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1.  Reference genomes and transcriptomes of Nicotiana sylvestris and Nicotiana tomentosiformis 
Genome Biology  2013;14(6):R60.
Nicotiana sylvestris and Nicotiana tomentosiformis are members of the Solanaceae family that includes tomato, potato, eggplant and pepper. These two Nicotiana species originate from South America and exhibit different alkaloid and diterpenoid production. N. sylvestris is cultivated largely as an ornamental plant and it has been used as a diploid model system for studies of terpenoid production, plastid engineering, and resistance to biotic and abiotic stress. N. sylvestris and N. tomentosiformis are considered to be modern descendants of the maternal and paternal donors that formed Nicotiana tabacum about 200,000 years ago through interspecific hybridization. Here we report the first genome-wide analysis of these two Nicotiana species.
Draft genomes of N. sylvestris and N. tomentosiformis were assembled to 82.9% and 71.6% of their expected size respectively, with N50 sizes of about 80 kb. The repeat content was 72-75%, with a higher proportion of retrotransposons and copia-like long terminal repeats in N. tomentosiformis. The transcriptome assemblies showed that 44,000-53,000 transcripts were expressed in the roots, leaves or flowers. The key genes involved in terpenoid metabolism, alkaloid metabolism and heavy metal transport showed differential expression in the leaves, roots and flowers of N. sylvestris and N. tomentosiformis.
The reference genomes of N. sylvestris and N. tomentosiformis represent a significant contribution to the SOL100 initiative because, as members of the Nicotiana genus of Solanaceae, they strengthen the value of the already existing resources by providing additional comparative information, thereby helping to improve our understanding of plant metabolism and evolution.
PMCID: PMC3707018  PMID: 23773524
2.  Molecular Linkage Mapping and Marker-Trait Associations with NlRPT, a Downy Mildew Resistance Gene in Nicotiana langsdorffii 
Nicotiana langsdorffii is one of two species of Nicotiana known to express an incompatible interaction with the oomycete Peronospora tabacina, the causal agent of tobacco blue mold disease. We previously showed that incompatibility is due to the hypersensitive response (HR), and plants expressing the HR are resistant to P. tabacina at all stages of growth. Resistance is due to a single dominant gene in N. langsdorffii accession S-4-4 that we have named NlRPT. In further characterizing this unique host-pathogen interaction, NlRPT has been placed on a preliminary genetic map of the N. langsdorffii genome. Allelic scores for five classes of DNA markers were determined for 90 progeny of a “modified backcross” involving two N. langsdorffii inbred lines and the related species N. forgetiana. All markers had an expected segregation ratio of 1:1, and were scored in a common format. The map was constructed with JoinMap 3.0, and loci showing excessive transmission distortion were removed. The linkage map consists of 266 molecular marker loci defined by 217 amplified fragment length polymorphisms (AFLPs), 26 simple-sequence repeats (SSRs), 10 conserved orthologous sequence markers, nine inter-simple sequence repeat markers, and four target region amplification polymorphism markers arranged in 12 linkage groups with a combined length of 1062 cM. NlRPT is located on linkage group three, flanked by four AFLP markers and one SSR. Regions of skewed segregation were detected on LGs 1, 5, and 9. Markers developed for N. langsdorffii are potentially useful genetic tools for other species in Nicotiana section Alatae, as well as in N. benthamiana. We also investigated whether AFLPs could be used to infer genetic relationships within N. langsdorffii and related species from section Alatae. A phenetic analysis of the AFLP data showed that there are two main lineages within N. langsdorffii, and that both contain populations expressing dominant resistance to P. tabacina.
PMCID: PMC3426812  PMID: 22936937
molecular markers; disease resistance; linkage mapping; AFLP; Nicotiana benthamiana
3.  Breeding systems, hybridization and continuing evolution in Avon Gorge Sorbus 
Annals of Botany  2013;111(4):563-575.
Background and Aims
Interspecific hybridization and polyploidy are key processes in plant evolution and are responsible for ongoing genetic diversification in the genus Sorbus (Rosaceae). The Avon Gorge, Bristol, UK, is a world ‘hotspot’ for Sorbus diversity and home to diploid sexual species and polyploid apomictic species. This research investigated how mating system variation, hybridization and polyploidy interact to generate this biological diversity.
Mating systems of diploid, triploid and tetraploid Sorbus taxa were analysed using pollen tube growth and seed set assays from controlled pollinations, and parent–offspring genotyping of progeny from open and manual pollinations.
Key Results
Diploid Sorbus are outcrossing and self-incompatible (SI). Triploid taxa are pseudogamous apomicts and genetically invariable, but because they also display self-incompatibility, apomictic seed set requires pollen from other Sorbus taxa – a phenomenon which offers direct opportunities for hybridization. In contrast tetraploid taxa are pseudogamous but self-compatible, so do not have the same obligate requirement for intertaxon pollination.
The mating inter-relationships among Avon Gorge Sorbus taxa are complex and are the driving force for hybridization and ongoing genetic diversification. In particular, the presence of self-incompatibility in triploid pseudogamous apomicts imposes a requirement for interspecific cross-pollination, thereby facilitating continuing diversification and evolution through rare sexual hybridization events. This is the first report of naturally occurring pseudogamous apomictic SI plant populations, and we suggest that interspecific pollination, in combination with a relaxed endosperm balance requirement, is the most likely route to the persistence of these populations. We propose that Avon Gorge Sorbus represents a model system for studying the establishment and persistence of SI apomicts in natural populations.
