小麦印度腥黑粉菌线粒体DNA提取及其ATP6基因在真菌遗传发育分析中的应用

小麦印度腥黑粉菌(Tilletia indica Mitra)是一种世界范围的重要检疫性有害真菌,该病原菌和近似种之间冬孢子的形态特征极为相似,遗传关系非常相近。为了从分子水平上探讨小麦印度腥黑粉菌和近似种之间线粒体基因序列的差异,从新鲜菌丝中提取总DNA,经两次氯化铯密度梯度超速离心分离线粒体DNA(mtDNA),提取的mtDNA纯度较高,可用于克隆、酶切分析和PCR扩增等分析。选取基因ATP(adenosine triphosphate)6的序列,并结合GenBank中相关种类的ATP6基因DNA序列进行了系统发育分析。结果表明,线粒体基因ATP6可用于科属水平的分类鉴定。     

小麦矮腥黑穗菌差异片段筛选与分子检测体系的建立

采取随机扩增DNA多态性(Random amplified polymorphic DNA,RAPD)引物介导的半特异PCR技术(RAPD primer mediated hemi-specific PCR,RM-PCR),在从不同地域征集的18个小麦矮腥黑穗菌(Tilletia controversa Kühn,TCK)菌株和29个小麦网腥黑穗病菌(Tilletia caries(DC)Tul,TCT)菌株的总基因组DNA中筛选鉴定出TCK独有的大小为1322bp差异基因组片段。根据该片段序列设计筛选出2对特异性引物CQUTCK2/CQUTCK3和CQUTCK4/CQUTCK5,均可以从18个TCK菌株的菌丝体和冬孢子DNA中稳定地扩增出747bp和200bp的单一靶带DNA,而在29个TCT菌株的菌丝体或冬孢子DNA均无任何扩增产物。以腥黑穗菌属通用引物对CQUK6/CQUK7为内置对照,可以确定被检样品是否含PCR抑制物质进而判断检测体系是否正确,同时有效地排除样品检测结果的假阳性和假阴性。采用建立的TCK特异PCR检测技术体系,实现简单而快速地鉴定小麦矮腥黑穗菌冬孢子或罹病小麦组织中侵染菌丝体的目的。     

贵州桐梓何首乌rDNA ITS序列分析

以改进的CTAB法对何首乌总基因组DNA进行提取,采用通用引物对不同来源的何首乌rDNA ITS序列进行PCR扩增、测序和序列分析.结果表明,何首乌rDNA完全序列片段长度共约652 bp,其中ITS1的长度为202 bp,5.8S的长度为161 bp,ITS2长度为232 bp,与其近缘种ITS序列间存在明显差异.其rDNA ITS序列在分子水平上为鉴别何首乌提供了参考依据.     

实时荧光PCR技术对小麦矮腥黑穗病菌的检测

通过对小麦矮腥黑穗病菌(TCK)及其近似种小麦网腥黑穗病菌(TCT)和小麦光腥黑穗病菌(TFL)的rDNA序列ETS区间测序比较分析,找出了TCK相对于TCT和TFL的特异性序列,并根据TCK的特异性序列设计了实时荧光PCR探针,利用实时荧光PCR技术成功实现了对TCK的检测。     

一种褐飞虱病原真菌的分子生物学鉴定

从田间褐飞虱Nilaparvata lugens( St(a)l)罹病虫体分离得到一株绿僵菌,以该菌为供试菌体,采用氯化苄法、CTAB法以及裂解液法分别提取了该菌的基因组DNA;以ITS1和ITS4为绿僵菌通用引物,对供试菌株的rDNA ITS序列进行PCR扩增、琼脂糖凝胶电泳检测和序列分析,并在核酸序列数据库中进行同...     

甘肃本地产当归、黄芪和大黄的rDNA ITS序列分析

本研究采用改良CTAB法分别提取18份甘肃本地产当归、黄芪和大黄基因组DNA,并用PCR分别扩增其ITS1-5.8S-ITS2序列、直接测序并作序列同源性比对分析。双向测序分析结果表明,甘肃6个不同产地当归rDNA的ITS1、5.8S和ITS2序列一致,片段长度分别为215bp、162bp和223bp;供试的黄芪ITS1、5.8S和ITS2序列分别为228bp、164bp和210bp;大黄ITS1、5.8S和ITS2序列分别为160bp、159bp和164bp。供试材料的ITS1-5.8S-ITS2核苷酸序列已提交GenBank。本研究为提供甘肃当归、黄芪和大黄指纹图谱鉴别的分子标记、其道地性药材的分子鉴定和品质评价提供参考。     

