Oligonucleotide probe set for identifying cabbage chromosomes, design method and application thereof

Abstract

The present invention relates to the field of cytology technology, and more specifically, to an oligonucleotide probe set, design method and application thereof for identifying cabbage chromosomes. The probe set design comprises the following steps: S1: obtaining the NR-TR set in the cabbage Korso reference genome; S2: statistically clustering the NR-TR set and obtaining cabbage chromosome TR array data; S3: obtaining a repetitive sequence cluster enriched in all cabbage chromosomes; S4: segmenting the repetitive sequence representative array of the repetitive sequence cluster obtained in step S3; S5: using bl astn to align the segmented sequences to the cabbage Korso reference genome, respectively, to obtain an oligonucleotide probe set covering 9 cabbage chromosomes. The present invention utilizes the cabbage Korso reference genome sequence information, designs a probe set for identifying cabbage chromosomes by a bioinformatics method, and establishes a technology for painting the cabbage genome, which can paint cabbage chromosome materials in batches.

Description

Technical Field

[0001] The present invention relates to the technical field of cytology, and more specifically to an oligonucleotide probe set for identifying cabbage chromosomes, a design method and an application thereof. Background Art

[0002] Cabbage (Brassica oleracea) is rich in nutrients and is one of the important cruciferous vegetables in my country. It is also one of the basic components of the "Yu's Triangle" (C genome). In most eukaryotes, centromeric DNA is composed of highly repetitive retrotransposon sequences and satellite sequences. Oligonucleotide fluorescence in situ hybridization technology is a new type of fluorescence in situ hybridization technology using single-stranded oligonucleotides as probes. These oligonucleotide probes are a new type of chromosome physical markers that can be developed from the genomes of sequenced species to identify centromeric repeat sequences from the reference genome of cabbage. The development of centromeric repeat sequence probes for genomic fluorescence in situ hybridization can effectively identify the Brassica C genome, and can achieve the identification of Brassica C genome materials and variants, the identification of the relationship between wild species and the Brassica genome, and the identification of Brassica C subgenomic chromosomes in allopolyploid materials, as well as the determination of the centromere position of each chromosome. It can be used for the creation of cabbage breeding materials and genetic research, which is of great significance. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides an oligonucleotide probe set for identifying cabbage chromosomes, a design method and its application, an oligonucleotide probe set developed based on cabbage repetitive sequences and a simple, low-cost and reliable fluorescence in situ hybridization method.

[0004] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0005] An oligonucleotide probe set for identifying cabbage chromosomes, including CY3 fluorescently labeled oP C1 and oP C2 Two sequences, the specific sequences are:

[0006] oP C1 :CY3-5'-TTTTCCGTTTGGGAATATGACAACTTCTTCGTCATTCTT-3';

[0007] oP C2 :CY3-5'-TGAAAGTGGGATAACTTCTTCATGCCAACTCCTATGAGAT-3'.

[0008] A method for designing an oligonucleotide probe set for identifying a cabbage chromosome comprises the following steps:

[0009] S1: Obtain tandem repeat sequences TR with repeat units greater than 500 bp in the reference genome of Brassica oleracea Korso, and remove redundancy from the obtained results to obtain a non-redundant TR array set, namely NR-TR;

[0010] S2: statistically clustering the NR-TR obtained in step S1 and obtaining the TR array data of the cabbage genome;

[0011] S3: detecting the distribution of homologous copies of the TR array sequence of the cabbage genome obtained in step S2 in the cabbage Korso reference genome, and obtaining a representative array of repeat sequences enriched in all chromosomes in the cabbage genome;

[0012] S4: cutting the repetitive sequence representative array obtained in step S3 into 40 bp fragments from the beginning, with a step length of 5 bp;

[0013] S5: The split sequences were aligned to the Brassica oleracea Korso reference genome using blastn, with parameters of pident=90 and qcovhsp=90, and the enrichment of these sequences on each chromosome of the Brassica oleracea genome was observed, and finally the sequences containing oP C1 and oP C2 Two sequenced Brassica oleracea genomic probe sets.

