木霉属补充DNA条形码筛选

木霉属真菌是一类重要的生物资源,在工农业、环境保护等方面具有较高经济价值,对其进行快速、准确的物种鉴定兼具理论意义和应用前景。以木霉属35个概念清晰的种为材料,选择ITS、rpb2和 tef1作为候选基因序列,利用TaxonGap对231个序列片段进行分析,将种内与种间序列差异以及序列获取难易程度作为评价指标,筛选该属的补充条形码片段。结果表明,rpb2具有适宜的种内与种间序列差异,其最小的种间差异（2.48%）,大于最大种内差异（1.8%）,种内、种间遗传距离存在明显的间隔区,并且该基因序列具有较高的PCR扩增与测序成功率（94.4%）;ITS和tef1基因序列的种内与种间序列差异之间存在交叉重叠。因此建议rpb2作为木霉属的补充DNA条形码序列。     

Applying DNA barcodes for identification of plant species in the family Araliaceae

An effective DNA marker in authentication of the family Araliaceae was screened out of the five DNA regions (matK, rbcL, ITS2, psbA-trnH and ycf5). In the present study, 1113 sequences of 276 species from 23 genera (Araliaceae) were collected from DNA sequencing and GenBank, in which 16 specimens were from 5 provinces in China and Japan. All of the sequences were assessed in the success rates of PCR amplifications, intra- and inter-specific divergence, DNA barcoding gaps and efficiency of identification. Compared with other markers, ITS2 showed superiority in species discrimination with an accurate identification of 85.23% and 97.29% at the species and genus levels, respectively, in plant samples from the 589 sequences derived from Araliaceae. Consequently, as one of the most popular phylogenetic markers, our study indicated that ITS2 was a powerful barcode for Araliaceae identification.     

ITS作为粒毛盘菌属DNA条形码的探索

以种内与种间差异以及PCR扩增和测序成功率为评价DNA条形码的重要指标,探索ITS序列作为粒毛盘菌属DNA条形码的可行性。结果表明,ITS成功区分了研究涉及的22个种,其PCR扩增和测序成功率为100%,该片段有望成为该属区分物种的DNA条形码。     

The Effectiveness of Three Regions in Mitochondrial Genome for Aphid DNA Barcoding: A Case in Lachininae

Background

The mitochondrial gene COI has been widely used by taxonomists as a standard DNA barcode sequence for the identification of many animal species. However, the COI region is of limited use for identifying certain species and is not efficiently amplified by PCR in all animal taxa. To evaluate the utility of COI as a DNA barcode and to identify other barcode genes, we chose the aphid subfamily Lachninae (Hemiptera: Aphididae) as the focus of our study. We compared the results obtained using COI with two other mitochondrial genes, COII and Cytb. In addition, we propose a new method to improve the efficiency of species identification using DNA barcoding.

Methodology/Principal Findings

Three mitochondrial genes (COI, COII and Cytb) were sequenced and were used in the identification of over 80 species of Lachninae. The COI and COII genes demonstrated a greater PCR amplification efficiency than Cytb. Species identification using COII sequences had a higher frequency of success (96.9% in “best match” and 90.8% in “best close match”) and yielded lower intra- and higher interspecific genetic divergence values than the other two markers. The use of “tag barcodes” is a new approach that involves attaching a species-specific tag to the standard DNA barcode. With this method, the “barcoding overlap” can be nearly eliminated. As a result, we were able to increase the identification success rate from 83.9% to 95.2% by using COI and the “best close match” technique.

Conclusions/Significance

A COII-based identification system should be more effective in identifying lachnine species than COI or Cytb. However, the Cytb gene is an effective marker for the study of aphid population genetics due to its high sequence diversity. Furthermore, the use of “tag barcodes” can improve the accuracy of DNA barcoding identification by reducing or removing the overlap between intra- and inter-specific genetic divergence values.     

