Development of peptide nucleic acid (PNA) microarray for identification of Panax species based on the nuclear ribosomal internal transcribed spacer (ITS) and 5.8S rDNA regions

This study describes the identification of Panax species using a peptide nucleic acid (PNA) microarray. P. ginseng, P. quienquefolius, and P. japonicus were distinguished from each other using 5 PNA probes designed based on three single nucleotide polymorphisms (SNPs) detected in internal transcribed spacer (ITS) and 5.8S rDNA regions. Signal intensity comparison between PNA and DNA microarrays revealed that the PNA microarray provides a significantly more stable and specific fluorescent signal intensity than the DNA microarray. Three Panax species identified by the PNA microarray were denoted as barcode numbers depending on their fluorescent signal patterns of each species using 5 PNA probes (PG-ITS-116, PG-ITS-414-1, PG-ITS-414-2, PG-ITS-425-1, and PG-ITS-425-2). P. ginseng, P. quinquefolius, and P. japonicus were denoted as ‘11010’, ‘00202’ and ‘00000’, respectively. The PNA microarray developed in this study will be useful for legitimizing the distribution of ginseng in domestic and foreign ginseng markets.     

Discriminating single-base difference miRNA expressions using microarray Probe Design Guru (ProDeG)

MicroRNAs (miRNA) are endogenous tissue-specific short RNAs that regulate gene expression. Discriminating each let-7 family member expression is especially important due to let-7's abundance and connection with development and cancer. However, short lengths (22 nt) and similarities between multiple sequences have prevented identification of individual members. Here, we present ProDeG, a computational algorithm which designs imperfectly matched sequences (previously yielding only noise levels in microarray experiments) for genome-wide microarray “signal” probes to discriminate single nucleotide differences and to improve probe qualities. Our probes for the entire let-7 family are both homogeneous and specific, verified using microarray signals from fluorescent dye-tagged oligonucleotides corresponding to the let-7 family, demonstrating the power of our algorithm. In addition, false let-7c signals from conventional perfectly-matched probes were identified in lymphoblastoid cell-line samples through comparison with our probe-set signals, raising concerns about false let-7 family signals in conventional microarray platform.     

Toxigenic algae and associated phycotoxins in two coastal embayments in the Ebro Delta (NW Mediterranean)

Harmful Algal Bloom (HAB) surveillance is complicated by high diversity of species and associated phycotoxins. Such species-level information on taxonomic affiliations and on cell abundance and toxin content is, however, crucial for effective monitoring, especially of aquaculture and fisheries areas. The aim addressed in this study was to determine putative HAB taxa and related phycotoxins in plankton from aquaculture sites in the Ebro Delta, NW Mediterranean. The comparative geographical distribution of potentially harmful plankton taxa was established by weekly field sampling throughout the water column during late spring–early summer over two years at key stations in Alfacs and Fangar embayments within the Ebro Delta. Core results included not only confirmed identification of HAB taxa that are common for the time period and geographical area, but also provided evidence of potentially new taxa. At least 25 HAB taxa were identified to species level, and an additional six genera were confirmed, by morphological criteria under light microscopy and/or by molecular genetics approaches involving qPCR and next generation DNA pyrosequencing. In particular, new insights were gained by the inclusion of molecular techniques, which focused attention on the HAB genera Alexandrium, Karlodinium, and Pseudo-nitzschia. Noteworthy is the discovery of Azadinium sp., a potentially new HAB species for this area, and Gymnodinium catenatum or Gymnodinium impudicum by means of light microscopy. In addition, significant amounts of the neurotoxin domoic acid (DA) were found for the first time in phytoplankton samples in the Ebro Delta. While the presence of the known DA-producing diatom genus Pseudo-nitzschia was confirmed in corresponding samples, the maximal toxin concentration did not coincide with highest cell abundances of the genus and the responsible species could not be identified. Combined findings of microscopic and molecular detection approaches underline the need for a synoptic strategy for HAB monitoring, which integrates the respective advantages and compensates for limitations of individual methods.     

