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1.  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
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.  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
4.  Autotetraploid plant regeneration by indirect somatic embryogenesis from leaf mesophyll protoplasts of diploid Gentiana decumbens L.f. 
Somaclonal variation, often manifested as the increased ploidy of plants observed following in vitro culture, can be advantageous in ornamental species or those used for secondary metabolite production. Polyploidy occurs especially when plantlets are produced by protoplast and callus cultures. Plants were regenerated from green leaf mesophyll protoplasts of diploid Gentiana decumbens L.f. through somatic embryogenesis. A yield of more than 9 × 105 protoplasts per gram of fresh weight was achieved by incubating fully expanded young leaves in an enzyme mixture containing 1.0% (w/v) cellulase and 0.5% (w/v) macerozyme. Protoplasts, cultured in agarose beads using a modified Murashige and Skoog medium, divided and formed microcalli, with the highest plating efficiency obtained on medium containing 2.0 mg l−1 1-naphthaleneacetic acid and 0.1 mg l−1 thidiazuron. Callus proliferation was also promoted by including thidiazuron in agar-solidified medium, while somatic embryogenesis was induced from microcalli on medium supplemented with 1.0 mg l−1 kinetin, 0.5 mg l−1 gibberellic acid, and 80 mg l−1 adenine sulfate. Flow cytometric analysis and chromosome counting revealed that all regenerants were tetraploid.
PMCID: PMC4471314  PMID: 26097374
Protoplast culture; Somaclonal variation; Gentian; Flow cytometry; Chromosome number
5.  High-resolution mapping reveals hundreds of genetic incompatibilities in hybridizing fish species 
eLife  2014;3:e02535.
Hybridization is increasingly being recognized as a common process in both animal and plant species. Negative epistatic interactions between genes from different parental genomes decrease the fitness of hybrids and can limit gene flow between species. However, little is known about the number and genome-wide distribution of genetic incompatibilities separating species. To detect interacting genes, we perform a high-resolution genome scan for linkage disequilibrium between unlinked genomic regions in naturally occurring hybrid populations of swordtail fish. We estimate that hundreds of pairs of genomic regions contribute to reproductive isolation between these species, despite them being recently diverged. Many of these incompatibilities are likely the result of natural or sexual selection on hybrids, since intrinsic isolation is known to be weak. Patterns of genomic divergence at these regions imply that genetic incompatibilities play a significant role in limiting gene flow even in young species.
eLife digest
In nature, closely related species often interbreed to produce hybrids. However, hybrids are often less fertile or unable to compete with parent species, making them less likely to thrive in the wild.
When the genomes of two different species are mixed, versions of genes that are meant to work together can become separated, which means that these genes do not work as well as they should. This reduces the hybrids' chances of survival, and a poor survival rate of hybrids is one barrier that keeps different species distinct, even though the species can interbreed.
Two species of swordtail fish live in the rivers in Mexico, and although they mostly live in different stretches of these rivers, the two species interbreed to produce hybrids in the regions where they overlap. These hybrids can outnumber the parental species in these ‘hybrid zones’, but the two species have remained separate in other parts of the rivers. Though some genetic incompatibilities that might keep the species distinct have previously been suggested, it is not known how many incompatibilities there are in these fish's genomes.
Schumer et al. have searched the genomes of wild hybrids between these species and found hundreds of genetic incompatibilities. These were identified by looking for species-specific pairs of genes that are found together more often than would be expected if there were no selection against hybrids. It is likely that many of these incompatibilities reduce the evolutionary fitness of the hybrid fish and Schumer et al. suggest that many could be the result of environmental pressures and the fish's mating preferences. Furthermore, Schumer et al. demonstrate that genes close to identified incompatibilities are more different between species, on average, than genes that are further away. When there is on-going interbreeding between two the species (as is the case with the swordtails), this finding is expected only if these incompatibilities reduce the hybrids' chances of finding mates or surviving.
The findings of Schumer et al. demonstrate how conflicts in the genomes of two species allow these species to remain distinct even when they live in overlapping environments and frequently interbreed. Future work will investigate how these genetic incompatibilities shape the hybrid populations that are found in the wild; and which incompatibilities are caused by poor survival of the hybrids or by the fish's mating preferences selecting against the hybrids.
PMCID: PMC4080447  PMID: 24898754
hybridization; speciation; hybrid fitness; genetic incompatibilities; other
6.  The chromosome content and genotype of two wheat cell lines and of their somatic fusion product with oat 
Planta  2010;231(5):1201-1210.
