Sergio Lucretti

Plant Genome Size Estimation by Flow Cytometry: Inter-laboratory Comparison

Flow cytometry is a convenient and rapid method that has been used extensively for estimation of nuclear genome size in plants. In contrast to general expectations, results obtained in different laboratories showed some striking discrepancies. The aim of this joint experiment was to test the reliability and reproducibility of methods. Care was taken to avoid a bias due to the quantity of DNA in the nucleus, the procedure for nuclei isolation or the type of instrument. Nuclear DNA content was estimated in nine plant species representing a typical range of genome size (2C = approx. 03-30 pg DNA). Each of the four laboratories involved in this study used a different buffer and/or procedure for nuclei isolation. Two laboratories used arc lamp-based instruments while the other two used laser-based instruments. The results obtained after nuclei staining with propidium iodide (a DNA intercalator) agreed well with those obtained using Feulgen densitometry. On the other hand, results obtained after staining with DAPI (binding preferentially to AT-rich regions) did not agree with those obtained using Feulgen densitometry. Small, but statistically significant, differences were found between data obtained with individual instruments. Differences between the same type of instruments were negligible, while larger differences were observed between lamp- and laserbased instruments. Ratios of fluorescence intensity obtained by laser instruments were higher than those obtained by lamp-based cytometers or by Feulgen densitometry. The results obtained in this study demonstrate that flow cytometry with DNA intercalators is a reliable method for estimation of nuclear genome size in plants. However, the study confirmed an urgent need for an agreement on standards. Given the small but systematic differences between different types of flow cytometers, analysis of very small differences in genome size should be made in the same laboratory and using the same instrument.

A high-yield procedure for isolation of metaphase chromosomes from root tips of Vicia faba L.

A new method is described for the isolation of large quantities of Vicia faba metaphase chromosomes. Roots were treated with 2.5 mM hydroxyurea for 18 h to accumulate meristem tip cells at the G1/S interface. After release from the block, the cells re-entered the cell cycle with a high degree of synchrony. A treatment with 2.5 μM amiprophos-methyl (APM) was used to accumulate mitotic cells in metaphase. The highest metaphase index (53.9%) was achieved when, 6 h after the release from the hydroxyurea block, the roots were exposed to APM for 4 h. The chromosomes were released from formaldehyde-fixed root tips by chopping with a scalpel in LB01 lysis buffer. Both the quality and the quantity of isolated chromosomes, examined microscopically and by flow cytometry, depended on the extent of the fixation. The best results were achieved after fixation with 6% formaldehyde for 30 min. Under these conditions, 1 · 106 chromosomes were routinely obtained from 30 root tips. The chromosomes were morphologically intact and suitable both for high-resolution chromosome studies and for flow-cytometric analysis and sorting. After the addition of hexylene glycol, the chromosome suspensions could be stored at 4° C for six months without any signs of deterioration.

Plant Chromosome Analysis and Sorting by Flow Cytometry

Abstract During the past decade, significant progress has been made in the development of methods for the preparation of plant chromosome suspensions suitable for flow cytometric analysis. In addition to successful classification of chromosomes (flow karyotyping), sorting of single chromosome types with a high degree of purity was reported in several plant species. Sorted chromosomes were used for the establishment of chromosome-specific DNA libraries and for gene mapping. The results confirmed the potential of plant flow cytogenetics and form a solid basis for further progress in this area. This article reviews its current status, analyzes major problems, and assesses future directions.

Development and Characterization of Microsatellite Markers from Chromosome 1-Specific DNA Libraries of Vicia Faba

An integrated approach has been developed for targeted retrieval of microsatellite markers from selected regions of the field bean (Vicia faba L.) genome. The procedure is based on a combination of advanced physical and genetic mapping techniques and includes the following steps: 1) flow-sorting of metaphase chromosomes, 2) construction of microsatellite-enriched chromosome-specific DNA libraries, 3) isolation of polymorphic microsatellite sequences from the libraries, 4) testing chromosome specificity of the microsatellites using polymerase chain reaction with purified fractions of individual chromosome types, and 5) integration of chromosome-specific markers into a genetic map. Several strategies for isolation of microsatellite clones were tested, including direct screening of non-enriched libraries with single or mixed probes and screening of the libraries after one or two rounds of enrichment. Finally, the usefulness of this approach was demonstrated by the retrieval of novel markers from a selected portion of the largest field been chromosome (No. 1).

