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Jaroslav Dolezel

Publications and source records attributed to Jaroslav Dolezel.

18 recordsLinked to original sources
PubMed2006

Advanced resources for plant genomics: a BAC library specific for the short arm of wheat chromosome 1B.

Common wheat (Triticum aestivum L., 2n = 6x = 42) is a polyploid species possessing one of the largest genomes among the cultivated crops (1C is approximately 17 000 Mb). The presence of three homoeologous genomes (A, B and D), and the prevalence of repetitive DNA make sequencing the wheat genome a daunting task. We have developed a novel 'chromosome arm-based' strategy for wheat genome sequencing to simplify this task; this relies on sub-genomic libraries of large DNA inserts. In this paper, we used a di-telosomic line of wheat to isolate six million copies of the short arm of chromosome 1B (1BS) by flow sorting. Chromosomal DNA was partially digested with HindIII and used to construct an arm-specific BAC library. The library consists of 65 280 clones with an average insert size of 82 kb. Almost half of the library (45%) has inserts larger than 100 kb, while 18% of the inserts range in size between 75 and 100 kb, and 37% are shorter than 75 kb. We estimated the chromosome arm coverage to be 14.5-fold, giving a 99.9% probability of identifying a clone corresponding to any sequence on the short arm of 1B. Each chromosome arm in wheat can be flow sorted from an appropriate cytogenetic stock, and we envisage that the availability of chromosome arm-specific BAC resources in wheat will greatly facilitate the development of ready-to-sequence physical maps and map-based gene cloning.

Chromosomes, Artificial, Bacterial↗
PubMed2006

Flow cytometric and microscopic analysis of the effect of tannic acid on plant nuclei and estimation of DNA content.

BACKGROUND AND AIMS: Flow cytometry (FCM) is extensively used to estimate DNA ploidy and genome size in plants. In order to determine nuclear DNA content, nuclei in suspension are stained by a DNA-specific fluorochrome and fluorescence emission is quantified. Recent studies have shown that cytosolic compounds may interfere with binding of fluorochromes to DNA, leading to flawed data. Tannic acid, a common phenolic compound, may be responsible for some of the stoichiometric errors, especially in woody plants. In this study, the effect of tannic acid on estimation of nuclear DNA content was evaluated in Pisum sativum and Zea mays, which were chosen as model species. METHODS: Nuclear suspensions were prepared from P. sativum leaf tissue using four different lysis buffers (Galbraith's, LB01, Otto's and Tris.MgCl2). The suspensions were treated with tannic acid (TA) at 13 different initial concentrations ranging from 0.25 to 3.50 mg mL-1. After propidium iodide (PI) staining, samples were analysed using FCM. In addition to the measurement of nuclei fluorescence, light scatter properties were assessed. Subsequently, a single TA concentration was chosen for each buffer and the effect of incubation time was assessed. Similar analyses were performed on liquid suspensions of P. sativum and Z. mays nuclei that were isolated, treated and analysed simultaneously. FCM analyses were accompanied by microscopic observations of nuclei suspensions. KEY RESULTS: TA affected PI fluorescence and light scatter properties of plant nuclei, regardless of the isolation buffer used. The least pronounced effects of TA were observed in Tris.MgCl2 buffer. Samples obtained using Galbraith's and LB01 buffers were the most affected by this compound. A newly described 'tannic acid effect' occurred immediately after the addition of the compound. With the exception of Otto's buffer, nuclei of P. sativum and Z. mays were affected differently, with pea nuclei exhibiting a greater decrease in fluorescence intensity. CONCLUSIONS: A negative effect of a secondary metabolite, TA, on estimation of nuclear DNA content is described and recommendations for minimizing the effect of cytosolic compounds are presented. Alteration in light scattering properties of isolated nuclei can be used as an indicator of the presence of TA, which may cause stoichiometric errors in nuclei staining using a DNA intercalator, PI.

Cell Nucleus↗
PubMed2006

Comparison of four nuclear isolation buffers for plant DNA flow cytometry.

