Flow cytometric analysis and sorting of plant mitotic chromosomes has been mastered by only a few laboratories worldwide. Yet, it has been contributing significantly to progress in plant genetics, including the production of genome assemblies and the cloning of important genes. The dissection of complex genomes by flow sorting into the individual chromosomes that represent small parts of the genome reduces DNA sample complexity and streamlines projects relying on molecular and genomic techniques. Whereas flow cytometric analysis, that is, chromosome classification according to fluorescence and light scatter properties, is an integral part of any chromosome sorting project, it has rarely been used on its own due to lower resolution and sensitivity as compared to other cytogenetic methods. To perform chromosome analysis and sorting, commercially available electrostatic droplet sorters are suitable. However, in order to resolve and purify chromosomes of interest the instrument must offer high resolution of optical signals as well as stability during long runs. The challenge is thus not the instrumentation, but the adequate sample preparation. The sample must be a suspension of intact mitotic metaphase chromosomes and the protocol, which includes the induction of cell cycle synchrony, accumulation of dividing cells at metaphase, and release of undamaged chromosomes, is time consuming and laborious and needs to be performed very carefully. Moreover, in addition to fluorescent staining chromosomal DNA, the protocol may include specific labelling of DNA repeats to facilitate discrimination of particular chromosomes. This review introduces the applications of chromosome sorting in plants, and discusses in detail sample preparation, chromosome analysis and sorting to achieve the highest purity in flow-sorted fractions, and their suitability for downstream applications.

Institute of Experimental Botany of the Czech Academy of Sciences Centre of the Region Haná for Biotechnological and Agricultural Research Olomouc Czech Republic

Italian National Agency for New Technologies Energy and Sustainable Economic Development Division of Biotechnology and Agroindustry Rome Italy

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Bartholdi MF. Flow cytogenetics. Pathobiology. 1990;58:118–28. PubMed

Boschman GA, Manders EM, Rens W, Slater R, Aten JA. Semi‐automated detection of aberrant chromosomes in bivariate flow karyotypes. Cytometry. 1992;13:469–77. PubMed

Kubaláková M, Vrána J, Číhalíková J, Šimková H, Doležel J. Flow karyotyping and chromosome sorting in bread wheat (Triticum aestivum L.). Theor Appl Genet. 2002;104:1362–72. PubMed

Cremer T, Lichter P, Borden J, Ward DC, Manuelidis L. Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosome‐specific library probes. Hum Genet. 1988;80:235–46. PubMed

Pinkel D, Landegent J, Collins C, Fuscoe J, Segraves R, Lucas J, et al. Fluorescence in situ hybridization with human chromosome‐specific libraries: detection of trisomy 21 and translocations of chromosome 4. Proc Natl Acad Sci U S A. 1988;85:9138–42. PubMed PMC

Lebo RV. Chromosome sorting and DNA sequence localization. Cytometry. 1982;3:145–54. PubMed

Van Dilla MA, Deaven LL. Construction of gene libraries for each human chromosome. Cytometry. 1990;11:208–18. PubMed

Požárková D, Koblížková A, Román B, Torres AM, Lucretti S, Lysák M, et al. Development and characterization of microsatellite markers from chromosome 1‐specific DNA libraries of Vicia faba . Biol Plant. 2002;45:337–45.

Wenzl P, Suchánková P, Carling J, Šimková H, Huttner E, Kubaláková M, et al. Isolated chromosomes as a new and efficient source of DArT markers for the saturation of genetic maps. Theor Appl Genet. 2010;121:465–74. PubMed

Tiwari VK, Wang S, Sehgal S, Vrána J, Friebe B, Kubaláková M, et al. SNP discovery for mapping alien introgressions in wheat. BMC Genomics. 2014;15:273. PubMed PMC

Wang H, Dai K, Xiao J, Yuan C, Zhao R, Doležel J, et al. Development of intron targeting (IT) markers specific for chromosome arm 4VS of Haynaldia villosa by chromosome sorting and next‐generation sequencing. BMC Genomics. 2017;18:167. PubMed PMC

