Rice genetic intelligent seed breeding vector transformation event GATV3-2890-2 and its application

Abstract

The present invention relates to the field of bioengineering technology, and in particular to a rice genetic intelligent seed breeding vector transformation event GATV3-2890-2 and its application. The flanking sequences provided by the present invention include: a right flank sequence as shown in SEQ ID NO.1 and a left flank sequence as shown in SEQ ID NO.2. The present invention also provides specific primers for detecting the flanking sequences. The above-mentioned specific primers are used to perform PCR amplification on a DNA sample of the rice to be tested, which can specifically indicate whether the T-DNA fragment of pC0309-KhvMaauMCMK5400 is inserted at the 5628243-5628280 bases of the 3' end of the F-box protein Os02g0200900 of rice chromosome 2. The present invention successfully realizes the detection and safety management of transgenic rice and its derivative lines.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and specifically to the safety assessment and detection of transgenic rice. The present invention relates to a flanking sequence of an exogenous insertion fragment of a rice genetic intelligent breeding technology (GAT) transformation event, specific primers thereof, and applications thereof; and in particular to a rice genetic intelligent breeding vector transformation event GATV3-2890-2 and applications thereof. Background Art

[0002] Genetic Automation Technology (GAT) is a novel hybrid seed breeding technology that can successfully utilize recessive nuclear male sterile lines. The core concept of GAT is to utilize modern biotechnology to tightly link crop pollen fertility restoration genes, pollen abortion genes, herbicide sensitivity genes, and selection marker genes in a specific sequence and orientation on a GAT vector. These genes are then introduced into recessive nuclear male sterile lines through high-throughput gene transformation technology, resulting in a large number of transformation events.

[0003] Conducting biosafety assessment of genetically modified crops is an important part of the regulation of genetically modified products. It includes identifying the specific insertion site of the transformation event in the host genome, judging the insertion inactivation or deletion of the host genome based on the insertion site, and further speculating on the impact of the transformation event on the host and the possible safety issues it may cause.

[0004] In the process of obtaining transformation events, the integration position of the exogenous insert in the host plant genome is random, and the insertion site flanking sequence formed by splicing the left and right end sequences of each exogenous insert with the host genome sequence is unique. Therefore, the insertion site flanking sequence is a unique identifier to distinguish different transformation events and is an important technical data for establishing a specific detection method for transgenic plant lines. The specific detection of transformation events is highly specific and can accurately identify different transgenic crop lines. At present, the isolation of flanking sequences of exogenous inserts is mainly based on PCR technology. The established specific detection methods include reverse PCR, exogenous linker-mediated PCR, semi-random primer PCR, whole genome resequencing technology, etc. Among them, thermal asymmetric staggered PCR (Tail-PCR), high-efficiency thermal asymmetric PCR (hiTail-PCR) or chromosome walking (Genome Walking) in semi-random primer PCR are currently commonly used methods. Summary of the Invention

[0005] The purpose of the present invention is to provide the insertion site and flanking sequences of the rice GAT transformation event GATV3-2890-2, and corresponding detection primers.

[0006] The rice GAT transformation event GATV3-2890-2 was disclosed in a conference abstract (The 9th International Conference on Botany, December 8-10, 2023, Characterization of GAT transformation events offered a promising option to GMS application in rice, Xiongxia Jin). The "2890-2" mentioned in the abstract of the conference paper refers to this transformation event GATV3-2890-2.

[0007] Specifically, the purpose of the present invention is to provide a flanking sequence of an exogenous vector inserted fragment of the rice genetic intelligent breeding technology (GAT) transformation event GATV3-2890-2, and to provide a DNA sequence for specific detection of the flanking sequence, such as a PCR amplification primer sequence.

[0008] In a first aspect, the present invention provides flanking sequences of exogenous insertion vectors for transgenic rice transformation events, wherein the right flanking sequence of the flanking sequence is shown as SEQ ID NO.1; the left flanking sequence of the flanking sequence is shown as SEQ ID NO.2.

