Dissection of the nuclear genome of barley by chromosome flow sorting

Isolation of mitotic chromosomes using flow cytometry is an attractive way to dissect nuclear genomes into their individual chromosomal components or portions of them. This approach is especially useful in plants with complex genomes, where it offers a targeted and hence economical approach to genome analysis and gene cloning. In several plant species, DNA of flow-sorted chromosomes has been used for isolation of molecular markers from specific genome regions, for physical mapping using polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH), for integration of genetic and physical maps and for construction of chromosome-specific DNA libraries, including those cloned in bacterial artificial chromosome vectors. Until now, chromosome analysis and sorting using flow cytometry (flow cytogenetics) has found little application in barley (2n = 14, 1C ∼ 5,100 Mbp) because of the impossibility of discriminating and sorting individual chromosomes, except for the smallest chromosome 1H and some translocation chromosomes with DNA content significantly different from the remaining chromosomes. In this work, we demonstrate that wheat–barley ditelosomic addition lines can be used to sort any arm of barley chromosomes 2H–7H. Thus, the barley genome can be dissected into fractions representing only about 6–12% of the total genome. This advance makes the flow cytogenetics an attractive tool, which may greatly facilitate genome analysis and gene cloning in barley.     

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.     

Reciprocal chromosome painting shows that genomic rearrangement between rat and mouse proceeds ten times faster than between humans and cats.

Reciprocal chromosome painting between mouse and rat using complete chromosome probe sets of both species permitted us to assign the chromosomal homology between these rodents. The comparative gene mapping data and chromosome painting have a better than 90% correspondence. The reciprocal painting results graphically show that mouse and rat have strikingly different karyotypes. At least 14 translocations have occurred in the 10-20 million years of evolution that separates these two species. The evolutionary rate of chromosome translocations between these two rodents appears to be up to 10 times greater than that found between humans and cats, or between humans and chimpanzees, where over the last 5-6 million years just one translocation has occurred. Outgroup comparison shows that the mouse genome has incorporated at least three times the amount of interchromosomal rearrangements compared to the rat genome. The utility of chromosome painting was also illustrated by the assignment of two new chromosome homologies between rat and mouse unsuspected by gene mapping: between mouse 11 and rat 20 and between mouse 17 and rat 6. We conclude that reciprocal chromosome painting is a powerful method, which can be used with confidence to chart the genome and predict the chromosome location of genes. Reciprocal painting combined with gene mapping data will allow the construction of large-scale comparative chromosome maps between placental mammals and perhaps other animals.     

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.     

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.     

An alternative to radiation hybrid mapping for large-scale genome analysis in barley

The presence of a monosomic gametocidal chromosome (GC) in a barley chromosome addition line of common wheat generates structural aberrations in the barley chromosome as well as in the wheat chromosomes of gametes lacking the GC. A collection of structurally aberrant barley chromosomes is analogous to a panel of radiation hybrid (RH) mapping and is valuable for high-throughput physical mapping. We developed 90 common wheat lines (GC lines) containing aberrant barley 7H chromosomes induced by a gametocidal chromosome, 2C. DNAs isolated from these GC lines provided a panel of 7H chromosomal fragments in a wheat genetic background, comparable with RH mapping panels in mammals. We used this 7H GC panel and the methodology for RH mapping to physically map PCR-based barley markers, SSRs and AFLPs, onto chromosome 7H, relying on polymorphism between the 7H chromosome and the wheat genome. We call this method GC mapping. This study describes a novel adaptation and combination of methods of inducing chromosomal rearrangements to produce physical maps of markers. The advantages of the presented method are similar to RH mapping in that non-polymorphic markers can be used and the mapping panels can be relatively easily obtained. In addition, mapping results are cumulative when using the same mapping set with new markers. The GC lines will be available from the National Bioresources Project-KOMUGI (). Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer.

A version of the polymerase chain reaction (PCR), termed degenerate oligonucleotide-primed PCR (DOP-PCR), which employs oligonucleotides of partially degenerate sequence, has been developed for genome mapping studies. This degeneracy, together with a PCR protocol utilizing a low initial annealing temperature, ensures priming from multiple (e.g., approximately 10(6) in human) evenly dispersed sites within a given genome. Furthermore, as efficient amplification is achieved from the genomes of all species tested using the same primer, the method appears to be species-independent. Thus, for the general amplification of target DNA, DOP-PCR has advantages over interspersed repetitive sequence PCR (IRS-PCR), which relies on the appropriate positioning of species-specific repeat elements. In conjunction with chromosome flow sorting, DOP-PCR has been applied to the characterization of abnormal chromosomes and also to the cloning of new markers for specific chromosome regions. DOP-PCR therefore represents a rapid, efficient, and species-independent technique for general DNA amplification.     

