BACKGROUND: The tall wheatgrass species Thinopyrum elongatum carries a strong fusarium head blight (FHB) resistance locus located on the long arm of chromosome 7 (7EL) as well as resistance to leaf and stem rusts, all diseases with a significant impact on wheat production. Towards understanding the contribution of Th. elongatum 7EL to improvement of disease resistance in wheat, the genomic sequence of the 7EL fragment present in the wheat Chinese Spring (CS) telosomic addition line CS-7EL was determined and the contribution and impact of 7EL on the rachis transcriptome during FHB infection was compared between CS and CS-7EL. RESULTS: We assembled the Th. elongatum 7EL chromosome arm using a reference-guided approach. Combining this assembly with the available reference sequence for CS hexaploid wheat provided a reliable reference for interrogating the transcriptomic differences in response to infection conferred by the 7EL fragment. Comparison of the transcriptomes of rachis tissues from CS and CS-7EL showed expression of Th. elongatum transcripts as well as modulation of wheat transcript expression profiles in the CS-7EL line. Expression profiles at 4 days after infection with Fusarium graminearum, the causal agent of FHB, showed an increased in expression of genes associated with an effective defense response, in particular glucan endo-1,3-beta-glucosidases and chitinases, in the FHB-resistant line CS-7EL while there was a larger increase in differential expression for genes associated with the level of fungal infection in the FHB-susceptible line CS. One hundred and seven 7EL transcripts were expressed in the smallest 7EL region defined to carry FHB resistance. CONCLUSION: 7EL contributed to CS-7EL transcriptome by direct expression and through alteration of wheat transcript profiles. FHB resistance in CS-7EL was associated with transcriptome changes suggesting a more effective defense response. A list of candidate genes for the FHB resistance locus on 7EL has been established.
- Klíčová slova
- Alien introgression, Disease resistance, Fusarium graminearum, Non-coding RNA, RNA-seq, Thinopyrum elongatum, Transcriptome, Triticum aestivum,
- MeSH
- chromozomy rostlin * genetika MeSH
- Fusarium * MeSH
- genomika MeSH
- lipnicovité * genetika mikrobiologie MeSH
- nemoci rostlin * genetika mikrobiologie MeSH
- odolnost vůči nemocem * genetika MeSH
- transkriptom MeSH
- Publikační typ
- časopisecké články MeSH
A genetic linkage map of dioecious garden asparagus (Asparagus officinalis L., 2n = 2x = 20) was constructed using F1 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.
- Klíčová slova
- Asparagus officinalis, FISH, SNPs, SSRs, flow-sorted chromosomes, genetic map, sex chromosome,
- Publikační typ
- časopisecké články 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.
- Klíčová slova
- Chromosome arm 4VS, Flow sorting, Genome zipper, Haynaldia villosa, Scaffold,
- MeSH
- chromozomy rostlin genetika MeSH
- druhová specificita MeSH
- genomika MeSH
- lipnicovité genetika MeSH
- mapování chromozomů MeSH
- pořadí genů genetika MeSH
- repetitivní sekvence nukleových kyselin genetika MeSH
- sekvenční analýza DNA * MeSH
- Publikační typ
- časopisecké články MeSH
Nuclear genomes of many important plant species are tremendously complicated to map and sequence. The ability to isolate single chromosomes, which represent small units of nuclear genome, is priceless in many areas of plant research including cytogenetics, genomics, and proteomics. Flow cytometry is the only technique which can provide large quantities of pure chromosome fractions suitable for downstream applications including physical mapping, preparation of chromosome-specific BAC libraries, sequencing, and optical mapping. Here, we describe step-by-step procedure of preparation of liquid suspensions of intact mitotic metaphase chromosomes and their flow cytometric analysis and sorting.
