Segmented PCR primer group for detecting consanguinity of saccharum arundinaceum chromosome 3 in sugarcane and application of segmented PCR primer group
By designing a segmented PCR primer set and detection method, the problem of detecting the distribution and structural variation of chromosome 3 in sugarcane was solved, achieving efficient and low-cost bloodline identification and chromosome structural variation research.
Patent Information
- Application Number
- CN202511368290.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-24
- Publication Date
- 2025-11-14
AI Technical Summary
Existing technologies are insufficient to accurately detect the distribution patterns and structural variations of chromosome 3 in sugarcane. Traditional cytological tools are inefficient, and the transmission of chromosomes in hybrid offspring is highly random.
A segmented PCR primer set was designed, including 15 pairs of specific primers covering the specific region of chromosome 3 of *Imperata cylindrica*. The presence of chromosome 3 of *Imperata cylindrica* in sugarcane was detected by PCR amplification and gel electrophoresis. A kit and detection method are provided.
It achieves highly sensitive and specific detection of chromosome 3 lineage in sugarcane, is simple and rapid, reduces costs, and provides accurate lineage identification and chromosome structural variation research support for sugarcane breeding.
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Figure CN120945112A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of molecular biology, and in particular to a segmented PCR primer set for detecting chromosome 3 lineage in sugarcane and its application. Background Technology
[0002] Spotted grass ( Tripidium arundinaceum *Saccharum spp.* is a perennial, tall, tufted herbaceous plant belonging to the genus *Saccharum* of the Poaceae family, and is an important close relative germplasm resource for sugarcane. Due to its excellent resistance to drought, disease, and poor soil conditions required for sugarcane breeding, it has attracted the attention of the sugarcane breeding community and has been included in breeding programs. Currently, some excellent multi-resistant germplasm or parental materials have been screened from it. However, the precise chromosomal fragments of *Saccharum spp.* chromosomes that have infiltrated into the sugarcane genetic background remain unclear. Studies have shown that the distribution of *Saccharum spp.* chromosomes in sugarcane backcrosses does not exhibit a clear pattern. However, with the advancement of backcross generations, the *Saccharum spp.* genetic composition in the backcross progeny is diluted generation by generation, making the use of traditional cytological tools inefficient. Furthermore, the flowering of sugarcane varieties is not universal; due to limitations in flowering, some *Saccharum spp.* chromosomes in backcross progeny fail to be passed on to offspring, which to some extent increases the randomness of the transmission of *Saccharum spp.* molecular marker sites to offspring. Therefore, research on detecting bloodline identification in hybrid progeny of *Saccharum spp.* in sugarcane and investigating chromosomal structural variations in *Saccharum spp.* is of great significance. Summary of the Invention
[0003] In view of this, the purpose of this invention is to provide a segmented PCR primer set for detecting the bloodline of chromosome 3 in sugarcane and its application. The primer set provided by this invention can accurately detect whether the offspring of sugarcane and *Imperata cylindrica* contain a specific region of chromosome 3 bloodline, providing technical support for the bloodline identification of offspring of *Imperata cylindrica* introduced from wild germplasm resources in sugarcane and basic research on chromosome structural variations in *Imperata cylindrica*.
