New record of Ascaridia nymphii (Secernentea: Ascaridiidae) from macaw parrot,Ara chloroptera,in China

Present study was performed to identify the species of ascarids from macaw parrot, Ara chloroptera, in China. Total 6 ascarids (3 males and 3 females) were collected in the feces of 3 macaws at Guangzhou Zoo in Guangdong Province, China. Their morphological characteristics with dimensions were observed under a light microscope, and their genetic characters were analyzed with the partial 18S rDNA, ITS rDNA and nad4 gene sequences, respectively. Results showed that all worms have no interlabia but male worms have two alate spicules, well-developed precloacal sucker and a tail with ventrolateral caudal alae and 11 pairs of papillae. The partial 18S rDNA, ITS rDNA and nad4 sequences were 831 bp, 1015 bp and 394 bp in length, respectively. They showed the highest similarity of 99.8% (18S rDNA) with Ascaridia nymphii, 93.8% identities (ITS rDNA) with A. columbae and 98.5% to 99.5% identities (nad4) with Ascaridia sp. from infected parrot. All Ascaridia nematodes from the macaws were clustered into one clade and formed monophyletic group of Ascaridia with A. columbae and A. galli in two phylogenetic trees. It is observed that the combining morphological and sequencing data from three loci, the present Ascaridia species was identified as Ascaridia nymphii, which is the first record of A. nymphii from macaw parrot in China.     

Secondary structure prediction for complete rDNA sequences (18S, 5.8S,and 28S rDNA) of Demodex folliculorum, and comparison of divergent domains structures across Acari

According to base pairing, the rRNA folds into corresponding secondary structures, which contain additional phylogenetic information. On the basis of sequencing for complete rDNA sequences (18S, ITS1, 5.8S, ITS2 and 28S rDNA) of Demodex, we predicted the secondary structure of the complete rDNA sequence (18S, 5.8S, and 28S rDNA) of Demodex folliculorum, which was in concordance with that of the main arthropod lineages in past studies. And together with the sequence data from GenBank, we also predicted the secondary structures of divergent domains in SSU rRNA of 51 species and in LSU rRNA of 43 species from four superfamilies in Acari (Cheyletoidea, Tetranychoidea, Analgoidea and Ixodoidea). The multiple alignment among the four superfamilies in Acari showed that, insertions from Tetranychoidea SSU rRNA formed two newly proposed helixes, and helix c3-2b of LSU rRNA was absent in Demodex (Cheyletoidea) taxa. Generally speaking, LSU rRNA presented more remarkable differences than SSU rRNA did, mainly in D2, D3, D5, D7a, D7b, D8 and D10.     

Chromosome mapping of repetitive sequences in four Serrasalmidae species (Characiformes)

The Serrasalmidae family is composed of a number of commercially interesting species, mainly in the Amazon region where most of these fishes occur. In the present study, we investigated the genomic organization of the 18S and 5S rDNA and telomeric sequences in mitotic chromosomes of four species from the basal clade of the Serrasalmidae family: Colossoma macropomum, Mylossoma aureum, M. duriventre, and Piaractus mesopotamicus, in order to understand the chromosomal evolution in the family. All the species studied had diploid numbers 2n = 54 and exclusively biarmed chromosomes, but variations of the karyotypic formulas were observed. C-banding resulted in similar patterns among the analyzed species, with heterochromatic blocks mainly present in centromeric regions. The 18S rDNA mapping of C. macropomum and P. mesopotamicus revealed multiple sites of this gene; 5S rDNA sites were detected in two chromosome pairs in all species, although not all of them were homeologs. Hybridization with a telomeric probe revealed signals in the terminal portions of chromosomes in all the species and an interstitial signal was observed in one pair of C. macropomum.     

Heterogeneity of the yeasts Zygowilliopsis californica: Z. californica var. dimennae comb. nov., stat. nov. and Z. californica var. fukushimae comb. nov., stat. nov.

A significant heterogeneity of the species Zygowilliopsis californica was revealed using RFLP-analysis of the PCR-amplified rDNA fragment spanning the 5.8S rRNA gene and the internal transcribed spacers ITS1 and ITS2. Phylogenetic analysis of the nucleotide sequences of ITS1 and ITS2 rDNA differentiated three varieties: Z. californica var. californica, Z. californica var. dimennae, and Z. californica var. fukushimae. The most variable was the ITS2 region, where 7–26 nucleotide substitutions were revealed. The varieties formed semisterile hybrids with meiotic segregation of control markers. The limits of the phylogenetic species concept are discussed.     

