Double-dominant SCAR marker for identifying allium fistulosum and allium fistulosum polluted by onion pollen and application of double-dominant SCAR marker

Abstract

The invention discloses a double-dominant SCAR marker for identifying allium fistulosum and allium fistulosum polluted by onion pollen and application of the double-dominant SCAR marker. The specific fragment length of the SCAR marker of an abnormal plant polluted by onion pollen is 1481 bp and 851 bp, the nucleotide sequences of the SCAR marker are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively, the specific fragment length of the SCAR marker of the allium fistulosum is 840 bp, and the nucleotide sequence of the SCAR marker is shown as SEQ ID NO.3; the invention also discloses a specific primer of the SCAR marker. When the marker determined by the method is used for verifying different varieties of green Chinese onions and abnormal plants of the green Chinese onions polluted by the onion pollen, the green Chinese onions and the abnormal plants of the green Chinese onions polluted by the onion pollen can be quickly and stably identified, and compared with a traditional seed purity identification method, the method is not influenced by natural environment and human factors, can be used for batch experiments, improves the identification efficiency, and has a wide application prospect. The invention provides a rapid and feasible method for solving the problem that allium fistulosum varieties are mixed due to the fact that allium fistulosum pollen is polluted by the allium fistulosum pollen cannot be identified in production.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a double dominant SCAR marker for distinguishing green onions and green onions contaminated by onion pollen and an application thereof. Background Art

[0002] Green onion ( Allium fistulosum Onion (L.) is a biennial or triennial herbaceous plant of the genus Allium, family Liliaceae. It promotes gastric secretion, aids digestion, and promotes blood circulation. It has an auxiliary therapeutic effect on symptoms such as influenza, headaches, and nasal congestion. It also has the effect of enhancing fibrinolytic activity and lowering blood lipids. It is mainly cultivated in Asia, with China's cultivated area of ​​about 570,000 hectares. Allium cepa L.) belongs to the Liliaceae family, Allium genus, and is a biennial plant. Onion has the effects of lowering cholesterol, softening blood vessels, benefiting the stomach and intestines, and resisting cold and killing bacteria. Onion is widely cultivated around the world. According to statistics, among all vegetable crops in the world, the planting area of ​​onion ranks second.

[0003] Because scallions and onions are widely cultivated in my country, professional seed production companies typically undertake the production of both onion and scallion seeds. This leads to the risk of scallion and onion flowering periods coinciding during actual planting, resulting in onion pollen contamination during scallion pollination. The main reasons are as follows: during cultivation, late-maturing scallion varieties and early-maturing onion varieties bloom together, and the distance between the reproductive isolation zones between the two species is not set properly, resulting in onion pollen contamination during scallion pollination. This phenomenon is very harmful and brings many uncertainties to scallion seed production. During the promotion of varieties in the market, if mixed seeds are used, not only will farmers suffer financial losses, but also the impure varieties can easily lead to disputes between seed production companies and the contracted seed production companies, thus affecting the healthy development of the scallion industry. According to the Seed Law of the People's Republic of China, seed production must comply with seed production technical regulations and seed inspection and quarantine regulations to ensure that seeds meet quality requirements and quarantine requirements such as clarity, purity, and germination rate. For example, national standards require that tomato parent seeds have a purity of 99% (GB 16715.3-2010) and cabbage parent seeds have a purity of 99% (GB 16715.3-2010). The Shandong Provincial Quality and Technical Supervision Bureau stipulates that the purity of onion stock seeds must be no less than 99% (DB37 / T 1543-2010). Therefore, in scientific research, production, and sales, seed purity identification is a very important step to ensure the authenticity of species and varieties.

[0004] Early identification methods relied on morphology. While this approach was simple, it had significant limitations. First, field phenotypic identification required long testing cycles, high workload, high costs, and susceptibility to environmental influences. Furthermore, with the advancement of breeding, morphological identification became difficult or impossible, necessitating a unified, standardized technical system for authenticating varieties. Therefore, the development of accurate and efficient identification methods was crucial to address the purity issues associated with onion pollen contamination in green onions.

