24406816 OR Advances in plant chromosome genomics Dotaz Zobrazit nápovědu
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 identification of causal genomic loci and their interactions underlying various traits in plants has been greatly aided by progress in understanding the organization of the nuclear genome. This provides clues to the responses of plants to environmental stimuli at the molecular level. Apart from other uses, these insights are needed to fully explore the potential of new breeding techniques that rely on genome editing. However, genome analysis and sequencing is not straightforward in the many agricultural crops and their wild relatives that possess large and complex genomes. Chromosome genomics streamlines this task by dissecting the genome to single chromosomes whose DNA is then used instead of nuclear DNA. This results in a massive and lossless reduction in DNA sample complexity, reduces the time and cost of the experiment, and simplifies data interpretation. Flow cytometric sorting of condensed mitotic chromosomes makes it possible to purify single chromosomes in large quantities, and as the DNA remains intact this process can be coupled successfully with many techniques in molecular biology and genomics. Since the first experiments with flow cytometric sorting in the late 1980s, numerous applications have been developed, and chromosome genomics has been having a significant impact in many areas of research, including the sequencing of complex genomes of important crops and gene cloning. This review discusses these applications, describes their contribution to advancements in plant genome analysis and gene cloning, and outlines future directions.
Chromosome numbers have been widely used to describe the most fundamental genomic attribute of an organism or a lineage. Although providing strong phylogenetic signal, chromosome numbers vary remarkably among eukaryotes at all levels of taxonomic resolution. Changes in chromosome numbers regularly serve as indication of major genomic events, most notably polyploidy and dysploidy. Here, we review recent advancements in our ability to make inferences regarding historical events that led to alterations in the number of chromosomes of a lineage. We first describe the mechanistic processes underlying changes in chromosome numbers, focusing on structural chromosomal rearrangements. Then, we focus on experimental procedures, encompassing comparative cytogenomics and genomics approaches, and on computational methodologies that are based on explicit models of chromosome-number evolution. Together, these tools offer valuable predictions regarding historical events that have changed chromosome numbers and genome structures, as well as their phylogenetic and temporal placements.
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.
- MeSH
- Brachypodium genetika MeSH
- chromozomy rostlin genetika MeSH
- genom rostlinný genetika MeSH
- hybridizace in situ fluorescenční MeSH
- ječmen (rod) genetika MeSH
- jednonukleotidový polymorfismus MeSH
- lipnicovité klasifikace genetika MeSH
- mapování chromozomů MeSH
- molekulární evoluce MeSH
- pořadí genů MeSH
- pšenice genetika MeSH
- rostlinné geny genetika MeSH
- rýže (rod) genetika MeSH
- Sorghum genetika MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Seabuckthorn (Hippophae rhamnoides) is a dioecious shrub commonly used in the pharmaceutical, cosmetic, and environmental industry as a source of oil, minerals and vitamins. In this study, we analyzed the transposable elements and satellites in its genome. We carried out Illumina DNA sequencing and reconstructed the main repetitive DNA sequences. For data analysis, we developed a new bioinformatics approach for advanced satellite DNA analysis and showed that about 25% of the genome consists of satellite DNA and about 24% is formed of transposable elements, dominated by Ty3/Gypsy and Ty1/Copia LTR retrotransposons. FISH mapping revealed X chromosome-accumulated, Y chromosome-specific or both sex chromosomes-accumulated satellites but most satellites were found on autosomes. Transposable elements were located mostly in the subtelomeres of all chromosomes. The 5S rDNA and 45S rDNA were localized on one autosomal locus each. Although we demonstrated the small size of the Y chromosome of the seabuckthorn and accumulated satellite DNA there, we were unable to estimate the age and extent of the Y chromosome degeneration. Analysis of dioecious relatives such as Shepherdia would shed more light on the evolution of these sex chromosomes.