BACKGROUND AND AIMS: DNA flow cytometry requires preparation of suspensions of intact nuclei, which are stained using a DNA-specific fluorochrome prior to analysis. Various buffer formulas were developed to preserve nuclear integrity, protect DNA from degradation and facilitate its stoichiometric staining. Although nuclear isolation buffers differ considerably in chemical composition, no systematic comparison of their performance has been made until now. This knowledge is required to select the appropriate buffer for a given species and tissue. METHODS: Four common lysis buffers (Galbraith's, LB01, Otto's and Tris.MgCl2) were used to prepare samples from leaf tissues of seven plant species (Sedum burrito, Oxalis pes-caprae, Lycopersicon esculentum, Celtis australis, Pisum sativum, Festuca rothmaleri and Vicia faba). The species were selected to cover a wide range of genome sizes (1.30-26.90 pg per 2C DNA) and a variety of leaf tissue types. The following parameters were assessed: forward (FS) and side (SS) light scatters, fluorescence of propidium iodide-stained nuclei, coefficient of variation of DNA peaks, presence of debris background and the number of nuclei released from sample tissue. The experiments were performed independently by two operators and repeated on three different days. KEY RESULTS: Clear differences among buffers were observed. With the exception of O. pes-caprae, any buffer provided acceptable results for all species. LB01 and Otto's were generally the best buffers, with Otto's buffer providing better results in species with low DNA content. Galbraith's buffer led to satisfactory results and Tris.MgCl2 was generally the worst, although it yielded the best histograms in C. australis. A combined analysis of FS and SS provided a 'fingerprint' for each buffer. The variation between days was more significant than the variation between operators. CONCLUSIONS: Each lysis buffer tested responded to a specific problem differently and none of the buffers worked best with all species. These results expand our knowledge on nuclear isolation buffers and will facilitate selection of the most appropriate buffer depending on species, tissue type and the presence of cytosolic compounds interfering with DNA staining.

Buffers↗
PubMed2006

Dissection of the nuclear genome of barley by chromosome flow sorting.

Isolation of mitotic chromosomes using flow cytometry is an attractive way to dissect nuclear genomes into their individual chromosomal components or portions of them. This approach is especially useful in plants with complex genomes, where it offers a targeted and hence economical approach to genome analysis and gene cloning. In several plant species, DNA of flow-sorted chromosomes has been used for isolation of molecular markers from specific genome regions, for physical mapping using polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH), for integration of genetic and physical maps and for construction of chromosome-specific DNA libraries, including those cloned in bacterial artificial chromosome vectors. Until now, chromosome analysis and sorting using flow cytometry (flow cytogenetics) has found little application in barley (2n = 14, 1C approximately 5,100 Mbp) because of the impossibility of discriminating and sorting individual chromosomes, except for the smallest chromosome 1H and some translocation chromosomes with DNA content significantly different from the remaining chromosomes. In this work, we demonstrate that wheat-barley ditelosomic addition lines can be used to sort any arm of barley chromosomes 2H-7H. Thus, the barley genome can be dissected into fractions representing only about 6-12% of the total genome. This advance makes the flow cytogenetics an attractive tool, which may greatly facilitate genome analysis and gene cloning in barley.

Cell Nucleus↗
PubMed2006

MK17, a specific marker closely linked to the gynoecium suppression region on the Y chromosome in Silene latifolia.

The aim of this work was to isolate new DNA markers linked to the Silene latifolia Y chromosome. To do this we created a chromosome-specific plasmid library after DOP-PCR amplification of laser-microdissected Y-chromosomes. The library screening led to the isolation of several clones yielding mostly to exclusive male specific hybridization signals. Subsequent PCR confirmed the Y-unique linkage for one of the sequences. This DNA sequence called MK17 has no homology to any known DNA sequence and it is not expressed. Based on PCR and Southern analyses, MK17 is present only in dioecious species of the Elisanthe section of the genus Silene (S. latifolia, S. dioica, and S. diclinis) and it is absent in related gynodioecious and hermaphroditic species. The mapping analysis using a panel of deletion mutants showed that MK17 is closely linked to the region controlling suppression of gynoecium development. Hence MK17 represents a valuable marker to isolate genes controlling the gynoecium development suppression on the Y chromosome of S. latifolia.

Base Sequence↗
PubMed2006

Nuclear DNA content variation among Central European Koeleria taxa.

