Probe, method for efficiently recognizing glycyrrhiza chromosome and application of method
By developing specific oligonucleotide probes and FISH technology, we have achieved efficient and accurate identification of licorice chromosomes, solved a problem in interspecific chromosome research of the genus Glycyrrhiza, and provided a tool for a deeper understanding of speciation mechanisms.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHIHEZI UNIVERSITY
- Filing Date
- 2026-03-11
- Publication Date
- 2026-05-12
AI Technical Summary
The chromosomes of plants in the genus Glycyrrhiza are small in size and highly similar, and lack strong and reliable chromosome markers, making it difficult to conduct research on chromosome evolution among species.
A set of specific oligonucleotide probes for amplifying licorice chromosomes 1-8 was developed, and chromosomes were stained using oligonucleotide fluorescence in situ hybridization (FISH). Highly efficient and accurate identification was achieved by using artificially designed non-genomic universal primer sequences and fluorescently modified probes.
It has achieved efficient and accurate identification of licorice chromosomes, solved a problem in the study of chromosome evolution among different species of the genus Glycyrrhiza, and provided a tool for a deeper understanding of interspecific differences and mechanisms in speciation.
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Figure CN122012788A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of bioinformatics and molecular cytogenetics, specifically a probe, a method for efficiently identifying licorice chromosomes, and its applications. Background Technology
[0002] Licorice is a plant belonging to the genus *Glycyrrhiza* of the subfamily Papilionoideae in the family Leguminosae. It possesses medicinal properties such as clearing heat and detoxifying, and harmonizing various herbs. It is often referred to as "one of the ten herbs used in traditional Chinese medicine" and "the national treasure." However, the genetic diversity of the *Glycyrrhiza* genus is rich and varied, and to date, we know very little about the differences in chromosome evolution and evolutionary trajectories among different species during licorice speciation. Cytogenetics is an important approach to studying chromosome evolution in species, but licorice chromosomes are generally small and highly similar, lacking strong and reliable chromosomal markers. This severely hinders the development of research on chromosome evolution among different species within the *Glycyrrhiza* genus.
[0003] Fluorescence in situ hybridization (FISH) is a key technique in plant molecular cytogenetics. Based on the principle of complementary base pairing in nucleic acids, this technique first labels nucleic acid fragments with known sequences as probes, then hybridizes them in situ with target nucleic acid sequences in cells or tissues. Finally, the distribution of specific sequences on chromosomes is visually displayed under a microscope by fluorescence signal detection, thus achieving precise sequence localization on chromosomes. Although FISH technology has been widely used in plant chromosome research, accurate chromosome identification remains a significant challenge for species with high ploidy, large chromosome numbers, and small morphology. The emergence of oligonucleotide fluorescence in situ hybridization technology has solved these problems.
[0004] This invention utilizes oligonucleotide fluorescence in situ hybridization technology to develop a set of markers that can efficiently identify chromosomes 1-8 of licorice, which helps to gain a deeper understanding of the differences in chromosome origin and evolution and speciation mechanisms during the speciation process of licorice. Summary of the Invention
[0005] The purpose of this invention is to develop a set of staining probes that can efficiently and accurately identify licorice chromosomes and to identify licorice chromosomes 1-8.
[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a set of primer combinations for amplifying a pool of licorice chromosome-specific oligonucleotide probes, comprising the following eight pairs of primers: The primer pair GF1 / GR1 for amplifying licorice chromosome 1 has the nucleotide sequence shown in SEQ ID NO:1 / SEQ ID NO:2; The primer pair GF2 / GR2 was used to amplify chromosome 2 of licorice, and the nucleotide sequence is shown in SEQ ID NO:3 / SEQ ID NO:4; The primer pair GF3 / GR3 was used to amplify chromosome 3 of licorice, and the nucleotide sequence is shown in SEQ ID NO:5 / SEQ ID NO:6; The primer pair GF4 / GR4 was used to amplify chromosome 4 of licorice, and the nucleotide sequence is shown in SEQ ID NO:7 / SEQ ID NO:8; The primer pair GF5 / GR5 was used to amplify chromosome 5 of licorice, and the nucleotide sequence is shown in SEQ ID NO:9 / SEQ ID NO:10; The primer pair GF6 / GR6 was used to amplify chromosome 6 of licorice, and the nucleotide sequence is shown in SEQ ID NO:11 / SEQ ID NO:12; The primer pair GF7 / GR7 was used to amplify chromosome 7 of licorice, and the nucleotide sequence is shown in SEQ ID NO:13 / SEQ ID NO:14; The primer pair GF8 / GR8 was used to amplify chromosome 8 of licorice, and the nucleotide sequence is shown in SEQ ID NO:15 / SEQ ID NO:16; The primer sequences are artificially designed non-genomic universal sequences that do not specifically bind to the licorice genome and are used to specifically amplify the oligonucleotide probe pool of the corresponding chromosome.
