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。     

DNA序列在悬钩子属植物分子系统学研究中的应用进展

悬钩子属植物种类繁多,类群复杂,而且多为多倍体和杂种。该文就近年来国内外有关DNA序列在悬钩子属植物分子系统学研究中的应用现状和进展进行了综述,并对中国悬钩子属植物系统发育研究进行了展望。研究认为:叶绿体DNA序列多应用非编码区,且多与ITS序列联合分析;核基因组中ITS序列应用最为广泛,主要用于研究悬钩子属空心莓组与木莓组的进化关系、栽培品种间亲缘关系及部分杂种和多倍体的起源等;在该属植物中发现了ITS个体内多态性,但未进行ITS假基因检测,其系统学应用价值需重新评价;低拷贝核基因只有GBSSI和LEAFY有相关应用。同时认为,悬钩子属植物系统学研究中应用的DNA序列及研究类群均较少,缺乏对整个悬钩子属全面而系统的研究。指出应进一步选择具有代表性的样本、筛选合适的DNA片段,并结合形态学、孢粉学和细胞学等手段对中国悬钩子属植物系统关系进行深入研究。     

DNA 序列在蕨类分子系统学研究中的应用

在分子系统学研究中, 目的基因或者基因片段的选择是最关键的一步, 由于进化速率的差异, 不同的DNA序列适用于不同分类阶元的系统发育研究。本文综述了目前蕨类分子系统发育研究中常用的DNA序列分析, 它们分别来自叶绿体基因组、核基因组和线粒体基因组, 着重阐明叶绿体基因在蕨类分子系统学研究中的应用。本文还简要介绍了分子系统学研究中常见的问题及解决方法(如内类群和外类群的选择, 适宜DNA片段的选择策略), 总结了目前蕨类植物分子系统学研究所取得的进展和研究现状, 展望了当今国际蕨类分子系统学的研究趋势。     

DNA序列在蕨类分子系统学研究中的应用

在分子系统学研究中,目的基因或者基因片段的选择是最关键的一步,由于进化速率的差异,不同的DNA序列适用于不同分类阶元的系统发育研究.本文综述了目前蕨类分子系统发育研究中常用的DNA序列分析,它们分别来自叶绿体基因组、核基因组和线粒体基因组,着重阐明叶绿体基因在蕨类分子系统学研究中的应用.本文还简要介绍了分子系统学研究中常见的问题及解决方法(如内类群和外类群的选择.适宜DNA片段的选择策略),总结了目前蕨类植物分子系统学研究所取得的进展和研究现状,展望了当今国际蕨类分子系统学的研究趋势.     

ITS假基因对栎属系统学研究的影响及其对分子系统学研究的启示

核糖体rDNA ITS是被子植物系统发育研究中应用最广泛的分子标记之一。以前人们认为同一物种中的ITS序列因致同进化而使不同拷贝高度一致,在分子系统学研究中常以ITS1-5.8S-ITS2序列作为构建系统进化树的基础。近年来,在对一些被子植物的研究中发现这段序列在同一物种中具有多态性,有些拷贝中的5．8S区不具编码功能,人们把含有不具编码功能5．8S区的ITS1-5．8S-ITS2序列定义为ITS假基因序列,它对同源基因致同进化的假设形成了新的挑战。在诸多应用ITS序列重建系统进化关系的研究中,栎属系统学研究因ITS假基因的发现而倍受关注。本文以栎属为例回顾了ITS假基因的发现过程,分析了其对该属系统学研究的影响,为分子生物学在植物系统进化研究中的应用提供一些新的参考。     

ITS 假基因对栎属系统学研究的影响及其对分子系统学研究的启示*

核糖体rDNA ITS 是被子植物系统发育研究中应用最广泛的分子标记之一。以前人们认为同一物种
中的ITS 序列因致同进化而使不同拷贝高度一致, 在分子系统学研究中常以ITS1- 518S- ITS2 序列作为构建
系统进化树的基础。近年来, 在对一些被子植物的研究中发现这段序列在同一物种中具有多态性, 有些拷
贝中的518S 区不具编码功能, 人们把含有不具编码功能518S 区的ITS1-51 8S- ITS2 序列定义为ITS 假基因序
列, 它对同源基因致同进化的假设形成了新的挑战。在诸多应用ITS 序列重建系统进化关系的研究中, 栎
属系统学研究因ITS 假基因的发现而倍受关注。本文以栎属为例回顾了ITS 假基因的发现过程, 分析了其
对该属系统学研究的影响, 为分子生物学在植物系统进化研究中的应用提供一些新的参考。     

