FISH and GISH analysis of the genomic relationships amongPanax species

Ginseng is a well-known medicinal plant that has been used as an anti-aging agent for many years in East Asia. In the genusPanax, only three species,P. ginseng (Oriental ginseng),P. quinquefolius (American ginseng) andP. notoginseng (Chinese ginseng), are currently considered to be important medicinal herbs. Despite the increase in their breeding value, molecular cytogenetic information on the species is not available. To analyze the genomic relationships among thePanax species, FISH (fluorescencein situ hybridization) and GISH (genomicin situ hybridization) techniques were applied. FISH analysis revealed that the 45S and 5S rRNA genes ofP. notoginseng (2n=2x=24) andP. ginseng (2n=4x=48) cluster on a single locus on different chromosomes, whileP. quinquefolius (2n=4x=48),P. japonicus (2n=4x=48), and Korean wild ginseng (2n =4x= 48) had one locus of the 45S rRNA gene and two loci of the 5S rRNA gene, respectively. GISH analysis using genomic DNA as a probe detected strong cross-hybridization of genomes betweenP. ginseng andP. quinquefolius. GISH analysis of other species showed weak or no distinct signals on the chromosomes. Our data revealed thatP. ginseng andP. quinquefolius showed the highest degree of homology, indicating that these species diverged in most recent years.     

Regulation and differential expression of protopanaxadiol synthase in Asian and American ginseng ginsenoside biosynthesis by RNA interferences

Asian ginseng (Panax ginseng) and American ginseng (Panax quinquefolium), are thought to be representative plant of Panax species, have important commercial value and are used in worldwide. Panax species produces triterpene saponins called ginsenosides, which are classified into two groups by the skeleton of aglycones, namely dammarane-type and oleanane-type. Dammarane-type ginsenosides dominate over oleanane-type not only in amount but also in structural varieties. Researches shows that the saponins content in American ginseng is higher than that in Asian ginseng, the higher part of ginsenosides is from dammarane-type biosynthesis. It has been proposed that protopanaxadiol derived from dammarenediol-II, is a key hydroxylation by cytochrome P450 for the biosynthesis of ginsenosides, and the gene number of protopanaxadiol synthase has been published independent in Asian ginseng (PgCYP716A47). However, little is known about genes involved in hydroxylation and glycosylation in American ginseng ginsenoside biosynthesis. Here, we first cloned and identified a P450 gene named PqD12H encoding enzymes catalyzed dammarenediol-II to protopanaxadiol by RT-PCR using degenerate primers designed based on sequence homology. In vitro, the ectopic expression of PqD12H in recombinant WAT21 yeast resulted in protopanaxadiol production after dammarenediol-II was added to the culture medium. In vivo, we established both PgCYP716A47 and PqD12H RNAi transgenic. The RT-PCR and HPLC analysis of the final products of protopanaxadiol and protopanaxatriol showed a result that declined level of protopanaxadiol-type and protopanaxatriol-type ginsenosides. It suggested that the P450 synthase content or expression in American ginseng exceed than in Asian ginseng. The result elucidated the evolution relationship of P450s and the reason of different saponins content among Panax species.     

Headspace Solid-Phase Micro-Extraction Gas Chromatography/Mass Spectrometry (HS-SPME-GC/MS)-Based Untargeted Metabolomics Analysis for Comparing the Volatile Components from 12 <i>Panax</i> Herbal Medicines