雀麦上雀麦腥黑粉菌Tilletia bromi的分子检测

雀麦是重要的牧草,近年来进口量激增。寄生于雀麦上的腥黑粉菌共有4种,即小麦矮腥黑穗菌Tilletia controversa(TCK)、雀麦腥黑粉菌T.bromi、T.bolayi和小麦网腥黑穗菌T.caries(TCT),根据冬孢子形态难以直接区分这些种类。本文在形态、自发荧光和萌发生理三方面的比较研究基础上,依据beta-微管蛋白tub2基因序列设计一套引物,转化为特异SCAR分子标记,建立了雀麦上T.bromi的菌丝基因组DNA的特异PCR检测方法和冬孢子的套式特异PCR检测方法,为病害提供了快速、可靠的检测方法。     

硬软蒺藜rDNA-ITS基因序列的测定和比较

用CTAB法提取总DNA,合成位于18 S rDNA和26S rDNA上的两条各20bp的引物,通过PCR扩增ITS的全序列,对PCR产物直接测序,分别获得了硬蒺藜(Tribulus terrestris L.)和软蒺藜(Atriples centralasiatica Iljin)的核糖体RNA基因-rDNA内转录间隔区(ribosomal DNA internal transcribed spacer,rDNA-ITS)的序列643 bp和607bp,其碱基总差异率为36.16%,其中,ITS1的碱基差异率为55.81%;5.8 S的碱基差异率为6.59%;ITS2的碱基差异率为56.77%.这种差异,以及基因序列本身,为硬软蒺藜的区别和种质资源鉴定提供了分子依据.     

苹果炭疽菌的分子鉴定与检测

测定苹果炭疽菌rDNA全序列,比对苹果炭疽菌和其它炭疽菌ITS序列以及构建系统关系树,发现苹果炭疽菌与胶孢炭疽菌的ITS序列相似性高达99．8％,并与胶孢炭疽菌聚在一起,可以明确苹果炭疽菌应属于胶孢炭疽菌。进一步的序列比对发现,苹果炭疽菌的18S rDNA3’端比其它胶孢炭疽菌多出一段379bp的序列,根据这一特有片段设计引物CgF1与通用引物ITS4配对,结果仅能从苹果炭疽菌中扩增出1232bp的特异性条带。用苹果炭疽菌接种离体苹果,以接种发病的病组织总DNA为模板,利用引物CgF1／ITS4进行PCR扩增,同样可以扩增出1232bp的特异性条带,而健康苹果组织DNA中未能扩增出任何条带,表明该方法可用于苹果炭疽菌的鉴定和快速检测。     

韩国赫坎按蚊复合体3成员种核糖体DNA内转录间隔2区的比较(英文)

本文对韩国中华按蚊、雷氏按蚊和八代按蚊核糖体DNA (rDNA)内转录间隔 2区 (ITS2 )序列进行了比较研究。用PCR扩增的rDNA ITS2片段直接测序 ,每种蚊测定 3个个体 ,结果显示 :韩国中华按蚊、雷氏按蚊和八代按蚊的rDNA ITS2序列长度分别为 4 6 8bp、 4 51bp和 4 53bp ,GC含量分别为 4 4 .87%、 4 6 .2 %和 4 5.7% ,3种按蚊序列差异范围为 12 .16 %— 30 .74 %。研究表明 ,rDNA ITS2序列差异可用于韩国中华按蚊、雷氏按蚊和八代按蚊的分子鉴别。     

Isolation of a species-specific mitochondrial DNA sequence for identification of Tilletia indica, the Karnal bunt of wheat fungus.

Mitochondrial DNA (mtDNA) from five isolates of Tilletia indica was isolated and digested with several restriction enzymes. A 2.3-kb EcoRI fragment was chosen, cloned, and shown to hybridize with total DNA restricted with EcoRI from T. indica and not from a morphologically similar smut fungus, Tilletia barclayana. The clone was partially sequenced, and primers were designed and tested under high-stringency conditions in PCR assays. The primer pair Ti1/Ti4 amplified a 2.3-kb fragment from total DNA of 17 T. indica isolates from India, Pakistan, and Mexico. DNA from 25 isolates of other smut fungi (T. barclayana, Tilletia foetida, Tilletia caries, Tilletia fusca, and Tilletia controversa) did not produce any bands, as detected by ethidium bromide-stained agarose gels and Southern hybridizations. The sensitivity of the assay was determined and increased by using a single nested primer in a second round of amplification, so that 1 pg of total mycelial DNA could be detected. The results indicated that the primers which originated from a cloned mtDNA sequence can be used to differentiate T. indica from other Tilletia species and have the potential to identify teliospores contaminating wheat seeds.     

