Knotweed (Reynoutria sp.) plants are known in the world mainly as invasive plants. However, it is known that their rhizomes or leaves contain secondary metabolites with biological activity. Our goal was to determine which of the three knotweed plants (Reynoutria japonica, Reynoutria × bohemica, and Reynoutria sachalinensis) is most suitable for seed growth stimulation. We tested alcoholic extracts of all three knotweed species by seed priming method on wheat germination and seedling characteristics, when 12 measured characteristics were monitored. Extracts from all three species of Reynoutria sp. generally showed an improvement in wheat germination and growth compared to the control. R. × bohemica appears to be the best source for stimulating wheat growth, as seedling vigor indexes I and II, R/S dry weight, shoot and seedling lengths, root, shoot, and seedling dry weights were significantly different (ANOVA, Duncan's test, α < 0.05). The plants Reynoutria spp. seem to be possible sources for the protection and stimulation of agriculture crops.

• Klíčová slova
• Polygonum, Alcoholic extract, Plant growth, Seed priming, Seedling vigor index,
• MeSH
• klíčení * fyziologie   MeSH
• listy rostlin * chemie   růst a vývoj   MeSH
• pšenice * růst a vývoj   MeSH
• rostlinné extrakty * farmakologie   chemie   MeSH
• semena rostlinná růst a vývoj   MeSH
• semenáček * růst a vývoj   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• rostlinné extrakty * MeSH

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.

• Klíčová slova
• Accumulation of metaphase cells, Chromosome isolation, Cytogenetic stocks, FISH, FISHIS, Flow cytometry and sorting, Hydroponic, Mitotic synchrony, Plants, Seedlings,
• MeSH
• chromozomy rostlin * MeSH
• chromozomy * MeSH
• cytogenetika MeSH
• karyotypizace MeSH
• průtoková cytometrie metody   MeSH
• suspenze MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• suspenze 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.

• Klíčová slova
• 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
• buněčný cyklus MeSH
• chromozomy rostlin * genetika   MeSH
• metafáze MeSH
• průtoková cytometrie MeSH
• rostliny * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Sex-chromosome systems tend to be highly conserved and knowledge about their evolution typically comes from macroevolutionary inference. Rapidly evolving complex sex-chromosome systems represent a rare opportunity to study the mechanisms of sex-chromosome evolution at unprecedented resolution. Three cryptic species of wood-white butterflies-Leptidea juvernica, L. sinapis and L. reali-have each a unique set of multiple sex-chromosomes with 3-4 W and 3-4 Z chromosomes. Using a transcriptome-based microarray for comparative genomic hybridisation (CGH) and a library of bacterial artificial chromosome (BAC) clones, both developed in L. juvernica, we identified Z-linked Leptidea orthologs of Bombyx mori genes and mapped them by fluorescence in situ hybridisation (FISH) with BAC probes on multiple Z chromosomes. In all three species, we determined synteny blocks of autosomal origin and reconstructed the evolution of multiple sex-chromosomes. In addition, we identified W homologues of Z-linked orthologs and characterised their molecular differentiation. Our results suggest that the multiple sex-chromosome system evolved in a common ancestor as a result of dynamic genome reshuffling through repeated rearrangements between the sex chromosomes and autosomes, including translocations, fusions and fissions. Thus, the initial formation of neo-sex chromosomes could not have played a role in reproductive isolation between these Leptidea species. However, the subsequent species-specific fissions of several neo-sex chromosomes could have contributed to their reproductive isolation. Then, significantly increased numbers of Z-linked genes and independent neo-W chromosome degeneration could accelerate the accumulation of genetic incompatibilities between populations and promote their divergence resulting in speciation.

• MeSH
• molekulární evoluce * MeSH
• motýli * genetika   MeSH
• pohlavní chromozomy * MeSH
• syntenie * MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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 9.47 Mb 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.

• MeSH
• Aegilops genetika   MeSH
• Basidiomycota růst a vývoj   patogenita   MeSH
• chromozomy rostlin MeSH
• fenotyp MeSH
• genetické markery MeSH
• genová introgrese MeSH
• jednonukleotidový polymorfismus MeSH
• listy rostlin genetika   mikrobiologie   MeSH
• mapování chromozomů MeSH
• nemoci rostlin genetika   mikrobiologie   MeSH
• odolnost vůči nemocem genetika   MeSH
• pšenice genetika   mikrobiologie   MeSH
• rekombinace genetická MeSH
• rostlinné geny MeSH
• šlechtění rostlin MeSH
• telomery genetika   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• genetické markery MeSH

BACKGROUND: The IWGSC strategy for construction of the reference sequence of the bread wheat genome is based on first obtaining physical maps of the individual chromosomes. Our aim is to develop and use the physical map for analysis of the organization of the short arm of wheat chromosome 5B (5BS) which bears a number of agronomically important genes, including genes conferring resistance to fungal diseases. RESULTS: A physical map of the 5BS arm (290 Mbp) was constructed using restriction fingerprinting and LTC software for contig assembly of 43,776 BAC clones. The resulting physical map covered ~ 99% of the 5BS chromosome arm (111 scaffolds, N50 = 3.078 Mb). SSR, ISBP and zipper markers were employed for anchoring the BAC clones, and from these 722 novel markers were developed based on previously obtained data from partial sequencing of 5BS. The markers were mapped using a set of Chinese Spring (CS) deletion lines, and F2 and RICL populations from a cross of CS and CS-5B dicoccoides. Three approaches have been used for anchoring BAC contigs on the 5BS chromosome, including clone-by-clone screening of BACs, GenomeZipper analysis, and comparison of BAC-fingerprints with in silico fingerprinting of 5B pseudomolecules of T. dicoccoides. These approaches allowed us to reach a high level of BAC contig anchoring: 96% of 5BS BAC contigs were located on 5BS. An interesting pattern was revealed in the distribution of contigs along the chromosome. Short contigs (200-999 kb) containing markers for the regions interrupted by tandem repeats, were mainly localized to the 5BS subtelomeric block; whereas the distribution of larger 1000-3500 kb contigs along the chromosome better correlated with the distribution of the regions syntenic to rice, Brachypodium, and sorghum, as detected by the Zipper approach. CONCLUSION: The high fingerprinting quality, LTC software and large number of BAC clones selected by the informative markers in screening of the 43,776 clones allowed us to significantly increase the BAC scaffold length when compared with the published physical maps for other wheat chromosomes. The genetic and bioinformatics resources developed in this study provide new possibilities for exploring chromosome organization and for breeding applications.

