SCAR (sequence characterized amplified region) marker for identifying purity of two allium plant seeds and application of SCAR marker

Abstract

The invention belongs to the technical field of biology, and particularly relates to an SCAR (sequence characterized amplified region) marker for identifying the purity of two allium plant seeds and application of the SCAR marker. Wherein the specific fragment length of the SCAR marker of the onion is 1388bp and 760bp, the nucleotide sequences of the onion are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2, the specific fragment length of the SCAR marker of the green Chinese onion is 760bp, and the nucleotide sequences of the green Chinese onion are respectively shown as SEQ ID NO: 3. The SCAR marker determined by the invention is strong in specificity and high in stability; through multiple biological repeated verification on 10 varieties of onion seeds and 10 varieties of green Chinese onion seeds which are wide in source, the results show that the onion seeds and the green Chinese onion seeds can be stably distinguished, the defects that a traditional morphological identification method is inaccurate, time-consuming and labor-consuming are overcome, the efficiency of identifying the purity of the onion seeds and the green Chinese onion seeds is remarkably improved, and the method is suitable for popularization and application. And a rapid and feasible method is provided for identifying the purity of the onion and green Chinese onion seeds in production.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a SCAR marker for identifying the purity of seeds of two Allium plants and application thereof. BACKGROUND

[0002] Onion (Allium cepa L.) belongs to the Allium genus of the Liliaceae family and is a biennial plant. Onion has multiple functions, such as reducing cholesterol, softening blood vessels, benefiting stomach and intestines, and resisting cold and sterilization. The planting area of onion ranks second among all vegetable crops. Chinese chives (Allium fistulosum L.) also belongs to the Allium genus of the Liliaceae family and is a biennial or triennial herb. Chinese chives can promote gastric juice secretion, aid digestion, promote blood circulation, and has auxiliary treatment effect on symptoms such as influenza, headache, and nasal congestion. In addition, Chinese chives has the functions of enhancing fibrinolysis activity and reducing blood lipids. Chinese chives is cultivated all over the world, especially in Asian regions. Allium cepa Allium fistulosum Since onion and Chinese chives are widely planted in China, seed producers usually need to undertake the seed planting production task of both crops, which leads to the risk of mixing onion and Chinese chives seeds in the actual production process. Seed purity identification before sale is an indispensable necessary link. The shapes and colors of onion and Chinese chives seeds are very similar: Chinese chives seeds are shield-shaped, angular, slightly flat, have few and regular wrinkles on the surface, and the umbilical part is concave. Onion seeds are also shield-shaped, but have more irregular wrinkles on the surface, and the umbilical part is concave. In addition, the seed surfaces of both are black, and it is difficult to distinguish them by observing the seed skin color and shape. After sowing, the seedling stage morphology is also very similar, and it is also difficult to distinguish them by relying on field morphological observation. Therefore, it is urgent to develop an accurate and efficient identification method to solve the purity problem caused by mechanical mixing of onion and Chinese chives seeds.

[0003] The development of molecular biology provides a new technical means based on DNA variation for plant genetic markers. Compared with traditional field identification methods, molecular markers have incomparable advantages. They exist in the form of DNA and can be detected in various tissues and development stages of plants, are not limited by seasons and environments, and do not have the problem of expression or not. At present, there is no research on using molecular marker method to solve the problem of mechanical mixing of onion and Chinese chives seeds. Therefore, it is of great significance to accurately and efficiently identify the purity of seeds by using molecular marker technology.

[0004] The development of molecular biology provides a new technical means based on DNA variation for plant genetic markers. Compared with traditional field identification methods, molecular markers have incomparable advantages. They exist in the form of DNA and can be detected in various tissues and development stages of plants, are not limited by seasons and environments, and do not have the problem of expression or not. At present, there is no research on using molecular marker method to solve the problem of mechanical mixing of onion and Chinese chives seeds. Therefore, it is of great significance to accurately and efficiently identify the purity of seeds by using molecular marker technology. SUMMARY

[0005] In order to solve the problems in the prior art, the application provides a SCAR marker for identifying the purity of seeds of two Allium plants and application thereof.

[0006] To achieve the above-mentioned purpose, the application adopts the following technical solutions: ​The first aspect of the present application provides a SCAR marker for identifying the purity of onion and Chinese onion seeds, characterized in that the specific fragments of the SCAR marker of onion have lengths of 1388 bp and 760 bp respectively, and the nucleotide sequences thereof are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively, and the specific fragment of the SCAR marker of Chinese onion has a length of 760 bp, and the nucleotide sequence thereof is shown in SEQ ID NO: 3.

