DNA条形码在淡水红藻中的应用评价——基于串珠藻科植物（英文）

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rbc L、UPA序列。结果表明,COI-5P、cox2-3 spacer、UPA以及rbc L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rbc L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。     

DNA条形码技术在大型红藻分子鉴定中的应用

本研究对浙江省枸杞岛贻贝养殖区大型海藻资源进行了调查,在形态学观察的基础上,选取93株红藻样品运用DNA分子条形码技术进行分子鉴定分析。采用的3个分子标记分别为rbcL(叶绿体基因组中1,5-二磷酸核酮糖羧化/加氧酶大亚基基因),COI(细胞色C氧化酶亚基I)和UPA(23S rRNA基因片段,通用质体扩增区)基因片段。通过实验,我们共扩增出rbcL基因片段79条,扩增成功率为84.95%;共扩增出COI基因片段63条,扩增成功率为67.74%;共扩增出UPA基因片段76条,扩增成功率为81.72%。3个分子标记的测序成功率分别为:rbcL基因78.49%,COI基因29.03%,UPA基因72.04%。COI基因的扩增效率和测序成功率均低于rbcL和UPA基因。基于3个基因构建(Neighbor-joining,N-J)系统进化树表明,rbcL基因可以将红藻样品归类为5目,6科,7属,12种;COI基因可以将样品归类为4目,5科,5属,5种;UPA基因可以将样品归类为6目,7科,8属,11种。rbcL、COI和UPA 3个基因结合可以将样品归类为7目8科11属21种。研究表明rbcL基因以及UPA基因较适合做为鉴定大型红藻的分子标记,COI基因做为红藻分子标记的潜力有待进一步开发和研究。     

中国秋海棠属 (秋海棠科) 植物的DNA条形码评价

秋海棠属植物种类繁多,形态变异多样,导致种类的系统放置混乱,近缘种类鉴定困难。利用DNA条形码实现物种快速准确的鉴定技术具有不受形态特征约束的优势,为秋海棠属植物的分类鉴定提供了新的方法。本研究选择4个DNA条形码候选片段（rbcL,matK,trnH psbA,ITS）对中国秋海棠属26种136个个体进行了分析。结果显示：叶绿体基因rbcL,matK和trnH psbA种内和种间变异小,对秋海棠属植物的鉴别能力有限;ITS/ITS2种内和种间变异大,在本研究中物种正确鉴定率达到100%/96%,可考虑作为秋海棠属DNA条形码鉴定的候选片段。研究结果支持中国植物条形码研究组建议将核基因ITS/ITS2纳入种子植物DNA条形码核心片段中的观点。     

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。     

中国秋海棠属（秋海棠科）植物的DNA条形码评价

秋海棠属植物种类繁多,形态变异多样,导致种类的系统放置混乱,近缘种类鉴定困难。利用DNA条形码实现物种快速准确的鉴定技术具有不受形态特征约束的优势,为秋海棠属植物的分类鉴定提供了新的方法。本研究选择4个DNA条形码候选片段（rbcL,matK,trnH-psbA,ITS）对中国秋海棠属26种136个个体进行了分析。结果显示：叶绿体基因rbcL,matK和trnH-psbA种内和种间变异小,对秋海棠属植物的鉴别能力有限：ITS／ITS2种内和种间变异大,在本研究中物种正确鉴定率达到100％／96％,可考虑作为秋海棠属DNA条形码鉴定的候选片段。研究结果支持中国植物条形码研究组建议将核基因ITS／ITS2纳人种子植物DNA条形码核心片段中的观点。     