- Klíčová slova
- Cell cycle synchronization, Chromosome isolation, Cytogenetic stocks, FISH, FISHIS, Flow cytometry and sorting, Metaphase accumulation, Plants,
- MeSH
- chromozomy rostlin * MeSH
- hybridizace in situ fluorescenční metody MeSH
- průtoková cytometrie metody MeSH
- rostliny genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- Klíčová slova
- Aegilops comosa, Aegilops markgrafii, Aegilops speltoides, Aegilops umbellulata, conserved orthologous set markers, flow cytometric chromosome sorting, fluorescence in situ hybridization,
- MeSH
- chromozomy rostlin genetika MeSH
- hybridizace in situ MeSH
- průtoková cytometrie MeSH
- pšenice genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Identification of causal mutations in barley and wheat is hampered by their large genomes and suppressed recombination. To overcome these obstacles, we have developed MutChromSeq, a complexity reduction approach based on flow sorting and sequencing of mutant chromosomes, to identify induced mutations by comparison to parental chromosomes. We apply MutChromSeq to six mutants each of the barley Eceriferum-q gene and the wheat Pm2 genes. This approach unambiguously identified single candidate genes that were verified by Sanger sequencing of additional mutants. MutChromSeq enables reference-free forward genetics in barley and wheat, thus opening up their pan-genomes to functional genomics.
- Klíčová slova
- Barley, Chromosome flow sorting, Gene cloning, MutChromSeq, Mutational genomics, Triticeae, Wheat,
- MeSH
- chromozomy rostlin * MeSH
- fenotyp MeSH
- ječmen (rod) genetika MeSH
- jednonukleotidový polymorfismus MeSH
- klonování DNA * MeSH
- mutace * MeSH
- pšenice genetika MeSH
- rostlinné geny * MeSH
- sekvenční analýza DNA MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- Klíčová slova
- chromosomes, flow sorting, optical mapping, physical map, sequencing, wheat,
- MeSH
- biotechnologie metody MeSH
- chromozomy rostlin genetika MeSH
- genom rostlinný * MeSH
- mapování chromozomů metody MeSH
- pšenice genetika MeSH
- sekvenční analýza DNA metody MeSH
- tandemové repetitivní sekvence MeSH
- umělé bakteriální chromozomy MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Identification of transgene insertion sites in plant genomes has practical implications for crop breeding and is a stepping stone to analyze transgene function. However, single copy sequences are not always easy to localize in large plant genomes by standard approaches. RESULTS: We employed flow cytometric chromosome sorting to determine chromosomal location of barley sucrose transporter construct in three transgenic lines of common wheat. Flow-sorted chromosomes were used as template for PCR and fluorescence in situ hybridization to identify chromosomes with transgenes. The chromosomes carrying the transgenes were then confirmed by PCR using DNA amplified from single flow-sorted chromosomes as template. CONCLUSIONS: Insertion sites of the transgene were unambiguously localized to chromosomes 4A, 7A and 5D in three wheat transgenic lines. The procedure presented in this study is applicable for localization of any single-copy sequence not only in wheat, but in any plant species where suspension of intact mitotic chromosomes suitable for flow cytometric sorting can be prepared.
- Klíčová slova
- Flow cytometric sorting, Hordeum vulgare, HvSUT1, Single chromosome amplification, Transgene localization, Triticum aestivum,
- Publikační typ
- časopisecké články MeSH
Flow cytometry enables chromosomes to be sorted into different groups based on their characteristics, such as relative DNA content and the presence of repetitive DNA sequences. Despite the recent progress in the analysis of plant genome organization and chromosome structure, there is a need for easy methods to assign DNA sequences to individual chromosomes. Here, we describe an easy way to allocate genes or DNA sequences to chromosomes in wheat using flow-sorted chromosomes combined with fluorescence in situ hybridization and PCR analyses.
- Klíčová slova
- Chromosomal allocation, Chromosome genomics, Chromosome sorting, FISH, Flow cytometry, Single chromosome amplification, Transgene, Wheat,
- MeSH
- chromozomy rostlin * MeSH
- hybridizace in situ fluorescenční metody MeSH
- polymerázová řetězová reakce metody MeSH
- průtoková cytometrie metody MeSH
- pšenice genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- Klíčová slova
- Triticum aestivum L. (bread wheat), chromosome sorting, flow cytometry, next-generation sequencing, sequence assembly, single-chromosome genomics, whole-genome amplification,
- MeSH
- chromozomy rostlin genetika MeSH
- DNA rostlinná chemie genetika MeSH
- genom rostlinný genetika MeSH
- genomika metody MeSH
- kontigové mapování metody MeSH
- kořeny rostlin genetika MeSH
- průtoková cytometrie MeSH
- pšenice genetika MeSH
- reprodukovatelnost výsledků MeSH
- rostlinné geny genetika MeSH
- sekvenční analýza DNA MeSH
- techniky amplifikace nukleových kyselin metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA rostlinná MeSH
