Flow sorting of mitotic chromosomes in common wheat (Triticum aestivum L.)

The aim of this study was to develop an improved procedure for preparation of chromosome suspensions, and to evaluate the potential of flow cytometry for chromosome sorting in wheat. Suspensions of intact chromosomes were prepared by mechanical homogenization of synchronized root tips after mild fixation with formaldehyde. Histograms of relative fluorescence intensity (flow karyotypes) obtained after the analysis of DAPI-stained chromosomes were characterized and the chromosome content of all peaks on wheat flow karyotype was determined for the first time. Only chromosome 3B could be discriminated on flow karyotypes of wheat lines with standard karyotype. Remaining chromosomes formed three composite peaks and could be sorted only as groups. Chromosome 3B could be sorted at purity >95% as determined by microscopic evaluation of sorted fractions that were labeled using C-PRINS with primers for GAA microsatellites and for Afa repeats, respectively. Chromosome 5BL/7BL could be sorted in two wheat cultivars at similar purity, indicating a potential of various wheat stocks for sorting of other chromosome types. PCR with chromosome-specific primers confirmed the identity of sorted fractions and suitability of flow-sorted chromosomes for physical mapping and for construction of small-insert DNA libraries. Sorted chromosomes were also found suitable for the preparation of high-molecular-weight DNA. On the basis of these results, it seems realistic to propose construction of large-insert chromosome-specific DNA libraries in wheat. The availability of such libraries would greatly simplify the analysis of the complex wheat genome.     

Analysis and sorting of rye (Secale cereale L.) chromosomes using flow cytometry.

Procedures for chromosome analysis and sorting using flow cytometry (flow cytogenetics) were developed for rye (Secale cereale L.). Suspensions of intact chromosomes were prepared by mechanical homogenization of synchronized root tips after mild fixation with formaldehyde. Histograms of relative fluorescence intensity obtained after the analysis of DAPI-stained chromosomes (flow karyotypes) were characterized and the chromosome content of the DNA peaks was determined. Chromosome 1R could be discriminated on a flow karyotype of S. cereale 'Imperial'. The remaining rye chromosomes (2R-7R) could be discriminated and sorted from individual wheat-rye addition lines. The analysis of lines with reconstructed karyotypes demonstrated a possibility of sorting translocation chromosomes. Supernumerary B chromosomes could be sorted from an experimental rye population and from S. cereale 'Adams'. Flow-sorted chromosomes were identified by fluorescence in situ hybridization (FISH) with probes for various DNA repeats. Large numbers of chromosomes of a single type sorted onto microscopic slides facilitated detection of rarely occurring chromosome variants by FISH with specific probes. PCR with chromosome-specific primers confirmed the identity of sorted fractions and indicated suitability of sorted chromosomes for physical mapping. The possibility to sort large numbers of chromosomes opens a way for the construction of large-insert chromosome-specific DNA libraries in rye.     

Flow cytometric sorting of maize chromosome 9 from an oat-maize chromosome addition line

Large numbers of maize chromosome 9 can be collected with high purity by flow cytometric sorting of chromosomes isolated from a disomic maize chromosome addition line of oat. Metaphase chromosome suspensions were prepared from highly synchronized seedling root-tips of an oat-maize chromosome-9 addition line (OM9) and its parental oat and maize lines. Chromosomes were stained with propidium iodide for flow cytometric analysis and sorting. Flow-karyotypes of the oat-maize addition line showed an extra peak not present in the parental oat line. This peak is due to the presence of a maize chromosome-9 pair within the genome of OM9. Separation of maize chromosome 9 by flow cytometric sorting of a chromosome preparation from a normal maize line was not possible because of its size similarity (DNA content) to maize chromosomes 6, 7 and 8. However, it is possible to separate maize chromosome 9 from oat chromosomes and chromatids. An average of about 6×10³ chromosomes of maize chromosome 9 can be collected by flow-sorting from chromosomes isolated from 30 root tips (ten seedlings) of the oat-maize addition line. Purity of the maize chromosome 9, sorted from the oat-maize chromosome addition line, was estimated to be more than 90% based on genomic in situ hybridization analysis. Sorting of individual chromosomes provides valuable genomic tools for physical mapping, library construction, and gene isolation. Received: 28 February 2000 / Accepted: 14 July 2000     

