Article

Interspecific Chloroplast Recombination in a Nicotiana Somatic Hybrid

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Abstract

Genetic recombination between chloroplasts of two flowering plant species, Nicotiana tabacum and Nicotiana plumbaginifolia, after somatic cell fusion is described. The parental lines differed in three cytoplasmic genetic markers. The N. tabacum mutant SR1-A15 was streptomycin-resistant, defective in chloroplast greening, and lincomycin-sensitive. The N. plumbaginifolia mutant LR400 was streptomycin-sensitive, normal green, and lincomycin-resistant. Streptomycin-resistant clones in cell culture are identified by their ability to form a green callus on a selective medium. Streptomycin resistance in the SR1-A15 mutant could not be expressed due to defective chloroplasts. Protoplasts of the two species were fused, and calli grown from the fused population were screened for the expression of streptomycin resistance from the SR1-A15 line as the result of interspecific chloroplast recombination. A somatic hybrid, pt14, expressed a new combination of the cytoplasmic genetic markers. In the pt14 chloroplast genome three N. tabacum and four N. plumbaginifolia parent specific restriction sites have been identified, indicating that the pt14 chloroplast genome contains at least six recombination sites.

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... Although recombination has been demonstrated in somatic hybrids (e.g. Medgyesy et al. 1985), evidence from natural populations is rare and based on limited data (Huang et al. 2001;Marshall et al. 2001;Erixon and Oxelman 2008;Bouillé et al. 2011;D'Alelio and Ruggiero 2015). Using hypothesis and data-driven analyses, we found evidence for at least one interspecific recombination event in Picea plastomes. ...
... To our knowledge, plastid fusion has not been observed directly in natural populations but has been demonstrated experimentally in somatic hybrids (e.g. Medgyesy et al. 1985). Once fused, recombination could occur between highly similar genomes through normal DNA replication and repair mechanisms (Day and Madesis 2007;Maréchal and Brisson 2010). ...
Article
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Plastid sequences are a cornerstone in plant systematic studies and key aspects of their evolution, such as uniparental inheritance and absent recombination, are often treated as axioms. While exceptions to these assumptions can profoundly influence evolutionary inference, detecting them can require extensive sampling, abundant sequence data, and detailed testing. Using advancements in high-throughput sequencing, we analyzed the whole plastomes of 65 accessions of Picea, a genus of ~35 coniferous forest tree species, to test for deviations from canonical plastome evolution. Using complementary hypothesis and data-driven tests, we found evidence for chimeric plastomes generated by interspecific hybridization and recombination in the clade comprising Norway spruce (P. abies) and ten other species. Support for interspecific recombination remained after controlling for sequence saturation, positive selection, and potential alignment artifacts. These results reconcile previous conflicting plastid-based phylogenies and strengthen the mounting evidence of reticulate evolution in Picea. Given the relatively high frequency of hybridization and biparental plastid inheritance in plants, we suggest interspecific plastome recombination may be more widespread than currently appreciated and could underlie reported cases of discordant plastid phylogenies.
... Although recombination has been demonstrated in somatic hybrids (e.g. Medgyesy et al. 1985), evidence from natural populations is rare and based on limited data (Marshall et al. 2001;Huang et al. 2001;Erixon & Oxelman 2008;Bouillé et al. 2011;D'Alelio & Ruggiero 2015). Using hypothesis and data-driven analyses, we found evidence for at least one interspecific recombination event in Picea plastomes. ...
... To our knowledge, plastid fusion has not been observed directly in natural populations but has been demonstrated experimentally in somatic hybrids (e.g. Medgyesy et al. 1985). Once fused, recombination could occur between highly similar genomes through normal DNA replication and repair mechanisms (Day & Madesis 2007;Maréchal & Brisson 2010). ...
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Plastid sequences are a cornerstone in plant systematic studies and key aspects of their evolution, such as uniparental inheritance and absent recombination, are often treated as axioms. While exceptions to these assumptions can profoundly influence evolutionary inference, detecting them can require extensive sampling, abundant sequence data, and detailed testing. Using advancements in high-throughput sequencing, we analyzed the whole plastomes of 65 accessions of Picea, a genus of ~35 coniferous forest tree species, to test for deviations from canonical plastome evolution. Using complementary hypothesis and data-driven tests, we found evidence for chimeric plastomes generated by interspecific hybridization and recombination in the clade comprising Norway spruce ( P. abies ) and ten other species. Support for interspecific recombination remained after controlling for sequence saturation, positive selection, and potential alignment artifacts. These results reconcile previous conflicting plastid-based phylogenies and strengthen the mounting evidence of reticulate evolution in Picea. Given the relatively high frequency of hybridization and biparental plastid inheritance in plants, we suggest interspecific plastome recombination may be more widespread than currently appreciated and could underlie reported cases of discordant plastid phylogenies.
... Potential for heteroplasmy (based on pollen screenings) was documented in 19/61 legume species examined (Corriveau and Coleman 1988;Zhang et al. 2003), and heteroplasmy has been directly documented in four legume genera: Astragalus (Lei et al. 2016), Cicer (Kumari et al. 2011), Medicago (Johnson and Palmer 1989;Lee et al. 1988), and Lens (Rajora and Mahon 1995). Plastid recombination is generally regarded as rare (Birky 1995), but several recent studies have highlighted potential cases of heteroplasmic recombination (Sullivan et al. 2017;Sancho et al. 2018), and this phenomenon has been documented in the laboratory (Medgyesy et al. 1985). We hesitate to attribute any of our observed conflict to FIGURE 2. The distribution of phylogenetic signal for three alternative topological hypotheses at the root of Leguminosae. ...
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Phylogenomic analyses have helped resolve many recalcitrant relationships in the angiosperm tree of life, yet phylogenetic resolution of the backbone of the Leguminosae, one of the largest and most economically and ecologically important families, remains poor due to generally limited molecular data and incomplete taxon sampling of previous studies. Here, we resolve many of the Leguminosae’s thorniest nodes through comprehensive analysis of plastome-scale data using multiple modified coding and noncoding datasets of 187 species representing almost all major clades of the family. Additionally, we thoroughly characterize conflicting phylogenomic signal across the plastome in light of the family’s complex history of plastome evolution. Most analyses produced largely congruent topologies with strong statistical support, and provided strong support for resolution of some long-controversial deep relationships among the early diverging lineages of the subfamilies Caesalpinioideae and Papilionoideae. The robust phylogenetic backbone reconstructed in this study establishes a framework for future studies on legume classification, evolution, and diversification. However, conflicting phylogenetic signal was detected and quantified at several key nodes that prevents the confident resolution of these nodes using plastome data alone.
... It has been believed that recombination rarely occurs in flowering plants due to the predominance of uniparental inheritance. However, evidence of intermolecular homologous recombination in flowering plants have been mounting [41,42]. There was no record of plastome recombination in Asparagaceae, however plastome studies examining the recombination in the taxa are completely lacking thus far. ...
Article
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Background: The genus Hosta is a group of economically appreciated perennial herbs consisting of approximately 25 species that is endemic to eastern Asia. Due to considerable morphological variability, the genus has been well recognized as a group with taxonomic problems. Chloroplast is a cytoplasmic organelle with its own genome, which is the most commonly used for phylogenetic and genetic diversity analyses for land plants. To understand the genomic architecture of Hosta chloroplasts and examine the level of nucleotide and size variation, we newly sequenced four (H. clausa, H. jonesii, H. minor, and H. venusta) and analyzed six Hosta species (including the four, H. capitata and H. yingeri) distributed throughout South Korea. Results: The average size of complete chloroplast genomes for the Hosta taxa was 156,642 bp with a maximum size difference of ~ 300 bp. The overall gene content and organization across the six Hosta were nearly identical with a few exceptions. There was a single tRNA gene deletion in H. jonesii and four genes were pseudogenized in three taxa (H. capitata, H. minor, and H. jonesii). We did not find major structural variation, but there were a minor expansion and contractions in IR region for three species (H. capitata, H. minor, and H. venusta). Sequence variations were higher in non-coding regions than in coding regions. Four genic and intergenic regions including two coding genes (psbA and ndhD) exhibited the largest sequence divergence showing potential as phylogenetic markers. We found compositional codon usage bias toward A/T at the third position. The Hosta plastomes had a comparable number of dispersed and tandem repeats (simple sequence repeats) to the ones identified in other angiosperm taxa. The phylogeny of 20 Agavoideae (Asparagaceae) taxa including the six Hosta species inferred from complete plastome data showed well resolved monophyletic clades for closely related taxa with high node supports. Conclusions: Our study provides detailed information on the chloroplast genome of the Hosta taxa. We identified nucleotide diversity hotspots and characterized types of repeats, which can be used for developing molecular markers applicable in various research area.
