Species-specific oligonucleotide probes for macroalgae: molecular discrimination of two marine fouling species of Enteromorpha (Ulvophyceae)

The green seaweeds Enteromorpha intestinalis and E. compressa are important fouling organisms commonly found in polluted and nutrient-enriched marine and brackish water habitats, where they are used in environmental monitoring. Discrimination of the two species is extremely difficult because of overlapping morphological characters. In this study a quick molecular method for species identification was developed based on the nuclear rDNA ITS2 sequence data of 54 E. intestinalis samples and 20 E. compressa samples from a wide geographical range. Oligonucleotide probes were designed for species-specific hybridization to dot-blots of the PCR-amplified ITS1, 5.8S gene and ITS2 fragment of both E. intestinalis and E. compressa. Specificity of the oligonucleotide probes was confirmed by tests with taxonomically diverse species that could morphologically be confused with E. intestinalis or E. compressa. This is the first use of species-specific probes for macroalgae. The restriction endonuclease NruI digested specifically the amplified PCR product from E. compressa into two fragments detectable on agarose gels, but no suitable restriction sites were identifiable in the PCR product of E. intestinalis.     

米尔顿姬小蜂rDNA ITS1和ITS2的序列分析及其分子鉴定

本研究测定了米尔顿姬小蜂Anselmella miltoni Girault的rDNA ITS1和ITS2序列,以探讨其分子鉴定方法。米尔顿姬小蜂的ITS1和ITS2侧翼区（18S和5.8S）序列相对稳定,ITS1和ITS2序列存在种间差异。根据18S rDNA部分序列,利用DNAMAN的Maximum Likelihood方法构建了与膜翅目其它科的系统发育树。根据米尔顿姬小蜂ITS1和ITS2序列设计了特异性引物,应用特异性引物对样品进行了PCR扩增,扩增效果理想,采用上述特异性引物可从单头米尔顿姬小蜂稳定地扩增出明显的目的DNA条带。因此,可以采用ITS1和ITS2区的特异性对米尔顿姬小蜂进行快速的分子鉴定。     

基于ITS条形码序列的刺桐姬小蜂分子鉴定

【目的】刺桐姬小蜂Quadrastichus erythrinae Kim体型小,传统的形态学鉴定方法难以快速准确识别。【方法】本研究测定了刺桐姬小蜂的rDNA ITS1和ITS2序列,根据18S rDNA部分序列,利用MEGA的最大相似法(Maximum Likehood)构建系统发育树。根据刺桐姬小蜂ITS1和ITS2序列设计了特异引物,应用特异引物对单只刺桐姬小蜂进行PCR扩增,可稳定地扩增出明显的目的DNA条带。【结果】研究表明,基于ITS基因的DNA条形码技术可以用于刺桐姬小蜂的快速准确鉴定。【结论】因此,采用ITS1和ITS2区的特异性引物可对刺桐姬小蜂进行快速分子鉴定。     

Differentiation of pathogenic and nonpathogenic isolates of <Emphasis Type="Italic">Geotrichum candidum</Emphasis> sensu Suprapta et al. (1995) on citrus fruit based on PCR-RFLP analysis of rDNA ITS and PCR using specific primers designed in polygalacturonase genes

Pathogenic and nonpathogenic isolates of Geotrichum candidum sensu Suprapta et al. (1995) that affect citrus fruit are indistinguishable morphologically. In this work, differentiation of the two pathogenicity types based on the internal transcribed spacer regions of ribosomal DNA (rDNA-ITS) and polygalacturonase (PG) genes was attempted. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of rDNA ITS and PCR using specific primers to PG genes from each type could clearly differentiate the two pathogenicity types, and the profiles by PCR-RFLP of rDNA ITS genes corresponded with those by type-specific PCR of PG genes. These results indicate that the two pathogenic types can be differentiated at a molecular level and that PG genes are alternatively useful for distinguishing between the two types.     

