ITS1: a DNA barcode better than ITS2 in eukaryotes?

A DNA barcode is a short piece of DNA sequence used for species determination and discovery. The internal transcribed spacer (ITS/ITS2) region has been proposed as the standard DNA barcode for fungi and seed plants and has been widely used in DNA barcoding analyses for other biological groups, for example algae, protists and animals. The ITS region consists of both ITS1 and ITS2 regions. Here, a large‐scale meta‐analysis was carried out to compare ITS1 and ITS2 from three aspects: PCR amplification, DNA sequencing and species discrimination, in terms of the presence of DNA barcoding gaps, species discrimination efficiency, sequence length distribution, GC content distribution and primer universality. In total, 85 345 sequence pairs in 10 major groups of eukaryotes, including ascomycetes, basidiomycetes, liverworts, mosses, ferns, gymnosperms, monocotyledons, eudicotyledons, insects and fishes, covering 611 families, 3694 genera, and 19 060 species, were analysed. Using similarity‐based methods, we calculated species discrimination efficiencies for ITS1 and ITS2 in all major groups, families and genera. Using Fisher's exact test, we found that ITS1 has significantly higher efficiencies than ITS2 in 17 of the 47 families and 20 of the 49 genera, which are sample‐rich. By in silico PCR amplification evaluation, primer universality of the extensively applied ITS1 primers was found superior to that of ITS2 primers. Additionally, shorter length of amplification product and lower GC content was discovered to be two other advantages of ITS1 for sequencing. In summary, ITS1 represents a better DNA barcode than ITS2 for eukaryotic species.     

The New Species Concept in Dermatophytes—a Polyphasic Approach

The dermatophytes are among the most frequently observed organisms in biomedicine, yet there has never been stability in the taxonomy, identification and naming of the approximately 25 pathogenic species involved. Since the identification of these species is often epidemiologically and ethically important, the difficulties in dermatophyte identification are a fruitful topic for modern molecular biological investigation, done in tandem with renewed investigation of phenotypic characters. Molecular phylogenetic analyses such as multilocus sequence typing have had to be tailored to accommodate differing the mechanisms of speciation that have produced the dermatophytes that are commonly seen today. Even so, some biotypes that were unambiguously considered species in the past, based on profound differences in morphology and pattern of infection, appear consistently not to be distinct species in modern molecular analyses. Most notable among these are the cosmopolitan bane of nails and feet, Trichophyton rubrum, and the endemic African agent of childhood tinea capitis, Trichophyton soudanense, which are effectively inseparable in all analyses. The molecular data require some reinterpretation of results seen in conventional phenotypic tests, but in most cases, phylogenetic insight is readily integrated with current laboratory testing procedures.     

Is Leadership a Reliable Concept in Animals? An Empirical Study in the Horse

Leadership is commonly invoked when accounting for the coordination of group movements in animals, yet it remains loosely defined. In parallel, there is increased evidence of the sharing of group decisions by animals on the move. How leadership integrates within this recent framework on collective decision-making is unclear. Here, we question the occurrence of leadership in horses, a species in which this concept is of prevalent use. The relevance of the three main definitions of leadership – departing first, walking in front travel position, and eliciting the joining of mates – was tested on the collective movements of two semi-free ranging groups of Przewalski horses (Equus ferus przewalskii). We did not find any leader capable of driving most group movements or recruiting mates more quickly than others. Several group members often displayed pre-departure behaviours at the same time, and the simultaneous departure of several individuals was common. We conclude that the decision-making process was shared by several group members a group movement (i.e., partially shared consensus) and that the leadership concept did not help to depict individual departure and leading behaviour across movements in both study groups. Rather, the different proxies of leadership produced conflicting information about individual contributions to group coordination. This study discusses the implications of these findings for the field of coordination and decision-making research.     

药用野生稻复合体ITS1和ITS2序列变异及其系统进化分析

通过PCR扩增并测序分析稻属药用野生稻复合体5个野生稻种基因组完整的ITS区及5.8S区,并与栽培稻ITS序列进行比较,构建分子系统进化树,探讨了稻属药用野生稻复合体内不同种间的亲缘关系和系统进化.结果表明,ITS1和ITS2均有较高的G/C含量,ITS1序列的长度多态性相对较高,ITS2序列的碱基突变频率较高.药用野生稻和高秆野生稻亲缘关系很近,而与栽培稻亲缘关系较远;短药野生稻、斑点野生稻、澳洲野生稻与药用野生稻亲缘关系渐近.处于进化的过渡阶段.     

