SSR (simple sequence repeat) marker construction method for DNA (deoxyribonucleic acid) fingerprint spectrum of Panjin rice

Abstract

The invention belongs to the technical field of rice variety identification and detection, and particularly relates to an SSR (simple sequence repeat) marker construction method for DNA (deoxyribonucleic acid) fingerprint spectrum of Panjin rice, which comprises the following steps: S1, extracting whole genome DNA in rice; s2, designing and synthesizing a specific primer; s3, carrying out PCR (Polymerase Chain Reaction) gene amplification; s4, amplifying a gene sequence through microchip electrophoresis detection; s5, synthesizing a DNA fingerprint spectrum database of the Panjin rice; s6, generating a barcode and a two-dimensional code of the disc-brocade rice variety; the method comprises the following steps: extracting rice genome DNA (deoxyribonucleic acid), designing and synthesizing specific primers, performing gene amplification by using a PCR (polymerase chain reaction) instrument, and performing gene sequence detection on amplified fragments by using a microchip electrophoresis instrument by using a Panjin rice DNA fingerprint database constructed on the basis of an SSR (simple sequence repeat) marking technology, so as to finally generate DNA fingerprints, bar codes and two-dimensional codes of each variety of Panjin rice; further, the adulteration phenomenon of the rice industry is reduced, and certain theoretical basis and technical support are provided for rice seed purity identification and rice variety protection.

Description

Technical Field

[0001] The invention belongs to the technical field of rice detection, in particular to a method for constructing an SSR marker of a DNA fingerprint of Panjin rice. Background Art

[0002] China is a country that mainly develops agriculture. The level of agricultural planting directly affects the economic level of the entire country. In today's society, my country's rice planting technology has developed rapidly and is at the forefront of the world. Rice is one of the most important food crops in my country and the main food source for about two-thirds of the world's population. The development of the rice planting 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 safety of agricultural biological resources. Rice planting is mainly distributed in three regions of our country: Northeast China, the Yangtze River Basin and the southeast coast. Among them, the output of indica rice in the south accounts for about 50%, japonica rice in the south accounts for 30%, and japonica rice in the north accounts for only about 20%. The growth period of rice in the north is longer, and the southern rice generally chooses varieties with a shorter growth period. The rice represented by the Northeast is better than the southern rice in taste and quality. The rice grains in the Northeast are full and round, and the rice is fragrant, sticky, oily and elastic when cooked. The rice grains in the south are slender and slightly less elastic. Due to the diversity of rice varieties, the quality of different varieties of rice varies greatly, and there is also a certain gap in price.

[0003] From the reality of Panjin, Panjin City, located on the coast of Bohai Sea and Liaohe River Delta, is a famous high-quality rice production base in my country. Panjin rice is not only complete in shape, white, transparent and shiny, but also has a strong aroma, soft and delicious taste, and is deeply loved by people. Panjin rice has been approved as a "national geographical indication product" by the General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China. It has been sold to more than 30 provinces and cities in China and exported to 15 countries and regions in five continents. The rice planting area in Panjin City is more than 1.7 million mu, and the output is nearly 1 million tons. Rice planting is an important part of Panjin's agriculture, and the income from rice planting accounts for a large proportion of farmers' income.

[0004] Due to the uniqueness of Panjin rice, in recent years, many merchants have been selling inferior products as good ones in order to make huge profits, resulting in a series of rice adulteration problems such as counterfeiting pure Panjin rice brands in the agricultural product market, non-Panjin rice varieties, rice grown in non-Panjin regions, and posting Panjin rice regional logos, making the entire Panjin rice market very chaotic, and rice farmers, rice processing companies and consumers have suffered certain economic losses. In the face of counterfeit rice in the rice market, rice processing companies and safety supervision departments are urged to effectively supervise and manage the adulteration phenomenon in the rice industry. It is very necessary to establish a set of fast, simple, practical and reliable Panjin rice variety identification methods. Therefore, a SSR marker construction method for Panjin rice DNA fingerprint is proposed to address the above problems. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, at least one technical problem raised in the background technology is solved.