Macas J, Gualberti G, Nouzová M, Samec P, Lucretti S, Doležel J. Construction of chromosome‐specific DNA libraries covering the whole genome of field bean (Vicia faba L.). Chromosome Res. 1996;4:531–9. PubMed

Šafář J, Šimková H, Kubaláková M, Číhalíková J, Suchánková P, Bartoš J, et al. Development of chromosome‐specific BAC resources for genomics of bread wheat. Cytogenet Genome Res. 2010;129:211–23. PubMed

Sánchez‐Martín J, Steuernagel B, Ghosh S, Herren G, Hurni S, Adamski N, et al. Rapid gene isolation in barley and wheat by mutant chromosome sequencing. Genome Biol. 2016;17:221. PubMed PMC

Thind AK, Wicker T, Šimková H, Fossati D, Moullet O, Brabant C, et al. Rapid cloning of genes in hexaploid wheat using cultivar‐specific long‐range chromosome assembly. Nat Biotechnol. 2017;35:793–6. PubMed

Martis MM, Zhou R, Haseneyer G, Schmutzer T, Vrána J, Kubaláková M, et al. Reticulate evolution of the rye genome. Plant Cell. 2013;25:3685–98. PubMed PMC

Mayer KFX, Martis M, Hedley PE, Šimková H, Liu H, Morris JA, et al. Unlocking the barley genome by chromosomal and comparative genomics. Plant Cell. 2011;23:1249–63. PubMed PMC

International Wheat Genome Sequencing Consortium (IWGSC) . A chromosome‐based draft sequence of the hexaploid bread wheat (Triticum aestivum) genome. Science. 2014;345:1251788. PubMed

Kreplak J, Madoui MA, Cápal P, Novák P, Labadie K, Aubert G, et al. A reference genome for pea provides insight into legume genome evolution. Nat Genet. 2019;51:1411–22. PubMed

Ruperao P, Chan CKK, Azam S, Karafiátová M, Hayashi S, Čížková J, et al. A chromosomal genomics approach to assess and validate the desi and kabuli draft chickpea genome assemblies. Plant Biotechnol J. 2014;12:778–86. PubMed

de Laat AMM, Blaas J. Flow‐cytometric characterization and sorting of plant chromosomes. Theor Appl Genet. 1984;67:463–7. PubMed

Arumuganathan K, Slattery JP, Tanksley SD, Earle ED. Preparation and flow cytometric analysis of metaphase chromosomes of tomato. Theor Appl Genet. 1991;82:101–11. PubMed

Wang ML, Leitch AR, Schwarzacher T, Heslop‐Harrison JS, Moore G. Construction of a chromosome‐enriched Hpall library from flow‐sorted wheat chromosomes. Nucleic Acids Res. 1992;20:1897–901. PubMed PMC

Eeckhaut T, Lakshmanan P, Deryckere D, Bockstaele E, Huylenbroeck J. Progress in plant protoplast research. Planta. 2013;238:991–1003. PubMed

Schwarzacher T, Wang ML, Leitch AR, Moore G, Heslop‐Harrison JS, Miller N. Flow cytometric analysis of the chromosomes and stability of a wheat cell‐culture line. Theor Appl Genet. 1997;94:91–7. PubMed

Conia J, Bergounioux C, Perennes C, Muller P, Brown S, Gadal P. Flow cytometric analysis and sorting of plant chromosomes from Petunia hybrida protoplasts. Cytometry. 1987;8:500–8. PubMed

Doležel J, Číhalíková J, Weiserová J, Lucretti S. Cell cycle synchronization in plant root meristems. Methods Cell Sci. 1999;21:95–107. PubMed

Lucretti S, Nardi L, Nisini PT, Moretti F, Gualberti G, Doležel J. Bivariate flow cytometry DNA/BrdUrd analysis of plant cell cycle. Methods Cell Sci. 1999;21:155–66. PubMed

Doležel J, Číhalíková J, Lucretti S. A high‐yield procedure for isolation of metaphase chromosomes from root tips of Vicia faba L. Planta. 1992;188:93–8. PubMed

Veuskens J, Marie D, Brown SC, Jacobs M, Negrutiu I. Flow sorting of the Y sex chromosome in the dioecious plant Melandrium album . Cytometry. 1995;21:363–73. PubMed

Neumann P, Lysák M, Doležel J, Macas J. Isolation of chromosomes from Pisum sativum L. hairy root cultures and their analysis by flow cytometry. Plant Sci. 1998;137:205–15.

