植物DNA条形码技术

DNA条形码技术是利用标准的、具有足够变异的、易扩增且相对较短的DNA片段在物种内的特异性和种间的多样性而创建的一种新的生物身份识别系统,从而实现对物种的快速自动鉴定。尽管这一技术在理论上和具体应用上仍存在很多争论。但DNA条形码概念自2003年由加拿大分类学家Paul Hebert首次提出后就在世界范围内受到了广泛关注。在植物类群中条形码的研究和应用尚处于探索阶段,稍落后于对动物类群的研究,这主要表现在：（1）DNA条形码的选择及其评价仍没有统一的标准：（2）对类群较全面的形态分类学修订和植物DNA条形码研究的结合十分缺乏：（3）以往研究在取样上尺度较大,而对具体类群的研究较少,一个科或一个属只用有限的种类作为代表,同一种内的取样个体数量也不足,这样虽然表面上看来利用选定的DNA条形码可以较容易地把代表物种区分开,但实际上目前建议的植物DNA条形码（例如由生命条形码咨询委员会植物工作组最近提出的rbcL和matK）由于其分子进化速率较慢,在种级水平上,特别是对于那些经历了适应辐射或快速进化的属来说,分辨率较低。而DNA条形码的应用主要集中在属内物种水平的鉴别,因此只有针对具体类群进行探索研究,发现进化速率较快、分辨率高且通用性好的条形码,才可能为建立完整的条形码数据库起到积极有效的作用。     

Testing four barcoding markers for species identification of Potamogetonaceae

The pondweeds (Potamogetonaceae) are among the most important plant groups in the aquatic environment. Owing to their high morphological and ecological diversity, species identification of this aquatic family remains problematic. DNA barcoding involves sequencing a standard DNA region and has been shown to be a powerful tool for species identification. In the present study, we tested four barcoding markers (rbcL, matK, internal transcribed spacer (ITS), and trnH-psbA) in 15 Potamogeton species and two Stuckenia species, representing most species of the Potamogetonaceae in China. The results show that all four regions can distinguish and support the newly proposed genera of Stuckenia from Potamogeton. Using ITS and trnH-psbA, significant interspecific genetic variability was shown. However, intraspecific genetic variability of trnH-psbA is high and so it is not suitable for barcoding in Potamogetonaceae. The ITS and matK regions showed good discrimination. However, matK was not easy to sequence using universal primers. The best performing single locus was ITS, making it a potentially useful DNA barcode in Potamogetonaceae.     

Testing four proposed barcoding markers for the identification of species within Ligustrum L.(Oleaceae)

DNA barcoding is a biological technique that uses short and standardized genes or DNA regions to facilitate species identification. DNA barcoding has been used successfully in several animal and plant groups. Ligustrum (Oleaceae) species occur widely throughout the world and are used as medicinal plants in China. Therefore, the accurate identification of species in this genus is necessary. Four potential DNA barcodes, namely the nuclear ribosomal internal transcribed spacer (ITS) and three chloroplast (cp) DNA regions (rbcL, marK, and trnH-psbA),were used to differentiate species within Ligustrum. BLAST, character-based method, tree-based methods and TAXONDNA analysis were used to investigate the molecular identification capabilities of the chosen markers for discriminating 92 samples representing 20 species of this genus. The results showed that the ITS sequences have the most variable information, followed by trnH-psbA, matK, and rbcL. All sequences of the four regions correctly identified the species at the genus level using BLAST alignment. At the species level, the discriminating power of rbcL, matK, trnH-psbA and ITS based on neighbor-joining (NJ) trees was 36.8%, 38.9%, 77.8%, and 80%,respectively. Using character-based and maximum parsimony (MP) tree methods together, the discriminating ability of trnH-psbA increased to 88.9%. All species could be differentiated using ITS when combining the NJ tree method with character-based or MP tree methods. Overall, the results indicate that DNA barcoding is an effective molecular identification method for Ligustrum species. We propose the nuclear ribosomal ITS as a plant barcode for plant identification and trnH-psbA as a candidate barcode sequence.     

植物DNA条形码促进系统发育群落生态学发展

系统发育群落生态学是近年兴起的一个重要牛态学研究分支,它以群落生态学为基础并引入了系统发育的分析方法,全面动态地反映了群落中物种内和物种间的相互作用关系,揭示了群落格局形成的生态学过程,研究了生物多样性的形成及维持机制.巴拿马BCI(Barro Colorado Island)样地的成功例子说明,在固定样地进行长期的群落生态与系统发育研究切实可行且极具意义;DNA条形码的快速兴起对这一研究发挥着重要作用.本文先列举了群落生态与系统发育综合分析能解决的群落系统发育结构、群落生态位结构、生物地理学和性状进化等生态学问题;接着介绍了标准植物DNA条形码以及利用片段组合(rbcL+matK+trnH-psbA)进行快速物种识别和近缘种区分、精确群落系统发育关系的构建以及群落生态学研究;随后提出DNA条形码研究在类群水平上需注意两片段的条形码组合(matK+rbcL)在同属种鉴别能力上的不足,而在较大尺度群落水平上需对实验设计进行优化.DNA条形码将为探讨物种多样性及其维持机理、系统发育beta多样性以及群落水平上功能性状进化研究提供新的思路.     