米尔顿姬小蜂rDNA ITS1和ITS2的序列分析及其分子鉴定

本研究测定了米尔顿姬小蜂Anselmella miltoni Girault的rDNA ITS1和ITS2序列,以探讨其分子鉴定方法。米尔顿姬小蜂的ITS1和ITS2侧翼区（18S和5.8S）序列相对稳定,ITS1和ITS2序列存在种间差异。根据18S rDNA部分序列,利用DNAMAN的Maximum Likelihood方法构建了与膜翅目其它科的系统发育树。根据米尔顿姬小蜂ITS1和ITS2序列设计了特异性引物,应用特异性引物对样品进行了PCR扩增,扩增效果理想,采用上述特异性引物可从单头米尔顿姬小蜂稳定地扩增出明显的目的DNA条带。因此,可以采用ITS1和ITS2区的特异性对米尔顿姬小蜂进行快速的分子鉴定。     

BONE BANKS—A New Concept in Procurement and Storage for Homologous Grafts

An unlimited supply of bones, obtained post-mortem and irradiated to make them sterile, is stored for as long as six months in a freezer, ready for use. After an immune response at first, bone thus treated is readily incorporated by the host in grafting procedures.     

基于ITS2序列的川贝母中成药的鉴定

润肺圣药川贝母资源匮乏,为国家三级保护植物.近年雾霾天气频发使得其资源需求加大,导致以川贝母入药的中成药掺假现象严重.中成药成分复杂,单利用普通的DNA条形码鉴定流程无法对其一次性完成鉴定,因此需结合单克隆分析逐一鉴定.本研究首先建立了包含贝母属208条ITS2序列的数据库;收集了市售20份川贝中成药,其中包含15份蛇胆川贝胶囊,4份川贝末胶囊和1份蛇胆川贝散.扩增其ITS2序列,对PCR产物进行克隆测序,对测序结果进行BLAST比对;同时本实验还选取了3份蛇胆川贝胶囊进行高通量测序,利用单克隆和二代测序相结合的混合测序方法对该3份中成药中川贝母再次鉴定.基于二代测序数据对中成药组分解析存在数据冗余现象,而单克隆结果与选取的克隆数量有关,二者结合可相互弥补缺陷.单克隆结果数据表明,15份蛇胆川贝胶囊中3份含有平贝母,10份含有伊贝母及14份含有黄花贝母等非标签成分,4份川贝末胶囊中有2份检测到川贝母,2份检测到平贝母,但同时3份中还检测到黄花贝母;1份蛇胆川贝散中仅检测到伊贝母与黄花贝母.二代测序结果表明,3份蛇胆川贝胶囊中主要检测出平贝母、伊贝母等非标签成分.通过比对二者序列,发现无论克隆还是二代测序数据均表明混合测序的3份蛇胆川贝胶囊中均不含川贝母;本研究表明,基于单克隆和二代测序辅助的方法可以准确对川贝母中成药进行鉴定.目前川贝母中成药掺假现象严重,平贝母和伊贝母是川贝母中成药的主要掺伪品,应加强对中成药市场的监管.     

A phylogenetic analysis of Momordica (Cucurbitaceae) in India based on karyo-morphology,nuclear DNA content and rDNA ITS1–5.8S–ITS2 sequences

The infrageneric delimitation of Momordica, a medicinally important genus of Cucurbitaceae, is ill-defined until date. Momordica chromosomes are extremely small and are difficult to stain and visualize because of the dense cytoplasmic background. We have conducted karyomorphometric analysis by EMA method in five Indian Momordica species, and the nuclear genome sizes were estimated by flow cytometry for the first time. The somatic chromosome numbers ranged from 2n = 18 to 56 in the species. We have resolved previously disputed chromosome numbers in M. cymbalaria and M. dioica as 2n = 18 (lowest) and 2n = 56, respectively. Chromosome counts in the other species were re-confirmed as 2n = 22 in M. charantia, 2n = 28 in M. cochinchinensis and 2n = 56 in M. subangulata. The largest genome size was recorded in M. cymbalaria (3.74 pg 2C⁻¹), while the smallest size (0.72 pg 2C⁻¹) was detected in M. charantia var. charantia. The nuclear genome sizes were analysed in comparison to chromosome numbers and total chromosome lengths of the species. Karyomorphometric indices showed comparable symmetric karyotypes in the species except in M. cymbalaria having tendency towards asymmetry. The UPGMA phenogram and principle component analysis based on nuclear DNA contents and karyomorphometric parameters demonstrated interspecies differences, intraspecific distinction within M. charantia varieties and highlighted distinction of M. cymbalaria. This study was further supported by the rDNA ITS sequence-based phylogenetic analysis which revealed the monophyletic origin of the Indian members of Momordica and clarified the intraspecies relationship among the studied members. As a whole, the study brought out new insights on species diversification within the genus Momordica in India and would benefit further studies on biosystematics and plant breeding programmes.

