5S rDNA Non-transcribed Spacer Research Articles

The improvement of the CAPS-marker for St, J and V subgenome identification in Triticeae tribe plants using the 5S non-transcribed spacer polymorphism

In the Triticeae tribe, several subgenomes (St, J, V and others) were described, and a subgenome-specific molecular marker development is an actual branch of studies. Non-transcribed spacers (NTS) of 5S rDNA are perspective to create species-specific or genome-specific molecular markers in closely related organisms. Early, the 5S rDNA NTS based CAPS marker (StJV-f/StJV-r primers with the SmiM1 enzyme digestion) was designed for identification of the St, J and V subgenomes. The V subgenome amplicons were differed from the St and J amplicons in length. The St amplicons were distinguishable from the J amplicons after the SmiM1 enzyme digestion (St fragments were digested, J fragments were not digested, which is a disadvantage, because a failure with the digestion of St amplicons can be mistaken for J amplicons). This article presents the results of the StJV marker improvement. Comparative analysis of restriction enzyme maps revealed that the Hpy166 II enzyme cuts the StJV amplicons of all studied subgenomes in such a way that different product patterns are obtained. Experimental testing confirmed this finding. Thus, the improved CAPS marker can serve as a promising tool for verifying samples in Triticeae plant collections, screening herbarium materials, as well as in the breeding process aimed at improving wheat by involving its wild relatives in traditional breeding programs efficiently.

Molecular characterization of the 5S rDNA non-transcribed spacer and reconstruction of phylogenetic relationships in Capsicum

Abstract Capsicum includes ca. 41 species of chili peppers. In this original report we PCR amplified, cloned, sequenced and characterized the 5S rDNA non-transcribed spacer -NTS- in 23 taxa of nine clades of Capsicum, divergent at geographical origin and fruit and chromosome traits, and compared the NTS features throughout Solanaceae. According to GC content, inner variability and regulatory elements, the NTS organizes into three distinct structural regions; genetic variability at the NTS in Capsicum and related genus clusters into defined taxa hierarchies. Based on the reconstruction of a maximum-likelihood phylogenetic tree and phylogenetic networks, NTS sequences of Capsicum and related taxa grouped into well recognized categories -genus, section, clade, species, variety-. An evolutionary scenario arose from combined genetic and phylogenetic NTS data, in which monophyly and lineage diversification over time of Capsicum are addressed. Our analysis is original to include all domesticated species of Capsicum prevailing in germplasm collections and breeding programs, together with a large group of wild taxa that demanded further genetic characterization. The NTS set up as a double purpose marker in Capsicum, to directly evaluate genetic variability and reconstruct phylogenetic relationships to a broad extent, and constitutes a valuable tool for germplasm characterization and evolutionary studies within Solanaceae.

A Comparative Study of 5S rDNA Non-Transcribed Spacers in Elaeagnaceae Species.

5S rDNA is organized as a cluster of tandemly repeated monomers that consist of the conservative 120 bp coding part and non-transcribed spacers (NTSs) with different lengths and sequences among different species. The polymorphism in the 5S rDNA NTSs of closely related species is interesting for phylogenetic and evolutional investigations, as well as for the development of molecular markers. In this study, the 5S rDNA NTSs were amplified with universal 5S1/5S2 primers in some species of the Elaeagnaceae Adans. family. The polymerase chain reaction (PCR) products of five Elaeagnus species had similar lengths near 310 bp and were different from Shepherdia canadensis (L.) Nutt. and Sh. argentea (Pusch.) Nutt. samples (260 bp and 215 bp, respectively). The PCR products were cloned and sequenced. An analysis of the sequences revealed that intraspecific levels of NTS identity are high (approximately 95–96%) and similar in the Elaeagnus L. species. In Sh. argentea, this level was slightly lower due to the differences in the poly-T region. Moreover, the intergeneric and intervarietal NTS identity levels were studied and compared. Significant differences between species (except E. multiflora Thunb. and E. umbellata Thunb.) and genera were found. Herein, a range of the NTS features is discussed. This study is another step in the investigation of the molecular evolution of Elaeagnaceae and may be useful for the development of species-specific DNA markers in this family.

