Diversity of the repetitive DNA fraction in Cestrum, the genus with the largest genomes within Solanaceae.

BACKGROUND: Cestrum species present large genomes (2 C = ~ 24 pg), a high occurrence of B chromosomes and great diversity in heterochromatin bands. Despite this diversity, karyotypes maintain the chromosome number 2n = 16 (except when they present B chromosomes), and a relative similarity in chromosome morphology and symmetry. To deepen our knowledge of the Cestrum genome composition, low-coverage sequencing data of C. strigilatum and C. elegans were compared, including cytogenomic analyses of seven species. METHODS AND RESULTS: Bioinformatics analyses showed retrotransposons comprising more than 70% of the repetitive fraction, followed by DNA transposons (~ 17%), but FISH assays using retrotransposon probes revealed inconspicuous and scattered signals. The four satellite DNA families here analyzed represented approximately 2.48% of the C. strigilatum dataset, and these sequences were used as probes in FISH assays. Hybridization signals were colocalized with all AT- and GC-rich sequences associated with heterochromatin, including AT-rich Cold-Sensitive Regions (CSRs). Although satellite probes hybridized in almost all tested species, a satDNA family named CsSat49 was highlighted because it predominates in centromeric regions. CONCLUSIONS: Data suggest that the satDNA fraction is conserved in the genus, although there is variation in the number of FISH signals between karyotypes. Except to the absence of FISH signals with probes CsSat1 and CsSat72 in two species, the other satellites occurred in species of different phylogenetic clades. Some satDNA sequences have been detected in the B chromosomes, indicating that they are rich in preexisting sequences in the chromosomes of the A complement. This comparative study provides an important advance in the knowledge on genome organization and heterochromatin composition in Cestrum, especially on the distribution of satellite fractions between species and their importance for the B chromosome composition.

Biosystematic studies of some Egyptian species of Cestrum (Solanaceae).

Cestrum is the second largest genus of family Solanaceae, after Solanum, distributed in warm to subtropical regions. Species of genus Cestrum are one of the most ethnopharmacological relevant plants, for their broad biological and pharmacological properties. There is a scarcity to taxonomical studies and identification of these plants in Egypt, thus, the objective of this study was to implement various morphological features, chemical markers and molecular tools to emphasize the taxonomical features of the different Cestrum species. Morphologically, the epidermal cells of C. diurnum, C. elegans and C. parqui were irregular with sinuate anticlinal wall patterns for both surfaces, while, C. nocturnum has anticlinal walls, sinuolate with polygonal to irregular epidermal cells on the abaxial surface. The species of Cestrum have hypostomatic leaves, except C. parqui that has amphistomatic leaves. The experimented species of Cestrum have Anomocytic and anisocytic stomata, while, C. elegans has a diacytic stomata. The morphologically identified Cestrum spp were molecular confirmed based on their ITS sequences, the sequences of C. diurnum, C. nocturnum, C. elegans and C. parqui were deposited on genbank with accession # MT742788.1, MT749390.1, MW091481.1 and MW023744.1, respectively. From the SCOT analyses, the four species of Cestrum were grouped into 2 clusters (I, II), cluster I contains C. elegans, C. nocturnum and C. parqui, while cluster II contains only C. diurnum with 100% polymorphism for all primers. From the GC-MS profile, the C. diurnum exhibited a diverse metabolic paradigm, ensuring their richness with different metabolites comparing to other experimented Cestrum species. Among the total resolved metabolites, 15-methyltricyclo 6.5.2-pentadeca-1,3,5,7,9, 11,13-heptene was the highly incident compound in C. elegans (35.89%) followed by C. parqui (21.81%) and C. diurnum (11.28%), while it absent on C. nocturnum. The compound, 2,2',6,6'-tetra-tert-butyl-4,4'-methylenediphenol was highly detected in C. elegans and C. dirunum with minor amounts in the other Cestrum species. Cypermethrin and 3-butynyl-2,2,5-trimethyl-1,3-dioxane-5-methanol were pivotally reported in C. nocturnum. Taken together, from molecular and metabolic markers, C. diurnum, C. parqui and C. elegans have higher proximity unlike to C. nocturnum.

Anatomical and genetic bases underlying the convergent evolution of fleshy and dry dehiscent fruits in Cestrum and Brugmansia (Solanaceae).

