Best practices in plant cytometry Flow cytometry (FCM) and flow cytometric sorting (FCS) systems ... more Best practices in plant cytometry Flow cytometry (FCM) and flow cytometric sorting (FCS) systems have developed as experimental tools of remarkable power and are enjoying an ever-increasing impact in the general field of biology. 1 Application of these tools to plant biology has developed more slowly given that the natural form of plants infrequently resembles that of the single cell suspension, prototypically the hematopoietic system that drove the original development of FCM/FCS. Nevertheless, these systems have had a profound influence at all levels of plant biology, from the study of single cells and subcellular organelles, to the behavior of populations of plants, and ultimately to the performance of ecosystems. It is safe to say their impact has not plateaued, as further applications of this unique technology are increasingly developed by innovative scientists around the world to address questions both in the basic sciences, and to increasingly confront emerging problems in the applied sector. For example, in addressing the challenges of sustainable production of sufficient food resources based on plant breeding involving ploidy-based approaches (e.g., induction of polyploidy) 2 for the needs of our future global citizens, FCM, and FCS systems will play central roles in this effort. The degree to which FCM and FCS systems have impacted plant biology and applied agricultural sciences must not be understated. The major applications of DNA FCM are ploidy level and genome size estimations, and cell cycle analysis/endoreplication (with the later included in a lower percentage of studies). Indeed, FCM is currently/ extensively and almost exclusively employed as the method of choice for measurement of plant genome sizes. 3,4 Measurements of this type impact agriculture in terms of ploidy estimation, with applications ranging from plant biotechnology, breeding and seed quality testing to taxonomy and population biology. They also impact the fundamental plant sciences in terms of biosystematics, ecology, evolution, genomics, and conservation, among other applications. One of the most startling observations of the angiosperms is the bandwidth occupied by genome size, which spans almost 2400-fold. Flow sorting of higher plant chromosomes has provided invaluable information regarding the organization of DNA sequences within plant species. It has also greatly facilitated the process of wholegenome sequencing by permitting subdivision of large genomes into samples comprising entire chromosomes or chromosome arms. 5 FCS methods applied to wall-less cells (protoplasts) expressing fluorescent proteins (FPs) in a cell type-specific manner have allowed elucidation of patterns of co-regulated gene expression and plant hormone gradients identification 6,7 within organized tissues, such as roots. 8,9
Before the recent advances in molecular biology and sequencing techniques enabled a deep knowledg... more Before the recent advances in molecular biology and sequencing techniques enabled a deep knowledge of the complete nucleotide sequence of many plant genomes, cytological and karyological studies have provided the first information concerning the genome organization of living beings. As stated by Figueroa and Bass (2010), plant cytogeneticists were among the earliest researchers who started to visualize genomes nearly a century before the first plant genome was sequenced (The Arabidopsis genome initiative 2000). In spite of the high development of next-generation sequencing approach and of the dramatic increase in available data regarding sequenced genomes, the cytological analysis of cell nucleus content remains a valuable tool for evolutionary studies and for structural and functional genomic research. Since Wilhelm Gottfried von Waldeyer-Hartz coined the term chromosome in 1888, many cytological techniques have been developed to disclose nucleus content and to allow a detailed des...
A bread wheat line (N11) and a disomic 2D(2R) substitution triticale line have been crossed and b... more A bread wheat line (N11) and a disomic 2D(2R) substitution triticale line have been crossed and backrossed four times. At each step electrophoretic selection for the seeds that possessed, simultaneously, the complete set of high molecular weight glutenin subunits of N11 and the two high molecular weight secalins of rye, present in the 2D(2R) line, was carried out. Molecular cytogenetic analyses of the BC4F5 generation have revealed that the selection carried out had produced a disomic addition line (2n=44). The pair of additional chromosomes consisted of the long arm of chromosome 1R (1RL) from rye fused with the satellite body of the wheat chromosome 6B. Rheological analyses revealed that the dough obtained by the new addition line had higher quality characteristics when compared with the two parents. The role of the two additional high molecular weight secalins, present in the disomic addition line, in influencing improved dough characteristics is discussed.