DNA Barcoding of Economically Important Mushrooms: A Case Study on Lethal Amanitas from China

Some species of the genus Amanita are economically important gourmet mushrooms, while others cause dramatic poisonings or even deaths every year in China and in many other countries. A DNA barcode is a short segment or a combination of short segments of DNA sequences that can distinguish species rapidly and accurately. To establish a standard DNA barcode for poisonous species of Amanita in China, three candidate markers, the large subunit nuclear ribosomal RNA (nLSU), the internal transcribed spacer (ITS), and the translation elongation factor 1 alpha (tef1 α) were tested using the eukaryotic general primers for their feasibility as barcodes to identify seven species of lethal fungi and two species of edible ones which can easily be confused with the lethal ones known from China. In addition, A.phalloides—a European and North American species closely related to one of the seven taxa, A.subjunquillea was also included. PCR amplification and sequencing success rate, intra and inter specific variation and rate of species identification were considered as main criteria for evaluation of the candidate DNA barcodes. Although the nLSU had high PCR and sequencing success rates (100% and 100% respectively), occasional overlapping occurred between the intra and inter specific variations. The PCR amplification and sequencing success rates of ITS were 100% and 85.7% respectively. ITS showed high sequence variation among species group and low variation within a given species. There was a relatively high PCR amplification and sequencing success rate for tef1 α (85.7% and 100% respectively), and its intra and inter specific variation was higher than that of ITS or nLSU. All three candidate markers showed hight species resolution. ITS and tef1 α had a more clearly defined barcode gap than nLSU. Our study showed that the tef1 α and nLSU can be proposed as supplementary barcodes for the genus Amanita, while ITS can be used as a primary barcode marker considering that the ITS region may become a universal barcode marker for the fungal kingdom.     

大型经济真菌的DNA条形码研究——以我国剧毒鹅膏为例

鹅膏属（Amanita）部分物种为重要食用真菌,而另外部分物种则是剧毒的,在我国及其他许多国家,每年都有因误食剧毒鹅膏而导致中毒甚至死亡的事件发生。DNA条形码是用一段或几段短的DNA序列来对物种进行快速、准确鉴定的方法。本研究选取三个候选片段,即核糖体大亚基（nLSU）、内转录间隔区（ITS）和翻译延长因子1-α（tef1-α）,使用真核生物通用引物,测试我国已知的7种剧毒鹅膏及2种易混的可食鹅膏,并将欧美分布的但与黄盖鹅膏（A.subjunquillea）亲缘关系密切的绿盖鹅膏（A.phalloides）纳入分析中。nLSU的PCR扩增和测序成功率均为100%,但种内和种间遗传变异偶有重叠。ITS的PCR扩增和测序成功率分别达到100%和85.7%,且具有高的种间变异和低的种内变异。tef1-α的PCR扩增和测序成功率分别达到85.7%和100%,种间和种内遗传分化均高于ITS和nLSU。三个片段的物种分辨率均较高,但与nLSU相比,ITS和tef1-α具有更为明显的barcode gap。鉴于ITS可能会成为真菌界的通用条码,故建议将ITS作为鹅膏属的核心条形码,tef1 α和nLSU作为该属的辅助条形码。     

DNA barcoding Chinese medicinal Bupleurum

We tested 4 markers, namely nuclear internal transcribed spacer 2 (ITS2), psbA-trnH, matK, and rbcL, to evaluate these candidate DNA barcodes for distinguishing Bupleuri radix (Chaihu) from its adulterants. 51 plant samples of Bupleurum representing 19 species were collected from different areas in China. Amplification and sequencing were attempted for all the 4 candidate barcode regions, whose validity was assessed in terms of the success rate of PCR amplification and sequencing, differential intra- and inter-specific divergences, DNA barcoding gap and the ability to discriminate species. The results showed that ITS2 had the best performance in identifying Bupleurum with an identification efficiency of 73.68%, which, after combining with psbA-trnH, increased to 83.33%. We further evaluated the efficiency of ITS2 for discriminating the species of Bupleurum using a large database from GenBank, which archived data of 223 samples from 74 species, and ITS2 successfully discriminated 64.13% of the samples at the species level. In conclusion, the ITS2 can serve as a potentially useful barcode for Bupleurum species, with psbA-trnH as a supplementary locus.     

