Scar marker for identifying onion pollen contamination of welsh onion and application thereof

Abstract

The application discloses a SCAR marker for identifying onion pollen contaminated scallion and application thereof, wherein the specific fragment length of the SCAR marker is 811 bp, the nucleotide sequence is shown as SEQ ID NO. 1, and the SCAR marker of the scallion has no target band; and the application further discloses specific primers of the SCAR marker. The marker determined by the application can be used for verifying different varieties of scallions and scallion hybrid plants (YZD) contaminated by onion pollen, and can quickly and stably identify the scallions, the scallion hybrid plants (YZD) contaminated by onion pollen, and is not influenced by natural environment and human factors compared with a traditional seed purity identification method, and can be used for batch experiments to improve identification efficiency, and provides a quick and feasible method for solving the problem that the scallion pollen is contaminated by onion pollen and thus causes mixed scallion varieties.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a SCAR marker for identifying onion pollen contamination in scallions and its application. Background Technology

[0002] scallions ( Allium fistulosum Allium tuberosum (L.) is a biennial or triennial herbaceous plant belonging to the genus Allium in the family Liliaceae. It promotes gastric juice secretion, aids digestion, and improves blood circulation. It also has auxiliary therapeutic effects on symptoms such as influenza, headache, and nasal congestion. Furthermore, it enhances fibrinolytic activity and lowers blood lipids. Cultivation is mainly in Asia, with China having a cultivation area of ​​approximately 570,000 hectares. Onion (… Allium cepa Onions (L.) belong to the Liliaceae family and the Allium genus. They are biennial plants. Onions have the effects of lowering cholesterol, softening blood vessels, benefiting the stomach and intestines, and resisting cold and killing bacteria. Onions are widely cultivated around the world. According to statistics, among all vegetable crops in the world, onions rank second in terms of planting area.

[0003] Because scallions and onions are widely cultivated in my country, the demand for their seeds is high. Professional seed production companies typically undertake the production of both onion and scallion seeds simultaneously. This leads to a risk that the flowering periods of scallions and onions may coincide during actual cultivation, resulting in scallion pollination by onion pollen. The main reasons are as follows: During cultivation, when late-maturing scallion varieties and early-maturing onion varieties flower at the same time, due to limited land and the need to reduce costs, the distance between the reproductive isolation zones of the two species may be improperly set, leading to scallion pollination by onion pollen. This phenomenon can cause significant harm and introduce many uncertainties into scallion seed production. Furthermore, in the market promotion of varieties, the use of mixed seeds not only causes economic losses for farmers but also easily leads to disputes between seed production companies and contracted seed production companies due to impure varieties, thus affecting the healthy development of the scallion industry. According to the Seed Law of the People's Republic of China, seed production shall comply with the technical regulations for seed production and the regulations for seed inspection and quarantine to ensure that seeds meet quality requirements such as purity, germination rate, and quarantine requirements. For example, the national standard requires that the purity of tomato parent seeds be 99% (GB 16715.3-2010), the purity of cabbage parent seeds be 99% (GB 16715.3-2010), and the Shandong Provincial Bureau of Quality and Technical Supervision stipulates that the purity of onion seed should not be less than 99% (DB37 / T 1543-2010). Therefore, ensuring the authenticity of species and varieties and conducting purity identification of seeds are very important aspects in scientific research, production, and sales.

[0004] In the early stages, morphological methods were used for identification. While simple, these methods had significant limitations. First, field phenotypic identification involved long experimental cycles, a large workload, high costs, and was easily affected by environmental factors. Second, the morphology of scallions contaminated with onion pollen was not significantly different from that of uncontaminated scallions, making it impossible to accurately determine whether they were contaminated. To avoid losses, there was an urgent need to develop accurate and efficient identification methods to solve the purity problem caused by onion pollen contamination in scallions.