The Development of Populus alba L. and Populus tremula L. Species Specific Molecular Markers Based on 5S rDNA Non-Transcribed Spacer Polymorphism

The Populus L. genus includes tree species that are botanically grouped into several sections. This species successfully hybridizes both in the same section and among other sections. Poplar hybridization widely occurs in nature and in variety breeding. Therefore, the development of poplar species’ specific molecular markers is very important. The effective markers for trees of the Aigeiros Duby section have recently been developed using the polymorphism of 5S rDNA non-transcribed spacers (NTSs). In this article, 5S rDNA NTS-based markers were designed for several species of the Leuce Duby section. The alb9 marker amplifies one fragment with the DNA matrix of P. alba and P. × canescens (natural hybrid P. alba × P. tremula). The alb2 marker works the same way, except for the case with Populus bolleana. In this case, the amplification of three fragments was observed. The tremu1 marker amplification was detected with the DNA matrix of P. tremula and P. × canescens. Thus, the developed markers may be applied as a useful tool for P. alba, P. tremula, P. × canescens, and P. bolleana identification in various areas of plant science such as botany, dendrology, genetics of populations, variety breeding, etc.

Development of the St/J and V Genome Specific Molecular Marker Based on 5s rDNA Polymorphism in Thinopyrum bessarabicum, Pseudoroegneria spicata, and Dasypyrum villosum

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.

Molecular genetic features of 5S rDNA nontranscribed spacer in Hippophae rhamnoides L.

Amplification of sea buckthorn Hippophae rhamnoides L. 5S rDNA nontranscribed spacer with coding border anneal primers showed existence of a single fragment. The fragment was cloned and sequenced. It was shown that length of the Hippophae rhamnoides L. 5S rDNA nontranscribed spacer is 807 bp. Analysis of the sequence allowed to detect a high homology with early described microsatellite locuses of Hippophae rhamnoides L., russian olive Elaeagnus angustifolia L., and Calligonum mongolicum Turcz. that include a (GA)9 motif. These results may be useful to study a ribosomal RNA gene organization.

Molecular diversity of Enteromorpha from the coast of Yantai: a dual-marker assessment

We collected nine Enteromorpha specimens from the coast of Yantai and evaluated their diversity based on analyses of their ITS (internal transcribed spacer) and 5S rDNA NTS (non-transcribed spacer) sequences. The ITS sequences showed slight nucleotide divergences between Enteromorpha linza and Enteromorpha prolifera. In contrast, multiple highly variable regions were found in the ITS region of Enteromorpha flexuosa. In general, there were more variable sites in the NTS region than in the ITS region in the three species. The variations in 5S rDNA NTS sequences indicated that the molecular diversity of Enteromorpha from the coast of Yantai is very high. However, a phylogenetic tree constructed using 5S rDNA NTS sequence data indicated that genetic differences were not directly related to geographical distribution.

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.

Chromosomal Localization of the 5S and 45S rDNA Sites and 5S rDNA Sequence Analysis in Nelumbo Species

The physical location of 45S and 5S rDNA sites by double-target fluorescence in situ hybridization (FISH) and 5S rDNA non-transcribed spacer (NTS) sequences was studied in five accessions of Nelumbo nucifera Gaertn. and one accession of N. nucifera subsp. lutea (Willd.). The chromosome number of all tested materials was 2n=16. The loci of 5S rDNA were close to the centromere regions of chromosome 3, and the loci of 45S rDNA were found on chromosomes 7 and 8 in all the six tested accessions, respectively. Similarly, 45S rDNAs were also located on chromosome 4 in four tested accessions. The 5S rRNA gene sequences were conserved and the NTS sequences were variable among the samples. N. nucifera subsp. lutea was the longest branch in the phylogenetic tree and clustered with N. nucifera cv. Liangzihu. N nucifera cv. Heilongjiang and N nucifera cv. Weishanhu showed a close relationship with each other. N nucifera cv. Dahe Lian was closer to N nucifera cv. Nihelu Lian than to other tested accessions. Analysis of the molecular karyotype and the 5S rRNA gene spacer sequence suggested the genetic diversity was limited within Nelumbo species and it seems suitable that American lotus was considered as the subspecies of N nucifera.