Traditionally globe artichoke and leafy cardoon have been cultivated for use as vegetables but th... more 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.
The phylogenetic relationships among theAegilops species belonging to the Sitopsis section were i... more The phylogenetic relationships among theAegilops species belonging to the Sitopsis section were investigated using RFLP (restriction-fragment-length polymorphism) analysis. Twenty-five probes, each of which hybridised to oneor more restriction fragments located in the B-genomechromosomes of cultivated wheats, were used. At least one and in most cases two fragments were located in every B genome chromosome arm. Adendrogram derived from a cluster analysis of the complete RFLP dataset showed a subdivision of the species belonging to the Sitopsis section into one group comprising the species of the Truncata subsection and another group comprised of the species of theEmarginata subsection. Dendrograms also were produced using RFLP data from loci located in different combinations of only three chromosomes, and some of these showed different subdivisions of the species. This demonstrates the importance in obtaining reliable classification data of using probes that detect loci evenly distri...
A number of leading crop species are either polyploid and/or hold a large genome as a result of t... more A number of leading crop species are either polyploid and/or hold a large genome as a result of the accumulation of highly repetitive sequences. These features hamper the assembly of complex genomes in spite of the fast development of new sequencing technologies (Next Generation Sequencing) and the availability of powerful bio-informatic tools. The chromosome approach, by enabling genome dissection into single chromosomes or chromosome arms via flow sorting, can contribute to reduce genome complexity. In all plants, this approach is restricted to species, or special cytogenetic stocks, as in the case of wheat, containing chromosome types that differ in size from the standard complement. We have developed an overall robust and easy method called FISHIS (Fluorescence In Situ Hybridization in Suspension) which overcomes the constrain of chromosome size and DNA content differences, allowing chromosome flow sorting on the base of fluorescent hybridization labeling of chromosomes in suspe...
Eukaryotic chromosomes can be studied either fixed on slides by use of cytogenetic techniques or ... more Eukaryotic chromosomes can be studied either fixed on slides by use of cytogenetic techniques or through flow-cytometry. The latter enables not only the chromosome characterization but allows for the sorting and manipulation of individual chromosomes out of a complete genome, provided that target chromosomes have flow-cytometric distinctive features (e.g. size and/or DNA base content). These requirements represent a major constraint when the chromosomes of a given species are similarly sized, a common feature in plants. In wheat, this constraint can be overcome using special aneuploid mutants, each containing single arm pairs for a given chromosome of the complement, thus keeping a balanced genome composition while showing a complement with chromosome parts which are half-sized in respect to standard autosomes. However, such genotypes are available in hexaploid wheat (Triticum aestivum) exclusively in the background of Chinese Spring, a non-elite reference variety. Notably, aneuploids are not available for the most part of plants and animals. In order to overcome this major hurdle we have developed a reliable, fast and cost-effective method for Fluorescence labeling and In situ Hybridization of chromosomes In Suspension (FISHIS). The method makes use of fluorescent oligonucleotides of simple repetitive DNA sequences (SSR) or short DNA fragments, as probes, and allows for specific chromosome flow-sorting based on the hybridization pattern determined by the FISHIS probe. We have successfully applied the FISHIS methodology for flow karyotyping and sorting highly pure, single-type chromosome fractions of commercial varieties of bread and durum wheat and other Triticeae species. Moreover, the complete chromosomal sets corresponding to the two genomes (A and B) of durum wheat have also been clearly separated by the same FISHIS approach. Given the ubiquitous occurrence in eukaryotic genomes of the type of sequences used as probes, the abundance of their variants and their overall chromosome-specific distribution, their use in FISHIS experiments provides simple and fast access to individual chromosomes of virtually any eukaryotic genome, paving the way for gaining knowledge of great potential impact on basic and applied research aspects.
