Segregation In Hybrids Research Articles

Transgressive Hybrids as Hopeful Monsters

The origin of novelty is a critical subject for evolutionary biologists. Early geneticists speculated about the sudden appearance of new species via special macromutations, epitomized by Goldschmidt’s infamous “hopeful monster”. Although these ideas were easily dismissed by the insights of the Modern Synthesis, a lingering fascination with the possibility of sudden, dramatic change has persisted. Recent work on hybridization and gene exchange suggests an underappreciated mechanism for the sudden appearance of evolutionary novelty that is entirely consistent with the principles of modern population genetics. Genetic recombination in hybrids can produce transgressive phenotypes, “monstrous” phenotypes beyond the range of parental populations. Transgressive phenotypes can be products of epistatic interactions or additive effects of multiple recombined loci. We compare several epistatic and additive models of transgressive segregation in hybrids and find that they are special cases of a general, classic quantitative genetic model. The Dobzhansky-Muller model predicts “hopeless” monsters, sterile and inviable transgressive phenotypes. The Bateson model predicts “hopeful” monsters with fitness greater than either parental population. The complementation model predicts both. Transgressive segregation after hybridization can rapidly produce novel phenotypes by recombining multiple loci simultaneously. Admixed populations will also produce many similar recombinant phenotypes at the same time, increasing the probability that recombinant “hopeful monsters” will establish true-breeding evolutionary lineages. Recombination is not the only (or even most common) process generating evolutionary novelty, but might be the most credible mechanism for sudden appearance of new forms.

Analysis of the meiotic segregation in intergeneric hybrids of tilapias.

Tilapia species exhibit a large ecological diversity and an important propensity to interspecific hybridisation. This has been shown in the wild and used in aquaculture. However, despite its important evolutionary implications, few studies have focused on the analysis of hybrid genomes and their meiotic segregation. Intergeneric hybrids between Oreochromis niloticus and Sarotherodon melanotheron, two species highly differentiated genetically, ecologically, and behaviourally, were produced experimentally. The meiotic segregation of these hybrids was analysed in reciprocal second generation hybrid (F2) and backcross families and compared to the meiosis of both parental species, using a panel of 30 microsatellite markers. Hybrid meioses showed segregation in accordance to Mendelian expectations, independent from sex and the direction of crosses. In addition, we observed a conservation of linkage associations between markers, which suggests a relatively similar genome structure between the two parental species and the apparent lack of postzygotic incompatibility, despite their important divergence. These results provide genomics insights into the relative ease of hybridisation within cichlid species when prezygotic barriers are disrupted. Overall our results support the hypothesis that hybridisation may have played an important role in the evolution and diversification of cichlids.

Analysis of Lateral Segregation Formed on DNA-Tethered Lipid Membranes by Varying Tether Lengths

We recently described a strategy to prepare DNA-tethered lipid membranes either to fixed DNA on a surface or to DNA displayed on a supported bilayer∗. An approach has been developed to improve the stability of the latter system, and with this it is possible to investigate the lateral segregation of DNA hybrids when different lengths are present (see Figure). The area fraction of the taller region is proportional to the mole percent of 48mer. The effects of DNA sequence (repeating vs. overlapping), length (72mer vs. 24mer) and salt concentration will be described. Starting from a theoretical model proposed by Qi et al.∗∗, the effect of population, length and affinity of DNA complexes is simulated and described. This model system captures some of the essential physics of synapse formation and is a step toward understanding lipid membrane behavior in a cell-to-cell junction.∗ Chung et al., Journal of Structural Biology, 168, 190-199 (2009)∗∗ Qi et al., Proc. Natl. Acad. Sci., 98, 6548 (2001)View Large Image | View Hi-Res Image | Download PowerPoint Slide

