A segmented PCR primer set for identifying blood relationship of chromosome 5 of erianthus arundinaceus and application thereof

Abstract

The application provides a segmented PCR primer group for identifying the blood relationship of chromosome 5 of Eragrostis poaeformis and application thereof, and belongs to the technical field of molecular biology. The segmented PCR primer group provided by the application comprises 16 primer groups, and the specific nucleotide sequences are shown as Seq_1-Seq_32. Each primer group comprises a forward primer and a reverse primer. The specific primer of the Eragrostis poaeformis chromosome 5 interval of the application at least comprises one pair of primers, the primer group and the method provided can quickly and comprehensively detect whether the hybrid offspring of sugarcane and Eragrostis poaeformis contains the blood relationship of the specific interval of the Eragrostis poaeformis chromosome 5, and provide technical support for the blood relationship identification of sugarcane and the breeding of sugarcane.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a segmented PCR primer set for identifying chromosome 5 lineage in sugarcane and its application. Background Technology

[0002] Sugarcane (Saccharum spp.) is widely cultivated in over 90 countries in tropical and subtropical regions, with China being the third largest sugarcane producer after Brazil and India. Sugarcane is a crucial raw material for producing food, feed, biofuels, and bioproducts, providing over 80% of the sugarcane for more than 100 countries worldwide. For a long time, sugarcane cultivars have suffered from narrow genetic backgrounds, varietal degeneration, and a lack of resistance to biotic and abiotic stresses. Global environmental change and increasing demand have further heightened the need for crop improvement. Utilizing wild genetic resources to improve varieties and enhance resistance to biotic and abiotic stresses while increasing biomass yield is a key objective of modern breeding.

[0003] *Imperata cylindrica*, a closely related wild species of sugarcane, is a key target for sugarcane breeders both domestically and internationally due to its numerous superior traits, including high biomass, robust growth, tolerance to poor soil and drought, resistance to pests and diseases, and wide adaptability. However, the precise chromosomal segments of *Imperata cylindrica* chromosomes infiltrated into the sugarcane genetic background remain unclear. Studies have shown that the clustering of backcross progeny from sugarcane containing *Imperata cylindrica* lineage does not exhibit a clear distribution pattern. As the backcross generations progress, the *Imperata cylindrica* genetic composition in the backcross progeny is diluted generation by generation, and some *Imperata cylindrica* molecular marker loci may be lost. Utilizing multi-locus molecular markers will help improve the efficiency of *Imperata cylindrica* lineage detection. Furthermore, Wang Qinnan et al. conducted pedigree evaluations on 10 parents of sugarcane containing *Imperata cylindrica* lineage, ultimately selecting Yacheng 11-31, Yacheng 06-140, and Yacheng 07-71 as productive parents. This indicates that there is a certain degree of phenotypic diversity in progeny materials containing different *Imperata cylindrica* chromosomal lineages. Therefore, developing multi-site molecular markers to detect the lineage of Imperata cylindrica in sugarcane and the specific chromosomal segments it contains is of great significance for improving the utilization efficiency of Imperata cylindrica. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a segmented PCR primer set for identifying the bloodline of chromosome 5 in sugarcane and its application. The primer set provided by this invention can quickly and accurately identify whether sugarcane samples contain the bloodline of chromosome 5 in sugarcane, providing technical support for sugarcane bloodline identification and sugarcane breeding.

