CN118773363A - Primers and methods for detecting genetically modified soybeans based on KASP technology - Google Patents

Abstract

The invention provides a primer and a method for detecting transgenic soybean LT32, progeny and derivative strains thereof based on a KASP technology. The method can rapidly and efficiently detect the genotype of the soybean, identify the editing type, has high specificity, high sensitivity, lower cost, short detection period and good application prospect.

Description

Primers and methods for detecting genetically modified soybeans based on KASP technology

Technical Field

The invention relates to the field of molecular biology, and in particular to a primer and a method for detecting genetically modified soybeans based on the KASP technology.

Background Art

Soybean is one of the most important economic crops in the world. Transgenic soybean LT32 is a transgenic soybean developed by the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences that tolerates oxadiazine and glufosinate herbicides. Its agronomic traits are also excellent, and its yield is comparable to that of its non-transgenic control soybean. Therefore, it is urgent to develop an efficient and low-cost method to distinguish homozygous and heterozygous transgenic soybean LT32 and its derivative lines.

KASP (competitive allele-specific PCR) technology is based on the specific matching of primer terminal bases to type SNPs and detect InDels (Insertions and Deletions). The advantages of this technology are: first, as long as two universal fluorescent probes, two universal quenching probes, and multiple SNP PCR primers targeting specific sites are synthesized, many sites can be tested; second, because fluorescent probes and quenching probes are very expensive, the KASP method can replace the fluorescent probes targeting the site with universal fluorescent probes compared to the Taqman method, which greatly saves costs. The KASP method is gradually becoming the main technical means for molecular-assisted breeding, fine positioning of trait genes, and seed resource identification.

The invention provides a primer and a method for detecting transgenic soybean based on KASP technology, which can distinguish homozygotes and heterozygotes in transgenic soybean LT32 and its progeny and derivative lines, save costs, and increase the accuracy and efficiency of selection.

Summary of the invention

The present invention aims to provide a primer and method for detecting transgenic soybean genotypes quickly and easily based on KASP technology, which has the characteristics of high specificity and high sensitivity, low cost, short detection cycle and good application prospect.

On the one hand, the present invention provides a KASP primer set for detecting the genotype of transgenic soybean, the primer set comprising a first specific primer, a second specific primer and a first universal primer, the nucleotide sequence of the first specific primer is shown in SEQ ID No.13, the nucleotide sequence of the second specific primer is shown in SEQ ID No.14; the nucleotide sequence of the first universal primer is shown in SEQ ID No.12.

In one embodiment, the transgenic soybean is LT32 and its progeny and derivative lines.

In the present invention, the transgenic soybean LT32 is also recorded in Chinese patent application CN117247937A. As recorded in CN117247937A, the transgenic soybean LT32 has been deposited in the China Center for Type Culture Collection with a deposit number of CCTCC No: P202320.

The receptor of transgenic soybean LT32 is soybean Tianlong No. 1.

The T-DNA sequence inserted into the transgenic soybean is shown as SEQ ID No.10, and the nucleotide sequence on the right side of the T-DNA insertion site is shown as SEQ ID No.11.

In one embodiment, the 5' ends of the first specific primer and the second specific primer are also connected to fluorescent tags, and the fluorescent tags are FAM, HEX, etc.; preferably, the fluorescent tag is FAM or HEX, and the base sequence of the FAM fluorescent tag is GAAGGTGACCAAGTTCATGCT, and the base sequence of the HEX fluorescent tag is GAAGGTCGGAGTCAACGGATT.

In another aspect, the present invention provides a method for detecting genetically modified soybeans, the method comprising the step of detecting a sample using the above-mentioned KASP primer set.

In one embodiment, the method further comprises the step of obtaining nucleic acid from the sample.

In one embodiment, the transgenic soybean is transgenic soybean LT32.

