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

Breeding of wheat adapted to new climatic conditions and resistant to diseases and pests is hindered by a limited gene pool due to domestication and thousands of years of human selection. Annual goatgrasses (Aegilops spp.) with M and U genomes are potential sources of the missing genes and alleles. Development of alien introgression lines of wheat may be facilitated by the knowledge of DNA sequences of Aegilops chromosomes. As the Aegilops genomes are complex, sequencing relevant Aegilops chromosomes purified by flow cytometric sorting offers an attractive route forward. The present study extends the potential of chromosome genomics to allotetraploid Ae. biuncialis and Ae. geniculata by dissecting their M and U genomes into individual chromosomes. Hybridization of FITC-conjugated GAA oligonucleotide probe to chromosomes suspensions of the two species allowed the application of bivariate flow karyotyping and sorting some individual chromosomes. Bivariate flow karyotype FITC vs. DAPI of Ae. biuncialis consisted of nine chromosome-populations, but their chromosome content determined by microscopic analysis of flow sorted chromosomes indicated that only 7Mb and 1Ub could be sorted at high purity. In the case of Ae. geniculata, fourteen chromosome-populations were discriminated, allowing the separation of nine individual chromosomes (1Mg, 3Mg, 5Mg, 6Mg, 7Mg, 1Ug, 3Ug, 6Ug, and 7Ug) out of the 14. To sort the remaining chromosomes, a partial set of wheat-Ae. biuncialis and a whole set of wheat-Ae. geniculata chromosome addition lines were also flow karyotyped, revealing clear separation of the GAA-rich Aegilops chromosomes from the GAA-poor A- and D-genome chromosomes of wheat. All of the alien chromosomes represented by individual addition lines could be isolated at purities ranging from 74.5% to 96.6% and from 87.8% to 97.7%, respectively. Differences in flow karyotypes between Ae. biuncialis and Ae. geniculata were analyzed and discussed. Chromosome-specific genomic resources will facilitate gene cloning and the development of molecular tools to support alien introgression breeding of wheat.

• Klíčová slova
• Aegilops biuncialis, Aegilops geniculata, chromosome flow sorting, flow karyotyping, genome dissecting,
• Publikační typ
• časopisecké články MeSH

BACKGROUND AND AIMS: Aegilops markgrafii (CC) and its natural hybrids Ae. triuncialis (U(t)U(t)C(t)C(t)) and Ae. cylindrica (D(c)D(c)C(c)C(c)) represent a rich reservoir of useful genes for improvement of bread wheat (Triticum aestivum), but the limited information available on their genome structure and the shortage of molecular (cyto-) genetic tools hamper the utilization of the extant genetic diversity. This study provides the complete karyotypes in the three species obtained after fluorescent in situ hybridization (FISH) with repetitive DNA probes, and evaluates the potential of flow cytometric chromosome sorting. METHODS: The flow karyotypes obtained after the analysis of 4',6-diamidino-2-phenylindole (DAPI)-stained chromosomes were characterized and the chromosome content of the peaks on the flow karyotypes was determined by FISH. Twenty-nine conserved orthologous set (COS) markers covering all seven wheat homoeologous chromosome groups were used for PCR with DNA amplified from flow-sorted chromosomes and genomic DNA. KEY RESULTS: FISH with repetitive DNA probes revealed that chromosomes 4C, 5C, 7C(t), T6U(t)S.6U(t)L-5C(t)L, 1C(c) and 5D(c) could be sorted with purities ranging from 66 to 91 %, while the remaining chromosomes could be sorted in groups of 2-5. This identified a partial wheat-C-genome homology for group 4 and 5 chromosomes. In addition, 1C chromosomes were homologous with group 1 of wheat; a small segment from group 2 indicated 1C-2C rearrangement. An extensively rearranged structure of chromosome 7C relative to wheat was also detected. CONCLUSIONS: The possibility of purifying Aegilops chromosomes provides an attractive opportunity to investigate the structure and evolution of the Aegilops C genome and to develop molecular tools to facilitate the identification of alien chromatin and support alien introgression breeding in bread wheat.