CULTIVATION‐INDEPENDENT SPECIES IDENTIFICATION OF DINOBRYON SPECIES (CHRYSOPHYCEAE) BY MEANS OF MULTIPLEX SINGLE‐CELL PCR1

With the discovery of a high molecular diversity of protists, a discrepancy between morphological and molecular species richness estimates became apparent. Solving the current concerns requires a comparative analysis of different sequences combined with morphological analyses of single cells originating from preserved field samples. We refined a single‐cell PCR (SC‐PCR) protocol for analyzing cells from field samples preserved with Lugol’s iodine solution. We linked microscopic screening with multiplex PCR targeting the SSU rDNA, internal transcribed spacer 1 (ITS1), 5.8S rDNA, internal transcribed spacer 2 (ITS2), and the mitochondrial cytochrome oxidase 1 (CO1) in a single PCR reaction. Using this method, we investigated the intraspecific molecular variation in Dinobryon populations originating from two lakes in the Salzkammergut area of Austria. All investigated genetic markers showed two separated clusters within the investigated populations of Dinobryon divergens O. E. Imhof, indicating a reproductive isolation of the two coexisting populations. Based on these findings, we describe a lineage, which is morphologically similar to D. divergens but, based on the molecular data, is reproductively isolated.     

Incomplete concerted evolution and reproductive isolation at the rDNA locus uncovers nine cryptic species within <Emphasis Type="Italic">Anopheles longirostris</Emphasis>from Papua New Guinea

Background  

Nuclear ribosomal DNA (rDNA) genes and transcribed spacers are highly utilized as taxonomic markers in metazoans despite the lack of a cohesive understanding of their evolution. Here we follow the evolution of the rDNA second internal transcribed spacer (ITS2) and the mitochondrial DNA cytochrome oxidase I subunit in the malaria mosquito Anopheles longirostris from Papua New Guinea (PNG). This morphospecies inhabits a variety of ecological environments indicating that it may comprise a complex of morphologically indistinguishable species. Using collections from over 70 sites in PNG, the mtDNA was assessed via direct DNA sequencing while the ITS2 was assessed at three levels - crude sequence variation through restriction digest, intragenomic copy variant organisation (homogenisation) through heteroduplex analysis and DNA sequencing via cloning.     

基于rDNA ITSl和ITS2序列的褐飞虱、白背飞虱和灰飞虱的分子鉴定

本研究测定了褐飞虱 Nilaparvata lugens、白背飞虱 Sogatella furcifera 和灰飞虱Laodelphax striatellus 的rDNA ITSl和ITS2的序列,以探讨这3种稻飞虱的分子鉴定方法.3种飞虱的ITSI和ITS2侧翼区(18S,5.8S和28S)序列相对稳定,但ITS1和ITS2序列在3种飞虱中变异较大.ITS1在所分析的438个位点中可变位点达294个,ITS2在分析的403个位点中可变位点为177个.根据3种飞虱rDNA的ITS1和ITS2序列设计了特异性引物,应用特异性引物对样品进行了PCR扩增,分析发现3种飞虱ITS1区的特异性引物扩增效果不理想.而ITS2区的特异性引物可以稳定地扩增出明显的目的DNA条带.因此,采用ITS2区的特异性引物可以对3种飞虱进行快速的分子鉴定.     

Molecular evidence for the synonymy of two species of Apatemon Szidat, 1928, A. gracilis (Rudolphi, 1819) and A. annuligerum (von Nordmann, 1832) (Digenea: Strigeidae) parasitic as metacercariae in British fishes

The current study examined rDNA (internal transcribed spacer regions, ITS1 and ITS2) and cytochrome c oxidase subunit 1 (CO1) sequence data of Apatemon annuligerum (originating from two geographical locations) and A. gracilis metacercariae (originating from four natural piscine hosts) to determine the systematic status of these two strigeid digeneans. With the exception of short repeat motifs, the ITS1 regions sequenced demonstrated no intra- or inter-specific sequence variation. ITS2 sequences were 292 bp and CO1 sequences 366 bp in length and identical for both nominal Apatemon species. These sequence data provide strong evidence that the two species are con-specific and that A. annuligerum should be regarded as a junior synonym of A. gracilis.     