COI基因作为丛赤壳科真菌DNA条形码的测试

以丛赤壳科33种76个菌株为材料,探讨COI基因作为该科DNA条形码的可能性。结果表明,该DNA片段存在较多内含子,为了获取某些种的短片段,需设计许多引物,PCR扩增与测序成功率低,难以达到便捷、快速的物种鉴定的目的。因此,COI不宜作为丛赤壳科的DNA条形码。对已获得的少数片段进行分析表明,该基因对丛赤壳科部分种具有较强的物种鉴别力。     

中国蜘蛛抱蛋属植物DNA条形码研究

利用植物DNA条形码候选序列mat K、psb A-trn H、psb K-psb I和rbc L对蜘蛛抱蛋属(Aspidistra)植物的19种104批样品进行扩增和测序,并采用相似性搜索算法(BLAST)对各序列的鉴定效率进行评价,得出蜘蛛抱蛋属物种鉴定的最佳序列。结果显示,psb K-psb I的物种鉴定成功率为88.7%,在单一序列中成功率最高。通过多序列组合鉴定效率的比较,发现组合序列的鉴定成功率明显高于单一序列,其中mat K+(psb K-psb I)组合的鉴定成功率高达100%,基于该序列组合构建蜘蛛抱蛋属植物的系统发育树,结果显示同一物种的样品聚集度较好,多表现为单系。研究结果表明mat K+(psb K-psb I)序列组合可作为蜘蛛抱蛋植物种鉴定的最佳条形码序列。     

The Internal Transcribed Spacer (ITS) Region and trnhH-psbA Are Suitable Candidate Loci for DNA Barcoding of Tropical Tree Species of India

Background

DNA barcoding as a tool for species identification has been successful in animals and other organisms, including certain groups of plants. The exploration of this new tool for species identification, particularly in tree species, is very scanty from biodiversity-rich countries like India. rbcL and matK are standard barcode loci while ITS, and trnH-psbA are considered as supplementary loci for plants.

Methodology and Principal Findings

Plant barcode loci, namely, rbcL, matK, ITS, trnH-psbA, and the recently proposed ITS2, were tested for their efficacy as barcode loci using 300 accessions of tropical tree species. We tested these loci for PCR, sequencing success, and species discrimination ability using three methods. rbcL was the best locus as far as PCR and sequencing success rate were concerned, but not for the species discrimination ability of tropical tree species. ITS and trnH-psbA were the second best loci in PCR and sequencing success, respectively. The species discrimination ability of ITS ranged from 24.4 percent to 74.3 percent and that of trnH-psbA was 25.6 percent to 67.7 percent, depending upon the data set and the method used. matK provided the least PCR success, followed by ITS2 (59. 0%). Species resolution by ITS2 and rbcL ranged from 9.0 percent to 48.7 percent and 13.2 percent to 43.6 percent, respectively. Further, we observed that the NCBI nucleotide database is poorly represented by the sequences of barcode loci studied here for tree species.

Conclusion

Although a conservative approach of a success rate of 60–70 percent by both ITS and trnH-psbA may not be considered as highly successful but would certainly help in large-scale biodiversity inventorization, particularly for tropical tree species, considering the standard success rate of plant DNA barcode program reported so far. The recommended matK and rbcL primers combination may not work in tropical tree species as barcode markers.     