... Absence of recombination between the plastid genomes suggested movement of intact organelles rather than naked, fragmented DNA (11,12). Indeed, chloroplasts rarely fuse (22), precluding recombination that enables new combinations of chloroplast genes. ...
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We report cell-to-cell movement of mitochondria through a graft junction. Mitochondrial movement was discovered in an experiment designed to select for chloroplast transfer from Nicotiana sylvestris into Nicotiana tabacum cells. The alloplasmic N. tabacum line we used carries Nicotiana undulata cytoplasmic genomes, and its flowers are male sterile due to the foreign mitochondrial genome. Thus, rare mitochondrial DNA transfer from N. sylvestris to N. tabacum could be recognized by restoration of fertile flower anatomy. Analyses of the mitochondrial genomes revealed extensive recombination, tentatively linking male sterility to orf293, a mitochondrial gene causing homeotic conversion of anthers into petals. Demonstrating cell-to-cell movement of mitochondria reconstructs the evolutionary process of horizontal mitochondrial DNA transfer and enables modification of the mitochondrial genome by DNA transmitted from a sexually incompatible species. Conversion of anthers into petals is a visual marker that can be useful for mitochondrial transformation.
... Later, as the early forms of gene transfer, protoplast cell fusion plants (i.e. cybrids) were developed in several laboratories [12][13][14][15][16]. ...
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In silico sequence diversities of four orthologous plant gsh1 genes and their anino acid translates of GSH1 proteins (Glutathione Synthase) were compared to the non-orthologous prokaryotic gshI/GSHI gene/protein of E. coli (NCBI # X03954). Primer pair was designed and transgene detection was carried out in two types of gshI-transgenic poplar clones (Populus x canescens) of ggs11 (cyt-ECS) and lgl6 (chl-ECS). Usefulness of genetic modification technologies (GMO) is indicated.
... Rapidly evolving sites could accumulate multiple mutations, which tend to be saturated and contribute to LBA [52][53][54] . Although the plastome has usually been considered a single linked locus, processes such as recombination, gene conversion, heteroplasmy, and incomplete lineage sorting may also cause a heterogeneous evolutionary rate among different plastid genes [55][56][57][58][59][60] . The concatenated plastome dataset composed of subsets with different evolutionary rates could generate well-supported but conflicting phylogenetic topologies 32,61 . ...
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Long-branch attraction (LBA) is a major obstacle in phylogenetic reconstruction. The phylogenetic relationships among Juniperus (J), Cupressus (C) and the Hesperocyparis-Callitropsis-Xanthocyparis (HCX) subclades of Cupressoideae are controversial. Our initial analyses of plastid protein-coding gene matrix revealed both J and C with much longer stem branches than those of HCX, so their sister relationships may be attributed to LBA. We used multiple measures including data filtering and modifying, evolutionary model selection and coalescent phylogenetic reconstruction to alleviate the LBA artifact. Data filtering by strictly removing unreliable aligned regions and removing substitution saturation genes and rapidly evolving sites could significantly reduce branch lengths of subclades J and C and recovered a relationship of J (C, HCX). In addition, using coalescent phylogenetic reconstruction could elucidate the LBA artifact and recovered J (C, HCX). However, some valid methods for other taxa were inefficient in alleviating the LBA artifact in J-C-HCX. Different strategies should be carefully considered and justified to reduce LBA in phylogenetic reconstruction of different groups. Three subclades of J-C-HCX were estimated to have experienced ancient rapid divergence within a short period, which could be another major obstacle in resolving relationships. Furthermore, our plastid phylogenomic analyses fully resolved the intergeneric relationships of Cupressoideae.
... In some cases, a manuscript can resemble a patchwork of two or more exemplars, with short sections from each interspersed. This is likely to prove very difficult to deal with, and has analogies in some cases of recombination where a large region of heteroduplex is produced between the recombining molecules and mismatches between the heteroduplex strands are resolved by the host repair machinery in different directions in different places 19 . This myghtty William duke of Northmandy That by just tytill And also by chyualery Conquered this land And kyng bycome And the kyng of Scotts he made his legeman (Bodleian Library, Bodley 131) ... with the rest of the text following the Kings of England II tradition. ...
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Frequently, letters, words, and sentences are used in undergraduate textbooks and the popular press as an analogy for the coding, transfer, and corruption of information in DNA.We discuss here how the converse can be exploited, by using programs designed for biological analysis of sequence evolution to uncover the relationships between different manuscript versions of a text.We point out similarities between the evolution of DNA and the evolution of texts.
... This could be advantageous in a region where and habitats are highly compartmentalized and variable steep rainfall clines, fire, and soil types across short geographical distances per growing season see (Fig. 5B) such as is typical in the CFR Lombard 2002, Linder 2003;Martínez-Cabrera 2012). Ostensibly, chloroplasts do not recombine (but see Medgyesy et al. 1985) and they would, therefore, adapt to rapid environmental changes slower than mitochondria would. Nevertheless, being able to maintain two (or more?) types of chloroplasts or mitochondria (heteroplasmy), implies that, should the circumstances require, such a plant could 'switch'. ...
... clearly not daughter plastids (Fig. 1). By overexpressing the membrane proteins in plants with plastids, in contrast to mitochondrial genomes (Rothenberg and Hanson, 1988), recombines very 208 rarely (Medgyesy et al., 1985;Clark et al., 1986). The genetic findings are consistent with the 209 absence of DNA in stromules and the rarity of interconnected plastids. ...
... The loss of GFP and loss of Str resistance in line A could be mediated by recombination of wild and transformed chloroplast DNA. The examples of chloroplast fusion and limited plastid gene transfer via recombination was described earlier (Medgyesy et al. 1985;Thanh and Medgyesy 1989). Maintenance of Sp resistance, but loss of Str resistance is difficult to explain by simple modification of aadA gene which encodes resistance to both antibiotics via an adenylyltransferase enzyme (EC 2.7.7.47). ...
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Key message: A new method based on mixing and wounding of callus tissue was used to transfer plastid or nuclear DNA between cells. Methods alternative to sexual hybridization can be powerful tools for crop improvement. We have developed a new hybridization technology based on wounding a mixed population of cells of two parents growing in vitro as callus ("cell grafting"), and have demonstrated the utility of this system for plastid or nuclear genome transfer. In our proof-of concept experiments, non-organized growing tissue (callus) from tobacco var. Samsun, carrying the nuclear marker genes nptII and uidA (GUS), and tobacco var. Petit Havana, carrying aadA and gfp genes in the plastid genome, were mixed together, wounded with a razor blade and placed for regeneration on selection medium containing both spectinomycin (aadA) and paromomycin (nptII). Plants with aadA and gfp positive plastids and nptII plus uidA positive nuclear background were produced. Molecular analysis confirmed the presence of all four genes in these plants. Morphology and ploidy level analysis confirmed the production of "diploid" plants similar to var. Samsun possessing transformed plastids from var. Petit Havana. Reciprocal crosses between the experimentally produced plants and wild type tobacco confirmed maternal inheritance of aadA and gfp and Mendelian inheritance of nptII and uidA. For transfer of nuclear traits between plants we used two nuclear-transformed parents with different selectable markers; one with nptII (paromomycin resistant), and another with aadA (spectinomycin resistant). Plants resistant to both antibiotics which also had different visible markers were produced.
