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.     

基于rDNA ITSl和ITS2序列的褐飞虱、白背飞虱和灰飞虱的分子鉴定

本研究测定了褐飞虱 Nilaparvata lugens、白背飞虱 Sogatella furcifera 和灰飞虱Laodelphax striatellus 的rDNA ITSl和ITS2的序列,以探讨这3种稻飞虱的分子鉴定方法.3种飞虱的ITSI和ITS2侧翼区(18S,5.8S和28S)序列相对稳定,但ITS1和ITS2序列在3种飞虱中变异较大.ITS1在所分析的438个位点中可变位点达294个,ITS2在分析的403个位点中可变位点为177个.根据3种飞虱rDNA的ITS1和ITS2序列设计了特异性引物,应用特异性引物对样品进行了PCR扩增,分析发现3种飞虱ITS1区的特异性引物扩增效果不理想.而ITS2区的特异性引物可以稳定地扩增出明显的目的DNA条带.因此,采用ITS2区的特异性引物可以对3种飞虱进行快速的分子鉴定.     

米尔顿姬小蜂rDNA ITS1和ITS2的序列分析及其分子鉴定

本研究测定了米尔顿姬小蜂Anselmella miltoni Girault的rDNA ITS1和ITS2序列,以探讨其分子鉴定方法。米尔顿姬小蜂的ITS1和ITS2侧翼区（18S和5.8S）序列相对稳定,ITS1和ITS2序列存在种间差异。根据18S rDNA部分序列,利用DNAMAN的Maximum Likelihood方法构建了与膜翅目其它科的系统发育树。根据米尔顿姬小蜂ITS1和ITS2序列设计了特异性引物,应用特异性引物对样品进行了PCR扩增,扩增效果理想,采用上述特异性引物可从单头米尔顿姬小蜂稳定地扩增出明显的目的DNA条带。因此,可以采用ITS1和ITS2区的特异性对米尔顿姬小蜂进行快速的分子鉴定。     

基于rDNA ITS1和ITS2序列的褐飞虱、白背飞虱和灰飞虱的分子鉴定

本研究测定了褐飞虱Nilaparvata lugens、白背飞虱Sogatella furcifera和灰飞虱Laodelphax striatellus的rDNA ITS1和ITS2的序列, 以探讨这3种稻飞虱的分子鉴定方法。3种飞虱的ITS1和ITS2侧翼区（18S, 5.8S和28S）序列相对稳定, 但ITS1和ITS2序列在3种飞虱中变异较大。 ITS1在所分析的438个位点中可变位点达294个, ITS2在分析的403个位点中可变位点为177个。根据3种飞虱rDNA的ITS1和ITS2序列设计了特异性引物, 应用特异性引物对样品进行了PCR扩增, 分析发现3种飞虱ITS1区的特异性引物扩增效果不理想, 而ITS2区的特异性引物可以稳定地扩增出明显的目的DNA条带. 因此, 采用ITS2区的特异性引物可以对3种飞虱进行快速的分子鉴定。     

水稻籼粳亚种间rDNA的ITS(internal transcribed spacer,ITS)序列分析

为探讨水稻亚种间的遗传背景及亲缘关系,运用PCR技术扩增并测序了水稻籼亚种的南京11号,9311、广陆矮4号三个品种和粳亚种的秋光、日本晴、爪哇稻三个品种的完整ITS区（包括5.8S区）。供试材料的5.8S rDNA的长度和G/C含量完全一致。ITSI的长度为193-195bp、G/C含量为72.31%-74.38;ITS2的长度为224-232bp,G/C含量为74.46%-76.86%。序列的相似性为94.4%-99.3%。CLUSTAL.W软件排序及分析表明;1）存在4个信息位点把6个品种分为籼粳两大类群;2）籼稻之间的ITS序列的同源性小于粳稻之间的同源性,由此可见,水稻核糖体DNA的ITS序列的变化规律与其传统的分类具有高度的一致性。     

福建青梅rDNA ITS区克隆与序列分析

利用PCR法对青梅ITS1、5.8S、ITS2序列扩增后克隆测序,用软件DNAMAN和MEGA3.1分析测序结果,研究18个福建青梅样品的核糖体ITS碱基序列差异。获得青梅18个样品rDNA中的ITS和5.8S完全序列,ITS1、5.8S和ITS2序列长度分别为223~224bp、164bp和241~246bp,5.8S较为保守。根据测序结果,以UPGMA法建立系统发生树,从分子水平说明18个样品间的变异程度,并将福建青梅差异较大的14条rDNA ITS序列登录GenBank,获得登录号:EF523482-EF523493、EF529435和EF529436。     

不同居群栽培牡丹rDNAITS区序列分析及鉴别

对我国四个地区牡丹主要栽培品种rDNA ITS区序列进行测定,研究各居群rDNA ITS序列特征及差异,建立不同地区主要牡丹栽培品种的区域性分子标记,参照GeneBank信息(登录号:U27692)利用Clustal X、MEGA 3.1分子进化遗传分析软件对测序结果排序.牡丹ITS全序列长度为652 bp,相对于GenBank中牡丹rDNA ITS全序列在448位少一个C碱基,其中ITS1为267 bp,5.8 S为163 bp,ITS2为222 bp,GC含量为55.7%～56.9%,共有30个变异位点,主要发生在ITS1、ITS2区,5.8S区也存在多个位点的变异.牡丹rDNA ITS区序列特征(SNPs)可用于不同地区主要牡丹栽培品种的鉴别.     