     

五种不同蛏类ITS2核苷酸序列分析

对5种不同蛏类的ITS2核苷酸序列进行扩增、测序及分析.结果表明:5种蛏类的509个序列位点中共有49个保守住点,402个变异位点,318个简约信息位点,84个单突变位点.5种不同蛏类遗传距离在1.194～3.173之间,总角截蛏和小刀蛏的遗传距离较近,而与其他3种蛏类的遗传距离较远,遗传距离在1.965～3.173之间.缢蛏、长竹蛏和细长竹蛏3种蛏之间的遗传距离较其他2种蛏近,遗传距离在1.194～1.318之间.同种蛏类不同个体之间的遗传距离很近,遗传差异很小.     

Concept of immunomics: a new frontier in the battle for gene function?

At the beginning of the 21^st century, biology will try to address the function of a large number of new genes. From the perspective of technologies applied today to functional genomics, this task appears to be more complex than the effort invested in the sequencing of the human genome. Conceptually, a high-throughput approach permitting correlation between newly discovered genes and functional properties of their protein products has yet to be developed. To address relationships between tens of thousands of genes and their cognate proteins, novel interdisciplinary technologies need to emerge. In this paper, a new idea of immunomics is presented and an experimental strategy is outlined to circumvent some of the restrictions associated with methodologies currently in use. It is proposed that cloned segments of genomic DNA are used for genetic immunization to obtain a large collection of antibodies, and to generate microarrays of these antibodies for tracing differentially expressed cellular proteins.     

裸子植物DNA条形码ITS2高通用性引物的筛选

DNA条形码技术是利用标准DNA片段进行准确快速鉴定物种的一种方法,理想的DNA条形码片段应具有高通用性。虽然核糖体DNA内部转录间隔区II（ITS2）被建议作为种子植物有效的DNA条形码,但目前裸子植物还没有通用性高的引物可用。为获得高通用性的ITS2引物,本研究基于裸子植物55个属的5．8S基因的保守序列区设计了3个正向引物,与已有的ITS反向引物组合,组成了7对ITS2引物进行通用性的评价。选取了裸子植物8目、12科和40属的56个种用于本文的研究。引物组合5．8SR／ITS4、5．8SRa／ITS4和5．8SF2／S3R因为在科水平评价中通用性低或者产生的PCR产物有双带,因而排除在全部物种水平上进一步评价。其余4对引物（GYM-5．8SF1／ITS4、GYM-5．8SFl／S3R、GYM-5．8SF2／ITS4和S2F／S3R）在56个物种的PCR检测中,均有100％的扩增率。基于PCR产物的亮度、序列质量和正反向引物覆盖率的综合评价,建议引物GYM_5．8SF2／ITS4作为裸子植物条形码片段ITS2最好的通用引物。     

Aggressive and Docile Colony Defence Patterns in Apis mellifera. A Retreater–Releaser Concept

Colony defence in Apis mellifera involves a variety of traits ranging from ‘aggressive’ (e.g. entrance guarding, recruitment of flying guards) to ‘docile’ (e.g. retreating into the nest) expression. We tested 11 colonies of three subspecies (capensis, scutellata, carnica) regarding their defensiveness. Each colony was selected as reportedly ‘aggressive’, ‘intermediate’ or ‘docile’ and consisted of about 10,000 bees. We applied three stimulation regimes (mechanical disturbance, exposure to alarm pheromones, and the combination of both) and measured their behaviours by tracking the rates of outflying bees at the entrance sites of the test hives. We provided evidence that for mechanical disturbances the test colonies resolved into two response types, if the ‘immediate’ defence response, assessed in the first minute of stimulation, was taken as a function of foraging: ‘releaser’ colonies allocated flying guards, ‘retreater’ colonies reduced the outside-hive activities. This division was observed irrespective of the subspecies membership and maintained in even roughly changing environmental conditions. However, if pheromone and mechanical stimulation were combined, the variety of colony defensiveness restricted to two further types irrespective of the subspecies membership: six of nine colonies degraded their rate of flying defenders with increasing foraging level, three of the colonies extended their ‘aggressiveness’ by increasing the defender rate with the foraging level. Such ‘super-aggressive’ colonies obviously are able to allocate two separate recruitment pools for foragers and flying defenders.     