Ultrafiltration and Microarray for Detection of Microbial Source Tracking Marker and Pathogen Genes in Riverine and Marine Systems

Pathogen identification and microbial source tracking (MST) to identify sources of fecal pollution improve evaluation of water quality. They contribute to improved assessment of human health risks and remediation of pollution sources. An MST microarray was used to simultaneously detect genes for multiple pathogens and indicators of fecal pollution in freshwater, marine water, sewage-contaminated freshwater and marine water, and treated wastewater. Dead-end ultrafiltration (DEUF) was used to concentrate organisms from water samples, yielding a recovery efficiency of >95% for Escherichia coli and human polyomavirus. Whole-genome amplification (WGA) increased gene copies from ultrafiltered samples and increased the sensitivity of the microarray. Viruses (adenovirus, bocavirus, hepatitis A virus, and human polyomaviruses) were detected in sewage-contaminated samples. Pathogens such as Legionella pneumophila, Shigella flexneri, and Campylobacter fetus were detected along with genes conferring resistance to aminoglycosides, beta-lactams, and tetracycline. Nonmetric dimensional analysis of MST marker genes grouped sewage-spiked freshwater and marine samples with sewage and apart from other fecal sources. The sensitivity (percent true positives) of the microarray probes for gene targets anticipated in sewage was 51 to 57% and was lower than the specificity (percent true negatives; 79 to 81%). A linear relationship between gene copies determined by quantitative PCR and microarray fluorescence was found, indicating the semiquantitative nature of the MST microarray. These results indicate that ultrafiltration coupled with WGA provides sufficient nucleic acids for detection of viruses, bacteria, protozoa, and antibiotic resistance genes by the microarray in applications ranging from beach monitoring to risk assessment.     

Using ITS2 secondary structure to create species-specific oligonucleotide probes for fungi

Oligonucleotide microarray based on ITS2 rDNA sequences would be extremely useful in identifying fungi within soil samples. However ITS2 contains phylogenetic information and duplication of sequences among taxa make false positive detections likely unless a way could be found to identify taxon-specific portions of the ITS2 sequence a priori. Examination of component ITS2 sequences suggested one method of identifying species-specific probes. Analysis of 168 fungal ITS2 sequences showed that all 168 ITS2 rRNA sequences could be folded to produce similar secondary structures of 3-4 loops. Unique probes occurred most often in the second loop. While the loop 2 sequence was unique in all taxa, there were partial congeneric and intergeneric duplicates. Evidence for a decrease in duplicates with increasing phylogenetic distance was mixed. From the evidence, 2 or 3 disjunct oligonucleotide probes from the loop 2 sequence might be sufficient to identify most fungal species. This combination appears minimally susceptible to false positives and conceivably could be extended to design probes to identify any eukaryotic species.     

Dynamics of marine bacterial and phytoplankton populations using multiplex liquid bead array technology

Heterotrophic bacteria and phytoplankton dominate the biomass and play major roles in the biogeochemical cycles of the surface ocean. Here, we designed and tested a fast, high‐throughput and multiplexed hybridization‐based assay to detect populations of marine heterotrophic bacteria and phytoplankton based on their small subunit ribosomal DNA sequences. The assay is based on established liquid bead array technology, an approach that is gaining acceptance in biomedical research but remains underutilized in ecology. End‐labelled PCR products are hybridized to taxon‐specific oligonucleotide probes attached to fluorescently coded beads followed by flow cytometric detection. We used ribosomal DNA environmental clone libraries (a total of 450 clones) and cultured isolates to design and test 26 bacterial and 10 eukaryotic probes specific to various ribotypes and genera of heterotrophic bacteria and eukaryotic phytoplankton. Pure environmental clones or cultures were used as controls and demonstrated specificity of the probes to their target taxa. The quantitative nature of the assay was demonstrated by a significant relationship between the number of target molecules and fluorescence signal. Clone library sequencing and bead array fluorescence from the same sample provided consistent results. We then applied the assay to a 37‐day time series of coastal surface seawater samples from the Southern California Bight to examine the temporal dynamics of microbial communities on the scale of days to weeks. As expected, several bacterial phylotypes were positively correlated with total bacterial abundances and chlorophyll a concentrations, but others were negatively correlated. Bacterial taxa belonging to the same broad taxonomic groups did not necessarily correlate with one another, confirming recent results suggesting that inferring ecological role from broad taxonomic identity may not always be accurate.     

