Chromosomes in the flow to simplify genome analysis

Nuclear genomes of human, animals, and plants are organized into subunits called chromosomes. When isolated into aqueous suspension, mitotic chromosomes can be classified using flow cytometry according to light scatter and fluorescence parameters. Chromosomes of interest can be purified by flow sorting if they can be resolved from other chromosomes in a karyotype. The analysis and sorting are carried out at rates of 10(2)-10(4) chromosomes per second, and for complex genomes such as wheat the flow sorting technology has been ground-breaking in reducing genome complexity for genome sequencing. The high sample rate provides an attractive approach for karyotype analysis (flow karyotyping) and the purification of chromosomes in large numbers. In characterizing the chromosome complement of an organism, the high number that can be studied using flow cytometry allows for a statistically accurate analysis. Chromosome sorting plays a particularly important role in the analysis of nuclear genome structure and the analysis of particular and aberrant chromosomes. Other attractive but not well-explored features include the analysis of chromosomal proteins, chromosome ultrastructure, and high-resolution mapping using FISH. Recent results demonstrate that chromosome flow sorting can be coupled seamlessly with DNA array and next-generation sequencing technologies for high-throughput analyses. The main advantages are targeting the analysis to a genome region of interest and a significant reduction in sample complexity. As flow sorters can also sort single copies of chromosomes, shotgun sequencing DNA amplified from them enables the production of haplotype-resolved genome sequences. This review explains the principles of flow cytometric chromosome analysis and sorting (flow cytogenetics), discusses the major uses of this technology in genome analysis, and outlines future directions.     

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.     

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.     

Dissecting large and complex genomes: flow sorting and BAC cloning of individual chromosomes from bread wheat

The analysis of the complex genome of common wheat (Triticum aestivum, 2n = 6x = 42, genome formula AABBDD) is hampered by its large size ( approximately 17 000 Mbp) and allohexaploid nature. In order to simplify its analysis, we developed a generic strategy for dissecting such large and complex genomes into individual chromosomes. Chromosome 3B was successfully sorted by flow cytometry and cloned into a bacterial artificial chromosome (BAC), using only 1.8 million chromosomes and an adapted protocol developed for this purpose. The BAC library (designated as TA-3B) consists of 67 968 clones with an average insert size of 103 kb. It represents 6.2 equivalents of chromosome 3B with 100% coverage and 90% specificity as confirmed by genetic markers. This method was validated using other chromosomes and its broad application and usefulness in facilitating wheat genome analysis were demonstrated by target characterization of the chromosome 3B structure through cytogenetic mapping. This report on the successful cloning of flow-sorted chromosomes into BACs marks the integration of flow cytogenetics and genomics and represents a great leap forward in genetics and genomic analysis.     

Advances in plant chromosome genomics

Next generation sequencing (NGS) is revolutionizing genomics and is providing novel insights into genome organization, evolution and function. The number of plant genomes targeted for sequencing is rising. For the moment, however, the acquisition of full genome sequences in large genome species remains difficult, largely because the short reads produced by NGS platforms are inadequate to cope with repeat-rich DNA, which forms a large part of these genomes. The problem of sequence redundancy is compounded in polyploids, which dominate the plant kingdom. An approach to overcoming some of these difficulties is to reduce the full nuclear genome to its individual chromosomes using flow-sorting. The DNA acquired in this way has proven to be suitable for many applications, including PCR-based physical mapping, in situ hybridization, forming DNA arrays, the development of DNA markers, the construction of BAC libraries and positional cloning. Coupling chromosome sorting with NGS offers opportunities for the study of genome organization at the single chromosomal level, for comparative analyses between related species and for the validation of whole genome assemblies. Apart from the primary aim of reducing the complexity of the template, taking a chromosome-based approach enables independent teams to work in parallel, each tasked with the analysis of a different chromosome(s). Given that the number of plant species tractable for chromosome sorting is increasing, the likelihood is that chromosome genomics – the marriage of cytology and genomics – will make a significant contribution to the field of plant genetics.     

