InDel marker for identifying onion cytoplasm genotype based on HRM (High Resolution Melting) technology and application of InDel marker

Abstract

The invention belongs to the technical field of onion breeding and molecular detection, and particularly relates to an InDel marker for identifying onion cytoplasm genotypes based on an HRM technology and application of the InDel marker. Specifically, the InDel molecular marker primer provided by the invention can be used for rapidly and efficiently detecting the onion cytoplasm genotype at high throughput, the detection speed is remarkably increased through a high-throughput PCR technology, and the time and labor cost is reduced. Therefore, high-throughput genotyping screening of onion field breeding materials is facilitated, and the method is particularly suitable for large-scale sample treatment. Meanwhile, an onion material with a known cytoplasm type is verified, the identification result is completely consistent with the phenotype, compared with an SCAR marker, the screening method is simpler and more convenient, electrophoresis is not needed after the PCR reaction is finished, the type of the onion cytoplasm can be determined only by analyzing a melting curve, and the method is efficient, accurate and low in cost. The technology can realize batch, automatic and standardized operation, and has good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of onion breeding and molecular detection, and particularly relates to an InDel marker for identifying onion cytoplasm genotype based on HRM technology and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Onion (Allium cepa L.), also known as jade onion, round onion, etc., is a biennial herbaceous plant belonging to the genus Allium of the Amaryllis family. It uses a fleshy bulb as an edible organ. It is rich in nutrients such as vitamins and anthocyanins. It has health benefits such as reducing and preventing thrombosis, lowering blood lipids and blood sugar, anti-oxidation, anti-aging, anti-inflammatory and anti-cancer. It is known as the queen of vegetables and is deeply loved by consumers. It is an important export-earning vegetable in my country. According to records, onions have a cultivation history of more than 5,000 years. It was not introduced to my country until modern times, and has only a cultivation history of more than 100 years in my country. Therefore, my country's onion germplasm resources are relatively scarce. In addition, the use of male sterility technology in onions is far behind other developed countries, resulting in the onion hybrid market being almost monopolized by foreign varieties. Therefore, it is extremely necessary to accelerate the selection and use of male sterile lines and their maintenance lines in onions.

[0004] Studies have reported that onion CMS includes CMS-S, CMS-T, petalized male sterility psf-A and other types, among which CMS-S is more common and widely used in production. In 1936, Jones and Emsweller in the United States first discovered S-type cytoplasmic male sterile plants in the "Italian Red" variety. Its male sterility is controlled by cytoplasmic genes (S) and nuclear genes (msms), belonging to the nuclear-cytoplasmic interaction type. After breeding, S-type cytoplasmic male sterile lines were obtained, and then onions became one of the earliest vegetable crops in the world to utilize hybrid vigor. Onion hybrid vigor is obvious, but its floral organs are small, the flowering period is not concentrated, the seed setting rate of single flowers is low, and the cost and difficulty of artificial emasculation are high. In addition, due to the presence of Ms genes or high frequencies of S-type cytoplasm, it is difficult to successfully obtain maintenance lines from some cultivated varieties. Therefore, in the actual breeding of onions, it is a very time-consuming and laborious task to select male sterile lines and their maintenance lines from a population of unknown cytoplasm types.

[0005] Molecular marker-assisted breeding using markers linked to cytoplasmic loci can identify the cytoplasm type of individual plants, eliminate S-type cytoplasm in fertile materials, and select maintenance lines only from individual plants with N-type cytoplasm, thereby reducing the number of test cross combinations and self-pollination plants, reducing workload, avoiding blindness, and improving selection efficiency. Havey et al. found that there is a highly variable region between trnT and trnL on the chloroplast DNA of onions, that is, there is a 100bp insertion sequence between the two genes in N-type cytoplasm compared with S-type cytoplasm. Based on this insertion sequence, SCAR markers (MS7 / MS8) that can identify onion S-type and N-type cytoplasm were developed. Sato found that there is a specific chloroplast gene insertion sequence upstream of the mitochondrial cob gene in the S-type cytoplasm of onions. Using this specific sequence, primers were designed for PCR amplification, and a 414bp specific fragment was amplified in the S-type cytoplasm, and a 180bp specific fragment was amplified in the N-type cytoplasm. This marker can be used to quickly and accurately identify onion S-type and N-type cytoplasm. Engelke et al. found that the mitochondrial gene orfA501 used to distinguish cytoplasmic fertility in chives also exists in onion mitochondria. Using the specific sequence of the chive atp9 gene to design primers, they successfully developed a specific PCR marker to distinguish the fertile N-type cytoplasm from the sterile cytoplasm (S or T type) of onions. Kim et al. found a chimeric open reading frame orf725 in the S-type and T-type cytoplasms, and developed a molecular marker that can distinguish three types of onion cytoplasm (N type, S type and T type) through a single PCR. Li Yongbo et al. obtained the flanking sequence of chive orfA501 by chromosome walking and developed the SCAR marker for identifying the onion cytoplasmic genotype. The marker amplified a 2175bp specific band and a 1053bp band in the onion sterile line, while only a 1053bp band was amplified in the maintenance line. Chen Li et al. used the sequence difference of orf393 between the S-type cytoplasmic male sterile line and the maintainer line to develop a stable and reliable SCAR molecular marker GXX393 for identifying onion cytoplasm types.