The mechanisms controlling evolutionary shifts between dry and fleshy fruits in angiosperms are poorly understood. In Solanaceae, Cestrum and Brugmansia represent cases of convergent evolution of fleshy and dry fruits, respectively. Here we study the anatomical and genetic bases of the independent origin of fleshy fruits in Cestrum and the reversion to dry dehiscent fruits in Brugmansia. We also characterize the expression of candidate fruit development genes, including ALCATRAZ/SPATULA, FRUITFULL, HECATE1/2/3, REPLUMLESS and SHATTERPROOF. We identify anatomical changes to establish developmental stages in the ovary-to-fruit transition in Cestrum nocturnum and Brugmansia suaveolens. We generate reference transcriptomes for both species, isolate homologs for all genes in the fruit genetic regulatory network (GRN) and perform gene expression analyses for ALC/SPT, FUL, HEC1/2/3, RPL and SHP throughout fruit development. Finally, we compare our results to expression patterns found in typical capsules of Nicotiana tabacum and berries of Solanum lycopersicum available in public repositories. We have identified homologous, homoplasious and unique anatomical features in C. nocturnum and B. suaveolens fruits, resulting in their final appearance. Expression patterns suggest that FUL, SHP and SPT might control homologous characteristics, while ALC and RPL likely contribute to homoplasious anatomical features. The fruit GRN changes considerably in these genera when compared to typical capsules and berries of Solanaceae, particularly in B. suaveolens, where expression of FUL2 and RPL1 is lacking.

IGS sequences in Cestrum present AT- and GC-rich conserved domains, with strong regulatory potential for 5S rDNA.

The 35S and 5S ribosomal DNA (rDNA) organized in thousands of copies in genomes, have been widely used in numerous comparative cytogenetic studies. Nevertheless, several questions related to the diversity and organization of regulatory motifs in 5S rDNA remain to be addressed. The 5S rDNA unit is composed of a conserved 120 bp length coding region and an intergenic spacer (IGS) containing potential regulatory motifs (Poly-T, AT-rich and GC-rich) differing in number, redundancy and position along the IGS. The Cestrum species (Solanaceae) have large genomes (about 10 pg/1C) and conserved 2n = 16 karyotypes. Strikingly, these genomes show high diversity of heterochromatin distribution, variability in 35S rDNA loci and the occurrence of B chromosomes. However, the 5S rDNA loci are highly conserved in the proximal region of chromosome 8. Comparison of seventy-one IGS sequences in plants revealed several conserved motifs with potential regulatory function. The AT- and GC-rich domains appeared highly conserved in Cestrum chromosomes. The 5S genic and the GC-rich IGS probe produced FISH signals in both A (pair 8) and B chromosomes. The GC-rich domain presented a strong potential for regulation because it may be associated with CpG islands organization, as well as to hairpin and loop organization. Another interesting aspect was the ability of AT- and GC-rich motifs to produce non-heterochromatic CMA/DAPI signals. While the length of the 5S rDNA IGS region varied in size between the Cestrum species, the individual sequence motifs seem to be conserved suggesting their regulatory function. The most striking feature was the conserved GC-rich domain in Cestrum, which is recognized as a signature trait of the proximal region of chromosome pair 8.

BAL31-NGS approach for identification of telomeres de novo in large genomes.

This article describes a novel method to identify as yet undiscovered telomere sequences, which combines next generation sequencing (NGS) with BAL31 digestion of high molecular weight DNA. The method was applied to two groups of plants: i) dicots, genus Cestrum, and ii) monocots, Allium species (e.g. A. ursinum and A. cepa). Both groups consist of species with large genomes (tens of Gb) and a low number of chromosomes (2n=14-16), full of repeat elements. Both genera lack typical telomeric repeats and multiple studies have attempted to characterize alternative telomeric sequences. However, despite interesting hypotheses and suggestions of alternative candidate telomeres (retrotransposons, rDNA, satellite repeats) these studies have not resolved the question. In a novel approach based on the two most general features of eukaryotic telomeres, their repetitive character and sensitivity to BAL31 nuclease digestion, we have taken advantage of the capacity and current affordability of NGS in combination with the robustness of classical BAL31 nuclease digestion of chromosomal termini. While representative samples of most repeat elements were ensured by low-coverage (less than 5%) genomic shot-gun NGS, candidate telomeres were identified as under-represented sequences in BAL31-treated samples.

Characterisation of an unusual telomere motif (TTTTTTAGGG)n in the plant Cestrum elegans (Solanaceae), a species with a large genome.