Mobile Antibiotic Resistance Encoding Elements Promote Their Own Diversity

Integrating conjugative elements (ICEs) are a class of bacterial mobile genetic elements that disseminate via conjugation and then integrate into the host cell genome. The SXT/R391 family of ICEs consists of more than 30 different elements that all share the same integration site in the host chromosome but often encode distinct properties. These elements contribute to the spread of antibiotic resistance genes in several gram-negative bacteria including Vibrio cholerae, the agent of cholera. Here, using comparative analyses of the genomes of several SXT/R391 ICEs, we found evidence that the genomes of these elements have been shaped by inter–ICE recombination. We developed a high throughput semi-quantitative method to explore the genetic determinants involved in hybrid ICE formation. Recombinant ICE formation proved to be relatively frequent, and to depend on host (recA) and ICE (s065 and s066) loci, which can independently and potentially cooperatively mediate hybrid ICE formation. s065 and s066, which are found in all SXT/R391 ICEs, are orthologues of the bacteriophage λ Red recombination genes bet and exo, and the s065/s066 recombination system is the first Red-like recombination pathway to be described in a conjugative element. Neither ICE excision nor conjugative transfer proved to be essential for generation of hybrid ICEs. Instead conjugation facilitates the segregation of hybrids and could provide a means to select for functional recombinant ICEs containing novel combinations of genes conferring resistance to antibiotics. Thus, ICEs promote their own diversity and can yield novel mobile elements capable of disseminating new combinations of antibiotic resistance genes.

Chromosome segregation in lymphocyte hybrids.

Chromosome segregation was studied in mouse/human and mouse/mouse T-lymphocyte hybrids, and in mouse/human, mouse/rabbit and mouse/rat B-lymphocyte hybrids. Chromosome loss was not random, it was moderate in mouse/mouse and mouse/rat lymphocyte hybrids, but extensive in mouse/human and mouse/rabbit lymphocyte hybrids. Chromosomes carrying genes coding for T-cell differentiation markers were preferentially retained in interspecific as well as intraspecific T-cell hybrids. In mouse/rabbit B-lymphocyte hybrids, only two of 750 cells from five independent mass cultures contained any rabbit chromosomes.

Morphological and molecular evidence for hybridization and introgression in Central European Arctium (Asteraceae)

Three species of burdock (Arctium lappa, A. tomentosum, and A. minus; Asteraceae), which commonly occur in sympatry in Central Europe, are thought to hybridize frequently, hence raising questions about their integrity. We surveyed mixed, monotypic, and/or single plant stands of these species in Central Germany to assess their extent of hybridization and introgression, and also the likelihood of insect host shifts between them. Individual plants were characterized by multivariate analyses of morphological and RAPD variation in combination with RAPD-based character-index-scores. All analyses recognized the three species as distinct units, but also identified a limited number of first (or early) generation hybrids between sympatric A. lappa and either A. tomentosum or A. minus, respectively, as well as one introgressant of A. tomentosum into A. lappa. While pre-zygotic isolation may partly account for the rarity of first generation crosses (F1's), the near absence of later generation hybrid segregants or introgressants likely reflects post-zygotic isolation due to the disruption of co-adapted gene complexes and/or habitat-mediated superiority shared by the ruderal parentals. As the tephritid fly Tephritis bardanae oviposits on both A. tomentosum (T) and A. minus (M), but not on A. lappa (L), the occurrence of L × M and L × T hybrids and/or introgressants may have important evolutionary consequences regarding the acquisition of a new host, i.e. A. lappa, by these insects.

Cytology and pollen grain fertility in creeping bentgrass interspecific and intergeneric hybrids

Creeping bentgrass (Agrostisstolonifera L. or A. palustris Huds.) is a highly outcrossing allotetraploid species. It can form hybrids with a number of other Agrostis species and Polypogon genus. However, cytology and pollen grain fertility of the creeping bentgrass interspecific and intergeneric hybrids are not well known. In this research, chromosome pairing behaviors during meiosis I in F1 and pollen viability of F1 hybrids, as well as seed set rate and seed germination rate of backcrosses were studied in hybrids between creeping bentgrass, and other bentgrass species and three species of Polypogon genus. Abnormal chromosome pairing, laggard chromosomes, and premature segregation in F1 hybrids were found. Pollen viability ranged from 1.6 to 48.5% amongst F1 hybrids, significantly lower than that of the parents (85.5–94.1%). Some hybrids produced pollens of different sizes within the same anther. Seed set following backcrosses using F1 hybrids as the male parent and creeping bentgrass as the recurrent parent was significantly lower than their parents. The study of chromosome paring behaviors and progeny fecundity are important in utilizing the alien genes to improve bio-stress and abio-stress resistance, and in assessing the potential transgene risks of creeping bentgrass.

