Primer group, kit and method for identifying consanguinity of saccharum arundinaceum chromosome 7 in sugarcane and application

Abstract

The invention provides a primer group, a kit and a method for identifying consanguinity of saccharum arundinaceum chromosome 7 in sugarcane and application, and belongs to the technical field of molecular biology. The primer group provided by the invention comprises 12 primer groups, each primer group comprises a forward primer and a reverse primer, and the specific nucleotide sequences are shown as Seq1 to Seq24. According to the primer group, the kit, the method and the application disclosed by the invention, the specific primer for every 30Mb of the saccharum arundinaceum chromosome 7 interval at least comprises one primer pair, and the primer group, the kit, the method and the application can be used for rapidly and comprehensively detecting whether the saccharum arundinaceum chromosome 7 specific interval consanguinity is contained in the sugarcane and saccharum arundinaceum filial generation; technical support is provided for hybridization generation consanguinity identification of wild germplasm resource saccharum arundinaceum introduced from sugarcane in China, saccharum arundinaceum chromosome structure variation and other basic researches.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology, in particular to a primer set for identifying the blood relationship of chromosome 7 of Erianthus arundinaceum, a kit, a method and application. BACKGROUND

[0002] Saccharum spp. is a C4 high photosynthetic efficiency economic crop widely planted in tropical and subtropical regions. According to the statistics of the Food and Agriculture Organization of the United Nations (FAO), the global sugar cane sugar yield accounts for more than 85% of the total supply of sugar. Therefore, the genetic improvement of sugar cane is crucial to the sustainable development of the sugar cane industry, and breeding high-yield, high-sugar, disease-resistant, high-stress-resistant sugar cane varieties is an important goal for sugar cane breeders.

[0003] The relative species of sugar cane, Erianthus arundinaceum, has high biomass, rough, tolerance to poor and drought, disease and pest resistance, and wide adaptability, etc. These excellent traits are just what sugar cane genetic improvement needs. Therefore, using Erianthus arundinaceum for distant hybridization of sugar cane has become one of the important methods to improve the resistance of sugar cane. At present, some sugar cane breeding bases have carried out research on distant hybridization of sugar cane and Erianthus arundinaceum, successfully introduced the blood relationship of Erianthus arundinaceum into the genetic background of sugar cane, and obtained a batch of true Erianthus arundinaceum hybrid offspring materials. Therefore, the accurate identification of the blood relationship of Erianthus arundinaceum will greatly improve the utilization efficiency of these materials containing the blood relationship of Erianthus arundinaceum. In the molecular breeding technology of crops, molecular markers are important technologies for identifying germplasm resources and evaluating genetic diversity. For example, in sugar beet, Wang Ying et al. used SSR molecular markers to efficiently evaluate the consistency of 12 sugar beet varieties. Using DNA molecular markers to identify the blood relationship of the hybrid offspring of sugar cane and Erianthus arundinaceum can quickly and clearly determine the Erianthus arundinaceum blood relationship contained in the hybrid offspring, providing a reference for further obtaining sugar cane hybrid offspring containing excellent Erianthus arundinaceum blood relationship, thereby effectively improving the utilization efficiency of Erianthus arundinaceum. SUMMARY

[0004] Therefore, the present application aims to provide a primer set for identifying the blood relationship of chromosome 7 of Erianthus arundinaceum, a kit, a method and application. The primer set, kit, method and application provided by the present application can quickly detect whether the hybrid offspring of sugar cane and Erianthus arundinaceum contains the blood relationship of a specific interval of chromosome 7 of Erianthus arundinaceum, and provide technical support for the blood relationship identification of the hybrid offspring of the introduced wild germplasm resource Erianthus arundinaceum of sugar cane and the basic research on the chromosomal structure variation of Erianthus arundinaceum.

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

[0006] A primer set for identifying the blood relationship of chromosome 7 of Erianthus arundinaceum, the primer set comprises 12 primer sets, each primer set comprises a forward primer and a reverse primer, and the specific nucleotide sequences are shown in Seq_1-Seq_24.