In one embodiment, the reaction conditions of the KASP reaction are: pre-denaturation at 94°C for 10 min; first step amplification reaction, denaturation at 94°C for 15 s; annealing and extension at 61°C to 55°C for 60 s, 10 Touch Down cycles, the temperature of annealing and extension is reduced by 0.6°C in each cycle; second step amplification reaction, denaturation at 95°C for 15 s, annealing and extension at 55°C for 60 s, 35 cycles.

In one embodiment, the method for detecting transgenic soybeans includes using Kraken software to detect the difference in fluorescence signals of PCR products, thereby identifying the genotype of each soybean to be tested.

In one embodiment, the method for detecting the genetically modified soybean KASP genotyping results includes LGC device reading, microplate reader reading, agarose gel electrophoresis and other methods. In this experiment, LGC device reading is preferred.

In one embodiment, if only the fluorescence carried by the first specific primer is detected in the soybean to be tested, the soybean to be tested is judged to be a transgenic homozygous; if only the fluorescence carried by the second specific primer is detected in the soybean to be tested, the soybean to be tested is judged to be a wild-type homozygous; if the fluorescence carried by the first specific primer and the second specific primer are simultaneously detected in the soybean to be tested, the soybean to be tested is judged to be a transgenic heterozygous.

In one embodiment, the sample to be tested is derived from a plant part of soybean, for example, leaves, pods, seeds or rhizomes.

In another aspect, the present invention provides a kit for detecting soybean genotypes to be tested, the kit comprising the above-mentioned KASP primer set.

In another aspect, the present invention provides use of the KASP primer set in detecting the genotype of transgenic soybean.

On the other hand, the present invention also provides the use of the above-mentioned KASP primer set in preparing a reagent or a kit for detecting the genotype of transgenic soybean.

In one embodiment, the soybean is the transgenic soybean mentioned above, and the transgenic soybean is LT32 and its progeny and derivative lines.

Advantageous Effects of the Invention

The present invention provides primers and a method for detecting transgenic soybeans and their progeny and derivative lines based on the KASP technology, which can detect multiple samples at a high throughput, greatly improve the detection efficiency and accuracy, and realize high-throughput, low-cost rapid detection of homozygous and heterozygous transgenic soybeans LT32 and their progeny and derivative lines, providing convenience for breeders to screen soybean varieties with excellent alleles and accelerating the breeding process.

The method of the present invention can be used to efficiently and at low cost identify whether the transgenic soybean LT32 and its progeny and derivative lines are transgenic homozygotes or heterozygotes. The method uses a universal fluorescent probe and does not need to synthesize a specific probe. Compared with the probe method fluorescent quantitative PCR, the cost is greatly reduced. The fluorescent scanning of the method is performed at the reaction end point, so PCR amplification of a large number of samples can be performed first, and then the fluorescent scanning detection is concentrated after the reaction is completed, realizing high-throughput detection, and its detection efficiency is 14 to 56 times that of the fluorescent quantitative PCR; gel electrophoresis is not required after the reaction is completed, and the reaction system construction has been automated, which not only saves the operator's time and manpower, but also effectively reduces the probability of error.

The following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. According to the drawings and the following detailed description of the preferred embodiments, various objects and advantages of the present invention will become apparent to those skilled in the art.

The sequence information involved in this application is as follows:

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Schematic diagram of the T-DNA insertion position of transgenic soybean and the genomic position of the designed KSAP primers. WT is the wild-type soybean genome, and the dark area is the region where the wild-type genome is replaced by the transgenic T-DNA insertion; LT32 is the transgenic soybean genome, and the dark part represents the sequence of the transgenic soybean T-DNA insertion; LT-F is the region designed for the first specific primer, TL-H is the region designed for the second specific primer, and TL-C is the region designed for the first universal primer;

Figure 2. Typing results of soybean tested by KASP primer set 1;

Figure 3. Typing results of soybean tested by KASP primer set 2;

Figure 4. Typing results of soybean tested by KASP primer set 3;

Figure 5. Genotyping results of the tested soybean using KASP primers LT-F, TL-H and TL-C.

