Pan-chloroplast genomes for accession-specific marker development in Hibiscus syriacus.

Hibiscus syriacus L. is a renowned ornamental plant. We constructed 95 chloroplast genomes of H. syriacus L. cultivars using a short-read sequencing platform (Illumina) and a long-read sequencing platform (Oxford Nanopore Technology). The following genome assembly, we delineate quadripartite structures encompassing large single-copy, small single-copy, and inverted repeat (IRa and IRb) regions, from 160,231 bp to 161,041 bp. Our comprehensive analyses confirmed the presence of 79 protein-coding genes, 30 tRNA genes, and 4 rRNA genes in the pan-chloroplast genome, consistent with prior research on the H. syriacus chloroplast genome. Subsequent pangenome analysis unveiled widespread genome sequence conservation alongside unique cultivar-specific variant patterns consisting of 193 single-nucleotide polymorphisms and 61 insertions or deletions. The region containing intra-species variant patterns, as identified in this study, has the potential to develop accession-specific molecular markers, enhancing precision in cultivar classification. These findings are anticipated to drive advancements in breeding strategies, augment biodiversity, and unlock the agricultural potential inherent in H. syriacus.

Meta-Transcriptomic Analysis Uncovers the Presence of Four Novel Viruses and Multiple Known Virus Genera in a Single Hibiscus rosa-sinensis Plant in Colombia.

Hibiscus is not native to Colombia but well suited to its arid soil and dry climates. A single hibiscus plant from Risaralda, showing black spots on upper and lower sides of its leaves, was collected for virome analysis using meta-transcriptomic high-throughput sequencing technology. Bioinformatic analysis identified 12.5% of the total reads in the Ribo-Zero cDNA library which mapped to viral genomes. BLAST searches revealed the presence of carlavirus, potexvirus, and of known members of the genera Betacarmovirus, Cilevirus, Nepovirus, and Tobamovirus in the sample; confirmed by RT-PCR with virus-specific primers followed by amplicon sequencing. Furthermore, in silico analysis suggested the possibility of a novel soymovirus, and a new hibiscus strain of citrus leprosis virus C2 in the mixed infection. Both RNA dependent RNA polymerase and coat protein gene sequences of the potex and carla viruses shared less than 72% nucleotide and 80% amino acid identities with any alphaflexi- and betaflexi-virus sequences available in GenBank, identifying three novel carlavirus and one potexvirus species in the Hibiscus rosa-sinensis plant. The detection of physalis vein necrosis nepovirus and passion fruit green spot cilevirus in hibiscus are also new reports from Colombia. Overall, the meta-transcriptome analysis identified the complex virome associated with the black spot symptoms on hibiscus leaves and demonstrated the diversity of virus genera tolerated in the mixed infection of a single H. rosa-sinensis plant.

A Breakthrough in Kitavirids: Genetic Variability, Reverse Genetics, Koch's Postulates, and Transmission of Hibiscus Green Spot Virus 2.

Hibiscus green spot virus 2 (HGSV-2), a member of the genus Higrevirus (family Kitaviridae), is a positive-stranded RNA virus associated with leprosis-like symptoms in citrus and green spots on leaves in hibiscus. HGSV-2 has only been reported in Hawaii, and while it is speculated that mites in the genus Brevipalpus might be responsible for its transmission, proper transmission assays have yet to be conducted. This study characterizes additional citrus and hibiscus isolates of HGSV-2 collected from two Hawaiian Islands. We constructed an infectious cDNA clone from a hibiscus isolate of HGSV-2 collected on Oahu and demonstrated its ability to infect several experimental hosts, including Phaseolus vulgaris, Nicotiana tabacum, and N. benthamiana, as well as natural hosts, Citrus reticulata and Hibiscus arnottianus. Bacilliform virions with varied sizes of 33 to 120 nm (length) and 14 to 70 nm (diameter) were observed in partially purified preparations obtained from agroinoculated leaves. Virus progeny from the infectious cDNA clone was found to be infectious after mechanical transmission to N. benthamiana and to cause local lesions. Finally, an isoline colony of the mite Brevipalpus azores had vector competence to transmit a citrus isolate of HGSV-2 collected from Maui to citrus and hibiscus plants, demonstrating the mite-borne nature of HGSV-2. The infectious cDNA clone developed in this study is the first reverse-genetics system for a kitavirid and will be fundamental to better characterize basic biology of HGSV-2 and its interactions with host plants and mite vectors.

