Primer group and method for detecting homozygote and heterozygote of transgenic maize LP026-2

Abstract

The invention belongs to the technical field of molecular biological detection, and particularly relates to a primer group and a method for detecting homozygote and heterozygote of transgenic maize LP026-2. The homozygote and heterozygote of the transgenic maize LP026-2 are distinguished by adopting a KASP (Competitive Allele Polymerase Chain Reaction) technology. According to the reaction, two possible genotypes of a detection sample in each reaction hole are subjected to two-color fluorescence reaction to reflect whether the genotype of the transgenic maize LP026-2 is a wild type, a homozygote or a heterozygote. The method has the advantages that high-throughput and high-sensitivity genotyping is realized, the method is suitable for LP026-2 derived varieties with different genetic backgrounds, the foreground selection requirements of large-scale samples in the backcross transformation process are met, and a technical template is provided for detection of other transgenic crops.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection technology, specifically relating to a primer set and method for detecting homozygous and heterozygous transgenic maize LP026-2. It employs competitive allele-specific PCR (KASP) technology to detect homozygous and heterozygous transgenic maize LP026-2, using dual-color fluorescence to detect the two possible genotypes in each well, achieving high-throughput and high-sensitivity genotyping. This method is applicable to LP026-2 derivative varieties with different genetic backgrounds, meets the needs of large-scale sample selection during backcrossing and breeding, and provides a technical template for the detection of other transgenic crops. Background Technology

[0002] Compared with traditional breeding techniques, transgenic breeding technology has significant advantages in terms of precision and efficiency. However, transgenic lines obtained directly through genetic transformation often cannot be directly applied to production due to unsatisfactory overall agronomic traits. Therefore, backcrossing is often used to introduce the target exogenous gene into a recipient parent with superior agronomic traits, thereby cultivating transgenic lines with production application value. In the backcrossing process of transgenic maize, prospective selection is required on a large number of samples to screen individuals carrying the target gene.

[0003] Traditional PCR methods (such as conventional PCR and quantitative real-time PCR) suffer from inherently low throughput, which is a major bottleneck because each round of testing relies on multi-well plates, limiting sample processing capacity. Furthermore, this process is time-consuming and cost-inefficient, involving tedious manual operations, low automation, and substantial reagent and time costs per test. Therefore, this relatively labor-intensive model is no longer sufficient to meet the high-throughput requirements of gene screening in terms of efficiency and scale, creating a demand for newer and more efficient genotype screening technologies.

[0004] Kompetitive allele specific PCR (KASP) is a fluorescence-based genotyping technique that uses dual-color fluorescence to detect two possible genotypes in each well of a sample. While offering advantages such as high throughput, low cost, and high automation, its application in transgenic detection is relatively limited, particularly lacking precise genotyping schemes for specific transformants (e.g., LP026-2). However, this technique requires the design of specific primer combinations to achieve target gene detection. Existing transgenic detection primers are mostly designed for single transformants, lacking versatility, and have not addressed the sensitivity reduction issue caused by competitive amplification in multiplex PCR. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention proposes a primer set and method for detecting homozygous and heterozygous transgenic maize LP026-2. The method employs competitive allele-specific PCR (KASP) to detect homozygous and heterozygous transgenic maize LP026-2, achieving high-throughput and high-sensitivity genotyping. It is applicable to LP026-2 derivative varieties with different genetic backgrounds, meets the needs for large-scale sample selection during backcrossing and breeding, and provides a technical template for the detection of other transgenic crops.

[0006] The technical solution adopted in this invention: In the first aspect, the primer set for detecting homozygotes and heterozygotes of transgenic maize LP026-2 includes a first forward primer, a second forward primer, and a universal reverse primer. The nucleotide sequence of the first forward primer is shown in SEQ ID NO.1, the nucleotide sequence of the second forward primer is shown in SEQ ID NO.2, and the nucleotide sequence of the universal reverse primer is shown in SEQ ID NO.3.

[0007] Secondly, the first forward primer SEQ ID NO.1 has a VIC fluorescent tag sequence 5'-GAAGGTCGGAGTCAACGGATTGCTCAGTATGAGGCCATGTTGC-3' added to its 5' end; the second forward primer SEQ ID NO.2 has a FAM fluorescent tag sequence 5'-GAAGGTGACCAAGTTCATGCTTGTCGTTTCCCGCCTTCAGT-3' added to its 5' end. Furthermore, a method for detecting homozygotes and heterozygotes of transgenic maize LP026-2 is provided, the steps of which include: (1) DNA extraction; (2) design and synthesis of specific primer sets; (3) preparation and amplification of KASP genotyping reaction system; (4) detection of fluorescence signal and genotyping in KASP reaction; (5) optional verification - conventional PCR and electrophoresis confirmation.

