A protocol is described for production of micrograms of DNA from single copies of flow‐sorted plant chromosomes. Of 183 single copies of wheat chromosome 3B, 118 (64%) were successfully amplified. ...Sequencing DNA amplification products using an Illumina HiSeq 2000 system to 10× coverage and merging sequences from three separate amplifications resulted in 60% coverage of the chromosome 3B reference, entirely covering 30% of its genes. The merged sequences permitted de novo assembly of 19% of chromosome 3B genes, with 10% of genes contained in a single contig, and 39% of genes covered for at least 80% of their length. The chromosome‐derived sequences allowed identification of missing genic sequences in the chromosome 3B reference and short sequences similar to 3B in survey sequences of other wheat chromosomes. These observations indicate that single‐chromosome sequencing is suitable to identify genic sequences on particular chromosomes, to develop chromosome‐specific DNA markers, to verify assignment of DNA sequence contigs to individual pseudomolecules, and to validate whole‐genome assemblies. The protocol expands the potential of chromosome genomics, which may now be applied to any plant species from which chromosome samples suitable for flow cytometry can be prepared, and opens new avenues for studies on chromosome structural heterozygosity and haplotype phasing in plants.
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 102–104 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.
Summary
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.
Significance Statement
Introgression of desirable genes from wild species (Aegilops sp.) into bread wheat (Triticum aestivum) is hampered by poor knowledge of Aegilops genome structure and lack of molecular tools. Here we used bivariate flow cytometry and microsatellite probes to discriminate and purify Aegilops chromosomes. DNA markers conserved in bread wheat and wild species will be useful in pre‐breeding programs to select chromosome segments carrying agronomically useful genes in T. aestivum – Aegilops recombinant lines.
► We describe preparation of plant chromosome DNA for next-generation sequencing. ► Chromosomes are purified by sorting using flow cytometry. ► Chromosomal DNA is amplified using a multiple ...displacement method. ► The amplified DNA is suitable for next-generation sequencing.
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.
A genetic linkage map of dioecious garden asparagus (
L., 2
= 2
= 20) was constructed using F
population, simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers. In total, 1376 ...SNPs and 27 SSRs were used for genetic mapping. Two resulting parental maps contained 907 and 678 markers spanning 1947 and 1814 cM, for female and male parent, respectively, over ten linkage groups representing ten haploid chromosomes of the species. With the aim to anchor the ten genetic linkage groups to individual chromosomes and develop a tool to facilitate genome analysis and gene cloning, we have optimized a protocol for flow cytometric chromosome analysis and sorting in asparagus. The analysis of DAPI-stained suspensions of intact mitotic chromosomes by flow cytometry resulted in histograms of relative fluorescence intensity (flow karyotypes) comprising eight major peaks. The analysis of chromosome morphology and localization of 5S and 45S rDNA by FISH on flow-sorted chromosomes, revealed that four chromosomes (IV, V, VI, VIII) could be discriminated and sorted. Seventy-two SSR markers were used to characterize chromosome content of individual peaks on the flow karyotype. Out of them, 27 were included in the genetic linkage map and anchored genetic linkage groups to chromosomes. The sex determining locus was located on LG5, which was associated with peak V representing a chromosome with 5S rDNA locus. The results obtained in this study will support asparagus improvement by facilitating targeted marker development and gene isolation using flow-sorted chromosomes.
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.
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.
The cereals are of enormous importance to mankind. Many of the major cereal species - specifically, wheat, barley, oat, rye, and maize - have large genomes. Early cytogenetics, genome analysis and ...genetic mapping in the cereals benefited greatly from their large chromosomes, and the allopolyploidy of wheat and oats that has allowed for the development of many precise cytogenetic stocks. In the genomics era, however, large genomes are disadvantageous. Sequencing large and complex genomes is expensive, and the assembly of genome sequence is hampered by a significant content of repetitive DNA and, in allopolyploids, by the presence of homoeologous genomes. Dissection of the genome into its component chromosomes and chromosome arms provides an elegant solution to these problems. In this review we illustrate how this can be achieved by flow cytometric sorting. We describe the development of methods for the preparation of intact chromosome suspensions from the major cereals, and their analysis and sorting using flow cytometry. We explain how difficulties in the discrimination of specific chromosomes and their arms can be overcome by exploiting extant cytogenetic stocks of polyploid wheat and oats, in particular chromosome deletion and alien addition lines. Finally, we discuss some of the applications of flow-sorted chromosomes, and present some examples demonstrating that a chromosome-based approach is advantageous for the analysis of the complex genomes of cereals, and that it can offer significant potential for the delivery of genome sequencing and gene cloning in these crops.