[0004] To achieve the above objectives, the present invention provides the following technical solution: A segmented PCR primer set for detecting chromosome 3 lineage in sugarcane, the primer set comprising: The nucleotide sequence is shown in the primer pair Chr03A_6615182_6615331 as shown in Seq_1~Seq_2; The nucleotide sequence is shown in the primer pair Chr03A_6707952_6708101 as shown in Seq_3~Seq_4; The nucleotide sequence is shown in the primer pair Chr03A_16870858_16871007 as shown in Seq_5~Seq_6; The nucleotide sequence is shown in the primer pair Chr03A_36879343_36879492 as shown in Seq_7~Seq_8; The nucleotide sequence is shown in Seq_9~Seq_10. Primer pair Chr03A_36909166_36909315; The nucleotide sequence is shown in Seq_11~Seq_12. Primer pair Chr03A_46161635_46161784; The nucleotide sequence is shown in Seq_13~Seq_14. Primer pair Chr03A_56134768_56134917; The nucleotide sequence is shown in Seq_15~Seq_16. Primer pair Chr03A_56812568_56812717; The nucleotide sequence is shown in Seq_17~Seq_18. Primer pair Chr03A_76146780_76146929; The nucleotide sequence is shown in the primer pair Chr03A_86348649_86348798 in Seq_19~Seq_20; The nucleotide sequence is shown in the primer pair Chr03A_86684289_86684438 as shown in Seq_21~Seq_22; The nucleotide sequence is shown in the primer pair Chr03A_96055692_96055841 in Seq_23~Seq_24; The nucleotide sequence is shown in the primer pair Chr03A_96148256_96148405 as shown in Seq_25~Seq_26; The nucleotide sequence is shown in the primer pair Chr03A_96329050_96329199 in Seq_27~Seq_28; The nucleotide sequence is shown in the primer pair Chr03A_96535855_96536004 as shown in Seq_29~Seq_30. In this invention, Seq_1~Seq_30 specifically refer to: Seq_1: 5'-CTGGTGTGGCAAGAAATTTGTTATC-3'; Seq_2: 5'-GCCATCCTTTCCTGCCTATAACT-3'; Seq_3: 5'-GCTCTAAGACATGTGACCTGATC-3'; Seq_4: 5'-CCAAAGCAGAGTAGAATATAGAGTTGC-3'; Seq_5: 5'-TCCTGGCGTTTATTAATAAGGAGGA-3'; Seq_6:5’-ACTCCAATTGTAGCCTAAACTTTAGT-3’; Seq_7:5’-AGATTCATAAATGTCCATAGATCACCCT-3’; Seq_8:5’-ATGCACCGACGCCTATAATGTAT-3’; Seq_9:5’-CACCAGCACAACACAACAAGAAT-3’; Seq_10:5’-CTGATTTCTGGATTCGGCGTCTTA-3’; Seq_11:5’-GCAGTCCCTTGAACAAACTTATAGC-3’; Seq_12:5’-TAGTTCAACAGTATTGCAATCGACAA-3’; Seq_13:5’-CATGAGCCAGCATTCTCTATTAAAGT-3’; Seq_14:5’-TAATTCGTTTCTGGCGTGTGTATG-3’; Seq_15:5’-TCGATCAATAAATGTCCTTCCAATTG-3’; Seq_16:5’-TCAAATCCACTTCAATTCCGTAGC-3’; Seq_17:5’-TCTTCCTCGTATGGTTTATGCTGT-3’; Seq_18:5’-ACACCGGTGCCTAATAATGCTG-3’; Seq_19:5’-TCAAGAGTTTAGCCATGCAAATGA-3’; Seq_20:5’-ACTACTGCTACTGTGTTCTGAGAAA-3’; Seq_21:5’-CAGGATCAGATTAGTAGGTGGGTATG-3’; Seq_22:5’-TCATTCTCTTCTAGCTCAGTTGC-3’; Seq_23:5’-ACTTGTTTGTGAGGAGGGAATCT-3’; Seq_24:5’-TGGAGTGTTTACAATTGAAGAAGGT-3’; Seq_25:5'-ACACTGTAAATTGGGTCATGAAGC-3'; Seq_26:5'-AACGCTCAGACCTGTTCAGATAG-3'; Seq_27:5'-TAGGAATGCTTGAAAGACTGGAATT-3'; Seq_28: 5'-GCCAATAAGTTACAAGAGGTTAACC-3'; Seq_29:5'-ACGTAGCAATAACACCTGAATGAC-3'; Seq_30: 5'-GCAATGATAAGGGCTTCTCTCTA-3'; In this invention, the amplification product of primer pair Chr03A_6615182_6615331 preferably corresponds to the position 6615182_6615331 on chromosome 3 of *Imperata cylindrica*; the amplification product of primer pair Chr03A_6707952_6708101 preferably corresponds to the position 6707952_6708101 on chromosome 3 of *Imperata cylindrica*; the amplification product of primer pair Chr03A_16870858_16871007 preferably corresponds to the position 16870858_16871007 on chromosome 3 of *Imperata cylindrica*; and the amplification product of primer pair Chr03A_36879343_36879492 preferably corresponds to the position 3 on chromosome 3 of *Imperata cylindrica*. The amplification products of primer pair Chr03A_36909166_36909315 correspond to the position of chromosome 3 of *Imperata