PMCID: PMC3605949  PMID: 23408832
Hybridization; evolution; polyploidy; apomixis; pseudogamy; self-incompatibility; Sorbus
4.  Heterologous Movement Protein Strongly Modifies the Infection Phenotype of Cucumber Mosaic Virus 
Journal of Virology  2002;76(7):3554-3557.
A hybrid virus (CMVcymMP) constructed by replacing the movement protein (MP) of cucumber mosaic cucumovirus (CMV) with that of cymbidium ringspot tombusvirus (CymRSV) was viable and could efficiently spread both cell to cell and long distance in host plants. The hybrid virus was able to move cell to cell in the absence of functional CP, whereas CP-deficient CMV was restricted to single inoculated cells. In several Chenopodium and Nicotiana species, the symptom phenotype of the hybrid virus infection was clearly determined by the foreign MP gene. In Nicotiana debneyi and Nicotiana tabacum cv. Xanthi, the hybrid virus could move systemically, contrary to CymRSV.
PMCID: PMC136044  PMID: 11884579
5.  Using maize as a model to study pollen tube growth and guidance, cross-incompatibility and sperm delivery in grasses 
Annals of Botany  2011;108(4):727-737.
In contrast to animals and lower plants such as mosses and ferns, sperm cells of flowering plants (angiosperms) are immobile and require transportation to the female gametes via the vegetative pollen tube cell to achieve double fertilization. The path of the pollen tube towards the female gametophyte (embryo sac) has been intensively studied in many intra- and interspecific crossing experiments with the aim of increasing the gene pool of crop plants for greater yield, improved biotic and abiotic stress resistance, and for introducing new agronomic traits. Many attempts to hybridize different species or genotypes failed due to the difficulty for the pollen tubes in reaching the female gametophyte. Detailed studies showed that these processes are controlled by various self-incompatible (intraspecific) and cross-incompatible (interspecific) hybridization mechanisms.
Understanding the molecular mechanisms of crossing barriers is therefore of great interest in plant reproduction, evolution and breeding research. In particular, pre-zygotic hybridization barriers related to pollen tube germination, growth, guidance and sperm delivery, which are considered the major hybridization controls in nature and thus also contribute to species isolation and speciation, have been intensively investigated. Despite this general interest, surprisingly little is known about these processes in the most important agronomic plant family, the Gramineae, Poaceae or grasses. Small polymorphic proteins and their receptors, degradation of sterility locus proteins and general compounds such as calcium, γ-aminobutyric acid or nitric oxide have been shown to be involved in progamic pollen germination, adhesion, tube growth and guidance, as well as sperm release. Most advances have been made in the Brassicaceae, Papaveraceae, Linderniaceae and Solanaceae families including their well-understood self-incompatibility (SI) systems. Grass species evolved similar mechanisms to control the penetration and growth of self-pollen to promote intraspecific outcrossing and to prevent fertilization by alien sperm cells. However, in the Poaceae, the underlying molecular mechanisms are still largely unknown.
We propose to develop maize (Zea mays) as a model to investigate the above-described processes to understand the associated intra- and interspecific crossing barriers in grasses. Many genetic, cellular and biotechnological tools including the completion of a reference genome (inbred line B73) have been established in the last decade and many more maize inbred genomes are expected to be available soon. Moreover, a cellular marker line database as well as large transposon insertion collections and improved Agrobacterium transformation protocols are now available. Additionally, the processes described above are well studied at the morphological level and a number of mutants have been described already, awaiting disclosure of the relevant genes. The identification of the first key players in pollen tube growth, guidance and burst show maize to be an excellent grass model to investigate these processes in more detail. Here we provide an overview of our current understanding of these processes in Poaceae with a focus on maize, and also include relevant discoveries in eudicot model species.
PMCID: PMC3170146  PMID: 21345919
Maize; male germline; sperm cell; interspecific crosses; self- and cross-incompatibility; pollen tube growth and guidance; fertilization; reproductive isolation
6.  Polyploidization Facilitates Biotechnological In Vitro Techniques in the Genus Cucumis 
Prezygotic interspecific crossability barrier in the genus Cucumis is related to the ploidy level of the species (cucumber (C. sativus), x = 7; muskmelon (C. melo) and wild Cucumis species, x = 12). Polyploidization of maternal plants helps hybridization among other Cucumis species by overcoming prezygotic genetic barriers. The main objective of this paper is to compare the results of several methods supporting interspecific crosses in cucumber without and with polyploidization (comparison between diploid (2x) and mixoploid (2x/4x) cucumber maternal plants). Mixoploid plants were obtained after in vivo and in vitro polyploidization by colchicine and oryzalin. Ploidy level was estimated by flow cytometry. Embryo rescue, in vitro pollination, and isolation of mesophyll protoplast were tested and compared. Positive effect of polyploidization was observed during all experiments presented by higher regeneration capacity of cultivated mixoploid cucumber embryos, ovules, and protoplasts. Nevertheless, the hybrid character of putative hybrid accessions obtained after cross in vivo and in vitro pollination was not confirmed.
PMCID: PMC3014699  PMID: 21234406
7.  Germplasm Modification and Its Potential for Finding New Sources of Resistance to Diseases 
Journal of Nematology  1986;18(1):18-22.
