云南美味牛肝菌ITS 区域结构特点

利用ITS 的通用引物(ITS5-ITS4) 对云南的美味牛肝菌( Boletus edulis) 子实体的DNA 进行PCR 扩增, 扩增产物回收后直接测序。序列的聚类分析表明, 在ITS1-5 . 8S rDNA-ITS2 区域, 云南的美味牛肝菌与欧洲的夏牛肝菌( B. aestivalis) 和铜色牛肝菌( B . aereus) 同源性较高, 但在ITS2 区域夏牛肝菌和铜色牛肝菌分别有一段美味牛肝菌没有的大小分别为73 bp 和26 bp 的特征序列。     

云南美味牛肝菌ITS区域结构特点

利用ITS的通用引物（ITS5-ITS4）对云南的美味牛肝菌（Boletus edulis）子实体的DNA进行PCR扩增,扩增产物回收后直接测序。序列的聚类分析表明,在ITS1—5．8S rDNA-ITS2区域,云南的美味牛肝菌与欧洲的夏牛肝菌（B．aestivalis）和铜色牛肝菌（B．aereus）同源性较高,但在ITS2区域夏牛肝菌和铜色牛肝菌分别有一段美味牛肝菌没有的大小分别为73bp和26bp的特征序列。     

三叶草斑潜蝇和美洲斑潜蝇的分子鉴定

以三叶草斑潜蝇Liriomyzatrifolii(Burgess)与美洲斑潜蝇LiriomyzasativaeBlanchand为材料,通过特异引物18SP1和5.8SP2,分别扩增出它们的rDNA非编码区ITS1的片段,根据序列分析和比较结果,重新设计出2条新的特异性引物Fly3和Flyus,并筛选到2个特异性内切酶,通过PCR和PCR产物的酶切分析,即可将这2种近缘昆虫区分开来。     

基于ITS条形码序列的刺桐姬小蜂分子鉴定

【目的】刺桐姬小蜂Quadrastichus erythrinae Kim体型小,传统的形态学鉴定方法难以快速准确识别。【方法】本研究测定了刺桐姬小蜂的rDNA ITS1和ITS2序列,根据18S rDNA部分序列,利用MEGA的最大相似法(Maximum Likehood)构建系统发育树。根据刺桐姬小蜂ITS1和ITS2序列设计了特异引物,应用特异引物对单只刺桐姬小蜂进行PCR扩增,可稳定地扩增出明显的目的DNA条带。【结果】研究表明,基于ITS基因的DNA条形码技术可以用于刺桐姬小蜂的快速准确鉴定。【结论】因此,采用ITS1和ITS2区的特异性引物可对刺桐姬小蜂进行快速分子鉴定。     

黑麦1R染色体特异性PCR引物的分子证据

根据黑麦（ＳｅｃａｌｅｃｅｒａｌｅＬ．）与小麦（ＴｒｉｔｉｃｕｍａｅｓｔｗｕｍＬ．）ｒＲＮＡ基因间隔区序列差异,按Ｋｏｅｂｎｅｒ设计的引物序列,合成了黑麦特异引物ＮＯＲ－Ｒ１。运用该引物对不同植物材料进行ＰＣＲ扩增,观察表明,含有黑麦１Ｒ染色体的植物均扩增出黑麦的特异带,但含有其他黑麦染色体的小麦种质,普通小麦品种及其近缘物种长穗偃麦草（Ａｇｒｏｐｙｒｏｎｅｌｏｎｇａｔｕｍ（Ｈｏｓｔ）Ｂｅａｕｖ）     