DETAILED DESCRIPTION

The present invention is further described below in conjunction with the embodiments. The following description is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change it into an equivalent embodiment with equivalent changes. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention falls within the protection scope of the present invention.

Example 1. Acquisition of transgenic soybean LT32 material and design of KASP primers

According to the flanking sequence of LT32 transgenic soybean disclosed in Chinese patent application CN117247937A, a series of KASP primers were designed using Primer3, and then the primers were evaluated using the NCBI online tool Primer-BLAST, and three groups of candidate KASP primers with high scores and good specificity were selected (as shown in the table below). The above primers were used to detect the DNA of transgenic soybean LT32 homozygous, transgenic soybean LT32 heterozygous, and non-transgenic soybean (Tianlong No. 1).

Preparation of DNA template: Prepare DNA of LT32 homozygote, LT32 heterozygote and non-transgenic soybeans respectively. DNA was extracted from LT32 homozygote and non-transgenic LT32 soybeans using CTAB extraction method, and transgenic hybrid DNA was extracted from backcross progeny materials with non-transgenic soybeans as recurrent parents and transgenic LT32 as donors.

Three sets of candidate KASP primers

Among them: gaaggtgaccaagttcatgct is the FAM fluorescent label sequence, and gaaggtcggagtcaacggatt is the HEX fluorescent label sequence.

The PCR reaction system is:

Components volume KASP Primer mix 5μL 2×KASP Master mix 0.5μL DNA template 30ng Total volume Add water to 10uμL

KASP Primer mix contains two specific primers and one universal primer (F, H and C), and their concentrations are 30μM, 12μM and 12μM respectively; KASP Master Mix contains the following components: universal FRET cassette fluorescent primer, ROX internal reference dye, Taq DNA polymerase, dNTP and MgCl2;

The PCR program was as follows: 95°C pre-denaturation, 10 min; 95°C, 15 s (denaturation); 61°C (-0.6°C/cycle) annealing for 60 s, 10 touchdown cycles; 95°C denaturation for 15 s, 55°C annealing for 60 s, 35 cycles. After amplification, the fluorescence signal was detected using the LGC PHERAstar instrument to check the typing status.

After the reaction is completed, the product is scanned for fluorescence, and the scanning results will appear in the form of a scatter plot. The genotype of the sample is determined based on the scatter plot: the horizontal axis of the graph represents the FAM fluorescence released by the product, and the vertical axis represents the HEX fluorescence released by the product. If significant FAM and HEX fluorescence can be detected, it indicates that the sample is a LT32 transgenic heterozygote; if only significant FAM fluorescence is detected but no significant HEX fluorescence is detected, it indicates that the sample is a LT32 transgenic homozygote; if only significant HEX fluorescence is detected but no FAM fluorescence is detected, it indicates that the sample is a wild type; if neither HEX fluorescence nor FAM fluorescence is detected, re-testing is required.

The results are shown in Figures 2 to 4. The three sets of candidate KASP primers (primer set 1 to primer set 3) could not effectively distinguish between heterozygous and homozygous transgenic soybean LT32.

In order to further design KASP primers that can be used to type LT32, the genomic sequences on the left and right sides of the T-DNA in the transgenic soybean LT32 were aligned to the reference genome using bioinformatics methods, including the inserted T-DNA sequence of the transgenic soybean LT32 (nucleotide sequence as shown in SEQ ID No. 10) and the sequence on the right side of the inserted T-DNA (nucleotide sequence as shown in SEQ ID No. 11); the applicant further analyzed the above sequences and found that the transgenic soybean LT32 had a partial base deletion due to the insertion of T-DNA. The inventor used this information to redesign a set of KASP primers LT-F, TL-H and TL-C, the sequences of which are shown in the following table:

LT-F gaaggtgaccaagttcatgctagattgtcgtttcccgccttcagt TL-H gaaggtcggagtcaacggatttgaagttgataaagtattaacc TL-C cgtgagaattgattttccaaca

Wherein: gaaggtgaccaagttcatgct is the FAM fluorescent tag sequence, gaaggtcggagtcaacggatt is the HEX fluorescent tag sequence. The positions of KASP primers LT-F, TL-H and TL-C are shown in Figure 1, where LT-F is the region designed for the first specific primer, TL-H is the region designed for the second specific primer, and TL-C is the region designed for the first universal primer.

