Construction method of disc jing rice DNA fingerprinting ssr marker
By constructing a DNA fingerprint of Panjin rice and using SSR labeling technology and microchip electrophoresis detection, barcodes and QR codes for rice varieties were generated, solving the problem of counterfeit genuine Panjin rice and achieving efficient and accurate variety identification and identity authentication.
Patent Information
- Application Number
- CN202410832646.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-26
- Publication Date
- 2026-01-06
- Estimated Expiration
- 2044-06-26
AI Technical Summary
Existing technologies cannot effectively solve the problem of identifying Panjin rice, resulting in a serious problem of counterfeit Panjin rice on the market.
By constructing a DNA fingerprint map of Panjin rice, using SSR marker technology, whole-genome DNA of rice was extracted, specific primers were designed, PCR gene amplification was performed, and microchip electrophoresis was used for detection to generate a DNA fingerprint map library of Panjin rice. Barcodes and QR codes for different varieties were also constructed to identify them.
This has enabled efficient and accurate identification of Panjin rice varieties, reduced the phenomenon of counterfeit genuine Panjin rice in the market, and ensured the safety and quality of the rice industry.
Smart Images
Figure CN119506401B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of rice detection technology, specifically a method for constructing SSR markers for DNA fingerprinting of Panjin rice. Background Technology
[0002] In today's society, my country's rice cultivation technology has developed rapidly, ranking among the world's leading levels. Rice is one of my country's most important food crops and a major food source for about two-thirds of the world's population. The development of the rice cultivation industry is not only related to my country's food security but also affects the income and quality of life of rice farmers, as well as the security of agricultural biological resources. Rice cultivation is mainly distributed in three regions of China: Northeast China, the Yangtze River Basin, and the Southeast Coast. Southern indica rice accounts for about 50% of the yield, southern japonica rice about 30%, and single-season japonica rice in the north only about 20%. Northern rice has a longer growing season, while southern rice generally uses varieties with shorter growing seasons. Rice from Northeast China, in particular, is considered to have better taste and quality than southern rice. Northeast rice grains are plump and round, fragrant, sticky, oily, and elastic when cooked, while southern rice grains are slender and less elastic. Due to the diversity of rice varieties, the quality of different varieties varies greatly, resulting in price differences.
[0003] Panjin, located on the coast of the Bohai Sea and in the Liaohe River Delta, is a renowned high-quality rice production base in my country. Panjin rice is not only known for its intact grains, pure white color, and glossy sheen, but also for its rich aroma, soft texture, and delicious taste, making it a favorite among consumers. Approved as a "National Geographical Indication Product" by the State Administration for Quality Supervision, Inspection and Quarantine, Panjin rice is sold to over 30 provinces and cities in China and exported to 15 countries and regions across five continents. Panjin has over 1.7 million mu (approximately 113,333 hectares) of rice cultivation area, producing nearly 1 million tons of rice. Rice cultivation is a crucial component of Panjin's agriculture, and income from rice farming constitutes a significant portion of farmers' income.
[0004] Due to the unique characteristics of Panjin rice, in recent years, the agricultural product market has seen a series of adulteration problems, including counterfeit Panjin rice brands, rice varieties grown outside of Panjin, and rice labeled with regional Panjin labels. Faced with counterfeit rice in the market, it is essential for rice processing enterprises and safety regulatory departments to effectively supervise and manage adulteration in the rice industry. Establishing a rapid, simple, practical, and reliable method for identifying Panjin rice varieties is crucial. Therefore, this paper proposes an SSR marker construction method for Panjin rice DNA fingerprinting to address the aforementioned issues. Summary of the Invention
[0005] In order to overcome the shortcomings of the prior art, at least one technical problem raised in the background art is solved.
[0006] The technical solution adopted by this invention to solve its technical problem is: a method for constructing SSR markers for DNA fingerprinting of Panjin rice, comprising the following steps:
[0007] S1. Extract whole genome DNA from rice;
[0008] S2. Design and synthesize specific primers;
[0009] S3, PCR gene amplification;
[0010] S4. Microchip electrophoresis gene sequence detection;
[0011] S5. Synthesize the DNA fingerprint library of Panjin rice;
[0012] S6. Generate barcodes and QR codes for Panjin rice varieties.