Quality control of ginseng currently is mainly based on ginsenoside analysis, but rarely focuses on the volatile organic components. In the current work, an untargeted metabolomics approach, by headspace solid-phase micro-extraction gas chromatography/mass spectrometry (HS-SPME-GC/MS), was elaborated and further employed to holistically compare the compositional difference of the volatile components simultaneously from 12 Panax herbal medicines, which included P. ginseng (PG), P. quinquefolius (PQ), P. notoginseng (PN), red ginseng (PGR), P. ginseng leaf (PGL), P. quinquefolius leaf (PQL), P. notoginseng leaf (PNL), P. ginseng flower (PGF), P. quinquefolius flower (PQF), P. notoginseng flower (PNF), P. japonicus (PJ), and P. japonicus var. major (PJvm). Chromatographic separation was performed on an HP-5MS elastic quartz capillary column using helium as the carrier gas, enabling good resolution within 1 h. We were able to characterize totally 259 volatile compounds, including 82 terpenes (T), 46 alcohols (Alc), 29 ketones (K), 25 aldehydes (Ald), 21 esters (E), and the others. By analyzing 90 batches of ginseng samples based on the untargeted metabolomics workflows, 236 differential ions were unveiled, and accordingly 36 differential volatile components were discovered. It is the first report that simultaneously compares the compositional difference of volatile components among 12 Panax herbal medicines, and useful information is provided for the quality control of ginseng aside from the well-known ginsenosides.     

High resolution microarray assay for rapid taxonomic assessment of Pseudo-nitzschia spp. (Bacillariophyceae) in the field

Deployable methods facilitating rapid microbial identification provide opportunities to respond quickly when pathogenic or toxin-producing organisms threaten water quality. We developed a microarray assay to streamline identification of microorganisms in the field, focusing on the harmful algal bloom diatom Pseudo-nitzschia. The assay employed electrochemical signal detection and a simplified protocol, allowing identification of specific taxa onboard research vessels within 7 h of water collection. Microarrays targeted the internal transcribed spacer (ITS1) region between the 18S and 28S ribosomal RNA genes using 307 oligonucleotide probes. The probes, ranging from broadly specific to unique, represented 118 Pseudo-nitzschia ribotypes from 15 species available at the time of assay development. Hybridization signals from multiple probes for each target strain were integrated using a novel algorithm for data analysis. Designated the ‘integrated sumscore data analysis’ (ISDA), the algorithm used probe specificity metrics for signal integration, with uniqueness corresponding to higher probe weight values. The integrated signals provided a ‘sumscore’ for each ribotype represented on the array, indicating its presence or absence in the sample. The algorithm was ‘trained’ by comparison with data from scanning electron microscopy, and cloning and sequence analysis of Pseudo-nitzschia ITS1 ribotypes from 7 laboratory cultures and a complex environmental sample. The ISDA provided correct identification and target sequences for all tested strains. Through design of custom probes (up to 12,000 on a microarray chip), this approach may be used to identify additional microbial taxa of interest and provides rapid, reliable shipboard assays for basic research or water quality monitoring.     

Chloroplast DNA variation of Panax (Araliaceae) in Nepal and its taxonomic implications

The restriction site and size variation of five PCR amplified fragments of noncoding chloroplast DNA (cpDNA) was examined in material from 13 populations ofPanax from Nepal and China. Fourteen restriction endonucleases produced 81 restriction site and length variations from the large single-copy region of cpDNA, 27 of which are polymorphic. The cpDNA dataset suggests two distinct groups ofPanax from Nepal (clades I and II). Clade I consists of two populations ofP. pseudoginseng subsp.pseudoginseng, and clade II is composed of material referable toP. pseudogingeng subsp.himalaicus (vars.himalaicus, angustifolius, andbipinnatifidus). The three accessions ofP. pseudoginseng subsp.japonicus andP. ginseng studied from China had cpDNA characters that differed from the HimalayanPanax. The highly distinctive cpDNA profile and morphology ofP. pseudoginseng subsp.pseudoginseng sensu Hara (1970) from central Nepal support its status as a separate species, which has an extremely restricted distribution.     

