Primer combination for identifying consanguinity of saccharum arundinaceum chromosome 2 contained in sugarcane and application of primer combination

Abstract

The invention provides a primer combination for identifying consanguinity of saccharum arundinaceum chromosome 2 and application of the primer combination, and belongs to the technical field of plant consanguinity identification. The primer combination provided by the invention comprises 14 primer pairs, and every 30Mb of specific primers in a chromosome 2 interval of saccharum arundinaceum at least comprises one primer pair. The method overcomes the defects of a traditional sugarcane and saccharum arundinaceum filial generation consanguinity detection and identification method, can accurately identify whether a sample to be detected contains a specific consanguinity interval of the saccharum arundinaceum chromosome 2, has the advantages of high sensitivity, strong specificity, low cost, simplicity, convenience, rapidness and the like, and can be widely applied to detection and identification of the consanguinity of the saccharum arundinaceum filial generation. The method can be used for blood relationship identification and high-throughput rapid detection of a specific interval of the saccharum arundinaceum chromosome 2 in a sugarcane and saccharum arundinaceum filial generation.

Description

Technical Field

[0001] This invention relates to the field of plant lineage identification technology, and in particular to a primer combination for identifying sugarcane containing chromosome 2 of Imperata cylindrica and its application. Background Technology

[0002] Sugarcane (Saccharum spp.) is an important sugar crop in my country. The narrow genetic background of modern sugarcane cultivars severely limits the efficiency of genetic improvement. Discovering and utilizing wild sugarcane germplasm resources is an effective way to improve sugarcane genetics. Tripidium arundinaceum is an important wild sugarcane germplasm, characterized by high tillering, disease resistance, and drought resistance. In recent years, breeders have gradually used it for hybridization, hoping to infiltrate its superior traits into sugarcane. However, the precise chromosomal segments of Tripidium arundinaceum chromosomes that have infiltrated into the sugarcane genetic background remain unclear.

[0003] Molecular markers can visualize the genomic structure of individuals or populations, reflecting genetic diversity and variability at the deoxyribonucleic acid (DNA) level. DNA molecular marker technology is simple to implement, highly efficient, and unaffected by tissue location, developmental stage, season, climate, or environment. It is a highly efficient technique for assessing the genetic diversity of germplasm resources and has been widely applied in research fields such as crop germplasm screening, genetic polymorphism analysis, and the localization of resistance genes. Hu Wenbin et al., based on reference genome data from dragon fruit, designed 10 specific primers for each chromosome pair, successfully developing 41 pairs of stable and efficient molecular markers. They used four of these primer pairs for variety identification with an accuracy rate of 100%. Guo Xiaojiao et al., using QTL mapping, discovered that different chromosome numbers contribute differently to panicle length in rice. In wheat, Qiu et al. also found the influence of different chromosome translocation lines on the superior quality of wheat. Therefore, different chromosome numbers carry different genes, resulting in significant differences in their phenotypic effects on crops. Based on previous work in bioinformatics, this invention patent designs specific primers according to the specific repetitive sequences of chromosome 2 of *Imperata cylindrica*. By combining the high sensitivity of PCR detection methods with the excellent amplification performance of rapid PCR mixtures, a rapid detection kit and detection method have been developed to identify the bloodline of sugarcane-*Imperata cylindrica* hybrid offspring containing a specific region of chromosome 2 of *Imperata cylindrica*. This aims to provide technical support for basic research on the bloodline identification of hybrid offspring of *Imperata cylindrica* introduced from wild germplasm resources of sugarcane in my country, as well as on chromosome structural variations in *Imperata cylindrica*. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a primer combination for identifying bloodlines containing chromosome 2 of *Imperata cylindrica* in sugarcane and its application. The primer combination provided by this invention can accurately identify bloodlines containing specific regions of chromosome 2 of *Imperata cylindrica*.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A primer set for identifying sugarcane containing chromosome 2 lineage of Imperata cylindrica, comprising 14 primer pairs, the specific names and sequences of which are shown below:

[0007] (1)Chr02A_5382939_5383088:

[0008] Seq_1: 5'-TTATCTCGTTGAGCACTATCCTTAG-3';