蒟蒻薯属(薯蓣科)植物DNA条形码研究

蒟蒻薯属(Tacca)植物种间在形态上差别不大,导致分类上存在一定的困难.DNA条形码是一种利用短的DNA标准片段来鉴别和发现物种的方法.本研究利用核基因ITS片段和叶绿体基因trn H-psbA,rbcL,matK片段对蒟蒻薯属6个种的DNA条形码进行研究,对4个DNA片段可用性,种内种间变异,barcode gap进行了分析,采用Tree-based和BBA两种方法比较不同序列的鉴定能力.结果显示:单片段ITS正确鉴定率最高,片段组合rbcL+matK正确鉴定率最高.支持CBOL植物工作组推荐的条码组合rbcL+matK可作为蒟蒻薯属物种鉴定的标准条码,建议ITS片段作为候选条码.丝须蒟蒻薯Tacca integrifolia采自西藏的居群与马来西亚居群形成了2个不同的遗传分支,且两者在形态上也存在一定的差异,很可能是一个新种.     

Molecular identification of species in Juglandaceae:A tiered method

DNA barcoding is a method of species identification and recognition using DNA sequence data. A tiered or multilocus method has been recommended for barcoding plant species. In this study, we sampled 196 individuals representing 9 genera and 54 species of Juglandaceae to investigate the utility of the four potential barcoding loci (rbcL, matK, trnH-psbA, and internal transcribed spacer (ITS)). Our results show that all four DNA regions are easy to amplify and sequence. In the four tested DNA regions, ITS has the most variable information, and rbcL has the least. At generic level, seven of nine genera can be efficiently identified by matK. At species level, ITS has higher interspecific p-distance than the trnH-psbA region. Difficult to align in the whole family, ITS showed heterogeneous variability among different genera. Except for the monotypic genera (Cyclocarya, Annamocarya, Platycarya), ITS appeared to have limited power for species identification within the Carya and Engelhardia complex, and have no power for Juglans or Pterocarya. Overall, our results confirmed that a multilocus tiered method for plant barcoding was applicable and practicable. With higher priority, matK is proposed as the first-tier DNA region for genus discrimination, and the second locus at species level should have enough stable variable characters.     

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.     

Comparison of four DNA barcodes in identifying certain medicinal plants of Lamiaceae

Many species in the family Lamiaceae have been widely used for the treatment of coronary heart disease,stroke,and other conditions,and authenticating each of these species has become an important topic...     

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.     

DNA barcodes for marine fungal identification and discovery

We employed DNA barcodes for identification of fungal species in marine sediments. Sediments were collected seasonally along the Southeast coast of India from which a culturable fungal library was constructed. All cultured species were morphologically documented using microscopical analysis. A maximum population density of 19.3 × 10³ CFU/g was recorded in monsoon and minimum of 3 × 10³ CFU/g in premonsoon season. Two-way analysis of variance suggests that the fungal community varied significantly between the seasons (F = 9.543, P < 0.001) and at various depths sampled (F = 4.655, P < 0.05). In total, 54 fungal species belonging to 13 different families were documented and all species were sequenced for internal transcribed spacer genes. Each species was represented by at least two specimens constituting a total of 171 specimens for DNA barcoding. Twelve species of a marine fungi were sequenced for the first time. Branching patterns of phylogenetic tree strongly supported the sequence variations within and between all species barcoded. Based on the pairwise distance model we suggest barcode gaps of 15 %, 21 %, 30 %, 35 % and 51 % for genera, family, order, class and phyla respectively.     

大白口蘑分离菌株的DNA鉴定

以松口蘑Tricholoma matsutake子实体为外类群,对大白口蘑T. giganteum 野生子实体及其组织分离菌丝进行ITS序列测序,通过DNAStar软件进行比较分析。结果表明大白口蘑ITS序列长度为589bp,松口蘑ITS序列长度为601bp,ITS1和ITS2呈现不同程度的种间多态性;ITS序列测定证实了大白口蘑野生子实体及其组织分离菌丝的同质性,并且ITS区序列在大白口蘑种内不同菌株间的变异程度很小,表明使用通用引物ITS4和ITS5,通过PCR扩增测序即可用于大白口蘑的种质鉴定。     

大白口蘑分离菌株的DNA鉴定

以松口蘑Tricholoma matsutake子实体为外类群,对大白口蘑T.giganteum野生子实体及其组织分离菌丝进行ITS序列测序,通过DNAStar软件进行比较分析。结果表明大白口蘑ITS序列长度为589bp,松口蘑ITS序列长度为601bp,ITS1和ITS2呈现不同程度的种间多态性;ITS序列测定证实了大白口蘑野生子实体及其组织分离菌丝的同质性,并且ITS区序列在大白口蘑种内不同菌株间的变异程度很小,表明使用通用引物ITS4和ITS5,通过PCR扩增测序即可用于大白口蘑的种质鉴定。     

维吾尔药材红豆杉植物基原研究

从分子学角度探明维吾尔药材红豆杉的植物基原,为维吾尔药材标准化与维吾尔药产业化提供科学依据。采用试剂盒法提取红豆杉属植物及药材基因组DNA,PCR扩增ITS2片段,双向测序,应用Mega 6.0软件分析序列,计算种内及种间遗传距离,构建NJ鉴别树。红豆杉属植物ITS2序列长度为229~231bp,GC含量为59.13%~60.26%;西藏红豆杉与红豆杉及南方红豆杉种内种间K2P距离为0,与东北红豆杉、命叶红豆杉及曼地亚红豆杉种间K2P距离分别为0.012、0.013、0.015。NJ树结果表明,西藏红豆杉与红豆杉及南方红豆杉无法区分,与东北红豆杉、命叶红豆杉可区分;曼地亚红豆杉与其母本东北红豆杉聚在一起,无法区分。根据ITS2序列鉴定结果,西藏红豆杉与红豆杉、南方红豆杉可作为药材的同一基原,东北红豆杉可与曼地亚红豆杉作为同一基原,为维吾尔药材的真伪鉴别及质量标准提升提供基原鉴定依据。