A simple and rapid method for isolating high-quality RNA from kenaf with high polysaccharide and polyphenol contents.

The extraction of high-quality RNA from kenaf is essential for genetic and molecular biology research. However, the presence of high levels of polysaccharide and polyphenol compounds in kenaf poses challenges for RNA isolation. We proposed a simplified, time-saving and cost-effective method for isolating high quantities of RNA from various kenaf tissues. This method exhibited superior efficiency in RNA isolation compared with the conventional cetyltrimethylammonium bromide method and demonstrated greater adaptability to different samples than commercial kits. Furthermore, the high-quality RNA obtained from this method was successfully utilized for RT-PCR, real-time RT-PCR and northern blot analysis. Moreover, this proposed protocol also enables the acquisition of both high-quality and -quantity gDNA through RNase A treatment. In addition, the effectiveness of this approach in isolating high-quality RNA from other plant species has been experimentally confirmed.

Two long read-based genome assembly and annotation of polyploidy woody plants, Hibiscus syriacus L. using PacBio and Nanopore platforms.

Improvements in long read DNA sequencing and related techniques facilitated the generation of complex eukaryotic genomes. Despite these advances, the quality of constructed plant reference genomes remains relatively poor due to the large size of genomes, high content of repetitive sequences, and wide variety of ploidy. Here, we developed the de novo sequencing and assembly of high polyploid plant genome, Hibiscus syriacus, a flowering plant species of the Malvaceae family, using the Oxford Nanopore Technologies and Pacific Biosciences Sequel sequencing platforms. We investigated an efficient combination of high-quality and high-molecular-weight DNA isolation procedure and suitable assembler to achieve optimal results using long read sequencing data. We found that abundant ultra-long reads allow for large and complex polyploid plant genome assemblies with great recovery of repetitive sequences and error correction even at relatively low depth Nanopore sequencing data and polishing compared to previous studies. Collectively, our combination provides cost effective methods to improve genome continuity and quality compared to the previously reported reference genome by accessing highly repetitive regions. The application of this combination may enable genetic research and breeding of polyploid crops, thus leading to improvements in crop production.

Physiological and transcriptional mechanisms associated with cadmium stress tolerance in Hibiscus syriacus L.

BACKGROUND: Cadmium (Cd) pollution of soils is a global concern because its accumulation in plants generates severe growth retardation and health problems. Hibiscus syriacus is an ornamental plant that can tolerate various abiotic stresses, including Cd stress. Therefore, it is proposed as a plant material in Cd-polluted areas. However, the molecular mechanisms of H. syriacus tolerance to Cd are not yet understood. RESULTS: This study investigated the physiological and transcriptional response of "Hongxing", a Cd2+-tolerant H. syriacus variety, grown on a substrate containing higher concentration of Cd (400 mg/kg). The Cd treatment induced only 28% of plant mortality, but a significant decrease in the chlorophyll content was observed. Malondialdehyde content and activity of the antioxidant enzymes catalase, peroxidase, and superoxide dismutase were significantly increased under Cd stress. Transcriptome analysis identified 29,921 differentially expressed genes (DEGs), including 16,729 down-regulated and 13,192 up-regulated genes, under Cd stress. Functional enrichment analyses assigned the DEGs mainly to plant hormone signal transduction, transport, nucleosome and DNA processes, mitogen-activated protein kinase signaling pathway, antioxidant process, fatty acid metabolism, and biosynthesis of secondary metabolites. Many MYB, EP2/ERF, NAC, WRKY family genes, and genes containing metal binding domains were up-regulated, implying that they are essential for the Cd-stress response in H. syriacus. The most induced genes were filtered out, providing valuable resources for future studies. CONCLUSIONS: Our findings provide insights into the molecular responses to Cd stress in H. syriacus. Moreover, this study offers comprehensive and important resources for future studies toward improving the plant Cd tolerance and its valorization in phytoremediation.

