Testing DNA barcoding in closely related groups of Lysimachia L. (Myrsinaceae)

It has been suggested that rbcL and matK are the core barcodes in plants, but they are not powerful enough to distinguish between closely related plant groups. Additional barcodes need to be evaluated to improve the level of discrimination between plant species. Because of their well-studied taxonomy and extreme diversity, we used Chinese Lysimachia (Myrsinaceae) species to test the performance of core barcodes (rbcL and matK) and two additional candidate barcodes (trnH-psbA and the nuclear ribosomal ITS); 97 accessions from four subgenus representing 34 putative Lysimachia species were included in this study. And many closely related species pairs in subgen. Lysimachia were covered to detect their discriminatory power. The inefficiency of rbcL and matK alone or combined in closely related plant groups was validated in this study. TrnH-psbA combined with rbcL + matK did not yet perform well in Lysimachia groups. In contrast, ITS, alone or combined with rbcL and/or matK, revealed high resolving ability in Lysimachia. We support ITS as a supplementary barcode on the basis of core barcode rbcL and matK. Besides, this study also illustrates several mistakes or underlying evolutionary events in Lysimachia detected by DNA barcoding.     

Are nuclear loci ideal for barcoding plants? A case study of genetic delimitation of two sister species using multiple loci and multiple intraspecific individuals

Considerable debate remains as to which DNA region should be used to barcode plants. Several different chloroplast (cp) DNA regions (rbcL, matK, and trnH-psbA) and nuclear ribosomal internal transcribed regions (ITS) have been suggested as suitable barcodes in plants. Recently, low-copy nuclear loci were also suggested to be potentially ideal barcode regions. The aim of the present study was to test the effectiveness of these proposed DNA fragments and five additional low-copy loci (CHS, DETl, COPl, PGICl, and RPS2; comprising both coding and non-coding regions) in barcoding closely related species. We examined the divergences within and between two species of Pugioniun (Brassicaceae). We failed to find any interspecific variation from three cpDNA fragments with which to discriminate the two species. However, a single base mutation in the internal transcribed spacer (ITS) could discriminate between the two species consistently. We found more variations among all individuals of the two species using each of the other five low-copy nuclear loci. However, only alleles from one locus (DET1) of the five low-copy loci related to flowering regulations was able to distinguish the sampled individuals into two species. We failed to amplify the corresponding fragments out of Brassicaceae using the designed DETl primers. We further discussed the discrimination power of different loci due to incomplete lineage sorting, gene flow, and species-specific evolution. Our results highlight the possibility of using the nuclear ITS as a core or complementary fragment to barcode recent diverged species.     

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.     

Are nuclear loci ideal for barcoding plants? A case study of genetic delimitation of two sister species using multiple loci and multiple intraspecific individuals

Considerable debate remains as to which DNA region should be used to barcode plants. Several different chloroplast (cp) DNA regions (rbcL, matK, and trnH–psbA) and nuclear ribosomal internal transcribed regions (ITS) have been suggested as suitable barcodes in plants. Recently, low-copy nuclear loci were also suggested to be potentially ideal barcode regions. The aim of the present study was to test the effectiveness of these proposed DNA fragments and five additional low-copy loci (CHS, DET1, COP1, PGIC1, and RPS2; comprising both coding and non-coding regions) in barcoding closely related species. We examined the divergences within and between two species of Pugionium (Brassicaceae). We failed to find any interspecific variation from three cpDNA fragments with which to discriminate the two species. However, a single base mutation in the internal transcribed spacer (ITS) could discriminate between the two species consistently. We found more variations among all individuals of the two species using each of the other five low-copy nuclear loci. However, only alleles from one locus (DET1) of the five low-copy loci related to flowering regulations was able to distinguish the sampled individuals into two species. We failed to amplify the corresponding fragments out of Brassicaceae using the designed DET1 primers. We further discussed the discrimination power of different loci due to incomplete lineage sorting, gene flow, and species-specific evolution. Our results highlight the possibility of using the nuclear ITS as a core or complementary fragment to barcode recent diverged species.     

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.     

On the monophyly of Dactylorhiza Necker ex Nevski (Orchidaceae): is Coeloglossum viride (L.) Hartman a Dactylorhiza?

Previous phylogenetic analyses of Orchidaceae subtribe Orchidinae resulted in the proposal to classify Coeloglossum viride (L.) Hartman within the genus Dactylorhiza in order to maintain its monophyly. In this paper, we report some results that contradict previous studies regarding the monophyly of the traditional Dactylorhiza and its phylogenetic relationship with Coeloglossum. Our results, which combine sequences of the internal and external transcribed spacers of the nuclear ribosomal DNA, support the monophyly of Dactylorhiza , with Coeloglossum being a sister clade. The position of C. viride in the phylogenetic tree, and the considerable morphological differences with respect to Dactylorhiza , incline us to retain both lineages as distinct genera.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 152 , 261–269.     