In vitro procedures are playing a major role in plant breeding. Embryo rescue, either through the culture of excised embryos derived from incompatible crosses or by means of ovule culture, has been a standard procedure for the introgression of genes conferring disease resistance into economically important plants. Somatic hybridization (i.e., protoplast fusion) has also been demonstrated to have some potential in obtaining hybrids that result from very wide interspecific and intergeneric crosses. Wide crosses have also been achieved by means of in vitro pollination of excised ovaries or ovules. Tissue culture-induced variability in regenerated plant (i.e., somaclonal variation) appears to be an effective way of obtaining undirected genetic change that can enhance disease resistance and yield and alter the growth habit of crops that are normally propagated vegetatively (e.g., potato) or by seed (e.g., tomato). In the near future, the isolation and sequencing of genes that confer resistance to specific plant pathogens will be possible, and transfer of this information between species will become a reality.
PMCID: PMC2618508  PMID: 19294132
plant tissue culture; somatic cell genetics; disease resistance
8.  Inhibition of phenylpropanoid biosynthesis increases cell wall digestibility, protoplast isolation, and facilitates sustained cell division in American elm (Ulmus americana) 
BMC Plant Biology  2012;12:75.
Protoplast technologies offer unique opportunities for fundamental research and to develop novel germplasm through somatic hybridization, organelle transfer, protoclonal variation, and direct insertion of DNA. Applying protoplast technologies to develop Dutch elm disease resistant American elms (Ulmus americana L.) was proposed over 30 years ago, but has not been achieved. A primary factor restricting protoplast technology to American elm is the resistance of the cell walls to enzymatic degradation and a long lag phase prior to cell wall re-synthesis and cell division.
This study suggests that resistance to enzymatic degradation in American elm was due to water soluble phenylpropanoids. Incubating tobacco (Nicotiana tabacum L.) leaf tissue, an easily digestible species, in aqueous elm extract inhibits cell wall digestion in a dose dependent manner. This can be mimicked by p-coumaric or ferulic acid, phenylpropanoids known to re-enforce cell walls. Culturing American elm tissue in the presence of 2-aminoindane-2-phosphonic acid (AIP; 10-150 μM), an inhibitor of phenylalanine ammonia lyase (PAL), reduced flavonoid content, decreased tissue browning, and increased isolation rates significantly from 11.8% (±3.27) in controls to 65.3% (±4.60). Protoplasts isolated from callus grown in 100 μM AIP developed cell walls by day 2, had a division rate of 28.5% (±3.59) by day 6, and proliferated into callus by day 14. Heterokaryons were successfully produced using electrofusion and fused protoplasts remained viable when embedded in agarose.
This study describes a novel approach of modifying phenylpropanoid biosynthesis to facilitate efficient protoplast isolation which has historically been problematic for American elm. This isolation system has facilitated recovery of viable protoplasts capable of rapid cell wall re-synthesis and sustained cell division to form callus. Further, isolated protoplasts survived electrofusion and viable heterokaryons were produced. Together, these results provide the first evidence of sustained cell division, callus regeneration, and potential application of somatic cell fusion in American elm, suggesting that this source of protoplasts may be ideal for genetic manipulation of this species. The technological advance made with American elm in this study has potential implications in other woody species for fundamental and applied research which require availability of viable protoplasts.
PMCID: PMC3464172  PMID: 22646730
Hydroxycinnamic acid; 2-aminoindane-2-phosphonic acid; Protoplast; Cell wall; Digestibility; American elm
9.  Dynamic Formation of Asexual Diploid and Polyploid Lineages: Multilocus Analysis of Cobitis Reveals the Mechanisms Maintaining the Diversity of Clones 
PLoS ONE  2012;7(9):e45384.
Given the hybrid genomic constitutions and increased ploidy of many asexual animals, the identification of processes governing the origin and maintenance of clonal diversity provides useful information about the evolutionary consequences of interspecific hybridization, asexuality and polyploidy. In order to understand the processes driving observed diversity of biotypes and clones in the Cobitis taenia hybrid complex, we performed fine-scale genetic analysis of Central European hybrid zone between two sexual species using microsatellite genotyping and mtDNA sequencing. We found that the hybrid zone is populated by an assemblage of clonally (gynogenetically) reproducing di-, tri- and tetraploid hybrid lineages and that successful clones, which are able of spatial expansion, recruit from two ploidy levels, i.e. diploid and triploid. We further compared the distribution of observed estimates of clonal ages to theoretical distributions simulated under various assumptions and showed that new clones are most likely continuously recruited from ancestral populations. This suggests that the clonal diversity is maintained by dynamic equilibrium between origination and extinction of clonal lineages. On the other hand, an interclonal selection is implied by nonrandom spatial distribution of individual clones with respect to the coexisting sexual species. Importantly, there was no evidence for sexually reproducing hybrids or clonally reproducing non-hybrid forms. Together with previous successful laboratory synthesis of clonal Cobitis hybrids, our data thus provide the most compelling evidence that 1) the origin of asexuality is causally linked to interspecific hybridization; 2) successful establishment of clones is not restricted to one specific ploidy level and 3) the initiation of clonality and polyploidy may be dynamic and continuous in asexual complexes.
PMCID: PMC3447977  PMID: 23028977
10.  Cytotype stability, facultative apomixis and geographical parthenogenesis in Ranunculus kuepferi (Ranunculaceae) 
Annals of Botany  2010;105(3):457-470.