Isolation of Chromosomes from Picea abies and their Analysis by Flow Cytometry

A high-yield method for preparation of suspensions of intact Norway spruce [Picea abies (L.) Karst.] chromosomes was developed for the first time. To accumulate meristem root tip cells at metaphase, actively growing roots were subjected to subsequent treatments with 0.625 mM hydroxyurea for 18 h and after 8 h recovery in distilled water with 0.05 % (m/v) colchicine for 8 h. These treatments resulted in 50 % metaphase indices. Synchronized root tips were fixed in 2 % formaldehyde for 10 min and chromosomes were released into a lysis buffer by mechanical homogenisation, producing 5 × 10⁵ chromosomes from 50 root tips, at average. The isolated chromosomes were morphologically intact and suitable for flow cytometric analysis. Flow karyotypes obtained after the analysis of DAPI-stained chromosomes indicated a possibility to sort at least three different chromosome types. This revised version was published online in July 2006 with corrections to the Cover Date.     

Swine chromosomes: flow sorting and spot blot hybridization

Flow cytometry analysis was applied to swine chromosomes prepared from phytohemagglutinin (PHA) stimulated peripheral blood lymphocytes. Flow karyotypes from both sexes and from t(3;7) translocation carrier females were obtained. A certain number of chromosome pairs could be assigned to various peaks. In fact, 13 peaks were observed for 18 autosomal pairs plus X and Y. Moreover, abnormalities owing to the t(3;7) translocation were readily observable. The number of base pairs for chromosomes associated with the various peaks was estimated by comparison with human flow karyotypes. The following four peaks were thus sorted: the peak assumed to represent the translocated chromosome 7 plus the normals associated with it; the corresponding peak from a normal swine; the peak assumed to contain among others the normal chromosome 7; and finally the peak corresponding to swine chromosome 1. Chromosomes of each peak were collected on Pall Biodyne membrane. Following appropriate denaturation and prehybridization, the four samples were hybridized with a human leucocyte antigen (HLA) class I 32P-labelled cDNA probe, representing most of the coding sequence of the HLA B7 gene. The results confirmed previous data from other techniques that assigned the swine MHC(SLA) to chromosome 7. Subsequently, sorted samples were hybridized with a porcine genomic Interferon alpha probe in order to confirm the mapping of this gene family on porcine chromosome 1.     

On-line sorting of human chromosomes by centromeric index,and identification of sorted populations by GTG-banding and fluorescent in situ hybridization

Summary Using slit-scan flow cytometry, the shape of human metaphase chromosomes, as expressed in their centromeric index (CI), and the DNA content of the chromosomes have been used as parameters in bivariate flow karyotyping. The resolution of the DNA vs CI flow karyogram of the larger chromosomes up to chromosome 13 is much higher than the resolution obtained in the DNA-based monovariate flow karyogram. Chromosome length appears to be an important factor in the resolution of the DNA vs CI-based flow karyogram. A method has been developed to obtain chromosomes in suspension that are long enough for adequate analysis. Several chromosomes that cannot be distinguished or are difficult to discriminate in the DNA-based karyogram can now be distinguished as individual peaks, e.g., chromosomes 1 and 2. The peak of chromosomes 9–12 can be separated into two peaks formed by chromosomes 9 and 11, and 10 and 12, respectively. The advantage of the system applied in this study is that the DNA vs CI analysis is performed on-line, allowing chromosomes to be sorted on the bases of their CI. Pulse shapes of the selected chromosomes can be recorded simultaneously with the transmission of the sorting command. The purity of the sorted fraction can be estimated from the offline inspection of these pulse shapes. Fractions of chromosome 1 have been sorted out on the basis of the CI information, centrifuged on slides, fixed and subsequently banded with trypsin and Giemsa or hybridized with the chromosome 1 specific probe, pUC 1.77. The observed purity under the selected conditions ranges from 80%–99% and is in accordance with the estimates of the purities made on the basis of the simultaneously recorded pulse shapes. Fixation of the chromosome suspension prior to flow cytometric analysis and sorting appears to be essential for the preservation of their morphology and has no adverse influence on the resolution of Giemsa banding or on the quality of in situ hybridization.     

