A double dominant scar marker for identifying allium fistulosum and allium fistulosum contaminated by onion pollen and application thereof

Abstract

The application discloses a double dominant SCAR marker for identifying Allium fistulosum and Allium fistulosum hybrid plants contaminated by onion pollen and application of the double dominant SCAR marker, wherein the specific fragments of the SCAR marker of the hybrid plants contaminated by onion pollen are 1481 bp and 851 bp, the nucleotide sequences are shown as SEQ ID NO. 1 and SEQ ID NO. 2 respectively, the specific fragment of the SCAR marker of Allium fistulosum is 840 bp, and the nucleotide sequence is shown as SEQ ID NO. 3; 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 Allium fistulosum and hybrid plants of Allium fistulosum contaminated by onion pollen, and Allium fistulosum hybrid plants contaminated by onion pollen can be identified quickly and stably. Compared with a traditional seed purity identification method, the application is not influenced by natural environment and human factors, can be used for batch experiments, improves identification efficiency, and provides a quick and feasible method for solving the problem of mixed Allium fistulosum varieties caused by the fact that Allium fistulosum pollen is contaminated by onion pollen.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a dual dominant SCAR marker for identifying scallions and scallion contamination by onion pollen, 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. Leeks promote gastric juice secretion, aid digestion, and improve blood circulation. They have an auxiliary therapeutic effect on symptoms such as influenza, headache, and nasal congestion. They also enhance fibrinolytic activity and lower blood lipids. Cultivation is mainly in Asia, with China having a cultivation area of ​​approximately 570,000 hectares. Onions (…) 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, professional seed production companies typically undertake the production of both onion and scallion seeds. 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, late-maturing scallion varieties and early-maturing onion varieties may flower at the same time, and if the distance between the reproductive isolation zones of the two species is not properly set, scallions may become contaminated with onion pollen during pollination. This phenomenon can cause significant harm, introducing many uncertainties into scallion seed production. 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, in scientific research, production, and sales, it is a very important step to identify the purity of seeds in order to ensure the authenticity of species and varieties.

[0004] In the early stage, people identify by morphological methods, which is simple, but there are great limitations. First, field phenotypic identification, test cycle is long, heavy workload, high cost, easy to be affected by the environment, etc. And with the development of breeding work, it is difficult or impossible to identify from the morphology, and it is urgent to seek a unified standard technical system for identifying the authenticity of varieties. Therefore, it is necessary to develop accurate and efficient identification methods to solve the purity problem of big onions caused by onion pollen pollution.

[0005] The development of molecular biology provides a new technical means based on DNA variation for plant genetic markers, i.e. molecular marker technology. Compared with other marker methods, molecular markers have strong advantages, which exist in the form of DNA, and can be detected in various tissues and development stages of plants, not limited by season and environment, and there is no problem of expression or not. CN110273020A (application publication number) discloses a SNP molecular marker for distinguishing summer orange and ordinary sweet orange. According to the 17856950 base sequence of the 4th chromosome of sweet orange, the primer is designed, the DNA of summer orange and ordinary sweet orange is extracted by CTAB method, and the result is analyzed on the gene scanning module (Gene Scan) and the gene genotyping dissolution curve module (Melt Curve Genotyping) on the LightCycler480 software. The results show that the molecular marker of the application can accurately distinguish summer orange and ordinary sweet orange [2] . At present, there is no research on solving the purity problem of big onion pollination caused by onion pollen pollution by using SCAR marker method. Therefore, this patent solves the problem of big onion pollination caused by onion pollen pollution by using molecular marker method, which has important significance for accurately and efficiently identifying the purity of big onion varieties. SUMMARY

[0006] The application provides a SCAR marker for identifying onion pollen contaminated scallion and application thereof, and the SCAR marker is obtained by amplification of specific primers designed according to the sequence of onion cell nucleus 898U and 898D disclosed in CN103981281A. The SCAR marker of the identified scallion and onion pollen contaminated hybrid (YZD) is used for PCR verification of 10 known scallion varieties and 10 scallion pollen contaminated hybrid (YZD) varieties, and the specific fragments with lengths of 1481 bp and 851 bp are amplified from the scallion pollen contaminated hybrid (YZD), and the specific fragment with a length of 840 bp is amplified from all non-hybrid scallions, and the amplification result is consistent with that of the scallion and the scallion pollen contaminated hybrid (YZD). The above identification method can be used for quickly and accurately identifying different varieties of scallions and scallion pollen contaminated hybrids (YZD), and provides a quick and feasible method for solving the problem of identification of scallion pollen contaminated by onion pollen and resulting in mixed scallion varieties.