[0006] The technical solution adopted by the present invention to solve the technical problem is: the SSR marker construction method of the Panjin rice DNA fingerprint map described in the present invention comprises the following steps: S1, extracting rice whole genome DNA; S2, design and synthesize specific primers; S3, PCR gene amplification; S4, microchip electrophoresis gene sequence detection; S5, synthetic Panjin rice DNA fingerprint library; S6. Generate barcode and QR code of Panjin rice varieties.

[0007] Preferably, the extraction of rice genomic DNA is to expand the rice, grind, sieve, and crush it into rice flour, directly crush the rice into rice flour, extract the genomic DNA in the rice by the CTAB method, and detect the effect of Panjin rice whole genome amplification by 1.0% agarose electrophoresis.

[0008] Preferably, the design and synthesis of specific primers utilizes Primer 5.0 software to design 48 pairs of specific amplification primers for SSR marker method, and the primer synthesis is completed by Shanghai Bioengineering.

[0009] Preferably, the PCR gene amplification utilizes 48 pairs of specific amplification primers to perform one-to-one amplification on 21 rice samples, and the parameters such as amplification temperature, number of cycles, and amplification system are designed.

[0010] 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 by the sequencer, compares and analyzes the numerical position corresponding to the standard sample with the position of the peak of each sample, and determines the size of the amplified fragment.

[0011] Preferably, the DNA fingerprint of Panjin rice is based on the results of rice primer amplification and microchip electrophoresis determination, and the characteristics of polymorphic fragments are recorded in the electrophoresis pattern of specific amplification of 48 pairs of primers, so as to establish a DNA fingerprint library of Panjin rice varieties.

[0012] Preferably, the barcode and QR code of Panjin rice varieties are generated by recording the number of polymorphic fragments by the number of amplified bands, encoding the DNA fingerprints of the 11 Panjin rice varieties, and then combining the basic information of the varieties to construct unique identity cards of the 11 main japonica rice varieties of Panjin rice, and using an online barcode generator and QR code generation software to express the constructed rice variety-specific identity information in the form of barcodes and QR codes.

[0013] The beneficial effects of the present invention are: 1. The present invention provides a method for constructing an SSR marker of a DNA fingerprint of Panjin rice, and constructs a DNA fingerprint library of Panjin rice based on SSR markers. The principle is a molecular marker technology based on specific primer PCR, also known as microsatellite DNA, which is a type of tandem repeat sequence of up to dozens of nucleotides composed of several nucleotides (generally 1 to 6) as repeating units. The sequences on both sides of each SSR are generally relatively conservative single copy sequences. The whole genome of rice contains a large number of repetitive sequences, which can be divided into tandem repeat sequences and interspersed repeat sequences according to the distribution form of the repetitive sequences in the genome. The morphological differences and trait characteristics between rice varieties are essentially caused by genetic differences. Therefore, using DNA molecular marker technology to identify the differences in genotypes between different varieties is one of the most efficient and accurate methods for identifying rice varieties. The DNA fingerprint of Panjin rice was constructed based on SSR markers. By extracting rice genomic DNA, designing and synthesizing specific primers, using a PCR instrument to amplify genes, and using a microchip electrophoresis instrument to detect the gene sequence of the amplified fragments, we finally generated a DNA fingerprint barcode for each variety of Panjin rice, thereby reducing adulteration in the rice industry.

[0014] 2. The present invention provides a method for constructing an SSR marker for a DNA fingerprint of Panjin rice. It is the first time in the world to construct a method for identifying Panjin rice varieties based on the SSR marker method. This method is used to establish a DNA fingerprint library of Panjin rice varieties, and to generate identity card QR codes and bar codes for the main varieties of Panjin rice. It is also the first time in the world to use microchip electrophoresis technology to identify different varieties of Panjin rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings: Figure 1 is a flow chart of the method of the present invention; Figure 2 It is a schematic diagram of the main rice varieties grown in Panjin area and non-Panjin rice samples of the present invention; Figure 3A schematic diagram of the basic information of primers for 48 pairs of SSR markers of the present invention; Figure 4 This is a schematic diagram of the microchip electrophoresis test results of the Panjin rice Red Beach No. 1 variety of the present invention; Figure 5 The microchip electrophoresis amplification diagrams of 11 kinds of Panjin rice of the present invention; Figure 6 The ID card barcode information diagram of the main rice varieties grown in Panjin area of ​​the present invention; DETAILED DESCRIPTION

[0016] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0017] See also Figure 1-6 As shown, a method for constructing an SSR marker of a Panjin rice DNA fingerprint comprises the following steps: S1, extracting rice whole genome DNA; S2, design and synthesize specific primers; S3, PCR gene amplification; S4, microchip electrophoresis gene sequence detection; S5, synthetic Panjin rice DNA fingerprint; S6. Generate barcode and QR code of Panjin rice varieties.