[0014] An application method of an oligonucleotide probe set for identifying cabbage chromosomes comprises the following steps:

[0015] A1. Cabbage pistil treatment: Collect fresh unopened inflorescences, fix and decolorize in Carnoy's solution for 12 hours, and store in 70% ethanol. Take the fixed material, peel the pistil in the prophase of mitosis from the bud, enzymatically hydrolyze at 37℃ for 12 hours, wash with ddH2O, and then hypotonic at room temperature for 4 hours;

[0016] A2. Preparation of slides of cabbage mitosis: Place the pistil on a clean slide, add 20 μL of 60% glacial acetic acid, smear the slide in a 37°C slide dryer, wash the slide with Carnoy's fixative, add 20 μL of 5% DAPI staining solution after drying, observe under a microscope, select the better slide, rinse off the cover glass and staining solution with running water, and dry the slide in a 55°C oven overnight;

[0017] A3. Chromosome denaturation: add 30 μL of 70% deionized formamide to the chromosome piece, cover with a cover glass, denature in a drying machine at 80°C for 5 min, immediately transfer to -20°C for gradient dehydration, 3 min each time, take out and dry at room temperature;

[0018] A4. Preparation of hybridization dye solution: add 0.5 μL of the oligonucleotide probe set solution for identifying cabbage chromosomes, 15 μL of 50% deionized formamide, 6 μL of 20% dextran sulfate, 3 μL of 10% 2×SSC solution, and 5 μL of ddH2O to each slide, totaling 30 μL, denature at 99°C for 10 min, quickly transfer to an ice-water mixture and place in a -20°C refrigerator for 10 min. The concentration of the cabbage chromosome probe set solution is 1 μg / μL;

[0019] A5. Hybridization: Add 30 μL of denaturing hybridization solution to each slide, cover with a cover slip, place in an 80°C slide drying machine for denaturation for 3 min, and hybridize at 37°C for 6 h;

[0020] A6. Staining: gently knock off the coverslip, wash three times in 2×SSC, 5 min each time, and finally rinse once with ddH2O running water, dry in the dark, add 20 μL of 5% DAPI staining solution prepared with anti-fluorescence quenching mounting medium to each slide, add coverslip, stain for 10 min, examine under a microscope, and take pictures.

[0021] Preferably, the 2×SSC buffer solution consists of 0.3 M trisodium citrate C6H5Na3O7·2H2O and 3 M NaCl.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The present invention analyzes tandem repeat sequences by bioinformatics methods, designs an oligonucleotide probe set for cabbage chromosomes, can effectively and quickly identify all chromosomes of cabbage (Brassica oleracea, CC, 2n=18), and establishes a technique for painting cabbage chromosomes, which can be used to paint cabbage chromosome materials in batches.

[0024] 2. The present invention provides an oligonucleotide probe set and a simple, low-cost and reliable fluorescence in situ hybridization method developed based on the repetitive sequences in the Brassica oleracea Korso reference genome. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The distribution of oligonucleotide probe sets for the cabbage chromosome in the Korso reference genome;

[0026] Figure 2 The results are shown in Figure 1, which are the staining results of the chromosomes of Brassica oleracea (Brassica oleracea, CC, 2n=18) using the oligonucleotide probe set for the chromosomes of Brassica oleracea. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] like Figure 1-2 A method for designing an oligonucleotide probe set for identifying cabbage chromosomes is shown in FIG.

[0029] Tandem Repeats Finder (TRF, v4.09) software was used to search the Brassica oleracea Korso reference genome ( https: / / figshare.com / authors / Zhangjun_Fei / 9345701 ) in which the repeating unit is larger than 500 bp, the TRF output results are de-redundant using TR-tookit software to obtain a non-redundant TR array (NR-TR) set. The obtained NR-TR is statistically clustered to obtain the TR array statistics of the cabbage chromosome.

[0030] Design method of cabbage chromosome oligonucleotide probe set: Use local blastn to detect the distribution of homologous copies of the above TR array sequence in the cabbage Korso reference genome. As a result, a representative array with a repeat unit length of 601bp was found, and the homologous copies of its repeat unit sequence were specifically and evenly enriched in the centromere region of all cabbage chromosomes. The full-length sequence of the obtained repeat sequence representative array was cut from scratch into oligonucleotide fragments with a length of 40bp and a step length of 5bp. The oligonucleotide sequences were submitted to local blastn for alignment to the cabbage Korso reference genome (parameters were pident=90, qcovhsp=90) to observe whether they were enriched in all cabbage chromosomes. If they were enriched in all cabbage chromosomes, they were retained, and if they were not enriched in some chromosomes, they were discarded. As a result, a cabbage chromosome oligonucleotide probe set containing two sequences was obtained, which was distributed in the centromere region of all cabbage chromosomes ( Figure 1 ), and further fluorescently labeled with CY3 to oP C1 and oP C2 , an oligonucleotide probe set for identifying the cabbage genome was obtained, as shown in Table 1.