PCR based molecular technique for identification and discrimination of quarantined and non-quarantined Tilletia sps

Polymerase chain reaction (PCR) based RAPD profiles, in conjunction with six primers, of Karnal bunt of wheat and rice bunt exhibiting distinct polymorphic DNA. A total of 84 RAPD loci were observed on polyacrylamide gel for both Tilletia sps. Out of 84, 16 loci were found monomorphic, while other 68 loci were unique. Usefulness of random primers was also checked with other seed borne fungal pathogens of wheat and rice. None of primers gave amplification with Magnaporthe grisea, a causative agent of rice blast. However, distinct RAPD profiles were obtained with Alternaria triticina, Fusarium monaliforme, Helminthosporium sativum and Rhizoctonia solani. These six arbitrary primers could distinguish T. indica, a quarantine fungal pathogen from a non-quarantine fungal pathogen, T. barclayana. The two Tilletia sps. could be discriminated not only on the basis of distinct RAPD profiles, but also by presence of few unique gene fragments amplified using all six primers.     

Phylogenetic analysis of Tilletia and allied genera in order Tilletiales (Ustilaginomycetes; Exobasidiomycetidae) based on large subunit nuclear rDNA sequences

The order Tilletiales (Ustilaginomycetes, Basidiomycota) includes six genera (Conidiosporomyces, Erratomyces, Ingoldiomyces, Neovossia, Oberwinkleria and Tilletia) and approximately 150 species. All members of Tilletiales infect hosts in the grass family Poaceae with the exception of Erratomyces spp., which occur on hosts in the Fabaceae. Morphological features including teliospore ornamentation, number and nuclear condition of primary basidiospores and ability of primary basidiospores to conjugate and form an infective dikaryon were studied in conjunction with sequence analysis of the large subunit nuclear rDNA gene (nLSU). Analysis based on nLSU data shows that taxa infecting hosts in the grass subfamily Pooideae form one well supported lineage. This lineage comprises most of the reticulate-spored species that germinate to form a small number of rapidly conjugating basidiospores and includes the type species Tilletia tritici. Two tuberculate-spored species with a large number of nonconjugating basidiospores, T. indica and T. walkeri, and Ingoldiomyces hyalosporus are also included in this lineage. Most of the species included in the analysis with echinulate, verrucose or tuberculate teliospores that germinate to form a large number (>30) of nonconjugating basidiospores infect hosts in the subfamilies Panicoideae, Chloridoideae, Arundinoideae and Ehrhartoideae. This group of species is more diverse than the pooid-infecting taxa and in general do not form well supported clades corresponding to host subfamily. The results of this work suggest that morphological characters used to segregate Neovossia, Conidiosporomyces and Ingoldiomyces from Tilletia are not useful generic level characters and that all included species can be accommodated in the genus Tilletia.     

Atomic force and electron microscopy of high molecular weight circular DNA complexes with synthetic oligopeptide trivaline

Intramolecular compact structures formed by high molecular weight circular superhelical DNA molecules due to interaction with synthetic oligopeptide trivaline (1) were studied by atomic force and electron microscopy. Three DNA preparations were used: plasmids pTbol, pRX10 and cosmid 27,877, with sizes 6,120 bp, 10,500 bp and 44,890 bp respectively. Plasmid pTbo1 and pRX10 preparations along with monomers contained significant amount of dimers and trimers. Main structures in all preparations observed were compact particles, which coincide in their appearance and compaction coefficient (3,5-3,7) with triple rings described earlier. The size and structure characteristics of triple rings and other compact particles on atomic force images in general coincide with those obtained by EM (2). AFM (3) images allow to get additional information about the ultrastructural organization and arrangement of DNA fibers within the compact structures. Along with triple rings in pTbol and pRX10-TVP complexes significant amount of compact structures were observed having the shape of two or three compact rings attached to each other by a region of compact fibre. Basing on the data of contour length measurements and the shape of the particles it was concluded that these structures were formed due to compaction of dimeric and trimeric circular DNA molecules. Structures consisting of several attached to each other triple rings were not found for pTbol, pRX10 monomers or cosmid preparations--TVP complexes where only single triple rings were observed. The conclusion is made that initiation of compact fibre formation within the circular molecules depends on the primary structure and for dimeric or trimeric circular molecules two or three compaction initiation points are present, located in each monomer unit within one circular DNA molecule. The nucleotide sequence dependent compaction mechanism providing independent compaction of portions of one circular molecule can be of interest for understanding of DNA compaction processes in vivo.     

