Spatial and temporal expression of the two sucrose synthase genes in maize: immunohistological evidence

Summary The DNAs of two diploid species of Gossypium, G. herbaceum var. africanum (A₁ genome) and G. raimondii (D₅ genome), and the allotetraploid species, G. hirsutum (A_h and D_h genomes), were characterized by kinetic analyses of single copy and repetitive sequences. Estimated haploid genome sizes of A₁ and D₅ were 1.04 pg and 0.68 pg, respectively, in approximate agreement with cytological observations that A genome chromosomes are about twice the size of D genome chromosomes. This differences in genome size was accounted for entirely by differences in the major repetitive fraction (0.56 pg versus 0.20 pg), as single copy fractions of the two genomes were essentially identical (0.41 pg for A₁ and 0.43 pg for D₅). Kinetic analyses and thermal denaturation measurements of single copy duplexes from reciprocal intergenomic hybridizations showed considerable sequence similarity between A₁ and D₅ genomes (77% duplex formation with an average thermal depression of 6 °C). Moreover, little sequence divergence was detectable between diploid single copy sequences and their corresponding genomes in the allotetraploid, consistent with previous chromosome pairing observations in interspecific F₁ hybrids.Journal paper No. 4461 of the Arizona Agricultural Experiment Station     

Ancient origin of the vacuolar H+-ATPase 69-kilodalton catalytic subunit superfamily

Recently, two distinct cDNA clones encoding the catalytic subunit of the vacuolar H⁺-ATPase (V-ATPase) were isolated from the allotetraploid cotton species Gossypium hirsutum L. cv Acala SJ-2 (Wilkins 1992, 1993). Differences in the nucleotide sequence of these clones were used as molecular markers to explore the organization and structure of the V-ATPase catalytic subunit genes in the A and D genomes of diploid and allotetraploid cotton species. Nucleotide sequencing of polymerase chain reaction (PCR) products amplified from G. arboreum (A₂, 2n=26), G. raimondii (D₅, 2n=26), and G. hirsutum cv Acala SJ-2 [(AD)₁, 2n=4x=52] revealed a V-ATPase catalytic subunit organization more complex than indicated hitherto in any species, including higher plants. In the genus Gossypium, the V-ATPase catalytic subunit genes are organized as a superfamily comprising two diverse but closely related multigene families, designated as vat69A and vat69B, present in both diploid and allotetraploid species. As expected, each vat69 subfamily is correspondingly more complex in the allotetraploid species due to the presence of both A and D alloalleles. Because of this, about one-half of the complex organization of V-ATPase catalytic subunit genes predates polyploidization and speciation of New World tetraploid species. Comparison of plant and fungal V-ATPase catalytic subunit gene structure indicates that introns accrued in the plant homologs following the bifurcation of plant and fungi but prior to the gene duplication event that gave rise to the vat69A and vat69B genes approximately 45 million years ago. The structural complexity of plant V-ATPase catalytic subunit genes is highly conserved, indicating the presence of at least ten introns dispersed throughout the coding region.     

Organization,inheritance and expression of acetohydroxyacid synthase genes in the cotton allotetraploid Gossypium hirsutum

The acetohydroxyacid synthase (AHAS) gene family of the cotton AD allotetraploid Gossypium hirsutum has been cloned and characterized. We have identified six different AHAS genes from an analysis of genomic clones and Southern blots of genomic DNA. Four of the six genes are organized as tandem pairs, in which the genes are separated by only 2–3 kb. Conservation of restriction fragment length polymorphisms between G. hirsutum and A-genome and D-genome-containing diploid cottons was sufficient to assign the single genes in clones A5 and A19 to the A and D subgenomes, respectively. Each diploid genome has one tandem pair, but in these cases we could not make specific subgenomic assignments. DNA and deduced amino acid sequences were determined for the A5 and A19 genes, and an AHAS cDNA clone isolated from a leaflibrary. The sequence of the A19 gene matches that of the cDNA clone, while the A5 gene is 97.8% similar. The four genes comprising the tandem pairs are much less similar to the cDNA clone. The deduced amino acid sequences of the mature polypeptides encoded by the A5 and A19 genes are collinear with the housekeeping forms of AHAS from Arabidopsis thaliana, Nicotiana tabacum and Brassica napus. The constitutive expression of A5 and A19 was confirmed with RNase protection assays and northern blots. We conclude that these genes encode the main house-keeping froms of AHAS in G. hirsutum. Among the four AHAS genes comprising the two tandem pairs, at least two are functional. These genes exhibit either low-level constitutive expression (one or both of the downstream genes of each pair), or highly specific expression in reproductive tissue (one or both of the upstream genes of each pair). The AHAS gene family of G. hirsutum is more complex than that of other plants so far examined.     

