Accurate identification of closely related Dendrobium species with multiple species-specific gDNA probes

About 63 species of Dendrobium are identified in China, making the identification of the origin of a particular Dendrobium species on the consumer market very difficult. We report evaluation of multiple species-specific probes screened from genomic DNA for closely related Dendrobium species identification, based on DNA array hybridization. Fourteen species-specific probes were screened from five closely related Dendrobium species, D. aurantiacum Kerr, D. officinale Kimura et Migo, D. nobile Lindl., D. chrysotoxum Lindl. and D. fimbriatum Hook., based on the SSH-Array technology we developed. Various commercial Dendrobium samples and unrelated samples were definitely identified. The specificity and accuracy of the multiple species-specific probes for species identification was assessed by identifying various commercial Dendrobium samples (Herba Dendrobii). Hybridization patterns of these multiple probes on digested genomic DNAs of Dendrobium species indicated that there are distinct polymorphic sequence fragment in the higher eukaryotes. This is the first report on detection and utilization of multiple species-specific probes of Dendrobium in whole genomic DNA, and this could be useful tools not only for a new technical platform for the closely related species identification but also for epidemiological studies on higher eukaryotes.     

Diversity Suppression-Subtractive Hybridization Array for Profiling Genomic DNA Polymorphisms

Genomlc DNA polymorphlsms are very useful for tracing genetic traits end studying biological diversity among species. Here, we present a method we call the ＂diversity suppresslon-subtractlve hybridization array＂ for effectively profiling genomlc DNA polymorphisms. The method first obtains the subtracted gDNA fragments between any two species by suppression subtraction hybridization （SSH） to establish e subtracted gDNA library, from which diversity SSH arrays are created with the selected subtracted clones. The diversity SSH array hybridizes with the DIG-labeled genomlc DNA of the organism to be assayed. Six closely related Dendrobium species were studied as model samples. Four Dendrobium species as testers were used to perform SSH. A total of 617 subtracted positive clones were obtained from four Dendrobium species, and the average ratio of positive clones was 80.3%. We demonstrated that the average percentage of polymorphlc fragments of palrwlse comparisons of four Dendrobium species was up to 42.4%. A dendrogram of the relatedness of six Dendrobium species was produced according to their polymorphic profiles. The results revealed that the diversity SSH array Is a highly effective platform for profiling genomlc DNA polymorphlsms and dendrograms.     

A gDNA Microarray for Genotyping Salvia Species

Salvia is an important genus from the Lamiaceae with approximately 1,000 species. This genus is distributed globally and cultivated for ornamental, culinary, and medicinal uses. We report the construction of the first fingerprinting array for Salvia species enriched with polymorphic and divergent DNA sequences and demonstrate the potential of this array for fingerprinting several economically important members of this genus. In order to generate the Salvia subtracted diversity array (SDA) a suppression subtractive hybridization (SSH) was performed between a pool of Salvia species and a pool of angiosperms and non-angiosperms to selectively isolate Salvia-specific sequences. A total of 285-subtracted genomic DNA (gDNA) fragments were amplified and arrayed. DNA fingerprints were obtained for fifteen Salvia genotypes including three that were not part of the original subtraction pool. Hierarchical cluster analysis indicated that the Salvia-specific SDA was capable of differentiating S. officinalis and S. miltiorrhiza from their closely related species and was also able to reveal genetic relationships consistent with geographical origins. In addition, this approach was capable of isolating highly polymorphic sequences from chloroplast and nuclear DNA without preliminary sequence information. Therefore, SDA is a powerful technique for fingerprinting non-model plants and for identifying new polymorphic loci that may be developed as potential molecular markers.     

Fingerprinting the Asterid species using subtracted diversity array reveals novel species-specific sequences

Background

Asterids is one of the major plant clades comprising of many commercially important medicinal species. One of the major concerns in medicinal plant industry is adulteration/contamination resulting from misidentification of herbal plants. This study reports the construction and validation of a microarray capable of fingerprinting medicinally important species from the Asterids clade.

