A transgenic intelligent sterile line material and detection primer and identification method of endogenous and exogenous genes in its offspring

Abstract

This invention provides primers and a method for detecting and identifying endogenous and exogenous genes in transgenic intelligent sterile lines and their progeny, belonging to the field of plant biotechnology. The detection primers of this invention include one forward primer (SEQ ID NO.1) and two reverse primers (SEQ ID NO.2 and SEQ ID NO.3). Using the primers of this invention, endogenous and exogenous genes in transgenic materials and their self-crossed, hybridized, and backcrossed progeny, as well as the genotypes composed of various endogenous and exogenous genes, can be distinguished simply by conventional PCR and PAGE gel electrophoresis. This improves the selection efficiency for target genotypes during the propagation and breeding of transgenic materials in their current and future generations. The method provided by this invention is simple to operate, rapid in typing, and provides intuitive and accurate results, showing broad application prospects in the field of plant breeding technology.

Description

Technical Field

[0001] This invention relates to the field of plant molecular genetics and breeding technology, specifically to a molecular marker for identifying endogenous and exogenous GMS2 genotypes in transgenic intelligent sterile rice lines and their self-pollinated, hybridized, and backcrossed offspring, and its application. Background Technology

[0002] Rice is one of the world's most important food crops and a major food crop in my country. Male sterility refers to the phenomenon where the male reproductive organs cannot produce normal pollen, while the female reproductive organs are normally developed and can accept normal pollen for fertilization and seed production. Rice is a self-pollinating crop. Utilizing male sterility to force rice to accept foreign pollen can improve rice yield, quality, and resistance by leveraging hybrid vigor, and can also achieve large-scale hybrid seed production, which is of great significance in the hybrid rice industry. In 2013, Hainan Bolian Rice Gene Technology Co., Ltd. used cobalt-60 to irradiate the indica rice material 9311 and screened to obtain a recessive nuclear male sterile material, named gms2. This mutant trait is caused by the deletion of the AACAGCTAC bases at positions 118-126 in the coding region of the GMS2 gene, resulting in the deletion of asparagine, serine, and tyrosine at positions 40, 41, and 42 of the protein encoded by the LOC_Os04g48490 gene.

[0003] Genetic Automation Technology (GAT) is a novel hybrid seed breeding technology that successfully utilizes recessive nuclear male sterile lines. Its core idea is to use modern biotechnology to construct pollen fertility restoration genes, pollen abortion genes, herbicide sensitivity genes, and selection marker genes in a specific sequence and direction, tightly linked on a GAT vector. This vector is then introduced into recessive nuclear male sterile lines via high-throughput gene transformation technology, resulting in numerous transformation events. However, multi-gene transformation often encounters problems such as partial gene transfer, inability of various transgenic elements to function simultaneously, and transgene silencing. Therefore, multi-gene transformation frequently fails to select transformation events that achieve all desired traits. Through screening of each functional element, an initial maintainer line is obtained where all elements function normally, creating a maintainer line for the recessive nuclear male sterile line. This maintainer line is used for the production of GAT-sterile lines and hybrids, thus successfully maintaining and propagating the recessive nuclear male sterile line, ultimately enabling its commercial utilization. In the process of developing GAT hybrid rice technology using the above-mentioned gms2 mutants and corresponding GMS2 mutant genes, how to effectively distinguish between exogenous GMS2 transgenic genes (i.e. fertility restorer genes), endogenous wild-type GMS2 genes, and endogenous mutant GMS2 genes in transgenic plants and their self-pollinated, hybridized, and backcrossed offspring has become a problem that needs to be overcome.

[0004] Genetically modified organism (GMO) detection can be broadly categorized into two types: nucleic acid-level detection and protein-level detection based on genetically modified plants (Querci M, Van den Bulcke M (2010) New approaches in GMO detection. Anal Bioanal Chem 396(6):1991-2002). Among these, nucleic acid-level detection is widely used due to its convenient detection, mature system, wide detection range, and high throughput (Elenis D, Kalogianni D (2008) Advances in molecular techniques for the detection and quantification of genetically modified organisms. Anal Bioanal Chem 392(3):347-54). Nucleic acid-level detection methods can be further classified according to their specific applications as follows: 1. Traditional qualitative PCR detection technology; 2. Quantitative real-time PCR detection technology; 3. Multiplex PCR detection technology; 4. Nucleic acid hybridization detection technology; 5. Gene chip detection technology, etc.

[0005] To address the challenge of effectively distinguishing exogenous GMS2 transgenic genes, endogenous wild-type genes, and mutant GMS2 genes and their combinations in transgenic plants and their self-crosses, hybrids, and backcrosses, this invention introduces SNPs into the exogenous GMS2 transgenic gene and designs primers based on the mutation sites of the GMS2 gene in gms2. This allows for qualitative genotyping of endogenous and exogenous genes in transgenic materials and their offspring using traditional PCR. This identification method is simple to operate, low in cost, and has controllable throughput, making it particularly suitable for the research and production needs of small and micro-enterprises. Summary of the Invention

[0006] The first objective of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide a molecular marker and primer combination for identifying endogenous and exogenous GMS2 genes in transgenic rice and its self-pollinated, hybridized, and backcrossed offspring.