Background and Aims
Asexual organisms are more widespread in previously glaciated areas than their sexual relatives (‘geographical parthenogenesis’). In plants, this pattern is probably dependent on reproductive isolation and stability of cytotypes within their respective distribution areas. Both partial apomixis and introgressive hybridization potentially destabilize the spatial separation of sexual and apomictic populations. The wide distribution of apomicts may be further enhanced by uniparental reproduction which is advantageous for colonization. These factors are studied in the alpine species Ranunculus kuepferi.
Geographical distribution, diversity and mode of reproduction of cytotypes were assessed using flow cytometry and flow cytometric seed screening on samples from 59 natural populations of Ranunculus kuepferi. Seed set of cytotypes was compared in the wild.
Key Results
Diploid sexuals are confined to the south-western parts of the Alps, while tetraploid apomicts dominate in previously glaciated and in geographically isolated areas despite a significantly lower fertility. Other cytotypes (3x, 5x and 6x) occur mainly in the sympatric zone, but without establishing populations. The tetraploids are predominantly apomictic, but also show a partial apomixis via an uncoupling of apomeiosis and parthenogenesis in the seed material. Both pseudogamy and autonomous endosperm formation are observed which may enhance uniparental reproduction.
Diploids occupy a glacial relic area and resist introgression of apomixis, probably because of a significantly higher seed set. Among the polyploids, only apomictic tetraploids form stable populations; the other cytotypes arising from partial apomixis fail to establish, probably because of minority cytotype disadvantages. Tetraploid apomicts colonize previously devastated and also distant areas via long-distance dispersal, confirming Baker's law of an advantage of uniparental reproduction. It is concluded that stability of cytotypes and of modes of reproduction are important factors for establishing a pattern of geographical parthenogenesis.
PMCID: PMC2826254  PMID: 20100695
Apomixis; flow cytometry; geographical parthenogenesis; glaciations; polyploidy; Ranunculus kuepferi
11.  A Stable Hybrid Containing Haploid Genomes of Two Obligate Diploid Candida Species 
Eukaryotic Cell  2013;12(8):1061-1071.
Candida albicans and Candida dubliniensis are diploid, predominantly asexual human-pathogenic yeasts. In this study, we constructed tetraploid (4n) strains of C. albicans of the same or different lineages by spheroplast fusion. Induction of chromosome loss in the tetraploid C. albicans generated diploid or near-diploid progeny strains but did not produce any haploid progeny. We also constructed stable heterotetraploid somatic hybrid strains (2n + 2n) of C. albicans and C. dubliniensis by spheroplast fusion. Heterodiploid (n + n) progeny hybrids were obtained after inducing chromosome loss in a stable heterotetraploid hybrid. To identify a subset of hybrid heterodiploid progeny strains carrying at least one copy of all chromosomes of both species, unique centromere sequences of various chromosomes of each species were used as markers in PCR analysis. The reduction of chromosome content was confirmed by a comparative genome hybridization (CGH) assay. The hybrid strains were found to be stably propagated. Chromatin immunoprecipitation (ChIP) assays with antibodies against centromere-specific histones (C. albicans Cse4/C. dubliniensis Cse4) revealed that the centromere identity of chromosomes of each species is maintained in the hybrid genomes of the heterotetraploid and heterodiploid strains. Thus, our results suggest that the diploid genome content is not obligatory for the survival of either C. albicans or C. dubliniensis. In keeping with the recent discovery of the existence of haploid C. albicans strains, the heterodiploid strains of our study can be excellent tools for further species-specific genome elimination, yielding true haploid progeny of C. albicans or C. dubliniensis in future.
PMCID: PMC3754532  PMID: 23709179
12.  Introgression of Swertia mussotii gene into Bupleurum scorzonerifolium via somatic hybridization 
BMC Plant Biology  2011;11:71.
The wild herb Swertia mussotii is a source of the anti-hepatitis compounds swertiamarin, mangiferin and gentiopicroside. Its over-exploitation has raised the priority of producing these compounds heterologously. Somatic hybridization represents a novel approach for introgressing Swertia mussotii genes into a less endangered species.
Protoplasts derived from calli of Bupleurum scorzonerifolium and S. mussotii were fused to produce 194 putative hybrid cell lines, of which three (all derived from fusions where the S. mussotii protoplasts were pre-treated for 30 s with UV light) later differentiated into green plants. The hybridity of the calli was confirmed by a combination of isozyme, RAPD and chromosomal analysis. The hybrid calli genomes were predominantly B. scorzonerifolium. GISH analysis of mitotic chromosomes confirmed that the irradiation of donor protoplasts increased the frequency of chromosome elimination and fragmentation. RFLP analysis of organellar DNA revealed that mitochondrial and chloroplast DNA of both parents coexisted and recombined in some hybrid cell lines. Some of the hybrid calli contained SmG10H from donor, and produced swertiamarin, mangiferin and certain volatile compounds characteristic of S. mussotii. The expression of SmG10H (geraniol 10-hydroxylase) was associated with the heterologous accumulation of swertiamarin.
Somatic hybrids between B. scorzonerifolium and S. mussotii were obtained, hybrids selected all contained introgressed nuclear and cytoplasmic DNA from S. mussotii; and some produced more mangiferin than the donor itself. The introgression of SmG10H was necessary for the accumulation of swertiamarin.
