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.     

江苏海域几种绿潮藻类rDNA ITS序列分析

于2009年5-7月定点采集了江苏海域绿潮藻类,测定、分析了这些藻类核糖体rDNA ITS序列,并进行分类鉴定。研究结果显示,ITS+5.8S序列片段长度为552-578 bp,其中ITS1序列部分长度为179-182 bp,5.8S序列全长为155-158 bp,ITS2序列全长为180-196 bp,序列的平均GC含量(contents)为61.6%-63.3%,不同ITS序列存在不同的插入/缺失位点。扩增得到27个序列中有7个不同序列,依据NCBI数据库相似性查找、系统进化关系及遗传距离等分析结果,确定有4个种类:浒苔(Ulva prolifeya),缘管浒苔(U.linza),石莼属(Ulva sp.)1种及盘苔属(Blidingia sp.)1种。分析表明,ITS序列可作为石莼科种类鉴定的标记。     

栽培种甘薯及其近缘野生种nrDNA ITS序列分析

采用核糖体DNA内转录间隔区(nrDNA ITS)序列比较分析了甘薯及其近缘野生种的遗传多样性及系统进化关系,首次报道了栽培种甘薯‘徐薯18’(Ipomoea batatas‘Xushu18’)及其近缘野生种I.triloba(DOM),I.cordatotriloba(MEX),I.nil(PER),I.nil(JPN),I.hederacea Jacq.(USA),I.hederacea Jacq.(HK)和种间杂交种67-1(I.batatas‘Xushu18’×I.hederacea Jacq.)及回交种(67-1×I.batatas‘Xushu18’)的nrDNA ITS序列。序列分析表明,栽培种甘薯及其近缘野生种nrDNA ITS序列长度为570~600bp。其中,ITS1序列为185~209 bp,GC含量为53.11%~61.83%;ITS2序列为214~226 bp,GC含量为61.21%~72.89%;5.8S序列均为165 bp,GC含量为54.55%~55.76%。此外,栽培种甘薯及其近缘野生种ITS序列信息位点均集中在ITS1和ITS2区;与其他甘薯属植物相比,I.wrightii ITS2的末端缺失了6~8个碱基。系统进化分析表明,栽培种甘薯‘徐薯18’(I.batatas‘Xushu18’)和野生种I.triloba、I.cordatotriloba、I.lacunosa、I.trifida的亲缘关系较近,与I.wrightii、I.pes-tigridis、I.grandifolia、I.nil、I.hederacea Jacq.、I.purpurea的亲缘关系较远;杂交后代与栽培种甘薯‘徐薯18’(I.batatas‘Xushu18’)亲缘关系较近,与野生种父本I.hederacea Jacq.的亲缘关系较远。     

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ITS序列的不同产地裂叶荆芥系统发育分析

采用PCR法扩增来自国内5个不同产地的裂叶荆芥ITS全序列,测序后以产自河北的裂叶荆芥代表中国裂叶荆芥与来自不同国家的裂叶荆芥ITS序列进行比较,构建分子系统发育树,探讨国内5个不同产地及不同国家的裂叶荆芥的亲缘关系和系统进化。结果表明:国内5个不同产地的裂叶荆芥ITS1和ITS2序列均有较高的G/C含量;5个产地的裂叶荆芥的扩增序列长度均为749bp,且序列完全相同;其中ITS1序列长231bp,5.8S序列长168bp,ITS2序列长236bp。中国裂叶荆芥与日本、韩国裂叶荆芥ITS序列一致性为100%,与美国裂叶荆芥ITS序列一致性为99.0%。与美国裂叶荆芥相比,中国裂叶荆芥ITS序列有7个碱基发生变异。来自不同国家的裂叶荆芥形成单系群和2个分支(中、日、韩3国为1个分支,美国单独形成1个分支)。ITS序列的一致性表明国内5个不同产地裂叶荆芥为同一个种。     

