Comparative Karyotype Analysis of Lycoris aurea Herb Populations Using Fluorochrome Banding and 45S and 5S rDNA-FISH

Summary

This protocol investigates the heterochromatin distribution in eight populations of Lycoris aurea (L′Her) Herb, using combined PI and DAPI chromosome staining, fluorescence in situ hybridization (FISH) with 18S-5.8-26S rRNA gene regions (45S rDNA regions), and 5S ribosomal DNA probes.

Abstract

To understand karyotype variation in eight populations, detailed karyotypes were meticulously established using chromosomal measurements, fluorescence bands, and rDNA FISH signals. The number of 45S rDNA sites varies from one to five pairs per population, with the most common number per karyotype being four pairs. The 45S rDNA locus is predominantly located in the short arms and terminal regions of chromosomes, while the 5S rDNA locus is found mainly in the short arm and the terminal or proximal regions. Populations HBWF, HNXN, HBBD, and HNZX showed a similar distribution of 45S rDNA sites, as did GXTL, HBFC, and SCLS, indicating a close relationship between populations with similar 45S rDNA site distributions. The karyotypes of all studied populations are symmetrical, comprising stable and metastable centromeres or exclusively stable centromeres. Scatter plots of MCA and CVCL effectively distinguish their karyotypic structures. The analysis includes six quantitative parameters (x, 2n, TCL, MCA, CVCL, CVCI). Additionally, the results indicate that PCoA based on these six parameters is a robust method for determining biological karyotype relationships among the eight populations. The chromosome number in Lycoris populations is x = 6-8. Based on the current study and literature, genomic differentiation of these populations is discussed in terms of genome size, heterochromatin, 45S and 5S rDNA sites, and karyotype asymmetry.

Introduction

Lycoris is a family that includes several important horticultural plants¹. Lycoris aurea (L'Hér.) Herb. is a traditional Chinese medicinal plant with a long history. Phytochemical analysis has shown that Lycoris aurea (L′Her.) Herb contains galantamine and other alkaloids, which can enhance acetylcholine sensitivity and have the potential to treat Alzheimer's disease as cholinesterase inhibitors².

In higher plants, karyotype analysis is crucial for integrating genetic and physical maps, with significant practical implications ....

Protocol

The reagents and equipment utilized in this study are detailed in the Table of Materials, while the probes employed are provided in Supplementary File 1.

1. Preparation of apical chromosomes

1. Take root by hydroponics. After removing the above-ground part of the bulb, place it in a tapered bottle filled with water, replacing the water every 3 days, following the method described by Song et al.³⁵.
2. Exc.......

Discussion

The preparation of Lycoris aurea (L'Hér.) Herb root chromosomes involves several critical steps: (1) cultivating roots via hydroponics, (2) treating root tips with saturated α-bromonaphthalene, (3) fixing roots using an alcohol-acetic acid solution, (4) performing enzymatic hydrolysis on root tips with an enzyme solution, and (5) thoroughly squashing the digested roots and drying the slides over an alcohol lamp flame.

Accurate chromosome measurement is essential for kary.......

Materials

Name	Company	Catalog Number	Comments
Alcohol	Sangon Biotech (Shanghai) Co., Ltd.	A500737-0005	(70%, 90%, 100%)
1× TNT	Sangon Biotech (Shanghai) Co., Ltd.	B548108	1×, 10×
2× SSC	Sangon Biotech (Shanghai) Co., Ltd.	B548109	2×, 20×
45S rDNA	Sangon Biotech (Shanghai) Co., Ltd.		TAMRA is added to both ends (5 'and 3' ends)
4'6-diamidino-2-phenylindole(DAPI)	Sangon Biotech (Shanghai) Co., Ltd.	E607303	20ml
5S rDNA	Sangon Biotech (Shanghai) Co., Ltd.		5 'end plus 6-FAM(FITC)
Adobe Photoshop software	Adobe Systems Incorporated	CS6
Alpha bromo-naphthalene	Sangon Biotech (Shanghai) Co., Ltd.	A602718	Saturation
Anti-burnout agent	Sangon Biotech (Shanghai) Co., Ltd.	Vectashield H-1000	10 mL
Biochemical incubator	Shanghai Yiheng Scientific Instrument Co., LTD	LRH-70
Cellulase	Sangon Biotech (Shanghai) Co., Ltd.	A426068	10 g
Citric acid	Sangon Biotech (Shanghai) Co., Ltd.	A501702	10 g
Deionized formamide (FAD)	Sangon Biotech (Shanghai) Co., Ltd.	A600211-0500	0.7
Dextran sulfate	Sangon Biotech (Shanghai) Co., Ltd.	A428229	10 mL
Fluorescence in situ hybridization instrument	USA/Abbott ThermoBrite	S500-24
Fluorescence microscope	Olympus China Co.ltd	BX60
Glacial acetic acid	Sangon Biotech (Shanghai) Co., Ltd.	A501931	500 mL
HCl	Aladdin Reagent Co. Ltd. (Shanghai)	H399657	500 mL
Ice machine	Dan Ding Shanghai International Trade Co., Ltd.	ST-70
Leica biological microscope	Germany Leica Instrument Co., LTD	DM6000B
Methyl alcohol	Sangon Biotech (Shanghai) Co., Ltd.	A601617	500 mL
MetMorph software	Molecular Devices	Version 7.35
Oven	Thermo Scientific™ Heratherm™	THM#51028152
Pectinase	Sangon Biotech (Shanghai) Co., Ltd.	A004297	10 g
Pepsin	Sangon Biotech (Shanghai) Co., Ltd.	1.07185	100 g
Propidium iodide(PI)	Sangon Biotech (Shanghai) Co., Ltd.	A425259	1 g
RNaseA	Sangon Biotech (Shanghai) Co., Ltd.	R4642	10 mg
Salmon sperm DNA(ssDNA)	Aladdin Reagent Co. Ltd. (Shanghai)	D119871	1 g
Sodium citrate	Aladdin Reagent Co. Ltd. (Shanghai)	S189183	10 g
Statistica for Windows 10.0			for statistical analysis