Method for detecting the copy number of inserted foreign genes in Setaria viridis or Setaria italica by digital PCR

Abstract

The present invention provides a method for detecting the copy number of exogenous gene insertion in Setaria viridis or foxtail millet by digital PCR, belonging to the field of biotechnology. The method uses the Sevir.3G057200 gene as an internal reference gene and Hyg the selectable marker gene as the exogenous gene, and uses the primer pairs 200-F / 200-R and Hyg-F / Hyg-R to detect the genomic DNA of transgenic Setaria viridis or transgenic foxtail millet, and to determine the copy number of exogenous gene insertion in transgenic Setaria viridis or transgenic foxtail millet. The method of the present invention has the characteristics of being simple, rapid, low sample consumption, efficient, and high-throughput, can improve the efficiency of screening transgenic lines of Setaria viridis or foxtail millet with low-copy insertion, and provides a new option for the detection method of the copy number of exogenous gene insertion in the research of transgenic Setaria viridis or transgenic foxtail millet.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a method for detecting the number of inserted copies of exogenous genes in foxtail grass or millet using digital PCR, and more particularly to a single-copy broomcorn millet gene capable of detecting the number of inserted copies of exogenous genes in broomcorn millet, and a method for detecting the number of inserted copies of exogenous genes in transgenic broomcorn millet using digital PCR. Background Art

[0002] Setaria viridis ( Setaria viridis ) belongs to the genus Setaria of the subfamily Panicideae of the Poaceae family, monocotyledons. It is a new type of transgenic model plant. Compared with the dicotyledonous model plant Arabidopsis thaliana, it has the advantages of short growth cycle, short plants, easy planting, small genome, diploid, and the ability to produce a large number of inbred seeds. It is an excellent monocotyledonous model plant. Because it originated in the tropics and has a C4 photosynthesis system, it is closely related to millet, corn, sorghum, sugarcane, coix and important energy grasses, and is also an important C4 plant model. At present, the whole genome sequencing and resequencing data of Setaria ME34 and A10 have been published, and more and more research institutions in China have begun to pay attention to it, especially millet ( Setaria italica Millet is also an annual plant of the genus Setaria, subfamily Paniculoideae, Poaceae. Setaria is the wild ancestor of millet. Millet and Setaria share essentially identical karyotypes, similar banding patterns, and a genome size of approximately 510 Mb. Setaria has a short growth cycle and high genetic transformation efficiency. Overexpressing or knocking out candidate millet genes in Setaria can rapidly verify their function and accelerate millet breeding research. Therefore, Setaria can serve as an important transgenic model plant for studying millet gene function.

[0003] In transgenic plant breeding research, the expression and genetic stability of exogenous genes are influenced by the copy number of the exogenous gene integrated into the recipient plant genome. When exogenous genes are inserted into the recipient plant genome at low copy numbers (1-2), they generally achieve efficient expression and stable inheritance. Therefore, in transgenic plant breeding research, transgenic lines with low exogenous gene copy numbers are often selected. However, these transgenic lines typically require screening from a large number of T0 generation transgenic lines, which is time-consuming. Therefore, simple, rapid, efficient, and high-throughput methods for detecting the copy number of inserted exogenous genes are crucial for transgenic plant breeding research.

[0004] Currently, Southern hybridization is the traditional method for detecting the copy number of foreign gene insertions in transgenic plants. While highly accurate, it also comes with high costs, long processing times, and the difficulty of quickly and easily testing large numbers of samples. In recent years, digital PCR (dPCR), due to its simplicity, speed, and efficiency, has been successfully applied to detect the copy number of foreign gene insertions in various transgenic crops. However, there are still no reports of using this PCR method to detect the copy number of foreign gene insertions in transgenic Setaria viridis. Therefore, there is an urgent need to establish a method for rapidly detecting the copy number of foreign gene insertions in transgenic Setaria viridis using dPCR. Summary of the Invention