The same method as above was used to distinguish the DNA of LT32 homozygotes, LT32 heterozygotes, and non-transgenics using KASP primers LT-F, TL-H, and TL-C. The results are shown in FIG5 . Each scattered point in FIG5 represents a test material, wherein “1” represents the homozygous wild-type soybean genotype, “2” represents the homozygous transgenic soybean LT32 genotype, “3” represents the heterozygous transgenic soybean LT32 genotype, and “N” represents the blank control. As shown in FIG5 , KASP primers LT-F, TL-H, and TL-C can distinguish the genotyping of transgenic soybean LT32, and can achieve good typing of transgenic homozygous, transgenic heterozygous, and wild homozygous genotype samples in transgenic soybean LT32 and its progeny and derivative lines.

Example 2. Typing results of KASP primer sets LT-F, TL-H and TL-C in samples

The KASP primer sets LT-F, TL-H and TL-C obtained in the above example were used to detect the genotype of the transgenic soybean sample to be tested, and the amplification system and procedure were referred to Example 1. A total of 64 samples were tested, of which the test results of 64 samples were consistent with the genotype information of the samples themselves, and the accuracy of the test results reached 100%. The results showed that the KASP primer sets LT-F, TL-H and TL-C can quickly and accurately distinguish the genotypes of transgenic soybean LT32 and its progeny and derivative lines, indicating that the high-throughput detection of samples using the method of the present invention has high accuracy and reliability.

Comparison of test results with sample sequencing information

Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims (10)

1. A KASP primer set for detecting transgenic soybean, wherein the primer set comprises a first specific primer, a second specific primer and a first universal primer; the nucleotide sequence of the first specific primer is shown as SEQ ID No.13, the nucleotide sequence of the second specific primer is shown as SEQ ID No.14, and the nucleotide sequence of the first universal primer is shown as SEQ ID No. 12.

2. The primer set of claim 1, wherein the transgenic soybean is LT32 and progeny or derived lines thereof.

3. The primer group of claim 2, wherein the T-DNA sequence of the transgenic soybean is shown in SEQ ID No.10, and the nucleotide sequence at the right side of the T-DNA insertion site is shown in SEQ ID No. 11.

4. A primer set according to any one of claims 1-3, wherein the 5' end of the first specific primer and/or the second specific primer is further linked to a fluorescent tag, e.g. FAM, HEX, etc.

5. The primer set of claim 4, wherein the fluorescent tag is selected from the group consisting of FAM and HEX.

6. A method for detecting the genotype of soybean to be tested, said method comprising the step of detecting a sample to be tested using the KASP primer set of any one of claims 1-5.

7. The method of claim 6, wherein the reaction conditions of the KASP reaction: pre-denaturation at 95 ℃ for 10min; the first step of amplification reaction, denaturation at 95 ℃ for 15s; annealing and extending at 61-55 ℃ for 60s,10 Touch Down cycles, wherein the annealing and extending temperature is reduced by 0.6 ℃ in each cycle; the second amplification step was 15s denatured at 95℃and annealed at 55℃and extended for 60s,35 cycles.

8. The method according to any one of claims 6 to 7, wherein the sample to be tested is derived from a plant part of soybean, such as a leaf, pod, seed or rhizome.

9. A kit for detecting soybean genotype, said kit comprising the KASP primer set of any one of claims 1-3.

10. Use of a KASP primer set according to any one of claims 1-4 for detecting the genotype of transgenic soybean or for the preparation of a reagent or kit for detecting the genotype of transgenic soybean.