[0005] The development of molecular biology has provided a new technology for plant genetic markers based on DNA variation, namely molecular marker technology. Compared with other marker methods, molecular markers have great advantages. They exist directly in the form of DNA and can be detected in all tissues and developmental stages of the plant body. They are not restricted by seasons or environments, and there is no problem of expression. CN110273020A (application publication number) discloses a SNP molecular marker for distinguishing between Citrus citrus summer orange and common sweet orange. The present invention designs primers based on the 17856950th base sequence of chromosome 4 of sweet orange, uses the CTAB method to extract DNA from Citrus citrus summer orange and common sweet orange, and analyzes the results on the genotyping melting curve module (Melt Curve Genotyping) and gene scanning module (Gene Scan) on the LightCycler480 software. The results show that the molecular marker of the present invention can accurately distinguish between Citrus citrus summer orange and common sweet orange. [2] Currently, no research has been found that publicly uses the SCAR marker method to solve the purity problem caused by onion pollen contamination during onion pollination. Therefore, this patent uses molecular markers to solve the problem of onion pollination being contaminated by onion pollen, thereby causing the mixing of onion varieties. This is of great significance for accurately and efficiently identifying the purity of onion varieties. Summary of the Invention

[0006] The present invention provides a SCAR marker for identifying onion pollen contamination in green onions and its application. Based on the sequences 898U and 898D in onion cell nuclei disclosed in CN103981281A, specific primers were designed for amplification to obtain a stable SCAR marker. Based on the identified SCAR markers for green onions and hybrid green onions contaminated with onion pollen (YZD), PCR validation was performed on 10 known green onion varieties and 10 hybrid green onion varieties contaminated with onion pollen (YZD). The hybrid green onions contaminated with onion pollen (YZD) all amplified specific fragments of 1481 bp and 851 bp, respectively, while all unhybridized green onions amplified a specific fragment of 840 bp. The amplification results were consistent with those for green onions and hybrid green onions contaminated with onion pollen (YZD). The above identification method can be used to quickly and accurately identify different varieties of green onions and hybrids (YZD) of green onions contaminated with onion pollen, providing a fast and feasible method for solving the problem of identifying green onion pollen contaminated by onion pollen in production, thereby causing the mixing of green onion varieties.

[0007] A double dominant SCAR marker for distinguishing green onions from green onions contaminated with onion pollen. The SCAR marker-specific fragments of the green onion hybrid (YZD) contaminated with onion pollen are 1481 bp and 851 bp long, respectively, and their nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2. The SCAR marker-specific fragment of green onions is 840 bp long, and its nucleotide sequence is shown in SEQ ID NO.3.

[0008] The present invention provides specific primers for distinguishing between green onions and green onions contaminated with onion pollen and a double dominant SCAR marker, the primer sequences of which are as follows: Forward primer 850-F: 5′-GACAGGTACACAGCATTTTGGC- 3′; Reverse primer 850-R: 5′-ATACACTTCTGGTGTCATATACTAG- 3′; The invention provides an application of a double dominant SCAR marker in identifying green onions and green onion hybrids (YZD) contaminated by onion pollen.

[0009] A method for identifying green onions and green onion hybrids contaminated with onion pollen (YZD), the specific steps of which are as follows: (1) Extract the total DNA of green onions and green onions contaminated with onion pollen (YZD); (2) performing PCR amplification using the above-mentioned specific primers to obtain PCR products; (3) The PCR product obtained in step (2) is tested and analyzed. If the test results of the PCR product show both 1481 bp and 851 bp SCAR marker specific fragment bands, the test sample is a hybrid strain (YZD) of green onion contaminated with onion pollen; if the test results of the PCR product show only an 840 bp SCAR marker specific fragment band, the test sample is green onion.

[0010] Furthermore, the PCR amplification reaction system in step (2) is: a 25 μL system, specifically: 10×Trans Fast® Taq Buffer 2.5 μL, 2.5 mM dNTPs 2 μL, Trans Fast® Taq DNA Polymerase 0.5 μL, 1 μL each of CDY-F and CDY-R primers, 1 μL DNA template, and Nuclease-free Water 17 μL; the reaction procedure is: 94°C pre-denaturation for 3 min, 94°C denaturation for 5 s, 58.3°C annealing for 15 s, 72°C extension for 20 s, 35 cycles, 72°C extension for 5 min, and storage at 4°C.

