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CN-122012788-A - Probe, method for efficiently identifying liquorice chromosome and application thereof

CN122012788ACN 122012788 ACN122012788 ACN 122012788ACN-122012788-A

Abstract

The invention discloses a probe, a method for efficiently identifying liquorice chromosomes and application thereof, belonging to the fields of bioinformatics and molecular cytogenetics. The invention utilizes bioinformatics to screen single copy oligonucleotide sequences of licorice No. 1-8 chromosomes, and constructs a chromosome specific oligonucleotide probe pool through a general primer sequence label splicing strategy designed artificially. The number 1-8 chromosomes of the liquorice can be identified efficiently and accurately through the multi-round FISH technology. The method solves the problem that accurate identification is difficult due to tiny liquorice chromosome shape and high similarity, has the advantages of high resolution, strong specificity, capability of identifying single chromosome and the like, and provides a powerful tool for subsequent liquorice chromosome karyotype analysis, evolution research and exploration of speciation mechanism.

Inventors

  • MENG ZHUANG
  • LI HONGBIN
  • ZHENG QIAN
  • SHI GUANGZHEN
  • WANG WEI
  • FENG XUEDONG
  • WU XIA
  • LI YUANHAO
  • LUO WENJUN
  • Xie Quanliang

Assignees

  • 石河子大学

Dates

Publication Date
20260512
Application Date
20260311

Claims (9)

  1. 1. A set of primer combinations for amplifying a licorice chromosome specific oligonucleotide probe pool, comprising 8 pairs of primers: the primer pair GF1/GR1 for amplifying the licorice chromosome 1 has a nucleotide sequence shown as SEQ ID NO. 1/SEQ ID NO. 2; the primer pair GF2/GR2 for amplifying the licorice chromosome 2 has a nucleotide sequence shown as SEQ ID NO 3/SEQ ID NO 4; The primer pair GF3/GR3 of the licorice chromosome 3 is amplified, and the nucleotide sequence is shown as SEQ ID NO. 5/SEQ ID NO. 6; The primer pair GF4/GR4 for amplifying the licorice chromosome 4 has a nucleotide sequence shown as SEQ ID NO. 7/SEQ ID NO. 8; The primer pair GF5/GR5 for amplifying the licorice chromosome 5 has a nucleotide sequence shown as SEQ ID NO 9/SEQ ID NO 10; the primer pair GF6/GR6 of the licorice chromosome 6 is amplified, and the nucleotide sequence is shown as SEQ ID NO. 11/SEQ ID NO. 12; the primer pair GF7/GR7 of the licorice chromosome 7 is amplified, and the nucleotide sequence is shown as SEQ ID NO. 13/SEQ ID NO. 14; The primer pair GF8/GR8 for amplifying the licorice chromosome 8 has a nucleotide sequence shown as SEQ ID NO. 15/SEQ ID NO. 16; wherein, the primer sequence is a non-genome universal sequence designed manually, does not generate specific combination with licorice genome, and is used for specifically amplifying an oligonucleotide probe pool of a corresponding chromosome.
  2. 2. The preparation method of the liquorice chromosome staining probe is characterized by comprising the following steps: (1) Constructing an oligonucleotide sequence pool, namely removing repeated sequences in a licorice genome, dividing the residual sequences into oligonucleotide sequences of 45bp, comparing the oligonucleotide sequences back to the genome, removing sequences with a plurality of matching sites, and constructing a single copy oligonucleotide sequence pool of licorice chromosomes 1-8; (2) The primer design and sequence synthesis are that the primer combination of claim 1 is selected and respectively distributed to licorice No. 1-8 chromosomes, and the primer sequences corresponding to the chromosomes are respectively spliced at two ends of a 45bp oligonucleotide sequence of the chromosome to form a full-length oligonucleotide sequence of a ' 5' -primer-45 bp sequence-primer-3 '; (3) And (3) probe amplification, namely, using the full-length oligonucleotide sequence pool constructed in the step (2) as a template, and carrying out PCR amplification by using the specific primer with fluorescent modification as set forth in claim 1 to obtain a chromosome staining probe with fluorescent modification.
  3. 3. The method of claim 2, wherein in step (1), the specific criteria for removing sequences having multiple matching sites is removal of sequences having homology of > 75%.
  4. 4. The method of claim 2, further comprising calculating Tm and hairpin Tm for each oligonucleotide using primers in step (1), leaving oligonucleotide sequences at dTm >10 ℃.
  5. 5. The method according to claim 2, wherein in the step (3), a probe having a green fluorescent modification is amplified for chromosome 1 to 4 of licorice and a probe having a red fluorescent modification is amplified for chromosome 5 to 8 of licorice.
  6. 6. A chromosome spread probe prepared by the method of any one of claims 2-5.
  7. 7. A method for efficiently identifying liquorice chromosomes, which is characterized in that a plurality of rounds of fluorescence in situ hybridization technology is adopted, and chromosome staining probes prepared by the method of any one of claims 2-5 are hybridized with liquorice metaphase chromosomes.
  8. 8. The method according to claim 7, wherein the hybridization step comprises preparing a hybridization solution comprising 3. Mu.L of 50% dextran sulfate, 7.5. Mu.L of deionized formamide, 1.5. Mu.L of 20 XSSC and 400ng of the oligonucleotide probe prepared in claim 2, thermally denaturing a metaphase chromosome-bearing slide at 85℃for 5 minutes, dehydrating by gradient of ethanol, dropping the hybridization solution, and hybridizing overnight in a 37℃incubator.
  9. 9. Use of a chromosome spread probe prepared according to the method of any one of claims 2 to 5, or the method of claim 7, in a licorice chromosome karyotyping assay, a licorice interspecies chromosome evolution study, or a licorice speciation mechanism study.