[0009] The right flank sequence is 512 bp in length. The 1st to 409th bases of the obtained sequence are located on chromosome 2 of the rice genome, and the 410th to 512th bases are completely identical to the partial sequence of the vector GATV3.

[0010] The left flank sequence is 1303bp long. By analyzing the left flank sequence, it was found that the obtained sequence from 1 to 356 is located in the rice genome sequence, which completely matches the published sequence on rice chromosome chr2 (AP014958.1 (5628280 to 5628635)); the 357 to 1303 positions are exactly the same as the partial sequence of the vector GATV3.

[0011] The above-mentioned right flank and left flank sequences are characteristic sequences of the rice GAT transformation event GATV3-2890-2, which can be used to distinguish the rice GAT transformation event GATV3-2890-2 from other transgenic / non-transgenic rice, as well as for qualitative detection and quantitative analysis of the rice GAT transformation event GATV3-2890-2.

[0012] The flanking sequences provided by the present invention were amplified using primer pairs shown in SEQ ID NOs. 11-12 and 9-10, respectively.

[0013] In a second aspect, the present invention provides primers for detecting the above-mentioned flanking sequences, wherein the nucleotide sequences of the primers are shown in SEQ ID NOs. 11-12 and the nucleotide sequences of the primers are shown in SEQ ID NOs. 9-10.

[0014] The use of the above-mentioned flanking sequences or primers in detecting or identifying transgenic rice and its derivative products; the transgenic rice is the rice GAT transformation event GATV3-2890-2.

[0015] Specifically, whether the T-DNA fragment of pC0309-KhvMaauMCMK5400 is inserted into the 5628243-5628280 bases at the 3' end of the F-box protein Os02g0200900 on chromosome 2 of the transgenic rice.

[0016] In a third aspect, the present invention provides a PCR detection reagent or kit, which includes: the above-mentioned primers.

[0017] The PCR detection reagent or kit provided by the present invention also includes: water, Taq DNA polymerase, dNTPs, PCR buffer, positive control substance and negative control substance.

[0018] In a fourth aspect, the present invention provides a method for detecting transgenic rice GATV3-2890-2, which detects whether the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 are simultaneously present in the DNA of a rice sample.

[0019] In the method provided by the present invention, the above-mentioned primers or the above-mentioned PCR detection reagent or kit are used to perform PCR amplification with the DNA of the sample to be tested as a template.

[0020] In the method provided by the present invention, based on the PCR amplification product, it is determined whether the T-DNA fragment of pC0309-KhvMaauMCMK5400 is inserted into the 5628243-5628280 bases at the 3' end of the F-box protein Os02g0200900 of chromosome 2 of the test sample;

[0021] If the primer pair shown in SEQ ID NO. 11-12 amplifies a target fragment of 512 bp, and the primer pair shown in SEQ ID NO. 9-10 amplifies a target fragment of 1303 bp, it means that the sample contains components derived from GATV3-2890-2.

[0022] pC0309-KhvMaauMCMK5400, referred to as GATV3 vector, has been disclosed in Chinese patent application number 202010379287.9.

[0023] In the method provided by the present invention, the PCR amplification program is: 93-95°C for 1-2.5 min; 93-95°C for 20-40 s; 50-60°C for 20-40 s; 70-73°C for 1-1.5 min; 70-73°C for 5-6 min; 23-27°C for 1.5-2.5 min, for 30-35 cycles.

[0024] The present invention is based on the genetically stable and agronomically excellent rice GAT transformation event GATV3-2890-2. The present invention clarifies the molecular characteristics of the rice GAT transformation event GATV3-2890-2 and advances the biosafety assessment of GATV3-2890-2. The present invention uses DNA from T0 generation plants of GATV3-2890-2 as a template, utilizes hiTail-PCR to isolate its T-DNA flanking sequences, and designs detection primers based on the left and right end sequences and left and right flanking sequences of the T-DNA. A method for specifically detecting the rice GAT transformation event GATV3-2890-2 is established, and the specificity and sensitivity of the method are simultaneously tested, providing a technical basis for the detection and identification of the rice GAT transformation event GATV3-2890-2 and its derivatives.

