Rice whole genome InDel molecular marker system based on capillary electrophoresis and application

Abstract

The invention provides a rice whole genome InDel molecular marker system based on capillary electrophoresis, the InDel molecular marker system comprises an InDel primer pair corresponding to 61 InDel molecular marker sites, and the nucleotide sequences of the InDel primer pair are as shown in SEQ ID NO. 1 to SEQ ID NO. 122. The invention also provides a preparation method of the rice whole genome InDel molecular marker system based on capillary electrophoresis. The invention further provides application of the rice whole genome InDel molecular marker system. The rice whole genome InDel molecular marker system can be applied to rice genetic map construction, rice molecular breeding and rice variety identification. Based on the high-precision characteristic of capillary gel electrophoresis (CGE) and the next-generation sequencing technology, a set of InDel molecular marker system covering 61 genome polymorphisms with the average difference fragment length of 8.4 bp of the whole rice genome is developed, and a new method is provided for the aspects of rice genetic diversity analysis, variety identification, map-based cloning and the like.

Description

Technical Field

[0001] This invention belongs to the field of rice molecular breeding technology, specifically relating to a rice whole genome InDel molecular marker system based on capillary electrophoresis and its application. Background Technology

[0002] InDel markers are molecular markers based on PCR fragment length polymorphism. They are simple to operate and provide reliable results, and are widely used in rice germplasm identification, map-based cloning, marker-assisted selection (MAS) breeding, and genetic diversity analysis. However, due to the limitations of conventional polyacrylamide gel electrophoresis, InDel markers with fragment length differences of less than 10 bp are difficult to apply.

[0003] Capillary gel electrophoresis (CGE) is an electrophoresis technique that uses a gel from a slab transferred to a capillary as a support. It is a product of the perfect combination of classical electrophoresis and modern microcolumn separation technology. Its analytical sensitivity has been improved to the point of detecting changes in a single base, and its separation efficiency reaches millions of theoretical plates. It can analyze fragments of all sizes, from resolving single nucleotide sequences to separating Mb of DNA. Analysis time has been reduced from hours to minutes and seconds. During analysis, the capillary is filled with gel and opened at both ends. This allows charged molecules within the gel to move to opposite ends of the capillary. Because different molecules have different size-to-charge ratios, they move at different rates within the tube, reaching the capillary endpoint at varying speeds. Capillary electrophoresis detects and separates different molecules based on this principle.

[0004] Based on the high precision of capillary gel electrophoresis (CGE) and next-generation sequencing technology, this invention develops a set of InDel markers covering 61 genomic polymorphisms with an average differential fragment length of 8.4 bp in the whole rice genome, adding a new method for rice genetic diversity analysis, variety identification, map-based cloning, and other fields. Summary of the Invention

[0005] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a rice whole genome InDel molecular marker system based on capillary electrophoresis and its application. This system is based on the high precision of capillary gel electrophoresis (CGE) and next-generation sequencing technology, and covers 61 genomic polymorphisms with an average differential fragment length of 8.4 bp in the whole rice genome. It provides a new method for rice genetic diversity analysis, variety identification, map-based cloning, and other aspects.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a rice whole-genome InDel molecular marker system based on capillary electrophoresis, characterized in that the InDel molecular marker system includes primer pairs corresponding to 61 InDel molecular marker sites, wherein the InDel primer pairs include CHR0105 primer pair, CHR0110 primer pair, CHR0115 primer pair, CHR0120 primer pair, CHR0125 primer pair, CHR0130 primer pair, CHR0135 primer pair, CHR0140 primer pair, and C... Primer pair HR0205, CHR0210, CHR0215, CHR0220, CHR0225, CHR0230, CHR0235, CHR0305, CHR0310, CHR0315, CHR0320, CHR0325, CHR0330, CHR0405, CHR0410, CHR0420, CHR0425, CHR0430 Primer pairs, CHR0435 primer pair, CHR0515 primer pair, CHR0520 primer pair, CHR0525 primer pair, CHR0605 primer pair, CHR0610 primer pair, CHR0620 primer pair, CHR0625 primer pair, CHR0630 primer pair, CHR0710 primer pair, CHR0715 primer pair, CHR0720 primer pair, CHR0725 primer pair, CHR0805 primer pair, CHR0810 primer pair, CHR0815 primer pair, CHR0820 primer pair, CHR0 Primer pairs 905, CHR0910, CHR0915, CHR0920, CHR1015, CHR1010, CHR1015, CHR1020, CHR1105, CHR1110, CHR1115, CHR1120, CHR1125, CHR1205, CHR1210, CHR1215, CHR1220, and CHR1225.