• Klíčová slova
• Ae. cylindrica, Ae. triuncialis, Aegilops markgrafii, COS, FISH, GISH, Poaceae, Triticum aestivum, conserved orthologous set markers, flow cytometric chromosome sorting, fluorescence in situ hybridization, genomic in situ hybridization, goatgrass, physical mapping, wheat,
• MeSH
• chromozomy rostlin genetika   MeSH
• genom rostlinný * MeSH
• hybridizace in situ fluorescenční MeSH
• indoly MeSH
• karyotyp MeSH
• karyotypizace MeSH
• konzervovaná sekvence genetika   MeSH
• lipnicovité genetika   MeSH
• metafáze MeSH
• průtoková cytometrie metody   MeSH
• pšenice genetika   MeSH
• sekvenční homologie nukleových kyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DAPI MeSH Prohlížeč
• indoly MeSH

BACKGROUND: Physical maps created from large insert DNA libraries, typically cloned in BAC vector, are valuable resources for map-based cloning and de novo genome sequencing. The maps are most useful if contigs of overlapping DNA clones are anchored to chromosome(s), and ordered along them using molecular markers. Here we present a novel approach for anchoring physical maps, based on sequencing three-dimensional pools of BAC clones from minimum tilling path. RESULTS: We used physical map of wheat chromosome arm 3DS to validate the method with two different DNA sequence datasets. The first comprised 567 genes ordered along the chromosome arm based on syntenic relationship of wheat with the sequenced genomes of Brachypodium, rice and sorghum. The second dataset consisted of 7,136 SNP-containing sequences, which were mapped genetically in Aegilops tauschii, the donor of the wheat D genome. Mapping of sequence reads from individual BAC pools to the first and the second datasets enabled unambiguous anchoring 447 and 311 3DS-specific sequences, respectively, or 758 in total. CONCLUSIONS: We demonstrate the utility of the novel approach for BAC contig anchoring based on mass parallel sequencing of three-dimensional pools prepared from minimum tilling path of physical map. The existing genetic markers as well as any other DNA sequence could be mapped to BAC clones in a single in silico experiment. The approach reduces significantly the cost and time needed for anchoring and is applicable to any genomic project involving the construction of anchored physical map.

• MeSH
• chromozomy rostlin * MeSH
• mapování chromozomů * MeSH
• rostliny genetika   MeSH
• umělé bakteriální chromozomy * MeSH
• vysoce účinné nukleotidové sekvenování * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: The banana family (Musaceae) includes genetically a diverse group of species and their diploid and polyploid hybrids that are widely cultivated in the tropics. In spite of their socio-economic importance, the knowledge of Musaceae genomes is basically limited to draft genome assemblies of two species, Musa acuminata and M. balbisiana. Here we aimed to complement this information by analyzing repetitive genome fractions of six species selected to represent various phylogenetic groups within the family. RESULTS: Low-pass sequencing of M. acuminata, M. ornata, M. textilis, M. beccarii, M. balbisiana, and Ensete gilletii genomes was performed using a 454/Roche platform. Sequence reads were subjected to analysis of their overall intra- and inter-specific similarities and, all major repeat families were quantified using graph-based clustering. Maximus/SIRE and Angela lineages of Ty1/copia long terminal repeat (LTR) retrotransposons and the chromovirus lineage of Ty3/gypsy elements were found to make up most of highly repetitive DNA in all species (14-34.5% of the genome). However, there were quantitative differences and sequence variations detected for classified repeat families as well as for the bulk of total repetitive DNA. These differences were most pronounced between species from different taxonomic sections of the Musaceae family, whereas pairs of closely related species (M. acuminata/M. ornata and M. beccarii/M. textilis) shared similar populations of repetitive elements. CONCLUSIONS: This study provided the first insight into the composition and sequence variation of repetitive parts of Musaceae genomes. It allowed identification of repetitive sequences specific for a single species or a group of species that can be utilized as molecular markers in breeding programs and generated computational resources that will be instrumental in repeat masking and annotation in future genome assembly projects.

• MeSH
• banánovníkovité klasifikace   genetika   MeSH
• DNA rostlinná analýza   genetika   MeSH
• fylogeneze MeSH
• genetická variace MeSH
• genom rostlinný * MeSH
• molekulární evoluce MeSH
• repetitivní sekvence nukleových kyselin * MeSH
• sekvenční analýza DNA MeSH
• výpočetní biologie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH

Nuclear genomes of human, animals, and plants are organized into subunits called chromosomes. When isolated into aqueous suspension, mitotic chromosomes can be classified using flow cytometry according to light scatter and fluorescence parameters. Chromosomes of interest can be purified by flow sorting if they can be resolved from other chromosomes in a karyotype. The analysis and sorting are carried out at rates of 10(2)-10(4) chromosomes per second, and for complex genomes such as wheat the flow sorting technology has been ground-breaking in reducing genome complexity for genome sequencing. The high sample rate provides an attractive approach for karyotype analysis (flow karyotyping) and the purification of chromosomes in large numbers. In characterizing the chromosome complement of an organism, the high number that can be studied using flow cytometry allows for a statistically accurate analysis. Chromosome sorting plays a particularly important role in the analysis of nuclear genome structure and the analysis of particular and aberrant chromosomes. Other attractive but not well-explored features include the analysis of chromosomal proteins, chromosome ultrastructure, and high-resolution mapping using FISH. Recent results demonstrate that chromosome flow sorting can be coupled seamlessly with DNA array and next-generation sequencing technologies for high-throughput analyses. The main advantages are targeting the analysis to a genome region of interest and a significant reduction in sample complexity. As flow sorters can also sort single copies of chromosomes, shotgun sequencing DNA amplified from them enables the production of haplotype-resolved genome sequences. This review explains the principles of flow cytometric chromosome analysis and sorting (flow cytogenetics), discusses the major uses of this technology in genome analysis, and outlines future directions.

• MeSH
• chromozomy chemie   genetika   MeSH
• fyzikální mapování chromozomů metody   MeSH
• genom lidský MeSH
• genomika metody   MeSH
• genová knihovna MeSH
• karyotyp MeSH
• lidé MeSH
• malování chromozomů metody   MeSH
• mitóza MeSH
• průtoková cytometrie metody   MeSH
• rostliny chemie   genetika   MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů metody   MeSH
• struktury chromozomu chemie   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Positional cloning in bread wheat is a tedious task due to its huge genome size and hexaploid character. BAC libraries represent an essential tool for positional cloning. However, wheat BAC libraries comprise more than million clones, which makes their screening very laborious. Here, we present a targeted approach based on chromosome-specific BAC libraries. Such libraries were constructed from flow-sorted arms of wheat chromosome 7D. A library from the short arm (7DS) consisting of 49,152 clones with 113 kb insert size represented 12.1 arm equivalents whereas a library from the long arm (7DL) comprised 50,304 clones of 116 kb providing 14.9x arm coverage. The 7DS library was PCR screened with markers linked to Russian wheat aphid resistance gene DnCI2401, the 7DL library was screened by hybridization with a probe linked to greenbug resistance gene Gb3. The small number of clones combined with high coverage made the screening highly efficient and cost effective.

• MeSH
• chromozomy rostlin genetika   MeSH
• fluorescence MeSH
• hybridizace nukleových kyselin genetika   MeSH
• karyotypizace MeSH
• klonování DNA metody   MeSH
• mikrosatelitní repetice genetika   MeSH
• mšice fyziologie   MeSH
• nemoci rostlin genetika   imunologie   parazitologie   MeSH
• polymerázová řetězová reakce MeSH
• přirozená imunita genetika   MeSH
• pšenice genetika   imunologie   parazitologie   MeSH
• rostlinné geny genetika   MeSH
• umělé bakteriální chromozomy genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cultivated chickpea is the third most important legume after field bean and garden pea worldwide. Despite considerable breeding towards improved yield and resistance to biotic and abiotic stresses, the production of chickpea remained stagnant, but molecular tools are expected to increase the impact of current improvement programs. As a first step towards this goal, various genetic linkage maps have been established and markers linked to resistance genes been identified. However, until now, only one linkage group (LG) has been assigned to a specific chromosome. In the present work, mitotic chromosomes were sorted using flow cytometry and used as template for PCR with primers designed for genomic regions flanking microsatellites. These primers amplify sequence-tagged microsatellite site markers. This approach confirmed the assignment of LG8 to the smallest chromosome H. For the first time, LG5 was linked to the largest chromosome A, LG4 to a medium-sized chromosome E, while LG3 was anchored to the second largest chromosome B. Chromosomes C and D could not be flow-sorted separately and were jointly associated to LG6 and LG7. By the same token, chromosomes F and G were anchored to LG1 and LG2. To establish a set of preferably diagnostic cytogenetic markers, the genomic distribution of various probes was verified using FISH. Moreover, a partial genomic bacterial artificial chromosome (BAC) library was constructed and putative single/low-copy BAC clones were mapped cytogenetically. As a result, two clones were identified localizing specifically to chromosomes E and H, for which no cytogenetic markers were yet available.