• Klíčová slova
• Chromosome 5BS, Genetic markers, Hexaploid wheat, Physical mapping, Sequencing, Synteny, Triticum aestivum,
• MeSH
• chléb * MeSH
• chromozomy rostlin genetika   MeSH
• fyzikální mapování chromozomů * MeSH
• polymerázová řetězová reakce MeSH
• pšenice genetika   MeSH
• umělé bakteriální chromozomy genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cereal crops such as wheat and maize have large repeat-rich genomes that make cloning of individual genes challenging. Moreover, gene order and gene sequences often differ substantially between cultivars of the same crop species. A major bottleneck for gene cloning in cereals is the generation of high-quality sequence information from a cultivar of interest. In order to accelerate gene cloning from any cropping line, we report 'targeted chromosome-based cloning via long-range assembly' (TACCA). TACCA combines lossless genome-complexity reduction via chromosome flow sorting with Chicago long-range linkage to assemble complex genomes. We applied TACCA to produce a high-quality (N50 of 9.76 Mb) de novo chromosome assembly of the wheat line CH Campala Lr22a in only 4 months. Using this assembly we cloned the broad-spectrum Lr22a leaf-rust resistance gene, using molecular marker information and ethyl methanesulfonate (EMS) mutants, and found that Lr22a encodes an intracellular immune receptor homologous to the Arabidopsis thaliana RPM1 protein.

• MeSH
• biotechnologie MeSH
• genetická vazba MeSH
• jedlá semena genetika   MeSH
• klonování DNA metody   MeSH
• pšenice genetika   MeSH
• rostlinné geny genetika   MeSH
• Publikační typ
• časopisecké články MeSH

The capacity of the bread wheat (Triticum aestivum) genome to tolerate introgression from related genomes can be exploited for wheat improvement. A resistance to powdery mildew expressed by a derivative of the cross-bread wheat cv. Tähti × T. militinae (Tm) is known to be due to the incorporation of a Tm segment into the long arm of chromosome 4A. Here, a newly developed in silico method termed rearrangement identification and characterization (RICh) has been applied to characterize the introgression. A virtual gene order, assembled using the GenomeZipper approach, was obtained for the native copy of chromosome 4A; it incorporated 570 4A DArTseq markers to produce a zipper comprising 2132 loci. A comparison between the native and introgressed forms of the 4AL chromosome arm showed that the introgressed region is located at the distal part of the arm. The Tm segment, derived from chromosome 7G, harbours 131 homoeologs of the 357 genes present on the corresponding region of Chinese Spring 4AL. The estimated number of Tm genes transferred along with the disease resistance gene was 169. Characterizing the introgression's position, gene content and internal gene order should not only facilitate gene isolation, but may also be informative with respect to chromatin structure and behaviour studies.

• Klíčová slova
• GenomeZipper, alien introgression, chromosome rearrangement, chromosome translocation, comparative analysis, linkage drag,
• MeSH
• Ascomycota patogenita   MeSH
• chléb MeSH
• chromozomy rostlin genetika   metabolismus   MeSH
• DNA rostlinná genetika   MeSH
• genetické markery MeSH
• mapování chromozomů MeSH
• mikrosatelitní repetice MeSH
• nemoci rostlin genetika   mikrobiologie   MeSH
• odolnost vůči nemocem MeSH
• počítačová simulace MeSH
• pšenice genetika   mikrobiologie   MeSH
• rostlinné geny MeSH
• sekvence nukleotidů MeSH
• translokace genetická MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH
• genetické markery 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

Bread wheat has a large and complex allohexaploid genome with low recombination level at chromosome centromeric and peri-centromeric regions. This significantly hampers ordering of markers, contigs of physical maps and sequence scaffolds and impedes obtaining of high-quality reference genome sequence. Here we report on the construction of high-density and high-resolution radiation hybrid (RH) map of chromosome 4A supported by high-density chromosome deletion map. A total of 119 endosperm-based RH lines of two RH panels and 15 chromosome deletion bin lines were genotyped with 90K iSelect single nucleotide polymorphism (SNP) array. A total of 2316 and 2695 markers were successfully mapped to the 4A RH and deletion maps, respectively. The chromosome deletion map was ordered in 19 bins and allowed precise identification of centromeric region and verification of the RH panel reliability. The 4A-specific RH map comprises 1080 mapping bins and spans 6550.9 cR with a resolution of 0.13 Mb/cR. Significantly higher mapping resolution in the centromeric region was observed as compared to recombination maps. Relatively even distribution of deletion frequency along the chromosome in the RH panel was observed and putative functional centromere was delimited within a region characterized by two SNP markers.

• Klíčová slova
• SNP iSelect array, Triticum aestivum, chromosome deletion bin map, endosperm radiation hybrid panel, radiation hybrid map, wheat chromosome 4A,
• Publikační typ
• časopisecké články MeSH