DNA barcoding of the fungal genus <Emphasis Type="Italic">Neonectria</Emphasis> and the discovery of two new species

To determine a suitable DNA barcode for the genus Neonectria, the internal transcribed spacer rDNA, β-tubulin, EF-1α, and RPB2 genes were selected as candidate markers. A total of 205 sequences from 19 species of the genus were analyzed. Intra- and inter-specific divergences and the ease of nucleotide sequence acquisition were treated as criteria to evaluate the feasibility of a DNA barcode. Our results indicated that any single gene among the candidate markers failed to serve as a successful barcode, while the combination of the partial EF-1α, and RPB2 genes recognized all species tested. We tentatively propose the combined partial EF-1α and RPB2 genes as a DNA barcode for the genus. During this study, two cryptic species were discovered, based on the combined data of morphology and DNA barcode information. We described and named these two new species N. ditissimopsis and N. microconidia.     

Is DNA barcoding child's play? Science education and the utility of DNA barcoding for the discrimination of UK tree species

We present the findings of a DNA barcoding study of the UK tree flora, implemented as part of an innovative, research‐based science education programme called ‘Tree School’. The UK tree flora comprises native and introduced species, and is a taxonomically diverse study group for the exploration of the potential and limitations of DNA barcoding. The children participating in the project collected voucher specimens and generated DNA barcode sequences from trees and shrubs found in the grounds and surrounding woodlands of a residential field centre in Dorset, UK. We assessed the potential of rbcL and matK markers for amplification and DNA sequencing success and for species discrimination among the 67 tree and shrub species included in this study. Although we achieved 100% PCR amplification and sequencing success for rbcL and matK, mononucleotide repeats affected sequence quality in matK for some taxonomic groups (e.g. Rosaceae). Species discrimination success ranged from 65% to 71% using tree‐based methods to 86% using BLASTN. The occurrence of known hybrids (diploid and polyploid) and their progenitors on the study site reduced the overall species discrimination success for both loci. This study demonstrates that, even in a floristic context, rbcL and matK alone are insufficient for the discrimination of UK tree species, especially where taxonomically complex groups are present. From a science education perspective, DNA barcoding represents a compelling and accessible platform for the engagement of non‐experts in ongoing research, providing an opportunity for them to contribute authentic scientific data to an international research campaign.     

A DNA mini‐barcode for land plants

Small portions of the barcode region – mini‐barcodes – may be used in place of full‐length barcodes to overcome DNA degradation for samples with poor DNA preservation. 591,491,286 rbcL mini‐barcode primer combinations were electronically evaluated for PCR universality, and two novel highly universal sets of priming sites were identified. Novel and published rbcL mini‐barcode primers were evaluated for PCR amplification [determined with a validated electronic simulation (n = 2765) and empirically (n = 188)], Sanger sequence quality [determined empirically (n = 188)], and taxonomic discrimination [determined empirically (n = 30 472)]. PCR amplification for all mini‐barcodes, as estimated by validated electronic simulation, was successful for 90.2–99.8% of species. Overall Sanger sequence quality for mini‐barcodes was very low – the best mini‐barcode tested produced sequences of adequate quality (B₂₀ ≥ 0.5) for 74.5% of samples. The majority of mini‐barcodes provide correct identifications of families in excess of 70.1% of the time. Discriminatory power noticeably decreased at lower taxonomic levels. At the species level, the discriminatory power of the best mini‐barcode was less than 38.2%. For samples believed to contain DNA from only one species, an investigator should attempt to sequence, in decreasing order of utility and probability of success, mini‐barcodes F (rbcL1/rbcLB), D (F52/R193) and K (F517/R604). For samples believed to contain DNA from more than one species, an investigator should amplify and sequence mini‐barcode D (F52/R193).     