Dynamic distribution patterns of ribosomal DNA and chromosomal evolution in Paphiopedilum, a lady's slipper orchid

BackgroundPaphiopedilum is a horticulturally and ecologically important genus of ca. 80 species of lady's slipper orchids native to Southeast Asia. These plants have long been of interest regarding their chromosomal evolution, which involves a progressive aneuploid series based on either fission or fusion of centromeres. Chromosome number is positively correlated with genome size, so rearrangement processes must include either insertion or deletion of DNA segments. We have conducted Fluorescence In Situ Hybridization (FISH) studies using 5S and 25S ribosomal DNA (rDNA) probes to survey for rearrangements, duplications, and phylogenetically-correlated variation within Paphiopedilum. We further studied sequence variation of the non-transcribed spacers of 5S rDNA (5S-NTS) to examine their complex duplication history, including the possibility that concerted evolutionary forces may homogenize diversity.Results5S and 25S rDNA loci among Paphiopedilum species, representing all key phylogenetic lineages, exhibit a considerable diversity that correlates well with recognized evolutionary groups. 25S rDNA signals range from 2 (representing 1 locus) to 9, the latter representing hemizygosity. 5S loci display extensive structural variation, and show from 2 specific signals to many, both major and minor and highly dispersed. The dispersed signals mainly occur at centromeric and subtelomeric positions, which are hotspots for chromosomal breakpoints. Phylogenetic analysis of cloned 5S rDNA non-transcribed spacer (5S-NTS) sequences showed evidence for both ancient and recent post-speciation duplication events, as well as interlocus and intralocus diversity.ConclusionsPaphiopedilum species display many chromosomal rearrangements - for example, duplications, translocations, and inversions - but only weak concerted evolutionary forces among highly duplicated 5S arrays, which suggests that double-strand break repair processes are dynamic and ongoing. These results make the genus a model system for the study of complex chromosomal evolution in plants.

Molecular variability in Brycon cf. pesu Muller and Troschel, 1845 (Characiformes: Characidae) from the Araguaia-Tocantins Basin

Brycon pesu is a small-sized fish distributed throughout the Amazon and Orinoco Basins and other coastal basins of northeastern South America. Brycon cf. pesu specimens from the Araguaia-Tocantins Basin are currently separated into two morphotypes, Brycon sp1 and Brycon sp2, owing to different coloration of their anal fin. Brycon sp2 has a reddish margin stripe on the anal fin which morphologically distinguishes it from Brycon sp1. In the present research, nuclear and mitochondrial markers were used to test the hypothesis that the Brycon sp1 and Brycon sp2 morphotypes are distinct species. Specimens from the two morphotypes were collected from the Lajeado Hydroelectric Plant and the Palmas River in the Araguaia-Tocantins Basin. Thirty-five loci obtained by the amplification of five inter-simple sequence repeat primers were analyzed but no species-specific bands were detected. Electrophoretic profiles obtained from 5S rDNA non-transcribed spacer amplification failed to show any differentiation in morphotypes. These results were corroborated by nucleotide sequence analysis of the mtDNA control region, in which 24 polymorphic nucleotide sites, representing a polymorphism rate of only 5%, were detected. The low rates of polymorphism detected by inter-simple sequence repeat, non-transcribed spacer and mtDNA D-loop markers strongly reject the hypothesis that the two morphotypes Brycon sp1 and Brycon sp2 represent distinct species within Brycon cf. pesu. Further studies are needed to obtain conclusive data on the notion that the coloration of the anal fin is an intraspecific polymorphism, possibly related to environmental factors.

