Independent chromosomal localization of two different size 5S rDNA of Allium victorialis var. platyphyllum by sequential fluorescence in situ hybridization in accordance with sequence polymorphism

5S rRNA gene repeat units in a species are usually organized as either one relatively close size with numbers of intraspecific variations in NTS region or two different sizes with completely different sequence in NTS. Allium victorialis var. platyphyllum revealed two different size products of approximately 0.39 kb and 0.51 kb with highly conserved coding region of 120 bp. However, an extra sequences of approximately 120 bp between at 324 and 443 bp in long NTS region revealed, besides the remaining sequences of two NTS regions of short and long size were highly conserved giving the identity of 94.9%. To identify whether two different size 5S rDNA are occupied by a mixed state as random repeat or an independent group by each size in a particular locus, two rounds of FISH was sequentially performed using two probes of independent different size 5S rDNA and additional probe of only extra sequences of 120 bp in long NTS. Due to the highly conserved coding regions of both 5S rDNA, two different size 5S rDNA were detected in 3 loci in short arm of chromosome 6, however, extra sequences of long NTS was shown only in one locus within detected 5S rDNA from all examined chromosomes and interphase cells. This independent localization of two different size 5S rDNA suggests that 5S rDNA may be organized as a tandem repeat with random positions in a molecular level, but of cytogenetic view in chromosomes and interphase cells, they are organized as an independent group in a significant loci consisting of own size by the patterns of nucleotide variations.     

Phylogeny of Cucurbitaceae species in Korea based on 5S rDNA non-transcribed spacer

The 5S rDNA coding region and its spacer have been successfully utilized in phylogenetic studies of plants. However, it has not been utilized in the phylogenetic analysis of Cucurbitaceae. Here, we obtained the 5S rDNA sequences of 12 Cucurbitaceae species by direct PCR or cloning. The 5S rDNA sequences ranged from 275 to 359 bp, and the coding regions of all species were 120 bp long, except for that of Cucurbita, which was 119 bp. Some genus-specific SNPs were observed in the coding regions of Cucurbita, Lagenaria, Melothria, and Tricosanthes. The GC content of the coding regions was generally higher than that of the NTS regions, and the difference in GC content between the coding and NTS regions varied among species, with Gynostemma pentaphyllum having the greatest difference of 20.3. The phylogenetic trees generated using maximum parsimony and maximum likelihood were congruent and well supported by the recently published classification of Cucurbitaceae. These results demonstrated the utility of the 5S rDNA sequence in inferring phylogenetic relationships among 12 Cucurbitaceae species, and its utility could be extended by using a greater number of species in future studies.     

Molecular database construction of rDNA and polyploidy identification by FISH on Achyranthes japonica (Miq.) Nakai

Numerous studies on Achyranthes japonica have been investigated on physiological and pharmacological interests, however, no information of molecular cytogenetic level has been introduced yet, which, in turn, it is very essential to construct the molecular database and polyploidy primarily for any further researches. In this study, full unit of 5S and partial unit of 45S rDNA including two ITS regions were analyzed with chromosomal loci of examined rRNA genes on mitotic chromosomes. From the sequence analysis of rDNA unit, only a few polymorphic sites revealed in both coding and non-coding regions of NTS, ITS 1 and 2 giving an idea that no inter-specific hybrids has been involved in A. japonica as highly conserved specie through the high evolutionary period. To identify the polyploidy of A. japonica which has been unclear due to the very small size and unspecific centromere, FISH analysis was carried out on mitotic chromosomes using analyzed 5S and pTa-71 for 45S rDNA. 2 loci of each 5S and 45S rDNA were confirmed on the short arm of different chromosomes which were assumed to be a pair of each rDNA by a very similar size. Thus, the analyzed sequence of rDNA with low polymorphic rates and the identified loci on a relative size chromosome suggest the polyploidy of A. japonica as highly conserved diploid specie.     

Molecular cytogenetic analysis of the Vigna species distributed in Korea

Vigna plants distributed in Korea were analyzed by molecular cytogenetic fluorescence in situ hybridization (FISH), genomic in situ hybridization (GISH) and rDNA ITS/NTS sequences. FISH revealed that variable 45S rRNA gene loci (one to four) were localized on the terminal regions of chromosomes, while two conserved 5S rRNA gene loci from all species examined, except for rice bean (single locus), were detected. FISH and GISH showed the characteristic organization of rRNA gene loci and genomic homology on the chromosomes, indicating their cytogenetic relatationships. ITS sequence revealed that there was considerable variation in length (190–207 bp in ITS1, 205–221 bp in ITS2) and nucleotide composition (7–67 bp). The 5S rRNA gene unit comprised coding region (118 bp) and extensive sequence heterogeneity (97–221 bp). Phylogenetic analysis of the ITS and NTS sequences demonstrated that the Vigna species are divided into two groups: angularis (V. angularis, V. umbellata, V. nakashimae and V. nipponensis) and unguiculata (V. unguiculata, V. sesquipedalis and V. vexillata). Sequence data also showed that mung bean was closer to the angularis group.     