A single species,two basic chromosomal numbers: case of Lygeum spartum (Poaceae)

Abstract

The existence of two chromosome numbers (2n?=?16 and 2n?=?40) in Lygeum spartum is confirmed in the Algerian steppe populations of Oran region. Chromosome counts were established for 11 Algerian populations and three supplementary populations from Italy, Spain and Greece. The karyotypes were characterized by chromosome markers obtained using fluorescence in situ hybridization and fluorochrome bandings. The organization of 5S and 18S-5.8S-26S (35S) rDNA was studied in both cytotypes. Six signals of 35S and 2 signals of 5S were observed in 2n?=?16 population, while 10 signals of 35S and 4 signals of 5S were detected in the population with 2n?=?40. All 35S loci were also strongly marked by chromomycin, but negatively stained by Hoechst, which indicates the presence of GC rich DNA in rDNA regions. The B chromosomes were found in both cytotypes, bearing a 35S locus in 2n?=?16 population. Genome size, determined by cytometry of 10 populations, ranged from 9.27?pg for 2n?=?16 to 26.63?pg for 2n?=?40 populations. The sequencing of plastid and nuclear DNA markers did not reveal major differences among 2n?=?16 and 2n?=?40 populations. However, given the differences between two cytotypes and based on their morphological and cytogenetic characteristics, the 2n?=?16 cytotype merits novel taxonomic treatment.     

Ribosomal DNA Comparisons of Globodera from Two Continents

Ribosomal DNA (rDNA) sequence data were compared for five species of Globodera, including G. rostochiensis, G. pallida, G. virginiae, and two undescribed Globodera isolates from Mexico collected from weed species and maintained on Solanum dulcamara. The rDNA comparisons included both internal transcribed spacers (ITS1 and ITS2), the 5.8S rRNA gene, and small portions of the 3'' end of the 18S gene and the 5'' end of the 28S gene. Phylogenetic analysis of the rDNA sequence data indicated that the two potato cyst nematodes, G. pallida and especially G. rostochiensis, are closely related to the Mexican isolates, whereas G. virginiae is relatively dissimilar to the others and more distantly related. The data are consistent with the thesis that Mexico is the center of origin for the potato cyst nematodes.     

FISH分析栽培高粱×拟高粱、甜高粱×拟高粱的染色体行为

采用荧光原位杂交技术对45S rDNA在栽培高粱×拟高粱、甜高粱×拟高粱F1的有丝分裂和减数分裂染色体进行定位研究。在有丝分裂中期染色体上2个杂种分别检测到2个杂交信号, 在减数分裂粗线期、终变期、中期Ⅰ染色体上45S rDNA位于一个二价体上, 说明这两个杂种携带45S rDNA的染色体为同源染色体。根据45S rDNA位点随细胞减数分裂过程的位置变化, 表明这两个杂种染色体配对行为正常, 平均构型为2n=2x=20(10Ⅱ), 证明45S rDNA可作为染色体的一个识别指标间接地观察细胞减数分裂过程染色体的变化行为。     

Detection of 5S and 25S rRNA genes in Sinapis alba, Raphanus sativus and Brassica napus by double fluorescence in situ hybridization

Different ribosomal RNA (5S and 25S) genes were investigated simultaneously by fluorescence in situ hybridization (FISH) in Sinapis alba, Raphanus sativus and Brassica napus. The chromosomes of S. alba carried four 5S and six 25S gene sites, and those of R. sativus four sites of each gene, respectively. These two species have one chromosome pair with both rDNA genes; the two are closely located on a short arm of S. alba, while in R. sativus one is distal on the short arm (5S) and the other more proximal on the long arm (25S). In B. napus we have confirmed 12sites of 25S rDNA. The detection of 5S rDNA genes revealed 14 signals on 12 chromosomes. Of these, six chromosomes had signals for both rDNA genes. The FISH with 5S rDNA probes detected two sites closely adjacent in four chromosomes of B napus. These results are discussed in relation to a probable homoeologous chromosome pair in B. oleracea. Received: 20 July 1999 / Accepted: 8 October 1999     

Analysis of Yeast-Like Symbiote Diversity in the Brown Planthopper (BPH), Nilaparvata lugens Stål, Using a Novel Nested PCR-DGGE Protocol