CAPS markers using mitochondrial consensus primers for molecular identification of Panax species and Korean ginseng cultivars (Panax ginseng C. A. Meyer)

Cleaved amplified polymorphic sequence (CAPS) marker system using mitochondrial consensus primers was applied for molecular identification of Korean ginseng cultivars (Panax ginseng). Initially, a total of 34 primers were tested to six Korean ginseng cultivars and two foreign Panax species, P. quinquefolius and P. notoginseng. In the polymerase chain reaction (PCR) amplification results, four primers (mt7, mt11, mt13, and mt18) generated co-dominant polymorphic banding patterns discriminating the Korean ginseng cultivars from P. quinquefolius and P. notoginseng. In the CAPS analysis results, the majority of the cleaved PCR products also yielded additional latent polymorphisms between the Korean ginseng cultivars and two foreign Panax species. Specific latent CAPS polymorphisms for cultivar Gopoong and Chunpoong were detected from internal region amplified with mt9 primer by treating HinfI and Tsp509I endonucleases, respectively. Sequencing analysis revealed that the length of amplified region of Korean ginseng cultivars was 2,179 bp, and those of P. quinquefolius and P. notoginseng were 2,178 and 2,185 bp, respectively. Blast search revealed that the amplified region was a mitochondrial cytochrome oxidase subunit 2 (cox2) gene intron II region. Nineteen single nucleotide polymorphisms (SNP) including each specific SNP for Gopoong and Chunpoong, and three insertion and deletion (InDel) polymorphisms were detected by sequence alignment. The CAPS markers developed in this study, which are specific to Gopoong and Chunpoong, and between the Korean ginseng cultivars and two foreign Panax species, will serve as a practical and reliable tool for their identification, purity maintenance, and selection of candidate lines and cultivars.     

A phylogenetic analysis ofPanax (Araliaceae): Integrating cpDNA restriction site and nuclear rDNA ITS sequence data

A phylogenetic analysis ofPanax was performed using restriction site variations of eight PCR-amplified chloroplast regions. Twenty populations were examined, representing 13 of the 14 species ofPanax. Aralia cordata was used as the outgroup. The 11 restriction endonucleases produced a total of 105 restriction sites and length variations from the large single-copy region of cpDNA. Forty restriction variations are polymorphic. The cpDNA tree is largely congruent with the nuclear ribosomal ITS phylogeny. Similar to the ITS tree, the cpDNA dataset suggests the following relationships: (1)P. trifolius from eastern North America is sister to the clade consisting of all otherPanax species; (2)P. ginseng andP. japonicus from eastern Asia form a clade withP. quinquefolius from eastern North America; (3) the HimalayanP. pseudoginseng is most closely related toP. stipuleanatus of southwestern China; and (4) the medicinally importantP. notoginseng forms a clade with the closely relatedP. bipinnatifidus, P. ginseng, P. japonicus, P. major, P. quinquefolius, P. sinensis, P. wangianus, andP. zingiberensis. Two biogeographic disjunctions are detectable withinPanax. One is the connection of the eastern North AmericanP. trifolius with the rest ofPanax species. The other is the more recent disjunction between the North AmericanP. quinquefolius and the eastern AsianP. ginseng andP. japonicus. The active orogenies caused by the collision of the Indian Plate with Asia may have facilitated the diversification ofPanax taxa in Asia in the late Tertiary.     

Development of a PNA probe for the detection of the toxic dinoflagellate Takayama pulchella

A peptide nucleic acid (PNA) probe was developed to detect the toxic dinoflagellate, Takayama pulchella TPXM, using fluorescent in situ hybridization (FISH) combined with epifluorescent microscopy and flow cytometry. The PNA probe was then used to analyze HAB samples from Xiamen Bay. The results indicated that the fluorescein phosphoramidite (FAM)-labeled probe (PNATP28S01) [Flu]-OO ATG CCA TCT CAA GA, entered the algal cells easily and bound to the target species specifically. High hybridization efficiency (nearly 100%) was observed. Detection by epifluorescence microscopy and flow cytometry gave comparable results. The fluorescence intensity of the PNA probe hybridized to T. pulchella cells was remarkably higher than that of two DNA probes used in this study and than the autofluorescence of the blank and negative control cells. In addition, the hybridization condition of the PNA probe was easier to control than DNA probes, and when applied to field-collected samples, the PNA probe showed higher binding efficiency to the target species than DNA probes. With the observed high specificity, binding efficiency, and detection signal intensity, the PNA probe will be useful for monitoring harmful algal blooms of T. pulchella.     