[0007] A double dominant SCAR marker for identifying scallion and onion pollen contaminated scallion, wherein the specific fragments of the SCAR marker of the scallion pollen contaminated hybrid (YZD) have lengths of 1481 bp and 851 bp, the nucleotide sequences of which are shown as SEQ ID NO. 1 and SEQ ID NO. 2, and the specific fragment of the SCAR marker of the scallion has a length of 840 bp, the nucleotide sequence of which is shown as SEQ ID NO. 3.

[0008] The application provides specific primers of the double dominant SCAR marker for identifying scallion and onion pollen contaminated scallion, and the sequences of the primers are as follows:

[0009] The forward primer 850-F is 5'-GACAGGTACACAGCATTTTGGC-3';

[0010] The reverse primer 850-R is 5'-ATACACTTCTGGTGTCATATACTAG-3';

[0011] The application provides application of the double dominant SCAR marker in identification of scallion, onion pollen contaminated hybrid (YZD) and the like.

[0012] A method for identifying scallion and onion pollen contaminated hybrid (YZD), and the specific operation steps are as follows:

[0013] (1) extracting total DNA of scallion and onion pollen contaminated hybrid (YZD);

[0014] (2) using the specific primers to perform PCR amplification to obtain a PCR product;

[0015] (3) detecting and analyzing the PCR product obtained in step (2), if the detection result of the PCR product simultaneously exists 1481 bp and 851 bp SCAR marker specific fragments, then the detection sample is the hybrid of Allium fistulosum contaminated by onion pollen (YZD); if the detection result of the PCR product only exists 840 bp SCAR marker specific fragment, then the detection sample is Allium fistulosum.

[0016] Further, the PCR amplification reaction system in step (2) is 25 μL, specifically: 10×Trans Fast Taq Buffer 2.5 μL, 2.5 mM dNTPs 2 μL, Trans Fast Taq DNA Polymerase 0.5 μL, CDY-F and CDY-R primers each 1 μL, DNA template 1 μL, Nuclease-free Water 17 μL; the reaction program is: 94℃ pre-denaturation 3 min, 94℃ denaturation 5 s, 58.3℃ annealing 15 s, 72℃ extension 20 s, 35 cycles, 72℃ extension 5 min, 4℃ preservation.

[0017] Further, the detection method of the PCR product in step (2) is: separating and analyzing the PCR product by 130-140 V voltage, 1.5% agarose gel electrophoresis for 40-45 min, bromide ethylene dyeing, and taking pictures for analysis by a gel imaging analysis system.

[0018] Beneficial effects:

[0019] (1) The present application determines specific SCAR marker fragments for identifying Allium fistulosum and the hybrid of Allium fistulosum contaminated by onion pollen (YZD), respectively. After biological repeated verification on 10 Allium fistulosum varieties and 10 hybrids of Allium fistulosum contaminated by onion pollen (YZD) from a wide range of sources, the results show that Allium fistulosum and the hybrid of Allium fistulosum contaminated by onion pollen (YZD) can be stably distinguished, and the marker determined in the present application has high specificity and high stability.

[0020] (2) The identification method of the application only needs to extract total DNA of the big green onions and the hybrid strains (YZD) of the big green onions contaminated by onion pollen, perform batch PCR amplification, and then perform gel imaging analysis, and these experiments are completed by instruments, compared with the traditional seed purity identification method, the method is not affected by natural environment and human factors, batch experiments can be performed, a large amount of manpower is saved, the tedious screening process of the conventional method is avoided, the complex operation of extracting mitochondrial DNA is also avoided, and the efficiency of identifying different varieties of big green onions and the hybrid strains (YZD) of the big green onions contaminated by onion pollen is significantly improved, thereby providing a fast and feasible method for solving the problem of mixed big green onion varieties caused by the contamination of big green onion pollen by onion pollen.