Diglyphus isaea (Hymenoptera: Eulophidae): a probable complex of cryptic species that forms an important biological control agent of agromyzid leaf miners

Diglyphus isaea ( Walker 1838 ) (Hymenoptera: Eulophidae) is a primary parasitoid of agromyzid leaf miners (Diptera: Agromyzidae) and has been commercialized as a biological control agent. Diglyphus isaea occurs throughout much of the world and different populations are morphologically identical. Using nuclear ribosomal internal transcribed spacer 1 (ITS1) sequences, we examined variation among Chinese populations of D. isaea . Phylogenetic analyses combined with an analysis of sympatry indicated that D. isaea consists of at least four species in mainland China. The results imply that ITS1 is an efficient marker for identifying congeneric species of parasitic waSPS, and that cryptic species could be common in temperate and subtropical regions.     

基于rDNA ITS1和ITS2序列的褐飞虱、白背飞虱和灰飞虱的分子鉴定

本研究测定了褐飞虱Nilaparvata lugens、白背飞虱Sogatella furcifera和灰飞虱Laodelphax striatellus的rDNA ITS1和ITS2的序列, 以探讨这3种稻飞虱的分子鉴定方法。3种飞虱的ITS1和ITS2侧翼区（18S, 5.8S和28S）序列相对稳定, 但ITS1和ITS2序列在3种飞虱中变异较大。 ITS1在所分析的438个位点中可变位点达294个, ITS2在分析的403个位点中可变位点为177个。根据3种飞虱rDNA的ITS1和ITS2序列设计了特异性引物, 应用特异性引物对样品进行了PCR扩增, 分析发现3种飞虱ITS1区的特异性引物扩增效果不理想, 而ITS2区的特异性引物可以稳定地扩增出明显的目的DNA条带. 因此, 采用ITS2区的特异性引物可以对3种飞虱进行快速的分子鉴定。     

Phylogenetic analysis of Allium subg. Melanocrommyum infers cryptic species and demands a new sectional classification

Allium subgenus Melanocrommyum (Alliaceae) from Eurasia comprises about 150 mostly diploid species adapted to arid conditions. The group is taxonomically complicated with different and contradictory taxonomic treatments, and was thought to include a considerable number of hybrid species, as the taxa show an admixture of assumed morphological key characters. We studied the phylogeny of the subgenus, covering all existing taxonomic groups and their entire geographic distribution. We analyzed sequences of the nuclear rDNA internal transcribed spacer region (ITS) for multiple individuals of more than 100 species. Phylogenetic analyses of cloned and directly sequenced PCR products confirmed the monophyly of the subgenus, while most sections were either para- or polyphyletic. The splits of the large sections are supported by differences in the anatomy of flower nectaries. ITS data (i) demand a new treatment at sectional level, (ii) do not support the hypotheses of frequent gene flow among species, (iii) indicate that multiple rapid radiations occurred within different monophyletic groups of the subgenus, and (iv) detected separately evolving lineages within three morphologically clearly defined species (cryptic species). In two cases these lineages were close relatives, while in Allium darwasicum they fall in quite different clades in the phylogenetic tree. Fingerprint markers show that this result is not due to ongoing introgression of rDNA (ITS capture) but that genome-wide differences between both lineages exist. Thus, we report one of the rare cases in plants where morphologically indistinguishable diploid species occurring in mixed populations are non-sister cryptic species.     

Discrimination of Aspergillus niger and other Aspergillus species belonging to section Nigri by PCR assays

Aspergillus species included in section Nigri are common in plant products and processed food, such as grapes, cereals, coffee and derivatives, particularly in warm and tropical climates. Two of these species, A. carbonarius and A. niger, are known to produce ochratoxin A (OTA), a potent nephrotoxin and carcinogenic to human (group 2B). Recognition of the several species of this section is difficult and requires considerable expertise using conventional methods based on morphological features. In this work we describe rapid, sensitive and robust assays based on the PCR technique to discriminate the main species included in section Nigri: A. japonicus, A. heteromorphus, A. ellipticus and the two morphologically indistinguishable species of the A. niger aggregate: A. niger and A. tubingensis. The species-specific primers have been designed on the basis of ITS (internal transcribed spacers of rDNA units) sequence comparisons obtained from several Aspergillus strains and have been tested in a number of strains from different origins and hosts. These PCR assays, based on multi-copy sequences, are highly sensitive and specific and represent a good tool for an early detection of OTA-producing Aspergillus species in order to prevent OTA from entering the food chain.     