Molecular organization of 5S rDNA in bitterlings (Cyprinidae)

Molecular organization and nucleotide sequences of the 5S rRNA gene and NTS were investigated in freshwater fish, bitterlings (Acheilognathinae), including 10 species/subspecies of four genera, Acheilognathus, Pseudoperilampus, Rhodeus, and Tanakia, to understand the evolutionary trait of 5S rDNA arrays. Southern hybridization analysis revealed a general trend with tandem repeats of 5S rDNA in all the examined bitterlings. Sequence analysis demonstrated a conserved 120 bp sequence of the 5S rRNA gene and a short NTS of 56–67 bp with two distinct portions, a conserved (5′-flanking portion; at positions −1 to −38) and a variable part (3′-flanking portion), in 6 of 10 species/subspecies examined. The conserved NTS region was most likely an external promoter so far observed in various vertebrates, whereas the variable NTS region could be divided into two types due to its nucleotide polymorphisms. Molecular phylogeny using the 5S rRNA gene and NTS sequences suggested the occurrence of 5S rDNA duplication before speciation and a concerted evolution for the gene and conserved NTS regions, but a birth-and-death process to maintain the variable NTS region. Thus, the 5S rDNA in the examined bitterlings might have evolved under a mixed process of evolution.     

5S rDNA genome regions of Lens species.

The length variability of the nontranscribed spacer (NTS) of the 5S rDNA repeats was analyzed in species of the genus Lens by means of PCR amplification. The NTS ranged from approximately 227 to approximately 952 bp. The polymorphism detected was higher than previous NTS polymorphisms described in this genus. Three NTS length variants from Lens culinaris subsp. culinaris and 2 from Lens culinaris subsp. orientalis were sequenced. The culinaris NTS fragment lengths were 239, 371, and 838 bp, whereas the orientalis ones were 472 bp and 506 bp, respectively. As a result of sequence similarities, 2 families of sequences were distinguished, 1 including the sequences of 838 and 506 bp, and others with the sequences of 239, 371, and 472 bp. The 1st family was characterized by the presence of a repeated sequence designated A, whereas the 2nd family showed a single A sequence and other repeated sequences designated B, C, and D. The presence of an (AT)n microsatellite was also observed in the 2nd family of sequences. The fragments, which included the 239-bp and 838-bp NTS sequences, as well as the intergenic spacer (IGS) of the 18S-5.8S-26S ribosomal DNA also from L. culinaris subsp. culinaris, were used to localize the nucleolar organizer region (NOR) and the 5S rDNA loci in the chromosomes of several species of the genus Lens by means of fluorescence in situ hybridization (FISH). The selective hybridization of the 2 NTS probes allowed us to distinguish between different 5S rDNA chromosomal loci.     

Molecular Organization of 5S rDNAs in Rajidae (Chondrichthyes): Structural Features and Evolution of Piscine 5S rRNA Genes and Nontranscribed Intergenic Spacers

The genomic and gene organisation of 5S rDNA clusters have been extensively characterized in bony fish and eukaryotes, providing general issues for understanding the molecular evolution of this multigene DNA family. By contrast, the 5S rDNA features have been rarely investigated in cartilaginous fish (only three species). Here, we provide evidence for a dual 5S rDNA gene system in the Rajidae by sequence analysis of the coding region (5S) and adjacent nontranscribed spacer (NTS) in five Mediterranean species of rays (Rajidae), and in a large number of piscine taxa including lampreys and bony fish. As documented in several bony fish, two functional 5S rDNA types were found here also in the rajid genome: a short one (I) and a long one (II), distinguished by distinct 5S and NTS sequences. That the ancestral piscine genome had these two 5S rDNA loci might be argued from the occurrence of homologous dual gene systems that exist in several fish taxa and from 5S phylogenetic relationships. An extensive analysis of NTS-II sequences of Rajidae and Dasyatidae revealed the occurrence of large simple sequence repeat (SSR) regions that are formed by microsatellite arrays. The localization and organization of SSR within the NTS-II are conserved in Rajiformes since the Upper Cretaceous. The direct correlation between the SSRs extension and the NTS length indicated that they might play a role in the maintenance of the larger 5S rDNA clusters in rays. The phylogenetic analysis indicated that NTS-II is a valuable systematic tool limited to distantly related taxa of Rajiformes. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Rafael Zardoya]     