Extracting DNA from museum bird eggs, and whole genome amplification of archive DNA

We present a comprehensive protocol for extracting DNA from egg membranes and other internal debris recovered from the interior of blown museum bird eggs. A variety of commercially available DNA extraction methods were found to be applicable. DNA sequencing of polymerase chain reaction (PCR) products for a 176‐bp fragment of mitochondrial DNA was successful for most egg samples (> 78%) even though the amount of DNA extracted (mean = 14.71 ± 4.55 ng/µL) was significantly less than that obtained for bird skin samples (mean = 67.88 ± 4.77 ng/µL). For PCR and sequencing of snipe (Gallinago) DNA, we provide eight new primers for the ‘DNA barcode’ region of COI mtDNA. In various combinations, the primers target a range of PCR products sized from 72 bp to the full ‘barcode’ of 751 bp. Not all possible combinations were tested with archive snipe DNA, but we found a significantly better success rate of PCR amplification for a shorter 176‐bp target compared with a larger 288‐bp fragment (67% vs. 39%). Finally, we explored the feasibility of whole genome amplification (WGA) for extending the use of archive DNA in PCR and sequencing applications. Of two WGA approaches, a PCR‐based method was found to be able to amplify whole genomic DNA from archive skins and eggs from museum bird collections. After WGA, significantly more archive egg samples produced visible PCR products on agarose (56.9% before WGA vs. 79.0% after WGA). However, overall sequencing success did not improve significantly (78.8% compared with 83.0%).     

锦葵科植物DNA条形码通用序列的筛选

对锦葵科植物样品的ITS、ITS2、rbcL、matK和psbA-trnH序列进行PCR扩增和测序, 比较各序列的扩增效率、测序成功率、种内和种间变异的差异以及barcoding gap图, 使用BLAST1和Nearest Distance方法评价不同序列的鉴定能力, 进而从这些候选序列中筛选出较适合锦葵科植物鉴别的DNA条形码序列。结果表明, ITS序列在采集的锦葵科植物11个种26个样品中的扩增成功率较高, 其种内、种间变异差异和barcoding gap较ITS2、psbA-trnH及rbcL序列具有更明显的优势, 且纳入60个属316个种共1 228个样品的网上数据后, 其鉴定成功率可达89.9%。psbA-trnH序列的扩增和测序成功率最高, 其鉴定成功率为63.2%, 并能鉴别一些ITS序列无法鉴别的种。实验结果表明, ITS和psbA-trnH是较适合鉴别锦葵科植物的DNA条形码序列组合。     

Next‐generation DNA barcoding: using next‐generation sequencing to enhance and accelerate DNA barcode capture from single specimens

DNA barcoding is an efficient method to identify specimens and to detect undescribed/cryptic species. Sanger sequencing of individual specimens is the standard approach in generating large‐scale DNA barcode libraries and identifying unknowns. However, the Sanger sequencing technology is, in some respects, inferior to next‐generation sequencers, which are capable of producing millions of sequence reads simultaneously. Additionally, direct Sanger sequencing of DNA barcode amplicons, as practiced in most DNA barcoding procedures, is hampered by the need for relatively high‐target amplicon yield, coamplification of nuclear mitochondrial pseudogenes, confusion with sequences from intracellular endosymbiotic bacteria (e.g. Wolbachia) and instances of intraindividual variability (i.e. heteroplasmy). Any of these situations can lead to failed Sanger sequencing attempts or ambiguity of the generated DNA barcodes. Here, we demonstrate the potential application of next‐generation sequencing platforms for parallel acquisition of DNA barcode sequences from hundreds of specimens simultaneously. To facilitate retrieval of sequences obtained from individual specimens, we tag individual specimens during PCR amplification using unique 10‐mer oligonucleotides attached to DNA barcoding PCR primers. We employ 454 pyrosequencing to recover full‐length DNA barcodes of 190 specimens using 12.5% capacity of a 454 sequencing run (i.e. two lanes of a 16 lane run). We obtained an average of 143 sequence reads for each individual specimen. The sequences produced are full‐length DNA barcodes for all but one of the included specimens. In a subset of samples, we also detected Wolbachia, nontarget species, and heteroplasmic sequences. Next‐generation sequencing is of great value because of its protocol simplicity, greatly reduced cost per barcode read, faster throughout and added information content.     

Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species

Background

The plant working group of the Consortium for the Barcode of Life recommended the two-locus combination of rbcL + matK as the plant barcode, yet the combination was shown to successfully discriminate among 907 samples from 550 species at the species level with a probability of 72%. The group admits that the two-locus barcode is far from perfect due to the low identification rate, and the search is not over.

Methodology/Principal Findings

Here, we compared seven candidate DNA barcodes (psbA-trnH, matK, rbcL, rpoC1, ycf5, ITS2, and ITS) from medicinal plant species. Our ranking criteria included PCR amplification efficiency, differential intra- and inter-specific divergences, and the DNA barcoding gap. Our data suggest that the second internal transcribed spacer (ITS2) of nuclear ribosomal DNA represents the most suitable region for DNA barcoding applications. Furthermore, we tested the discrimination ability of ITS2 in more than 6600 plant samples belonging to 4800 species from 753 distinct genera and found that the rate of successful identification with the ITS2 was 92.7% at the species level.

Conclusions

The ITS2 region can be potentially used as a standard DNA barcode to identify medicinal plants and their closely related species. We also propose that ITS2 can serve as a novel universal barcode for the identification of a broader range of plant taxa.     

Rapid Mitochondrial Genome Evolution through Invasion of Mobile Elements in Two Closely Related Species of Arbuscular Mycorrhizal Fungi

Arbuscular mycorrhizal fungi (AMF) are common and important plant symbionts. They have coenocytic hyphae and form multinucleated spores. The nuclear genome of AMF is polymorphic and its organization is not well understood, which makes the development of reliable molecular markers challenging. In stark contrast, their mitochondrial genome (mtDNA) is homogeneous. To assess the intra- and inter-specific mitochondrial variability in closely related Glomus species, we performed 454 sequencing on total genomic DNA of Glomus sp. isolate DAOM-229456 and we compared its mtDNA with two G. irregulare isolates. We found that the mtDNA of Glomus sp. is homogeneous, identical in gene order and, with respect to the sequences of coding regions, almost identical to G. irregulare. However, certain genomic regions vary substantially, due to insertions/deletions of elements such as introns, mitochondrial plasmid-like DNA polymerase genes and mobile open reading frames. We found no evidence of mitochondrial or cytoplasmic plasmids in Glomus species, and mobile ORFs in Glomus are responsible for the formation of four gene hybrids in atp6, atp9, cox2, and nad3, which are most probably the result of horizontal gene transfer and are expressed at the mRNA level. We found evidence for substantial sequence variation in defined regions of mtDNA, even among closely related isolates with otherwise identical coding gene sequences. This variation makes it possible to design reliable intra- and inter-specific markers.     

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.     

Testing DNA barcodes in closely related species of Curcuma (Zingiberaceae) from Myanmar and China

The genus Curcuma L. is commonly used as spices, medicines, dyes and ornamentals. Owing to its economic significance and lack of clear‐cut morphological differences between species, this genus is an ideal case for developing DNA barcodes. In this study, four chloroplast DNA regions (matK, rbcL, trnH‐psbA and trnL‐F) and one nuclear region (ITS2) were generated for 44 Curcuma species and five species from closely related genera, represented by 96 samples. PCR amplification success rate, intra‐ and inter‐specific genetic distance variation and the correct identification percentage were taken into account to assess candidate barcode regions. PCR and sequence success rate were high in matK (89.7%), rbcL (100%), trnH‐psbA (100%), trnL‐F (95.7%) and ITS2 (82.6%) regions. The results further showed that four candidate chloroplast barcoding regions (matK, rbcL, trnH‐psbA and trnL‐F) yield no barcode gaps, indicating that the genus Curcuma represents a challenging group for DNA barcoding. The ITS2 region presented large interspecific variation and provided the highest correct identification rates (46.7%) based on BLASTClust method among the five regions. However, the ITS2 only provided 7.9% based on NJ tree method. An increase in discriminatory power needs the development of more variable markers.