Characterization of Fusarium Oxysporum Isolates Obtained from Wax Gourd and Chieh-qua in China by Pathogenicity, RAMs and Sequence Analysis of the rDNA Internal Transcribed Spacers (ITS1 and ITS2)

Wax gourd (Benincasa hispida Thumb. Cogn) is called white gourd, winter melon, Chinese preserving melon, Chinese squash, and don kwa. It has been cultivated in China for over 2 300 years. It probably

Phylogeny of Ptychostomum (Bryaceae,Musci) inferred from sequences of nuclear ribosomal DNA internal transcribed spacer (ITS) and chloroplast rps4

The phylogeny of Ptychostomum was first spacer （ITS） region of the nuclear ribosomal （nr） DNA DNA rps4 sequences. Maximum parsimony, maximum undertaken based on analysis of the internal transcribed and by combining data from nrDNA ITS and chloroplast likelihood, and Bayesian analyses all support the conclusion that the reinstated genus Ptychostomum is not monophyletic. Ptychostomum funkii （Schwagr.） J. R. Spence （≡ Bryum funkii Schwaigr.） is placed within a clade containing the type species of Bryum, B. argenteum Hedw. The remaining members of Ptychostomum investigated in the present study constitute another well-supported clade. The results are congruent with previous molecular analyses. On the basis of phylogenetic evidence, we agree with transferring B. amblyodon Mull. Hal. （≡ B. inclinatum （Brid.） Turton≡ Bryum archangelicum Bruch ＆ Schimp.）, Bryum lonchocaulon Mull. Hal., Bryum pallescens Schleich. ex Schwaigr., and Bryum pallens Sw. to Ptychostomum.

Application of the first internal transcribed spacer(ITS-1)of ribosomal DNA as a molecular marker to population analysis in farrer's scallop Chlamys farreri

Sequence variation of the first internal transcribed spacer of ribosomal DNA （ ITS - 1 ） was examined and its application to the study of genetic variation was explored in four populations of farter＇ s scallop Chlamys farreri. ITS - 1 fragments, with a length of about 300 bp,of 78 individuals collected from Dalian, Qingdao, Yantai in China and Korea respectively were amplified via PCR, cloned and sequenced. Intra-genomic variation was examined by sequencing several clones of single individuals. Alignment and polymorphism analysis detected 44 haplotypes and 50 polymorphic sites which consist of 30 substitutions and 20 indels, indicating a high level of polymorphisms. Sequence analysis also showed a very low level of intra-individual variation. All these features validated the feasibility of application of ITS - 1 fragment to population analysis. Polymorphism analysis showed that the Korea sample has the richest genetic variation, followed by Yantai and Qingdao samples. AMOVA （analysis of molecular variance） showed that the majority （96.26%） of genetic variation was distributed within populations and 3.74% resulted from among populations, but with P 〈 0.05 （ = 0.042）, indicating that the populations in this study have significant divergence. This output was basically concordant with the result arising from RAPD data and different from that from mitochondrial 16S rDNA sequence data. Discussion on this inconsistency was made accordingly.

The homology analysis of internal transcribed spacer sequence of ribosomal DNA in common dermatophytes

In order to analyze the sequences of the internal transcribed spacer （ITS） including the 5.8 S ribosomal DNA （rDNA） of common dermatophytes, so as to obtain a rapid and accurate method to identify the species of dermatophytes and to establish the phylogenetic tree of these species to understand their relationship, 16 strains of dermatophytes were collected and preliminarily identified by morphological characteristics. General primers for fungi ITS1 and ITS4 were used to amplify the ITS rDNA of each strains with PCR. The PCR products after purification were sequenced directly and were analyzed through internet. In the results, 11 strains were identified by means of morphological features, among which 5 strains were Trichophyton, 5 strains were Microsporum and 1 was Epidermophytoa, which was consistent with the results by molecular biology. In the 5 unidentifiable strains, 1 strain was proved to be Chrysosporium by molecular biology. These strains studied could be divided into 3 different classes as indicated in the analysis of the phylogenetic tree of the sequences in ITS, which were quite different from those of morphological classification. It is evident from the above observations that the molecular method of analysis on the ITS sequences is a rapid, highly sensitive and accurate approach for the detection of dematophyte species, however, it still exhibits some limitations needing the supplementation with morphological identification.