[0018] See also Figure 1 As shown, the extraction of rice genomic DNA is to expand the rice, grind, sieve, and crush it into rice powder, crush the rice into rice powder, use the CTAB method to extract the genomic DNA in the rice, and detect the effect of Panjin rice whole genome amplification by 1.0% agarose electrophoresis; the rice seeds were collected from the Liaoning Salt-Alkali Land Utilization Research Institute, Panjin Northern Agricultural Technology Development Co., Ltd. and Panjin Xingfudao Ecological Agriculture Technology Development Co., Ltd., and three of the three major rice varieties promoted by the Panjin Municipal Government were donated free of charge, namely Red Beach No. 1, Salt Jing 939, Salt Jing 219 rice varieties, and other 8 most commonly planted Panjin rice varieties. 10 varieties of non-Panjin rice were purchased through online shopping and supermarkets, totaling 21 rice samples, including 20 samples of japonica rice and 1 sample of indica rice; The rice was expanded, ground, sieved and crushed into rice powder. The genomic DNA in the rice was extracted by CTAB method. The genomic DNA of the rice was detected by 1.0% agarose gel. See also Figure 4 As shown in the figure, the design and synthesis of specific primers used 48 pairs of specific amplification primers designed by Primer 5.0 software for SSR marker method, and the primer synthesis was completed by Shanghai Bioengineering. The microchip electrophoresis of primer RM1 for Red Beach No. 1 rice sample and primer RM13 for Koshihikari rice are shown in the figure. Figure 4 As shown, Figure 4 The results showed that products were obtained by amplification at 75 bp and 106 bp for RM1 and at 50 bp, 68 bp and 122 bp for RM13.

[0019] See also Figure 1-5 As shown, the PCR gene amplification uses 48 pairs of specific amplification primers to amplify 21 rice samples one by one, and the parameters such as amplification temperature, number of cycles, and amplification system are designed. The PCR amplification PCR reaction system is: 10μmol primers 1.0 μL each, 50 ng / μL DNA template 1.0 μL, 1 U / μL 2*San Taq PCRMIX 10.0 μL, dd H2O 7.0 μL, and the total reaction volume is 20.0 μL. The 49 pairs of primers used for SSR markers were synthesized by Shanghai Biotechnology (Table 2). The reaction program is: 94 ℃ pre-denaturation for 5 min; each cycle of 94 ℃ pre-denaturation for 1 min, 58 ℃ annealing for 1 min, 72 ℃ extension for 1 min, 35 cycles; and finally 72 ℃ extension for 8 min.

[0020] See also Figure 1-6 As shown, the microchip electrophoresis gene sequence detection uses a microchip electrophoresis instrument from Shimadzu Corporation to detect the amplification effect, and performs data analysis on the raw data obtained by the sequencer, compares and analyzes the position of the molecular weight standard in each channel with the position of the peak of each sample, and determines the size of the amplified fragment.

[0021] See also Figure 5As shown, the DNA fingerprint of Panjin rice is based on the results of rice primer amplification and microchip electrophoresis measurement, and the characteristics of polymorphic fragments are recorded in the electrophoresis pattern of specific amplification of 48 pairs of primers, so as to establish a DNA fingerprint library of Panjin rice varieties. The experiment is based on the results of rice primer amplification and microchip electrophoresis measurement, and the characteristics of polymorphic fragments are recorded in the electrophoresis pattern of specific amplification of 48 pairs of primers. The DNA fingerprints of Panjin rice varieties are encoded, and then combined with the basic information of the varieties, the unique identity cards of 11 varieties of Panjin rice are constructed, and the online barcode generator and QR code generation software are used to express the constructed rice variety-specific identity information in the form of barcode and QR code. According to the results of 21 rice samples amplified by 48 pairs of primers and microchip electrophoresis measurement, the number of polymorphic fragments is recorded with the number of amplified bands, and the DNA fingerprint library of 11 Panjin rice varieties is constructed. For example, the fingerprint of Red Beach No. 1 Panjin rice counts the amplification products of 48 SSR markers in the variety and the fragment sizes directly read by microchip electrophoresis, as well as the number of read fragments.