Flow karyotyping and chromosome sorting in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Previously, we reported on the development of procedures for chromosome analysis and sorting using flow cytometry (flow cytogenetics) in bread wheat. That study indicated the possibility of sorting large quantities of intact chromosomes, and their suitability for analysis at the molecular level. However, due to the lack of sufficient differences in size between individual chromosomes, only chromosome 3B could be sorted into a high-purity fraction. The present study aimed to identify wheat stocks that could be used to sort other chromosomes. An analysis of 58 varieties and landraces demonstrated a remarkable reproducibility and sensitivity of flow cytometry for the detection of numerical and structural chromosome changes. Changes in flow karyotype, diagnostic for the presence of the 1BL·1RS translocation, have been found and lines from which translocation chromosomes 5BL·7BL and 4AL·4AS-5BL could be sorted have been identified. Furthermore, wheat lines have been identified which can be used for sorting chromosomes 4B, 4D, 5D and 6D. The ability to sort any single arm of the hexaploid wheat karyotype, either in the form of a ditelosome or a isochromosome, has also been demonstrated. Thus, although originally considered recalcitrant, wheat seems to be suitable for the development of flow cytogenetics and the technology can be applied to the physical mapping of DNA sequences, the targeted isolation of molecular makers and the construction of chromosome- and arm-specific DNA libraries. These approaches should facilitate the analysis of the complex genome of hexaploid bread wheat.     

Flow sorting and microcloning of maize chromosome 1

Flow sorting maize chromosome 1 and construction of the first chromosome 1 DNA Lambda library are described. Maize metaphase chromosome suspensions were prepared from synchronized seedling root tip cells of the maize hybrid line Seneca 60 and stained with propidium iodide for flow karyotyping and sorting. The observed flow karyotype was very similar to the predicted flow karyotype constructed based on published values for the relative chromosome sizes of Seneca 60. The estimated size of chromosomes from the peak for the chromosome 1 matched the expected size of maize chromosome 1. The peak for the chromosome 1 was well resolved from other peaks on the flow karyotype. An average of 7 x 10(3) chromosomes of chromosome 1 could be produced from 10 root tips. About 0.6 million chromosomes of maize chromosome 1 were sorted and pooled based on the cytogram of fluorescent pulse area Vs fluorescent pulse width and stored at -20 degrees C in the freezer. DNA isolated from sorted chromosomes was good quality of more than 100 kb in size. Chromosome 1 DNA was partially digested with BamHI, dephosphorylated and ligated with arms of BamHI digested Lambda Dash vector. A total of 1.2 x 10(5) independent recombinants with the average insert size 12.6 kb was obtained. This library covered approximately 90% of maize chromosome 1. Hybridization of cloned fragments with labeled maize genomic DNA showed that the high, middle, or low copy number DNA sequences presented in the different phage clones. PCR (polymerase chain reaction) using chromosome-specific primers confirmed the specificity of this library. The individual chromosome library is useful in plant genome mapping and gene isolation.     

Flow Cytometric Analysis and Chromosome Sorting of Barley (Hordeum vulgare L.)

Flow cytometric analysis was systematically performed to optimize the concentration and duration of hydroxyurea (DNA synthesis inhibitor) and trifluralin (metaphase blocking reagent) treatments for synchronizing the cell cycle and accumulating metaphase chromosomes in barley root tips. A high metaphase index (76.5% in the root tip meristematic area) was routinely achieved. Seedlings of about 1.0-cm length were treated with 1.25 mM hydroxyurea for 14 h to synchronize the root tip meristem cells at the S/G2 phase. After rinsing with hydroxyurea, the seedlings were incubated in a hydroxyurea-free solution for 2 h and were treated with 1 M trifluralin for 4 h to accumulate mitotic cells in the metaphase. The consistent high metaphase index depended on the uniform germination of seeds prior to treatment. High-quality and high-quantity isolated metaphase chromosomes were suitable for flow cytometric analysis and sorting. Flow karyotypes of barley chromosomes were established via univariate and bivariate analysis. A variation of flow karyotypes was detected among barley lines. Two single chromosome types were identified and sorted. Bivariate analysis showed no variation among barley individual chromosomes in AT and GC content.     