[0006] Compared with traditional methods such as "test cross-backcross, self-pollination", the development and application of these markers effectively avoid the blindness of the screening of the maintenance line and improve the selection efficiency. However, the inventors found that these markers are all based on PCR, and then electrophoresis is performed to determine the type of cytoplasm. Although it is much faster than field identification, it can only detect up to 96 samples each time. For a large number of sample detections, the detection efficiency is low, the amplification results are not very stable, and it has certain limitations. InDel (insertion-deletion insertion / deletion) refers to the insertion or deletion of nucleotide fragments of different sizes in the sequence of the same site of the genome, and SNP (single nucleotide polymorphism) refers to the replacement, transversion, deletion and insertion of a single nucleotide in the genome. Indel / SNP markers have the advantages of good genetic stability, high polymorphism, and simple typing system. Therefore, the development of InDel markers for identifying onion cytoplasmic genotypes can conveniently and accurately identify the genotype of candidate individuals, thereby effectively accelerating the breeding process and reducing breeding costs. Summary of the invention

[0007] In view of the above-mentioned prior art, after long-term technical and practical exploration, the present invention provides an InDel marker for identifying onion cytoplasm genotypes based on HRM (high resolution melting curve) technology and its application, which is used to assist in the breeding of onion male sterile lines and matching maintenance lines, and has the advantages of rapidity, high efficiency and high throughput, thus playing an important role in the detection of a large number of onion cytoplasm type groups. Based on the above research results, the present invention is completed.

[0008] Specifically, the present invention is achieved through the following technical solutions:

[0009] The first aspect of the present invention provides an application of an InDel molecular marker or a substance for detecting the InDel molecular marker in any of the following cases, wherein the InDel molecular marker may be a DNA molecule having a nucleotide sequence of SEQ ID NO.1;

[0010] A1) Application in identifying or assisting in identifying onion cytoplasm types;

[0011] A2) Use in the preparation of a product for identifying or assisting in identifying the type of onion cytoplasm;

[0012] A3) Application in screening onion male sterile lines and their maintenance lines;

[0013] A4) Application in the preparation of products for screening onion male sterile lines and their maintainers;

[0014] A5) Application in onion breeding;

[0015] A6) Use in preparing onion breeding products.

[0016] Furthermore, in the applications A1) and A2), the onion cytoplasm type can be identified as S type or N type. In particular, the technical solution of the present invention is particularly suitable for quickly identifying the cytoplasm types of a large number of onion populations.

[0017] Furthermore, the substance for detecting the InDel molecular marker may be a primer for amplifying an onion genomic DNA fragment containing the InDel molecular marker;

[0018] The second aspect of the present invention provides a product for identifying or assisting in identifying the type of onion cytoplasm, wherein the product is selected from any one of the following:

[0019] B1) the above primers;

[0020] B2) a reagent containing the primer described in B1);

[0021] B3) A kit comprising the primers described in B1) and / or the reagents described in B2).

[0022] The third aspect of the present invention provides a method for identifying or assisting in identifying the type of onion cytoplasm, the method comprising detecting whether the onion genome contains the above-mentioned InDel molecular marker, and identifying or assisting in determining the type of onion cytoplasm based on whether the onion genome contains the InDel molecular marker.