[0007] The nucleotide sequences of the forward and reverse primers of the CHR0105 primer pair are shown in SEQ ID NO.1-2;

[0008] The nucleotide sequences of the forward and reverse primers of the CHR0110 primer pair are shown in SEQ ID NO.3-4;

[0009] The nucleotide sequences of the forward and reverse primers of the CHR0115 primer pair are shown in SEQ ID NO.5-6;

[0010] The nucleotide sequences of the forward and reverse primers of the CHR0120 primer pair are shown in SEQ ID NO.7-8;

[0011] The nucleotide sequences of the forward and reverse primers of the CHR0125 primer pair are shown in SEQ ID NO. 9-10;

[0012] The nucleotide sequences of the forward and reverse primers of the CHR0130 primer pair are shown in SEQ ID NO.11-12;

[0013] The nucleotide sequences of the forward and reverse primers of the CHR0135 primer pair are shown in SEQ ID NO.13-14;

[0014] The nucleotide sequences of the forward and reverse primers of the CHR0140 primer pair are shown in SEQ ID NO.15-16;

[0015] The nucleotide sequences of the forward and reverse primers of the CHR0205 primer pair are shown in SEQ ID NO.17-18;

[0016] The nucleotide sequences of the forward and reverse primers of the CHR0210 primer pair are shown in SEQ ID NO.19-20;

[0017] The nucleotide sequences of the forward and reverse primers of the CHR0215 primer pair are shown in SEQ ID NO.21-22;

[0018] The nucleotide sequences of the forward and reverse primers of the CHR0220 primer pair are shown in SEQ ID NO. 23-24;

[0019] The nucleotide sequences of the forward and reverse primers of the CHR0225 primer pair are shown in SEQ ID NO.25-26;

[0020] The nucleotide sequences of the forward and reverse primers of the CHR0230 primer pair are shown in SEQ ID NO.27-28;

[0021] The nucleotide sequences of the forward and reverse primers of the CHR0235 primer pair are shown in SEQ ID NO.29-30;

[0022] The nucleotide sequences of the forward and reverse primers of the CHR0305 primer pair are shown in SEQ ID NO.31-32;

[0023] The nucleotide sequences of the forward and reverse primers of the CHR0310 primer pair are shown in SEQ ID NO.33-34;

[0024] The nucleotide sequences of the forward and reverse primers of the CHR0315 primer pair are shown in SEQ ID NO.35-36;

[0025] The nucleotide sequences of the forward and reverse primers of the CHR0320 primer pair are shown in SEQ ID NO.37-38;

[0026] The nucleotide sequences of the forward and reverse primers of the CHR0325 primer pair are shown in SEQ ID NO.39-40;

[0027] The nucleotide sequences of the forward and reverse primers of the CHR0330 primer pair are shown in SEQ ID NO.41-42;

[0028] The nucleotide sequences of the forward and reverse primers of the CHR0405 primer pair are shown in SEQ ID NO.43-44;

[0029] The nucleotide sequences of the forward and reverse primers of the CHR0410 primer pair are shown in SEQ ID NO.45-46;

[0030] The nucleotide sequences of the forward and reverse primers of the CHR0420 primer pair are shown in SEQ ID NO.47-48;

[0031] The nucleotide sequences of the forward and reverse primers of the CHR0425 primer pair are shown in SEQ ID NO.49-50;

[0032] The nucleotide sequences of the forward and reverse primers of the CHR0430 primer pair are shown in SEQ ID NO. 51-52;

[0033] The nucleotide sequences of the forward and reverse primers of the CHR0435 primer pair are shown in SEQ ID NO. 53-54;

[0034] The nucleotide sequences of the forward and reverse primers of the CHR0515 primer pair are shown in SEQ ID NO. 55-56;

[0035] The nucleotide sequences of the forward and reverse primers of the CHR0520 primer pair are shown in SEQ ID NO. 57-58;

[0036] The nucleotide sequences of the forward and reverse primers of the CHR0525 primer pair are shown in SEQ ID NO.59-60;

[0037] The nucleotide sequences of the forward and reverse primers of the CHR0605 primer pair are shown in SEQ ID NO. 61-62;

[0038] The nucleotide sequences of the forward and reverse primers of the CHR0610 primer pair are shown in SEQ ID NO. 63-64;

[0039] The nucleotide sequences of the forward and reverse primers of the CHR0620 primer pair are shown in SEQ ID NO. 65-66;

[0040] The nucleotide sequences of the forward and reverse primers of the CHR0625 primer pair are shown in SEQ ID NO. 67-68;

[0041] The nucleotide sequences of the forward and reverse primers of the CHR0630 primer pair are shown in SEQ ID NO. 69-70;

[0042] The nucleotide sequences of the forward and reverse primers of the CHR0710 primer pair are shown in SEQ ID NO.71-72;

[0043] The nucleotide sequences of the forward and reverse primers of the CHR0715 primer pair are shown in SEQ ID NO. 73-74;