Somatic hybridization seeks to genetically combine phylogenetically distant parents. An effective system has been established in bread wheat (Triticum aestivum L.) involving protoplasts from a non-totipotent cell line adapted to in vitro culture (T1) in combination with totipotent protoplasts harvested from embryogenic calli (T2). Here, we report the karyotype and genotype of T1 and T2. Line T1 carries nine A (A-genome of wheat), seven B (B-genome of wheat) and eight D (D-genome of wheat) genome chromosomes, while T2 cells have 12 A, 10 B and 12 D genome chromosomes. Rates of chromosome aberration in the B- and D-genomes were more than 25%, higher than in the A-genome. DNA deletion rates were 55.6% in T1 and 19.4% in T2, and DNA variation rates were 8.3% in T1 and 13.9% in T2. Rate of DNA elimination was B- > D- > A-genome in both T1 and T2. The same set of cytological and genetic assays was applied to a derivative of the somatic fusion between protoplasts of T1, T2 and oat (Avena sativa L.). The regenerant plants were near euploid with respect to their wheat complement. Six wheat chromosome arms—4AL, 3BS, 4BL, 3DS, 6DL and 7DL—carried small introgressed segments of oat chromatin. A genotypic analysis of the hybrid largely confirmed this cytologically-based diagnosis.
Electronic supplementary material
The online version of this article (doi:10.1007/s00425-010-1113-1) contains supplementary material, which is available to authorized users.
PMCID: PMC2840659  PMID: 20204401
Chromosome content and genotype; Common wheat; Oat; Somatic hybrid
7.  Chromosome elimination, addition and introgression in intertribal partial hybrids between Brassica rapa and Isatis indigotica 
Annals of Botany  2009;103(7):1039-1048.
Background and Aims
Partial hybrids with female-parent-type phenotypes and chromosome numbers but altered genomic compositions have been reported in wide crosses of several plants. In order to introgress desirable genes from a wild relative, Isatis indigotica (a dye and medicinal plant; 2n = 14), into Brassica crops, intertribal sexual hybridizations were carried out with B. rapa (2n = 20), and the resulting hybrids and their progenies were characterized.
Using genomic in situ hybridization (GISH) and amplified fragment length polymorphism (AFLP), chromosomal/genomic components of the hybrids and their progenies were analysed.
Key Results
Many hybrid plants were obtained from the mature seeds harvested from the B. rapa × I. indigotica cross, and these exhibited different morphological traits. However, the majority of them did not survive and only three plants grew to maturity. These three hybrids showed poor growth and much smaller stature than the two parents, but had some morphological traits and chemical composition of I. indigotica. One plant had 2n = 10, the haploid chromosome number of B. rapa, and was absolutely sterile. The other two plants had 20 and 22 somatic chromosomes and were male sterile but produced seeds following pollinations with B. rapa. All back-cross progenies over several generations maintained a B. rapa-type phenotype and also displayed some variations in morphological characters and fatty acid compositions. They were all 2n = 20 and showed good seed-set. The hybrid with 2n = 22 produced some progeny plants with 2n = 21 and 2n = 22. GISH detected two chromosomes of I. indigotica in the hybrid with 2n = 22 but none in the one with 2n = 20. AFLP bands specific for I. indigotica, novel for two parents or absent in B. rapa, were detected in the two hybrids and their progenies. These progeny plants were novel B. rapa types with an altered genomic constitution or alien additions.
Complete or partial chromosome elimination and diploidization with genomic rearrangements were considered to lead to the formation of partial hybrids in this cross.
PMCID: PMC2707916  PMID: 19258339
Brassica rapa; Isatis indigotica; intertribal hybridization; partial hybrids; chromosome elimination; alien addition; introgression; genomic in situ hybridization (GISH); amplified fragments length polymorphism (AFLP)
8.  Overexpression of the Synthetic Chimeric Native-T-phylloplanin-GFP Genes Optimized for Monocot and Dicot Plants Renders Enhanced Resistance to Blue Mold Disease in Tobacco (N. tabacum L.) 
The Scientific World Journal  2014;2014:601314.
To enhance the natural plant resistance and to evaluate the antimicrobial properties of phylloplanin against blue mold, we have expressed a synthetic chimeric native-phylloplanin-GFP protein fusion in transgenic Nicotiana tabacum cv. KY14, a cultivar that is highly susceptible to infection by Peronospora tabacina. The coding sequence of the tobacco phylloplanin gene along with its native signal peptide was fused with GFP at the carboxy terminus. The synthetic chimeric gene (native-phylloplanin-GFP) was placed between the modified Mirabilis mosaic virus full-length transcript promoter with duplicated enhancer domains and the terminator sequence from the rbcSE9 gene. The chimeric gene, expressed in transgenic tobacco, was stably inherited in successive plant generations as shown by molecular characterization, GFP quantification, and confocal fluorescent microscopy. Transgenic plants were morphologically similar to wild-type plants and showed no deleterious effects due to transgene expression. Blue mold-sensitivity assays of tobacco lines were performed by applying P. tabacina sporangia to the upper leaf surface. Transgenic lines expressing the fused synthetic native-phyllopanin-GFP gene in the leaf apoplast showed resistance to infection. Our results demonstrate that in vivo expression of a synthetic fused native-phylloplanin-GFP gene in plants can potentially achieve natural protection against microbial plant pathogens, including P. tabacina in tobacco.