Introduction of Alien Host–parasite Complexes in a Natural Environment and the Symbiota Concept

When a species is introduced in a new territory its parasites may also follow, thus forming a sort of biotic unit. We propose the term symbiota to indicate this host–parasite complex. This concept is at the basis of the present research in which 11 species of alien and native freshwater fish were collected by electrofishing in the Po river area (Northern Italy). The alien host fish Ictalurus melas and Silurus glanis were parasitized by only one monogenean species each. Lepomis gibbosus, of North American origin, hosted four monogeneans and Carassius carassius three. All these parasites should be considered as alien species, except those collected from C. carassius that should be considered in part natives and in part aliens.     

荒川库蠓rDNA ITS2的序列测定与分析

库蠓种类繁多,给人畜健康带来巨大危害.荒川库蠓(Culicoides arakawae)是卡氏住白虫的重要传播媒介,引起鸡的卡氏住白虫病,导致蛋鸡产蛋量下降、肉鸡体重下降甚至死亡(Li et al.,2000),给养鸡业带来巨大经济损失.如何对各种媒介库蠓进行正确的区分或鉴定,是摆在各位寄生虫学家面前的具体问题.目前,寄生虫的分类鉴别主要依据形态学特征,一旦遇到形态学相似的近缘种就非常棘手.     

长江白甲鱼ITS2序列结构和群体遗传多样性

对长江白甲鱼(Onychostoma sima)样本核DNA进行PCR扩增,获得白甲鱼核DNA上编码核糖体5.8SrRNA和28SrRNA基因的部分序列和完整的ITS2序列(876bp)。运用DNA分析软件对白甲鱼2个驯养群体(重庆水产研究所长寿湖珍稀鱼类繁育中心及涪陵鱼种场)进行了遗传多样性分析。结果表明:该序列平均T、C、A和G碱基组成为22%、32.5%、29.6%和15.8%,颠换Tv=40,转换Ts=10,转换和颠换比值为R=Ts/Tv=0.25。40个体都是单倍型,单倍型多样度为H=1.000,平均核苷酸差异系数K=5.978,核苷酸多样性Pi=0.0682。中性检验及聚类分析表明,两个群体没有分化成单一的群体,两个驯养群体遗传多样性高,种质质量良好。     

鲽科10种鱼类核糖体ITS2序列比较分析

为了解鲽科鱼类ITS2序列的多态性特征,本研究获得了鲽科10种鱼类310条ITS2序列,长度在419 ~ 486 bp之间。种内序列长度差异最小的为粒鲽（Clidoderma asperrima）（419 ~ 420 bp）和太平洋拟庸鲽（Hippoglossoides elassodon）（419 ~ 420 bp）,其次为北岩鲽（Lepidopsetta polyxystra）（447 ~ 452 bp）和刺黄盖鲽（Limanda aspera）（457 ~ 463 bp）,松木高眼鲽（Cleisthenes pinetorum）（452 ~ 462 bp）和圆斑星鲽（Verasper variegatus）（465 ~ 479 bp）的种内序列差异分别为10 bp和14 bp;其余4种鱼类根据长度差异（14 ~ 32 bp）分型为长（A型）、短（B型）序列类型,同时检测存在重组类型（R型）,其中长度差异最大的是钝吻黄盖鲽（Pseudopleuronectes yokohamae）（454 ~ 486 bp）,其次为尖吻黄盖鲽（P. herzensteini）（433 ~ 458 bp）、虫鲽（Eopsetta grigorjewi）（420 ~ 439 bp）、星突江鲽（Platichthys stellatus）（466 ~ 480 bp）。通过双参数模型（K2P）计算遗传距离可见,种内遗传距离多集中于0.002 ~ 0.027之间,仅星突江鲽和尖吻黄盖鲽因类型差异导致较高数值（0.043和0.053）;不同物种间遗传距离在0.046 ~ 0.180之间。10种鱼类ITS2的GC含量为63.95% ~ 70.16%;9种鱼类的二级结构均为具有5个分支（HelixⅠ ~ Ⅴ）的闭合环状结构,仅圆斑星鲽中由于存在Helix Ⅳ变异形成Helix Ⅳ-a和Helix Ⅳ-b而具有6个分支。基于ITS2构建的鲽科10种鱼类的系统进化树显示,不同种鱼类的克隆序列均单独聚支。序列的多态性特征分析表明,在具有不同序列类型的虫鲽、星突江鲽、尖吻黄盖鲽和钝吻黄盖鲽4种鲽科鱼类中,ITS2以非协同进化的方式存在,而其他6种鱼类为协同进化;虽然存在种间K2P遗传距离小于种内的个例,但ITS2在属间不同物种的区分上具有适用性。本研究结果丰富了鲽形目鱼类的ITS2数据,也将为鱼类的核糖体RNA序列多态性的研究提供科学依据。     

