双壳纲动物核糖体RNA 185-ITS1序列及其在分子系统发育研究中的应用

测定了双壳纲不同科、属、种及种内共11个个体的核糖体RNA 18S-ITS1序列。结果表明,该序列在种间存在很高的多态性,长度从558bp到784bp不等,碱基差异百分比在10.7％～61.7％之间,ITS1序列同源性很低,有片段的插入与缺失。种间18S部分序列碱基差异百分比在0.9％～23.7％之间,变化主要是碱基的转换。用邻接法(NJ)构建了8个种的18S部分序列(约240bp)的系统发育树,与传统形态学分类结果相符。马氏珠母贝(Pinctada martensi)4个不同地域个体间的序列差异百分比在0.6％～1.9％之间。分析指出:18S基因可以作为双壳纲动物高阶元系统发育的分子标记;ITS1序列种间变化很大,可以应用于该纲物种的分类及鉴别,在亲缘关系相近种及种内变异相对较小,但核苷酸变异位点信息量丰富,可用于属内种间、亚种和群体间的遗传多样性研究。     

帘蛤科贝类rDNA内转录间隔区序列的研究

根据18SrDNA、5．8SrDNA和28SrDNA保守序列设计引物,应用聚合酶链式反应（PCR）扩增了文蛤（Meretrix meretrix L．）、青蛤（Cyclina sinensis G）、硬壳蛤（Mercenaria mercenaria L．）和江户布目蛤（Protothaca jedoensis L．）4种帘蛤科贝类的第一内转录间隔区（ITS1）和第二内转录间隔区（ITS2）序列,并进行了测序。结果表明,文蛤、青蛤、硬壳蛤和江户布目蛤的ITS1扩增产物大小分别为978bp、663bp、757bp和942bp,GC含量分别为61．55％、60．78％、62．48％和64．86％～64．97％,其中ITS1序列长度分别为900bp、585bp、679bp和864bp,是迄今已报道双壳贝类中变化范围最大的,GC含量分别为61．67％、61．03％、63．03％和65．51％～65．62％,江户布目蛤种内ITS1序列有个体差异;ITS2扩增产物大小分别为644bp、618～620bp、593bp和513～514bp,GC含量分别为61．18％、61．29％～61．81％、62．73％和61．48％61．60％,其中ITS2序列长度分别为412bp、386～388bp、361bp和281～282bp,GC含量分别为65．29％、65．21％～66．06％、67．87％和67．38％～67．62％,青蛤和江户布目蛤种内ITS2序列有个体差异。4种蛤ITS1和ITS2序列种间差异很大,有明显的长度多态性,ITS2种间序列相似度73．0％～89．1％,与ITS1的种间序列相似度48．7％～81．5％相比略高。此外,在4种蛤ITS1和ITS2序列中各发现2个与rRNA加工有关的保守区。通过对ITS1和ITS2序列的组装获得了4种蛤5．8SrRNA基因完整序列,序列长度都是157bp,GC含量57．96％～58．60％,4种蛤5．8SrRNA基因相对保守,种间序列差异度0-6．0％,共有10个变异位点,其中转换4处,颠换6处,硬壳蛤和江户布目蛤5．8SrRNA基因序列完全相同。以ITS2序列（包含5．8SrRNA和28SrRNA基因部分序列）为标记,调用北极蛤科的Arctica islandica相应序列数据作外群,构建了帘蛤科贝类的系统发育树,其拓扑结构显示江户布目蛤与硬壳蛤亲缘关系最近,青蛤与其他3物种的亲缘关系最远。     

老芒麦和垂穗披碱草rDNA-ITS序列分析

分析了老芒麦和垂穗披碱草rDNA-ITS序列,为这两个种的鉴别提供分子指纹图谱,为系统发育提供分子生物学依据。结果表明,22份材料的ITS、ITS1、5.8S、ITS2序列长度均相同,依次为604bp、222bp、164bp、218bp,GC含量依次为62.25%～63.08%,62.16%～63.06%,59.76%,64.22%～65.60%。除5.8S外,碱基位点都有不同程度的变化,一些变异位点有明显的种性变异规律,可作为老芒麦和垂穗披碱草种质DNA指纹特异鉴别位点。7个ITS序列的同源性98.7%～99.8%,遗传分歧0.2～1.3,具有保守性,为非近期分化类群。系统发育分析表明不同来源地的同种材料,差异主要表现在DNA变化快慢及碱基替换数,但相同的种归为一类,进一步反映了种内的遗传稳定性。     

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.     