植物DNA条形码研究进展

DNA条形码(DNA barcoding)已成为近5年来国际上生物多样性研究的热点,即通过使用短的标准DNA片段,对物种进行快速、准确的识别和鉴定.该技术在动物研究中已得到广泛的应用,所采用的标准片段是线粒体COI基因中约650 bp长的一段.然而在植物中DNA条形码的研究进展相对缓慢,目前尚处于对所提议的各片段比较和评价阶段,还未获得一致的标准片段.由于植物中线粒体基因组进化速率较慢,因此条形码片段主要在叶绿体基因组上进行选择,被提议的编码基因片段主要有rpoB,rpoCI,matK,rbcL,UPA,非编码区片段有tmH-psbA,atpF-atpH,psbK-psbI,此外还有核基因ITS.已有的研究表明以上任何一个单片段都不足以区分所有植物物种,因而不同的研究组相继提出了不同的片段组合方案,目前被广泛讨论的组合主要有5种.本文综述了DNA条形码序列的优点、标准、工作流程、分析方法和存在的争议,重点论述了植物条形码研究中被提议的各序列片段和组合的研究现状.     

藻类DNA条形码研究进展

DNA barcode,又称为DNA条形码,是指利用短的标准DNA序列的核苷酸多样性进行物种的鉴定和快速识别.目前该方法在动物分类研究中应用广泛,其中线粒体的细胞色素c氧化酶亚基1(cytochrome c oxidase subunit 1,COI或cox 1)基因中的约700bp长度的一段被用来作为标准DNA片段.在陆地植物条形码研究中,生命-植物条形码联盟会(Consortium for the Barcode of Life-Plant Working Group,CBOL-Plant Working Group)近期推荐将植物叶绿体中的两个基因片段rbcL+ matK作为初步的陆生植物条形码,此组合能在70％的程度上进行植物物种的鉴别.在海藻的分类研究中,DNA条形码的应用较少,已有的研究主要集中在硅藻、红藻和褐藻,尚没有学者明确提出适合藻类的DNA条形码.总结了能够作为藻类DNA条形码的序列特点、应用流程及分析方法,综述了DNA条形码在藻类中的研究现状和存在的问题,展望了藻类DNA条形码的应用前景.     

DNA条形码在中国金合欢属中的应用

根据形态特征难以准确地辨别金合欢属植物,DNA条形码技术提供了一种准确地鉴定物种的方法。本文利用条形码技术对中国金合欢属物种的序列(psbA trnH、matK、rbcL和ITS)及其不同组合进行比较,通过计算种内和种间变异进行barcoding gap分析,运用Wilcoxon秩和检验比较不同序列的变异性,构建系统树。结果表明：4个片段均存在barcoding gap,ITS序列种间变异率较psbA trnH、rbcL和matK序列有明显优势,单片段ITS正确鉴定率最高,ITS+rbcL片段联合条码的正确鉴定率最高,因此我们认为ITS片段或条形码组合ITS+rbcL是金合欢属的快速鉴别最理想的条码。     

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18SrDNA、26SrDNA和ITS序列,叶绿体基因组的rbcL序列,线粒体基因组的cox9．-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18SrDNA与rbcLFF列既可用于种问关系分析,又可用于更高水平分析的构建系统树。这些分子枥对己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。     

利用DNA条形码技术识别植物物种

DNA条形码技术能够快速、准确地识别物种,对于开展基础性的分类学研究和应用性的生物多样性研究极为重要.本文对鼎湖山20 hm²大样地183个植物物种进行DNA条形码测序.结果表明： 单个条形码片段时, psbA-trnH的综合成功率最高(75%),其次是matK(70%)和rbcL(56%);片段组合时,matK+rbcL+psbA-trnH三片段组合的物种水平识别率在87%以上,随后是matK+psbA-trnH(85%)、rbcL+psbA-trnH(83%)和matK+rbcL(81%).综合了亚热带波多黎各的LFDP样地(143个种)和热带巴拿马的BCI样地(296个种)以及圭亚那的Nouragues样地(254个种)3个森林类型的研究结果,评价DNA条形码各片段在4个森林样地的通用性.在热带和亚热带地区的森林样地中,各片段测序成功率分别为rbcL(93%,95.1%)、psbA-trnH(91.5%,94.6%)和matK(68.5%,79.7%).在植物类群水平上,核心条形码片段matK+rbcL组合的物种准确识别率不高,只在局部群落中表现较为理想;而三位点DNA条形码片段组合在热带和亚热带森林样地中综合成功率可达84%和90%.     