[0007] The second objective of this invention is to provide a method and its application for distinguishing endogenous and exogenous GMS2 genes in transgenic rice and its self-pollinated, hybridized, and backcrossed offspring using the above-mentioned marker and primer combinations and for identifying plant genotypes.

[0008] A third objective of this invention is to provide a method for predicting the pollen fertility of transgenic rice materials and their self-pollinated, hybridized, and backcrossed offspring using the aforementioned molecular markers.

[0009] The present invention provides a molecular marker for identifying endogenous and exogenous GMS2 genes in transgenic rice, which is obtained by primer amplification with nucleotide sequences as shown in SEQ ID NO.1-3.

[0010] This invention provides the application of the above-mentioned molecular markers in identifying endogenous and exogenous GMS2 genotypes in transgenic rice.

[0011] This invention provides an application for predicting the pollen fertility of transgenic rice materials and their self-pollinated, hybridized, and backcrossed offspring using the aforementioned molecular markers.

[0012] This invention provides the application of the aforementioned molecular markers, primer combinations, or kits in crop breeding or germplasm resource improvement.

[0013] The crops mentioned include, but are not limited to, rice, corn, soybeans, wheat, barley, millet, and sorghum.

[0014] The present invention provides a primer combination for detecting endogenous and exogenous GMS2 genotypes in transgenic rice and its self-pollinated, hybridized, and backcrossed offspring, containing primers with nucleotide sequences as shown in SEQ ID NO.1-3.

[0015] Kits containing the aforementioned primer combinations fall within the scope of protection of this invention.

[0016] The present invention includes forward primer F1: GAGCTCGGAGCCGTAGGA, forward primer F2: GTGAGCACGAGGCAGGTG, and reverse primer R: CCGGAGTACTCGCTGTAC, as shown in SEQ ID NO.1.

[0017] The primer combination described in this invention was designed and screened to target the deletion of the AACAGCTAC bases at positions 118-126 of the coding region of the wild-type GMS2 gene (whose sequence information is described in a Chinese patent application, patent application number: 202010491115.0), as well as the SNP polymorphism (C / G) at position 174 of the coding region with the exogenous GMS2 transgene (i.e., the fertility restoration gene), and the SNP polymorphism (AG / TC) at positions 304 and 305 with the exogenous gene. The two SNPs in the exogenous GMS2 transgene were artificially introduced and are synonymous mutations. Since wild-type varieties contain the endogenous gene corresponding to this transgene, this application introduces such SNPs for gene modification without altering the encoded amino acid sequence to distinguish between the endogenous genes in the plant genome and the introduced exogenous genes. The location of the introduced SNPs in this invention is ensured to be within 300 bp of the sequence difference site that causes functional changes (i.e., the gms2 mutation site), facilitating primer design and control of PCR product size.

[0018] Primer F1 can only pair with exogenous GMS2 transgenes with modified codons, primer F2 can only pair with wild-type GMS2 genes, and primer R can pair with both wild-type and mutant endogenous genes. Furthermore, an artificial mismatch mutation C→A is introduced at the fourth base of the 3' end of primer F1 to improve the relative recognition ability of F1 and R for the target sequence.

[0019] Furthermore, the present invention provides a method for detecting endogenous and exogenous GMS2 genes and genotypes in transgenic materials and their self-crossing, hybridization, and backcrossing offspring. First, genomic DNA is extracted from the sample to be tested, and PCR is performed using the forward primers shown in SEQ ID NO.1 and SEQ ID NO.2 and the reverse primer shown in SEQ ID NO.3. The size of the PCR amplification product is analyzed, and the genotype of the sample to be tested is determined based on the electrophoretic bands.

[0020] When the target band is only 92bp, it indicates that the tested material is wild-type, does not contain exogenous GMS2 transgene, and has a male fertile phenotype.

[0021] When the target band is only 83bp, it indicates that the material being tested is a mutant, does not contain exogenous GMS2 transgene, and has a male sterility phenotype.

[0022] When the target band is amplified with both 83bp and 92bp bands, it indicates that the tested material is heterozygous, does not contain exogenous GMS2 transgene, and has a male fertile phenotype.

[0023] When the target band is amplified with both 83bp and 222bp bands, it indicates that the tested material is a transgenic plant with an endogenous mutant GMS2 gene, containing exogenous GMS2 transgene, and can be used for subsequent hybridization and breeding.

[0024] When the target band is amplified with both 92bp and 222bp bands, it indicates that the tested material is a transgenic plant of wild-type GMS2 gene, containing exogenous GMS2 transgene, and cannot be used for subsequent hybridization and breeding.

[0025] When the target band is amplified simultaneously with bands of 83bp, 92bp, and 222bp, it indicates that the tested material is a transgenic plant with a heterozygous GMS2 gene, containing exogenous GMS2 transgene, and cannot be used for subsequent hybridization and breeding.