A preliminary analysis of phylogenetic relationships of Arundinaria and related genera based on nucleotide sequences of nrDNA （ITS region） and cpDNA （trnL-F intergenic spacer）

Phylogenetic relationships of Arundinaria and related genera (Pleioblastus, Pseudosasa, Oligostachyum, Bashania, Clavinodum, etc.) were assessed by analyzing the sequences of the nrDNA internal transcribed spacer (ITS) and the cpDNA trnL-F intergenic spacer (IGS). Comparison with trnL-F IGS sequence, the ITS region provided the higher number of parsimony informative characters, and the interspecific variation of the ITS sequence was higher than that of the trnL-F IGS sequence.The tree obtained by combining both sets of data showed that the species sampled in Arundinaria and the related genera were monophyletic and divided into two clades. The relationships and positioning of all the taxa surveryed (including A. oleosa, A. hsienchuensis, A. chino, A. amara, A. yixingensis, A. amabilis, A. fortunei, A. pygmaea, A. gramineus, A. fargesii, A. faberi, A. hupehense, Pseudosasa japonica cv. Tsutsumiana, P. japonica and Brachystachyum densiflorum) were also discussed. The results from the sequences were broadly consistent with morphological characters, appearing all these taxa sampled belong to the genus of Arundinaria. The topologies of the trees generated from individual data and the combined data were similar.

基于ITS和ISSR标记的灵芝遗传多样性和群体结构分析

以来自不同地理区域的48个灵芝种质资源为试材,采用ITS和ISSR分子标记的方法,研究了灵芝遗传多样性,以期利用遗传信息数据较理想地显示出不同灵芝菌株的遗传多样性。结果表明:通过进行ITS序列扩增测序,将48个灵芝菌株分为赤芝(35个)、无柄灵芝(11个)、四川灵芝(1个)、古巴栓孔菌(1个)。5条ISSR引物共扩增得到53条条带,多态性条带比率为96.23%,Nei′s基因多样性为0.27,Shannon′s信息指数为0.43。ISSR标记遗传相似系数约为0.70,将48个灵芝菌株分为3组,其中第1组为11个无柄灵芝,第2组为菌株29“盆景1”,其他菌株为第3组,说明2种标记结合分析能够更加准确分析不同菌株间的亲缘关系。菌株16和17同为赤芝,且ISSR分子标记遗传相似系数达0.98,推测可能为同一品种,为生产中出现的同物异名现象。该研究选取的用于PCR扩增的ISSR引物,多态性较好、稳定性较高,能有效鉴别出无柄灵芝与其他灵芝,并揭示种质的遗传多样性和群体遗传结构,可为灵芝种质资源保护及优质种质材料选育提供参考依据,以期更好地推动灵芝产业健康发展。

Sequence differences of rDNA-ITS2 and species-diagnostic PCR assay of Anopheles sinensis and Anopheles anthropophagus from China

Anopheles sinensis and An. anihropophagus are two morphologically indistinguishable yet genetically and behaviorally distinct mosquito species that vary dramatically in their importance as vectors of malaria inChina. The sequence of the ribosomal DNA internal transcribed spacer 2 (ITS2) was determined for bothspecies to assess the species differentiation. The lengths of ITS2 was 468 hp for An. sinensis and 452 hp forAn. anthropophagus. Interspecies difference in sequence was 28. 8%. Intraspecies sequence divergence wasnegligible. A PCR method was developed for distinguishing the two species based on the species-specific variations of the ITS2 sequences. The method would amplify a diagnostic fragment in length of 425 hp for An.sinensis and 253 hp for An. anthropophagus. Field collections from 12 localities in 10 provinces of China weretested. In 440 mosquitoes identified by adult morphological characteristics as An. sinensis, 291 (66. 2 % ) wereidentified later as An. sinensis and 56 (12. 7 % ) as An. anthropophagus, the remaining 93 (22. 1 % ) were notamplified by PCR. The results showed that the morphological characteritics of adult was not reliable for fieldidentification. The PCR assay was a simple, fast and reliable method for species identification.

11株螨虫分离株的ITS－2序列分析与系统关系研究

为探讨动物体上一些常见寄生螨虫的分类地位，对11株螨虫分离株的核糖体第二内部转录间隔区（ITS-2）基因序列进行测定，并从GenBank下载21条螨虫的ITS-2序列，用UPGMA法构建分子系统树。序列分析结果显示：6株疥螨分离株ITS－2基因全长均为361bp，含有335个保守性位点，15个变异位点和9个简约信息位点；序列间的同源性为96．9％～99．7％。5株足螨分离株ITS－2基因存在长度上的变异（225～232bp），序列中包括184个保守性位点，44个变异位点，9个简约信息位点；分离自黄牛和奶牛的4株足螨分离株的ITS-2序列同源性为92．4％～97．3％，而熊猫足螨分离株同黄牛、奶牛足螨分离株的同源性较低（78．7％～82．6％）。UPGMA显示：32株螨虫分离株分成2个支系，第1个支系包括疥螨科的疥螨属Sarcoptes和背肛螨属Notoedres；第2个支系包括痒螨科的足螨属Chorioptes和痒螨属Psoroptes。从分析结果来看，笔者支持疥螨的单种说法；而足螨属中分离自熊猫的足螨和痒螨属中分离自水牛的痒螨的分类地位还有待进一步探讨。