Yeast-like symbiotes (YLS) are endosymbionts that are intimately associated with the growth, development, reproduction of their host, the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae). However, it is unclear how many species of YLS are found within N. lugens, and how they are related to each other. Traditional methods or simple amplification based on 18S rDNA sequence does not reliably identify new species quickly and efficiently. Therefore, a novel nested PCR-denaturing gradient gel electrophoresis (DGGE) strategy was developed in this article to analyze the YLS of brown planthopper using a nested PCR protocol that involved the 18S rDNA gene and the 5.8S–ITS gene using fungal universal primers. The nested PCR protocol was developed as follows: firstly, the 18S rDNA gene, and 5.8S–ITS gene were amplified using fungal universal primers. Subsequently, these products were used as a template in a second PCR with primers ITS1GC–ITS2, ITS1FGC–ITS2, and NFGC-NR, which was suitable for DGGE. Using this highly specific molecular approach, we found several previously detected fungi: Noda, Pichia guilliermondii, Candida sp., and some previously undetected fungi, such as Saccharomycetales sp., Debaryomyces hansenii, and some uncultured fungi. In conclusion, the nested PCR system developed in this study, coupled with DGGE fingerprinting, offers a new tool for uncovering fungal endosymbiont diversity within planthoppers.     

Localization of 45S and 5S rDNA sites and karyotype of Chrysanthemum and its related genera by fluorescent in situ hybridization

The phylogenetic relationships among Chrysanthemum and its related genera (Anthemideae, Asteraceae) is poorly understood. In the present study, these relationships were investigated using 45S and 5S ribosomal DNA (rDNA)-targeted fluorescent in situ hybridization. The results showed that there were two 45S rDNA signals present in Crossostephium chinense, four 45S rDNA signals in Cercidiphyllum japonicum, Artemisia sieversiana, Artemisia annua and Artemisia absinthium, six 45S rDNA signals in Chrysanthemum boreale and Pyrethrum parthenium, eight 45S rDNA signals in Chrysanthemum nankingense, Chrysanthemum dichrum, Chrysanthemum lavandulifolium and Tanacetum vulgare, and ten 45S rDNA signals in Ajania przewalskii. For the 5S rDNA locus, two 5S rDNA signals were present in C. nankingense, C. dichrum, C. lavandulifolium, C. boreale, C. japonicum, C. chinense and P. parthenium, four in A. sieversiana, A. annua, A. absinthium and A. przewalskii, and six 5S in T. vulgare. In addition, karyotypes of the 12 species were investigated. From this study, we inferred that Chrysanthemum was closely related to Ajania, and that Chrysanthemum species originating from China and Japan may have evolved differently. These findings add a new level to the understanding of the phylogenetic relationships of Chrysanthemum and related genera.     

Phylogenetic Analysis and Karyotype Evolution in the Genus Clivia(Amaryllidaceae)

Phylogenetic relationships of five taxa of Clivia, one probablenew species plus four recognized species, and three outgroupspecies were studied using sequences of the nuclear ribosomal5S non-transcribed spacer and the internal transcribed spacer(ITS) of 45S rDNA. Analysis of the data sets separately generatedsome well-supported groupings and congruent phylogenies. Cliviaminiata and C. gardenii are closely related. ‘Robust Gardenii’,the putative new species, is a sister clade of this group. Clivianobilis is distantly related to these three taxa and C. caulescensoccupies an intermediate position between the two groups. Chromosomelocations and distribution patterns of the 5S nuclear ribosomalgene in the species of Clivia were investigated using fluorescenceinsitu hybridization (FISH). In all species, only one pair of5S rDNA signals was observed. These were located on the shortarm of chromosome 8, at the position of the interstitial C-bands.The phylogenies obtained from the DNA sequences together withthe chromosome data accumulated here and previously publishedinformation on the location of the 45S rDNA sites have beenused to postulate evolutionary trends in Clivia chromosomes.Copyright 2001 Annals of Botany Company Clivia, chromosome evolution, 45S and 5S rDNA, ITS, FISH, molecular phylogeny     

Do polyploids require proportionally less rDNA loci than their corresponding diploids? Examples from Artemisia subgenera Absinthium and Artemisia (Asteraceae,Anthemideae)

