Primer group and kit for detecting consanguinity of saccharum arundinaceum chromosome 4 in sugarcane and application of primer group and kit

Abstract

The invention provides a primer group and a kit for detecting consanguinity of a saccharum arundinaceum chromosome 4 in sugarcane and application of the primer group and the kit, and belongs to the technical field of molecular biology. The primer group provided by the invention comprises 24 pairs of primers, and the sequences of the primers are shown as Seq1 to Seq48. The primer group, the kit and the application thereof provided by the invention can be used for rapidly detecting whether a sugarcane sample to be detected contains the specific interval consanguinity of the saccharum arundinaceum chromosome 4, have the characteristics of low cost and high sensitivity, and are of great significance in saccharum arundinaceum consanguinity detection in sugarcane filial generations, sugarcane breeding and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology, in particular to a primer set for detecting the Erianthus arundinaceus chromosome 4 blood relationship in sugarcane, a kit and application thereof. BACKGROUND

[0002] Sugarcane is a C4 high photosynthetic efficiency economic crop widely planted in tropical and subtropical regions, and is the most important sugar-producing crop. The statistics of the Food and Agriculture Organization of the United Nations (FAO) shows that the global sugarcane sugar yield accounts for more than 85% of the total sugar supply. Therefore, sugarcane variety improvement is crucial to the sustainable development of the sugarcane industry, and sugarcane breeding experts all over the world are committed to breeding excellent varieties with high yield, high sugar, disease resistance and high stress resistance.

[0003] The Erianthus arundinaceus, a close relative of sugarcane, has excellent field traits such as high biomass, rough growth, tolerance to poor soil and drought, resistance to diseases and pests, and wide adaptability, making the utilization of Erianthus arundinaceus distant hybridization a research hotspot for domestic and foreign sugarcane breeders. At present, breeders have carried out research on sugarcane and Erianthus arundinaceus distant hybridization, successfully introduced some chromosomes of Erianthus arundinaceus into sugarcane, and the backcross generations have been advanced to BC4, and excellent hybrid parents and varieties containing Erianthus arundinaceus blood relationship have been obtained. It is extremely important to further detect whether the Erianthus arundinaceus blood relationship is successfully introduced into the offspring of sugarcane and Erianthus arundinaceus using modern molecular biology techniques to improve the efficiency of sugarcane hybrid breeding. Molecular marker method is a stable and reliable method for evaluating genetic diversity of parents, which has been widely used in genetic resource evaluation of various crops including sugarcane. Wei Zhongyan et al. carried out rapid genotyping of 599 soybeans by SNP molecular markers, laying a foundation for accurate evaluation of soybean germplasm resources and improving soybean breeding efficiency. Therefore, rapid and accurate identification of specific chromosome blood relationship of sugarcane and Erianthus arundinaceus will help to determine whether the hybrid offspring contains Erianthus arundinaceus blood relationship, and combining with the existing agronomic traits phenotype will greatly improve the utilization process of Erianthus arundinaceus in sugarcane breeding. SUMMARY

[0004] Therefore, the present application aims to provide a primer set for detecting the Erianthus arundinaceus chromosome 4 blood relationship in sugarcane, a kit and application thereof. The primer set, the kit and the application thereof provided by the present application can rapidly detect whether the sugarcane sample to be tested contains the specific interval blood relationship of Erianthus arundinaceus chromosome 4, which has important significance for blood relationship detection of sugarcane hybrid offspring and sugarcane breeding.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0006] A primer set for detecting the Erianthus arundinaceus chromosome 4 blood relationship in sugarcane, the primer set comprising 24 pairs of primers, specifically as follows:

[0007] (1) Chr04A_1258540_1258689:

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

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

[0010] (2) Chr04A_7888667_7888816:

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

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

[0013] (3) Chr04A_8236752_8236901:

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

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

[0016] (4) Chr04A_13501234_13501383:

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

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

[0019] (5) Chr04A_18596400_18596549:

[0020] Seq_9: 5'-CATGAGTAACAACCACATTCTATGC-3'; Seq_10: 5'-TGAACTCATTAACCATTGCTACAGAAA-3';