[0005] In response to the above problems, the present invention provides a single-copy Setaria viridis gene and a method for detecting the number of inserted copies of exogenous genes in Setaria viridis by digital PCR technology using the single-copy Setaria viridis gene.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] A method for detecting the number of inserted copies of exogenous genes in foxtail grass or foxtail millet using digital PCR is provided, wherein the Sevir.3G057200 gene is used as an internal reference gene. Hyg The selection marker gene is an exogenous gene, namely the selection marker gene hygromycin phosphotransferase gene (hygromycin phosphotransferase, Hyg ), using primers 200-F / 200-R and Hyg-F / Hyg-R, to detect the genomic DNA of transgenic Setaria or transgenic millet, and to determine the copy number of the exogenous gene inserted in the transgenic Setaria or transgenic millet;

[0008] The Sevir.3G057200 gene is a single-copy gene in Setaria viridis. The corresponding gene number of this gene in foxtail millet is Seita.3G056300, which is also a single-copy gene in foxtail millet. The nucleotide sequence homology of the two genes is 99.3%, and the primer pair sequences of the two genes are completely identical. The sequence of the Sevir.3G057200 gene in Setaria viridis is shown in SEQ ID NO: 7; the sequence of the Seita.3G056300 gene in foxtail millet is shown in SEQ ID NO: 8.

[0009] Primer pair sequence for detecting the internal reference gene Sevir.3G057200:

[0010] Forward primer 200-F is: 5′-GTTCCGCACCAACATCTACTCCTAC-3′, as shown in SEQ ID NO: 1;

[0011] The reverse primer 200-R is: 5′-GTTGATGACGCTGCTCCCTTCC-3′, as shown in SEQ ID NO: 2;

[0012] Probe 200-P is: VIC-CCTACTTCCTGGTGACCAAGCACGC-MGB, as shown in SEQ ID NO: 3;

[0013] Detection Hyg Primer pairs and probe sequences for screening marker genes:

[0014] Forward primer Hyg-F is: 5'-GTCAAGACCAATGCGGAGCA-3', as shown in SEQ ID NO: 4;

[0015] The reverse primer Hyg-R is: 5'-CCCAATACGAGGTCGCCAAC-3', as shown in SEQ ID NO: 5;

[0016] The probe Hyg-P is: FAM-TCGAAGTAGCGCGTCTGCTGCTCCA-BHQ1, as shown in SEQ ID NO: 6;

[0017] The method for determining the copy number of the exogenous gene inserted in transgenic foxtail grass or transgenic millet is as follows:

[0018] When the copy number ratio of the exogenous gene to the internal reference gene is about 0.5 (i.e., when the copy number ratio of the exogenous gene to the internal reference gene is 0.26-0.74), the transgenic foxtail grass or transgenic millet has a single copy insertion;

[0019] When the copy number ratio of the exogenous gene to the internal reference gene is about 1 (when the copy number ratio of the exogenous gene to the internal reference gene is 0.75-1.25), the transgenic foxtail grass or transgenic millet has a double copy insertion;

[0020] When the copy number ratio of the exogenous gene to the internal reference gene is about 1.5 (when the copy number ratio of the exogenous gene to the internal reference gene is 1.26-1.74), the transgenic foxtail grass or transgenic millet has three copies inserted;

[0021] When the copy number ratio of the exogenous gene to the internal reference gene is about 2 (when the copy number ratio of the exogenous gene to the internal reference gene is 1.75-2.25), the transgenic foxtail grass or transgenic millet has four copies inserted;

[0022] The same can be applied to other insertion copy numbers of transgenic foxtail grass or transgenic millet, such as five-copy insertion, six-copy insertion, etc.

[0023] Furthermore, the method uses the genomic DNA of transgenic foxtail grass or transgenic millet as a DNA template and uses the primer pair RA1-F / RA1-R and the primer pair Hyg-F / Hyg-R to perform digital PCR detection.

[0024] Furthermore, the reaction system of digital PCR is: reaction premix, 200-F, 200-R, Hyg-F, Hyg-R, probe 200-P, probe Hyg-P, DNA template and nuclease-free water.