... In some cases, they nuclear genome can also recombine. Chloroplast genome might also recombine under strong selective pressure, but it is a very rare event [261]. An example of CMS generated by somatic hybridization is that obtained by Atanassov et al. [13] from protoplast fusion of Nicotiana tabacum and Nicotiana alata, two sexually incompatible species. ...
Thesis
Nucleo-cytoplasmic male sterility (CMS) systems are natural systems controlled by two genetic factors: a cytoplasmic determinant causing male sterility and a nuclear determinant able to restore fertility. The Ogura CMS system is widely used for the production of hybrid seeds in cultivated Brassicas. The restorer locus of the Ogura system (Rfo) includes three highly related genes encoding PentatricoPeptide Repeat (PPR) proteins. Two of the most important questions regarding restorers of fertility concern the molecular mechanisms by which PPR restorers counter sterility, and the evolutionary relationships between related PPR genes found at restorer loci. The present study launched a functional analysis of the Rfo restorer protein (PPRB) and a study of the evolution of the Rfo locus in radish. A structure-function analysis of PPRB was undertaken in order to identify the protein domains or residues that are essential for impairing ORF138 accumulation. A functional test for the restorer activity was developed, based on the relative quantification of ORF138 in transgenic roots. For the evolutionary study the sequence of an allele of the Rfo locus obtained from a non-restorer genotype was determined. This allele carries two PPR genes closely related to those of the restorer sequence. The comparison of the two allele sequences showed that the Rfo locus evolved rapidly that intergenic and intragenic recombination operated during its evolution. Overall, the results described in this thesis contributes to a better understanding of the structural and biochemical features important for Rfo restoration activity and sheds light on the peculiar evolution of the Rfo locus.
... As plastids undergo fusion less readily than mitochondria (see below), and within-cell ptDNA heteroplasmy appears limited, the current picture is that ptDNA recombination between different ''chlorotypes'' over cellular timescales is comparatively limited (Greiner, 2012;Tonti-Filippini et al., 2017). Limited recombination has been observed between different plastid types in cells (Medgyesy et al., 1985;Thanh and Medgyesy, 1989;Day and Madesis, 2007), with segregation separating rather than homogenizing these populations. Recombination-dependent replication of ptDNA has been suggested to account for diversity in ptDNA structure (Ruhlman et al., 2017), and bioinformatic studies have suggested that rearrangements of gene order in ptDNA may provide adaptive advantages (Cui et al., 2006). ...
Article
Mitochondria and plastids form dynamic, evolving populations physically embedded in the fluctuating environment of the plant cell. Their evolutionary heritage has shaped how the cell controls the genetic structure and the physical behaviour of its organelle populations. While the specific genes involved in these processes are gradually being revealed, the governing principles underlying this controlled behaviour remain poorly understood. As the genetic and physical dynamics of these organelles are central to bioenergetic performance and plant physiology, this challenges both fundamental biology and strategies to engineer better-performing plants. This article will review current knowledge of the physical and genetic behaviour of mitochondria and chloroplasts in plant cells. An overarching hypothesis is proposed, whereby organelles face a tension between genetic robustness and individual control and responsiveness, and different species resolve this tension in different ways. As plants are immobile and therefore subject to fluctuating environments, their organelles are proposed to favour individual responsiveness, sacrificing genetic robustness. Several notable features of plant organelle dynamics including mtDNA recombination and plastid/mitochondrial differences may be explained by this hypothesis. Finally, the article highlights how tools from quantitative and systems biology can help shed light on the plethora of open questions in this field.
... Evidence is beginning to demonstrate that recombination in animal mtDNA is possible, at least in certain taxa (reviewed in Rokas et al. 2003). However, recombination has long been accepted in plant organellar genomes (Medgyesy et al. 1985;Barr et al. 2005;Fritsch et al. 2014), although mtDNA is more often found to undergo recombination than ptDNA. The mtDNA in plants is much larger than their animal counterparts (at least an order of magnitude), contains many repeat regions, and is now known to exist as multiple, distinct chromosomes, and not as single, circular molecules (Alverson et al. 2011;termed "cytoploid" in Sloan et al. 2012;Sloan 2013;Wu et al. 2015;Ramsey and Mandel 2019). ...
Article
Organellar genomes are considered to be strictly uniparentally-inherited. Uniparental inheritance allows for cytonuclear coevolution and the development of highly coordinated cytonuclear interactions. Yet, instances of biparental inheritance have been documented across eukaryotes. Biparental inheritance in otherwise uniparentally-inherited organelles is termed leakage (maternal or paternal) and allows for the presence of multiple variants of the same organellar genome within an individual, called heteroplasmy. It is unclear what, if any, evolutionary consequences are placed on nuclear and/or organellar genomes due to heteroplasmy. One way of accessing cytonuclear interactions and potential coevolution is through calculating cytonuclear linkage disequilibrium, or the non-random association of alleles between nuclear and organellar genomes. Patterns of cytonuclear LD (cnLD) can indicate positive or negative cytonuclear selection, coevolution between the nuclear and organellar genomes, non-traditional organellar inheritance, or instances of ancestral heteroplasmy. In plants, cytonuclear interactions have been shown to play a role in cytoplasmic male sterility (CMS) which occurs in gynodioecious species and is associated with leakage. We used the gynodioecious species, Daucus carota L. spp. carota, or wild carrot, to investigate cnLD. We genotyped a total of 265 individuals from two regions of the United States at 15 nuclear microsatellites, the mitochondrial genes cox1 and atp9, and an intergenic region between trnS and trnG (StoG) in the plastid genome to calculate nuclear-nuclear LD (nucLD), cnLD, and organellar LD (i.e. within the mtDNA and between mtDNA and ptDNA) within the two regions. We were further able to identify cox1 and StoG heteroplasmy and calculate some of the same LD measures within heteroplasmic and homoplasmic (non-heteroplasmic) datasets. We used a Z-transformation test to demonstrate that heteroplasmic individuals display significantly higher levels of cnLD within both regions. In spite of this, within and between organellar LD is low to moderate. Given these patterns of LD in two regions of the US in which gene flow has been shown to occur between crop and wild carrot, we suggest that heteroplasmy is an evolutionary mechanism which permits the maintenance of cnLD while also acting to disrupt organellar LD.
... Meanwhile, the functional enrichment analysis showed that PDGFA was involved in MAPK signaling pathway, pathways in cancer, and transcriptional dysregulation in cancer. TMC8 plays an important role in the transmembrane channel-like domain [33,34], and its mutation is associated with high-risk HPV infection and HNSCC survival risk [33]. YIPF4 is a new cell-binding molecule of papillomavirus E5 [35], which is involved in regulating membrane dynamics in the endomembrane system [36]. ...
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Head and neck squamous cell carcinoma (HNSCC), one of the most common cancers with high morbidity and mortality rates worldwide, has a poor prognosis. The transcriptome sequencing data of 500 patients with HNSCC in the TCGA dataset were assessed to find biomarkers associated with HNSCC prognosis so as to improve the prognosis of patients with HNSCC. The patients were divided into the training and testing sets. A model of six mRNAs (FRMD5, PCMT1, PDGFA, TMC8, YIPF4, ZNF324B) that could predict patient prognosis was identified in the training set using the Cox regression analysis. According to this model, the patients were divided into highrisk and low-risk groups. The Kaplan-Meier analysis showed that the high-risk group showed significantly shorter overall survival time compared with the low-risk group in both training and testing sets. The receiver operating characteristic analysis further confirmed high sensitivity and specificity for the model, which was more accurate compared with some known biomarkers in predicting HNSCC prognosis. Moreover, the model was applicable to patients of different ages, genders, clinical stages, tumor locations, smoking history, and human papillomavirus (HPV) status, as well as to microarray dataset. This model could be used as a novel biomarker for the prognosis of HNSCC and a significant tool for guiding the clinical treatment of HNSCC. The risk score acquired from the model might contribute to improving outcome prediction and management for patients with HNSCC, indicating its clinical significance.