Constructing plant radiation hybrid panels

Radiation hybrid (RH) mapping, a somatic cell genetic technique, has been developed in animal systems as a general approach for the construction of long-range physical maps of chromosomes. This statistical method relies on X-ray induced breakage of chromosomes to determine the physical distance between markers, as well as their order on the chromosome. The method can be applied to single chromosomes or across the whole genome. The generation of plant (barley) radiation hybrids and their culture in vitro is described here. PCR-based marker systems are used to verify hybrid status and to demonstrate genome coverage. RH panels of the type generated can be used for physical mapping, map-based cloning, or sequence contig assembly. RH resources will greatly aid the physical characterisation of crop plants with large genomes.     

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.     

Nonisotopic in situ hybridization and plant genome mapping: the first 10 years.

Nonisotopic in situ hybridization (ISH) was introduced in plants in 1985. Since then the technique has been widely used in various areas of plant genome mapping. ISH has become a routine method for physical mapping of repetitive DNA sequences and multicopy gene families. ISH patterns on somatic metaphase chromosomes using tandemly repeated sequences provide excellent physical markers for chromosome identification. Detection of low or single copy sequences were also reported. Genomic in situ hybridization (GISH) was successfully used to analyze the chromosome structure and evolution of allopolyploid species. GISH also provides a powerful technique for monitoring chromatin introgession during interspecific hybridization. A sequential chromosome banding and ISH technique was developed. The sequential technique is very useful for more precise and efficient mapping as well as cytogenetic determination of genomic affinities of individual chromosomes in allopolyploid species. A critical review is made on the present resolution of the ISH technique and the future outlook of ISH research is discussed.     

Consequences of unique double-stranded breaks in yeast chromosomes: death or homozygosis

We have developed a system in which a unique double-stranded break (DSB) can be introduced into a yeast chromosome during mitotic growth. The recognition site for the endonuclease I-SceI was inserted at different places in the yeast genome in haploid and diploid cells expressing this endonuclease. Induction of the break in haploids results in cell death if no intact copy of the cleaved region is present in the cell. If such a copy is provided on a plasmid, as an ectopic gene duplication, or on a homologous chromosome, the break can be repaired. Repair results in two identical copies in the genome of the locus which has been cut. We call this phenomenon homozygotization by reference to diploids heterozygous for the cut site in which repair leads to homozygosis at this site. We have compared the efficiencies of repair in the various topological situations examined, and conclude that some mechanism must search for regions of homology to both sides of the DSB and that repair is successful only if the homologies are provided by the same template molecule.     

Improving gene replacement by intracellular formation of linear homologous DNA

BACKGROUND: Gene targeting is a potential tool for gene therapy but is limited by the low rate of homologous recombination. Using highly homologous linear DNA improves gene targeting frequency but requires microinjection into nuclear cells to be effective. Because transfection of circular DNA is more efficient than transfection of linear DNA and adaptable to viral vectors, we developed a system for the intracellular release of linear fragments from circular plasmids. METHODS: Only one cutting site inside the "donor" DNA was not convenient because it led to integration of exogenous sequences into the target. So we constructed several "donor" plasmids containing the homologous sequences flanked by two I-Sce I recognition sites. Expression of I-Sce I allowed intracellular delivery of "ends-out" (replacement) vectors. We compared the efficiency of different constructions to correct a mutated gfp target. RESULTS: Co-transfection of "donor" plasmids and an I-Sce I expression vector into CHO cells enhanced the correction of an extrachromosomal mutated gfp target by at least 10 times. Maximum correction was observed with the greatest homology size and maximum effect of I-Sce I was obtained when the long hemi-sites of the duplicated I-Sce I sites were contiguous to the homologous sequence. Unexpectedly, the reverse orientation of I-Sce I sites provided little or no effect, probably due to the asymmetrical activity of the I-Sce I meganuclease. CONCLUSIONS: Releasing homologous DNA fragments with I-Sce I enhances gene replacement. This work provides the basis for the future design of viral vectors for gene replacement.     

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.     

BioNano genome mapping of individual chromosomes supports physical mapping and sequence assembly in complex plant genomes

The assembly of a reference genome sequence of bread wheat is challenging due to its specific features such as the genome size of 17 Gbp, polyploid nature and prevalence of repetitive sequences. BAC‐by‐BAC sequencing based on chromosomal physical maps, adopted by the International Wheat Genome Sequencing Consortium as the key strategy, reduces problems caused by the genome complexity and polyploidy, but the repeat content still hampers the sequence assembly. Availability of a high‐resolution genomic map to guide sequence scaffolding and validate physical map and sequence assemblies would be highly beneficial to obtaining an accurate and complete genome sequence. Here, we chose the short arm of chromosome 7D (7DS) as a model to demonstrate for the first time that it is possible to couple chromosome flow sorting with genome mapping in nanochannel arrays and create a de novo genome map of a wheat chromosome. We constructed a high‐resolution chromosome map composed of 371 contigs with an N50 of 1.3 Mb. Long DNA molecules achieved by our approach facilitated chromosome‐scale analysis of repetitive sequences and revealed a ~800‐kb array of tandem repeats intractable to current DNA sequencing technologies. Anchoring 7DS sequence assemblies obtained by clone‐by‐clone sequencing to the 7DS genome map provided a valuable tool to improve the BAC‐contig physical map and validate sequence assembly on a chromosome‐arm scale. Our results indicate that creating genome maps for the whole wheat genome in a chromosome‐by‐chromosome manner is feasible and that they will be an affordable tool to support the production of improved pseudomolecules.     