[0026] The reference sequence for the 83bp band is shown in SEQ ID NO.4, the reference sequence for the 92bp band is shown in SEQ ID NO.5, and the reference sequence for the 222bp band is shown in SEQ ID NO.6.

[0027] The transgenic material used in the embodiments of the present invention is transgenic rice. Those skilled in the art should understand that, due to the differences in rice varieties, the predicted PCR product sequence when designing primers can only be used as a reference sequence. The sequences of products amplified from different varieties may be completely identical to the reference sequence, or they may have some base differences from the reference sequence, but such differences usually do not affect the use of the marker.

[0028] Furthermore, the PCR reaction system was: Biomiga's 2×Bench Top TM 6 μL of Taq Master Mix, 0.5 μL each of two forward and one reverse primers (10 μM), 0.5 μL of 10% DMSO, 50 ng of template DNA, and add ddH2O to a final volume of 10 μL.

[0029] The PCR reaction conditions were: 94℃ for 5 min; 94℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s, for a total of 35 cycles; then 72℃ for 5 min, and finally 16℃ for 1 min.

[0030] In an embodiment of the present invention, the length of PCR amplification products is determined by PAGE gel electrophoresis. The PAGE gel electrophoresis conditions are: 6% PAGE gel, U = 2000V, I = 200mA, P = 85W, electrophoresis for 1 hour.

[0031] This invention also provides a method for predicting the pollen fertility of transgenic rice and its self-pollinated, hybridized, and backcrossed offspring using the aforementioned markers. The genotype of the GMS2 gene in transgenic plants is identified using the aforementioned method for detecting endogenous and exogenous GMS2 genes and genotypes in transgenic materials and their self-pollinated, hybridized, and backcrossed offspring. Transgenic plants carrying exogenous GMS2 genes and / or endogenous wild-type GMS2 genes have fertile pollen, while transgenic plants carrying only endogenous mutant GMS2 genes have sterile pollen. Specifically, the primers described in SEQ ID NO. 1-3 of this invention are used to amplify the test sample in the same PCR reaction system. If only an 83bp band appears in the amplification product, it indicates that the corresponding plant pollen is sterile; if a 92bp or 222bp band appears in the amplification band pattern, it indicates that the corresponding plant pollen is fertile.

[0032] Compared with existing technologies, this invention has the following advantages: This invention designs and develops a short, highly specific three-primer molecular marker. Using this marker, only a simple PCR reaction and PAGE gel electrophoresis are required. It can be used not only for identifying endogenous and exogenous GMS2 genotypes in transgenic materials and their progeny, but also for transferring endogenous and exogenous GMS2 genes from the aforementioned transgenic materials to other rice germplasms during breeding. Furthermore, it can be used to track and eliminate corresponding endogenous and exogenous genes in various rice germplasms. This identification method is simple to operate, has controllable throughput, provides accurate and intuitive experimental results, and requires inexpensive experimental equipment and primer synthesis. Attached Figure Description

[0033] Figure 1 This is the spectrum of the transition vector pC1300.

[0034] Figure 2 It is used to construct plant pollen abortion gene element expression cassettes, seed screening element expression cassettes, and plant male fertility restoration gene element final vector pC1300-C490MK map.

[0035] Figure 3 This is a schematic diagram of the primer design for the molecular markers of this invention. R is the reverse primer, which can match the gms2 mutant gene, wild-type gene, and exogenous transgene of GMS2, ​​respectively. F1 is the forward primer, which can only match the exogenous GMS2 transgene. F2 is also a forward primer, which can match both the mutant gene and the wild-type gene. The arrows indicate mismatched bases introduced into the primers. The AACAGCTAC mutation site and the SNP sites of the endogenous and exogenous transgenes are marked with boxes.

[0036] Figure 4 This is a PAGE gel electrophoresis image showing the genotype identification of T0 generation plants of the GMS2 homozygous mutant transformed with the exogenous GMS2 gene against the background of Zhonghua 11. Lane 1 is the negative control ddH2O, lane 2 is the control Zhonghua 11, lane 3 is the GMS2 homozygous mutant, lane 4 is the GMS2 exogenous gene plasmid, and lanes 5-7 are T0 generation single plants. The PCR product size markers are shown on the right side of the gel image.

[0037] Figure 5 These are pollen fertility identification images of T0 generation plants of the gms2 homozygous mutant transformed with the exogenous GMS2 gene in the Zhonghua 11 background. Among them, A is the iodine staining image of wild-type Zhonghua 11 pollen, B is the iodine staining image of gms2 mutant pollen, and C is the iodine staining image of gms2 mutant pollen transformed with plasmid of intelligent sterile line. Detailed Implementation

[0038] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0039] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the embodiments are all commercially available.

[0040] Example 1: Preparation of transgenic materials

[0041] The intelligent sterile line vector adopts a segmented construction strategy based on expression cassettes, followed by unit assembly. First, it is constructed in the transition vector pC1300 (… Figure 1 Plant pollen abortion gene element expression cassettes, seed selection element expression cassettes, and plant male fertility restoration gene element expression cassettes were constructed and verified by enzyme digestion and sequencing. The expression cassettes were then spliced ​​into the final vector to obtain the vector pC1300-C490MK. Figure 2 ).