Effect of growth regulators on ginsenoside production in the cell culture of two ginseng species

Plants belonging to the genus Panax produce ginsenosides that possess pharmacological properties. The ability to synthesize these compounds is preserved in some cultured cells of ginseng. In this work, we used suspension cell cultures of two species of ginseng: Panax japonicus var. repens C. A. Mey and P. ginseng C. A. Mey. The first culture was grown on MS medium supplemented with α-NAA. After one subculturing, cell biomass increased 5–6 times with the level of ginsenosides being equal to 2.5–3.0% of dry weight. The second culture was grown on the same medium supplemented with 2,4-D. In this case, biomass increment was 3–5-fold, and ginsenosides were produced in trace amounts. Substitution of 2,4-D for NAA in suspension cell culture of P. japonicus brought about deterioration of growth characteristics, but the content and composition of ginsenosides was not changed. In the suspension cell culture of P. ginseng, substitution of NAA for 2,4-D decreased the rate of biomass accumulation and increased the extent of cell aggregation, with total content of ginsenosides increasing 25 times and their assortment being complete.     

Targeting Species-Specific Low-Affinity 16S rRNA Binding Sites by Using Peptide Nucleic Acids for Detection of Legionellae in Biofilms

Using fluorescence in situ hybridization to detect bacterial groups has several inherent limitations. DNA probes are generally used, targeting sites on the 16S rRNA. However, much of the 16S rRNA is highly conserved, with variable regions often located in inaccessible areas where secondary structures can restrict probe access. Here, we describe the use of peptide nucleic acid (PNA) probes as a superior alternative to DNA probes, especially when used for environmental samples. A complex bacterial genus (Legionella) was studied, and two probes were designed, one to detect all species and one targeted to Legionella pneumophila. These probes were developed from existing sequences and are targeted to low-binding-affinity sites on the 16S rRNA. In total, 47 strains of Legionella were tested. In all cases, the Legionella spp. PNA probe labeled cells strongly but did not bind to any non-Legionella species. Likewise, the specific L. pneumophila PNA probe labeled only strains of L. pneumophila. By contrast, the equivalent DNA probes performed poorly. To assess the applicability of this method for use on environmental samples, drinking-water biofilms were spiked with a known concentration of L. pneumophila bacteria. Quantifications of the L. pneumophila bacteria were compared using PNA hybridization and standard culture methods. The culture method quantified only 10% of the number of L. pneumophila bacteria found by PNA hybridization. This illustrates the value of this method for use on complex environmental samples, especially where cells may be in a viable but noncultivable state.     

Classical and molecular cytogenetics and DNA content in Maihuenia and Pereskia (Cactaceae)

