External Transcribed Spacer Research Articles

Comparative analysis and classification of highly divergent mouse rDNA units based on their intergenic spacer (IGS) variability.

Ribosomal DNA (rDNA) repeat units are organized into tandem clusters in eukaryotic cells. In mice, these clusters are located on at least eight chromosomes and show extensive variation in the number of repeats between mouse genomes. To analyze intra- and inter-genomic variation of mouse rDNA repeats, we selectively isolated 25 individual rDNA units using Transformation-Associated Recombination (TAR) cloning. Long-read sequencing and subsequent comparative sequence analysis revealed that each full-length unit comprises an intergenic spacer (IGS) and a ∼13.4 kb long transcribed region encoding the three rRNAs, but with substantial variability in rDNA unit size, ranging from ∼35to ∼46 kb. Within the transcribed regions of rDNA units, we found 209 variants, 70 of which are in external transcribed spacers (ETSs); but the rDNA size differences are driven primarily by IGS size heterogeneity, due to indels containing repetitive elements and some functional signals such as enhancers. Further evolutionary analysis categorized rDNA units into distinct clusters with characteristic IGS lengths; numbers of enhancers; and presence/absence of two common SNPs in promoter regions, one of which is located within promoter (p)RNA and may influence pRNA folding stability. These characteristic features of IGSs also correlated significantly with 5'ETS variant patterns described previously and associated with differential expression of rDNA units. Our results suggest that variant rDNA units are differentially regulated and open a route to investigate the role of rDNA variation on nucleolar formation and possible associations with pathology.

Nuclear DNA-based phylogenetic analysis of Neocinnamomum species

AbstractNeocinnamomum plants are evergreen shrubs or small trees belonging to the Neocinnamomeae tribe of the Lauraceae family. Their seeds are rich in fatty acids, and their leaves are often used in traditional Chinese medicine. Presently, only a few studies have been performed on Neocinnamomum plants; therefore, the genome-based phylogeny among Neocinnamomum species has not been determined, which limits the germplasm innovation of this genus. In this study, by the Illumina (next-generation sequencing) and third-generation sequencing technologies, the whole genomes of seven Neocinnamomum species samples were sequenced, their nuclear DNA (nrDNA) sequences were assembled and characterized, and their phylogeny was reconstructed. The results revealed four hypervariable regions (i.e. transcribed spacer regions) in the nrDNA sequences, among which the highest degree of variation was observed in the external transcribed spacer (ETS) region localized behind the 26S gene. A total of 27 insertions/deletions and 184 single-nucleotide polymorphisms, both localized mainly in the ETS and internal transcribed spacer regions, were identified. Phylogenetic trees were constructed based on the nrDNA sequences using the maximum likelihood (ML) and Bayesian inference (BI) methods with Caryodaphnopsis henryi as the outgroup. The ML tree divided the seven Neocinnamomum species into four clades. Clade I consisted of Neocinnamomum caudatum var. macrocarpum and Neocinnamomum caudatum, clade II included Neocinnamomum delavayi and Neocinnamomum mekongense, clade III included Neocinnamomum fargesii and a branch species of N. delavayi and clade IV included Neocinnamomum lecomtei, constituting a monophyletic and basal group. The BI tree shared the same topological structure as the ML tree, and all the support values of the BI tree were one except for that of one Neocinnamomum species (0.98). The results of this study provide new evidence regarding the phylogenetic evolution of the Neocinnamomum plants.

45S rDNA Diversity In Natura as One Step towards Ribosomal Heterogeneity in Arabidopsis thaliana.

The keystone of ribosome biogenesis is the transcription of 45S rDNA. The Arabidopsis thaliana genome contains hundreds of 45S rDNA units; however, they are not all transcribed. Notably, 45S rDNA units contain insertions/deletions revealing the existence of heterogeneous rRNA genes and, likely, heterogeneous ribosomes for rRNAs. In order to obtain an overall picture of 45S rDNA diversity sustaining the synthesis of rRNAs and, subsequently, of ribosomes in natura, we took advantage of 320 new occurrences of Arabidopsis thaliana as a metapopulation named At66, sampled from 0 to 1900 m of altitude in the eastern Pyrenees in France. We found that the 45S rDNA copy number is very dynamic in natura and identified new genotypes for both 5' and 3' External Transcribed Spacers (ETS). Interestingly, the highest 5'ETS genotype diversity is found in altitude while the highest 3'ETS genotype diversity is found at sea level. Structural analysis of 45S rDNA also shows conservation in natura of specific 5'ETS and 3'ETS sequences/features required to control rDNA expression and the processing of rRNAs. In conclusion, At66 is a worthwhile natural laboratory, and unraveled 45S rDNA diversity represents an interesting starting material to select subsets for rDNA transcription and alter the rRNA composition of ribosomes both intra- and inter-site.