DNA content of wheat monosomics at interphase estimated by flow cytometry

 Two complete, independently maintained sets of 21 monosomic wheat lines derived from cv. ‘Chinese Spring’ were analyzed for their DNA content at the G1 stage with flow cytometry. The DNA content of individual chromosomes was estimated by subtracting the value of a monosomic line from that of euploid wheat. Our data show that the estimated 2C DNA of individual wheat chromosomes in 21 monosomics at the G1 stage ranges from about 0.58 pg in chromosome 1D to approximately 1.12 pg in chromosome 3A. The A genome (2C=6.15 pg) seems to contain more DNA than the B (2C=6.09 pg) and D (2C=5.05 pg) genomes. Analysis of variance showed significant differences (α=0.01) in DNA content both among homoeologous groups and among genomes. Our estimates of interphase DNA content of wheat chromosomes from monosomic lines were poorly correlated to the chromosome sizes at metaphase (r=0.622, P≤0.01). This poor correlation might be due to differential coiling among chromosomes during cell division, possible bias of fluorochrome binding to heterochromatin, or heterogeneity among monosomic lines. Finally, flow cytometry may aid but cannot replace cytological checks in aneuploid maintenance. Received: 21 January 1997 / Accepted: 23 June 1997     

Exploring the tertiary gene pool of bread wheat: sequence assembly and analysis of chromosome 5Mg of Aegilops geniculata

Next‐generation sequencing (NGS) provides a powerful tool for the discovery of important genes and alleles in crop plants and their wild relatives. Despite great advances in NGS technologies, whole‐genome shotgun sequencing is cost‐prohibitive for species with complex genomes. An attractive option is to reduce genome complexity to a single chromosome prior to sequencing. This work describes a strategy for studying the genomes of distant wild relatives of wheat by isolating single chromosomes from addition or substitution lines, followed by chromosome sorting using flow cytometry and sequencing of chromosomal DNA by NGS technology. We flow‐sorted chromosome 5M^g from a wheat/Aegilops geniculata disomic substitution line [DS5M^g (5D)] and sequenced it using an Illumina HiSeq 2000 system at approximately 50 × coverage. Paired‐end sequences were assembled and used for structural and functional annotation. A total of 4236 genes were annotated on 5M^g, in close agreement with the predicted number of genes on wheat chromosome 5D (4286). Single‐gene FISH indicated no major chromosomal rearrangements between chromosomes 5M^g and 5D. Comparing chromosome 5M^g with model grass genomes identified synteny blocks in Brachypodium distachyon, rice (Oryza sativa), sorghum (Sorghum bicolor) and barley (Hordeum vulgare). Chromosome 5M^g‐specific SNPs and cytogenetic probe‐based resources were developed and validated. Deletion bin‐mapped and ordered 5M^g SNP markers will be useful to track 5M‐specific introgressions and translocations. This study provides a detailed sequence‐based analysis of the composition of a chromosome from a distant wild relative of bread wheat, and opens up opportunities to develop genomic resources for wild germplasm to facilitate crop improvement.     

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.     

FISHIS: Fluorescence In Situ Hybridization in Suspension and Chromosome Flow Sorting Made Easy

The large size and complex polyploid nature of many genomes has often hampered genomics development, as is the case for several plants of high agronomic value. Isolating single chromosomes or chromosome arms via flow sorting offers a clue to resolve such complexity by focusing sequencing to a discrete and self-consistent part of the whole genome. The occurrence of sufficient differences in the size and or base-pair composition of the individual chromosomes, which is uncommon in plants, is critical for the success of flow sorting. We overcome this limitation by developing a robust method for labeling isolated chromosomes, named Fluorescent In situ Hybridization In suspension (FISHIS). FISHIS employs fluorescently labeled synthetic repetitive DNA probes, which are hybridized, in a wash-less procedure, to chromosomes in suspension following DNA alkaline denaturation. All typical A, B and D genomes of wheat, as well as individual chromosomes from pasta (T. durum L.) and bread (T. aestivum L.) wheat, were flow-sorted, after FISHIS, at high purity. For the first time in eukaryotes, each individual chromosome of a diploid organism, Dasypyrum villosum (L.) Candargy, was flow-sorted regardless of its size or base-pair related content. FISHIS-based chromosome sorting is a powerful and innovative flow cytogenetic tool which can develop new genomic resources from each plant species, where microsatellite DNA probes are available and high quality chromosome suspensions could be produced. The joining of FISHIS labeling and flow sorting with the Next Generation Sequencing methodology will enforce genomics for more species, and by this mightier chromosome approach it will be possible to increase our knowledge about structure, evolution and function of plant genome to be used for crop improvement. It is also anticipated that this technique could contribute to analyze and sort animal chromosomes with peculiar cytogenetic abnormalities, such as copy number variations or cytogenetic aberrations.     