PMCID: PMC3980785  PMID: 24778589
9.  High frequency regeneration via direct somatic embryogenesis and efficient Agrobacterium- mediated genetic transformation of tobacco 
Plant Signaling & Behavior  2013;8(6):e24354.
A direct somatic embryogenesis protocol was developed for four cultivars of Nicotiana species, by using leaf disc as an explant. Direct somatic embryogenesis of Nicotiana by using BAP and IAA has not been investigated so far. This method does not require formation of callus tissues which leads to somaclonal variations. The frequency of somatic embryogenesis was strongly influenced by the plant growth hormones. The somatic embryos developing directly from explant tissue were noticed after 6 d of culture. Somatic embryogenesis of a high frequency (87–96%) was observed in cultures of the all four genotypes (Nicotiana tabacum, N. benthamiyana, N. xanthi, N. t cv petihavana). The results showed that the best medium for direct somatic embryogenesis was MS supplemented with 2.5 mg/l, 0.2 mg/l IAA and 2% sucrose. Subculture of somatic embryos onto hormone free MS medium resulted in their conversion into plants for all genotypes. About 95% of the regenerated somatic embryos germinated into complete plantlets. The plants showed morphological and growth characteristics similar to those of seed-derived plants. Explants were transformed using Agrobacterium tumifacious LBA4404 plasmid pCAMBIA1301 harboring the GUS gene. The regenerated transgenic plants were confirmed by PCR analysis and histochemical GUS assay. The transformation efficiency obtained by using the Agrobacterium- mediated transformation was more than 95%. This method takes 6 wk to accomplish complete transgenic plants through direct somatic embryogenesis. The transgenic plantlets were acclimatized successfully with 98% survival in greenhouse and they showed normal morphological characteristics and were fertile. The regeneration and transformation method described herein is very simple, highly efficient and fast for the introduction of any foreign gene directly in tobacco through direct somatic embryogenesis.
PMCID: PMC3906319  PMID: 23518589
Agrobacterium tumefacien; direct somatic embryogenesis; GUS; micropropagation; Nicotiana; regeneration; tobacco; transformation
10.  Construction of Whole Genome Radiation Hybrid Panels and Map of Chromosome 5A of Wheat Using Asymmetric Somatic Hybridization 
PLoS ONE  2012;7(7):e40214.
To explore the feasibility of constructing a whole genome radiation hybrid (WGRH) map in plant species with large genomes, asymmetric somatic hybridization between wheat (Triticum aestivum L.) and Bupleurum scorzonerifolium Willd. was performed. The protoplasts of wheat were irradiated with ultraviolet light (UV) and gamma-ray and rescued by protoplast fusion using B. scorzonerifolium as the recipient. Assessment of SSR markers showed that the radiation hybrids have the average marker retention frequency of 15.5%. Two RH panels (RHPWI and RHPWII) that contained 92 and 184 radiation hybrids, respectively, were developed and used for mapping of 68 SSR markers in chromosome 5A of wheat. A total of 1557 and 2034 breaks were detected in each panel. The RH map of chromosome 5A based on RHPWII was constructed. The distance of the comprehensive map was 2103 cR and the approximate resolution was estimated to be ∼501.6 kb/break. The RH panels evaluated in this study enabled us to order the ESTs in a single deletion bin or in the multiple bins cross the chromosome. These results demonstrated that RH mapping via protoplast fusion is feasible at the whole genome level for mapping purposes in wheat and the potential value of this mapping approach for the plant species with large genomes.
PMCID: PMC3398029  PMID: 22815731
11.  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
12.  Development and Functional Analysis of Novel Genetic Promoters Using DNA Shuffling, Hybridization and a Combination Thereof 
PLoS ONE  2012;7(3):e31931.
Development of novel synthetic promoters with enhanced regulatory activity is of great value for a diverse range of plant biotechnology applications.