黑粉菌属某些种的核rDNA ITS2序列分析

本文报道从某些黑粉菌的黑粉孢子粉末中提取核糖体脱氧核酸(rDNA)后,用聚合酶链式反应(PCR)的方法,对其转录间区(ITS2)片断进行了扩增,核苷酸序列测定和分析,其结果表明异形黑粉菌(Ustilago anomala)与其它3种近似黑粉菌,科尔达黑粉菌(U. cordae),柳叶刺黑粉菌(U. bungeana)和网孢黑粉菌(U. reticulata)的rDNA ITS2序列区别很大,在被测定的190个碱基对中,差别达69个位点以上(26.3-36.3%),异形黑粉菌与这三种黑粉菌亲缘关系较远,在中国作者原定名为异形黑粉菌的种与在欧洲取材的科尔达黑粉菌相似程度较高.因此将在中国已报道的异形黑粉菌更正为科尔达黑粉菌,而柳叶刺黑粉菌与科尔达黑粉菌近似程度也较高,将柳叶刺黑粉菌作为科尔达黑粉菌的异名.     

普通小麦基因组最可能的4个供体的ITS1和ITS2序列及其亲缘关系

对普通小麦（ＴｒｉｔｉｃｕｍａｅｓｔｉｖｕｍＬ．）基因组（ＡＡＢＢＤＤ）最可能的供体－Ｔ．ｕｒａｔｒｔｕＴｈｕｍ．（ＡＡ）、Ｔ．ｍｏｎｏｃｃｕｍｖａｒ．ｂｏｅｏｔｉｃｕｍ（Ｂｏｉｓｓ．）ＭＫ（ＡＡ）、ＡｅｇｉｌｏｐｓｓｐｅｌｔｏｉｄｅｓＴａｕｓｃｈ．和Ａｅ．ｔａｕｓｃｈｉｉ（Ｃｏｓｓ．（ＤＤ）的核糖体ＲＮＡ基因ＩＴＳ区进行了ＰＣＲ扩增和克隆,并测定了ＩＴＳ１和ＩＴＳ２的ＤＮＡ序列,讨论和纠正了前人     

海参核糖体基因间隔区2(ITS2)的克隆及序列分析

为探讨刺参科海参和海参科海参的系统进化关系,本研究通过PCR技术获取19种刺参科和海参科海参的ITS2序列,从NCBI上获取瓜参(C. salma)的ITS2序列。结果表明ITS2序列具有长度多态性,从318 bp (绿刺参)到591 bp (白尼参属)。海参属的ITS2序列长度多态性高,ITS2的GC含量从56.7%(糙海参)到70.6%(瓜参)。海参ITS2序列保守性不高,仅有48个保守位点,其余均为变异位点。基于ITS2的系统进化树结果显示进化树主要分成两支,一支包括海参科的4个属:海参属、白尼参属、辐肛参属和格皮氏海参属。辐肛参属和格皮氏海参属为姐妹关系,二者聚在一起后与白尼参属聚为一支,随后再与海参属聚在一起。白尼参属和辐肛参属为单系,海参属为复系。另一支为C. salma和刺参科。梅花参属与刺参属聚为一支后,再与仿刺参属聚在一起,3个属都是单系。在20种海参中,S. naso与B. argus的遗传距离最大(6.415)。刺参属中,S. monotuberculatus和S. horrens遗传距离最近(0.012),海参属中,糙海参与H. fuscopunctata的遗传距离最大(3.24)。本研究为从分子水平上研究海参科和刺参科之间的系统进化关系奠定了基础。