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。     

基于ITS序列分析钩苞大丁草九个居群的亲缘关系

该研究对我国西南地区钩苞大丁草(Gerbera delavayi)9个居群rDNA ITS序列进行PCR的扩增和检测序列,并以非洲菊(G.jamesonii)的ITS序列作为外类群,比较了序列之间的差异,同时分析了钩苞大丁草不同居群在地理距离与遗传距离之间的关系,构建了NJ系统发育树。结果表明:(1)钩苞大丁草9个居群的ITS序列全长介于600~700 bp之间,平均长度约为657 bp,其中,ITS1长度为243~246 bp,(G+C)含量为45.67%~46.80%之间,5.8S长度191~193 bp,(G+C)含量为58.60%~58.61%之间,ITS2长度为220~221 bp,(G+C)含量为57.00%~57.45%之间;ITS序列共有22个变异位点,ITS1序列(17个)、5.8S序列(2个)以及ITS2序列(3个)上均有变异。(2)地理距离与遗传距离有正相关(r2=0.652),序列间遗传分化距离为0.001 1~0.024 3,其中普洱居群与其他居群间遗传距离最大。(3)钩苞大丁草9个居群分成三个分支,普洱居群单独成支,丽江和洱源居群聚为一支,富源、武定、德昌、石林、新平和开远6个居群聚为一支。rDNA ITS序列可以用于钩苞大丁草群体遗传研究的分析,该研究结果为其保护性开发提供了参考依据。     

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。     

金针菇rDNA序列结构特征分析

rDNA序列中的ITS作为DNA barcoding广泛应用于真菌的系统发育与物种辅助鉴定,IGS被认为可以用于种内水平不同菌株的鉴别。食用菌中还没有完整的rDNA序列的报道。本研究采用二代和三代测序技术分别对金针菇单核菌株“6-3”进行测序,用二代测序的数据对三代测序组装得到的基因组序列进行修正,得到一个在基因完整性、连续性和准确性均较好的基因组序列,对比Fibroporia vaillantii rDNA序列,获得金针菇完整的rDNA序列。金针菇rDNA序列结构分析表明,它有8个rDNA转录单元,长度均为5 903bp,有9个基因间隔区,其长度有较大差异,3 909-4 566bp。rDNA转录单元中,各元件的序列长度分别为：18S rDNA 1 796bp、ITS1 234bp、5.8S rDNA 173bp、ITS2 291bp、28S rDNA 3 410bp。基因间间隔区中,IGS1 1 351-1 399bp、5S rDNA 124bp、IGS2 2 435-3 092bp。金针菇的5S、5.8S、18S、28S rDNA序列准确性得到转录组数据的验证,也得到系统发育分析结果的支持。多序列比对发现,不同拷贝的基因间间隔区序列（IGS1和IGS2）存在丰富的多态性,多态性来源于SNP、InDel和TRS（串联重复序列）,而TRS来源于重复单元的类型和数量。9个基因间间隔区之间,IGS1只有少量的SNP和InDel,IGS2不仅有SNP和InDel,还有TRS。本研究结果提示,在应用IGS进行种内水平不同菌株之间的鉴别时,需要选取不同拷贝之间的保守IGS序列。     

蓼属头状蓼组rDNA-ITS的序列扩增及分析

以贵州境内蓼属头状蓼组6种(含1变种)植物为材料,对其rDNA的内转录间隔区(ITS)序列进行PCR扩增,得到6种植物的ITS序列,分别为:赤胫散2个居群(Polygonum runcinatum var.sinense,GenBank登录号FJ606887、FJ648802),平卧蓼(P.strindbergii,GenBank登录号FJ648803 ),尼泊尔蓼(P.nepalense,GenBank登录号FJ648804),羽叶蓼(P.runcinatum,GenBank登录号FJ648805),火炭母(P.chinense,GenBank登录号FJ648806)和头花蓼(P.capitatum,GenBank登录号FJ648807).其中赤胫散与平卧蓼的ITS序列为首次报道.序列分析结果表明,蓼属头状蓼组6种植物ITS序列总长度为661～666 bp,ITS1区序列长度为243～246 bp,5.8 S rDNA区序列长度165 bp,ITS2区序列长度253～258 bp,6种植物的差异主要集中在ITS1和ITS2区.聚类分析显示,6种头状蓼组植物具有共同起源,结果支持赤胫散从羽叶蓼变种上升为独立物种.     