Segregation Distortion in Hybrids Between the Bogota and USA Subspecies of Drosophila pseudoobscura

We show that, contrary to claims in the literature, "sterile" males resulting from the cross of the Bogota and USA subspecies of Drosophila pseudoobscura are weakly fertile. Surprisingly, these hybrid males produce almost all daughters when crossed to females of any genotype (pure Bogota, pure USA, hybrid F1). Several lines of evidence suggest that this sex ratio distortion is caused by sex chromosome segregation distortion in hybrid males. We genetically analyze this normally cryptic segregation distortion and show that it involves several regions of the Bogota X chromosome that show strong epistatic interactions with each other. We further show that segregation distortion is normally masked within the Bogota subspecies by autosomal suppressors. Our analysis shows that the genetic basis of hybrid segregation distortion is similar to that of hybrid male sterility between the same subspecies. Indeed the severity of segregation distortion is correlated with the severity of sterility among hybrids. We discuss the possibility that hybrid sterility in this paradigmatic case of incipient speciation is caused by segregation distortion.

RAPD analysis of grapevine hybrids and cultivars

Utilization of the Randomly Amplified Polymorphic DNA (RAPD) technique as a molecular marker was tested to investigate the relationships between some representative grapevine cultivars and hybrids established at the Department of Genetics and Plant Breeding (CUB), to distinguish clones as well as to characterize various hybrids between species or cultivars and their parents. Vitis vinifera cultivars were easily and successfully distinguished by the RAPD technique and they were grouped according to the traditional taxonomic classification. RAPD patterns of the examined Pinot gris clones proved to be completely identical. Number of generations was reflected by the value of genetic distance of the examined hybrids. Genetic identity of parents and their offsprings was influenced by the selection applied in the process of plant breeding. Parental phenotypic and morphologic characteristics showed high degree of segregation in hybrids, but RAPD analysis revealed that their genetic similarity is considerable. The three Vitis anntrensis clones were properly discriminated from every cultivar and hybrid of Vitis vinifera, i.e. hybrids are much closer to the cultivated grapevine than to V. anzurensis due to the phenotypic selection carried out during the life-cycle of one or two generations.

Comparative fitness of a wild squash species and three generations of hybrids between wild×virus-resistant transgenic squash

We compared some fitness components of the wild squash species Cucurbita pepo spp. ovifera var. texana (C. texana) and three generations of hybrids (F1, BC1, and BC2) between C. texana and commercial transgenic squash CZW-3 over three consecutive years under field conditions of low (LDP) and high disease pressure (HDP) by Cucumber mosaic virus (CMV), Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus (WMV). Transgenic squash CZW-3 expresses the coat protein (CP) genes of CMV, ZYMV, and WMV, and is resistant to these three aphid-borne viruses. Across all HDP trials, transgenic BC1 and BC2 hybrids expressing the three CP genes grew more vigorously, displayed resistance to CMV, ZYMV, and WMV, and produced a greater number of mature fruits and viable seeds than nontransgenic hybrid segregants and C. texana. Transgenic F1 hybrids behaved similarly to BC1 and BC2 hybrids but grew less vigorously than C. texana. In contrast, across all LDP trials, C. texana outperformed the transgenic and nontransgenic hybrid segregants. Further, only one back cross was necessary to recover individuals with most of the C. texana characteristics and yet maintain virus resistance. Our data suggest that C. texana acquiring CP transgenes upon hybridization and introgression could have a selective advantage if CMV, ZYMV, and WMV are severely limiting the growth and reproductibility of wild squash populations.

Single and joint gene segregation in intraspecific hybrids of brown trout ( Salmo trutta L.) lineages

Brown trout ( Salmo trutta L.) stocking practices in French Mediterranean rivers often result in artificial secondary contact and introgression between substantially differentiated genomes. Single and joint segregation at five protein and four microsatellite loci were analysed in two back-crosses between hybrid females (resulting from domestic×Mediterranean genitors) and hatchery males in order to test whether there is genetic incompatibility and selective phenomena between the genomes. Three crosses between hatchery genitors were performed and followed in the same time to measure and compare survival among back-cross (2) and hatchery (3) families. Only one of 23 single segregation tests ( LDH-5 for family 2) was significant with an excess of allele of the domestic origin in the F 1 hybrid. Out of 70 joint segregation tests, only six were significant. One segregation corresponded to “weak” associations involving one microsatellite locus ( Strutta-24) and one enzyme ( FBP-1). One case ( Strutta-24 and Strutta-12) was clearly caused by differential maternal transmission of alleles. Even if the question of a breakdown of fitness is only addressed in the hatchery environment, these results showed the existence of events during meiosis, which have affected the allelic transmission for hybrids of the two genomes.