[0007] In certain embodiments, the primer set is specifically:

[0008] (1) Chr07A_11572915_11573064:

[0009] Seq_1: 5'-TTGCAACCTCATCCTTACAGTGA-3';

[0010] Seq_2: 5'-AGGCCTCACACTATTTCACTATGT-3';

[0011] (2) Chr07A_19076247_19076396:

[0012] Seq_3: 5'-TCTGTTAGACTATGGACATGAGCTT-3';

[0013] Seq_4: 5'-AGCAGTGGAGGAGTATAAGTGTTG-3';

[0014] (3) Chr07A_32084586_32084735:

[0015] Seq_5: 5'-TCCTTCAATACGATGCGATGAGT-3';

[0016] Seq_6: 5'-TGTTGATGATGATGCTGACTGTG-3';

[0017] (4) Chr07A_40212990_40213139:

[0018] Seq_7: 5'-TCTATTCAGCACGACCATTCATGA-3';

[0019] Seq_8: 5'-GCTATATAGGAGATACACCATGTTGCT-3';

[0020] (5) Chr07A_44532481_44532630:

[0021] Seq_9: 5'-ACACTTGAATGGAAAGCTGAGAGA-3';

[0022] Seq_10: 5'-ACATCTACTCAAGGAATTGGCCATT-3';

[0023] (6) Chr07A_48150382_48150531:

[0024] Seq_11 : 5 '-CACTATCAACAACCACAACCGT-3 ';

[0025] Seq_12: 5'-CGGGTGATGAGCTTCTAGGTG-3';

[0026] (7) Chr07A_57473314_57473463:

[0027] Seq_13: 5 '-CGATGTCTCTCAAGTCTCCACC-3 ';

[0028] Seq_14: 5'-AGCTACGATGACTGGCTTGAAGA-3';

[0029] (8) Chr07A_71049442_71049591:

[0030] Seq_15: 5 '-GCGAGCAATAATTAGCCACATCA-3 ';

[0031] Seq_16: 5'-TCCTACTTATGGATCAAGCTGCTATT-3';

[0032] (9) Chr07A_76623593_76623742:

[0033] Seq_17: 5 '-TGGTTGTATCTTCTAAGTTCTATGCTAGT-3 ';

[0034] Seq_18: 5'-GGTCTAGGATTAGTTGTAGTGTCTTGT-3';

[0035] (10) Chr07A_77864313_77864462:

[0036] Seq_19: 5 '-AGCATATAGTGGAGTATTGGAGTTTATCT-3 ';

[0037] Seq_20: 5'-TGGCTACCGAGTATTCCAAGAAA-3';

[0038] (11) Chr07A_80038156_80038305:

[0039] Seq_21: 5 '-CATGTTGCTGAATTCACTGGTGC-3 ';

[0040] Seq_22: 5 -TGTCATGCCAATACCACTGTCAG-3 ';

[0041] (12) Chr07A_90392296_90392445:

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

[0043] Seq_24: 5 -AGCTTGAGTTTGAACACTTCTGT-3 '.

[0044] In certain embodiments, the amplified product of Chr07A_11572915_11573064 corresponds to position 11572915_11573064 of chromosome 7; the amplified product of Chr07A_19076247_19076396 corresponds to position 19076247_19076396 of chromosome 7; the amplified product of Chr07A_32084586_32084735 corresponds to position 32084586_32084735 of chromosome 7; the amplified product of Chr07A_40212990_40213139 corresponds to position 40212990_40213139 of chromosome 7; the amplified product of Chr07A_44532481_44532630 corresponds to position 44532481_44532630 of chromosome 7; the amplified product of Chr07A_48150382_48150531 corresponds to position 48150382_48150531 of chromosome 7; the amplified product of Chr07A_57473314_57473463 corresponds to position 57473314_57473463 of chromosome 7; the amplified product of Chr07A_62076414_62076563 corresponds to position 62076414_62076563 of chromosome 7; the amplified product of Chr07A_76623593_76623742 corresponds to position 76623593_76623742 of chromosome 7; the amplified product of Chr07A_77864313_77864462 corresponds to position 77864313_77864462 of chromosome 7; the amplified product of Chr07A_80038156_80038305 corresponds to position 80038156_80038305 of chromosome 7; the amplified product of Chr07A_90392296_90392445 corresponds to position 90392296_90392445 of chromosome 7.

[0045] In the present application, each 30 Mb interval-specific primer of chromosome 7 of Saccharum officinarum contains at least one pair of primer pairs. The primer set of the present application can comprehensively cover the specific interval of chromosome 7 of Saccharum officinarum.