ABSTRACT The “chromosome approach”, or isolating chromosomes via flow sorting according their dif... more ABSTRACT The “chromosome approach”, or isolating chromosomes via flow sorting according their different sizes, resolves genome complexity by focusing sequencing to a small and meaningful part of the whole genome. Plant chromosomes are not so variable in dimensions to permit a direct flow-sorting of single type ones, and this approach is rather limited to plants where cytogenetic mutants by chromosome size are available. A new reliable, fast and cost effective method for Fluorescence In Situ Hybridization In Suspension (FISHIS) of plant chromosomes has been developed that overcomes this limitation allowing chromosome sorting based on total DNA content (size) and a FISH specific chromosome labeling pattern. FISHIS relies on alkaline DNA denaturation and on chromosome hybridization with readily available synthetic, fluorescent labeled, simple sequence repeats (SSR) as probe. By using SSR probes with a known and distinctive labeling patterns, FISHIS lets flow sorting homeologus genomes and several chromosomes from commercial varieties of bread and durum wheat and other Triticeae.
ABSTRACT Chromosomes and nuclei are mostly studied by FISH (Fluorescent In Situ Hybridization) an... more ABSTRACT Chromosomes and nuclei are mostly studied by FISH (Fluorescent In Situ Hybridization) and microscope observation on slide preparations. They can be analyzed and effectively separated according their DNA content by flow sorting, and this possibility has been used to develop the chromosome approach, the main way to get isolated single chromosomes and nuclei for genomics. An innovative strategy for precisely labeling individual chromosomes in suspension would improve the resolution power of the chromosome approach allowing a new way of characterizing, and therefore flow-sorting, an all new set of chromosomes. A new method called FISHIS, or Fluorescent In Situ Hybridization in Suspension, has been developed which combines the high-throughput and preparative capabilities of flow cytometry and the specificity of FISH labeling required to sort pure samples of chromosomes and nuclei which are similar to each others by DNA content. The standard FISH protocol relies on the heat denaturation of the probe and target DNAs and their annealing under sufficiently stringent conditions to allow a specific and reproducible hybridization. If treated this way, chromosomes in suspension are inclined to dissolve and/or clump together when exposed to the harsh heat denaturation settings required for FISH. FISHIS is an easy method that removes any washing and heat denaturation standard steps, replaced by an alkaline DNA denaturation. Syntetized fluorescent labeled DNA simple sequence repeats (e.g. SSR) are used as one of the standard probes, further simplifying the process for FISHIS labeling. In less than two hours, FISHIS can effectively label chromosomes in suspension allowing to discriminate, purify and flow sort whole homeologus genomes and a number of chromosomes belonging to several Triticeae species (e.g. T. aestivum, T. turgidum, T. monococcum, T. urartu, D. villosum, and Triticale)
The large size and complex polyploid nature of many genomes has often hampered genomics developme... more 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 flowsorted 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 Perennial wheatgrass species of the genus Thinopyrum possess several appealing attribute... more ABSTRACT Perennial wheatgrass species of the genus Thinopyrum possess several appealing attributes for wheat improvement, contributing to tolerance to biotic and abiotic stresses, as well as to quality and even to yield increase. Major genes or QTLs underlying such traits have been identified on numerous chromosomes of both diploid (Th. elongatum and Th. bessarabicum) and polyploid (mainly Th. intermedium and Th. ponticum) representatives of the genus, having different genome origin (E, J, St/S) and involving several homoeologous groups. Thinopyrum chromosomes sharing homoeology with wheat group 7 chromosomes turned to be particularly rich in beneficial genes; among them, a Th. ponticum group 7 chromosome referred to as 7Ag or 7el has been extensively targeted in various successful attempts of harnessing its attractive gene content. A survey of the several wheat translocation/recombinant lines involving this chromosome in the background of both bread and durum wheat is given. Such lines are described as highly valuable tools for a variety of studies, from development of integrated genetic and physical maps, to the analysis of structural and functional characteristics associated with defined alien chromosome subregions. The validity of Th. ponticum group 7 transfers as breeding materials (notable genes and traits including Lr19, Sr25, Fusarium head blight resistance, yellow pigment content, and even yield) is also highlighted. Finally, examples are given of pyramiding of group 7 Thinopyrum genes through ‘precision’ breeding strategies of chromosome engineering, which, efficiently aided by current genetic, cytogenetic and genomic (or, collectively, ‘cytogenomic’) technologies, enable a multifaceted and sustainable improvement of the wheat crop based on the use of the wealth of natural genetic resources of its related gene pools.