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

[0006] A segmented PCR primer set for identifying chromosome 5 lineage in sugarcane, comprising 16 primer sets, with specific nucleotide sequences as shown in Seq_1~Seq_2 (Chr05A_3654658_3654807); Seq_3~Seq_4 (Chr05A_13255127_13255276); Seq_5~Seq_6 (Chr05A_13623777_13623926); and Seq_7~Seq_8 (Chr... 05A_19855186_19855335; specific nucleotide sequences are shown in Seq_9~Seq_10 as Chr05A_31139660_31139809; specific nucleotide sequences are shown in Seq_11~Seq_12 as Chr05A_41719808_41719957; specific nucleotide sequences are shown in Seq_13~Seq_14 as Chr05A_47116008_47116157; specific nucleotide sequences are shown in Seq_15~Seq_16 as Chr05A_47530334_475 30483; specific nucleotide sequences are shown in Seq_17~Seq_18 as Chr05A_55122419_55122568; specific nucleotide sequences are shown in Seq_19~Seq_20 as Chr05A_57957698_57957847; specific nucleotide sequences are shown in Seq_21~Seq_22 as Chr05A_64673377_64673526; specific nucleotide sequences are shown in Seq_23~Seq_24 as Chr05A_74321110_74321259; specific nucleotide sequences are shown in S The nucleotide sequences shown in eq_25~Seq_26 are Chr05A_74639055_74639204; the specific nucleotide sequences shown in eq_27~Seq_28 are Chr05A_103694448_103694597; the specific nucleotide sequences shown in eq_29~Seq_30 are Chr05A_111320492_111320641; the specific nucleotide sequences shown in eq_31~Seq_32 are Chr05A_120873438_120873587; each primer set includes a forward primer and a reverse primer.

[0007] In this invention, Seq_1 to Seq_50 are specifically as follows:

[0008] (1)Chr05A_3654658_3654807:

[0009] Seq_1:5’-TCGCTGATTCCAACTACACTGAG-3’;

[0010] Seq_2:5’-CCAGCTAAGAAGACACTGATCGA-3’;

[0011] (2)Chr05A_13255127_13255276:

[0012] Seq_3:5’-AGATTGTTACGGTTGTAGAATGGG-3’;

[0013] Seq_4:5’-CCAACACATCTCGTCAACAGAC-3’;

[0014] (3)Chr05A_13623777_13623926:

[0015] Seq_5:5’-CCAAATCAAGATGAAGGGTTGCA-3’;

[0016] Seq_6:5’-GTGACCATGAATACTTGAACACGAT-3’;

[0017] (4)Chr05A_19855186_19855335:

[0018] Seq_7:5’-TGTTGCGTAATACTCTCTTGCTTT-3’;

[0019] Seq_8:5’-TGAATGATATGACTCAAATGCTTCAGT-3’;

[0020] (5)Chr05A_31139660_31139809:

[0021] Seq_9:5’-TCGAACTTTGGATAAGCTTGGTTAG-3’;

[0022] Seq_10:5’-CACATGCACTTAGACACAGAGT-3’;

[0023] (6)Chr05A_41719808_41719957:

[0024] Seq_11:5’-TCATCTCATTACAAAGTCTGATCAGT-3’;

[0025] Seq_12:5’-CAACTAGCCAATGGATTTCACAAGT-3’;

[0026] (7)Chr05A_47116008_47116157:

[0027] Seq_13:5’-ACCATTCAAGATAGCTCAGGAACC-3’;

[0028] Seq_14:5’-TGAATGAGGTCCCTGAGGTTGAA-3’;

[0029] (8)Chr05A_47530334_47530483:

[0030] Seq_15:5’-TGCTGTTACCAAGATTTGCAAGG-3’;

[0031] Seq_16:5’-GTTCATCATAGTCTACCACAACAGC-3’;

[0032] (9)Chr05A_55122419_55122568:

[0033] Seq_17:5’-ATGTCTGTAGATTTGGAGTACACCC-3’;

[0034] Seq_18:5’-GATGTTGCTAAGTGGATGTATCAGG-3’;

[0035] (10)Chr05A_57957698_57957847:

[0036] Seq_19:5’-CGGACTGCTAGGCTAATCATACT-3’;

[0037] Seq_20:5’-AGATCAAGGAACATGTAGTAGTGCT-3’;

[0038] (11)Chr05A_64673377_64673526:

[0039] Seq_21:5’-GGGAAGACAGAGATCAAAGAACGA-3’;

[0040] Seq_22:5’-CAATGCGTCAATCCGATCTTCAG-3’;

[0041] (12)Chr05A_74321110_74321259:

[0042] Seq_23: 5'-TGAGACCAAATGCTACTAGTGTCAA-3';