Variability of Indian Isolates of Tilletia indica Assessed by Pathogenicity and Molecular Markers

Twenty isolates of Tilletia indica collected from sites in North and North‐western India showed pathogenic variation on 18 host differentials. Sixteen aggressive pathotypes were identified on the basis of percent coefficient of infection (PCI). Two major clusters were apparent in the dendrogram; cluster 1 comprised 13 isolates and cluster two consisted of seven isolates. One of the isolate Kashipur had a high PCI on most of the host differentials compared to other isolates. Polymerase chain reaction‐based random amplified polymorphic DNA (PCR – RAPD) analysis also divided isolates into two major clusters, one comprising of 5 isolates collected from hill and foot‐hill sites and another group comprising of 15 isolates collected from plain sites. Thus, the clusters identified based on PCI did not match closely with those identified by molecular analysis based on RAPD. Although diversity among the isolates of T. indica was absent in the rDNA‐ITS region, our study based on pathogenicity and molecular markers confirms the existence of great diversity in the pathogen, also shifting of ‘hot spot’ areas from one place to another within Karnal bunt prevailing areas.     

中国野生稻及栽培稻核糖体DNA第一转录间隔区序列分析及其系统学意义

用 PCR技术从产于我国的 3种野生稻和亚洲栽培稻的 2个亚种中特异地扩增和测序了 r DNA的第一转录间隔区。普通野生稻 (Oryza rufipogon)、药用野生稻 (O.officinalis)、疣粒野生稻 (O.granu-lata)和栽培稻的两个亚种 (O.sativa ssp.indica,O.sativa ssp.japonica)的 ITS1序列为 1 93bp、1 94bp、2 1 8bp、1 94bp和 1 94bp,它们的 G/ C含量为 69.3%～ 72 .7% ,序列中位点趋异率为 1 .5%～ 1 0 .6%。序列的相似性比较和简约性分支分析的结果表明 ,普通野生稻与栽培稻的两个亚种之间的亲缘关系最为密切 ;药用野生稻与普通野生稻和与栽培稻的两个亚种的相似性都为 82 % ,说明它与 AA基因组有一定的亲缘关系 ;疣粒野生稻与普通野生稻、药用野生稻和栽培稻两个亚种的亲缘关系相对较远 ,它在稻属中可能是一个系统地位较独特的类群。以 ITS1序列构建的 3种野生稻和 2个栽培稻亚种的系统发育关系与前人用同工酶、叶绿体 DNA、线粒体 DNA和核 DNA资料重建的稻属的系统发育关系基本一致     

小麦印度腥黑粉菌与近似种的形态学比较*

利用光学显微镜和扫描电镜对小麦印度腥黑粉菌及其近似种的形态学特征进行了系统研究。T. indica和T. horrida不同菌株冬孢子大小变化范围均较大。在所研究的种中,T. indica与T. walkeri最近似,前者冬孢子大小平均值比后者略大,分别为:38.35?5.92祄和32.86?1.53祄。T. indica与其它具有疣状或刺状突起的腥黑粉菌:T. horrida, T. barclayana, T. setariae, T. opaca, T. sumatii和T. savilei等区别明显,T. indica冬孢子大小平均值明显大于这些腥黑粉菌,前者大于30祄,后者则小于30祄, T. indica孢壁纹饰与这些腥黑粉菌也有一定区别。应用光学显微镜和扫描电镜,能将T. indica与除T. walkeri之外的其它近似种区别开,但在区别T. indica与T. walkeri方面则有一定的局限性。     

Cryptic long internal repeat sequences in the ribosomal DNA ITS1 gene of the dinoflagellate Cochlodinium polykrikoides (dinophyceae): a 101 nucleotide six-repeat track with a palindrome-like structure

Extremely long PCR fragments were generated by PCR amplification of ITS and 5.8S rDNA from Cochlodinium polykrikoides against other dinoflagellates. These patterns were consistent among geographically different isolates of C. polykrikoies. DNA sequencing reactions revealed that the PCR products were 1,166 bp in length and consisted of 813 bp of ITS1, 160 bp of 5.8S rDNA and 193 bp of ITS2. Thus, the long length was caused mainly by the long ITS1 sequence. Cryptically, the ITS1 contained a tract of 101 bp that occurs six times in tandem. The six repeated elements had identical nucleotide sequences. ITS1, therefore, separated three distinct regions: the 5' end (122 bp), the six parallel repeats (606 bp), and the 3' region (85 bp). Interestingly, both the single and six-repeat sequences should be palindrome-like sequences. In inferred secondary structures, both repeat sequences formed a long helical structure. This is the first reported discovery of comparatively long internal repeats in the ITS1 of dinoflagellates.     

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。