Metcalfe CJ, Li J, Giorgi D, Doležel J, Piperidis N, Aitken KS. Flow cytometric characterisation of the complex polyploid genome of Saccharum officinarum and modern sugarcane cultivars. Sci Rep. 2019;9:19362. PubMed PMC

Vrána J, Cápal P, Šimková H, Karafiátová M, Čížková J, Doležel J. Flow analysis and sorting of plant chromosomes. Curr Protoc Cytom. 2016;78:5.3.1–5.3.43. PubMed

Doležel J, Lucretti S, Schubert I. Plant chromosome analysis and sorting by flow cytometry. Crit Rev Plant Sci. 1994;13:275–309.

Doležel J, Vrána J, Šafář J, Bartoš J, Kubaláková M, Šimková H. Chromosomes in the flow to simplify genome analysis. Funct Integr Genomics. 2012;12:397–416. PubMed PMC

Zwyrtková J, Šimková H, Doležel J. Chromosome genomics uncovers plant genome organization and function, Biotechnol Adv. 2021;46:107659. PubMed

Hoagland DR, Arnon DI. The water‐culture method for growing plants without soil. Circular (California agricultural experiment station). Volume 347. Berkeley: University of California, College of Agriculture, Agricultural Experiment Station; 1938.

Koç A, Wheeler LJ, Mathews CK, Merrill GF. Hydroxyurea arrests DNA replication by a mechanism that preserves basal dNTP pools. J Biol Chem. 2004;279:223–30. PubMed

Molnár I, Kubaláková M, Šimková H, Cseh A, Molnár‐Láng M, Doležel J. Chromosome isolation by flow sorting in Aegilops umbellulata and Ae. comosa and their allotetraploid hybrids Ae. biuncialis and Ae. geniculata . PLoS One. 2011;6:e27708. PubMed PMC

Molnár I, Vrána J, Farkas A, Kubaláková M, Cseh A, Molnár‐Láng M, et al. Flow sorting of C‐genome chromosomes from wild relatives of wheat Aegilops markgrafii, Ae. triuncialis and Ae. cylindrica, and their molecular organization. Ann Bot. 2015;116:189–200. PubMed PMC

Molnár I, Kubaláková M, Šimková H, Farkas A, Cseh A, Megyeri M, et al. Flow cytometric chromosome sorting from diploid progenitors of bread wheat, T. urartu, Ae. speltoides and Ae. tauschii . Theor Appl Genet. 2014;127:1091–104. PubMed

Said M, Kubaláková M, Karafiátová M, Molnár I, Doležel J, Vrána J. Dissecting the complex genome of crested wheatgrass by chromosome flow sorting. Plant Genome. 2019;12:180096. PubMed

Moreno R, Castro P, Vrána J, Kubaláková M, Cápal P, García V, et al. Integration of genetic and cytogenetic maps and identification of sex chromosome in garden asparagus (Asparagus officinalis L.). Front Plant Sci. 2018;9:1068. PubMed PMC

Zatloukalová P, Hřibová E, Kubaláková M, Suchánková P, Šimková H, Adoración C, et al. Integration of genetic and physical maps of the chickpea (Cicer arietinum L.) genome using flow‐sorted chromosomes. Chrom Res. 2011;19:729–39. PubMed

Grosso V, Farina A, Gennaro A, Giorgi D, Lucretti S. Flow sorting and molecular cytogenetic identification of individual chromosomes of Dasypyrum villosum L. (H. villosa) by a single DNA probe. PLoS One. 2012;7:e50151. PubMed PMC