Sandwich hybridization probes for the detection of Pseudo-nitzschia (Bacillariophyceae) species: An update to existing probes and a description of new probes

New sandwich hybridization assay (SHA) probes for detecting Pseudo-nitzschia species (P. arenysensis, P. fraudulenta, P. hasleana, P. pungens) are presented, along with updated cross-reactivity information on historical probes (SHA and FISH; fluorescence in situ hybridization) targeting P. australis and P. multiseries. Pseudo-nitzschia species are a cosmopolitan group of diatoms that produce varying levels of domoic acid (DA), a neurotoxin that can accumulate in finfish and shellfish and transfer throughout the food web. Consumption of infected food sources can lead to illness in humans (amnesic shellfish poisoning; ASP) and marine wildlife (domoic acid poisoning; DAP). The threat of human illness, along with economic loss from fishery closures has resulted in the implementation of monitoring protocols and intensive ecological studies. SHA probes have been instrumental in some of these efforts, as the technique performs well in complex heterogeneous sample matrices and has been adapted to benchtop and deployable (Environmental Sample Processor) platforms. The expanded probe set will enhance future efforts towards understanding spatial, temporal and successional patterns in species during bloom and non-bloom periods.     

Microarray Analysis of Microbial Virulence Factors

Hybridization with oligonucleotide microchips (microarrays) was used for discrimination among strains of Escherichia coli and other pathogenic enteric bacteria harboring various virulence factors. Oligonucleotide microchips are miniature arrays of gene-specific oligonucleotide probes immobilized on a glass surface. The combination of this technique with the amplification of genetic material by PCR is a powerful tool for the detection of and simultaneous discrimination among food-borne human pathogens. The presence of six genes (eaeA, slt-I, slt-II, fliC, rfbE, and ipaH) encoding bacterial antigenic determinants and virulence factors of bacterial strains was monitored by multiplex PCR followed by hybridization of the denatured PCR product to the gene-specific oligonucleotides on the microchip. The assay was able to detect these virulence factors in 15 Salmonella, Shigella, and E. coli strains. The results of the chip analysis were confirmed by hybridization of radiolabeled gene-specific probes to genomic DNA from bacterial colonies. In contrast, gel electrophoretic analysis of the multiplex PCR products used for the microarray analysis produced ambiguous results due to the presence of unexpected and uncharacterized bands. Our results suggest that microarray analysis of microbial virulence factors might be very useful for automated identification and characterization of bacterial pathogens.     

Reticulate evolution and phylogeography in Asarum sect. Asiasarum (Aristolochiaceae) documented in internal transcribed spacer sequences (ITS) of nuclear ribosomal DNA

Phylogenetic analyses were performed for the taxonomically complicated group, Asarum sect. Asiasarum, using internal transcribed spacers (ITS) of nuclear ribosomal DNA. Direct sequences for 99 samples of a total of 14 taxa and geographic races and cloning analyses for 32 of these samples provided new insights that extensive reticulate evolution has occurred in this group. Eight taxa had slight or no polymorphism of the ITS sequences. On the other hand, the other five taxa had polymorphic ITS sequences composed of two ribotypes that were completely the same or almost the same as the sequences recognized in the taxa with only slight or no polymorphism, and were probably of diploid hybrid origin and to have retained their parental ribotypes. In terms of biogeographic implications, at least four interactions including migration, hybridization, and introgression, were presumed between the Japanese Archipelago and the continents, two times via a southern route, from the Korean Peninsula, and two times via a northern route, from Sakhalin or directly from the Eurasian continent.     

Ribosomal Tag Pyrosequencing of DNA and RNA Reveals “Rare” Taxa with High Protein Synthesis Potential in the Sediment of a Hypersaline Lake in Western Australia

Little is known about the potential activity of microbial communities in hypersaline sediment ecosystems. Ribosomal tag libraries of DNA and RNA extracted from the sediment of Lake Strawbridge (Western Australia) revealed bacterial and archaeal operational taxonomic units (OTUs) with high RNA/DNA ratios providing evidence for the presence of ‘rare’ but potentially “active” taxa. Among the ‘rare’ bacterial taxa Halomonas, Salinivibrio and Idiomarina showed the highest protein synthesis potential. Rare but ‘active’ archaeal OTUs were related to the KTK 4A cluster and the Marine-Benthic-Groups B and D. We present the first molecular analysis of the microbial diversity and protein synthesis potential of rare microbial taxa in a hypersaline sediment ecosystem.     