cylindrica*, preferably 36909166_36909315; the amplification products of primer pair Chr03A_46161635_46161784 correspond to the position of chromosome 3 of *Imperata cylindrica*, preferably 46161635_46161784; the amplification products of primer pair Chr03A_56134768_56134917 correspond to the position of chromosome 3 of *Imperata cylindrica*, preferably 56134768_56134917; the amplification products of primer pair Chr03A_56812568_56812717... The preferred position for the amplification product of primer pair Chr03A_76146780_76146929 on chromosome 3 is 76146780_76146929; the preferred position for the amplification product of primer pair Chr03A_86348649_86348798 on chromosome 3 is 86348649_86348798; the preferred position for the amplification product of primer pair Chr03A_86684289_86684438 on chromosome 3 is 86684289_86684438; the preferred position for the amplification product of primer pair Chr03A_960556 on chromosome 3 is 86684289_86684438; the preferred position for the amplification product of primer pair Chr03A_960556 on chromosome 3 is 86812568_56812717; the preferred position for the amplification product of primer pair Chr03A_86348649_86348798 on chromosome 3 is 86684289_86684438; the preferred position for the amplification product of primer pair Chr03A_960556 on chromosome 3 is 86684289_86684438. The preferred position of the amplification product of primer pair 92_96055841 corresponding to chromosome 3 of *Imperata cylindrica* is 96055692_96055841; the preferred position of the amplification product of primer pair Chr03A_96148256_96148405 corresponding to chromosome 3 of *Imperata cylindrica* is 96148256_96148405; the preferred position of the amplification product of primer pair Chr03A_96329050_96329199 corresponding to chromosome 3 of *Imperata cylindrica* is 96329050_96329199; the preferred position of the amplification product of primer pair Chr03A_96535855_96536004 corresponding to chromosome 3 of *Imperata cylindrica* is 96535855_96536004.
[0005] In this invention, the primer set contains at least one pair of primers specific to the 3rd chromosome region of *Imperata cylindrica* for every 30 Mb. The primer set of this invention can comprehensively cover the specific region of 3rd chromosome of *Imperata cylindrica*.
[0006] In this invention, the amplification products of the primer pairs are preferably 146bp to 150bp in size.
[0007] The present invention also provides the application of the primer set described in the above technical solution in detecting or identifying the bloodline of chromosome 3 in sugarcane.
[0008] The present invention also provides a kit comprising the primer set described in the above technical solution.
[0009] In this invention, positive sample DNA and negative sample DNA are also preferably included.
[0010] In this invention, the positive sample DNA is preferably leaf DNA containing chromosome 3 lineage of Imperata cylindrica; the negative sample DNA is preferably leaf DNA not containing chromosome 3 lineage of Imperata cylindrica.
[0011] The present invention also provides the application of the kit described in the above technical solution in detecting or identifying chromosome 3 lineage in sugarcane.
[0012] This invention also provides a method for detecting or identifying chromosome 3 lineage in sugarcane, comprising the following steps: (1) Extract genomic DNA from the sample to be tested; (2) Using the extracted genomic DNA as a template, perform PCR amplification using the primer set or the kit described in the above technical solution; (3) The PCR amplification products were detected by gel electrophoresis; (4) Analyze the bands in the gel electrophoresis.