[0008] Preferably, in step (1), high-quality genomic DNA is extracted from the maize leaf or seed tissue to be tested.

[0009] Specifically, the primer design process in step (2) is as follows: based on the boundary sequence of the exogenous insertion fragment of transgenic maize LP026-2, a combination of transformant-specific primers is designed. The nucleotide sequence of the LP026-2 insertion site is shown in SEQ ID NO.7, and the sequence resulting from the T-DNA insertion is: CAGAGTGAT.

[0010] Preferably, the specific process of step (3) is as follows: the DNA template, primer set and KASP reaction solution are mixed in a certain proportion to construct the reaction system. The system includes 2×KASP Master Mix; primer working solution (Primer Mix); template DNA; sterile ultrapure water. The reaction conditions are: 94℃, hot start for 15 min; first step amplification reaction, denaturation at 94℃ for 20 seconds, annealing and extension at 61℃~55℃ for 60 seconds, 10 TouchDown cycles, with the annealing and extension temperature decreasing by 0.6℃ in each TouchDown cycle; second step amplification reaction, denaturation at 94℃ for 20 seconds, annealing and extension at 55℃ for 60 seconds, 26 cycles; optional step: fluorescence signal stabilization at 37℃ for 1 min.

[0011] Furthermore, the result determination process of step (4) is as follows: after the reaction is completed, the product is scanned for fluorescence and the results are analyzed.

[0012] In step (5), the primers used for optional verification-conventional PCR are: the nucleotide sequence of primer 128-genome-F1 at the 3' end of the genome insertion site is as described in SEQ ID NO.4; the nucleotide sequence of primer 128-genome-R1 at the 5' end of the genome insertion site is as described in SEQ ID NO.5; and the nucleotide sequence of primer TDNA-PAR-R at the 5' end of the T-DNA region is as described in SEQ ID NO.6.

[0013] PCR amplification was performed to validate the KASP genotyping results, especially for samples with ambiguous genotyping results. Electrophoresis was then performed after amplification. DNA molecular weight standards were used as a reference.

[0014] Compared with existing technologies, this invention has the following advantages: Firstly, it is the first to apply KASP technology to the detection of homozygous and heterozygous genotypes in transgenic maize LP026-2, overcoming the throughput limitations of traditional PCR. This method enables high-efficiency and low-cost detection of whether the genotype of transgenic maize LP026-2 is homozygous or heterozygous. Secondly, the specific design of the three primers achieves simultaneous detection of transformation events and heterozygous insertion site sequences through competitive amplification, improving genotyping accuracy. Furthermore, it is applicable to LP026-2 derivative varieties with different genetic backgrounds, providing a technical template for the detection of other transgenic crops. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of primer multiplex amplification fluorescence typing (distribution of heterozygous and homozygous signals).

[0016] Figure 2 The results of amplification and typing using ordinary primers alone (showing LP026-2 specificity and homozygous discrimination). Detailed Implementation

[0017] The technical solutions of the embodiments of this application will be further described clearly and completely below with reference to the accompanying drawings. It should be noted that the described embodiments are only some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0018] This embodiment details the specific technical solution implementation steps for the detection of LP026-2 transformants according to the present invention, including the standardized operation procedures of the primer set design module, sample detection module, and verification module.

[0019] Example 1: KASP genotyping confirmation for homozygous and heterozygous transgenic maize LP026-2 was based on the LP026-2 transformant-specific insertion boundary sequence, the nucleotide sequence of which is shown in SEQ ID NO.7 and its T-DNA segment structure. Primers were designed using Primer Premier 5.0 software. Priority considerations for primer design included primer specificity, annealing temperature (Tm value controlled between 58–62℃), GC content (40%–60%), amplicon length (80–200 bp), and avoiding primer dimers or hairpin structures.

[0020] After designing several candidate primer pairs, preliminary screening was conducted using single-primer amplification experiments. The 20 μL reaction system contained: 10 ng template DNA, 1× PCR Buffer, 0.2 mM dNTPs, 0.2 μM primers, and 1 U Taq DNA polymerase. The reaction program was: 95℃ pre-denaturation for 5 min; 35 cycles of 95℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s; and a final extension at 72℃ for 5 min. The amplified products were detected by 2% agarose gel electrophoresis. Primers with high amplification efficiency and single bands were selected, resulting in three specific primers: a first forward primer, a second forward primer, and a universal reverse primer. The nucleotide sequence of the first forward primer is shown in SEQ ID NO.1, the second forward primer in SEQ ID NO.2, and the universal reverse primer in SEQ ID NO.3.