... Most somatic hybrid plants contain one or the other plastid genome, evidently as a result of segregation of different plastid types during regeneration (Hanson et al., 1985). By imposing selection for drug resistances carried by each of the parental plastid genomes, Medgyesy et al. (1985) were able to obtain somatic hybrids carrying both resistance genes, resulting from recombination of plastid genomes originally separate in different organelles. However, the frequency of recombinant plastids was extremely low. ...
Article
Green fluorescent stroma filled tubules (stromules) emanating from the plastid surface were observed in transgenic plants containing plastid-localized green fluorescent protein (GFP). These transgenic tobacco plants were further investigated by epifluorescence and confocal laser scanning microscopy (CSLM) to identify developmental and/or cell type specific differences in the abundance and appearance of stromules and of plastids. Stromules are rarely seen on chlorophyll-containing plastids in cell types such as trichomes, guard cells or mesophyll cells of leaves. In contrast, they are abundant in tissues that contain chlorophyll-free plastids, such as petal and root. The morphology of plastids in roots and petals is highly dynamic, and plastids are often elongated and irregular. The shapes, size, and position of plastids vary in particular developmental zones of the root. Furthermore, suspension cells of tobacco exhibit stromules on virtually every plastid with two major forms of appearance. The majority of cells show a novel striking ‘octopus- or millipede-like’ structure with plastid bodies clustered around the nucleus and with long thin stromules of up to at least 40 (micro)m length stretching into distant areas of the cell. The remaining cells have plastid bodies distributed throughout the cell with short stromules. Photobleaching experiments indicated that GFP can flow through stromules and that the technique can be used to distinguish interconnected plastids from independent plastids.
... Plastid recombination is generally regarded as rare (Birky 1995), but several recent studies have highlighted potential cases of heteroplasmic http://mc.manuscriptcentral.com/systbiol recombination (Sullivan et al. 2017;Sancho et al. 2018), and this phenomenon has been documented in the laboratory (Medgyesy et al. 1985). We hesitate to attribute any of our observed conflict to such causes, as explicit documentation of heteroplasmic recombination is a challenging task, beyond the scope of this study. ...
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Phylogenomic analyses have helped resolve many recalcitrant relationships in the angiosperm tree of life, yet phylogenetic resolution of the backbone of the Leguminosae, one of the largest and most economically and ecologically important families, remains poor due to generally limited molecular data and incomplete taxon sampling of previous studies. Here, we resolve many of the Leguminosae's thorniest nodes through comprehensive analysis of plastome-scale data using multiple modified coding and noncoding datasets of 187 species representing almost all major clades of the family. Additionally, we thoroughly characterize conflicting phylogenomic signal across the plastome in light of the family's complex history of plastome evolution. Most analyses produced largely congruent topologies with strong statistical support, and provided strong support for resolution of some long-controversial deep relationships among the early diverging lineages of the subfamilies Caesalpinioideae and Papilionoideae. The robust phylogenetic backbone reconstructed in this study establishes a framework for future studies on legume classification, evolution, and diversification. However, conflicting phylogenetic signal was detected and quantified at several key nodes that prevents the confident resolution of these nodes using plastome data alone.
... Highly asymmetric hybrids to which the irradiated parent almost exclusively contributed the cytoplasmic DNA and the other parent donated both cytoplasmic and nuclear DNA (i.e., the socalled cybrids, obtained by the donor-recipient method) proved to be a useful tool in producing interspecific mitochondrial recombinants and provided a fast method of transferring whole plasmons or selected cytoplasmic traits from one species to another (Kumashiro and Kubo 1986;. Cytoplasmic recombinants are practically impossible to obtain by sexual hybridization because in Nicotiana, as in most other genera, the cytoplasmic DNA is inherited unilaterally through the maternal lineage, save for some rare exceptions (Medgyesy et al. 1985;Horlow et al. 1990). ...
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There are more than 80 naturally occurring relatives of cultivated tobacco (Nicotiana tabacum L.) in the genus Nicotiana. In this review, we examine how and to what extent these natural germplasm resources have been utilized in hybridization and introgression experiments over the past century. To date, more than 400 interspecific Nicotiana hybrids have been reported. We focus on individual Nicotiana species involved in interspecific hybrids with cultivated tobacco produced by sexual and asexual methods, including the recently discovered grafting method. Problems related to the hybridization of N. tabacum with other species, namely, cross-incompatibility, maternal phenotypes in hybrid offspring, interspecific incongruity, lethality of juvenile hybrids, and sterility of viable hybrids, are reviewed. Among the 58 interspecific hybrids involving N. tabacum reported thus far, 25 were also reported as somatic hybrids and two were obtained only as somatic hybrids. Thirty-six sterile sexual F1 hybrids were converted to fertile or partly fertile allopolyploids. Sixteen Nicotiana species have been deployed as a source of usable traits that were introgressed into N. tabacum, offering resistance to or tolerance of pathogens or pests. The mechanisms of introgression, such as alien addition and substitution, as well as the barriers and limitations of introgression, including erratic inheritance and adverse linkages, are discussed. Thirty-one Nicotiana species used as sources of cytoplasmic male sterility in N. tabacum have produced multiple alloplasmics; most showed negative effects of alien cytoplasm but a few have been deployed successfully in hybrid cultivars of N. tabacum.
... These results confirmed that in weck jar no 5 there was at least 1 plant with both celery and carrot chloroplasts, which thus was a true asymmetric somatic hybrid between the two species. Many somatic hybrids contain mitochondria or plastid sequences from both parents in species like chicory , citrus (Guo et al., 2004), Nicotiana (Medgyesy et al., 1985), Petunia (Kumar et al., 1982), Brassica (Sundberg and Glimelius, 1991), etc. ...
... rarely occurs in angiosperms because of the predominance of uniparental inheritance. Nevertheless, evidence of intermolecular homologous recombination in flowering plants has been reported 32,33 . To date, studies screening plastome recombination in the taxa are entirely lacking. ...
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Adrinandra megaphylla Hu is a medicinal plant belonging to the Adrinandra genus, which is well-known for its potential health benefits due to its bioactive compounds. This study aimed to assemble and annotate the chloroplast genome of A. megaphylla as well as compare it with previously published cp genomes within the Adrinandra genus. The chloroplast genome was reconstructed using de novo and reference-based assembly of paired-end reads generated by long-read sequencing of total genomic DNA. The size of the chloroplast genome was 156,298 bp, comprised a large single-copy (LSC) region of 85,688 bp, a small single-copy (SSC) region of 18,424 bp, and a pair of inverted repeats (IRa and IRb) of 26,093 bp each; and a total of 51 SSRs and 48 repeat structures were detected. The chloroplast genome includes a total of 131 functional genes, containing 86 protein-coding genes, 37 transfer RNA genes, and 8 ribosomal RNA genes. The A. megaphylla chloroplast genome indicated that gene content and structure are highly conserved. The phylogenetic reconstruction using complete cp sequences, matK and trnL genes from Pentaphylacaceae species exhibited a genetic relationship. Among them, matK sequence is a better candidate for phylogenetic resolution. This study is the first report for the chloroplast genome of the A. megaphylla .
... We also revealed that N. knightiana is more closely related to N. rustica than N. paniculata, which can be further corroborated by the distribution of indels highlighted in the present study. The paternal inheritance of plastid genomes was observed in Nicotiana under certain stressed conditions (Medgyesy, Fejes & Maliga, 1985;Medgyesy, Páy & Márton, 1986;Thang & Medgyesy, 1989;Avni & Edelman, 1991;Ruf, Karcher & Bock, 2007;Thyssen, Svab & Maliga, 2012;) Such low-frequency paternal leakage of plastids via pollen was suggested to be universal in plants with strict maternal plastid inheritance (Azagiri & Maliga, 2007). Thus, we expect that the plastids in the putative parents of N. rustica are maternally inherited. ...
