5S rDNA Loci Research Articles

Comparative karyotype analysis provides cytogenetic evidence for the origin of sweetpotato.

The origin of hexaploid sweetpotato [Ipomoea batatas (L.) Lam.] remains controversial. Comparative karyotype analysis is particularly useful in determining species relationships and the origin of polyploid species. In previous study, we developed a set of oligo probes and identified all chromosomes of Ipomoea nil, a model diploid Ipomoea species. Here, we found that this set of oligo probes could be used to identify all chromosomes of sweetpotato and its wild relatives with different ploidy. Karyotypes based on individually identified chromosomes were established and the number and position of 5S and 35S rDNA loci were determined for these Ipomoea species. Comparison of their karyotypes revealed distinct variations in the karyotypic parameters. Karyological relationships among these species were revealed by principal coordinate analysis (PCoA) based on six quantitative parameters (x, 2n, TCL, MCA, CVCL and CVCI). These results show that I. trifida is the most closely related diploid species to sweetpotato, and other diploid species could be excluded from consideration as its possible diploid ancestor. In addition, our study also provides cytogenetic evidence for the segmental allopolyploid hypothesis of sweetpotato origin.

Karyotype diversity of Polybia (gr. occidentalis) species complex (Hymenoptera: Vespidae): Taxonomic and evolutionary implications

AbstractPolybia is a common wasp genus found in most of South America. Polybia (gr. occidentalis) encompasses several species that are difficult to identify because of their similar morphologies. Our goal was to cytogenetically characterize three species belonging to Polybia (gr. occidentalis) and discuss the importance of cytogenetic data for integrative taxonomy. Polybia colonies were sampled from different regions of Brazil to perform classical and molecular cytogenetic analyses. Polybia paulista and Polybia sp. 1 showed 2n = 34 and 18S rDNA loci on two chromosomes, whereas Polybia sp. 2 showed 2n = 40 and 18S rDNA clusters on four chromosomes. The microsatellites GA(15), GAG(10), CAA(10), TTAGG(6), and TCAGG(6) showed similar distributions among the species, forming blocks in the euchromatic regions of the chromosomes, whereas CGG(10) did not yield any positive markings. In contrast, TAT(10) hybridized on the centromeric heterochromatin, showing differences in the number of marked chromosomes among the species. Therefore, it may potentially be a species‐specific cytotaxonomic marker in this group of wasps, but this feature needs further investigation. Fluorochromes evidenced that AT‐rich DAPI+ sequence distribution was coincident with heterochromatin, while the distribution of CG‐rich CMA3+ sequences was coincident with the 18S rDNA region. The present study revealed differences in diploid number, heterochromatin content, 18S rDNA sites, and microsatellite patterns between morphologically similar species, demonstrating the usefulness of cytotaxonomy in studying species complexes.

The histone chaperones ASF1 and HIRA are required for telomere length and 45S rDNA copy number homeostasis.

Genome stability is significantly influenced by the precise coordination of chromatin complexes that facilitate the loading and eviction of histones from chromatin during replication, transcription, and DNA repair processes. In this study, we investigate the role of the Arabidopsis H3 histone chaperones ANTI-SILENCING FUNCTION 1 (ASF1) and HISTONE REGULATOR A (HIRA) in the maintenance of telomeres and 45S rDNA loci, genomic sites that are particularly susceptible to changes in the chromatin structure. We find that both ASF1 and HIRA are essential for telomere length regulation, as telomeres are significantly shorter in asf1a1b and hira mutants. However, these shorter telomeres remain localized around the nucleolus and exhibit a comparable relative H3 occupancy to the wild type. In addition to regulating telomere length, ASF1 and HIRA contribute to silencing 45S rRNA genes and affect their copy number. Besides, ASF1 supports global heterochromatin maintenance. Our findings also indicate that ASF1 transiently binds to the TELOMERE REPEAT BINDING 1 protein and the N terminus of telomerase in vivo, suggesting a physical link between the ASF1 histone chaperone and the telomere maintenance machinery.

Unambiguous chromosome identification reveals the factors impacting irregular chromosome behaviors in allotriploid AAC Brassica.