Flow karyotyping is the classification of mitotic metaphase chromosomes by flow cytometry accordi... more Flow karyotyping is the classification of mitotic metaphase chromosomes by flow cytometry according to DNA content and AT:GC ratio. The analysis is performed at high speed and chromosomes that are resolved as single peaks on the resulting frequency distributions (flow ...
Sugarcane (Saccharum spp.) is a globally important crop for sugar and bioenergy production. Its h... more Sugarcane (Saccharum spp.) is a globally important crop for sugar and bioenergy production. Its highly polyploid, complex genome has hindered progress in understanding its molecular structure. Flow cytometric sorting and analysis has been used in other important crops with large genomes to dissect the genome into component chromosomes. Here we present for the first time a method to prepare suspensions of intact sugarcane chromosomes for flow cytometric analysis and sorting. Flow karyotypes were generated for two S. officinarum and three hybrid cultivars. Five main peaks were identified and each genotype had a distinct flow karyotype profile. The flow karyotypes of S. officinarum were sharper and with more discrete peaks than the hybrids, this difference is probably due to the double genome structure of the hybrids. Simple Sequence Repeat (SSR) markers were used to determine that at least one allelic copy of each of the 10 basic chromosomes could be found in each peak for every genot...
Best practices in plant cytometry Flow cytometry (FCM) and flow cytometric sorting (FCS) systems ... more Best practices in plant cytometry Flow cytometry (FCM) and flow cytometric sorting (FCS) systems have developed as experimental tools of remarkable power and are enjoying an ever-increasing impact in the general field of biology. 1 Application of these tools to plant biology has developed more slowly given that the natural form of plants infrequently resembles that of the single cell suspension, prototypically the hematopoietic system that drove the original development of FCM/FCS. Nevertheless, these systems have had a profound influence at all levels of plant biology, from the study of single cells and subcellular organelles, to the behavior of populations of plants, and ultimately to the performance of ecosystems. It is safe to say their impact has not plateaued, as further applications of this unique technology are increasingly developed by innovative scientists around the world to address questions both in the basic sciences, and to increasingly confront emerging problems in the applied sector. For example, in addressing the challenges of sustainable production of sufficient food resources based on plant breeding involving ploidy-based approaches (e.g., induction of polyploidy) 2 for the needs of our future global citizens, FCM, and FCS systems will play central roles in this effort. The degree to which FCM and FCS systems have impacted plant biology and applied agricultural sciences must not be understated. The major applications of DNA FCM are ploidy level and genome size estimations, and cell cycle analysis/endoreplication (with the later included in a lower percentage of studies). Indeed, FCM is currently/ extensively and almost exclusively employed as the method of choice for measurement of plant genome sizes. 3,4 Measurements of this type impact agriculture in terms of ploidy estimation, with applications ranging from plant biotechnology, breeding and seed quality testing to taxonomy and population biology. They also impact the fundamental plant sciences in terms of biosystematics, ecology, evolution, genomics, and conservation, among other applications. One of the most startling observations of the angiosperms is the bandwidth occupied by genome size, which spans almost 2400-fold. Flow sorting of higher plant chromosomes has provided invaluable information regarding the organization of DNA sequences within plant species. It has also greatly facilitated the process of wholegenome sequencing by permitting subdivision of large genomes into samples comprising entire chromosomes or chromosome arms. 5 FCS methods applied to wall-less cells (protoplasts) expressing fluorescent proteins (FPs) in a cell type-specific manner have allowed elucidation of patterns of co-regulated gene expression and plant hormone gradients identification 6,7 within organized tissues, such as roots. 8,9