Ilex danxiaensis (Aquifoliaceae), a Distinct New Tree Species Endemic to Danxia Mountain in Guangdong Province, China, Based on Molecular and Morphological Evidence

Many holly species are dominant species in evergreen broad-leaved forests, and they play an important role in the formation and maintenance of forest ecosystems in tropical and subtropical regions. Here, a new holly species (Ilex danxiaensis K. W. Xu & Q. Fan) of Aquifoliaceae was discovered from Guangdong, China, when conducting biodiversity investigations in the Danxia landform. We inferred the phylogenetic position of the new species based on sequences from three nuclear markers, namely internal transcribed spacer (ITS), external transcribed spacer (ETS), and chloroplast glutamine synthetase gene (nepGS). The results demonstrated a close relationship among the new species and members of I. sect. Lioprinos. The inferred phylogenetic position of the new species is also supported by morphological evidence that all of these species are characterized by having axillary cymes inflorescences and abaxially 1-sulcate pyrenes. However, the new species can be distinguished from others by having puberulent branchlets, petioles, peduncles, and pedicel; (1–)2–3-flowered cymes of male inflorescences; 1-flowered cymes of female inflorescences; ciliate sepals, 4-merous flowers, pink or white female flowers; and 4 pyrenes.

Nucleolar URB1 ensures 3' ETS rRNA removal to prevent exosome surveillance.

The nucleolus is the most prominent membraneless condensate in the nucleus. It comprises hundreds of proteins with distinct roles in the rapid transcription of ribosomal RNA (rRNA) and efficient processing within units comprising a fibrillar centre and a dense fibrillar component and ribosome assembly in a granular component1. The precise localization of most nucleolar proteins and whether their specific localization contributes to the radial flux of pre-rRNA processing have remained unknown owing to insufficient resolution in imaging studies2-5. Therefore, how these nucleolar proteins are functionally coordinated with stepwise pre-rRNA processing requires further investigation. Here we screened 200 candidate nucleolar proteins using high-resolution live-cell microscopy and identified 12 proteins that are enriched towards the periphery of the dense fibrillar component (PDFC). Among these proteins, unhealthy ribosome biogenesis 1 (URB1) is a static, nucleolar protein that ensures 3' end pre-rRNA anchoring and folding for U8 small nucleolar RNA recognition and the subsequent removal of the 3' external transcribed spacer (ETS) at the dense fibrillar component-PDFC boundary. URB1 depletion leads to a disrupted PDFC, uncontrolled pre-rRNA movement, altered pre-rRNA conformation and retention of the 3' ETS. These aberrant 3' ETS-attached pre-rRNA intermediates activate exosome-dependent nucleolar surveillance, resulting in decreased 28S rRNA production, head malformations in zebrafish and delayed embryonic development in mice. This study provides insight into functional sub-nucleolar organization and identifies a physiologically essential step in rRNA maturation that requires the static protein URB1 in the phase-separated nucleolus.

Time to update the sectional classification ofIlex(Aquifoliaceae): New insights fromIlexphylogeny, morphology, and distribution

AbstractResolving the infrageneric classification of species‐rich genera has been challenging in plant taxonomy.IlexL. is a subcosmopolitan genus with over 600 species of dioecious trees and shrubs. Many classification systems based on morphological data have been proposed during the past 250 years. However, these systems (such as Loesener's and Galle's systems) may not truly reflectIlex's evolutionary trajectories because most of those system's infrageneric hierarchies are not monophyletic. In this study, we reconstructed a phylogeny ofIlexL. comprising 15 moderately to highly supported clades using rigorously identified samples (202 species) and closely authenticated gene sequences of three nuclear genes [internal transcribed spacer (ITS), external transcribed spacer (ETS), andnepGS]. The newly generated phylogenetic tree resembles essentially that of the nuclear tree of Manen et al., but shows conspicuous topological differences with the phylogeny of Yao et al. Closely scrutinizing morphological variation and distributional patterns of 202 species, this study found that most lineages ofIlexidentified herein are well defined by a particular trait or a combination of morphological and distributional traits, displaying phylogeny–morphology–distribution conformity that has seldom been uncovered in previous studies. Given the general phylogeny–morphology–distribution conformity revealed in this genus, we put forward an updated sectional classification system forIlexthat temporarily contains 14 sections. The new classification will provide a robust framework for studying the evolution and diversification of this ecologically and economically important genus.