基于核rDNA的ITS序列在种子植物系统发育研究中的应用

种子植物核rDNA是高度重复的串联序列,由于同步进化的力量．大多数物种中这些重复单位间已发生纯合或接近纯合。5．8S rDNA把核rDNA的内转录间隔区分为ITS1和ITS2两部分．在被子植物中ITS1的长度为165～298bp,ITS2的长度为177～266bp,而在裸子植物中ITS片段较长。且其长度变化主要由ITS1的长度变异所致。可对这两个片段PCR产物进行直接测序或克隆测序。由于ITS序列变异较快．能够提供较丰富的变异位点和信息位点,已成为被子植物较低分类阶元的系统发育和分类研究中的重要分子标记,为探讨多倍体复合体网状进化关系,异源多倍体的起源提供了重要的系统学信息．但它一般不适合科以上水平的系统学研究。裸子植物中ITS片段较长,重复序列间的纯合程度不同,测序比较困难．因此对探讨裸子植物系统发育和分类受到了一定的限制,但近年来有所发展。     

不同产区太子参的rDNA ITS区序列的比较

使用1对引物18SPl和26SP2对采自14个产区的太子参[Pseudostellaria heterophylla(Miq．)Pax ex Pax et Hoffm．]进行ITS基因的PCR扩增和测序。序列分析结果表明,14个产区太子参的ITSl片段长度为219—222bp,ITS2片段长度为235～236bp,5．8S片段长度为155—157bp.除江苏宜兴,江苏句容马梗,江苏南京老鹰山和江苏溧阳等4个产区的ITS序列碱基完全一致外,其他10个产区的ITS序列则有不同的变异,碱基变异数目(包括5．8S编码区)为1—17个。使用UPGMA法重建系统发生树,从分子生物学角度说明了它们的变异程度,为利用ITS区序列的差异鉴别不同产区的太子参提供了依据。     

乳白石蒜rDNA-ITS序列分析及种内系统发育研究

对来源于不同产地的11个乳白石蒜(Lycoris albiflora Koidz.)种源的rDNA-ITS序列进行了扩增、纯化、克隆、酶切和测序,并对各种源的rDNA-ITS序列长度、G+C含量、碱基差异和遗传距离进行了比较分析,构建了系统发育树.结果表明,乳白石蒜11个种源的rDNA-ITS序列具有较高的同源性,但不同种源间的ITS序列长度和碱基变异较大;乳白石蒜rDNA-ITS序列总长度约为700 bp,共有318个变异位点;ITS1、ITS2和5.8S rDNA片段长度分别为222～245、240～252和163 bp;ITS序列中的G+C含量均明显高于A+T含量,ITS1和ITS2片段中的G+C含量分别为53.8%～70.5%和63.4%～73.4%;碱基变异类型多为颠换、转换、插入和缺失.种源间的遗传距离差异较大,其中浙江天目山种源(TM3)与浙江宁波种源(NB2)的遗传距离最小(0.000),其他种源间遗传距离为0.067～0.323.基于ITS序列分析结果可将11个乳白石蒜种源聚为3大类,第1类包括采自浙江天目山(TM1和TM2)和浙江宁波(NB3)的3个种源,花和花丝多为白色;第2类包含来源于不同产地的7个种源,花多为乳白色或乳黄色;第3类仅有采自浙江兰溪(LX)的1个种源,花色变异较大.研究结果表明,乳白石蒜种内有丰富的遗传变异,种源间的ITS序列差异与花的特征变化一致,但与地理分布并不相关;rDNA-ITS序列分析可用于乳白石蒜的亲缘关系、物种鉴别和遗传多样性研究.     