[0025] The beneficial effects of the present invention are:

[0026] (1) This invention discloses for the first time the flanking sequences of the insertion site of the exogenous gene of the rice genetic intelligent breeding technology (GAT) transformation event GATV3-2890-2 in the rice genome;

[0027] (2) This invention confirms for the first time the origin of the different bases in the flanking sequences of the insertion site of the exogenous gene in the rice genetic intelligent breeding technology (GAT) transformation event GATV3-2890-2 in the rice genome, and determines the junction site sequence where the exogenous vector is inserted into the rice genome sequence;

[0028] (3) Using the flanking sequences provided by the present invention, a specific qualitative detection method for the rice genetic intelligent breeding technology (GAT) transformation event GATV3-2890-2 was established for the first time;

[0029] (4) The present invention is applicable to the detection, monitoring and safety management of the rice genetic intelligent breeding technology (GAT) transformation event GATV3-2890-2 generation and its derivative lines. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0031] Figure 1 This is the phenotype diagram of the rice GAT transformation event GATV3-2890-2 sprayed with 5x imazethapyr in Example 1 of the present invention; wherein 0d is before spraying, 14d is 14 days after spraying, WT is the wild type, CK+ is the positive control (imazethapyr-resistant plant), and 2890-2 is GATV3-2890-2.

[0032] Figure 2 This is a phenotypic diagram of the rice GAT transformation event GATV3-2890-2 sprayed with 3 g / L bentazon in Example 1 of the present invention; wherein 0d is before spraying, 7d is 7 days after spraying, 14d is 14 days after spraying, WT is the wild type, CK+ is the positive control (benzon-sensitive mutant), and 2890-2 is GATV3-2890-2.

[0033] Figure 3 This is a pollen fertility and seed fluorescence diagram of the rice GAT transformation event GATV3-2890-2 in Example 1 of the present invention; wherein ZH11 is Zhonghua 11, 2890-2 (T0) is the T0 generation of GATV3-2890-2, and 2890-2 (T1) is the T1 generation of GATV3-2890-2.

[0034] Figure 4 This is a hiTail-PCRIII electrophoresis diagram of the right border flanking sequence of the rice GAT transformation event GATV3-2890-2 in Example 2 of the present invention; wherein M is a marker; ddH2O is double-distilled water; ZH11 is the non-transgenic japonica rice Zhonghua 11; 9311 is the non-transgenic indica rice 9311; P is the GATV3 vector plasmid; and 2890-2 is the rice GAT transformation event GATV3-2890-2.

[0035] Figure 5 Schematic diagram of the integration site of the rice GAT transformation event GATV3-2890-2 in the rice genome in Example 3 of the present invention; wherein, the T-DNA of the rice GAT transformation event GATV3-2890-2 is inserted into the 3' end of the F-box protein Os02g0200900 on chromosome 2 of the rice genome at bases 5628243-5628280.

[0036] Figure 6This is a specific qualitative PCR amplification diagram of the rice GAT transformation event GATV3-2890-2 in Example 5 of the present invention; wherein: WT is a non-transgenic rice gene DNA template; 2890-2 is a genomic DNA template of the rice GAT transformation event GATV3-2890-2. DETAILED DESCRIPTION

[0037] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0038] Example 1 Obtaining Rice GAT Transformation Event GATV3-2890-2

[0039] In this example, based on the genetically intelligent breeding technology GAT, pollen fertility restorer genes, pollen abortion genes, herbicide-sensitive genes, and selection marker genes were tightly linked in a specific order and orientation on the vector pC0309-KhvMaauMCMK5400 (GAT) (referred to as the GATV3 vector in this invention). The specific sequence and construction method of the vector pC0309-KhvMaauMCMK5400 were described in reference to Chinese patent application number 202010379287.9, "A genetically intelligent seed breeding system for hybrid crop breeding and its application," and the homozygous recessive male sterility gene was successfully introduced into the rice plant Zhonghua 11 (ZH11). Oscyp704b2-3 GATV3-2890-2 is one of the transformation events, which has been disclosed in the conference abstract (The 9th International Conference on Botany, Dec. 8-10, 2023, Characterization of GAT transformation events offered a promising option to GMS application in rice, Xiongxia Jin), of which 2890-2 is the current transformation event.

