGS变异程度较小,ITS变异程度极高;而欧洲群体IGS的变异程度较大,多态性高,ITS变异程度非常小。RAMS系统分析表明,中国、日本和非洲的同宗配合蜜环菌属于一个系统发育系,该发育系同欧洲和北美的异宗配合种的两个发育系分属3个不同的独立进化分支。据此建议欧洲和北美的异宗配合蜜环菌应作为蜜环菌的两个亚种。     

同宗配合蜜环菌与欧美异宗配合蜜环菌狭义种的分子系统学关系

从亚洲、欧洲和北美收集18个蜜环菌狭义种菌株,PCR扩增其rDNA的IGS和ITS区域,用AluI、HaeIII、HinfI和TagI四种限制性内切酶进行酶切,同时用随机扩增微卫星(RAMS)多态性,对蜜环菌狭义种的进行了遗传多样性和分子系统学分析。结果蜜环菌狭义种的IGS和ITS-RFLP类型比以前报道的更多,亚洲、欧洲和北美菌株都具有比较明显的特征片段,通过ITS和IGS图谱可将三个大陆的菌株区分开,其中IGS-HinfI图谱能完全区分3个大陆的菌株。RFLP数据的系统学分析表明,该种存在明显的大陆遗传分化,亚、欧、北美的群体分属三个相互独立的进化系,北美群体IGS变异程度较小,ITS变异程度极高;而欧洲群体IGS的变异程度较大,多态性高,ITS变异程度非常小。RAMS系统分析表明,中国、日本和非洲的同宗配合蜜环菌属于一个系统发育系,该发育系同欧洲和北美的异宗配合种的两个发育系分属3个不同的独立进化分支。据此建议欧洲和北美的异宗配合蜜环菌应作为蜜环菌的两个亚种。     

采用PCR-RFLP技术在不同水平上鉴定大豆根瘤菌

采用16S rRNA基因PCR扩增与限制性酶切片段多态性分析(RFLP)技术对选自弗氏中华根瘤菌(S.fredii)、大豆慢生根瘤菌(B.japonicum)和埃氏慢生根瘤菌(B.elkanii)的19株代表菌进行了比较分析,根据用3种限制性内切酶的RFLP分析结果,可将供试菌株分为S.fredii,B.japonicum, B.elkanii Ⅱ和B.elkanii Ⅱa等4种基因型。各类菌株之间没有交叉,因此本研究采用的PCR-RFLP技术不失为一种快速鉴别大豆根瘤菌的新方法。采用本技术已将分离自中国的22株快生菌和19株慢生菌分别鉴定为S.fredii和B.japonicum。对供试参比菌株和野生型菌株进行的16S～23S基因间隔DNA(IGS)的PCR-RFLP分析结果表明：S.fredii和B.japonicum菌株的IGS长度不同,所有供试S.fredii菌株的IGS为2.1 kb,而供试B.japonicum菌株则为2.0 kb。依据RFLP的差异,可将来自中国两个不同地区的S.fredii株区分为2个基因型,而来自中国东北黑龙江地区的19株B.japonicum菌株则可分为11个基因型。对上述野生型菌株还进行了REP-PCR和ERIC-PCR分析并确定其具有菌株水平的特异性。     

IGS基因及RAPD标记分析在加特隐球酵母基因分类中的应用

根据ITS1-5.8S-ITS2区域的特异核酸序列变化,加特隐球酵母Cryptococcus gattii(≡新型隐球酵母加特变种Cryptococcu neoformans var.gattii)可分为6种基因型。本研究通过检测加特隐球酵母的IGS基因,发现其IGS序列有着更丰富的变异和信息位点。通过结合加特隐球酵母RAPD(随机扩增的多态性DNA)图谱比较研究,与IGS和ITS的序列分析结果大体一致,说明新近发现的加特隐球酵母ITS8型确实有别于以前报道过的其他加特隐球酵母ITS基因型。研究证明IGS1及IGS2基因片段分析可以作为加特隐球酵母基因分类鉴定中有效的辅助鉴别的分子生物学方法,联合多种基因分类鉴定的方法可以更有效地揭示新型隐球酵母加特变种种内不同基因亚型间的遗传进化关系。     

香菇菌株分子鉴别技术的分辨率比较

本研究运用酯酶同工酶、RAPD、IGS1和IGS24种方法鉴别2个野生香菇菌株和19个栽培香菇菌株,并比较这4种鉴别方法的分辨率,结果表明:16条酯酶同工酶带中有15条具有多态性,将21个供试菌株分成11个类型,分辨率为0.92;10个随机扩增引物共扩增出86个DNA片段,其中95.3%具有多态性,将21个供试菌株分成16个类型,分辨率为0.97;IGS1将21个供试菌株分成7个类型,分辨率为0.81;IGS2将21个供试菌株分成7个类型,分辨率为0.73。这4种鉴别方法中RAPD的分辨率最高,与这4种鉴别方法的综合分析结果相同,因此RAPD可以作为香菇菌株鉴别的可靠依据。     