- MeSH
- chromozomy rostlin * MeSH
- DNA rostlinná genetika MeSH
- fylogeneze MeSH
- genom rostlinný MeSH
- Hippophae genetika MeSH
- molekulární evoluce MeSH
- pohlavní chromozomy * MeSH
- satelitní DNA * MeSH
- sekvenční analýza DNA metody MeSH
- transpozibilní elementy DNA * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The human bed bug Cimex lectularius is one of the most prevalent human ectoparasites in temperate climate zones. The cytogenetic features of this resilient pest include holokinetic chromosomes, special chromosome behavior in meiosis, and numerical variation of chromosomes, where the diploid number ranges from 26 + X1 X2 Y to 26 + X1-20 Y. It is desirable to assess the nuclear DNA content of various cytotypes for a further detailed study of the C. lectularius genome. Detailed knowledge of the DNA content of this parasite could also clarify the origin of additional chromosomes. The average nuclear genome size C. lectularius with 2n = 26 + X1 X2 Y is 2C = 1.94 pg for males and 1.95 pg for females. There is a significant correlation between genome size and the number of chromosomes, but in some specimens with additional chromosomes, nuclear genome size decreases or remains average. Several species used as the internal reference standard were tested for further investigations of genome size in C. lectularius, and the plant Solanum pseudocaspicum turned out to be the most suitable. © 2019 International Society for Advancement of Cytometry.
- MeSH
- barvení a značení MeSH
- buněčné jádro chemie genetika MeSH
- cytogenetika MeSH
- délka genomu * MeSH
- indoly MeSH
- meióza genetika MeSH
- metafáze genetika MeSH
- mitóza genetika MeSH
- pohlavní chromozomy * MeSH
- propidium MeSH
- průtoková cytometrie MeSH
- štěnice genetika MeSH
- zastoupení bazí MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We report a comprehensive toolkit that enables targeted, specific modification of monocot and dicot genomes using a variety of genome engineering approaches. Our reagents, based on transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, are systematized for fast, modular cloning and accommodate diverse regulatory sequences to drive reagent expression. Vectors are optimized to create either single or multiple gene knockouts and large chromosomal deletions. Moreover, integration of geminivirus-based vectors enables precise gene editing through homologous recombination. Regulation of transcription is also possible. A Web-based tool streamlines vector selection and construction. One advantage of our platform is the use of the Csy-type (CRISPR system yersinia) ribonuclease 4 (Csy4) and tRNA processing enzymes to simultaneously express multiple guide RNAs (gRNAs). For example, we demonstrate targeted deletions in up to six genes by expressing 12 gRNAs from a single transcript. Csy4 and tRNA expression systems are almost twice as effective in inducing mutations as gRNAs expressed from individual RNA polymerase III promoters. Mutagenesis can be further enhanced 2.5-fold by incorporating the Trex2 exonuclease. Finally, we demonstrate that Cas9 nickases induce gene targeting at frequencies comparable to native Cas9 when they are delivered on geminivirus replicons. The reagents have been successfully validated in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), Medicago truncatula, wheat (Triticum aestivum), and barley (Hordeum vulgare).
- MeSH
- genetické inženýrství metody MeSH
- geneticky modifikované rostliny genetika MeSH
- ječmen (rod) genetika MeSH
- pšenice genetika MeSH
- RNA rostlin genetika MeSH
- rostlinné proteiny genetika MeSH
- sekvence CRISPR genetika MeSH
- Solanum lycopersicum genetika MeSH
- TALENs genetika MeSH
- Publikační typ
- časopisecké články MeSH
Significant advances in chromosome preparation and other techniques have greatly increased the potential of plant cytogenetics in recent years. Increase in longitudinal resolution using DNA extended fibers as well as new developments in imaging and signal amplification technologies have enhanced the ability of FISH to detect small gene targets. The combination of fluorescence in situ hybridization with immunocytochemistry allows the investigation of cell events, chromosomal rearrangements and chromatin features typical for plant nuclei. Chromosome manipulation techniques using microdissection and flow sorting have accelerated the analysis of complex plant genomes. Together, the different cytogenetic approaches are invaluable for the unravelling of detailed structures of plant chromosomes, which are of utmost importance for the study of genome properties, DNA replication and gene regulation. In this technical review, different cytogenetic approaches are discussed for the analysis of plant chromosomes, with a focus on mitotic chromosomes.