Core Ideas
Flow sorting enabled to dissect the genome of crested wheatgrass into chromosomes.
This is a powerful approach to access the complex genome of a wheat wild relative.
Chromosome genomics ...will support alien introgression breeding of the crop.
Wheatgrass (Agropyron sp.) is a potential source of beneficial traits for wheat improvement. Among them, crested wheatgrass A. cristatum (L.) Gaertn. comprises a complex of diploid, tetraploid, and hexaploid forms with the basic genome P, with some accessions carrying supernumerary B chromosomes (Bs). In this work, we applied flow cytometry to dissect the complex genome of crested wheatgrass into individual chromosomes to facilitate its analysis. Flow karyotypes obtained after the analysis of 4ʹ,6‐diamidino‐2‐phenylindole (DAPI)‐stained mitotic chromosomes of diploid and tetraploid accessions consisted of three peaks, each corresponding to a group of two or three chromosomes. To improve the resolution, bivariate flow karyotyping after fluorescent labeling of chromosomes with fluorescein isothiocyanate (FITC)‐conjugated probe (GAA)7 microsatellite was applied and allowed discrimination and sorting of P genome chromosomes from wheat–crested wheatgrass addition lines. Chromosomes 1P–6P and seven telomeric chromosomes could be sorted at purities ranging from 81.7 to 98.2% in disomics and from 44.8 to 87.3% in telosomics. Chromosome 7P was sorted at purities reaching 50.0 and 39.5% in diploid and tetraploid crested wheatgrass, respectively. In addition to the whole complement chromosomes (A), Bs could be easily discriminated and sorted from a diploid accession at 95.4% purity. The sorted chromosomes will streamline genome analysis of crested wheatgrass, facilitating gene cloning and development of molecular tools to support alien introgression into wheat.
Monitoring alien introgressions in crop plants is difficult due to the lack of genetic and molecular mapping information on the wild crop relatives. The tertiary gene pool of wheat is a very ...important source of genetic variability for wheat improvement against biotic and abiotic stresses. By exploring the 5Mg short arm (5MgS) of Aegilops geniculata, we can apply chromosome genomics for the discovery of SNP markers and their use for monitoring alien introgressions in wheat (Triticum aestivum L).
The short arm of chromosome 5Mg of Ae. geniculata Roth (syn. Ae. ovata L.; 2n = 4x = 28, UgUgMgMg) was flow-sorted from a wheat line in which it is maintained as a telocentric chromosome. DNA of the sorted arm was amplified and sequenced using an Illumina Hiseq 2000 with ~45x coverage. The sequence data was used for SNP discovery against wheat homoeologous group-5 assemblies. A total of 2,178 unique, 5MgS-specific SNPs were discovered. Randomly selected samples of 59 5MgS-specific SNPs were tested (44 by KASPar assay and 15 by Sanger sequencing) and 84% were validated. Of the selected SNPs, 97% mapped to a chromosome 5Mg addition to wheat (the source of t5MgS), and 94% to 5Mg introgressed from a different accession of Ae. geniculata substituting for chromosome 5D of wheat. The validated SNPs also identified chromosome segments of 5MgS origin in a set of T5D-5Mg translocation lines; eight SNPs (25%) mapped to TA5601 T5DL · 5DS-5MgS(0.75) and three (8%) to TA5602 T5DL · 5DS-5MgS (0.95). SNPs (gsnp_5ms83 and gsnp_5ms94), tagging chromosome T5DL · 5DS-5MgS(0.95) with the smallest introgression carrying resistance to leaf rust (Lr57) and stripe rust (Yr40), were validated in two released germplasm lines with Lr57 and Yr40 genes.
This approach should be widely applicable for the identification of species/genome-specific SNPs. The development of a large number of SNP markers will facilitate the precise introgression and monitoring of alien segments in crop breeding programs and further enable mapping and cloning novel genes from the wild relatives of crop plants.
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