[0040] Based on the various elements of the GAT vector, the transformation event GATV3-2890-2 was tested for herbicide phenotype, pollen fertility, and seed fluorescence. The results were as follows: (1) According to the positive / negative herbicide screening, the transformation event GATV3-2890-2 showed a high resistance phenotype when sprayed with 5x imidazolinone solution, indicating that the expression cassette of the maintainer screening element works efficiently and can be used for the purification of the GAT maintainer line. Figure 1 When sprayed with 3g / L bentazon solution, the phenotype was highly sensitive, indicating that the herbicide sensitive element expression cassette was highly efficient and sensitive and could be used for purification of GAT sterile lines. Figure 2 (2) Using potassium iodide staining, the fertility of the pollen of the transformation event GATV3-2890-2 was tested. The ratio of aborted pollen to fertile pollen was 1:1, indicating that the working efficiency of the restoration gene element and the pollen abortion gene element was high, which enabled the maintenance line to maintain a heterozygous state. Figure 3 (3) According to the results of pollen fertility identification, the self-fertilized seeds will also show a 1:1 separation when observed under a 560-595nm excitation light microscope, that is, 50% of the seeds contain the GAT vector and show deep red fluorescence; 50% of the seeds do not contain the GAT vector and have no fluorescence, indicating that the fluorescent protein expression can work normally and can be used for mechanical seed sorting, see Figure 3 The above functions are present in both T1 and T2, proving that they can be stably inherited from generation to generation.

[0041] In summary, the single-copy transformation event GATV3-2890-2, in which all elements function normally, represents an excellent initial maintainer line with genetic stability. Self-pollination of this maintainer line allows for the propagation of sterile and maintainer lines, which can be widely applied to hybrid rice, improving the efficiency of hybrid rice breeding.

[0042] Example 2 Amplification of the Right Border Flanking Sequence of Rice GAT Transformation Event GATV3-2890-2

[0043] (1) Extraction of rice genomic DNA using TPS method

[0044] ① Grinding: Take a 3-4 cm long young leaf (1-2 cm long old leaf) of the transgenic rice GAT transformation event GATV3-2890-2 obtained in Example 1, place it in a 2 ml centrifuge tube, add 800 μl of TPS extract, add steel balls, and grind for 120 seconds using a cell disruptor (grinding machine);

[0045] ②After grinding, place in a 75°C water bath and incubate for 30 min;

[0046] ③ Centrifuge at 13,000 rpm for 10 min and transfer the supernatant (about 500 µl) to another 1.5 ml centrifuge tube;

[0047] ④ Add two volumes of pre-cooled anhydrous ethanol or an equal volume of isopropanol, mix gently, and place in a -30°C refrigerator for 2-3 hours (or place in a 4°C refrigerator overnight; or place in a -80°C refrigerator for 1-2 hours) until the DNA precipitates;

[0048] ⑤ Centrifuge at 13,000 rpm for 5 minutes, discard the supernatant, and place the centrifuge tube upside down on the table to air dry;

[0049] ⑥ After air drying, dissolve the sample in 200µl of 1×TE solution or sterile ddH2O. Check its integrity by 1.0% agarose gel electrophoresis and determine the DNA concentration by a micro-UV spectrophotometer. Store in a 4°C refrigerator until ready for use.

[0050] (2) Isolation of T-DNA right flank sequence using hiTail-PCR

[0051] Referring to the high-efficiency thermal asymmetric PCR (hiTail-PCR) method of Liu et al. (2007), three specific primers (GATV3-RB-F1 to F3) were designed based on the right border (RB) sequence of the GATV3 (pC0309-KhvMaauMCMK5400) plasmid map. They were combined with the degenerate primers LAD1-1, LAD1-3, and AC1 to isolate the right flank sequence of the T-DNA. The specific primer sequences are shown in Table 1.

[0052]

[0053] N is (A / T / G / C), A or T or G or C; V is (G / C / A), V is G or C or A.

