Chromosome Sorting Research Articles

Dissection of a rapidly evolving wheat resistance gene cluster by long-read genome sequencing accelerated the cloning of Pm69

Gene cloning in repeat-rich polyploid genomes remains challenging. Here, we describe a strategy for overcoming major bottlenecks in cloning of the powdery mildew resistance gene (R-gene) Pm69 derived from tetraploid wild emmer wheat. A conventional positional cloning approach was not effective owing to suppressed recombination. Chromosome sorting was compromised by insufficient purity. A Pm69 physical map, constructed by assembling Oxford Nanopore Technology (ONT) long-read genome sequences, revealed a rapidly evolving nucleotide-binding leucine-rich repeat (NLR) R-gene cluster with structural variations. A single candidate NLR was identified by anchoring RNA sequencing reads from susceptible mutants to ONT contigs and was validated by virus-induced gene silencing. Pm69 is likely a newly evolved NLR and was discovered in only one location across the wild emmer wheat distribution range in Israel. Pm69 was successfully introgressed into cultivated wheat, and a diagnostic molecular marker was used to accelerate its deployment and pyramiding with other R-genes.

Y chromosome sequence and epigenomic reconstruction across human populations

Recent advances in long-read sequencing technologies have allowed the generation and curation of more complete genome assemblies, enabling the analysis of traditionally neglected chromosomes, such as the human Y chromosome (chrY). Native DNA was sequenced on a MinION Oxford Nanopore Technologies sequencing device to generate genome assemblies for seven major chrY human haplogroups. We analyzed and compared the chrY enrichment of sequencing data obtained using two different selective sequencing approaches: adaptive sampling and flow cytometry chromosome sorting. We show that adaptive sampling can produce data to create assemblies comparable to chromosome sorting while being a less expensive and time-consuming technique. We also assessed haplogroup-specific structural variants, which would be otherwise difficult to study using short-read sequencing data only. Finally, we took advantage of this technology to detect and profile epigenetic modifications among the considered haplogroups. Altogether, we provide a framework to study complex genomic regions with a simple, fast, and affordable methodology that could be applied to larger population genomics datasets.

Mammalian Chromosome Analysis and Sorting by Flow Cytometry.

The analysis of chromosomes by flow cytometry is termed flow cytogenetics, and it involves the analysis and sorting of single mitotic chromosomes in suspension. The study of flow karyograms provides insight into chromosome number and structure to provide information on chromosomal DNA content and can enable the detection of deletions, translocations, or any forms of aneuploidy. Beyond its clinical applications, flow cytogenetics greatly contributed to the Human Genome Project through the ability to sort pure populations of chromosomes for gene mapping, cloning, and the construction of DNA libraries. Maximizing the potential of these important applications of flow cytogenetics relies on precise instrument setup and optimal sample processing, both of which impact the accuracy and quality of the data that are generated. This article is a compilation of the existing protocols that describe the stepwise methodology of accumulating, isolating, and staining metaphase chromosomes to prepare single-chromosome suspensions for flow cytometric analysis and sorting. Although the chromosome preparation protocols have remained largely unchanged, cytometer technology has advanced dramatically since these protocols were originally developed. Advances in cytometry technologies offer new and exciting approaches for understanding and monitoring chromosomal aberrations, but the hallmark of these protocols remains their simplicity in methodologies and reagent requirements and the accuracy of data resolvable to every chromosome of the cell. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Mitotic block and cell harvesting Basic Protocol 2: Propidium iodide isolation Support Protocol 1: Swelling test Basic Protocol 3: MgSO4 low-molecular-weight isolation Basic Protocol 4: Polyamine high-molecular-weight isolation Support Protocol 2: Molecular-weight determination of chromosomal DNA Basic Protocol 5: Chromosome analysis and sorting.

Nitrous Oxide-Induced Metaphase Arrest: A Technique for Somatic Chromosome Analysis.

