A rice indica-japonica type identification method based on indel in gene promoter region

Abstract

The application belongs to the technical field of biological type identification, and particularly relates to a rice indica-japonica type identification method based on a gene promoter region indel. OsARF13 The promoter region of the gene has a presence rate of more than 80% in indica rice, and almost no presence in japonica rice, so that the indica-japonica type of rice can be distinguished through the difference. Based on the technical advantages, the application further provides a primer pair for detecting the indel marker, a product and application thereof in identifying the indica-japonica type of rice. The whole process is not only accurate, but also only needs 3-4 hours, the cost of reagents and equipment used is extremely low, the cost of single detection can be controlled below 0.1 yuan, and the economic threshold of variety identification is greatly reduced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological type identification, and particularly relates to a rice indica-japonica type identification method based on a gene promoter region indel. BACKGROUND

[0002] As one of the most important food crops in the world, rice variety identification is of great significance to agricultural production, breeding and market supervision. In particular, through variety identification, the purity of rice seeds can be ensured, hybridization or adulteration can be prevented, and the excellent traits of the variety can be maintained, which is crucial for stabilizing rice yield and quality.

[0003] Rice variety identification technology has evolved from traditional morphological methods to molecular marker methods, including Cheng's index method and molecular marker methods. Cheng's index method has obvious shortcomings such as long detection period (requires several days or even weeks of field observation), susceptibility to environmental factors and operator experience, difficulty in distinguishing indica-japonica intermediate types or hybrid offspring, and the need for a large number of seeds for seed purity identification, which occupies land resources and manpower. SSR molecular marker method and SNP molecular marker method require high-throughput equipment and large amounts of data, which are not suitable for small-scale or rapid detection needs. Although inDel marker method has the advantages of simple operation, low detection cost and fast detection speed, there is no report on rice indica-japonica type identification using this method. SUMMARY

[0004] The purpose of the present application is to provide a rice indica-japonica type identification method based on a gene promoter region indel, which has the characteristics of simple operation, rapidness and accurate results.

[0005] The present application provides an indel marker for distinguishing rice indica-japonica types, which is located at position 5329530-35333147 of rice chromosome 4; the genome version of the rice is IRGSP-1.0; the nucleotide sequence of the indel marker is shown in SEQ ID NO. 2.

[0006] Preferably, the indel marker is located in the promoter region of the OsARF13 gene of rice;

[0007] The nucleotide sequence of the OsARF13 gene is shown in SEQ ID NO. 1.

[0008] The present application provides a primer pair for detecting the indel marker described in the above technical solution, which comprises OsARF13-indel-1-F and OsARF13-indel-1-R.

[0009] The nucleotide sequence of the OsARF13-indel-1-F is shown as SEQ ID NO. 3.

[0010] The nucleotide sequence of the OsARF13-indel-1-R is shown as SEQ ID NO. 4.

[0011] The application provides a product for detecting the indel marker in the above technical solution, and the product comprises the primer pair in the above technical solution.

[0012] Preferably, the product comprises a kit.

[0013] The application provides an application of the indel marker, the primer pair or the product in the above technical solution in identifying indica and japonica types of rice.

[0014] Preferably, the identification comprises auxiliary identification.

[0015] The application provides a method for identifying indica and japonica types of rice based on an indel in a promoter region of a gene, comprising the following steps:

[0016] Using the above technical solution, the primer is used for PCR amplification reaction with the genomic DNA of the rice to be detected as a template, and the PCR amplification product is detected.

[0017] When the PCR amplification product is a 1000-2000bp band, the rice to be detected is determined as indica type.

[0018] When the PCR amplification product is a 200-250bp band, the rice to be detected is determined as japonica type.

[0019] Beneficial effects:

[0020] The application provides an indel marker for distinguishing indica and japonica types of rice, and the indel marker is located at 5329530-35333147 of the 4th chromosome of rice. OsARF13 The indel marker is located in a promoter region of a gene, and the existence rate of the indel marker in indica rice is as high as 80% or more, while the indel marker is almost not contained in japonica rice.