The major irregular chromosome pairing and mis-segregation were detected during meiosis through unambiguous chromosome identification and found that allotriploid Brassica can undergo meiosis successfully and produce mostly viable aneuploid gametes. Triploids have played a crucial role in the evolution of species by forming polyploids and facilitating interploidy gene transfer. It is widely accepted that triploids cannot undergo meiosis normally and predominantly produce nonfunctional aneuploid gametes, which restricts their role in species evolution. In this study, we demonstrated that natural and synthetic allotriploid Brassica (AAC), produced by crossing natural and synthetic Brassica napus (AACC) with Brassica rapa (AA), exhibits basically normal chromosome pairing and segregation during meiosis. Homologous A chromosomes paired faithfully and generally segregated equally. Monosomic C chromosomes were largely retained as univalents and randomly entered daughter cells. The primary irregular meiotic behaviors included associations of homoeologs and 45S rDNA loci at diakinesis, as well as homoeologous chromosome replacement and premature sister chromatid separation at anaphase I. Preexisting homoeologous arrangements altered meiotic behaviors in both chromosome irregular pairing and mis-segregation by increasing the formation of A-genomic univalents and A-C bivalents, as well as premature sister chromatid separation and homologous chromosome nondisjunction. Meiotic behaviors depended significantly on the genetic background and heterozygous homoeologous rearrangement. AAC triploids mainly generated aneuploid gametes, most of which were viable. These results demonstrate that allotriploid Brassica containing an intact karyotype can proceed through meiosis successfully, broadening our current understanding of the inheritance and role in species evolution of allotriploid.

Cytogenetic analysis and visualization of genetic relationships in wild lilies.

Lilies are highly regarded for their ornamental appeal and striking flowers, which are of significant importance in horticulture. Understanding the genetic makeup of these plants is crucial for breeding and developing new cultivars. This study presents a comprehensive cytogenetic analysis of 45S and 5S rDNA loci in 34 wild Lilium species. To reveal the genetic relationships within the genus, advanced visualization methods, such as heatmaps and 3D network plots, were utilized. The results of this study identified both conserved and divergent genetic features, which offer insights into the evolutionary history and potential genetic compatibility of these species. Notably, the clustering of species based on rDNA locus patterns highlights the need for potential taxonomic re-evaluation and reveals candidates for cross-breeding. This integrated approach emphasizes the importance of combining cytogenetic data with traditional morphological classifications to refine our understanding of the Lilium species. Future research should expand the range of analyzed species, incorporate additional molecular markers to further elucidate genetic relationships, and support the development of resilient and diverse ornamental crops. The findings of this study provide a novel framework for genetic analysis of Lilium, offering valuable insights for both scientific understanding and practical breeding programs.

The Genome Organization of 5S rRNA Genes in the Model Organism Tribolium castaneum and Its Sibling Species Tribolium freemani.

5S ribosomal DNAs (rDNAs) are arranged in tandem and are often under-represented in genome assemblies. In the present study, we performed a global and in-depth analysis of the 5S rDNAs in the model insect Tribolium castaneum and its closely related species Tribolium freemani. To accomplish this goal, we used our recently published genome assemblies based on Nanopore and PacBio long-read sequencing. Although these closely related species share the 5S rRNA gene sequence with high homology, they show a different organization of the 5S rDNA locus. Analysis of 5S rDNA arrays in T. castaneum revealed a typical tandemly repeated organization characterized by repeat units consisting of the 121 bp long 5S rRNA gene and the 71 bp long nontranscribed spacer (NTS). In contrast, T. freemani showed a much more complex organization of 5S rDNA arrays characterized by two patterns. The first is based on the association of 5S rRNA gene with arrays of a satellite DNA, representing the NTS sequence of the 5S rDNA genes in T. freemani. The second, more complex type is characterized by a somewhat less frequent occurrence of the 5S rRNA gene and its association with longer satellite DNA arrays that are regularly interrupted by Jockey-like retrotransposons. This organization, in which the ribosomal gene is associated with two completely different repetitive elements such as satellite DNAs and retrotransposons, suggests that the 5S rRNA gene, regardless of its crucial function in the genome, could be a subject of extremely dynamic genomic rearrangements.