[0021] (6) Chr04A_26002164_26002313:

[0022] Seq_11 : 5 '-CGTCGTACTAACTGATGGAATAATCT-3 ';

[0023] Seq_12: 5'-ATTCTGTTCTTGTTCCAGTCGATTG-3';

[0024] (7) Chr04A_35713506_35713655:

[0025] Seq_13: 5'-TCCTGAGTCATCTGTGCCTGAAT-3'; Seq_14: 5'-CTTCCAGGAGCTAGGTGTTACAT-3';

[0026] (8) Chr04A_36809690_36809839:

[0027] Seq_15: 5'-CTCCTTGCCAAGTGTTCGTACTC-3'; Seq_16: 5'-TGGCTACATCTCCATTGTCCAAC-3';

[0028] (9) Chr04A_40381618_40381767:

[0029] Seq_17: 5'-ACTACAATTAACTTCAATTCTATCCTAAGC-3'; Seq_18: 5'-TGAATACTGTGTTATTGATGTTTGATTCAG-3';

[0030] (10) Chr04A_42985488_42985637:

[0031] Seq_19: 5'-AAACAGATGCATTTATGTGGTGTTCT-3';

[0032] Seq_20: 5'-GCTACTCTTTGGTGAATGGTCCA-3';

[0033] (11) Chr04A_44488847_44488996:

[0034] Seq_21: 5'-AGGAACCATTGTATAGACTCTTAAGGT-3';

[0035] Seq_22: 5'-TCTTTAGATGATACAACATTTCTCACGAG-3';

[0036] (12) Chr04A_53297004_53297153:

[0037] Seq_23: 5 '-ACTTCTATCGATTGAGTATGCATGAC-3 ';

[0038] Seq_24: 5 '-GGCCAACGTATATTCCTGGTATTG-3 ';

[0039] (13) Chr04A_56309870_56310019:

[0040] Seq_25: 5 '-GTCGTGATCTATGAGGATGGAAATT-3 ';

[0041] Seq_26: 5 '-TAAAGTATTAATGGTGCATAGTTCTAAGC-3 ';

[0042] (14) Chr04A_57242396_57242545:

[0043] Seq_27: 5 '-CCTTGCATACTCTGGACCTGATT-3 '; Seq_28: 5 '-TTCGTTTATAATATGCATGTTCTCACTCT-3 ';

[0044] (15) Chr04A_65804190_65804339:

[0045] Seq_29: 5 '-AGTCCAATCAACACTCTTAGTCTATAC-3 ';

[0046] Seq_30: 5 '-GGTGTAGATGTAGTTGAAGAAGCC-3 ';

[0047] (16) Chr04A_73020762_73020911:

[0048] Seq_31: 5 '-TGTTTGGTGTGGAGTCTAATTAACT-3 ';

[0049] Seq_32: 5 '-AGTCCACAATCTACCAAGGTCTC-3 ';

[0050] (17) Chr04A_76086468_76086617:

[0051] Seq_33: 5 '-CCACATCATCGATAAAGTTACAGTGA-3 ';

[0052] Seq_34: 5'-TGGCTAGATGAACGGATGGATTAG-3';

[0053] (18) Chr04A_80549538_80549687:

[0054] Seq_35: 5'-TGAGGATAATTCGCAGCATATATGG-3';

[0055] Seq_36: 5'-TCTACTTGGAATCCTCAGGTTGT-3';

[0056] (19) Chr04A_87104902_87105051:

[0057] Seq_37: 5'-ACAACACCGTACTGCCATTACT-3';

[0058] Seq_38: 5'-ACCACTAAGATATCGATAACAACATTTCA-3';

[0059] (20) Chr04A_92147482_92147631:

[0060] Seq_39: 5'-GAAATGGTGCGTAGTTTATCGTCAT-3';

[0061] Seq_40: 5'-CTGCAAGCTCAAAGACCATATATCA-3';

[0062] (21) Chr04A_101233074_101233223:

[0063] Seq_41: 5'-AATTGCGTTGCCTGTAGTTGTAG-3';

[0064] Seq_42: 5'-TTGATGTAGGGTTAGTTGAGGCC-3';