Kopecký D, Martis M, Číhalíková J, Hřibová E, Vrána J, Bartoš J, et al. Flow sorting and sequencing meadow fescue chromosome 4F. Plant Physiol. 2013;163:1323–37. PubMed PMC

Lysák MA, Číhalíková J, Kubaláková M, Šimková H, Künzel G, Doležel J. Flow karyotyping and sorting of mitotic chromosomes of barley (Hordeum vulgare L.). Chrom Res. 1999;7:431–44. PubMed

Gualberti G, Doležel J, Macas J, Lucretti S. Preparation of pea (Pisum sativum L.) chromosome and nucleus suspensions from single root tips. Theor Appl Genet. 1996;92:744–51. PubMed

Kubaláková M, Valárik M, Bartoš J, Vrána J, Číhalíková J, Molnár‐Láng M, et al. Analysis and sorting of rye (Secale cereale L.) chromosomes using flow cytometry. Genome. 2003;46:893–905. PubMed

Giorgi D, Farina A, Grosso V, Lucretti S. A new and “open access” chromosome approach to complex genomes: flow sorting of FISH labeled chromosome in suspension. Options Méd. 2014;110:605–11.

Abrouk M, Balcárková B, Šimková H, Komínková E, Martis MM, Jakobson I, et al. The in silico identification and characterization of a bread wheat/Triticum militinae introgression line. Plant Biotechnol J. 2017;15:249–56. PubMed PMC

Akpinar BA, Yuce M, Lucas S, Vrána J, Burešová V, Doležel J, et al. Molecular organization and comparative analysis of chromosome 5B of the wild wheat ancestor Triticum dicoccoides . Sci Rep. 2015;5:10763. PubMed PMC

Kubaláková M, Kovářová P, Suchánková P, Číhalíková J, Bartoš J, Lucretti S, et al. Chromosome sorting in tetraploid wheat and its potential for genome analysis. Genetics. 2005;170:823–9. PubMed PMC

Kovářová P, Navrátilová A, Macas J, Doležel J. Chromosome analysis and sorting in Vicia sativa using flow cytometry. Biol Plant. 2007;51:43–8.

Vláčilová K, Ohri D, Vrána J, Číhalíková J, Kubaláková M, Kahl G, et al. Development of flow cytogenetics and physical genome mapping in chickpea (Cicer arietinum L.). Chromosome Res. 2002;10:695–706. PubMed

Lee JH, Arumuganathan K, Kaeppler SM, Papa CM, Kaeppler HF. Cell synchronization and isolation of metaphase chromosomes from maize (Zea mays L.) root tips for flow cytometric analysis and sorting. Genome. 1996;39:697–703. PubMed

Kato A. Air drying method using nitrous oxide for chromosome counting in maize. Biotech Histochem. 1999;74:160–6. PubMed

Doležel J, Binarová P, Lucretti S. Analysis of nuclear DNA content in plant cells by flow cytometry. Biol Plant. 1989;31:113–20.

Šimková H, Číhalíková J, Vrána J, Lysák MA, Doležel J. Preparation of HMW DNA from plant nuclei and chromosomes isolated from root tips. Biol Plant. 2003;46:369–73.

Petrovská B, Jeřábková H, Chamrád I, Vrána J, Lenobel R, Uřinovská J, et al. Proteomic analysis of barley cell nuclei purified by flow sorting. Cytogenet Genome Res. 2014;143:78–86. PubMed

Giorgi D, Farina A, Grosso V, Gennaro A, Ceoloni C, Lucretti SFISHIS. Fluorescence in situ hybridization in suspension and chromosome flow sorting made easy. PLoS One. 2013;8:e57994. PubMed PMC

Doležel J, Lucretti S. High‐resolution flow karyotyping and chromosome sorting in Vicia faba lines with standard and reconstructed karyotypes. Theor Appl Genet. 1995;90:797–802. PubMed

Lucretti S, Doležel J, Schubert I, Fuchs J. Flow karyotyping and sorting of Vicia faba chromosomes. Theor Appl Genet. 1993;85:665–72. PubMed