[0009] Seq_2: 5'-GTTGAACAAGACAGTACTCATGCT-3';

[0010] (2)Chr02A_15228184_15228333:

[0011] Seq_3: 5'-AATTTGGTGGATGAATAGAAGATGGA-3';

[0012] Seq_4: 5'-TACTTCTCTATAGTTCATTGCCAGCA-3';

[0013] (3)Chr02A_15385351_15385500:

[0014] Seq_5: 5'-CCATCATTCATCAATAGGTGTTGGG-3';

[0015] Seq_6: 5'-TCCGTGTATTTCTAGCATAACTCCA-3':

[0016] (4)Chr02A_15940369_15940518:

[0017] Seq_7: 5'-GTTGGTGTTGAGGTTGGTATTAAAT-3';

[0018] Seq_8: 5'-CATCCATATGTGTGTCCTTTGTTC-3';

[0019] (5)Chr02A_25206017_25206166:

[0020] Seq_9:5’-ATAACTGAGCTTGTATTGTGCATAGA-3’;

[0021] Seq_10:5’-TACCAGGAACAAGATGCACTACA-3’;

[0022] (6)Chr02A_35490726_35490875:

[0023] Seq_11:5’-TGAAATGACTACCTGATATCTCATCAA-3’;

[0024] Seq_12:5’-TGAGACTGGTATCAAGCTAGAACC-3’;

[0025] (7)Chr02A_40499476_40499625:

[0026] Seq_13:5’-ACCATCTACTTACTGCTACGACTAC-3’;

[0027] Seq_14:5’-GTATCAAGCAACATCTCAACACAGT-3’;

[0028] (8)Chr02A_53484931_53485080:

[0029] Seq_15:5’-CATATAGCTTTGTTCATATTAGGCTGATG-3’;

[0030] Seq_16:5’-ATCAATGAGCTGAGGTACCATTTGT-3’;

[0031] (9)Chr02A_57737745_57737894:

[0032] Seq_17:5’-ATGGCAAGGATCCGAGTTATTATGA-3’;

[0033] Seq_18:5’-AGACTTAGAGTATGTGGATAATGAGCC-3’;

[0034] (10)Chr02A_69111689_69111838:

[0035] Seq_19:5’-ATGGTCAAGTGGCTAAGTAATTGA-3’;

[0036] Seq_20:5’-AGCCAATCATCACTATCACAAAGG-3’;

[0037] (11)Chr02A_87475754_87475903:

[0038] Seq_21:5’-CCTAGAGACTTGGATCCACGAA-3’;

[0039] Seq_22:5’-ACAAGCATCCAATTCATACCACTTT-3’;

[0040] (12)Chr02A_94878142_94878291:

[0041] Seq_23:5’-AATCAATGATCATGTAGGACCTTAGAG-3’;

[0042] Seq_24:5’-CGTCACTGTGGAGGTTTGTCTTA-3’;

[0043] (13)Chr02A_100601923_100602072:

[0044] Seq_25:5’-TTACATAATACGCTTCTCACATGTTATATG-3’;

[0045] Seq_26:5’-TACTTACCTAATGTCCCTGATAGAAGAA-3’;

[0046] (14)Chr02A_110776583_110776732;

[0047] Seq_27:5’-CGGCTAGTCATATGCAGATAACTACT-3’;