[0005] The development of molecular biology has provided a new technical means for plant genetic markers based on DNA variation, namely molecular marker technology. Compared with other marker methods, molecular markers have strong advantages. They exist directly in the form of DNA and can be detected in all tissues and developmental stages of the plant, without being limited by season or environment, and there is no issue of expression. CN110273020A (application publication number) discloses an SNP molecular marker for distinguishing between summer oranges and common sweet oranges. Based on the 17,856,950th base sequence of chromosome 4 of sweet orange, primers were designed, and DNA from summer oranges and common sweet oranges was extracted using the CTAB method. The results were analyzed using the genotyping and gene scanning modules of LightCycler480 software. The results show that the molecular marker of this invention can accurately distinguish between summer oranges and common sweet oranges. Currently, there is no research on using molecular markers to solve the purity problem of scallion pollination caused by onion pollen contamination. Therefore, molecular markers can be used to solve the problem of scallion pollination caused by onion pollen contamination, which leads to the mixing of scallion varieties. This is of great significance for accurately and efficiently identifying the purity of scallion varieties. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a SCAR marker for identifying onion pollen contamination in scallions and its application. Based on the sequences of 898U and 898D in onion cell nuclei disclosed in CN103981281A, this invention designs specific primers for amplification, obtaining stable SCAR markers. Based on the identified SCAR markers in scallions and onion pollen-contaminated hybrids (YZD), this invention performs PCR verification on 10 known scallion varieties and 10 onion pollen-contaminated hybrids (YZD). All onion pollen-contaminated hybrids (YZD) amplified a specific fragment of 811 bp, while all unhybridized scallions showed no target band. The amplification results are consistent with those of scallions and onion pollen-contaminated hybrids (YZD). The above identification method can quickly and accurately distinguish between different varieties of scallions and scallion hybrids contaminated with onion pollen (YZD), providing a fast and feasible method for solving the problem of scallion varietal mixing caused by onion pollen contamination in production.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0008] A SCAR marker for identifying onion pollen contamination in scallions, wherein the specific fragment of the SCAR marker is 811 bp in length and its nucleotide sequence is shown in SEQ ID NO.1.

[0009] This invention provides specific primers for identifying SCAR markers in scallions contaminated by onion pollen, the primer sequences of which are as follows:

[0010] Forward primer 804-F: 5'-CACGGATTCGATTGTTAAATCGT- 3';

[0011] Reverse primer 804-R: 5'-ACACACACACAGAGTGAGAAATTTTATA-3';

[0012] This invention provides an application of SCAR markers in identifying scallions, scallion plants or seeds contaminated with onion pollen.

[0013] A method for identifying SCAR markers in scallions contaminated with onion pollen, characterized by the following specific operational steps:

[0014] (1) Extract total DNA from the plant or seed to be tested;

[0015] (2) PCR amplification was performed using the specific primers described above to obtain PCR products;

[0016] (3) Detect and analyze the PCR product obtained in step (2). If the PCR product detection result shows a band of 811bp SCAR-labeled specific fragment, then the test sample is a scallion contaminated with onion pollen (YZD); if the PCR product detection result does not show a band of SCAR-labeled specific fragment, then the test sample is a scallion.

[0017] Further, the PCR amplification reaction system in step (2) is a 25 μL system, specifically: 2.5 μL of 10×Trans Fast® Taq Buffer, 2 μL of 2.5 mM dNTPs, 0.5 μL of Trans Fast® Taq DNA Polymerase, 1 μL each of CDY-F and CDY-R primers, 1 μL of DNA template, and 17 μL of Nuclease-free Water; the reaction program is: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 5 s, 54.1℃ annealing for 15 s, 72℃ extension for 15 s, 25 cycles, 72℃ extension for 5 min, and storage at 4℃.

[0018] Furthermore, the detection method of the PCR product in step (2) is as follows: the PCR product is separated and analyzed by electrophoresis at 120-125 V voltage, 1.5% agarose gel for 40-45 min, stained with ethidium bromide, and photographed and analyzed by a gel imaging analysis system.

[0019] Beneficial effects:

[0020] (1) The present invention can be used for identification at both the plant stage and the seed stage. When a suspicious plant is found at the plant stage, identification is carried out, which avoids the economic loss caused by incorrect rejection due to uncertainty, and also avoids the difficulty of identification in the later stage after a large number of hybrids are mixed into the scallion seeds.

[0021] (2) The present invention has identified a specific SCAR marker fragment for the identification of scallions and scallion hybrids (YZD) contaminated with onion pollen. After multiple biological replication verifications on 10 scallion varieties from a wide range of sources and 10 scallion hybrids (YZD) contaminated with onion pollen, the results showed that they can stably distinguish between scallions and scallion hybrids contaminated with onion pollen. The marker identified in the present invention has high specificity and high stability.

[0022] (3) The identification method of the present invention only requires extracting the total DNA of scallions and scallion hybrids (YZD) contaminated with onion pollen, performing batch PCR amplification, and then performing gel imaging analysis. These experiments are all performed by the instrument. Compared with the traditional seed purity identification method, it is not affected by natural environment and human factors, and batch experiments can be carried out, saving a lot of manpower. It not only avoids the cumbersome screening process of conventional methods, but also avoids the complicated operation of extracting mitochondrial DNA. It significantly improves the identification of different varieties of scallions and scallion hybrids (YZD) contaminated with onion pollen. It provides a fast and feasible method for solving the problem of scallion pollen contamination by onion pollen in production, which causes scallion variety mixing. Attached Figure Description