Chromosomal segregation and fertility in Robertsonian chromosomal heterozygotes of the house musk shrew (Suncus murinus, Insectivora, Soricidae).

Crucial to our understanding of chromosomal variation and evolution in mammals are detailed studies of chromosomal heterozygotes, with analyses of chromosomal segregation and chromosome-derived infertility. We studied segregation and fertility in hybrids between karyotypic races of the house musk shrew Suncus murinus. These individuals were heterozygous for up to five Robertsonian fusions (Rbs) and an insertion of heterochromatin in an autosome. All variant chromosomes showed Mendelian segregation and all Rbs segregated independently of each other in the progeny of double heterozygotes. Litter size in single and even multiple Rb heterozygotes was no smaller than that in the less fertile parental strain. The effects of genetic background were more important in determining litter size than Rb heterozygosity for the shrews that we examined.

Characterization of Somatic Hybrids between Tetraploid Solanum tuberosum L. and Dihaploid S. acaule

Electrofusion was performed to produce somatic hybrids between Solanum tuberosum (2n=4x=48) and dihaploid S. acaule (2n=2x=24). Among the 18 regenerated plants obtained, 14 were found to be hexaploid hybrids based on RAPD analysis and cytological observation. In order to identify their cytoplasmic genome constitution, RFLP analysis of chloroplast and mitochondrial DNA was carried out. Six and eight hybrids were found to have S. tuberosum and S. acaule chloroplast genomes, respectively, suggesting that the chloroplast genomes of the somatic hybrids segregated randomly. In addition, two regenerated plants derived from the same callus had different chloroplast genomes, indicating that the segregation of the chloroplast genome occurred before shoot formation from callus. Concerning the mitochondrial genome constitution, 13 hybrids harbored the genome of S. tuberosum type and the others the S. acaule type genome. This mode of segregaion is obviously in favor of S. tuberosum mitochondrial genome. Regarding cytoplasmic genome segregation in somatic hybrids, the mitochondrial genome was not associated with the chloroplast genome. For the cytoplasmic genomes of both species, no hybrid was observed with recombinant or heterogeneous genome from both parents. In most of the somatic hybrids, some characters such as plant habit, shape of foliage and shape of tuber, were generally similar to those of S. tuberosum. The high level of resistance to potato virus X (PVX) detected in dihaploid S. acaule and somatic hybrids, suggests that the resistance to PVX is incorporated by fusion with S. acaule.

Extinction of Albumin Gene Expression in a Panel of Human Chromosome 2 Microcell Hybrids

Expression of the serum albumin gene is extinguished in rat hepatoma microcell hybrids that retain mouse chromosome 1. These data define atrans-dominant extinguisher locus,Tse-2,on mouse chromosome 1. To localize the human TSE2 locus, we prepared and characterized rat/human microcell hybrids that contained either human chromosome 1 or chromosome 2, the genetic homologues of mouse chromosome 1. Rat hepatoma microcell hybrids retaining a derivative human chromosome 1 [der 1 t(1;17)(p34.3;q11.2)] expressed their serum albumin genes at levels similar to those of parental hepatoma cells. In contrast, microcell transfer of human chromosome 2 into rat hepatoma recipients produced karyotypically heterogeneous collections of hybrid clones, some of which displayed dramatic albumin extinction phenotypes. For example, albumin mRNA levels in several extinguished microcell hybrids were reduced at least 500-fold, similar to albumin mRNA levels in hepatoma × fibroblast whole-cell hybrids. Expression of several other liver genes, including α1-antitrypsin, aldolase B, alcohol dehydrogenase, and phosphoenolpyruvate carboxykinase, was also affected in some of the microcell hybrids, but expression of these genes was not concordant with expression of albumin. Hybrid segregants were prepared from the albumin-extinguished hybrids, and reexpression of albumin mRNA and protein was observed in sublines that had lost or fragmented human chromosome 2. Finally, expression of mRNAs encoding the liver-enrichedtransactivators HNF-1, HNF-4, HNF-3α, and HNF-3β was not affected in any of the chromosome 2-containing hybrids. These data define and map a genetic locus on human chromosome 2 that extinguishes albumin gene expression intrans,and they suggest that TSE2-mediated extinction is independent of HNF-1, -4, -3α, and -3β expression.

Identification of human tumour suppressor genes by monochromosome transfer: rapid growth-arrest response mapped to 9p21 is mediated solely by the cyclin-D-dependent kinase inhibitor gene, CDKN2A (p16INK4A).