[0023] Furthermore, the method is carried out using HRM technology, including: based on the above-mentioned InDel-labeled amplification primers, using the genomic DNA of the onion material to be tested as a template, amplifying the target sequence through PCR and high-resolution melting curve analysis, and using the difference in melting curve peaks to detect and determine the cytoplasm type of the onion material to be tested.

[0024] The fourth aspect of the present invention provides a DNA molecule, wherein the DNA molecule is selected from any one or more of the following:

[0025] C1) a DNA molecule having a nucleotide sequence of SEQ ID NO.1;

[0026] C2) a DNA molecule having a nucleotide sequence of SEQ ID NO.2;

[0027] C3) A DNA molecule whose nucleotide sequence is SEQ ID NO.3.

[0028] Beneficial technical effects achieved by one or more of the above technical solutions:

[0029] (1) The InDel molecular marker primers provided by the above technical solution can detect onion cytoplasmic genotypes quickly, efficiently and with high throughput, significantly improving the detection speed through high-throughput PCR technology and reducing time and labor costs. This is extremely beneficial for high-throughput genotyping screening of onion field breeding materials, and is particularly suitable for large-scale sample processing.

[0030] (2) The onion materials with known cytoplasmic types were verified, and the identification results were completely consistent with the phenotype. Compared with the SCAR marker, the screening method is simpler and does not require electrophoresis after the PCR reaction. The 96 system can analyze the melting curve to determine the type of onion cytoplasm, which is efficient, accurate and low-cost. This technology can realize batch, automatic and standardized operation and has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0032] Figure 1 This is a melting curve diagram of the HRM marker in Example 1 of the present invention.

[0033] Figure 2 This is a melting curve diagram of the HRM marker in Example 1 of the present invention in a population with known cytoplasmic genotypes.

[0034] Figure 3 This is the verification of the HRM marker detection results using mature SCAR molecular markers in Example 2 of the present invention. Among them: 1-24, 25-48, 49-72, 73-93 are 922, 926, 935 and 938 from the OP population respectively; 94 is the positive control 211, the cytoplasm type is S; 95 is the positive control 212, the cytoplasm type is N; 96 is the no-template control NTC; M is the DNA molecular weight standard DL2000. DETAILED DESCRIPTION

[0035] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0036] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention.

[0037] The present invention is further described in conjunction with specific examples. The following examples are only for explaining the present invention and are not intended to limit the content thereof. If the specific experimental conditions are not specified in the examples, they are usually carried out according to conventional conditions or the conditions recommended by the reagent company; the reagents and consumables used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0038] The invention provides an InDel molecular marker and corresponding amplification primer for rapidly and accurately identifying the types of onion cell nuclei in a large population, which can be used to assist in the breeding of onion male sterile lines and matching maintenance lines, and has the characteristics of high throughput, low cost and high accuracy, thus playing an important role in the detection of a large number of onion cell nuclear genotype populations.

[0039] Specifically, in a typical embodiment of the present invention, an InDel molecular marker or a substance for detecting the InDel molecular marker is provided for any of the following uses, wherein the InDel molecular marker may be a DNA molecule having a nucleotide sequence of SEQ ID NO.1;

[0040] A1) Application in identifying or assisting in identifying onion cytoplasm types;

[0041] A2) Use in the preparation of a product for identifying or assisting in identifying the type of onion cytoplasm;

[0042] A3) Application in screening onion male sterile lines and their maintenance lines;

[0043] A4) Application in the preparation of products for screening onion male sterile lines and their maintainers;

[0044] A5) Application in onion breeding;

[0045] A6) Use in preparing onion breeding products.

[0046] In the applications A1) and A2), the onion cytoplasm type can be identified as S type or N type. Specifically, the nucleotide sequence in the S type cytoplasm is shown in SEQ ID NO.2. Compared with the S type cytoplasm, orf393 lacks 36 bases (i.e., InDel molecular marker) in the N type cytoplasm, and its nucleotide sequence is shown in SEQ ID NO.3. In particular, the technical solution of the present invention is particularly suitable for rapidly identifying the cytoplasm types of a large number of onion populations.

[0047] In another specific embodiment of the present invention, the substance for detecting the InDel molecular marker may be a primer for amplifying an onion genomic DNA fragment containing the InDel molecular marker.