Abstract

Fluorescent in situ hybridisation (FISH) of 5S and 18S-5.8S-26S ribosomal DNA was carried out in two species of the genus Artemisia, belonging to the subgenera Artemisia (A. medioxima) and Absinthium (A. lagocephala), each one showing both low and high ploidy levels (2x, 4x and 16x, and 2x and 6x, respectively). Both species have a base chromosome number of x = 9. Linkage of both rDNA genes has been observed confirming previous results. Diploid A. lagocephala (2n = 18) shows three rDNA loci, and the hexaploid six. Also in A. medioxima, the number of rDNA loci does not increase in the proportion given by the ploidy level, and a relative loss is found. In this species, the diploid population shows two rDNA loci, the tetraploid four, and the hexaidecaploid has around 20. The results evidence a relative loss of rDNA loci and heterochromatin, a phenomenon that is more pronounced at higher ploidy levels. Nevertheless, the DAPI banding pattern of A. lagocephala does not follow this trend, as it shows a spectacular increase of heterochromatic bands at the hexaploid level. These results are discussed in the light of possible chromosome restructuring and gene silencing mechanisms that take place during polyploidy, and more especially allopolyploid formation.     

Characterization of Japanese flounder karyotype by chromosome bandings and fluorescence in situ hybridization with DNA markers

The chromosomes of Japanese flounder, Paralichthys olivaceus, were examined by conventional differential staining methods including G-, Q-, C-, silver (Ag)-, fluorochrome, and replication R-bandings and by fluorescence in situ hybridization (FISH) with 5S and 18S rDNAs and telomeric DNA as probes. Replication R-banding substantially made it possible to identify 24 homologous pairs by their RBG-banding pattern and relative length. Both rDNA loci were mapped to chromosome 1, where 5S and 18S rDNA loci were located at the centromeric region and secondary constriction, respectively. C-banding revealed that both rDNA loci were heterochromatic, and 18S rDNA loci were positive for chromomycin A₃ but negative for 4′,6-diamidino-2-phenylindole (DAPI) staining. Telomeric FISH signals were observed at all chromosome ends and at the interstitial region of some chromosomes. The observed results were discussed in relation to the karyotype evolution in the order Pleuronectiformes.     

Karyotype,chromosomal characteristics of multiple rDNA clusters and intragenomic variability of ribosomal ITS2 in Caryophyllaeides fennica (Cestoda)

Karyotype and chromosomal characteristics, i.e. number and location of ribosomal DNA (rDNA) clusters, and sequence variation of the ribosomal internal transcribed spacer 2 (ITS2) were studied in a monozoic (unsegmented) tapeworm, Caryophyllaeides fennica (Caryophyllidea), using conventional and Ag-staining, fluorescent in situ hybridization (FISH) with 18S rDNA probe, and PCR amplification, cloning and sequencing of the complete ribosomal ITS2 spacer. The karyotype of this species was composed of ten pairs of metacentric (m) chromosomes (2n = 20). All chromosomes except the pair No. 2 displayed DAPI-positive heterochromatin in centromeric regions. In addition, two distinct interstitial DAPI-positive bands were identified on chromosome pair No. 7. FISH with 18S rDNA probe revealed four clusters of major ribosomal genes situated in the pericentromeric region of the short arms in two pairs of metacentric chromosomes Nos. 8 and 9. Hybridization signals were stronger in the pair No. 8, indicating a higher amount of rDNA repeats at this nucleolar organizer region (NOR). Analysis of 15 ITS2 rDNA sequences (five recombinant clones from each of three individuals) showed 13 structurally different ribotypes, distinguished by 26 nucleotide substitutions and variable numbers and combinations of short repetitive motifs that allowed sorting the sequences into four ITS2 variants. These results contribute to recently published evidence for the intraindividual ribosomal ITS sequence variability in basal tapeworms with multiple rDNA loci and imply that both phenomena may be mutually linked.     

Phylogenetic Relationships Among Selected Heteroderoidea Based on 18S and ITS Ribosomal DNA

In a study of relationships among selected cyst-forming and noncyst-forming species of Heteroderoidea, combined sequences comprised of DNA from part of the conserved 18S ribosomal RNA gene (rDNA) plus the complete ITS rDNA segment were more similar to analyses based on the ITS data alone than to analyses based on the 18S data alone. One of the two noncyst-forming species, Ekphymatodera thomasoni, grouped with cyst-forming species of Heteroderoidea. Bilobodera flexa, also a noncyst-forming species, was separated from all the other taxa by a long branch. Afenestrata koreana, with a weakly sclerotized cyst, grouped closely with H. bifenestra. These observations suggest that phylogenetic analyses using molecular data may aid in our understanding of the evolution of cyst formation in nematodes, including the possibility of secondary loss. The usefulness of molecular phylogenetic analyses in nematodes may depend more on the particular selection of taxa than on mere addition of data from additional genes.     