[0048] Seq_28:5’-ATGATTGTCCGATGGCTATATAAATATACT-3’。

[0049] In this invention, the amplification product of primer pair (1) is preferably located at position 5382939-5383088 on chromosome 2; the amplification product of primer pair (2) is preferably located at position 15228184-15228333 on chromosome 2; the amplification product of primer pair (3) is preferably located at position 15385351-15385500 on chromosome 2; and the amplification product of primer pair (4) is preferably located at position 15385351-15385500 on chromosome 2. The preferred positions are 15940369_15940518; the preferred positions of the amplification products of primer pair (5) corresponding to chromosome 2 of *Imperata cylindrica* are 25206017_25206166; the preferred positions of the amplification products of primer pair (6) corresponding to chromosome 2 of *Imperata cylindrica* are 35490726_35490875; the preferred positions of the amplification products of primer pair (7) corresponding to chromosome 2 of *Imperata cylindrica* are 40499476_40499625; primers The preferred position of the amplification product of primer pair (8) corresponding to chromosome 2 of *Imperata cylindrica* is 53484931_53485080; the preferred position of the amplification product of primer pair (9) corresponding to chromosome 2 of *Imperata cylindrica* is 57737745_57737894; the preferred position of the amplification product of primer pair (10) corresponding to chromosome 2 of *Imperata cylindrica* is 69111689_69111838; the preferred position of the amplification product of primer pair (11) corresponding to chromosome 2 of *Imperata cylindrica* is... The amplification products of primer pair (12) are preferably located at the position of chromosome 2 of *Imperata cylindrica* 94878142_94878291; the amplification products of primer pair (13) are preferably located at the position of chromosome 2 of *Imperata cylindrica* 100601923_100602072; the amplification products of primer pair (14) are preferably located at the position of chromosome 2 of *Imperata cylindrica* 110776583_110776732.

[0050] In this invention, the amplification products of the 14 primer pairs are preferably 146bp to 150bp in size.

[0051] The present invention also provides the application of the primer combination described in the above technical solution in identifying or assisting in the identification of chromosome 2 lineage in sugarcane.

[0052] The present invention also provides the application of the primer combination described in the above technical solution in the preparation of products for identifying or assisting in the identification of chromosome 2 lineage in sugarcane.

[0053] In this invention, the product preferably includes, but is not limited to, a reagent kit.

[0054] In this invention, the kit preferably also includes Rapid Taq Master Mix, a positive control, and a negative control.

[0055] In this invention, the positive control is preferably sugarcane leaf DNA containing chromosome 2 lineage; the negative control is preferably sugarcane leaf DNA not containing chromosome 2 lineage.

[0056] The present invention also provides an application of the primer combination described in the above technical solution in sugarcane-assisted breeding.

[0057] This invention also provides a method for identifying or assisting in the identification of chromosome 2 lineage in sugarcane, comprising the following steps:

[0058] (1) Extract genomic DNA from the sample to be tested;

[0059] (2) Using the genomic DNA in (1) as a template, perform PCR amplification using the primer combination described in the above technical solution to obtain the amplification product;

[0060] (3) Perform gel electrophoresis on the PCR products in (2) and count the electrophoresis results;

[0061] (4) Based on the electrophoresis results in (3), determine whether the sample to be tested contains a specific region of chromosome 2 of the variegated chromosomal lineage.

[0062] In this invention, the preferred criterion for determining the method is: if a specific band appears at the 146bp to 150bp position on the gel, it can be determined that the sugarcane material to be tested contains the bloodline of chromosome 2 of Imperata cylindrica; otherwise, it does not contain the bloodline of chromosome 2 of Imperata cylindrica.

[0063] In this invention, the PCR reaction system 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 each of 10 μM upstream and downstream primers, and the remainder is ddH2O.

[0064] In this invention, the preferred PCR amplification program is: 95°C pre-denaturation for 3 min; 95°C denaturation for 30 s, 60°C annealing for 20 s, 72°C extension for 15 s, 32 cycles; 72°C final extension for 3 min; and storage at 12°C.

[0065] Beneficial Technical Effects: This invention provides a primer set for identifying the presence of chromosome 2 kinship in sugarcane and its application. The primer set provided by this invention contains 14 primer pairs, with at least one primer pair for each 30Mb region specific to chromosome 2 kinship. This invention overcomes the shortcomings of traditional methods for detecting and identifying kinship in sugarcane-citrus hybrid offspring. Using the primer set and method provided by this invention, it is possible to accurately identify whether the sample contains a specific kinship region of chromosome 2 kinship. It has the advantages of high sensitivity, strong specificity, low cost, simplicity, and speed, and can be used for identifying kinship in specific regions of chromosome 2 kinship in sugarcane-citrus hybrid offspring as well as for high-throughput rapid detection. Attached Figure Description

[0066] Figure 1 Electrophoresis diagram for bloodline identification of specific regions of chromosome 2 of Banmao. Lane M is the relative molecular mass of 100bp DNA gradient standard, lane 1 is nuclease-free sterile water, lanes 2-7 are sugarcane leaf samples of Baldila, LA-Purple, Np-X, SES208, Zhongzhe No. 1 and ROC22 respectively, and lanes 8-10 are sugarcane leaf samples of Hainan 92-77, Yunnan 2012-3 and Yacheng 05-164 respectively.