Uniparental silencing of 5S rRNA genes in plant allopolyploids - insights from Cardamine (Brassicaceae).

While the phenomenon of uniparental silencing of 35S rDNA in interspecific hybrids and allopolyploids is well documented, there is a notable absence of information regarding whether such silencing extends to the 5S RNA component of ribosomes. To address this gap in knowledge, we analyzed the 5S and 35S rDNA expression in Cardamine (Brassicaceae) allopolyploids, namely C. × insueta (2n = 3x = 24, genome composition RRA), C. flexuosa (2n = 4x = 32, AAHH), and C. scutata (2n = 4x = 32, PPAA) which share a common diploid ancestor (AA). We employed high-throughput sequencing of transcriptomes and genomes and phylogenetic analyses of 5S rRNA variants. The genomic organization of rDNA was further scrutinized through clustering and fluorescence in situ hybridization. In the C. × insueta allotriploid, we observed uniparental dominant expression of 5S and 35S rDNA loci. In the C. flexuosa and C. scutata allotetraploids, the expression pattern differed, with the 35S rDNA being expressed from the A subgenome, whereas the 5S rDNA was expressed from the partner subgenome. Both C. flexuosa and C. scutata but not C. × insueta showed copy and locus number changes. We conclude that in stabilized allopolyploids, transcription of ribosomal RNA components occurs from different subgenomes. This phenomenon appears to result in the formation of chimeric ribosomes comprising rRNA molecules derived from distinct parental origins. We speculate that the interplay of epigenetic silencing and rDNA rearrangements introduces an additional layer of variation in multimolecule ribosomal complexes, potentially contributing to the evolutionary success of allopolyploids.

Insight into chromatin compaction and spatial organization in rice interphase nuclei.

Chromatin organization and its interactions are essential for biological processes, such as DNA repair, transcription, and DNA replication. Detailed cytogenetics data on chromatin conformation, and the arrangement and mutual positioning of chromosome territories in interphase nuclei are still widely missing in plants. In this study, level of chromatin condensation in interphase nuclei of rice (Oryza sativa) and the distribution of chromosome territories (CTs) were analyzed. Super-resolution, stimulated emission depletion (STED) microscopy showed different levels of chromatin condensation in leaf and root interphase nuclei. 3D immuno-FISH experiments with painting probes specific to chromosomes 9 and 2 were conducted to investigate their spatial distribution in root and leaf nuclei. Six different configurations of chromosome territories, including their complete association, weak association, and complete separation, were observed in root meristematic nuclei, and four configurations were observed in leaf nuclei. The volume of CTs and frequency of their association varied between the tissue types. The frequency of association of CTs specific to chromosome 9, containing NOR region, is also affected by the activity of the 45S rDNA locus. Our data suggested that the arrangement of chromosomes in the nucleus is connected with the position and the size of the nucleolus.

Karyotype Description and Comparative Chromosomal Mapping of 5S rDNA in 42 Species.

This study was conducted to evaluate the 5S rDNA site number, position, and origin of signal pattern diversity in 42 plant species using fluorescence in situ hybridization. The species were selected based on the discovery of karyotype rearrangement, or because 5S rDNA had not yet been explored the species. The chromosome number varied from 14 to 160, and the chromosome length ranged from 0.63 to 6.88 μm, with 21 species having small chromosomes (<3 μm). The chromosome numbers of three species and the 5S rDNA loci of nineteen species are reported for the first time. Six 5S rDNA signal pattern types were identified. The 5S rDNA varied and was abundant in signal site numbers (2-18), positions (distal, proximal, outside of chromosome arms), and even in signal intensity. Variation in the numbers and locations of 5S rDNA was observed in 20 species, whereas an extensive stable number and location of 5S rDNA was found in 22 species. The potential origin of the signal pattern diversity was proposed and discussed. These data characterized the variability of 5S rDNA within the karyotypes of the 42 species that exhibited chromosomal rearrangements and provided anchor points for genetic physical maps.

Molecular cytogenetic characterization of 9 populations of four species in the genus Polygonatum (Asparagaceae).