Before the recent advances in molecular biology and sequencing techniques enabled a deep knowledg... more Before the recent advances in molecular biology and sequencing techniques enabled a deep knowledge of the complete nucleotide sequence of many plant genomes, cytological and karyological studies have provided the first information concerning the genome organization of living beings. As stated by Figueroa and Bass (2010), plant cytogeneticists were among the earliest researchers who started to visualize genomes nearly a century before the first plant genome was sequenced (The Arabidopsis genome initiative 2000). In spite of the high development of next-generation sequencing approach and of the dramatic increase in available data regarding sequenced genomes, the cytological analysis of cell nucleus content remains a valuable tool for evolutionary studies and for structural and functional genomic research. Since Wilhelm Gottfried von Waldeyer-Hartz coined the term chromosome in 1888, many cytological techniques have been developed to disclose nucleus content and to allow a detailed des...
A bread wheat line (N11) and a disomic 2D(2R) substitution triticale line have been crossed and b... more A bread wheat line (N11) and a disomic 2D(2R) substitution triticale line have been crossed and backrossed four times. At each step electrophoretic selection for the seeds that possessed, simultaneously, the complete set of high molecular weight glutenin subunits of N11 and the two high molecular weight secalins of rye, present in the 2D(2R) line, was carried out. Molecular cytogenetic analyses of the BC4F5 generation have revealed that the selection carried out had produced a disomic addition line (2n=44). The pair of additional chromosomes consisted of the long arm of chromosome 1R (1RL) from rye fused with the satellite body of the wheat chromosome 6B. Rheological analyses revealed that the dough obtained by the new addition line had higher quality characteristics when compared with the two parents. The role of the two additional high molecular weight secalins, present in the disomic addition line, in influencing improved dough characteristics is discussed.
Traditionally globe artichoke and leafy cardoon have been cultivated for use as vegetables but th... more 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.
The phylogenetic relationships among theAegilops species belonging to the Sitopsis section were i... more The phylogenetic relationships among theAegilops species belonging to the Sitopsis section were investigated using RFLP (restriction-fragment-length polymorphism) analysis. Twenty-five probes, each of which hybridised to oneor more restriction fragments located in the B-genomechromosomes of cultivated wheats, were used. At least one and in most cases two fragments were located in every B genome chromosome arm. Adendrogram derived from a cluster analysis of the complete RFLP dataset showed a subdivision of the species belonging to the Sitopsis section into one group comprising the species of the Truncata subsection and another group comprised of the species of theEmarginata subsection. Dendrograms also were produced using RFLP data from loci located in different combinations of only three chromosomes, and some of these showed different subdivisions of the species. This demonstrates the importance in obtaining reliable classification data of using probes that detect loci evenly distri...
A number of leading crop species are either polyploid and/or hold a large genome as a result of t... more A number of leading crop species are either polyploid and/or hold a large genome as a result of the accumulation of highly repetitive sequences. These features hamper the assembly of complex genomes in spite of the fast development of new sequencing technologies (Next Generation Sequencing) and the availability of powerful bio-informatic tools. The chromosome approach, by enabling genome dissection into single chromosomes or chromosome arms via flow sorting, can contribute to reduce genome complexity. In all plants, this approach is restricted to species, or special cytogenetic stocks, as in the case of wheat, containing chromosome types that differ in size from the standard complement. We have developed an overall robust and easy method called FISHIS (Fluorescence In Situ Hybridization in Suspension) which overcomes the constrain of chromosome size and DNA content differences, allowing chromosome flow sorting on the base of fluorescent hybridization labeling of chromosomes in suspe...
