Flow Cytometric Analysis and Sorting in Plant Genomics

doi:10.11983/CBB24107

Abstract

Abstract: Flow cytometry is a high-throughput technology that allows for the simultaneous and rapid detection of multiple physical and biological characteristics of individual particles. With the significant reduction in sequencing costs, flow cytometry is playing an increasingly prominent role in high-throughput sample acquisition for plant genomics. Taking rice and soybean as examples, this paper describes in detail the application of flow cytometry for fine sorting of plant cell nuclei and the subsequent ATAC-seq and RNA-seq experiments and analysis process, which provides a preferred tool for efficient mining of genes for agrobiological breeding. The key techniques and common problems in the experimental operation, such as the precautions for cell nuclei preparation, the balance between sorting purity and efficiency, and the debugging method for single-cell sorting, were also analyzed and suggested to provide references for the plant scientists in applying flow cytometry to carry out genomics research.

Key words: plants, flow cytometry, nuclei, sorting, ATAC-seq, RNA-seq

Chunjiao Xia, Yunguang Li, Shu Xia, Wei Pang, Chunli Chen. Flow Cytometric Analysis and Sorting in Plant Genomics[J]. Chinese Bulletin of Botany, 2024, 59(5): 774-782.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB24107

https://www.chinbullbotany.com/EN/Y2024/V59/I5/774

Figures/Tables 3

References 42

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Names	Components	Applications
Galbraith’s (Galbraith et al., 1983)	45 mmol∙L^-1 MgCl₂, 30 mmol∙L^-1 sodium citrate, 20 mmol∙L^-1 MOPS, 0.1% (v/v) TritonX-100, pH7.0	Arabidopsis thaliana, Glycine max
LB01 (Dpooležel et al., 1989)	15 mmol∙L^-1 Tris, 2 mmol∙L^-1 Na₂EDTA, 0.5 mmol∙L^-1 spermine, 80 mmol∙L^-1 KCl, 20 mmol∙L^-1 NaCl, 0.1% (v/v) TritonX-100, 15 mmol∙L^-1 β-mercaptoethanol, pH7.0-8.0	Oryza sativa, Chrysanthemum indicum, Solanum lycopersicum
Otto’s (Otto, 1992; Dolezel and Gohde, 1995)	OTTO I: 100 mmol∙L^-1 citric acid, 0.5% (v/v) Tween-20, pH2.0- 3.0; OTTO II: 400 mmol∙L^-1 Na₂HPO₄·12H₂O, pH8.0-9.0	Ranunculus japonicus, O. sativa
Tris-MgCl₂ (Pfosser et al., 1995)	200 mmol∙L^-1 Tris, 4 mmol∙L^-1 MgCl₂·6H₂O, 0.5% (v/v) TritonX- 100, pH7.5	Centaurea cyanus, Celtis au- stralis
GPB (Loureiro et al., 2007)	0.5 mmol∙L^-1spermine, 30 mmol∙L^-1 sodium citrate, 20 mmol∙L^-1 MOPS, 80 mmol∙L^-1 KCl, 20 mmol∙L^-1 NaCl, 0.5% (v/v) TritonX- 100, pH7.0	O. sativa, Actinidia chinensis
WPB (Loureiro et al., 2007)	0.2 mol∙L^-1 Tris-HCl, 4 mmol∙L^-1 MgCl₂∙6H₂O, 2 mmol∙L^-1 EDTA Na₂·2H₂O, 86 mmol∙L^-1 NaCl, 10 mmol∙L^-1 sodium pyrosulfite, 1% PVP-10, 1% (v/v) TritonX-100, pH7.5	Vitis vinifera, Quercus robur
PVPK12-mGB2 (Zhang and Feng, 2023)	30 mmol∙L^-1 sodium citrate, 45 mmol∙L^-1 MgCl₂, 20 mmol∙L^-1 MO- PS, 20 mmol∙L^-1 NaCl, 20 mmol∙L^-1 EDTA Na₂·2H₂O, 0.1% (v/v) TritonX-100, 0.5% (v/v) Tween-20, 10 μL·mL^-1 β-mercaptoethanol, 1%-2% PVPK12, pH7.0	Silicone fast-drying plant ma- terials

Names	Components	Applications
Galbraith’s (Galbraith et al., 1983)	45 mmol∙L^-1 MgCl₂, 30 mmol∙L^-1 sodium citrate, 20 mmol∙L^-1 MOPS, 0.1% (v/v) TritonX-100, pH7.0	Arabidopsis thaliana, Glycine max
LB01 (Dpooležel et al., 1989)	15 mmol∙L^-1 Tris, 2 mmol∙L^-1 Na₂EDTA, 0.5 mmol∙L^-1 spermine, 80 mmol∙L^-1 KCl, 20 mmol∙L^-1 NaCl, 0.1% (v/v) TritonX-100, 15 mmol∙L^-1 β-mercaptoethanol, pH7.0-8.0	Oryza sativa, Chrysanthemum indicum, Solanum lycopersicum
Otto’s (Otto, 1992; Dolezel and Gohde, 1995)	OTTO I: 100 mmol∙L^-1 citric acid, 0.5% (v/v) Tween-20, pH2.0- 3.0; OTTO II: 400 mmol∙L^-1 Na₂HPO₄·12H₂O, pH8.0-9.0	Ranunculus japonicus, O. sativa
Tris-MgCl₂ (Pfosser et al., 1995)	200 mmol∙L^-1 Tris, 4 mmol∙L^-1 MgCl₂·6H₂O, 0.5% (v/v) TritonX- 100, pH7.5	Centaurea cyanus, Celtis au- stralis
GPB (Loureiro et al., 2007)	0.5 mmol∙L^-1spermine, 30 mmol∙L^-1 sodium citrate, 20 mmol∙L^-1 MOPS, 80 mmol∙L^-1 KCl, 20 mmol∙L^-1 NaCl, 0.5% (v/v) TritonX- 100, pH7.0	O. sativa, Actinidia chinensis
WPB (Loureiro et al., 2007)	0.2 mol∙L^-1 Tris-HCl, 4 mmol∙L^-1 MgCl₂∙6H₂O, 2 mmol∙L^-1 EDTA Na₂·2H₂O, 86 mmol∙L^-1 NaCl, 10 mmol∙L^-1 sodium pyrosulfite, 1% PVP-10, 1% (v/v) TritonX-100, pH7.5	Vitis vinifera, Quercus robur
PVPK12-mGB2 (Zhang and Feng, 2023)	30 mmol∙L^-1 sodium citrate, 45 mmol∙L^-1 MgCl₂, 20 mmol∙L^-1 MO- PS, 20 mmol∙L^-1 NaCl, 20 mmol∙L^-1 EDTA Na₂·2H₂O, 0.1% (v/v) TritonX-100, 0.5% (v/v) Tween-20, 10 μL·mL^-1 β-mercaptoethanol, 1%-2% PVPK12, pH7.0	Silicone fast-drying plant ma- terials