[0031] Table 1

[0032]

[0033] Depend on Figure 1 It can be seen that the oligonucleotide probe set covers all chromosomes of Cabbage and is in an enriched state.

[0034] Example 2: Application method of an oligonucleotide probe set for identifying cabbage chromosomes

[0035] A1. Cabbage pistil treatment: Collect fresh unopened inflorescences, fix and decolorize in Carnoy's solution (absolute ethanol: glacial acetic acid = 3:1) for 12 hours, and store in 70% ethanol. Take the fixed material, peel the pistil in the prophase of mitosis from the bud, enzymatically hydrolyze at 37℃ for 12 hours, wash with ddH2O, and hypotonic at room temperature for 4 hours.

[0036] A2. Preparation of slides of cabbage mitosis: Place the pistil on a clean slide, add 20 μL of 60% glacial acetic acid, smear the slides in a 37°C slide dryer, wash the slides with Carnoy's fixative, add 20 μL of 5% DAPI stain (prepared in ddH2O) after drying, observe under a microscope, select the better slides, rinse off the coverslip with running water, and dry the slides in a 55°C oven overnight.

[0037] A3. Chromosome denaturation: add 30 μL of 70% deionized formamide on the chromosome piece, cover with a cover slip and denature in a drying machine at 80°C for 5 min, then immediately transfer to -20°C for gradient dehydration (75%, 85%, 100%), 3 min per level, and take out to dry at room temperature.

[0038] A4. Preparation of hybridization dye solution: 0.5 μL of the above cabbage chromosome probe set solution, 15 μL of 50% deionized formamide, 6 μL of 20% dextran sulfate, 3 μL of 10% 2×SSC solution, and 5 μL of ddH2O were added to each slide, totaling 30 μL, and denatured at 99°C for 10 min, and quickly transferred to an ice-water mixture and placed in a -20°C refrigerator for 10 min. The concentration of the cabbage chromosome probe set solution is 1 μg / μL.

[0039] A5. Hybridization: Add 30 μL of denaturing hybridization solution to each slide, cover with a coverslip, place in an 80°C slide drying machine for denaturation for 3 minutes, and hybridize at 37°C for 6 hours.

[0040] A6. Staining: Knock off the cover glass, wash 3 times in 2×SSC, 5 min each time, and finally rinse once with ddH2O running water, dry in the dark, add 20 μL of 5% DAPI staining solution (prepared with anti-fluorescence quenching sealing agent) to each slide, add cover glass, stain for 10 min, examine under a microscope, and take pictures ( Figure 2 ).

[0041] The staining results of the chromosomes of Brassica oleracea (Brassica oleracea, CC, 2n=18) were identified by the oligonucleotide probe set of the chromosomes of Brassica oleracea. Figure 2As shown, (a) (blue) is DAPI staining of cabbage (kale, CC, 2n = 18) chromosomes (the dye solution does not contain the oligonucleotide probe set of cabbage chromosomes), and 18 blue signal chromosomes can be clearly observed; (b) (red) is the staining result of the oligonucleotide probe set of cabbage chromosomes on the centromere position of cabbage (kale, CC, 2n = 18) chromosomes, and 18 chromosome centromeres marked with red signals can be clearly observed; (c) is a dyeing composite image of the oligonucleotide probe set of cabbage chromosomes identifying cabbage (kale, CC, 2n = 18) chromosomes, and 18 blue chromosomes mixed with red centromere signals can be clearly observed, which indicates that the oligonucleotide probe set of cabbage chromosomes designed by the present invention can efficiently and accurately identify the centromere positions of all chromosomes of cabbage (kale, CC, 2n = 18).