[0042] A C-to-G synonymous mutation was introduced at position 174 of the coding region of the exogenous GMS2 transgene, which is the expression cassette of the male fertility restoration gene element. Synonymous mutations from AG to TC were introduced at positions 304 and 305. The introduction and deletion of these artificial mismatches facilitate the construction of vectors for the exogenous GMS2 transgene as a fertility restoration gene element in GAT hybridization breeding technology. Both SNPs are located within 300 bp of the AACAGCTAC base deletion mutation in the GMS2 gene (MSU Osa1Release 7Annotation, LOC_Os04g48490), which facilitates primer design and product detection (see [link to product description]). Figure 3 Using the gms2 (AACAGCTAC base deletion at positions 118-126 of the GMS2 gene) sterile mutant with a genetic background of Zhonghua 11 as the genetic transformation recipient material, the vector carrying the exogenous GMS2 transgene was transformed into rice callus tissue by Agrobacterium-mediated genetic transformation. Hygromycin resistance was used for screening, and positive transgenic plants were obtained after differentiation and rooting.

[0043] Example 2: PCR primers and method for identifying endogenous and exogenous genes in transgenic rice.

[0044] 1. Primer design

[0045] Based on the differences between the wild-type GMS2 (SEQ ID NO. 7) and the GMS2 mutant gene sequence in the 9311 genome sequence and the GMS2 transgenic sequence (i.e., the GMS2 described in Example 1 with C / G and AG / TC SNPs introduced at positions 174, 304, and 305 of the coding region, respectively, and its nucleotide sequence is shown in SEQ ID NO. 8), a combination of three primers was designed:

[0046] F1:GAGCTCGGAGCCGTAGGA(SEQ ID NO.1),

[0047] F2:GTGAGCACGAGGCAGGTG (SEQ ID NO.2),

[0048] R: CCGGAGTACTCGCTGTAC (SEQ ID NO. 3).

[0049] 2. Amplified Fragment Analysis

[0050] Using the above primer combinations to amplify rice materials, when the amplified target band is only 92 bp, it indicates that the tested material is wild-type, does not contain exogenous GMS2 transgene, and has a male fertile phenotype; when the amplified target band is only 83 bp, it indicates that the tested material is mutant, does not contain exogenous GMS2 transgene, and has a male sterile phenotype; when the amplified target band contains both 83 bp and 92 bp bands, it indicates that the tested material is heterozygous, does not contain exogenous GMS2 transgene, and has a male fertile phenotype; when the amplified target band contains both 83 bp and 222 bp bands... When the target band is 92 bp and 222 bp simultaneously, it indicates that the tested material is a transgenic plant with an endogenous mutant GMS2 gene, containing exogenous GMS2 transgene, and can be used for subsequent hybridization and conversion. When the target band is 83 bp, 92 bp, and 222 bp simultaneously, it indicates that the tested material is a transgenic plant with a heterozygous GMS2 gene, containing exogenous GMS2 transgene, and cannot be used for subsequent hybridization and conversion. The nucleotide sequences of the 83 bp, 92 bp, and 222 bp bands are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

[0051] Example 3: Identification of the genotype of self-crossed offspring of transgenic material using the molecular markers of the present invention.

[0052] 1. Experimental Materials

[0053] The T1 generation segregating population of the single-copy transgenic plant obtained in Example 1.

[0054] 2. Extraction of rice genomic DNA

[0055] Rice genomic DNA was extracted using the CTAB method. The specific steps are as follows: At the seedling stage, 3 cm long rice leaves were collected and ground in 800 μL of extraction buffer [1.5% (w / v) CTAB, 1.05 mol / L NaCl, 75 mmol / L Tris-HCl (pH 8.0), 15 mmol / L EDTA (pH 8.0)]. The mixture was collected in a 1.5 mL centrifuge tube. The tube was incubated at 65°C for 30 min, occasionally inverting to mix. 800 μL of chloroform:isoamyl alcohol (24:1 v / v) was added, and the mixture was inverted to mix for 15 min. The tube was centrifuged at 12000 rpm for 10 min at room temperature. 450 μL of the supernatant was aspirated and transferred to a new 1.5 mL centrifuge tube. Two volumes of 95% ethanol were added, and the mixture was mixed. The tube was then precipitated at -20°C for 30 min. The tube was centrifuged at 12000 rpm for 15 min. The 95% ethanol was discarded, and the precipitate was washed with 75% ethanol. Discard the 75% ethanol, dry the product, and then add 200 μL of sterile ddH2O to dissolve the DNA.

[0056] 3. PCR amplification and detection

[0057] The DNA of the material described in this example was amplified by PCR using the specific primer combination (F1, F2, R) obtained from Example 2. The PCR reaction system was: Biomiga 2×Bench Top TM 6 μL of Taq Master Mix, 0.5 μL each of one forward and two reverse primers (10 μM), 0.5 μL of 10% DMSO, 50 ng of template DNA, and add ddH2O to a final volume of 10 μL.