Procedures to arrest metaphase chromosomes are used for determining chromosome numbers, chromosomal aberrations, and natural chromosome variation, as well as chromosome sorting. Here is described a technique of nitrous oxide gas treatment of freshly harvested root tips that is highly effective at producing an excellent mitotic index together with well-spread chromosomes. The details of the treatment and equipment used are provided. The metaphase spreads can be used directly for determining chromosome numbers or for in situ hybridization to reveal chromosomal features.

Flow Cytometric Analysis and Sorting of Plant Chromosomes.

Flow cytometry offers a unique way of analyzing and manipulating plant chromosomes. During a rapid movement in a liquid stream, large populations can be classified in a short time according to their fluorescence and light scatter properties. Chromosomes whose optical properties differ from other chromosomes in a karyotype can be purified by flow sorting and used in a range of applications in cytogenetics, molecular biology, genomics, and proteomics. As the samples for flow cytometry must be liquid suspensions of single particles, intact chromosomes must be released from mitotic cells. This protocol describes a procedure for preparation of suspensions of mitotic metaphase chromosomes from meristem root tips and their flow cytometric analysis and sorting for various downstream applications.

Chromosome analysis and sorting.

Flow cytometric analysis and sorting of plant mitotic chromosomes has been mastered by only a few laboratories worldwide. Yet, it has been contributing significantly to progress in plant genetics, including the production of genome assemblies and the cloning of important genes. The dissection of complex genomes by flow sorting into the individual chromosomes that represent small parts of the genome reduces DNA sample complexity and streamlines projects relying on molecular and genomic techniques. Whereas flow cytometric analysis, that is, chromosome classification according to fluorescence and light scatter properties, is an integral part of any chromosome sorting project, it has rarely been used on its own due to lower resolution and sensitivity as compared to other cytogenetic methods. To perform chromosome analysis and sorting, commercially available electrostatic droplet sorters are suitable. However, in order to resolve and purify chromosomes of interest the instrument must offer high resolution of optical signals as well as stability during long runs. The challenge is thus not the instrumentation, but the adequate sample preparation. The sample must be a suspension of intact mitotic metaphase chromosomes and the protocol, which includes the induction of cell cycle synchrony, accumulation of dividing cells at metaphase, and release of undamaged chromosomes, is time consuming and laborious and needs to be performed very carefully. Moreover, in addition to fluorescent staining chromosomal DNA, the protocol may include specific labelling of DNA repeats to facilitate discrimination of particular chromosomes. This review introduces the applications of chromosome sorting in plants, and discusses in detail sample preparation, chromosome analysis and sorting to achieve the highest purity in flow‐sorted fractions, and their suitability for downstream applications.

In Memoriam: Mortimer L. Mendelsohn: (1 December, 1925-8 January, 2020).

In Memoriam: Mortimer L. Mendelsohn: (1 December, 1925-8 January, 2020).

Incomplete Sterility of Chromosomal Hybrids: Implications for Karyotype Evolution and Homoploid Hybrid Speciation.

Heterozygotes for major chromosomal rearrangements such as fusions and fissions are expected to display a high level of sterility due to problems during meiosis. However, some species, especially plants and animals with holocentric chromosomes, are known to tolerate chromosomal heterozygosity even for multiple rearrangements. Here, we studied male meiotic chromosome behavior in four hybrid generations (F1–F4) between two chromosomal races of the Wood White butterfly Leptidea sinapis differentiated by at least 24 chromosomal fusions/fissions. Previous work showed that these hybrids were fertile, although their fertility was reduced as compared to crosses within chromosomal races. We demonstrate that (i) F1 hybrids are highly heterozygous with nearly all chromosomes participating in the formation of trivalents at the first meiotic division, and (ii) that from F1 to F4 the number of trivalents decreases and the number of bivalents increases. We argue that the observed process of chromosome sorting would, if continued, result in a new homozygous chromosomal race, i.e., in a new karyotype with intermediate chromosome number and, possibly, in a new incipient homoploid hybrid species. We also discuss the segregational model of karyotype evolution and the chromosomal model of homoploid hybrid speciation.

In Memoriam: Marvin A. Van Dilla: 1919-2019.

© 2020 International Society for Advancement of Cytometry.