Rapid proliferation and nucleolar organizer targeting centromeric retrotransposons in cotton

Centromeric chromatin in most eukaryotes is composed of highly repetitive centromeric retrotransposons and satellite repeats that are highly variable even among closely related species. The evolutionary mechanisms that underlie the rapid evolution of centromeric repeats remain unknown. To obtain insight into the evolution of centromeric repeats following polyploidy, we studied a model diploid progenitor (Gossypium raimondii, D‐genome) of the allopolyploid (AD‐genome) cottons, G. hirsutum and G. barbadense. Sequence analysis of chromatin‐immunoprecipitated DNA showed that the G. raimondii centromeric repeats originated from retrotransposon‐related sequences. Comparative analysis showed that nine of the 10 analyzed centromeric repeats were absent from the centromeres in the A‐genome and related diploid species (B‐, F‐ and G‐genomes), indicating that they colonized the centromeres of D‐genome lineage after the divergence of the A‐ and D‐ ancestral species or that they were ancestrally retained prior to the origin of Gossypium. Notably, six of the nine repeats were present in both the A‐ and D‐subgenomes in tetraploid G. hirsutum, and increased in abundance in both subgenomes. This finding suggests that centromeric repeats may spread and proliferate between genomes subsequent to polyploidization. Two repeats, Gr334 and Gr359 occurred in both the centromeres and nucleolar organizer regions (NORs) in D‐ and AD‐genome species, yet localized to just the NORs in A‐, B‐, F‐, and G‐genome species. Contained within is a story of an established centromeric repeat that is eliminated and allopolyploidization provides an opportunity for reinvasion and reestablishment, which broadens our evolutionary understanding behind the cycles of centromeric repeat establishment and targeting.     

Alloplasmic male sterility in AD allotetraploid Gossypium hirsutum upon replacement of its resident A cytoplasm with that of D species G. harknessii

Summary Alloplasmic male sterile (cms) and restoration-of-fertility (Rf) lines of the AD allotetraploid Gossypium hirsutum were earlier derived from the presumed introgression of the cytoplasm of the D species G. harknessii. To confirm that this happened and address its significance, cytoplasms of the maternal progenitor, backcross intermediates, derived breeding lines, related A, D, and F species, and a synthetic AD tetraploid were examined by agarose and polyacrylamide gel electrophoresis of 140 restriction enzyme fragments of chloroplast DNA. Length mutations of 10–50 nucleotides predominate over site loss/gain mutations. Chloroplast DNA is maternally inherited and that of G. harknessii has been maintained in the cms lines for at least 13 successive generations without detectable alteration. Chloroplast DNA divergence is consistent with current nuclear genome classification and shows that the A progenitor was the maternal parent of the AD tetraploids. As predicted from incompatability models of cms, the degree of male sterility in alloplasmic Gossypium tetraploids is correlated with the extent of evolutionary divergence of their cytoplasms. It is suggested that the A genome in the AD tetraploids dominates those nuclear-cytoplasm interactions reflected by male fertility.     

Production of fertile somatic hybrids of Gossypium hirsutum?+?G. bickii and G. hirsutum?+?G. stockii via protoplast fusion

Fertile somatic hybrids were obtained via symmetric electrofusion of protoplasts from two combinations of tetraploid cotton (G. hirsutum cv. Coker 201, AD genome) and diploid wild cottons G. bickii (G genome) and G. stockii (E genome), respectively. Observation by morphological, flow cytometric analysis, chromosome counting and RAPD analysis of the tested hybrids of Coker 201 + G. bickii and Coker 201 + G. stockii confirmed the regenerated plants as hybrid status. Cytological investigation of the metaphase root-tip cells revealed there were 78 chromosomes in the hybrids. Flow cytometric analysis showed the tested plants had a relative DNA contents close to the total DNA contents of the two parents. RAPD analysis revealed the hybrids contained specific genomic fragments from both fusion partners, further confirmed their hybridity. The morphology of the hybrids was intermediate between the two fusion partners. The hybrid plants were successfully transferred to the soil, and they bloomed and set bolls. It is sure that the new hexaploids developed by cell fusion would contribute to cotton breeding through backcrossing with the elite genotypes of G. hirsutum.     