Microcell transfer of intact normal human chromosomes into immortal mouse and hamster fibroblast cell lines has revealed growth suppressive activity associated with a small sub-set of the human complement. Here, we describe the results of a detailed study aimed at identifying the gene or genes responsible for the rapid growth-arrest response obtained with human chromosome-9. Initially, STS-PCR deletion mapping of segregants arising in monochromosome transfer experiments was used successfully to localize the active sub-chromosomal region to 9p21. Subsequent fine-structure deletion mapping of previously uniformative hybrid segregants, employing additional markers between D9S162 and D9S171, provided strong evidence that the cyclin-dependent kinase (cdk) inhibitor gene CDKN2A (p16INK4A) was solely responsible for the chromosome-9 effect; 9p21 microdeletions in a significant proportion of segregant clones were restricted to a single CDKN2A exon. Transfection experiments with CDKN2A and CDKN2B cDNA expression vectors, using mouse A9 cells and three human malignant melanoma cell lines as recipients, provided further evidence in support of this hypothesis. Collectively, our results indicate that expression of human CDKN2A (controlled either by its natural regulatory elements, or by a cytomegalovirus promoter) is incompatible with in vitro proliferation in immortalized rodent cells and in human melanoma cell lines. The rapidity of the growth inhibitory effects of CDKN2A was inconsistent with a mode of action involving induction of replicative cell senescence via telomerase repression, but was consistent with a mechanism based on cell cycle arrest through cdk inhibition. The study described here has generated a panel of microdeleted monochromosome-9 donor hybrids which may prove valuable in functional investigations aimed at identifying other important tumour suppressor genes located on human chromosome-9.

Mapping eight new polymorphisms in 11q13 in the vicinity of multiple endocrine neoplasia type 1: identification of a new distal recombinant

Multiple endocrine neoplasia type 1 (MEN 1) is an autosomal dominant disorder that predisposes affected individuals to neoplasms of the parathyroid glands, endocrine pancreas, anterior pituitary, and carcinoids. The MEN1 locus has been localized by family studies to 11q13, flanked by markers PGA and D11S97. Eight new polymorphisms located in three separate radiation-reduced somatic cell hybrid segregation groups were developed. The order of the new markers, within the context of previously described loci, was determined by linkage analysis on the Venezuelan reference pedigree. Four independent MEN1 families, consisting of 57 affected individuals, and 70 individuals at-risk for the disease were genotyped. Sixteen people inherited a chromosome that shows recombination between a linked marker and the disease. The nearest proximal and distal markers that show recombination with the disease are D11S822 and GSTP1, respectively, thereby narrowing the candidate region for MEN1 by 50% on the distal side. Using these loci in haplotype analysis, an accurate presymptomatic molecular diagnostic test has been developed. These new markers in 11q13 linked to MEN1 also facilitate the genetic and physical characterization of this very gene-rich region.

Tissue-specific extinguisher loci in the human genome: a screening study based on random marking and transfer of human chromosomes.

Expression of many liver-specific genes is extinguished when cultured hepatoma cells are fused with fibroblasts, but liver genes can be reexpressed in hybrid segregants that have lost fibroblast chromosomes. To map extinguisher loci involved in this process, hepatoma microcell hybrids retaining single fibroblast chromosomes have been employed. Two different, transdominant loci that affect liver gene expression have been defined in this way. To determine whether other monochromosomal extinction phenotypes could be observed, we inserted a selectable marker into many human chromosomal sites and transferred the marked human chromosomes into rat hepatoma recipient cells by microcell fusion. Nearly 200 microcell hybrid clones were isolated and screened for expression of liver-specific mRNAs. Most liver transcripts continued to be expressed. However, PEPCK mRNA was extinguished in 12 hybrid clones. Some of these hybrids contained human TSE1, the previously characterized extinguisher locus on chromosome 17, but others contained a novel extinguishing function that mapped to human chromosome 14. The implications of these findings are discussed.