Methodology/Principal Findings

Pooled genomic DNA of 104 non-asterid angiosperm and non-angiosperm species was subtracted from pooled genomic DNA of 67 asterid species. Subsequently, 283 subtracted DNA fragments were used to construct an Asterid-specific array. The validation of Asterid-specific array revealed a high (99.5%) subtraction efficiency. Twenty-five Asterid species (mostly medicinal) representing 20 families and 9 orders within the clade were hybridized onto the array to reveal its level of species discrimination. All these species could be successfully differentiated using their hybridization patterns. A number of species-specific probes were identified for commercially important species like tea, coffee, dandelion, yarrow, motherwort, Japanese honeysuckle, valerian, wild celery, and yerba mate. Thirty-seven polymorphic probes were characterized by sequencing. A large number of probes were novel species-specific probes whilst some of them were from chloroplast region including genes like atpB, rpoB, and ndh that have extensively been used for fingerprinting and phylogenetic analysis of plants.

Conclusions/Significance

Subtracted Diversity Array technique is highly efficient in fingerprinting species with little or no genomic information. The Asterid-specific array could fingerprint all 25 species assessed including three species that were not used in constructing the array. This study validates the use of chloroplast genes for bar-coding (fingerprinting) plant species. In addition, this method allowed detection of several new loci that can be explored to solve existing discrepancies in phylogenetics and fingerprinting of plants.     

Generation of subspecies level-specific microbial diagnostic microarrays using genes amplified from subtractive suppression hybridization as microarray probes

The generation of microarray probes with specificity below the species level is an ongoing challenge, not least because the high-throughput detection of microorganisms would be an efficient means of identifying environmentally relevant microbes. Here, we describe how suppression subtractive hybridization (SSH) can be applied to the production of microarray probes that are useful for microbial differentiation at the subspecies level. SSH was used to initially isolate unique genomic sequences of nine Salmonella strains, and these were validated in quadruplicate by microarray analysis. The results obtained indicate that a large group of genes subtracted by SSH could serve together, as one probe, for detecting a microbial subspecies. Similarly, the whole microbial genome (not subjected to SSH) can be used as a species-specific probe. The detailed methods described herein could be used and adapted for the estimation of any cultivable bacteria from different environments.     

Subtracted Diversity Array Identifies Novel Molecular Markers Including Retrotransposons for Fingerprinting Echinacea Species

Echinacea, native to the Canadian prairies and the prairie states of the United States, has a long tradition as a folk medicine for the Native Americans. Currently, Echinacea are among the top 10 selling herbal medicines in the U.S. and Europe, due to increasing popularity for the treatment of common cold and ability to stimulate the immune system. However, the genetic relationship within the species of this genus is unclear, making the authentication of the species used for the medicinal industry more difficult. We report the construction of a novel Subtracted Diversity Array (SDA) for Echinacea species and demonstrate the potential of this array for isolating highly polymorphic sequences. In order to selectively isolate Echinacea-specific sequences, a Suppression Subtractive Hybridization (SSH) was performed between a pool of twenty-four Echinacea genotypes and a pool of other angiosperms and non-angiosperms. A total of 283 subtracted genomic DNA (gDNA) fragments were amplified and arrayed. Twenty-seven Echinacea genotypes including four that were not used in the array construction could be successfully discriminated. Interestingly, unknown samples of E. paradoxa and E. purpurea could be unambiguously identified from the cluster analysis. Furthermore, this Echinacea-specific SDA was also able to isolate highly polymorphic retrotransposon sequences. Five out of the eleven most discriminatory features matched to known retrotransposons. This is the first time retrotransposon sequences have been used to fingerprint Echinacea, highlighting the potential of retrotransposons as based molecular markers useful for fingerprinting and studying diversity patterns in Echinacea.     