[0048] In another specific embodiment of the present invention, the primer is a primer based on HRM technology, and its nucleotide sequence is as follows:

[0049] AcHRM-F: 5'-CATCGCCTCAGTCGGTGATT-3' (SEQ ID NO.4);

[0050] AcHRM-R: 5'-CGACGTAGGCGAGTCTGAAG-3' (SEQ ID NO. 5).

[0051] In another specific embodiment of the present invention, a product for identifying or assisting in identifying the type of onion cytoplasm is provided, wherein the product is selected from any one of the following:

[0052] B1) the above primers;

[0053] B2) a reagent containing the primer described in B1);

[0054] B3) A kit comprising the primers described in B1) and / or the reagents described in B2).

[0055] In another specific embodiment of the present invention, a method for identifying or assisting in identifying the type of onion cytoplasm is provided, the method comprising detecting whether the onion genome contains the above-mentioned InDel molecular marker, and identifying or assisting in determining the type of onion cytoplasm based on whether the onion genome contains the InDel molecular marker.

[0056] In another specific embodiment of the present invention, the method is carried out using HRM technology, including: based on the above-mentioned InDel-labeled amplification primers, using the genomic DNA of the onion material to be tested as a template, amplifying the target sequence by PCR and high-resolution melting curve analysis, and detecting and judging the cytoplasm type of the onion material to be tested by using the difference in melting curve peaks.

[0057] In another specific embodiment of the present invention, the method comprises:

[0058] S1. Extracting the total DNA of the onion material to be tested;

[0059] S2, using the sample DNA as a template, performing PCR amplification;

[0060] S3. The cytoplasmic type of the onion material to be tested can be distinguished according to the Tm value of the HRM melting curve.

[0061] In step S2, the PCR reaction system is: 20 ng genomic DNA, 2× 480 High Resolution Melting Master Mix 10 μL, upstream and downstream primers (10 uM) 1 μL each, MgCl2 1.6 μL, add ddH2O to make up to 20 μL;

[0062] The PCR amplification reaction procedure included: 94°C pre-denaturation for 6 min, followed by 45 cycles of 94°C denaturation for 10 sec, 58°C annealing for 15 sec, and 72°C extension for 10 sec; the amplification product HRM detection procedure was: 95°C for 1 min, 40°C for 1 min, 65°C for 1 s, and during the process of heating from 65°C to 95°C, the temperature was increased by 15 times·℃ -1 The fluorescence information was collected at a frequency of 100 °C and the temperature was finally lowered to 40 °C. After the reaction was complete, a high-resolution melting curve analysis was performed.

[0063] Furthermore, in step S3, the InDel molecular marker is used to determine the type of onion cytoplasm. If a melting curve peak of 85°C appears in the DNA amplification of the onion material to be tested, the cytoplasm type is S-type, and if a melting curve peak of 84°C appears, the onion material to be tested is N-type cytoplasm.

[0064] InDel molecular markers and detection methods based on HRM technology can be used to assist in the breeding of onion cytoplasmic male sterile lines and their maintenance lines. Through fluorescent quantitative PCR amplification, the cytoplasm type of the onion material to be tested can be distinguished according to the Tm value of the HRM melting curve. If the Tm value is 84°C, the cytoplasm of the onion to be tested is N-type; when the Tm value is 85°C, the cytoplasm of the onion to be tested is S-type. Using the InDel molecular markers and detection methods based on HRM technology provided by the present invention, individual genotypes can be determined in the onion seedling stage, so that early selection and early elimination can be achieved, which has important practical value for improving selection efficiency and saving breeding costs.

[0065] In another specific embodiment of the present invention, a DNA molecule is provided, wherein the DNA molecule is selected from any one or more of the following:

[0066] C1) a DNA molecule having a nucleotide sequence of SEQ ID NO.1;

[0067] C2) a DNA molecule having a nucleotide sequence of SEQ ID NO.2;

[0068] C3) A DNA molecule whose nucleotide sequence is SEQ ID NO.3.

[0069] In the above-mentioned applications, products and methods of the present invention, the product may be a (detection) kit.

[0070] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0071] Example 1 Establishment of onion cytoplasm HRM molecular marker and detection method (I) Obtaining InDel marker primer sequence based on HRM technology

[0072] Based on the differential sequences of the open reading frame orf393 in the onion CMS-S mitochondrial genome sequence (GenBank accession: KU318712) in the S-type cytoplasmic male sterile line and its maintainer line, specific primers were designed using Primer Premier 5.0 and Primer 3.0 (http: / / bioinfo.ut.ee / primer3-0.4.0 / primer3 / input.htm), consisting of an upstream primer (AcHRM-F) and a downstream primer (AcHRM-R). The primer nucleotide sequences are shown in SEQ ID NO.4 and SEQ ID NO.5 in the sequence listing.