Characterization of Variability in Some Pathogenic Species of Alternaria Based on the Nucleotide Sequence of Ribosomal DNA

The internal transcribed spacer (ITS) sequences within the ribosomal DNA (rDNA) region were targeted to delineate genetic variability among eight Alternaria species that cause economically important diseases in crops. The rDNA regions of Alternaria species comprising of rRNA genes and the ITS regions were cloned and sequenced. Phylogenetic relationship based on the rDNA sequences and PCR-RFLP of amplified rDNA sequences clustered eight species of Alternaria into three major groups. A. macrospora and A. helianthi accumulated wide genetic variations and are distantly related to rest of the six species which formed two major groups. Group I comprised of three species viz., A. dianthicola, A. brassicae and A. citri, while group II had A. longipes, A. porri and A. alternata. Incorporation of unique stretches of nucleotides and single nucleotide substitutions within relatively conserved ITS1 and ITS2 regions led to clustering of the members of Alternaria species in each group. The divergent sequences within the ITS regions can be employed to design species-specific PCR primer for use in molecular diagnostics.     

Molecular evidence of natural hybridization between Fasciola hepatica and F. gigantica.

Nucleotide sequences of two regions, cytochrome c-oxidase subunit 1 (CO1) and NADH dehydrogenase subunit 1 (ND1) of the mitochondrial DNA and two regions, internal-transcribed spacer 2 (ITS2) and the D2 region in the 28S rDNA (28S) of the nuclear DNA were obtained from five Korean worms of the genus Fasciola in order to elucidate their taxonomic status. The CO1 and ND1 regions are all monomorphic in the Korean worms and similar to those of F. gigantica. On the other hand, the ITS2 and D2 regions were found to be polymorphic; that is, out of five worms, two possessed a F. gigantica-type sequence, one, a F. hepatica-type sequence and two possessed sequences of both types indicating an existence of different alleles at the loci. It should be noted that these variations of the ITS2 and D2 regions co-occur at the same individual worms. This was confirmed by sequencing five to six cloned PCR products for each worm. The present study strongly suggests interspecific cross-hybridization between the two species coexisting in Korea.     

Identification by polymerase chain reaction (PCR) of Marshallagia marshalli and Ostertagia gruehneri from Svalbard reindeer

A polymerase chain reaction (PCR) assay to identify two common abomasal nematodes Marshallagia marshalli and Ostertagia gruehneri of Svalbard reindeer was developed. Species-specific PCR primers were designed from internal transcribed spacer (ITS)-2 sequences of rDNA and validated using morphologically identified adult male and female nematodes. Using the species-specific primers, a 110 bp fragment was amplified from M. marshalli and its minor morph Marshallagia occidentalis and a 149 bp fragment was amplified from Ostertagia gruehneri and its minor morph Ostertagia arctica. No PCR products were amplified from the third rare species, Teladorsagia circumcincta, or DNA from the reindeer host. The assay provides a useful tool to estimate species composition for both sexes in this nematode community.     

Sequence analysis of ribosomal and mitochondrial genes of the giant liver fluke Fascioloides magna (Trematoda: Fasciolidae): intraspecific variation and differentiation from Fasciola hepatica

Complete sequences of ribosomal and mitochondrial genes of the giant liver fluke Fascioloides magna are presented. In particular, small subunit (18S) and internal transcribed spacers (ITS1 and ITS2) of the ribosomal gene (rDNA), as well as cytochrome c oxidase subunit I (cox1) and nicotinamide dehydrogenase subunit I (nad1) of the mitochondrial DNA (mtDNA), were analyzed. The 18S and ITS sequences were compared with previously published sequences of the liver fluke Fasciola hepatica. Fixed interspecific genetic differences were determined that allow molecular differentiation of F. magna and F. hepatica using either the PCR-RFLP method or PCR amplification of species-specific DNA regions. Additionally, intraspecific sequence polymorphism of the complete cox1 and nad1 mitochondrial genes in geographically distinct F. magna populations was determined. Based on the sequence divergences, short (< 500 bp) variable regions suitable for broader biogeographical studies of giant liver fluke were designed.     