BACKGROUND: QTL cloning for the discovery of genes underlying polygenic traits has historically been cumbersome in long-lived perennial plants like Populus. Linkage disequilibrium-based association mapping has been proposed as a cloning tool, and recent advances in high-throughput genotyping and whole-genome resequencing enable marker saturation to levels sufficient for association mapping with no a priori candidate gene selection. Here, multiyear and multienvironment evaluation of cell wall phenotypes was conducted in an interspecific P. trichocarpa x P. deltoides pseudo-backcross mapping pedigree and two partially overlapping populations of unrelated P. trichocarpa genotypes using pyrolysis molecular beam mass spectrometry, saccharification, and/ or traditional wet chemistry. QTL mapping was conducted using a high-density genetic map with 3,568 SNP markers. As a fine-mapping approach, chromosome-wide association mapping targeting a QTL hot-spot on linkage group XIV was performed in the two P. trichocarpa populations. Both populations were genotyped using the 34 K Populus Infinium SNP array and whole-genome resequencing of one of the populations facilitated marker-saturation of candidate intervals for gene identification. RESULTS: Five QTLs ranging in size from 0.6 to 1.8 Mb were mapped on linkage group XIV for lignin content, syringyl to guaiacyl (S/G) ratio, 5- and 6-carbon sugars using the mapping pedigree. Six candidate loci exhibiting significant associations with phenotypes were identified within QTL intervals. These associations were reproducible across multiple environments, two independent genotyping platforms, and different plant growth stages. cDNA sequencing for allelic variants of three of the six loci identified polymorphisms leading to variable length poly glutamine (PolyQ) stretch in a transcription factor annotated as an ANGUSTIFOLIA C-terminus Binding Protein (CtBP) and premature stop codons in a KANADI transcription factor as well as a protein kinase. Results from protoplast transient expression assays suggested that each of the polymorphisms conferred allelic differences in the activation of cellulose, hemicelluloses, and lignin pathway marker genes. CONCLUSION: This study illustrates the utility of complementary QTL and association mapping as tools for gene discovery with no a priori candidate gene selection. This proof of concept in a perennial organism opens up opportunities for discovery of novel genetic determinants of economically important but complex traits in plants.
- MeSH
- alely MeSH
- buněčná stěna genetika MeSH
- celulosa metabolismus MeSH
- fenotyp MeSH
- genetická vazba MeSH
- genotyp MeSH
- jednonukleotidový polymorfismus MeSH
- lignin biosyntéza MeSH
- lod skóre MeSH
- lokus kvantitativního znaku MeSH
- mapování chromozomů MeSH
- Populus genetika MeSH
- rostlinné geny * MeSH
- rostlinné proteiny chemie genetika MeSH
- sekvence nukleotidů MeSH
- sekvenční seřazení MeSH
- transkripční faktory chemie genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
BACKGROUND: Recent advances in genomics indicate functional significance of a majority of genome sequences and their long range interactions. As a detailed examination of genome organization and function requires very high quality genome sequence, the objective of this study was to improve reference genome assembly of banana (Musa acuminata). RESULTS: We have developed a modular bioinformatics pipeline to improve genome sequence assemblies, which can handle various types of data. The pipeline comprises several semi-automated tools. However, unlike classical automated tools that are based on global parameters, the semi-automated tools proposed an expert mode for a user who can decide on suggested improvements through local compromises. The pipeline was used to improve the draft genome sequence of Musa acuminata. Genotyping by sequencing (GBS) of a segregating population and paired-end sequencing were used to detect and correct scaffold misassemblies. Long insert size paired-end reads identified scaffold junctions and fusions missed by automated assembly methods. GBS markers were used to anchor scaffolds to pseudo-molecules with a new bioinformatics approach that avoids the tedious step of marker ordering during genetic map construction. Furthermore, a genome map was constructed and used to assemble scaffolds into super scaffolds. Finally, a consensus gene annotation was projected on the new assembly from two pre-existing annotations. This approach reduced the total Musa scaffold number from 7513 to 1532 (i.e. by 80%), with an N50 that increased from 1.3 Mb (65 scaffolds) to 3.0 Mb (26 scaffolds). 89.5% of the assembly was anchored to the 11 Musa chromosomes compared to the previous 70%. Unknown sites (N) were reduced from 17.3 to 10.0%. CONCLUSION: The release of the Musa acuminata reference genome version 2 provides a platform for detailed analysis of banana genome variation, function and evolution. Bioinformatics tools developed in this work can be used to improve genome sequence assemblies in other species.