Dated Phylogeny of Astragalus Section Stereothrix (Fabaceae) and Allied Taxa in the Hypoglottis Clade.

The Astragalus subgenus Hypoglottis Bunge, which consists of several sections, is one of the taxonomically most complicated groups in the genus. The Astragalus section Stereothrix Bunge belongs to this subgenus and is a significant element of the Irano-Turanian floristic region. A molecular phylogenetic analysis of this section and its closely related taxa using nuclear ribosomal DNA internal transcribed spacers (ITS) and external transcribed spacer (ETS) regions as well as plastid matK sequences were conducted. Parsimony analyses and Bayesian phylogenetic inference revealed that the section is not monophyletic in its current form, as some taxa belonging to closely related sections such as Hypoglottidei DC. and the Malacothrix Bunge group within the sect. Stereothrix render it paraphyletic. Moreover, species groups belonging to sect. Stereothrix are placed in different clades within the phylogenetic tree of subgenus Hypoglottis, which indicates polyphyly, i.e., multiple independent origins of taxa placed in the sect. Stereothrix. Molecular dating of the group estimated an age of 3.62 (1.73-5.62) My for this assemblage with the major diversification events happening during the last 2 My. Many species groups separated only within the last 0.5 to 1 My. Based on morphological and molecular data, we discuss the phylogenetic relationships of the groups and synonymy of species. In addition, the included taxa of sect. Hypoglottidei are not monophyletic and include species belonging to sects. Hololeuce, Koelziana, Malacothrix, Onobrychoideae, and Ornithodpodium group within the sect. Stereothrix taxa. We conclude that only an analysis including all groups and nearly all species of the sections within the Hypoglottis clade can finally result in an new evolutionary-based system for these taxa.

Upon heat stress processing of ribosomal RNA precursors into mature rRNAs is compromised after cleavage at primary P site in Arabidopsis thaliana

ABSTRACT Transcription and processing of 45S rRNAs in the nucleolus are keystones of ribosome biogenesis. While these processes are severely impacted by stress conditions in multiple species, primarily upon heat exposure, we lack information about the molecular mechanisms allowing sessile organisms without a temperature-control system, like plants, to cope with such circumstances. We show that heat stress disturbs nucleolar structure, inhibits pre-rRNA processing and provokes imbalanced ribosome profiles in Arabidopsis thaliana plants. Notably, the accuracy of transcription initiation and cleavage at the primary P site in the 5’ETS (5’ External Transcribed Spacer) are not affected but the levels of primary 45S and 35S transcripts are, respectively, increased and reduced. In contrast, precursors of 18S, 5.8S and 25S RNAs are rapidly undetectable upon heat stress. Remarkably, nucleolar structure, pre-rRNAs from major ITS1 processing pathway and ribosome profiles are restored after returning to optimal conditions, shedding light on the extreme plasticity of nucleolar functions in plant cells. Further genetic and molecular analysis to identify molecular clues implicated in these nucleolar responses indicate that cleavage rate at P site and nucleolin protein expression can act as a checkpoint control towards a productive pre-rRNA processing pathway.

Mouse Oocytes, A Complex Single Cell Transcriptome.

Germinal vesicle (GV) stage is a critical transition point from growth to maturation in mammalian oocyte development. During the following meiotic maturation, active RNA degradation and absence of transcription significantly reprofile the oocyte transcriptome to determine oocyte quality. Oocyte RNA-seq has revealed transcriptome differences between two defined phases of GV stage, namely non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) phases. In addition, oocyte RNA-seq has identified a variety of dysregulated genes upon genetic mutation or environmental perturbation. Historically, due to the low amount of RNA per oocyte, a few (20–200) oocytes were needed for a regular library construction in bulk RNA-seq. In recent years, development of single cell sequencing allows detailing the transcriptome of individual oocytes. Here in this study, different RNA-seq datasets from single and bulk of mouse oocytes are compared, and single oocyte RNA-seq (soRNA-seq) shows higher reproducibility. In addition, soRNA-seq better illustrates developmental progression of GV oocytes, revealing more complex gene changes than traditional views. Specially, an elevated level of ribosomal RNA 5′-ETS (5′ external transcribed spacer) has been shown to highly correlate with SN property. This study further demonstrates that UMI (unique molecular identifiers) based and other deduplication methods are limited in their ability to improve the precision of the soRNA-seq datasets. Finally, this study proposes that external spike-in molecules are useful for normalizing samples of different transcriptome sizes. A list of stable genes has been identified during oocyte maturation that are comparable to external spike-in molecules. These findings highlight the advantage of soRNA-seq, and have established ways for better clustering and cross-stage normalization, which can provide more insight into the biological features of oocyte maturation.

