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.

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

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.

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.

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.

Molecular phylogenetic analyses based on the complete plastid genomes and nuclear sequences reveal Daphne(Thymelaeaceae)to be non-monophyletic as current circumscription

The diverse members of the genus Daphne are prized for their fragrant flowers.Despite being promising ornamental plants in many countries,genetic information of Daphne is scarce.In this study,the plastomes of four species and one variety of Daphne were sequenced and analyzed.The plastomes were typical and contained a pair of inverted repeat(IR)regions that separated the large single-copy(LSC)region from the small single-copy(SSC)region.With a length ranging from 132,869 bp(D.genkwa)to 174,773 bp(D.championii),106 to 141 genes were predicted.Comparative plastome analysis of the newly sequenced plastomes with four publicly available Daphne plastomes identified an expansion of the IRs,sequence variations,and mutational hotspots.Phylogenetic analyses indicated that the genus Daphne in its current circumscription is polyphyletic.Daphne genkwa was nested within the genus Wikstroemia,while D.championii was well resolved as sister to Edgeworthia.These findings concurred with results from our study that used nuclear ribosomal internal transcribed spacer sequence data.The conflicts on the molecular placement of D.championii and D.genkwa and the present taxonomic classification in Daphne suggest that a new intergeneric classification system of Daphneae warrants consideration.

基于ISSR-PCR体系鉴别樟芝单核体交配型

通过原生质体单核化技术获得樟芝单核体,基于内转录间隔区(internal transcribed spacer,ITS)序列进行鉴定,采用14个引物对简单重复序列区间(inter-simple sequence repeats,ISSR)进行多态性扩增,筛选条带清晰、重复性好的引物用于樟芝单核体的交配型鉴定。结果表明,通过原生质体单核化技术获得31个樟芝单核体,经ITS序列分析确定获得的单核体为樟芝。以单核体S2、S10、S14、S27的DNA为模板,对14条ISSR引物进行初步筛选,得到4个条带清晰、重复性好的引物P7、P9、P21、P25用于交配型鉴定。单核体S9和S25为同一交配型,两者与S2为不同交配型,通过镜检进一步验证S9、S25可以与S2形成具有锁状联合的双核菌株。该方法可明显缩短樟芝单核体交配型的鉴定时间。

基于SCAR标记和DNA条形码技术的苍术基原鉴别研究

目的开发出能同时鉴别北苍术和关苍术的分子标记方法,并探究不同种质资源苍术的遗传进化关系。方法对不同地区北苍术Atractylodes chinensis(Bunge)Koidz及关苍术A.japonica Koidz.ex Kitam基因组DNA的差异片段进行测序,结合SRAP、ISSR、DAMD分子标记方法,优化PCR反应体系,筛选并转换成特异性标记,同时,采用条形码方法分析种间序列差异。结果通过SRAP、ISSR、DAMD三种分子标记方法的PCR扩增,共筛选出198对能稳定扩增且重现性好的引物,转换出7对能稳定、快速鉴别北苍术和关苍术的SCAR引物。条形码方法检测出北苍术ITS2序列长度为454 bp,关苍术ITS2序列长度为453 bp,与其他苍术属植物之间遗传距离较远。NJ树结果显示,北苍术、关苍术及其他苍术属植物均各自聚为一支,表现出良好的单系性。依据ITS2二级结构,4种苍术属植物在螺旋区的茎环数目、大小、位置均有明显差异,可以直观地进行区分。结论所开发的特异性SCAR标记为苍术属植物优良品种的筛选提供了新方法,DNA条形码能稳定、准确鉴别北苍术。

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。

葡萄座腔菌Botryosphaeria dothidea胞内miRNA及siRNA的鉴定与分析

通过ITS-rDNA检测和形态学典型特征观察确定葡萄座腔菌Botryosphaeria dothidea的遗传背景,并对其胞内小RNA测序.新预测10个miRNAs和298个siRNAs,并分析了预测的miRNAs和siRNAs的序列信息、结构、长度分布、碱基偏好性、表达情况,为在分子水平了解葡萄座腔菌发育和致病机制提供了理论基础.

