Segmented PCR (polymerase chain reaction) primer and kit for detecting consanguinity of saccharum arundinaceum chromosome 9 in sugarcane and application of segmented PCR primer and kit

Abstract

The invention provides segmented PCR primers and a kit for detecting consanguinity of a saccharum arundinaceum chromosome 9 in sugarcane and application of the segmented PCR primers and the kit, and belongs to the technical field of molecular biology. The segmented PCR primer provided by the invention comprises 14 primer pairs, and the sequences of the primer pairs are shown as Seq1 to Seq28. According to the invention, at least one pair of specific primers is included in every 30Mb of the saccharum arundinaceum chromosome 9 interval and comprehensively covers the saccharum arundinaceum chromosome 9, and the provided segmented PCR primers can rapidly and comprehensively detect or identify whether the sugarcane and saccharum arundinaceum filial generation contains the specific interval consanguinity of the saccharum arundinaceum chromosome 9; technical support is provided for hybridization generation consanguinity identification of wild germplasm resource saccharum arundinaceum introduced from sugarcane in China and basic research on saccharum arundinaceum chromosome structure variation and the like, and the method has important significance on sugarcane breeding.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a segmented PCR primer, kit, and application for detecting chromosome 9 lineage in sugarcane. 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 increased the need for crop improvement. Utilizing wild genetic resources to improve varieties and resist biotic and abiotic stresses while increasing biomass is a key objective of modern breeding. *Imperata cylindrica*, a close relative of sugarcane, possesses excellent field traits such as high biomass, robust growth, tolerance to poor soil and drought, resistance to pests and diseases, and wide adaptability. Therefore, *Imperata cylindrica* has become one of the important wild germplasm resources for sugarcane breeders both domestically and internationally to improve sugarcane traits.

[0003] The polyploidy and heterogeneous nature of sugarcane cells, its large genome, and high chromosome number have hindered research in the molecular and cytogenetic aspects of sugarcane, particularly in terms of chromosome composition and genetic mechanisms. Furthermore, the transmission of chromosomes from parents to offspring during distant hybridization in sugarcane is complex. Cytological studies of intergenus hybrids in sugarcane have revealed various complex transmission patterns of chromosomes in distant hybrids, including n+n, 2n+n, and n+2n, a phenomenon known as "unbalanced inheritance" where gamete numbers often fluctuate. Simultaneously, the infiltration of bloodlines from different wild relatives further complicates the identification and analysis of the composition of sugarcane-Imperata cylindrica hybrids. Therefore, developing efficient DNA molecular markers is crucial for further research into whether sugarcane hybrids contain Imperata cylindrica bloodlines, which is essential for sugarcane breeding. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a segmented PCR primer, kit, and application for detecting the lineage of chromosome 9 in sugarcane. The segmented PCR primer provided by this invention can rapidly detect or identify whether the offspring of sugarcane and *Imperata cylindrica* contain a specific region of chromosome 9 lineage, providing technical support for basic research on the lineage identification of hybrid offspring of *Imperata cylindrica* introduced from wild germplasm resources of sugarcane in my country and on chromosome structural variations of *Imperata cylindrica*.

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

[0006] A segmented PCR primer for detecting chromosome 9 lineage in sugarcane, comprising 14 primer pairs, specifically:

[0007] (1)Chr09A_5660084_5660233:

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

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

[0010] (2)Chr09A_15255725_15255874:

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

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

[0013] (3)Chr09A_25288837_25288986:

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

[0015] Seq_6: 5'-GCTTAGCTACCAGTCACACTCTT-3';

[0016] (4)Chr09A_35398511_35398660:

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

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

[0019] (5)Chr09A_35491480_35491629:

[0020] Seq_9: 5'-GCTTGTAGGTGCTGAAATAGATGC-3';

[0021] Seq_10: 5'-TCTGAAGAACCTATGCTAGTCCC-3';

[0022] (6)Chr09A_45070709_45070858:

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

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

[0025] (7)Chr09A_45640443_45640592:

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

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

[0028] (8)Chr09A_55120302_55120451:

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

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

[0031] (9)Chr09A_65029530_65029679:

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

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

[0034] (10)Chr09A_75573538_75573687:

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

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

[0037] (11)Chr09A_85046234_85046383:

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

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

[0040] (12)Chr09A_85706112_85706261:

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

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

[0043] (13)Chr09A_95562646_95562795:

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

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

[0046] (14)Chr09A_95764480_95764629:

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

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

[0049] In this invention, the amplification position of Chr09A_5660084_5660233 is preferably position 5660084_5660233 on chromosome 9 of *Imperata cylindrica*; the amplification position of Chr09A_15255725_15255874 is preferably position 15255725_15255874 on chromosome 9 of *Imperata cylindrica*; the amplification position of Chr09A_25288837_25288986 is preferably position 25288837_25288986 on chromosome 9 of *Imperata cylindrica*; the amplification position of Chr09A_35398511_35398660 is... The preferred amplification positions are: 35398511-35398660 on chromosome 9 of *Imperata cylindrica*; 35491480-35491629 on chromosome 9 of *Imperata cylindrica*; 45070709-45070858 on chromosome 9 of *Imperata cylindrica*; and 45640443-45640592 on chromosome 9 of *Imperata cylindrica*. The preferred amplification location for Chr09A_55120302_55120451 is position 55120302_55120451 on chromosome 9; the preferred amplification location for Chr09A_65029530_65029679 is position 65029530_65029679 on chromosome 9; the preferred amplification location for Chr09A_75573538_75573687 is position 75573538_75573687 on chromosome 9; the preferred amplification location for Chr09A_85046234_85046383 is... The preferred amplification positions are: positions 85046234-85046383 on chromosome 9 of *Imperata cylindrica*; positions 85706112-85706261 on chromosome 9 of *Imperata cylindrica*; positions 95562646-95562795 on chromosome 9 of *Imperata cylindrica*; and positions 95764480-95764629 on chromosome 9 of *Imperata cylindrica*.

[0050] In this invention, the amplification product size of the segmented PCR primers is preferably 146bp to 150bp.

[0051] The present invention also provides a kit comprising the segmented PCR primers described in the above technical solution.

[0052] In this invention, the kit preferably includes positive sample DNA and negative sample DNA; the positive sample DNA is preferably sugarcane leaf DNA containing chromosome 9 lineage; the negative sample DNA is preferably sugarcane leaf DNA not containing chromosome 9 lineage.

[0053] The present invention also provides the application of the segmented PCR primers or the kits described in the above technical solutions in detecting chromosome 9 lineage in sugarcane.

[0054] The present invention also provides the application of the segmented PCR primers or the kits described in the above technical solutions in sugarcane breeding.

[0055] The present invention also provides the application of the segmented PCR primers or the kits described in the above technical solutions in the study of chromosome structural variations in *Imperata cylindrica*.

[0056] This invention also provides a method for detecting chromosome 9 lineage in sugarcane, comprising the following steps:

[0057] Genomic DNA was extracted from the sugarcane samples to be tested;

[0058] The extracted genomic DNA was amplified by PCR using the segmented PCR primers or the kits described in the above technical solutions.

[0059] The amplification products were subjected to gel electrophoresis, and the results were determined.

[0060] In this invention, the preferred criterion for determining the result is: if an amplification band appears in the PCR amplification product at the position of 146bp to 150bp, the sample to be tested contains a bloodline of a specific region of chromosome 9; if no amplification band appears in the PCR amplification product at the position of 146bp to 150bp, the sample to be tested does not contain a bloodline of a specific region of chromosome 9.

[0061] Beneficial Technical Effects: This invention provides a segmented PCR primer and kit for detecting chromosome 9 lineage in sugarcane, along with its applications. The segmented PCR primers provided by this invention include 14 primer pairs, with sequences shown in Seq_1 to Seq_28. This invention utilizes at least one pair of primers specific to each 30Mb region of chromosome 9 in sugarcane, comprehensively covering chromosome 9. The provided segmented PCR primers can rapidly and comprehensively detect or identify whether sugarcane-sugarcane hybrids contain specific regions of chromosome 9 lineage. This provides technical support for the identification of lineage in hybrids of introduced wild germplasm resources of *Imperata cylindrica* in my country, as well as basic research on chromosome structural variations in *Imperata cylindrica*, and is also of great significance for sugarcane breeding. Attached Figure Description

[0062] Figure 1 Electrophoresis images for bloodline identification of specific regions of chromosome 9 of *Imperata cylindrica*. In these images, M represents the relative molecular mass of a 100bp DNA gradient standard, 1 represents nuclease-free sterile water, 2-7 represent sugarcane leaf samples of *Bardila*, LA-Purple, Np-X, SES208, Zhongzhe No. 1, and ROC22, respectively, and 8-10 represent sugarcane leaf samples of Hainan 92-77, Yunnan 2012-3, and Yacheng 05-164, respectively.

[0063] Figure 2 Electrophoretic images of different segments of chromosome 9 in materials from Yacheng 01-92 and Yacheng 01-36 were obtained by detecting blood relations using 14 primer pairs. Detailed Implementation

[0064] 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.