基于香菇菌株rDNA-ITS序列的系统发育分析

根据真菌核糖体通用引物ITS1和ITS4扩增出13个福建袋栽香菇主要菌株的5.8S rDNA、ITS序列,对该序列进行测序后,得到完整的5.8S rDNA、ITS序列,将该序列提交NCBI并获得登录号,对该序列进行比对分析并构建了系统发育树,从分子水平对香菇菌株进行了区分鉴定,结果显示13个菌株可以明显的分成2丛,而其他菌株又可以从一丛中延伸出几个亚丛。     

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。     

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.     

PCR-based Specific Detection of Ustilaginoidea virens and Ephelis japonica

A PCR‐based technique for detection of clavicipitaceous pathogens in rice and related grasses was developed. The target pathogens were Ustilaginoidea virens, which causes rice false smut, and Ephelis japonica, which causes rice udbatta disease and black choke in grasses. To design specific primers, a comparison was made on genetic diversity on the rDNA internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene of U. virens, Ephelis japonica, as well as some other clavicipitaceous fungi. Each fungus was successfully detected by using a specific primer set with high sensitivity. Species‐specific primers designed here were capable of detecting these pathogens in plant tissues. The PCR detection was consistent with conventional histological observation. This nested PCR assay was sensitive and reliable for the detection of U. virens and E. japonica, and thus can be a used to study disease cycles and early prediction of false smut and udbatta‐disease incidence in fields.     

Species-specific PCR primers for Pythium developed from ribosomal ITS1 region

AIMS: To develop a specific method for distinguishing and detecting Pythium species. METHODS AND RESULTS: Twenty PCR primers were designed from the sequences of the rDNA internal transcribed spacer 1 (ITS1) region from 34 Pythium species. The specificity of these forward primers paired with ITS2 or ITS4 and reverse universal primers was tested. Five species-specific primers were obtained, other primers showed different specificity to Pythium species. The specific amplifications enabled nine Pythium species to be differentiated. Specific detection of Pythium aphanidermatum from infested plants and P. dimorphum from soil was demonstrated. CONCLUSIONS: A method for identifying nine Pythium species using specific PCR amplification was achieved. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of its rapidness and ease, the results of PCR amplified with different primers can be a powerful method for identifying Pythium species and detecting or monitoring the target fungus directly from plant material, soil and water samples.     

Discrimination of <Emphasis Type="Italic">Tricholoma</Emphasis> species by species-specific ITS primers

The ITS region of ectomycorrhizal fungi was analyzed, and species-specific PCR primers were designed for 8 ectomycorrhizal Tricholoma species. Although a high degree of intraspecific homology was observed, interspecific variation was sufficient to design species-specific primers based on sequence of the ITS region. PCR amplification with the specific primers generated fragments of the expected sizes from DNA extracted from the strains of each species but gave no amplified products from the strains of the other 16 species in eight genera. These results suggest that sequence of the ITS region is appropriate to be used for species-level identification of ectomycorrhizal fungi.     

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.     

Molecular tools for isolate and community studies of Pyrenomycete fungi

The Pyrenomycetes, defined physiologically by the formation of a flask-shaped fruiting body present in the sexual form, are a monophyletic group of fungi that consist of a wide diversity of populations including human and plant pathogens. Based on sequence analysis of 18S ribosomal DNA (rDNA), rDNA regions conserved among the Pyrenomycetes but divergent among other organisms were identified and used to develop selective PCR primers and a highly specific primer set. The primers presented here were used to amplify large portions of the 18S rDNA as well as the entire internal transcribed spacer (ITS) region (ITS 1, 5.8S rDNA, and ITS 2). In addition to database searches, the specificity of the primers was verified by PCR amplification of DNA extracted from pure culture isolates and by sequence analysis of fungal rDNA PCR-amplified from environmental samples. In addition, denaturing gradient gel electrophoresis (DGGE) analyses were performed on closely related Colletotrichum isolates serving as a model pathogenic genus of the Pyrenomycetes. Although both ITS and 18S rDNA DGGE analyses of Colletotrichum were consistent with a phylogeny established from sequence analysis of the ITS region, DGGE analysis of the ITS region was found to be more sensitive than DGGE analysis of the 18S rDNA. This study introduces molecular tools for the study of Pyrenomycete fungi by the development of two specific primers, demonstration of the enhanced sensitivity of ITS-DGGE for typing of closely related isolates and application of these tools to environmental samples.     