斑玉蕈种质资源评价及遗传多样性分析

利用体细胞不亲和性、核糖体基因转录间隔区域(internal transcribed spacer,ITS)序列和简单序列重复区间(inter-simple sequence repeat,ISSR)对67株斑玉蕈种质资源进行鉴定评价,并分析遗传多样性。结果表明,67株斑玉蕈菌株被分为15组,组内无拮抗反应,组间拮抗反应明显。褐色品系和白色品系间拮抗反应明显。采用邻接法(neighbor-joinging,NJ)构建系统进化树,将其分为3个类群,种内遗传距离为0~0.079 4,平均遗传距离为0.005 5±0.001 6。11条ISSR引物,扩增出51个多态性片段,菌株间Nei’s遗传距离为0.034 7~0.627 2。经非加权算数平均配对法(unweighted pair-group method with arithmetic mean,UPGMA)聚类分析,将其分为3个大类群,类群I中分为6个小类群。

基于ITS和ISSR的灵芝种质资源遗传多样性分析

为明确吉林栽培的灵芝(Ganoderma lucidum)种质资源的遗传多样性,以吉林省18个主栽灵芝菌株为试验材料,通过应用ITS(Internal transcribed spacer)序列分析及拮抗试验,并与ISSR(Inter-simple sequence repeat)分子标记相结合对其进行鉴定和遗传多样性分析。基于ITS序列构建的系统发育树表明,18种灵芝菌株均与赤芝(Ganoderma lingzhi)聚为一类,并细分为4个类群;79.7%的灵芝菌株之间存在拮抗现象,基于ITS序列分析及拮抗试验结果,菌株被分为7个类群;ISSR遗传多样性分析结果表明,18种灵芝菌株的遗传相似系数在0.3824~0.9744,平均为0.6529,当相似系数约0.679时,其亦可被分为7个类群。综上,基于ISSR分子标记的分类结果与基于ITS序列分析+拮抗试验结果的分类一致,18种灵芝菌株表现出明显的遗传多样性。

中国香菇自然种质的rDNA遗传多样性分析

测定了全国范围内的59个野生香菇菌株rDNA的ITS区域的序列。所得序列的总长度在723-727个碱基之间,G+C含量也基本稳定在57.5%左右。用Phylip3.6软件包对59个ITS序列进行DNA数据分析。结果表明59个菌株被划分到3个不同的谱系中,其中谱系Ⅰ包括11个菌株,分布较为均衡,涉及东北、西北、西南和东部沿海等地区的8个省份;谱系Ⅱ包括14菌株,以西北、西南的4个省份为主;谱系Ⅲ包括34个菌株,覆盖范围最广,除东北和西北高原地区外,其它均有涉及。地缘关系分析表明以陕、甘为主的西北高原地区的菌株涉及Ⅰ、Ⅱ两个谱系;以闽、浙、赣和台湾为主的东部沿海地区的菌株涉及Ⅰ、Ⅲ两个谱系;而以云、贵、川为主的西南地区的菌株则覆盖到了所有的3个谱系。这三个地区的香菇遗传多样性最丰富,西南地区尤为突出,是香菇种质资源保护与利用的重点区域。

苏皖产大戟属药用植物rDNA的ITS序列分析

目的研究苏皖产大戟属内6种药用植物的ITS长度的变异,为探讨大戟属植物的系统演化关系和大戟属植物鉴定提供DNA分子证据.方法利用PCR技术对大戟属植物的rDNA ITS区碱基序列进行测定.结果这6种大戟属植物的ITS1的长度范围为255～262 bp,ITS2的长度范围为214～236 bp.运用Mega2软件进行的系统分析得到大戟属内6种植物的系统进化树.这一分析结果与来自形态学的研究结果相吻合.结论此法可用于大戟属植物种间及真伪品鉴别.

弓形虫ITS及5.8S序列的PCR扩增、克隆及分析

通过对国内来源于不同宿主的ZS人株、SH人株、CN猪株、QH绵羊株4个弓形虫虫株,以及国际标准强毒株RH株的核糖体DNA内转录间隔区(ITS)及5 8SDNA序列进行PCR扩增、克隆、测序和序列分析,旨在对国内不同宿主间弓形虫虫株的遗传变异情况进行分析和验证,为分子遗传学和分子诊断学研究提供资料。结果显示:QH绵羊株、ZS人株、SH人株、CN猪株的ITS及5 8S序列完全一致,且与GenBank上注册RH株的ITS及5 8S序列也一致;仅实验室传代保存的RH株的ITS2序列与其它4株的ITS2有2个碱基的差异。结果表明ITS可作为分子标记用于弓形虫与其它原虫的种间鉴定,但不适合用于弓形虫种内遗传变异的研究。