小麦族中含St染色体组物种的特异分子标记的建立

以拟鹅观草(Pseudoroegneria spicata)、偏凸山羊草(Aegilops ventricosa)、二倍体簇毛麦(Dasypyrum villosum)、荆州黑麦(Secale cereale cv. Jingzhou rye)、普通小麦中国春(Chinese Spring)等15个物种为材料, 用200条10碱基随机引物进行RAPD分析, 筛选到拟鹅观草基因组中1个542 bp的特异DNA片段(GenBank登录号为DQ992032), 命名为OPH11542。根据OPH11542设计特异引物, 对小麦族物种进行PCR扩增, 发现拟鹅观草可以扩增出OPH11542以及分子量分别为742 bp (GenBank登录号为DQ992033, 记为OPH11742)和743 bp (GenBank登录号为EF014218, 记为OPH11743)的DNA片段, 而其他材料均未扩增出这3个片段。经序列比对结合多个软件的分析结果认为该3个片段为同一类新重复序列。利用特异引物对15份含St染色体的物种进行扩增, 发现含StY染色体组的物种均能扩增出OPH11742或OPH11743, 而含StH染色体组的物种均能扩增出OPH11542。这表明St染色体组在与其它染色体组组合形成多倍体的过程中往往会出现不同程度的重组或修饰。OPH11542、OPH11742和OPH11743可以作为检测St染色体的分子标记。     

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.     

微卫星跨物种交叉PCR扩增的一个新问题:扩增非同源产物

微卫星已被广泛应用于群体遗传学、生态学和进化生物学研究。然而,一些物种微卫星尚未克隆。为了节省时间和经费,研究人员往往使用一个物种已发表的微卫星引物扩增其近缘物种的微卫星。该研究对属于3个不同科(Clariidae、Heteropneustidae 和Pimelodidae)的7个鲶鱼物种的微卫星跨物种PCR扩增产物进行了序列分析,研究发现扩增非同源（non-orthologous）产物是微卫星跨物种PCR扩增的一个新问题。该研究共采用4对胡子鲶微卫星座位引物对7个鲶鱼物种进行了跨物种PCR扩增。对获得的204个PCR产物的序列分析结果表明,两对微卫星座位引物扩增了所有7个物种的同源特异产物。而其他两个座位的引物扩增了特异但非同源的多态产物,对近缘物种的扩增也获得类似结果。另外,除胡子鲶等位基因大小异源同型(size homoplasy)的特征不明显外,其他物种在3个微卫星座位都具有这一非常明显的特征。这些数据表明,微卫星跨物种间交叉扩增能产生非同源产物;等位基因大小异源同型与微卫星座位本身有关,而与物种间的亲缘关系无明显的相关性。微卫星跨物种扩增产生的非同源产物和等位基因大小异源同型将使系统发育、群体遗传学和进化研究明显复杂化。因此,在应用微卫星跨物种交叉扩增数据以前,最好对跨物种交叉扩增产物进行测序验证。     

5种杂交粳稻亲本SCAR标记的建立及其在真伪纯度鉴定中的应用

选用36个随机引物对"寒丰A"、"寒丰B"、"8204A"、"8204B"、"R161"等5份杂交粳稻亲本材料进行RAPD扩增,对其中特异RAPD标记片段进行克隆和测序.根据获得的特异DNA序列设计序列特征扩增区(SCAR)特异的引物,将18个RAPD标记转化成6个稳定的SCAR标记.用这些SCAR标记对亲本和杂种F1代单株进行检测,实验室检测种子纯度的结果与海南田间种植的结果基本一致.此外,应用水稻细胞质雄性不育特异的1对PCR引物,分辨出2对不育系/保持系亲本:"寒丰A"与"寒丰B"、"8204A"与"8204B".     