Using the Figwort mosaic virus full-length transcript promoter (F) and the sub-genomic transcript promoter (FS) sequences, we generated two single shuffled promoter libraries (LssF and LssFS), two multiple shuffled promoter libraries (LmsFS-F and LmsF-FS), two hybrid promoters (FuasFScp and FSuasFcp) and two hybrid-shuffled promoter libraries (LhsFuasFScp and LhsFSuasFcp). Transient expression activities of approximately 50 shuffled promoter clones from each of these libraries were assayed in tobacco (Nicotiana tabacum cv. Xanthi) protoplasts. It was observed that most of the shuffled promoters showed reduced activity compared to the two parent promoters (F and FS) and the CaMV35S promoter. In silico studies (computer simulated analyses) revealed that the reduced promoter activities of the shuffled promoters could be due to their higher helical stability. On the contrary, the hybrid promoters FuasFScp and FSuasFcp showed enhanced activities compared to F, FS and CaMV 35S in both transient and transgenic Nicotiana tabacum and Arabidopsis plants. Northern-blot and qRT-PCR data revealed a positive correlation between transcription and enzymatic activity in transgenic tobacco plants expressing hybrid promoters. Histochemical/X-gluc staining of whole transgenic seedlings/tissue-sections and fluorescence images of ImaGene Green™ treated roots and stems expressing the GUS reporter gene under the control of the FuasFScp and FSuasFcp promoters also support the above findings. Furthermore, protein extracts made from protoplasts expressing the human defensin (HNP-1) gene driven by hybrid promoters showed enhanced antibacterial activity compared to the CaMV35S promoter.
Both shuffled and hybrid promoters developed in the present study can be used as molecular tools to study the regulation of ectopic gene expression in plants.
PMCID: PMC3303778  PMID: 22431969
13.  Autoimmune Response as a Mechanism for a Dobzhansky-Muller-Type Incompatibility Syndrome in Plants 
PLoS Biology  2007;5(9):e236.
Epistatic interactions between genes are a major factor in evolution. Hybrid necrosis is an example of a deleterious phenotype caused by epistatic interactions that is observed in many intra- and interspecific plant hybrids. A large number of hybrid necrosis cases share phenotypic similarities, suggesting a common underlying mechanism across a wide range of plant species. Here, we report that approximately 2% of intraspecific crosses in Arabidopsis thaliana yield F1 progeny that express necrosis when grown under conditions typical of their natural habitats. We show that several independent cases result from epistatic interactions that trigger autoimmune-like responses. In at least one case, an allele of an NB-LRR disease resistance gene homolog is both necessary and sufficient for the induction of hybrid necrosis, when combined with a specific allele at a second locus. The A. thaliana cases provide insights into the molecular causes of hybrid necrosis, and serve as a model for further investigation of intra- and interspecific incompatibilities caused by a simple epistatic interaction. Moreover, our finding that plant immune-system genes are involved in hybrid necrosis suggests that selective pressures related to host–pathogen conflict might cause the evolution of gene flow barriers in plants.
Author Summary
Hybridization brings together genetic material from different genomes. Sometimes, the novel combinations of genes are deleterious in the offspring, even though the genes were innocuous, or even beneficial, in their parents. Such “genetic incompatibilities” have been observed in crosses within and between species in plants, animals, and fungi, and could contribute to the maintenance of population or species boundaries. We have investigated a highly deleterious genetic incompatibility called hybrid necrosis that is observed in many plant taxa. Using different wild strains of Arabidopsis thaliana as a model, we show that hybrid necrosis is often associated with inappropriate activation of the plant immune system—effectively plant autoimmunity. We identified a gene in one strain that triggers necrosis when combined with a second locus from another strain. The product of this gene is an NB-LRR protein, the most common type of plant disease resistance protein. This finding raises the possibility that selective pressure exerted by pathogens can promote rapid evolution of gene variants that might provide benefits to the parent lineage but can cause serious problems for hybrid progeny.
Sometimes, genes that are innocuous in the parents are deleterious when combined in the offspring. Here, some genes involved in hybrid necrosis in plants have been identified.
PMCID: PMC1964774  PMID: 17803357
14.  A New Synthetic Allotetraploid (A1A1G2G2) between Gossypium herbaceum and G. australe: Bridging for Simultaneously Transferring Favorable Genes from These Two Diploid Species into Upland Cotton 
PLoS ONE  2015;10(4):e0123209.
Gossypium herbaceum, a cultivated diploid cotton species (2n = 2x = 26, A1A1), has favorable traits such as excellent drought tolerance and resistance to sucking insects and leaf curl virus. G. australe, a wild diploid cotton species (2n = 2x = 26, G2G2), possesses numerous economically valuable characteristics such as delayed pigment gland morphogenesis (which is conducive to the production of seeds with very low levels of gossypol as a potential food source for humans and animals) and resistance to insects, wilt diseases and abiotic stress. Creating synthetic allotetraploid cotton from these two species would lay the foundation for simultaneously transferring favorable genes into cultivated tetraploid cotton. Here, we crossed G. herbaceum (as the maternal parent) with G. australe to produce an F1 interspecific hybrid and doubled its chromosome complement with colchicine, successfully generating a synthetic tetraploid. The obtained tetraploid was confirmed by morphology, cytology and molecular markers and then self-pollinated. The S1 seedlings derived from this tetraploid gradually became flavescent after emergence of the fifth true leaf, but they were rescued by grafting and produced S2 seeds. The rescued S1 plants were partially fertile due to the existence of univalents at Metaphase I of meiosis, leading to the formation of unbalanced, nonviable gametes lacking complete sets of chromosomes. The S2 plants grew well and no flavescence was observed, implying that interspecific incompatibility, to some extent, had been alleviated in the S2 generation. The synthetic allotetraploid will be quite useful for polyploidy evolutionary studies and as a bridge for transferring favorable genes from these two diploid species into Upland cotton through hybridization.