Genetic assessment improves conservation efforts for the critically endangered oceanic island endemic Hibiscus liliiflorus.

Hibiscus liliiflorus, endemic to the Indian Ocean island of Rodrigues, is one of the rarest plant species in the world; only 2 wild individuals remain. Previously, when 4 wild individuals remained, the Mauritian Wildlife Foundation (MWF) in Rodrigues propagated cuttings of them in their nursery, then planted seedlings produced in the nursery into 3 outplanted populations on the island. Our goals were to: 1) assess whether all 4 original wild genotypes are represented in the MWF nursery; 2) determine whether ex situ living collections at international botanical gardens maintain unique genotypes of H. liliiflorus; 3) assess whether nursery individuals have crossed or self-fertilized to produce seed and quantify their relative contributions to outplanted populations; and 4) provide recommendations for future conservation actions. We used a 2b-RADseq approach to produce 2,711 genome-wide single nucleotide polymorphisms (SNPs) from 98 samples. Genotype identity analysis, principal component analysis, and model-based clustering in STRUCTURE found 4 genotypes extant in Rodrigues but no unique genotypes in ex situ botanic garden collections. Only 3 genotypes are represented in the MWF nursery; the one remaining genotype is represented by an extant wild individual. Parentage analysis showed that seeds produced in the MWF nursery resulted from both self-fertilization and crossing between genotypes, a result supported by internal relatedness and hybrid index calculations. Each outplanted population is dominated by a subset of parental genotypes, and we propose actions to balance the parental contributions to outplanted populations. Our study highlights how genetic assessments of ex situ conservation projects help conserve critically endangered species.

Spatiotemporal process of long-distance seed dispersal in a pantropically distributed sea hibiscus group.

Long-distance dispersal (LDD) of seeds plays an essential role in the migration of plants to a new habitat and maintaining gene flow among geographically isolated populations. Pantropical plants with sea-drifted seeds, which have one of the largest distributions in all flowering plants, have achieved their global distribution by LDD. However, the spatiotemporal processes to achieve the wide distribution and the role of LDD in it have not yet been elucidated. In this study, we conducted phylogenomic analyses on the plastome, genome-wide nuclear SNP, and low-copy gene data of Hibiscus tiliaceus and its relatives. The dated phylogeny suggested that global expansion started approximately 4 million years ago (Ma), and species diversification occurred 1 Ma. Plastome phylogeny confirmed the nonmonophyly of the haplotypes in the two widely distributed coastal species, H. tiliaceus and H. pernambucensis. In contrast, genome-wide nuclear SNP phylogenies demonstrated clear genetic segregation among species and/or geographical regions. Ancestral polymorphisms in chloroplast genomes shared among widely distributed species have remained below the range of rapid expansion and speciation of marginal populations. This study demonstrated that the LDD of sea-drifted seeds contributed to the rapid expansion and pantropical distribution of sea hibiscus in the last few million years, and adaptation to local environment or isolation by regional effect after LDD promoted speciation, suppressing gene flow.

Genome-Wide Analysis of the MADS-Box Gene Family in Hibiscus syriacus and Their Role in Floral Organ Development.

Hibiscus syriacus belongs to the Malvaceae family, and is a plant with medicinal, edible, and greening values. MADS-box transcription factor is a large family of regulatory factors involved in a variety of biological processes in plants. Here, we performed a genome-wide characterization of MADS-box proteins in H. syriacus and investigated gene structure, phylogenetics, cis-acting elements, three-dimensional structure, gene expression, and protein interaction to identify candidate MADS-box genes that mediate petal developmental regulation in H. syriacus. A total of 163 candidate MADS-box genes were found and classified into type I (Mα, Mß, and Mγ) and type II (MIKC and Mδ). Analysis of cis-acting elements in the promoter region showed that most elements were correlated to plant hormones. The analysis of nine HsMADS expressions of two different H. syriacus cultivars showed that they were differentially expressed between two type flowers. The analysis of protein interaction networks also indicated that MADS proteins played a crucial role in floral organ identification, inflorescence and fruit development, and flowering time. This research is the first to analyze the MADS-box family of H. syriacus and provides an important reference for further study of the biological functions of the MADS-box, especially in flower organ development.

