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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.

Traditionally globe artichoke and leafy cardoon have been cultivated for use as vegetables but these crops are now finding multiple new roles in applications ranging from paper production to cheese preparation and biofuel use, with interest in their functional food potential. So far, their chromosome complements have been poorly investigated and a well-defined karyotype was not available. In this paper, a detailed karyo-morphological analysis and molecular cytogenetic studies were conducted on globe artichoke (Cynara cardunculus Linnaeus, 1753 var. scolymus Fiori, 1904) and leafy cardoon (C. cardunculus Linneaus, 1753 var. altilis De Candolle, 1838). Fluorescent In Situ Hybridization In Suspension (FISHIS) was applied to nuclei suspensions as a fast method for screening of labelling probes, before metaphase spread hybridization. Classic Fluorescent In Situ Hybridization (FISH) on slide, using repetitive telomeric and ribosomal sequences and Simple Sequence Repeats (SSRs) oligonucleotide as probes, identified homologous chromosome relationships and allowed development of molecular karyotypes for both varieties. The close phylogenetic relationship between globe artichoke and cardoon was supported by the very similar karyotypes but clear chromosomal structural variation was detected. In the light of the recent release of the globe artichoke genome sequencing, these results are relevant for future anchoring of the pseudomolecule sequence assemblies to specific chromosomes. In addition, the DNA content of the two crops has been determined by flow cytometry and a fast method for standard FISH on slide and methodological improvements for nuclei isolation are described. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Flow cytometric estimation of nuclear DNA content was performed in six plant species employing three fluorochromes showing different DNA base preferences: propidium iodide (no base preference). 4',6-diamidino-2-phenylindole (DAPI; AT preference), and mithramycin (GC preference). Nuclei isolated from human leukocytes were used as a primary reference standard. While nuclear DNA contents estimated using propidium iodide were in agreement with published data obtained using other techniques, the values obtained using fluorochromes showing base preference were significantly different. It was found that the differences were caused by the differences in overall AT/GC ratios, and by the species-specific differences in binding of these fluorochromes to DNA. It was concluded that nuclear DNA content estimations performed with fluorochromes showing base preference should be interpreted with caution even when AT/GC ratios of the reference and the sample are equal. The use of intercalating, dyes (e.g. propidium iodide) is recommended for this purpose. On the other hand, comparison of the staining behaviour of intercalating dyes with that of dyes showing base preference may give additional information on chromatin structural differences and arrangement of molecule pairs in DNA. [ABSTRACT FROM AUTHOR]

Abstract: Phyllostictine A is a powerful toxin produced by Phyllosticta cirsii, a potential mycoherbicide of Cirsium arvense. To support its potential use as a natural herbicide, toxin production has been studied using different media and cultural conditions. The toxin content in the crude extracts has been determined by using a HPLC method set up for this purpose. Furthermore, its phytotoxicity has been evaluated on tobacco protoplasts by flow cytometric analysis, and on C. arvense protoplasts, by fluorescence microscopy. The best cultural conditions found allowed to produce more than 28mgml−1 of toxin in culture filtrate. The pure metabolite proved to have rapid dose-dependant toxic effects on host and no-host plant protoplasts. [Copyright &y& Elsevier]

One of the most important goals in the genetic improvement of lemon in the Mediterranean area is certainly the obtainment of new genotypes tolerant or resistant to mal secco disease. Two lemon cybrids (one diploid and one autotetraploid) with an intermediate degree of resistance to mal secco disease, spontaneously obtained by symmetric protoplast fusion between ‘Valencia’ orange and ‘Femminello’ lemon, were used as mother plants in backcrosses with a diploid clone of ‘Femminello’ lemon tolerant to the mal secco disease (LTMS) to improve tolerance and fruit quality. Since ‘Femminello’ lemon normally reproduces apomictically by nucellar embryony, an embryo-rescue technique was applied 105 days after pollination to recover zygotic embryos. Sixty-six plantlets were regenerated as follows: 20 from the 4nX2n cross and 46 from the 2nX2n cross. In order to distinguish zygotic embryos from nucellars, flow cytometry was applied on all the 66 plantlets; and a modified amplified fragment-length polymorphism (AFLP) technique and microsatellite analysis were carried out on a sample of 20 seedlings (six triploids and 14 diploids). Here, we report the usefulness and reliability of the tested techniques and discuss the results obtained. Flow cytometry (FCM) was useful only in inter-ploid crosses, identifying six triploids, four of which were also detected by simple sequence repeat (SSR) analysis and only two by modified AFLP. Among the 14 tested diploids, five zygotic genotypes were identified by SSR and four by modified AFLP. In our hands, the SSR technique appeared to be more suitable than modified AFLP. [Copyright &y& Elsevier]