[0011] Furthermore, the detection method of the PCR product in step (2) is as follows: the PCR product is separated and analyzed by electrophoresis on a 1.5% agarose gel at a voltage of 130-140 V for 40-45 min, stained with ethidium bromide, and photographed and analyzed using a gel imaging analysis system.

[0012] Beneficial effects: (1) The present invention has respectively determined specific SCAR marker fragments for distinguishing green onions and green onion hybrids contaminated by onion pollen (YZD). After multiple biological replications of 10 green onion varieties from a wide range of sources and 10 green onion hybrids contaminated by onion pollen (YZD), the results showed that green onions and green onion hybrids contaminated by onion pollen can be stably distinguished. The markers determined by the present invention have strong specificity and high stability.

[0013] (2) The identification method of the present invention only requires extracting the total DNA of green onions and green onion hybrids (YZD) contaminated by onion pollen, performing batch PCR amplification, and then performing gel imaging analysis. These experiments are all completed by instrument operation. Compared with the traditional seed purity identification method, it is not affected by the natural environment and human factors, and batch experiments can be carried out, saving a lot of manpower. It not only avoids the tedious screening process of conventional methods, but also avoids the complex operation of extracting mitochondrial DNA, significantly improving the efficiency of identifying different varieties of green onions and green onion hybrids (YZD) contaminated by onion pollen, and providing a fast and feasible method for solving the problem of the mixing of green onion varieties caused by the inability to identify green onion pollen contaminated by onion pollen in production.

[0014] (3) The present invention has respectively determined the specific SCAR marker fragments for identifying green onions and green onion hybrids (YZD) contaminated with onion pollen, which can effectively overcome technical errors, such as: failure to add DNA or primers, etc., resulting in the absence of bands in green onions and green onion hybrids (YZD) contaminated with onion pollen. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The total DNA electrophoresis results of green onions are shown in Figure 1. A1-A10 are the total DNA electrophoresis results of green onion varieties: Yuanzang, Tianguang Yiben, Xiarentian, Changbao, Changyue, Chunwei, Jiwanchu Yiben, Jiabao, Jizang, and Tianbao, respectively. M is the molecular weight standard DL15000. Figure 2 The total DNA electrophoresis results of a hybrid strain (YZD) of green onions contaminated with onion pollen are shown. B1-B10 are the total DNA electrophoresis results of Yuanzang × Jinqiu, Tianguang Yiben × Binyu, Xiarentian × Xiuyuwan, Changbao × Jinxing, Changyue × Fuxing, Chunwei × Guijin, Jiwanchou Yiben × Jinqiu, Jiabao × Guijin, Jizang × Binyu, and Tianbao × Xiuyuwan, respectively. M is the molecular weight standard DL15000. Figure 3 The PCR amplification test results of green onions and hybrid strains (YZD) of green onions contaminated with onion pollen are shown in Figure 1-10, respectively, for the green onions Yuanzang, Tianguang Yiben, Xiarentian, Changbao, Changyue, Chunwei, Jiwanchou Yiben, Jiabao, Jizang, and Tianbao; the PCR amplification test results of the green onions Yuanzang×Jinqiu, Tianguang Yiben×Binyu, Xiarentian×Xiuyuwan, Changbao×Jinxing, Changyue×Fuxing, Chunwei×Guijin, Jiwanchou Yiben×Jinqiu, Jiabao×Guijin, Jizang×Binyu, and Tianbao×Xiuyuwan are shown in Figure 11-20, respectively, for the green onions hybrid strains (YZD) contaminated with onion pollen are 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.

[0016] Figure 4 This is a graph showing the growth of green onions and green onion hybrids (YZD) contaminated with onion pollen after mixing in the field. DETAILED DESCRIPTION In order to enable those skilled in the art to better understand the technical solutions in this application, the present invention is further described below in conjunction with embodiments. The described embodiments are only part of the embodiments of this application, not all of them, and the present invention is not limited to the following embodiments.

[0017] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.