PMCID: PMC4400159  PMID: 25879660
15.  X Chromosome Control of Meiotic Chromosome Synapsis in Mouse Inter-Subspecific Hybrids 
PLoS Genetics  2014;10(2):e1004088.
Hybrid sterility (HS) belongs to reproductive isolation barriers that safeguard the integrity of species in statu nascendi. Although hybrid sterility occurs almost universally among animal and plant species, most of our current knowledge comes from the classical genetic studies on Drosophila interspecific crosses or introgressions. With the house mouse subspecies Mus m. musculus and Mus m. domesticus as a model, new research tools have become available for studies of the molecular mechanisms and genetic networks underlying HS. Here we used QTL analysis and intersubspecific chromosome substitution strains to identify a 4.7 Mb critical region on Chromosome X (Chr X) harboring the Hstx2 HS locus, which causes asymmetrical spermatogenic arrest in reciprocal intersubspecific F1 hybrids. Subsequently, we mapped autosomal loci on Chrs 3, 9 and 13 that can abolish this asymmetry. Combination of immunofluorescent visualization of the proteins of synaptonemal complexes with whole-chromosome DNA FISH on pachytene spreads revealed that heterosubspecific, unlike consubspecific, homologous chromosomes are predisposed to asynapsis in F1 hybrid male and female meiosis. The asynapsis is under the trans- control of Hstx2 and Hst1/Prdm9 hybrid sterility genes in pachynemas of male but not female hybrids. The finding concurred with the fertility of intersubpecific F1 hybrid females homozygous for the Hstx2Mmm allele and resolved the apparent conflict with the dominance theory of Haldane's rule. We propose that meiotic asynapsis in intersubspecific hybrids is a consequence of cis-acting mismatch between homologous chromosomes modulated by the trans-acting Hstx2 and Prdm9 hybrid male sterility genes.
Author Summary
Genomes of newly emerging species restrict their gene exchange with related taxa in order to secure integrity. Hybrid sterility is one of the reproductive isolation mechanisms restricting gene flow between closely related, sexually reproducing organisms. We showed that hybrid sterility between two closely related mouse subspecies is executed by a failure of meiotic synapsis of orthologous chromosomes in F1 hybrid males. The asynapsis of orthologous chromosomes occurred in meiosis of male and female hybrids, though only males were sterile due to trans-acting male-specific hybrid sterility genes. We located one of the two major hybrid sterility genes to a 4.7 Mb interval on Chromosome X, showed that it controls male sterility by modulating the extent of meiotic asynapsis and using the inter-subspecific chromosome substitution strains we refuted the simple interpretation of dominance theory of Haldane's rule. A new working hypothesis posits male sterility of mouse inter-subsubspecific F1 hybrids as a consequence of meiotic chromosome asynapsis caused by the cis-acting mismatch between orthologous chromosomes modulated by the trans-acting hybrid male sterility genes.
PMCID: PMC3916230  PMID: 24516397
16.  Instability of chromosome number and DNA methylation variation induced by hybridization and amphidiploid formation between Raphanus sativus L. and Brassica alboglabra Bailey 
BMC Plant Biology  2010;10:207.
Distant hybridization can result genome duplication and allopolyploid formation which may play a significant role in the origin and evolution of many plant species. It is unclear how the two or more divergent genomes coordinate in one nucleus with a single parental cytoplasm within allopolyploids. We used cytological and molecular methods to investigate the genetic and epigenetic instabilities associated with the process of distant hybridization and allopolyploid formation, measuring changes in chromosome number and DNA methylation across multiple generations.
F1 plants from intergeneric hybridization between Raphanus sativus L. (2n = 18, RR) and Brassica alboglabra Bailey (2n = 18, CC) were obtained by hand crosses and subsequent embryo rescue. Random amplification of polymorphic DNA (RAPD) markers were used to identify the F1 hybrid plants. The RAPD data indicated that the hybrids produced specific bands similar to those of parents and new bands that were not present in either parent. Chromosome number variation of somatic cells from allotetraploids in the F4 to F10 generations showed that intensive genetic changes occurred in the early generations of distant hybridization, leading to the formation of mixopolyploids with different chromosome numbers. DNA methylation variation was revealed using MSAP (methylation-sensitive amplification polymorphism), which showed that cytosine methylation patterns changed markedly in the process of hybridization and amphidiploid formation. Differences in cytosine methylation levels demonstrated an epigenetic instability of the allopolyploid of Raphanobrassica between the genetically stable and unstable generations.