[0021] Based on the aforementioned technical advantages, this invention also provides primer pairs for detecting the indel marker, the product, and its application in identifying indica and japonica rice varieties. By using the rice genomic DNA as a template, performing PCR amplification using the primers described above, and detecting the PCR product, and subsequently analyzing the PCR product, the indica and japonica rice varieties can be determined efficiently and accurately. The entire process can be completed within 3-4 hours, and the cost of the reagents and equipment used is extremely low, with the cost per test controlled below 0.1 yuan per grain, significantly reducing the economic barrier to variety identification. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Electrophoresis diagram of the product amplified with OsARF13-indel-1 provided by this invention;

[0024] Figure 2 Electrophoresis diagram of the product amplified with OsARF13-indel-2 provided by this invention;

[0025] Figure 3 Electrophoresis diagram of the product amplified with OsARF13-indel-3 provided by this invention;

[0026] Figure 4 Sequence alignment diagrams of different rice varieties provided by this invention;

[0027] Figure 5 This is a schematic diagram showing the location of the indel provided by the present invention;

[0028] Figure 6 The frequency of indel provided by this invention Figure 1 ;

[0029] Figure 7 The frequency of indel provided by this invention Figure 2 . Detailed Implementation

[0030] The InDel marker described in this invention is located in rice. OsARF13 The promoter region of the gene is specifically located at position 5329530-35333147 on chromosome 4 of rice; the genome version of the rice is IRGSP-1.0; OsARF13The nucleotide sequence of the gene (containing promoter) is shown in SEQ ID NO. 1, specifically: 5'- AATCGAAGGGCCAAAAAAC CTCCAATCCTCCATGCATGTTATGTGTTAAAACGAGACCAAATGCAGTCTCTCTTTAGTAAACGATCTGATCATGA ATTTCTCCATTGCACGAAGGGTTTTCTTACTTTTCCCAATGCTCAAGGTAAAATGCTAAATGGTGAAATTTAACTC CAAAGCTTGTTAATAAACCGTGTAGATCGACATCCTTCTGTCATCCCCGAATAGTACGGGCTGCTGGATTTACATA AGGTCTTGTGTAGTTCCTAATTTTTTTTCCGAAAAACGTCACATCGACTCTATGAATATATGTTTGAAGCATTAAA TAATGATAAAATAAAAAACTAATTGCACAGTTCGTATGGAAATCGCGAGACGAATCTTTTGAGCCTAATTAGTCCA TGATTAGCCATAAGTGCTATAGTAACCTACATGTGCTAATGACATATTAATTAGGCTTAATAAATTCGTCTCGCGG TTTCCAGGGTTACAAAATTAGTTTTTTCATTCGTGTCCGAAAACCCCTTCCGACATCCAGTCAAACGTCCGATATA ATGTGACACCCAAATTTTCTTTTTTTGAACTAAACAAGGCCTAAATATTCGCACAGTAAACACAGTGCATATATAT AACTACACTACTGGATGTCAACTTTGAACTGCACTGTCATACAAATGTTTTCCCTGCTTGTTCTTCAATTTGGCCT AATGAGGGTATACTCTCCAAATTAACCCTTCCCCACAAATTAACTATGAACTACATATTTATGGCATTGTATCACA AAATTATAGATTTAATATCAATTTTCTCATAAAACTACAGCTTTGTAACTTTAGAAAACAATAGTTATATATGGTC TGAACTCTAAAATATATAGTTATTATTTATAACTTTATGACTAAGTATGTAGTTTTGTGATAAATGTCCTACATCT ATAGCTTTGTGATAATTTTGGTGCAAAATCTTTAGTTTGATTGAATACAGATGAAGGCTGATTGGAACATAAATTT GTTCATACTATGAACAAATAGATTAAAATACAAATGAAGGATAGTAATTTAGAACACAGATTACATTTTATTGGAA