SCAR (sequence characterized amplified region) marker for identifying pollution of onion pollen to green Chinese onion and application of SCAR marker

Abstract

The invention discloses an SCAR marker for identifying onion pollen pollution and application thereof, the specific fragment length of the SCAR marker is 811bp, the nucleotide sequence of the SCAR marker is as shown in SEQ ID NO.1, and the SCAR marker of the onion has no target band; the invention also discloses a specific primer of the SCAR marker. When the marker determined by the method is used for verifying the allium fistulosum of different varieties and the allium fistulosum YZD polluted by the onion pollen, the allium fistulosum and the allium fistulosum YZD polluted by the onion pollen can be rapidly and stably identified, compared with a traditional seed purity identification method, the method is not influenced by natural environment and human factors, batch experiments can be carried out, the identification efficiency is improved, and the method is suitable for large-scale popularization and application. 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 onion pollen in production.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a SCAR marker for identifying Allium fistulosum polluted by Allium cepa pollen and its application. Background Art

[0002] Allium fistulosum L. is a biennial or triennial herbaceous plant of the Liliaceae family and the Allium genus. Allium fistulosum can promote gastric juice secretion, aid digestion, promote blood circulation, and has an adjuvant therapeutic effect on symptoms such as influenza, headache, and nasal congestion. It also has the effects of enhancing fibrinolytic activity and reducing blood lipid. Its cultivation is mainly in Asia, and the cultivation area in China is about 570,000 hectares. Allium cepa L. is a biennial plant of the Liliaceae family and the Allium genus. Allium cepa has the effects of lowering cholesterol, softening blood vessels, benefiting the stomach and intestines, and resisting cold and sterilization. Allium cepa is widely cultivated worldwide. According to statistics, among all vegetable crops in the world, the planting area of Allium cepa ranks second.

[0003] Since Allium fistulosum and Allium cepa are widely cultivated in China and the demand for seeds is large, professional seed production companies generally undertake the seed production of both Allium cepa and Allium fistulosum at the same time. This leads to the risk that the flowering periods of Allium fistulosum and Allium cepa may coincide during actual planting, resulting in the situation where Allium fistulosum is polluted by Allium cepa pollen during pollination. The main reasons are as follows: During the cultivation process, the late-maturing variety of Allium fistulosum and the early-maturing variety of Allium cepa coincide in the flowering period. Due to the relatively tight land use, in order to reduce costs, the distance of the reproductive isolation zone between the two species is set unreasonably, resulting in the phenomenon that Allium fistulosum is polluted by Allium cepa pollen during pollination. This phenomenon will cause great harm and bring many uncertain factors to the seed production of Allium fistulosum. During the promotion process of varieties in the market, if mixed seeds are used, on the one hand, it will bring economic losses to farmers, and on the other hand, impure varieties are likely to lead to disputes between seed production companies and commissioned seed production companies, thus affecting the healthy development of the Allium fistulosum industry. According to the Seed Law of the People's Republic of China, seed production shall implement seed production technical regulations and seed inspection and quarantine regulations 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 parental seeds is 99% (GB 16715.3-2010), the purity of Chinese cabbage parental seeds is 99% (GB 16715.3-2010), and the Shandong Provincial Bureau of Quality and Technical Supervision stipulates that the purity of Allium fistulosum original seeds is not less than 99% (DB37 / T1543-2010). Therefore, ensuring the authenticity of species and varieties and conducting purity identification of seeds is a very important link in scientific research, production, and sales.

[0004] In the early days, people used morphological methods for identification. Although the method is simple, it has great limitations. First, field phenotypic identification has a long test cycle, a large workload, high cost, and is easily affected by the environment. Second, the plant and fruit morphology of green onions contaminated by onion pollen is not much different from that of green onions that are not contaminated, and it is impossible to accurately determine whether they are contaminated. In order to avoid losses, it is urgent to develop accurate and efficient identification methods to solve the purity problem caused by green onions contaminated by onion pollen.