Sequence polymorphisms in ribosomal RNA genes and variations in chromosomal loci of Oenothera odorata and O. laciniata

Numerous studies on Oenothera species have been investigated for the physiological and ecological characteristics. However, no such an information based on molecular cytogenetic has yet been introduced, in turn, is very essential for identifying sequence polymorphisms of rRNA genes with their loci on mitotic phases for further biological researches. In this study, sequence variations of rRNA genes in Oenothera odorata and O. laciniata were examined to identify informative factors as unique or repeat sequences in intra- and inter-specific variations. Intra-specific variation revealed that the sequences of the rRNA genes including the spacer regions were highly conserved revealing only a few variations. From the inter-specific variation, spacer regions of species were completely different as (1) non-homologous sequences in NTS and (2) different type repeat sequences in ITS 1, 2 and 5.8S rRNA, whereas the remaining coding regions were highly conserved. FISH was carried out on mitotic phases using the 5S rDNA of the analyzed sequences. From the interphase and metaphase chromosomes of the examined species, two loci of 5S rDNA in O. odorata and four loci in O. laciniata were confirmed on the telomeric region of the short arm. Due to the small size and unclear centromere of the chromosomes, karyotype could not be completed. However, we confirmed that the chromosomes are organized by meta- and acrocentric chromosomes and the chromosomes with identified loci were assumed to be paired by the location of loci at the telomeric region on the short arm with relative lengths.     

Molecular characterization of major and minor rDNA repeats and genetic variability assessment in different species of mahseer found in North India

Relationship among the mahseer species (Family: Cyprinidae) has long been debated in fish systematics. Present study concentrates on the nature of the phylogenetic relationship among the five mahseer species using the sequence of major ribosomal DNA (45S rDNA). We have covered rDNA sequence of approximately 5.2 kb per individual, 26.0 kb per species and 130.0 kb as a whole. We also characterized the 45S and 5S rDNA regions with respect to their nucleotide composition. For phylogenetic analyses, nucleotide sequences were divided into four datasets. First and second datasets contained 18S rDNA and ITS1 sequence, whereas third and fourth datasets consisted of ITS2 and complete 18S-ITS1-5.8S-ITS2-28S, respectively. The NJ tree was constructed for all the datasets. The mahseer species under study formed a monophyletic group well separated from the outgroup species. Similarly, the individuals of Neolissochilus hexagonolepis form monophyletic group with Tor species, indicating Neolissochilus as a sister genus of Tor. The findings from the present study provide greater insights into taxonomic status of mahseer, and set the stage for future investigations dealing with phylo-geography, taxonomy, conservation and co-evolution within this interesting and important group of fish.     

Development of the St/J and V Genome Specific Molecular Marker Based on 5s rDNA Polymorphism in <Emphasis Type="Italic">Thinopyrum bessarabicum</Emphasis>, <Emphasis Type="Italic">Pseudoroegneria spicata</Emphasis>, and <Emphasis Type="Italic">Dasypyrum villosum</Emphasis>

Nontranscribed spacers (NTS) of 5S rDNA are often polymorphic in closely related species and even in the same genome. The polymorphism of 5S rDNA NTS was shown between genomes St, J, and V of Triticeae species Thinopyrum bessarabicum, Pseudoroegneria spicata, and Dasypyrum villosum, respectively. A molecular genetic marker was designed based on the 5S rDNA NTS polymorphism that allows identification of the St, J, and V genomes. We designed a pair of primers that correspond to the conserved regions of 5S rDNA NTS between the genomes studied. The PCR amplicon length is 158 bp, 171 bp, and 172 bp for V, St, and J genomes, respectively. The fragment of the St genome is characterized by the SmiM I restriction site that enables its differentiation from the J genome fragment that lacks this site. The developed marker showed its efficiency for verification of germplasm accessions and the study of allopolyploids.     

Regular distribution of length heterogeneities within non-transcribed spacer regions of cloned and genomic rDNA of Saccharomyces cerevisiae.