[0043] Seq_24: 5'-CAGAACTGCCGTCATCTCCTTAT-3';

[0044] (13)Chr05A_74639055_74639204:

[0045] Seq_25: 5'-TCCTTTGGCTCTACTTCAGGAAAC-3';

[0046] Seq_26: 5'-CCTGCTTGTTGATTTGCTGAGTA-3';

[0047] (14)Chr05A_103694448_103694597:

[0048] Seq_27: 5'-TGCCATCGTCAGAAGTTCTAATGA-3';

[0049] Seq_28: 5'-AGTTCGATTCCTTTCTAGCTCCAT-3';

[0050] (15)Chr05A_111320492_111320641:

[0051] Seq_29: 5'-AGTCATCGAATTCAGCCACTACAA-3';

[0052] Seq_30: 5'-ATCCTCAGCACTTTGGGCAGTAT-3';

[0053] (16)Chr05A_120873438_120873587:

[0054] Seq_31: 5'-GAATTTAGTTATTATCATCGCTTCTGTTCT-3';

[0055] Seq_32: 5'-ACAGATAAAGTTACCATCGTCAACCT-3'.

[0056] In this invention, the amplification product size of the primer set is preferably 146bp to 150bp.

[0057] In this invention, each 30Mb of primers specific to the 5th chromosome of *Imperata cylindrica* contains at least one pair. The primer set of this invention can comprehensively cover the specific region of 5th chromosome of *Imperata cylindrica*.

[0058] In this invention, the two sets of numbers following the names of the 16 primer pairs indicate the location of the amplification product of that primer pair on chromosome 5 of the sclerotium.

[0059] The present invention also provides the application of the primer set described in the above technical solution in identifying the bloodline of chromosome 5 in sugarcane.

[0060] The present invention also provides an application of the primer set described in the above technical solution in sugarcane breeding.

[0061] The present invention also provides a kit, preferably comprising the primer set, PCR reaction solution, and control as described in the above technical solution.

[0062] In this invention, the control samples preferably include a positive control and a negative control; the positive control is preferably total DNA from leaves containing a specific region of chromosome 5 of Imperata cylindrica, and the negative control is preferably total DNA from leaves without a specific region of chromosome 5 of Imperata cylindrica.

[0063] In this invention, the PCR reaction solution preferably includes a rapid PCR mixture and nuclease-free sterile water.

[0064] In this invention, the preferred PCR reaction system of the kit is as follows: each 25 μL reaction system contains 12.5 μL of 2×RapidTaq 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; the preferred PCR reaction system is one reaction system prepared for each primer pair.

[0065] In this invention, the preferred PCR amplification program for the kit 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℃.

[0066] The present invention also provides the application of the kit described in the above technical solution in identifying the lineage of chromosome 5 in sugarcane.

[0067] The present invention also provides an application of the reagent kit described in the above technical solution in sugarcane breeding.

[0068] The present invention also provides a method for identifying chromosome 5 lineage in sugarcane, the method comprising PCR amplification of genomic DNA of the sample to be tested using the primer set or the kit described in the above technical solution.

[0069] In this invention, the preferred criterion for judgment is: if an amplification band appears in the PCR amplification product at the position of 146bp to 150bp, then the sample to be tested contains a bloodline of a specific region of chromosome 5 of the 'Pissula' chromosome; if no amplification band appears in the PCR amplification product at the position of 146bp to 150bp, then the sample to be tested does not contain a bloodline of a specific region of chromosome 5 of the 'Pissula' chromosome.