GCGTGATGCGAATACTTCATGGCAATAATCTAGCCGATCAAGTTATTGCCAAAGATTACGCGTGATAAATAGAGAT GGGTACGTAGTAGACCAGATTTAGACAAGCCTAGTCCATTTATTTTATATTTGTCCAAGGCAAGTCTGGGCTTCAA TGAAGATACGAATTGGTGCTAGTTGTTACCATGCATAATAAGGGCATATACAAAGCTAGAGTCTAATATGAGCTCT CTATATTAATTAATATCACTAGAGACTATCCTTCTAGTAGAGACAATAAGATTTCTAATCCATTAATACACTAAAT ATTTTTTTATTATACTTTTTTCCTTTTCTCCACCCCTCATGCAACAAACTTTCCGTTAATAAACGCTAAGAGTTGA CTCTGAGCCGTTGTCATGCATAACAACGATGTTTCTCCTTCCTTCCCTCTCTTTCTTCCATGTCACCAAATTTACC TACATGACAACGAAGAGAGCCCGTTATTAGACACCATTGTACATGCCCTAATAGCCTCATAGCAAATGAGTCGAAG GCAACGTTTCTCAACCCAGGAACATTGTGCATGTCTTAGGAATACAGAACATAGAGATATTTTTGTCCTCGTGTAG AAATATTCGCAAGTAACTATTGAATGTTTCTCGACATGAGAACTTTGTGTTGTGGTGGTGAAAATGATCACCAAAT GCTGTATTGGCTTCATACGGAATGGGATTAATTAATTGTTTAGCTGGCATTCCGCAACAAGGTTTGCAATACAGTA ATAATGTGTGCAAAAGGTACTACTCCGTCCCAGAAAAAACGAATTCCTATCTACGAATCTAGACACACATATATCC AGATTCGTAGATAGGATTTGTCTTTTTTGAGACGGAGGGGTGAATTAATTATGAATTGTGGGCCAGATGTCATCCA TGAACGACATGTTATATCACGTATCTGTGCTAGTTGTGTACTCCTTCTGTCAAAAAAAAAAGCCAACCTAGGTGGG GATGGGACACTAACTAGGACAACGAATCTAGACATACATAATGAATCTGGACATGTATGTCTAGATTCGTTGTCCT ATGTGGTGCCCATCCCCATCTAGGTTGTTTTTTTTCAACTTTTTTGCTATTTTACATTGTAAATTATTTCTAAAGG TCACACTATTAAGGGGTTGTTTGGTTCAGAACTTCAATTTGCCATGTTTCACCTTAGACGTGCCACACTATCTTAA TCTTATGGCGGGCTCATCACCATAGTTATAACTTGTTACACTTTTTAGCACCTTGTTCTATGCTTTATCTGTCCCG AAATAAATAAAATCAGTACGGGATGTGACATACTTTCTATCTAGATTCACGATACTAGAATATTGTCATATCACGT ACTAGTTTTGTTTATTTTGGAAGATCGGGAGTACGTCATAGAGTGAATTGTTTGTCTAACTTAATCACAAGTGCAG CTTACTATAATTTAGTGGTCCTAAAAAGTGTGGCAAGTGAAATTTGTTTTAAACCGTATAATAAGATGGCTATGTG AAACTAATTTGCAGGTTTTCCTCCTATCTCAAAGGAGAATTCTTGGTCGTGCTTATCTCCTCGGAATAAAGAAAAG AAAAGAAAAGCAACGTGGGAGAATTGAGACGTGCGGGTTGTTGGCAACTGAATCGCGCCATTGCCCGTGTAAATTT GGTTGGTCTTTCTTGGCCGGCCCGGTAAAAAAATAATTCATTAAAGACAGACAGGGGAAATTAGAAAAGTGTGTAC AAATTTAAAGACAGGTCGCTTCCCTTGGCCTTAATTTGGGCACGGGGGAGACTAAATTCTCGGAGAAGGAAGGTGT GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACGGCGCGCCGCGTGCGACG gtactagtactttcttattgatattcatgcatttacttgcaattaatggaaaaaaaattcagaaatgcacttactaatcggtctctagttctcaatgccatatatactcaattatactatatgtatagttgtaaaagtactttcttattgatattcatgcatttacttgcaattaatggaaaaagaaattcagaaatgcacgtactaaccggtctctagttctcaatgccatatatactcaattatactatatgtatagttgtaaaagtactttcttattgatattcatgcatttacctgcaattaatggaaaaaattcagaaatgcacttactaaccggtctctagttctcaatgccatatatactcaattatactatatgtatagttgtctggttaccctttgttctctgcaattcgtgctgctcattatttgtcgatgaacaaaagccatattcaatagagtctcctgctctccttcattttttcctgactggattgtcattttctacatcaaccgatttagaagtaactatgttttttttgcagagatacgatgtacttcctccgtttcacaatataagtcattctagcatttcccacatttaaatatgaatgtggaaaatcctagaatgacttacattgtaaaacagagggagtaatatatttttatcagccacctgtttactgactgtttgtttactgttttctacag A GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG 3'. In SEQ ID NO. 1, the double underlined capital bold part represents the promoter sequence; the capital bold part represents the 5' UTR; the capital bold underlined part represents the 3' UTR; the capital part represents the exon sequence; and the lowercase part is the intron sequence.