[0073] AcHRM-F: 5'-CATCGCCTCAGTCGGTGATT-3' (SEQ ID NO.4);

[0074] AcHRM-R: 5'-CGACGTAGGCGAGTCTGAAG-3' (SEQ ID NO. 5).

[0075] The above primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0076] (II) Detection of InDel markers based on HRM technology

[0077] 1. Onion genomic DNA extraction

[0078] Onion seedling leaves were taken and the total onion genomic DNA was extracted using the quick plant genomic DNA extraction system of Tiangen Biochemical Technology (Beijing) Co., Ltd. The extraction method was referred to the instruction manual. After extraction, the DNA quality and concentration were detected using 1% agarose gel electrophoresis and ultra-micro nucleic acid analyzer.

[0079] 2. High Resolution Melting (HRM) Amplification

[0080] Using sample DNA as template, 96 fluorescence quantitative PCR instrument (Roche) for amplification. The PCR reaction system was: 20 ng genomic DNA, 2× 480 High Resolution Melting Master Mix 10 μL, upstream and downstream primers (10 uM) 1 μL each, MgCl2 1.6 μL, add ddH2O to make up to 20 μL; PCR reaction conditions are: 94℃ pre-denaturation for 6 min, 94℃ denaturation for 10 sec, 58℃ annealing for 15 sec, 72℃ extension for 10 sec for 45 cycles; then 95℃ for 1 min, 40℃ for 1 min, 65℃ for 1 s, and in the process of heating from 65℃ to 95℃, 15 times·℃ -1 The fluorescence information was collected at a frequency of 100 °C and then cooled to 40 °C. 96 system software to generate melting curves. The melting curve results are shown in the attached Figure 1 .

[0081] 3. Results and analysis

[0082] The nucleotide sequence of the InDel molecular marker in the S-type cytoplasm is shown in SEQ ID NO.1. Compared with the S-type cytoplasm, orf393 lacks 36 bases in the N-type cytoplasm, and its nucleotide sequence is shown in SEQ ID NO.2. This deletion mutation causes a difference in the melting curve. The cytoplasm type of the onion material to be tested can be distinguished according to the high or low Tm value of the HRM melting curve. After PCR amplification of the specific sequence region of SEQ ID NO.1, the amplified fragment size of the S-type cytoplasm onion material is 218bp, corresponding to a melting temperature of 85°C, and the amplified fragment size of the N-type cytoplasm onion material is 182bp, corresponding to a melting temperature of 84°C.

[0083] (III) Validation results of the molecular marker in a population with known cytoplasmic genotypes

[0084] In order to verify the feasibility of the marker in onion molecular marker-assisted breeding, high-resolution melting curve (HRM) amplification was performed using onion materials with known cytoplasmic genotypes, and the detection was performed according to the steps in Example 1. The cytoplasmic genotype of the sample was determined based on the melting curve peak. The melting curve results are shown in Figure 2 The melting curve peaks of S-type cytoplasmic onion materials were all 85°C, and the melting curve peaks of N-type cytoplasmic onion materials were all 84°C. The judgment results were completely consistent with the known cytoplasmic types.

[0085] Example 2 The SCAR molecular marker was used to verify the detection results of the InDel molecular marker.

[0086] Four groups of OP populations from different sources, 922 (24 samples), 926 (24 samples), 935 (24 samples) and 938 (21 samples), were selected as onion materials. The sterile lines with known cytoplasm types and their corresponding maintainer lines 249-1 and 250-1 were used as positive controls. The consistency of the identification results of the above OP populations was verified by comparing the SCAR molecular marker with the InDel molecular marker.

[0087] High-resolution melting curve (HRM) amplification was performed on the four groups of OP populations from different sources. The corresponding melting temperature of the onion individuals with cytoplasm type S was 85°C; the corresponding melting temperature of the onion individuals with cytoplasm type N was 84°C. The identification results are summarized in Table 1.