Comparative Analysis of the nrDNA Repeat Unit of Manila Clam Ruditapes philippinarum and Quahog Mercenaria mercenaria

Ruditapes philippinarum and Mercenaria mercenaria are economically important bivalve species. The complete ribosomal DNA (rDNA) unit sequences of R. philippinarum and M. mercenaria, with as-sembled rDNA unit lengths of 12,910 and 12,100 bp, respectively, were obtained in this study for the first time. The rDNA unit structural organisation was similar to that in other eukaryotes, in-cluding the following elements in order: 18S rRNA-internal transcribed spacer 1 (ITS1); 5.8S rRNA-ITS2-28S rRNA-intergenic spacer (IGS) (3′ external transcribed spacer (ETS); non-transcribed spacer (NTS)-5′ ETS). The genetic differences between R. philippinarum and M. mercenaria were mainly attributable to non-coding regions (ITS1, ITS2 and IGS), especially the IGS region. The boundaries of putative 3′ ETS, NTS and 5′ ETS were confirmed. Seven and three sub-repeat fragments were found in R. philippinarum and M. mercenaria, respectively. These frag-ments ranged from 4 to 154 bp in length, and were located at the NTS and 5′ ETS regions. Five and six cytosine–guanine (CpG) islands were detected in R. philippinarum and M. mercenaria, respec-tively, and these covered 85.58% and 79.29% of the entire IGS sequence, respectively. The phylo-genetic tree was constructed based on Veneridae ITS and 18S rRNA sequences using the maxi-mum likelihood (ML) method. The ML tree based on ITS revealed that species within the same genus clearly clustered together with relatively high supporting values, and all the genera were recovered as monophyletic. The phylogenetic analyses using 18S rRNA provided a weaker phy-logenetic signal than ITS.

Histone acetylation of 45S rDNA correlates with disrupted nucleolar organization during heat stress response in Zea mays L.

The role of the nucleolus in plant response to heat stress remains largely obscure. Our current efforts focused on exploring the underlying mechanism by which nucleolar disorganization is regulated in heat stressed-maize lines. Here, two maize lines, a heat-sensitive line, ZD958, and a heat-tolerant line, ZDH, were submitted to heat stress for investigating their association with the nucleolar disruption. Immunofluorescence staining showed that nucleolar disruption increased with prolonged treatment time. After heat treatment, a significant change in nucleolus organization was observed in the ZD958 line, but the ZDH line showed mild alteration. Moreover, actinomycin D (ActD)-induced nucleolus fission led to inhibition of maize growth under the normal condition. The ZD958 line exhibited a significant increase in the level of H3K9ac and H4K5ac of the 45S rDNA accompanied by a higher transcription of the 5'-external transcribed spacer (ETS) region, while the line ZDH showed a slight increase in histone acetylation levels and the transcriptional initiation at this site after heat treatment. To our knowledge, this is the first report providing a comparative insight between heat stress, rDNA histone modifications, and nucleolus disintegration in a heat-tolerant ZDH compared with a heat-sensitive line ZD958. Our investigation might assist maize breeders in obtaining heat-tolerant lines by targeting nucleoli using epigenetics.

Hoya pyrifolia (Apocynaceae), a new species from south-western Yunnan, China.

Hoya pyrifolia, a new species of Apocynaceae from Yunnan Province, China, is described and illustrated. Results from phylogenetic analyses, based on combined DNA fragments of the nuclear ribosomal external transcribed spacer (ETS), intergeneric transcribed spacer (ITS) and three plastid DNA fragments (matK, psbA-trnH and trnT-trnL), showed that the new species was nested within a clade, including Hoya species distributed in the subtropical foothills of the Himalayas and the Tibet-Sichuan Plateau. Morphologically, the new species can be distinguished from its close relatives by its pyriform and slightly pubescent leaves, as well as the 4-flowered inflorescences.