Biased mitochondrial segregation, independent of cell type used for fusion and of hybrid nuclear DNA content, was found in Brassica napus (+) B. oleracea somatic hybrids

To determine whether the cell type used for protoplast fusion influenced the mitochondrial segregation in somatic hybrids, mtDNA analysis was performed in Brassica napus (+) B. oleracea hybrid plants obtained from four different fusion combinations of hypocotyl and mesophyll protoplasts. In these fusions B. oleracea mitochondria were combined with B. campestris mitochondria since the B. napus cultivar had a cytoplasm derived from B campestris. Two mitochondrial gene probes, atp9 and atpA, were used to analyse mtDNA in the regenerated hybrids. In total, 96 plants were analysed. In all fusion categories, an overall biased segregation of mtDNA demonstrating a B. campestris specific pattern was found. No significant differences in segregation pattern of atp9 or atpA were found between the fusion categories. Thus, no major influence of the cell type used for protoplast fusion on mitochondrial segregation in the hybrids was found. Furthermore, nuclear DNA content analyses were available for 74 hybrids and we investigated whether the mitochondrial segregation patterns were different in hybrids exhibiting different ploidy levels. No significant difference in mitochondrial segregation was found between symmetric hybrids and hybrids with higher or lower ploidy levels. The overall strong bias of hybrids exhibiting a B. campestris RFLP pattern indicates that inherent differences of mitochondrial genomes from the different species exist, which affect the segregation in biparental cytoplasms.

Studies on Backcross Progeny of N. rustica * N. tabacum Interspecific Hybrids I. Cytological analysis.

The mammoth gene in Nicotiana tabacum causes the tobacco plant, which is otherwise day-neutral to flower under short photoperiodic conditions. The mammoth gene has been transferred to the genome of N. rustica through interspecific hybridization followed by repeated backcrossing to N. rustica. The expression and segregation of the mammoth gene was complex in the N. rustica genetic background. Plants with abnormal floral expressions and altered responses to environmental factors such as photoperiod and thermoperiod frequently appeared in the rustica-tabacum hybrid population.Three different flowering responses characterized the segregating populations. A small proportion of rustica-tabacum mammoth hybrids remained non flowering with continued vegetative growth for an indefinite period of time irrespective of photoperiod or thermoperiod. Nine percent of plants in the segregating backcross progeny expressed flower reversion phenomenon which was characterized by the appearance of terminal flowers with abnormal floral morphology, later reverting to normal inflorescence as in N. rustica. The remainder of the hybrid segregants were identical to N. rustica in floral morphology as well as in their responses to photoperiod. The segregation of plants with altered flowering expressions among the backcross progeny was highly heterogeneous.Cytological investigations were carried out both at mitosis and meiosis to find out whether the heterogeneous segregation of plants with altered flowering expressions could be related to abnormal chromosome segregations. The studies revealed variation in chromosome number at mitosis in flower revertant and non-flowering plants. The studies also revealed cytological aberrations in the hybrid progenies segregating for flower revertant and normal plants at meiosis. The results indicate that their genomes are at an unstable state and it was concluded that the heterogeneous segregation in the backcross hybrid progeny with altered responses to environmental factors and abnormal floral expressions may at least in part be due to chromosome instabilities.

Studies on Backcross Progeny of N. rustica * N. tabacum Interspecific Hybrids II. Genomic DNA analysis.

The mammoth gene of Nicotiana tabacum has been transferred to N. rustica genome through interspecific hybridization followed by repeated backcrossing to N. rustica. Our previous studies have revealed that the expression and segregation of the mammoth gene was more complex in the N. rustica genetic background than in N. tabacum. Plants with abnormal floral expressions such as non flowering (NF) and flower revertants (FR) with altered responses to environmental factors such as photoperiod and thermoperiod frequently appeared in the backcross progeny of rustica-tabacum hybrids. The segregation of plants for FR and NF characters among the progeny was highly heterogeneous and chromosome instabilities were found both at mitosis and meiosis.Characterization of the genomic DNA of rustica-tabacum hybrid segregants (FR and NF) and the parental species (N. tabacum and N. rustica) was carried out in the present study using thermal denaturation and analytical density gradient analyses. Differences were observed in the DNA composition among the hybrid plants segregating for abnormal flowering expressions, FR and NF, as well as the parental species. Variation was observed in the mole fraction of 5-methyl deoxycytidine in the genomic DNA of the hybrid progenies and the parental species. Variation in the amount of satellite DNA fraction was also found between the hybrid plants, FR and NF and the recurrent parental species N. rustica. The differences in the buoyant densities, satellite DNA fraction, 5-methyl deoxycytidine contents between FR, NF and N. rustica suggests reorganization of genomes of the hybrid progenies following interspecific hybridization. The variation could result in part from the abnormal segregation of chromosomes and chromosome aberrations frequently found in the backcross progeny of rustica-tabacum hybrids at mitosis and meiosis.