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Species of the genus Nicotiana (Solanaceae), commonly referred to as tobacco plants, are often cultivated as non-food crops and garden ornamentals. In addition to the worldwide production of tobacco leaves, they are also used as evolutionary model systems due to their complex development history, tangled by polyploidy and hybridization. Here, we assembled the plastid genomes of five tobacco species: N. knightiana, N. rustica, N. paniculata, N. obtusifolia and N. glauca. De novo assembled tobacco plastid genomes had the typical quadripartite structure, consisting of a pair of inverted repeat (IR) regions (25,323–25,369 bp each) separated by a large single-copy (LSC) region (86,510 – 86,716 bp) and a small single-copy (SSC) region (18,441–18,555 bp). Comparative analyses of Nicotiana plastid genomes with currently available Solanaceae genome sequences showed similar GC content, gene content, codon usage, simple sequence repeats, oligonucleotide repeats, RNA editing sites, and substitutions. We identified 20 highly polymorphic regions, mostly belonging to intergenic spacer regions (IGS), which could be suitable for the development of robust and cost-effective markers for inferring the phylogeny of the genus Nicotiana and family Solanaceae. Our comparative plastid genome analysis revealed that the maternal parent of the tetraploid N. rustica was the common ancestor of N. paniculata and N. knightiana, and the later species is more closely related to N. rustica. Relaxed molecular clock analyses estimated the speciation event between N. rustica and N. knightiana appeared 0.56 Ma (HPD 0.65–0.46). Biogeographical analysis supported a south-to-north range expansion and diversification for N. rustica and related species, where N. undulata and N. paniculata evolved in North/Central Peru, while N. rustica developed in Southern Peru and separated from N. knightiana, which adapted to the Southern coastal climatic regimes. We further inspected selective pressure on protein-coding genes among tobacco species to determine if this adaptation process affected the evolution of plastid genes. These analyses indicate that four genes involved in different plastid functions, including DNA replication (rpoA) and photosynthesis (atpB, ndhD and ndhF), came under positive selective pressure as a result of specific environmental conditions. Genetic mutations in these genes might have contributed to better survival and superior adaptations during the evolutionary history of tobacco species. Keywords: Mutational hotspots, Nicotiana, plastid genomes, positive selection, substitutions, speciation
... Given the highly active recombination system functioning in plastids could there be a relationship between the presence of two plastome genotypes and genomic instability? Extensive recombination demonstrated in somatic hybrids and male sterile lines of Nicotiana [260][261][262] and through the incorporation of foreign sequences using plastid transformation strategies leaves little room for the notion that different plastome genotypes cannot recombine. Further evidence for ptDNA recombination has been documented in interspecific somatic hybrids of Solanum [263] and Brassica [264], and in wild populations of lodgepole pine [265]. ...
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The plastid genome (plastome) has proved a valuable source of data for evaluating evolutionary relationships among angiosperms. Through basic and applied approaches, plastid transformation technology offers the potential to understand and improve plant productivity, providing food, fiber, energy, and medicines to meet the needs of a burgeoning global population. The growing genomic resources available to both phylogenetic and biotechnological investigations is allowing novel insights and expanding the scope of plastome research to encompass new species. In this chapter, we present an overview of some of the seminal and contemporary research that has contributed to our current understanding of plastome evolution and attempt to highlight the relationship between evolutionary mechanisms and the tools of plastid genetic engineering.
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Chloroplast nucleoids are large, compact nucleoprotein structures containing multiple copies of the plastid genome. Studies on structural and quantitative changes of plastid DNA (ptDNA) during leaf development are scarce and have produced controversial data. We have systematically investigated nucleoid dynamics and ptDNA quantities in mesophyll of Arabidopsis, tobacco, sugar beet, and maize from the early post-meristematic stage until necrosis. DNA of individual nucleoids was quantified by DAPI-based supersensitive epifluorescence microscopy. Nucleoids occurred in scattered, stacked or ring-shaped arrangements and in recurring patterns during leaf development remarkably similar between the species studied. Nucleoids per organelle varied from few in meristematic plastids to> 30 in mature chloroplasts (corresponding to about 20-750 nucleoids per cell). Nucleoid ploidies ranged from haploid to> 20-fold even within individual organelles, with average values between 2.6- and 6.7-fold and little changes during leaf development. DNA quantities per organelle increased gradually from about a dozen plastome copies in tiny plastids of apex cells to 70-130 copies in chloroplasts of about 7 μm diameter in mature mesophyll tissue, and from about 80 plastome copies in meristematic cells to 2,600-3,300 copies in mature diploid mesophyll cells without conspicuous decline during leaf development. Pulsed-field electrophoresis, restriction of high-molecular weight DNA from chloroplasts and gerontoplasts, and CsCl equilibrium centrifugation of single- and double-stranded ptDNA revealed no noticeable fragmentation of the organelle DNA during leaf development, implying that plastid genomes in mesophyll tissues are remarkably stable until senescence.
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Extensive progress has been made in the last couple of years in the fields of plant cell genetics and gene technology. Techniques of asexual recombination of genomes, chromosomes or genes have been improved and applied to more species including cultivars of various crop plants. Scientific areas where particularly large steps forward have been taken are (1) establishment of new hybrids by protoplast fusion, (2) induction of somaclonal variation, (3) elucidation of the structure and function of mobile genetic elements (transposons) and (4) genetic engineering via construction and application of different vector systems.
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Somatic hybridization can be used to produce various combinations of organellar and nuclear genomes [20]. Furthermore, the fact that organelle populations from both fusion partners are present, at least initially in the same heterokaryon, creates an opportunity for interspecific recombination in the organelle genomes. This has made protoplast fusion a useful approach to produce hybrids for both compatible [23, 25, 26] and incompatible species [1, 7].
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Plant organ, tissue and cell culture procedures have developed rapidly in the half century since the pioneering efforts of Laibach, Gautheret, Nobecourt and White (1). Micropropagation may generally be defined as in vitro regeneration of plants from organs, tissues, cells, or protoplasts. Availability of micropropagation techniques vary widely among species. Within species, micropropagation protocols vary with objectives.
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Mutants resistant to different antibiotics have proved to be extremely useful in genetic studies on unicellular organisms like Chlamydomonas and yeast. Many of these mutations are uniparentally inherited and have been located in either chloroplast or mitochondrial genomes. Using these mutants, segregation and recombination of organelles could be investigated in lower eukaryotes. Cell genetic techniques are now sophisticated enough to allow similar studies on higher plants, but the study of organelle segregation and recombination in heteroplasmic cells obtained by protoplast fusion requires the use of selectable, cytoplasmically coded mutations. Such mutants have been available only in a small number of flowering plants. In Nicotiana, the model genus for cell genetics, plastid mutant plants have been produced resistant to streptomycin (Maliga et al. 1973) and spectinomycin (Fluhr et al. 1985), each carrying a single nucleotide change at a different position in the 16S rRNA gene (Etzold et al. 1987; Fromm et al. 1987). Lincomycin-resistant mutants (Cséplö and Maliga 1982, 1984; Cséplö et al. 1984) carry point mutations in the 23S rRNA gene (Cséplö et al. 1988). Using photomixotrophic cultures, a terbutryn-resistant plastid mutant has also been isolated (Cséplö et al. 1985) which possessed a point mutation in the 32-kDa protein gene (Páy et al. 1988). Resistant calli and seedlings formed in the presence of the drugs are normal green, in contrast to the complete bleaching of sensitive ones, giving an excellent visual marker. Antibiotic selection has been shown to have a principal role in the selection of rare genetic events such as chloroplast recombination or transformation (Medgyesy et al. 1985; Sváb et al. 1990).
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Transposition events have created chloroplast-DNA-derived ribosomal protein pseudogenes in chloroplast (Bowman et al 1988, Shimada and Sugiura 1989) and mitochondrial (eg Moon et al 1988) genomes of some higher plants. Examining the role of homologous recombination in the evolutionary fate of these ribosomal protein genes and their pseudogenes, can offer some insight into the possible influence of homologous recombination on the evolution of organelle genomes in general.