[0021] The primer combination was further validated using a dual PCR amplification system. Two pairs of primers were added to the dual system simultaneously, and the reaction conditions were the same as above. After amplification, agarose gel electrophoresis was used for detection, and the bands were clearly genotyped and consistent with the expected results.

[0022] Example 2 describes a method for detecting homozygous and heterozygous transgenic maize LP026-2, using KASP technology for genotyping confirmation. The specific steps are as follows: 1. DNA Extraction: Genomic DNA was extracted from plant samples using the SDS method. The specific steps were as follows: 100 mg of plant tissue was ground in liquid nitrogen, and 500 μL of SDS extraction buffer (100 mM Tris-HCl pH 8.0, 50 mM EDTA pH 8.0, 500 mM NaCl, 1.25% SDS) was added. After mixing, the mixture was incubated at 65°C for 30 min. 150 μL of 5 M potassium acetate was added, and the mixture was incubated on ice for 15 min. The mixture was then centrifuged at 12000 rpm for 15 min at 4°C. The supernatant was collected, and an equal volume of isopropanol was added to precipitate the DNA. After washing with 70% ethanol, the DNA was dissolved in 50 μL of TEL buffer. The DNA concentration was determined using NanoDrop.

[0023] 2. Preparation of the amplification system: Prepare a mixed stock solution of the three primers (first forward primer LP026-1-KASP-CNVs-VIC, second forward primer LP026-1-KASP-CNVs-FAM, and universal reverse primer LP026-1-KASP-CNVs-C). The preparation method is as follows: mix the stock solutions (100 μM) of each primer in an equimolar ratio, and then dilute with sterile ultrapure water to the working concentration (usually the final working concentration of each primer is ~0.7 μM in the Primer Mix). The working solution should be aliquoted and stored at -20℃ protected from light. Aliquoting method: It is recommended to use an automated liquid handling workstation or a repeat pipette to aliquot 2.5 μL of the mixture of Master Mix and 0.014 μL of primer working solution into each well of a 384-well plate, and then add 1.4 μL of template DNA. Each plate should have at least 3 negative controls (NTC, water instead of template DNA) and 3 positive controls (one homozygous, one heterozygous, and one wild-type sample with known genotypes).

[0024] 3. PCR Amplification: The reaction mixture consisted of 25 μL of 1× PCR Buffer, 0.2 mM dNTPs, 0.4 μM forward and reverse primers, 1 U Taq DNA polymerase, and 20 ng template DNA. The reaction program was as follows: 95℃ pre-denaturation for 5 min; 35 cycles: 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s; and 72℃ final extension for 5 min. After amplification, electrophoresis was performed on a 1.5% agarose gel.

[0025] To verify the specificity of the detection system, 88 LP026-2 BC2 generation samples were selected for testing, and negative controls (non-GMO samples) and positive controls (LP026-2 standard) were set up.

[0026] Further, fluorescence signals were collected and analyzed after amplification, and the genotype of each sample was determined based on signal clustering. Immediately after the amplification process, the reaction plate was briefly centrifuged to collect the condensate at the bottom of the wells; the reaction plate was then placed in a fluorescence detector, and the FAM and VIC / HEX channels were excited separately to collect the fluorescence signal values ​​of each well; cluster analysis was automatically performed using the instrument's software (see attached). Figure 1 .

[0027] Optional validation for transgenic maize LP026-2 - routine PCR and electrophoresis confirmation.

[0028] Using the primer screening process of Example 1, three optional validation-conventional PCR primers were obtained: 128-genome-F1 (3' end primer for the genome insertion site), 128-genome-R1 (5' end primer for the genome insertion site), and TDNA-PAR-R (5' end boundary primer for the T-DNA region). The nucleotide sequence of the 128-genome-F1 primer for the genome insertion site is shown in SEQ ID NO.4; the nucleotide sequence of the 128-genome-R1 primer for the genome insertion site is shown in SEQ ID NO.5; and the nucleotide sequence of the TDNA-PAR-R primer for the T-DNA region is shown in SEQ ID NO.6.

[0029] The KASP genotyping results were validated, especially for samples with ambiguous genotyping results. The main reaction system and amplification procedure are shown in the table below.

[0030] PCR reaction system (20 μL)

[0031] PCR amplification procedure:

[0032] After amplification, electrophoresis was performed. 5 μL of PCR product was mixed with 1 μL of 6× Loading Buffer and electrophoresed on a 1.5% agarose gel (containing nucleic acid dye), using DNA molecular weight standards as a reference. The bands were observed and recorded using a UV gel imaging system. Figure 2 ).