Barcoding diatoms: exploring alternatives to COI-5P

Diatoms are a diverse lineage with species that can be difficult to identify or cryptic, but DNA barcoding, a molecular technique, can assist identification and facilitate studies of speciation and biogeography. The most common region used for DNA barcoding, COI-5P, can distinguish diatom species, but has not displayed universality (i.e., successful PCR amplification from diverse taxa). Therefore, we have assessed the following alternative markers: ～1400bp of rbcL; 748bp at the 3' end of rbcL (rbcL-3P); LSU D2/D3 and UPA. Sellaphora isolates were used to determine each marker's ability to discriminate among closely related species and culture collection material was utilized to explore further marker universality. All of the alternative markers investigated have greater universality than COI-5P. Both full and partial (3P) rbcL regions had the power to discriminate between all species, but rbcL-3P can be sequenced more easily. LSU D2/D3 could distinguish between all but the most closely related species (96%), whereas UPA only distinguished 20% of species. Our observations suggest that rbcL-3P should be used as the primary marker for diatom barcoding, while LSU D2/D3 should be sequenced as a secondary marker to facilitate environmental surveys.     

Molecular systematics of four endemic Batrachospermaceae (Rhodophyta) species in China with multilocus data

Four endemic Batrachospermaceae species in China, Batrachospermum hongdongense Xie & Feng, Batrachospermum longipedicellatum Hua & Shi, Kumanoa curvata (Shi) Vis et al., and Kumanoa intorta (Jao) Entwisle et al., were analyzed with multilocus data in order to assess their phylogenetic placement. Multilocus molecular data from one nuclear gene (the ribosomal SSU), one chloroplast gene (rbcL), and two mitochondrial genes (COI and the cox2-3 spacer) as well as morphological studies were used to determine the phylogenetic relationships of these endemic species within the Batrachospermales. Batrachospermum hongdongense and B. longipedicellatum had previously been placed in Batrachospermum section Batrachospermum, but based on the data from this study they should be transferred into section Helminthoidea. Kumanoa curvata and K. intorta clearly belong to the newly established genus Kumanoa.     

Comparison of three organelle markers for phylogeographic inference in Batrachospermum helminthosum (Batrachospermales, Rhodophyta) from North America

Phylogeographic signal provided by the newly developed 23S plastid rRNA marker (universal plastid amplicon, UPA) and the cytochrome oxidase subunit I gene marker (COI) in the freshwater red alga Batrachospermum helminthosum, throughout its range in North America, was investigated. These markers were compared in individuals from a previous study using the cytochrome oxidase 2–3 spacer region ( cox 2–3), which has yielded the most useful data to date with 13 haplotypes among geographic locations. Five haplotypes were resolved for the UPA, differing by only one to two base pairs (bp), and we conclude that this marker may be more appropriate for studying interspecific variation. In contrast, the COI gene revealed 16 haplotypes, differing from one to 44 base pairs or up to 6.6% sequence variation. The intraspecific variation of COI in this taxon is much greater than that reported thus far for marine red algae (generally <5 bp). The intraspecific variation within B. helminthosum is in accord with levels shown in Batrachospermum macrosporum (48 bp within distant locations in Brazil). The COI gene is comparable to the cox 2–3 spacer for phylogeographic studies as the haplotype networks were similar and showed the same geographic patterns. To our knowledge, this is the first comparison of these three regions for phylogeographic research in the red algae.     

Contrasting intra versus interspecies DNA sequence variation for representatives of the Batrachospermales (Rhodophyta): Insights from a DNA barcoding approach