Nuclear 28S rDNA phylogeny supports the basal placement of Noctiluca scintillans (Dinophyceae; Noctilucales) in dinoflagellates

Noctiluca scintillans (Macartney) Kofoid et Swezy, 1921 is an unarmoured heterotrophic dinoflagellate with a global distribution, and has been considered as one of the ancestral taxa among dinoflagellates. Recently, 18S rDNA, actin, α-, β-tubulin, and Hsp90-based phylogenies have shown the basal position of the noctilucids. However, the relationships of dinoflagellates in the basal lineages are still controversial. Although the nuclear rDNA (e.g. 18S, ITS-5.8S, and 28S) contains much genetic information, DNA sequences of N. scintillans rDNA molecules were insufficiently characterized as yet. Here the author sequenced a long-range nuclear rDNA, spanning from the 18S to the D5 region of the 28S rDNA, of N. scintillans. The present N. scintillans had a nearly identical genotype (>99.0% similarity) compared to other Noctiluca sequences from different geographic origins. Nucleotide divergence in the partial 28S rDNA was significantly high (p<0.05) as compared to the 18S rDNA, demonstrating that the information from 28S rDNA is more variable. The 28S rDNA phylogeny of 17 selected dinoflagellates, two perkinsids, and two apicomplexans as outgroups showed that N. scintillans and Oxyrrhis marina formed a clade that diverged separately from core dinoflagellates.     

Comparisons of Isolates of Heterodera avenae using 2-D PAGE Protein Patterns and Ribosomal DNA

Six geographic isolates of Heterodera avenae, including two isolates each from Sweden, Australia, and the United States, were compared on the basis of 2-D PAGE protein patterns and the complete DNA sequence for the two internal transcribed ribosomal DNA spacers (rDNA ITS1 and ITS2) and the 5.8S rRNA gene. The protein pattern data and rDNA ITS sequence data both indicated that the Swedish Gotland strain of H. avenae differed markedly from the rest of the isolates. Protein patterns for the Australia isolates differed more from a Swedish strict H. avenae isolate and isolates from Oregon and Idaho, than the two U.S. isolates and the Swedish strict H. avenae isolate differed from each other. Except for the Gotland strain isolate, the rDNA ITS sequences were highly conserved among all of the H. avenae isolates, just as we earlier found them to be conserved among species of the schachtii group of Heterodera.     

Genetic diversity and phylogenetic relations of salmon trematode Nanophyetus japonensis

Nanophyetiasis is the severe zoonotic disease caused by parasitic worms from the genus Nanophyetus. Humans and carnivorous animals become infected when they ingest raw fish containing metacercariae, especially Pacific salmonids. Nanophyetiasis is detected in limited geographical areas which include the coastal regions of the North Pacific: the United States of America, Russian Federation and Japan. Despite the epidemiological significance, Nanophyetus species have not been well studied genetically. In this research, we for the first time explored genetic diversity of Nanophyetus japonensis from Japan in comparison with those of related species, N. salmincola from North America and N. schikhobalowi from the Russian Far East, based on sequence variation in the nuclear ribosomal gene family (18S, ITS1–5.8S–ITS2 and 28S) and mitochondrial nad1 gene, encoding subunit I of the respiratory chain NADH dehydrogenase. The results confirmed the independent species status for the compared flukes, demonstrated a greater genetic similarity of Asian species between themselves than each of them with the North American one, suggesting that N. japonensis and N. schikhobalowi are close sister species, and also revealed discrepancy between the levels of morphological and genetic differentiation.     

Chromosome identification and karyotype analysis of Podophyllum hexandrum Roxb. ex Kunth using FISH

Podophyllum hexandrum is an important high altitude medicinal plant from Himalaya. Somatic chromosomes of this species were studied to delineate and physical mapping of repetitive rDNA sites to provide landmarks in chromosome identification. The karyotype formula of this species was found to be 6m + 2sm + 2st + 2t with secondary constriction in the chromosome 1 and 7. The FISH analysis of rDNA sites showed 4 sites for 18S rDNA and 2 sites for 5S rDNA. The chromosome number 1, 2, 5 and 6 can be identified based on 18S rDNA sites in their short arm and chromosome 1 and 2 can be identified by 5S rDNA site in the centromere region. The estimated genome size of this plant is 16.07 pg (1C).