BACKGROUND AND AIMS: Polyploidization plays an important role in the evolution of many plant genera, including Koeleria. The knowledge of ploidy, chromosome number and genome size may enable correct taxonomic treatment when other features are insufficient as in Koeleria. Therefore, these characteristics and their variability were determined for populations of six central European Koeleria taxa. METHODS: Chromosome number analysis was performed by squashing root meristems, and ploidy and 2C nuclear DNA content were estimated by flow cytometry. KEY RESULTS: Three diploids (K. glauca, K. macrantha var. macrantha and var. pseudoglauca), one tetraploid (K. macrantha var. majoriflora), one decaploid (K. pyramidata) and one dodecaploid (K. tristis) were found. The 2C nuclear DNA content of the diploids ranged from 4.85 to 5.20 pg. The 2C DNA contents of tetraploid, decaploid and dodecaploid taxa were 9.31 pg, 22.89 pg and 29.23 pg, respectively. The DNA content of polyploids within the K. macrantha aggregate (i.e. within K. macrantha and K. pyramidata) was smaller than the expected multiple of the diploid genome (K. macrantha var. macrantha). Geography-correlated variation of DNA content was found for some taxa. Czech populations of K. macrantha var. majoriflora had a 5.06% smaller genome than the Slovak ones. An isolated eastern Slovakian population of K. tristis revealed 8.04% less DNA than populations from central Slovakia. In central and north-west Bohemia, diploid and tetraploid cytotypes of K. macrantha were sympatric; east from this region diploid populations, and towards the west tetraploid populations were dominant. CONCLUSIONS: Remarkable intra-specific inter-population differences in nuclear DNA content were found between Bohemian and Pannonian populations of Koeleria macrantha var. majoriflora and between geographically isolated central and eastern Slovakian populations of K. tristis. These differences occur over a relatively small geographical scale.

Cell Nucleus↗
PubMed2006

PRINS on plant chromosomes.

The introduction of primed in situ labeling (PRINS) into plant cytogenetics provided a novel means for fast and highly specific visualization of DNA sequences on chromosomes and in interphase nuclei. Although the technique does not reach the sensitivity of fluorescence in situ hybridization that is needed for detection of single-copy targets, it is superior in its speed and simplicity. Thus, the main applications of PRINS include fluorescent labeling of repeated DNA sequences, such as ribosomal DNA and satellite repeats, which are used to discriminate individual chromosome types within karyotypes and to assess the purity of chromosome fractions separated by flow-sorting. Recently, an application of PRINS for the discrimination of sequence subfamilies of satellite repeats has been developed that takes advantage of the sensitivity of Taq polymerase to mismatches between 3'-end of the primer and the template sequences. This approach allows the distinguishing of sequences that differ in only a few nucleotides and has proved to be valuable for studies of satellite DNA evolution in plants.

Chromosomes, Plant↗
PubMed2006

The bacterial artificial chromosome (BAC) library of the narrow-leafed lupin (Lupinus angustifolius L.).

The narrow-leafed lupin possesses valuable traits for environment-friendly agriculture and for the production of unconventional agricultural products. Despite various genetic and environmental studies, the breeding of improved cultivars has been slow due to the limited knowledge of its genomic structure. Further advances in genomics require, among other things, the availability of a genomic DNA library with large inserts. We report here on the construction of the first DNA library cloned in a BAC (bacterial artificial chromosome) vector from diploid Lupinus angustifolius L. cv. Sonet. The high molecular weight DNA used for its preparation was isolated from interphase nuclei that were purified by flow cytometry. The library comprises 55,296 clones and is ordered in 144x384-well microtitre plates. With an average insert size of 100 kb, the library represents six haploid genome equivalents. Thanks to the purification of the nuclei by flow cytometry, contamination with chloroplast DNA and mitochondrial DNA was negligible. The availability of a BAC library opens avenues for the development of a physical contig map and positional gene cloning, as well as for the analysis of the plant's genome structure and evolution.

Chromosomes, Artificial, Bacterial↗
PubMed2005

Chromosome sorting in tetraploid wheat and its potential for genome analysis.