根肿病菌核糖体基因ITS区段的克隆测序及其在检测中的应用

应用真菌核糖体基因ITS区段通用引物ITS1和ITS4,对十字花科蔬菜根肿病菌(Plasmodiophorabrassicae)rDNA进行PCR扩增,并将扩增到的目的片段克隆到pGEM-T载体上。对重组克隆进行测序和碱基编码结构特点分析,结果表明:十字花科蔬菜根肿病菌ITS1区长141bp,5 8S区长160bp,ITS2区长187bp,rDNA总长为488bp.根据此序列设计一对根肿病菌特异性寡聚核苷酸引物(前引物:5'AGGTGAACCTGCGGAAGGAT3'和后引物:5'TTCAGCGGGTAATCCTACCT3'),应用聚合酶链式反应(polymerasechainreaction,PCR)技术,对分离自十字花科蔬菜(白菜、青菜、甘蓝、芥蓝、花椰菜等)根肿病菌全基因组DNA进行特异性扩增试验。试验结果表明,该对引物能从十字花科根肿病菌全基因组DNA中扩增到约500bp长度的分子片段,而对照菌株和健康对照则无扩增产物。该实验结果对有效追踪根肿病菌在田间的发生动态监测和预测预报提供了重要的信息和手段。

广西山药炭疽病病原菌的鉴定与ITS序列分析

本文对山药炭疽病在广西的危害、症状特点以及病原菌的鉴定进行了研究。2005年从广西5个病区采集的25个标样均分离到类似的分离物,根据病原菌的形态特征和致病性,并结合其rDNA-ITS区域的序列分析,将广西山药炭疽病的病原菌鉴定为胶孢炭疽菌(Colletotrichum gloeosporioides)。

用ITS序列研究杨属各组之间的系统发育关系

ITS sequences of 15 representative species of five sections in the genus Populus L. were determined. By using direct sequencing of PCR product, it was found that the fragments of internal transcribed spacers （ITS） are about 594 bp in length. The length of ITS1 and ITS2 is about 220 bp and 210 bp, respectively, while that of 5.8s is 164 bp. Its G+C content is about 69.0%. The number of phylogenetically informative loci is higher in ITS2 than in ITS1. Transversion and transition are two main factors that drive the ITS evolution, and more insertions and deletions occurred in ITS2. Taking Salix matsudana Koidz. and Salix suchowensis Cheng as outgroups, phylogenetic analysis of ITS sequences using PAUP 4.0 software indicated that Populus is monophyletic group and can be divided into two main clades. One is the section Leuce , and the other is the remaining sections.

百合枯萎病病原鉴定与ITS序列分析

对从百合枯萎病病株上分离得到的病原菌进行形态特征、致病性以及核糖体DNA-ITS序列分析。结果表明,该菌在PDA培养基上气生菌丝白色绒毛状,小型分生孢子卵圆形或椭圆形,大型分生孢子镰刀形。克隆分析菌株的核糖体DNA-ITS区域序列,分离菌与GenBank中尖孢镰刀菌的ITS序列同源性最高达99.8%,仅有1个碱基的差异,进一步证实嵩明百合种植基地百合枯萎病的病原菌为尖孢镰刀菌(Fusarium oxysporum)。

广义青篱竹属(Arundinaria)核糖体DNAITS序列及亲缘关系研究

利用PCR扩增产物直接测序的方法分析广义青篱竹属 (Arundinaria)中有关争议类群的代表种或模式种 (毛竹为外类群 )等 18种竹种的核糖体DNA内转录间隔区 (InternalTranscribedSpacers ,ITS)序列。通过最简约性分析产生的ITS系统发育树表明 ,供试竹种形成一个自然的单系类群 ,这说明广义青篱竹属中这些不同的类群归属青篱竹属是合理的。 17种竹种可聚为 2大分支 :其中斑苦竹 (A .oleosa)、仙居苦竹 (A .hsienchuensis)、茶秆竹 (A .amabilis)、长叶苦竹 (A .chino)、苦竹 (A .amara)、宜兴苦竹 (A .yixingensis)、菲白竹 (A .fortunei)、翠竹 (A .pygmaea)为一个分支 ;而大明竹 (A .graminea)、巴山木竹 (A .fargesii)、冷箭竹 (A .faberi)、凤竹 (A .hupehense)、鼓节矢竹 (Pseudosasajaponicacv.Tsutsumiana)、矢竹 (Pseudosasajaponica)、短穗竹 (Brachystachyumdensiflorum)、肿节竹 (A .oedogonata)、少穗竹 (A .sulcata)组合在另一分支。ITS系统发育树还表明 ,大明竹与巴山木竹、鼓节矢竹与矢竹、少穗竹与短穗竹和肿节竹关系极为密切 ,均得到较高的Bootstrap(分别为 99%、10 0 %和 87% )的支持 ;茶秆竹与仙居苦竹关系非常密切 ,茶秆竹可归隶到青篱竹属中 ;翠竹和菲白竹关系密切 。