Ng BL, Fu B, Graham J, Hall C, Thompson S. Chromosome analysis using benchtop flow analysers and high speed cell sorters. Cytometry. 2019;95:323–31. PubMed PMC

Barcellona ML, Cardiel G, Gratton E. Time‐resolved fluorescence of DAPI in solution and bound to polydeoxynucleotides. Biochem Biophys Res Commun. 1990;170:270–80. PubMed

Wang Y, Schellenberg H, Walhorn V, Toensing K, Anselmetti D. Binding mechanism of fluorescent dyes to DNA characterized by magnetic tweezers. Mater Today: Proceedings. 2017;4:S218–S225. PubMed

Fantes JA, Green DK, Sharkey A. Chromosome sorting by flow cytometry. In: Gosden JR, editor. Chromosome analysis protocols. methods in molecular biology. Volume 29. Totowa, NJ: Humana Press; 1994. PubMed

Lucretti S, Doležel J. Bivariate flow karyotyping in broad bean (Vicia faba). Cytometry. 1997;28:236–42. PubMed

Lee JH, Arumuganathan K, Chung YS, Kim KY, Chung WB, Bae KS, et al. Flow cytometric analysis and chromosome sorting of barley (Hordeum vulgare L). Mol Cells. 2000;10:619–25. PubMed

Macas J, Doležel J, Gualberti G, Pich U, Schubert I, Lucretti S. Primer‐induced labelling of pea and field bean chromosomes in situ and in suspension. BioTechnigues. 1995;19:402–8. PubMed

Pich U, Meister A, Macas J, Doležel J, Lucretti S, Schubert I. Primed in situ labelling facilitates flow sorting of similar sized chromosomes. Plant J. 1995;7:1039–44.

Ma Y, Lee JH, Li LC, Uchiyama S, Ohmido N, Fukui K. Fluorescent labeling of plant chromosomes in suspension by FISH. Genes Genet Syst. 2005;80:35–9. PubMed

Molnár I, Vrána J, Burešová V, Cápal P, Farkas A, Darkó É, et al. Dissecting the U, M, S and C genomes of wild relatives of bread wheat (Aegilops spp.) into chromosomes and exploring their synteny with wheat. Plant J. 2016;88:452–67. PubMed

Lucretti S, Giorgi D, Farina A, Grosso V. FISHIS: a new way in chromosome flow sorting makes complex genomes more accesible. In: Tuberosa R, Graner A, Frison E, editors. Genomics of plant genetic resources. Volume 1. Cham, Switzerland: Springer; 2014. p. 319–48.

Tsõmbalova J, Karafiátová M, Vrána J, Kubaláková M, Peuša H, Jakobson I, et al. A haplotype specific to north European wheat (Triticum aestivum L.). Genet Resour Crop Evol. 2017;64:653–64.

Cápal P, Endo TR, Vrána J, Kubaláková M, Karafiátová M, Komínková E, et al. The utility of flow sorting to identify chromosomes carrying a single copy transgene in wheat. Plant Methods. 2016;12:24. PubMed PMC

Gerlach WL, Bedbrook JR. Cloning and characterization of ribosomal RNA genes from wheat and barley. Nucleic Acids Res. 1979;7:1869–85. PubMed PMC

Conia J, Muller P, Brown S, Bergounioux C, Gadal . Monoparametric models of flow cytometric karyotypes with spreadsheet software. Theor Appl Genet. 1989;77:295–303. PubMed

Doležel J. KARYOSTAR: microcomputer program for modelling of monoparametric flow karyotypes. Biológia. 1991;46:1059–64.