串珠藻目植物的系统发育-基于rbcL序列的证据

世界范围内报道的全部串珠藻目种类均生活于淡水中,而在淡水红藻中,70%约有130种属于串珠藻目。研究以目前获取的来自世界各大洲串珠藻目植物43种的rbcL基因序列,结合其形态和生物地理特征,构建了该目的系统发育关系,以期探讨整个串珠藻目植物的系统发育关系及发生途径,进而为研究该目以至淡水红藻的起源提供基本资料。运用PAUP*4.0b10和MrBayes 3.0b4等软件对43种串珠藻目植物的叶绿体DNA rbcL基因序列进行系统发育分析,探讨了其主要分类群的系统演化关系。用最大简约法、邻接法和贝叶斯分析方法构建的系统树基本一致,结果显示:(1)基于分子数据分析结果显示,红索藻目植物均独立于串珠藻目植物,构成一个单独的分支,支持红索藻目的建立。(2)鱼子菜科属于串珠藻目植物中较为进化的类群。(3)串珠藻属扭曲组与杂生组的差异度较小,结合其形态特点,倾向于将杂生组并入扭曲组。(4)串珠藻科属于串珠藻目中最大的科,包括较多的种类,其系统关系也较为复杂。因此,串珠藻科系统发育关系的明确有待于进一步结合更多的分子数据和形态学特征加以分析研究。       

DNA条形码在淡水红藻中的应用评价——基于串珠藻科植物

研究采用4种DNA序列, 分析了各片段序列特征以及在串珠藻科植物中种属水平的鉴定能力, 包括线粒体COI-5P、cox2-3 spacer序列, 以及叶绿体rbcL、UPA序列。结果表明, COI-5P、cox2-3 spacer、UPA以及rbcL序列的PCR扩增成功率分别为96%、100%、96%和98%。其中, COI-5P、cox2-3 spacer和UPA的片段大小符合标准DNA条形码的判定标准, 即片段大小在300—800 bp, 能够通过单对引物双向测序获得。系统发育分析的结果显示, 这4种DNA片段在串珠藻属植物的鉴定中能够鉴定大部分的种类, 但COI-5P、cox2-3 spacer以及rbcL序列均不能将两种中国特有种洪洞串珠藻B. hongdongense和长柄串珠藻B. longipedicellatum与弧形串珠藻B. arcuatum分开。在种水平的鉴定中, UPA基因的种间差异最大, 显示了较好的分离效果, 在串珠藻科植物的鉴定中可以作为一个标准的DNA条形码。     

植物分子系统学近五年的研究进展概况

本文综述了与分子系统学发展密切相关的4个因素：1.分子生物学方法的不断改进;2.基因组的全序列测定;3.用于分子系统学研究的基因种类不断增加,对这些基因进化规律的认识不断深入;4.化石DNA的研究。本文还阐述了核基因及叶绿体基因在系统学研究中的应用,例举了rbcL、matk、18s rDNA和ITS序列分析在植物系统发育研究中取得的重要成果,同时提出了分子系统学研究中应注意的一些问题。     

基于叶绿体DNA trnL内含子序列数据的檀香目科间系统发育关系的研究

应用叶绿体DNAtrnL内含子序列分析檀香目科间的系统发育关系。取样研究的檀香目个体的trnL内含子序列长度在科间呈现较大差异(从291bp到587bp)。最大简约性分析产生的严格一致树与以前已发表的基于其它基因的檀香目的分子系统学研究结果大体一致。香芙木属(铁青树科)是最早分支出的类群：桑寄生科、槲寄生科分别表现为单系类群,檀香科为并系;桑寄生科和槲寄生科并不具密切亲缘关系,槲寄生科从檀香科内衍生出来。本研究表明,具相对高的核苷酸替换率的叶绿体DNAtrnL内含子序列可为高等级类群系统发育关系的研究提供更多的信息位点。     

Application of DNA barcodes in Hedyotis L.(Spermacoceae, Rubiaceae)

The potential application of DNA barcodes of plastid (matK, trnH-psbA, petD, and rbcL) and nuclear (internal transcribed spacer (ITS) of rDNA) DNA regions was investigated for 25 Hedyotis taxa. The ITS showed the best species discrimination by resolving 23 of the species as exclusive lineages with no shared alleles between any of the 24 distinct species (H. Assimilis and H. Mellii are not supported as distinct species based on our molecular and morphological data). Conversely, rbcL performed the worst and only resolved 10 of the species as exclusive lineages, and 10 species with shared alleles. Using ITS has the advantage of high PCR amplification success and it provides good intra- and interspecific variation distribution patterns. The most powerful plastid markers were petD and trnH-psbA, but we could amplify and sequence trnH-psbA for only 83% of the accessions sampled. Combination of ITS and petD performed extremely well, with all 24 of the distinct species resolved as exclusive lineages and no shared alleles between any of the distinct species. We therefore recommend ITS, or a combination of ITS and petD, as the standard DNA barcode in Hedyotis, but acknowledge that there are no shared alleles between distinct species for marK and rbcL combined.     