[0044] The nucleotide sequences of the forward and reverse primers of the CHR0720 primer pair are shown in SEQ ID NO.75-76;

[0045] The nucleotide sequences of the forward and reverse primers of the CHR0725 primer pair are shown in SEQ ID NO.77-78;

[0046] The nucleotide sequences of the forward and reverse primers of the CHR0805 primer pair are shown in SEQ ID NO.79-80;

[0047] The nucleotide sequences of the forward and reverse primers of the CHR0810 primer pair are shown in SEQ ID NO. 81-82;

[0048] The nucleotide sequences of the forward and reverse primers of the CHR0815 primer pair are shown in SEQ ID NO. 83-84;

[0049] The nucleotide sequences of the forward and reverse primers of the CHR0820 primer pair are shown in SEQ ID NO. 85-86;

[0050] The nucleotide sequences of the forward and reverse primers of the CHR0905 primer pair are shown in SEQ ID NO. 87-88;

[0051] The nucleotide sequences of the forward and reverse primers of the CHR0910 primer pair are shown in SEQ ID NO. 89-90;

[0052] The nucleotide sequences of the forward and reverse primers of the CHR0915 primer pair are shown in SEQ ID NO. 91-92;

[0053] The nucleotide sequences of the forward and reverse primers of the CHR0920 primer pair are shown in SEQ ID NO. 93-94;

[0054] The nucleotide sequences of the forward and reverse primers of the CHR1015 primer pair are shown in SEQ ID NO. 95-96;

[0055] The nucleotide sequences of the forward and reverse primers of the CHR1010 primer pair are shown in SEQ ID NO. 97-98;

[0056] The nucleotide sequences of the forward and reverse primers of the CHR1015 primer pair are shown in SEQ ID NO. 99-100;

[0057] The nucleotide sequences of the forward and reverse primers of the CHR1020 primer pair are shown in SEQ ID NO.101-102;

[0058] The nucleotide sequences of the forward and reverse primers of the CHR1105 primer pair are shown in SEQ ID NO. 103-104;

[0059] The nucleotide sequences of the forward and reverse primers of the CHR1110 primer pair are shown in SEQ ID NO. 105-106;

[0060] The nucleotide sequences of the forward and reverse primers of the CHR1115 primer pair are shown in SEQ ID NO. 107-108;

[0061] The nucleotide sequences of the forward and reverse primers of the CHR1120 primer pair are shown in SEQ ID NO. 109-110;

[0062] The nucleotide sequences of the forward and reverse primers of the CHR1125 primer pair are shown in SEQ ID NO. 111-112;

[0063] The nucleotide sequences of the forward and reverse primers of the CHR1205 primer pair are shown in SEQ ID NO. 113-114;

[0064] The nucleotide sequences of the forward and reverse primers of the CHR1210 primer pair are shown in SEQ ID NO. 115-116;

[0065] The nucleotide sequences of the forward and reverse primers of the CHR1215 primer pair are shown in SEQ ID NO. 117-118;

[0066] The nucleotide sequences of the forward and reverse primers of the CHR1220 primer pair are shown in SEQ ID NO. 119-120;

[0067] The nucleotide sequences of the forward and reverse primers of the CHR1225 primer pair are shown in SEQ ID NO.121-122.

[0068] This invention also provides a method for identifying rice varieties using the aforementioned capillary electrophoresis-based whole-genome InDel molecular marker system, the method comprising the following steps:

[0069] S1. Extract the genomic DNA of the rice to be tested;

[0070] S2. The rice genomic DNA obtained in S1 was amplified by PCR using InDel primers to obtain the PCR amplification product.

[0071] S3. Capillary gel electrophoresis analysis was performed on the PCR amplification products obtained in S2.

[0072] S4. Determine the types of samples to be tested based on the diversity of electrophoretic bands.

[0073] Preferably, the rice genomic DNA extraction in S1 is performed using the SDS method.

[0074] Preferably, the PCR amplification reaction system described in S2 is as follows: 2 μL 10×Buffer, 0.2 μL dNTP (10 mmol / L), 1 μL forward primer (5 μmol / L), 1 μL reverse primer (5 μmol / L), 1 μL rice genomic DNA to be tested, 0.15 μL Taq DNA polymerase (5 U / μL), and ddH2O to a final volume of 20 μL;

[0075] Preferably, the PCR amplification reaction program is as follows: pre-denaturation at 94℃ for 3 min; denaturation at 94℃ for 30 s, annealing at 65℃ for 20 s, extension at 72℃ for 15 s, for 35 cycles; and final extension at 72℃ for 5 min.