[0046] The present application also provides a primer set as described in the above technical solutions for identifying the chromosome 7 of Saccharum officinarum.

[0047] The present application also provides a kit comprising the primer set as described in the above technical solutions.

[0048] In some embodiments, the Rapid Taq Master Mix, nuclease-free sterile water are also preferably included.

[0049] In some embodiments, the positive sample DNA is preferably total DNA of sugarcane leaves containing the Chromosome 7 of Eremochloa ophiuroides blood relationship, and the negative sample DNA is preferably total DNA of sugarcane leaves not containing the Chromosome 7 of Eremochloa ophiuroides blood relationship.

[0050] The application also provides a kit as described in the above technical solution for identifying the Chromosome 7 of Eremochloa ophiuroides blood relationship in sugarcane.

[0051] The application also provides a method for identifying the Chromosome 7 of Eremochloa ophiuroides blood relationship in sugarcane, which comprises performing PCR amplification on the genomic DNA of a sample to be tested using the primer set as described in the above technical solution or the kit as described in the above technical solution.

[0052] In some embodiments, the PCR amplification is preferably performed by preparing a reaction system for each primer pair: 2x Rapid Taq Master Mix 12.5 μL, 50 ng / μL DNA template 2.5 μL, 10 μM of the upstream and downstream primers each 2.5 μL, and the rest is ddH2O in each 25 μL reaction system.

[0053] In some embodiments, the amplification procedure of the PCR amplification is preferably 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; and 12℃ storage.

[0054] In some embodiments, the method preferably further comprises performing gel electrophoresis after the PCR amplification and analyzing and judging the electrophoresis results.

[0055] In some embodiments, the extraction method of the genomic DNA of the sample to be tested preferably includes but is not limited to the CTAB method.

[0056] In some embodiments, the gel electrophoresis is preferably low-melting-point agarose gel electrophoresis with a mass-volume ratio of 2.0%, and the electrophoresis is performed at 120 V for 30 min.

[0057] In some embodiments, the judgment criteria of the method are preferably as follows: if the PCR amplification product appears an amplification band at the position of 146 bp-150 bp, the sample to be tested contains the Chromosome 7 of Eremochloa ophiuroides specific interval blood relationship; and if the PCR amplification product does not appear an amplification band at the position of 146 bp-150 bp, the sample to be tested does not contain the Chromosome 7 of Eremochloa ophiuroides specific interval blood relationship.

[0058] Beneficial technical effects: the application provides a primer group, a kit, a method and an application for identifying Erianthus arundinaceus chromosome 7 in sugarcane, the primer group includes 12 primer groups, each primer group includes a forward primer and a reverse primer, and specific nucleotide sequences are shown in Seq_1-Seq_24. The specific primer of each 30Mb Erianthus arundinaceus chromosome 7 interval contains at least one primer pair, the primer group, the kit, the method and the application can quickly and comprehensively detect whether the hybrid offspring of Erianthus arundinaceus and sugarcane contains Erianthus arundinaceus chromosome 7 specific interval blood, and provide technical support for the blood identification of the hybrid offspring of the wild germplasm resource Erianthus arundinaceus introduced in China and the basic research on the chromosomal structure variation of Erianthus arundinaceus. BRIEF DESCRIPTION OF DRAWINGS

[0059] Figure 1 It is an electrophoretogram for identifying the specific interval blood of Erianthus arundinaceus chromosome 7, wherein M is a 100bp DNA ladder standard relative molecular mass, lane 1 is nuclease-free sterile water, lanes 2-7 are sugarcane leaf samples of Bala, LA-Purple, Np-X, SES208, Zhensu No.1 and ROC22 in turn, and lanes 8-10 are sugarcane leaf samples of Hainan 92-77, Yunnan 2012-3 and Yacheng 05-164 in turn.

[0060] Figure 2 It is an electrophoretogram for detecting the blood of different segments of Erianthus arundinaceus chromosome 7 in Yacheng 01-92 and Yacheng 01-36 materials by 12 primer groups. DETAILED DESCRIPTION

[0061] In order to better understand the present application, the content of the present application will be further illustrated below in combination with examples, but the content of the present application is not limited to the following examples only. 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.

[0062] Materials: see Table 1.