Flow Cytometric Analysis and Chromosome Sorting of Barley (Hordeum vulgare L.)

Flow cytometric analysis was systematically performed to optimize the concentration and duration of hydroxyurea (DNA synthesis inhibitor) and trifluralin (metaphase blocking reagent) treatments for synchronizing the cell cycle and accumulating metaphase chromosomes in barley root tips. A high metaphase index (76.5% in the root tip meristematic area) was routinely achieved. Seedlings of about 1.0-cm length were treated with 1.25 mM hydroxyurea for 14 h to synchronize the root tip meristem cells at the S/G2 phase. After rinsing with hydroxyurea, the seedlings were incubated in a hydroxyurea-free solution for 2 h and were treated with 1 microM trifluralin for 4 h to accumulate mitotic cells in the metaphase. The consistent high metaphase index depended on the uniform germination of seeds prior to treatment. High-quality and high-quantity isolated metaphase chromosomes were suitable for flow cytometric analysis and sorting. Flow karyotypes of barley chromosomes were established via univariate and bivariate analysis. A variation of flow karyotypes was detected among barley lines. Two single chromosome types were identified and sorted. Bivariate analysis showed no variation among barley individual chromosomes in AT and GC content.     

Flow sorting of wheat chromosome arms from the ditelosomic line 7BL

Flow cytometric analysis confirmed that root tip cells can be synchronized with 1.25 mM hydroxyurea (DNA synthesis inhibitor) for 12 h and 1 μM trifluralin (metaphase blocking reagent) treatment for 5 h. Chromosome suspensions prepared from homogenized tissue were suitable for chromosome sorting. A flow karyotypic histogram showed that the genome of common wheat (Triticum aestivum L.) ‘Chinese Spring’ was divided into 4 chromosome peaks, but the 7BL ditelosomic line had an additional chromosome peak. PCR amplification of sorted chromosome arms indicated that the extra chromosome peak consisted of 7BL telosomics. Some technical details of sample preparation and parameter setting for flow cytometric analysis are described.     

Metaphase chromosome accumulation and flow karyotypes in rice (Oryza sativa L.) root tip meristem cells.

Highly efficient cell synchronization and metaphase chromosome accumulation in rice root tip cells were achieved. Flow cytometric analysis was performed for obtaining optimal parameters to synchronize the cell cycles. High mitotic indices (about 57.6% in root tip meristemic area) were obtained by treating seedlings with 0.5 cm length using 0.5 mM hydroxyurea at 30 degrees C for 4 h, incubating in a hydroxyurea-free solution for 30 min, and then treating with 0.3 microM trifluralin for 3 h. After trifluralin treatment, incubation in distilled water for 15 min reduced chromosome clumping on metaphase spread. Uniformity of seed germination at the time of treatment is a critical parameter for obtaining high metaphase index. Isolated rice chromosomes were suitable for flow cytometric analysis and chromosome sorting. The morphology of flow sorted metaphase chromosomes was intact.     

Verification of flow cytometorically-sorted X- and Y-bearing porcine spermatozoa and reanalysis of spermatozoa for DNA content using the fluorescence in situ hybridization (FISH) technique

Flow cytometric sperm sorting based on X and Y sperm DNA difference has been established as the only effective method for sexing the spermatozoa of mammals. The standard method for verifying the purity of sorted X and Y spermatozoa has been to reanalyze sorted sperm aliquots. We verified the purity of flow-sorted porcine X and Y spermatozoa and accuracy of DNA reanalysis by fluorescence in situ hybridization (FISH) using chromosome Y and 1 DNA probe. Eight ejaculates from 4 boars were sorted according to the Beltsville Sperm Sexing method. Porcine chromosome Y- and chromosome 1-specific DNA probes were used on sorted sperm populations in combination with FISH. Aliquots of the sorted sperm samples were reanalyzed for DNA content by flow cytometry. The purity of the sorted X-bearing spermatozoa was 87.4% for FISH and 87.0% for flow cytometric reanalysis; purity for the sorted Y-bearing spermatozoa was 85.9% for FISH and 84.8% for flow cytometric reanalysis. A total of 4,424 X sperm cells and 4,256 Y sperm cells was examined by FISH across the 8 ejaculates. For flow cytometry, 5,000 sorted X spermatozoa and 5,000 Y spermatozoa were reanalyzed for DNA content for each ejaculate. These results confirm the high purity of flow sorted porcine X and Y sperm cells and the validity of reanalysis of DNA in determining the proportions of X- and Y-sorted spermatozoa from viewing thousands of individual sperm chromosomes directly using FISH.     