ITS primers with enhanced specificity for basidiomycetes - application to the identification of mycorrhizae and rusts

We have designed two taxon-selective primers for the internal transcribed spacer (ITS) region in the nuclear ribosomal repeat unit. These primers, ITS1-F and ITS4-B, were intended to be specific to fungi and basidiomycetes, respectively. We have tested the specificity of these primers against 13 species of ascomycetes, 14 of basidiomycetes, and 15 of plants. Our results showed that ITS4-B, when paired with either a 'universal' primer ITS1 or the fungal-specific primer ITS1-F, efficiently amplified DNA from all basidiomycetes and discriminated against ascomycete DNAs. The results with plants were not as clearcut. The ITS1-F/ITS4-B primer pair produced a small amount of PCR product for certain plant species, but the quantity was in most cases less than that produced by the 'universal' ITS primers. However, under conditions where both plant and fungal DNAs were present, the fungal DNA was amplified to the apparent exclusion of plant DNA. ITS1-F/ITS4-B preferential amplification was shown to be particularly useful for detection and analysis of the basidiomycete component in ectomycorrhizae and in rust-infected tissues. These primers can be used to study the structure of ectomycorrhizal communities or the distribution of rusts on alternate hosts.     

牛肝菌属卷边组Boletus sect. Appendiculati物种的分子识别

依据牛肝菌属卷边组Boletus sect. Appendiculati内7个物种的核糖体基因rDNA内转录间隔区（internal transcribed spacer,ITS）序列比对,设计5对ITS特异引物,分别用于卷边牛肝菌Boletus appendiculatus与亚卷边牛肝菌B. subappendiculatus、卷边牛肝菌与拟桃红牛肝菌B. pseudoregius、华靛牛肝菌B. roseoflavus与卷边牛肝菌B.?appendiculatus、华靛牛肝菌与华美牛肝菌B. speciosus、拟桃红牛肝菌与华美牛肝菌的相互识别。ITS区段的PCR扩增结果表明,5对ITS特异引物皆成功扩增出可用于辨别这些近缘物种的目的条带。但未能设计出ITS特异引物,以识别华靛牛肝菌与桃红牛肝菌B. regius两个近缘物种。     

Identification of yeast species from orange fruit and juice by RFLP and sequence analysis of the 5.8S rRNA gene and the two internal transcribed spacers

Yeast isolates from orange fruit and juice in a spontaneous fermentation were identified and classified by two molecular techniques. The first was analysis of the restriction pattern generated from the polymerase chain reaction (PCR)-amplified 5.8S rRNA gene and the two internal transcribed spacers (ITS) using specific primers. The second technique was sequence analysis of the ITS regions using the same two primers. Nine different restriction profiles were obtained from the size of the PCR products and the restriction analyses with three endonucleases (CfoI, HaeIII and HinfI). These groups were identified as Candida tropicalis, Clavispora lusitaniae, Hanseniaspora uvarum, Pichia anomala, Pichia fermentans, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Saccharomyces unisporus, and Trichosporon asahii. Checking against identification according to morphological, physiological and biochemical traits corroborated this molecular identification. A total concordance was found in the identification with PCR-restriction fragment length polymorphism of the ITS region after analysing certified yeast strains from two different culture collections. Consequently, a rapid and reliable identification of the yeast populations was achieved by using molecular techniques.     

A Multiplex-PCR approach to identification of the Brazilian intermediate hosts of Schistosoma mansoni

Due to difficulties concerning morphological identification of planorbid snails of the genus Biomphalaria, and given a high variation of characters and in the organs with muscular tissue, we designed specific polymerase chain reaction (PCR) primers for Brazilian snail hosts of Schistosoma mansoni from available sequences of internal transcribed spacer 2 (ITS2) of the ribosomal RNA gene. From the previous sequencing of the ITS2 region, one primer was designed to anchor in the 5.8S conserved region and three other species-specific primers in the 28S region, flanking the ITS2 region. These four primers were simultaneously used in the same reaction (Multiplex-PCR), under high stringency conditions. Amplification of the ITS2 region of Biomphalaria snails produced distinct profiles (between 280 and 350 bp) for B. glabrata, B. tenagophila and B. straminea. The present study demonstrates that Multiplex-PCR of ITS2-DNAr showed to be a promising auxiliary tool for the morphological identification of Biomphalaria snails, the intermediate hosts of S. mansoni.     

rDNA ITS sequence of Rhizopus oryzae: its application to classification and identification of lactic acid producers

Rhizopus oryzae is an important organism for its production of organic acids such as lactic acid, fumaric acid, etc. To date, there were no easy methods to classify strains according to their acid production. The sequences of the ribosomal RNA-encoding DNA (rDNA) internal transcribed spacer (ITS) region of 64 strains of R. oryzae were analyzed and found to conserve mutations correspond to acid production. We have devised a way to use these mutations for a novel method to identify lactic-acid-producing Rhizopus oryzae, by designing specific polymerase chain reaction (PCR) primers on them. Touch down PCR using these primers amplified the ITS DNA of lactic acid producers specifically. By this method, we could isolate lactic acid producing strains from Indonesian fermented foods.