[0076] Preferably, the capillary gel electrophoresis analysis described in S3 uses a FragmentAnalyser 5200 capillary electrophoresis apparatus and a DNF-900 kit provided by Agilent Technologies. The detection conditions are as follows: sample injection voltage is 5-7.5 kV, time is 10 s; pre-test voltage is 5-8 kV, time is 30 s; capillary electrophoresis separation voltage is 5-8 kV, time is 60 min to 80 min.

[0077] Preferably, the basis for identifying rice varieties in S4 is that the probability of InDel mutations of the same length but different insertion or deletion base sequences occurring at the same locus on the biological genome is extremely small. Therefore, the InDel marker has strong genetic stability between generations. If different samples of the same variety show different types of bands in the electrophoresis diagram, it indicates that the test samples are mixed or have insufficient purity. The InDel marker electrophoresis band pattern of the same variety should be the same. If multiple InDel locus markers have different electrophoresis band patterns, it indicates that there are genetic differences between the test samples, which may be different varieties.

[0078] This invention also provides an application of the rice whole genome InDel molecular marker system based on capillary electrophoresis, which can be applied to rice genetic map construction, rice molecular breeding, and rice variety identification.

[0079] Compared with the prior art, the present invention has the following advantages:

[0080] 1. Based on the high precision of capillary gel electrophoresis and next-generation sequencing technology, this invention has developed an InDel molecular marker system covering 61 genomic polymorphisms in the rice genome with an average of only 8.4 bp of insertions or deletions, providing a new method for rice genetic diversity analysis, variety identification, map-based cloning, and other aspects.

[0081] 2. Due to the limitations of conventional polyacrylamide gel electrophoresis, InDel markers with small fragment length differences are difficult to apply. This invention develops an InDel molecular marker system with a small average number of inserted or deleted bases, which has important reference value for the construction of genetic maps, molecular breeding, and variety identification of rice germplasm resources.

[0082] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0083] Figure 1 This is an electrophoresis diagram of five InDel primer pairs (CHR0105, CHR0110, CHR0115, CHR0120, and CHR0125) used in Example 3 of this invention for three rice varieties: Zhonghua 11, Huanghuazhan, and Nipponbare.

[0084] Figure 2 This is an electrophoresis image of 11 rice varieties, namely He Guang Simiao, Hua Jing, Gui Ye Feng, E Feng Simiao, Jing Gui Simiao, Guang Ai Zhan, Yue Nong Simiao, Jia Fu Zhan, Wu Shan Simiao, Huang Guang You Zhan, and Chang Li Xiang, using the InDel primer pair CHR0320 of Example 3 of this invention. Detailed Implementation

[0085] Example 1

[0086] This embodiment describes the construction of a rice whole-genome InDel molecular marker system based on capillary electrophoresis.

[0087] Based on the publicly available rice database RiceVarMap v2.0, within a 0.1Mb ​​range before and after every 5Mb of rice chromosomes, SNPs that are neither A / T nor G / C, do not contain repeating units within 50bp before and after them, and have a GC content between 30% and 70% in the 50bp region before and after them were selected as candidate Indel markers. The initial number of rice Indel markers published from the RiceVarMap v2.0 database is 790.

[0088] Example 2

[0089] In this embodiment, rice genomic DNA was used as a template to detect the effectiveness of the 790 Indel markers obtained in Example 1 by PCR amplification. The detection method is as follows:

[0090] 1. Extracting rice genomic DNA

[0091] DNA was extracted from rice seedlings using the SDS method. The extracted DNA samples were dissolved in TE buffer (10 mM Tris-HCl, pH 8.0, 10 mM EDTA), and their mass and concentration were determined. The samples were then diluted to a final concentration of 20 mg / L. -1 Store at -20℃ for later use.

[0092] The method for extracting DNA from rice seedlings includes the following steps: Rice leaves are taken and placed in a mortar pre-cooled with liquid nitrogen. Liquid nitrogen is added and the mixture is ground into powder. The powder is then transferred to a 1.5 mL centrifuge tube. 700 μL of 20% SDS solution preheated to 65°C is added to the tube, mixed, and incubated at 65°C for 30 min, shaking every 5 min during the incubation process. 200 μL of 5 mol / L KAc solution is added, and the mixture is agitated 30–50 times. The tube is then placed in an ice bath at -20°C for 30 min. After timing, the tube is centrifuged at 11000 rpm for 15 min, and the supernatant is transferred to another centrifuge tube. This process is repeated once. Then, 0.7 mL of isopropanol is added, and the mixture is agitated 30–50 times. The tube is then precipitated at -20°C for 30 min. The tube is then centrifuged again at 11000 rpm for 5 min, the supernatant is discarded, and the precipitate is washed with 1 mL of 70% ethanol solution. The genomic DNA precipitate is recovered, dried, and dissolved in TE buffer. The DNA integrity is then detected by electrophoresis.