PMCID: PMC3098146  PMID: 21513581
13.  Seven of eight species in Nicotiana section Suaveolentes have common factors leading to hybrid lethality in crosses with Nicotiana tabacum 
Annals of Botany  2010;106(2):267-276.
Background and Aims
Reproductive isolation is a mechanism that separates species, and is classified into two types: prezygotic and postzygotic. Inviability of hybrids, or hybrid lethality, is a type of postzygotic isolation and is observed in some plant species, including Nicotiana species. Previous work has shown that the Q chromosome, which belongs to the S subgenome of N. tabacum, encodes one or more genes leading to hybrid lethality in some crosses.
Interspecific crosses of eight wild species were conducted in section Suaveolentes (which consists of species restricted to Australasia and Africa) with the cultivated species Nicotiana tabacum. Hybrid seedlings were cultivated at 28, 34 or 36 °C, and PCR and chromosome analysis were performed.
Results and Conclusions
Seven of eight wild species produced inviable hybrids after crossing. Hybrid lethality, which was observed in all crosses at 28 °C, was Type II lethality, with the characteristic symptoms of browning of hypocotyl and roots; lethality was suppressed at elevated temperatures (34 or 36 °C). Furthermore, one or more genes on the Q chromosome of N. tabacum were absolutely responsible for hybrid lethality, suggesting that many species of section Suaveolentes share the same factor that triggers hybrid lethality by interaction with the genes on the Q chromosome. Exceptionally, only one wild species, N. fragrans, produced 100 % viable hybrids after crossing with N. tabacum, suggesting that N. fragrans has no factor triggering hybrid lethality.
PMCID: PMC2908168  PMID: 20519236
Hybrid lethality; interspecific cross; Nicotiana section Suaveolentes; Q chromosome; reproductive isolation; tobacco
14.  Plant Regeneration and Somatic Embryogenesis from Immature Embryos Derived through Interspecific Hybridization among Different Carica Species 
Plant regeneration and somatic embryogenesis through interspecific hybridization among different Carica species were studied for the development of a papaya ringspot virus-resistant variety. The maximum fruit sets were recorded from the cross of the native variety C. papaya cv. Shahi with the wild species C. cauliflora. The highest hybrid embryos were recorded at 90 days after pollination and the embryos were aborted at 150 days after pollination. The immature hybrid embryos were used for plant regeneration and somatic embryogenesis. The 90-day-old hybrid embryos from the cross of C. papaya cv. Shahi × C. cauliflora showed the highest percentage of germination, as well as plant regeneration on growth regulators free culture medium after 7 days pre-incubation on half-strength MS medium supplemented with 0.2 mg/L BAP, 0.5 mg/L NAA and 60 g/L sucrose. The 90-day-old hybrid embryos from the cross of C. papaya cv. Shahi × C. cauliflora produced maximum callus, as well as somatic embryos when cultured on half-strength MS medium containing 5 mg/L 2,4-D, 100 mg/L glutamine, 100 mg/L casein hydrolysate and 60 g/L sucrose. The somatic embryos were transferred into half-strength MS medium containing 0.5 mg/L BAP and 0.2 mg/L NAA and 60 g/L sucrose for maturation. The highest number of regenerated plants per hybrid embryo (10.33) was recorded from the cross of C. papaya cv. Shahi × C. cauliflora. Isoenzyme and dendrogram cluster analysis using UPGMA of the regenerated F1 plantlets confirmed the presence of the hybrid plantlets.
PMCID: PMC3546739  PMID: 23235330
Carica species; hybridization; plant regeneration; somatic embryogenesis
15.  Hypersensitive Response-Like Reaction Is Associated with Hybrid Necrosis in Interspecific Crosses between Tetraploid Wheat and Aegilops tauschii Coss 
PLoS ONE  2010;5(6):e11326.
Hybrid speciation is classified into homoploid and polyploid based on ploidy level. Common wheat is an allohexaploid species that originated from a naturally occurring interploidy cross between tetraploid wheat and diploid wild wheat Aegilops tauschii Coss. Aegilops tauschii provides wide naturally occurring genetic variation. Sometimes its triploid hybrids with tetraploid wheat show the following four types of hybrid growth abnormalities: types II and III hybrid necrosis, hybrid chlorosis, and severe growth abortion. The growth abnormalities in the triploid hybrids could act as postzygotic hybridization barriers to prevent formation of hexaploid wheat.
Methodology/Principal Findings
Here, we report on the geographical and phylogenetic distribution of Ae. tauschii accessions inducing the hybrid growth abnormalities and showed that they are widely distributed across growth habitats in Ae. tauschii. Molecular and cytological characterization of the type III necrosis phenotype was performed. The hybrid abnormality causing accessions were widely distributed across growth habitats in Ae. tauschii. Transcriptome analysis showed that a number of defense-related genes such as pathogenesis-related genes were highly up-regulated in the type III necrosis lines. Transmission electron microscope observation revealed that cell death occurred accompanied by generation of reactive oxygen species in leaves undergoing type III necrosis. The reduction of photosynthetic activity occurred prior to the appearance of necrotic symptoms on the leaves exhibiting hybrid necrosis.