[0063] Table 1: Erianthus arundinaceus and sugarcane clones

[0064]

[0065]

[0066] The main reagents used in the present application are as follows: (all chemical reagents are analytical pure)

[0067] (1) CTAB extraction buffer: 100 mM Tris-HCl (pH = 8.0), 20 mM EDTA-Na2, 1.4 M NaCl, 2% CTAB (mass / volume), 0.1% beta-mercaptoethanol (volume / volume) was added before use;

[0068] (2) 1x TAE buffer: 242 g Tris, 37.2 g EDTA-Na2, 57.1 mL glacial acetic acid, and purified water to 1 L;

[0069] (3) PCR amplification reagents were purchased from Nanjing Nuowezan Biotechnology Co., Ltd.

[0070] The instruments used in the present application are mainly as follows:

[0071] (1) PCR amplifier: Singapore T100 TM Thermal Cycler PCR amplifier;

[0072] (2) Electrophoresis instrument: DYY-6C electrophoresis instrument from Beijing Liyi Biological Technology Co., Ltd.;

[0073] (3) Centrifuge: German Heraeus Pico 17 high-speed centrifuge;

[0074] (4) Gel imaging system: American Omega Fluor plus Gel imaging system;

[0075] (5) Spectrophotometer: Monar (Suzhou) Biological Technology Co., Ltd. Eva 3200 ultramicro nucleic acid protein detector.

[0076] Example 1

[0077] 1. Sugarcane leaf total DNA extraction

[0078] (1) Prepare tools: clean and sterilize the mortar and scissors, wherein the scissors are sterilized with 75% alcohol, and the mortar is sterilized by burning with anhydrous ethanol, and then pre-cooled on ice for use;

[0079] (2) Process the sample: select fresh and healthy young leaves of Puller, 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 small pieces, pour liquid nitrogen into the pre-cooled mortar, grind into fine powder, generally to the best color change from dark green to light green and white, put into a 2 mL centrifuge tube, about 1 / 3 of the volume of the tube;

[0080] (3) Cell lysis: The prepared 2x CTAB was preheated in a 65°C water bath for 30 min. 900 μL of the preheated CTAB extraction solution and 100 μL of anhydrous ethanol were added to the powder, which was mixed quickly and uniformly in a water bath at 65°C for 0.5-1 h. The mixture was taken out every 10 min and mixed by inverting to ensure complete cell lysis.

[0081] (4) Chloroform extraction: After water bath, the mixture was centrifuged for 10 min. An equal volume of a phenol: chloroform: isoamyl alcohol (25:24:1) lower phase solution was added to the supernatant, which was mixed thoroughly on a shaker. The precipitate was removed by centrifugation for 10 min at 12,000 rpm. An equal volume of chloroform: isoamyl alcohol (24:1) solution was added, and the mixture was mixed several times and centrifuged for 10 min at 12,000 rpm.

[0082] (5) DNA precipitation: An equal volume of 3M NaAc (pH 5.2) solution and an equal volume of pre-cooled isoamyl alcohol solution were added to the supernatant, which was mixed thoroughly and placed in a -20°C environment for 1 h.

[0083] (6) DNA washing: After precipitation, the mixture was centrifuged for 10 min. 1 mL of 75% ethanol solution was added to rinse the precipitate twice. The first rinse was centrifuged for 5 min, and the second rinse was performed after the first rinse. Preferably, pre-cooled ethanol solution was used.

[0084] (7) DNA purification: After rinsing, the mixture was centrifuged for 10 min to retain the precipitate. The ethanol was allowed to evaporate at room temperature. Finally, 100 μL of TE Buffer and 1 μL of RNase A were added, and the mixture was purified at 37°C for 30 min to remove RNA.

[0085] (8) After detecting the DNA quality and concentration using a nucleic acid protein detector, the DNA was stored at 4°C or -20°C for long-term preservation.

[0086] 2. Primer design

[0087] Firstly, Repeatmasker software was used to screen the whole genome of LA-Purple (DNA can be sequenced after DNA extraction by the above method, the same below), Np-X and Hainan92-77, respectively. K-mers method was used to cut the 7th chromosome of the three sets of genomes, and the genome cutting was completed by Python script. The bwa software was used to align the cut fragments, and the sequence set of 150bp fragments specific to Hainan92-77 was screened. Primer3 was used to design primers for the Hainan92-77 specific sequence set, and the default values of the software were used for the primer design parameters, and some modifications were made, in which the modification conditions were as follows: the fragment size was about 17bp-23bp, the primer output quantity was 1, and the target product was 146bp-150bp. The filtered sequences were designed into primers. The specific primer sequences of the 7th chromosome of Hainan92-77 are shown in Table 2.