[0021] (3) The application respectively determines specific SCAR marker fragments for identifying big green onions and hybrid strains (YZD) of the big green onions contaminated by onion pollen, so that technical errors, such as not adding DNA or primers, can be effectively overcome, and no band of the big green onions and the hybrid strains (YZD) of the big green onions contaminated by onion pollen is caused. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is a total DNA electrophoresis result graph of big green onions; wherein A1-A10 are total DNA electrophoresis results of big green onion varieties: Yuanzang, Tianguang Yiben, Xiaren Tian, Changbao, Changyue, Chunwei, Jiluan Chua Yiben, Jiabao, Jizang, Tianbao, and M is a molecular weight standard DL15000;

[0023] Figure 2 It is a total DNA electrophoresis result graph of hybrid strains (YZD) of the big green onions contaminated by onion pollen; wherein B1-B10 are total DNA electrophoresis results of Yuanzang x Jinqiu, Tianguang Yiben x Binyu, Xiaren Tian x Xiuyumar, Changbao x Jinxing, Changyue x Fuxing, Chunwei x Guijin, Jiluan Chua Yiben x Jinqiu, Jiabao x Guijin, Jizang x Binyu, and Tianbao x Xiuyumar, and M is a molecular weight standard DL15000;

[0024] Figure 3 It is a PCR amplification detection result of big green onions and hybrid strains (YZD) of the big green onions contaminated by onion pollen; wherein 1-10 are PCR amplification detection results of big green onions Yuanzang, Tianguang Yiben, Xiaren Tian, Changbao, Changyue, Chunwei, Jiluan Chua Yiben, Jiabao, Jizang, and Tianbao, 11-20 are PCR amplification results of big green onion hybrid strains (YZD) of the big green onions contaminated by onion pollen, and M is a molecular weight standard DL2000.

[0025] Figure 4 It is a growth graph of big green onions and hybrid strains (YZD) of the big green onions contaminated by onion pollen after being mixed in the field. DETAILED DESCRIPTION

[0026] In order to better understand the technical solutions in the present application, the present application will be further described below in conjunction with embodiments. The described embodiments are only some of the embodiments of the present application, and the present application is not limited by the following embodiments.

[0027] The following examples facilitate better understanding of the present application, but do not limit the present application. In the following examples, the experimental methods are conventional methods unless otherwise specified. In the following examples, the test materials used are commercially available from conventional biochemical reagent stores unless otherwise specified.

[0028] The experimental materials, reagents and instruments used in the present application are as follows:

[0029] 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 at a later stage.

[0030] Reagents used: polysaccharide polyphenol plant genome DNA extraction kit (DP360) was purchased from Tian Gen Bio-Science Co., Ltd. (Beijing), 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 Zonnuo Biotech Co., Ltd.; primers were synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd.

[0031] Instruments used: C1000 Touch Thermal cycler PCR instrument, GelDoc go gel imaging analysis system were purchased from BIO-RAD company.

[0032] Example 1: Obtaining of SCAR markers of Allium fistulosum and Allium fistulosum hybrid plants (YZD) contaminated by onion pollen

[0033] According to the complete sequence of onion cell nucleus 898U, 898D disclosed in CN103981281A, the primer before and after design is analyzed by DNA MAN software. The results show that: according to the designed primer for amplification, finally the stable molecular marker SCAR marker is obtained. Sequencing, the sample of big onion hybrid contaminated by onion pollen (YZD) has two target bands, the length is 1481 bp and 851 bp respectively, the nucleotide sequences are shown as SEQ ID NO. 1 and SEQ ID NO. 2; the target band of big onion is 840 bp, and the nucleotide sequence is shown as SEQ ID NO. 3.

[0034] Example 2 Application of big onion, big onion hybrid contaminated by onion pollen (YZD) SCAR marker

[0035] 1, Extraction and detection of total DNA of big onion, big onion hybrid contaminated by onion pollen (YZD)

[0036] First, obtain the identification material of big onion, big onion hybrid contaminated by onion pollen (YZD), then use polysaccharide polyphenol plant genome DNA extraction kit (DP360) to extract total DNA of different varieties of big onion, big onion hybrid contaminated by onion pollen (YZD), the specific operation steps are shown in the kit instruction manual. 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 confirmed that the quality of the extracted total DNA sample is reliable, and the next step test can be carried out.