The characterization of unusual telomere sequence sheds light on patterns of telomere evolution, maintenance and function. Plant species from the closely related genera Cestrum, Vestia and Sessea (family Solanaceae) lack known plant telomeric sequences. Here we characterize the telomere of Cestrum elegans, work that was a challenge because of its large genome size and few chromosomes (1C 9.76 pg; n = 8). We developed an approach that combines BAL31 digestion, which digests DNA from the ends and chromosome breaks, with next-generation sequencing (NGS), to generate data analysed in RepeatExplorer, designed for de novo repeats identification and quantification. We identify an unique repeat motif (TTTTTTAGGG)n in C. elegans, occurring in ca. 30 400 copies per haploid genome, averaging ca. 1900 copies per telomere, and synthesized by telomerase. We demonstrate that the motif is synthesized by telomerase. The occurrence of an unusual eukaryote (TTTTTTAGGG)n telomeric motif in C. elegans represents a switch in motif from the 'typical' angiosperm telomere (TTTAGGG)n . That switch may have happened with the divergence of Cestrum, Sessea and Vestia. The shift in motif when it arose would have had profound effects on telomere activity. Thus our finding provides a unique handle to study how telomerase and telomeres responded to genetic change, studies that will shed more light on telomere function.

Heterozygote excess through life history stages in Cestrum miradorense Francey (Solanaceae), an endemic shrub in a fragmented cloud forest habitat.

Comparisons of genetic diversity and population genetic structure among different life history stages provide important information on the effect of the different forces and micro-evolutionary processes that mould diversity and genetic structure after fragmentation. Here we assessed genetic diversity and population genetic structure using 32 allozymic loci in adults, seeds, seedlings and juveniles of eight populations of the micro-endemic shrub Cestrum miradorense in a highly fragmented cloud forest in central-eastern Mexico. We expected that due to its long history or rarity, this species may have endured the negative effects of fragmentation and would show moderate to high levels of genetic diversity. High genetic diversity (H(e) = 0.445 ± 0.03), heterozygote excess (F(IT) = -0.478 ± 0.034, F(IS) = -0.578 ± 0.023) and low population differentiation (F(ST) = 0.064 ± 0.011) were found. Seeds had higher genetic diversity (H(e) = 0.467 ± 0.05) than the later stages (overall mean for adults, seedlings and juveniles H(e) = 0.438 ± 0.08). High gene flow was observed despite the fact that the fragmentation process began more than 100 years ago. We conclude that the high genetic diversity was the result of natural selection, which favours heterozygote excess in all stages, coupled with a combination of a reproductive system and seed/pollen dispersal mechanisms that favour gene flow.

Two faces of Solanaceae telomeres: a comparison between Nicotiana and Cestrum telomeres and telomere-binding proteins.

While most Solanaceae genera (e.g.Solanum, Nicotiana) possess Arabidopsis-type telomeres of (TTTAGGG)n maintained by telomerase, the genera Cestrum, Vestia and Sessea (Cestrum group) lack these telomeres. Here we show that in the Cestrum-group the activity of telomerase has been lost. Nevertheless, proteins binding the single-stranded G-rich strand of the Arabidopsis-type and related human-type (TTAGGG)n telomeric sequences are present in nuclear extracts of both Nicotiana and Cestrum species. These proteins may have a role in telomere function or other cellular activities. In addition to characterizing DNA binding specificity and molecular weights of these proteins, we searched in both N. tabacum (tobacco) and C. parqui for the presence of POT1-like proteins, involved in telomere capping and telomerase regulation. Analysis of POT1-like proteins available on public databases and cloned by us from C. parqui, revealed the N-terminal OB folds typical for this protein family and a novel, plant-specific conserved C-terminal OB-fold domain (CTOB). We propose that CTOB is involved in protein-protein interactions.

Differential effects of NAA and 2,4-D in reducing floret abscission in cestrum (Cestrum elegans) cut flowers are associated with their differential activation of Aux/IAA homologous genes.