[0025] Furthermore, the reaction premix is ​​a reaction premix for droplet digital PCR.

[0026] Furthermore, the digital PCR reaction system was as follows: 10.00 µL of reaction premix, 1.80 µL of 200-F at a concentration of 10.00 µM, 1.80 µL of 200-R at a concentration of 10.00 µM, 1.80 µL of Hyg-F at a concentration of 10.00 µM, 1.80 µL of Hyg-R at a concentration of 10.00 µM, 0.50 µL of probe 200-P at a concentration of 10.00 µM, 0.50 µL of probe Hyg-P at a concentration of 10.00 µM, 1 µL of DNA template at a concentration of 30.00 ng / µL, and 0.80 µL of nuclease-free water, for a total volume of 20.00 µL.

[0027] Furthermore, the amplification program of digital PCR was as follows: 95°C for 10 min; 95°C for 30 s, 56.4-69.6°C for 1 min, 45 cycles; and 98°C for 10 min.

[0028] Furthermore, the amplification program of digital PCR was: 95°C for 10 min; 95°C for 30 s, 62°C for 1 min, 45 cycles; 98°C for 10 min.

[0029] Furthermore, after the digital PCR reaction is completed, the copy numbers of the exogenous gene and the internal reference gene are obtained, and the copy number ratio of the exogenous gene to the internal reference gene in the transgenic foxtail grass or transgenic millet is calculated. The ratio is used to determine the inserted copy number of the exogenous gene in the transgenic foxtail grass or transgenic millet genome.

[0030] Furthermore, the copy numbers of the exogenous gene and the internal reference gene were obtained by placing the chip into a biochip analyzer to read the FAM and VIC fluorescence signals and performing fluorescence data analysis using QuantDrop Software.

[0031] Furthermore, the genomic DNA of the transgenic foxtail grass or transgenic millet is extracted using a MEGA plant genomic kit.

[0032] The beneficial effects of the method of the present invention for detecting the copy number of exogenous gene insertion in Setaria viridis or millet using digital PCR are as follows:

[0033] The single copy of the foxtail grass gene or millet gene provided by the present invention can be used as an internal reference gene for detecting the copy number of the exogenous gene inserted in the transgenic foxtail grass or transgenic millet, and is the most commonly used screening marker in transgenic foxtail grass or transgenic millet. Hyg The gene is an exogenous gene, and digital PCR technology is used to achieve rapid high-throughput detection of the number of inserted copies of the exogenous gene in transgenic Setaria viridis.

[0034] The present invention provides a method for high-throughput detection of the number of inserted copies of exogenous genes in transgenic Setaria or transgenic millet using digital PCR technology. The method has the characteristics of simplicity, rapidity, low sample requirements, high efficiency, and high throughput. It can improve the efficiency of large-scale screening of transgenic strains and provide a new option for detecting the number of inserted copies of exogenous genes in transgenic Setaria or transgenic millet breeding research.

[0035] The method provided by the present invention for detecting the number of inserted copies of foreign genes in foxtail grass or millet by using digital PCR can be used for all Hyg The copy number detection of transgenic foxtail grass or transgenic millet for screening markers; the nucleotide sequence homology of the Sevir.3G057200 gene in the present invention and its corresponding gene Seita.3G056300 in millet is 99.3%, and the primer pair sequences of the two are completely consistent; therefore, the method provided by the present invention is also applicable to Hyg The method is to detect the copy number of genetically modified millet with screening markers; the method has the advantages of absolute quantification, high accuracy, simplicity and rapidity, small sample requirement, and batch detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the alignment result of the Setaria viridis gene Sevir.3G057200 in the genome in Example 1 of the present invention;

[0037] Figure 2 This is the result of exploring the PCR amplification conditions of the Setaria viridis internal reference gene and the exogenous gene in Example 2 of the present invention; in the figure, M is the DL 2000 Marker, the unit is bp, and the molecular weights from top to bottom are 2000, 1000, 750, 500, 250 and 100; labels 1 to 8 represent PCR products at annealing temperatures of 56.4°C, 58.0°C, 60.0°C, 62.0°C, 64.0°C, 66.0°C, 68.0°C and 69.6°C, respectively;

