Effects of parental ploidy level and genetic divergence on chromosome elimination and chloroplast segregation in somatic hybrids within Brassicaceae

Summary Chromosome and organelle segregation after the somatic hybridization of related species with different degrees of genetic divergence were studied by comparing the interspecific somatic hybrids Brassica oleracea (CC) (+) B. campestris (AA), B. napus (AACC) (+) B. oleracea (CC) B. napus (AACC) (+) B. nigra (BB) and B. napus (AACC) (+) B. juncea (AABB) with the intergeneric somatic hybrids B. napus (AACC) (+) Raphanus sativus (RR) and B. napus (AACC) (+) Eruca sativa (EE). Within each combination, some hybrids were found whose DNA content was equal to the sum of parental chromosomes, others had a relatively higher DNA content and in most of the cases, some had a relatively lower content. However, the frequency distribution in these three classes differed significantly between the combinations. A positive correlation between the frequency of hybrids with eliminated chromosomes and the genetic distance between the species in each combination was found. Furthermore, by combining species with different ploidy levels we found a significantly higher degree of chromosome elimination compared to combinations of species with the same ploidy level. In the B. napus (+) B. Nigra, B. napus (+) R. sativus and B. napus (+) E. sativa combinations chromosomes from the B, R and E genomes appeared to be preferentially sorted out, as indicated by the fact that some of the nuclear markers from these genomes were missing in 7–46% of the plants, whereas no plants were lacking B. napus nuclear markers. Fertile hybrids were found in all but the B. napus (+) R. sativus fusion combination; the latter hybrids were male sterile, but female fertile. Hybrids between the A and C genomes were more fertile than hybrids obtained between the distantly related AC and B, R or E genomes, respectively. Analysis of the chloroplast RFLP pattern revealed that chloroplasts in the B. oleracea (+) B. campestris hybrids segregated randomly. A slightly biased segregation, favouring B. napus chloroplasts, was found in the B. napus (+) B. oleracea combination, whereas B. napus chloroplasts were strongly selected for in the B. napus (+) B. juncea, B. napus (+) B. nigra, B. napus (+) R. sativus and B. napus (+) E. sativa somatic hybrids.     

Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants

Flow cytometric estimation of nuclear DNA content was performed in six plant species employing three fluorochromes showing different DNA base preferences: propidium iodide (no base preference), 4',6-diamidino-2-phenylindole (DAPI; AT preference), and mithramycin (GC preference). Nuclei isolated from human leukocytes were used as a primary reference standard. While nuclear DNA contents estimated using propidium iodide were in agreement with published data obtained using other techniques, the values obtained using fluorochromes showing base preference were significantly different. It was found that the differences were caused by the differences in overall AT/GC ratios, and by the species-specific differences in binding of these fluorochromes to DNA. It was concluded that nuclear DNA content estimations performed with fluorochromes showing base preference should be interpreted with caution even when AT/GC ratios of the reference and the sample are equal. The use of intercalting dyes (e.g. propidium iodide) is recommended for this purpose. On the other hand, comparison of the staining behaviour of intercalating dyes with that of dyes showing base preference may give additional information on chromatin structural differences and arrangement of molecule pairs in DNA.     

Analysis of chloroplast and mitochondrial segregation in three different combinations of somatic hybrids produced within Brassicaceae

Summary Mitochondrial and chloroplast DNA were characterized in three different combinations of somatic hybrids produced between different species within Brassicaceae. The fusions were made between B. campestris and B. oleracea, B. napus and B. nigra and between B. napus and Eruca sativa. The combinations represent interspecific hybridizations, but the phylogenetic distance between the species used in each instance is different. Whereas the B. campestris (+) B. oleracea and the B. napus (+)B. nigra hybrids are both examples of intrageneric hybrids, B. campestris is more closely related to B. oleracea than B. napus is to B. nigra. The fusion of B. napus and E. sativa represents an intergeneric hybridization. Since hybrids were produced with reproducible and uniform fusion and culture methods, a comparison of chloroplast and mitochondrial segregation and mitochondrial DNA (mt-DNA) rearrangements could be made between the combinations. The segregation of both chloroplasts and mitochondria was biased in the B. napus (+)B. nigra and the B. napus (+)E. sativa combination. The nonrandom segregation of chloroplasts and mitochondria could be due to the different ploidy levels of the fusion partners and/or reflect differences in organelle replication rate. Furthermore, segregation of mitochondria was correlated to the differences in phylogenetic distance between the species used in the fusions. However, mitochondrial segregation, in contrast to chloroplast segregation, could in all combinations also have been affected by the cell type used as protoplast source in the fusions. All different chloroplast types could be established within each combination. Hybrids containing chloroplast from one parent together with mitochondria from the other parent were found in two of the combinations, although the majority of the hybrids had mt-DNA that was altered compared to the parental species. The rearranged mt-DNA found in most hybrids was an effect of the heteroplasmic state following protoplast fusion rather than of the tissue culture methods, since no mt-DNA rearrangements were found in B. napus plants regenerated from protoplast culture. The mtDNA restriction patterns of the hybrids with rearranged mt-DNA indicated that specific regions of the mt-DNA were involved in the rearrangements following protoplast fusion.     