[0058] The PCR reaction conditions were: 94℃, 5 min; 94℃, 30 s, 60℃, 30 s, 72℃, 30 s, for a total of 35 cycles; 72℃, 5 min, 16℃, 1 min.

[0059] The amplified products were detected by 6% PAGE gel electrophoresis under the following conditions: U = 2000V, I = 200mA, P = 85W, for 1 hour. After electrophoresis, the gel was stained with 0.1% AgNO3 and observed and photographed under a viewing lamp.

[0060] 4. Pollen iodine staining

[0061] Anthers from mature spikelets were placed on a glass slide, broken, stained with iodine-potassium iodide solution (0.6% KI, 0.3% I2), and examined under a regular tabletop microscope.

[0062] 5. Results and Analysis

[0063] like Figure 4As shown, the four T0 generation plants in lanes 5-7 were classified into three genotypes. Lane 1, the negative control ddH2O, showed no amplification band. Lane 2, the wild-type control Zhonghua 11, amplified only a 92bp band. Lane 3, the homozygous GMS2 mutant, amplified only an 83bp band. Lane 4, the control GMS2 exogenous gene plasmid, amplified only a 222bp band. Lane 5 amplified both 83bp and 222bp bands, indicating that this plant carries both the GMS2 mutant gene and the exogenous transgene. Lane 6 amplified 83bp, 92bp, and 222bp bands, indicating that this plant carries the GMS2 mutant gene, the endogenous GMS2 gene, and the exogenous transgene. Lane 7 amplified only an 83bp band, indicating that this plant contains only the GMS2 mutant gene.

[0064] The control group, Zhonghua 11, can form normal fertile pollen. Figure 5 (A) The control gms2 mutant cannot form normal pollen ( Figure 5 (B), fertile pollen and sterile pollen from a single plant carrying both the gms2 mutant gene and the exogenous restorer gene segregated in a 1:1 ratio. Figure 5 (C).

[0065] Example 4: Transgenic GMS2 and sterile lines of the gms2 mutant gene were created using the molecular markers of this invention.

[0066] GMS2 is a rice GMS2 gene mutant with a deletion of the AACAGCTAC bases at positions 118-126 of the rice GMS2 gene, resulting in the deletion of asparagine, serine, and tyrosine at positions 40, 41, and 42 of the protein encoded by the LOC_Os04g48490 gene (Patent Application No.: 202010491115.0). The GMS2 mutant was crossed, backcrossed, and self-crossed with a fertile recipient. Molecular markers were used for selection based on the GMS2 gene and genetic background during this process, ultimately obtaining a recessive nuclear male-sterile line carrying the homozygous GMS2 gene in the target recipient background. The specific conversion steps are as follows, using H28B as an example:

[0067] 1. The F1 generation is obtained by crossing the recipient parent, such as H28B, as the male parent with gms2.

[0068] 2. Use F1 as the female parent and the recipient parent, such as H28B, to backcross and obtain BC1F1.

[0069] 3. Plant BC1F1 and use primers with sequences such as SEQ ID NO.1-3 to detect the GMS2 genotype. Select plants with the GMS2 heterozygous genotype, i.e., those that can simultaneously amplify 83bp and 222bp bands.

[0070] 4. Using a set of molecular markers (such as 100 or 200, etc.) that are polymorphic between the GMS2 mutant and the recurrent parent genome and are evenly distributed (can be, but not limited to, SSR, SNP, INDEL, EST, RFLP, AFLP, RAPD, SCAR, etc.), the genetic background of the single plants selected in step 3 is identified, and plants with high genotype similarity to the recurrent parent (such as greater than 88% similarity, or 2% selection rate, etc.) are selected.

[0071] 5. Use the plants selected in step 4 and the recipient parent, such as H28B, to backcross and obtain BC2F1.

[0072] 6. Plant BC2F1, repeat steps 3 and 4, select plants with heterozygous GMS2 genotype and high genetic background recovery rate (e.g., greater than 98%, or 2% selection rate), and harvest them from crossbred BC2F2.

[0073] 7. Plant BC2F2, repeat steps 3 and 4, and select the heterozygous GMS2 genotype plants with the highest genetic background homozygosity. Harvest these plants from the cross BC2F3. The homozygous GMS2 genotype plants that segregate from the BC2F3 progeny are the GMS2 recessive nuclear male sterile lines. BC2F3 is used to preserve the germplasm resources of the GMS2 recessive nuclear male sterile lines. The recessive nuclear male sterile lines are named with the letter G. For example, in this embodiment, the GMS2 homozygous recessive nuclear male sterile line H28B is named H28G.

[0074] The above example uses H28B as a breeding example, but it is not limited to H28B and can be any rice material.

