A method for optimizing multiplex PCR amplification primer system for cotton SSR marker detection

Abstract

The application provides a multiplex PCR amplification primer system optimization method for cotton SSR marker detection, which comprises the following steps: synthesizing 10 pairs of cotton SSR primers, taking extracted cotton genome DNA to be detected as a template, performing multiplex PCR amplification by using the primers, performing capillary electrophoresis detection on the amplification products, according to the analysis result of the detection data, grouping the primers which can detect products into one group and grouping the primers which cannot detect products into another group, respectively performing second amplification according to the grouping, uniformly mixing and detecting the amplification products, detecting products by using 10 pairs of primers, and finally determining 10 pairs of primers and grouping them into two multiplex combinations. The application adopts a step-by-step elimination method, divides 10 pairs of primers into two multiplex amplification combinations through twice amplification detection, reduces the amplification times of the 10 pairs of primers from 10 times to 2 times compared with single amplification, simultaneously reduces the use amount of primers and mix reagents, saves the cost, improves the sample detection efficiency, and is suitable for cotton variety identification and genetic analysis.

Description

Technical Field

[0001] This invention belongs to the field of molecular-assisted breeding technology, specifically relating to an optimization method for multiplex PCR amplification primer system for SSR marker detection in cotton. Background Technology

[0002] Variety authenticity verification is the process of identifying the true identity of a variety, providing data support for combating counterfeit seeds in the seed market and protecting breeders' intellectual property rights. Commonly used methods include field plot planting identification and DNA molecular marker identification. Field plot planting identification is accurate but time-consuming, while DNA molecular marker identification is fast and accurate.

[0003] The combination of multiplex PCR and capillary electrophoresis is a highly efficient method for detecting SSR markers. This method comprises two stages: PCR amplification and SSR primer amplification product detection. In the SSR primer amplification product detection stage, Wu Yuzhen et al., based on the primer amplification range and allele distribution characteristics of cotton primers, divided 60 primer pairs into 6 groups of 10 primer pairs each, forming a ten-pair fluorescent capillary electrophoresis combination. This significantly improved the efficiency of cotton authenticity detection, and this combination has now become the standard combination for different laboratories, facilitating the sharing of detection data among them. In the PCR amplification stage, multiplex PCR amplification has been widely used in species identification, maize transgenic detection, and maize purity identification; however, optimization of the 60-pair cotton multiplex PCR amplification system is currently rarely reported. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an optimization method for multiplex PCR amplification primer system for cotton SSR marker detection, which addresses the shortcomings of the prior art. This method adopts a stepwise elimination method, which divides 10 pairs of primers into two multiplex amplification combinations through two amplification detections. Compared with single amplification, this method reduces the amount of primers, mixes and other reagents used, saves costs and improves sample detection efficiency.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: an optimization method for multiplex PCR amplification primer system for cotton SSR marker detection, comprising the following steps: S1. Synthesize multiplex PCR amplification primer pairs for cotton SSR marker detection; The multiplex PCR amplification primer pairs are PC01-F and PC01-R, PC02-F and PC02-R, PC13-F and PC13-R, PC19-F and PC19-R, PC20-F and PC20-R, PC26-F and PC26-R, PC28-F and PC28-R, PC08-F and PC08-R, PC17-F and PC17-R, and PC27-F and PC27-R. S2. Extract genomic DNA from the cotton sample to be tested as a template, and perform multiplex PCR amplification using the multiplex PCR amplification primers synthesized in S1 to obtain the amplification product. S3. Take 1 μL of the amplification product obtained in S2, dilute it 40 times with ddH2O to obtain a diluent, mix the diluent with Liz-500 molecular weight internal standard and deionized formamide, centrifuge for 10s, and then perform capillary electrophoresis detection and read the detection data after denaturation, cooling and centrifugation in sequence. S4. Analyze the detection data read in S3 and divide the multiplex PCR amplification primer pairs into two groups of primer pairs. The first group of primer pairs includes PC01, PC02, PC13, PC19, PC20, PC26, and PC28, and the second group of primer pairs includes PC08, PC17, and PC27. S5. Perform a second multiplex PCR amplification using the two primer pairs described in S4. Mix the two amplification products in equal volumes, perform capillary electrophoresis detection, and read the detection data. Both primer pairs can amplify the results. The nucleotide sequence of PC01-F in S1 is 5'-NED-CCACCAGCCTTACCTTATACGC-3'; the nucleotide sequence of PC01-R is shown in SEQ ID NO:1; the nucleotide sequence of PC02-F is 5'-FAM-AGTCCTCCCACTATTCGAAGCT-3'; the nucleotide sequence of PC02-R is shown in SEQ ID NO:2; the nucleotide sequence of PC13-F is 5'-NED-ACCATCCTTGCCGAGTAACCTG-3'; the nucleotide sequence of PC13-R is shown in SEQ ID NO:3; the nucleotide sequence of PC19-F is 5'-VIC-CCAGCCACTCGGAGATCCTTG-3'; the nucleotide sequence of PC19-R is shown in SEQ ID NO:4; the nucleotide sequence of PC20-F is 5'-PET-ACCACTTTGTCCACCTGTCCAC-3'; the nucleotide sequence of PC20-R is shown in SEQ ID NO:3. The nucleotide sequence of PC26-F is 5'-FAM-TTCCATGAGGCTATCCACAAGC-3'; the nucleotide sequence of PC26-R is shown in SEQ ID NO:6; the nucleotide sequence of PC28-F is 5'-PET-ACAAGCGCGTCTGCCATATCC-3'; the nucleotide sequence of PC28-R is shown in SEQ ID NO:7; the nucleotide sequence of PC08-F is 5'-VIC-GGCCTCCCATCTTTATCCAATGT-3'; the nucleotide sequence of PC08-R is shown in SEQ ID NO:8; the nucleotide sequence of PC17-F is 5'-PET-CGCCACATGACCCCACCATC-3'; the nucleotide sequence of PC17-R is shown in SEQ ID NO:9; the nucleotide sequence of PC27-F is 5'-FAM-GCGGGCCACCTAATGATGATTG-3'; the nucleotide sequence of PC27-R is shown in SEQ ID NO:10.

