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.

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 Epidermaphyton, 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.

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。

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.

An improved PCR method for direct identification of Porphyra(Bangiales,Rhodophyta) using conchocelis based on a RUBISCO intergenic spacer

An improved method of PCR in which the small segment of conchocelis is amplified directly without DNA extraction was used to amplify a RUBISCO intergenic spacer DNA fragment from nine species of red algal genus Porphyra(Bangiales,Rhodophyta),including Porphyra yezoensis(Jiangsu,China),P.haitanensis(Fujian,China),P.oligospermatangia(Qingdao,China),P.katadai(Qingdao,China),P.tenera(Qingdao,China),P.suborboculata(Fujian,China),P.pseudolinearis(Kogendo,Korea),P.linearis(Devon,England),and P.fallax(Seattle,USA).Standard PCR and the method developed here were both conducted using primers specific for the RUBISCO spacer region,after which the two PCR products were sequenced.The sequencing data of the amplicons obtained using both methods were identical,suggesting that the improved PCR method was functional.These findings indicate that the method developed here may be useful for the rapid identification of species of Porphyra in a germplasm bank.In addition,a phylogenetic tree was constructed using the RUBISCO spacer and partial rbcS sequence,and the results were in concordant with possible alternative phylogenies based on traditional morphological taxonomic characteristics,indicating that the RUBISCO spacer is a useful region for phylogenetic studies.

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。

洋山港宏病毒组分析揭示CRISPR-Cas系统病毒靶标序列的特异性

目的 为了探究CRISPR-Cas系统的靶标特点。方法 运用了包括BALST在内的多种生物信息学分析技术和方法对洋山港宏病毒组数据进行了分析。结果 洋山港宏病毒组数据集中共有25 391条双链DNA病毒序列,其中238条序列上的265个开放读码框(ORF)与134条规律间隔短回文重复(CRISPR)间隔序列产生了315个匹配。经注释后获得了128个ORF和135个匹配的功能信息,占比前5位的依次为终止酶(terminase)、衣壳蛋白(capsid protein)、门蛋白(portal protein)、肽酶(peptidase)和DNA甲基化转移酶(DNA methyltransferase)。匹配多在病毒特定功能基因的保守域或关键结构域中。这表明,CRISPR-Cas系统发挥免疫功能时表现出针对病毒特定基因、功能和结构域的靶标特异性。此外,属于Class 1门类下的Type_I型系统的CRISPR-Cas间隔序列匹配数量远高于其他类型系统,占总数的89.0%。结论 本文的研究结果揭示了CRISPR-Cas系统的靶标偏好,增进了对其的认识,为更好地理解病毒-宿主免疫互作机制提供了新的证据和线索。

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。

多杀性巴氏杆菌CRISPR-Cas系统生物信息学分析

【目的】了解多杀性巴氏杆菌中CRISPR-Cas系统的结构特征及cas基因的分布,为不同CRISPR亚型的应用及多杀巴氏杆菌的抗病毒进化研究提供基础。【方法】通过CRISPRCasfinder在线网站筛选多杀性巴氏杆菌基因组中的CRISPR簇和cas基因;通过生物信息学方法对各亚型系统中repeat和spacer序列保守性、repeat二级结构、cas基因核苷酸序列保守性、CRISPR簇相似性等进行分析。【结果】在选取的19株多杀性巴氏杆菌基因组中均存在Ⅰ-F、Ⅱ-C、Ⅲ-A亚型CRISPR系统。在相同亚型的CRISPR系统中repeat序列有着较保守的回文重复序列,但在不同CRISPR亚型中repeat序列差异较大。Ⅱ-C亚型的tracrRNA可以与crRNA形成保守的结构。在各亚型CRISPR系统中,新获取的spacer序列可以插入到CRISPR簇的5′-端、3′-端或中间位置。通过对各菌株spacer序列分析发现,在菌株内或菌株间的CRISPR序列中均存在着相同的spacer序列,这可能与细菌在遗传进化过程中repeat-spacer发生序列重组或基因水平转移产生的趋同进化有关。【结论】19株多杀性巴氏杆菌中含有Ⅰ-F、Ⅱ-C、Ⅲ-A 3种CRISPR-Cas系统,不同CRISPR亚型中repeat序列差异较大,spacer特征性重复的分布可能与多杀性巴氏杆菌在遗传进化过程中repeat-spacer发生序列重组有关。