[0007] The second aspect of the present application provides a primer set for detecting the SCAR marker of the first aspect, which comprises a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO: 8, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 9.

[0008] The third aspect of the present application provides a reagent for identifying the purity of onion and Chinese onion seeds, which comprises the primer set of the second aspect.

[0009] The fourth aspect of the present application provides a kit for identifying the purity of onion and Chinese onion seeds, which comprises the primer set of the second aspect or the reagent of the third aspect.

[0010] The fifth aspect of the present application provides the use of at least one of the following (a1) to (a4) in identifying the purity of onion and Chinese onion seeds: (a1) the SCAR marker of the first aspect; (a2) the primer set of the second aspect; (a3) the reagent of the third aspect; (a4) the kit of the fourth aspect.

[0011] The sixth aspect of the present application provides a method for identifying the purity of onion and Chinese onion seeds, which comprises identifying the onion and Chinese onion seeds by using any one of the following (b1) to (b3): (b1) the primer set of the second aspect; (b2) the reagent of the third aspect; (b3) the kit of the fourth aspect.

[0012] In some embodiments of the present application, the method comprises the following steps: performing PCR amplification on the sample to be tested by using the primer set, and identifying according to the characteristics of the amplified fragments; the sample to be tested comprises onion seeds and Chinese onion seeds; and the identification method is agarose gel electrophoresis or first-generation sequencing.

[0013] In some embodiments of the present application, the varieties of the onion include Diqiu, Hongye No. 3, Dabao, Jinxing, Fuxing, Guijin, Quanzhouhuang, Fuhong, Ziyang and Ziyu.

[0014] In some embodiments of the present invention, the varieties of scallions include Yuanzang, Tianguang Yiben, Xiarentian, Changbao, Changyue, Chunwei, Jiwanchou Yiben, Jiabao, Jizang, and Tianbao.

[0015] In some embodiments of the present invention, the PCR amplification reaction system includes: DNA polymerase, reaction buffer, amplification primers, DNA of the sample to be tested, and sterile water.

[0016] In some embodiments of the present invention, the PCR amplification reaction system is a 25 μL system, specifically comprising: 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.

[0017] In some embodiments of the present invention, the PCR amplification reaction conditions are as follows: 94°C pre-denaturation for 3 min, 94°C denaturation for 5 s, 50°C annealing for 15 s, 72°C extension for 20 s, 35 cycles, 72°C extension for 5 min, and storage at 4°C.

[0018] In some embodiments of the present invention, the identification method is agarose gel electrophoresis. If the electrophoretic detection of the sample shows an electrophoretic band of 1388bp and 760bp, the sample is an onion; if a 760bp electrophoretic band is shown, the sample is a scallion. Alternatively, the identification method is first-generation sequencing. If the sequence of the electrophoretic recovered fragment is as shown in SEQ ID NO: 1 and SEQ ID NO: 2, then the sample to be tested is an onion; if the sequence of the electrophoretic recovered fragment is as shown in SEQ ID NO: 3, then the sample to be tested is a scallion.

[0019] The beneficial effects of the above technical solutions are as follows: (1) This invention identifies specific SCAR marker fragments for the identification of onion and scallion seeds, which can effectively overcome technical errors, such as the absence of DNA or primers, resulting in no bands for onion and scallion seeds. The SCAR markers identified in this invention have high specificity and stability. After multiple biological replications on 10 varieties of onion seeds and 10 varieties of scallion seeds from a wide range of sources, the results show that they can stably distinguish between onion and scallion seeds.

[0020] (2) The identification method of the present invention only requires extracting total DNA from onions and scallions for 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 and significantly improving the efficiency of identifying the purity of onion and scallion seeds. It provides a fast and feasible method for identifying the purity of onion and scallion seeds in production. Attached Figure Description