Sixty‐five accessions of the species‐rich freshwater red algal order Batrachospermales were characterized through DNA sequencing of two regions: the mitochondrial cox1 gene (664 bp), which is proposed as the DNA barcode for red algae, and the UPA (universal plastid amplicon) marker (370 bp), which has been recently identified as a universally amplifying region of the plastid genome. upgma phenograms of both markers were consistent in their species‐level relationships, although levels of sequence divergence were very different. Intraspecific variation of morphologically identified accessions for the cox1 gene ranged from 0 to 67 bp (divergences were highest for the two taxa with the greatest number of accessions; Batrachospermum helminthosum and Batrachospermum macrosporum); while in contrast, the more conserved universal plastid amplicon exhibited much lower intraspecific variation (generally 0–3 bp). Comparisons to previously published mitochondrial cox2–3 spacer sequences for B. helminthosum indicated that the cox1 gene and cox2–3 spacer were characterized by similar levels of sequence divergence, and phylogeographic patterns based on these two markers were consistent. The two taxa represented by the largest numbers of specimens (B. helminthosum and B. macrosporum) have cox1 intraspecific divergence values that are substantially higher than previously reported, but no morphological differences can be discerned at this time among the intraspecific groups revealed in the analyses. DNA barcode data, which are based on a short fragment of an organellar genome, need to be interpreted in conjunction with other taxonomic characters, and additional batrachospermalean taxa need to be analyzed in detail to be able to draw generalities regarding intraspecific variation in this order. Nevertheless, these analyses reveal a number of batrachospermalean taxa worthy of more detailed DNA barcode study, and it is predicted that such research will have a substantial effect on the taxonomy of species within the Batrachospermales in the future.     

Molecular prospecting for cryptic species of nematodes: mitochondrial DNA versus internal transcribed spacer

DNA sequence divergence at internal transcribed spacer regions (ITS-1 and ITS-2) was compared with divergence at mitochondrial cox1 or nad4 loci in pairs of congeneric nematode species. Mitochondrial sequences accumulate substitutions much more quickly than internal transcribed spacer, the difference being most striking in the most closely related species pairs. Thus, mitochondrial DNA may be the best choice for applications in which one is using sequence data on small numbers of individuals to search for potential cryptic species. On the other hand, internal transcribed spacer remains an excellent tool for DNA diagnostics (quickly distinguishing between known species) owing to its lower level of intraspecific polymorphism.     

Comparative analysis of three species populations of Profundulus (Teleostei: Cyprinodontiformes) using two genetic markers

Background and aims: The fish Family Profundulidae is endemic in south-southeast Mexico, with five known species. The species-level analysis of the genus based on morphological characters is insufficient and complex due to increased phenotypic and intraspecific variation. This variation might be owed to the existence of yet undescribed forms. Materials and methods: In the present study, the genetic composition of three species of the State of Chiapas, Mexico, is compared by examining different populations of the species Profundulus candalarius, Profundulus labialis, and Profundulus punctatus. Results: We identified specimens from 16 localities and sequenced mitochondrial DNA representing partial regions of the cytochrome b and cox1 genes. Conclusion: The results suggest an extreme divergence of the populations of P. labialis from El Sabinal.     

Hypnea species (Gigartinales,Rhodophyta) from the southeastern coast of Brazil based on molecular studies complemented with morphological analyses,including descriptions of Hypnea edeniana sp. nov. and H. flava sp. nov.

The red algal genus Hypnea (Gigartinales) has a wide geographical distribution along tropical and subtropical coasts around the world. The relatively simple and plastic morphology, often influenced by the conditions of its habitat, complicates the identification of Hypnea species. Therefore, the number and status of some species remain in doubt. Molecular studies have been performed to supplement traditional studies based on morphology, mainly for Hypnea species occurring in Asia. In the present study, sequence data from the DNA barcode COI-5P for 114 samples from the southeastern coast of Brazil, indicated the occurrence of six taxa. Additionally, sequence data from the UPA and rbcL markers for representatives of each of those taxa confirmed the existence of six different species. After morphological analysis and comparison with sequences available in GenBank, these species were named as follows: H. aspera, H. cervicornis, H. cf. musciformis, H. spinella, and two new species, H. flava Nauer, Cassano & M.C. Oliveira and H. edeniana Nauer, Cassano & M.C. Oliveira. Hypnea cervicornis, often considered as a later synonym of H. spinella, should be considered as a distinct species based on morphology and divergence of the three molecular markers used. Hypnea aspera is a new record for the Atlantic Ocean.     

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.