Our results showed that chromosome instability occurred in the early generations of allopolyploidy and then the plants were reverted to largely euploidy in later generations. During this process, DNA methylation changed markedly. These results suggest that, epigenetic mechanisms play an important role in intergeneric distant hybridization, probably by maintaining a genetic balance through the modification of existing genetic materials.
PMCID: PMC2956556  PMID: 20849584
17.  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
18.  Genetic Diversity and Genomic Plasticity of Cryptococcus neoformans AD Hybrid Strains 
G3: Genes|Genomes|Genetics  2012;2(1):83-97.
Natural hybridization between two strains, varieties, or species is a common phenomenon in both plants and animals. Although hybridization may skew established gene pools, it generates population diversity efficiently and sometimes results in the emergence of newly adapted genotypes. Cryptococcus neoformans, which causes the most frequent opportunistic fungal infection in immunocompromised hosts, has three serotypes: A, D, and AD. Serotype-specific multilocus sequence typing and serotype-specific comparative genome hybridization were applied to investigate the genetic variability and genomic organization of C. neoformans serotype AD isolates. We confirm that C. neoformans serotype AD isolates are hybrids of serotype A and D strains. Compared with haploid strains, most AD hybrid isolates exhibit unique multilocus sequence typing genotypes, suggesting that multiple independent hybridization events punctuated the origin and evolutionary trajectory of AD hybrids. The MATa alleles from both haploid and AD hybrid isolates group closely to form a cluster or subcluster in both the serotype A and D populations. The rare and unique distribution of MATa alleles may restrict sexual reproduction between isolates of opposite mating types. The genetic diversity of the serotype D population, including haploid strains and serotype D genomes of the AD hybrid, is significantly greater than that of serotype A, and there are signatures of recombination within the serotype D population. Given that MATa isolates are relatively rare, both opposite-sex and same-sex mating may contribute to genetic recombination of serotype D in nature. Extensive chromosome loss was observed in AD hybrid isolates, which results in loss of heterozygosity in the otherwise-heterozygous AD hybrid genome. Most AD hybrid isolates exhibit hybrid vigor and are resistant to the antifungal drug FK506. In addition, the C. neoformans AD hybrid genome is highly dynamic, with continuous chromosome loss, which may be a facile route for pathogen evolution through which genotypic and phenotypic variation is generated.
PMCID: PMC3276195  PMID: 22384385
19.  A New Synthetic Amphiploid (AADDAA) between Gossypium hirsutum and G. arboreum Lays the Foundation for Transferring Resistances to Verticillium and Drought 
PLoS ONE  2015;10(6):e0128981.
Gossypium arboreum, a cultivated cotton species (2n = 26, AA) native to Asia, possesses invaluable characteristics unavailable in the tetraploid cultivated cotton gene pool, such as resistance to pests and diseases and tolerance to abiotic stresses. However, it is quite difficult to transfer favorable traits into Upland cotton through conventional methods due to the cross-incompatibility of G. hirsutum (2n = 52, AADD) and G. arboreum. Here, we improved an embryo rescue technique to overcome the cross-incompatibility between these two parents for transferring favorable genes from G. arboreum into G. hirsutum. Our results indicate that MSB2K supplemented with 0.5 mgl-1 kinetin and 250 mg-1 casein hydrolysate is an efficient initial medium for rescuing early (3 d after pollination) hybrid embryos. Eight putative hybrids were successfully obtained, which were further verified and characterized by cytology, molecular markers and morphological analysis. The putative hybrids were subsequently treated with different concentrations of colchicine solution to double their chromosomes. The results demonstrate that four putative hybrid plants were successfully chromosome-doubled by treatment with 0.1% colchicine for 24 h and become amphiploid, which were confirmed by cytological observation, self-fertilization and backcrossing. Preliminary assessments of resistance at seedling stage indicate that the synthetic amphiploid showed highly resistant to Verticillium and drought. The synthetic amphiploid between G. hirsutum × G. arboreum would lay the foundation for developing G. arboreum-introgressed lines with the uniform genetic background of G. hirsutum acc TM-1, which would greatly enhance and simplify the mining, isolation, characterization, cloning and use of G. arboreum-specific desirable genes in future cotton breeding programs.
PMCID: PMC4465178  PMID: 26061996
20.  Different genome-specific chromosome stabilities in synthetic Brassica allohexaploids revealed by wide crosses with Orychophragmus 
Annals of Botany  2009;104(1):19-31.
Background and Aims
In sexual hybrids between cultivated Brassica species and another crucifer, Orychophragmus violaceus (2n = 24), parental genome separation during mitosis and meiosis is under genetic control but this phenomenon varies depending upon the Brassica species. To further investigate the mechanisms involved in parental genome separation, complex hybrids between synthetic Brassica allohexaploids (2n = 54, AABBCC) from three sources and O. violaceus were obtained and characterized.