Validation of a cotton-specific gene, Sad1, used as an endogenous reference gene in qualitative and real-time quantitative PCR detection of transgenic cottons

Genetically modified (GM) cotton lines have been approved for commercialization and widely cultivated in many countries, especially in China. As a step towards the development of reliable qualitative and quantitative PCR methods for detecting GM cottons, we report here the validation of the cotton (Gossypium hirsutum) endogenous reference control gene, Sad1, using conventional and real-time (RT)-PCR methods. Both methods were tested on 15 different G. hirsutum cultivars, and identical amplicons were obtained with all of them. No amplicons were observed when DNA samples from three species of genus Gossypium, Arabidopsis thaliana, maize, and soybean and others were used as amplified templates, demonstrating that these two systems are specific for the identification and quantification of G. hirsutum. The results of Southern blot analysis also showed that the Sad1 gene was two copies in these 15 different G. hirsutum cultivars. Furthermore, one multiplex RT-quantitative PCR employing this gene as an endogenous reference gene was designed to quantify the Cry1A(c) gene modified from Bacillus thuringiensis (Bt) in the insect-resistant cottons, such as Mon531 and GK19. The quantification detection limit of the Cry1A(c) and Sad1 genes was as low as 10 pg of genomic DNA. These results indicat that the Sad1 gene can be used as an endogenous reference gene for both qualitative and quantitative PCR detection of GM cottons.     

DNA evidence for multiple origins of intergeneric allopolyploids in annualMicroseris (Asteraceae)

Seventy populations of North American annualMicroseris, Stebbinsoseris, andUropappus species were examined for chloroplast and nuclear ribosomal DNA restriction site variability to determine the origin of the allotetraploid speciesS. heterocarpa andS. decipiens. Previously identified chloroplast DNA restriction site variants were used in concert with restriction site variation forNco I in the nuclear-encoded ribosomal DNA repeat. The presence of two, mutually exclusive restriction site gains were observed in diploid populations ofM. douglasii; these same variants were also found in populations of allotetraploidS. heterocarpa, indicating mutiple origins of this species from different maternal diploid populations ofM. douglasii. Variation in the rDNA repeat between the diploid annual species and the putative paternal genome ofU. lindleyi was found to be additive inS. heterocarpa. A similar relationship was observed for the origin ofS. decipiens; cpDNA restriction site variants found inM. bigelovii andM. douglasii were present inS. decipiens. The rDNANco I variants also were additive in this purported allotetraploid. These results confirm the reticulate evolutionary pattern inStebbinsoseris and provide another example of multiple origins of intergeneric allopolyploids.     

Restriction fragment length polymorphism analysis of CCDD genome species of the genus Oryza L.

Restriction fragment length polymorphisms (RFLPs) were studied in fourteen accessions of CCDD genome allotetraploid wild rice species (Oryza latifolia, O. alta and O. grandiglumis). Fourteen nuclear RFLP markers previously mapped in AA genome-cultivated rice were used as probes. A phylogenetic tree, constructed by parsimony analysis based on RFLPs, grouped the accessions according to their geographic origin from Central or South America. Oryza alta, O. grandiglumis and one accession of O. latifolia grouped together as a subgroup, and our results suggested that the three taxa should be considered as populations of a single complex species. Duplicate loci, representing the two constituent genomes of the allotetraploid, were observed for most RFLP markers. By comparing RFLPs from the allotetraploids with those from a CC genome diploid wild species (O. officinalis), it was possible to detect RFLPs specific for both the CC and DD genomes of the allotetraploid. In inter-accession F2 populations, independent segregation of RFLP markers for CC and DD genomes was observed.     

Genetic differentiation,polyploidization and hybridization in northern EuropeanDactylorhiza (Orchidaceae): Evidence from allozyme markers

Taxa endemic to North-western Europe are rare, but the orchid genusDactylorhiza contains several species restricted to this area. Evidence from morphological and cytological studies have indicated that some species may have arisen recently and may be of hybrid origin. In the present report, I use allozymes to characterize the genomes in various species ofDactylorhiza and evaluate the possibilities for rapid evolutionary change in the genus. Allotetraploid species have evolved repeatedly from two principal diploid ancestral lineages. These lineages include extant diploid and autotetraploid species, from which allotetraploid derivatives may still arise. It is suggested that allotetraploidization dominates over introgression as speciation mechanism in the genus. The more common and widespread allotetraploid species could be characterized by their allozyme characters over considerable distances, indicating that each of them may have a unique origin and that they have spread from their ancestral populations to the present distribution areas. However, it is also possible that some allotetraploid species contain local populations that have been independently derived from the ancestral lineages.     