[0021] Figure 1 The image shows the electrophoresis results of total DNA from onions in an embodiment of the present invention; where A1-A10 are the electrophoresis results of total DNA from onion varieties Earth, Red Leaf No. 3, Dabao, Jinxing, Fuxing, Guijin, Quanzhou Yellow, Fuhong, Ziyang, and Ziyu, respectively, and M is the molecular weight standard DL15000; Figure 2 The image shows the electrophoresis results of total DNA from scallions in an embodiment of the present invention; where B1-B10 are the electrophoresis results of total DNA from scallion varieties Yuanzang, Tianguang Yiben, Xiarentian, Changbao, Changyue, Chunwei, Jiwanchou Yiben, Jiabao, Jizang, and Tianbao, respectively, and M is the molecular weight standard DL15000; Figure 3 The results of PCR amplification of onion seeds and scallion seeds by primer group A in this embodiment of the invention are shown. A1-A10 are the PCR amplification detection results of Onion Earth, Red Leaf No. 3, Dabao, Jinxing, Fuxing, Guijin, Quanzhou Yellow, Fuhong, Ziyang and Ziyu, respectively. B1-B10 are the PCR amplification detection results of Scallion Yuanzang, Tianguang Yiben, Xiarentian, Changbao, Changyue, Chunwei, Jiwanchou Yiben, Jiabao, Jizang and Tianbao, respectively. Figure 4 The results of PCR amplification of onion seeds and scallion seeds by primer group B in this embodiment of the invention are shown. A1-A10 are the PCR amplification detection results of Onion Earth, Red Leaf No. 3, Dabao, Jinxing, Fuxing, Guijin, Quanzhou Yellow, Fuhong, Ziyang and Ziyu, respectively. B1-B10 are the PCR amplification detection results of Scallion Yuanzang, Tianguang Yiben, Xiarentian, Changbao, Changyue, Chunwei, Jiwanchou Yiben, Jiabao, Jizang and Tianbao, respectively. Figure 5 The results of PCR amplification detection of onion seeds by primer set C in this embodiment of the invention are shown; where A1-A10 are the PCR amplification detection results of Onion Earth, Red Leaf No. 3, Dabao, Jinxing, Fuxing, Guijin, Quanzhou Yellow, Fuhong, Ziyang and Ziyu, respectively. Figure 6 The results of PCR amplification detection of scallion seeds by primer group C in this embodiment of the invention are shown below; B1-B10 are the PCR amplification detection results of scallion Yuanzang, Tianguang Yiben, Xiarentian, Changbao, Changyue, Chunwei, Jiwanchou Yiben, Jiabao, Jizang, and Tianbao, respectively. Detailed Implementation

[0022] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0023] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, 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, and / or combinations thereof.

[0024] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0025] The experimental materials, reagents, and instruments used in this invention are as follows: Experimental materials: Onion Earth, Red Leaf No. 3, Dabao, Jinxing, Fuxing, Guijin, Quanzhou Yellow, Fuhong, Ziyang, and Ziyu seeds; and scallion Yuanzang, Tianguang Yiben, Xiarentian, Changbao, Changyue, Chunwei, Jiwanchou Yiben, Jiabao, Jizang, and Tianbao seeds were all commercially available.

[0026] 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.

[0027] Instruments used: C1000 Touch Thermal cycler PCR instrument and GelDoc go gel imaging analysis system were purchased from BIO-RAD.

[0028] Example 1: Screening and application of SCAR markers in onions and scallions 1. Cultivation of onion and scallion seeds Seeds of various onion varieties were collected, including Onion Earth, Red Leaf No. 3, Dabao, Jinxing, Fuxing, Guijin, Quanzhou Yellow, Fuhong, Ziyang, Ziyu, and Scallion Yuanzang, Tianguang Yiben, Xiarentian, Changbao, Changyue, Chunwei, Jiwanchou Yiben, Jiabao, Jizang, and Tianbao. 100 plump, uniformly sized seeds from each variety were selected and surface-sterilized by soaking in 75% alcohol for 2 minutes. The seeds were then rinsed 2-3 times with pure water to remove the alcohol. Afterward, the seeds were soaked in warm water at 30-40℃ for 4 hours. Following soaking, the seeds were placed on a damp gauze in a 21℃ constant temperature incubator for germination. Germination occurred after 3 days, and small sprouts of 2-3 cm appeared after 6 days. Small sprouts and leaves were cut from the onion and scallion sprouts to obtain identification materials for the onion and scallion seeds. These were then numbered and kept for later use.

[0029] 2. Extraction and detection of total DNA from onions and scallions Total DNA was extracted from different varieties of onions and scallions using the Polysaccharide-Polyphenol Plant Genomic DNA Extraction Kit (DP360). Specific operating procedures are detailed in the kit's instruction manual. 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 , Figure 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.

[0030] 3. Primer design and synthesis The laboratory retrieved the complete sequence of the skp1-Ms gene (GenBank: KM065384.1) of onion (Allium spp.) from the National Center for Biotechnology Information database and designed primers. The sequences of the three primer sets are shown in Table 1.

[0031] Table 1 Primer Sequences

[0032] The primer sequences shown in Table 1 were synthesized by Sangon Biotech (Shanghai) Co., Ltd., purified by PAGE, and dissolved in sterile water to form a 100mM preservation solution. The working solution was diluted 10 times before use.

[0033] 4. PCR amplification 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 forward and reverse primers, 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, 50℃ annealing for 15 s, 72℃ extension for 20 s, 35 cycles, followed by a 72℃ extension for 5 min. PCR products were stored at 4℃.