Estimating Genetic Analysis Using Half Diallel Cross Underlying Kenaf (Hibiscus cannabinus L.) Fibre Yield in Tropical Climates.

Kenaf (Hibiscus cannabinus L.) is a natural fibre crop that can be used for a variety of purposes and has various applications in industry. Despite this, its potential has not been fully exploited because of low yields and a narrow genetic base, limiting hybrids' development. Based on this background, eight kenaf mutants and one commercial cultivar were selected and crossed in a half-diallel for general and specific combining abilities (GCA and SCA) to get the desired results done in this investigation. The 36 hybrid offspring and their parental lines were tested in the field over two environments. Diallel results based on Griffing B method 2 indicated significant differences for all characters studied except for GCA in top diameter and plant height and top diameter SCA, indicating the existence of both additive and nonadditive gene actions for the inheritance of the traits. The amplitude of GCA variation was much higher than that of SCA variation for all parameters except top diameter and node number, showing the additive gene's prevalence and the likelihood of genetic advancement through selection. In both conditions, Hayman and Jinks graphical studies demonstrated that partial dominance controlled various fibre yield component parameters such as plant height, middle diameter, stick weight, and fibre weight. On the other hand, fibre yield and the majority of physical features indicated either dominance or overdominance gene action. Plant height, base diameter, core diameter, middle diameter, fresh stem weight, and stick weight all strongly positively correlated with fibre yield. These traits also had a higher proportion of additive effects, a moderate narrow-sense heritability, and a higher baker ratio, indicating successful indirect selection for fibre yield. The parents P1, P3, and P4 had the most dominant alleles for most of the features, while the parents P2, P7, and P9 had the most recessive alleles. The hybrids P1 × P4, P1 × P9, P2 × P3, P2 × P5, P4 × P6, P4 × P7, P4 × P9, P5 × P8, and P7 × P9 outperformed the parents in terms of heterotic responses and showed that they have a lot of genetic potential for kenaf enhancement in tropical climates.

Integrated Methylome and Transcriptome Analysis Provides Insights into the DNA Methylation Underlying the Mechanism of Cytoplasmic Male Sterility in Kenaf (Hibiscus cannabinus L.).

Cytoplasmic male sterility (CMS) is widely exploited in hybrid seed production. Kenaf is an important fiber crop with high heterosis. The molecular mechanism of kenaf CMS remains unclear, particularly in terms of DNA methylation. Here, using the anthers of a kenaf CMS line (P3A) and its maintainer line (P3B), comparative physiological, DNA methylation, and transcriptome analyses were performed. The results showed that P3A had considerably lower levels of IAA, ABA, photosynthetic products and ATP contents than P3B. DNA methylome analysis revealed 650 differentially methylated genes (DMGs) with 313 up- and 337 down methylated, and transcriptome analysis revealed 1788 differentially expressed genes (DEGs) with 558 up- and 1230 downregulated genes in P3A compared with P3B. Moreover, 45 genes were characterized as both DEGs and DMGs, including AUX,CYP, BGL3B, SUS6, AGL30 and MYB21. Many DEGs may be regulated by related DMGs based on methylome and transcriptome studies. These DEGs were involved in carbon metabolism, plant hormone signal transduction, the TCA cycle and the MAPK signaling pathway and were shown to be important for CMS in kenaf. These results provide new insights into the epigenetic mechanism of CMS in kenaf and other crops.

Near-Hexaploid and Near-Tetraploid Aneuploid Progenies Derived from Backcrossing Tetraploid Parents Hibiscus syriacus × (H. syriacus × H. paramutabilis).