[0065] (22) Chr04A_110004459_110004608:

[0066] Seq_43: 5'-AGGATTAGCAGTTGGAAGCATCA-3';

[0067] Seq_44: 5'-ATTTGGCATTAACAAGCATCAGAAGA-3';

[0068] (23) Chr04A_110690412_110690561:

[0069] Seq_45: 5'-TGATTGTGAGTAGTGGTCCAAATG-3';

[0070] Seq_46: 5'-TCAGAACCTAGATCAATAAAGTGTTGTC-3';

[0071] (24) Chr04A_120456547_120456696:

[0072] Seq_47: 5'-CTGGTGTTCCCTGTACTAGTCAG-3';

[0073] Seq_48: 5'-GCTGTGTACTTGACTTAACGAGGA-3'.

[0074] In the present application, the PCR amplification product size of the primer set is 146bp-150bp.

[0075] In the present application, each 30Mb of the specific primer of the Chromosome 4 of Erianthus arundinaceus interval contains at least one pair. The primer set of the present application can comprehensively cover the specific interval of the Chromosome 4 of Erianthus arundinaceus.

[0076] In the present application, the two groups of numbers behind the name of the 24 primer pairs are the location of the amplification product of the primer pair on the Chromosome 4 of Erianthus arundinaceus.

[0077] The present application also provides a kit comprising the primer set, PCR reaction solution, negative control and positive control in the above technical solution.

[0078] In the present application, the PCR reaction solution preferably comprises rapid PCR mixture and nuclease-free sterile water.

[0079] In the present application, the negative control is preferably total DNA of sugarcane leaf without Chromosome 4 of Erianthus arundinaceus blood relationship, and the positive control is preferably total DNA of sugarcane leaf containing Chromosome 4 of Erianthus arundinaceus blood relationship.

[0080] In the present application, the PCR reaction system of the kit is preferably: 2x RapidTaq Master Mix 12.5uL, 50ng / uL DNA template 2.5uL, 10uM of upstream and downstream primers 2.5uL each, and the rest is ddH2O in each 25uL reaction system; the PCR reaction system is preferably one reaction system for each primer pair.

[0081] In the present application, the PCR amplification procedure of the kit is preferably: 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; 12℃ storage.

[0082] The present application also provides application of the primer set in the technical solution or the kit in the technical solution in detecting the Erianthus arundinaceus chromosome 4 blood relationship.

[0083] The present application also provides application of the primer set in the technical solution or the kit in the technical solution in sugarcane breeding.

[0084] The present application also provides a method for detecting the Erianthus arundinaceus chromosome 4 blood relationship, which comprises using the primer set in the technical solution or the kit in the technical solution to perform PCR amplification on the genomic DNA of a sample to be detected.

[0085] In the present application, if the PCR amplification product appears an amplification band at the position of 146bp-150bp, the sample to be detected contains the Erianthus arundinaceus chromosome 4 specific interval blood relationship; if the PCR amplification product does not appear an amplification band at the position of 146bp-150bp, the sample to be detected does not contain the Erianthus arundinaceus chromosome 4 specific interval blood relationship.

[0086] Beneficial technical effects: the present application provides a primer set, a kit and application thereof for detecting the Erianthus arundinaceus chromosome 4 blood relationship, wherein the primer set comprises 24 pairs of primers, and the sequences are shown in Seq_1-Seq_48. The primer set, the kit and the application thereof provided by the present application can quickly detect whether the sample to be detected contains the Erianthus arundinaceus chromosome 4 specific interval blood relationship, have the characteristics of low cost and high sensitivity, and have important significance for detecting the Erianthus arundinaceus blood relationship in the hybrid offspring of sugarcane, sugarcane breeding and the like. BRIEF DESCRIPTION OF DRAWINGS

[0087] Figure 1 is an electrophoresis map for identifying the Erianthus arundinaceus chromosome 4 specific interval blood relationship, wherein M is a 100bp DNA ladder standard relative molecular mass, lane 1 is ddH2O, 2 is Balar, 3 is LA-Purple, 4 is Np-X, 5 is SES208, 6 is Zhensu No.1, 7 is ROC22, 8 is Hainan 92-77, 9 is Yunnan 2012-3, 10 is Yacheng 05-164;