Flow cytometric analysis of nuclear DNA content in Musa

 Nuclear genome size variation was studied in Musa acuminata (A genome), Musa balbisiana (B genome) and a range of triploid clones differing in genomic constitution (i.e. the relative number of A and B genomes). Nuclear DNA content was estimated by flow cytometry of nuclei stained by propidium iodide. The A and B genomes of Musa differ in size, the B genome being smaller by 12% on average. No variation in genome size was found among the accessions of M. balbisiana (average genome size 537 Mbp). Small, but statistically significant, variation was found among the subspecies and clones of M. acuminata (ranging from 591 to 615 Mbp). This difference may relate to the geographical origin of the individual accessions. Larger variation in genome size (8.8%) was found among the triploid Musa accessions (ranging from 559 to 613 Mbp). This variation may be due to different genomic constitutions as well as to differences in the size of their A genomes. It is proposed that a comparative analysis of genome size in diploids and triploids may be helpful in identifying putative diploid progenitors of cultivated triploid Musa clones. Statistical analysis of data on genome size resulted in a grouping which agreed fairly well with the generally accepted taxonomic classification of Musa. Received: 11 May 1998 / Accepted: 29 September 1998     

Physiological studies and determination of chromosome number in Maca,Lepidium Meyenii (Brassicaceae)

Experiments based on four accessions of maca (Lepdium meyenii) disclosed higher developmental rates in plants grown in neutral pH (6.6) soil when compared with those grown in acidic soil (5.3). Photoperiod response studies revealed similar growth rate for plants grown under either long day or short day condition. Plants in the field and growth chambers completed their life cycle in 11 months or less, therefore maca can be considered an annual crop. These results suggest that the range of adaptation of maca is not as narrow as previously believed, and therefore it can be successfully produced outside its natural habitat. Chromosome counts and predominance of bivalents in diakinesis and metaphase I disclosed that maca is a disomic octoploid of 2n=8x=64 chromosomes. Field and growth chamber observations and morphological uniformity of the plants within accessions indicate that maca relies mainly on self-fertilization for its reproduction.     

Diploid Brassica napus somatic hybrids: characterization of nuclear and organellar DNA

Summary Five somatic hybrids between Brassica campestris and B. oleracea were obtained. Molecular, morphological and cytological information all suggest that the resynthesized B. napus plants were hybrids. All five plants were diploid (2n=38) and had mainly bivalents at meiosis. Seedset was low after selfing but normal after crossing with B. napus. Molecular proof of the hybrid nature of these plants was obtained by hybridization of a rDNA repeat to total DNA. Analysis of chloroplast DNA restriction patterns revealed that all hybrids had chloroplasts identical to the B. oleracea parent. The analysis of mitochondrial DNA indicated that three hybrids had restriction patterns identical to those of B. campestris, and the other two had restriction patterns similar to those of B. oleracea. The 11.3 kb plasmid present in mitochondria of the B. campestris parent was also found in mitochondria of all five hybrids. This suggests that the plasmid from a B. campestris type of mitochondria was transferred into mitochondria of a B. oleracea type.     

Analysis of chromatin, nuclear DNA and organelle composition in somatic hybrids between Solanum tuberosum and Solanum sanctae-rosae

A protoplast fusion strategy has been applied to advance aspects of a potato breeding programme. A sub-population of somatic hybrids, selected for agronomic potential, between tetraploid Solanum tuberosum cv. Brodick and a diploid EBN2 accession, S. sanctae-rosae was subjected to detailed molecular analysis. This study reports the use of simple sequence repeats (SSRs) to identify nuclear hybrid genomes and PCR and DNA-DNA analysis to determine organelle composition in somatic hybrids derived from these parents. SSR analysis revealed somatic hybrids containing the genetic background of S. tuberosum cv. Brodick with some specific markers from S. sanctae-rosae. One somatic hybrid contained the chloroplasts derived from S. sanctae-rosae, and several hybrids had detectable RFLP mitochrondrial DNA profiles, indicating genetic re-arrangements. We also examined the use of DNase I sensitivity to the genomic and ribosomal RNA sequences in these somatic hybrids as an indicator of changes in chromatin structure. Chromatin and DNAse I analysis showed differential sensitivity to increasing levels of nuclease; DNA from several somatic hybrids was found to be resistant to DNase I compared to the parental plants. The significance of the findings to somatic cell genetics and plant breeding studies is discussed. Received: 6 July 1999 / Accepted: 29 February 2000     