[0013] Preferably, the extraction of rice genomic DNA involves topdressing, grinding, sieving, and pulverizing rice into rice flour. The rice is directly pulverized into rice flour, and the genomic DNA in the rice is extracted using the CTAB method. The amplification effect of the whole genome of Panjin rice is detected by 1.0% agarose gel electrophoresis.
[0014] Preferably, the specific primers designed and synthesized utilize Primer 5.0 software to design 48 pairs of specific amplification primers for SSR labeling, and the primer synthesis is completed by Shanghai Sangon Biotech.
[0015] Preferably, the PCR gene amplification utilizes 48 pairs of specific amplification primers for one-to-one amplification of 21 rice samples, and parameters such as amplification temperature, cycle number, and amplification system are designed.
[0016] Preferably, the microchip electrophoresis gene sequence detection uses a microchip electrophoresis instrument from Shimadzu Corporation of Japan to detect the amplification effect, and performs data analysis on the raw data obtained from the sequencer, comparing the numerical positions corresponding to the standard samples with the positions of the peak values of each sample to determine the size of the amplified fragment.
[0017] Preferably, the DNA fingerprint of Panjin rice is determined by using the results of rice primer amplification and microchip electrophoresis, with the electrophoretic pattern of specific amplification of 48 pairs of primers recording the characteristics of polymorphic fragments, thereby establishing a DNA fingerprint library of Panjin rice varieties.
[0018] Preferably, the generation of Panjin rice variety barcodes and QR codes involves encoding the DNA fingerprints of 11 Panjin rice varieties by recording the number of polymorphic fragments based on the number of amplified bands, and then combining this with the basic information of the varieties to construct unique identity cards for the 11 main cultivated japonica rice varieties of Panjin rice. The unique identity information of the rice varieties is then expressed in the form of barcodes and QR codes using an online barcode generator and QR code generation software.
[0019] The beneficial effects of this invention are:
[0020] 1. This invention provides a method for constructing SSR markers for Panjin rice DNA fingerprinting. Based on SSR markers, a Panjin rice DNA fingerprint library is constructed. The principle is a molecular marker technology based on specific primer PCR, also known as microsatellite DNA. SSRs are tandem repeat sequences of up to tens of nucleotides, composed of repeating units of a few nucleotides (generally 1-6). The sequences flanking each SSR are generally relatively conserved single-copy sequences. The rice genome contains a large number of repetitive sequences, which can be classified into tandem repeats and scattered repeats based on their distribution within the genome. The morphological differences and phenotypic characteristics between rice varieties are essentially caused by genetic differences. Therefore, using DNA molecular marker technology to identify genotypic differences between different varieties is one of the most efficient and accurate methods for rice variety identification. By constructing a DNA fingerprint map of Panjin rice based on SSR markers, the process involves extracting rice genomic DNA, designing and synthesizing specific primers, performing gene amplification using a PCR instrument, detecting the gene sequence of the amplified fragments using a microchip electrophoresis instrument, and finally generating a DNA fingerprint barcode for each variety of Panjin rice, thereby reducing adulteration in the rice industry.
[0021] 2. This invention provides an SSR marker construction method for the DNA fingerprint spectrum of Panjin rice, which is the world's first method for identifying Panjin rice varieties based on the SSR marker method. This method was used to establish a DNA fingerprint spectrum library of Panjin rice varieties and generate QR codes and barcodes for the main varieties of Panjin rice. It is also the world's first use of microchip electrophoresis technology to identify different varieties of Panjin rice. Attached Figure Description
[0022] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:
[0023] Figure 1 This is a flowchart of the method of the present invention;
[0024] Figure 2 This is a schematic diagram of the main rice varieties cultivated in Panjin area and rice samples from outside Panjin, as presented in this invention.