[0037] 2, Design and synthesis of primer

[0038] According to the complete sequence of onion cell nucleus 898U, 898D disclosed in CN103981281A, the primer before and after design is analyzed by SnapGene software: 850-F: 5'-GACAGGTACACAGCATTTTGGC-3'; 850-R: 5'-ATACACTTCTGGTGTCATATACTAG-3', the degenerate primer synthesis is responsible by Sheng Wu Bioengineering (Shanghai) Co., Ltd., and PAGE purification.

[0039] The primer sequence used for the SCAR marker of big onion hybrid contaminated by onion pollen (YZD) and the primer sequence used for the SCAR marker of big onion are as follows:

[0040] Forward primer 850-F: 5'-GACAGGTACACAGCATTTTGGC-3';

[0041] Reverse primer 850-R: 5'-ATACACTTCTGGTGTCATATACTAG-3'.

[0042] 3. PCR amplification

[0043] PCR amplification was performed on a BIO-RAD C1000 Touch Thermal cycler PCR instrument. The detection system was 25 μL system: 10x Trans Fast® Taq Buffer was 2.5 μL, 2.5 mM dNTPs was 2 μL, Trans Fast® Taq DNA Polymerase was 0.5 μL, CDY-F and CDY-R was 1 μL each, DNA template was 1 μL, Nuclease-free Water was 17 μL; the reaction program was: 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 the PCR product was stored at 4°C.

[0044] 4. Detection and analysis of PCR product

[0045] The PCR product was separated by electrophoresis at 130 V (constant voltage) for 45 min on a 1.5% agarose gel, stained with ethidium bromide, and photographed for analysis by a gel imaging system. The results are shown in Figure 3 .

[0046] The gel imaging system photographing results are as follows: if the PCR test detection result simultaneously exists 1481 bp and 851 bp target bands, then the detection result is an onion pollen contaminated hybrid (YZD) sample of Welsh onion; if the PCR test detection result only has an 840 bp target band, then the detection result is a Welsh onion sample. The detection results of different varieties of Welsh onion and onion pollen contaminated hybrid (YZD) of Welsh onion are shown in Table 1. After a plurality of biological repeated tests on Welsh onion (10 varieties) and onion pollen contaminated hybrid (10 varieties) of Welsh onion with a wide variety of sources, the results showed that different varieties of Welsh onion and onion pollen contaminated hybrid (YZD) of Welsh onion can be stably distinguished.

[0047] Table 1 Detection results of different varieties of Welsh onion and onion pollen contaminated hybrid (YZD) of Welsh onion

[0048]

[0049] Note: ++ means both 1481 bp and 851 bp bands are present; - means only 840 bp band is present.

Claims

1. A dominant SCAR marker for discriminating between Allium fistulosum and A. fistulosum contaminated with onion pollen, characterized in that, The specific fragment length of the SCAR marker of the onion pollen contaminated allium fistulosum hybrid plant is 1481 bp and 851 bp, respectively, and the nucleotide sequences are shown as SEQ ID NO. 1 and SEQ ID NO. 2, respectively, and the specific fragment length of the SCAR marker of the allium fistulosum is 840 bp, and the nucleotide sequence is shown as SEQ ID NO.

3.

2. The dominant SCAR marker for distinguishing between Allium fistulosum and A. fistulosum contaminated with A. cepa pollen according to claim 1, characterized in that, The specific primers of the SCAR marker are as follows: Forward primer 850-F: 5'-GACAGGTACACAGCATTTTGGC-3'; Reverse primer 850-R: 5'-ATACACTTCTGGTGTCATATACTAG-3'.

3. The SCAR marker of claim 1 or 2 is used for identifying allium fistulosum and onion pollen contaminated allium fistulosum plants.

4. Use 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) Use the specific primers of claim 2 to perform PCR amplification to obtain PCR products; (3) Detect and analyze the PCR products obtained in step (2), if the detection results of the PCR products simultaneously exist 1481 bp and 851 bp SCAR marker specific fragments, then the detection sample is an onion pollen contaminated hybrid plant of allium fistulosum; If the detection results of the PCR products only exist 840 bp SCAR marker specific fragments, then the detection sample is allium fistulosum.

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

6. Use according to claim 4, characterized in that, The detection method of the PCR product of step (2) is: separate and analyze the PCR product by 130-140 V voltage, 1.5% agarose gel electrophoresis for 40-45 min, bromide ethylene dyeing, and gel imaging analysis system photographing analysis.

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