枇杷属内栽培种和部分野生种ITS序列分析

对不同栽培区的25种普通枇杷品种以及7种枇杷属野生种的ITS序列进行扩增并测序,采用邻接法和最大简约法进行系统发育树的构建并对枇杷属内不同种间的遗传关系进行了分析。结果表明:枇杷属植物ITS序列ITS1+5.8S rDNA+ITS2总长度为592 bp或594 bp,长度变化发生在ITS2。所有样本的ITS1和5.8S rDNA长度一样,都是223 bp和168 bp;而ITS2为201 bp或203 bp。5种枇杷属野生种的ITS序列长度为594 bp,包括栎叶枇杷、大渡河枇杷、南亚枇杷、南亚枇杷窄叶变种和大瑶山枇杷;其余2种枇杷属野生种(麻栗坡枇杷、小叶枇杷)和普通枇杷栽培种的ITS序列长度都为592 bp。所有样本ITS序列的GC含量为64.2%~64.5%,其中ITS1为64.1%~65.5%,ITS2为68.1%~72.6%。对所有样本的ITS序列比对产生44个可变位点,其中38个为简约信息位点,其中11个位于ITS1,5个位于5.8S rDNA,22个位于ITS2。最大的种间序列差异为7.7%,最小的种间差异发生在麻栗坡枇杷和小叶枇杷之间,仅为0.2%。普通枇杷种内的ITS序列差异很低,25种普通枇杷栽培种之间的序列差异为0~1.5%。所研究的枇杷属植物可分为3个分支。分支Ⅰ包括所有普通枇杷品种,分支Ⅱ包含5种野生枇杷种,包括栎叶枇杷、大渡河枇杷、南亚枇杷、南亚枇杷窄叶变种和大瑶山枇杷;分支Ⅲ由2个野生枇杷种(麻栗坡枇杷、小叶枇杷)组成。该研究结果表明ITS序列对枇杷种间鉴定和系统发育分析具有一定意义,但对普通枇杷栽培种间的鉴定作用不大。     

我国赫坎按蚊复合体成员种的rDNA-ITS2区序列差异及系统发育分析

测定了我国赫坎按蚊复合体 9成员种的核糖体DNA第二内转录间隔区 (rDNA ITS2 )序列 ,根据序列差异分析各蚊种间的系统发育关系。结果显示 :( 1 )ITS2区序列最长的是中华按蚊 ( 4 6 8bp) ,最短的是克劳按蚊和赫坎按蚊 ( 4 36bp) ;GC含量为 4 4 9%～ 4 6 8% ;( 2 )发现该复合体 4成员种的ITS2区序列存在种内个体间差异 ,幅度为 0～ 3 8% ,明显小于种间差异 ;( 3)将各蚊种的ITS2区序列进行同源排序比较 ,发现其变异大多是简单重复单元的拷贝数不同 ;种间差异性最大的是克劳按蚊与嗜人按蚊( 32 3% ) ,最小的是贵阳按蚊与凉山按蚊 ( 9 0 % )平均差异率为 2 2 3% ;( 4 )根据ITS2区序列特征 ,用 3种方法构建的树状图拟合一致。以上结果表明赫坎按蚊复合体各成员种rDNA ITS2序列在种内非常保守 ,以种间序列差异分析为基础的分子鉴别技术是甄别蚊种分类地位混淆和错误的有效方法。     

福建橄榄18S-26S rRNA及其ITS片段的克隆与序列分析

利用18S-26S rRNA基因及其ITS片段的PCR扩增、克隆及测序分析,对福建14个橄榄品种进行分子标记及遗传分类。序列分析结果表明:14个品种可分为4个类别,以1号品种为参照,3、4、6、7、8、9、11号品种的ITS1、ITS2和5.8S共634个碱基序列完全一致,与1号仅ITS2上有1个碱基差异,2号和1号品种的ITS1和5.8S序列完全一致,仅ITS2上有2个碱基差异,归为第一类;5号和14号的序列完全一致,与1号在ITS1和ITS2上各有1个碱基差异,归为第二类;10号和12号序列完全一致,与1号在ITS1上有2个碱基差异,归为第三类;13号品种5.8S的序列与1号相同,但在ITS1上有1个碱基差异,在ITS2上有24个碱基差异,归为第四类。使用DNAMAN软件建立的同源关系树说明了不同橄榄品种的变异程度。将橄榄18S-26S rRNA基因及其ITS序列登陆GenBank,登陆号:DQ517524。关键词:橄榄;ITS序列;克隆;测序     