[0031] The nucleotide sequence of the indel marker is shown as SEQ ID NO. 2, specifically 5'-CGTACTAGTTTTGTTTATTTTGGAAGATCGGGAGTACGTCATAGAGTGAATTGTTTGTCTAACTTAATCACA-3'.

[0032] In order to further illustrate the present application, the scheme provided by the present application is described in detail below in combination with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0033] The rice varieties used in the present application are shown in Table 1.

[0034] Table 1 Rice varieties used in the experiments of the present application

[0035]

[0036] The components of the TPS lysis solution are shown in Table 2:

[0037] Table 2 Components of the TPS lysis solution

[0038]

[0039] Preparation before experiment:

[0040] The promoter sequences of OsARF13 of three japonica rice (Nipponbare NIP-RAP, ZH11 and Kitaake) and three indica rice (9311, MH63 and ZS97) were found in the website respectively, and the website instructions are as follows:

[0041] Nipponbare NIP-RAP: https: / / rapdb.dna.affrc.go.jp / index.html. Version: IGRSP-1.0 2023-03-15; ZH11: http: / / ricerc.sicau.edu.cn / RiceRC / download / downloadBefore; Kitaake: Oryza sativa Kitaake v3.1 https: / / phytozome-next.jgi.doe.gov / ;

[0042] 9311: http: / / ricerc.sicau.edu.cn / RiceRC / download / downloadBefore; MH63: http: / / rice.hzau.edu.cn / cgi-bin / rice_rs3 / download_ext (http: / / rice.hzau.edu.cn / rice_rs3 / ); ZS97: http: / / rice.hzau.edu.cn / cgi-bin / rice_rs3 / download_ext.

[0043] The above sequences were aligned using the Alignment software, and the results are shown in GCGCGCCGCGTGCGACG .

[0044] Results GCGCGCCGCGTGCGACG It can be seen that there is an indel of the sequence shown in SEQ ID NO. 2 in indica rice.

[0045] The genomic DNA of 3000 rice varieties (707 japonica and 1615 indica) was aligned at https: / / cgm.sjtu.edu.cn / 3kricedb / index.php. That is, the position region of the promoter indel was determined by sequence alignment: that is, if there is an indel, the gene will show a vacancy. Based on this, 3000 rice varieties were aligned to determine the frequency of indels in indica and japonica rice, and the results are shown in GCGCGCCGCGTGCGACG , GCGCGCCGCGTGCGACG and GCGCGCCGCGTGCGACG .

[0046] In combination with GCGCGCCGCGTGCGACG , GCGCGCCGCGTGCGACG and GCGCGCCGCGTGCGACG , it can be seen that the sequence shown in SEQ ID NO. 2 is almost non-existent in japonica rice, but the existence rate is as high as more than 80% in indica rice.

[0047] Example 1

[0048] A rice indica-japonica type identification method based on the promoter region indel of a gene, the steps are:

[0049] (1) Extraction of rice genomic DNA (TPS method):

[0050] The rice samples in Table 1 were used as detection objects, and the specific method was as follows:

[0051] 1. Add sample and lysis: Take 2-3 pieces of 0.5 cm rice leaves into a 2 mL sterile centrifuge tube, add 700 μL of TPS lysis solution (or adjust according to the sample amount, generally 6-10 μL of TPS solution per mg of leaves), and put it into a grinder at 60HZ for 2 min to obtain a mixture;

[0052] 2. Water bath extraction: Place the centrifuge tube in a 65°C water bath for 30-60 minutes (invert and mix every 10 minutes for 3-4 times);

[0053] 3. Protein removal (optional, to improve purity): Add 600 μL of chloroform / isopentanol / ethanol mixture (volume ratio 400:20:80), mix thoroughly (invert 10-15 times), and centrifuge at 12000 rpm for 10 minutes at room temperature. Carefully pipette the supernatant (about 500 μL) into a new 1.5 mL centrifuge tube;

[0054] 4. DNA precipitation: Add an equal volume of isopropanol (about 500 μL) to the supernatant, mix well, and place it at -20°C for 10 minutes. Then, centrifuge at 12000 rpm for 10 minutes at room temperature, and carefully discard the supernatant;

[0055] 5. DNA washing: Add 500 μL of 70% ethanol, centrifuge at 12000 rpm for 5 minutes at room temperature, discard the supernatant, and repeat the washing once. Place the centrifuge tube on a clean bench to dry (about 5-10 minutes, avoid complete drying);

[0056] 6. DNA dissolution: Add 30-50 μL of sterile double distilled water (ddH2O) to dissolve the DNA;

[0057] 7. DNA preservation: The DNA solution can be used immediately for PCR or stored at -20°C. Before storage, check the DNA concentration and purity (OD260 / 280 should be between 1.8 and 2.0).