SYNTHESIS OF MOLECULAR RESEARCH ON BATRACHOSPERMUM HELMINTHOSUM (RHODOPHYTA) FROM STREAM REACHES IN EASTERN NORTH AMERICA

Vis, M. L., Hall, M. M., Machesky, N. J. & Miller, E. J. Department of Environmental and Plant Biology, Ohio University, Athens, OH 45701 USA The freshwater red alga Batrachospermum helminthosum was collected from eleven streams throughout the species range in eastern North America as follows: three stream reaches from Ohio, and one each from Michigan, Indiana, Tennessee, Louisiana, North Carolina, Connecticut, Rhode Island and Massachusetts. The molecular marker technique of inter-simple sequence repeats (ISSR) and sequence data from the plastid encoded rubisco large subunit gene (rbcL), the mitochondrial COX2-COX3 gene spacer region, and the nuclear region of ITS1-5.8S rDNA-ITS2 were employed to examine biogeographic trends in this alga. Analysis of the rbcL sequence revealed 5 genotypes with one genotype representing individuals from seven stream reaches. Data from the ISSR molecular markers gave a distinct banding pattern for each of 165 individuals examined. ISSR results showed all individuals within a reach clustered together but did not provide well-defined groupings based on stream reach. The sequence data for the COX2-COX3 gene spacer was invariant among individuals from a stream reach. The individuals from Connecticut, Rhode Island and 2 Ohio stream reaches were identical and similarly the individuals from the North Carolina and another Ohio location did not vary in sequence so that seven genotypes were recorded among the individuals from the eleven stream reaches. Analysis of the ITS1-5.8S rDNA-ITS2 region showed sequence variation not only among individuals from different streams but also among individuals from the same reach. The utility and congruency of these data sets to answer biogeographic questions will be discussed.     

Variation of nuclear and mitochondrial DNAs in Korean and Chinese isolates of Clonorchis sinensis

We compared the DNA sequence difference of isolates of Clonorchis sinensis from one Korean (Kimhae) and two Chinese areas (Guangxi and Shenyang). The sequences of nuclear rDNA (18S, internal transcribed spacer 1 and 2: ITS1 and ITS2) and mitochondrial DNA (cytochrome c oxidase subunit 1: cox1) were compared. A very few intraspecific nucleotide substitution of the 18S, ITS1, ITS2 and cox1 was found among three isolates of C. sinensis and a few nucleotide insertion and deletion of ITS1 were detected. The 18S, ITS1, ITS2 and cox1 sequences were highly conserved among three isolates. These findings indicated that the Korean and two Chinese isolates are similar at the DNA sequence level.     

Use of Nuclear and Mitochondrial Single Nucleotide Polymorphisms to Characterize English Walnut (Juglans regia L.) Genotypes

English walnut (Juglans regia L.) is the most economically important species, for both food and timber, of the 21 species belonging to the genus Juglans. This study was undertaken to analyze and compare DNA sequences of the mitochondrial cytochrome oxidase subunit II (COX2) and ribosomal DNA (rDNA) genes in the molecular characterization of 30 English walnut genotypes. rDNA sequences revealed the presence of 402 variations, including 101 in 3′ ends of 18S, 21 in internal transcribed spacer 1(ITS1), 170 in ITS2, 30 in 5.8S, and 80 in 5′ ends of 28S regions. Cox2 intron I sequences showed 769 variable positions and GG insertion/deletion at 3′ end regions. Based on single nucleotide polymorphism markers of rDNA and cox2 intron I sequences, an amplification refractory mutation system was used to fingerprint 18 out of 30 walnut genotypes. The findings revealed that the cox2 intron I region, either alone or in conjunction with rDNA, could be used effectively in identifying these walnut genotypes.     

A Conserved Motif in the 5.8S Ribosomal RNA (rRNA) Gene is a Useful Diagnostic Marker for Plant Internal Transcribed Spacer (ITS) Sequences

The nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) region has become an important nuclear locus for molecular systematic investigations of angiosperms at the intergenic and interspecific levels. Universal PCR primers are positioned on the conserved rRNA genes (18S, 5.8S, 26S) to amplify the entire ITS spacer region. Recent reports of fungal and algal contaminants, first described as plant ITS sequences, stress the need for diagnostic markers specific for the angiosperm ITS region. This report describes a conserved 14 base pair (bp) motif in the 5.8S rRNA gene that can be used to differentiate between flowering plants, bryophytes, and several orders of algae and fungi, including common plant pathogenic and non-pathogenic fungi. A variant of the motif (found in fungi and algae) contains a convenient EcoRI restriction site that has several applications for eliminating problematic contaminants from plant ITS preparations.     