BACKGROUND AND AIMS: A previous study showed that the relative effectiveness of 2,4-dichlorophenoxyacetic acid (2,4-D) compared with that of 1-naphthaleneacetic acid (NAA) in reducing floret bud abscission in cestrum (Cestrum elegans) cut flowers was due to its acropetal transport. The aim of the present study was to examine if the differential effect of these auxins on floret abscission is reflected in the expression of Aux/IAA genes in the floret abscission zone (AZ). METHODS: cDNAs were isolated by PCR-based cloning from the floret AZ of auxin-treated cut flowers. The expression patterns of the cDNAs in various tissues and the effect of indole-3-acetic acid (IAA), applied with or without cycloheximide, on their expression in the floret AZ were examined by northern blot analysis. The regulation of transcript accumulation in the floret AZ in response to NAA or 2,4-D was measured by real-time PCR during auxin pulsing of cut flowers and vase life, concomitantly with floret abscission. KEY RESULTS: Six isolated cDNAs were identified to represent Aux/IAA homologous genes, designated as Cestrum elegans (Ce)-IAA1 to Ce-IAA6. Four Ce-IAA genes were characterized as early auxin-responsive genes (ARGs), and two (Ce-IAA1 and Ce-IAA5) as late ARGs. Only Ce-IAA5 was AZ-specific in floret buds. A temporal regulation of Ce-IAA transcript levels in the floret AZ was found, with 2,4-D inducing higher expression levels than NAA in floret buds. These Ce-IAA expression levels were negatively correlated with floret abscission. CONCLUSIONS: The differential transport characteristics of NAA and 2,4-D in cestrum cut flowers were reflected in differential activation of the Ce-IAA genes identified in the floret AZ. Therefore, Aux/IAA genes can be used as molecular markers to measure auxin activity, which reflects free auxin level in the AZ. Two of the identified genes, Ce-IAA1 and Ce-IAA5, may also have a regulatory role in abscission.

The occurrence of different Bs in Cestrum intermedium and C. strigilatum (Solanaceae) evidenced by chromosome banding.

In this study, we examine the morphology, mitotic stability, meiotic behavior and the composition of heterochromatin of B chromosomes in Cestrum intermedium and C. strigilatum. The results showed that B chromosome number shows intraindividual variation in the root meristem, which seems to lead to a slight rate of B elimination in this somatic tissue. B chromosomes in both species were similar in size and shape, but differed with regard to the type, size and distribution of heterochromatin. Possible evolutionary pathways for B chromosome origin in Cestrum are discussed.

The signature of the Cestrum genome suggests an evolutionary response to the loss of (TTTAGGG)n telomeres.

The genus Cestrumin the Solanaceae family is unusual in lacking Arabidopsis-type telomeres (TTTAGGG)n, although short interstitial telomeric sequences (ITSs) occur scattered throughout the genome in both orientations. To isolate candidate telomeric sequences in Cestrum we assumed that some of the ITSs were residues of the original telomeres and that they may still be located in the vicinity of present-day telomeres. Three sequence types associated with ITSs were cloned and characterized; these were termed NA3G, BR23 and A/T-rich minisatellite. These high copy number sequences are dispersed across the genome and clustered at a number of chromosomal loci. Their association with ITSs, which can act as recombination hotspots, might indicate past recombination and chromosomal fusion events, processes that may have contributed to the large size of Cestrum chromosomes. The sequences are frequently arranged as NA3G-ITS-BR23 blocks embedded in an A/T-rich minisatellite array. The A/T-rich minisatellite is of particular interest because the consensus 5'-T(4-5)AGCAG-3' might be a derivative of "typical" eukaryotic telomeric sequence motifs. The sequence is abundant at the end of some chromosomes in C. parqui and is found not only in Cestrum but also in the closely related genera Sessea and Vestia, which also lack Arabidopsis-type telomeric sequences. However, the sequence is absent from the Solanaceae genera investigated that are outside the group, including the closely related genus Streptosolen, which all have the Arabidopsis-type telomere. The data indicate that the A/T rich minisatellite might have evolved in response to the loss of Arabidopsis-type telomeres.

The absence of Arabidopsis-type telomeres in Cestrum and closely related genera Vestia and Sessea (Solanaceae): first evidence from eudicots.

Using slot-blot and fluorescent in situ hybridization (FISH), we found no evidence for the presence of the Arabidopsis-type telomeric sequence (TTTAGGG)n at the chromosome termini in any of the Cestrum species we investigated. Probing for the human-type telomere (TTAGGG)n also revealed no signal. However, polymerase chain reaction experiments indicated that there are short lengths of the sequence TTTAGGG dispersed in the genome but that these sequences are almost certainly too short to act as functional telomeres even if they were at the chromosome termini. An analysis of related genera Vestia and Sessea indicates that they too lack the Arabidopsis-type telomere, and the sequences were lost in the common ancestor of these genera. We found that the Cestrum species investigated had particularly large mean chromosome sizes. We discuss whether this is a consequence of alternative telomere end maintenance systems.