[0038] Figure 3The PCR identification results of the transgenic Setaria viridis in Example 3 of the present invention; in the figure, M is DL 2000 Marker, the unit is bp, and the molecular weights from top to bottom are 2000, 1000, 750, 500, 250 and 100; the numbers 1 to 24 represent transgenic Setaria viridis SV-1 to SV-24 respectively; "-" represents Hyg Negative control, “+” represents Hyg Positive control;

[0039] Figure 4 is the Sevir.3G057200 gene in Example 4 of the present invention and Hyg Digital PCR scatter plot of genes; blue scatter plots represent Hyg The number of fluorescent wells of Sevir.3G057200 gene is shown in green; the number of fluorescent wells of Sevir.3G057200 gene is shown in red. Hyg Gray scattered dots represent the number of wells with no signal. DETAILED DESCRIPTION

[0040] The technical solutions in the embodiments of the present invention are described clearly and completely below. Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. The present invention is further described in detail below in conjunction with specific embodiments to facilitate understanding by those skilled in the art.

[0041] In the examples disclosed below, if specific techniques or conditions are not specified, the experiments were performed according to those described in literature in the field (e.g., Molecular Cloning: A Laboratory Manual, 3rd edition, by Sambrook et al., translated by Huang Peitang et al., Science Press) or according to the product instructions. Reagents used without manufacturer's information are commercially available.

[0042] Example 1 Determination of internal reference genes

[0043] The bioinformatics analysis of the copy number of the Sevir.3G057200 gene in the Setaria genome specifically includes the following steps:

[0044] On the Phytozome website (https: / / phytozome-next.jgi.doe.gov / ), the nucleotide sequence of the Sevir.3G057200 gene was used as the query sequence to search the Setaria genome and obtain the alignment results. Figure 1The comparison results showed that the Sevir.3G057200 gene was a single-copy gene with no other homologous genes with similar sequences. Therefore, it could be used as an internal reference gene for detecting the copy number of inserted exogenous genes in transgenic Setaria viridis.

[0045] The Sevir.3G057200 gene in foxtail millet corresponds to Seita.3G056300. The Sevir.3G057200 gene in Setaria viridis shares 99.3% sequence identity with the Seita.3G056300 gene in foxtail millet. The sequence of the Sevir.3G057200 gene in Setaria viridis is shown in SEQ ID NO: 7, while the sequence of the Seita.3G056300 gene in foxtail millet is shown in SEQ ID NO: 8.

[0046] Since the gene sequence homology between the Sevir.3G057200 gene in Setaria viridis and the Seita.3G056300 gene in millet is as high as 99.3%, and both are single-copy genes, the primers and probes designed based on the Sevir.3G057200 gene in Setaria viridis are also suitable for amplifying and detecting the Seita.3G056300 gene in millet (i.e., the primer pair sequence is completely consistent with the sequence in the corresponding Seita.3G056300 gene in millet).

[0047] At the same time, since the commonly used screening marker genes in transgenic foxtail grass and transgenic millet are Hyg Genes can Hyg The gene is used as an exogenous screening marker gene, and both foxtail millet and Setaria viridis are diploid plants. Foxtail millet is also an annual plant of the genus Setaria in the subfamily Panicoideae of the Poaceae family. Setaria viridis is the wild ancestor of foxtail millet. Foxtail millet and Setaria viridis have basically the same karyotype, similar banding patterns, and a genome size of approximately 510M. Therefore, the method for detecting the number of inserted copies of exogenous genes in Setaria viridis of the present invention can be directly applied to detecting the number of inserted copies of exogenous genes in foxtail millet. In summary, the method for detecting the number of inserted copies of exogenous genes in Setaria viridis using digital PCR of the present invention can be directly used to detect the number of inserted copies of exogenous genes in transgenic foxtail millet.