[0054] The hiTail-PCR technique was used to amplify the right flanking sequence of the exogenous vector insertion site in the rice GAT transformation event GATV3-2890-2 using a three-stage hiTail-PCR reaction. The first hiTail-PCR reaction used two long random primers (LAD1-1 and LAD1-3 mixed in equal proportions) in combination with the specific primer GATV3-RB-F1. Genomic DNA from the rice GAT transformation event GATV3-2890-2 was used as a template for PCR amplification. ddH2O, non-transgenic rice lines Zhonghua 11 and 9311, and the GATV3 plasmid served as controls. The product from the first stage PCR amplification reaction was diluted 40-fold and used as a template in the second stage Tail-PCR reaction using the primer combination AC1 / GATV3-RB-F2. The product from the second stage PCR amplification reaction was diluted 10-fold and used as a template in the third stage reaction using the primer combination AC1 / GATV3-RB-F3. The amplified products of the second and third stage reactions were separated by 1.0% agarose gel electrophoresis, and the specific band 211 bp was selected for sequencing (e.g. Figure 4). The PCR reaction system and procedure are shown in Tables 2 and 3.

[0055]

[0056]

[0057] Example 3 Integration Site of T-DNA of Rice GAT Transformation Event GATV3-2890-2 in the Rice Genome

[0058] The specific band PCR product amplified by hiTail-PCR was sequenced to obtain a 211bp right border fusion sequence, as shown in SEQ ID NO. 13. Analysis of the sequence using the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) and the GATV3 vector sequence revealed that positions 1 to 60 completely matched the vector right border sequence, and positions 61 to 211 were located in the rice genome sequence, completely matching the published sequence on rice chromosome chr2 (AP014958.1 (5628093 to 5628243). This indicates that the 3' end of the T-DNA of the rice GAT transformation event GATV3-2890-2 was inserted into the 3' end of the F-box protein (Os02g0200900) on chromosome 2 of the rice genome at base 5628243 (as shown in the figure). Figure 5 ).

[0059] Example 4 Amplification of the Left Border Flanking Sequence of Rice GAT Transformation Event GATV3-2890-2

[0060] According to the insertion site information obtained in Example 3, a forward primer LB-F2 was designed based on a partial sequence of the vector GATV3, the sequence of which is shown in SEQ ID NO. 9 (5'-GCAATGAATATGCTGCCATCC-3'). A reverse primer G2R1 (2890-2) was designed based on a published sequence on rice chromosome 2, the sequence of which is shown in SEQ ID NO. 10 (5'-ACCCAGACATTGGTCCTTGC-3'). This primer pair was used to amplify the left flank sequence of the rice GAT transformation event GATV3-2890-2. The amplified product was sequenced, and the resulting sequence was shown in SEQ ID NO. 2, which was 1303 bp in length. Analysis of SEQ ID NO. 2 revealed that the obtained sequence from positions 1 to 356 was located in the rice genome sequence and completely matched the published sequence on rice chromosome chr2 (AP014958.1 (5628280 to 5628635)); positions 357 to 1303 were identical to a partial sequence of the vector GATV3. This means that the 5' end of the T-DNA of the rice GAT transformation event GATV3-2890-2 was inserted into the rice chromosome 2 at base 5628280 (e.g., Figure 5 The T-DNA of the rice GAT transformation event GATV3-2890-2 was inserted into the rice chromosome 2 at bases 5628243-5628280, resulting in a 36-bp deletion on rice chromosome chr2 (AP014958.1 (5628244 to 5628279), the sequence of which is shown in SEQ ID NO. 14).

[0061] Example 5 Specific PCR Detection Method for Rice GAT Transformation Event GATV3-2890-2

[0062] According to Examples 3 and 4, the right and left flanking sequences of the rice GAT transformation event GATV3-2890-2 were obtained. Specific primers were designed on rice chromosome chr2 and the exogenous vector, respectively. The specific primer sequences are shown in Table 4. PCR amplification was performed using genomic DNA from the progeny of the rice GAT transformation event GATV3-2890-2 as a template and the wild type (WT) as a control. The specific PCR reaction system is shown in Table 5. The amplification program was: 94°C for 2 minutes; 94°C for 30 seconds; 55°C for 30 seconds; 72°C for 1 minute; 72°C for 5 minutes; 25°C for 2 minutes, for 30-35 cycles. PCR products were detected using 1.0% agarose gel (see Table 5). Figure 6 ).