[0088] Figure 2 is an electrophoresis map for detecting the Erianthus arundinaceus chromosome 4 blood relationship in Yacheng 01-92 and Yacheng 01-36 materials by 24 pairs of primers. DETAILED DESCRIPTION

[0089] In order to better understand the present application, the content of the present application is further illustrated below in combination with examples, but the content of the present application is not limited to only the following examples. The materials, reagents and the like used in the examples and test examples of the present application can be obtained from commercial channels unless otherwise specified, and the methods used in the examples and test examples of the present application are conventional methods unless otherwise specified.

[0090] Example 1

[0091] 1. Materials and reagents

[0092] 1.1 Materials

[0093] As shown in Table 1.

[0094] Table 1 Sample information

[0095]

[0096]

[0097] 1.2 Reagents

[0098] The main reagents used in the present application are as follows: (all chemical reagents are analytical pure)

[0099] (1) CTAB extraction buffer: 100 mM Tris-HCl (pH = 8.0), 20 mM EDTA-Na2, 1.4 M NaCl, mass / volume ratio 2% CTAB, 0.1% β-mercaptoethanol is added before use;

[0100] (2) 1 × TAE buffer: 242 g Tris, 37.2 g EDTA-Na2, 57.1 mL glacial acetic acid, add pure water to 1 L;

[0101] (3) PCR amplification reagent is purchased from Nanjing Nuowezan Biotechnology Co., Ltd.

[0102] The instruments used in the present application are mainly as follows:

[0103] (1) PCR amplifier: Singapore T100 TM Thermal Cycler PCR amplifier;

[0104] (2) Electrophoresis instrument: DYY-6C electrophoresis instrument of Beijing Liyi Biological Technology Co., Ltd.;

[0105] (3) Centrifuge: Germany Heraeus Pico 17 high-speed centrifuge;

[0106] (4) Gel imaging system: American Omega Fluor plus Gel imaging system;

[0107] (5) Spectrophotometer: Eva 3200 ultramicro nucleic acid protein detector of Monar (Suzhou) Biotechnology Co., Ltd.

[0108] 2. Sugarcane leaf total DNA extraction

[0109] (1) Prepare tools: clean and disinfect the mortar and scissors, wherein the scissors are disinfected with 75% alcohol, and the mortar is disinfected by burning with anhydrous ethanol and then pre-cooled on ice for use;

[0110] (2) Process the sample: select fresh and healthy young leaves of Bala, LA-Purple, Np-X, SES208, Zhongzhe No. 1, ROC22, Hainan 92-77, Yunnan 2012-3 and Yacheng 05-164 in the seedling stage planted in the planting resource garden of Fusui base of Guangxi University, disinfect the leaf surface with 75% alcohol cotton ball, remove the leaf veins, cut the leaves into pieces, put them into the pre-cooled mortar, pour liquid nitrogen to grind them into fine powder, generally until the color changes from dark green to light green and white, put them into a 2 mL centrifuge tube, about 1 / 3 of the volume of the tube;

[0111] (3) Cell lysis: the prepared 2x CTAB is preheated in a 65°C water bath for 30 min, 900 μL of preheated CTAB extract and 100 μL of anhydrous ethanol are added to the powder, quickly shake and mix, 65°C water bath for 0.5-1 h, take it out every 10 min, mix it by inverting, and make sure that the cells are fully lysed;

[0112] (4) Chloroform extraction: after water bath, centrifuge for 10 min, add an equal volume of phenol: chloroform: isopropyl alcohol (25:24:1) lower phase solution to the supernatant, mix well on the shaker, remove the precipitate by centrifugation for 10 min, add an equal volume of chloroform: isopropyl alcohol (24:1) solution, mix well several times, and centrifuge for 10 min each time at 12000 rpm;

[0113] (5) DNA precipitation: add 3M NaAc (pH=5.2) solution with a volume of 1 / 10 of the supernatant in the previous step and an equal volume of pre-cooled isopropyl alcohol solution, mix well, and place it in -20°C for 1 h;

[0114] (6) Wash the DNA: after precipitation, centrifuge for 10 min, add 1 mL of 75% ethanol solution to rinse the precipitate twice, centrifuge for 5 min after the first rinse, and then rinse again, preferably using pre-cooled ethanol solution;

[0115] (7) Purification of DNA: After centrifugation for 10 min to retain the precipitate, wait for ethanol to evaporate at room temperature, and finally add 100 μL of TE Buffer and 1 μL of RNase A, and purify at 37°C for 30 min. Remove the RNA.