We studied cacti species of the subfamilies Pereskioideae (five species of the southern clade) and both species of Maihuenioideae using molecular cytogenetic techniques and DNA content. Mitotic chromosomes were analyzed for Pereskia aculeata, P. bahiensis, P. grandifolia, P. nemorosa, P. sacharosa, Maihuenia poeppigii, and M. patagonica, using the Feulgen stain, CMA/DAPI fluorescent chromosome banding, fluorescence in situ hybridization (FISH, probes of 5S rDNA and pTa71 for 18-5.8-26S rDNA), and DNA content by flow cytometry technique. The karyotypes were highly symmetrical, most of the pairs being metacentric (m). CMA/DAPI banding revealed the presence of CMA⁺/DAPI^? bands associated with NORs in the first m pair of all species. The co-localization of 18-5.8-26S rDNA loci with CMA⁺/DAPI^?/NORs blocks allowed the identification of homeologous chromosome pairs between species of both subfamilies. FISH using probe 5S rDNA was applied for the first time in both subfamilies. Diploid species had always one m pair carrying 5S rDNA genes, with pericentromeric location in different chromosome pairs. In the tetraploid cytotype of M. patagonica, the 5S rDNA probe hybridized to two pairs. The 2C DNA content obtained by FC varied twofold (from 1.85 to 2.52 pg), with significant differences between species. Mean chromosome length, karyotype formula, percentage of heterochromatin position of 5S rDNA locus, and nuclear Cx DNA content vary among Maihuenia and Pereskia species and allowed to differentiate them. Both genera are closely related and that the differences found are not strong enough to separate Maihuenioideae from Pereskioideae.     

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.     

A taxonomic issue of two species of Trissolcus (Hymenoptera: Platygastridae) parasitic on eggs of the brown-winged green bug,Plautia stali (Hemiptera: Pentatomidae): resurrection of T. plautiae,a cryptic species of T. japonicus revealed by morphology,reproductive isolation and molecular evidence

Trissolcus plautiae (Watanabe) is known as a major egg parasitoid of the brown-winged green bug Plautia stali Scott, which is a serious pest of various fruit trees in Japan. Although T. plautiae was synonymized with T. japonicus (Ashmead) in 1981, both scientific names have been used for the same egg parasitoid of P. stali for about the past 30 years because of their taxonomic confusion. To promote an effective IPM program for P. stali using its egg parasitoids, we attempted to resolve the confusion by the use of a variety of methods. On detailed observation of adult morphology, we found that sublateral setae on the T1 are present in T. plautiae and absent in T. japonicus, and that this morphological difference is corroborated by more subtle differences between the two species. This finding supports the view that they are different species. The view was also supported by the results of mating experiments to determine the reproductive isolation of T. plautiae from T. japonicus and DNA analysis of these two species. We conclude that T. plautiae is a cryptic species of T. japonicus and resurrect T. plautiae from T. japonicus stat. rev.     

Molecular authentication of ginseng cultivars by comparison of internal transcribed spacer and 5.8S rDNA sequences

Molecular authentication among three Panax species and within cultivars and accessions of P. ginseng was investigated using the DNA sequence in the ribosomal ITS1–5.8S–ITS2 region. Four single-nucleotide polymorphisms were identified between P. ginseng and other Panax species. In the electrophoresis profile, obtained after digestion with the enzyme TaqI, three fingerprinting patterns were obtained from cultivars and accessions of Panax species. Consequently, this authentication procedure based upon the restriction fragment length polymorphism in the ribosomal ITS1–5.8S–ITS2 region can now be utilized to differentiate these Panax species as well as major Korean cultivars such as Gopoong and Kumpoong from other cultivars and accessions in Panax species at the DNA level. O. T. Kim and K. H. Bang contributed equally to this paper.     

Identification of indicator microorganisms using a standardized PNA FISH method.

A standardized fluorescent in situ hybridization (FISH) method using Peptide Nucleic Acid (PNA) probes for analysis of gram-negative and gram-positive bacteria, as well as yeast, has been developed. Fluorescently labeled PNA probes targeting specific rRNA sequences of Escherichia coli, Pseudomonas aeruginosa, Staphyloccocus aureus, Salmonella were designed, as well as PNA probes targeting eubacteria and eucarya. These PNA probes were evaluated by PNA FISH using 27 bacterial and 1 yeast species, representing both phylogenetically closely related species, as well as species important to both clinical and industrial settings. The S. aureus and P. aeruginosa PNA probes did not cross react with any of the organisms tested, whereas the E. coli PNA probe, as expected from sequence data, also detected Shigella species. The Salmonella PNA probe reacted with all of the 13 Salmonella strains, representing the 7 subspecies of Salmonella, however, it is also complementary to a few other bacterial species. The eubacteria- and eucarya-specific PNA probes detected all bacterial species and one yeast species, respectively. The general applicability of the PNA FISH method made simultaneous identification of multiple species, both gram-negative and gram-positive, in a mixed population an attractive possibility never accomplished using DNA probes. Four color images using differently labeled PNA probes showed simultaneous identification of E. coli, P. aeruginosa, S. aureus and Salmonella, thereby demonstrating the potential of multiplex FISH for various diagnostic applications within both clinical and industrial microbiology.     