[0075] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. sequence list <110> Hainan Bolian Rice Gene Technology Co., Ltd. <120> Primers and identification method for detecting endogenous and exogenous genes in transgenic intelligent sterile lines and their offspring. <130> KHP211123765.8 <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <400> 1 gagctcggag ccgtagga 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <400> 2 gtgagcacga ggcaggtg 18 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <400> 3 ccggagtact cgctgtac 18 <210> 4 <211> 83 <212> DNA <213> Artificial Sequence <400> 4 gtgagcacga ggcaggtgac cgtgctccgg ctgttgatct cgtcgcacac cttggtctgg 60 gagaggtaca gcgagtactc cgg 83 <210> 5 <211> 92 <212> DNA <213> Artificial Sequence <400> 5 gtgagcacga ggcaggtgac cgtgctccgg ctgttgatct cgtcgcacac cttggtctgg 60 gagaggtagc tgttgtacag cgagtactcc gg 92 <210> 6 <211> 222 <212> DNA <213> Artificial Sequence <400> 6 gagctcggag ccgtaggaga gggagtgcag cttcttggtg tcgtagtagt cgaggagggt 60 gaggaggcgg agcgcgttct tgatgtcggc gagggagagg ttggagacga gggaggacat 120 ggcgccgttg gtgagcacga ggcaggtcac cgtgctccgg ctgttgatct cgtcgcacac 180 cttggtctgg gagaggtagc tgttgtacag cgagtactcc gg 222 <210> 7 <211> 1296 <212> DNA <213> Artificial Sequence <400> 7 atggccgcca ccgaccgccg cctgctcttc ctcctggccg cctccctcgc cgtcgcggcg 60 gtgagctccc acaacatcac ggacatcctc gacggctacc cggagtactc gctgtacaac 120 agctacctct cccagaccaa ggtgtgcgac gagatcaaca gccggagcac ggtcacctgc 180 ctcgtgctca ccaacggcgc catgtcctcc ctcgtctcca acctctccct cgccgacatc 240 aagaacgcgc tccgcctcct caccctcctc gactactacg acaccaagaa gctgcactcc 300 ctcagcgacg gctccgagct caccaccacg ctgtaccaga ccaccggcga cgcctccggt 360 aacatgggcc acgtcaacat caccaacctg cgcggcggca aggttgggtt cgcctccgcc 420 gcgcccggct ccaagttcca ggccacctac accaagtccg tcaagcagga gccgtacaac 480 ctctccgttc ttgaggtctc cgaccccatc accttccccg gcctcttcga ctccccgtcg 540 gccgcgtcga ccaacctcac cgcgcttctt gagaaggccg ggtgcaagca gttcgcgcgg 600 ctcatcgtgt cgtccggggt gatgaagatg taccaggcgg ccatggacaa ggcgctgacg 660 ctgttcgcgc ccaacgacga cgcgttccag gccaagggcc tgccggatct gagcaagctg 720 accagcgccg agctggtgac gcttctgcag taccacgcct tgccgcagta cgcgcccaag 780 gcgtcgctca agaccatcaa gggccacatc cagaccctgg cctccaccgg agcgggtaag 840 tacgacctct ccgtcgtcac taagggcgac gacgtgtcca tggacaccgg catggacaag 900 tcccgcgtcg cgtccaccgt gctggacgac accccgacgg ttatccacac ggtggacagc 960 gtgctgctgc cgccagagct cttcggtggc gcaccttccc ccgcgccggc gcccggaccg 1020 gcaagcgatg tgccagccgc ttctcccgcg ccagaaggct cctcgccggc gccctccccc 1080 aaggcggcgg gcaagaagaa aaagaagggc aagtcgcctt cccattcccc acccgcgcct 1140 ccggccgaca cgcctgacat gtcgcccgcc gacgcgcccg cgggagaaga ggctgcagac 1200 aaagccgaga agaagaacgg cgccaccgcg gcggccacga gcgttgcggc cactgtggcc 1260 tccgccgccg ctctgctcgc cgcgtcgttc ttgtga 1296 <210> 8 <211> 1296 <212> DNA <213> Artificial Sequence <400> 8 atggccgcca ccgaccgccg cctgctcttc ctcctggccg cctccctcgc cgtcgcggcg 60 gtgagctccc acaacatcac ggacatcctc gacggctacc cggagtactc gctgtacaac 120 agctacctct cccagaccaa ggtgtgcgac gagatcaaca gccggagcac ggtgacctgc 180 ctcgtgctca ccaacggcgc catgtcctcc ctcgtctcca acctctccct cgccgacatc 240 aagaacgcgc tccgcctcct caccctcctc gactactacg acaccaagaa gctgcactcc 300 ctctccgacg gctccgagct caccaccacg ctgtaccaga ccaccggcga cgcctccggt 360 aacatgggcc acgtcaacat caccaacctg cgcggcggca aggttgggtt cgcctccgcc 420 gcgcccggct ccaagttcca ggccacctac accaagtccg tcaagcagga gccgtacaac 480 ctctccgttc ttgaggtctc cgaccccatc accttccccg gcctcttcga ctccccgtcg 540 gccgcgtcga ccaacctcac cgcgcttctt gagaaggccg ggtgcaagca gttcgcgcgg 600 ctcatcgtgt cgtccggggt gatgaagatg taccaggcgg ccatggacaa ggcgctgacg 660 ctgttcgcgc ccaacgacga cgcgttccag gccaagggcc tgccggatct gagcaagctg 720 accagcgccg agctggtgac gcttctccag taccacgcct tgccgcagta cgcgcccaag 780 gcgtcgctca agaccatcaa gggccacatc cagaccctgg cctccaccgg agcgggtaag 840 tacgacctct ccgtcgtcac taagggcgac gacgtgtcca tggacaccgg catggacaag 900 tcccgcgtcg cgtccaccgt gctggacgac accccgacgg ttatccacac ggtggacagc 960 gtgctgctgc cgccagagct cttcggtggc gcaccttccc ccgcgccggc gccgggaccg 1020 gcaagcgatg tgccagccgc ttctcccgcg ccagaaggct cctcgccggc gccctccccc 1080 aaggcggcgg gcaagaagaa aaagaagggc aagtcgcctt cccattcccc acccgcgcct 1140 ccggccgaca cgcctgacat gtcgcccgcc gacgcgcccg cgggagaaga ggctgccgac 1200 aaagccgaga agaagaacgg cgccaccgcg gcggccacga gcgttgcggc cactgtggcc 1260 tccgccgccg ctctgctcgc cgcgtcgttc ttgtga 1296