Hibiscus syriacus, azalea, is an important woody ornamental shrub planted throughout many temperate and subtropical regions of the world. However, flower size is smaller in this species than some of its relatives. To increase flower size, interspecific hybridization has been used, and such hybrid cultivars are usually characterized by larger flowers, increased vigor, diverse leaf shapes, and reduced fertility. Our earlier studies have shown that these hybrid cultivars could backcross with H. syriacus when used as male parents. To understand the breeding potential of these hybrid cultivars, two popular tetraploid hybrid cultivars, 'Lohengrin' and 'Resi', were used as pollen parents to backcross several tetraploid H. syriacus cultivars. As a result, 28.76% and 64.4% of 'Lohengrin' and 'Resi' progenies exhibited larger flowers than both of their parents. Interestingly, 14 of 18 progenies of 'Resi' were putative hexaploids, whereas 19 tested 'Lohengrin' progenies were tetraploid. Because putative hexaploid progenies were only observed among progenies of 'Resi', this hybrid cultivar appears to produce unreduced gametes. In addition, among the 14 putative hexaploids derived from 'Resi', 11 had larger flowers than both of their parents and their tetraploid siblings (p < 0.05). The 45S rDNA and 5S rDNA locus segregation among those BC1F1 progenies was tested by fluorescent in situ hybridization (FISH), and the wide range of 45S rDNA signal numbers among siblings indicated that these aneuploids resulted from unequal segregation or chromosome rearrangement. Chromosome counting confirmed aneuploidy among BC1F1 progenies. Ploidy diversity and aneuploidy have been known to contribute to various elements of morphological diversity, such as larger flower size and reduced fertility, which are important in ornamental plant breeding. The present study demonstrated the breeding potential of interspecific Hibiscus cultivars for increasing ploidy level and flower size.

Detectability of Hibiscus Mealybug, Nipaecoccus viridis (Hemiptera: Pseudoccocidae), DNA in the Mealybug Destroyer, Cryptolaemus montrouzieri (Coleoptera: Coccinellidae), and Survey of Its Predators in Florida Citrus Groves.

The Hibiscus mealybug, Nipaecoccus viridis (Newstead), has recently established in Florida citrus and become a pest of concern given secondary pest outbreaks associated with management of citrus greening disease. Chemical controls used to manage other citrus arthropod pests are not as effective against N. viridis due to its waxy secretions, clumping behavior, and induced cellular changes to host plant tissue which increase microhabitats. Populations of this mealybug pest are regulated by natural enemies in its native region, but it remains unclear if resident natural enemies in Florida citrus could similarly suppress N. viridis populations. This investigation: 1) established species-specific primers for N. viridis based on the mitochondrial gene Cytochrome-oxidase 1 (COI), 2) determined duration of N. viridis DNA detectability in a known predator, the mealybug destroyer (Cryptolaemus montrouzieri Mulsant), by using identified primers in molecular gut content analysis, and 3) screened field-collected predators for the presence of N. viridis DNA. The detection rate of N. viridis DNA was >50% at 36 h after adult C. montrouzieri feeding but DNA was no longer detectable by 72 h after feeding. Field-collected predators were largely comprised of spiders, lacewings, and C. montrouzieri. Spiders, beetles (primarily C. montrouzieri), and juvenile lacewings were the most abundant predators of N. viridis, with 17.8, 43.5, and 58.3 of field-collected samples testing positive for N. viridis DNA, respectively. Our results indicate that Florida citrus groves are hosts to abundant predators of N. viridis and encourage the incorporation of conservation or augmentative biological control for management of this pest.

Combining ability and gene action for yield improvement in kenaf (Hibiscus cannabinus L.) under tropical conditions through diallel mating design.

Nine morphologically distinct kenaf genotypes were hybridized to produce 36 hybrids following a half diallel mating design. The combining ability and gene action of 15 yield and yield components were assessed in hybrids and their parents across two environments. Except for the mid diameter and plant height traits, there were highly significant differences (p ≤ 0.01) between the environments and the interaction of genotype and environment. Additive gene effects were considerable for the inheritance of these traits, and the expression of these additive genes was heavily influenced by the environment. Significant differences were found for all studied traits for GCA except top diameter and SCA except plant height and top diameter, implying the presence of both additive and non-additive gene action for the inheritance of the concerned characters. For all features except top diameter and number of nodes, the magnitude of GCA variation was significantly higher than that of SCA variance, indicating the additive gene's predominance. The parental lines P1, P3 and P4 were outstanding general combiners for fiber yield and yield-related parameters. Considering combining ability and genetic analysis study, the crosses P1 × P4, P1 × P9, P2 × P3, P2 × P5, P4 × P6, P4 × P7, P4 × P9, P5 × P8, and P7 × P9 were found promising for their heterotic response to higher fiber yield, stick yield, seed yield and could be for future improvement in kenaf breeding programmes.