ITS2 ribosomal DNA sequence variation of the bumblebee,Bombus ardens (hymenoptera: Apidae)

The bumblebee species,Bombus, is an invaluable natural resource for greenhouse pollination. Low levels of genetic variation ofBombus ardens have been reported in a previous mitochondrial (mt) gene study. In this study, we sequenced the complete internal transcribed spacer 2 (ITS2) of the nuclear rDNA obtained from 100B. ardens individuals collected from several Korean localities, in an effort to assess its usefulness in characterizing the genetic diversity and relationships among populations of B. ardens. The ITS2 sequences ofB. ardens were shown to be longest among known insects, ranging in size from 1,971–1,984 bp. The sequences harbor four duplicated repeats-≈27 bp repeats, ≈20 bp repeats, ≈33 bp repeats, and ≈34 bp repeats-which have never before been reported in other insect ITS2 rDNA. The maximum sequence divergence of 1.01% among 96 sequence types confirmed the applicability of this molecule to the study of intraspecific variation, revealing higher sequence variation as compared to the previously studied mt COI gene. Overall, a very high per generation migration ratio (Nm = 5.83 ≈ infinite) and a very low level of genetic fixation (FST =0 –0.08) were noted to exist among populations ofB. ardens. The high estimation of gene flow among most populations-in particular, between the remote island Ulleungdo and several inland populations-suggest that historical events may be more responsible for the contemporary population structure of B. ardens. The finding of the lowest genetic diversity (π) in the population on Ulleungdo Island (π = 0.007434) may be reflective of a relatively small population size and the geographical isolation of the population as compared with other inland populations.     

Study on Sequences of Ribosomal DNA Internal Transcribed Spacers of Clams Belonging to the Veneridae Family (Mollusca: Bivalvia)

The first and second internal transcribed spacer (ITS1 and ITS2) regions of the ribosomal DNA from four species, Meretrix meretrix L., Cyclina sinensis G., Mercenaria mercenaria L., and Protothaca jedoensis L., belonging to the family Veneridae were amplified by PCR and sequenced. The size of the ITS1 PCR amplification product ranged from 663 bp to 978 bp, with GC contents ranging from 60.78% to 64.97%. The size of the ITS1 sequence ranged from 585 bp to 900 bp, which is the largest range reported thus far in bivalve species, with GC contents ranging from 61.03% to 65.62%. The size of the ITS2 PCR amplification product ranged from 513 bp to 644 bp, with GC contents ranging from 61.29% to 62.73%. The size of the ITS2 sequence ranged from 281 bp to 412 bp, with GC contents ranging from 65.21% to 67.87%. Extensive sequence variation and obvious length polymorphisms were noted for both regions in these species, and sequence similarity of ITS2 was higher than that of ITS1 across species. The complete sequences of 5.8S ribosomal RNA gene were obtained by assembling ITS1 and ITS2 sequences, and the sequence length in all species was 157 bp. The phylogenetic tree of Veneridae clams was reconstructed using ITS2-containing partial sequences of both 5.8S and 28S ribosomal DNA as markers and the corresponding sequence information in Arctica islandica as the outgroup. Tree topologies indicated that P. jedoensis shared a close relationship with M. mercenaria and C. sinensis, a distant relationship with other species.     