Oligonucleotide Array for Identification and Detection of Pythium Species

A DNA array containing 172 oligonucleotides complementary to specific diagnostic regions of internal transcribed spacers (ITS) of more than 100 species was developed for identification and detection of Pythium species. All of the species studied, with the exception of Pythium ostracodes, exhibited a positive hybridization reaction with at least one corresponding species-specific oligonucleotide. Hybridization patterns were distinct for each species. The array hybridization patterns included cluster-specific oligonucleotides that facilitated the recognition of species, including new ones, belonging to groups such as those producing filamentous or globose sporangia. BLAST analyses against 500 publicly available Pythium sequences in GenBank confirmed that species-specific oligonucleotides were unique to all of the available strains of each species, of which there were numerous economically important ones. GenBank entries of newly described species that are not putative synonyms showed no homology to sequences of the spotted species-specific oligonucleotides, but most new species did match some of the cluster-specific oligonucleotides. Further verification of the specificity of the DNA array was done with 50 additional Pythium isolates obtained by soil dilution plating. The hybridization patterns obtained were consistent with the identification of these isolates based on morphology and ITS sequence analyses. In another blind test, total DNA of the same soil samples was amplified and hybridized on the array, and the results were compared to those of 130 Pythium isolates obtained by soil dilution plating and root baiting. The 13 species detected by the DNA array corresponded to the isolates obtained by a combination of soil dilution plating and baiting, except for one new species that was not represented on the array. We conclude that the reported DNA array is a reliable tool for identification and detection of the majority of Pythium species in environmental samples. Simultaneous detection and identification of multiple species of soilborne pathogens such as Pythium species could be a major step forward for epidemiological and ecological studies.     

Discrimination of Six Pratylenchus Species Using PCR and Species-Specific Primers

A PCR-based assay for identification of six species of Pratylenchus common in California is described. In this assay, five forward species-specific primers were designed from the internal variable portion of the D3 expansion region of the 26S rDNA and were each used with a single, common reverse primer. The optimized species-specific primers produced unique amplicons from their respective target and did not amplify DNA from other Pratylenchus species. With this assay we were able to identify single females to species level. This method obviates the need for subsequent RFLP or sequence analysis of the PCR product and can be used as a rapid diagnostic tool in epidemiological and management studies.     

Molecular confirmation of Dicrocoelium dendriticum in the Himalayan ranges of Pakistan

Lancet liver flukes of the genus Dicrocoelium (Trematoda: Digenea) are recognised parasites of domestic and wild herbivores. The aim of the present study was to confirm the species identity of Dicrocoeliid flukes collected from the Chitral valley in the Himalayan ranges of Pakistan. The morphology of 48 flukes belonging to eight host populations was examined; but overlapping traits prevented accurate species designation. Phylogenetic comparison of published D. dendriticum ribosomal cistron DNA, and cytochrome oxidase-1 (COX-1) mitochondrial DNA sequences with those from D. chinensis was performed to assess within and between species variation and re-affirm the use of species-specific single nucleotide polymorphism markers. PCR and sequencing of 34 corresponding fragments of ribosomal DNA and 14 corresponding fragments of mitochondrial DNA from the Chitral valley flukes, revealed 10 and 4 unique haplotypes, respectively. These confirmed for the first time the molecular species identity of Pakistani lancet liver flukes as D. dendriticum. This work provides a preliminary illustration of a phylogenetic approach that could be developed to study the ecology, biological diversity, and epidemiology of Dicrocoeliid lancet flukes when they are identified in new settings.     