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The presence of cytoplasmic male sterility (CMS) in oilseed crops and its importance in developing F1 hybrids has led to a plethora of research activity in cytoplasmic genome manipulations. This has involved both traditional breeding methodologies and, relatively more recent, protoplast technology. The backcross substitution of the nucleus of one species into the cytoplasmic background of the alien species has been utilized extensively in Brassica to produce alloplasmic lines. Many of these alloplasmic lines are CMS. Alternatively, the protoplast fusion provides a possibility of producing any desired nuclear/cytoplasmic combination and, more significantly, any chloroplast/ mitochondrial combination within the cytoplasm. The resultant cytoplasmic hybrids are referred to as cybrids. The ability to substitute specific chloroplast populations or plastomes independent of the mitochondrial genome (chondriome) or vice versa is, with a few exceptions, a technique exclusive to the protoplast fusion. This is in comparison to the backcross substitution, where only whole cytoplasm can be transferred.
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The chloroplast and mitochondrial genome (plastome and chondriome) have a considerable impact on crop characteristics of breeding importance. Their role in the energy household of the plant may be directly linked to growth, harvest time, and grain yield. They can also determine such defined traits as pathotoxin resistance, herbicide resistance, temperature tolerance, and male sterility. A notable aspect of plant breeding is that nucleocytoplasmic heterosis might have an important role in hybrid vigor (discussed by Srivastava 1983). Furthermore, the need for broadening the cytoplasmic diversity within a crop species is of a general importance in itself since the hard lesson learnt from the universal use of the Texas male sterile cytoplasm, which turned out to be susceptible to the fungus causing southern corn leaf blight disease in corn.
Article
The species tree paradigm that dominates current molecular systematic practice infers species trees from collections of sequences under assumptions of the multispecies coalescent (MSC), i.e., that there is free recombination between the sequences and no (or very low) recombination within them. These coalescent genes (c-genes) are thus defined in an historical rather than molecular sense, and can in theory be as large as an entire genome or as small as a single nucleotide. A debate about how to define c-genes centers on the contention that nuclear gene sequences used in many coalescent analyses undergo too much recombination, such that their introns comprise multiple c-genes, violating a key assumption of the MSC. Recently a similar argument has been made for the genes of plastid (e.g., chloroplast) and mitochondrial genomes, which for the last 30 or more years have been considered to represent a single c-gene for the purposes of phylogeny reconstruction because they are non-recombining in a historical sense. Consequently, it has been suggested that these genomes should be analyzed using coalescent methods that treat their genes-over 70 protein-coding genes in the case of most plastid genomes (plastomes)-as independent estimates of species phylogeny, in contrast to the usual practice of concatenation, which is appropriate for generating gene trees. However, although recombination certainly occurs in the plastome, as has been recognized since the 1970's, it is unlikely to be phylogenetically relevant. This is because such historically effective recombination can only occur when plastomes with incongruent histories are brought together in the same plastid. However, plastids sort rapidly into different cell lineages and rarely fuse. Thus, because of plastid biology, the plastome is a more canonical c-gene than is the average multi-intron mammalian nuclear gene. The plastome should thus continue to be treated as a single estimate of the underlying species phylogeny, as should the mitochondrial genome. The implications of this long-held insight of molecular systematics for studies in the phylogenomic era are explored.
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In recent years, several methods for somatic gene transfer in plants have been developed (see Bajaj 1993). If the gene of interest has been cloned, advanced transformation systems based on viral or bacterial vectors provide an efficient way of introducing it into a desired recipient genome (Potrykus 1990). Unfortunately, the genetic and biochemical basis of many economically important traits such as disease resistance, stress tolerance, yield increase, etc. are largely unknown, and these traits might be subjected to complex regulatory mechanisms which will complicate the molecular biological approaches to elucidating their genetic and biochemical basis. Therefore, although very powerful cloning methodologies are available, the isolation and characterization of these traits should be anticipated on a relatively long-term basis. For the transfer of genes which are not available as cloned DNA sequences or for transferring “blocks” of genes of agronomic interest, somatic cell hybridization via protoplast fusion represents a good alternative method (Negrutiu et al. 1989a, b; Glimelius et al. 1991).
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Brassica species, particularly B. rapa (syn. B. campestris), B. oleracea, and B. napus, have been extensively used in protoplast fusion experiments, for several reasons. These species have great economic value, because they include important vegetables (cabbage, broccoli, cauliflower, brussels sprouts, kale, etc., in B. oleracea) as well as oilseeds (in B. napus, B. rapa). Brassica is also a relatively tractable genus in vitro. There are substantial variations in regenerability from expiants and protoplasts both within and among species (with B. rapa still largely recalcitrant), but the good response of many B. oleracea and B. napus lines favors recovery of plants from fusion products. Furthermore, protoplast fusion is well suited as a solution to several horticultural and agronomic problems encountered with Brassica crops.
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In higher plants, sexual reproduction generally prevents any exchange of cytoplasmic information between the two parents, and chloroplastic as well as mitochondrial genomes are maternally inherited. However, induced fusion of isolated protoplasts followed by regeneration of entire plants allows the mixing in the same cell of organelles and the screening or selection in resulting individuals of recombined forms of cytoplasmic genetic characteristics. These forms are called cybrids, for cytoplasmic hybrids. The originality of cybrids resides in their possibilities to associate in the regenerated plant, after a more or less stochastic sorting-out process, chloroplasts from one parent with mitochondria from the other, or to create new cytoplasmic genomes resulting from interparental recombinations.
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Plant breeders have, for a long time, been tempted to cross very different species to create new living forms. Interspecific crosses have been extensively used and in some cases produced new species as, for example, triticale. To overcome sexual barriers, in vitro fertilization and in vitro embryo or ovary culture are technical refinements which have been used on a relatively limited scale, because each combination between two species brings specific problems. Protoplast fusion was proposed very early as a possible way to produce new hybrid forms (Kuster, 1909) by radically eliminating sexual barriers. Nevertheless it was known that interspecific incompatibilities occur not only before and during the fertilization process, but also later, when the embryo and the endosperm co-differentiate in the maternal tissues, and even later, when the young plantlet develops after germination.
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The reasons for subjecting the tomato eultivar, Lycopersicon esculentum, to numerous cell and tissue culture studies have been twofold: agronomic improvement of this important crop species and exploitation of tomatO’s abundant classical genetics for basic research purposes. Several years ago we began experiments utilizing protoplasts of Lycopersicon species, with the goals of developing methods to culture and regenerate plants, influencing transmission of genomes following cell fusion, and genetically analyzing plants resulting from protoplast or fusion product regeneration. At the time, little information was available about the requirements for successful culture and regeneration of members of the tomato genus. We looked to the facile protoplast culture and fusion of other Solanaceous species, Nicotiana and Petunia, as guides, as well as the arduous but effective methods for potato protoplasts developed by Shepard et al. (1980). Here we will summarize our knowledge of Lycopersicon protoplast culture and fusion and the analysis of regenerated plants.
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Cichorium intybus is a member of the Compositae and has attractive blue flowers. Chicory is the ‘traditional’ western European winter salad, but as a result of recent technical progress which has led to reliable ways of forcing roots to give firm and clean chicons, chicory is now cultivated all over the world. Since most countries are only starting chicory production, one can predict an increasing demand for this very tasty and easy to use salad. Chicory breeding will also become more active with the identification of new objectives, which the application of novel in vitro techniques, including the use of isolated protoplasts, may help to achieve (see Schoofs and de Langhe 1988).
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Interspecific and intergeneric hybridization has been successfully employed for plant improvement. However, due to incompatibility barriers, many sexual crosses are not possible. Moreover, cytoplasmic genetic elements are known to be transmitted maternally in most higher plants (Gillham 1978). This limits, to some extent, the induction of variability in the cytoplasmic genome after crossing. With recent advancement in genetic engineering and biotechnology research (see Bajaj 1986), plant breeders are now able to combine classical and new technologies for plant improvement. Somatic hybridization overcomes the sexual incompatibility and uniparental inheritance of cytoplasmic traits, and provides a wide range of nuclear cytoplasmic genetic combinations.