[0063]

[0064]

[0065] The results show that:

[0066] (1) G2F1(2890-2) / G2R1(2890-2) combination: The rice GAT transformation event GATV3-2890-2 template and wild type (WT) can both amplify the 801bp target band (e.g. Figure 6 ) is consistent with expectations and indicates that the rice GAT transformation event GATV3-2890-2 is a heterozygous line;

[0067] (2) G2F1(2890-2) / RB-R2 combination: The rice GAT transformation event GATV3-2890-2 template can expand the right border of the insertion vector and the rice genome fusion sequence 512bp target band, the obtained sequence is shown in SEQ ID NO.1, while the wild type (WT) failed to expand the target band (as shown in Figure 6 ), which was consistent with the expected result, indicating that the rice GAT transformation event GATV3-2890-2 contained a GAT vector insert.

[0068] (3) LB-F2 / G2R1(2890-2) combination: The rice GAT transformation event GATV3-2890-2 template was able to amplify the 1303bp target band of the fusion sequence of the insertion vector and the rice genome at the left border, and the obtained sequence was shown in SEQ ID NO.2, while the wild type (WT) failed to amplify the target band (as shown in Figure 6 ), which was consistent with the expected result, indicating that the rice GAT transformation event GATV3-2890-2 contained the GAT vector insert;

[0069] The above results indicate that the primer combinations LB-F2 / G2R1(2890-2) and G2F1(2890-2) / RB-R2 can amplify the left and right boundary sequences of the rice GAT transformation event GATV3-2890-2 and its transgenic lines (derivatives), while no specific fusion bands could be amplified in non-transgenic or other transgenic rice varieties. These primers can be used to identify the rice GAT transformation event GATV3-2890-2 generation and its derivative sequences.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for detecting rice GAT transformation event GATV3-2890-2, characterized in that: Detecting whether the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 are simultaneously present in the rice sample DNA; The rice GAT transformation event GATV3-2890-2 is a T-DNA fragment of pC0309-KhvMaauMCMK5400 inserted into the 5628243-5628280 bases at the 3' end of the F-box protein Os02g0200900 on chromosome 2.

2. The method according to claim 1, characterized in that PCR amplification is performed using the primers or a PCR detection reagent or kit including the primers and the DNA of the sample to be tested as a template; the nucleotide sequences of the primers are shown in SEQ ID NOs. 11-12 and SEQ ID NOs. 9-10.

3. The method according to claim 2, characterized in that Based on the PCR amplification product, it was determined whether the T-DNA fragment of pC0309-KhvMaauMCMK5400 was inserted into the 5628243-5628280 bases at the 3' end of the chromosome 2 F-box protein Os02g0200900 of the test sample.

4. The method according to claim 2 or 3, characterized in that The PCR amplification program was as follows: 93-95°C for 1-2.5 min; 93-95°C for 20-40 s; 50-60°C for 20-40 s; 70-73°C for 1-1.5 min; 70-73°C for 5-6 min; 23-27°C for 1.5-2.5 min, for 30-35 cycles.

5. Use of flanking sequences or primers thereof in detecting or identifying products of rice GAT transformation event GATV3-2890-2; said detection or identification comprising: Detecting whether the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 are simultaneously present in the rice sample DNA; The rice GAT transformation event GATV3-2890-2 is a T-DNA fragment of pC0309-KhvMaauMCMK5400 inserted into the 5628243-5628280 bases at the 3' end of the F-box protein Os02g0200900 on chromosome 2; The right wing sequence of the flanking sequence is shown as SEQ ID NO.1; the left wing sequence of the flanking sequence is shown as SEQ ID NO.2; The nucleotide sequences of the primers are shown in SEQ ID NOs. 11-12 and SEQ ID NOs. 9-10.

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