Genomic in situ hybridization, amplified fragment length polymorphism (AFLP) and single-strand conformation polymorphism (SSCP) were used to explore chromosomal/genomic components and rRNA gene expression of the complex hybrids and their progenies.
Key Results
Complex hybrids with variable fertility exhibited phenotypes that were different from the female allohexaploids and expressed some traits from O. violaceus. These hybrids were mixoploids (2n = 34–46) and retained partial complements of allohexaploids, including whole chromosomes of the A and B genomes and some of the C genome but no intact O. violaceus chromosomes; AFLP bands specific for O. violaceus, novel for two parents and absent in hexaploids were detected. The complex hybrids produced progenies with chromosomes/genomic complements biased to B. juncea (2n = 36, AABB) and novel B. juncea lines with two genomes of different origins. The expression of rRNA genes from B. nigra was revealed in all allohexaploids and complex hybrids, showing that the hierarchy of nucleolar dominance (B. nigra, BB > B. rapa, AA > B. oleracea, CC) in Brassica allotetraploids was still valid in these plants.
The chromosomes of three genomes in these synthetic Brassica allohexaploids showed different genome-specific stabilities (B > A > C) under induction of alien chromosome elimination in crosses with O. violaceus, which was possibly affected by nucleolar dominance.
PMCID: PMC2706731  PMID: 19403626
Synthetic Brassica allohexaploids; Orychophragmus violaceus; intergeneric hybrids; genomic in situ hybridization; amplified fragment length polymorphism; single-strand conformation polymorphism; chromosome elimination; chromosome stability; nucleolar dominance
21.  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
22.  Production of bacterial blight resistant lines from somatichybridization between Oryza sativa L. and Oryza meyeriana L.*  
Novel bacterial blight (BB) resistance gene(s) for rice was (were) introduced into a cultivated japonica rice variety Oryza sativa (cv. 8411), via somatic hybridization using the wild rice Oryza meyeriana as the donor of the resistance gene(s). Twenty-nine progenies of somatically hybridized plants were obtained. Seven somatically hybridized plants and their parents were used for AFLP (amplified fragment length polymorphism) analysis using 8 primer pairs. Results confirmed that these plants were somatic hybrids containing the characteristic bands of both parents. The morphology of the regenerated rice showed characters of both O. sativa and O. meyeriana. Two somatic hybrids showed highest BB resistance and the other 8 plants showed moderate resistance. The new germplasms with highest resistance have been used in the rice breeding program for the improvement of bacterial blight resistance.
PMCID: PMC1388724  PMID: 15362190
Oryza sativa L.; Oryza meyeriana L.; Somatic hybridization; Rice bacterial blight resistance
23.  Production of Polyploids and Unreduced Gametes in Lilium auratum × L. henryi Hybrid 
Intergenomic F1 hybrids between L. auratum x L. henryi and their BC1 progeny were investigated through genomic in situ hybridization technique (GISH) to determine their potential value in lily breeding. We confirmed that F1 intergenomic hybrids possessed a set of chromosomes (x=12) from both parents and that flowers of the F1 auratum × henryi hybrid showed an intermediate morphological phenotype. Pollen size, viability and germination ability were measured through microscopic observations. F1 intergenomic hybrids produced a relevant frequency of 2n-gametes, which were successfully used to perform crosses with Oriental hybrids, resulting in the triploid Oriental Auratum Henryi (OAuH) hybrid. Twenty BC1 plants were generated by crossing between four different Oriental hybrid cultivars and F1 AuH hybrids using an in vitro embryo rescue technique, after which the genome constitution and chromosome composition were analyzed by GISH. All plants were triploid, showing 12 from female parents (diploid Oriental hybrid) and 24 from male parents (diploid F1 AuH hybrid). Overall, 16 out of 20 BC1 progeny possessed recombinant chromosomes with 1-5 crossover sites per plant. Cytological analysis of 20 BC1 plants by GISH verified that the occurrence of 2n pollen formation in all F1 AuH hybrids was derived from the FDR (first division restitution) mechanism, in which the genome composition of all BC1 plants possess 12 Oriental + 12 L. auratum + 12 L. henryi chromosomes. Allotriploids derived from the AuH hybrid were used as female for crossing with the diploid Oriental hybrid cultivar 'Sorbonne' and considerable numbers of plants (0-6.5 plants per ovary) were only obtained when female OAuH (BC1) triploids were used. Taken together, the results of this study indicate that production and analysis of F1 AuH hybrids and their progeny through sexual polyploidization can be useful for efficient creation of important horticultural traits.
PMCID: PMC3729011  PMID: 23904793
Allotriploid; Polyploidization; Homoeologous recombination; Interspecific hybrid; 2n-gamete.