Flow karyotyping and sorting of Vicia faba chromosomes

SummaryChromosome suspensions were prepared from formaldehyde-fixed, synchronized Vicia faba root tips. After staining with the DNA intercalating fluorochrome propidium iodide, the suspensions were analysed with a flow cytometer. The resulting histograms of integral fluorescence intensity contained peaks similar to those of theoretical V.faba flow karyotypes. From V. Faba cv ‘Inovec’ (2n = 12) only one peak, corresponding to a single chromosome type (metacentric chromosome), could be discriminated. However, it was found that the peak also contained doublets of acrocentric chromosomes. Bivariate analysis of fluorescence pulse area (chromosome DNA content) and fluorescence pulse width (chromosome length) was necessary to distinguish the metacentric chromosome. To achieve a high degree of purity, a two-step sorting protocol was adopted. During a working day, more than 25 000 metacentric chromosomes (corresponding to 0.2 μg DNA) were sorted with a purity of more than 90%. Such chromosomes are suitable for physical gene mapping by in situ hybridization or via the polymerase chain reaction (PCR) and allow the construction of chromosome-specific DNA libraries. While it was only possible to distinguish and sort one chromosome type from V. Faba cv ‘Inovec’ with the standard karyotype, it was possible to sort with a high degree of purity five out of six chromosome types of the line EFK of V. Faba, which has six pairs of morphologically distinct chromosomes. This result confirmed the possibility of using reconstructed karyotypes to overcome existing problems with the discrimination and flow sorting of individual chromosome types in plants.

Cell cycle synchronization in plant root meristems

The analysis of structure and metabolism of a cell at a defined phase of cell cycle is often difficult because cell cycle progression in somatic tissues is asynchronous and only a fraction of cells are cycling. An elegant solution to obtain populations of cells enriched for single stage of the cell cycle is to impose the synchrony artificially. Different systems have been used to obtain synchronized populations of plant cells, including suspension-cultured cells, leaf mesophyll protoplasts and root tip meristems. Root tips have been frequently used in a variety of studies ranging from chromosome analysis to cell cycle and its regulation. Seedlings with actively growing roots may be obtained in most plant species, they are easy to handle, the experimental system is well defined, reproducible and can be easily modified for different species. This paper describes a protocol for cell cycle synchronization in root tips of Vicia faba, which is based on the use of DNA synthesis inhibitor hydroxyurea [18]. Modifications of the protocol for Pisum sativum, Medicago sativa, Hordeum vulgare, Secale cereale, Triticum aestivum, and Zea mays are also given. Flow cytometric data indicate that about 90% of root tip cells are synchronized. On average, mitotic indices exceeding 50% are obtained with the method. Synchronized cells may be accumulated at metaphase using a mitotic spindle inhibitor to achieve metaphase indices exceeding 50%.

Inheritance of parental genomes in progenies of Poa pratensis L. from sexual and apomictic genotypes as assessed by RAPD markers and flow cytometry

Abstract Moving gene(s) responsible for the apomictic trait into crop plants that naturally reproduce through a sexual process would open up new areas in plant breeding and agricultural systems. Kentucky bluegrass (Poa pratensis L.) is one of the most important forage and turf grasses in temperate climates. It reproduces through facultative aposporous parthenogenesis, but the reproductive behaviour ranges naturally from nearly obligate apomixis to complete sexuality. In addition to apomictic reproduction, sexual hybridization may take place. Selfing may also occur, and occasionally reduced egg cells may develop through parthenogenesis generating (poly)haploids. The inheritance of parental genomes was assessed in Kentucky bluegrass progenies by employing RAPD markers in combination with flow cytometry (FCM). Nine progenies from different crosses carried out between completely sexual and highly apomictic genotypes were evaluated in order to probe the reproductive behaviour of the mother plants and to distinguish the different classes of aberrant plants. Not only were maternals and balanced BII hybrids recorded, but so were (poly)triploid BIII hybrids, selfs, and (poly)haploids. The application of these techniques demonstrated that FCM analysis accurately distinguishes the n, 2n, and 3n ploidy levels of progenies, and that RAPD markers unequivocally recognize progenies of apomictic and hybrid origin. The occurrence of aneusomaty was documented in one of the selected sexual genotypes, whose crossed progeny plants manifested two distinct classes of ploidy. The nomenclature BI was adopted to refer to hybrids with a hypodiploid nuclear condition. On the whole, the FCM analysis confirmed most of the RAPD data. The combined evaluation of DNA markers and DNA contents proved to be an efficient screening tool for scoring maternal plants, assessing the genetic origin of aberrant plants, and quantifying the inheritance of parental genomes in Kentucky bluegrass. Hybrid populations from sexual×apomictic matings that segregate for the mode of reproduction represent a valuable basis for attempting to identify molecular markers linked to the apomixis gene(s).