Genome-wide study of the GRAS gene family in Hibiscus hamabo Sieb. et Zucc and analysis of HhGRAS14-induced drought and salt stress tolerance in Arabidopsis.

GRAS proteins are widely distributed plant-specific transcription factors. In this study, we identified 59 GRAS proteins (HhGRASs) from the genomic and transcriptomic datasets of Hibiscus hamabo Sieb. et Zucc. These proteins were phylogenetically divided into nine subfamilies. RNA-seq analysis revealed that most HhGRASs were expressed in response to abiotic stresses. Results from quantitative real-time PCR analysis of nine selected HhGRASs suggested that HhGRAS14 was significantly upregulated under multiple abiotic stresses; therefore, this gene was selected for further study. Silencing HhGRAS14 in H. hamabo reduced the tolerance to drought and salt stress, while overexpression in Arabidopsis thaliana significantly increased the tolerance to drought and salt and reduced the sensitivity to abscisic acid (ABA). In summary, we analyzed the GRAS family of proteins in semi-mangrove plants for the first time and identified a gene that responds to drought and salt stress, which provided the basis for a comprehensive analysis of GRAS genes and insight into the abiotic stress response mechanism in H. hamabo.

Genome-Wide Identification and Characterization of NAC Family in Hibiscus hamabo Sieb. et Zucc. under Various Abiotic Stresses.

NAC transcription factor is one of the largest plant gene families, participating in the regulation of plant biological and abiotic stresses. In this study, 182 NAC proteins (HhNACs) were identified based on genomic datasets of Hibiscus hamabo Sieb. et Zucc (H. hamabo). These proteins were divided into 19 subfamilies based on their phylogenetic relationship, motif pattern, and gene structure analysis. Expression analysis with RNA-seq revealed that most HhNACs were expressed in response to drought and salt stress. Research of quantitative real-time PCR analysis of nine selected HhNACs supported the transcriptome data's dependability and suggested that HhNAC54 was significantly upregulated under multiple abiotic stresses. Overexpression of HhNAC54 in Arabidopsis thaliana (A. thaliana) significantly increased its tolerance to salt. This study provides a basis for a comprehensive analysis of NAC transcription factor and insight into the abiotic stress response mechanism in H. hamabo.

Nucleo-cytoplasmic interactions affect the 5' terminal transcription of mitochondrial genes between the isonuclear CMS line UG93A and its maintainer line UG93B of kenaf (Hibiscus cannabinus).

Gene expression and translation in plant mitochondria remain poorly understood due to the complicated transcription of its mRNA. In this study, we report the 5' and 3' RNA extremities and promoters of five mitochondrial genes, atp1, atp4, atp6, atp9, and cox3. The results reveal that four genes (atp1, atp4, atp6, and cox3) are transcribed from multiple initiation sites but with a uniform transcript at the 3' end, indicating that heterogeneity of the 5' end is a common feature in the transcription of kenaf mitochondrial genes. Furthermore, we found that the transcription initiation sites of these four genes are significantly different in UG93A, UG93B, and the F1 hybrid. These data indicate that nuclear loci and unknown transcription factors within the mitochondria of different cytoplasmic types may be involved in mitochondrial transcription. Promoter architecture analysis showed that the promoter core sequences are conserved in the kenaf mitochondrial genome but are highly divergent, suggesting that these elements are essential for the promoter activity of mitochondrial genes in kenaf. Our results reveal that the heterogeneity of the 5' end and uniformity at the 3' end are common transcriptional features of mitochondrial genes. These data provide essential information for understanding the transcription of mitochondrial genes in kenaf and can be used as a reference for other plants.