[0005] The development of molecular biology provides 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 various tissues and developmental stages of the plant body. They are not restricted by seasons and environments, and there is no problem of expression or not. CN110273020A (application publication number) discloses a SNP molecular marker for distinguishing 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 of citrus summer orange and common sweet orange, and analyzes the results on the genotyping melt 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 summer orange from common sweet orange. At present, there is no research on using molecular markers to solve the purity problem caused by onion pollen contamination during onion pollination. Therefore, molecular markers can be used 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] In order to solve the above technical problems, the present invention provides a SCAR marker for identifying onion pollen contamination of green onions and its application. The present invention designs specific primers for amplification based on the sequences of 898U and 898D of onion nuclei disclosed in CN103981281A to obtain a stable SCAR marker. Based on the determined SCAR markers of green onions and hybrid strains (YZD) contaminated by onion pollen, the present invention performs PCR verification on 10 known green onion varieties and 10 hybrid strains (YZD) contaminated by onion pollen. The hybrid strains (YZD) contaminated by onion pollen of green onions all amplify a specific fragment with a length of 811bp, while all unhybridized green onions have no target bands, and the amplification results are consistent with green onions and hybrid strains (YZD) contaminated by onion pollen of green onions. 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] To solve the above technical problems, the present invention provides the following technical solutions:

[0008] A SCAR marker for identifying Welsh onion contaminated by onion pollen, 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] The present invention provides specific primers for a SCAR marker for identifying Welsh onion contaminated by onion pollen, and the primer sequences are as follows:

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

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

[0012] The present invention provides an application of a SCAR marker in identifying Welsh onion, plants or seeds of Welsh onion contaminated by onion pollen.

[0013] An application method of a SCAR marker for identifying Welsh onion contaminated by onion pollen, characterized in that the specific operation steps are as follows:

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

[0015] (2) Perform PCR amplification using the above specific primers to obtain a PCR product;

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

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

[0018] Further, the detection method for 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 120 - 125V for 40 - 45 minutes, stained with ethidium bromide, and photographed and analyzed with a gel imaging analysis system.

[0019] Beneficial effects:

[0020] (1) The present invention can be used for identification during both the plant stage and the seed stage. Identifying suspicious plants during the plant stage can avoid both the economic losses caused by incorrect elimination due to uncertainty and the increased difficulty in later identification when a large number of hybrids are mixed into scallion seeds.

[0021] (2) The present invention has determined specific SCAR marker fragments for the identification of scallions and hybrid plants (YZD) contaminated with onion pollen. Through multiple biological replicates of 10 scallion varieties and 10 hybrid plants (YZD) contaminated with onion pollen from a wide range of sources, the results show that scallions and hybrid plants contaminated with onion pollen can be stably distinguished. The markers determined by the present invention have strong specificity and high stability.

[0022] (3) The identification method of the present invention only requires extracting the total DNA of scallions and hybrid plants (YZD) contaminated with onion pollen, performing batch PCR amplification, and then performing gel imaging analysis. These experiments are all completed by instrument operation. Compared with traditional seed purity identification methods, it is not affected by natural environment and human factors, etc., can conduct batch experiments, saves a large amount of manpower, not only avoids the cumbersome screening process of conventional methods, but also avoids the complex operation of extracting mitochondrial DNA, and significantly improves the identification of different varieties of scallions and hybrid plants (YZD) contaminated with onion pollen, providing a rapid and feasible method for solving the problem of scallion variety mixing caused by scallion pollen being contaminated with onion pollen in production. Description of the drawings

[0023] Figure 1 It is the electrophoresis result diagram of the total DNA of scallions; among them, B1 - B10 are respectively the electrophoresis results of the total DNA of scallion varieties: Yuanzang, Tian Guang Yi Ben, Xia Ren Tian, Chang Bao, Chang Yue, Chun Wei, Ji Wan Chou Yi Ben, Jia Bao, Ji Zang, Tian Bao, and M is the molecular weight standard DL15000;