Generation of microdissected DNA probes from metaphase chromosomes when chromosome identification by routine staining is impossible.

Application of microdissected DNA libraries and DNA probes in numerous and various modern molecular cytogenetic studies showed them as an efficient and reliable tool in the analysis of chromosome reorganization during karyotypic evolution and in the diagnosis of human chromosome pathology. An important advantage of DNA probe generation by metaphase chromosome microdissection followed by sequence-independent polymerase chain reaction in comparison with the method of DNA probe generation using chromosome sorting is the possibility of DNA probe preparation from chromosomes of an individual sample without cell line establishment for the production of a large number of metaphase chromosomes. One of the main requirements for successful application of this technique is a possibility for identification of the chromosome of interest during its dissection and collection of its material from metaphase plates spread on the coverslip. In the present study, we developed and applied a technique for generation of microdissected DNA probes in the case when chromosome identification during microdissection appeared to be impossible. The technique was used for generation of two sets of Whole Chromosome Paints (WCPs) from all chromosomes of two species of free-living flatworms in the genus Macrostomum, M. mirumnovem and M. cliftonensis. The single-copy chromosome technique including separate collection of all chromosomes from one metaphase plate allowed us to generate WCPs that painted specifically the original chromosome by Chromosome In Situ Suppression Hybridization (CISS-Hybridization). CISS-Hybridization allowed identifying the original chromosome(s) used for DNA probe generation. Pooled WCPs derived from homologous chromosomes increased the intensity and specificity of chromosome painting provided by CISS-Hybridization. In the result, the obtained DNA probes appeared to be good enough for application in our studies devoted to analysis of karyotypic evolution in the genus Macrostomum and for analysis of chromosome rearrangements among the worms of laboratory cultures of M. mirumnovem.

A flow cytometry-based analysis to establish a cell cycle synchronization protocol for Saccharum spp.

Modern sugarcane is an unusually complex heteroploid crop, and its genome comprises two or three subgenomes. To reduce the complexity of sugarcane genome research, the ploidy level and number of chromosomes can be reduced using flow chromosome sorting. However, a cell cycle synchronization (CCS) protocol for Saccharum spp. is needed that maximizes the accumulation of metaphase chromosomes. For flow cytometry analysis in this study, we optimized the lysis buffer, hydroxyurea(HU) concentration, HU treatment time and recovery time for sugarcane. We determined the mitotic index by microscopic observation and calculation. We found that WPB buffer was superior to other buffers for preparation of sugarcane nuclei suspensions. The optimal HU treatment was 2 mM for 18 h at 25 °C, 28 °C and 30 °C. Higher recovery treatment temperatures were associated with shorter recovery times (3.5 h, 2.5 h and 1.5 h at 25 °C, 28 °C and 30 °C, respectively). The optimal conditions for treatment with the inhibitor of microtubule polymerization, amiprophos-methyl (APM), were 2.5 μM for 3 h at 25 °C, 28 °C and 30 °C. Meanwhile, preliminary screening of CCS protocols for Badila were used for some main species of genus Saccharum at 25 °C, 28 °C and 30 °C, which showed that the average mitotic index decreased from 25 °C to 30 °C. The optimal sugarcane CCS protocol that yielded a mitotic index of >50% in sugarcane root tips was: 2 mM HU for 18 h, 0.1 X Hoagland’s Solution without HU for 3.5 h, and 2.5 μM APM for 3.0 h at 25 °C. The CCS protocol defined in this study should accelerate the development of genomic research and cytobiology research in sugarcane.

Flow Sorting Enrichment and Nanopore Sequencing of Chromosome 1 From a Chinese Individual.

Sorting of individual chromosomes by Flow Cytometry (flow-sorting) is an enrichment method to potentially simplify genome assembly by isolating chromosomes from the context of the genome. We have recently developed a workflow to sequence native, unamplified DNA and applied it to the smallest human chromosome, the Y chromosome. Here, we modify improve upon that workflow to increase DNA recovery from chromosome sorting as well as sequencing yield. We apply it to sequence and assemble the largest human chromosome - chromosome 1 - of a Chinese individual using a single Oxford Nanopore MinION flow cell. We generate a selective and highly continuous assembly whose continuity reaches into the order of magnitude of the human reference GRCh38. We then use this assembly to call candidate structural variants against the reference and find 685 putative novel SV candidates. We propose this workflow as a potential solution to assemble structurally complex chromosomes, or the study of very large plant or animal genomes that might challenge traditional assembly strategies.