To characterize the chromosomes of the four species of Polygonatum Miller, 1754, used in traditional Chinese medicine, P.cyrtonema Hua, 1892, P.kingianum Collett et Hemsley, 1890, P.odoratum (Miller, 1768) Druce, 1906, and P.sibiricum Redouté, 1811, and have an insight into the karyotype variation of the genus Polygonatum, fluorescence in situ hybridization (FISH) with 5S and 45S rDNA oligonucleotide probes was applied to analyze the karyotypes of 9 populations of the four species. Detailed molecular cytogenetic karyotypes of the 9 populations were established for the first time using the dataset of chromosome measurements and FISH signals of 5S and 45S rDNA. Four karyotype asymmetry indices, CVCI, CVCL, MCA and Stebbins' category, were measured to elucidate the asymmetry of the karyotypes and karyological relationships among species. Comparison of their karyotypes revealed distinct variations in the karyotypic parameters and rDNA patterns among and within species. The basic chromosome numbers detected were x = 9, 11 and 13 for P.cyrtonema, x = 15 for P.kingianum, x = 10 and 11 for P.odoratum, and x = 12 for P.sibiricum. The original basic chromosome numbers of the four species were inferred on the basis of the data of this study and previous reports. All the 9 karyotypes were of moderate asymmetry and composed of metacentric, submetacentric and subtelocentric chromosomes or consisted of two of these types of chromosomes. Seven populations have one locus of 5S rDNA and two loci of 45S rDNA, and two populations added one 5S or 45S locus. The karyological relationships among the four species revealed by comparison of rDNA patterns and PCoA based on x, 2n, TCL, CVCI, MCA and CVCL were basically accordant with the phylogenetic relationships revealed by molecular phylogenetic studies. The mechanisms of both intra- and inter-specific dysploidy in Polygonatum were discussed based on the data of this study and literature.

Karyological Study of Acanthocephalus lucii (Echinorhynchida): The Occurrence of B Chromosomes in Populations from PCB-Polluted Waters

In this study, we performed a cytogenetic analysis of Acanthocephalus lucii specimens from three sites with different levels of environmental pollution. Standard and fluorochrome staining (CMA3/DAPI), fluorescence in situ hybridization (FISH) with 18S rDNA and histone H3 probes, and silver impregnation were performed. Chromosome complements of 2n = 7/8 (male/female), n = 1m + 2sm + 1a (X), and CMA3-positive bands in all chromosomes were found in all three populations. FISH revealed one 18S rDNA locus on the X chromosome and one locus of H3 histone genes on the first chromosome pair. At the intraspecific level, the populations differed in the presence of supernumerary B chromosomes, which were found in all specimens from Zemplínska Šírava and in 89.4% of specimens from the Laborec River, but not at the reference site. The first two sites are considered to be water bodies with high toxin contamination. Based on this fact, we assume an increased frequency of chromosome breaks leading to the formation of DNA fragments that have the potential to form B chromosomes. The present results add to the very limited data on the organization of multigene families in the genome of Acanthocephala and suggest a possible causal link between water pollution and the occurrence of B chromosomes in fish parasites.

Evaluation of the paired-Cas9 nickase and RNA-guided FokI genome editing tools in precise integration of an anti-CD52 bicistronic monoclonal antibody expression construct at Chinese hamster ovary cells 18S rDNA locus