ITS2条形码对白及属植物的初步分析研究

本研究通过ITS2条形码对云南白及本地种的分布情况初步了解,并对药用白及和云南本地种进行分析研究。选取滇东南、滇东北、滇西北、滇西的材料,进行DNA提取、PCR、测序,MEGA 5.1软件进行序列比对、NJ系统聚类树构建,并预测ITS2二级结构。结果表明:药用白及和本地种ITS2序列之间存在明显差异,在NJ树上分别聚为两类。ITS2可以区分药用白及和本地种,且可以作为初步分析药用白及真伪的潜在条形码。

嗜群血蜱和长角血蜱ITS-2、COⅠ和COⅡ基因序列变异与亲缘关系分析

本研究旨在阐明长角血蜱与嗜群血蜱间的亲缘关系。采用聚合酶链式反应(PCR)技术从长角血蜱和嗜群血蜱基因组DNA中扩增得到核糖体第二内部转录间隔区基因(ITS-2)片段、线粒体细胞色素氧化酶亚基Ⅰ基因(COⅠ)片段和线粒体细胞色素氧化酶亚基II基因(COⅡ)片段,并进行测序,进而分析其基因变异与系统发育。结果表明,长角血蜱和嗜群血蜱的ITS-2、COⅠ和COⅡ基因片段的大小存在差异,长角血蜱3种基因分别为361、479和647bp,嗜群血蜱基因分别为354、474和661bp。长角血蜱与嗜群血蜱间ITS-2、COⅠ和COⅡ基因的相似性分别为84.2%、89.0%和88.4%。以3种基因构建的NJ进化树中,长角血蜱和嗜群血蜱均聚类。因此,认为长角血蜱和嗜群血蜱间ITS-2、COI和COII基因间变异较小,它们是血蜱属中亲缘关系较近的2个有效种,支持传统形态学的分类地位。

长耳珠母贝核rRNA基因ITS-2序列分析

以相应引物聚合酶链式反应(PCR)扩增长耳珠母贝(Pinctadachemnitzi)核核糖体RNA基因第2转录间隔区(ITS 2),PCR产物大小约500bp,经克隆、测序,所得序列包含5.8S和28SrRNA基因部分序列和ITS 2全序列,4种碱基含量分别为27.11%(A)、24.95%(G)、21.26%(T)和26.68%(C)。5个克隆的DNA序列差异为0.0%—0.4%,不存在个体内变异。对其潜在应用进行了讨论。

rDNA-ITS2应用于赤眼蜂分子鉴定的研究

本研究通过对拟澳洲赤眼蜂 Trichogramma confusum、甘蓝夜蛾赤眼蜂 T. brassicae、广赤眼蜂 T.evanescens、食胚赤眼蜂 T. embryophagum,及松毛虫赤眼蜂 T. dendrolimi的 6个地理种群的 r DNA- ITS2进行克隆测序 ,又运用软件 DNAStar的 Meg Align程序对不同赤眼蜂属间、赤眼蜂属种间、同种不同地理种群之间以及同一赤眼蜂个体不同拷贝之间的 ITS2序列的遗传分歧及相似性进行了分析。结果表明 :赤眼蜂属与外群 ITS2序列的遗传相似性很低、赤眼蜂属内不同种之间 ITS2序列保守性适中、种内或不同地理种群之间以及同一个体不同 ITS2拷贝间非常保守。说明