[0088] Table 1 InDel molecular marker identification results

[0089]

[0090]

[0091] The developed SCAR molecular marker that can accurately identify the onion cytoplasm type was used to identify the cytoplasm genotype of the above four groups of OP populations from different sources. The specific method of SCAR molecular marker detection of onion cytoplasm genotype refers to the authorized invention patent ZL201410153951.2, as follows:

[0092] PCR primers:

[0093] OC2175-F: 5'-ATGCCACTTCTGCTTCCTCATATCGT-3' (SEQ ID NO. 6);

[0094] OC2175-R: 5'-CCAACGATTGCCAAGCATTAGGCACTGAC-3' (SEQ ID NO. 7).

[0095] The above primers were used to amplify DNA from OP populations of different origins. The PCR products were analyzed by 1% agarose gel electrophoresis to detect polymorphism. The samples with cytoplasmic genotype N only amplified a fragment of 1053 bp, while the samples with cytoplasmic genotype S amplified two bands of 2175 bp and 1053 bp ( Figure 3 ). It can be seen that the judgment result of the present invention is completely consistent with the detection result of InDel molecular markers.

[0096] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to perform equivalent replacements on parts thereof. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention. Although the above describes the specific implementation modes of the present invention in conjunction with the accompanying drawings, it is not intended to limit the protection scope of the present invention. Those skilled in the art should understand that, based on the technical solution of the present invention, various modifications or deformations that can be made by those skilled in the art without creative labor are still within the protection scope of the present invention.

Claims

1. An application of an InDel molecule marker or a substance for detecting the InDel molecule marker in any of the following, wherein the InDel molecule marker is a DNA molecule having a nucleotide sequence of SEQ ID NO.1; A1) Application in identifying or assisting in identifying onion cytoplasm types; A2) Use in the preparation of a product for identifying or assisting in identifying the type of onion cytoplasm; A3) Application in screening onion male sterile lines and their maintenance lines; A4) Application in the preparation of products for screening onion male sterile lines and their maintainers; A5) Application in onion breeding; A6) Use in preparing onion breeding products.

2. The use according to claim 1, characterized in that: In the applications A1) and A2), the onion cytoplasm type is identified as S type or N type, the nucleotide sequence in the S type cytoplasm is shown as SEQ ID NO.2, and the nucleotide sequence in the N type cytoplasm is shown as SEQ ID NO.

3.

3. The use according to claim 1, characterized in that: The substance for detecting the InDel molecular marker is a primer for amplifying an onion genomic DNA fragment containing the InDel molecular marker.

4. The use according to claim 3, characterized in that: The primers are primers based on HRM technology, and their nucleotide sequences are shown in SEQ ID NO.4 and SEQ ID NO.

5.

5. A product for identifying or assisting in identifying the type of onion cytoplasm, characterized in that: The product is selected from any of the following: B1) The primer for use according to claim 3 or 4; B2) a reagent containing the primer described in B1); B3) A kit comprising the primers described in B1) and / or the reagents described in B2).

6. A method for identifying or assisting in identifying the type of onion cytoplasm, characterized in that: The method comprises detecting whether the onion genome contains the InDel molecular marker used in claim 1, and identifying or assisting in determining the onion cytoplasm type according to whether the onion genome contains the InDel molecular marker.

7. The method according to claim 6, characterized in that The method adopts HRM technology, including: based on the InDel-labeled amplification primers, taking the genomic DNA of the onion material to be tested as a template, amplifying the target sequence through PCR and high-resolution melting curve analysis, and using the difference in melting curve peaks to detect and judge the cytoplasm type of the onion material to be tested.

8. The method according to claim 7, characterized in that The method comprises: S1. Extracting the total DNA of the onion material to be tested; S2, using the sample DNA as a template, performing PCR amplification; S3. The cytoplasmic type of the onion material to be tested can be distinguished according to the Tm value of the HRM melting curve.

9. The method according to claim 8, characterized in that In step S3, if the melting curve peak of the DNA amplified onion material to be tested appears at 85°C, the cytoplasm type is S-type, and if the melting curve peak appears at 84°C, the cytoplasm type of the onion material to be tested is N-type.

10. A DNA molecule, characterized in that The DNA molecule is selected from any one or more of the following: C1) a DNA molecule having a nucleotide sequence of SEQ ID NO.1; C2) a DNA molecule having a nucleotide sequence of SEQ ID NO.2; C3) A DNA molecule whose nucleotide sequence is SEQ ID NO.3.

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