CONSPECIFICITY AND INDO-PACIFIC DISTRIBUTION OF SYMBIODINIUM GENOTYPES (DINOPHYCEAE) FROM GIANT CLAMS

We previously reported the occurrence of genetically‐diverse symbiotic dinoflagellates (zooxanthellae) within and between 7 giant clam species (Tridacnidae) from the Philippines based on the algal isolates' allozyme and random amplified polymorphic DNA (RAPD) patterns. We also reported that these isolates all belong to clade A of the Symbiodinium phylogeny with identical 18S rDNA sequences. Here we extend the genetic characterization of Symbiodinium isolates from giant clams and propose that they are conspecific. We used the combined DNA sequences of the internal transcribed spacer (ITS)1, 5.8S rDNA, and ITS2 regions (rDNA‐ITS region) because the ITS1 and ITS2 regions evolve faster than 18S rDNA and have been shown to be useful in distinguishing strains of other dinoflagellates. DGGE of the most variable segment of the rDNA‐ITS region, ITS1, from clonal representatives of clades A, B, and C showed minimal intragenomic variation. The rDNA‐ITS region shows similar phylogenetic relationships between Symbiodinium isolates from symbiotic bivalves and some cnidarians as does 18S rDNA, and that there are not many different clade A species or strains among cultured zooxanthellae (CZ) from giant clams. The CZ from giant clams had virtually identical sequences, with only a single nucleotide difference in the ITS2 region separating two groups of isolates. These data suggest that there is one CZ species and perhaps two CZ strains, each CZ strain containing individuals that have diverse allozyme and RAPD genotypes. The CZ isolated from giant clams from different areas in the Philippines (21 isolates, 7 clam species), the Australian Great Barrier Reef (1 isolate, 1 clam species), Palau (8 isolates, 7 clam species), and Okinawa, Japan (1 isolate, 1 clam species) shared the same rDNA‐ITS sequences. Furthermore, analysis of fresh isolates from giant clams collected from these geographical areas shows that these bivalves also host indistinguishable clade C symbionts. These data demonstrate that conspecific Symbiodinium genotypes, particularly clade A symbionts, are distributed in giant clams throughout the Indo‐Pacific.     

临床常见镰刀菌的鉴别

目的从分子生物学角度寻找一种快速准确鉴定临床常见镰刀菌的方法。方法将受试镰刀菌接种于PDA培养基,观察其菌落及镜下形态,在此基础上PCR扩增受试镰刀菌的rDNA ITS并测其序列,在GenBank核酸序列数据库进行同源序列搜索及分析。选择限制性内切酶Dra Ⅱ和Cfr13 Ⅰ进行RFLP。设计了茄病镰刀菌的种特异性引物Sol1、Sol2,初步验证其特异性。结果形态学鉴定结果显示,茄病镰刀菌所占比例最高,除2株串珠镰刀菌外,其余镰刀菌ITS序列分析的结果与形态学鉴定结果一致。茄病、层生和串珠镰刀菌的Dra Ⅱ、Cfr13 I酶切带形互不相同。用Sol1、Sol2扩增受试菌的rDNA ITS,只有茄病镰刀菌为阳性。结论rDNA ITS序列测定及其PCR-RFLP可用于初步鉴别几种临床常见镰刀菌,合适的种特异性引物可以初步快速鉴定茄病镰刀菌。     

Primers are designed for amplification and direct sequencing of ITS region of rDNA from Myxomycetes

Four new primers were designed, based on comparison of Physarum polycephalum sequences retrieved from Genbank (primers PHYS-5 and PHYS-4) and our own sequences (primers PHYS-3 and PHYS-2), to amplify the ITS regions of rDNA, including the 5.8S gene segment from Lamproderma species. Sequencing analysis shows that Lamproderma contains ITS1-5.8S-ITS2 regions of approximately 900 bp, which is similar in size to most eukaryotes. However, the corresponding region in another common myxomycete, Fuligo septica, is more than 2000 bp due to the presence of large direct-repeat motifs in ITS1. Myxomycete rDNA ITS regions are interesting both as phylogenetic markers in taxonomic studies and as model sequences for molecular evolution.