[0070] Beneficial Technical Effects: This invention provides a segmented PCR primer set for identifying chromosome 5 lineage in sugarcane and its application. The segmented PCR primer set includes 16 primer sets, with specific nucleotide sequences shown in Seq_1 to Seq_32. Each primer set includes a forward primer and a reverse primer. Each 30Mb segmented primer set for chromosome 5 lineage in this invention contains at least one primer pair. The provided primer set and method can rapidly and comprehensively detect whether sugarcane and *Imperata cylindrica* hybrids contain a specific segment of chromosome 5 lineage, providing technical support for sugarcane lineage identification and sugarcane breeding. Attached Figure Description

[0071] Figure 1 This is an electrophoresis image of a specific region of chromosome 5 of *Imperata cylindrica* for bloodline identification. In the image, M represents the relative molecular mass of a 100bp DNA gradient standard. Lane 1 is ddH2O, lane 2 is badila, lane 3 is LA-Purple, lane 4 is Np-X, lane 5 is SES208, lane 6 is Zhongzhe 01, lane 7 is ROC22, lane 8 is Hainan 92-77, lane 9 is Yunnan 2012-3, and lane 10 is Yacheng 05-164.

[0072] Figure 2 Electrophoretic images of different segments of chromosome 5 in the Yacheng 01-92 and Yacheng 01-36 materials were obtained by detecting blood relations using 16 primer pairs. Detailed Implementation

[0073] 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.

[0074] Materials and reagents:

[0075] 1. Materials: As shown in Table 1.

[0076] Table 1 Sampling Information

[0077]

[0078] 2. Main reagents: (All chemical reagents are of analytical grade)

[0079] (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;

[0080] (2) 1×TAE buffer: 242g Tris, 37.2g EDTA-Na2, 57.1mL glacial acetic acid, add pure water to make up to 1L;

[0081] (3) PCR amplification reagents were purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0082] The main instruments used in this invention are as follows:

[0083] (1) PCR amplification instrument: Singapore T100 TM Thermal Cycler PCR Amplification Instrument;

[0084] (2) Electrophoresis apparatus: DYY-6C electrophoresis apparatus from Beijing Liuyi Biotechnology Co., Ltd.;

[0085] (3) Centrifuge: Heraeus Pico 17 high-speed centrifuge from Germany;

[0086] (4) Gel imaging system: Omega Fluor (USA) plus Gel imaging system;

[0087] (5) Spectrophotometer: Mona (Suzhou) Biotechnology Co., Ltd. Eva 3200 Ultra-micro Nucleic Acid and Protein Detector.

[0088] Example 1

[0089] 1. Total DNA extraction from sugarcane leaves

[0090] (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.

[0091] (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.

[0092] (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.

[0093] (4) Chloroform extraction: After water bath, take out and centrifuge for 10 min. Add an equal volume of phenol:chloroform:isoamyl alcohol (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 (24:1) solution to the supernatant, shake several times to mix, and centrifuge for 10 min. The centrifugation speed is 12000 rpm each time.

[0094] (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.

[0095] (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.

[0096] (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.

[0097] (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.

[0098] 2. Primer design

[0099] 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 5 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 *Imperata cylindrica*-specific 150bp sequence set 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 17-23bp, primer output quantity of 1, and target product size of 146-150bp. The filtered sequences were then used to design primers. Specific primer sequences and amplification sequences are shown in Table 2.

[0100] Table 2. Primer sequences and amplification sequences for specific regions of chromosome 5 of *Imperata cylindrica*.

[0101]

[0102]

[0103]

[0104] 3. PCR amplification

[0105] 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×RapidTaq 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.

[0106] 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℃.

[0107] 4. Electrophoresis detection

[0108] 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% in 1×TAE electrophoresis buffer at a constant voltage of 120V for 30 min. 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 5 of *Imperata cylindrica*; the absence of PCR amplification bands indicated that the sample did not contain a specific region of chromosome 5 of *Imperata cylindrica*. The amplified products were sent to a sequencing company for analysis, and the specific sequences are shown in Table 2.

[0109] 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 5 of *Imperata cylindrica*. Samples 8-10 show PCR product amplification bands at around 150bp, indicating that these samples contain blood relations corresponding to specific segments of chromosome 5 of *Imperata cylindrica*. Figure 1 The absence of PCR product amplification bands in samples 2-7 (negative controls) indicates that these samples do not contain bloodlines from a specific region of chromosome 5. Figure 1 The absence of PCR product restriction bands in sample 1 (sterile water without nuclease) indicates that the experiment was not contaminated. Overall, this invention can effectively distinguish the bloodline of chromosome 5 of *Imperata cylindrica* from other bloodline materials that do not contain specific regions of *Imperata cylindrica*.