Chromosomal Locations of a Non-LTR Retrotransposon, Menolird18, in Cucumis melo and Cucumis sativus, and Its Implication on Genome Evolution of Cucumis Species

Mobile elements are major regulators of genome evolution through their effects on genome size and chromosome structure in higher organisms. Non-long terminal repeat (non-LTR) retrotransposons, one of the subclasses of transposons, are specifically inserted into repetitive DNA sequences. While studies on the insertion of non-LTR retrotransposons into ribosomal RNA genes and other repetitive DNA sequences have been reported in the animal kingdom, studies in the plant kingdom are limited. Here, using FISH, we confirmed that Menolird18, a member of LINE (long interspersed nuclear element) in non-LTR retrotransposons and found in Cucumis melo, was inserted into ITS and ETS (internal and external transcribed spacers) regions of 18S rDNA in melon and cucumber. Beside the 18S rDNA regions, Menolird18 was also detected in all centromeric regions of melon, while it was located at pericentromeric and sub-telomeric regions in cucumber. The fact that FISH signals of Menolird18 were found in centromeric and rDNA regions of mitotic chromosomes suggests that Menolird18 is a rDNA and centromere-specific non-LTR retrotransposon in melon. Our findings are the first report on a non-LTR retrotransposon that is highly conserved in 2 different plant species, melon and cucumber. The clear distinction of chromosomal localization of Menolird18 in melon and cucumber implies that it might have been involved in the evolutionary processes of the melon (C. melo) and cucumber (C. sativus) genomes.

NEW DISTRIBUTION REPORT ON THE ALIEN SPECIES ARTEMISIA VERLOTIORUM LAMOTTE (ASTERACEAE-ANTHEMIDEAE) FROM GILGIT-BALTISTAN REGION OF PAKISTAN

Artemisia verlotiorum Lamotte (Asteraceae), a species described from Europe but native to East Asia is an alien and/or invasive species grow naturally in many European regions, North and South Africa, South America, Australia, New Zealand and Western Asia. In continuance to our work on Northeastern Pakistani Artemisia, here we report the first local occurrence of verlotiorum Lamotte. from Gilgit-Baltistan region of Pakistan by investigating its geographical distribution and phylogenetic analysis. To date, we have observed the species in stony landscapes of Ghizer district of Gilgit-Baltistan. Phylogenetic analysis of Northeastern Pakistani A. verlotiorum with maximum likelihood approach using its internal transcribed spacer (ITS) and external transcribed spacer (ETS) of nuclear ribosomal DNA (nrDNA) sequences showed its resemblance with other Artemisia species reported from different parts of the world. This species needs to be included in the rare plant species list in the flora of Pakistan and immediate conservative approaches should be adopted to impede its extinction from the country.

Genetic Diversity and Evolutionary Relationships of Chinese Pepper Based on nrDNA Markers

Chinese pepper, referring to Zanthoxylum bungeanum Maxim. and Zanthoxylum armatum DC. species, is an important spice crop that has long attracted people’s interest due to its extensive application in Asian cuisine to improve taste. Numerous cultivars have been developed during the long history of domestication and cultivation. However, little to no information is available on the genetic diversity and evolutionary relationships of Chinese pepper cultivars and their historical diversification has not been clarified. Herein, we sequenced two nrDNA non-coding region markers, the external transcribed spacer (ETS) and the internal transcribed spacer 2 (ITS2), to assess genetic diversity and phylogenetic relationships among 39 cultivated and wild populations of Chinese pepper from eight provinces and to address the question of ancient demographic trends which were probably influenced by changing climate during evolutionary history. In total, 31 haplotypes were identified based on 101 polymorphism sites. Our results revealed relatively high level of genetic variation despite long-term cultivation of this crop. AMOVA revealed that genetic variation existed predominantly within provinces rather than among provinces. The genetic structure result based on haplotype network analysis largely reflected historical records, which suggested a Gansu origin for Chinese pepper and an ancient west-to-east spread of Chinese pepper circulating in China. We also provided evidence that changing Pleistocene climates had shaped the demographic trends of Chinese pepper. Taken together, our findings not only suggest that Chinese pepper is a dynamic genetic system that responds to evolutionary forces, but it also provides a fundamental genetic profile for the conservation and responsible exploitation of the extant germplasm of Chinese pepper and for improving the genetic basis for breeding the cultivars.