Isozyme variation and species relationships in peanut and its wild relatives (Arachis L. — Leguminosae)

Summary Arachis hypogaea (peanut or groundnut) is an AABB allotetraploid whose precise ancestry is not yet clear. Its closest diploid relatives are the annual and perennial wild species included with it in the section Arachis. Variation in these species for 11 different enzymes was studied by starch-gel electrophoresis. Differences attributed to at least 13 genetic loci were found among eight enzymes, while three enzymes appeared uniform throughout the section. Values for Nei's genetic distance were calculated for all pairs of species and were used to estimate relationships. All diploid species, apart from two whose validity had previously been questioned, could be distinguished by their overall zymotypes, but few contained unique alleles. When species were grouped by their mean genetic distances, they formed two clusters, which agreed reasonably well with the division of the section into annual versus perennial species. The single B-genome species was an outlier within the annual group. A. hypogaea showed fixed heterozygosity at four loci (in ssp. hypogaea) or six loci (in ssp. fastigiata), which agrees with previous conclusions that the peanut is an allotetraploid. None of the diploids included in this survey could be conclusively identified as donors of either the A or the B genome to the tetraploids. The two subspecies of A. hypogaea differed consistently in two of the thirteen putative loci studied. This may call into question the simple hypothesis that A. hypogaea originated from just two diploid species.     

Phylogenetic diversity and relationship among Gossypium germplasm using SSRs markers

Gossypium species represent a vast resource of genetic multiplicity for the improvement of cultivated cotton. To determine genetic diversity and relationships within a diverse collection of Gossypium, we employed 120 SSR primers on 20 diploid species representing seven basic genome groups of the genus Gossypium, five AD allotetraploid cotton accessions while T. populnea served as an outgroup species. Out of 120 SSR primers, 49 pairs are polymorphic, which produced a total of 99 distinct alleles with an average of 2.0 alleles per primer pair. A total of 1139 major SSR bands were observed. Genetic similarities among all the diploid species ranged from 0.582 (between G. herbaceum and G. trilobum) up to 0.969 (between G. arboreum and G. herbaceum). Phylogenetic trees based on genetic similarities were consistent with known taxonomic relationships. The results also indicated that G. raimondii is the closest living relative of the ancestral D-genome donor of tetraploid species and the A-genome donor is much similar to the present-day G. herbaceum and G. arboreum. Ancient tetraploid cotton species were formed by hybridizing and chromosome doubling between them, then different tetraploid cotton species appeared by further geographical and genetic isolation and separating differentiation. The results showed that SSRs could be an ideal means for the identification of the genetic diversity and relationship of cotton resources at the genomic level.     

Stylosanthes sp. aff.S. scabra: a putative diploid progenitor ofStylosanthes scabra (Fabaceae)

Stylosanthes sp. aff.S. scabra is an undescribed taxon showing affinities with the allotetraploid speciesS. scabra, but distinct in a number of attibutes. Several collections show potential as forage for clay soils in northern Australia. Twelve accessions have been analysed using STS (sequence-tagged-sites) as genetic markers, and they all displayed STS phenotypes of typical diploid species. Taking into account their morphological similarities, the STS analysis provides strong evidence thatStylosanthes sp. aff.S. scabra might be a diploid progenitor of the allotetraploidS. scabra. This speculation was supported by cytological examinations. Somatic chromosome numbers of two of these accessions were counted and both were found to be diploid (2n = 20). The level of polymorphism among the 12Stylosanthes sp. aff.S. scabra accessions, estimated using randomly amplified polymorphic DNA (RAPD) as markers, was 7.8%, and the dissimilarity value betweenStylosanthes sp. aff.S. scabra andS. viscosa (the other putative progenitor ofS. scabra) was 89%.     