IntroductionThe aim of this study was to compare two CRISPR/Cas9-based orthogonal strategies, paired-Cas9 nickase (paired-Cas9n) and RNA-guided FokI (RFN), in targeting 18S rDNA locus in Chinese hamster ovary (CHO) cells and precisely integrating a bicistronic anti-CD52 monoclonal antibody (mAb) expression cassette into this locus. MethodsT7E1 and high-resolution melt (HRM) assays were used to compare the ability of mentioned systems in inducing double-strand break (DSB) at the target site. Moreover, 5′- and 3′-junction polymerase chain reactions (PCR) were used to verify the accuracy of the targeted integration of the mAb expression cassette into the 18S rDNA locus. Finally, anti-CD52 mAb gene copy number was measured and, its expression was analyzed using ELISA and western blot assays. ResultsOur results indicated that both paired-Cas9n and RFN induced DSB at the target site albeit RFN performance was slightly more efficient in HRM analysis. We also confirmed that the anti-CD52 mAb cassette was accurately integrated at the 18S rDNA locus and the mAb was expressed successfully in CHO cells. ConclusionTaken together, our findings elucidated that both paired-Cas9n and RFN genome editing tools are promising in targeting the 18S rDNA locus. Site specific integration of the bicistronic anti-CD52 mAb expression cassette at this locus in the CHO-K1 cells was obtained, using RFN. Moreover, proper expression of the anti-CD52 mAb at the 18S rDNA target site can be achieved using the bicistronic internal ribosome entry site (IRES)-based vector system.

Phaseolus vulgaris mutants reveal variation in the nuclear genome.

Phaseolus vulgaris L. (common bean) is an essential source of proteins in the human diet worldwide. Bean breeding programs to increase genetic diversity based on induced mutagenesis have a long tradition in Bulgaria. Common bean varieties with high productivity, wide environmental adaptability, good nutritional properties, and improved disease resistance have been successfully developed. In this study, we aimed to investigate selected nuclear genome features, such as the genome size, the number and chromosomal distribution of 5S and 35S rDNA loci by using the fluorescence in situ hybridization (FISH), as well as the level of DNA damage in some local Bulgarian accessions and mutants of P. vulgaris. Flow cytometry analyses revealed no significant differences in genome size between analyzed lines except for one of the analyzed mutants, M19. The value of genome size 2C DNA is about 1.37 pg2C -1 for all lines, whereas it is 1.42 pg2C-1 for M19. The chromosome number remains the same (2n=22) for all analyzed lines. Results of FISH analyses showed that the number of 5S rDNA was stable among accessions and mutant lines (four loci), while the number of 35S rDNA loci was shown as highly polymorphic, varying between ten and sixteen, and displaying differences in the size and location of 35S rDNA loci between analyzed genotypes. The cell cycle profile was different for the analyzed genotypes. The results revealed that wide variation in genome organization and size as well as DNA damage characterizes the analyzed genetic resources of the common bean.

Extrachromosomal DNA-mediated glyphosate resistance in Italian ryegrass.

An Italian ryegrass population from Arkansas, USA developed glyphosate resistance due to 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene amplification. The plants in this population with approximately 70 EPSPS copies were used in the present study for the physical mapping of amplified copies of EPSPS gene to determine the possible mechanism of EPSPS gene amplification conferring glyphosate resistance in Italian ryegrass. Fluorescence in situ hybridization (FISH) analysis of glyphosate resistant (GR) Italian ryegrass plants with approximately 70 EPSPS copies displayed EPSPS hybridization signals randomly on most of the metaphase chromosomes. Glyphosate susceptible (GS) Italian ryegrass plants with one EPSPS copy displayed single prominent EPSPS hybridization signal, which was co-localized with 5S rDNA locus along with few additional signals on the outside of chromosomes. Pulsed-field gel electrophoresis (PFGE) followed by DNA blot using EPSPS gene as a probe identified a prominent EPSPS hybridization around the 400 kb region in GR DNA samples, but not in GS DNA samples. We report the extrachromosomal DNA-mediated glyphosate resistance in Italian ryegrass. Physical mapping of amplified copies of EPSPS gene in Italian ryegrass by FISH gives us a clue that the amplified copies of EPSPS gene may be present in the extrachromosomal DNA elements. Further analysis by PFGE followed by DNA blotting revealed that the extrachromosomal DNA containing EPSPS is approximately 400 kb similar in size with that of eccDNA replicon in Amaranthus palmeri. © 2023 Society of Chemical Industry.