Taking these results together strongly suggests that an autoimmune response might be triggered by intergenomic incompatibility between the tetraploid wheat and Ae. tauschii genomes in type III necrosis, and that genetically programmed cell death could be regarded as a hypersensitive response-like cell death similar to that observed in Arabidopsis intraspecific and Nicotiana interspecific hybrids. Only Ae. tauschii accessions without such inhibiting factors could be candidates for the D-genome donor for the present hexaploid wheat.
PMCID: PMC2892878  PMID: 20593003
16.  The mode of inheritance in tetraploid cut roses 
Tetraploid hybrid tea roses (Rosa hybrida) represent most of the commercial cultivars of cut roses and form the basis for breeding programmes. Due to intensive interspecific hybridizations, modern cut roses are complex tetraploids for which the mode of inheritance is not exactly known. The segregation patterns of molecular markers in a tetraploid mapping population of 184 genotypes, an F1 progeny from a cross of two heterozygous parents, were investigated for disomic and tetrasomic inheritance. The possible occurrence of double reduction was studied as well. We can exclude disomic inheritance, but while our observations are more in line with a tetrasomic inheritance, we cannot exclude that there is a mixture of both inheritance modes. Two novel parental tetraploid linkage maps were constructed using markers known from literature, combined with newly generated markers. Comparison with the integrated consensus diploid map (ICM) of Spiller et al. (Theor Appl Genet 122:489–500, 2010) allowed assigning numbers to each of the linkage groups of both maps and including small linkage groups. So far, the possibility of using marker-assisted selection in breeding of tetraploid cut roses and of other species with a tetrasomic or partly tetrasomic inheritance, is still limited due to the difficulties in establishing marker-trait associations. We used these tetraploid linkage maps to determine associations between markers, two morphological traits and powdery mildew resistance. The knowledge on inheritance and marker-trait associations in tetraploid cut roses will be of direct use to cut rose breeding.
Electronic supplementary material
The online version of this article (doi:10.1007/s00122-012-1855-1) contains supplementary material, which is available to authorized users.
PMCID: PMC3397129  PMID: 22526522
17.  Development of somatic hybrids Solanum × michoacanum Bitter. (Rydb.) (+) S. tuberosum L. and autofused 4xS. × michoacanum plants as potential sources of late blight resistance for potato breeding 
Plant Cell Reports  2013;32(8):1231-1241.
Key message
Phytophthora infestansresistant somatic hybrids ofS. × michoacanum(+)S. tuberosumand autofused 4xS. × michoacanumwere obtained. Our material is promising to introgress resistance fromS. × michoacanuminto cultivated potato background.
Solanum × michoacanum (Bitter.) Rydb. (mch) is a wild diploid (2n = 2x = 24) potato species derived from spontaneous cross of S. bulbocastanum and S. pinnatisectum. This hybrid is a 1 EBN (endosperm balance number) species and can cross effectively only with other 1 EBN species. Plants of mch are resistant to Phytophthora infestans (Mont) de Bary. To introgress late blight resistance genes from mch into S. tuberosum (tbr), genepool somatic hybridization between mch and susceptible diploid potato clones (2n = 2x = 24) or potato cultivar Rywal (2n = 4x = 48) was performed. In total 18,775 calli were obtained from postfusion products from which 1,482 formed shoots. The Simple Sequence Repeat (SSR), Cleaved Amplified Polymorphic Sequences (CAPS) and Random Amplified Polymorphic DNA (RAPD) analyses confirmed hybrid nature of 228 plants and 116 autofused 4xmch. After evaluation of morphological features, flowering, pollen stainability, tuberization and ploidy level, 118 somatic hybrids and 116 autofused 4xmch were tested for late blight resistance using the detached leaf assay. After two seasons of testing three somatic hybrids and 109 4xmch were resistant. Resistant forms have adequate pollen stainability for use in crossing programme and are a promising material useful for introgression resistance from mch into the cultivated potato background.
PMCID: PMC3713256  PMID: 23525760
Potato species; Protoplast electrofusion; Phytophthora infestans resistance; Somatic hybridization
18.  Interspecific and interploidal gene flow in Central European Arabidopsis (Brassicaceae) 
Effects of polyploidisation on gene flow between natural populations are little known. Central European diploid and tetraploid populations of Arabidopsis arenosa and A. lyrata are here used to study interspecific and interploidal gene flow, using a combination of nuclear and plastid markers.
Ploidal levels were confirmed by flow cytometry. Network analyses clearly separated diploids according to species. Tetraploids and diploids were highly intermingled within species, and some tetraploids intermingled with the other species, as well. Isolation with migration analyses suggested interspecific introgression from tetraploid A. arenosa to tetraploid A. lyrata and vice versa, and some interploidal gene flow, which was unidirectional from diploid to tetraploid in A. arenosa and bidirectional in A. lyrata.
Interspecific genetic isolation at diploid level combined with introgression at tetraploid level indicates that polyploidy may buffer against negative consequences of interspecific hybridisation. The role of introgression in polyploid systems may, however, differ between plant species, and even within the small genus Arabidopsis, we find very different evolutionary fates when it comes to introgression.