Genome-wide Analysis of Basic Helix-Loop-Helix Family Genes and Expression Analysis in Response to Drought and Salt Stresses in Hibiscus hamabo Sieb. et Zucc.

The basic helix-loop-helix (bHLH) family of transcription factors is one of the most significant and biggest in plants. It is involved in the regulation of both growth and development, as well as stress response. Numerous members of the bHLH family have been found and characterized in woody plants in recent years. However, no systematic study of the bHLH gene family has been published for Hibiscus hamabo Sieb. et Zucc. In this research, we identified 162 bHLH proteins (HhbHLHs) from the genomic and transcriptomic datasets of H. hamabo, which were phylogenetically divided into 19 subfamilies. According to a gene structural study, the number of exon-introns in HhbHLHs varied between zero and seventeen. MEME research revealed that the majority of HhbHLH proteins contained three conserved motifs, 1, 4, and 5. The examination of promoter cis-elements revealed that the majority of HhbHLH genes had several cis-elements involved in plant growth and development and abiotic stress responses. In addition, the overexpression of HhbHLH2 increased salt and drought stress tolerance in Arabidopsis.

Gene count from target sequence capture places three whole genome duplication events in Hibiscus L. (Malvaceae).

BACKGROUND: The great diversity in plant genome size and chromosome number is partly due to polyploidization (i.e. genome doubling events). The differences in genome size and chromosome number among diploid plant species can be a window into the intriguing phenomenon of past genome doubling that may be obscured through time by the process of diploidization. The genus Hibiscus L. (Malvaceae) has a wide diversity of chromosome numbers and a complex genomic history. Hibiscus is ideal for exploring past genomic events because although two ancient genome duplication events have been identified, more are likely to be found due to its diversity of chromosome numbers. To reappraise the history of whole-genome duplication events in Hibiscus, we tested three alternative scenarios describing different polyploidization events. RESULTS: Using target sequence capture, we designed a new probe set for Hibiscus and generated 87 orthologous genes from four diploid species. We detected paralogues in > 54% putative single-copy genes. 34 of these genes were selected for testing three different genome duplication scenarios using gene counting. All species of Hibiscus sampled shared one genome duplication with H. syriacus, and one whole genome duplication occurred along the branch leading to H. syriacus. CONCLUSIONS: Here, we corroborated the independent genome doubling previously found in the lineage leading to H. syriacus and a shared genome doubling of this lineage and the remainder of Hibiscus. Additionally, we found a previously undiscovered genome duplication shared by the /Pavonia and /Malvaviscus clades (both nested within Hibiscus) with the occurrences of two copies in what were otherwise single-copy genes. Our results highlight the complexity of genomic diversity in some plant groups, which makes orthology assessment and accurate phylogenomic inference difficult.

Distribution of FISH oligo-5S rDNA and oligo-(AGGGTTT)3 in Hibiscus mutabilis L.

Hibiscus exhibits high variation in chromosome number both within and among species. The Hibiscus mutabilis L. karyotype was analyzed in detail using fluorescence in situ hybridization (FISH) with oligonucleotide probes for (AG3T3)3 and 5S rDNA, which were tested here for the first time. In total, 90 chromosomes were counted in prometaphase and metaphase, and all exhibited similarly intense (AG3T3)3 signals at both ends. (AG3T3)3 showed little variation and thus did not allow discrimination among H. mutabilis chromosomes, but its location at both ends confirmed the integrity of each chromosome, thus contributing to accurate counting of the numerous, small chromosomes. Oligo-5S rDNA marked the proximal/distal regions of six chromosomes: weak signals on chromosomes 7 and 8, slightly stronger signals on chromosomes 15 and 16, and very strong signals on chromosomes 17 and 18. Therefore, 5S rDNA could assist in chromosome identification in H. mutabilis. Metaphase chromosome lengths ranged from 3.00 to 1.18 µm, indicating small chromosomes. The ratios of longest to shortest chromosome length in prometaphase and metaphase were 2.58 and 2.54, respectively, indicating karyotype asymmetry in H. mutabilis. These results provide an exact chromosome number and a physical map, which will be useful for genome assembly and contribute to molecular cytogenetics in the genus Hibiscus.