Flow karyotyping and sorting of Vicia faba chromosomes

Summary Chromosome suspensions were prepared from formaldehyde-fixed, synchronized Vicia faba root tips. After staining with the DNA intercalating fluorochrome propidium iodide, the suspensions were analysed with a flow cytometer. The resulting histograms of integral fluorescence intensity contained peaks similar to those of theoretical V.faba flow karyotypes. From V. Faba cv Inovec (2n = 12) only one peak, corresponding to a single chromosome type (metacentric chromosome), could be discriminated. However, it was found that the peak also contained doublets of acrocentric chromosomes. Bivariate analysis of fluorescence pulse area (chromosome DNA content) and fluorescence pulse width (chromosome length) was necessary to distinguish the metacentric chromosome. To achieve a high degree of purity, a two-step sorting protocol was adopted. During a working day, more than 25 000 metacentric chromosomes (corresponding to 0.2 g DNA) were sorted with a purity of more than 90%. Such chromosomes are suitable for physical gene mapping by in situ hybridization or via the polymerase chain reaction (PCR) and allow the construction of chromosome-specific DNA libraries. While it was only possible to distinguish and sort one chromosome type from V. Faba cv Inovec with the standard karyotype, it was possible to sort with a high degree of purity five out of six chromosome types of the line EFK of V. Faba, which has six pairs of morphologically distinct chromosomes. This result confirmed the possibility of using reconstructed karyotypes to overcome existing problems with the discrimination and flow sorting of individual chromosome types in plants.     

Flow Karyotyping of Wheat Addition Line “T240” with a Haynaldia villosa 6VS Telosome

To construct a chromosome-specific DNA library of chromosome 6VS from Haynaldia villosa, a wheat alien chromosome addition line T240 with a 6VS chromosome arm and its parental common wheat cv. CA921 were used to optimize protocols for preparing chromosome suspension amenable to flow sorting of the 6VS chromosome. Our results showed that root tips incubated sequentially in Hogland’s solution containing 1.25 mM hydroxyurea (DNA synthesis inhibitor) for 18 h, a hydroxyurea-free period for 2 h, and 1 μM trifluralin for 4 h (metaphase blocking reagent) increased the metaphase index (MI) by up to 62 % . Many metaphase chromosomes were released to form a chromosome suspension when root tips fixed in 2 % paraformaldehyde were treated in a homogenizer at 9,500 rpm for 10–15 s. Most of the released chromosomes had intact morphology. The background solution of chromosome suspension was clear and relatively few of cell debris and chromosome clumps. Univariate flow karyotypes were established with chromosome suspension flow through FACS Vantage 2000 SE flow cytometer. The flow karyotype of CA921 consisted of four chromosome peaks, whereas that for T240 had a fifth peak. This fifth peak in T240 contained the telosome, which was further confirmed to be 6VS through fluorescence in situ hybridization.     

Chromosome sorting in tetraploid wheat and its potential for genome analysis

This study evaluates the potential of flow cytometry for chromosome sorting in durum wheat (Triticum turgidum Desf. var. durum, 2n = 4x = 28). Histograms of fluorescence intensity (flow karyotypes) obtained after the analysis of DAPI-stained chromosomes consisted of three peaks. Of these, one represented chromosome 3B, a small peak corresponded to chromosomes 1A and 6A, and a large peak represented the remaining 11 chromosomes. Chromosomes sorted onto microscope slides were identified after fluorescence in situ hybridization (FISH) with probes for GAA microsatellite, pSc119.2, and Afa repeats. Genomic distribution of these sequences was determined for the first time in durum wheat and a molecular karyotype has been developed for this crop. Flow karyotyping in double-ditelosomic lines of durum wheat revealed that the lines facilitated sorting of any arm of the wheat A- and B-genome chromosomes. Compared to hexaploid wheat, flow karyotype of durum wheat is less complex. This property results in better discrimination of telosomes and high purities in sorted fractions, ranging from 90 to 98%. We have demonstrated that large insert libraries can be created from DNA purified using flow cytometry. This study considerably expands the potential of flow cytogenetics for use in wheat genomics and opens the possibility of sequencing the genome of this important crop one chromosome arm at a time.     