PMCID: PMC3247304  PMID: 22126410
19.  Speciation towards tetraploidization after intermediate processes of non-sexual reproduction 
Polyploidy, hybridization and variation in mating systems are central issues for a deeper understanding of animal evolution. The Iberian species Squalius alburnoides represents an example combining all three phenomena. Previous studies showed that S. alburnoides populations are mainly composed of triploid and diploid hybrid forms (mainly females), and that the tetraploid forms are rare or absent. Both populations from the Douro drainage reveal a distinct scenario: tetraploid individuals represent 85.6–97.5% of the population, with no sex ratio bias observed. Based on the flow cytometry measurements of blood and spermatozoa cells, microsatellite loci and experimental crosses, we describe here, for the first time, two symmetric allotetraploid populations (CCAA) that resumed normal meiosis after undergoing intermediate processes of non-sexual reproduction to give rise to a new sexually reproducing polyploid species. Prezygotic (habitat selection and assortative mating) and postzygotic mechanisms (nonviable embryos) are responsible for the reproductive isolation from other forms of the S. alburnoides complex (e.g. CA, CAA). This example illustrates how hybrid polyploid complexes may lead to speciation.
PMCID: PMC2606740  PMID: 18522919
allopolyploid speciation; non-sexual reproduction; microsatellites; tetraploidization
20.  Assessing the bioconfinement potential of a Nicotiana hybrid platform for use in plant molecular farming applications 
BMC Biotechnology  2013;13:63.
The introduction of pharmaceutical traits in tobacco for commercial production could benefit from the utilization of a transgene bioconfinement system. It has been observed that interspecific F1Nicotiana hybrids (Nicotiana tabacum × Nicotiana glauca) are sterile and thus proposed that hybrids could be suitable bioconfined hosts for biomanufacturing. We genetically tagged hybrids with green fluorescent protein (GFP), which was used as a visual marker to enable gene flow tracking and quantification for field and greenhouse studies. GFP was used as a useful proxy for pharmaceutical transgenes.
Analysis of DNA content revealed significant genomic downsizing of the hybrid relative to that of N. tabacum. Hybrid pollen was capable of germination in vitro, albeit with a very low frequency and with significant differences between plants. In two field experiments, one each in Tennessee and Kentucky, we detected outcrossing at only one location (Tennessee) at 1.4%. Additionally, from 50 hybrid plants at each field site, formation of 84 and 16 seed was observed, respectively. Similar conclusions about hybrid fertility were drawn from greenhouse crosses. In terms of above-ground biomass, the hybrid yield was not significantly different than that of N. tabacum in the field.
N. tabacum × N. glauca hybrids show potential to contribute to a bioconfinement- and biomanufacturing host system. Hybrids exhibit extremely low fertility with no difference of green biomass yields relative to N. tabacum. In addition, hybrids are morphologically distinguishable from tobacco allowing for identity preservation. This hybrid system for biomanufacturing would optimally be used where N. glauca is not present and in physical isolation of N. tabacum production to provide total bioconfinement.
PMCID: PMC3750662  PMID: 23914736
Gene flow; Male-sterility; Pharming; Bioconfinement; Nicotiana; Green fluorescent protein (GFP); Plant-made-pharmaceuticals (PMPs)
21.  Genome size variation in diploid and tetraploid wild wheats 
AoB Plants  2010;2010:plq015.
Low but significant intraspecific genome size variations were found in diploid and tetraploid wild wheats. This limited variation is not correlated with geographical and climate variables. It can be concluded that the genome size of Triticum species is generally stable, despite of the presence of many potentially active retroelements.
Background and aims
Intra- and interspecific variations of C-values and the relationship between habitat factors and genome size were studied in natural populations of diploid and tetraploid wild wheats.
The 1C nuclear DNA content of 376 individual plants representing 41 populations of diploid and tetraploid wild wheats was determined by flow cytometry (FCM) and correlated with geographical and bioclimate variables.
Principal results
Based on analysis of variance, significant differences between diploid and tetraploid Triticum species were found. Differences among populations of T. boeoticum and T. dicoccoides were also statistically significant and argue for isolation between populations, except for T. araraticum. However, the variation among individuals of the same population was not statistically significant. Maximum genome size differences among populations for T. boeoticum (0.143 pg; 2.32 %), T. dicoccoides (0.314 pg; 2.49 %) and T. araraticum (0.116 pg; 0.98 %) argue for genome constancy in these species. There was no significant correlation between intra-population variance and geographical and bioclimate variables for T. boeoticum and T. dicoccoides. In contrast to the limited genome size variation at the intraspecific level, the interspecific variation was large: ∼0.5 pg/1C (8 %) at the diploid level (T. boeoticum vs. T. urartu) and ∼1 pg/1C (9.7 %) at the tetraploid level (T. dicoccoides vs. T. araraticum).
Low intraspecific genome size variation occurs in diploid and tetraploid wild wheats, and this limited variation is not correlated with geographical and climate variables. However, interspecific variation is significant at the diploid and tetraploid level. It can be concluded that the genome size of wild self-fertilizing Triticum species is generally stable, despite the presence of many potentially active retroelements. In natural habitats, it is very difficult to distinguish wild wheats from each other. However, all four species can be distinguished easily, quickly and unambiguously by using the FCM technique.
PMCID: PMC2992354  PMID: 22476073
22.  Domestication, Genomics and the Future for Banana 
Annals of Botany  2007;100(5):1073-1084.
Cultivated bananas and plantains are giant herbaceous plants within the genus Musa. They are both sterile and parthenocarpic so the fruit develops without seed. The cultivated hybrids and species are mostly triploid (2n = 3x = 33; a few are diploid or tetraploid), and most have been propagated from mutants found in the wild. With a production of 100 million tons annually, banana is a staple food across the Asian, African and American tropics, with the 15 % that is exported being important to many economies.