[0023] Figure 1The image shows the electrophoresis results of total DNA from scallions; where A1-A10 are the total DNA electrophoresis results of scallion varieties: Yuanzang, Tianguang Yiben, Xiarentian, Changbao, Changyue, Chunwei, Jiwanchou Yiben, Jiabao, Jizang, and Tianbao, respectively, and M is the molecular weight standard DL15000;

[0024] Figure 2 The image shows the electrophoresis results of total DNA from scallion hybrids (YZD) contaminated with onion pollen. B1-B10 represent the total DNA electrophoresis results of the following scallion strains: Yuanzang × Jinqiu, Tianguang Yiben × Binyu, Shimorenda × Xiuyuwan, Changbao × Jinxing, Changyue × Fuxing, Chunwei × Guijin, Jiwanchou Yiben × Jinqiu, Jiabao × Guijin, Jizang × Binyu, and Tianbao × Xiuyuwan. M represents the molecular weight standard DL15000.

[0025] Figure 3 The results of PCR amplification detection of scallions and scallion hybrids (YZD) contaminated with onion pollen are shown. Among them, 1-10 are the PCR amplification detection results of scallion Yuanzang, Tianguang Yiben, Xiarentian, Changbao, Changyue, Chunwei, Jiwanchou Yiben, Jiabao, Jizang, and Tianbao, respectively. 11-20 are the PCR amplification results of scallion hybrids (YZD) contaminated with onion pollen: Yuanzang × Jinqiu, Tianguang Yiben × Binyu, Xiarentian × Xiuyuwan, Changbao × Jinxing, Changyue × Fuxing, Chunwei × Guijin, Jiwanchou Yiben × Jinqiu, Jiabao × Guijin, Jizang × Binyu, and Tianbao × Xiuyuwan. M is the molecular weight standard DL2000.

[0026] Figure 4 This image shows the growth of scallions and scallion hybrids (YZD) contaminated with onion pollen in the field. Detailed Implementation

[0027] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0029] The experimental materials, reagents, and instruments used in this invention are as follows:

[0030] Experimental materials: The scallion varieties were Yuanzang, Tianguang Yiben, Shimonita, Changbao, Changyue, Chunwei, Jiwanchou Yiben, Jiabao, Jizang, and Tianbao; the scallion hybrids (YZD) contaminated with onion pollen were: Yuanzang × Jinqiu, Tianguang Yiben × Binyu, Shimonita × Xiuyuwan, Changbao × Jinxing, Changyue × Fuxing, Chunwei × Guijin, Jiwanchou Yiben × Jinqiu, Jiabao × Guijin, Jizang × Binyu, and Tianbao × Xiuyuwan. All of these varieties were commercially available.

[0031] Reagents used: Polysaccharide and polyphenol plant genomic DNA extraction kit (DP360) was purchased from Tiangen Biotech (Beijing) Co., Ltd.; PCR detection kit (Trans Fast® Taq DNA Polymerase), 10×Trans Fast® Taq Buffer, DNA molecular weight MakerDL2000, and DNA molecular weight MakerDL15000 were purchased from Beijing TransGen Biotech Co., Ltd.; primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0032] Instruments used: C1000 Touch Thermal cycler PCR instrument and GelDoc go gel imaging analysis system were purchased from BIO-RAD.

[0033] Example 1: Obtaining the SCAR marker for scallions and scallion hybrids contaminated with onion pollen (YZD).

[0034] In the database of the National Center for Biotechnology Information (NCBI) National Center for Biotechnology Information available at https: / / www.ncbi.nlm.nih.gov / Download the FASTA format sequence of the skp1-Ms allele from [source missing]. Based on the complete sequences of onion cell nuclei 898U and 898D published in CN103981281A, alignment analysis was performed using DNAMAN software, and primers were designed using SnapGene software. Results showed that amplification using the designed primers yielded a stable SCAR molecular marker. Sequencing revealed a target band (811 bp) in the YZD hybrid scallion sample contaminated with onion pollen; if no target band was detected, the sample was considered a scallion sample.

[0035] Example 2: Application of SCAR markers in scallions and scallion hybrids contaminated with onion pollen (YZD).

[0036] 1. Extraction and detection of total DNA from scallions and scallion hybrids contaminated with onion pollen (YZD).

[0037] Total DNA was extracted from different varieties of scallions and scallion hybrids (YZD) contaminated with onion pollen using the Polysaccharide-Polyphenol Plant Genomic DNA Extraction Kit (DP360). Specific operating procedures are detailed in the kit instructions. DNA quality was checked using 0.8% agarose gel electrophoresis. The results of the extracted total DNA agarose gel electrophoresis are shown below. Figure 1 , 2 The bands were clearly visible. The purity and concentration of the extracted DNA were determined using an ultra-micro spectrophotometer. The DNA showed a significant absorption peak at OD260, with the OD260 / OD280 ratio between 1.7 and 1.9. This confirms that the extracted total DNA sample is of reliable quality and can be used for further experiments.