Flow cytogenetics and chromosome sorting

This review of flow cytogenetics and chromosome sorting provides an overview of general information in the field and describes recent developments in more detail. From the early developments of chromosome analysis involving single parameter or one color analysis to the latest developments in slit scanning of single chromosomes in a flow stream, the field has progressed rapidly and most importantly has served as an important enabling technology for the human genome project. Technological innovations that advanced flow cytogenetics are described and referenced. Applications in basic cell biology, molecular biology, and clinical investigations are presented. The necessary characteristics for large number chromosome sorting are highlighted. References to recent review articles are provided as a starting point for locating individual references that provide more detail. Specific references are provided for recent developments.     

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.     

Dissecting the U,M, S and C genomes of wild relatives of bread wheat (Aegilops spp.) into chromosomes and exploring their synteny with wheat

Goat grasses (Aegilops spp.) contributed to the evolution of bread wheat and are important sources of genes and alleles for modern wheat improvement. However, their use in alien introgression breeding is hindered by poor knowledge of their genome structure and a lack of molecular tools. The analysis of large and complex genomes may be simplified by dissecting them into single chromosomes via flow cytometric sorting. In some species this is not possible due to similarities in relative DNA content among chromosomes within a karyotype. This work describes the distribution of GAA and ACG microsatellite repeats on chromosomes of the U, M, S and C genomes of Aegilops, and the use of microsatellite probes to label the chromosomes in suspension by fluorescence in situ hybridization (FISHIS). Bivariate flow cytometric analysis of chromosome DAPI fluorescence and fluorescence of FITC‐labelled microsatellites made it possible to discriminate all chromosomes and sort them with negligible contamination by other chromosomes. DNA of purified chromosomes was used as a template for polymerase chain reation (PCR) using Conserved Orthologous Set (COS) markers with known positions on wheat A, B and D genomes. Wheat–Aegilops macrosyntenic comparisons using COS markers revealed significant rearrangements in the U and C genomes, while the M and S genomes exhibited structure similar to wheat. Purified chromosome fractions provided an attractive resource to investigate the structure and evolution of the Aegilops genomes, and the COS markers assigned to Aegilops chromosomes will facilitate alien gene introgression into wheat.     

Flow cytogenetics

Classical cytogenetics is often tedious and many efforts have been made to develop other methods of chromosome analysis, among which flow karyotyping has recently emerged. Although less efficient than banding techniques to identify each chromosome, flow cytometry offers the opportunity of analyzing large quantities of chromosomes at a very high rate, resulting in a flow karyotype. Even if the initial aim of this technique, namely clinical diagnosis, has not been reached, another major application has emerged, namely chromosome sorting. This method is unique for isolating a set of purified chromosomes from most cells grown in culture in sufficient amount to perform experiments using molecular biology techniques. Significant results have been already obtained either through the construction of chromosome-specific libraries or in the assignment of DNA probes to particular sorted chromosomes.     

Current status and the future of fluorescence in situ hybridization (FISH) in plant genome research.

Fluorescence in situ hybridization (FISH), which allows direct mapping of DNA sequences on chromosomes, has become the most important technique in plant molecular cytogenetics research. Repetitive DNA sequence can generate unique FISH patterns on individual chromosomes for karyotyping and phylogenetic analysis. FISH on meiotic pachytene chromosomes coupled with digital imaging systems has become an efficient method to develop physical maps in plant species. FISH on extended DNA fibers provides a high-resolution mapping approach to analyze large DNA molecules and to characterize large genomic loci. FISH-based physical mapping provides a valuable complementary approach in genome sequencing and map-based cloning research. We expect that FISH will continue to play an important role in relating DNA sequence information to chromosome biology. FISH coupled with immunoassays will be increasingly used to study features of chromatin at the cytological level that control expression and regulation of genes.     

Flow cytometric chromosome sorting in plants: The next generation

Genome analysis in many plant species is hampered by large genome size and by sequence redundancy due to the presence of repetitive DNA and polyploidy. One solution is to reduce the sample complexity by dissecting the genomes to single chromosomes. This can be realized by flow cytometric sorting, which enables purification of chromosomes in large numbers. Coupling the chromosome sorting technology with next generation sequencing provides a targeted and cost effective way to tackle complex genomes. The methods outlined in this article describe a procedure for preparation of chromosomal DNA suitable for next-generation sequencing.     