Claims

1. A molecular marker for identifying endogenous and exogenous GMS2 genes in transgenic intelligent sterile line rice materials and their selfed, hybridized, backcrossed progeny, characterized in that, obtained by amplification with primers having nucleotide sequences as shown in SEQ ID NO. 1-3.

2. A primer combination for detecting the genotype of endogenous and exogenous GMS2 genes in transgenic intelligent sterile line rice material and its self-cross, cross, backcross progeny, characterized in that, containing primers having nucleotide sequences as shown in SEQ ID NO. 1-3.

3. A kit containing the primer combination of claim 2.

4. Use of the molecular marker of claim 1 or the primer combination of claim 2 or the kit of claim 3 in rice breeding or germplasm improvement; the use is that after extracting genomic DNA from a rice sample to be tested, the primer combination as shown in SEQ ID NO. 1-3 is used for PCR amplification, the amplification product is electrophoresed, and the genotype of the transgenic rice material is determined according to the electrophoresis bands; when the amplified target band is only a 92bp band, it indicates that the detected rice material is wild type, does not contain exogenous GMS2 transgene, and has a male fertile phenotype; when the amplified target band is only an 83bp band, it indicates that the detected rice material is mutant type, does not contain exogenous GMS2 transgene, and has a male sterile phenotype; when the amplified target band is both an 83bp and a 92bp band, it indicates that the detected rice material is hybrid type, does not contain exogenous GMS2 transgene, and has a male fertile phenotype; when the amplified target band is both an 83bp and a 222bp band, it indicates that the detected rice material is a transgenic plant of endogenous mutant GMS2 gene, contains exogenous GMS2 transgene, and can be used for subsequent hybrid breeding; when the amplified target band is both a 92bp and a 222bp band, it indicates that the detected rice material is a transgenic plant of wild type GMS2 gene, contains exogenous GMS2 transgene, and cannot be used for subsequent hybrid breeding; when the amplified target band is both an 83bp, a 92bp and a 222bp band, it indicates that the detected rice material is a transgenic plant of hybrid type GMS2 gene, contains exogenous GMS2 transgene, and cannot be used for subsequent hybrid breeding.

5. Use of the molecular marker of claim 1 or the primer combination of claim 2 or the kit of claim 3 in identifying endogenous gene GMS2 and exogenous gene GMS2 and different genotype combinations of endogenous and exogenous genes in a transgenic intelligent sterile line rice and its self-cross, hybrid, backcross offspring; the use is that after extracting genomic DNA from a rice sample to be tested, the primer combination as shown in SEQ ID NO. 1-3 is used for PCR amplification, the amplification product is electrophoresed, and the genotype of the transgenic rice material is determined according to the electrophoresis bands; when the amplified target band is only a 92bp band, it indicates that the detected rice material is wild type, does not contain exogenous GMS2 transgene, and has a male fertile phenotype; when the amplified target band is only an 83bp band, it indicates that the detected rice material is mutant type, does not contain exogenous GMS2 transgene, and has a male sterile phenotype; when the amplified target band is both an 83bp and a 92bp band, it indicates that the detected rice material is hybrid type, does not contain exogenous GMS2 transgene, and has a male fertile phenotype; When the amplified target bands are 83bp and 222bp bands, it indicates that the detected rice material is a transgenic plant of endogenous mutant GMS2 gene, containing exogenous GMS2 transgene, which can be used for subsequent hybrid breeding; When the amplified target bands are 92bp and 222bp bands, it indicates that the detected rice material is a transgenic plant of wild type GMS2 gene, containing exogenous GMS2 transgene, which cannot be used for subsequent hybrid breeding; When the amplified target bands are 83bp, 92bp and 222bp bands, it indicates that the detected rice material is a transgenic plant of hybrid type GMS2 gene, containing exogenous GMS2 transgene, which cannot be used for subsequent hybrid breeding.