明党参和川明参种间遗传分化和系统关系的分子标记和ITS序列分析

为了阐明明党参（Changium smyrnioides）和川明参（Chuanminshen violaceum）种间遗传分化和系统关系，利用RAPD、ISSR和ITs序列分析方法对明党参和川明参不同居群，以及芹亚科（Apioideae）6个族的一些代表种进行研究．RAPD和ISSR分析表明，明党参与川明参种间在全基因组水平出现明显的遗传分化．ITS序列分析则表明，明党参的6个居群在ITS序列上稍有差异，ITS-1的GC含量为58．3％～59．2％，ITS2的GC含量为54．3％～56．6％．ITS-1的长度范围为215～219bp，ITS-2的长度范围为224～227bp．川明参与明党参种间的ITS序列也出现明显分化．芹亚科6个族一些代表种的ITS序列系统发育分析结果支持明党参置于美味芹族（SmyrnieaeKoch）的分类地位．在ITS系统树上，川明参与明党参为姐妹类群的置信度为100％，因此，提出将川明参置于美味芹族的分类学处理．

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.

应用5S rDNA间隔序列分析鉴定体细胞杂种

Three intergeneric somatic hybrids (wheat(+) Haynaldia villosa, wheat(+) Bromus inermis, wheat(+) Agropyron elongatum) and one interfamily somatic hybrid between Vitis vinifera and Bupleurum scorzonerifollium were analyzed by PCR with two kinds of primers.The results showed that in the electrophoresis pattern of the PCR products the somatic hybrids had the characteristic bands of two parents and (or) new bands.This research reveals that PCR analysis with 5S rDNA spacer sequence primers can be used for the identification of somatic hybrids at the molecular level and it is a good method because of its simplicity and good reproducibility.

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。

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)。

肠炎沙门氏菌2种rDNA 16s～23s基因间隔区序列分析

采用凝胶分离ＰＣＲ产物的方法，对肠炎沙门氏菌中２种ｒＤＮＡ１６ｓ～２３ｓ基因间隔区进行克隆和序列分析．肠炎沙门氏菌小ｒＤＮＡ１６ｓ～２３ｓ基因间隔区，全长３５４个碱基对，包含１个谷氨酸ｔＲＮＡ基因．大ｒＤＮＡ１６ｓ～２３ｓ基因间隔区，全长５１８个碱基对，其中包含异亮氨酸和丙氨酸２个ｔＲＮＡ基因．它们分别与大肠杆菌的２个ｒＲＮＡ操纵子ｒｎＥ和ｒｒｎＨ有较高的序列相似性．肠炎沙门氏菌大ｒＤＮＡ１６ｓ～２３ｓ基因间隔区比大肠杆菌ｒＤＮＡ对应的区域长７１个碱基对，这一插入片段很可能是沙门氏菌属ｒＤＮＡ的一个特征序列．

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。

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.

5S-rRNA基因间区序列变异用于金银花药材道地性研究初探

目的 研究金银花药材道地性形成的基因基础。方法 用 SDS法提取金银花 L onicera japonica不同居群、外类群细毡毛忍冬 L.similis和山银花 L.confusa的总 DNA,进行 5 S- r RNA基因间区的 PCR扩增和测序 ,并用软件 Mega进行分析。结果 L onicera L.属植物 5 S- r RNA基因间区约 2 10 bp,其中 G+C含量较高 ,达 70 %左右 ,不同居群的 L.japonica碱基序列有差别 ,通过测序可以进行鉴别。L.confusa与 L.japonica间的遗传距离较大。结论 种间 5 S- r RNA基因间区的序列差异大于种内 ;道地药材之间的遗传距离较小 ;道地与非道地药材之间的遗传距离大于道地药材之间的遗传距离。