24.  Microspore culture reveals complex meiotic behaviour in a trigenomic Brassica hybrid 
BMC Plant Biology  2015;15:173.
Development of synthetic allohexaploid Brassica (2n = AABBCC) would be beneficial for agriculture, as allelic contributions from three genomes could increase hybrid vigour and broaden adaptation. Microspore culture of a near-allohexaploid hybrid derived from the cross (B. napus × B. carinata) × B. juncea was undertaken in order to assess the frequency and distribution of homologous and homoeologous crossovers in this trigenomic hybrid. SNP and SSR molecular markers were used to detect inheritance of A, B and C genome alleles in microspore-derived (MD) progeny. SNP allele copy number was also assessed. The MD progeny were also compared to progeny derived by self-pollination and open-pollination for fertility (estimated by self-pollinated seed set and pollen viability) and DNA ploidy (measured by flow cytometry).
In the trigenomic hybrid, homologous chromosome pairs Aj-An, Bj-Bc and Cn-Cc had similar meiotic crossover frequencies and segregation to that previously observed in established Brassica species, as demonstrated by marker haplotype analysis of the MD population. Homoeologous pairing between chromosomes A1-C1, A2-C2 and A7-C6 was detected at frequencies of 12–18 %, with other homoeologous chromosome regions associating from 8 % (A3-C3) to 0–1 % (A8-C8, A8-C9) of the time. Copy number analysis revealed eight instances of additional chromosomes and 20 instances of chromosomes present in one copy in somatically doubled MD progeny. Presence of chromosome A6 was positively correlated with self-pollinated seed set and pollen viability in the MD population. Many MD progeny were unable to produce self-pollinated seed (76 %) or viable pollen (53 %), although one MD plant produced 198 self-pollinated seeds. Average fertility was significantly lower in progeny obtained by microspore culture than progeny obtained by self-pollination or open-pollination, after excluding MD progeny which had not undergone chromosome doubling.
Based on SNP data analysis of the microspore-derived progeny, crossover frequency per chromosome in the allohexaploid hybrid was found to be similar to that in established Brassica species, suggesting that the higher chromosome number did not significantly disrupt cellular regulation of meiosis. SNP allele copy number analysis revealed the occurrence not only of homoeologous duplication/deletion events but also other cryptic duplications and deletions that may have been the result of mitotic instability. Microspore culture simplified the assessment of chromosome behaviour in the allohexaploid hybrid but yielded progeny with lower fertility and a greater range of ploidy levels compared to progeny obtained by self- or open-pollination.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-015-0555-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4493989  PMID: 26152188
Allohexaploid; Brassica; SNP chip; Chromosome behaviour; Allele copy number analysis; Interspecific hybrids
25.  An orange fluorescent protein tagging system for real-time pollen tracking 
BMC Research Notes  2013;6:383.
Monitoring gene flow could be important for future transgenic crops, such as those producing plant-made-pharmaceuticals (PMPs) in open field production. A Nicotiana hybrid (Nicotiana. tabacum × Nicotiana glauca) shows limited male fertility and could be used as a bioconfined PMP platform. Effective assessment of gene flow from these plants is augmented with methods that utilize fluorescent proteins for transgenic pollen identification.
We report the generation of a pollen tagging system utilizing an orange fluorescent protein to monitor pollen flow and as a visual assessment of transgene zygosity of the parent plant. This system was created to generate a tagged Nicotiana hybrid that could be used for the incidence of gene flow. Nicotiana tabacum ‘TN 90’ and Nicotiana glauca were successfully transformed via Agrobacterium tumefaciens to express the orange fluorescent protein gene, tdTomato-ER, in pollen and a green fluorescent protein gene, mgfp5-er, was expressed in vegetative structures of the plant. Hybrids were created that utilized the fluorescent proteins as a research tool for monitoring pollen movement and gene flow. Manual greenhouse crosses were used to assess hybrid sexual compatibility with N. tabacum, resulting in seed formation from hybrid pollination in 2% of crosses, which yielded non-viable seed. Pollen transfer to the hybrid formed seed in 19% of crosses and 10 out of 12 viable progeny showed GFP expression.
The orange fluorescent protein is visible when expressed in the pollen of N. glauca, N. tabacum, and the Nicotiana hybrid, although hybrid pollen did not appear as bright as the parent lines. The hybrid plants, which show limited ability to outcross, could provide bioconfinement with the benefit of detectable pollen using this system. Fluorescent protein-tagging could be a valuable tool for breeding and in vivo ecological monitoring.
PMCID: PMC3856485  PMID: 24070251
Gene flow; Male-sterility; Plant made pharmaceuticals; Bioconfinement; Nicotiana; Green fluorescent protein (GFP); Orange fluorescent protein (OFP)

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