Aegilops umbellulata introgression carrying leaf rust and stripe rust resistance genes Lr76 and Yr70 located to 9.47-Mb region on 5DS telomeric end through a combination of chromosome sorting and sequencing.

Lr76 and Yr70 have been fine mapped using the sequence of flow-sorted recombinant 5D chromosome from wheat-Ae. umbellulata introgression line. The alien introgression has been delineated to 9.47-Mb region on short arm of wheat chromosome 5D. Leaf rust and stripe rust are among the most damaging diseases of wheat worldwide. Wheat cultivation based on limited number of rust resistance genes deployed over vast areas expedites the emergence of new pathotypes warranting a continuous deployment of new resistance genes. In this paper, fine mapping of Aegilops umbellulata-derived leaf rust and stripe rust resistance genes Lr76 and Yr70 is being reported. We flow sorted and paired-end sequenced 5U chromosome of Ae. umbellulata, recombinant chromosome 5D (5DIL) from wheat-Ae. umbellulata introgression line pau16057 and 5DRP of recurrent parent WL711. Chromosome 5U reads were mapped against the reference Chinese Spring chromosome 5D sequence, and alien-specific SNPs were identified. Chromosome 5DIL and 5DRP sequences were de novo assembled, and alien introgression-specific markers were designed by selecting 5U- and 5D-specific SNPs. Overall, 27 KASP markers were mapped in high-resolution population consisting of 1404 F5 RILs. The mapping population segregated for single gene each for leaf rust and stripe rust resistance. The physical order of the SNPs in pau16057 was defined by projecting the 27 SNPs against the IWGSC RefSeq v1.0 sequence. Based on this physical map, the size of Ae. umbellulata introgression was determined to be 9.47Mb on the distal most end of the short arm of chromosome 5D. This non-recombining alien segment carries six NB-LRR encoding genes based on NLR annotation of assembled chromosome 5DIL sequence and IWGSC RefSeq v1.1 gene models. The presence of SNPs and other sequence variations in these genes between pau16057 and WL711 suggested that they are candidates for Lr76 and Yr70.

Sequencing and Assembling Genomes and Chromosomes of Cereal Crops.

Next generation sequencing (NGS) and assembly are key pieces in the success of cereal crops genomes sequencing. One interesting strategy for reducing the complexity in large genomes, the case of several cereal crops, is the sequencing of individual chromosomes. This has been done with success by flow cytometric chromosome sorting followed by sequencing using available next generation (high throughput) sequencing platforms. In this chapter, methodologies for sequencing and assembly of flow sorted chromosomes and whole genomes in cereal crops, with special emphasis on the case of the International Wheat Genome Sequencing Consortium (IWGSC), are reviewed.