[0065] The materials, reagents, and instruments used in this invention are as follows:

[0066] Materials: As shown in Table 1.

[0067] Table 1 Clones of Imperata cylindrica and sugarcane

[0068]

[0069]

[0070] Reagents: (All chemical reagents are of analytical grade)

[0071] (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;

[0072] (2) 1×TAE buffer: 242g Tris, 37.2g EDTA-Na2, 57.1mL glacial acetic acid, add pure water to make up to 1L;

[0073] (3) PCR amplification reagents were purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0074] instrument:

[0075] (1) PCR amplification instrument: Singapore T100TM Thermal Cycler PCR Amplification Instrument;

[0076] (2) Electrophoresis apparatus: DYY-6C electrophoresis apparatus from Beijing Liuyi Biotechnology Co., Ltd.;

[0077] (3) Centrifuge: Heraeus Pico 17 high-speed centrifuge from Germany;

[0078] (4) Gel imaging system: Omega Fluor (USA) plus Gel imaging system;

[0079] (5) Spectrophotometer: Mona (Suzhou) Biotechnology Co., Ltd. Eva 3200 Ultra-micro Nucleic Acid and Protein Detector.

[0080] Example 1

[0081] 1. Total DNA extraction from sugarcane leaves

[0082] (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.

[0083] (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.

[0084] (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.

[0085] (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.

[0086] (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.

[0087] (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.

[0088] (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.

[0089] (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.

[0090] 2. Primer design

[0091] 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 9 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 primers are shown in Table 2.

[0092] Table 2 Primer sequences for molecular markers on specific regions of chromosome 9 of *Imperata cylindrica*.

[0093]

[0094]

[0095] 3. PCR amplification

[0096] 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.

[0097] 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℃.

[0098] 4. Electrophoresis detection

[0099] 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 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 product amplification bands in the electrophoresis detection image indicated that the sample contained a specific region of chromosome 9 of the 'Pistacia chinensis' lineage; the absence of PCR product amplification bands indicated that the sample did not contain a specific region of chromosome 9 of the 'Pistacia chinensis' lineage.

[0100] Gel 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 9. In the figure, samples 8-10 show PCR product amplification bands at around 150bp, indicating that these samples contain blood relations corresponding to specific intervals of chromosome 9. 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 9. 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 9 of *Imperata cylindrica* from other bloodline materials that do not contain specific regions of *Imperata cylindrica*.

[0101] Experiment 1 used primer set detection to identify bloodline materials containing different segments of chromosome 9 of the variegated iris.

[0102] 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.

[0103] Genomic DNA containing chromosome 9 of *Imperata cylindrica* chromosome 9 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.

[0104] Accuracy of primer pairs 1-14 for different segments of chromosome 9 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 9 lineage. Results showed that the target bands were amplified in all the above clones, indicating that primer pairs 1-14 have good accuracy and can be used to detect different segments of chromosome 9 lineage in high-generation sugarcane-*Imperata cylindrica* hybrids.