A genetic diversity study of endangered Psiadia species endemic from Mauritius Island using PCR markers

The genus Psiadia Jacq. represents the most important indigenous genus, by the number of species present, in the Mascarene archipelago (Mauritius, Reunion, Rodrigues), and is a typical example of adaptive radiation in oceanic islands. The Mauritius species are used in traditional pharmacopoeia for their expectorant properties, and most of them are heavily threatened. Molecular genetic relationships between representatives of eight endangered endemic Psiadia species from Mauritius, conserved in Le Mondrain Reserve, and P. dentata (Cass.) DC, endemic from Reunion island, were studied. The absence of length variations of the 5s rDNA non-transcribed spacer demonstrated the recent common origin of all the species surveyed. RAPD analysis revealed a relatively high intra-specific variability in accordance with the outcrossing mode of reproduction of Psiadia species. Moreover, RAPD analysis showed the existence of four major phenetic groups: (A) P. arguta (Pers.) Voigt, P. dentata, (B) P. penninervia D. C., P. terebinthina A.J. Scott, P. lithospermifolia (Lam.) Cordem, (C) P. viscosa (Lam.) A.J. Scott, P. canescens A.J. Scott, P. cataractae A.J. Scott, and (D) P. pollicina A.J. Scott. These groups were consistent with the chemical composition of the essential oils of the species as well as with their floral characteristics, based on literature. A molecular germplasm database for Psiadia species was established, which will allow further characterisation of new samples being introduced in Le Mondrain Reserve for conservation purpose.

Length and sequence heterogeneity in 5S rDNA of Populus deltoides.

The 5S rRNA genes and their associated non-transcribed spacer (NTS) regions are present as repeat units arranged in tandem arrays in plant genomes. Length heterogeneity in 5S rDNA repeats was previously identified in Populus deltoides and was also observed in the present study. Primers were designed to amplify the 5S rDNA NTS variants from the P. deltoides genome. The PCR-amplified products from the two accessions of P. deltoides (G3 and G48) suggested the presence of length heterogeneity of 5S rDNA units within and among accessions, and the size of the spacers ranged from 385 to 434 bp. Sequence analysis of the non-transcribed spacer (NTS) revealed two distinct classes of 5S rDNA within both accessions: class 1, which contained GAA trinucleotide microsatellite repeats, and class 2, which lacked the repeats. The class 1 spacer shows length variation owing to the microsatellite, with two clones exhibiting 10 GAA repeat units and one clone exhibiting 16 such repeat units. However, distance analysis shows that class 1 spacer sequences are highly similar inter se, yielding nucleotide diversity (pi) estimates that are less than 0.15% of those obtained for class 2 spacers (pi = 0.0183 vs. 0.1433, respectively). The presence of microsatellite in the NTS region leading to variation in spacer length is reported and discussed for the first time in P. deltoides.

Evolution of 5S rDNA unit arrays in the plant genus Nicotiana (Solanaceae).

Nicotiana tabacum (tobacco, Solanaceae) has two 5S ribosomal DNA (rDNA) families, one of unit length approximately 646 bp and the other -430 bp, that differ in the length of the 5S rDNA non-transcribed spacer (NTS). The long 5S rDNA family, found on the T genome of tobacco and in Nicotiana tomentosiformis, contains a GC-rich subregion that is absent in the short family. We designed primers for this subregion and generated a probe that we used against a range of Nicotiana and related Solanaceous species. We demonstrated the presence of the GC-rich subregion in a range of Nicotiana species, but it was absent in Nicotiana sylvestris, Nicotiana longiflora, and two closely related genera, Petunia and Solanum. These data suggest that this subregion of the NTS is likely to have evolved with the genus Nicotiana. The absence of the subregion in N. sylvestris and N. longiflora is likely to have arisen by a deletion event in the evolution of section alatae. We demonstrate patterns of evolution in the 5S rDNA unit cluster in relation to a phylogenetic reconstruction of species relationships in section tomentosae. Nicotiana glutinosa diverged early from the section and contains a 5S rDNA family based on a 550-bp unit. After this divergence, 430- and 650-bp rDNA unit families evolved. The 650-bp family is found in all species of tomentosae (except N. glutinosa) and in tobacco. The 430-bp family within tomentosae includes the GC-rich subregion and is thus unrelated to the 430-bp family in N. sylvestris. Nicotiana setchellii is unusual in that it has three 5S rDNA loci, including one locus that is exceptionally large. This species, unique to tomentosae, has a very abundant 900-bp unit family. It is possible that this 900-bp family occurs on this one large locus. In N. tomentosa and N. kawakamii, the 650-bp family is predominant, whereas N. tomentosiformis and N. otophora have only the 650-bp family. There is no clear relationship between the number of 5S families and the number of 5S rDNA loci. Certainly the replacement of 5S rDNA units, perhaps by gene conversion, has occurred repeatedly in the evolution of genus Nicotiana.