Localization of seed protein genes on flow-sorted field bean chromosomes.

Chromosomes from reconstructed field bean (Vicia faba L.) karyotypes were flow-sorted and the DNA was used for the physical localization of seed storage and nonstorage (USP) protein genes using PCR with sequence specific primers. The data were confirmed and refined by using DNA of microisolated chromosomes of other karyotypes as the target for PCR. The specificity of the PCR products was proved by restrictase digestion into fragments of predicted length or by reamplification using ‘nested’ primers. The genes are located within defined regions of chromosome I (USP=unknown seedprotein genes), II (vicilin genes, legumin B3 genes), III (legumin B4 genes), IV (pseudogenes ψ1) and V (legumin A genes and pseudogenes ψ1). Except for the pseudogene derived from the sequence of legumin B4 gene, all members of each gene family are located in one chromosome region exclusively. This approach proved to be useful for localizing genes that cannot be mapped genetically (due to the lack of allelic variants) and might be applied to integrate physical and genetic maps.

Preparation of pea (Pisum sativum L.) chromosome and nucleus suspensions from single root tips.

A high-yield method for the isolation of intact nuclei and chromosomes in suspension from a variable number of pea root tips (1–10) has been developed. This procedure is based on a two-step cell-cycle synchronization of root-tip meristems to obtain a high mitotic index, followed by formaldehyde fixation and mechanical isolation of chromosomes and nuclei by homogenization. In the explant, up to 50% of metaphases were induced through a synchronization of the cell cycle at the G1/S interface with hydroxyurea (1.25 mM), followed, after a 3-h release, by a block in metaphase with amiprophos-methyl (10 μM). The quality and quantity of nuclei and chromosomes were related to the extent of the fixation. Best results were obtained after a 30-min fixation with 2% and 4% formaldehyde for nuclei and chromosomes, respectively. The method described here allowed the isolation of nuclei and chromosomes, even from a single root tip, with a yield of 1×105/root and 1.4×105/root, respectively. Isolated suspensions were suitable for flow cytometric analysis and sorting and PRINS labelling with a rDNA probe.

FISHIS: fluorescence in situ hybridization in suspension and chromosome flow sorting made easy.

The large size and complex polyploid nature of many genomes has often hampered genomics development, as is the case for several plants of high agronomic value. Isolating single chromosomes or chromosome arms via flow sorting offers a clue to resolve such complexity by focusing sequencing to a discrete and self-consistent part of the whole genome. The occurrence of sufficient differences in the size and or base-pair composition of the individual chromosomes, which is uncommon in plants, is critical for the success of flow sorting. We overcome this limitation by developing a robust method for labeling isolated chromosomes, named Fluorescent In situ Hybridization In suspension (FISHIS). FISHIS employs fluorescently labeled synthetic repetitive DNA probes, which are hybridized, in a wash-less procedure, to chromosomes in suspension following DNA alkaline denaturation. All typical A, B and D genomes of wheat, as well as individual chromosomes from pasta (T. durum L.) and bread (T. aestivum L.) wheat, were flow-sorted, after FISHIS, at high purity. For the first time in eukaryotes, each individual chromosome of a diploid organism, Dasypyrum villosum (L.) Candargy, was flow-sorted regardless of its size or base-pair related content. FISHIS-based chromosome sorting is a powerful and innovative flow cytogenetic tool which can develop new genomic resources from each plant species, where microsatellite DNA probes are available and high quality chromosome suspensions could be produced. The joining of FISHIS labeling and flow sorting with the Next Generation Sequencing methodology will enforce genomics for more species, and by this mightier chromosome approach it will be possible to increase our knowledge about structure, evolution and function of plant genome to be used for crop improvement. It is also anticipated that this technique could contribute to analyze and sort animal chromosomes with peculiar cytogenetic abnormalities, such as copy number variations or cytogenetic aberrations.