裸子植物DNA条形码ITS2高通用性引物的筛选

DNA条形码技术是利用标准DNA片段进行准确快速鉴定物种的一种方法,理想的DNA条形码片段应具有高通用性。虽然核糖体DNA内部转录间隔区II（ITS2）被建议作为种子植物有效的DNA条形码,但目前裸子植物还没有通用性高的引物可用。为获得高通用性的ITS2引物,本研究基于裸子植物55个属的5．8S基因的保守序列区设计了3个正向引物,与已有的ITS反向引物组合,组成了7对ITS2引物进行通用性的评价。选取了裸子植物8目、12科和40属的56个种用于本文的研究。引物组合5．8SR／ITS4、5．8SRa／ITS4和5．8SF2／S3R因为在科水平评价中通用性低或者产生的PCR产物有双带,因而排除在全部物种水平上进一步评价。其余4对引物（GYM-5．8SF1／ITS4、GYM-5．8SFl／S3R、GYM-5．8SF2／ITS4和S2F／S3R）在56个物种的PCR检测中,均有100％的扩增率。基于PCR产物的亮度、序列质量和正反向引物覆盖率的综合评价,建议引物GYM_5．8SF2／ITS4作为裸子植物条形码片段ITS2最好的通用引物。     

Species and Strain Identification of the Predatory Mite Euseius Finlandicus by RAPD-PCR and ITS Sequences

The variation within and between Finnish Euseius finlandicus populations was investigated by RAPD-PCR and ITS sequence analyses. Resin DNA extraction was found to be a suitable method for samples of single mites used in PCR. The banding patterns from 24 RAPD primers and 10 primer pairs were very similar and reproducible in all specimens of the predatory mite studied. However, the E. finlandicus K-strain could be distinguished from organophosphate-resistant predatory mites (R-strain), since almost all of them produced a 1,400 bp RAPD-PCR product, which was missing or very rare in other strains studied. Another RAPD band of ca. 680 bp was in turn much more common in other mites of E. finlandicus than in the K-strain mites. Mite specific primers were designed and used to follow the survival of the R-strain released on apple trees. The 680 bp band obtained with specific primers was specific to the species E. finlandicus mites studied, including those that had been negative with RAPD primers. The 1,400 bp specific primers could be used as a marker for following the survival of R-strain mites on apple trees. At the species level it was possible to distinguish adults and eggs of E. finlandicus from Anthoseius rhenanus and Phytonemus pallidus by RAPD-PCR. In addition, a band at 480bp was found to correspond to DNA of the predatory mite Phytoseius macropilis, when both specific primer pairs were used together. It was not possible to amplify the ITS region of E. finlandicus rDNA using several primer pairs that work in other mites and aphids. However, a basidiomycete rDNA sequence was amplified with one of these ITS primer pairs in K-strain mites. Finally, it was found that fungal rDNA-specific primers amplified an ITS region of ca. 650 bp in several strains of E. finlandicus. Internal primers, designed to amplify the central part of the 650 bp product, successfully amplified this product from all the mites.     

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.     

Molecular detection of native and invasive marine invertebrate larvae present in ballast and open water environmental samples collected in Puget Sound

Non-native marine species have been and continue to be introduced into Puget Sound via several vectors including ship's ballast water. Some non-native species become invasive and negatively impact native species or near shore habitats. We present a new methodology for the development and testing of taxon specific PCR primers designed to assess environmental samples of ocean water for the presence of native and non-native bivalves, crustaceans and algae. The intergenic spacer regions (IGS; ITS1, ITS2 and 5.8S) of the ribosomal DNA were sequenced for adult samples of each taxon studied. We used these data along with those available in Genbank to design taxon and group specific primers and tested their stringency against artificial populations of plasmid constructs containing the entire IGS region for each of the 25 taxa in our study, respectively. Taxon and group specific primer sets were then used to detect the presence or absence of native and non-native planktonic life-history stages (propagules) from environmental samples of ballast water and plankton tow net samples collected in Puget Sound. This methodology provides an inexpensive and efficient way to test the discriminatory ability of taxon specific oligonucleotides (PCR primers) before creating molecular probes or beacons for use in molecular ecological applications such as probe hybridizations or microarray analyses. This work addresses the current need to develop molecular tools capable of diagnosing the presence of planktonic life-history stages from non-native marine species (potential invaders) in ballast water and other environmental samples.     