Ribosomal DNA Copy Number Variation is Coupled with DNA Methylation Changes at the 45S rDNA Locus

ABSTRACT The human ribosomal DNA (rDNA) copy number (CN) has been challenging to analyse, and its sequence has been excluded from reference genomes due to its highly repetitive nature. The 45S rDNA locus encodes essential components of the cell, nevertheless rDNA displays high inter-individual CN variation that could influence human health and disease. CN alterations in rDNA have been hypothesized as a possible factor in autism spectrum disorders (ASD) and were shown to be altered in Schizophrenia patients. We tested whether whole-genome bisulphite sequencing can be used to simultaneously quantify rDNA CN and measure DNA methylation at the 45S rDNA locus. Using this approach, we observed high inter-individual variation in rDNA CN, and limited intra-individual copy differences in several post-mortem tissues. Furthermore, we did not observe any significant alterations in rDNA CN or DNA methylation in Autism Spectrum Disorder (ASD) brains in 16 ASD vs 11 control samples. Similarly, no difference was detected when comparing neurons form 28 Schizophrenia (Scz) patients vs 25 controls or oligodendrocytes from 22 Scz samples vs 20 controls. However, our analysis revealed a strong positive correlation between CN and DNA methylation at the 45S rDNA locus in multiple tissues. This was observed in brain and confirmed in small intestine, adipose tissue, and gastric tissue. This should shed light on a possible dosage compensation mechanism that silences additional rDNA copies to ensure homoeostatic regulation of ribosome biogenesis.

Oligo-FISH of Populus simonii Pachytene Chromosomes Improves Karyotyping and Genome Assembly.

Poplar was one of the first woody species whose individual chromosomes could be identified using chromosome specific painting probes. Nevertheless, high-resolution karyotype construction remains a challenge. Here, we developed a karyotype based on the meiotic pachytene chromosome of Populus simonii which is a Chinese native species with many excellent traits. This karyotype was anchored by oligonucleotide (oligo)-based chromosome specific painting probes, a centromere-specific repeat (Ps34), ribosomal DNA, and telomeric DNA. We updated the known karyotype formula for P. simonii to 2n = 2x = 38 = 26m + 8st + 4t and the karyotype was 2C. The fluorescence in situ hybridization (FISH) results revealed some errors in the current P. simonii genome assembly. The 45S rDNA loci were located at the end of the short arm of chromosomes 8 and 14 by FISH. However, they were assembled on pseudochromosomes 8 and 15. In addition, the Ps34 loci were distributed in every centromere of the P. simonii chromosome in the FISH results, but they were only found to be present in pseudochromosomes 1, 3, 6, 10, 16, 17, 18, and 19. Our results reveal that pachytene chromosomes oligo-FISH is a powerful tool for constructing high-resolution karyotypes and improving the quality of genome assembly.

Expanding the Chromosomal Evolution Understanding of Lygaeioid True Bugs (Lygaeoidea, Pentatomomorpha, Heteroptera) by Classical and Molecular Cytogenetic Analysis.

The Lygaeoidea comprise about 4660 species in 790 genera and 16 families. Using standard chromosome staining and FISH with 18S rDNA and telomeric (TTAGG)n probes, we studied male karyotypes and meiosis in 10 species of Lygaeoidea belonging to eight genera of the families Blissidae, Cymidae, Heterogastridae, Lygaeidae, and Rhyparochromidae. Chromosome numbers were shown to range from 12 to 28, with 2n = 14 being predominant. All species have an XY system and all but one have a pair of m-chromosomes. The exception is Spilostethus saxatilis (Lygaeidae: Lygaeinae); in another species of Lygaeinae, Thunbergia floridulus, m-chromosomes were present, which represents the first finding for this subfamily. All species have an inverted sequence of sex chromosome divisions ("post-reduction"). The 18S rDNA loci were observed on one or both sex chromosomes in Kleidocerys resedae and Th. floridulus, respectively (Lygaeidae), while on an autosomal bivalent in all other species. The rDNA loci tended to be close to the end of the chromosome. Using (TTAGG)n-FISH, we were able to show for the first time that the Lygaeoidea lack the canonical "insect" telomere motif (TTAGG)n. We speculate that this ancestral motif is absent from the entire infraorder Pentatomomorpha being replaced by some other telomere repeat motif sequences.

Nuclear DNA content and comparative FISH mapping of the 5s and 45s rDNA in wild and cultivated populations of Physalis peruviana L.