4. PGT-M FOR DE NOVO MUTATIONS – HAPLOTYPE DETERMINATION USING MORPHOLOGICALLY POOR EMBRYOS

Introduction Couples requesting PGT-M involving a de novo mutation pose a challenge when the PGT-M test used incorporates linkage analysis, as parental samples cannot be used to determine the mutant and normal allele haplotypes. In our experience, de novo mutations represent approximately 6-7% of clinical referrals, and thus it is important to have strategies in place to be able to offer PGT-M for these patients. Historically, we have employed two main strategies for these couples. If the mutation in question was paternal, we would perform single sperm analysis to determine the mutant haplotype. When the mutation was maternal, we would perform single chromosome sorting to determine the mutant haplotype. We would then confirm the phase when we obtained samples from clinical embryos. More recently, we began to question if determining phase prior to a PGT-M cycle was necessary, as we were confirming the phase as part of the cycle. We decided to evaluate if it was quicker and simpler to aim to determine phase directly from the embryos created as part of the clinical PGT-M. Materials and methods Haplotype determination was performed for the cases, utilising biopsies from clinically usable embryos. In addition, in order to obtain enough sample numbers to determine phase, when there were less than 4-5 embryos biopsied, we also requested samples from embryos with poor morphological development. Karyomapping was the PGT-M test performed, in combination with a method to detect the mutation from MDA amplifications from the biopsied samples. A grandparental DNA reference was available in all cases to evaluate SNP coverage prior. Results We obtained 3-7 samples from each of 14 cases to determine mutant and normal haplotypes. On 8/14 (57%) occasions, there were not enough samples from clinically usable embryos to provide us with the required sample numbers, so additional samples from embryos with poor morphological development were requested. Phase was established in all 14 cases. In all 12/14 (86%) cases the de novo mutation had a grandpaternal origin consistent with previous cases showing 89% of these de novo mutations have arisen in a sperm. Conclusions This strategy proved successful in all 14 cases, allowing PGT-M to proceed without any delay. Additional samples from morphologically poor embryos were required 57% of the time, in order to obtain the necessary sample numbers to determine the mutant haplotype. This strategy can be employed to facilitate fast access to Karyomapping based PGT-M for cases involving de novo mutations, with a back-up strategy of using the above mentioned other methods if sample numbers are lacking.

Genetic analysis by chromosome sorting and painting: phylogenetic and diagnostic applications.

Chromosome sorting from fluid suspensions of metaphase chromosomes using a fluorescence-activated cell sorter has been used for a number of years to produce chromosome-specific genomic libraries and other reagents for chromosome mapping. Improved techniques for fluorescence in situ hybridisation and the amplification and labelling of sorted chromosomes using degenerate oligonucleotide-primed PCR have led to the widespread use of chromosome painting both for the resolution of complex chromosome aberrations and for the study of karyotype evolution by cross-species reciprocal chromosome painting. The chromosomes of a large number of different species have been sorted and used to make chromosome-specific paints and already new data challenging results of earlier phylogenetic studies have been obtained. Sorted chromosomes provide the resource for multicolour chromosome analysis of all chromosomes simultaneously. Such reagents are now available for all human and mouse chromosomes and are proving particularly useful in the analysis of cancer chromosomes.

Methodology for Y Chromosome Capture: A complete genome sequence of\xa0 Y chromosome using flow cytometry, laser microdissection and magnetic streptavidin-beads

This study is a comparison of the efficiency of three technologies used for Y chromosome capture and the next-generation sequencing (NGS) technologies applied for determining its whole sequence. Our main findings disclose that streptavidin–biotin magnetic particle-based capture methodology offers better and a deeper sequence coverage for Y chromosome capture, compared to chromosome sorting and microdissection procedures. Moreover, this methodology is less time consuming and the most selective for capturing only Y chromosomal material, in contrast with other methodologies that result in considerable background material from other, non-targeted chromosomes. NGS results compared between two platforms, NextSeq 500 and SOLID 5500xl, produce the same coverage results. This is the first study to explore a methodological comparison of Y chromosome capture and genetic analysis. Our results indicate an improved strategy for Y chromosome research with applications in several scientific fields where this chromosome plays an important role, such as forensics, medical sciences, molecular anthropology and cancer sciences.

Identification of genetic linkage group 1-linked sequences in Japanese eel (Anguilla japonica) by single chromosome sorting and sequencing.