[0116] (8) After detecting the quality and concentration of DNA using a nucleic acid protein detector, store it at 4°C or -20°C for long-term preservation.

[0117] 3. Primer design

[0118] First, the Repeatmasker software was used to mask the whole genome repeat sequences of the LA-Purple genome (DNA can be extracted by the above DNA extraction method and sequenced, the same below), Np-X genome of the Cleavers species, and Hainan 92-77 genome of the Knapweed. Using the K-mers method, the three sets of genomes were cut on chromosome 4, and the genome cutting was completed by Python script. The bwa software was used to align the cut fragments, and the sequence set of 150 bp fragments specific to Knapweed was screened. Primer3 was used to design primers for Knapweed-specific sequence sets, and the software default values were used for primer design parameters with some modifications. The modified conditions are as follows: the fragment size is about 17 bp to 23 bp, the primer output quantity is 1, and the target product is 146 bp to 150 bp; the filtered sequence is designed into a primer. The specific primer sequences are shown in Table 2.

[0119] Table 2: Primer sequences and amplification sequences of specific intervals of chromosome 4 of Knapweed

[0120]

[0121]

[0122]

[0123] 3. PCR amplification

[0124] Each primer pair was prepared with a reaction system, and the PCR reaction system was as follows: each 25 μL reaction system contained 2x RapidTaq Master Mix 12.5 μL, 50 ng / μL DNA template 2.5 μL, 10 μM upper and lower primers 2.5 μL each, and the rest was ddH2O.

[0125] After adding the reagents in the above reaction system to the PCR tube, mix well, centrifuge at 8000 rpm for 5 s, and the amplification program is as follows: 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; 12°C storage.

[0126] 4. Electrophoresis detection

[0127] After the amplification reaction, 10 μL of PCR product was taken for low-melting-point agarose gel electrophoresis with a mass-volume ratio of 2.0%. In the 1xTAE electrophoresis buffer environment, 120V constant voltage electrophoresis was performed for 30 min, and then a gel imaging system was used for observation and photography. The appearance of the PCR product amplification band in the electrophoresis detection map indicated that the sample contained the blood relationship of the specific interval of the Saccharum arundinaceum chromosome 4; the absence of the PCR product amplification band indicated that the sample did not contain the blood relationship of the specific interval of the Saccharum arundinaceum chromosome 4. The amplification product was sent to a sequencing company for detection, and the specific sequence is shown in Table 2.

[0128] The electrophoresis detection results are shown in Table 2. Figure 1 Each electrophoresis map is a specific identification of the blood relationship of different segment positions of the Saccharum arundinaceum chromosome 4. In the map, samples 8-10 appear PCR product amplification bands at about 150bp positions, indicating that these samples contain the blood relationship of the specific interval of the Saccharum arundinaceum chromosome 4. Figure 1 Samples 2-7 (negative control) in Table 2 do not appear PCR product amplification bands, indicating that these samples do not contain the blood relationship of the specific interval of the Saccharum arundinaceum chromosome 4. Figure 1 Sample 1 (nuclease-free sterile water) in Table 2 does not appear PCR product amplification bands, indicating that the experiment is not contaminated, and the overall application can effectively distinguish the Saccharum arundinaceum chromosome 4 blood relationship from other materials that do not contain the Saccharum arundinaceum specific interval blood relationship.

[0129] Test Example 1 uses primer groups to detect materials containing Saccharum arundinaceum chromosome 4 blood relationship

[0130] It is reported (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.) that the genomic DNA of Yacheng 01-92 and Yacheng 01-36 materials contains Saccharum arundinaceum chromosome 4 blood relationship.