[0048] Example 2 Exploration of PCR amplification conditions

[0049] Since the commonly used screening marker gene in transgenic Setaria viridis is Hyg Genes, therefore selection Hyg The screening marker gene was used as the exogenous gene in transgenic Setaria viridis, and the Sevir.3G057200 gene was used as the internal reference gene for detection. The PCR amplification conditions of the internal reference gene and the exogenous gene primers were explored, including the following specific steps:

[0050] 1) Primer sequence

[0051] The primer pair sequences for detecting the internal reference gene Sevir.3G057200 include:

[0052] Forward primer 200-F is: 5′-GTTCCGCACCAACATCTACTCCTAC-3′, as shown in SEQ ID NO: 1;

[0053] The reverse primer 200-R is: 5′-GTTGATGACGCTGCTCCCTTCC-3′, as shown in SEQ ID NO: 2;

[0054] Detection Hyg The primer pair sequences for screening marker genes include:

[0055] The forward primer Hyg-F is: 5′-GTCAAGACCAATGCGGAGCA-3′, as shown in SEQ ID NO: 4;

[0056] The reverse primer Hyg-R is: 5′-CCCAATACGAGGTCGCCAAC-3′, as shown in SEQ ID NO: 5;

[0057] 2) Sevir.3G057200 gene primers and Hyg Exploration of PCR amplification conditions for gene primers

[0058] Using DNA from a transgenic Setaria viridis plant as a template, gradient PCR reactions were performed using the 200-F / 200-R primer pair and the Hyg-F / Hyg-R primer pair, respectively.

[0059] The PCR reaction system for detecting the internal reference gene Sevir.3G057200 was as follows: Green Master Mix 12.50 µL, 200-F (10.00 µM) 1.00 µL, 200-R (10.00 µM) 1.00 µL, DNA template (30.00 ng / µL) 1.00 µL, and nuclease-free water 9.50 µL, with a total volume of 25.00 µL.

[0060] The PCR reaction system for detecting exogenous genes was as follows: Green Master Mix 12.50 µL, Hyg-F (10.00 µM) 1.00 µL, Hyg-R (10.00 µM) 1.00 µL, DNA template (30.00 ng / µL) 1.00 µL, and nuclease-free water 9.50 µL, with a total volume of 25.00 µL.

[0061] The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 30 s, annealing for 30 s, and extension at 72°C for 30 s; a final extension at 72°C for 7 min, and storage at 4°C; the annealing temperatures used were 56.4°C, 58.0°C, 60.0°C, 62.0°C, 64.0°C, 66.0°C, 68.0°C, and 69.6°C, respectively.

[0062] After the PCR amplification reaction, 5 μL of the corresponding PCR products were taken and electrophoresed on a 2% agarose gel. Hyg The PCR products of 83bp and 143bp were respectively amplified bands of the target gene. The remaining PCR products were sent for sequencing. The sequencing results were consistent with those of Sevir.3G057200 gene and Hyg The nucleotide sequences of the genes were compared.

[0063] The electrophoresis results are as follows Figure 2 As shown, when the annealing temperatures were 56.4℃, 58.0℃, 60.0℃ and 62.0℃, the Sevir.3G057200 gene and Hyg The target amplified bands were obtained for all genes, but the bands were brightest when the annealing temperature was 62.0℃, and there were no non-specific bands. Hyg The PCR product of the gene was sequenced and compared with the Sevir.3G057200 gene of Setaria viridis and Hyg The sequences of the genes are consistent, so the 200-F / 200-R primer pair can be used for the detection of the Sevir.3G057200 gene of Setaria viridis, and the Hyg-F / Hyg-R primer pair can be used for the detection of exogenous genes. Hyg The annealing temperature of the genes was determined to be 62.0°C.