Isolated chromosomes as a new and efficient source of DArT markers for the saturation of genetic maps

We describe how the diversity arrays technology (DArT) can be coupled with chromosome sorting to increase the density of genetic maps in specific genome regions. Chromosome 3B and the short arm of chromosome 1B (1BS) of wheat were isolated by flow cytometric sorting and used to develop chromosome- and chromosome arm-enriched genotyping arrays containing 2,688 3B clones and 384 1BS clones. Linkage analysis showed that 553 of the 711 polymorphic 3B-derived markers (78%) mapped to chromosome 3B, and 59 of the 68 polymorphic 1BS-derived markers (87%) mapped to chromosome 1BS, confirming the efficiency of the chromosome-sorting approach. To demonstrate the potential for saturation of genetic maps, we constructed a consensus map of chromosome 3B using 19 mapping populations, including some that were genotyped with the 3B-enriched array. The 3B-derived DArT markers doubled the number of genetic loci covered. The resulting consensus map, probably the densest genetic map of 3B available to this date, contains 939 markers (779 DArTs and 160 other markers) that segregate on 304 genetically distinct loci. Importantly, only 2,688 3B-derived clones (probes) had to be screened to obtain almost twice as many polymorphic 3B markers (510) as identified by screening approximately 70,000 whole genome-derived clones (269). Since an enriched DArT array can be developed from less than 5 ng of chromosomal DNA, a quantity which can be obtained within 1 h of sorting, this approach can be readily applied to any crop for which chromosome sorting is available.     

RATT: Rapid Annotation Transfer Tool

Second-generation sequencing technologies have made large-scale sequencing projects commonplace. However, making use of these datasets often requires gene function to be ascribed genome wide. Although tool development has kept pace with the changes in sequence production, for tasks such as mapping, de novo assembly or visualization, genome annotation remains a challenge. We have developed a method to rapidly provide accurate annotation for new genomes using previously annotated genomes as a reference. The method, implemented in a tool called RATT (Rapid Annotation Transfer Tool), transfers annotations from a high-quality reference to a new genome on the basis of conserved synteny. We demonstrate that a Mycobacterium tuberculosis genome or a single 2.5 Mb chromosome from a malaria parasite can be annotated in less than five minutes with only modest computational resources. RATT is available at http://ratt.sourceforge.net.     

荧光标记的染色体原位杂交及其在人类基因组研究中的应用

FISH技术是80年代开始发展起来的一种新的定位技术.在人类基因组研究中得到了广泛的应用.通过中期染色体的FISH可以进行SCP,Cosmid和YAC的染色体定位,嵌合克隆的鉴别;通过间期核的FISH可以在50kb的分辨率下进行基因作图;最新的研究进展已可以进行伸展的染色质丝(chromatin fibre)的FISH,直接测量基因的长度,从而达到高精度基因作图的目的.总之,随着FISH技术本身的发展,它将在人类基因组研究中发挥更大的作用.     

Markerless gene replacement in Escherichia coli stimulated by a double-strand break in the chromosome.

A simple and efficient gene replacement method, based on the recombination and repair activities of the cell, was developed. The method permits the targeted construction of markerless deletions, insertions and point mutations in the Escherichia coli chromosome. A suicide plasmid, carrying the mutant allele and the recognition site of meganuclease I- Sce I, is inserted into the genome by homologous recombination between the mutant and the wild-type (wt) alleles. Resolution of this cointegrate by intramolecular recombination of the allele pair results in either a mutant or a wt chromosome which can be distinguished by allele-specific PCR screening. The resolution process is stimulated by introducing a unique double-strand break (DSB) into the chromosome at the I- Sce I site. Cleavage by the nuclease not only enhances the frequency of resolution by two to three orders of magnitude, but also selects for the resolved products. The DSB-stimulated gene replacement method can be used in recombination-proficient E.coli cells, does not require specific growth conditions, and is potentially applicable in other microorganisms. Use of the method was demonstrated by constructing a 17-bp and a 62-kb deletion in the MG1655 chromosome. Cleavage of the chromosome induces the SOS response but does not lead to an increased mutation rate.     

Physical Map of the Saccharomyces Cerevisiae Genome at 110-Kilobase Resolution

A physical map of the Saccharomyces cerevisiae genome is presented. It was derived by mapping the sites for two restriction endonucleases, SfiI and NotI, each of which recognizes an 8-bp sequence. DNA-DNA hybridization probes for genetically mapped genes and probes that span particular SfiI and NotI sites were used to construct a map that contains 131 physical landmarks--32 chromosome ends, 61 SfiI sites and 38 NotI sites. These landmarks are distributed throughout the non-rDNA component of the yeast genome, which comprises 12.5 Mbp of DNA. The physical map suggests that those genes that can be detected and mapped by standard genetic methods are distributed rather uniformly over the full physical extent of the yeast genome. The map has immediate applications to the mapping of genes for which single-copy DNA-DNA hybridization probes are available.