[0024] Figure 2Electrophoresis result diagram of total DNA of miscellaneous plants (YZD) of scallions contaminated by onion pollen; among them, B1 - B10 are the electrophoresis results of total DNA of Yuanzang × Jinqiu, Tianguangyiben × Binyu, Xiarentian × Xiuyuwan, Changbao × Jinxing, Changyue × Fuxing, Chunwei × Guijin, Jiwanchouyiben × Jinqiu, Jiabao × Guijin, Jizang × Binyu, Tianbao × Xiuyuwan respectively, and M is the molecular weight standard DL15000;

[0025] Figure 3 PCR amplification detection results of scallions and miscellaneous plants (YZD) of scallions contaminated by onion pollen; among them, 1 - 10 are the PCR amplification detection results of scallions Yuanzang, Tianguangyiben, Xiarentian, Changbao, Changyue, Chunwei, Jiwanchouyiben, Jiabao, Jizang, Tianbao respectively, and 11 - 20 are the PCR amplification results of the varieties of miscellaneous plants (YZD) of scallions contaminated by onion pollen: Yuanzang × Jinqiu, Tianguangyiben × Binyu, Xiarentian × Xiuyuwan, Changbao × Jinxing, Changyue × Fuxing, Chunwei × Guijin, Jiwanchouyiben × Jinqiu, Jiabao × Guijin, Jizang × Binyu, Tianbao × Xiuyuwan, and M is the molecular weight standard DL2000.

[0026] Figure 4 Growth diagram of scallions and miscellaneous plants (YZD) of scallions contaminated by onion pollen after being mixed in the field. Detailed implementation manners

[0027] The present invention will be further described in detail below in combination with the detailed implementation manners. The given embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0028] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

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

[0030] Experimental materials: The scallion varieties are Yuanzang, Tianguangyiben, Xiarentian, Changbao, Changyue, Chunwei, Jiwanchouyiben, Jiabao, Jizang, Tianbao; Miscellaneous plants (YZD) of scallions contaminated by onion pollen: Yuanzang × Jinqiu, Tianguangyiben × Binyu, Xiarentian × Xiuyuwan, Changbao × Jinxing, Changyue × Fuxing, Chunwei × Guijin, Jiwanchouyiben × Jinqiu, Jiabao × Guijin, Jizang × Binyu, Tianbao × Xiuyuwan, and all these varieties are commercially available.

[0031] Reagents used: The Polysaccharide and Polyphenol Plant Genomic DNA Extraction Kit (DP360) was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The PCR Detection Kit (Trans Taq DNA Polymerase), 10×Trans TaqBuffer, DNA Molecular Weight Marker DL2000, and DNA Molecular Weight Marker DL15000 were purchased from Beijing TransGen Biotech Co., Ltd.; The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0032] Instruments used: The C1000 Touch Thermal cycler PCR instrument and GelDoc go Gel Imaging Analysis System were purchased from BIO-RAD.

[0033] Example 1: Obtaining SCAR Markers for Welsh Onion and the Hybrid Strain (YZD) Contaminated with Onion Pollen

[0034] In the National Center for Biotechnology Information database ( National Center for Biotechnology Information, website: https: / / www.ncbi.nlm.nih.gov / ), the FASTA format sequence of its skp1-Ms allele was downloaded. According to the complete sequences of onion nucleus 898U and 898D disclosed in CN103981281A, alignment analysis was performed using DNAMAN software, and forward and reverse primers were designed using SnapGene software. The results showed that: amplification was carried out according to the designed primers, and finally a stable molecular marker, the SCAR marker, was obtained. After sequencing, the sample of the hybrid strain (YZD) of Welsh onion contaminated with onion pollen had a target band with a length of 811 bp; when no target band was detected in the detection result, the detection result was a Welsh onion sample.