Species composition and toxicity of the genus Pseudo-nitzschia in Taiwan Strait,including P. chiniana sp. nov. and P. qiana sp. nov.

In a field survey in the Taiwan Strait during April 2016, the species composition and the domoic acid production of the diatom genus Pseudo-nitzschia were investigated. A total of 80 strains of Pseudo-nitzschia were established, and species identification was determined based on a combination of morphological and molecular data. Fourteen taxa were recognized, i.e., P. americana, P. brasiliana, P. calliantha, P. cuspidata, P. galaxiae, P. lundholmiae, P. multiseries, P. multistriata, P. pseudodelicatissima, P. pungens var. aveirensis, P. pungenus var. pungens and P. sabit, as well as two novel species P. chiniana C.X. Huang & Yang Li and P. qiana C.X. Huang & Yang Li. Morphologically, P. chiniana is characterized by striae comprising one or two rows of poroids, and valve ends that are normally dominated by two rows of poroids within each stria. Whereas P. qiana is unique by having a narrow valve width (1.3–1.5 μm) and sharply pointed valve ends. Both taxa constitute their own monophyletic lineage in the phylogenetic analyses inferred from LSU and ITS2 rDNA, and are well differentiated from other Pseudo-nitzschia species. Pseudo-nitzschia chiniana forms a group with P. abrensis and P. batesiana in LSU and ITS trees, whereas P. qiana is sister to P. lineola. When comparing ITS2 secondary structure, five CBCs and seven HCBCs are recognized between P. chiniana and P. abrensis, and four CBCs and ten HCBCs between P. chiniana and P. batesiana. Two CBCs and eight HCBCs are found between P. qiana with P. lineola. The ability of the strains to produce domoic acid was assessed, including a potential toxin induction by the presence of brine shrimps. Results revealed production of domoic acid in six strains belonging to three species. Without presence of brine shrimps, cellular DA (pDA) was detected in four P. multiseries strains (1.6 ± 0.3, 26.6 ± 2.7, 68.3 ± 4.2 and 56.9 ± 4.7 fg cell⁻¹, separately), one strain of P. pseudodelicatissima (0.8 ± 0.2 fg cell⁻¹) and one strain of P. lundholmiae (2.5 ± 0.4 fg cell⁻¹). In the presence of brine shrimps, pDA contents increased significantly (p < 0.05) in P. lundholmiae (strain MC4218) and P. multiseries (strain MC4177), from 2.5 ± 0.4 to 8.9 ± 0.7 and 1.6 ± 0.3 to 37.2 ± 2.5 fg cell⁻¹ respectively.     

Assessment of candidate plant DNA barcodes using the Rutaceae family

DNA barcoding is a rapidly developing frontier technology that is gaining worldwide attention.Here,seven regions (psbA-trnH,matK,ycf5,rpoC1,rbcL,ITS2,and ITS) with potential for use as DNA barcodes were tested for their ability to identify 300 samples of 192 species from 72 genera of the family Rutaceae.To evaluate each barcode’s utility for species authentication,PCR amplification efficiency,genetic divergence,and barcoding gaps were assessed.We found that the ITS2 region exhibited the highest inter-specific divergence,and that this was significantly higher than the intra-specific variation in the "DNA barcoding gap" assessment and Wilcoxon two-sample tests.The ITS2 locus had the highest identification efficiency among all tested regions.In a previous study,we found that ITS2 was able to discriminate a wide range of plant taxa,and here we confirmed that ITS2 was also able to discriminate a number of closely related species.Therefore,we propose that ITS2 is a promising candidate barcode for plant species identification.     

Development of an oligonucleotide microarray for the detection and monitoring of marine dinoflagellates