Intergenic spacer (IGS) polymorphism: a new genetic marker for differentiation of Toxoplasma gondii strains and Neospora caninum

The region between the 28S and 18S rRNA genes, including the intergenic spacer (IGS) region and the 5S rRNA gene, from 32 strains of Toxoplasma gondii and the NC1 strain of Neospora caninum was amplified and used for DNA sequencing and/or restriction fragment length polymorphism (RFLP) analysis. The 5S rDNA sequences from 20 strains of T. gondii were identical. The IGS region between the 5S and 18S rRNA genes (nontranscribed spacer 2 or NTS 2) showed 10 nucleotide variations. Six of the 10 variant positions correlated with the murine virulence of the strains. Intraspecific polymorphisms distinguished the virulent strains of zymodemes 5, 6, and 8 from other virulent strains (in zymodeme 1). RFLP methods (IGS-RFLP) were developed and used to characterize the virulent and avirulent patterns among 29 T. gondii strains. Sequence diversity of 19.8% was found between T. gondii and N. caninum when comparing a region of 919 bp at the 3' end of NTS 2. The sequence variation in ribosomal IGS could therefore be a useful marker for Toxoplasma strain identification and for distinguishing N. caninum from T. gondii.     

Assessing the potential of regions of the nuclear and mitochondrial genome to develop a "molecular tool box" for the detection and characterization of Phytophthora species

Four different intergenic regions of mitochondrial DNA (mt-IGS), a fragment of the intergenic spacer (IGS) region of the rDNA (rDNA-IGS), and a fragment of the ras-related protein (Ypt1) gene were amplified and sequenced from a panel of 31 Phytophthora species representing the most significant forest pathogens and the breadth of diversity in the genus. Over 80 kbp of novel sequences were generated and alignments showed very variable (introns and non-coding regions) as well as conserved coding regions. The mitochondrial DNA regions had an AT/GC ratio ranging from 67.2 to 89.0% and were appropriate for diagnostic development and phylogeographic analysis. The IGS fragment was less variable but still appropriate to discriminate amongst some important forest pathogens. The introns of the Ypt1 gene were sufficiently polymorphic for the development of molecular markers for almost all Phytophthora species, with more conserved flanking coding regions appropriate for the design of Phytophthora genus-specific primers. In general, phylogenetic analysis of the sequence alignments grouped species in clades that matched those based on the ITS regions of the rDNA. In many cases the resolution was improved over ITS but in other cases sequences were too variable to align accurately and yielded phylograms inconsistent with other data. Key studies on the intraspecific variation and primer specificity remain. However the research has already yielded an enormous dataset for the identification, detection and study of the molecular evolution of Phytophthora species.     

Typing method for N2-fixing bacteria based on PCR-F — application to the characterization of Frankia strains

DNA sequences of an intergenic spacer (IGS) and parts of genes in the nif cluster were amplified by the polymerase chain reaction (PCR) using two primers derived from nifD -and nifK -conserved sequences. The PCR products were cleaved by ten 4–base cutting restriction enzymes and the restriction patterns were used as fingerprints to type Frankia strains. The feasability of this PCR-RFLP method for typing Frankia strains was investigated on Frankia reference strains belonging mainly to the Elaeagnaceae infectivity group but also on new Frankia isolates and on other N₂-fixing microorganisms. By modulating the stringency of the amplifications, we showed the method allowed to target either Frankia strains or the whole N₂-fixing microbial community. DNA digestion patterns were used to estimate the sequence divergence between the Frankia nifD-K fragment. The estimated relationships deduced from these genotypic data correlated well with established Frankia taxonomic schemes.     

RAPD patterns,nrDNA ITS sequences and morphological patterns inSilene sectionSedoideae (Caryophyllaceae)

Hierarchical patterns inSilene sect.Sedoideae were investigated using random amplified polymorphic DNA (RAPD), nucleotide sequences of the internal transcribed spacer (ITS) regions of the 18S–28S nuclear ribosomal DNA, and discrete morphological characters. All data sets firmly supported the species recognized. The RAPD data offered the best resolution at the intraspecific level, supporting the current intraspecific classifications ofS. sedoides andS. integripetala. The ITS sequences and the morphological data gave poor resolution within species, and the three data sets disagreed about the relationships among species. The signal from the RAPD data was strongest and remained when the total data set was analysed. The three data sets all support an amphiploid origin ofS. aegaea, with the strongest evidence from the ITS sequences. Incongruences among data sets as well as merits and shortcomings of each are discussed. The robustness of the results can be evaluated using perturbations of data, i.e., bootstrap and jackknife of taxa and characters. These methods should not be taken as methods of statistical inference at the taxonomic level, because unbiased sampling appears impossible. RAPD data, however, come close to being suitable for statistical estimation of hierarchies at the genome level, but several methodological problems have to be solved.     