Molecular organization of 5S rDNA in fishes of the genusBrycon

There are few reports on the genomic organization of 5S rDNA in fish species. To characterize the 5S rDNA nucleotide sequence and chromosomal localization in the Neotropical fishes of the genus Brycon, 5S rDNA copies from seven species were generated by PCR. The nucleotide sequences of the coding region (5S rRNA gene) and the nontranscribed spacer (NTS) were determined, revealing that the 5S rRNA genes were highly conserved, while the NTSs were widely variable among the species analyzed. Moreover, two classes of NTS were detected in each species, characterized by base substitutions and insertions–deletions. Using fluorescence in situ hybridization (FISH), two 5S rDNA chromosome loci that could be related to the two 5S rDNA NTS classes were observed in at least one of the species studied. 5S rDNA sequencing and chromosomal localization permitted the characterization of Brycon spp. and suggest a higher similarity among some of them. The data obtained indicate that the 5S rDNA can be an useful genetic marker for species identification and evolutionary studies.Key words: Brycon, FISH, nontranscribed spacer, nucleotide sequence, 5S rDNA.

Molecular organization of 5S rDNA in fishes of the genus <i>Brycon</i>

There are few reports on the genomic organization of 5S rDNA in fish species. To characterize the 5S rDNA nucleotide sequence and chromosomal localization in the Neotropical fishes of the genus Brycon, 5S rDNA copies from seven species were generated by PCR. The nucleotide sequences of the coding region (5S rRNA gene) and the nontranscribed spacer (NTS) were determined, revealing that the 5S rRNA genes were highly conserved, while the NTSs were widely variable among the species analyzed. Moreover, two classes of NTS were detected in each species, characterized by base substitutions and insertions-deletions. Using fluorescence in situ hybridization (FISH), two 5S rDNA chromosome loci that could be related to the two 5S rDNA NTS classes were observed in at least one of the species studied. 5S rDNA sequencing and chromosomal localization permitted the characterization of Brycon spp. and suggest a higher similarity among some of them. The data obtained indicate that the 5S rDNA can be an useful genetic marker for species identification and evolutionary studies.

The molecular diversity of the 5S rRNA gene in barley (Hordeum vulgare).

The 5S rRNA genes from several accessions of cultivated barley, Hordeum vulgare L., were amplified by the polymerase chain reaction, cloned, and sequenced. Analysis of the aligned sequences, followed by principal coordinate analysis, support the recognition of at least two distinct classes of 5S rDNA genes. The short repeat class corresponds to the 300-bp tandem repeat defined by E.V. Ananiev as containing several TAG repeating units. The long repeat class contains long tandem repeats and lacks the TAG repeating unit. Sequences in each class can be further subdivided, with the long repeat class containing two groups and the short repeat class containing two and possibly three groups. These results suggest that in cultivated barley the sequence diversity found within the 5S rDNA nontranscribed spacer region may be encoded by three or more loci and may be useful for phylogenetic analyses provided that orthology can be established.