[0093] 2. Primer design:

[0094] Based on the 790 rice Indel markers obtained in Example 1, specific primers were designed, and the preferred parameter settings were as follows:

[0095] The length of the PCR product is preferably 100bp to 200bp, and more preferably 150bp;

[0096] The primer length is preferably 19bp to 25bp, and more preferably 22bp;

[0097] The preferred Tm value is 57℃~64℃, and more preferably 61℃.

[0098] To obtain as many primers as possible, it is preferable to design primers with a GC content in the range of 40% to 60%.

[0099] The primers used in this invention are preferably synthesized by Shanghai Sangon Biotech Co., Ltd.

[0100] 3. PCR amplification

[0101] Using the rice genomic DNA obtained in step 1 as a template, different primers obtained in step 2 were selected to perform PCR amplification on the rice genomic DNA to obtain PCR amplification products.

[0102] The PCR reaction system consisted of: 2 μL 10×Buffer, 0.2 μL dNTP (10 mmol / L), 1 μL forward primer (5 μmol / L), 1 μL reverse primer (5 μmol / L), 1 μL template DNA, 0.15 μL Taq DNA polymerase (5 U / μL), and ddH2O to a final volume of 20 μL.

[0103] The PCR amplification reaction program was as follows: pre-denaturation at 94℃ for 3 min; denaturation at 94℃ for 30 s, annealing at 65℃ for 20 s, extension at 72℃ for 15 s, for 35 cycles; and final extension at 72℃ for 5 min.

[0104] 4. Capillary gel electrophoresis detection

[0105] In this embodiment, capillary gel electrophoresis was performed using an Agilent FragmentAnalyser 5200 capillary electrophoresis system and a DNF-900 kit. The Conditioning bottle, Gel 1 bottle, Gel 2 bottle, and Waste bottle used below are all kits provided with the instrument. The kit includes: dsDNA 905 separating gel, insertion dye, 5× capillary washing solution, 5× 930 dsDNA Inlet Buffer, 35bp and 500bp Markers, DNA Ladder in the range of 75bp to 400bp, mineral oil, and 1× TE dilution buffer.

[0106] The steps for capillary gel electrophoresis detection are as follows:

[0107] (1) Prepare the dsDNA 905 separating gel and the insertion dye mixture. Allow the gel and dye to reach room temperature. Add 1.5 μL of insertion dye to every 20 mL of dsDNA 905 separating gel. After mixing, pour the mixture into a bottle labeled Gel 1 and insert the tube into the bottom of the bottle. For the first plate, run the Full Condition program, which requires 25 mL of dye-mixed gel. After that, run the GelPrime Only program, which requires 5 mL of dye-mixed gel per plate.

[0108] (2) Take out the 5× capillary cleaning solution that has been placed at room temperature, add 20 mL of 5× capillary cleaning solution to every 80 mL of ultrapure water, shake to mix well, and pour into a bottle labeled Conditioning. Insert the tube into the bottom of the bottle.

[0109] (3) Pour the ultrapure water into a bottle labeled Gel 2 and insert the tube into the bottom of the bottle; discard the waste liquid in the Waste bottle;

[0110] (4) Preparation of Inlet Buffer: Take out 5×930dsDNA Inlet Buffer from the refrigerator and let it reach room temperature. Add 20mL of 5×930dsDNA Inlet Buffer to every 80mL of ultrapure water and shake to mix. Add the prepared Inlet Buffer to the 96-well plate of the first layer (B), adding 1.1mL to each well. Place an empty 1mL deep-well 96-well plate into the drawer of the second layer (W) as a waste plate.

[0111] (5) Remove the 35bp and 500bp markers from the refrigerator and allow them to reach room temperature. Shake the liquid to ensure it is thoroughly mixed. Then, pipette 30μL into each well of the sample plate and cover with 20μL of mineral oil.

[0112] (6) Open the Fragment Analyzer software, click Solution levels, select Utilities—Solution Levels, and enter the solution volume in each bottle;

[0113] (7) Take the 75bp-400bp DNA Ladder out of the refrigerator and let it reach room temperature. Then, aspirate 24μL of the 75bp-400bp DNA Ladder and add it to the H12 well of the sample plate.

[0114] (8) Take a clean 96-well plate and aspirate 22 μL of 1×TE dilution buffer into each well of the plate; aspirate 2 μL of the PCR amplification product obtained in step 3 into each well of the plate, and use a pipette to repeatedly aspirate and mix the mixture thoroughly. Then check each well of the plate to ensure that there are no air bubbles at the bottom.

[0115] (9) Set the experimental parameters from the main interface of the Fragment Analyzer5200 instrument control software: 35bp / 500bp Marker injection voltage is 5-7.5kV and time is 10s; pre-test voltage is 5-8kV and time is 30s; capillary electrophoresis separation voltage is 5-8kV and time is 60min-80min; then save the method and run the experiment.