Flow cytometric determination of DNA content in malignant and benign bone tumours

The nuclear DNA content of 38 malignant and 25 benign bone tumours was measured by flow cytometry. The specimens were taken either from biopsies or from surgical specimens. Seventeen of 26 primary malignant bone tumours were aneuploid, 15 had a single aneuploid DNA content, and 2 had a biclonal abnormality. Thirteen of 15 osteosarcomas were aneuploid, but only 2 of 6 chondrosarcomas showed an aneuploid DNA content. Six of 12 metastatic malignant bone tumours were also aneuploid. All 25 benign tumours had a diploid DNA content. Cell cycle analysis showed that the proportion of cells in S- and G2M-phases was higher in the malignant compared to benign tumours, indicating a higher proliferative activity. The increase was statistically significant (p less than 0.05) both in diploid and in aneuploid tumours. Among five tumours studied after chemotherapy, four displayed a marked hyperdiploid abnormality. Aneuploidy and high proliferative activity both were highly associated with malignant bone tumours, suggesting that DNA flow cytometry may be an adjunct in the assessment of malignancy of bone tumours.     

Flow cytometric measurement of nuclear DNA content inCapsicum (Solanaceae)

Nuclei were isolated from leaf tissue of differentCapsicum species and the relative fluorescence intensity was measured by flow cytometry after propidium iodide staining.Pisum sativum nuclei with known nuclear genome size (9.07 pg) were used as internal standard to determine nuclear DNA content of the samples in absolute units. The 2C DNA contents ranged between 7.65 pg inC. annuum and 9.72 pg inC. pubescens, and the general mean of the genus was 8.42 pg. These values correspond, respectively, to 1C genome size of 3.691 (C. annuum), 4.690 (C. pubescens) and 4.063 (general mean) Mbp. In general, white-flowered species proved to have less DNA, with the exception ofC. praetermissum, which displayed a 2C DNA content of 9.23 pg. It was possible to divide the studied species into three main groups according to their DNA content, and demonstrate differences in DNA content within two of the three species complexes established on the basis of morphological traits.     

Regional localization of DNA sequences on chromosome 21 using somatic cell hybrids.

We have used a panel of Chinese hamster X human somatic cell hybrids, each containing various portions of chromosome 21 as the only detectable human chromosome component, for regional mapping of cloned, chromosome 21-derived DNA sequences. Thirty unique and very low-repeat sequences were mapped to the short arm and three sections of the long arm. Three unique sequences map to the proximal part of the terminal band 21q22.3, and five to the distal part of this band. Some of these may represent parts of gene sequences that may be relevant to the pathogenesis of Down syndrome, as 21q22 is the area required to be present in triplicate for the full clinical picture.     

Simultaneous flow cytometric detection of nuclear DNA and tumor-associated antigens in lung cancers

Flow cytometric (FCM) determinations of DNA index were found to be insufficient to distinguish the presence of tumor cells from normal ones in neoplastic tissues obtained from 29 patients with lung cancer. Therefore, the DNA and tumor-associated antigen (TAA) contents of cultured human lung cancer cells were simultaneously analyzed using FCM to assess whether this dual technique would help in distinguishing tumor cells from normal ones. For the study, cells from PC-10 (a squamous cell carcinoma line), PC-3 (an adenocarcinoma line) and PC-6 (a small cell carcinoma line) were mixed with normal peripheral lymphocytes. The TAAs studied were carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCCA) and neuron-specific enolase (NSE). The alcohol-fixed cells were treated with the respective primary TAA, followed by fluorescein-isothiocyanate-conjugated secondary antibody; the cellular DNA was then stained using propidium iodide. Red and green fluorescences were measured simultaneously by FCM. The results showed CEA mainly in PC-3 cells, SCC in PC-10 cells and NSE in PC-6 cells; thus, each cell type had a relatively specific TAA. DNA content and cell size analyses differentiated neoplastic cells from normal lymphocytes for PC-3 and PC-10 cells, but not for PC-6 cells. Simultaneous FCM analyses of DNA and the TAA specific for the individual cell type made it possible to distinguish all tumor cell types from normal lymphocytes.     

Flow cytometric determination of nuclear DNA content during differentiation (spherulation and germination) of the myxomycete Physarum polycephalum

Summary Using flow cytometry, spherulating nuclei of Physarum isolated at the beginning of spherule wall formation were found to exhibit a DNA content corresponding to the G₂ phase of the cell cycle, although 8% lower. Before the first mitosis after spherule germination, a very slight incorporation of ³H thymidine into DNA was observed that was too weak to correspond to S phase, strongly suggesting that nuclei are stopped in G₂ phase inside the spherules. The lower value of nuclear DNA content found using flow cytometry of germinating spherules may not be related to DNA quantity, but may be due to a difference in chromatin organization during growth or spherulation, resulting in interference with the staining.     