关于棉属四倍体种起源问题的过氧化物酶同工酶研究

本文采用聚丙烯酰胺凝胶垂直板电泳和等电聚焦技术,对棉属(Gossypium)A基因组2个二倍体种、D基因组10个二倍体野生种和四倍体2(AD)基因组的3个种进行过氧化物酶同工酶酶谱分析。种间酶谱关系符合形态学,细胞学和遗传学的研究结果,但G.gossypioides,G.thurberi和G.trilobum的酶谱与D基因组其他种有较大差异却与A基因组相似。由二倍体种酶液组成的体外人工混合体与自然四倍体的比较分析表明,四倍体棉种G.darwinii,G.barbadense和G.hirsutum是A基因组和D基因组的异质组合,G.raimondii而不是G.thurberi或G.trilobum为四倍体种祖先基因组的最可能的D亚基因组供体。对过氧化物酶同工酶分析为棉属种间亲缘关系和四倍体起源的研究提供生化遗传依据的可行性进行了阐述。     

Entire nucleotide sequences of Gossypium raimondii and G. arboreum mitochondrial genomes revealed A‐genome species as cytoplasmic donor of the allotetraploid species

• Cotton (Gossypium spp.) is commonly grouped into eight diploid genomic groups, designated A–G and K, and an allotetraploid genomic group, AD. Gossypium raimondii (D₅) and G. arboreum (A₂) are the putative contributors to the progenitor of G. hirsutum (AD₁), the economically important fibre‐producing cotton species.
• Mitochondrial DNA from week‐old etiolated seedlings was extracted from isolated organelles using discontinuous sucrose density gradient method. Mitochondrial genomes were sequenced, assembled, annotated and analysed in orderly.
• Gossypium raimondii (D₅) and G. arboreum (A₂) mitochondrial genomes were provided in this study. The mitochondrial genomes of two diploid species harboured circular genome of 643,914 bp (D₅) and 687,482 bp (A₂), respectively. They differ in size and number of repeat sequences, both contain illuminating triplicate sequences with 7317 and 10,246 bp, respectively, demonstrating dynamic difference and rearranged genome organisations. Comparing the D₅ and A₂ mitogenomes with mitogenomes of tetraploid Gossypium species (AD₁, G. hirsutum; AD₂, G. barbadense), a shared 11 kbp fragment loss was detected in allotetraploid species, three regions shared by G. arboreum (A₂), G. hirsutum (AD₁) and G. barbadense (AD₂), while eight regions were specific to G. raimondii (D₅). The presence/absence variations and gene‐based phylogeny supported that A‐genome is a cytoplasmic donor to the progenitor of allotetraploid species G. hirsutum and G. barbadense.
• The results present structure variations and phylogeny of Gossypium mitochondrial genome evolution.

     

Use of single-primer DNA amplifications in genetic studies of peanut (Arachis hypogaea L.)

A recent approach to detecting genetic polymorphism involves the amplification of genomic DNA using single primers of arbitrary sequence. When separated electrophoretically in agarose gels, the amplification products give banding patterns that can be scored for genetic variation. The objective of this research was to apply these techniques to cultivated peanut (Arachis hypogaea L.) and related wild species to determine whether such an approach would be feasible for the construction of a genetic linkage map in peanut or for systematic studies of the genus. Two peanut cultivars, 25 unadapted germplasm lines of A. hypogaea, the wild allotetraploid progenitor of cultivated peanut (A. monticola), A. glabrata (a tetraploid species from section Rhizomatosae), and 29 diploid wild species of Arachis were evaluated for variability using primers of arbitrary sequence to amplify segments of genomic DNA. No variation in banding pattern was observed among the cultivars and germplasm lines of A. hypogaea, whereas the wild Arachis species were uniquely identified with most primers tested. Bands were scored (+/–) in the wild species and the PAUP computer program for phylogenetic analysis and the HyperRFLP program for genetic distance analysis were used to generate dendrograms showing genetic relationships among the diploid Arachis species evaluated. The two analyses produced nearly identical dendrograms of species relationships. In addition, approximately 100 F₂ progeny from each of two interspecific crosses were evaluated for segregation of banding patterns. Although normal segregation was observed among the F₂ progeny from both crosses, banding patterns were quite complex and undesirable for use in genetic mapping. The dominant behavior of the markers prevented the differentiation of heterozygotes from homozygotes with certainty, limiting the usefulness of arbitrary primer amplification products as markers in the construction of a genetic linkage map in peanut.     