[0064] Example 3 PCR identification of positive transgenic Setaria viridis plants

[0065] Using the method in Example 2 Hyg The PCR identification of transgenic Setaria plants using gene primers specifically includes the following steps:

[0066] Leaves of 24 transgenic Setaria plants (numbered SV-1 to SV-24) were collected and their genomic DNA was extracted using the MEGA Plant Genomic DNA Rapid Extraction Kit (D3187-02) as a DNA template. The genomic DNA of wild-type Setaria was used as a template. Hyg Negative control, containing Hyg The genomic DNA of transgenic Setaria viridis Hyg Positive control, using the method in Example 2 through Hyg-F / Hyg-RHyg The PCR experiment of the gene was performed to exclude the negative transgenic plants of Setaria viridis. The PCR reaction amplification system and reaction conditions were the same as those in Example 2. Hyg The PCR amplification system and reaction conditions of the gene were the same, and the annealing temperature was 62.0°C.

[0067] After the PCR amplification reaction is completed, 5 μL of the corresponding PCR product is electrophoresed on a 2% agarose gel. If a target band of about 143 bp appears, it is a positive transgenic Setaria plant. If no target band appears, it is a negative transgenic plant. The PCR test results of 24 transgenic Setaria plants are shown in Figure 3 Among them, the plants numbered SV-1~SV-6, SV-8~SV-10, SV-13~SV-19, SV-21 and SV-23 were all transgenic positive plants, totaling 18 plants; the plants numbered SV-7, SV-11, SV-12, SV-20, SV-22 and SV-24 were all transgenic negative plants, totaling 6 plants.

[0068] Example 4 Digital PCR Detection

[0069] The method for detecting the number of inserted copies of exogenous genes in Setaria viridis using digital PCR comprises the following specific steps:

[0070] 1) Digital PCR primers and probes

[0071] Primer pair and probe sequences for detecting the internal reference gene Sevir.3G057200:

[0072] Forward primer 200-F is: 5′-GTTCCGCACCAACATCTACTCCTAC-3′, as shown in SEQ ID NO: 1;

[0073] The reverse primer 200-R is: 5′-GTTGATGACGCTGCTCCCTTCC-3′, as shown in SEQ ID NO: 2;

[0074] Probe 200-P is: VIC-CCTACTTCCTGGTGACCAAGCACGC-MGB, as shown in SEQ ID NO: 3;

[0075] Detection Hyg Primer pairs and probe sequences for screening marker genes:

[0076] Forward primer Hyg-F is: 5'-GTCAAGACCAATGCGGAGCA-3', as shown in SEQ ID NO: 4;

[0077] The reverse primer Hyg-R is: 5'-CCCAATACGAGGTCGCCAAC-3', as shown in SEQ ID NO: 5;

[0078] The probe Hyg-P is: FAM-TCGAAGTAGCGCGTCTGCTGCTCCA-BHQ1, as shown in SEQ ID NO: 6.

[0079] 2) DNA template extraction

[0080] From the 18 transgenic-positive plants screened in Example 3, 9 plants (SV-1 to SV-6 and SV-8 to SV-10) were selected as test samples, and the transgenic-negative plant SV-7 was used as a negative control. The corresponding genomic DNA was extracted using the MEGA Plant Genomic Kit and used as a DNA template.

[0081] 3) Digital PCR method to detect the number of inserted copies of exogenous genes in the test sample

[0082] Sevir.3G057200 gene was used as the internal reference gene. Hyg The gene is an exogenous gene, and the number of inserted copies of the exogenous gene in the transgenic Setaria genome is detected by digital PCR experiments. Three replicates are set for each sample to be tested, and the DNA of the transgenic negative plant SV-7 is used as a negative control. The MicroDrop droplet digital PCR system was used, and the reaction system was as follows: droplet digital PCR premix 10.00 µL, 200-F (10.00 µM) 1.80 µL, 200-R (10.00 µM) 1.80 µL, Hyg-F (10.00 µM) 1.80 µL, Hyg-R (10.00 µM) 1.80 µL, probe 200-P (10.00 µM) 0.50 µL, probe Hyg-P (10.00 µM) 0.50 µL, DNA template (30.00 ng / µL) 1 µL, and nuclease-free water 0.80 µL, for a total volume of 20.00 µL.