Flow cytometric analysis of electronic nuclear volume and DNA content in normal mouse tissues

Light scatter is used in flow cytometry for identification of cells based on their size and/or granularity. However, forward light scatter is not an accurate measure of cell size. The measurement of Electronic Volume (EV) by Coulter principle is more accurate. However, EV cannot be measured on most of the commercially available flow cytometers. We have described the development and applications of a flow cytometer that can simultaneously measure Electronic Nuclear Volume (ENV) and DNA content. In the present study we have used a commercially available NPE Quanta for measuring EV and DNA content of different normal mice tissues. Fresh/frozen or formalin fixed-paraffin embedded tissues from mice were processed for isolation of nuclei, which were then analyzed for EV versus DNA content. By using these two parameters, distinct sub-populations were identified in liver, thymus, small intestine and bone marrow. Dual parametric analysis of EV versus DNA content can be a valuable technique for identification of sub-populations in heterogeneous cell mixtures such as those of complex tissues like bone marrow, intestine and tumors. The methods established are rapid and can provide valuable data for identification and characterization of sub-populations for cell cycle analysis by flow cytometry.     

Fluorescence in situ hybridization to interphase cell nuclei in suspension allows flow cytometric analysis of chromosome content and microscopic analysis of nuclear organization

Summary Fluorescence hybridization to interphase nuclei in liquid suspension allows quantification of chromosome-specific DNA sequences using flow cytometry and the analysis of the three-dimensional positions of these sequences in the nucleus using fluorescence microscopy. The three-dimensional structure of nuclei is substantially intact after fluorescence hybridization in suspension, permitting the study of nuclear organization by optical sectioning. Images of the distribution of probe and total DNA fluroescence within a nucleus are collected at several focal planes by quantitative fluorescence microscopy and image processing. These images can be used to reconstruct the three-dimensional organization of the target sequences in the nucleus. We demonstrate here the simultaneous localization of two human chromosomes in an interphase nucleus using two probe labeling schemes (AAF and biotin). Alternatively, dual-beam flow cytometry is used to quantify the amount of bound probe and total DNA content. We demonstrate that the intensity of probe-linked fluorescence following hybridization is proportional to the amount of target DNA over a 100-fold range in target content. This was shown using four human/hamster somatic cell hybrids carrying different numbers of human chromosomes and diploid and tetraploid human cell lines hybridized with human genomic DNA. We also show that populations of male, female, and XYY nuclei can be discriminated by measuring their fluores-cence intensity following hybridization with a Y-chromosome-specific repetitive probe. The delay in the increase in Y-specific fluorescence until the end of S-phase is consistent with the results recorded in previous studies indicating that these sequences are among the last to replicate in the genome. A chromosome-17-specific repetitive probe is used to demonstrate that target sequences as small as one megabase (Mb) can be detected using fluorescence hybridization and flow cytometry.     

Comparative flow cytometric estimation of nuclear DNA content in oil palm (Elaeis guineensis Jacq) tissue cultures and seed-derived plants

Flow cytometric analysis performed on two different crosses of dura×pisifera oil palm gave an accurate estimation of nuclear DNA content. The genome size of Elaeis guineensis was found to be 2C=3.76±0.09 pg and therefore ca. 3.4×10⁹ bp. Embryogenic calli and plants showed the same ploidy level, but the measured 2C DNA values differed significantly. No variation in the ploidy level between three different types of calli originating from foliar explants, namely nodular compact callus, fast-growing callus and friable callus was observed. Since fast-growing callus (FGC), already identified as a source of `mantled' phenotype variants, did not show any difference in their ploidy level, these results are consistent with the hypothesis of an epigenetic origin for this type of somaclonal variant. Received: 17 February 1997 / Revision received: 13 May 1997 / Accepted: 22 May 1997     

DNA content and chromosome number of a heteroploid human tumour cell line

Summary In this paper investigations concerning the relation between variability of chromosome number and variability of DNA content within the cells of a tumour stemline are reported. A highly heteroploid human tumour cell line was used, which was derived from a chondrosarcoma.Flow cytometrical and scanning cytophotometrical measurements confirmed the heteroploid nature of the original cell line and of several subclones. Measurement of the DNA content per metaphase showed a linear relation between chromosome number and DNA content of heteroploid cells. This finding is discussed with regard to its implications for the mechanism of heteroploidy in tumour cells.Supported by grant no. 28-394 of the Praeventiefonds, 's-Gravenhage, The Netherlands