Identification of Diploid <Emphasis Type="Italic">Stylosanthes seabrana</Emphasis> Accessions from Existing Germplasm of <Emphasis Type="Italic">S. scabra</Emphasis> Utilizing Genome-Specific STS Markers and Flow Cytometry,and Their Molecular Characterization

Stylosanthes seabrana (Maass and ‘t Mannetje) (2n = 2x = 20), commonly known as Caatinga stylo, is an important tropical perennial forage legume. In nature, it largely co-exist with S. scabra, an allotetraploid (2n = 4x = 40) species, sharing a very high similarity for morphological traits like growth habit, perenniality, fruit shape and presence of small appendage at the base of the pod or loment. This makes the two species difficult to distinguish morphologically, leading to chances of contamination in respective germplasm collections. In present study, 10 S. seabrana accessions were discovered from the existing global germplasm stock of S. scabra represented by 48 diverse collections, utilizing sequence-tagged-sites (STS) genome-specific markers. All the newly identified S. seabrana accessions displayed STS phenotypes of typical diploid species. Earlier reports have conclusively indicated S. seabrana and S. viscosa as two diploid progenitors of allotetraploid S. scabra. With primer pairs SHST3F3/R3, all putative S. seabrana yielded single band of ~550 bp and S. viscosa of ~870 bp whereas both of these bands were observed in allotetraploid S. scabra. Since SHST3F3/R3 primer pairs are known to amplify single or no band with diploid and two bands with tetraploid species, the amplification patterns corroborated that all newly identified S. seabrana lines were diploid in nature. Flow cytometric measurement of DNA content of the species, along with distinguishing morphological traits such as flowering time and seedling vigour, which significantly differ from S. scabra, confirmed all identified lines as S. seabrana. These newly identified lines exhibited high level of similarity among themselves as revealed by RAPD and STS markers (>92% and 80% respectively). Along with the enrichment in genetic resources of Stylosanthes, these newly identified and characterized accessions of S. seabrana can be better exploited in breeding programs targeted to quality.     

Alcohol dehydrogenase isozymes inGossypium hirsutum and its putative diploid progenitors: The biochemical consequences of enzyme multiplicity

Electrophoretic variation in alcohol dehydrogenase (ADH) was examined in tetraploidGossypium hirsutum and its putative diploid progenitorsG. ramondii, G. herbaceum, and a close relative,G. arboreum. All the diploids had three isozymes, while strains of the tetraploidG. hirsutum had either 4 or 6. Each isozyme was eluted from starch gels and significant differences in activity were noted between several of the isozymes relative to pH, substrate, temperature and salinity. This suggests that an increase in enzyme heterozygosity can result in higher levels of developmental homeostasis, but it depends on the isozyme alleles involved. Michigan Agricultural Experiment Station Journal Article No. 10379.     

Genome composition of asymmetric hybrids in relation to the phylogenetic distance between the parents. Nucleus-chloroplast interaction

Summary A series of fusion experiments were performed between protoplasts of a cytoplasmic albino mutant of tomato, Lycopersicon esculentum (ALRC), and gamma-irradiated protoplasts of L. hirsutum and the Solanum species S. commersonii, S. etuberosum and S. nigrum. These species were chosen for their different phylogenetic relationships to tomato. In all fusion combinations except from those between ALRC and S. nigrum, green calli were selected as putative fusion products and shoots regenerated from them. They were subsequently analyzed for their morphology, nuclear DNA composition and chloroplast DNA origin. The hybrids obtained between ALRC and L. hirsutum contained the chloroplasts of L. hirsutum and had the flower and leaf morphology of L. esculentum. After Southern blot analysis, using 13 restriction fragment length polymorphisms (RFLPs) randomly distributed over all chromosomes, all hybrids showed L. esculentum hybridization patterns. No chromosomes of L. hirsutum were found. These results indicate that these hybrids were true cybrids.The putative asymmetric hybrids, obtained with S. commersonii and S. etuberosum, showed phenotypic traits of both parents. After hybridization with species-specific repetitive nuclear DNA probes it was found that nuclear material of both parents was present in all plants. In the case of S. nigrum, which combination has the greatest phylogenetic distance between the fusion parents, no hybrid plants could be obtained. The chloroplast DNA of all hybrid plants was of the donor type suggesting that chloroplast transfer by asymmetric protoplast fusion can overcome problems associated with large phylogenetic distances between parental plants.