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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.
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.
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 for plant biology. This analysis allows for chromosomal-level genome characterization, clarification of cellular taxonomic relationships between populations, identification of genetic aberrations, and understanding of chromosomal evolution trends3,4,5. Typically, a karyotype description includes chromosome number, absolute and relative chromosome lengths, primary and secondary constriction locations, heterochromatic fragment distribution, rDNA site numbers and locations, and other DNA sequence characteristics, as well as karyotype asymmetry6,7,8. Among karyotype parameters, asymmetry is determined by variations in chromosome length (interchromosomal asymmetry) and centromere position (intrachromosomal asymmetry). Karyotype asymmetry is a significant feature reflecting chromosome morphology and is widely used in plant cell taxonomy9,10,11,12.
Often, the absence of chromosome markers limits karyotype analysis and impedes the identification of individual chromosomes. In recent years, Giemsa staining, fluorescence banding, and fluorescence in situ hybridization (FISH) have become popular in plant chromosome analysis. Dual fluorescent staining methods, such as CMA (chromomycin A3) / DAPI (4, 6-diamino-2-phenylindole) staining and PI (propyl iodide) / DAPI staining (known as CPD staining), reveal GC-rich and AT-rich heterochromatic regions on chromosomes4,13. During metaphase or pachytene of plant mitosis, repeated DNA sequences or long DNA segments can generate specific signals in plant populations through FISH hybridization14,15,16.
Fluorescent bands and FISH signals provide useful markers for chromosome identification. By measuring chromosome length, analyzing fluorescence banding characteristics, and comparing FISH signal differences, detailed molecular cytogenetic karyotypes of different plant germplasms or populations can be constructed. These karyotypes effectively display chromosome morphology in various germplasms or populations, enabling comparisons of heterochromatin distribution and DNA sequence localization and encouraging further research4,8,17. Molecular cytogenetic karyotype comparisons can offer valuable insights into the phylogenetic relationships and chromosomal evolution of related populations8,14,18,19.
The Lycoris genus, a diverse and intriguing group within the Amaryllis family, is known for its perennial bulbs. Comprising approximately 20 species, 15 of which are endemic to China, it showcases rich biodiversity. This genus is found exclusively in warm temperate and subtropical regions of East Asia, including southwest China, southern Korea, and Japan, with a few populations extending to northern India and Nepal20. Early cytogenetic studies primarily involved chromosome counting to establish the genus's basic chromosome number and to describe chromosome morphology in specific populations, focusing largely on Lycoris radiata21. The total chromosome numbers observed in this genus range from 12 to 33 or 44, representing diploid, abnormal diploid, triploid, tetraploid, and aneuploid levels, respectively. The basic chromosome numbers, x, are 6, 7, 8, and 11.
Lycoris aurea (L'Hér.) Herb, a notable species within the Lycoris genus, is widely distributed throughout China22. It thrives in relaxed, moist environments and reproduces through small bulbs. These bulbs, containing lycorine and galantamine-two key medicinal compounds-have been used in traditional Chinese medicine for centuries. Lycoris aurea also captivates horticulturalists with its striking red flowers in the fall and evergreen leaves in the winter. However, the morphological similarity across all studied Lycoris aurea (L'Hér.) Herb populations present a significant challenge for chromosomal identification using conventional cytological methods.
Cloning of FISH, fosmid, or BAC (bacterial artificial chromosomes) with repetitive DNA sequences and oligonucleotide probes on metaphase or pachydermatous chromosomes has been used for karyotype analysis23,24,25, comparative cytogenetic analysis26,27, cytogenetic map construction28,29, and chromosome-specific assays30. Recently, the FISH technique has been applied to the chromosome analysis of Lycoris populations31. Although the chromosome number and karyotype vary in Lycoris populations, the total chromosome number remains constant, with three basic types observed: the central centromere chromosome (M-), the distal centromere chromosome (T-), and the proximal centromere chromosome (A-). Additionally, diverse chromosome shapes and sizes are observed in natural populations32.
RNA fluorescence in situ hybridization (FISH) is useful for detecting chromosomal changes, such as centromere fusion, inversion, gene amplification, and fragment deletion. Fluorescence In Situ Hybridization (RNA-FISH) technology enables high-resolution visual analysis of chromosomes to detect and identify multiple complex changes, including the fusion of chromosome central regions, formation of new chromosome structures, reversal of chromosomal regions, amplification of genes or gene fragments, and gene sequence loss on specific chromosome regions33. Five of the 500 clones showed strong FISH signals on the centromere region of the central centromere chromosome (M-) but not on the distal centromere chromosome (T-)34. The unique FISH signal distribution pattern on each chromosome enabled the identification of individual chromosomes, which had previously been challenging in Lycoris populations using traditional staining methods31. Currently, there are few studies on the molecular cytogenetics of Lycoris aurea (L'Hér.) Herb, emphasizing the urgent need for further research in this field.
In this study, eight well-differentiated metaphase chromosomes of Lycoris aurea (L'Hér.) Herb populations were prepared using enzyme immersion and flame drying (EMF) methods. CPD staining and 45S and 5S rDNA probes were used for FISH identification. Detailed molecular cytogenetic karyotypes of these populations were constructed using combined data from chromosomal measurements, fluorescent bands, and 45S and 5S rDNA FISH signals. Six different karyotypic asymmetry indices were calculated for each population to identify karyotypic relationships among them. Evaluation of the molecular cytogenetic karyotype data provided significant insights into the genomic differentiation and evolutionary relationships of the eight populations, potentially reshaping the understanding of Lycoris chromosomes.
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
2. CPD staining
3. Fluorescence in situ hybridization
4. Karyotype analysis
By employing the meticulous enzyme immersion and flame drying (EMF) method, scattered and well-differentiated metaphase chromosomes of Lycoris aurea (L'Hér.) Herb were obtained, and the cytogenetic chromosome karyotype of Lycoris aurea was constructed (Figure 1). The metaphase chromosomes with the highest degree of condensation are unsuitable for karyotype analysis due to the reduced morphological differences. However, since the total length of haploid chromosomes ...
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...
The authors have declared that no competing interests exist.
This work was supported by the Natural Science Foundation of China (32070367).
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 |
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