[0116] The analysis revealed that 61 InDel markers exhibited high polymorphism in rice varieties, with an average of 8.4 bp of inserted or deleted bases. These markers can be used for variety identification and genetic diversity analysis. Primer sequences for the 61 InDel markers are shown in Table 1.

[0117] Table 161 nucleotide sequences of InDel-tagged primers

[0118]

[0119]

[0120] Example 3

[0121] This example demonstrates the detection of InDel molecular marker primer diversity among different rice varieties.

[0122] Figure 1 This is an electrophoresis image of five InDel primer pairs (CHR0105, CHR0110, CHR0115, CHR0120, and CHR0125) amplifying three rice varieties: Zhonghua 11, Huanghuazhan, and Nipponbare. "1-4" represent Zhonghua 11, "5-8" represent Huanghuazhan, and "9-12" represent Nipponbare. According to the RiceVarMap v2.0 database, the number of bases inserted or deleted at the Indel sites of primer pairs CHR0105, CHR0110, CHR0115, CHR0120, and CHR0125 are 15bp, 4bp, 6bp, 4bp, and 3bp, respectively. Figure 1The electrophoresis results yielded the following conclusions: (1) The PCR products of primer pairs CHR0105, CHR0110, CHR0115, CHR0120, and CHR0125 showed single electrophoretic bands and good polymorphism between indica and japonica rice (Huang Huazhan is indica rice, while Zhonghua 11 and Nipponbare are japonica rice); (2) The electrophoretic band patterns of Huang Huazhan in the five primer pairs were different from those of Zhonghua 11 and Nipponbare, and the electrophoretic band patterns of the PCR products of primer pair CHR0120 in Zhonghua 11 and Nipponbare were different, indicating that Huang Huazhan and Nipponbare and Zhonghua 11 have large genetic differences, while Nipponbare and Zhonghua 11 have small genetic differences; (3) The amplification products of primers CHR0110 in Zhonghua 11 and Huang Huazhan showed differences in electrophoretic band patterns within the variety, indicating that the experimental samples were impure or mixed.

[0123] Figure 2 This is an electrophoresis image of 11 rice varieties amplified by the InDel primer pair CHR0320: He Guang Simiao, Hua Jing, Gui Ye Feng, E Feng Simiao, Jing Gui Simiao, Guang Ai Zhan, Yue Nong Simiao, Jia Fu Zhan, Wu Shan Simiao, Huang Guang You Zhan, and Chang Li Xiang. The numbers "1-5" represent He Guang Simiao, "6-10" Hua Jing, "11-15" Gui Ye Feng, "16-20" E Feng Simiao, "21-25" Jing Gui Simiao, "26-30" Guang Ai Zhan, "31-35" Yue Nong Simiao, "36-40" Jia Fu Zhan, "41-45" Wu Shan Simiao, "46-50" Huang Guang You Zhan, and "50-55" Chang Li Xiang. According to the RiceVarMap v2.0 database, the number of inserted or deleted bases at this Indel site is 6 bp. Figure 2 The electrophoresis results lead to the following conclusions: the PCR products of the CHR0320 primer pair show a single band and good polymorphism among indica rice varieties; the PCR amplification products of Heguang Simiao, Efeng Simiao, Guang'ai Zhan, Wushan Simiao, and Changlixiang have the same band pattern, while the PCR amplification products of Huajing, Guiyefeng, Jingui Simiao, Yue Nong Simiao, Jiafu Zhan, and Huangguangyou Zhan have the same band pattern.

[0124] The rice whole genome InDel molecular marker system based on capillary electrophoresis of the present invention can be applied to rice genetic map construction, rice molecular breeding and rice variety identification.