枇杷属植物4个种的花序性状多样性研究

在相同的种植和管理条件下,对普通枇杷、栎叶枇杷、大瑶山枇杷和南亚枇杷等4个种210份种质资源的8个花序性状多样性进行鉴定,旨在为进一步的分类鉴定、利用研究提供参考依据。结果表明:(1)枇杷属种质资源的花序支轴姿态、花序支轴密度、花序支轴数、花序形状、花序长度、花序宽度、花瓣颜色、花冠直径Shannon-Weaver多样性指数1.352～2.951,存在丰富的多样性。(2)不同来源地的种质资源花序性状多样性水平差异较大,多样性指数最高的为云南(2.108),其次是贵州(1.962)、四川(1.953)、福建(1.900),美国的最低(1.137)。(3)云南、贵州的枇杷种质资源花序长度、宽度显著或极显著地大于其他地区;野生资源的花序长度、花序宽度和花冠直径均极显著地大于地方品种、选育品种。     

基于rDNAITS序列分析Alternaria与相似属的关系及A.leucanthemi的分类地位

对Alternaria,Stemphylium,Ulocladium,Nimbya,Embellisia等五个形态相似属的代表性种进行了5.8S/ITS区段序列测定,连同从GenBank下载的相关有性型Pleospora,Lewia的同项资料,由Neighbor-joining方法构建系统发育树。序列对比结果显示,所有参试的菌株/种的5.8SrDNA序列保守程度较高,ITS序列变化较大,ITS1比ITS2变化更大。由5.8S/ITS构建的系统发育树可以足够将供试菌区分到属;系统发育树显示Alternaria,Ulocladium,Nimbya和Embellisia系统学关系较近,又存在一定的分化;Stemphylium聚在相对较远的分支上,与上述四属关系较远,相对独立进化;有性型的Pleospora与无性型的Stemphylium被聚到一个分支,这种不同阶段菌株在遗传基础上的一致,充分证明所用方法及所选测序的rDNA区段对本类群真菌的分类很有价值。Alternarialeucanthemi位于系统发育树的外侧,较为独立进化,与Alternaria的其它种亲缘关系较远,却与Stemphyliumspp.和Bipolarisspp.关系较近,对其目前的分类地位提出质疑。     

海洋喇叭虫rRNA基因18S-ITS1序列及其系统发育分析

海洋喇叭虫Maristentor dinoferus 1996年在关岛(Guam)的珊瑚暗礁上被发现,至今尚未阐明其确切的系统发育地位.克隆到的海洋喇叭虫的18S-ITS1-5.8S rDNA序列包括222 bp的18S序列,77 bp的ITS1序列和22 bp的5.8S序列.比较分析了纤毛虫主要类群的ITS1序列后得出:短的ITS1序列可能是异毛类纤毛虫的特征.根据18S序列,利用邻接法构建,最大简约法和最大似然法构建系统发育树.其拓扑结构显示海洋喇叭虫属于异毛纲纤毛虫,但并不隶属喇叭虫科,应予以新的分类地位.     

Phylogenetic relationships among 28 spirotrichous ciliates documented by rDNA

The contiguous sequence of the SSU rDNA, ITS 1, 5.8S, ITS 2, and approximately 1370 bp at the 5(') end of the LSU rDNA was determined in 25 stichotrichs, one oligotrich, and two hypotrichs. Maximum parsimony, neighbor-joining, and quartet-puzzling analyses were used to construct individual phylogenetic trees for SSU rDNA, for LSU rDNA, and ITS 1+5.8S+ITS 2, as well as for all these components combined. All trees were similar, with the greatest resolution obtained with the combined components. Phylogenetic relationships were largely consistent with classical taxonomy, with notable disagreements. DNA sequences indicate that Oxytricha granulifera and Oxytricha longa are rather distantly related. The oligotrich, Halteria grandinella, is placed well within the order Stichotrichida. Uroleptus pisces and Uroleptus gallina probably belong to different genera. Holosticha polystylata (family Holostichidae) and Urostyla grandis (family Urostylidae) are rather closely related. These rDNA sequence analyses imply the need for some modifications of classical taxonomic schemes.     

Pseudo-nitzschia pungens (Bacillariophyceae): A cosmopolitan diatom species?