A length difference of about 50 bp in the EcoRI fragment B of the rDNA from two different strains of Saccharomyces cerevisiae has been mapped in detail by sequencing of cloned fragments. This 2.4 kb EcoRI fragment contains the start of the 35S rRNA gene at one end and the 5S rRNA gene in the middle flanked by non-transcribed spacers, NTS1 and NTS2. The difference appeared as short deletions or insertions in five regularly spaced regions within the 1 kb NTS1, 3' to the 5S rRNA gene. The same regions of heterogeneities were displayed when all available sequence data of the NTS1 were compared. Four of the variable regions are located 160-170 bp apart, indicating that they might represent linker sequences between phased nucleosomes. Two variant clones, differing in the length of one subfragment of NTS1, were isolated for each strain. In both cases these represented the major variants among chromosomal NTS1 as revealed by sequencing of genomic fragments.     

Identification ofEnterococcus sp. from midgut of silkworm based on biochemical and 16S rDNA sequencing analysis

Enterococcus sp. was isolated from the midgut of silkworm against the germination ofNosema bombycis spores. Identification was based on the biochemical characteristics, 16S rDNA sequences analysis and species-specific probes ofEnterococcus spp. The isolated strains fermented sorbitol and arabinose but did not ferment raffinose.Enterococcus sp. was clustered together withEnterococcus mundtii ATCC 43188 and 100% sequence homology was found by 16S rDNA sequences BLAST analysis and constructing the phylogenetic tree. Comparison of the sequences of the 16S rDNA species-specific probes ofEnterococcus spp. with the 16S rDNA sequence of isolate revealed similar segment to the species-specific probe ofE. mundtii. So, we can make conclusion the 16S rDNA segment ofEnterococcus sp. can hybridise with species-specific probe ofE. mundtii. Enterococcus mundtii was detected for the first time in the intestine of silkworm.     

gvpA-C间隔区在微囊藻株系分型中的应用

从暴发水华的水体分离微囊藻(Microcystis)株,并依据gvpA-C间隔区和16S rDNA序列对其分型和比较。在gvpA-C间隔区序列中,有的类型具有一段172—176 bp的额外序列。以不包括额外序列的0.27 kb序列相比较,不同类型之间差异碱基位点达50余个。在16S rDNA碱基变异集中的0.69 kb序列中,不同类型之间有差异的碱基位点共有8个。与16S rDNA序列相比,微囊藻gvpA-C间隔区序列具有高度变异性,并且其PCR扩增可一步完成,不受其他细菌污染,因而可用于微囊藻株系分型。由于横向转移,2种分型存在交叉关系。此外,原本含有或不含有额外序列的微囊藻gvpA-C间隔区序列在系统进化树中分别聚成一簇。     

Satellite DNA derived from 5S rDNA in Physalaemus cuvieri (Anura, Leiuperidae)

In the present study, we describe for the first time a family of 190-bp satellite DNA related to 5S rDNA in anurans and the existence of 2 forms of 5S rDNA, type I (201 bp) and type II (690 bp). The sequences were obtained from genomic DNA of Physalaemus cuvieri from Palmeiras, State of Bahia, Brazil. Analysis of the nucleotide sequence revealed that the satellite DNA obtained by digestion with EcoRI, called PcP190EcoRI, is 70% similar to the coding region of type I 5S rDNA and 66% similar to the coding region of type II 5S rDNA. Membrane hybridization and PCR amplification of the sequence showed that PcP190EcoRI is tandemly repeated. The satellite DNA as well as type I and type II 5S rDNA were localized in P. cuvieri chromosomes by fluorescent in situ hybridization. The PcP190EcoRI sequence was found in the centromeres of chromosomes 1-5 and in the pericentromeric region of chromosome 3. Type I 5S rDNA was detected in chromosome 3, coincident with the site of PcP190EcoRI. Type II 5S rDNA was located interstitially in the long arm of chromosome 5. None of these sequences co-localized with nucleolar organizer regions. Our data suggests that this satellite DNA originates from the 5S ribosomal multigene family, probably by gene duplication, nucleotide divergence and sequence dispersion in the genome.     

5S rDNA variation and its phylogenetic inference in the genus <Emphasis Type="Italic">Leporinus</Emphasis> (Characiformes: Anostomidae)

5S rDNA sequences have proven to be valuable as genetic markers to distinguish closely related species and also in the understanding of the dynamic of repetitive sequences in the genomes. In the aim to contribute to the knowledge of the evolutionary history of Leporinus (Anostomidae) and also to contribute to the understanding of the 5S rDNA sequences organization in the fish genome, analyses of 5S rDNA sequences were conducted in seven species of this genus. The 5S rRNA gene sequence was highly conserved among Leporinus species, whereas NTS exhibit high levels of variations related to insertions, deletions, microrepeats, and base substitutions. The phylogenetic analysis of the 5S rDNA sequences clustered the species into two clades that are in agreement with cytogenetic and morphological data.