[0007] Secondly, the present invention provides a method for preparing a licorice chromosome staining probe, characterized by comprising the following steps: (1) Constructing an oligonucleotide sequence pool: Remove repetitive sequences from the licorice genome and divide the remaining sequences into 45bp oligonucleotide sequences; Align the oligonucleotide sequences back to the genome, remove sequences with multiple matching sites, and construct a single-copy oligonucleotide sequence pool for licorice chromosomes 1-8. (2) Primer design and sequence synthesis: The primer combination described in the first aspect is selected and assigned to licorice chromosomes 1-8 respectively; the primer sequence corresponding to each chromosome is spliced to both ends of the 45bp oligonucleotide sequence of the chromosome to form a full-length oligonucleotide sequence with the structure "5'-primer-45bp sequence-primer-3'"; the primers are artificially designed non-genomic universal primer sequences that do not specifically bind to the licorice genome; (3) Probe amplification: Using the full-length oligonucleotide sequence pool constructed in step (2) as a template, PCR amplification is performed using the specific primers described in the first aspect with fluorescent modification to obtain chromosome staining probes with fluorescent modification.
[0008] Preferably, in step (1), the specific criterion for removing sequences with multiple matching sites is: removing sequences with homology > 75%.
[0009] Preferably, step (1) further includes using Primer to calculate the Tm and hairpin Tm of each oligonucleotide, and retaining oligonucleotide sequences with dTm>10℃.
[0010] Preferably, in step (3), probes modified with green fluorescence are amplified for chromosomes 1-4 of licorice, and probes modified with red fluorescence are amplified for chromosomes 5-8 of licorice.
[0011] Thirdly, the present invention provides a chromosome staining probe prepared by the method for preparing the licorice chromosome staining probe described in the second aspect.
[0012] Fourthly, the present invention provides a method for efficiently identifying licorice chromosomes, which employs multi-round fluorescence in situ hybridization technology and uses chromosome-smearing probes prepared by the method described in the second aspect to hybridize with licorice metaphase chromosomes.
[0013] Preferably, the hybridization step includes: preparing a hybridization solution comprising: 3 μL of 50% dextran sulfate, 7.5 μL of deionized formamide, 1.5 μL of 20×SSC and 400 ng of the oligonucleotide probe prepared according to claim 2; heat-denaturing a slide carrying metaphase chromosomes at 85°C for 5 minutes, followed by ethanol gradient dehydration, adding the hybridization solution, and hybridizing overnight in a 37°C incubator.
[0014] Fifthly, the present invention provides the application of the chromosome staining probe prepared by the method described in the second aspect, or the method described in the fourth aspect, in the analysis of licorice chromosome karyotype, the study of chromosome evolution among species of the genus Glycyrrhiza, or the study of the speciation mechanism of Glycyrrhiza.
[0015] Compared with the prior art, the beneficial effects of the present invention are: This invention develops markers that can efficiently and accurately identify chromosomes 1-8 of licorice. Through multiple rounds of FISH, it identifies the eight chromosomes in a single licorice cell. This technology solves the problem of the small and difficult-to-distinguish morphology of licorice chromosomes and is a valuable tool for the study of chromosome and karyotype evolution in the genus Glycyrrhiza. Attached Figure Description
[0016] Figure 1Hybridization results of the developed licorice chromosome staining probe in Ural licorice; signal diagrams of chromosome 1 modified with FAM (a1, a5); chromosome 2 modified with FAM (a2, a6); chromosome 3 modified with FAM (a3, a7); chromosome 4 modified with FAM (a4, a8); chromosome 5 modified with TAMRA (a9, a13); chromosome 6 modified with TAMRA (a10, a14); chromosome 7 modified with TAMRA (a11, a15); and chromosome 8 modified with TAMRA (a12, a16). Detailed Implementation
[0017] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features described therein can be combined with each other. Example 1: Development of a marker for efficiently identifying licorice chromosomes 1. Oligonucleotide pool design: Repeat Masker was used to remove repetitive sequences from the genome, and the remaining sequences were divided into 45bp oligonucleotide sequences. Each oligonucleotide sequence was aligned back to the Ural licorice genome, and all sequences with multiple matching sites (homology >75%) were filtered out. Primer was used to calculate the Tm and hairpin Tm of each oligonucleotide, and oligonucleotide sequences with dTm >10℃ were retained to construct a single-copy oligonucleotide sequence pool for licorice chromosomes 1-8.