Use of hybridization melting kinetics for detecting Phytophthora species using three-dimensional microarrays: demonstration of a novel concept for the differentiation of detection targets

Microarray-based detection is limited by variable and inconsistent hybridization intensities across the diversity of probes used in each array. In this paper, we introduce a novel concept for the differentiation of detection targets using duplex melting kinetics. A microarray assay was developed on a PamChip microarray enabling the differentiation of target Phytophthora species using the melting kinetics of probe-target duplexes. In the majority of cases the hybridization kinetics of target and non-target duplexes differed significantly. Analysis of the melting kinetics of duplexes formed by probes with target and non-target DNA was found to be an effective method for determining specific hybridization and was independent of fluctuations in hybridization signal intensity. This form of analysis was more robust than the traditional approach based on hybridization intensity, and enabled the detection of individual Phytophthora species and mixtures thereof.     

Identifying Fishes through DNA Barcodes and Microarrays

Background

International fish trade reached an import value of 62.8 billion Euro in 2006, of which 44.6% are covered by the European Union. Species identification is a key problem throughout the life cycle of fishes: from eggs and larvae to adults in fisheries research and control, as well as processed fish products in consumer protection.

Methodology/Principal Findings

This study aims to evaluate the applicability of the three mitochondrial genes 16S rRNA (16S), cytochrome b (cyt b), and cytochrome oxidase subunit I (COI) for the identification of 50 European marine fish species by combining techniques of “DNA barcoding” and microarrays. In a DNA barcoding approach, neighbour Joining (NJ) phylogenetic trees of 369 16S, 212 cyt b, and 447 COI sequences indicated that cyt b and COI are suitable for unambiguous identification, whereas 16S failed to discriminate closely related flatfish and gurnard species. In course of probe design for DNA microarray development, each of the markers yielded a high number of potentially species-specific probes in silico, although many of them were rejected based on microarray hybridisation experiments. None of the markers provided probes to discriminate the sibling flatfish and gurnard species. However, since 16S-probes were less negatively influenced by the “position of label” effect and showed the lowest rejection rate and the highest mean signal intensity, 16S is more suitable for DNA microarray probe design than cty b and COI. The large portion of rejected COI-probes after hybridisation experiments (>90%) renders the DNA barcoding marker as rather unsuitable for this high-throughput technology.

Conclusions/Significance

Based on these data, a DNA microarray containing 64 functional oligonucleotide probes for the identification of 30 out of the 50 fish species investigated was developed. It represents the next step towards an automated and easy-to-handle method to identify fish, ichthyoplankton, and fish products.     