This study evaluates the potential of flow cytometry for chromosome sorting in durum wheat (Triticum turgidum Desf. var. durum, 2n = 4x = 28). Histograms of fluorescence intensity (flow karyotypes) obtained after the analysis of DAPI-stained chromosomes consisted of three peaks. Of these, one represented chromosome 3B, a small peak corresponded to chromosomes 1A and 6A, and a large peak represented the remaining 11 chromosomes. Chromosomes sorted onto microscope slides were identified after fluorescence in situ hybridization (FISH) with probes for GAA microsatellite, pSc119.2, and Afa repeats. Genomic distribution of these sequences was determined for the first time in durum wheat and a molecular karyotype has been developed for this crop. Flow karyotyping in double-ditelosomic lines of durum wheat revealed that the lines facilitated sorting of any arm of the wheat A- and B-genome chromosomes. Compared to hexaploid wheat, flow karyotype of durum wheat is less complex. This property results in better discrimination of telosomes and high purities in sorted fractions, ranging from 90 to 98%. We have demonstrated that large insert libraries can be created from DNA purified using flow cytometry. This study considerably expands the potential of flow cytogenetics for use in wheat genomics and opens the possibility of sequencing the genome of this important crop one chromosome arm at a time.

Cell Cycle↗
PubMed2005

Plant DNA flow cytometry and estimation of nuclear genome size.

BACKGROUND: DNA flow cytometry describes the use of flow cytometry for estimation of DNA quantity in cell nuclei. The method involves preparation of aqueous suspensions of intact nuclei whose DNA is stained using a DNA fluorochrome. The nuclei are classified according to their relative fluorescence intensity or DNA content. Because the sample preparation and analysis is convenient and rapid, DNA flow cytometry has become a popular method for ploidy screening, detection of mixoploidy and aneuploidy, cell cycle analysis, assessment of the degree of polysomaty, determination of reproductive pathway, and estimation of absolute DNA amount or genome size. While the former applications are relatively straightforward, estimation of absolute DNA amount requires special attention to possible errors in sample preparation and analysis. SCOPE: The article reviews current procedures for estimation of absolute DNA amounts in plants using flow cytometry, with special emphasis on preparation of nuclei suspensions, stoichiometric DNA staining and the use of DNA reference standards. In addition, methodological pitfalls encountered in estimation of intraspecific variation in genome size are discussed as well as problems linked to the use of DNA flow cytometry for fieldwork. CONCLUSIONS: Reliable estimation of absolute DNA amounts in plants using flow cytometry is not a trivial task. Although several well-proven protocols are available and some factors controlling the precision and reproducibility have been identified, several problems persist: (1) the need for fresh tissues complicates the transfer of samples from field to the laboratory and/or their storage; (2) the role of cytosolic compounds interfering with quantitative DNA staining is not well understood; and (3) the use of a set of internationally agreed DNA reference standards still remains an unrealized goal.

Cell Nucleus↗
PubMed2005

The origin, evolution and proposed stabilization of the terms 'genome size' and 'C-value' to describe nuclear DNA contents.

BACKGROUND: Perusing the literature on nuclear 'genome size' shows that the term is not stabilized, but applied with different meanings. It is used for the DNA content of the complete chromosome complement (with chromosome number n), for which others use 'C-value', but also for the DNA content of the monoploid chromosome set only (with chromosome number x). Reconsideration of the terminology is required. AIM: Our purpose is to discuss the currently unstable usage of the terms 'genome size' and 'C-value', and to propose a new unified terminology which can describe nuclear DNA contents with ease and without ambiguity. PROPOSALS: We argue that there is a need to maintain the term genome size in a broad sense as a covering term, because it is widely understood, short and phonetically pleasing. Proposals are made for a unified and consensual terminology. In this, 'genome size' should mean the DNA content based on chromosome number x and n, and should be used mainly in a general sense. The necessary distinction of the kinds of genome sizes is made by the adjectives 'monoploid' and the neology 'holoploid'. 'Holoploid genome size' is a shortcut for the DNA content of the whole chromosome complement characteristic for the individual (and by generalization for the population, species, etc.) irrespective of the degree of generative polyploidy, aneuploidies, etc. This term was lacking in the terminology and is for reasons of linguistic consistency indispensable. The abbreviated terms for monoploid and holoploid genome size are, respectively, Cx-value and C-value. Quantitative data on genome size should always indicate the C-level by a numerical prefix, such as 1C, 1Cx, 2C, etc. The proposed conventions cover general fundamental aspects relating to genome size in plants and animals, but do not treat in detail cytogenetic particularities (e.g. haploids, hybrids, etc.) which will need minor extensions of the present scheme in a future paper.