Molecular phylogeny of Artemisia (Asteraceae-Anthemideae) with emphasis on undescribed taxa from Gilgit-Baltistan (Pakistan) based on nrDNA (ITS and ETS) and cpDNA (psbA-trnH) sequences

Background – Gilgit-Baltistan, the Northeast region of Pakistan, is well known for its floristic diversity, including members of the genus Artemisia. Artemisia is a large, taxonomically complex genus including ~500 species of both herbs and shrubs. This study was conducted to determine the phylogenetic position of ten undescribed Artemisia taxa from northern Pakistan, using nrDNA internal transcribed spacer (ITS), external transcribed spacer (ETS) and cpDNA intergenic spacer (psbA-trnH) regions.Methods – The phylogenetic relationships of 28 taxa of Artemisia using separate and combined data sets of sequences of three markers (ITS, ETS and psbA-trnH) were analysed with maximum parsimony, maximum likelihood, and Bayesian approaches. Key results – The results resolve northeastern Pakistani Artemisia, which represent five morphologically defined subgenera, into ten major clades. Subgenera Artemisia and Absinthium are shown to be polyphyletic, while Dracunculus, Pacifica and Tridentatae appear monophyletic. All species of subgenus Seriphidium are retrieved in a single clade that also includes annual species from subgenus Artemisia. In the Flora of Pakistan, Seriphidium is described as a separate genus but in this study, Seriphidium fell within the genus Artemisia. In addition, on the basis of phylogenetic analysis, we present evidence that ten as-yet undescribed taxa are present in northeastern Pakistan based on newly recognized three groups (Groups I, II and III) of taxa within the genus Artemisia. One undescribed taxon from group I was placed within the subgenus Dracunculus clade and the remaining nine taxa from groups II and III were placed in the subgenus Absinthium clade. Morphological studies coupled with modern molecular techniques may lead to a new infrageneric classification of the genus Artemisia. It will also clarify and characterize the undescribed taxa reported in this study.

ETS and plastid sequence data indicate a spontaneous origin of Scandinavian betony, Betonica officinalis L.

Betony (Betonica officinalis L.) is one of the rarest and most spectacular plants in the Scandinavian flora. A long-term question has been whether it is spontaneous or introduced, or whether it comprises both spontaneous and introduced populations. This study aimed to answer this question by analyzing sequence data from the nuclear external transcribed spacer (ETS) region and three regions of the plastid genome, the trnT-trnL intergenic spacer (IGS) region, tRNA-Leu (trnL) intron, and the trnS-trnG IGS. Altogether 41 samples from 11 European countries were analyzed. A unique duplication in the trnT-trnL IGS was detected in material from Skåne (southern Sweden), the "Skåne-duplication." Populations with this duplication are united on a moderately supported branch in the phylogeny based on plastid sequences. A distinct heath genotype from Yorkshire was discovered in the phylogeny based on plastid sequences and in a comparative cultivation. Phylogeny based on ETS sequences does not support any Scandinavian group, whereas a principal coordinates analysis ordination based on variable ETS positions indicated a spontaneous origin for all Scandinavian populations, which comprise a genetically well-defined subgroup of the species, most closely related to other spontaneous populations from adjacent parts of continental parts of northern Europe. Seven possible naturally occurring localities remain in Scandinavia, five in central Skåne, southernmost Sweden, and two on the southwestern part of the Danish island of Lolland.

The ribosomal intergenic spacer (IGS) in the potato and tobacco cyst nematodes, Globodera pallida, G. rostochiensis and G. tabacum