Harmful Algal Blooms (HABs), mainly caused by dinoflagellates and diatoms, have great economic and sanitary implications. An important contribution for the comprehension of HAB phenomena and for the identification of risks related to toxic algal species is given by the monitoring programs. In the microscopy-based monitoring methods, harmful species are distinguished through their morphological characteristics. This can be time consuming and requires great taxonomic expertise due to the existence of morphologically close-related species. The high throughput, automation possibility and specificity of microarray-based detection assay, makes this technology very promising for qualitative detection of HAB species. In this study, an oligonucleotide microarray targeted to the ITS1-5.8S-ITS2 rDNA region of nine toxic dinoflagellate species/clades was designed and evaluated. Two probes (45-47 nucleotides in length) were designed for each species/clade to reduce the potential for false positives. The specificity and sensitivity of the probes were evaluated with ITS1-5.8S-ITS2 PCR amplicons obtained from 20 dinoflagellates cultured strains. Cross hybridization experiments confirmed the probe specificity; moreover, the assay showed a good sensitivity, allowing the detection of up to 2 ng of labeled PCR product. The applicability of the assay with field samples was demonstrated using net concentrated seawater samples, un-spiked or spiked with known amounts of cultured cells. Despite the general application of microarray technology for harmful algae detection is not new, a peculiar group of target species/clades has been included in this new-format assay. Moreover, novelties regarding mainly the probes and the target rDNA region have allowed sensitivity improvements in comparison to previously published studies.     

Morphology,phylogeny and azaspiracid profile of Azadinium poporum (Dinophyceae) from the China Sea

Azadinium poporum is a small dinoflagellate from the family Amphidomataceae which is known for the production potential of azaspiracid toxins. A. poporum has been recorded from European and Korean waters. Here we present the first report of its occurrence along the coast of China. Morphology of Chinese A. poporum is similar to those from Europe and Korea. Several stalked pyrenoids surrounded by a starch sheath were revealed with light microscopy and confirmed by transmission electron microscopy. Among 25 strains from the China Sea we identified two distinct ribotypes (referred to as ribotypes B and C). ITS sequences of strains within the same ribotype are identical, whereas ribotype B and C differ from each other at 11 positions (98.3% similarity). A. poporum ribotypes B and C type differ from European strains (referred to as ribotype A) at 16 and 15 positions (97.5% and 97.7% similarity). The ITS region pairwise distance within A. poporum ranged from 0.017 to 0.022. Among all three ribotypes, no hemi-compensatory based changes were found within helix III of ITS indicating that they are conspecific. Azaspiracid profiles were analyzed for six strains and turned out to be unexpectedly diverse. Whereas no AZAs could be detected for one strain, another strain was found to contain a m/z 348 fragment type AZA previously found in a Korean Isolate and traces of two other unknown AZAs of higher masses. A third strain produced a novel AZA with a molecular mass of 871 Da. Three strains were found to contain considerable amounts of toxic AZA-2 as the sole AZA, a finding that might elegantly explain the detection of AZA-2 in sponges in the Sea of Japan and which underline the risk potential of A. poporum blooms with subsequent shellfish intoxication episodes for the Asian Pacific.     

Cryptic speciation in Protoceratium reticulatum (Dinophyceae): Evidence from morphological,molecular and ecophysiological data

The cosmopolitan, potentially toxic dinoflagellate Protoceratium reticulatum possesses a fossilizable cyst stage which is an important paleoenvironmental indicator. Slight differences in the internal transcribed spacer ribosomal DNA (ITS rDNA) sequences of P. reticulatum have been reported, and both the motile stage and cyst morphology of P. reticulatum display phenotypic plasticity, but how these morpho-molecular variations are related with ecophysiological preferences is unknown. Here, 55 single cysts or cells were isolated from localities in the Northern (Arctic to subtropics) and Southern Hemispheres (Chile and New Zealand), and in total 34 strains were established. Cysts and/or cells were examined with light microscopy and/or scanning electron microscopy. Large subunit ribosomal DNA (LSU rDNA) and/or ITS rDNA sequences were obtained for all strains/isolates. All strains/isolates of P. reticulatum shared identical LSU sequences except for one strain from the Mediterranean Sea that differs in one position, however ITS rDNA sequences displayed differences at eight positions. Molecular phylogeny was inferred using maximum likelihood and Bayesian inference based on ITS rDNA sequences. The results showed that P. reticulatum comprises at least three ribotypes (designated as A, B, and C). Ribotype A included strains from the Arctic and temperate areas, ribotype B included strains from temperate regions only, and ribotype C included strains from the subtropical and temperate areas. The average ratios of process length to cyst diameter of P. reticulatum ranged from 15% in ribotype A, 22% in ribotype B and 17% in ribotype C but cyst size could overlap. Theca morphology was indistinguishable among ribotypes. The ITS-2 secondary structures of ribotype A displayed one CBC (compensatory change on two sides of a helix pairing) compared to ribotypes B and C. Growth response of one strain from each ribotype to various temperatures was examined. The strains of ribotypes A, B and C exhibited optimum growth at 15 °C, 20 °C and 20–25 °C, respectively, thus corresponding to cold, moderate and warm ecotypes. The profiles of yessotoxins (YTXs) were examined for 25 strains using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The parent compound yessotoxin (YTX) was produced by strains of ribotypes A and B, but not by ribotype C strains, which only produced the structural variant homoyessotoxin (homoYTX). Our results support the notion that there is significant intra-specific variability in Protoceratium reticulatum and the biogeography of the different ribotypes is consistent with specific ecological preferences.     