Cell Nucleus↗
PubMed2004

Construction of a subgenomic BAC library specific for chromosomes 1D, 4D and 6D of hexaploid wheat.

The analysis of the hexaploid wheat genome (Triticum aestivum L., 2 n=6 x=42) is hampered by its large size (16,974 Mb/1C) and presence of three homoeologous genomes (A, B and D). One of the possible strategies is a targeted approach based on subgenomic libraries of large DNA inserts. In this work, we purified by flow cytometry a total of 10(7) of three wheat D-genome chromosomes: 1D, 4D and 6D. Chromosomal DNA was partially digested with HindIII and used to prepare a specific bacterial artificial chromosome (BAC) library. The library (designated as TA-subD) consists of 87,168 clones, with an average insert size of 85 kb. Among these clones, 53% had inserts larger than 100 kb, only 29% of inserts being shorter than 75 kb. The coverage was estimated to be 3.4-fold, giving a 96.5% probability of identifying a clone corresponding to any sequence on the three chromosomes. Specificity for chromosomes 1D, 4D and 6D was confirmed after screening the library pools with single-locus microsatellite markers. The screening indicated that the library was not biased and gave an estimated coverage of sixfold. This is the second report on BAC library construction from flow-sorted plant chromosomes, which confirms that dissecting of the complex wheat genome and preparation of subgenomic BAC libraries is possible. Their availability should facilitate the analysis of wheat genome structure and evolution, development of cytogenetic maps, construction of local physical maps and map-based cloning of agronomically important genes.

Chromosome Mapping↗
PubMed2004

Flow cytogenetics and plant genome mapping.

The application of flow cytometry and sorting (flow cytogenetics) to plant chromosomes did not begin until the mid-1980s, having been delayed by difficulties in preparation of suspensions of intact chromosomes and discrimination of individual chromosome types. These problems have been overcome during the last ten years. So far, chromosome analysis and sorting has been reported in 17 species, including major legume and cereal crops. While chromosome classification by flow cytometry (flow karyotyping) may be used for quantitative detection of structural and numerical chromosome changes, chromosomes purified by flow sorting were found to be invaluable in a broad range of applications. These included physical mapping using PCR, high-resolution cytogenetic mapping using FISH and PRINS, production of recombinant DNA libraries, targeted isolation of markers, and protein analysis. A great potential is foreseen for the use of sorted chromosomes for construction of chromosome and chromosome-arm-specific BAC libraries, targeted isolation of low-copy (genic) sequences, high-throughput physical mapping of ESTs and other DNA sequences by hybridization to DNA arrays, and global characterization of chromosomal proteins using approaches of proteomics. This paper provides a comprehensive review of the methodology and application of flow cytogenetics, and assesses its potential for plant genome analysis.

Chromosomes, Plant↗
PubMed2004

Dissecting large and complex genomes: flow sorting and BAC cloning of individual chromosomes from bread wheat.

The analysis of the complex genome of common wheat (Triticum aestivum, 2n = 6x = 42, genome formula AABBDD) is hampered by its large size ( approximately 17 000 Mbp) and allohexaploid nature. In order to simplify its analysis, we developed a generic strategy for dissecting such large and complex genomes into individual chromosomes. Chromosome 3B was successfully sorted by flow cytometry and cloned into a bacterial artificial chromosome (BAC), using only 1.8 million chromosomes and an adapted protocol developed for this purpose. The BAC library (designated as TA-3B) consists of 67 968 clones with an average insert size of 103 kb. It represents 6.2 equivalents of chromosome 3B with 100% coverage and 90% specificity as confirmed by genetic markers. This method was validated using other chromosomes and its broad application and usefulness in facilitating wheat genome analysis were demonstrated by target characterization of the chromosome 3B structure through cytogenetic mapping. This report on the successful cloning of flow-sorted chromosomes into BACs marks the integration of flow cytogenetics and genomics and represents a great leap forward in genetics and genomic analysis.

Chromosome Mapping↗
PubMed2004

Creation of a BAC resource to study the structure and evolution of the banana (Musa balbisiana) genome.