栀子栽培品种与近缘种的ITS2序列分析

【目的】栀子属植物是重要的观赏、药用、色素和油用资源植物,有着悠久的栽培历史。由于生长环境的不同,以及长期的人工栽培和选育,使其习性、叶的形状、花的形态、果实的形状及大小等发生诸多变异,使得栀子栽培群体的变异类型丰富,并形成了一些较为稳定的品种类型。开展栀子栽培品种和近缘种核糖体DNA内部转录间隔区2(nrDNA ITS2)序列变异程度、遗传距离和亲缘关系研究,为栀子属植物品种鉴定、遗传育种及多样性保护提供科学依据。【方法】以保存于江西省林业科学院中药资源圃内的栀子18个栽培品种和3个近缘种共38个样品为试验材料,进行聚合酶链式反应(PCR)直接测序法,对样品进行ITS2片段扩增和双向测序,所得序列用Codon Code Aligner v6.0.1软件拼接后,采用Lasergene Edit Seq 11.1软件进行平均碱基组成百分比统计,用MEGA7.0软件进行分析比对,获得序列长度、GC含量以及变异位点,基于K2P模型分析遗传距离,用UPGMA法构建系统聚类树。【结果】序列变异程度分析显示,狭叶栀子和栀子的ITS2序列长度均为347 bp,GC含量为66.86%,大黄栀子序列长度为355 bp,GC含量为67.61%,栀子栽培品种序列长度除‘燃叶’、‘金福水栀’为348 bp外,其他品种序列长度和栀子一致,均为347 bp,栀子栽培品种与近缘种间的平均GC含量相差不明显,GC含量为66.28%~67.15%,其中‘白蟾’GC含量最低,‘雀舌栀子’和‘银边雀舌’GC含量最高。所有样本ITS2序列比对共产生12个变异位点,10个碱基插入,其中8个碱基插入是大黄栀子所独有的,且存在5个特异的变异位点,一部分表型特征有共性的品种之间有共同的变异位点,如植株矮小、叶片小而窄长的品种‘雀舌栀子’和‘银边雀舌’在50 bp处,果实相对较大的品种‘分关1号’、‘金福水栀’和‘金元’在84 bp处等。一些品种具有特异的变异位点,如‘白蟾’(133 bp处)、‘小白蟾’(229 bp处)等。序列遗传距离分析显示,栀子属种间遗传距离普遍小于近缘种和品种间的遗传距离,大于栀子栽培品种之间的遗传距离,所有样本平均遗传距离为0.0055,大黄栀子和‘白蟾’间遗传距离最大,为0.0206。聚类分析结果显示,栀子属乔木树种大黄栀子单独成一支,与栀子属其它种和栀子栽培品种亲缘关系最远,狭叶栀子和野生栀子所有个体总体表现出较近的亲缘关系。【结论】分析结果表明,3种栀子属植物在ITS2序列长度和GC含量上表现出较高的保守性,研究发现的部分品种共同变异位点以及部分品种特异变异位点将对栀子品种鉴定有较大的应用价值。部分聚类结果和以表型数据为基础的数量分类结果类似,但并没有像数量分类结果那样以花重瓣、大果、小果等性状形成明显的系统分枝,研究结果对栀子品种起源、资源保护和种质创新等方面有重要的指导价值。

白前及其混伪品的ITS2分子鉴定

目的:采用ITS2序列鉴定白前及其易混伪品,为准确鉴别白前和保证临床用药的安全性提供参考。方法:白前与其混伪品的ITS2序列从Gen Bank数据库中获取,然后使用MEGA 6.06软件进行种内、种间序列变异分析、构建邻接系统树(NJ树)并计算遗传距离,运用ITS2数据库相似性搜索引擎预测ITS2二级结构。结果:白前ITS2序列长度在246~270 bp范围内,种内变异少且种间存在明显差异,与所有混伪品种间遗传距离为0.026~0.309。NJ树显示白前独聚一支,另外白前有明显ITS2二级结构特征,可与其混伪品明显区分。结论:ITS2序列能够有效地区分白前与其混伪品,为白前的用药安全提供技术保障。

眼蚤属(Ophthalmopsylla)两蚤种的rDNA ITS2序列研究

目的:研究伏河眼蚤异常亚种Ophthalmopsylla volgensis extrema(Ioffet Scalon,1953)和长突眼蚤Oph-thalmopsylla kiritschenkoi(Wagner,1930)rDNA ITS2序列,为蚤类分子系统发育的研究提供基础资料。方法:PCR扩增两蚤种rDNA ITS2片段并测序,比较两者差异。结果:伏河眼蚤异常亚种rDNA ITS2序列长372 bp,长突眼蚤长380 bp;两蚤种间共有30处碱基不同,包括16处缺失/插入、4处转换、10处颠换,种内无差异。结论:rDNA ITS2序列可用作两蚤种鉴别的分子标记。

放养型和野生型柞蚕的核糖体基因转录间隔区Ⅱ(ITS-2)的克隆与序列比较

核糖体基因转录间隔区Ⅱ(ITS-2)在物种间甚至种内群体间均表现出较高的差异,被用于种内或近缘种的研究。测定了放养型和野生型柞蚕(Antheraea pernyi)的rDNA ITS-2全长序列(GenBank登录号:GU073314、GU073315),并分析了二者之间的遗传差异。放养型和野生型柞蚕的rDNA ITS-2全长分别为1 111、1 112 bp,2个序列的GC含量达到61%,相似性高达98%。序列比对后得到1 117 bp的对齐序列,共鉴定变异位点19个,其中转换位点8个,无颠换,有11处发生核苷酸的插入/缺失。基于K2P模型计算的2条rDNA ITS-2序列的遗传距离为0.007,这一数值与估算的鳞翅目昆虫rDNA ITS-2序列种内平均遗传距离(0.009)基本一致。