Molecular characterization of two natural hybrids from the Iberian Peninsula: <Emphasis Type="Italic">Ranunculus × luizetii</Emphasis> and <Emphasis Type="Italic">R. × peredae</Emphasis> (Ranunculaceae)

Two nothospecies, Ranunculus × luizetii and R. × peredae (Ranunculaceae), were analyzed and discussed. For this purpose, Amplified Fragment Length Polymorphism (AFLP) markers, nuclear rDNA sequences (ITS1, 5.8S and ITS2) and pollen viability were conducted. The profiles of these hybrid samples were compared to their putative progenitors. Several additive polymorphic sites detected in the ITS sequences of the hybrid samples (R. × luizetii and R. × peredae) also confirmed their derived origins from ribotypes of their parental taxa (R. parnassiifolius subsp. parnassiifolius × R. pyrenaeus; R. amplexicaulis × R. cabrerensis subsp. cabrerensis, respectively). Despite the lack of exclusive AFLP markers reported in both hybrids, presumably due to effects of introgression, the concerted evolution of many rDNA polymorphisms towards either of the parental ribotypes indicated their ancient origin. Pollen fertility estimation in R. × luizetii presented a mean value of 60.58%, which showed that hybrid samples are well established and fertile. However, a larger difference was observed in R. × peredae, where the mean value of pollen fertility was very low (18.91%).     

A new chromosome nomenclature system for oat (Avena sativa L. and A. byzantina C. Koch) based on FISH analysis of monosomic lines

Fluorescent in situ hybridization (FISH) with multiple probes was used to analyze mitotic and meiotic chromosome spreads of Avena sativa cv ‘Sun II’ monosomic lines, and of A. byzantina cv ‘Kanota’ monosomic lines from spontaneous haploids. The probes used were A. strigosa pAs120a (a repetitive sequence abundant in A-genome chromatin), A. murphyi pAm1 (a repetitive sequence abundant in C-genome chromatin), A. strigosa pITS (internal transcribed spacer of rDNA) and the wheat rDNA probes pTa71 (nucleolus organizer region or NOR) and pTa794 (5S). Simultaneous and sequential FISH employing pairs of these probes allowed the identification and genome assignation of all chromosomes. FISH mapping using mitotic and meiotic metaphases facilitated the genomic and chromosomal identification of the monosome in each line. Of the 17 ‘Sun II’ lines analyzed, 13 distinct monosomic lines were found, corresponding to four monosomes of the A-genome, five of the C-genome and four of the D-genome. In addition, 12 distinct monosomic lines were detected among the 20 ‘Kanota’ lines examined, corresponding to six monosomes of the A-genome, three of the C-genome and three of the D-genome. The results show that 19 chromosomes out of 21 of the complement are represented by monosomes between the two genetic backgrounds. The identity of the remaining chromosomes can be deduced either from one intergenomic translocation detected on both ‘Sun II’ and ‘Kanota’ lines, or from the single reciprocal, intergenomic translocation detected among the ‘Sun II’ lines. These results permit a new system to be proposed for numbering the 21 chromosome pairs of the hexaploid oat complement. Accordingly, the A-genome contains chromosomes 8A, 11A, 13A, 15A, 16A, 17A and 19A; the C-genome contains chromosomes 1C, 2C, 3C, 4C, 5C, 6C and 7C; and the D-genome consists of chromosomes 9D, 10D, 12D, 14D, 18D, 20D and 21D. Moreover, the FISH patterns of 16 chromosomes in ‘Sun II’ and 15 in ‘Kanota’ suggest that these chromosomes could be involved in intergenomic translocations. By comparing the identities of individually translocated chromosomes in the two hexaploid species with those of other hexaploids, we detected different types of intergenomic translocations.     

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.