[0083] The reaction amplification program was as follows: 95°C for 10 min; 95°C for 30 s, 62°C for 1 min, 45 cycles; and 98°C for 10 min.

[0084] 4) Result analysis

[0085] After the reaction is completed, the obtained chip is placed in a biochip analyzer to read the FAM and VIC fluorescence signals, and QuantDrop Software is used to analyze the fluorescence data to obtain the copy number (copy / µL) of the exogenous gene and the internal reference gene in the corresponding DNA template. The copy number ratio of the exogenous gene to the internal reference gene in the DNA template is calculated, and the copy number of the exogenous gene in the Setaria genome is determined by the ratio.

[0086] Since Setaria viridis is a diploid plant, the copy number ratio of a single-copy inserted exogenous gene fragment to the internal reference gene fragment is approximately 0.5 (i.e., the general copy number ratio of a single-copy inserted exogenous gene fragment to the internal reference gene fragment is 0.26-0.74), the copy number ratio of a double-copy inserted exogenous gene fragment to the internal reference gene fragment is approximately 1 (i.e., the general copy number ratio of a double-copy inserted exogenous gene fragment to the internal reference gene fragment is 0.75-1.25), the copy number ratio of a triple-copy inserted exogenous gene fragment to the internal reference gene fragment is approximately 1.5 (i.e., the general copy number ratio of a double-copy inserted exogenous gene fragment to the internal reference gene fragment is 1.26-1.74), the copy number ratio of a quadruple-copy inserted exogenous gene fragment to the internal reference gene fragment is approximately 2 (i.e., the general copy number ratio of a double-copy inserted exogenous gene fragment to the internal reference gene fragment is 1.75-2.25), and the copy numbers of multiple-copy exogenous gene fragments such as pentacopy and hexacopy are similar.

[0087] The digital PCR scatter plots of 9 transgenic positive Setaria plants in this example are shown in Figure 4 The copy number detection results are shown in Table 1. The copy number ratios of SV-1, SV-4 and SV-5 in the transgenic positive plants are close to 0.5, indicating single copy insertions; the copy number ratios of SV-2, SV-3, SV-8 and SV-9 in the transgenic positive plants are all close to 1, indicating double copy insertions; the copy number ratios of SV-6 and SV-10 in the transgenic positive plants are close to 2, indicating four copy insertions. Two internal reference genes RA1 The results were consistent with those obtained with Sevir.3G057200, indicating that both genes can be used as internal reference genes for detecting the copy number of exogenous gene insertions in transgenic Setaria viridis.

[0088] Table 1 Results of digital PCR detection of the number of inserted copies of exogenous genes in transgenic Setaria viridis

[0089]

[0090] Depend on Figure 4As can be seen from Table 1, three transgenic-positive plants (SV-1, SV-4, and SV-5) had a copy number ratio of approximately 0.5, indicating single-copy insertions; four transgenic-positive plants (SV-2, SV-3, SV-8, and SV-9) had a copy number ratio of approximately 1, indicating double-copy insertions; and two transgenic-positive plants (SV-6 and SV-10) had a copy number ratio of approximately 2, indicating quadruple-copy insertions. These results demonstrate that the method for detecting the copy number of exogenous gene insertions in Setaria viridis using digital PCR in this example offers the advantages of absolute quantification, high accuracy, simplicity, rapidity, low sample requirements, and batch testing capabilities. This method can be used to rapidly screen plants with low-copy insertions of exogenous genes from a large number of transgenic plants, saving significant time and cost for the breeding of superior transgenic strains.