[0125] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A rice whole-genome InDel molecular marker system based on capillary electrophoresis, characterized in that, The InDel molecular marker system includes 61 InDel molecular marker sites corresponding to InDel primer pairs. These InDel primer pairs include the following primer pairs: CHR0105, CHR0110, CHR0115, CHR0120, CHR0125, CHR0130, CHR0135, CHR0140, CHR0205, CHR0210, CHR0215, and CHR022... Primer pair 0, CHR0225, CHR0230, CHR0235, CHR0305, CHR0310, CHR0315, CHR0320, CHR0325, CHR0330, CHR0405, CHR0410, CHR0420, CHR0425, CHR0430, CHR0435, CHR0515 Primer pair CHR0520, CHR0525, CHR0605, CHR0610, CHR0620, CHR0625, CHR0630, CHR0710, CHR0715, CHR0720, CHR0725, CHR0805, CHR0810, CHR0815, CHR0820, CHR0905, CHR09 Primer pairs: 10, CHR0915, CHR0920, CHR1015, CHR1010, CHR1015, CHR1020, CHR1105, CHR1110, CHR1115, CHR1120, CHR1125, CHR1205, CHR1210, CHR1215, CHR1220, and CHR1225. The nucleotide sequences of the forward and reverse primers of the CHR0105 primer pair are shown in SEQ ID NO.1-2; The nucleotide sequences of the forward and reverse primers of the CHR0110 primer pair are shown in SEQ ID NO.3-4; The nucleotide sequences of the forward and reverse primers of the CHR0115 primer pair are shown in SEQ ID NO.5-6; The nucleotide sequences of the forward and reverse primers of the CHR0120 primer pair are shown in SEQ ID NO.7-8; The nucleotide sequences of the forward and reverse primers of the CHR0125 primer pair are shown in SEQ ID NO. 9-10; The nucleotide sequences of the forward and reverse primers of the CHR0130 primer pair are shown in SEQ ID NO.11-12; The nucleotide sequences of the forward and reverse primers of the CHR0135 primer pair are shown in SEQ ID NO.13-14; The nucleotide sequences of the forward and reverse primers of the CHR0140 primer pair are shown in SEQ ID NO.15-16; The nucleotide sequences of the forward and reverse primers of the CHR0205 primer pair are shown in SEQ ID NO.17-18; The nucleotide sequences of the forward and reverse primers of the CHR0210 primer pair are shown in SEQ ID NO.19-20; The nucleotide sequences of the forward and reverse primers of the CHR0215 primer pair are shown in SEQ ID NO.21-22; The nucleotide sequences of the forward and reverse primers of the CHR0220 primer pair are shown in SEQ ID NO. 23-24; The nucleotide sequences of the forward and reverse primers of the CHR0225 primer pair are shown in SEQ ID NO.25-26; The nucleotide sequences of the forward and reverse primers of the CHR0230 primer pair are shown in SEQ ID NO.27-28; The nucleotide sequences of the forward and reverse primers of the CHR0235 primer pair are shown in SEQ ID NO.29-30; The nucleotide sequences of the forward and reverse primers of the CHR0305 primer pair are shown in SEQ ID NO.31-32; The nucleotide sequences of the forward and reverse primers of the CHR0310 primer pair are shown in SEQ ID NO.33-34; The nucleotide sequences of the forward and reverse primers of the CHR0315 primer pair are shown in SEQ ID NO. 35-36; The nucleotide sequences of the forward and reverse primers of the CHR0320 primer pair are shown in SEQ ID NO.37-38; The nucleotide sequences of the forward and reverse primers of the CHR0325 primer pair are shown in SEQ ID NO.39-40; The nucleotide sequences of the forward and reverse primers of the CHR0330 primer pair are shown in SEQ ID NO.41-42; The nucleotide sequences of the forward and reverse primers of the CHR0405 primer pair are shown in SEQ ID NO.43-44; The nucleotide sequences of the forward and reverse primers of the CHR0410 primer pair are shown in SEQ ID NO.45-46; The nucleotide sequences of the forward and reverse primers of the CHR0420 primer pair are shown in SEQ ID NO.47-48; The nucleotide sequences of the forward and reverse primers of the CHR0425 primer pair are shown in SEQ ID NO.49-50; The nucleotide sequences of the forward and reverse primers of the CHR0430 primer pair are shown in SEQ ID NO. 51-52; The nucleotide sequences of the forward and reverse primers of the CHR0435 primer pair are shown in SEQ ID NO. 53-54; The nucleotide sequences of the forward and reverse primers of the CHR0515 primer pair are shown in SEQ ID NO. 55-56; The nucleotide sequences of the forward and reverse primers of the CHR0520 primer pair are shown in SEQ ID NO. 57-58; The nucleotide sequences of the forward and reverse primers of the CHR0525 primer pair are shown in SEQ ID NO.59-60; The nucleotide sequences of the forward and reverse primers of the CHR0605 primer pair are shown in SEQ ID NO. 61-62; The nucleotide sequences of the forward and reverse primers of the CHR0610 primer pair are shown in SEQ ID NO. 63-64; The nucleotide sequences of the forward and reverse primers of the CHR0620 primer pair are shown in SEQ ID NO. 65-66; The nucleotide sequences of the forward and reverse primers of the CHR0625 primer pair are shown in SEQ ID NO. 67-68; The nucleotide sequences of the forward and reverse primers of the CHR0630 primer pair are shown in SEQ ID NO. 69-70; The nucleotide sequences of the forward and reverse primers of the CHR0710 primer pair are shown in SEQ ID NO.71-72; The nucleotide sequences of the forward and reverse primers of the CHR0715 primer pair are shown in SEQ ID NO. 73-74; The nucleotide sequences of the forward and reverse primers of the CHR0720 primer pair are shown in SEQ ID NO.75-76; The nucleotide sequences of the forward and reverse primers of the CHR0725 primer pair are shown in SEQ ID NO.77-78; The nucleotide sequences of the forward and reverse primers of the CHR0805 primer pair are shown in SEQ ID NO.79-80; The nucleotide sequences of the forward and reverse primers of the CHR0810 primer pair are shown in SEQ ID NO. 81-82; The nucleotide sequences of the forward and reverse primers of the CHR0815 primer pair are shown in SEQ ID NO. 83-84; The nucleotide sequences of the forward and reverse primers of the CHR0820 primer pair are shown in SEQ ID NO. 85-86; The nucleotide sequences of the forward and reverse primers of the CHR0905 primer pair are shown in SEQ ID NO. 87-88; The nucleotide sequences of the forward and reverse primers of the CHR0910 primer pair are shown in SEQ ID NO. 89-90; The nucleotide sequences of the forward and reverse primers of the CHR0915 primer pair are shown in SEQ ID NO. 91-92; The nucleotide sequences of the forward and reverse primers of the CHR0920 primer pair are shown in SEQ ID NO. 93-94; The nucleotide sequences of the forward and reverse primers of the CHR1015 primer pair are shown in SEQ ID NO. 95-96; The nucleotide sequences of the forward and reverse primers of the CHR1010 primer pair are shown in SEQ ID NO. 97-98; The nucleotide sequences of the forward and reverse primers of the CHR1015 primer pair are shown in SEQ ID NO. 99-100; The nucleotide sequences of the forward and reverse primers of the CHR1020 primer pair are shown in SEQ ID NO.101-102; The nucleotide sequences of the forward and reverse primers of the CHR1105 primer pair are shown in SEQ ID NO. 103-104; The nucleotide sequences of the forward and reverse primers of the CHR1110 primer pair are shown in SEQ ID NO. 105-106; The nucleotide sequences of the forward and reverse primers of the CHR1115 primer pair are shown in SEQ ID NO. 107-108; The nucleotide sequences of the forward and reverse primers of the CHR1120 primer pair are shown in SEQ ID NO. 109-110; The nucleotide sequences of the forward and reverse primers of the CHR1125 primer pair are shown in SEQ ID NO. 111-112; The nucleotide sequences of the forward and reverse primers of the CHR1205 primer pair are shown in SEQ ID NO. 113-114; The nucleotide sequences of the forward and reverse primers of the CHR1210 primer pair are shown in SEQ ID NO. 115-116; The nucleotide sequences of the forward and reverse primers of the CHR1215 primer pair are shown in SEQ ID NO. 117-118; The nucleotide sequences of the forward and reverse primers of the CHR1220 primer pair are shown in SEQ ID NO. 119-120; The nucleotide sequences of the forward and reverse primers of the CHR1225 primer pair are shown in SEQ ID NO.121-122.

