Most cited article - PubMed ID 28202009
Development of intron targeting (IT) markers specific for chromosome arm 4VS of Haynaldia villosa by chromosome sorting and next-generation sequencing
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.
- Keywords
- Accumulation of metaphase cells, Chromosome isolation, Cytogenetic stocks, FISH, FISHIS, Flow cytometry and sorting, Hydroponic, Mitotic synchrony, Plants, Seedlings,
- MeSH
- Chromosomes, Plant * MeSH
- Chromosomes * MeSH
- Cytogenetics MeSH
- Karyotyping MeSH
- Flow Cytometry methods MeSH
- Suspensions MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Suspensions MeSH
Genomics studies in wild species of wheat have been limited due to the lack of references; however, new technologies and bioinformatics tools have much potential to promote genomic research. The wheat-Haynaldia villosa translocation line T6VS·6AL has been widely used as a backbone parent of wheat breeding in China. Therefore, revealing the genome structure of translocation chromosome 6VS·6AL will clarify how this chromosome formed and will help to determine how it affects agronomic traits. In this study, chromosome flow sorting, NGS sequencing and Chicago long-range linkage assembly were innovatively used to produce the assembled sequences of 6VS·6AL, and gene prediction and genome structure characterization at the molecular level were effectively performed. The analysis discovered that the short arm of 6VS·6AL was actually composed of a large distal segment of 6VS, a small proximal segment of 6AS and the centromere of 6A, while the collinear region in 6VS corresponding to 230-260 Mb of 6AS-Ta was deleted when the recombination between 6VS and 6AS occurred. In addition to the molecular mechanism of the increased grain weight and enhanced spike length produced by the translocation chromosome, it may be correlated with missing GW2-V and an evolved NRT-V cluster. Moreover, a fine physical bin map of 6VS was constructed by the high-throughput developed 6VS-specific InDel markers and a series of newly identified small fragment translocation lines involving 6VS. This study will provide essential information for mining of new alien genes carried by the 6VS·6AL translocation chromosome.
- Keywords
- Chicago long-range linkage assembly, InDel markers, genome annotation, physical bin map, wheat-Haynaldia villosa translocation line T6VS·6AL,
- MeSH
- Chromosomes, Plant genetics MeSH
- Poaceae genetics MeSH
- Triticum * genetics MeSH
- Plant Breeding * MeSH
- Translocation, Genetic MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
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.
- Keywords
- DNA amplification, DNA isolation, cell cycle synchronization, gene mapping and cloning, genome sequencing, liquid chromosome suspension, marker development, mitotic metaphase chromosomes, repetitive DNA labelling,
- MeSH
- Cell Cycle MeSH
- Chromosomes, Plant * genetics MeSH
- Metaphase MeSH
- Flow Cytometry MeSH
- Plants * genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
BACKGROUND: Haynaldia villosa (H. villosa) has been recognized as a species potentially useful for wheat improvement. The availability of its genomic sequences will boost its research and application. RESULTS: In this work, the short arm of H. villosa chromosome 4V (4VS) was sorted by flow cytometry and sequenced using Illumina platform. About 170.6 Mb assembled sequences were obtained. Further analysis showed that repetitive elements accounted for about 64.6% of 4VS, while the coding fraction, which is corresponding to 1977 annotated genes, represented 1.5% of the arm. The syntenic regions of the 4VS were searched and identified on wheat group 4 chromosomes 4AL, 4BS, 4DS, Brachypodium chromosomes 1 and 4, rice chromosomes 3 and 11, and sorghum chromosomes 1, 5 and 8. Based on genome-zipper analysis, a virtual gene order comprising 735 gene loci on 4VS genome was built by referring to the Brachypodium genome, which was relatively consistent with the scaffold order determined for Ae. tauschii chromosome 4D. The homologous alleles of several cloned genes on wheat group 4 chromosomes including Rht-1 gene were identified. CONCLUSIONS: The sequences provided valuable information for mapping and positional-cloning genes located on 4VS, such as the wheat yellow mosaic virus resistance gene Wss1. The work on 4VS provided detailed insights into the genome of H. villosa, and may also serve as a model for sequencing the remaining parts of H. villosa genome.
- Keywords
- Chromosome arm 4VS, Flow sorting, Genome zipper, Haynaldia villosa, Scaffold,
- MeSH
- Chromosomes, Plant genetics MeSH
- Species Specificity MeSH
- Genomics MeSH
- Poaceae genetics MeSH
- Chromosome Mapping MeSH
- Gene Order genetics MeSH
- Repetitive Sequences, Nucleic Acid genetics MeSH
- Sequence Analysis, DNA * MeSH
- Publication type
- Journal Article MeSH