• MeSH
• chromozomy rostlin genetika   MeSH
• Cicer genetika   MeSH
• cytogenetika metody   MeSH
• DNA rostlinná genetika   MeSH
• genetická vazba MeSH
• genetické markery MeSH
• genom rostlinný MeSH
• hybridizace in situ fluorescenční MeSH
• mapování chromozomů metody   MeSH
• polymerázová řetězová reakce MeSH
• průtoková cytometrie MeSH
• umělé bakteriální chromozomy MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH
• genetické markery MeSH

BACKGROUND: Rye (Secale cereale L.) belongs to tribe Triticeae and is an important temperate cereal. It is one of the parents of man-made species Triticale and has been used as a source of agronomically important genes for wheat improvement. The short arm of rye chromosome 1 (1RS), in particular is rich in useful genes, and as it may increase yield, protein content and resistance to biotic and abiotic stress, it has been introgressed into wheat as the 1BL.1RS translocation. A better knowledge of the rye genome could facilitate rye improvement and increase the efficiency of utilizing rye genes in wheat breeding. RESULTS: Here, we report on BAC end sequencing of 1,536 clones from two 1RS-specific BAC libraries. We obtained 2,778 (90.4%) useful sequences with a cumulative length of 2,032,538 bp and an average read length of 732 bp. These sequences represent 0.5% of 1RS arm. The GC content of the sequenced fraction of 1RS is 45.9%, and at least 84% of the 1RS arm consists of repetitive DNA. We identified transposable element junctions in BESs and developed insertion site based polymorphism markers (ISBP). Out of the 64 primer pairs tested, 17 (26.6%) were specific for 1RS. We also identified BESs carrying microsatellites suitable for development of 1RS-specific SSR markers. CONCLUSION: This work demonstrates the utility of chromosome arm-specific BAC libraries for targeted analysis of large Triticeae genomes and provides new sequence data from the rye genome and molecular markers for the short arm of rye chromosome 1.

• MeSH
• chromozomy rostlin genetika   MeSH
• DNA rostlinná genetika   MeSH
• fyzikální mapování chromozomů MeSH
• genetické markery MeSH
• genom rostlinný * MeSH
• genová knihovna MeSH
• hybridizace in situ fluorescenční MeSH
• molekulární sekvence - údaje MeSH
• repetitivní sekvence nukleových kyselin MeSH
• rostlinné geny MeSH
• sekvence nukleotidů MeSH
• sekvenční homologie nukleových kyselin MeSH
• umělé bakteriální chromozomy MeSH
• výpočetní biologie MeSH
• žito genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH
• genetické markery MeSH

BACKGROUND: Flow cytometry facilitates sorting of single chromosomes and chromosome arms which can be used for targeted genome analysis. However, the recovery of microgram amounts of DNA needed for some assays requires sorting of millions of chromosomes which is laborious and time consuming. Yet, many genomic applications such as development of genetic maps or physical mapping do not require large DNA fragments. In such cases time-consuming de novo sorting can be minimized by utilizing whole-genome amplification. RESULTS: Here we report a protocol optimized in barley including amplification of DNA from only ten thousand chromosomes, which can be isolated in less than one hour. Flow-sorted chromosomes were treated with proteinase K and amplified using Phi29 multiple displacement amplification (MDA). Overnight amplification in a 20-microlitre reaction produced 3.7 - 5.7 micrograms DNA with a majority of products between 5 and 30 kb. To determine the purity of sorted fractions and potential amplification bias we used quantitative PCR for specific genes on each chromosome. To extend the analysis to a whole genome level we performed an oligonucleotide pool assay (OPA) for interrogation of 1524 loci, of which 1153 loci had known genetic map positions. Analysis of unamplified genomic DNA of barley cv. Akcent using this OPA resulted in 1426 markers with present calls. Comparison with three replicates of amplified genomic DNA revealed >99% concordance. DNA samples from amplified chromosome 1H and a fraction containing chromosomes 2H - 7H were examined. In addition to loci with known map positions, 349 loci with unknown map positions were included. Based on this analysis 40 new loci were mapped to 1H. CONCLUSION: The results indicate a significant potential of using this approach for physical mapping. Moreover, the study showed that multiple displacement amplification of flow-sorted chromosomes is highly efficient and representative which considerably expands the potential of chromosome flow sorting in plant genomics.

• MeSH
• chromozomy rostlin genetika   MeSH
• DNA rostlinná genetika   MeSH
• fyzikální mapování chromozomů metody   MeSH
• genetické markery MeSH
• ječmen (rod) genetika   MeSH
• jednonukleotidový polymorfismus * MeSH
• polymerázová řetězová reakce MeSH
• průtoková cytometrie MeSH
• techniky amplifikace nukleových kyselin metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH
• genetické markery MeSH