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The plasma membrane of plant cells is surrounded by a cellulose wall and adjacent cells are connected with a pectin-rich matrix. Disconnection of plant cells and removal of the cell wall experimentally, by either a mechanical or an enzymatic process, results in the production of single, `naked’ plant cells termed `isolated protoplasts’. Protoplasts thus can be considered as special individual plant cells with fully exposed outer plasma membrane which is the only barrier between the external environment and the interior of the living cells. However, plant protoplasts cannot serve as the botanical analog of animal cells, but they should be viewed as injured cells (see Section 2.3.2) that have to go through a repair process before they are capable of sustained division.
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Green plants and photosynthetic organisms are unique among eukaryotes since they possess three genomes: the nuclear, the mitochondrial and the plastid. The first indication that genes were present in the cytoplasm was discovered by the inheritance of certain traits that did not follow Mendel’s rules. Even though these observations were made as early as the beginning of the 20th century, it was not until the 1960s that convincing physical evidence was obtained regarding presence of unique DNA in chloroplasts (Chun et al. 1963; Sager and Ishida 1963) and mitochondria (Luck and Reich 1964). Since then, the rapid development in molecular techniques has contributed enormously to our knowledge about genome organization, gene content and gene structure. For details of the genetics and molecular biology of the organelles in higher plants, the readers are referred to review articles in the field (Lonsdale 1989; Gray 1990; Bogorad and Vasil 1991; Herrmann 1992).
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The terms symmetric and asymmetric hybrids refer to the nuclear constitution of fusion products. Symmetric hybrids are defined as consisting of complete sets of chromosomes from both the parents, while asymmetric ones result from a preferential or unidirectional loss of chromosomes belonging to one of the fusion partners. Since protoplasts can be isolated from almost any plant species and readily induced to fuse in all possible combinations, it was expected and even accepted that fusion technology was going to make important contributions in both basic and applied research. This matter was critically evaluted by Harms (1983 a), Burgess (1984) and reviewed in detail by Gleba and Sytnik (1984) and Horn et al. (1986).
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Plastome (plastid genome) engineering has grown up and got smarter for the transgene expression. Plastid transformation has profound benefits over nuclear transformation, includes a higher level of transgene expression, integration via homologous recombination, transgene containment, lack of gene silencing, and position effect. Substantial and fruitful progress has been achieved in plastome engineering of vegetable crops through the use of improved regeneration/selection procedures, plastid transformation vectors with efficient promoters, and 3/, 5/regulatory sequences. Plastid transformation technology developed for vegetable crops being used as a platform for the production of industrially important proteins and some of the genes of agronomic importance has been stably integrated and expressed in plastome. Although great progress has been accomplished in the plastid transformation of vegetable crops, still it is restricted to few species because of the unavailability of whole plastome sequencing. In this review, the author focus on the technology, progress, and advancements in plastid transformation of vegetable plants such as lettuce, tomato, potato, cabbage, cauliflower, eggplant, carrot, soybean, and bitter melon are reviewed. The conclusions, future prospects, and expansion of plastid transformation technology to other vegetable crops for genetic improvement and production of edible vaccines are proposed.
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Few pan-genomic studies have been conducted in plants, and none of them have focused on the intraspecific diversity and evolution of their plastid genomes. We address this issue in Brachypodium distachyon and its close relatives B. stacei and B. hybridum, for which a large genomic data set has been compiled. We analyze inter- and intraspecific plastid comparative genomics and phylogenomic relationships within a family-wide framework. Major indel differences were detected between Brachypodium plastomes. Within B. distachyon, we detected two main lineages, a mostly Extremely Delayed Flowering (EDF+) clade and a mostly Spanish (S+) – Turkish (T+) clade, plus nine chloroplast capture and two plastid DNA (ptDNA) introgression and micro-recombination events. Early Oligocene (30.9 million yr ago (Ma)) and Late Miocene (10.1 Ma) divergence times were inferred for the respective stem and crown nodes of Brachypodium and a very recent Mid-Pleistocene (0.9 Ma) time for the B. distachyon split. Flowering time variation is a main factor driving rapid intraspecific divergence in B. distachyon, although it is counterbalanced by repeated introgression between previously isolated lineages. Swapping of plastomes between the three different genomic groups, EDF+, T+, S+, probably resulted from random backcrossing followed by stabilization through selection pressure.
Article
Cytoplasmic organelles are inherited in a nonMendelian fashion in all eukaryotic organisms investigated. Among the seed plants, plastids can be inherited strictly from the female parent, strictly from the male parent, or biparentally. Most flowering plants studied to date exhibit maternal plastid inheritance, but approximately one-third of the genera investigated display biparental plastid inheritance to some degree. Among the gymnosperms, paternal plastid inheritance is the rule in the conifers, whereas the other groups appear to have maternal plastid inheritance, although they have been less well studied. Mitochondrial inheritance is predominantly maternal in the seed plants, except for a few coniferous families where it is predominantly paternal. The advent of recombinant DNA technology has allowed restriction fragment length polymorphisms to be used as molecular markers, and has stimulated much research in organelle inheritance and its application to studies of population genetics and phylogenetic biology. This report emphasizes the various mechanisms by which organelles are, or are not, transmitted among the seed plants in order that researchers directly or indirectly involved with organelle inheritance may better understand the potential and the limitations of their investigations. A summary and discussion of the possible evolutionary significance of the various patterns of cytoplasmic inheritance among the seed plants are also included.
Article
In the majority of higher plants there is maternal inheritance of cytoplasmic organelles and, as a consequence, there are few opportunities for the study of the effects on plant phenotype of having cytoplasm initially containing organelles of both parents. Now the availability of somatic plant protoplasts, which can be fused together and suitably cultured to produce somatic hybrid plants, is enabling the effects of such hybrid cytoplasms to be investigated in higher plants exhibiting maternal inheritance. A very wide range of cytoplasmic genetic diversity, including mitochondrial and chloroplast recombinants, can be produced by such somatic hybridizations, and a theoretical model is presented to show the origins of this wide range of cytoplasmic diversity. Cybrids produced by such protoplast fusions have been shown to be of importance in plant breeding especially in relation to transfer of cytoplasmic male sterility and herbicide resistance. Protoplast fusion, including the fusion of gametic and somatic protoplasts, is also enabling the study of the inheritance of cytoplasmic controlled traits in higher plants.
Article
In living organisms, DNA molecules are included in structures that allow their transmission from generation to generation as well as exchanges with other DNA molecules. The designation “chromosome” is reserved for DNA molecules that are consistently found in all individuals of the same species, while plasmids or episomes are used to designate those whose presence is inconstant. Sexual reproduction, which should rather be referred to as “gametic reproduction”, involves the fusion of two gametes to form an “egg” or zygote with a double number (diploid) of chromosomes. In all cases, meiosis must produce haploid cells that contain a complete set of chromosomes. Many microorganisms have a clonal mode of reproduction that may be exclusive or, on the contrary, associated to some degree with sexual reproduction. Clonal reproduction also concerns certain multicellular organisms where it may be exclusive or associated with sexual reproduction.
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Differences in the distribution of Ava I and BstEII restriction sites in the chloroplast DNA (cpDNA) of Chlamydomonas eugametos and C. moewusii have been used to detect extensive cpDNA recombination in the hybrid progeny of these interfertile algae. In the present study, the inheritance of these restriction-site differences was tested for recombination with nonmendelian genetic markers for resistance to streptomycin and erythromycin in interspecific crosses and in hybrid backcrosses to C. moewusii. Most of the restriction-pattern markers appear linked to the antibiotic-resistance markers, thus supporting the chloroplast localization of the resistance markers. The streptomycin marker, in particular, shows perfect coordinate inheritance with an Ava I band containing one cpDNA fragment and a BstEII band containing two comigrating cpDNA fragments. Molecular hybridization experiments using DNA from the Ava I band as a probe show sequence homology between this DNA, the two comigrating BstEII fragments, and cpDNA fragments from C. reinhardtii containing the genes for 16S rRNA. The results show the feasibility of using C. eugametos-C. moewusii hybrids to identify cpDNA sequences that either contain or are closely linked to nonmendelian genetic markers.