[0035] Example 2: Application of SCAR Markers for Welsh Onion and the Hybrid Strain (YZD) Contaminated with Onion Pollen

[0036] 1. Extraction and Detection of Total DNA from Welsh Onion and the Hybrid Strain (YZD) Contaminated with Onion Pollen

[0037] For the obtained identification materials of Welsh onion and the hybrid strain (YZD) of Welsh onion contaminated with onion pollen, the total DNA of different varieties of Welsh onion and the hybrid strain (YZD) of Welsh onion contaminated with onion pollen was extracted using the Polysaccharide and Polyphenol Plant Genomic DNA Extraction Kit (DP360). The specific operation steps are shown in the kit instruction manual. The quality of the DNA was checked using 0.8% agarose gel, and the detection results of the extracted total DNA by agarose gel electrophoresis are shown in Figure 1 、 2, the visible bands are clear; the purity and concentration of the extracted DNA are detected by a ultra-micro spectrophotometer. The DNA has a significant absorption peak at OD260, and the ratio of OD260 / OD280 is between 1.7 - 1.9. It is confirmed that the quality of the extracted total DNA sample is reliable and can be used for the next experiment.

[0038] 2. Primer Design and Synthesis

[0039] Based on the complete sequences of onion nucleus 898U and 898D disclosed in CN103981281A, the present invention designs primers before and after the differential sequences through SnapGene software: 804-F: 5'-CACGGATTCGATTGTTAAATCGT-3'; 804-R: 5'-ACACACACACAGAGTGAGAAATTTTATA-3'. Sangon Biotech (Shanghai) Co., Ltd. is responsible for the synthesis of degenerate primers and PAGE purification.

[0040] The primer sequences for the SCAR marker nucleotide sequence SEQ ID NO.1 of the hybrid strain (YZD) of Chinese onion contaminated by onion pollen are 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 is carried out on a C1000 Touch Thermal cycler PCR instrument of BIO-RAD company. The detection system is a 25μL system: 10×Trans The volume of Taq Buffer is 2.5μL, the volume of 2.5mM dNTPs is 2μL, Trans The volume of Taq DNA Polymerase is 0.5μL, the volume of CDY-F and CDY-R is 1μL each, the volume of DNA template is 1μL, and the volume of Nuclease-free Water is 17μL; the reaction procedure is: pre-denaturation at 94℃ for 3min, denaturation at 94℃ for 5s, annealing at 54.1℃ for 15s, extension at 72℃ for 15s, 25 cycles, extension at 72℃ for 5min, and the PCR product is 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, stained with ethidium bromide, and photographed and analyzed using a gel imaging system. The results are shown in Figure 3 .

[0047] The results of photographing with the gel imaging system are as follows: If an 811 bp target band is detected in the PCR test result, then the test result is a sample of a hybrid plant (YZD) of Chinese chive contaminated with onion pollen; if no target band is detected in the PCR test result, then the test result is a Chinese chive sample. The test results of different varieties of Chinese chive and hybrid plants (YZD) of Chinese chive contaminated with onion pollen are shown in Table 1. After conducting multiple biological replicate tests on Chinese chives from a wide range of varieties (10 varieties) and hybrid plants (10 varieties) of Chinese chive contaminated with onion pollen, the results showed that different varieties of onion and Chinese chive seeds could be stably distinguished.

[0048] Table 1 Test results of different varieties of Chinese chive and hybrid plants (YZD) of Chinese chive contaminated with onion pollen

[0049]

[0050]

[0051] Note: + indicates the presence of an 811 bp band; - indicates the absence of a band.

Claims

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

1.

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

3. Use of the SCAR marker according to claim 1 or 2 in identifying plants or seeds of Chinese onion contaminated by onion pollen.

4. The application according to claim 3, characterized in that, The specific operation steps are as follows: (1) Extract the total DNA of the sample to be detected; (2) Perform PCR amplification using the specific primers described in claim 2 to obtain a PCR product; (3) Detect and analyze the PCR product obtained in step (2). If the bands of the specific fragment of the 811 bp SCAR marker are present in the detection result of the PCR product, then the detected sample is a hybrid plant of Chinese onion contaminated by onion pollen; if no bands of the specific fragment of the SCAR marker are present in the detection result of the PCR product, then the detected sample is Chinese onion.

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

6. The application according to claim 4, characterized in that, The detection method of the PCR product in step (2) is: separating and analyzing the PCR product by electrophoresis on a 1.5% agarose gel at a voltage of 120-125 V for 40-45 min, staining with ethidium bromide, and photographing and analyzing with a gel imaging analysis system.

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