2. A method for identifying rice varieties using the InDel molecular marker system based on capillary electrophoresis of the whole rice genome as described in claim 1, the method comprising the following steps: S1. Extract the genomic DNA of the rice to be tested; S2. Use InDel primers to perform PCR amplification on the rice genomic DNA to be tested obtained in S1 to obtain PCR amplification products. S3. Capillary gel electrophoresis analysis was performed on the PCR amplification products obtained in S2. S4. Determine the variety of samples to be tested based on the diversity of electrophoretic bands obtained in S3.

3. The method according to claim 2, characterized in that, The rice genomic DNA extraction described in S1 was performed using the SDS method.

4. The method according to claim 2, characterized in that, The PCR amplification reaction system described in S2 is as follows: 2 μL 10×Buffer, 0.2 μL 10 mmol / L dNTP, 1 μL 5 μmol / L forward primer, 1 μL 5 μmol / L reverse primer, 1 μL rice genomic DNA to be tested, 0.15 μL 5 U / μL Taq DNA polymerase, and ddH2O to a final volume of 20 μL. The PCR amplification reaction program is as follows: pre-denaturation at 94℃ for 3 min; denaturation at 94℃ for 30 s, annealing at 65℃ for 20 s, extension at 72℃ for 15 s, for 35 cycles; and final extension at 72℃ for 5 min.

5. The method according to claim 2, characterized in that, The detection conditions for capillary gel electrophoresis analysis described in S3 are as follows: sample injection voltage is 5-7.5kV, time is 10s; pre-test voltage is 5-8kV, time is 30s; capillary electrophoresis separation voltage is 5-8kV, time is 60min-80min.

6. An application of the rice whole-genome InDel molecular marker system based on capillary electrophoresis as described in claim 1, characterized in that, The Indel molecular marker system is applied to rice genetic map construction, rice molecular breeding, and variety identification.

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