[0018] 2. Primer design and synthesis: This invention employs a "universal primer-specific sequence" splicing strategy. First, eight pairs of universal primer sequences were artificially designed, as shown in Table 1. These primer sequences have no specific binding sites in the licorice genome. During the bioinformatics synthesis stage, these eight pairs of universal primers were "tagged" and assigned to chromosomes 1-8, i.e., the first pair of primers was spliced to both ends of a 45bp oligonucleotide sequence on chromosome 1, the second pair was spliced to both ends of an oligonucleotide sequence on chromosome 2, and so on. This resulted in a full-length oligonucleotide sequence with a "5'-primer-45bp sequence-primer-3'" structure. This design avoids the tedious process of designing complex primers for each chromosome individually and ensures the specificity of amplification. The constructed full-length oligonucleotide sequences of licorice chromosomes 1-8 were sent to a company for artificial synthesis. The primers were also sent to a company for synthesis and fluorescent modification.
[0019] Table 1. Licorice-specific probe primer sequences
[0020] 3. Preparation of Chromosome Staining Probes: Using PCR technology, the full-length oligonucleotide sequences of artificially synthesized licorice chromosomes 1-8 were used as templates and amplified using specific primers with fluorescent modifications (see Table 1). Through PCR, the sequences in the template pool were amplified in large quantities, and fluorescent groups were introduced to obtain licorice chromosome staining probes (i.e., oligonucleotide probes) with different fluorescent modifications. Chromosomes 1-4 were amplified using corresponding primer pairs to produce green fluorescent probes (e.g., those modified with FAM), and chromosomes 5-8 were amplified using corresponding primer pairs to produce red fluorescent probes (e.g., those modified with TAMRA).
[0021] Example 2: Fluorescence in situ hybridization (FISH) detection 1. Preparation of metaphase chromosomes: Young root tips of licorice were pretreated by soaking in a 25 ppm actinomycete ketone solution for 2 hours to enrich the concentrated chromosomes, and then fixed with a fixative (anhydrous ethanol: glacial acetic acid = 3:1). After fixation, the root cap and elongation zone were removed, and the cell walls were digested using a compound enzyme solution (containing 4% R-10 cellulase, 2% pectinase, 2% RS cellulase and 1% Y-23 pectinase). Finally, chromosome slides were prepared using the liquid nitrogen cryogenic knocking method.
[0022] 2. Hybridization: The hybridization solution was prepared with the following components: 3 μL 50% dextran sulfate, 7.5 μL deionized formamide, 1.5 μL 20×SSC, and 400 ng of the corresponding oligo probe for the chromosome prepared in Example 1. A slide containing metaphase chromosomes was heat-denatured at 85°C for 5 minutes, followed by dehydration at -20°C using a gradient of 70% and 100% ethanol. After dehydration, the hybridization solution was added dropwise to the slide, and hybridization was incubated overnight at 37°C.
[0023] 3. Washing and Restaining: After hybridization, the slide was washed three times in 2×SSC solution for 5 minutes each time, and then washed once in 1×PBS solution for 5 minutes. After rinsing with distilled water and air drying, DAPI staining solution was added for chromosome counterstaining, and a coverslip was placed on top.
[0024] 4. Image acquisition and multi-round sequential FISH recognition strategy: Because chromosomes 1-4 of the licorice plant are labeled with green fluorescence (FAM) and chromosomes 5-8 with red fluorescence (TAMRA), simultaneous hybridization cannot distinguish specific chromosomes within a group (e.g., chromosomes 1 and 2 are both green). Therefore, this invention employs a multi-round sequential FISH strategy for accurate identification. (1) First round of hybridization: Take a glass slide of licorice metaphase chromosomes and use a probe mixture targeting only chromosome 1 (green) and chromosome 5 (red) for hybridization. After acquiring images, record the position and signal of chromosomes 1 and 5.
[0025] (2) Probe elution: After image acquisition, place the slide in 1×PBS and elute twice for 5 minutes each time; then elute twice in 2×SSC for 15 minutes each time to completely remove the fluorescent probe from the previous round, and then air dry.
[0026] (3) Second round of hybridization: Hybridization was performed using a probe mixture targeting chromosome 2 (green) and chromosome 6 (red), images were acquired and eluted.
[0027] (4) Subsequent rounds: The third round (No. 3, No. 7) and the fourth round (No. 4, No. 8) of hybridization are carried out in sequence.
[0028] (5) Image synthesis and analysis: Multiple rounds of FISH images from the same cell field of view are overlaid and analyzed. Since each chromosome has a unique signal appearance order in different rounds, accurate identification of chromosomes 1-8 in the same cell is achieved.
[0029] Results analysis: such as Figure 1 As shown, the signals on each chromosome are clear and highly specific, with no cross-hybridization signals. Images were acquired using a fluorescence microscope to observe and record the distribution of the oligo probes on the chromosomes (see...). Figure 1 (where a1-a4 and a9-a12 show specific signals for different chromosomes), through multiple rounds of hybridization, chromosomes 1-8 within the same cell can be clearly identified. This method can overcome interference from chromosome morphological similarities and achieve accurate identification of single chromosomes.