奥利亚罗非鱼与尼罗罗非鱼rDNA内转录间隔区序列特征

核糖体DNA内转录间隔区(internal transcribed spacers,ITS)是经常被用作种和种群水平系统研究的分子序列.本文分离了奥利亚罗非鱼(Oreochromis aureus)、尼罗罗非鱼(O.niloticus)内转录间隔区,包括部分185序列,ITS1、5.8S、ITS2全序列及部分28S序列.4尾奥利亚罗非鱼的10个克隆序列分析表明,其存在长度不同的a、b两种类型ITS1.a型长为536 bp,GC含量为69.96%;b型长为520 bp,GC含量为69.04%～69.42%.4尾尼罗罗非鱼的10个克隆序列分析表明,其只存在a型ITS1,长为536～540 bp,GC含量为69.42%～70.19%.与b型ITS1相比,a型ITS1在16～31 nt有16 bp片段(GGCCCGCCTCGGCGC)的插入.奥利亚罗非鱼和尼罗罗非鱼共20条ITS序列中,5.8S长度均为157 bp,GC含量为56.69%～57.96%;ITS2为408 bp,GC含量为72.79%～74.26%.奥利亚罗非鱼和尼罗罗非鱼ITS区序列相似性高达98.2%,表明这两种罗非鱼亲缘关系很近.此外,本文对14尾奥利亚罗非鱼、15尾尼罗罗非鱼以及15尾奥尼罗非鱼[O.aureus(♂)×O.niloticus(♀)]ITS1的扩增结果显示,奥利亚罗非鱼均有a、b两种类型ITS1;15尾尼罗罗非鱼中1尾为a、b两类型ITS1,14尾为a型ITS1;15尾奥尼罗非鱼中则有6尾具有a、b两类型ITS1,9尾为单一的a型ITS1.分析表明,奥利亚罗非鱼在ITS1这个位点一致性高,但尼罗罗非鱼中有1尾混杂了奥利亚罗非鱼的基因,同时也说明分子生物学手段应用于种质鉴定比形态学手段更为精确.     

Multiple Divergent ITS1 Copies Were Identified in Single Tomato Genome Using DGGE Analysis

The intra-genomic variation in the internal transcribed spacer (ITS) region has led to misleading conclusions in the evolutionary analysis of plants; understanding this variation is critical for correct evolutionary analysis based on ITS sequences. To reveal the ITS variation in tomato, entire copies of ITS1 sequences within tomato species were separated using denaturing gradient gel electrophoresis (DGGE) and DNA sequence analysis. ITS1 copies varied significantly in sequence composition, but not in sequence length within the same tomato cultivar. DNA sequence similarity of the ITS1 copies was 77–100 %. Moreover, AT and GC contents in ITS1 copies from each tomato cultivar were significantly different, ranging from 50.4 to 64.3 % for GC and from 35.7 to 49.6 % for AT. However, the length variation of ITS1 was insignificant, ranging from 279 to 282 bp. Multiple copies of divergent ITS1 present in the tomato genome indicate that some copies may be paralogues. In conclusion, DGGE technique is a reliable and novel approach to reveal the entire ITS copy variation and the possible evolutionary relationship of tomato.     

Genetic variation in 16S-23S rDNA internal transcribed spacer regions and the possible use of this genetic variation for molecular diagnosis of Bacteroides species

The structural variation in 16S-23S rDNA internal transcribed spacer regions (ITS) among Bacteroides species was assessed by PCR amplification and sequencing analysis, and its possible use for molecular diagnosis of these species was evaluated. Ninety strains of the genus Bacteroides, including the species B. distasonis, B. eggerthii, B. fragilis, B. ovatus, B. thetaiotaomicron, B. uniformis and B. vulgatus, produced one to three ITS amplification products with sizes ranging from 615 to 810 bp. Some Bacteroides strains could be differentiated at species level on the basis of ITS amplification patterns and restriction fragment length polymorphism (RFLP) analysis using a four-nucleotide-recognizing enzyme, Msp I. The results of sequence analysis of ITS amplification products revealed genes for Ile-tRNA and Ala-tRNA in all strains tested. The nucleotide sequence, except for that in tRNA-coding regions, was highly variable and characteristic for each species, but a common sequence among B. fragilis, B. thetaiotaomicron and B. ovatus was observed. A digoxigenin-labeled oligonucleotide probe (named FOT1), which was designed from this conserved sequence, specifically hybridized to the ITS amplification products from B. fragilis, B. thetaiotaomicron and B. ovatus. These results suggest that the ITS region is a useful target for the development of rapid and accurate techniques for identification of Bacteroides species.     