Application of DNA barcodes in Hedyotis L. (Spermacoceae,Rubiaceae)

The potential application of DNA barcodes of plastid (matK, trnH–psbA, petD, and rbcL) and nuclear (internal transcribed spacer (ITS) of rDNA) DNA regions was investigated for 25 Hedyotis taxa. The ITS showed the best species discrimination by resolving 23 of the species as exclusive lineages with no shared alleles between any of the 24 distinct species (H. assimilis and H. mellii are not supported as distinct species based on our molecular and morphological data). Conversely, rbcL performed the worst and only resolved 10 of the species as exclusive lineages, and 10 species with shared alleles. Using ITS has the advantage of high PCR amplification success and it provides good intra- and interspecific variation distribution patterns. The most powerful plastid markers were petD and trnH–psbA, but we could amplify and sequence trnH–psbA for only 83% of the accessions sampled. Combination of ITS and petD performed extremely well, with all 24 of the distinct species resolved as exclusive lineages and no shared alleles between any of the distinct species. We therefore recommend ITS, or a combination of ITS and petD, as the standard DNA barcode in Hedyotis, but acknowledge that there are no shared alleles between distinct species for matK and rbcL combined.     

Molecular relationships in Encephalartos (Zamiaceae, Cycadales) based on nucleotide sequences of nuclear ITS 1&2, rbcL, and genomic ISSR fingerprinting

The cycad genus Encephalartos is restricted to Africa and is threatened with extinction in most of its range. Total DNA was extracted from 51, i.e., 78 %, of the described species of Encephalartos. The accessions were sampled from the furthest western occurrence of the genus in Nigeria, via Sudan and Uganda, to southern South Africa. The sequences of nuclear ribosomal internal transcribed spacer regions 1 and 2 (ITS 1&2), the chloroplast encoded rbcL gene, and ISSR genomic fingerprinting were employed to resolve the molecular history and the relationships within the genus. Sequence alignment, as well as ISSR fingerprinting, data show low genetic variation among all analysed accessions, indicating diversification within the Pliocene/Pleistocene. ITS 1&2 data agree well with morphological and geographical characters and resolved three major genetic clusters with overlapping distribution ranges in eastern South Africa. This area, that contains the largest diversity of genotypes of Encephalartos, may have served as a Pliocene/Pleistocene refugium.     

A combined approach using ISSR and ITS analysis for the characterization of Artemisia halodendron from Horqin sandy land,northern China

Artemisia halodendron is the most dominant and constructive species among the Horqin sandy land. We evaluated its genetic variation and phylogenetic analysis within and among its populations sampled from six different habitats gradient from the Horqin sandy land in the northeast of China by using inter-simple sequence repeat polymorphism (ISSR) and the nuclear ribosomal internal transcribed spacer (ITS) molecular markers. The results showed that eight ISSR primers generated 84 bands, of which 48 (57.14%) were polymorphic. The highest genetic diversity was observed in the inter-dune lowland population, whereas the lowest diversity was found in the mobile dune population. The AMOVA analysis revealed a relatively high level of genetic variation within its populations. The alignment of 124 ITS sequences showed 401 variable sites and the GC content ranged from 54.02% to 57.32%. The ITS tree revealed the existence of three major clades, and that the semi-mobile dune population was more closely related to the inter-dune lowland population.     

Sequence variability in two mitochondrial DNA regions and internal transcribed spacer among three cestodes infecting animals and humans from China

Sequence variability in two mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 4 (nad4), and internal transcribed spacer (ITS) of rDNA among and within three cestodes, Spirometra erinaceieuropaei, Taenia multiceps and Taenia hydatigena, from different geographical origins in China was examined. A portion of the cox1 (pcox1), nad4 genes (pnad4) and the ITS (ITS1+5.8S rDNA+ITS2) were amplified separately from individual cestodes by polymerase chain reaction (PCR). Representative amplicons were subjected to sequencing in order to estimate sequence variability. While the intra-specific sequence variations within each of the tapeworm species were 0-0.7% for pcox1, 0-1.7% for pnad4 and 0.1-3.6% for ITS, the inter-specific sequence differences were significantly higher, being 12.1-17.6%, 18.7-26.2% and 31-75.5% for pcox1, pnad4 and ITS, respectively. Phylogenetic analyses based on the pcox1 sequence data revealed that T. multiceps and T. hydatigena were more closely related to the other members of the Taenia genus, and S. erinaceieuropaei was more closely related to the other members of the Spirometra genus. These findings demonstrated clearly the usefulness of mtDNA and rDNA sequences for population genetic studies of these cestodes of socio-economic importance.