[0067] Figure 2 Electrophoretic images of different segments of chromosome 2 in Yacheng 01-92 and Yacheng 01-36 materials were obtained by detecting blood relations using 14 primer pairs. Detailed Implementation

[0068] 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.

[0069] Example 1

[0070] 1. Materials, Reagents and Instruments

[0071] 1.1 Materials

[0072] As shown in Table 1.

[0073] Table 1 Sample Information

[0074]

[0075] 1.2 Reagents

[0076] (1) CTAB extraction buffer: 100mM Tris-HCl (pH 8.0), 20mM EDTA-Na2, 1.4M NaCl, 2% CTAB by volume, add 0.1% β-mercaptoethanol by volume before use;

[0077] (2) 1×TAE buffer: 242g Tris, 37.2g EDTA-Na2, 57.1mL glacial acetic acid, add pure water to make up to 1L;

[0078] (3) PCR amplification reagents were purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0079] 1.3 Instruments

[0080] (1) PCR amplification instrument: Singapore T100TM Thermal Cycler PCR amplification instrument;

[0081] (2) Electrophoresis apparatus: DYY-6C electrophoresis apparatus from Beijing Liuyi Biotechnology Co., Ltd.;

[0082] (3) Centrifuge: Heraeus Pico 17 high-speed centrifuge from Germany;

[0083] (4) Gel imaging system: Omega Fluor plus gel imaging system (USA);

[0084] (5) Spectrophotometer: Mona (Suzhou) Biotechnology Co., Ltd. Eva 3200 Ultra-micro Nucleic Acid and Protein Detector.

[0085] 2. Methods and Results

[0086] 2.1 Total DNA extraction from sugarcane leaves

[0087] (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 them on ice to cool before use.

[0088] (2) Sample processing: Fresh and healthy young leaves of seedlings of Badila, LA-Purple, Np-X, SES208, Zhongzhe No. 1, Xintai Sugar No. 22 (ROC22), 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.

[0089] (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 h to 1 h. The mixture was taken out every 10 minutes and inverted to mix, ensuring that the cells were fully lysed.

[0090] (4) Chloroform extraction: After water bath, take out and centrifuge for 10 min. Add an equal volume of phenol:chloroform:isoamyl alcohol (volume ratio of 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 of 24:1) solution to the supernatant, shake several times to mix, centrifuge for 10 min, and the centrifugation speed is 12000 rpm each time.

[0091] (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.

[0092] (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.

[0093] (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.

[0094] (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.

[0095] 2.2 Primer Design

[0096] 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 2 of the three genomes was then cut using the K-mers method, with the genome cutting performed via a Python script. The cut fragments were aligned using bwa software, and a set of 150bp fragments 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 of 1, and target product size of 146bp–150bp. The filtered sequences were then used to design primers. Specific primer names, primer sequences, and amplification products are shown in Table 2.

[0097] Table 2. Primer sequences and amplification sequences for specific regions of chromosome 2 of *Imperata cylindrica*.

[0098]

[0099]

[0100]

[0101] 2.3 PCR amplification

[0102] The reaction systems prepared for each primer pair are shown in Table 3.

[0103] Table 3 PCR reaction system

[0104]

[0105] After adding the reagents in Table 3 to the PCR tube, mix well, centrifuge at 8000 rpm for 5 seconds, and then the amplification program 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; store at 12℃.

[0106] 2.4 Electrophoresis detection

[0107] After the amplification reaction, 10 μL of the 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 120V for 30 min in 1×TAE electrophoresis buffer. The gel was then observed and photographed using a gel imaging system. The presence of PCR amplification bands in the electrophoresis results indicated that the sample contained a specific region of chromosome 2 (Pseudomonas aeruginosa); the absence of PCR amplification bands indicated that the sample did not contain a specific region of chromosome 2 (Pseudomonas aeruginosa). The amplified products were sent to a sequencing company for sequencing, and the specific sequences are shown in Table 2.