[0042] Only the preferred embodiments of the present invention are described in detail above, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge scope of ordinary technicians in this field without departing from the purpose of the present invention, and various changes should be included in the protection scope of the present invention. SEQUENCE LISTING <110> Zhengzhou University Horticulture Research Institute, Henan Academy of Agricultural Sciences Henan Kaiyuan Agricultural Development Co., Ltd. <120> Oligonucleotide probe set for identifying cabbage chromosomes, design method and application thereof <130> 2022 <160> 2 <170> PatentIn version 3.3 <210> 1 <211> 40 <212> DNA <213> Artificial Sequence <400> 1 ttttcctgtt tgggaatatg acaacttctt cgtcattctt 40 <210> 2 <211> 40 <212> DNA <213> Artificial Sequence <400> 2 tgaaagtggg ataacttctt catgccaact cctatgagat 40

Claims

1. An oligonucleotide probe set for identifying cabbage chromosomes, characterized in that: Including CY3 fluorescently labeled oP C1 and oP C2 Two sequences, the specific sequences are: on C1 :CY3-5'-TTTTCCTGTTTGGGAATATGACAACTTCTTCGTCATTCTT-3'; on C2 :CY3-5'-TGAAAGTGGGATAACTTCTTCATGCCAACTCCTATGAGAT-3'。 2. A method for designing an oligonucleotide probe set for identifying cabbage chromosomes as claimed in claim 1, characterized in that: The following steps are involved: S1: Obtain the tandem repeat sequences TR with repeat units greater than 500 bp in the reference genome of Brassica oleracea Korso, and remove redundancy from the obtained results to obtain a non-redundant TR array set, namely NR-TR; S2: statistically clustering the NR-TR obtained in step S1 and obtaining the TR array data of the cabbage genome; S3: detecting the distribution of homologous copies of the TR array sequence of the cabbage genome obtained in S2 in the cabbage Korso reference genome, and obtaining a representative array of repeat sequences enriched in all chromosomes in the cabbage genome; S4: cutting the repetitive sequence representative array obtained in step S3 into 40 bp fragments from the beginning, with a step length of 5 bp; S5: The split sequences were aligned to the Brassica oleracea Korso reference genome using blastn, with parameters of pident=90 and qcovhsp=90, and the enrichment of these sequences on each chromosome of the Brassica oleracea genome was observed, and finally the sequences containing oP C1 and oP C2 Two sequenced Brassica oleracea genomic probe sets.

3. A method for using the oligonucleotide probe set for identifying cabbage chromosomes according to claim 1, characterized in that , including the following steps: A1. Cabbage pistil treatment: Collect fresh unopened inflorescences, fix and decolorize in Carnoy's solution for 12 hours, and store in 70% ethanol; take the fixed material, peel the pistil in the early stage of mitosis from the bud, enzymatically hydrolyze at 37℃ for 12 hours, wash with ddH2O, and then hypotonic at room temperature for 4 hours; A2. Preparation of slides of cabbage mitosis: Place the pistil on a clean slide, add 20 μL of 60% glacial acetic acid, smear the slide in a 37°C slide dryer, wash the slide with Carnoy's fixative, add 20 μL of 5% DAPI staining solution after drying, observe under a microscope, select the better slide, rinse off the cover glass and staining solution with running water, and dry the slide in a 55°C oven overnight; A3. Chromosome denaturation: add 30 μL of 70% deionized formamide to the chromosome piece, cover with a cover glass, denature in a drying machine at 80°C for 5 min, immediately transfer to -20°C for gradient dehydration, 3 min each time, take out and dry at room temperature; A4. Preparation of hybridization dye solution: add 0.5 μL of the oligonucleotide probe set solution for identifying cabbage chromosomes, 15 μL of 50% deionized formamide, 6 μL of 20% dextran sulfate, 3 μL of 10% 2×SSC solution, and 5 μL of ddH2O to each slide, totaling 30 μL, denature at 99°C for 10 min, quickly transfer to an ice-water mixture and place in a -20°C refrigerator for 10 min; the concentration of the cabbage chromosome probe set solution is 1 μg / μL; A5. Hybridization: Add 30 μL of denaturing hybridization solution to each slide, cover with a cover slip, place in an 80°C slide drying machine for denaturation for 3 minutes, and hybridize at 37°C for 6 hours; A6. Staining: gently knock off the coverslip, wash three times in 2×SSC, 5 min each time, and finally rinse once with ddH2O running water, dry in the dark, add 20 μL of 5% DAPI staining solution prepared with anti-fluorescence quenching mounting medium to each slide, cover the slide, stain for 10 min, examine under a microscope, and take pictures.

4. The method for using the oligonucleotide probe set for identifying cabbage chromosomes according to claim 3, characterized in that: The 2×SSC buffer solution consists of 0.3 M trisodium citrate C 6 H 5 Na 3 O 7 · 2H 2 O and 3 M NaCl.

5. Use of the oligonucleotide probe set as claimed in claim 1 in identifying cabbage chromosomes.

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