[0088] Table 2 Primer sequences of specific interval molecular markers of the 7th chromosome of Hainan92-77

[0089]

[0090]

[0091] 3. PCR amplification

[0092] Each primer pair was prepared into a reaction system, and the PCR reaction system was as follows: 2x RapidTaq Master Mix 12.5μL, 50ng / μL DNA template 2.5μL, 10μM upstream and downstream primers 2.5μL each, and the rest was ddH2O in each 25μL reaction system.

[0093] After adding the reagents in the above reaction system into the PCR tube, mix well, centrifuge at 8000rpm for 5s, and the amplification program is as follows: 95℃ pre-denaturation for 3min; 95℃ denaturation for 30s, 60℃ annealing for 20s, 72℃ extension for 15s, 32 cycles; 72℃ final extension for 3min; 12℃ storage.

[0094] 4. Electrophoresis detection

[0095] After the amplification reaction, 10μL PCR product was taken for low-melting-point agarose gel electrophoresis with a mass-volume ratio of 2.0%, in the environment of 1xTAE electrophoresis buffer, 120V constant voltage electrophoresis for 30min, then observed and photographed by gel imaging system. The appearance of PCR product amplification band in the electrophoresis detection diagram indicates that the sample contains Hainan92-77 specific interval of the 7th chromosome; the absence of PCR product amplification band indicates that the sample does not contain Hainan92-77 specific interval of the 7th chromosome.

[0096] The electrophoresis detection results are as follows: Figure 1As shown in the figure, each electrophoresis image is a specific identification of blood relations at different segments of chromosome 7. Samples 8-10 show PCR product amplification bands at around 150bp, indicating that these samples contain blood relations corresponding to specific segments of chromosome 7. 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 7. 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 7 of *Imperata cylindrica* from other bloodline materials that do not contain specific regions of *Imperata cylindrica*.

[0097] Experiment 1 used primer set detection to identify bloodline materials containing different segments of chromosome 7 of the variegated iris.

[0098] 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 chromosome 7 lineage of Tripidium arundinaceumin sugarcane.

[0099] Genomic DNA containing chromosome 7 of *Imperata cylindrica* chromosome 7 was extracted from the BC1 progeny 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.

[0100] Accuracy of primer pairs 1-12 for different segments of chromosome 7 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 7 lineage. Results showed that the target bands were amplified in all the clones, indicating that primer pairs 1-12 have good accuracy and can be used to detect different segments of chromosome 7 lineage in high-generation sugarcane-*Imperata cylindrica* hybrids.

[0101] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. A primer set for identifying the lineage of chromosome 7 in sugarcane, characterized in that, The primer set includes 12 primer sets, each of which includes a forward primer and a reverse primer, and the specific nucleotide sequences are shown in Seq_1 to Seq_24.

2. The application of the primer set described in claim 1 in identifying the lineage of chromosome 7 in sugarcane.

3. A reagent kit, characterized in that, Includes the primer set described in claim 1.

4. The reagent kit according to claim 3, characterized in that, It also includes Rapid Taq Master Mix and nuclease-free sterile water.

5. The use of the kit according to claim 3 or 4 in identifying chromosome 7 lineage in sugarcane.

6. A method for identifying the lineage of chromosome 7 in sugarcane, characterized in that, The method includes PCR amplification of the genomic DNA of the sample to be tested using the primer set of claim 1 or the kit of any one of claims 3 to 4.

7. The method according to claim 6, characterized in that, The method also includes performing gel electrophoresis after PCR amplification and analyzing and judging the electrophoresis results.

8. The method according to claim 6, characterized in that, The methods for extracting genomic DNA from the samples to be tested include, but are not limited to, the CTAB method.

9. The method according to claim 7, characterized in that, The gel electrophoresis was performed using a low-melting-point agarose gel with a mass-to-volume ratio of 2.0%, at a constant voltage of 120V for 30 minutes.

10. The method according to any one of claims 6 to 9, characterized in that, The judgment criteria of the method 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 7 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 7 of the variegated iris.

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