Eukaryotic chromosomes can be studied either fixed on slides by use of cytogenetic techniques or ... more Eukaryotic chromosomes can be studied either fixed on slides by use of cytogenetic techniques or through flow-cytometry. The latter enables not only the chromosome characterization but allows for the sorting and manipulation of individual chromosomes out of a complete genome, provided that target chromosomes have flow-cytometric distinctive features (e.g. size and/or DNA base content). These requirements represent a major constraint when the chromosomes of a given species are similarly sized, a common feature in plants. In wheat, this constraint can be overcome using special aneuploid mutants, each containing single arm pairs for a given chromosome of the complement, thus keeping a balanced genome composition while showing a complement with chromosome parts which are half-sized in respect to standard autosomes. However, such genotypes are available in hexaploid wheat (Triticum aestivum) exclusively in the background of Chinese Spring, a non-elite reference variety. Notably, aneuploids are not available for the most part of plants and animals. In order to overcome this major hurdle we have developed a reliable, fast and cost-effective method for Fluorescence labeling and In situ Hybridization of chromosomes In Suspension (FISHIS). The method makes use of fluorescent oligonucleotides of simple repetitive DNA sequences (SSR) or short DNA fragments, as probes, and allows for specific chromosome flow-sorting based on the hybridization pattern determined by the FISHIS probe. We have successfully applied the FISHIS methodology for flow karyotyping and sorting highly pure, single-type chromosome fractions of commercial varieties of bread and durum wheat and other Triticeae species. Moreover, the complete chromosomal sets corresponding to the two genomes (A and B) of durum wheat have also been clearly separated by the same FISHIS approach. Given the ubiquitous occurrence in eukaryotic genomes of the type of sequences used as probes, the abundance of their variants and their overall chromosome-specific distribution, their use in FISHIS experiments provides simple and fast access to individual chromosomes of virtually any eukaryotic genome, paving the way for gaining knowledge of great potential impact on basic and applied research aspects.
ABSTRACT The “chromosome approach”, or isolating chromosomes via flow sorting according their dif... more ABSTRACT The “chromosome approach”, or isolating chromosomes via flow sorting according their different sizes, resolves genome complexity by focusing sequencing to a small and meaningful part of the whole genome. Plant chromosomes are not so variable in dimensions to permit a direct flow-sorting of single type ones, and this approach is rather limited to plants where cytogenetic mutants by chromosome size are available. A new reliable, fast and cost effective method for Fluorescence In Situ Hybridization In Suspension (FISHIS) of plant chromosomes has been developed that overcomes this limitation allowing chromosome sorting based on total DNA content (size) and a FISH specific chromosome labeling pattern. FISHIS relies on alkaline DNA denaturation and on chromosome hybridization with readily available synthetic, fluorescent labeled, simple sequence repeats (SSR) as probe. By using SSR probes with a known and distinctive labeling patterns, FISHIS lets flow sorting homeologus genomes and several chromosomes from commercial varieties of bread and durum wheat and other Triticeae.