The potato cyst nematodes Globodera pallida and G. rostochiensis (PCN), and tobacco cyst nematode (TCN), G. tabacum, are the most important parasitic nematodes of potato and tobacco worldwide. Ribosomal DNA provides useful molecular data for diagnostics, the study of polymorphisms and for evolutionary research in eukaryotic organisms including nematodes. Here we present data on the structure and organization of a rarely studied part of the intergenic spacer (IGS) region of the PCN and TCN genome of cyst nematodes. This region has shown potential for diagnostic purposes and population studies in other organisms including nematodes. In nematodes, the ribosomal RNA gene cluster comprises three genes: 5.8S, 18S and 28S rRNA, which are separated by spacer regions: the intergenic spacer (IGS), non-transcribed spacer (NTS), externally transcribed spacer (EST) and the internally transcribed spacer (ITS). The intergenic spacer (IGS) region consists of an external transcribed spacer (ETS) and a non-transcribed spacer (NTS) which is located between the 28S of one repeat and the 18S gene of the next repeat within the rRNA genes cluster. In this study, the first flanking portion of the IGS was amplified, cloned and sequenced from PCN and TCN. Primers were then designed to amplify the whole IGS sequence. PCR amplification of IGS from G. tabacum, G. pallida, and G. rostochiensis yielded respectively: a single amplicon of 3 kb, three amplicons sized 2.5, 2.6 and 2.9 kb, and two amplicons sized 2.8 and 2.9 kb. Results showed that Globodera spp. has more than one variant copy of the IGS, with both long and short repetitive DNA elements. An approximately 400 bp long region without any internal repetitive elements, were identified in a position between the two repetitive regions suggesting that there is a 5S gene in the IGS of these species.

Phylogeny of the Neotropical legume genera Zygia and Marmaroxylon and close relatives

AbstractThe Neotropical legume genera Zygia and Marmaroxylon have traditionally been considered closely related and are sometimes treated as congeners. They have been referred to the mimosoid tribe Ingeae based on their possession of flowers with basally fused stamen filaments. However, their systematic status and position have not been analysed in a phylogenetic context. This study provides the first molecular phylogeny of Zygia and Marmaroxylon and closely related mimosoid legume genera, particularly from the tribe Ingeae. DNA sequence data were derived from the nuclear external transcribed spacer (ETS) and the internal transcribed spacer (ITS), and from the chloroplast psbA‐trnH and trnL‐trnF regions, and were analysed using Bayesian inference. In all, 29 species of Zygia and 7 species of Marmaroxylon were included in the study along with representative species from the closely related genera Abarema, Acacia s.str., Archidendropsis, Calliandra, Chloroleucon, Cojoba, Faidherbia, Havardia, Hydrochorea, Inga, Leucochloron, Macrosamanea, Pithecellobium, Pseudosamanea, Samanea, Senegalia, Vachellia and Zapoteca. The results show that neither Zygia nor Marmaroxylon are monophyletic as presently circumscribed. Furthermore, these two genera are not monophyletic together. None of the nine presently recognized sections of Zygia that contain more than one species are shown to be monophyletic. Two of the monospecific sections of Zygia, sect. Ingopsis (Z. inundata) and sect. Pseudocojoba (Z. sabatieri), were found in a clade together with species of Inga. Marmaroxylon ocumarense and M. magdalenae, previously treated as synonyms (as Zygia), are not conspecific, and are found in a clade with Macrosamanea and in a clade with species of Abarema and Hydrochorea, respectively. Our results also show the Inga alliance to be non‐monophyletic. In correspondence with the results presented, all species referred to Marmaroxylon, except for M. ocumarense and M. magdalenae, are here included in Zygia, and Z. inundata is treated in Inga, in which genus it was first described.

Genome skimming provides well resolved plastid and nuclear phylogenies, showing patterns of deep reticulate evolution in the tropical carnivorous plant genus Nepenthes (Caryophyllales)

Nepenthes is a genus of carnivorous plants consisting of ~160 species that are distributed in the paleotropics. Molecular systematics has so far not been able to resolve evolutionary relationships of most species because of the limited genetic divergence in previous studies. In the present study, we used a genome-skimming approach to infer phylogenetic relationships on the basis of 81 plastid genes and the highly repetitive rRNA (external transcribed spacer (ETS)–26S) for 39 accessions representing 34 species from eight sections. Maximum-likelihood analysis and Bayesian inference were performed separately for the nuclear and the plastid datasets. Divergence-time estimations were conducted on the basis of a relaxed molecular-clock model, using secondary calibration points. The phylogenetic analyses of the nuclear and plastid datasets yielded well resolved and supported phylogenies. Incongruences between the two datasets were detected, suggesting multiple hybridisation events or incomplete lineage sorting in the deeper and more recent evolutionary history of the genus. The inclusion of several known and suspected hybrids in the phylogenetic analysis provided insights into their parentage. Divergence-time estimations placed the crown diversification of Nepenthes in the early Miocene, c. 20 million years ago. This study showed that genome skimming provides well resolved nuclear and plastid phylogenies that provide valuable insights into the complex evolutionary relationships of Nepenthes.