秦巴山区三种人参属药用植物叶绿体基因组特征分析

为明确珠子参、羽叶三七和秀丽假人参3种药用植物叶绿体基因组特征与系统发育关系,该文以秦巴山区3种人参属药用植物为研究对象,运用生物信息学技术,分析其叶绿体基因组特征及密码子使用偏好性,并探讨三者之间的亲缘关系。结果表明:(1)3种人参属药用植物的叶绿体基因组为典型的四分体结构,序列全长为 156 071～156 104 bp,总 GC 含量为 38.10%,基因组大小相似度较高。(2)均注释到 133 个基因,包括 88 个蛋白编码基因、37 个tRNA基因和 8 个 rRNA 基因。(3)3种人参属药用植物叶绿体密码子使用偏好性相似,密码子第 3 位碱基以 A/U 结尾为主,密码子使用模式在受到突变影响的同时,主要受到自然选择的影响。(4)系统发育结果显示,3种人参属药用植物的亲缘关系较近,并且秀丽假人参同羽叶三七亲缘关系更近。综上认为,秀丽假人参与珠子参基源植物之间存在近缘关系,这项发现对于珠子参中药材的资源开发利用和分子鉴定,以及进一步研究人参属物种的分类、系统发育和进化机制提供了重要依据。     

Diversified chromosomal distribution of tandemly repeated sequences revealed evolutionary trends in Secale (Poaceae)

Genomic in situ hybridization (GISH) with Secale cereale cv. ‘Jingzhou rye’ DNA as a probe to chromosomes of hexaploid triticale line Fenzhi-1 revealed that not only were all chromosomes of rye strongly hybridized along the entire chromosome length, but there were also stronger signals in terminal or subtelomeric regions. This pattern of hybridization signals is referred to as GISH banding. After GISH banding, sequential fluorescene in situ hybridizaion (FISH) with tandem repeated sequence pSc200 and pSc250 as probes showed that the chromosomal distribution of pSc200 is highly coincident with the GISH banding pattern, suggesting that GISH banding revealed chromosomal distribution of pSc200 in rye. In addition, FISH using pSc200 and pSc250 as probes to chromosomes of 11 species of the genus Secale and two artificial amphiploids (Triticum aestivum-S. strictum subsp. africanum amphiploid and Aegilops tauschii-S. silvestre amphiploid) showed that (1) the chromosomal distribution of pSc200 and pSc250 differed greatly in Secale species, and the trend towards an increase in pSc200 and pSc250 binding sites from wild species to cultivated rye suggested that pSc200 and pSc250 sequences gradually accumulated during Secale evolution; (2) the chromosomal distribution of pSc200 and pSc250 presented polymorphism on homologous chromosomes, suggesting that the same species has two heterogeneous homologous chromosomes; (3) the intensity and number of hybridization signals varied differently on chromosomes between pSc200 and pSc250, suggesting that each repetitive family evolved independently.     

Distribution of repetitive DNA sequences in chromosomes of five opisthorchid species (Trematoda, Opisthorchiidae)

Genomes of opisthorchid species are characterized by small size, suggesting a reduced amount of repetitive DNA in their genomes. Distribution of repetitive DNA sequences in the chromosomes of five species of the family Opisthorchiidae (Opisthorchis felineus 2n = 14 (Rivolta, 1884), Opisthorchis viverrini 2n = 12 (Poirier, 1886), Metorchis xanthosomus 2n = 14 (Creplin, 1846), Metorchis bilis 2n = 14 (Braun, 1890), Clonorchis sinensis 2n = 14 (Cobbold, 1875)) was studied with C- and AgNOR-banding, generation of microdissected DNA probes from individual chromosomes and fluorescent in situ hybridization on mitotic and meiotic chromosomes. Small-sized C-bands were discovered in pericentric regions of chromosomes. Ag-NOR staining of opisthorchid chromosomes and FISH with ribosomal DNA probe showed that karyotypes of all studied species were characterized by the only nucleolus organizer region in one of small chromosomes. The generation of DNA probes from chromosomes 1 and 2 of O. felineus and M. xanthosomus was performed with chromosome microdissection followed by DOP-PCR. FISH of obtained microdissected DNA probes on chromosomes of these species revealed chromosome specific DNA repeats in pericentric C-bands. It was also shown that microdissected DNA probes generated from chromosomes could be used as the Whole Chromosome Painting Probes without suppression of repetitive DNA hybridization. Chromosome painting using microdissected chromosome specific DNA probes showed the overall repeat distribution in opisthorchid chromosomes.