Description

Probe, method for efficiently identifying liquorice chromosome and application thereof Technical Field The invention belongs to the fields of bioinformatics and molecular cytogenetics, and in particular relates to a probe, a method for efficiently identifying liquorice chromosomes and application thereof. Background Glycyrrhrizae radix is plant of Glycyrrhiza (Glycyrrhiza L.) of Papilionaceae (Papilionoideae) of Leguminosae, and has effects of clearing heat, detoxicating, and harmonizing various drugs. The Chinese medicinal composition is called as "Jiufangcao" and "GuoLao". However, the genetic diversity of the licorice plant is rich and the variety is complex, so far, little is known about the inter-species chromosome evolution difference and evolution track in the licorice species formation process. Cytogenetics is an important approach for species chromosome evolution research, but liquorice has the defects of small general chromosome morphology, high similarity and lack of powerful and reliable chromosome markers, which seriously hinders the development of related researches on liquorice interspecies chromosome evolution. Fluorescence In Situ Hybridization (FISH) is a key technology in plant molecular cytogenetics. The technology is based on the nucleic acid base complementary pairing principle, firstly, a nucleic acid fragment with a known sequence is marked as a probe, then the probe is hybridized with a target nucleic acid sequence in cells or tissues in situ, and finally, the distribution condition of a specific sequence on a chromosome is visually displayed under a microscope through fluorescent signal detection, so that the accurate positioning of the sequence on the chromosome is realized. Although FISH technology has found widespread use in plant chromosome research, accurate chromosome identification still presents a major challenge for species with high ploidy, high chromosome number, and small morphology. The advent of oligonucleotide fluorescent in situ hybridization techniques has addressed these challenges. The invention develops a set of markers capable of efficiently identifying licorice No. 1-8 chromosomes by utilizing an oligonucleotide fluorescence in situ hybridization technology, which is helpful for deeply knowing the inter-species chromosome origin evolution difference and speciation mechanism in the licorice speciation process. Disclosure of Invention The invention aims to develop a set of dyeing probes capable of efficiently and accurately identifying liquorice chromosomes and used for identifying liquorice No. 1-8 chromosomes. In order to achieve the above object, the present invention is realized by the following technical scheme: in a first aspect, the present invention provides a set of primer combinations for amplifying a licorice chromosome specific oligonucleotide probe pool, comprising 8 pairs of primers as follows: the primer pair GF1/GR1 for amplifying the licorice chromosome 1 has a nucleotide sequence shown as SEQ ID NO. 1/SEQ ID NO. 2; the primer pair GF2/GR2 for amplifying the licorice chromosome 2 has a nucleotide sequence shown as SEQ ID NO 3/SEQ ID NO 4; The primer pair GF3/GR3 of the licorice chromosome 3 is amplified, and the nucleotide sequence is shown as SEQ ID NO. 5/SEQ ID NO. 6; The primer pair GF4/GR4 for amplifying the licorice chromosome 4 has a nucleotide sequence shown as SEQ ID NO. 7/SEQ ID NO. 8; The primer pair GF5/GR5 for amplifying the licorice chromosome 5 has a nucleotide sequence shown as SEQ ID NO 9/SEQ ID NO 10; the primer pair GF6/GR6 of the licorice chromosome 6 is amplified, and the nucleotide sequence is shown as SEQ ID NO. 11/SEQ ID NO. 12; the primer pair GF7/GR7 of the licorice chromosome 7 is amplified, and the nucleotide sequence is shown as SEQ ID NO. 13/SEQ ID NO. 14; The primer pair GF8/GR8 for amplifying the licorice chromosome 8 has a nucleotide sequence shown as SEQ ID NO. 15/SEQ ID NO. 16; wherein, the primer sequence is a non-genome universal sequence designed manually, does not generate specific combination with licorice genome, and is used for specifically amplifying an oligonucleotide probe pool of a corresponding chromosome. In a second aspect, the invention provides a preparation method of a licorice chromosome staining probe, which is characterized by comprising the following steps: (1) Constructing an oligonucleotide sequence pool, namely removing repeated sequences in a licorice genome, dividing the residual sequences into oligonucleotide sequences of 45bp, comparing the oligonucleotide sequences back to the genome, removing sequences with a plurality of matching sites, and constructing a single copy oligonucleotide sequence pool of licorice chromosomes 1-8; (2) The primer design and sequence synthesis comprises the steps of selecting the primer combination in the first aspect, respectively distributing the primer combination to licorice No. 1-8 chromosomes, respectively splicing primer sequences corresponding to t