Japanese eel (Anguilla japonica) constitutes one of the most important food fish in Japan; accordingly, genome sequencing and linkage mapping have been conducted for the purpose of artificial cultivation. In the next stage, integration of genomic sequences within linkage groups (LG) is required to construct higher-resolution genetic markers for quantitative trait loci mapping and selective breeding of beneficial traits in farming. In order to identify LG1-linked scaffolds from the draft genome assembly (323,776 scaffolds) reported previously, we attempted to isolate chromosomes corresponding to LG1 by flow sorting and subsequent analyses. Initially, single chromosomes were randomly collected by chromosome sorting and subjected to whole-genome amplification (WGA). A total of 60 WGA samples were screened by PCR with primers for a known LG1-linked scaffold, and five positive WGA samples were sequenced by next-generation sequencing (NGS). Following reference mapping analysis of the NGS reads, four of the five WGA samples were found to be enriched by LG1-linked sequences. These samples were cytogenetically assigned to chromosome 5 by fluorescence in situ hybridization. Using blastn searches with 82,081 contigs constructed from the NGS reads of the four WGA samples as queries, 2323 scaffolds were identified as putative LG1-linked scaffolds from the draft genome assembly. The total length of the putative LG1-linked scaffolds was 99.0 Mb, comparable to the estimated DNA amounts of chromosome 5 (91.1 Mb). These results suggest that the methodology developed herein is applicable to isolate specific chromosome DNAs and integrate unanchored scaffold sequences onto a particular LG and chromosome even in teleost fishes, in which isolation of specific chromosomes by flow sorting is generally difficult owing to similar morphologies, sizes, and GC-contents among chromosomes in the genome. The putative LG1-linked scaffolds of Japanese eel contain a total of 6833 short tandem repeats which will be available for higher-resolution linkage mapping.

Homologue-specific chromosome sequencing characterizes translocation junctions and permits allelic assignment.

Chromosome translocations can be detected by cytogenetic analysis, but it is hard to characterize the breakpoints at the sequence level. Chromosome sorting by flow cytometry produces flow karyotypes that enable the isolation of abnormal chromosomes and the generation of chromosome-specific DNA. In this study, a derivative chromosome t(9; 14) and its homologous normal chromosomes 9 and 14 from the Ishikawa 3-H-12 cell line were sorted to collect homologue-specific samples. Chromosome sequencing identified the breakpoint junction in the der(9) at 9p24.3 and 14q13.1 and uncovered the formation of a fusion gene, WASH1–NPAS3. Amplicon sequencing targeted for neighbouring genes at the fusion breakpoint revealed that the variant frequencies correlate with the allelic copy number. Sequencing of sorted chromosomes permits the assignment of allelic variants and can lead to the characterization of abnormal chromosomes. We show that allele-specific chromosome sequencing of homologues is a robust technique for distinguishing alleles and this provides an efficient approach for the comprehensive analysis of genomic changes.

Development of intron targeting (IT) markers specific for chromosome arm 4VS of Haynaldia villosa by chromosome sorting and next-generation sequencing

BackgroundHaynaldia villosa (L.) Schur (syn. Dasypyrum villosum L. Candargy, 2n = 14, genome VV) is the tertiary gene pool of wheat, and thus a potential resource of genes for wheat improvement. Among other, wheat yellow mosaic (WYM) resistance gene Wss1 and a take-all resistance gene were identified on the short arm of chromosome 4 V (4VS) of H. villosa. We had obtained introgressions on 4VS chromosome arm, with the objective of utilizing the target genes. However, monitoring these introgressions has been a daunting task because of inadequate knowledge as to H.villosa genome, as reflected by the lack of specific markers.ResultsThis study aims to develop 4VS-specific markers by combination of chromosome sorting and next-generation sequencing. The short arm of chromosome 4VS of H.villosa was flow-sorted using a FACSVantage SE flow cytometer and sorter, and then sequenced by Illumina sequencing. The sequence of H. villosa 4VS was assembled by the software Hecate, and then was compared with the sequence assemblies of wheat chromosome arms 4AL, 4BS and 4DS and Ae. tauschii 4DS, with the objectives of identifying exon-exon junctions and localizing introns on chromosome 4VS of H. villosa. The intron length polymorphisms suitable for designing H. villosa primers were evaluated with criteria. Consequently, we designed a total of 359 intron targeting (IT) markers, among which 232 (64.62%) markers were specific for tracing the 4VS chromatin in the wheat background.ConclusionThe combination of chromosome sorting and next-generation sequencing to develop specific IT markers for 4VS of H. villosa has high success rate and specificity, thus being applicable for the development of chromosome-specific markers for alien chromatin in wheat breeding.