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.     

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.     

Rapid and reliable high-throughput methods of DNA extraction for use in barcoding and molecular systematics of mushrooms

We present two methods for DNA extraction from fresh and dried mushrooms that are adaptable to high-throughput sequencing initiatives, such as DNA barcoding. Our results show that these protocols yield ∼85% sequencing success from recently collected materials. Tests with both recent (<2 year) and older (>100 years) specimens reveal that older collections have low success rates and may be an inefficient resource for populating a barcode database. However, our method of extracting DNA from herbarium samples using small amount of tissue is reliable and could be used for important historical specimens. The application of these protocols greatly reduces time, and therefore cost, of generating DNA sequences from mushrooms and other fungi vs. traditional extraction methods. The efficiency of these methods illustrates that standardization and streamlining of sample processing should be shifted from the laboratory to the field.     

Disproportionate abundance between ectomycorrhizal root tips and their associated mycelia

Extensive knowledge of various ectomycorrhizal fungal communities has been obtained over the past 10 years based on molecular identification of the fungi colonizing fine roots. In contrast, only limited information exists about the species composition of ectomycorrhizal hyphae in soil. This study compared the ectomycorrhizal external mycelial community with the adjacent root-tip community in a Danish beech forest. Sand-filled in-growth mesh bags were used to trap external mycelia by incubating the mesh bags in the soil for 70 days. The adjacent ectomycorrhizal root-tip communities were recorded at the times of insertion and retrieval of the mesh bags. Ectomycorrhizal fungi were identified by sequencing the internal transcribed spacer region. In total, 20, 31 and 24 ectomycorrhizal species were recorded from the two root-tip harvests and from the mesh bags, respectively. Boletoid species were significantly more frequent as mycelia than as root tips, while russuloid and Cortinarius species appeared to be less dominant as mycelia than as root tips. Tomentella species were equally frequent as root tips and as mycelia. These discrepancies between the root-tip and the mycelial view of the ectomycorrhizal fungal community are discussed within the framework of ectomycorrrhizal exploration types.     

Using DNA-based techniques to identify hybrids among linear-leaved Potamogeton plants collected in China

Itis well known that interspecific hybrids occur in the genus Potamogeton. The linear-leaved Potamogeton species commonly have highly variable morphological characteristics. Their hybrids often show similar vegetative characters to their parental species and their identification based solely on morphology is not always conclusive. In order to clarify whether there are any hybrids from the linear-leaved Potamogeton plants collected in China, we used internal transcribed spacers (ITS) of nuclear ribosomal DNA and chloroplast rbcL gene sequences to identify the hybrids. Using ITS sequence additivity, we identified four hybrids, namely R orientalis (Rpusillus × P.oxyphyllus),P.pusillus × P. berchtoldii, P. foliosus × P. octandrus, and P. cristatus × P. octandrus. The latter three hybrids should be considered as new hybrids in Potamogeton. The maternal parents of the four hybrids were confirmed using chloroplast rbcL gene sequences.     

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。     

Detection of the antimicrobial peptide gene in different Amaranthus species

Using primers to amplify the gene AMP2 in Amaranthus caudatus, we found the gene to be present in seven other species of the Amaranthus genus (A. albus, A. cruentus, A. blitum, A. hybridus, A. hypochondriacus, A. retroflexus and A. tricolor), in which it had not been described previously. The PCR products were sequenced and it was established that all the sequences were identical, except for two polymorphisms. These single nucleotide polymorphisms occurred at nucleotide positions 45 and 246. This exchange of one nucleotide for another was manifested in an amino acid change in both cases. Due to the fact that both polymorphisms lay outside the region encoding the chitin-binding peptide domain, which is crucial for antimicrobial peptide function, they will not likely affect the proper functioning of the peptide. With the exception of the above-mentioned polymorphisms, all sequences were identical to the sequence of the AMP2 gene that codes for the A. caudatus Ac-AMP2 (antimicrobial peptide isolated from Amaranthus caudatus seeds). The detection of sequences with high degree of sequence similarity to A. caudatus AMP2 gene leads us to the assumption that an antimicrobial peptide could also be produced by other amaranth species.     

A fast SNP identification and analysis of intraspecific variation in the medicinal Panax species based on DNA barcoding

Medicinal plants of the Panax genus belonging to Araliaceae family are well-known, rare plants used as tonics in traditional Chinese medicine and have been described in the Chinese Pharmacopoeia. Because of the high price and the huge human demand, these commercial products often contain adulterants. In this study, 377 sequences from four species were analyzed. Single nucleotide polymorphisms (SNPs) were detected and patterns of intragenomic variation in internal transcribed spacer 2 (ITS2) from the four Panax species were studied. Intraspecific variations were analyzed based on three typical DNA barcodings (ITS2, matK and psbA-trnH). Results from this study revealed that intraspecific genetic distances in Panax ginseng and Panax quinquefolius were quite low (0–0.002) and the multi-copy ITS2 could be considered a single locus in the genomes of these two species. Five stable SNPs were detected in ITS2 region to identify the Panax medicinal species. Considering the mixed powder of P. ginseng and P. quinquefolius, double peaks could be clearly examined at SNP positions and the height of the peaks could indicate the mixed ratio roughly. Our findings indicate that SNP-based molecular barcodes could be developed as a routine method for the identification of the Panax genus with closely related species and the mixed powder P. ginseng and P. quinquefolius.     

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。     

Characterization of Genotypes of Enterocytozoon bieneusi in Immunosuppressed and Immunocompetent Patient Groups

ABSTRACT. A retrospective phylogenetic analysis was performed on isolates of Enterocytozoon bieneusi to characterize the genotypes in different patient cohorts. Fifty-seven isolates, collected from patients living in Malawi and the Netherlands, were classified by age and immune status of the hosts. Sequence analysis of the internal transcribed spacer (ITS) region identified 16 genotypes; nine have not previously been described. Genotypes K and D were most prevalent among patient groups, whereas genotype C was restricted to transplantation patients receiving immunosupressives and genotype B showed a predisposition toward patients living with HIV/AIDS. Different genotypes showed more dispersion among isolates from Malawi compared with those from the Netherlands. A constructed map estimating the genealogy of the ITS region reveals a dynamic evolutionary process between the genotypes.