Physalis peruviana L. often known as goldenberry, has increased its commercial growth in the international market in recent years due to its nutritional value and antioxidant potential. This situation has enabled countries such as Peru to increase their production in order to meet the global demand. However, investigations about the genetic diversity of cultivated and wild populations of goldenberry are still in their early stages. FISH mapping of 5s and 45s rDNA loci and flow cytometry estimation of nuclear DNA content were used to assess genetic differences between wild and cultivated goldenberry populations from Ayacucho and Cajamarca. The majority of metaphases had six 5s rDNA sites for all populations and two and four 45s rDNA sites for the cultivated and wild populations, respectively. We were able to characterize nine different types of chromosomes based on their morphology, fluorescence, rDNA location, and conservation across populations by analyzing the chromosomes that contained rDNA. Furthermore, cultivated populations had more nuclear DNA (13.262±0.087 pg) than wild populations (12.955±0.086 pg). The results show genetic differences between wild and cultivated populations of goldenberry at molecular cytogenetic level as well as in genome size. These findings establish a precedent for future cytogenetic and genomic studies in goldenberry populations, enabling future breeding programs.

Non-Rabl chromosome organization in endoreduplicated nuclei of barley embryo and endosperm tissues.

Rabl organization is a type of interphase chromosome arrangement with centromeres and telomeres clustering at opposite nuclear poles. Here, we analyzed nuclear morphology and chromosome organization in cycling and endoreduplicated nuclei isolated from embryo and endosperm tissues of developing barley seeds. We show that endoreduplicated nuclei have an irregular shape, less sister chromatid cohesion at 5S rDNA loci, and a reduced amount of centromeric histone CENH3. While the chromosomes of the embryo and endosperm nuclei are initially organized in Rabl configuration, the centromeres and telomeres are intermingled within the nuclear space in the endoreduplicated nuclei with an increasing endoreduplication level. Such a loss of chromosome organization suggests that Rabl configuration is introduced and further reinforced by mitotic divisions in barley cell nuclei in a tissue- and seed age-dependent manner.

Living on the edge: morphological, karyological and genetic diversity studies of the Hungarian Plantago maxima populations and established ex situ collection

BackgroundThe analysis of genetic diversity of protected plant species can greatly support conservation efforts. Plantago maxima Juss. ex Jacq. is a perennial species distributed along the Eurasian steppe. The westernmost range edge of the species’ distribution is located in the Pannonian basin, in Hungary where it is represented by a few, fragmented and highly endangered populations. We studied population diversity of all Hungarian range edge, natural populations, and one established ex situ population. One population from the centre of distribution (Kazakhstan) was implemented in the cpDNA haplotype study to compare the peripheral vs. central populations. We performed morphometric trait-based analysis, chromosome studies (morphometric analyses and FISH) and genetic diversity evaluations using inter simple sequence repeats (ISSR) and cpDNA trnL-trnF to evaluate differences between the in situ and ex situ populations as well as central vs. peripheral populations.ResultsOur results showed no obvious morphological differences among the in situ and ex situ populations in the period between 2018 and 2020. One ex situ subpopulation develops flowers three years in a row from 2019, which is a favourable indicator of the introduction success. Hungarian populations are exclusively diploids (2n = 2x = 12). The karyogram consists of 5 metacentric and 1 acrocentric chromosome pair. Plantago maxima has one 35S and two 5S rDNA loci, located on the acrocentric chromosome pair. Eight variable ISSR primers yielded 100 fragments, of which 74.6% were polymorphic (mean He = 0.220). A high level of genetic variation within population was observed (92%) while the genetic differentiation among the populations was only 8%. STRUCTURE analysis revealed that the largest Kunpeszér population separated from the rest of the Hungarian populations, indicating a high rate of admixture among the other ones. Based on the trnL-trnF sequence analysis the Hungarian populations represent a single haplotype, which can indicate a reduced diversity due to isolation and recent population decline. By contrast, Kazakh population represents a distinct haplotype compared to the Hungarian samples.ConclusionsThe present study draws the attention to the high conservation value of the Plantago maxima populations from the westernmost range edge of the species’ distribution.