Isolation of genomic DNA suitable for community analysis from mature trees adapted to arid environment

Isolation of intact and pure genomic DNA (gDNA) is essential for many molecular biology applications. It is difficult to isolate pure DNA from mature trees of hot and dry desert regions because of the accumulation of high level of polysaccharides, phenolic compounds, tannins etc. We hereby report the standardized protocol for the isolation and purification of gDNA from seven ecologically and medically important tree species of Combretaceae viz. Anogeissus (Anogeissus sericea var. nummularia, Anogeissus pendula, and Anogeissus latifolia) and Terminalia (Terminalia arjuna, Terminalia bellirica, Terminalia catappa and Terminalia chebula). This method involves (i) washing the sample twice with Triton buffer (2%) then (ii) isolation of gDNA by modified-CTAB (cetyl trimethyl ammonium bromide) method employing a high concentration (4%) of PVP (Polyvinylpyrrolidone) and 50 mM ascorbic acid, and (iii) purification of this CTAB-isolated gDNA by spin-column. gDNA isolated by modified CTAB or spin-column alone were not found suitable for PCR amplification. The Triton washing step is also critical. The quality of DNA was determined by the A₂₆₀/A₂₈₀ absorbance ratio. gDNA was also observed for its intactness by running on 0.8% agarose gel. The suitability of extracted DNA for PCR was tested by amplification with RAPD primers, which was successful. Further, rbcLa (barcoding gene) was amplified and sequenced to check the quality of extracted gDNA for its downstream applications.     

DNA Probes Show Genetic Variation in Cyanobacterial Symbionts of the Azolla Fern and a Closer Relationship to Free-Living Nostoc Strains than to Free-Living Anabaena Strains

Twenty-two isolates of Anabaena azollae derived from seven Azolla species from various geographic and ecological sources were characterized by DNA-DNA hybridization. Cloned DNA fragments derived from the genomic sequences of three different A. azollae isolates were used to detect restriction fragment length polymorphism among all symbiotic anabaenas. DNA clones were radiolabeled and hybridized against southern blot transfers of genomic DNAs of different isolates of A. azollae digested with restriction endonucleases. Eight DNA probes were selected to identify the Anabaena strains tested. Two were strain specific and hybridized only to A. azollae strains isolated from Azolla microphylla or Azolla caroliniana. One DNA probe was section specific (hybridized only to anabaenas isolated from Azolla ferns representing the section Euazolla), and five other probes gave finer discrimination among anabaenas representing various ecotypes of Azolla species. These cloned genomic DNA probes identified 11 different genotypes of A. azollae isolates. These included three endosymbiotic genotypes within Azolla filiculoides species and two genotypes within both A. caroliniana and Azolla pinnata endosymbionts. Although we were not able to discriminate among anabaenas extracted from different ecotypes of Azolla nilotica, Azolla mexicina, Azolla rubra and Azolla microphylla species, each of the endosymbionts was easily identified as a unique genotype. When total DNA isolated from free-living Anabaena sp. strain PCC7120 was screened, none of the genomic DNA probes gave detectable positive hybridization. Total DNA of Nostoc cycas PCC7422 hybridized with six of eight genomic DNA fragments. These data imply that the dominant symbiotic organism in association with Azolla spp. is more closely related to Nostoc spp. than to free-living Anabaena spp.     

Large-Restriction-Fragment Polymorphism Analysis of Mycobacterium chelonae and Mycobacterium terrae Isolates

Mycobacterium chelonae and Mycobacterium terrae were reported to be frequently present in the environment of the Mycobacterium bovis BCG trial area in south India. Six isolates of M. chelonae and four isolates of M. terrae obtained from different sources in this area were analyzed by pulsed-field gel electrophoresis (PFGE) to examine large-restriction-fragment (LRF) polymorphism using the chromosomal DNA digested with DraI and XbaI restriction enzymes. With the exception of one isolate of M. terrae, DNA from all other isolates could be digested with DraI and XbaI and resulted in separable fragments. Visual comparison of the LRFs showed a unique pattern for each of the isolates tested. A computer-assisted dendrogram of the percent similarity demonstrated a high degree of genetic diversity in this group of isolates. This study demonstrates that species of nontuberculous mycobacteria, particularly M. chelonae and M. terrae, can be successfully typed by their LRF pattern using PFGE, which does not require species-specific DNA probes.     