[0108] Electrophoresis results as follows Figure 1 As shown. Figure 1 The electrophoresis diagram shows the specific identification of blood relations at different segments of chromosome 2 of *Imperata cylindrica*. In the diagram, the samples show PCR product amplification bands at approximately 150 bp in lanes 8-10, indicating that these samples contain blood relations corresponding to specific segments of chromosome 2 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 in a specific region of chromosome 2. Figure 1 The absence of PCR product restriction bands in lane 1 (sterile water without nuclease) indicates that the experiment was not contaminated. Overall, this kit can effectively distinguish the bloodline of chromosome 2 of *Imperata cylindrica* from other bloodline materials that do not contain specific regions of *Imperata cylindrica*.

[0109] Example 1 used primer sets to detect bloodline materials that had been identified as containing different segments of chromosome 2 of the variegated iris.

[0110] According to reports (Fan Yu, Zehuai Yu, Jin Chai, et al. Intergeneric chromosome-specific painting reveals differential chromosomal transmission from Tripidium arundinaceumin 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 Tripidium arundinaceumin sugarcane progeny.

[0111] Genomic DNA containing chromosome 2 of *Imperata cylindrica* chromosome 2 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.

[0112] Accuracy of primer pairs 1-14 for different segments of chromosome 2 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 2 lineage. Results showed that the target bands were amplified in all the clones, indicating that primer pairs 1-14 have good accuracy and can be used to detect different segments of chromosome 2 lineage in high-generation sugarcane-*Imperata cylindrica* hybrids.

[0113] 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 primer combination for identifying sugarcane containing chromosome 2 lineage, characterized in that, The primer combination contains 14 primer pairs, the specific names and sequences of which are shown below: (1)Chr02A_5382939_5383088: Seq_1: 5'-TTATCTCGTTGAGCACTATCCTTAG-3'; Seq_2: 5'-GTTGAACAAGACAGTACTCATGCT-3'; (2)Chr02A_15228184_15228333: Seq_3: 5'-AATTTGGTGGATGAATAGAAGATGGA-3'; Seq_4: 5'-TACTTCTCTATAGTTCATTGCCAGCA-3'; (3)Chr02A_15385351_15385500: Seq_5: 5'-CCATCATTCATCAATAGGTGTTGGG-3'; Seq_6: 5'-TCCGTGTATTTCTAGCATAACTCCA-3': (4)Chr02A_15940369_15940518: Seq_7: 5'-GTTGGTGTTGAGGTTGGTATTAAAT-3'; Seq_8: 5'-CATCCATATGTGTGTCCTTTGTTC-3'; (5)Chr02A_25206017_25206166: Seq_9: 5'-ATAACTGAGCTTGTATTGTGCATAGA-3'; Seq_10: 5'-TACCAGGAACAAGATGCACTACA-3'; (6)Chr02A_35490726_35490875: Seq_11: 5'-TGAAATGACTACCTGATATCTCATCAA-3'; Seq_12: 5'-TGAGACTGGTATCAAGCTAGAACC-3'; (7)Chr02A_40499476_40499625: Seq_13: 5'-ACCATCTACTTACTGCTACGACTAC-3'; Seq_14: 5'-GTATCAAGCAACATCTCAACACAGT-3'; (8)Chr02A_53484931_53485080: Seq_15: 5'-CATATAGCTTTGTTCATATTAGGCTGATG-3'; Seq_16: 5'-ATCAATGAGCTGAGGTACCATTTGT-3'; (9)Chr02A_57737745_57737894: Seq_17: 5'-ATGGCAAGGATCCGAGTTATTATGA-3'; Seq_18: 5'-AGACTTAGAGTATGTGGATAATGAGCC-3'; (10)Chr02A_69111689_69111838: Seq_19:5’-ATGGTCAAGTGGCTAAGTAATTGA-3’; Seq_20:5’-AGCCAATCATCACTATCACAAAGG-3’; (11)Chr02A_87475754_87475903: Seq_21:5’-CCTAGAGACTTGGATCCACGAA-3’; Seq_22:5’-ACAAGCATCCAATTCATACCACTTT-3’; (12)Chr02A_94878142_94878291: Seq_23:5’-AATCAATGATCATGTAGGACCTTAGAG-3’; Seq_24:5’-CGTCACTGTGGAGGTTTGTCTTA-3’; (13)Chr02A_100601923_100602072: Seq_25:5’-TTACATAATACGCTTCTCACATGTTATATG-3’; Seq_26:5’-TACTTACCTAATGTCCCTGATAGAAGAA-3’; (14)Chr02A_110776583_110776732; Seq_27:5’-CGGCTAGTCATATGCAGATAACTACT-3’; Seq_28:5’-ATGATTGTCCGATGGCTATATAAATATACT-3’。 2. The primer combination according to claim 1, characterized in that, The amplification product of primer pair (1) corresponds to the position 5382939_5383088 on chromosome 2 of *Imperata cylindrica*; the amplification product of primer pair (2) corresponds to the position 15228184_15228333 on chromosome 2 of *Imperata cylindrica*; the amplification product of primer pair (3) corresponds to the position 15385351_15385500 on chromosome 2 of *Imperata cylindrica*; the amplification product of primer pair (4) corresponds to the position 15385351_15385500 on chromosome 2 of *Imperata cylindrica*. 15940369_15940518; the amplification product of primer pair (5) corresponds to the position of chromosome 2 of *Imperata cylindrica* 25206017_25206166; the amplification product of primer pair (6) corresponds to the position of chromosome 2 of *Imperata cylindrica* 35490726_35490875; the amplification product of primer pair (7) corresponds to the position of chromosome 2 of *Imperata cylindrica* 40499476_40499625; primer pair (8) The amplification product of primer pair (9) corresponds to the position of chromosome 2 of *Imperata cylindrica* 53484931_53485080; the amplification product of primer pair (9) corresponds to the position of chromosome 2 of *Imperata cylindrica* 57737745_57737894; the amplification product of primer pair (10) corresponds to the position of chromosome 2 of *Imperata cylindrica* 69111689_69111838; the amplification product of primer pair (11) corresponds to the position of chromosome 2 of *Imperata cylindrica* 87. 475754_87475903; the amplification product of primer pair (12) corresponds to the position of chromosome 2 of *Imperata cylindrica* 94878142_94878291; the amplification product of primer pair (13) corresponds to the position of chromosome 2 of *Imperata cylindrica* 100601923_100602072; the amplification product of primer pair (14) corresponds to the position of chromosome 2 of *Imperata cylindrica* 110776583_110776732.