The first bacterial artificial chromosome (BAC) library of the banana species Musa balbisiana 'Pisang Klutuk Wulung' (PKW BAC library) was constructed and characterized. One improved and one novel protocol for nuclei isolation were employed to overcome problems caused by high levels of polyphenols and polysaccharides present in leaf tissues. The use of flow cytometry to purify cell nuclei eliminated contamination with secondary metabolites and plastid DNA. Furthermore, the usefulness of the inducible pCC1BAC vector to obtain a higher amount of BAC DNA was demonstrated. The PKW BAC library represents nine haploid genome equivalents of M. balbisiana and its mean insert size is 135 kb. It consists of two sublibraries, of which the first one (SN sublibrary with 24,960 clones) was prepared according to an improved standard nuclei isolation protocol, whereas the second (FN sublibrary with 11,904 clones) was obtained from flow-sorted nuclei. Screening with 12 RFLP probes, which were genetically anchored to 8 genetic linkage groups of the banana species Musa acuminata, revealed an average of 11 BAC clones per probe, thus confirming the genome coverage estimated based on the insert size, as well as a high level of conservation between the two species of Musa. Localization of selected BAC clones to mitotic chromosomes using FISH indicated that the BAC library represented a useful resource for cytogenetic mapping. As the first step in map-based cloning of a genetic factor that is involved in the activation of integrated pararetroviral sequences of Banana streak virus (BSV), the BSV expressed locus (BEL) was physically delimited. The PKW BAC library represents a publicly available tool, and is currently used to reveal the integration and activation mechanisms of BSV sequences and to study banana genome structure and evolution.

Cell Nucleus↗
PubMed2003

Duplicative transfer of a MADS box gene to a plant Y chromosome.

Y chromosomes carry genes with functions in male reproduction and often have few other loci. Their evolution and the causes of genetic degeneration are of great interest. In addition to genetic degeneration, the acquisition of autosomal genes may be important in Y chromosome evolution. We here report that the dioecious plant Silene latifolia harbors a complete MADS box gene, SlAP3Y, duplicated onto the Y chromosome. This gene has no X-linked homologs but only an autosomal paralog, SlAP3A, and sequence divergence suggests that the duplication is a quite old event that occurred soon after the evolution of the sex chromosomes. Evolutionary sequence analyses using homologs of closely related species, including hermaphroditic Silene conica and dioecious Silene dioica and Silene diclinis, suggest that both SlAP3A and SlAP3Y genes encode functional proteins. Indeed, quantitative RT-PCR and in situ hybridization analyses showed that SlAP3A is expressed specifically in developing petals, but SlAP3Y is much more strongly expressed in developing stamens. The S. conica homolog, ScAP3A, is expressed in developing petals, suggesting subfunctionalization with evolution of male-specific functions, possibly due to evolutionary change in regulatory elements. Our results suggest that the acquisition of autosomal genes is an important event in the evolution of plant Y chromosomes.

Chromosomes, Plant↗
PubMed2002

Chromosome sorting and PCR-based physical mapping in pea (Pisum sativum L.).

Three pea lines with reconstructed karyotypes were used for analysis and subsequent purification of individual chromosome types using flow cytometry and sorting. The lines JI 145, JI 146, and JI 148 possess defined chromosomal translocations allowing discrimination of three to four chromosome types from each line based on the different sizes of translocation chromosomes. Whereas only two chromosomes could be sorted from standard (wild-type) karyotype, a combined use of these lines allowed sorting of six out of the seven types of pea chromosomes. Chromosomes were identified and purity of flow-sorted fractions was assessed using fluorescence in-situ hybridization with a PisTR-B probe that was previously shown to give labelling patterns characteristic for each chromosome type. The fractions of flow-sorted chromosomes were of very high purity (> 95%) and proved to be suitable for detection of gene and marker sequences using PCR with specific primers. Three fractions containing chromosomes 27, 72 and a pool of all remaining chromosomes (1, 3, 4, 5, 6) flow-sorted from the line JI 148 were then used for PCR-based physical localization of genetic markers selected from linkage groups IV and VII. These experiments enabled assignment of the linkage groups IV and VII to chromosomes 4 and 7, respectively.

Base Sequence↗