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Chloroplasts of Nicotiana tabacum SR1 were transferred into Nicotiana plumbaginifolia by protoplast fusion. The protoplasts of the organelle donor were irradiated with different lethal doses using a (60)Co source, to facilitate the elimination of their nuclei from the fusion products. After fusion induction, clones derived from fusion products and containing streptomycin-resistant N. tabacum SR1 chloroplasts were selected by their ability to green on a selective medium. When N. tabacum protoplasts were inactivated by iodoacetate instead of irradiation, the proportion of N. plumbaginifolia nuclear segregant clones was low (1-2%). Irradiation markedly increased this value: Using 50, 120, 210 and 300 J kg(-1) doses, the frequency of segregant clones was 44, 57, 84 and 70 percent, respectively. Regeneration of resistant N. plumbaginifolia plants with SR1 chloroplasts indicated that plastids can be rescued from the irradiated cells by fusion with untreated protoplasts. Resistant N. plumbaginifolia plants that were regenerated (43 clones studied) had diploid (2n = 2X = 20) or tetraploid chromosome numbers and were identical morphologically to parental plants. The absence of aneuploids suggests that in these clones irradiation resulted in complete elimination of the irradiated N. tabacum nuclei. Resistance is inherited maternally (five clones tested). The demonstration of chloroplast transfer and the presence of N. tabacum plastids in the N. plumbaginifolia plants was confirmed by chloroplast DNA fragmentation patterns after EcoRI digestion.
Article
THE possibility of producing mutant plants of economic value by tissue culture techniques has attracted considerable interest and discussion1-6, and was confirmed by the regeneration of plants from mutant cell lines1. The isolation of cytoplasmic mutants (all of those studied so far have carried Mendelian traits1,2) can be of practical importance; for example, male sterile ones in maize may be used for producing hybrid seeds. We report here, however, properties of a non-Mendelian, cytoplasmic mutant.
Article
Our previous studies indicated that fusion products with one functional nucleus but organelles of the two fusion partners (i.e. heteroplastomic cybrids) could be obtained by fusing X-irradiated (cytoplasmic donor) with non-irradiated (recipient) Nicotiana protoplasts. The present report deals with the analysis of mitochondria in cybrid populations resulting from the fusion of donor Nicotiana tabacum protoplasts with recipient protoplasts having a N. Sylvestris nucleus but chloroplasts of an alien Nicotiana species, and exhibiting cytoplasmic male sterility. The two fusion parents showed significant differences in restriction patterns of their chloroplast and mitochondrial DNA. Four groups of cybrid plants were obtained by this fusion. All had N. sylvestris nuclei but contained either donor or recipient chloroplasts and had either sterile or fertile anthers. There was no correlation between anther fertility and chloroplasts type. The mitochondrial DNA restriction patterns of sterile cybrids were similar to the respective patterns of the sterile fusion partner while the mitochondrial DNA restriction patterns of the fertile cybrids were similar to the respective patterns of the fertile fusion partner. The results indicate an independent assortment of chloroplasts and mitochondria from the heteroplastomic fusion products.
Covalently closed circular chloroplast DNA (ctDNA) molecules have been isolated from pea, bean, spinach, lettuce, corn and oat plants by ethidium bromide/cesium chloride density-gradient centrifugation. As much as 30–40% of the total ctDNA could be isolated as closed circular DNA molecules and up to 80% of the total ctDNA was found in the form of circular molecules.The size of pea, spinach, lettuce, corn and oat ctDNA relative to an internal standard (ΦX174 replicative form II monomer DNA) was determined by electron microscopy. The ctDNAs showed significant differences in their sizes, and their molecular weights ranged from 85.4 · 106 for corn ctDNA to 96.7 · 106 for lettuce ctDNA. Each of these ctDNAs contained 3–4% of the circular molecules as circular dimers and 1–2% of the circular molecules as catenated dimers.The molecular complexity of these ctDNAs was studied by renaturation kinetics using T4 DNA as a standard. The molecular weights of the unique sequence of the ctDNAs ranged from 83.7 · 106 for oat ctDNA to 93.1 · 106 for lettuce ctDNA, which are in excellent agreement with the sizes of the circular ctDNA molecules. No repeating sequences were detected in any of the ctDNAs.The ctDNAs from pea, lettuce, corn, and spinach were studied by thermal denaturation using T4 DNA as a standard. All of the ctDNAs melted more broadly than T4 DNA and they all had a distinctly different . For example, the of pea ctDNA was 1°C below the of T4 DNA and the of corn was 1.9°C higher than the of T4 DNA.
Article
Sixteen different mature interspecific parasexual hybrids, produced by fusing leaf protoplasts of Nicotiana glauca (G) and N. langsdorffii (L), were analyzed for fraction I protein (ribulose-1,5-bisphosphate carboxylase/oxygenase) which consists of large subunit polypeptides coded by chloroplast DNA and small subunit polypeptides coded by nuclear DNA. All the hybrids showed the combined small subunits of both parents, thus confirming the hybridity of each of the fusion products. Fourteen of the hybrids displayed the large subunit electrofocusing pattern characteristic of only one parent (eight L and six G). From one hybrid callus, two plants were regenerated, of which one had exclusively L-type large subunit and the other had exclusively G. A single plant retained a mixture of L ang G chloroplast DNA's; this later yielded six F2 progeny from one branch, all of which were G type, and three asexual progeny from another branch, all of which had the L-type pattern. In all, 46 F2 progeny and 8 different F3s were analyzed and each of these, with few if any exceptions, showed the same single subunit type as the F1 and F2 parent hybrid plants. Reasons for the rapid sorting out of the chloroplast types are discussed.
Article
Covalently closed circular choloroplast DNA (ctDNA) molecules have been isolated from pea, bean, spinach, lettuce, corn and oat plants by ethidium bromide/cesium choloride density-gradient entrifugation. As much as 30-40% of the total ctDNA could be isolated as closed circular DNA molecules and up to 80% of the total ctDNA was found in the form of circular molecules. The size of pea, spinach, lettuce, corn and oat ctDNA relative to an internal standard (phiX174 replicative form II monomer DNA) was determined by electron microscopy. The ctDNAs showed significant differences in their sizes, and their molecular weights ranged from 85.4 - 10(6) for corn ctDNA to 96.7 - 10(6) for lettuce ctDNA. Each of these ctDNAs contained 3-4% of the circular molecules as circular dimers and 1-2% of the circular molecules as catenated dimes. The molecular complexity of these ctDNAs was studied by renaturation kinetics using T4 DNA as a standard. The molecular weights of the unique sequences of the ctDNAs ranged from 83.7 - 10(6) for oat ctDNA to 93.1 - 10(6) for lettuce ctDNA, which are in excellent agreement with the sizes of the circular ctDNA molecules...
Article
All of the PstI restriction fragments of the chloroplast DNA of Nicotiana tabacum have been cloned in the plasmid vector pBR322. The cloned fragment sizes range from 0.8 to 26 kb, are stable, and can be amplified by chloramphenicol with varying efficiencies. Using these clones we have detailed a PstI physical map of the tobacco chloroplast genome. Selected clones of SalI, BamHI and PstI fragments were used to localize the map positions of the alpha, beta, and epsilon subunits of the chloroplast ATPase coupling factor, the large subunit of ribulosediphosphate carboxylase and the 32-kDal membrane protein. The gene products of these clones were characterized by RNA transcript sizing, immunoprecipitation of maxicell-directed protein synthesis, and hybrid-arrested translation.
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