[0018] The experimental materials, reagents and instruments used in the present invention are as follows: Experimental materials: Allium fistulosum varieties are Yuanzang, Tianguang Yiben, Xiaren Tian, Changbao, Changyue, Chunwei, Jiluancha Yiben, Jiabao, Jizang, Tianbao; Allium fistulosum hybrid plants (YZD) contaminated by onion pollen (Yuanzang x Jinqiu, Tianguang Yiben x Binyu, Xiaren Tian x Xiuyumar, Changbao x Jinxing, Changyue x Fuxing, Chunwei x Guijin, Jiluancha Yiben x Jinqiu, Jiabao x Guijin, Jizang x Binyu, Tianbao x Xiuyumar. Allium fistulosum varieties and onion varieties are purchased from the market, and hybrid plants are obtained by hybridization of Allium fistulosum and onion in the later stage.

[0019] Reagents used: polysaccharide polyphenol plant genome DNA extraction kit (DP360) was purchased from Tian Gen Biochemical Technology (Beijing) Co., Ltd., PCR detection kit (Trans Fast Taq DNA Polymerase), 10x Trans Fast Taq Buffer, DNA molecular weight Marker DL2000, DNA molecular weight Marker DL15000 were purchased from Beijing Zonking Biotechnology Co., Ltd.; Primers were synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd.

[0020] Instruments used: C1000 Touch Thermal cycler PCR instrument, GelDoc go gel imaging analysis system were purchased from BIO-RAD company.

[0021] Example 1: Obtaining of SCAR marker of Allium fistulosum and Allium fistulosum hybrid plants (YZD) contaminated by onion pollen According to the complete sequence of onion nucleus 898U and 898D disclosed in CN103981281A, the primers were designed by DNAMAN software and SnapGene software. The results showed that stable molecular markers SCAR markers were obtained by amplification with the designed primers. Sequencing showed that the Allium fistulosum hybrid plants (YZD) sample had two target bands with lengths of 1481 bp and 851 bp, respectively, and the nucleotide sequences were shown as SEQ ID NO. 1 and SEQ ID NO. 2; the target band of Allium fistulosum was 840 bp, and the nucleotide sequence was shown as SEQ ID NO. 3.

[0022] Example 2: Application of SCAR marker of Allium fistulosum and Allium fistulosum hybrid plants (YZD) contaminated by onion pollen 1. Extraction and detection of total DNA of Allium fistulosum and Allium fistulosum hybrid plants (YZD) contaminated by onion pollen Firstly, the identification materials of Allium fistulosum and Allium fistulosum hybrid plants (YZD) contaminated by onion pollen are obtained, and then the total DNA of different varieties of Allium fistulosum and Allium fistulosum hybrid plants (YZD) contaminated by onion pollen is extracted by a polysaccharide and polyphenol plant genomic DNA extraction kit (DP360), and the specific operation steps are shown in the kit instruction. The quality of the extracted total DNA is checked by 0.8% agarose gel, and the agarose gel electrophoresis detection result of the extracted total DNA is shown in Figure 1 、 2 It can be seen that the band is clear; the purity and concentration of the extracted DNA are detected by ultramicro spectrophotometer, and the DNA has a significant absorption peak at OD260, and the ratio of OD260 / OD280 is between 1.7-1.9. It is proved that the quality of the extracted total DNA sample is reliable, and the next step test can be carried out.

[0023] 2. Design and synthesis of primer According to the complete sequence of onion nucleus 898U and 898D disclosed in CN103981281A, the primers before and after the differential sequence are designed by SnapGene software: 850-F: 5'-GACAGGTACACAGCATTTTGGC-3'; 850-R: 5'-ATACACTTCTGGTGTCATATACTAG-3', and the degenerate primer synthesis is responsible by Sheng Wu Bioengineering (Shanghai) Co., Ltd., and PAGE purification.

[0024] The primer sequence used for the SCAR marker of Allium fistulosum hybrid plants (YZD) contaminated by onion pollen and the primer sequence used for the SCAR marker of Allium fistulosum are as follows: Forward primer 850-F: 5'-GACAGGTACACAGCATTTTGGC-3'; Reverse primer 850-R: 5'-ATACACTTCTGGTGTCATATACTAG-3'.