[0033] Results analysis: The results of the verification detection system showed that the genotype determination results of KASP fluorescence detection and agarose gel electrophoresis were consistent, with a concordance rate of 100%, indicating that the designed primers and detection system have high specificity for LP026-2 transformants.

[0034] The results of KASP genotyping for transgenic maize LP026-2 are typically displayed as scatter plots, where the X-axis represents FAM signal intensity and the Y-axis represents VIC / HEX signal intensity. Specific interpretation criteria: Wild-type (WT) homozygotes: Data points cluster on the Y-axis (strong VIC / HEX signal, weak FAM signal). LP026-2 homozygotes: Data points cluster on the X-axis (strong FAM signal, weak VIC / HEX signal). Heterozygotes: Data points cluster on the diagonal between the X and Y axes (both FAM and VIC / HEX signals are strong). Invalid response / negative control: Data points cluster near the origin (both signals are weak), with some areas showing weaker FAM / HEX signals. Figure 1 The image shows a distinct green bright spot near the Y-axis and a distinct blue bright spot near the X-axis, along with the distribution of different genotypes in the 96-well plate. Optional validation for transgenic maize LP026-2 is achieved through routine PCR and electrophoresis: if only one 766 bp band (genomic primer amplification product) appears, the product is identified as wild-type (WT); if only one 475 bp band appears (transformant-specific primer amplification product), the product is identified as homozygous for LP026-2; if both 766 bp and 475 bp bands appear simultaneously, the product is identified as heterozygous, as shown in the attached image. Figure 2 The stripe is correctly positioned and clearly visible.

[0035] Although preferred embodiments of this application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this application.

[0036] Obviously, those skilled in the art can make various modifications and variations to this application without departing from the spirit and scope of this application. Therefore, if such modifications and variations fall within the scope of the claims of this application and their equivalents, this application also intends to include such modifications and variations.

Claims

1. A primer set for detecting homozygous and heterozygous transgenic maize LP026-2, characterized in that, The primer set includes a first forward primer, a second forward primer, and a universal reverse primer. The nucleotide sequence of the first forward primer is shown in SEQ ID NO.1, the nucleotide sequence of the second forward primer is shown in SEQ ID NO.2, and the nucleotide sequence of the universal reverse primer is shown in SEQ ID NO.

3.

2. The primer set according to claim 1, characterized in that, The first forward primer has a VIC fluorescent tag sequence 5'-GAAGGTCGGAGTCAACGGATTGCTCAGTATGAGGCCATGTTGC-3' added to its 5' end; the second forward primer has a FAM fluorescent tag sequence 5'-GAAGGTGACCAAGTTCATGCTTGTCGTTTCCCGCCTTCAGT-3' added to its 5' end.

3. A method for detecting homozygous and heterozygous transgenic maize LP026-2 using the primer set described in claim 1, characterized in that, The steps include: (1) DNA extraction; (2) design and synthesis of the primer set described in claim 1; (3) preparation and amplification of the KASP typing detection reaction system; (4) fluorescence signal detection and genotype interpretation in KASP technology; (5) optional verification - conventional PCR and electrophoresis confirmation.

4. The method according to claim 3, characterized in that, The specific process of step (3) is as follows: DNA template, primers and KASP reaction solution are mixed to construct a reaction system; the system includes 2×KASP Master Mix; Primer Mix working solution; template DNA; sterile ultrapure water; the reaction conditions are: 94℃, hot start for 15 min; the first step amplification reaction, denaturation at 94℃ for 20 seconds, annealing and extension at 61℃~55℃ for 60 seconds, 10 TouchDown cycles, the annealing and extension temperature is reduced by 0.6℃ in each TouchDown cycle; the second step amplification reaction, denaturation at 94℃ for 20 seconds, annealing and extension at 55℃ for 60 seconds, 26 cycles; optional step: fluorescence signal stabilization at 37℃ for 1 min.

5. The method according to claim 3, characterized in that, In step (5), the primers used for optional verification-conventional PCR are: 128-genome-F1, 128-genome-R1, and TDNA-PAR-R, which are primers for the 3' end of the genome insertion site and the 5' end of the T-DNA region. The nucleotide sequence of the 3' end primer 128-genome-F1 is shown in SEQ ID NO.

4. The nucleotide sequence of the 5' end primer 128-genome-R1 is shown in SEQ ID NO.

5. The nucleotide sequence of the 5' end primer TDNA-PAR-R is shown in SEQ ID NO.6.

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