[0013] In this invention, the PCR amplification reaction system in step (2) is preferably prepared as one reaction system for each primer pair: each 25 μL reaction system contains 12.5 μL of 2× Rapid Taq Master Mix, 2.5 μL of 50 ng / μL DNA template, 2.5 μL of 10 μM upstream and downstream primers, and the remainder is ddH2O.
[0014] In this invention, the preferred amplification program for PCR amplification in step (2) is: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 60℃ annealing for 20 s, 72℃ extension for 15 s, 32 cycles; 72℃ final extension for 3 min; storage at 12℃.
[0015] In this invention, the gel electrophoresis detection in step (3) is preferably performed using low-melting-point agarose with a mass-to-volume ratio of 2.0%.
[0016] In this invention, the analytical gel electrophoresis bands are preferably determined by the presence of a specific band at the 146bp~150bp position on the gel, which indicates that the sugarcane material being tested contains the bloodline of chromosome 3 of Imperata cylindrica; otherwise, it does not contain the bloodline of chromosome 3 of Imperata cylindrica.
[0017] Beneficial Technical Effects: This invention provides a segmented PCR primer set for detecting chromosome 3 lineage in sugarcane and its application. The primer set provided by this invention includes 15 primer pairs, with nucleotide sequences shown in Seq_1~Seq_30. The primer set, kit, and method of this invention can accurately detect whether sugarcane-jumbo hybrids contain a specific region of chromosome 3 lineage. It has advantages such as high sensitivity, strong specificity, low cost, and simplicity and speed, providing technical support for the identification of lineage in hybrids of sugarcane and wild *Imperata cylindrica*, as well as basic research on chromosome structural variations in *Imperata cylindrica*. Attached Figure Description
[0018] Figure 1 Electrophoresis diagram for bloodline identification of specific regions of chromosome 3 of *Imperata cylindrica*. Lane M represents the relative molecular mass of 100bp DNA gradient standard, lane 1 represents nuclease-free sterile water, lanes 2-7 represent sugarcane leaf samples of *Bardila*, LA-Purple, Np-X, SES208, Zhongzhe No. 1, and Xintai Sugar No. 22, respectively, and lanes 8-10 represent sugarcane leaf samples of Hainan 92-77, Yunnan 2012-3, and Yacheng 05-164, respectively. Figure 2 Electrophoretic images of different segments of chromosome 3 in the Yacheng 01-92 and Yacheng 01-36 materials were obtained by detecting the bloodline of 15 primer pairs. Detailed Implementation
[0019] To better understand the present invention, the following embodiments further illustrate the content of the present invention, but the content of the present invention is not limited to the following embodiments. Unless otherwise specified, the materials, reagents, etc., used in the embodiments and experimental examples of the present invention can be obtained commercially; unless otherwise specified, the methods used in the embodiments and experimental examples of the present invention are conventional methods.
[0020] The main reagents used in this invention are as follows: (All chemical reagents are of analytical grade) (1) CTAB extraction buffer: 100mM Tris-HCl (pH=8.0), 20mM EDTA-Na2, 1.4M NaCl, 2% CTAB by volume, and 0.1% β-mercaptoethanol by volume before use; (2) 1×TAE buffer: 242g Tris, 37.2g EDTA-Na2, 57.1mL glacial acetic acid, add pure water to make up to 1L; (3) PCR amplification reagents were purchased from Nanjing Novizan Biotechnology Co., Ltd.