[0110] Comparative Example 1 used primer set detection to identify bloodline materials containing different segments of chromosome 5 of *Imperata cylindrica*.

[0111] 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.

[0112] Genomic DNA containing chromosome 5 of *Imperata cylindrica* chromosome 5 was extracted from the BC1 offspring of the *Imperata cylindrica* hybridization, specifically from the Yacheng 01-92 and Yacheng 01-36 materials. 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.

[0113] Accuracy of specific primer pairs for different segments of chromosome 5 of *Imperata cylindrica* (1-16) was determined. PCR detection was performed using sugarcane containing *Imperata cylindrica* chromosome 5 lineage confirmed by FISH and *Imperata cylindrica* BC1 generation materials Yacheng 01-92 and Yacheng 01-36. The results showed that ( Figure 2 The target bands were amplified in all the above clones, indicating that primer pair 1-16 has good accuracy and can be used to detect different segments of chromosome 5 in high-generation sugarcane and Imperata cylindrica hybrids.

[0114] 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 identifying chromosome 5 lineage in sugarcane, characterized in that, The segmented PCR primer set includes 16 primer sets, with specific nucleotide sequences as shown in Seq_1~Seq_2: Chr05A_3654658_3654807; as shown in Seq_3~Seq_4: Chr05A_13255127_13255276; as shown in Seq_5~Seq_6: Chr05A_13623777_13623926; and as shown in Seq_7~Seq_8: Chr05A_19855186_1985533. 5; Specific nucleotide sequences are shown in Seq_9~Seq_10 as Chr05A_31139660_31139809; Specific nucleotide sequences are shown in Seq_11~Seq_12 as Chr05A_41719808_41719957; Specific nucleotide sequences are shown in Seq_13~Seq_14 as Chr05A_47116008_47116157; Specific nucleotide sequences are shown in Seq_15~Seq_16 as Chr05A_47530334_47530483; Specific nucleotide sequences Examples of nucleotide sequences include: Chr05A_55122419_55122568 (Seq_17-Seq_18); Chr05A_57957698_57957847 (Seq_19-Seq_20); Chr05A_64673377_64673526 (Seq_21-Seq_22); Chr05A_74321110_74321259 (Seq_23-Seq_24); and Chr05A_74321110_74321259 (Seq_25-Seq_26). Seq_26 shows Chr05A_74639055_74639204; specific nucleotide sequences are shown in Seq_27~Seq_28 as Chr05A_103694448_103694597; specific nucleotide sequences are shown in Seq_29~Seq_30 as Chr05A_111320492_111320641; specific nucleotide sequences are shown in Seq_31~Seq_32 as Chr05A_120873438_120873587; each primer set includes a forward primer and a reverse primer.

2. The primer set according to claim 1, characterized in that, The amplification product size of the primer set is 146bp to 150bp.

3. The application of the primer set according to claim 1 or 2 in identifying chromosome 5 lineage in sugarcane.

4. The application of the primer set according to claim 1 or 2 in sugarcane breeding.

5. A reagent kit, characterized in that, Includes the primer set, PCR reaction solution, and control as described in claim 1 or 2.

6. The reagent kit according to claim 5, characterized in that, The control standards include a positive control and a negative control; the positive control is the total DNA of leaves containing a specific region of chromosome 5 of Imperata cylindrica, and the negative control is the total DNA of leaves without a specific region of chromosome 5 of Imperata cylindrica.

7. The reagent kit according to claim 5, characterized in that, The PCR reaction solution includes a rapid PCR mixture and nuclease-free sterile water.

8. The use of the kit according to any one of claims 5 to 7 in identifying chromosome 5 lineage in sugarcane.

9. The application of the kit according to any one of claims 5 to 7 in sugarcane breeding.

10. A method for identifying the lineage of chromosome 5 in sugarcane, characterized in that, The method includes PCR amplification of the genomic DNA of the sample to be tested using the primer set according to claim 1 or 2 or the kit according to any one of claims 5 to 7.

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