[0131] The genomic DNA of the real Saccharum arundinaceum hybrid BC1 progeny Yacheng 01-92 and Yacheng 01-36 materials containing Saccharum arundinaceum chromosome 4 was extracted by the CTAB method, and the OD 260 / OD 280The DNA quality with the ratio between 1.6 and 1.8 meets the experimental requirements, the concentration is determined and diluted to 50 ng / μL for sub-packaging as the template DNA.

[0132] Accuracy detection of different segment specific primer pairs 1-24 of Saccharum spontaneum chromosome 4. PCR detection was carried out using the sugarcane with Saccharum spontaneum chromosome 4 bloodline verified by FISH and the BC1 generation material Yacheng 01-92 and Yacheng 01-36. Figure 2 ), the above clone materials can all amplify the target band, which indicates that the primer pair 1-24 has good accuracy and can be used for detecting the different segment bloodline of chromosome 4 of the high generation sugarcane and Saccharum spontaneum hybrid offspring.

[0133] The above only is the preferred embodiment of the present application, it should be pointed out that, for the ordinary skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A primer set for detecting the chromosomal ancestry of Erianthus arundinaceus chromosome 4, characterized in that, The primer set includes 24 pairs of primers, which are specifically as follows: (1) Chr04A_1258540_1258689: Seq_1: 5’-AGGGCCTTAGTTTGAAATCTATCTATT-3’; Seq_2: 5’-AGGATCAGGTCAAATTAGCTTACC-3’; (2) Chr04A_7888667_7888816: Seq_3: 5’-AGCTACTGTACATTGAAGAAATTCTAGC-3’; Seq_4: 5’-CCAATCTGGCCGAATTTCCATAG-3’; (3) Chr04A_8236752_8236901: Seq_5: 5’-ACATCCTGATTCCTGAATATAGCCA-3’; Seq_6: 5’-ACACAACATTCTAAGACGTAAAGGC-3’; (4) Chr04A_13501234_13501383: Seq_7: 5’-AATGTCTGATTCGTCTACTCTTGCA-3’; Seq_8: 5’-GATCAATTGCGTTCAGAACTACAG-3’; (5) Chr04A_18596400_18596549: Seq_9: 5’-CATGAGTAACAACCACATTCTATGC-3’; Seq_10: 5’-TGAACTCATTAACCATTGCTACAGAAA-3’; (6) Chr04A_26002164_26002313: Seq_11: 5’-CGTCGTACTAACTGATGGAATAATCT-3’; Seq_12: 5’-ATTCTGTTCTTGTTCCAGTCGATTG-3’; (7) Chr04A_35713506_35713655: Seq_13: 5’-TCCTGAGTCATCTGTGCCTGAAT-3’; Seq_14: 5’-CTTCCAGGAGCTAGGTGTTACAT-3’; (8) Chr04A_36809690_36809839: Seq_15: 5’-CTCCTTGCCAAGTGTTCGTACTC-3’; Seq_16: 5’-TGGCTACATCTCCATTGTCCAAC-3’; (9) Chr04A_40381618_40381767: Seq_17: 5’-ACTACAATTAACTTCAATTCTATCCTAAGC-3’; Seq_18: 5’-TGAATACTGTGTTATTGATGTTTGATTCAG-3’; (10) Chr04A_42985488_42985637: Seq_19:5’-AAACAGATGCATTTATGTGGTGTTCT-3’;Seq_20:5’-GCTACTCTTTGGTGAATGGTCCA-3’; (11)Chr04A_44488847_44488996: Seq_21:5’-AGGAACCATTGTATAGACTCTTAAGGT-3’;Seq_22:5’-TCTTTAGATGATACAACATTTCTCACGAG-3’;(12)Chr04A_53297004_53297153: Seq_23:5’-ACTTCTATCGATTGAGTATGCATGAC-3’; Seq_24:5’-GGCCAACGTATATTCCTGGTATTG-3’; (13)Chr04A_56309870_56310019: Seq_25:5’-GTCGTGATCTATGAGGATGGAAATT-3’; Seq_26:5’-TAAAGTATTAATGGTGCATAGTTCTAAGC-3’;(14)Chr04A_57242396_57242545: Seq_27:5’-CCTTGCATACTCTGGACCTGATT-3’; Seq_28:5’-TTCGTTTATAATATGCATGTTCTCACTCT-3’;(15)Chr04A_65804190_65804339: Seq_29:5’-AGTCCAATCAACACTCTTAGTCTATAC-3’;Seq_30:5’-GGTGTAGATGTAGTTGAAGAAGCC-3’; (16)Chr04A_73020762_73020911: Seq_31:5’-TGTTTGGTGTGGAGTCTAATTAACT-3’; Seq_32:5’-AGTCCACAATCTACCAAGGTCTC-3’; (17)Chr04A_76086468_76086617: Seq_33:5’-CCACATCATCGATAAAGTTACAGTGA-3’;Seq_34:5’-TGGCTAGATGAACGGATGGATTAG-3’; (18)Chr04A_80549538_80549687: Seq_35:5’-TGAGGATAATTCGCAGCATATATGG-3’; Seq_36:5’-TCTACTTGGAATCCTCAGGTTGT-3’; (19)Chr04A_87104902_87105051: Seq_37: 5'-ACAACACCGTACTGCCATTACT-3'; Seq_38: 5'-ACCACTAAGATATCGATAACAACATTTCA-3'; (20)Chr04A_92147482_92147631: Seq_39: 5'-GAAATGGTGCGTAGTTTATCGTCAT-3'; Seq_40: 5'-CTGCAAGCTCAAAGACCATATATCA-3'; (21)Chr04A_101233074_101233223: Seq_41: 5'-AATTGCGTTGCCTGTAGTTGTAG-3'; Seq_42: 5'-TTGATGTAGGGTTAGTTGAGGCC-3'; (22)Chr04A_110004459_110004608: Seq_43: 5'-AGGATTAGCAGTTGGAAGCATCA-3'; Seq_44: 5'-ATTTGGCATTAACAAGCATCAGAAGA-3'; (23)Chr04A_110690412_110690561: Seq_45: 5'-TGATTGTGAGTAGTGGTCCAAATG-3'; Seq_46: 5'-TCAGAACCTAGATCAATAAAGTGTTGTC-3'; (24)Chr04A_120456547_120456696: Seq_47: 5'-CTGGTGTTCCCTGTACTAGTCAG-3'; Seq_48: 5'-GCTGTGTACTTGACTTAACGAGGA-3'.