[0025] Figure 3 This is a schematic diagram illustrating the basic information of the 48 pairs of SSR-labeled primers of the present invention;
[0026] Figure 4 This is a schematic diagram of the microchip electrophoresis detection results of Panjin Red Beach No. 1 rice variety according to the present invention;
[0027] Figure 5 This is a schematic diagram of the microchip electrophoresis detection results of Panjin rice Koshihikari variety according to the present invention;
[0028] Figure 6 This is a barcode information diagram of the main rice varieties cultivated in Panjin area according to the present invention; Detailed Implementation
[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0030] Please see Figure 1-6 As shown, a method for constructing SSR markers for the DNA fingerprint of Panjin rice includes the following steps:
[0031] S1. Extract whole genome DNA from rice;
[0032] S2. Design and synthesize specific primers;
[0033] S3, PCR gene amplification;
[0034] S4. Microchip electrophoresis gene sequence detection;
[0035] S5. Synthesized DNA fingerprint of Panjin rice;
[0036] S6. Generate barcodes and QR codes for Panjin rice varieties.
[0037] Please see Figure 1As shown, the extraction of rice genomic DNA involved topdressing, grinding, sieving, and pulverizing rice into rice flour. The genomic DNA was extracted using the CTAB method, and the amplification effect of the whole genome of Panjin rice was detected by 1.0% agarose gel electrophoresis. The rice seeds were collected free of charge from the Liaoning Provincial Institute of Saline-Alkali Land Utilization, Panjin Northern Agricultural Technology Development Co., Ltd., and Panjin Xingfudao Ecological Agriculture Technology Development Co., Ltd. These seeds included three of the three major rice varieties promoted by the Panjin Municipal Government: Honghaitan No. 1, Yanjing 939, and Yanjing 219, as well as eight other commonly grown Panjin rice varieties. Ten non-Panjin rice varieties, totaling 21 samples, were purchased online and from supermarkets, including 20 samples of japonica rice and 1 sample of indica rice.
[0038] Rice was topdressed, ground, sieved, and pulverized into rice flour. Genomic DNA was extracted from the rice using the CTAB method, and the rice genomic DNA was detected using a 1.0% agarose gel electrophoresis.
[0039] Please see Figure 3 As shown, the specific primers were designed and synthesized using Primer 5.0 software, which designs 48 pairs of specific amplification primers for SSR labeling. Primer synthesis was performed by Shanghai Sangon Biotech. Microchip electrophoresis images of primer RM1 (for Red Beach No. 1 rice sample) and primer RM13 (for Koshihikari rice sample) are shown below. Figure 4-5 As shown, Figure 4-5 The results showed that RM1 was amplified at 75bp and 106bp and RM13 was amplified at 50bp, 68bp and 122bp to obtain products.
[0040] Please see Figure 1-6 As shown, the PCR gene amplification utilized 48 pairs of specific amplification primers for one-to-one amplification of 21 rice samples. Parameters such as amplification temperature, cycle number, and amplification system were designed. The PCR reaction system consisted of: 1.0 μL of each 10 μmol primer, 1.0 μL of 50 ng / µL DNA template, 10.0 μL of 1 U / µL 2*SanTaqPCRMIX, and 7.0 μL of ddH2O, for a total reaction volume of 20.0 μL. The 49 pairs of primers used for SSR labeling were synthesized by Shanghai Sangon Biotech. The reaction program was: 94℃ pre-denaturation for 5 min; each cycle consisting of 94℃ pre-denaturation for 1 min, 58℃ annealing for 1 min, and 72℃ extension for 1 min, for 35 cycles; and a final extension at 72℃ for 8 min.
[0041] Please see Figure 1-6 As shown, the microchip electrophoresis gene sequence detection uses Shimadzu's microchip electrophoresis instrument to detect the amplification effect, and performs data analysis on the raw data obtained from the sequencer, comparing and analyzing the position of the molecular weight standard in each channel with the position of the peak value of each sample to determine the size of the amplified fragment.