[0105] 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 for detecting chromosome 9 lineage in sugarcane, characterized in that, The segmented PCR primers include 14 primer pairs, specifically: (1) Chr09A_5660084_5660233: Seq_1: 5’-TGAGGTTAAATAATTGTCTTCCATCAGC-3’; Seq_2: 5’-TCAAAGTATCATCAACAAAGCTGCC-3’; (2) Chr09A_15255725_15255874: Seq_3: 5’-GGTGTTTGGATGAATGCGTTTGT-3’; Seq_4: 5’-GAATCCGGGCAATACAACCTCAG-3’; (3) Chr09A_25288837_25288986: Seq_5: 5’-CCCTTATTCCATGGTTACTTACGT-3’; Seq_6: 5’-GCTTAGCTACCAGTCACACTCTT-3’; (4) Chr09A_35398511_35398660: Seq_7: 5’-GGATGCATGCGTTCAATCATATATG-3’; Seq_8: 5’-GGAGAAGAGAGTGATAGATCGATGAA-3’; (5) Chr09A_35491480_35491629: Seq_9: 5’-GCTTGTAGGTGCTGAAATAGATGC-3’; Seq_10: 5’-TCTGAAGAACCTATGCTAGTCCC-3’; (6) Chr09A_45070709_45070858: Seq_11: 5’-CAACTGCAATGGAATGTTCAGGG-3’; Seq_12: 5’-AGTGAACCTCAATACCTTCTTCCT-3’; (7) Chr09A_45640443_45640592: Seq_13: 5’-ATCATGGATAAATTCTATCGGTTCATCAT-3’; Seq_14: 5’-TCATCTCAACTAATACTCCATTGGCT-3’; (8) Chr09A_55120302_55120451: Seq_15: 5’-GCTACAAGGAGTCATAACAACACTG-3’; Seq_16: 5’-CAAGCTTGATGATTACCAGTTCTTTCT-3’; (9) Chr09A_65029530_65029679: Seq_17: 5’-AGGAAGTTGAAGCTTGTAAATGTATTC-3’; Seq_18: 5’-TTGTTGATGACTCGGTATACTAAAGGT-3’; (10) Chr09A_75573538_75573687: Seq_19:5’-CCAATCCTCTCCGATATTTACTGCT-3’; Seq_20:5’-TACACGAGAGGACATGACATTGC-3’; (11)Chr09A_85046234_85046383: Seq_21:5’-CAGAAGCAGTCATCACCAACGTT-3’; Seq_22:5’-GTGGATGTCGTGGAAGGCATAAC-3’; (12)Chr09A_85706112_85706261: Seq_23:5’-CGCCTTTCACCTCTATGTACGTA-3’; Seq_24:5’-AGAGACTAAGGTTGAAGGATTTGTC-3’; (13)Chr09A_95562646_95562795: Seq_25:5’-CGTCATATTTAATTCCCATTGCTCCT-3’; Seq_26:5’-ACTGGCTTATGTACTACAAACAGAGA-3’; (14)Chr09A_95764480_95764629: Seq_27:5’-AATGTCTACGAGAGGGATATAGGTGA-3’; Seq_28:5’-CACTACTTCGACCACCATTTCTT-3’。 2. The segmented PCR primers according to claim 1, characterized in that, The amplification position of Chr09A_5660084_5660233 is position 5660084_5660233 on chromosome 9 of *Imperata cylindrica*; the amplification position of Chr09A_15255725_15255874 is position 15255725_15255874 on chromosome 9 of *Imperata cylindrica*; the amplification position of Chr09A_25288837_25288986 is position 25288837_25288986 on chromosome 9 of *Imperata cylindrica*; the amplification position of Chr09A_35398511_35398660 is... The amplification positions of Chr09A_35491480_35491629 on chromosome 9 are: positions 35491480_35491629; positions 45070709_45070858 on chromosome 9; positions 45640443_45640592 on chromosome 9; Ch... The amplification position of r09A_55120302_55120451 is position 55120302_55120451 on chromosome 9; the amplification position of Chr09A_65029530_65029679 is position 65029530_65029679 on chromosome 9; the amplification position of Chr09A_75573538_75573687 is position 75573538_75573687 on chromosome 9; the amplification position of Chr09A_85046234_85046383 is... The amplified positions of Chr09A_85706112_85706261 and Chr09A_95562646_95562795 on chromosome 9 are the same as those of Chr09A_95764480_95764629.

3. The segmented PCR primers according to claim 1, characterized in that, The amplification products of the segmented PCR primers are all 146bp to 150bp in size.

4. A reagent kit, characterized in that, The kit includes the segmented PCR primers as described in any one of claims 1 to 3.

5. The reagent kit according to claim 4, characterized in that, The kit also includes positive sample DNA and negative sample DNA.

6. The use of any of the segmented PCR primers according to claims 1 to 3 or any of the kits according to claims 4 to 5 in detecting chromosome 9 lineage in sugarcane.

7. The application of any of the segmented PCR primers according to claims 1 to 3 or any of the kits according to claims 4 to 5 in sugarcane breeding.

8. The use of any of the segmented PCR primers according to claims 1 to 3 or any of the kits according to claims 4 to 5 in the study of chromosome structural variations in *Imperata cylindrica*.

9. A method for detecting chromosome 9 lineage in sugarcane, characterized in that, Includes the following steps: Genomic DNA was extracted from the sugarcane samples to be tested; The extracted genomic DNA was amplified by PCR using the segmented PCR primers as described in any one of claims 1 to 3 or the kit as described in any one of claims 4 to 5. The amplification products were subjected to gel electrophoresis, and the results were determined.

10. The method according to claim 9, characterized in that, The criteria for judging the results are as follows: if the PCR amplification product shows an amplification band at the position of 146bp to 150bp, then the sample to be tested contains a bloodline of a specific region of chromosome 9 of the variegated iris. If no amplification band appears in the PCR amplification product at the position of 146bp to 150bp, the sample to be tested does not contain the bloodline of the specific region of chromosome 9 of the variegated iris.

Images