There are well over a thousand domesticated Musa cultivars and their genetic diversity is high, indicating multiple origins from different wild hybrids between two principle ancestral species. However, the difficulty of genetics and sterility of the crop has meant that the development of new varieties through hybridization, mutation or transformation was not very successful in the 20th century. Knowledge of structural and functional genomics and genes, reproductive physiology, cytogenetics, and comparative genomics with rice, Arabidopsis and other model species has increased our understanding of Musa and its diversity enormously.
There are major challenges to banana production from virulent diseases, abiotic stresses and new demands for sustainability, quality, transport and yield. Within the genepool of cultivars and wild species there are genetic resistances to many stresses. Genomic approaches are now rapidly advancing in Musa and have the prospect of helping enable banana to maintain and increase its importance as a staple food and cash crop through integration of genetical, evolutionary and structural data, allowing targeted breeding, transformation and efficient use of Musa biodiversity in the future.
PMCID: PMC2759213  PMID: 17766312
Musa; banana; plantain; genome evolution; biodiversity; selection; plant breeding
23.  A high density genetic map of tobacco (Nicotiana tabacum L.) obtained from large scale microsatellite marker development 
Tobacco (Nicotiana tabacum L.) is a species in the large family of the Solanaceae and is important as an agronomic crop and as a model system in plant biotechnology. Despite its importance, only limited molecular marker resources are available that can be used for genome analysis, genetic mapping and breeding. We report here on the development and characterization of 5,119 new and functional microsatellite markers and on the generation of a high-resolution genetic map for the tetraploid tobacco genome. The genetic map was generated using an F2 mapping population derived from the intervarietal cross of Hicks Broadleaf × Red Russian and merges the polymorphic markers from this new set with those from a smaller set previously used to produce a lower density map. The genetic map described here contains 2,317 microsatellite markers and 2,363 loci, resulting in an average distance between mapped microsatellite markers which is less than 2 million base pairs or 1.5 cM. With this new and expanded marker resource, a sufficient number of markers are now available for multiple applications ranging from tobacco breeding to comparative genome analysis. The genetic map of tobacco is now comparable in marker density and resolution with the best characterized genomes of the Solanaceae: tomato and potato.
Electronic supplementary material
The online version of this article (doi:10.1007/s00122-011-1578-8) contains supplementary material, which is available to authorized users.
PMCID: PMC3114088  PMID: 21461649
24.  Embryonic Stem Cells Contribute to Mouse Chimeras in the Absence of Detectable Cell Fusion 
Cloning and Stem Cells  2008;10(2):231-247.
Embryonic stem (ES) cells are capable of differentiating into all embryonic and adult cell types following mouse chimera production. Although injection of diploid ES cells into tetraploid blastocysts suggests that tetraploid cells have a selective disadvantage in the developing embryo, tetraploid hybrid cells, formed by cell fusion between ES cells and somatic cells, have been reported to contribute to mouse chimeras. In addition, other examples of apparent stem cell plasticity have recently been shown to be the result of cell fusion. Here we investigate whether ES cells contribute to mouse chimeras through a cell fusion mechanism. Fluorescence in situ hybridization (FISH) analysis for X and Y chromosomes was performed on dissociated tissues from embryonic, neonatal, and adult wild-type, and chimeric mice to follow the ploidy distributions of cells from various tissues. FISH analysis showed that the ploidy distributions in dissociated tissues, notably the tetraploid cell number, did not differ between chimeric and wild-type tissues. To address the possibility that early cell fusion events are hidden by subsequent reductive divisions or other changes in cell ploidy, we injected Z/EG (lacZ/EGFP) ES cells into ACTB-cre blastocysts. Recombination can only occur as the result of cell fusion, and the recombined allele should persist through any subsequent changes in cell ploidy. We did not detect evidence of fusion in embryonic chimeras either by direct fluorescence microscopy for GFP or by PCR amplification of the recombined Z/EG locus on genomic DNA from ACTB-cre::Z/EG chimeric embryos. Our results argue strongly against cell fusion as a mechanism by which ES cells contribute to chimeras.
PMCID: PMC2981375  PMID: 18338954
25.  Molecular Analysis of Hypervirulent Somatic Hybrids of the Entomopathogenic Fungi Beauveria bassiana and Beauveria sulfurescens 
Protoplast fusion of diauxotrophic mutants of a Beauveria bassiana entomopathogenic strain (Bb28) and a Beauveria sulfurescens toxinogenic strain (Bs2) produced hybrids which were significantly different from the parents in pathogenicity. Some of the hybrids were hypervirulent and killed insects more quickly than the Bb28 strain, probably because these hybrids had acquired the toxic activity of the Bs2 strain. By using six nuclear genes and a telomeric fingerprint probe, the molecular structures of the hybrids were studied. The results demonstrated the occurrence of parasexual events. Hybrids appeared to be diploid or aneuploid, with portions of the genome being heterozygous. A mitochondrial molecular marker indicated homoplasmy of the hybrids and inheritance of mitochondria from strain Bs2 or Bb28. The pathogenicities and the ploidies of the hybrids remained stable after passage through the host insect, showing that somatic hybridization provides an attractive method for the genetic improvement of biocontrol efficiency in the genus Beauveria.
PMCID: PMC124676  PMID: 9435064

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