Meister AK. A WIN software for modelling flow karyotypes of dual labelled chromosomes. 2014; unpublished (armin.meister@t-online.de)

Vrána J, Kubaláková M, Šimková H, Číhalíková J, Lysák MA, Doležel J. Flow‐sorting of mitotic chromosomes in common wheat (Triticum aestivum L.). Genetics. 2000;156:2033–41. PubMed PMC

Doležel J, Kubaláková M, Bartoš J, Macas J. Flow cytogenetics and plant genome mapping. Chrom Res. 2004;12:77–91. PubMed

Beseda T, Mascher M, Petrovská B, Himmelbach A, Šimková H, Šebela M, Stein N, Doležel J. Molecular organization of mitotic metaphase chromosomes in higher plants. Paper presented at: Abstracts of the International Conference “Plant and Animal Genome XXVI”. W753. Scherago International, Inc., San Diego, 2018. https://pag.confex.com/pag/xxvi/meetingapp.cgi/Paper/27520

Petrovská B, Šebela M, Doležel J. Discovering the world of plant nuclear proteins. In: Rose RJ, editor. Molecular cell biology of the growth and differentiation of plant cells. Boca Raton, FL: CRC Press; 2016. p. 22–36.

Valárik M, Bartoš J, Kovářová P, Kubaláková M, de Jong H, Doležel J. High‐resolution FISH on super‐stretched flow‐sorted plant chromosomes. Plant J. 2004;37:940–50. PubMed

Endo TR, Kubaláková M, Vrána J, Doležel J. Hyperexpansion of wheat chromosomes sorted by flow cytometry. Genes Genet Syst. 2014;89:181–5. PubMed

Binarová P, Hause B, Doležel J, Dráber P. Association of g‐tubulin with kinetochore/centromeric region of plant chromosomes. Plant J. 1998;14:751–7.

Ten Hoopen R, Manteuffel R, Doležel J, Malysheva L, Schubert I. Evolutionary conservation of kinetochore protein sequences in plants. Chromosoma. 2000;109:482–9. PubMed

Xing L, Hu P, Liu J, Witek K, Zhou S, Xu J, et al. Pm21 from Haynaldia villosa encodes a CC‐NBS‐LRR protein conferring powdery mildew resistance in wheat. Mol Plant. 2018;11:874–8. PubMed

Dracatos PM, Bartoš J, Elmansour H, Singh D, Karafiátová M, Zhang P, et al. The coiled‐coil NLR Rph1, confers leaf rust resistance in barley cultivar Sudan. Plant Physiol. 2019;179:1362–72. PubMed PMC

Hiebert CW, Moscou MJ, Hewitt T, Steuernagel B, Hernández‐Pinzón I, Green P, et al. Stem rust resistance in wheat is suppressed by a subunit of the mediator complex. Nat Commun. 2020;11:1123. PubMed PMC

Orman‐Ligeza B, Borrill P, Chia T, Chirico M, Doležel J, Drea S, et al. LYS3 encodes a prolamin‐box‐binding transcription factor that controls embryo growth in barley and wheat. J Cereal Sci. 2020;93:102965. PubMed PMC

Muñoz‐Amatriaín M, Moscou MJ, Bhat PR, Svensson JT, Bartoš J, Suchánková P, et al. An improved consensus linkage map of barley based on flow‐sorted chromosomes and single nucleotide polymorphism markers. Plant Genome. 2011;4:238–49.

Neumann P, Požárková D, Vrána J, Doležel J, Macas J. Chromosome sorting and PCR‐based physical mapping in pea (Pisum sativum L.). Chrom Res. 2002;10:63–71. PubMed

Macas J, Doležel J, Lucretti S, Pich U, Meister A, Fuchs J, et al. Localization of seed protein genes on flow‐sorted field bean chromosomes. Chrom Res. 1993;1:107–15. PubMed

Cápal P, Blavet N, Vrána J, Kubaláková M, Doležel J. Multiple displacement amplification of the DNA from single flow‐sorted plant chromosome. Plant J. 2015;84:838–44. PubMed

Šimková H, Svensson JT, Condamine P, Hřibová E, Suchánková P, Bhat PR, et al. Coupling amplified DNA from flow‐sorted chromosomes to high‐density SNP mapping in barley. BMC Genomics. 2008;9:294. PubMed PMC

Lieberman‐Aiden E, van Berkum NL, Williams L, Imakaev M, Ragoczy T, Telling A, et al. Comprehensive mapping of long‐range interactions reveals folding principles of the human genome. Science. 2009;326:289–93. PubMed PMC

Flow Cytometric Analysis and Sorting of Plant Chromosomes

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