Differentiation of Two Pathogens of Powdery Mildew Disease in Flowering Dogwood (Cornus florida) by PCR-mediated Method Based on ITS Sequences

Two fungi, Phyllactinia guttata and Erysiphe pulchra were identified as the pathogens of powdery mildew of flowering dogwood ( Cornus florida ). The objective of this research was to identify and distinguish the two fungi by developing species-specific primers. The internal transcribed spacer (ITS) universal primers and a series of species-specific primers designed from the ITS regions were used to evaluate and validate the two fungi causing powdery mildew in dogwood. Four primer pairs showed specificity to P. guttata and three to E. pulchra . These species-specific primer pairs can be used as molecular markers to provide diagnostic tools for detection and differentiation of the two powdery mildew pathogens in flowering dogwood.     

Diversity measures in environmental sequences are highly dependent on alignment quality--data from ITS and new LSU primers targeting basidiomycetes

The ribosomal DNA comprised of the ITS1-5.8S-ITS2 regions is widely used as a fungal marker in molecular ecology and systematics but cannot be aligned with confidence across genetically distant taxa. In order to study the diversity of Agaricomycotina in forest soils, we designed primers targeting the more alignable 28S (LSU) gene, which should be more useful for phylogenetic analyses of the detected taxa. This paper compares the performance of the established ITS1F/4B primer pair, which targets basidiomycetes, to that of two new pairs. Key factors in the comparison were the diversity covered, off-target amplification, rarefaction at different Operational Taxonomic Unit (OTU) cutoff levels, sensitivity of the method used to process the alignment to missing data and insecure positional homology, and the congruence of monophyletic clades with OTU assignments and BLAST-derived OTU names. The ITS primer pair yielded no off-target amplification but also exhibited the least fidelity to the expected phylogenetic groups. The LSU primers give complementary pictures of diversity, but were more sensitive to modifications of the alignment such as the removal of difficult-to align stretches. The LSU primers also yielded greater numbers of singletons but also had a greater tendency to produce OTUs containing sequences from a wider variety of species as judged by BLAST similarity. We introduced some new parameters to describe alignment heterogeneity based on Shannon entropy and the extent and contents of the OTUs in a phylogenetic tree space. Our results suggest that ITS should not be used when calculating phylogenetic trees from genetically distant sequences obtained from environmental DNA extractions and that it is inadvisable to define OTUs on the basis of very heterogeneous alignments.     

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.     

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。     

Differentiation of sourdough yeast species by a novel species-specific PCR assay

The objective of this study was to develop species-specific primer pairs based on the internal transcribed spacer region (ITS) of the ribosome DNA for species identification of the frequently found sourdough yeast species. Species-specific primers were designed by the alignment of sourdough yeast ITS sequences, which were then employed for PCR using the template DNA of sourdough yeast strains. PCR primers were shown to be specific for the following species: Issatchenkia orientalis (Candida krusei), C. humilis, Kazachstania exigua (C. holmii), Pichia anomala and P. subpelliculosa. Therefore, we conclude that our novel species-specific primers could be used to rapidly and accurately identify the most frequent sourdough yeast species using a PCR-based assay.     

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.     

基于诊断引物的赤眼蜂鉴定和检测技术

通过对松毛虫赤眼蜂Tichogramma dendrolimi Matsumura和玉米螟赤眼蜂T.ostriniae Pang et Chen（膜翅目：赤眼蜂科）核内可转录第二间隔区（简称：ITS2）的克隆、测序,并获取和分析了GenBank中已登录的同源序列,然后设计了松毛虫赤蜂的特异引物以用于该蜂的分子鉴定和检测,经过反复筛选发现：采用鉴定引物通过PCR扩增不仅可以区分鑫头样品,单头样品（雌蜂或雄峰）,而且可鉴定幼期虫和卵,这用传统方法是无法办到的。该鉴定技术比基于形态学鉴定检测技术用来鉴定了从中国大陆不同地域和寄主上采集到的12个样品,结果表明：该方法可用于赤眼蜂田间分子监测和实验室拟寄生行为研究。