Identification of Dekkera bruxellensis (Brettanomyces) from Wine by Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes

A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification of Brettanomyces is described. The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA of Dekkera bruxellensis. The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy. The results obtained from testing 127 different yeast strains, including 78 Brettanomyces isolates from wine, show that the spoilage organism Brettanomyces belongs to the species D. bruxellensis and that the new method is able to identify Brettanomyces (D. bruxellensis) with 100% sensitivity and 100% specificity.     

Isolation and characterization of a species-specific DNA probe for Candida albicans.

As a model for the isolation of species-specific sequences of DNA, we isolated and characterized a species-specific DNA fragment from the Candida albicans genome. We used a series of differential colony hybridization experiments, in which a C. albicans genomic library was hybridized with genomic DNA probes from related organisms to minimize the number of potentially specific C. albicans DNA fragments to be tested. Six clones were tested by dot blot analysis, and one of them, a 1348 bp EcoRI DNA fragment, was confirmed as specific for C. albicans. This species-specific fragment could be utilized as a DNA probe for rapid, sensitive, and specific diagnosis of C. albicans DNA in clinical specimens.     

西藏鸢尾兰属(兰科)植物5个新记录种

通过对西藏兰科鸢尾兰属植物进行的系统调查、标本采集、查阅、整理和鉴定,发现了西藏鸢尾兰属植物5个新记录种,分别为:长苞鸢尾兰(Oberonia longibracteata Lindl.)、短耳鸢尾兰(O. falconeri Hook. f.)、裂唇鸢尾兰(O. pyrulifera Lindl.)、条裂鸢尾兰(O. jenkinsiana Griff. ex Lindl.)和长裂鸢尾兰(O. anthropophora Lindl.),并附有这些新记录种的特征照片。凭证标本保存于西藏高原生态研究所标本室(XZE)。目前西藏鸢尾兰属植物共有14种,编写出西藏鸢尾兰属植物的分种检索表,以期达到对该区鸢尾兰属植物资源鉴定识别和有效保护的目的。     

Molecular Characterization of Yarrowia lipolytica and Candida zeylanoides Isolated from Poultry

Yeast isolates from raw and processed poultry products were characterized using PCR amplification of the internally transcribed spacer (ITS) 5.8S ribosomal DNA region (ITS-PCR), restriction analysis of amplified products, randomly amplified polymorphic DNA (RAPD) analysis, and pulsed-field gel electrophoresis (PFGE). ITS-PCR resulted in single fragments of 350 and 650 bp, respectively, from eight strains of Yarrowia lipolytica and seven strains of Candida zeylanoides. Digestion of amplicons with HinfI and HaeIII produced two fragments of 200 and 150 bp from Y. lipolytica and three fragments of 350, 150, and 100 bp from C. zeylanoides, respectively. Although these fragments showed species-specific patterns and confirmed species identification, characterization did not enable intraspecies typing. Contour-clamped heterogeneous electric field PFGE separated chromosomal DNA of Y. lipolytica into three to five bands, most larger than 2 Mbp, whereas six to eight bands in the range of 750 to 2,200 bp were obtained from C. zeylanoides. Karyotypes of both yeasts showed different polymorphic patterns among strains. RAPD analysis, using enterobacterial repetitive intergenic sequences as primers, discriminated between strains within the same species. Cluster analysis of patterns formed groups that correlated with the source of isolation. For ITS-PCR, extraction of DNA by boiling yeast cells was successfully used.     

Development of Primers to O-Antigen Biosynthesis Genes for Specific Detection of Escherichia coli O157 by PCR