Plant Chromosome Analysis and Sorting by Flow Cytometry

During the past decade, significant progress has been made in the development of methods for the preparation of plant chromosome suspensions suitable for flow cytometric analysis. In addition to successful classification of chromosomes (flow karyotyping), sorting of single chromosome types with a high degree of purity was reported in several plant species. Sorted chromosomes were used for the establishment of chromosome-specific DNA libraries and for gene mapping. The results confirmed the potential of plant flow cytogenetics and form a solid basis for further progress in this area. This article reviews its current status, analyzes major problems, and assesses future directions.     

Preparation of pea (Pisum sativum L.) chromosome and nucleus suspensions from single root tips

A high-yield method for the isolation of intact nuclei and chromosomes in suspension from a variable number of pea root tips (1–10) has been developed. This procedure is based on a two-step cell-cycle synchronization of root-tip meristems to obtain a high mitotic index, followed by formaldehyde fixation and mechanical isolation of chromosomes and nuclei by homogenization. In the explant, up to 50% of metaphases were induced through a synchronization of the cell cycle at the G₁/S interface with hydroxyurea (1.25 mM), followed, after a 3-h release, by a block in metaphase with amiprophos-methyl (10 M). The quality and quantity of nuclei and chromosomes were related to the extent of the fixation. Best results were obtained after a 30-min fixation with 2% and 4% formaldehyde for nuclei and chromosomes, respectively. The method described here allowed the isolation of nuclei and chromosomes, even from a single root tip, with a yield of 1×10⁵/root and 1.4×10⁵/root, respectively. Isolated suspensions were suitable for flow cytometric analysis and sorting and PRINS labelling with a rDNA probe.     

Chromosome isolation by flow sorting in Aegilops umbellulata and Ae. comosa and their allotetraploid hybrids Ae. biuncialis and Ae. geniculata

This study evaluates the potential of flow cytometry for chromosome sorting in two wild diploid wheats Aegilops umbellulata and Ae. comosa and their natural allotetraploid hybrids Ae. biuncialis and Ae. geniculata. Flow karyotypes obtained after the analysis of DAPI-stained chromosomes were characterized and content of chromosome peaks was determined. Peaks of chromosome 1U could be discriminated in flow karyotypes of Ae. umbellulata and Ae. biuncialis and the chromosome could be sorted with purities exceeding 95%. The remaining chromosomes formed composite peaks and could be sorted in groups of two to four. Twenty four wheat SSR markers were tested for their position on chromosomes of Ae. umbellulata and Ae. comosa using PCR on DNA amplified from flow-sorted chromosomes and genomic DNA of wheat-Ae. geniculata addition lines, respectively. Six SSR markers were located on particular Aegilops chromosomes using sorted chromosomes, thus confirming the usefulness of this approach for physical mapping. The SSR markers are suitable for marker assisted selection of wheat-Aegilops introgression lines. The results obtained in this work provide new opportunities for dissecting genomes of wild relatives of wheat with the aim to assist in alien gene transfer and discovery of novel genes for wheat improvement.     

Separation of large quantities of chinese hamster chromosomes by velocity sedimentation at unit gravity followed by flow sorting (FACS II)

Separation of large quantities of isolated metaphase chromosomes of Chinese hamster cells was performed by velocity sedimentation at unit gravity in a specially designed sedimentation chamber. This simple and easy technique results in chromosome fractions of relatively high purity as determined by flow cytometry and microscopy. Up to 10¹⁰ chromosomes can be processed depending upon the size of the sedimentation device, and enrichments up to 10 times of individual chromosomes were achieved. In addition, further chromosome purification was performed by fluorescence activated flow sorting using fractions, pre-enriched at unit gravity. The flow sorted chromosomal fractions were pure according to flow cytometric analyses. The combination of l g sedimentation and flow-sorting opens the possibility for preparative chromosome sorting by reducing the flow sorting time considerably.