[0022] See also Figure 6 As shown, the barcode and QR code of Panjin rice varieties are generated by recording the number of polymorphic fragments with the number of amplified bands, encoding the DNA fingerprints of 11 Panjin rice varieties, and then combining the basic information of the varieties to construct the unique identity cards of the 11 varieties of Panjin rice, and using an online barcode generator and QR code generation software to express the constructed rice variety-specific identity information in the form of barcodes and QR codes. Referring to the requirements of the national technical standards "GB / T18347-20011 Barcode" and "GB / T 184-2000 Quick Response Matrix Code", the online barcode and QR code generation software is used to generate unique barcodes and QR codes for the identity cards of the 11 varieties of Panjin rice. The barcode and QR code of the Red Beach No. 1 variety of Panjin rice are shown in the figure below. Figure 3 and Figure 4 As shown, use Code1A in the barcode generator to generate the ID card barcode of the Red Beach No. 1 variety, and finally use the QR code generation software to generate the corresponding ID card QR code.

[0023] The above shows and describes 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, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A method for constructing a SSR marker of a Panjin rice DNA fingerprint, characterized in that: A method for constructing an SSR marker of a Panjin rice DNA fingerprint comprises the following steps: S1, extract rice genomic DNA; S2, design and synthesize specific primers; S3, PCR gene amplification; S4, microchip electrophoresis gene sequence detection; S5, synthetic Panjin rice DNA fingerprint library; S6. Generate barcode and QR code of Panjin rice varieties.

2. The method for constructing a SSR marker of a Panjin rice DNA fingerprint according to claim 1, characterized in that: The method of extracting rice genomic DNA is to expand rice, grind, sieve and crush it into rice powder, extract genomic DNA from rice by CTAB method, and detect the effect of Panjin rice whole genome amplification by 1.0% agarose electrophoresis.

3. The method for constructing a SSR marker of a Panjin rice DNA fingerprint according to claim 1, characterized in that: The specific primers designed and synthesized utilized Primer 5.0 software to design 48 pairs of specific amplification primers for SSR marker method, and the primer synthesis was completed by Shanghai Bioengineering.

4. The method for constructing a SSR marker of a Panjin rice DNA fingerprint according to claim 1, characterized in that: The PCR gene amplification utilizes 48 pairs of specific amplification primers to perform one-to-one amplification on 21 rice samples, and designs parameters such as amplification temperature, cycle number, and amplification system.

5. The method for constructing a SSR marker of a Panjin rice DNA fingerprint according to claim 1, characterized in that: The microchip electrophoresis gene sequence detection uses a microchip electrophoresis instrument from Shimadzu Corporation to detect the amplification effect, and performs data analysis on the raw data obtained by the sequencer, compares and analyzes the position of the molecular weight standard in each channel with the position of each sample peak, and determines the size of the amplified fragment.

6. The method for constructing a SSR marker of a Panjin rice DNA fingerprint according to claim 1, characterized in that: The DNA fingerprint of Panjin rice is based on the results of rice primer amplification and microchip electrophoresis determination, and the characteristics of polymorphic fragments are recorded in the electrophoresis map of specific amplification of 48 pairs of primers, so as to establish a DNA fingerprint library of Panjin rice varieties.

7. The method for constructing a SSR marker of a Panjin rice DNA fingerprint according to claim 1, characterized in that: The barcode and QR code of Panjin rice varieties are generated by encoding the DNA fingerprints of 11 varieties of Panjin rice by recording the number of polymorphic fragments with the number of amplified bands, and then combining the basic information of the varieties to construct the unique identity cards of the 11 varieties of Panjin rice. The unique identity information of the 11 varieties of Panjin rice is expressed in the form of barcodes and QR codes using an online barcode generator and QR code generation software.

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