[0021] The main instruments used in this invention are as follows: (1) PCR amplification instrument: Singapore T100 TM Thermal Cycler PCR Amplification Instrument; (2) Electrophoresis apparatus: DYY-6C electrophoresis apparatus from Beijing Liuyi Biotechnology Co., Ltd.; (3) Centrifuge: Heraeus Pico 17 high-speed centrifuge from Germany; (4) Gel imaging system: Omega Fluor (USA) plus Gel imaging system; (5) Spectrophotometer: Mona (Suzhou) Biotechnology Co., Ltd. Eva 3200 Ultra-micro Nucleic Acid and Protein Detector.
[0022] The materials used in this invention are shown in Table 1: Table 1 Clones of Imperata cylindrica and sugarcane Example 1 1. Total DNA extraction from sugarcane leaves (1) Prepare tools: Clean and disinfect the mortar and scissors. The scissors are disinfected with 75% alcohol, while the mortar is disinfected by burning with anhydrous ethanol. After that, place it on ice to cool before use. (2) Sample processing: Fresh and healthy young leaves of seedlings of Badila, LA-Purple, Np-X, SES208, Zhongzhe No. 1, Xintai Sugar No. 22, Hainan 92-77, Yunnan 2012-3 and Yacheng 05-164 planted in the planting resource nursery of Guangxi University Fusui Base were selected. The leaf surface was disinfected by wiping with 75% alcohol cotton balls. After removing the leaf veins, the leaves were chopped and placed in a pre-cooled mortar. Liquid nitrogen was poured in and the leaves were quickly ground into fine powder. Generally, the best time is when the color changes from dark green to light green and white. The powder was placed in a 2mL centrifuge tube, which occupies about 1 / 3 of the tube volume. (3) Cell lysis: The prepared 2×CTAB was preheated in a water bath at 65°C for 30 minutes. 900 μL of the preheated CTAB extract and 100 μL of anhydrous ethanol were added to the powder. The mixture was quickly shaken and mixed. The mixture was then placed in a water bath at 65°C for 0.5 to 1 hour. The mixture was taken out every 10 minutes and inverted to mix, ensuring that the cells were fully lysed. (4) Chloroform extraction: After water bath, take out and centrifuge for 10 min. Add an equal volume of phenol:chloroform:isoamyl alcohol (volume ratio: 25:24:1) lower phase separation solution to the supernatant, mix thoroughly on a shaker, remove the precipitate by centrifugation for 10 min, add an equal volume of chloroform:isoamyl alcohol (volume ratio: 24:1) solution to the supernatant, shake several times to mix, centrifuge for 10 min, and the centrifugation speed is 12000 rpm each time; (5) Precipitate DNA: Add 1 / 10 of the volume of the supernatant from the previous step of 3M NaAc (pH=5.2) solution and an equal volume of pre-cooled isopropanol solution, mix them thoroughly, and let them stand at -20℃ for 1 hour. (6) Washing DNA: After precipitation, centrifuge for 10 min, add 1 mL of 75% ethanol solution to rinse the precipitate twice. After the first rinse, centrifuge for 5 min and then rinse a second time. It is best to use pre-cooled ethanol solution. (7) Purify DNA: After rinsing, centrifuge for 10 min and retain the precipitate. Let it stand at room temperature until the ethanol evaporates. Finally, add 100 μL of TE Buffer and 1 μL of RNase A and purify in a water bath at 37°C for 30 min to remove RNA. (8) After testing the DNA quality and concentration with a nucleic acid protein detector, store at 4°C for later use, or store at -20°C for long-term storage.
[0023] 2. Primer design First, the genomes of the tropical species *LA-Purple* (obtained by sequencing DNA extracted using the total DNA extraction method described above, hereinafter the same), *Dendrobium nobile* Np-X, and *Imperata cylindrica* Hainan 92-77 were masked for whole-genome repetitive sequences using Repeatmasker software. Chromosome 3 of the three genomes was then cut using the K-mers method, with the cutting performed via a Python script. The cut fragments were aligned using bwa software, and a 150bp sequence set specific to *Imperata cylindrica* was selected. Primers were designed for the *Imperata cylindrica*-specific sequence set using Primer3. The primer design parameters used the software's default values with some modifications, including: fragment size approximately 17bp~23bp, primer output quantity 1, and target product 146bp~150bp. The filtered sequences were then used to design primers. Specific primer sequences are shown in Table 2.