Intraspecific diversity of the Cassava Green Mite Mononychellus progresivus (Acari: Tetranychidae) using comparisons of mitochondrial and nuclear ribosomal DNA sequences and cross-breeding

Intraspecific diversity in Cassava Green Mite Mononychellus progresivus Doreste was examined using individuals collected in Benin and the Congo and in Columbia and Brazil. Comparisons were based on mitochondrial and ribosomal DNA sequences and the results of several cross-breeding experiments. Sequence variation was examined in a total of 1139 base pairs (bp) constituting the ITS2 ribosomal DNA (805 bp) and a fragment of the Cytochrome Oxidase I (COI) gene (334 bp). Sequence divergence is low, ranging from 0% to 2.1% for COI and from 0% to 0.4% for ITS2. Inter-strain comparisons have shown that the two African populations appear to be identical. They were similar to the Colombian population while the Brazilian population was clearly different. The data support the hypothesis of a single introduction of the species in the two African populations. Crossing experiments have shown partial hybrid sterility, suggesting a genetic incompatibility consistent with differences detected by sequence data. The results show the usefulness of molecular markers as a tool for determining taxonomic status and dispersion paths in spider mites.     

Preliminary Analysis of Length and GC Content Variation in the Ribosomal First Internal Transcribed Spacer (ITS1) of Marine Animals

Length and guanine–cytosine (GC) content of the ribosomal first internal transcribed spacer (ITS1) were compared across a wide variety of marine animal species, and its phylogenetic utility was investigated. From a total of 773 individuals representing 599 species, we only failed to amplify the ITS1 sequence from 87 individuals by polymerase chain reaction with universal ITS1 primers. No species was found to have an ITS1 region shorter than 100 bp. In general, the ITS1 sequences of vertebrates were longer (318 to 2,318 bp) and richer in GC content (56.8% to 78%) than those of invertebrates (117 to 1,613 bp and 35.8% to 71.3%, respectively). Specifically, gelatinous animals (Cnidaria and Ctenophora) were observed to have short ITS1 sequences (118 to 422 bp) with lower GC content (35.8% to 61.7%) than the other animal taxa. Mollusca and Crustacea were diverse groups with respect to ITS1 length, ranging from 108 to 1,118 and 182 to 1,613 bp, respectively. No universal relationship between length and GC content was observed. Our data indicated that ITS1 has a limited utility for phylogenetic analysis as obtaining confident sequence alignment was often impossible between different genera of the same family and even between congeneric species.     

串珠藻目分子系统学研究进展

串珠藻目(Batrachospermales)是淡水红藻中最主要的类群。近年来, 应用DNA序列分析探讨串珠藻目的系统发育, 并与传统的形态学和生态学特征相结合, 为串珠藻目系统学研究拓展了新的思路。本文回顾了串珠藻目的建立及其所含类群的研究历史, 归纳了目前在串珠藻目系统发育与进化研究中常用的分子标记方法, 其中包括核基因组的18S rDNA、26S rDNA和ITS序列, 叶绿体基因组的rbcL序列, 线粒体基因组的cox2-3序列, 以及新兴的ISSR技术, 并对各种分子标记的特点及适用范围做了评述。结果表明, ITS序列多适用于种群分化及相近种间遗传分析, ISSR标记适用于种下分类群间及同一种群不同个体间基因多态性分析, cox2-3序列在一定程度上也可用于同一种群不同个体间的基因多态性分析, 而18S rDNA 与rbcL序列既可用于种间关系分析, 又可用于更高水平分析的构建系统树。这些分子标记已被证明在研究串珠藻目系统地理、物种起源和散布机制方面有着广泛的应用前景。同时, 本文对串珠藻目分子系统学研究的最新进展也进行了概述,并对今后的研究方向做了展望。