白色念珠菌ITS2的实时荧光定量PCR快速检测方法的建立及评价

目的建立一种快速鉴定白色念珠菌的实时荧光定量PCR检测方法。方法在白色念珠菌核糖体RNA编码基因(r DNA)中的内转录间隔区2(ITS2)上设计特异性引物和Taq Man-MGB探针,通过实时荧光定量PCR分析系统建立白色念珠菌的检测方法,对其敏感度、重复性和特异性进行方法学评价,并在模拟白色念珠菌血症的血标本中初步探讨其应用价值。结果所建方法对白色念珠菌纯菌液的准确检测下限为101CFU/ml,相应的批内、批间变异系数(CV)分别为1.57%和2.20%。对135株白色念珠菌临床分离株、其他非白念菌株、细菌临床分离株和血液标本进行检测,结果显示该方法特异性为100%。另外,对于模拟白色念珠菌血症的血标本,该方法的最低检测下限为102CFU/ml。结论所建方法具有特异性和敏感度高、重复性好的特性,可适用于临床上白色念珠菌引起的血流感染的快速鉴定。

基于DNA条形码(ITS2)的中药材防风与其混伪品的鉴定

目的通过ITS2序列分析,对防风及其易混伪品进行鉴定,确保用药安全。方法从Gen Bank数据库下载中药材防风与其混伪品的DNA条形码ITS2序列,利用MEGA 6.06软件进行比对分析,计算GC含量及变异位点、种内、种间遗传距离,并构建NJ系统发育树,应用ITS2数据库网站预测其ITS2二级结构。结果序列分析与遗传距离结果显示,防风与其混伪品存在明显差异,基于ITS2序列的NJ树及其ITS2二级结构均可以将防风与其混伪品进行区分。结论 ITS2能够准确鉴定防风及其易混伪品,可用于中药材的快速鉴别。

不对称荧光PCR扩增白念珠菌ITS-2基因单链DNA实验

目的比较在限制性引物不同稀释比例条件下,不对称荧光聚合酶链反应(PCR)扩增白念珠菌5.8S rDNA与28S rDNA间内转录间区2(ITS-2)基因的单链DNA(ssDNA)的实际效果,探讨其体系优化方法。方法以常规PCR为对照,将20μmol/L的正向引物(P1)按照一定比例预稀释后,分别与20μmol/L的荧光标记反向引物(P2')组成含不同P1水平的引物对,采用不对称荧光PCR扩增真菌ITS-2,并对PCR产物进行电泳和测序分析,比较各实验组扩增ssDNA的效果。结果不对称荧光PCR成功扩增出白念珠菌ITS-2基因的ssDNA,随着限制性引物水平的降低,ITS-2基因的双链DNA(dsDNA)条带逐渐变淡,而ssDNA条带逐渐变亮;在限制性引物稀释比例达到1∶90~1∶100时,ds DNA条带已经模糊不清,而ssDNA条带则达到最亮。提示在此扩增体系条件下,ssDNA拷贝数最多,为不对称荧光PCR扩增白念珠菌ITS-2基因ssDNA的最佳扩增条件。PCR产物测序也表明,dsDNA上方的条带为ITS-2基因的ssDNA。结论通过优化限制性引物水平,不对称荧光PCR可以高效地扩增白念珠菌ITS-2基因的ssDNA。

利用ITS2序列识别盾蚧科昆虫

[目的]盾蚧科隶属于半翅目蚧总科,大多数种类是危害很严重的经济害虫。由于其外部形态特征简单,导致识别该类群物种相对困难。利用DNA条形码为解决这个问题提供有效的方法。[方法]共采集了5个省47个样本,属于7属11个种,并从GeneBank下载了4属5个物种的序列。采用distance-based方法和tree-based方法对ITS2基因片段是否可以作为盾蚧科DNA条形码的补充做了分析。[结果]虽然ITS2没有Barcode gap存在,然而所有的分支都表现出了明显的单系群,表现出较高的物种识别能力。同时,Best Match(BM)和Best closed match(BCM)也表现出了超过90%的识别能力。[结论]这些结果证实ITS2在盾蚧科物种的识别有效性方面具有较高的应用价值。