Genetic, reproductive and morphological variation were studied in 193 global strains of the marine diatom species Pseudo-nitzschia pungens (Grunow ex Cleve) Hasle to assess potential intraspecific variation and biogeographic distribution patterns. Genetic differentiation between allo- and sympatric strains was investigated using the ITS1–5.8S–ITS2 rDNA region. Three ITS clades were found. Clones of opposite mating type were sexually compatible within clades I or II, and viable F1 hybrid offspring were produced in crosses between them. The molecular differences between these clades were correlated with slight but consistent morphological differences. At present, nothing can be said about morphology and mating behavior for clade III clones because only ITS data were available. The three ITS clades showed different geographic distributions. Clade II was restricted to the NE Pacific, whereas clones belonging to clade III originated from geographically widely separated areas (Vietnam, China and Mexico). ITS clade I was recovered in all locations studied: the North Sea (Belgium, The Netherlands, France), the eastern and western N Atlantic (Spain, Canada), the NW and S Pacific (Japan, New Zealand) and the NE Pacific (Washington State). Clade I thus appears to be globally distributed in temperate coastal areas and provides the first strong evidence to date for the global distribution of a biologically, genetically and morphologically defined diatom species.     

Phylogenetic relationships on 14 morphologically similar species of Pucciniastrum in Japan based on rDNA sequence data

We analyzed the phylogenetic relationships of 49 specimens comprising 14 morphologically similar species of Pucciniastrum distributed in Japan based on the sequence data of the large subunit rDNA (D1/D2), 5.8S rDNA, and internal transcribed spacer (ITS) regions. Neighbor-joining and parsimony analyses generated six major groups for both the D1/D2 and ITS regions. Pucciniastrum circaeae and P. epilobii formed a single group. P. hydrangeae-petiolaris, P. coryli, P. fagi, P. hikosanense, P. tiliae, and P. boehmeriae were each a distinct clade, and P. fagi formed a close relationship with P. hikosanense. However, these analyses did not support the monophyly of the following species: P. kusanoi, P. actinidiae, P. corni, P. styracinum, P. yoshinagai, and P. miyabeanum. Contribution no. 201, Laboratory of Plant Parasitic Mycology, Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan     

Phylogenetic relationships of Pythium and Phytophthora species based on ITS rDNA, cytochrome oxidase II and beta-tubulin gene sequences

Fifty-eight isolates representing 39 Pythium species and 17 isolates representing nine Phytophthora species were chosen to investigate intra- and intergeneric relationships with sequence analysis of three genomic areas. The internal transcribed spacer regions (ITS1 and ITS2), including the 5.8S gene of the ribosomal DNA were PCR amplified with the universal primers ITS1 and ITS4. On the other hand 563 bp of the cytochrome oxidase II (cox II) gene was amplified with the primer pair FM66 and FM58 for Pythium and FM75 and FM78 for Phytophthora. The 658 bp partial beta-tubulin gene was amplified with the forward primer BT5 and reverse primer BT6. Maximum parsimony analysis of the three DNA regions revealed four major clades, reflective of sporangial morphology. Clade 1 was composed of Pythium isolates that bear filamentous to lobulate sporangia. Clade 2 represents Pythium isolates that bear globose to spherical zoosporangia or spherical hyphal swellings. Meanwhile Phytophthora isolates were lumped into Clade 3 wherein the papillate, semipapillate and nonpapillate species occupied separate subclades. Lastly, Clade 4 was composed of Pythium species that bear subglobose sporangia resembling the papillate sporangia observed in Phytophthora. Hence a number of species (Ph. undulata, P. helicoides, P. ostracodes, P. oedochilum and P. vexans) have been proposed to be the elusive intermediate species in the Pythium-to-Phytophthora evolutionary line.     

Sequence heterogeneity in the internal transcribed spacers of two rat ribosomal DNA clones

The sequence of one cloned rat rDNA segment is determined, encompassing the 3'-terminal part of 18 S rDNA (231 bp), ITS 1 (1072 bp), 5.8 S rDNA (156 bp), ITS 2 (771 bp) and the 5'-terminal part of 28 S rDNA (210 bp). Comparison with a distinct clone of the same rDNA segment reveals the identity of the rRNA gene sequences and considerable heterogeneity in both ITS 1 and ITS 2. The heterogeneities include mainly single base-pair changes (23 deletions or insertions and 14 substitutions) distributed all along the ITS sequences.