[0091] All other parts not described in detail are prior art. Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Those skilled in the art can also derive other embodiments based on this embodiment without inventiveness, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for detecting the copy number of inserted foreign genes in Setaria viridis or foxtail millet by digital PCR, characterized in that, The method uses the Sevir.3G057200 gene as an internal reference gene and Hyg a selection marker gene as a foreign gene, uses the primer pairs 200-F / 200-R and Hyg-F / Hyg-R, and detects the genomic DNA of transgenic foxtail millet or transgenic millet by digital PCR, and determines the copy number of foreign gene insertion in transgenic foxtail millet or transgenic millet; Primer pair sequences for detecting the reference gene Sevir.3G057200: Forward primer 200-F is shown as SEQ ID NO: 1; Reverse primer 200-R is shown as SEQ ID NO: 2; Probe 200-P is shown as SEQ ID NO: 3; Detection Hyg Primer pairs and probe sequences for screening marker genes: Forward primer Hyg-F is shown as SEQ ID NO: 4; Reverse primer Hyg-R is shown as SEQ ID NO: 5; Probe Hyg-P is shown as SEQ ID NO: 6; The method for determining the copy number of foreign gene insertions in transgenic Setaria viridis or transgenic Setaria italica is as follows: When the copy number ratio of the foreign gene to the reference gene is 0.26 - 0.74, the transgenic Setaria viridis or transgenic Setaria italica has a single-copy insertion; When the copy number ratio of the foreign gene to the reference gene is 0.75 - 1.25, the transgenic Setaria viridis or transgenic Setaria italica has a double-copy insertion; When the copy number ratio of the foreign gene to the reference gene is 1.26 - 1.74, the transgenic Setaria viridis or transgenic Setaria italica has a triple-copy insertion; When the copy number ratio of the foreign gene to the reference gene is 1.75 - 2.25, the transgenic Setaria viridis or transgenic Setaria italica has a quadruple-copy insertion; And so on for other insertion copy numbers of transgenic Setaria viridis or transgenic Setaria italica.

2. The method according to claim 1, wherein The method uses the genomic DNA of transgenic Setaria viridis or transgenic Setaria italica as the DNA template, and performs digital PCR detection using the primer pairs RA1-F / RA1-R and Hyg-F / Hyg-R.

3. The method according to claim 2, wherein The reaction system for digital PCR is: reaction premix, 200-F, 200-R, Hyg-F, Hyg-R, probe 200-P, probe Hyg-P, DNA template, and nuclease-free water.

4. The method according to claim 3, characterized in that, The reaction premix is a reaction premix for droplet digital PCR.

5. The method according to any one of claims 2 - 4, characterized in that, The reaction system for digital PCR is: 10.00 µL of reaction premix, 1.80 µL of 200-F with a concentration of 10.00 µM, 1.80 µL of 200-R with a concentration of 10.00 µM, 1.80 µL of Hyg-F with a concentration of 10.00 µM, 1.80 µL of Hyg-R with a concentration of 10.00 µM, 0.50 µL of probe 200-P with a concentration of 10.00 µM, 0.50 µL of probe Hyg-P with a concentration of 10.00 µM, 1 µL of DNA template with a concentration of 30.00 ng / µL, and 0.80 µL of nuclease-free water, with a total volume of 20.00 µL.

6. The method according to any one of claims 1 to 4, characterized in that, The amplification program for digital PCR is: 95°C for 10 min; 95°C for 30 s, 56.4 - 69.6°C for 1 min, 45 cycles; 98°C for 10 min.

7. The method according to any one of claims 1-4, characterized in that, The amplification program for digital PCR is: 95°C for 10 min; 95°C for 30 s, 62°C for 1 min, 45 cycles; 98°C for 10 min.

8. The method according to any one of claims 1 to 4, characterized in that After the digital PCR reaction is completed, the copy numbers of the exogenous gene and the reference gene are obtained, and the ratio of the copy numbers of the exogenous gene to the reference gene in transgenic Setaria viridis or transgenic foxtail millet is calculated. The insertion copy number of the exogenous gene in the genome of transgenic Setaria viridis or transgenic foxtail millet is determined by the ratio.

9. The method according to any one of claims 1-4, characterized in that, The copy numbers of the exogenous gene and the reference gene are obtained by putting the chip into a biochip analyzer to read the fluorescence signal and performing fluorescence data analysis.

10. The method according to any one of claims 1-4, characterized in that, The genomic DNA of transgenic Setaria viridis or transgenic foxtail millet is obtained by extraction using a plant genome kit.

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