2. A kit characterized in that, It includes the primer set, PCR reaction solution, negative control, and positive control as described in claim 1.

3. The kit of claim 2, wherein The PCR reaction system includes rapid PCR mixture and nuclease-free sterile water.

4. The kit of claim 2, wherein The negative control was total DNA from sugarcane leaves without chromosome 4 lineage, and the positive control was total DNA from sugarcane leaves containing chromosome 4 lineage.

5. The kit according to any one of claims 2 to 4, characterized in that The PCR reaction system of the kit is as follows: 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 forward and reverse primers, and the remainder is ddH2O; the PCR reaction system is prepared as one reaction system for each primer pair.

6. The kit according to any one of claims 2 to 4, characterized in that The PCR amplification program of the kit 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℃.

7. The use of the primer set according to claim 1 or the kit according to any one of claims 2 to 6 in detecting chromosome 4 lineage in sugarcane.

8. The application of the primer set according to claim 1 or the kit according to any one of claims 2 to 6 in sugarcane breeding.

9. A method for detecting chromosome 4 lineage in sugarcane, characterized in that, The method involves PCR amplification of the genomic DNA of the sample to be tested using the primer set described in claim 1 or the kit described in any one of claims 2 to 6.

10. The method according to claim 9, characterized in that, If the PCR amplification product shows an amplification band at the position of 146bp to 150bp, the sample to be tested contains a bloodline of a specific region of chromosome 4 of the genus Imperata. If the PCR amplification product does not show an amplification band at the position of 146bp to 150bp, the sample to be tested does not contain a bloodline of a specific region of chromosome 4 of the genus Imperata.

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