Determination of genetic diversity among Indian isolates of Rhizoctonia bataticola causing dry root rot of chickpea

Genetic diversity among 27 isolates (23 from chickpea and 4 from other host crops) of Rhizoctonia bataticola representing 11 different states of India was determined by random amplified polymorphic DNA (RAPD), internal transcribed spacer restriction fragment length polymorphism (ITS-RFLP) and ITS sequencing. The isolates showed variability in virulence test. Unweighted paired group method with arithmetic average cluster analysis was used to group the isolates into distinct clusters. The clusters generated by RAPD grouped all the isolates into six categories at 40% genetic similarity. High level of diversity was observed among the isolates of different as well as same state. Some of the RAPD (OPN 4, OPN 12, and OPN 20) markers clearly distinguished majority of the isolates into the area specific groups. The ITS I, 5.8rDNA and ITS II regions of 11 isolates representing different RAPD groups were amplified with primers ITS 1 and ITS 4 and digested with seven restriction enzymes. The restriction enzymes DraI, MboI, RsaI, and AluI were found to be suitable for differentiating the isolates into five categories by showing isolate specific ITS-RFLP patterns. The isolates were variable in their nucleotide sequences of the ITS regions. This is the first study on genetic diversity among chickpea isolates of R. bataticola.     

甘肃省冬虫夏草菌的RAPD和ITS序列分析

目的对甘肃省冬虫夏草分离菌株进行鉴定和系统发育研究。方法采用随机扩增多态性DNA（RAPD）分析和核糖体（rDNA）内部转录间隔区（ITS）序列分析。结果共分离到18个菌株,菌株间的遗传相似性系数为0.623～1.000,遗传分化距离为0.000～0.018,每个菌株的序列与中国被毛孢的序列一致性均高于98%且18个菌株（G＋C）%的差别最大值是4.7%。结论 18个分离菌株均为中国被毛孢;多样性分析表明,不同区域样本间的遗传分化较大;同一地点样本间的遗传分化很小;同一材料不同部位获得的菌株在RAPD标记上差异无统计学意义,两技术对相同的供试菌株所反映的遗传分化不尽相同,表明两技术用于冬虫夏草遗传多样性和种类鉴定上各有它们的优势和具体选择。     

Genetic diversity of root-nodulating bacteria isolated from pea (Pisum sativum) in subtropical regions of China

Diversity of 42 isolates from effective nodules of Pisum sativum in different geographical regions of China were studied using 16S rRNA gene RFLP patterns, 16S rRNA sequencing, 16S–23S rRNA inter-genic spacer (IGS) region RFLP patterns and G-C rich random amplified polymorphic DNA (RAPD). The isolates were distributed in two groups on the basis of their 16S rRNA gene RFLP patterns. The 16S rRNA gene sequences of strains from 16S rRNA gene RFLP patterns group I were very closely related (identities higher than 99.5%) to Rhizobium leguminosarum USDA 2370. Group II consisting of WzP3 and WzP15 was closely related to Rhizobium etli CFN42. The analysis of the 16S–23S IGS RFLP pat-terns divided the isolates into 18 genotypes and four groups. Group I was clustered with R. legumino-sarum USDA2370. Group II consisted of YcP2, YcP3 and CqP7. The strains of group III were distributed abroad. Group IV consisted of WzP3, WzP15 and R. etli CFN42. RAPD divided the isolates into nine clusters in which group IV only consisted of YcP2 and the strains of group V and IX were from Wenzhou and Xiantao, respectively. This assay demonstrated the geographical effect on genetic diversity of pea rhizobia.     

Presence of a novel 16S-23S rRNA gene intergenic spacer insert in Bradyrhizobium canariense strains

Seven slow-growing bacterial strains isolated from root nodules of yellow serradella (Ornithopus compressus) that originated from Asinara Island on North Western Sardinia in Italy were characterized by partial 16S rRNA gene and intergenic spacer (ITS) sequencing as well as amplified fragment length polymorphism (AFLP) genomic fingerprinting. The results indicated that the O. compressus isolates belong to the Bradyrhizobium canariense species. The analysis of ITS sequences divided the branch of B. canariense strains into two statistically separated groups (ITS clusters I and II). All the strains in ITS cluster I showed the presence of unique oligonucleotide insert TTAGAGACTTAGGTTTCTK. This insert was neither found in other described species of the family Rhizobiaceae nor in any other bacterial families and can be used as a natural and high selective genetic marker for ITS cluster I of B. canariense strains. ITS grouping of O. compressus isolates was supported by the unweighted pair group method with arithmetic averages cluster analysis of their AFLP patterns, suggesting that the strains of ITS cluster II were genetically closer to each other than to isolates from the ITS cluster I. A taxonomic importance is supposed of the revealed 19 bp ITS insert for an intraspecific division within high heterogeneous B. canariense species.