[0042] Please see Figure 1-6 As shown, the DNA fingerprinting of Panjin rice is based on the results of rice primer amplification and microchip electrophoresis. The electrophoretic patterns obtained from the specific amplification of 48 primer pairs record the characteristics of polymorphic fragments, thereby establishing a DNA fingerprinting library for Panjin rice varieties. The experiment, based on the results of rice primer amplification and microchip electrophoresis, recorded the characteristics of polymorphic fragments from the specific amplification of 48 primer pairs, encoding the DNA fingerprints of Panjin rice varieties. Combined with the basic information of the varieties, unique identification codes for 11 Panjin rice varieties were constructed. These unique identification information was expressed in the form of barcodes and QR codes using an online barcode generator and QR code generation software. Based on the results of amplification using 48 primer pairs and microchip electrophoresis of 21 rice samples, the number of polymorphic fragments was recorded by the number of amplified bands, thus constructing a DNA fingerprinting library for 11 Panjin rice varieties. For example, the fingerprint spectrum of Panjin rice No. 1 from Honghai Beach was used to analyze the amplification products of 48 SSR markers in the variety and the size and number of fragments read directly by microchip electrophoresis.
[0043] Please see Figure 6 As shown, the generation of barcodes and QR codes for Panjin rice varieties involves encoding the DNA fingerprints of 11 Panjin rice varieties by recording the number of polymorphic fragments based on the number of amplified bands. This, combined with basic variety information, constructs unique identification codes for each of the 11 Panjin rice varieties. Using an online barcode generator and QR code generation software, the constructed unique identity information of the rice varieties is expressed in barcode and QR code form. Following the requirements of the national technical standards GB / T18347-20011 Barcode and GB / T184-2000 Fast Response Matrix Code, the online barcode and QR code generation software generates unique barcodes and QR codes for each of the 11 Panjin rice varieties. The barcode and QR code for the Honghaitan No. 1 variety of Panjin rice are shown below. Figure 6 As shown, the barcode for the Red Beach No. 1 variety is generated using Code1A in the barcode generator. Finally, the corresponding QR code is generated using QR code generation software.
[0044] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.
Claims
1. A method for constructing a DNA fingerprinting SSR marker of Panjin rice, characterized by comprising the following steps: It comprises the following steps: S1, extracting the genomic DNA of the rice in Panjin; S2, designing and synthesizing 48 pairs of specific primers, as shown in the following table: ； S3, PCR gene amplification; S4, microchip electrophoresis gene sequence detection; S5, synthesizing the DNA fingerprint library of the rice in Panjin; S6, generating the bar code and two-dimensional code of the rice varieties in Panjin.
2. The method according to claim 1, wherein the method is characterized by: The extraction of the genomic DNA of the rice in Panjin is to grind the rice into rice powder, and then extract the genomic DNA in the rice by using the CTAB method. The whole genome amplification effect of the rice in Panjin is detected by 1.0% agarose electrophoresis.
3. The method according to claim 1, wherein the method is characterized by: The design and synthesis of the specific primers utilize 48 pairs of specific amplification primers for SSR marker method designed by Primer 5.0 software, and the primer synthesis is completed by Shanghai Generay Biotech Co., Ltd.
4. The method according to claim 1, wherein the method is characterized by: The PCR gene amplification utilizes 48 pairs of specific amplification primers for one-to-one amplification of 21 rice samples, and the amplification temperature, cycle number, and amplification system parameters are designed. The 21 rice samples are shown in the following table: 。 5. The method according to claim 1, wherein the method is characterized by: The microchip electrophoresis gene sequence detection utilizes the microchip electrophoresis instrument of Shimadzu Corporation to detect the amplification effect, and the raw data obtained by the sequencer are analyzed. The positions of the molecular weight markers in each channel are compared with the positions of the peak values of each sample for comparative analysis to determine the size of the amplified fragments.
6. The method according to claim 1, wherein the method is characterized by: The synthesis of the DNA fingerprint library of the rice in Panjin is based on the results of the primer amplification and microchip electrophoresis determination. The characteristics of the polymorphic fragments are recorded in the electrophoretogram of the specific amplification of 48 pairs of primers to establish the DNA fingerprint library of the rice varieties in Panjin.
7. The method according to claim 1, wherein the method is characterized by: The generation of the bar code and two-dimensional code of the rice varieties in Panjin is to record the number of polymorphic fragments by the number of amplified bands, encode the DNA fingerprints of 11 rice varieties in Panjin, and then combine the basic information of the varieties to construct the identity card of 11 rice varieties in Panjin. Online bar code generator and two-dimensional code generation software are used to express the identity information of 11 rice varieties in Panjin in the form of bar code and two-dimensional code. The 11 rice varieties in Panjin are shown in the following table: 。