[0058] Different varieties of rice genomic DNA are obtained by the above method.

[0059] (2) Design primer set one: OsARF13-indel-1, primer parameters as shown in Table 3;

[0060] Table 3 Parameters of OsARF13-indel-1

[0061]

[0062] (3) PCR amplification

[0063] Using the different varieties of rice DNA group in step (1) as the template, and using the primer OsARF13-indel-1 in step (2), construct the PCR amplification system and perform amplification.

[0064] The PCR amplification system is shown in Table 4, and the PCR amplification program is shown in Table 5.

[0065] Table 4 PCR amplification system

[0066]

[0067] Table 5 PCR amplification program

[0068]

[0069] After the PCR reaction was completed, 5 μL of the product was taken and detected by 1% agarose gel electrophoresis. The results are as follows. GCGCGCCGCGTGCGACG As shown (in) GCGCGCCGCGTGCGACG In the table, the numerical designation corresponds to the serial number of the rice variety in Table 1.

[0070] Combination GCGCGCCGCGTGCGACG The results show that the OsARF13-indel-1 primer detected large bands for indica rice and small bands for non-indica rice, making them easy to distinguish.

[0071] Comparative Example 1

[0072] The only difference from Example 1 is that OsARF13-indel-1 in Example 1 is replaced by OsARF13-indel-2 in Comparative Example 1. The parameters of OsARF13-indel-2 are shown in Table 6.

[0073] Table 6 Parameters of OsARF13-indel-2

[0074]

[0075] After the PCR reaction was completed, 5 μL of the product was taken and detected by 1% agarose gel electrophoresis. The results are as follows. GCGCGCCGCGTGCGACG As shown (in) GCGCGCCGCGTGCGACG In the table, the numerical designation corresponds to the serial number of the rice variety in Table 1.

[0076] Combination GCGCGCCGCGTGCGACG The results showed that the PCR products of indica rice and japonica rice were not significantly different, and it was not possible to clearly distinguish between the different types.

[0077] Comparative Example 2

[0078] The only difference from Example 1 is that in Comparative Example 2, OsARF13-indel-3 is used instead of OsARF13-indel-1 in Example 1. The parameters of OsARF13-indel-3 are shown in Table 7.

[0079] Table 7 Parameters of OsARF13-indel-3

[0080]

[0081] After the PCR reaction was completed, 5 μL of the product was taken and detected by 1% agarose gel electrophoresis. The results are as follows. GCGCGCCGCGTGCGACG As shown (in) GCGCGCCGCGTGCGACG In the table, the numerical designation corresponds to the serial number of the rice variety in Table 1.

[0082] Combination GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTGCGACG GCGCGCCGCGTG The results showed that both indica and japonica rice had bands, and the PCR products were slightly different in size.

[0083] After amplifying the rice DNA in Table 1 using OsARF13-indel-1, OsARF13-indel-2, and OsARF13-indel-3 primers, it was found that the bands of indica and japonica rice detected by OsARF13-indel-1 primers were significantly different and easily distinguishable; the PCR products of indica and japonica rice detected by OsARF13-indel-2 primers showed low distinguishability; and OsARF13-indel-3 primers showed bands of both indica and japonica rice with some differences, but the bands were similar, requiring careful control of electrophoresis time. Therefore, based on the above results, OsARF13-indel-1 primer is the preferred primer for distinguishing between indica and japonica rice.

[0084] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for auxiliary identification of indica and japonica rice varieties, characterized in that, Includes the following steps: Using rice genomic DNA as a template, PCR amplification was performed using primer pairs, and the PCR amplification products were detected. The primer pair comprises: an F primer with a nucleotide sequence as shown in SEQ ID NO. 3 and an R primer with a nucleotide sequence as shown in SEQ ID NO. 4; When a PCR amplification product detects a band of 1000-2000bp, the rice being tested is determined to be of the indica type. When the PCR amplification product does not detect a 1000-2000bp band but detects a 200-250bp band, the rice to be tested is determined to be japonica type.

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