[0024] Table 2. Primer sequences and amplification products for specific regions of chromosome 3 of *Imperata cylindrica*. 3. PCR amplification Each primer pair was prepared as a separate reaction system. The PCR reaction system consisted of the following components per 25 μL reaction volume: 12.5 μL of 2×Rapid Taq Master Mix, 2.5 μL of 50 ng / μL DNA template, 2.5 μL each of 10 μM forward and reverse primers, and the remainder being ddH2O.
[0025] After adding the reagents from the above reaction system to the PCR tube, mix well, centrifuge at 8000 rpm for 5 seconds, and then perform the amplification program as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 60℃ annealing for 20 s, 72℃ extension for 15 s, 32 cycles; 72℃ final extension for 3 min; store at 12℃.
[0026] 4. Electrophoresis detection After the amplification reaction, 10 µL of PCR product was subjected to low-melting-point agarose gel electrophoresis at a volume ratio of 2.0%. Electrophoresis was performed at a constant voltage of 120 V for 30 min in 1×TAE electrophoresis buffer. The gel was then observed and photographed using a gel imaging system. The presence of PCR product amplification bands in the electrophoresis detection image indicated that the sample contained a specific region of chromosome 3 of the 'Pistacia chinensis' lineage; the absence of PCR product amplification bands indicated that the sample did not contain a specific region of chromosome 3 of the 'Pistacia chinensis' lineage.
[0027] Electrophoresis results as follows Figure 1 As shown in the figure. Each electrophoresis image is a specific identification of blood relations at different segments of chromosome 3 of *Imperata cylindrica*. In the image, the samples show PCR product amplification bands at approximately 150 bp in lanes 8-10, indicating that these samples contain blood relations corresponding to a specific segment of chromosome 3 of *Imperata cylindrica*. Figure 1 No PCR product amplification bands were observed in lanes 2-7 (negative control), indicating that these samples do not contain bloodlines for a specific region of chromosome 3. Figure 1 No PCR product restriction bands were observed in lane 1 (sterile water without nuclease), indicating that the experiment was not contaminated. Overall, this invention can effectively distinguish the bloodline of chromosome 3 of *Imperata cylindrica* from other bloodline materials that do not contain specific regions of *Imperata cylindrica*.
[0028] Experiment 1 used primer set detection to identify bloodline materials containing different segments of chromosome 3 of *Imperata cylindrica*. According to reports (Fan Yu, Zehuai Yu, Jin Chai, et al. Intergeneric chromosome-specific painting reveals differential chromosomal transmission from Tripidium arundinaceum(in sugarcane progeny, Journal of Integrative Agriculture, 2024, 23, 11: 3751-3762.), the genomic DNA of materials Yacheng 01-92 and Yacheng 01-36 contains the bloodline of chromosome 3 of Imperata cylindrica.
[0029] Genomic DNA containing chromosome 3 of *Imperata cylindrica* chromosome 3 was extracted from the BC1 offspring of the *Imperata cylindrica* hybridization, specifically from the materials Yacheng 01-92 and Yacheng 01-36. Its OD... 260 / OD 280 DNA with a ratio between 1.6 and 1.8 meets the experimental requirements. The concentration is determined and diluted to 50 ng / μL for aliquoting as template DNA.
[0030] Accuracy of primer pairs 1-15 for different segments of chromosome 3 in *Imperata cylindrica* was determined. PCR detection was performed using sugarcane and *Imperata cylindrica* BC1 generation materials, Yacheng 01-92 and Yacheng 01-36, which were confirmed by FISH to contain chromosome 3 lineage. Results showed that the target band was amplified in all of these clones, indicating that primer pairs 1-15 have good accuracy and can be used to detect different segments of chromosome 3 lineage in high-generation sugarcane-*Imperata cylindrica* hybrids.