染色体分选技术在植物学研究中的应用

介绍了染色体分选技术的基本原理和样品处理的基本流程,对根尖分生区采用同步化处理,制备染色体悬浮液,最后通过流式细胞仪的分析与收集获得纯度高、数量多的目标染色体.综述了染色体分选技术在植物学研究中的主要应用,包括物理图谱的构建、DNA分子标记的开发、以及复杂多倍体植物的基因组测序等.通过染色体分选技术的不断完善与发展,应...     

First survey of the wheat chromosome 5A composition through a next generation sequencing approach

Wheat is one of the world's most important crops and is characterized by a large polyploid genome. One way to reduce genome complexity is to isolate single chromosomes using flow cytometry. Low coverage DNA sequencing can provide a snapshot of individual chromosomes, allowing a fast characterization of their main features and comparison with other genomes. We used massively parallel 454 pyrosequencing to obtain a 2x coverage of wheat chromosome 5A. The resulting sequence assembly was used to identify TEs, genes and miRNAs, as well as to infer a virtual gene order based on the synteny with other grass genomes. Repetitive elements account for more than 75% of the genome. Gene content was estimated considering non-redundant reads showing at least one match to ESTs or proteins. The results indicate that the coding fraction represents 1.08% and 1.3% of the short and long arm respectively, projecting the number of genes of the whole chromosome to approximately 5,000. 195 candidate miRNA precursors belonging to 16 miRNA families were identified. The 5A genes were used to search for syntenic relationships between grass genomes. The short arm is closely related to Brachypodium chromosome 4, sorghum chromosome 8 and rice chromosome 12; the long arm to regions of Brachypodium chromosomes 4 and 1, sorghum chromosomes 1 and 2 and rice chromosomes 9 and 3. From these similarities it was possible to infer the virtual gene order of 392 (5AS) and 1,480 (5AL) genes of chromosome 5A, which was compared to, and found to be largely congruent with the available physical map of this chromosome.     

Cytologically integrated physical restriction fragment length polymorphism maps for the barley genome based on translocation breakpoints

We have developed a new technique for the physical mapping of barley chromosomes using microdissected translocation chromosomes for PCR with sequence-tagged site primers derived from >300 genetically mapped RFLP probes. The positions of 240 translocation breakpoints were integrated as physical landmarks into linkage maps of the seven barley chromosomes. This strategy proved to be highly efficient in relating physical to genetic distances. A very heterogeneous distribution of recombination rates was found along individual chromosomes. Recombination is mainly confined to a few relatively small areas spaced by large segments in which recombination is severely suppressed. The regions of highest recombination frequency (相似文献   

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.     

Quantification of the DNA content of structurally abnormal X chromosomes and X chromosome aneuploidy using high resolution bivariate flow karyotyping

Quantification of the Hoechst and chromomycin A3 fluorescence intensities of mitotic human chromosomes isolated from karyotypically normal and abnormal cells was performed with a dual beam flow cytometer. The resultant flow karyotypes contain information about the relative DNA content and base composition of chromosomes and their relative frequencies in the mitotic cell sample. The relative copy number of X and Y chromosomes was determined for 38 normal males and females and 6 cell lines with X or Y chromosome aneuploidy. Flow karyotype diagnoses corresponded with conventional cytogenetic results in all cases. We show that chromosome DNA content can be derived from peak position in Hoechst vs. chromomycin flow karyotypes. These values are linearly related to propidium iodide staining intensity as measured with flow cytometry and to the binding of gallocyanin chrome alum to phosphate groups as measured with slide-based scanning photometry. Cell lines with deleted or dicentric X chromosomes ranging in length from 0.53 to 1.95 times normal were analyzed by using flow cytometry. The measured difference in DNA content between a normal X and each of the structurally abnormal chromosomes was linearly correlated to the difference predicted from cytogenetics and/or probe analyses. Deletions of 3-5 Mb, which were at and below the detection limits of conventional cytogenetics, could be quantified by flow karyotyping in individuals with X-linked diseases such as Duchenne muscular dystrophy, choroideremia, and ocular albinism/ichthyosis. The results show that the use of flow karyotyping to quantify the size of restricted regions of the genome can complement conventional cytogenetics and other physical mapping techniques in the study of genetic disorders.