[0034] 5. Detection and analysis of PCR products 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 images were taken and analyzed using a gel imaging system. Results are shown below. Figures 3 to 6 .

[0035] Depend on Figure 3 It can be seen that the amplification bands of scallion and onion seeds are almost identical under primer set A, indicating that primer set A cannot accurately distinguish between scallion and onion seeds. Figure 4 It can be seen that primer set B amplified more non-specific bands in scallion and onion seeds, indicating that primer set B could not accurately distinguish between scallion and onion seeds. Figures 5 to 6 As can be seen, primer set C yielded two clear bands for onion and one clear band for scallion, effectively distinguishing between 10 varieties of onion seeds and 10 varieties of scallion seeds. The amplified bands from primer set C were recovered via gel electrophoresis and TA cloning was performed. The bacterial culture was then sent to Sangon Biotech Co., Ltd. for sequencing, yielding stable SCAR molecular markers for onion and scallion, respectively. Sequencing revealed two target bands for onion, with lengths of 1388 bp and 760 bp, their nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; the target band for scallion was 760 bp, with its nucleotide sequence shown in SEQ ID NO: 3.

[0036] Therefore, primer set C was determined to be the specific identification primer for onion seeds and scallion seeds, and the identification criteria of the gel imaging system were clarified: if the PCR test result shows two bands of 1388 bp and 760 bp, the test result is an onion sample; if the PCR test result shows only one band of 760 bp, the test result is a scallion sample.

[0037] The results of the detection of different varieties of onion and scallion using primer group C are shown in Table 2. After multiple biological replication experiments on onion (10 varieties) and scallion (10 varieties) from a wide range of varieties, the results showed that different varieties of onion and scallion seeds could be stably distinguished.

[0038] Table 2. Detection results of different varieties of onions and scallions

[0039] Note: ++ indicates the simultaneous presence of two stripes, 1388bp and 760bp; + indicates the presence of a single 760bp stripe. Finally, it should be noted that the above description is merely 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 foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A SCAR marker for identifying the purity of onion and scallion seeds, characterized in that, The SCAR marker-specific fragments of onions are 1388 bp and 760 bp in length, and their nucleotide sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The SCAR marker-specific fragment of scallions is 760 bp in length, and its nucleotide sequence is shown in SEQ ID NO: 3, respectively.

2. The primer set for detecting the SCAR-labeled material as described in claim 1, characterized in that, The primer set includes a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO: 8, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:

9.

3. A reagent for identifying the purity of onion and scallion seeds, characterized in that, Includes the primer set as described in claim 2.

4. A reagent kit for identifying the purity of onion and scallion seeds, characterized in that, Includes the primer set as described in claim 2 or the reagent as described in claim 3.

5. The application of at least one of the following (a1) to (a4) in determining the purity of onion and scallion seeds: (a1) The SCAR mark as described in claim 1; (a2) The primer set as described in claim 2; (a3) The reagent according to claim 3; (a4) The kit according to claim 4.

6. A method for identifying the purity of onion and scallion seeds, characterized in that, This includes identifying onion and scallion seeds using any of the methods shown in (b1) to (b3) below: (b1) The primer set as described in claim 2; (b2) The reagent as described in claim 3; (b3) The kit according to claim 4.

7. The method according to claim 6, characterized in that, Includes the following steps: The primer set was used to perform PCR amplification on the sample to be tested, and the amplified fragments were identified based on their characteristics. The samples to be tested included onion and scallion seeds; The identification method is agarose gel electrophoresis or first-generation sequencing.

8. The method according to claim 7, characterized in that, The PCR amplification reaction system includes: DNA polymerase, reaction buffer, amplification primers, DNA from the sample to be tested, and sterile water.

9. The method according to claim 7, characterized in that, The PCR amplification reaction conditions were as follows: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 5 s, 50℃ annealing for 15 s, 72℃ extension for 20 s, 35 cycles, 72℃ extension for 5 min, and storage at 4℃.

10. The method according to claim 7, characterized in that, The identification method is agarose gel electrophoresis. If the electrophoretic detection of the sample shows an electrophoretic band of 1388bp and 760bp, the sample is an onion; if it shows a 760bp electrophoretic band, the sample is a scallion. Alternatively, the identification method is first-generation sequencing. If the sequence of the electrophoretic recovered fragment is as shown in SEQ ID NO: 1 and SEQ ID NO: 2, then the sample to be tested is an onion; if the sequence of the electrophoretic recovered fragment is as shown in SEQ ID NO: 3, then the sample to be tested is a scallion.

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