[0031] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A segmented PCR primer set for detecting chromosome 3 lineage in sugarcane, characterized in that, The primer set includes: The nucleotide sequence is shown in the primer pair Chr03A_6615182_6615331 as shown in Seq_1 to Seq_2; The nucleotide sequence is shown in the primer pair Chr03A_6707952_6708101 as shown in Seq_3~Seq_4; The nucleotide sequence is shown in the primer pair Chr03A_16870858_16871007 as shown in Seq_5~Seq_6; The nucleotide sequence is shown in the primer pair Chr03A_36879343_36879492 as shown in Seq_7~Seq_8; The nucleotide sequence is shown in Seq_9~Seq_10. Primer pair Chr03A_36909166_36909315; The nucleotide sequence is shown in Seq_11~Seq_12. Primer pair Chr03A_46161635_46161784; The nucleotide sequence is shown in Seq_13~Seq_14 for the primer pair Chr03A_56134768_56134917; The nucleotide sequence is shown in Seq_15~Seq_16. Primer pair Chr03A_56812568_56812717; The nucleotide sequence is shown in Seq_17~Seq_18. Primer pair Chr03A_76146780_76146929; Nucleotide sequences are shown in the primer pairs Chr03A_86348649_86348798 as shown in Seq_19~Seq_20; Nucleotide sequences are shown in the primer pair Chr03A_86684289_86684438 as shown in Seq_21~Seq_22; The nucleotide sequence is shown in the primer pair Chr03A_96055692_96055841 in Seq_23-Seq_24; The nucleotide sequence is shown in the primer pair Chr03A_96148256_96148405 as shown in Seq_25~Seq_26; The nucleotide sequence is shown in the primer pair Chr03A_96329050_96329199 in Seq_27 to Seq_28; The nucleotide sequence is shown in the primer pair Chr03A_96535855_96536004 in Seq_29 to Seq_30.
2. The application of the primer set according to claim 1 in detecting or identifying chromosome 3 lineage in sugarcane.
3. A reagent kit, characterized in that, The kit includes the primer set as described in claim 1.
4. The reagent kit according to claim 3, characterized in that, It also includes positive sample DNA and negative sample DNA.
5. The use of the kit according to claim 3 or 4 in detecting or identifying chromosome 3 lineage in sugarcane.
6. A method for detecting or identifying chromosome 3 lineage in sugarcane, characterized in that, Includes the following steps: (1) Extract genomic DNA from the sample to be tested; (2) Using the extracted genomic DNA as a template, perform PCR amplification using the primer set described in claim 1 or the kit described in claims 3-4; (3) The PCR amplification products were detected by gel electrophoresis; (4) Analyze the bands in the gel electrophoresis.
7. The method according to claim 6, characterized in that, The PCR amplification reaction system in step (2) is prepared as one reaction system for each primer pair: each 25 μL reaction system contains 12.5 μL of 2×Rapid Taq Master Mix, 2.5 μL of 50 ng / μL DNA template, 2.5 μL each of 10 μM upstream and downstream primers, and the remainder is ddH2O.
8. The method according to claim 6, characterized in that, The PCR amplification program in step (2) is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 60℃ annealing for 20 s, 72℃ extension for 15 s, 32 cycles; 72℃ final extension for 3 min; storage at 12℃.
9. The method according to claim 6, characterized in that, The gel electrophoresis detection in step (3) is performed using low-melting-point agarose at a mass-volume ratio of 2.0%.
10. The method according to any one of claims 6 to 9, characterized in that, The analysis of gel electrophoresis bands is based on the presence of a specific band at the 146bp to 150bp position on the gel. If such a band appears, it indicates that the sugarcane material being tested contains chromosome 3 lineage; otherwise, it does not.