ABSTRACT Chromosomes and nuclei are mostly studied by FISH (Fluorescent In Situ Hybridization) an... more ABSTRACT Chromosomes and nuclei are mostly studied by FISH (Fluorescent In Situ Hybridization) and microscope observation on slide preparations. They can be analyzed and effectively separated according their DNA content by flow sorting, and this possibility has been used to develop the chromosome approach, the main way to get isolated single chromosomes and nuclei for genomics. An innovative strategy for precisely labeling individual chromosomes in suspension would improve the resolution power of the chromosome approach allowing a new way of characterizing, and therefore flow-sorting, an all new set of chromosomes. A new method called FISHIS, or Fluorescent In Situ Hybridization in Suspension, has been developed which combines the high-throughput and preparative capabilities of flow cytometry and the specificity of FISH labeling required to sort pure samples of chromosomes and nuclei which are similar to each others by DNA content. The standard FISH protocol relies on the heat denaturation of the probe and target DNAs and their annealing under sufficiently stringent conditions to allow a specific and reproducible hybridization. If treated this way, chromosomes in suspension are inclined to dissolve and/or clump together when exposed to the harsh heat denaturation settings required for FISH. FISHIS is an easy method that removes any washing and heat denaturation standard steps, replaced by an alkaline DNA denaturation. Syntetized fluorescent labeled DNA simple sequence repeats (e.g. SSR) are used as one of the standard probes, further simplifying the process for FISHIS labeling. In less than two hours, FISHIS can effectively label chromosomes in suspension allowing to discriminate, purify and flow sort whole homeologus genomes and a number of chromosomes belonging to several Triticeae species (e.g. T. aestivum, T. turgidum, T. monococcum, T. urartu, D. villosum, and Triticale)
The large size and complex polyploid nature of many genomes has often hampered genomics developme... more 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 flowsorted 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 Perennial wheatgrass species of the genus Thinopyrum possess several appealing attribute... more ABSTRACT Perennial wheatgrass species of the genus Thinopyrum possess several appealing attributes for wheat improvement, contributing to tolerance to biotic and abiotic stresses, as well as to quality and even to yield increase. Major genes or QTLs underlying such traits have been identified on numerous chromosomes of both diploid (Th. elongatum and Th. bessarabicum) and polyploid (mainly Th. intermedium and Th. ponticum) representatives of the genus, having different genome origin (E, J, St/S) and involving several homoeologous groups. Thinopyrum chromosomes sharing homoeology with wheat group 7 chromosomes turned to be particularly rich in beneficial genes; among them, a Th. ponticum group 7 chromosome referred to as 7Ag or 7el has been extensively targeted in various successful attempts of harnessing its attractive gene content. A survey of the several wheat translocation/recombinant lines involving this chromosome in the background of both bread and durum wheat is given. Such lines are described as highly valuable tools for a variety of studies, from development of integrated genetic and physical maps, to the analysis of structural and functional characteristics associated with defined alien chromosome subregions. The validity of Th. ponticum group 7 transfers as breeding materials (notable genes and traits including Lr19, Sr25, Fusarium head blight resistance, yellow pigment content, and even yield) is also highlighted. Finally, examples are given of pyramiding of group 7 Thinopyrum genes through ‘precision’ breeding strategies of chromosome engineering, which, efficiently aided by current genetic, cytogenetic and genomic (or, collectively, ‘cytogenomic’) technologies, enable a multifaceted and sustainable improvement of the wheat crop based on the use of the wealth of natural genetic resources of its related gene pools.
Flow karyotyping is the classification of mitotic metaphase chromosomes by flow cytometry accordi... more Flow karyotyping is the classification of mitotic metaphase chromosomes by flow cytometry according to DNA content and AT:GC ratio. The analysis is performed at high speed and chromosomes that are resolved as single peaks on the resulting frequency distributions (flow ...
Sugarcane (Saccharum spp.) is a globally important crop for sugar and bioenergy production. Its h... more Sugarcane (Saccharum spp.) is a globally important crop for sugar and bioenergy production. Its highly polyploid, complex genome has hindered progress in understanding its molecular structure. Flow cytometric sorting and analysis has been used in other important crops with large genomes to dissect the genome into component chromosomes. Here we present for the first time a method to prepare suspensions of intact sugarcane chromosomes for flow cytometric analysis and sorting. Flow karyotypes were generated for two S. officinarum and three hybrid cultivars. Five main peaks were identified and each genotype had a distinct flow karyotype profile. The flow karyotypes of S. officinarum were sharper and with more discrete peaks than the hybrids, this difference is probably due to the double genome structure of the hybrids. Simple Sequence Repeat (SSR) markers were used to determine that at least one allelic copy of each of the 10 basic chromosomes could be found in each peak for every genot...
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Papers by Debora Giorgi