3. The primer combination according to claim 1, characterized in that, The amplification products of the 14 primer pairs were all 146bp to 150bp in size.

4. The use of any primer combination according to claims 1 to 3 in identifying or assisting in the identification of bloodlines on chromosome 2 of Imperata cylindrica in sugarcane.

5. The use of a primer combination according to any one of claims 1 to 3 in the preparation of a product for identifying or assisting in the identification of chromosome 2 lineage in sugarcane.

6. The application according to claim 5, characterized in that, The products include, but are not limited to, reagent kits.

7. The application according to claim 6, characterized in that, The kit also includes Rapid Taq MasterMix, a positive control, and a negative control.

8. The application of any primer combination according to claims 1 to 3 in sugarcane breeding.

9. A method for identifying or assisting in the identification of chromosome 2 lineage in sugarcane, characterized in that, Includes the following steps: (1) Extract genomic DNA from the sample to be tested; (2) Using the genomic DNA in (1) as a template, perform PCR amplification using any of the primer combinations described in claims 1 to 3 to obtain the amplification product; (3) Perform gel electrophoresis on the PCR products in (2) and count the electrophoresis results; (4) Based on the electrophoresis results in (3), determine whether the sample to be tested contains a specific region of chromosome 2 of the variegated chromosomal lineage.

10. The method according to claim 7, characterized in that, The judgment criteria of the method are as follows: if a specific band appears at the position of 146bp to 150bp on the gel, it can be determined that the sugarcane material to be tested contains the bloodline of chromosome 2 of Imperata cylindrica. Conversely, they do not have the bloodline of chromosome 2 of the variegated spur.

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