6. A method for detecting the genotype of the transgenic smartsterile line rice material GMS2, characterized in that, After extracting the genomic DNA of the to-be-tested rice sample, the primer combination shown in SEQ ID NO. 1-3 is used for PCR amplification, and the amplified product is electrophoresed to determine the genotype of the transgenic rice material according to the electrophoretic bands; When the amplified target band is only a 92bp band, it indicates that the detected rice material is wild type, does not contain exogenous GMS2 transgene, and has a male fertile phenotype; When the amplified target band is only an 83bp band, it indicates that the detected rice material is mutant type, does not contain exogenous GMS2 transgene, and has a male sterile phenotype; When the amplified target bands are 83bp and 92bp bands, it indicates that the detected rice material is hybrid type, does not contain exogenous GMS2 transgene, and has a male fertile phenotype; When the amplified target bands are 83bp and 222bp bands, it indicates that the detected rice material is a transgenic plant of endogenous mutant GMS2 gene, containing exogenous GMS2 transgene, which can be used for subsequent hybrid breeding; When the amplified target bands are 92bp and 222bp bands, it indicates that the detected rice material is a transgenic plant of wild type GMS2 gene, containing exogenous GMS2 transgene, which cannot be used for subsequent hybrid breeding; When the amplified target bands are 83bp, 92bp and 222bp bands, it indicates that the detected rice material is a transgenic plant of hybrid type GMS2 gene, containing exogenous GMS2 transgene, which cannot be used for subsequent hybrid breeding.

7. A method for identifying endogenous and exogenous GMS2 genes in a transgenic smart sterile line rice material, characterized in that, After extracting the genomic DNA of the to-be-tested rice sample, the primer combination shown in SEQ ID NO. 1-3 is used for PCR amplification, and the amplified product is electrophoresed to determine whether the GMS2 gene in the transgenic material is an endogenous or exogenous gene according to the electrophoretic band size; When the amplified target band is only a 92bp band, it indicates that the detected rice material is wild type, does not contain exogenous GMS2 transgene, and has a male fertile phenotype; When the amplified target band is only an 83bp band, it indicates that the detected rice material is mutant type, does not contain exogenous GMS2 transgene, and has a male sterile phenotype; When the amplified target bands are 83bp and 92bp bands, it indicates that the detected rice material is hybrid type, does not contain exogenous GMS2 transgene, and has a male fertile phenotype; When the amplified target band is 83bp and 222bp at the same time, it indicates that the detected rice material is a transgenic plant of endogenous mutant GMS2 gene, containing exogenous GMS2 transgene, which can be used for subsequent hybrid breeding; When the amplified target band is 92bp and 222bp at the same time, it indicates that the detected rice material is a transgenic plant of wild type GMS2 gene, containing exogenous GMS2 transgene, which cannot be used for subsequent hybrid breeding; When the amplified target band is 83bp, 92bp and 222bp at the same time, it indicates that the detected rice material is a transgenic plant of hybrid type GMS2 gene, containing exogenous GMS2 transgene, which cannot be used for subsequent hybrid breeding.

8. The method of claim 6 or 7, wherein, The PCR reaction system was: Biomiga's 2×BenchTop TM 5 μL of Taq Master Mix, 0.5 μL each of two 10 μM forward and one reverse primer, 0.5 μL of 10% DMSO, 50 ng of template DNA, and ddH2O to a final volume of 10 μL; PCR reaction conditions: 94℃ for 5 min; 94℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s, for a total of 35 cycles; followed by 72℃ for 5 min, and finally 16℃ for 1 min.

9. A method of predicting transgenic smart-storiless rice, characterized by, The genotype of GMS2 gene in transgenic rice is identified by the method of any one of claims 6-8. When the amplified target band is only 92bp, it indicates that the detected rice material is wild type, does not contain exogenous GMS2 transgene, and has a male fertile phenotype; When the amplified target band is only 83bp, it indicates that the detected rice material is mutant type, does not contain exogenous GMS2 transgene, and has a male sterile phenotype; When the amplified target band is 83bp and 92bp at the same time, it indicates that the detected rice material is hybrid type, does not contain exogenous GMS2 transgene, and has a male fertile phenotype; When the amplified target band is 83bp and 222bp at the same time, it indicates that the detected rice material is a transgenic plant of endogenous mutant GMS2 gene, containing exogenous GMS2 transgene, which can be used for subsequent hybrid breeding; When the amplified target band is 92bp and 222bp at the same time, it indicates that the detected rice material is a transgenic plant of wild type GMS2 gene, containing exogenous GMS2 transgene, which cannot be used for subsequent hybrid breeding; When the amplified target band is 83bp, 92bp and 222bp at the same time, it indicates that the detected rice material is a transgenic plant of hybrid type GMS2 gene, containing exogenous GMS2 transgene, which cannot be used for subsequent hybrid breeding.

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