The chemical composition of each O-antigen subunit in gram-negative bacteria is a reflection of the unique DNA sequences within each rfb operon. By characterizing DNA sequences contained with each rfb operon, a diagnostic serotype-specific probe to Escherichia coli O serotypes that are commonly associated with bacterial infections can be generated. Recently, from an E. coli O157:H7 cosmid library, O-antigen-positive cosmids were identified with O157-specific antisera. By using the cosmid DNAs as probes, several DNA fragments which were unique to E. coli O157 serotypes were identified by Southern analysis. Several of these DNA fragments were subcloned from O157-antigen-positive cosmids and served as DNA probes in Southern analysis. One DNA fragment within plasmid pDS306 which was specific for E. coli O157 serotypes was identified by Southern analysis. The DNA sequence for this plasmid revealed homology to two rfb genes, the first of which encodes a GDP-mannose dehydratase. These rfb genes were similar to O-antigen biosynthesis genes in Vibrio cholerae and Yersinia enterocolitica serotype O:8. An oligonucleotide primer pair was designed to amplify a 420-bp DNA fragment from E. coli O157 serotypes. The PCR test was specific for E. coli O157 serotypes. PCR detected as few as 10 cells with the O157-specific rfb oligonucleotide primers. Coupled with current enrichment protocols, O157 serotyping by PCR will provide a rapid, specific, and sensitive method for identifying E. coli O157.     

Use of Rolling Circle Amplification to Rapidly Identify Species of Cladophialophora Potentially Causing Human Infection

The genus Cladophialophora comprises etiologic agents of disease in immunocompetent patients, ranging from mild cutaneous colonization to cerebral encephalitis, in addition to saprobic species. Due to the high degree of phenotypic similarity between closely related species of the genus, identification problems are imminent. In the present study, we described rapid and sensitive rolling circle amplification (RCA) method based on species-specific padlock probes targeted for the internal transcribed spacer regions of rDNA. ITS regions of 12 Cladophialophora species were sequenced, and subsequently, 10 specific padlock probes were designed for the detection of single nucleotide polymorphisms. The majority of circularizable padlock probes were designed based on single nucleotide polymorphisms (SNPs), while for C. bantiana, C. immunda and C. devriesii were characterized by two or more nucleotides. Individual species-specific probes correctly identified in all ten Cladophialophora species correctly by visualization on 1.2 % agarose gels used to verify specificity of probe-template binding; no cross-reactivity was observed. Simplicity, sensitivity, robustness and low costs provide RCA a distinct place among isothermal techniques for DNA diagnostics. However, restriction and specificity and sensitivity should be lowered and increased, respectively, to be useful for a wide variety of clinical applications.     

Locked Nucleic Acid Pentamers as Universal PCR Primers for Genomic DNA Amplification

Background

Multiplexing technologies, which allow for simultaneous detection of multiple nucleic acid sequences in a single reaction, can save a lot of time, cost and labor compared to traditional single reaction detection methods. However, the multiplexing method currently used requires precise handiwork and many complicated steps, making a new, simpler technique desirable. Oligonucleotides containing locked nucleic acid residues are an attractive tool because they have strong affinities for their complementary targets, they have been used to avoid dimer formation and mismatch hybridization and to enhance efficient priming. In this study, we aimed to investigate the use of locked nucleic acid pentamers for genomic DNA amplification and multiplex genotyping.

Results

We designed locked nucleic acid pentamers as universal PCR primers for genomic DNA amplification. The locked nucleic acid pentamers were able to prime amplification of the selected sequences within the investigated genomes, and the resulting products were similar in length to those obtained by restriction digest. In Real Time PCR of genomic DNA from three bacterial species, locked nucleic acid pentamers showed high priming efficiencies. Data from bias tests demonstrated that locked nucleic acid pentamers have equal affinities for each of the six genes tested from the Klebsiella pneumoniae genome. Combined with suspension array genotyping, locked nucleic acid pentamer-based PCR amplification was able to identify a total of 15 strains, including 3 species of bacteria, by gene- and species-specific probes. Among the 32 species used in the assay, 28 species and 50 different genes were clearly identified using this method.

Conclusion

As a novel genomic DNA amplification, the use of locked nucleic acid pentamers as universal primer pairs in conjunction with suspension array genotyping, allows for the identification of multiple distinct genes or species with a single amplification procedure. This demonstrates that locked nucleic acid pentamer-based PCR can be utilized extensively in pathogen identification.