[0038] 2. Primer design and synthesis

[0039] Based on the complete sequences of onion cell nuclei 898U and 898D disclosed in CN103981281A, primers were designed before and after the differential sequences using SnapGene software: 804-F: 5'-CACGGATTCGATTGTTAAATCGT- 3'; 804-R: 5'-ACACACACACAGAGTGAGAAATTTTATA- 3'. The degenerate primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and purified by PAGE.

[0040] The primer sequence used for the SCAR-tagged nucleotide sequence SEQ ID NO.1 of the scallion hybrid (YZD) contaminated with onion pollen is as follows:

[0041] Forward primer 804-F: 5'-CACGGATTCGATTGTTAAATCGT- 3';

[0042] Reverse primer 804-R: 5'-ACACACACACAGAGTGAGAAATTTTATA-3';

[0043] 3. PCR amplification

[0044] PCR amplification was performed on a BIO-RAD C1000 Touch Thermal Cycler PCR instrument. The detection system consisted of a 25 μL volume: 2.5 μL of 10× Trans Fast® Taq Buffer, 2 μL of 2.5 mM dNTPs, 0.5 μL of Trans Fast® Taq DNA Polymerase, 1 μL each of CDY-F and CDY-R, 1 μL of DNA template, and 17 μL of Nuclease-free Water. The reaction program was as follows: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 5 s, 54.1℃ annealing for 15 s, 72℃ extension for 15 s, 25 cycles, followed by a 72℃ extension for 5 min. PCR products were stored at 4℃.

[0045] 4. Detection and analysis of PCR products

[0046] The PCR products were separated by electrophoresis on a 1.5% agarose gel at a constant voltage of 120 V for 45 min. Ethidium bromide staining was performed, and the gel was photographed and analyzed using a gel imaging system. The results are shown below. Figure 3 .

[0047] The results of the gel imaging system are as follows: If the PCR test result shows an 811bp target band, the result is a sample of scallion contaminated with onion pollen (YZD); if the PCR test result does not detect any target band, the result is a scallion sample. The results of different varieties of scallion and scallion contaminated with onion pollen (YZD) are shown in Table 1. After multiple biological replication experiments on scallion (10 varieties) from a wide range of sources and scallion contaminated with onion pollen (10 varieties), the results show that different varieties of scallion and scallion contaminated with onion pollen can be stably distinguished.

[0048] Table 1. Detection results of different varieties of scallions and scallion hybrids contaminated with onion pollen (YZD).

[0049]

[0050] Note: + indicates the presence of an 811bp band; - indicates the absence of a band.

Claims

1. A SCAR marker for identifying onion pollen contamination in scallions, characterized in that, The SCAR-tagged specific fragment is 811 bp in length, and its nucleotide sequence is shown in SEQ ID NO.

1.

2. The SCAR marker for identifying onion pollen contamination in scallions according to claim 1, characterized in that, The specific primers for the SCAR marker are as follows: Forward primer 804-F: 5'-CACGGATTCGATTGTTAAATCGT- 3'; Reverse primer 804-R: 5'-ACACACACACAGAGTGAGAAATTTTATA-3'.

3. The application of the SCAR marker as described in claim 1 or 2 in identifying scallion plants or seeds contaminated with onion pollen.

4. The application according to claim 3, characterized in that, The specific operating steps are as follows: (1) Extract total DNA from the sample to be tested; (2) PCR amplification is performed using the specific primers described in claim 2 to obtain PCR products; (3) Detect and analyze the PCR product obtained in step (2). If the PCR product detection result shows a band of 811 bp SCAR-labeled specific fragment, then the test sample is a hybrid of scallion contaminated by onion pollen; if the PCR product detection result does not show a band of SCAR-labeled specific fragment, then the test sample is a scallion.

5. The application according to claim 4, characterized in that, The PCR amplification reaction system in step (2) is a 25 μL system, specifically: 2.5 μL of 10×Trans Fast® Taq Buffer, 2 μL of 2.5 mM dNTPs, 0.5 μL of Trans Fast® Taq DNA Polymerase, 1 μL each of 804-F and 804-R primers, 1 μL of DNA template, and 17 μL of Nuclease-free Water; the reaction program is: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 5 s, 54.1℃ annealing for 15 s, 72℃ extension for 15 s, 25 cycles, 72℃ extension for 5 min, and storage at 4℃.

6. The application according to claim 4, characterized in that, The detection method for the PCR products in step (2) is as follows: the PCR products are separated and analyzed by electrophoresis at 120-125 V voltage, 1.5% agarose gel for 40-45 min, stained with ethidium bromide, and photographed and analyzed by a gel imaging analysis system.

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