Molecular characterisation and development of rapid molecular methods to identify species of Gracilariaceae from the Atlantic coast of Morocco

In Gracilariaceae, species identification is traditionally based on gross morphology; therefore the taxonomic status of terete individuals remains frequently problematic due to the lack of diagnostic characters to identify specimens. Different morphospecies have been recorded along the Atlantic coast of Morocco; however, no clear diagnostic characters were available to discriminate between terete species. Rapid molecular techniques have been developed recently to resolve many taxonomic problems and to re-assess the global diversity and biogeography in algae. In this study, molecular markers were used as DNA barcoding to characterise species. The sequence of the Rubisco spacer allowed identification of six species of Gracilariaceae: Gracilaria gracilis, Gracilaria dura, Gracilaria conferta, Gracilaria vermiculophylla, Gracilaria multipartita and Gracilariopsis longissima. In order to identify species with certainty, two simple and rapid methods based on the amplification of rDNA ITS and PCR-RFLP of the large subunit of the Rubisco were developed.     

DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer

Oomycete species occupy many different environments and many ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak. The use of DNA for oomycete species identification is well established, but DNA barcoding with cytochrome c oxidase subunit I (COI) is a relatively new approach that has yet to be assessed over a significant sample of oomycete genera. In this study we have sequenced COI, from 1205 isolates representing 23 genera. A comparison to internal transcribed spacer (ITS) sequences from the same isolates showed that COI identification is a practical option; complementary because it uses the mitochondrial genome instead of nuclear DNA. In some cases COI was more discriminative than ITS at the species level. This is in contrast to the large ribosomal subunit, which showed poor species resolution when sequenced from a subset of the isolates used in this study. The results described in this paper indicate that COI sequencing and the dataset generated are a valuable addition to the currently available oomycete taxonomy resources, and that both COI, the default DNA barcode supported by GenBank, and ITS, the de facto barcode accepted by the oomycete and mycology community, are acceptable and complementary DNA barcodes to be used for identification of oomycetes.     

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.     

Evaluation of six candidate DNA barcoding loci in Ficus (Moraceae) of China

Ficus, with about 755 species, diverse habits and complicated co‐evolutionary history with fig wasps, is a notoriously difficult group in taxonomy. DNA barcoding is expected to bring light to the identification of Ficus but needs evaluation of candidate loci. Based on five plastid loci (rbcL, matK, trnH‐psbA, psbK‐psbI, atpF‐atpH) and a nuclear locus [internal transcribed spacer (ITS)], we calculated genetic distances and DNA barcoding gaps individually and in combination and constructed phylogenetic trees to test their ability to distinguish the species of the genus. A total of 228 samples representing 63 putative species in Ficus (Moraceae) of China were included in this study. The results demonstrated that ITS has the most variable sites, greater intra‐ and inter‐specific divergences, the highest species discrimination rate (72%) and higher primer universality among the single loci. It is followed by psbK‐psbI and trnH‐psbA with moderate variation and considerably lower species discrimination rates (about 19%), whereas matK, rbcL and atpF‐atpH could not effectively separate the species. Among the possible combinations of loci, ITS + trnH‐psbA performed best but only marginally improved species resolution over ITS alone (75% vs. 72%). Therefore, we recommend using ITS as a single DNA barcoding locus in Ficus.     

核糖体DNA的内转录间隔区序列标记在真菌分类鉴定中的应用

传统的真菌分类主要根据真菌菌株的形态特征、生长特性与生理生化指标进行,而分子生物学技术的发展提升了真菌分类鉴定研究的手段。真菌核糖体DNA内转录间隔区(ITS)在进化上比编码区快,种内的不同菌株之间高度保守,但在种间变化极大,故可为真菌学的研究提供丰富的遗传信息。简要综述了ITS序列分析技术在真菌分类鉴定中的应用现状、相关问题及前景。     

Variability of Ribosomal DNA ITS-2 and Its Utility in Detecting Genetic Relatedness of Pearl Oyster

The objective of this study was to detect interspecific and intraspecific genetic variations of the second internal transcribed spacer of ribosomal DNA (ITS-2), and explore the feasibility of using it as a molecular marker phylogenetic analyses and species identification among pearl oysters. ITS-2 sequences of 6 pearl oysters were amplified via polymerase chain reaction. The amplified DNA fragments were about 500 bp, spanning the partial sequences of 5.8S and 28S rRNA genes. The GC contents of all species used in this study were higher than the AT contents. The variations of sequences involved substitutions as well as insertions/deletions and were mainly concentrated in spacer regions. Sequences of about 30-bp in spacer regions showed no variations among 5 Pincatda species. Intraindividual and intraspecific polymorphisms of ITS-2 sequences were detected in some species; the interspecific variability was significantly larger than the variability within species, and the variability at the genus level was higher than that at the species level. Both neighbor-joining and parsimony analyses of ITS-2 sequences revealed the distinguishable species boundary of 6 pearl oysters, and indicated that P. chemnitzi and P. nigra were the closely related species, as were P. maxima and P. margaritifera. The findings revealed that ITS-2 sequences could be an appropriate tool for phylogenetic study of pearl oysters.