[0025] 3. PCR amplification PCR amplification was performed on a BIO-RAD C1000 Touch Thermal Cycler. The detection system used a 25 μL system: 2.5 μL of 10× TransFast® Taq Buffer, 2 μL of 2.5 mM dNTPs, 0.5 μL of TransFast® 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°C initial denaturation for 3 min, followed by 35 cycles of denaturation at 94°C for 5 s, annealing at 58.3°C for 15 s, and extension at 72°C for 20 s, followed by extension at 72°C for 5 min. PCR products were stored at 4°C.

[0026] 4. Detection and analysis of PCR products The PCR products were separated by electrophoresis on 1.5% agarose gel at 130 V (constant voltage) for 45 min, stained with ethidium bromide, and photographed and analyzed with a gel imaging system. Figure 3 .

[0027] The results of taking photos on the gel imaging system are as follows: if the PCR test results show both 1481 bp and 851 bp target bands, the test result is a hybrid (YZD) sample of green onion contaminated with onion pollen; if the PCR test result shows only the 840 bp target band, the test result is a green onion sample. The test results of different varieties of green onion and hybrid (YZD) green onion contaminated with onion pollen are shown in Table 1. After multiple biological replicate tests on green onions (10 varieties) from a wide range of varieties and hybrid (10 varieties) green onions contaminated with onion pollen, the results showed that different varieties of green onions and hybrid (YZD) green onions contaminated with onion pollen can be stably distinguished.

[0028] Table 1 Detection results of different varieties of green onions and green onion hybrids (YZD) contaminated with onion pollen

[0029] Note: ++ indicates the presence of two bands, 1481 bp and 851 bp; - indicates the presence of a single band, 840 bp.

Claims

1. A double dominant SCAR marker for distinguishing green onions from green onions contaminated with onion pollen, characterized in that: The SCAR marker-specific fragments of the onion hybrids contaminated by onion pollen are 1481 bp and 851 bp long, respectively, and their nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The SCAR marker-specific fragment of the onion is 840 bp long, and its nucleotide sequence is shown in SEQ ID NO.

3.

2. A double dominant SCAR marker for distinguishing green onions from green onions contaminated with onion pollen according to claim 1, characterized in that: The specific primers for the SCAR marker are as follows: Forward primer 850-F: 5′-GACAGGTACACAGCATTTTGGC- 3′; Reverse primer 850-R: 5'-ATACACTTCTGGTGTCATATACTAG -3'.

3. Use of the SCAR marker according to claim 1 or 2 in distinguishing green onions from green onion plants contaminated with onion pollen.

4. The use according to claim 3, characterized in that: The specific steps are as follows: (1) Extract the total DNA of the sample to be tested; (2) performing PCR amplification using the specific primers described in claim 2 to obtain a PCR product; (3) Detecting and analyzing the PCR product obtained in step (2). If the detection results of the PCR product show both 1481 bp and 851 bp SCAR marker-specific fragment bands, the test sample is a hybrid strain of green onion contaminated by onion pollen; If the PCR product detection result only contains a band of the 840 bp SCAR marker specific fragment, then the test sample is green onion.

5. The use according to claim 4, characterized in that: The PCR amplification reaction system in step (2) is: 25 μL system, specifically: 10×Trans Fast® Taq Buffer 2.5 μL, 2.5mM dNTPs 2 μL, Trans Fast® Taq DNA Polymerase 0.5 μL, 804-F and 804-R primers 1 μL each, DNA template 1 μL, Nuclease-free Water 17 μL; the reaction procedure is: 94°C pre-denaturation for 3 min, 94°C denaturation for 5 s, 58.3°C annealing for 15 s, 72°C extension for 20 s, 35 cycles, 72°C extension for 5 min, and storage at 4°C.

6. The use according to claim 4, characterized in that: The detection method of the PCR product in step (2) is as follows: the PCR product is separated and analyzed by electrophoresis on a 1.5% agarose gel at a voltage of 130-140 V for 40-45 min, stained with ethidium bromide, and photographed and analyzed using a gel imaging analysis system.

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