[0030] The embodiments described above are merely illustrative of specific implementations of the present invention, and while the descriptions are detailed, they should not be construed as limiting the scope of protection of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.
Claims
1. A primer combination for amplifying a pool of licorice chromosome-specific oligonucleotide probes, characterized in that, Includes the following 8 pairs of primers: The primer pair GF1 / GR1 for amplifying licorice chromosome 1 has the nucleotide sequence shown in SEQ ID NO:1 / SEQ ID NO:2; The primer pair GF2 / GR2 was used to amplify chromosome 2 of licorice, and the nucleotide sequence is shown in SEQ ID NO:3 / SEQ ID NO:4; The primer pair GF3 / GR3 was used to amplify chromosome 3 of licorice, and the nucleotide sequence is shown in SEQ ID NO:5 / SEQ ID NO:6; The primer pair GF4 / GR4 was used to amplify chromosome 4 of licorice, and the nucleotide sequence is shown in SEQ ID NO:7 / SEQ ID NO:8; The primer pair GF5 / GR5 was used to amplify chromosome 5 of licorice, and the nucleotide sequence is shown in SEQ ID NO:9 / SEQ ID NO:10; The primer pair GF6 / GR6 was used to amplify chromosome 6 of licorice, and the nucleotide sequence is shown in SEQ ID NO:11 / SEQ ID NO:12; The primer pair GF7 / GR7 was used to amplify chromosome 7 of licorice, and the nucleotide sequence is shown in SEQ ID NO:13 / SEQ ID NO:14; The primer pair GF8 / GR8 was used to amplify chromosome 8 of licorice, and the nucleotide sequence is shown in SEQ ID NO:15 / SEQ ID NO:16; The primer sequences are artificially designed non-genomic universal sequences that do not specifically bind to the licorice genome and are used to specifically amplify the oligonucleotide probe pool of the corresponding chromosome.
2. A method for preparing a licorice chromosome staining probe, characterized in that, Includes the following steps: (1) Constructing an oligonucleotide sequence pool: Remove repetitive sequences from the licorice genome and divide the remaining sequences into 45bp oligonucleotide sequences; Align the oligonucleotide sequences back to the genome, remove sequences with multiple matching sites, and construct a single-copy oligonucleotide sequence pool for licorice chromosomes 1-8. (2) Primer design and sequence synthesis: The primer combination described in claim 1 is selected and assigned to licorice chromosomes 1-8 respectively; the primer sequence corresponding to each chromosome is spliced to both ends of the 45bp oligonucleotide sequence of the chromosome to form a full-length oligonucleotide sequence with the structure "5'-primer-45bp sequence-primer-3'". (3) Probe amplification: Using the full-length oligonucleotide sequence pool constructed in step (2) as a template, PCR amplification is performed using the specific primers of claim 1 with fluorescent modification to obtain chromosome staining probes with fluorescent modification.
3. The preparation method according to claim 2, characterized in that, In step (1), the specific criteria for removing sequences with multiple matching sites are: removing sequences with homology > 75%.
4. The preparation method according to claim 2, characterized in that, Step (1) also includes using Primer to calculate the Tm and hairpin Tm of each oligonucleotide, and retaining oligonucleotide sequences with dTm>10℃.
5. The preparation method according to claim 2, characterized in that, In step (3), probes modified with green fluorescence are amplified for chromosomes 1-4 of licorice, and probes modified with red fluorescence are amplified for chromosomes 5-8 of licorice.
6. The chromosome staining probe prepared by the method according to any one of claims 2-5.
7. A method for efficiently identifying licorice chromosomes, characterized in that, The chromosome-smearing probe prepared by the method described in any one of claims 2-5 was hybridized with the metaphase chromosome of licorice using a multi-round fluorescence in situ hybridization technique.
8. The method as described in claim 7, characterized in that, The hybridization step includes: preparing a hybridization solution, the components of which are: 3 μL of 50% dextran sulfate, 7.5 μL of deionized formamide, 1.5 μL of 20×SSC and 400 ng of the oligonucleotide probe prepared according to claim 2; heat-denaturing a glass slide carrying metaphase chromosomes at 85°C for 5 minutes, dehydrating it with an ethanol gradient, adding the hybridization solution, and hybridizing overnight in a 37°C incubator.
9. The application of the chromosome staining probe prepared by the method according to any one of claims 2-5, or the method according to claim 7, in the analysis of licorice chromosome karyotype, the study of chromosome evolution among species of licorice, or the study of the speciation mechanism of licorice.