[0006] Preferably, the reaction system for multiplex PCR amplification in S2 is: 3 μL DNA, 10 μL 2×Taq Plus Master Mix, 1 μL primer working solution, and ddH2O to a final volume of 20 μL; the primer working solution comprises: 40 μmol / L PC01-F 0.05μL, 40μmol / L PC01-R 0.05μL, 40μmol / L PC02-F 0.05μL, 40μmol / L PC02-R 0.05μL, 40μmol / LPC13-F 0.05μL, 40μmol / L PC13-R 0.05μL, 40μmol / L PC19-F 0.05μL, 40μmol / L PC19-R0.05μL, 40μmol / L PC20-F 0.05μL, 40μmol / L PC20-R 0.05μL, 40μmol / L PC26-F 0.05μL, 40μmol / L PC26-R 0.05μL, 40μmol / L PC28-F 0.05μL, 40μmol / L PC28-R 0.05μL, 40μmol / L PC08-F 0.05μL, 40μmol / L PC08-R 0.05μL, 40μmol / L PC17-F 0.05μL, 40μmol / L PC17-R0.05μL, 40μmol / L PC27-F 0.05μL and 40μmol / L PC27-R 0.05μL; The reaction program for the multiplex PCR amplification was as follows: 94℃ for 5 min; 94℃ for 45 s, 60℃ for 45 s, 72℃ for 45 s, 32 cycles; 72℃ for 7 min; and stored at 4℃.

[0007] Preferably, the denaturation condition in S3 is: denaturation at a temperature of 95°C for 5 minutes; the cooling condition is: cooling at a temperature of 4°C for 10 minutes.

[0008] Preferably, the volume ratio of the diluent, Liz-500 molecular weight internal standard, and deionized formamide in S3 is 1 μL: 0.15 μL: 8.85 μL.

[0009] Compared with the prior art, the present invention has the following advantages: This invention employs a stepwise elimination method, combining multiplex PCR with capillary electrophoresis. Through two amplification detections, 10 pairs of primers are divided into two multiplex amplification combinations. Compared with single amplification, this reduces the amount of primers, mixes, and other reagents used, saves costs, and improves sample detection efficiency. It is suitable for cotton variety identification and genetic analysis.

[0010] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0011] Figure 1 This is a schematic diagram of the multiple combination construction process in Embodiment 1 of the present invention.

[0012] Figure 2 This is a comparison chart of capillary electrophoresis detection effects under different PCR amplification modes in Example 1 of the present invention; wherein, a is single amplification with 10 pairs of primers; b is 10-fold amplification with 10 pairs of primers; and c is grouped multiplex amplification with 10 pairs of primers. Detailed Implementation

[0013] Example 1

[0014] This example uses New Tower Cotton No. 4 collected from the seed market.

[0015] The optimization process for the multiplex PCR amplification primer system for cotton SSR marker detection in this embodiment is as follows: Figure 1 As shown, the specific method includes the following steps: S1. Synthesize multiplex PCR amplification primer pairs for cotton SSR marker detection; The primer pairs for multiplex PCR amplification are PC01-F and PC01-R, PC02-F and PC02-R, PC13-F and PC13-R, PC19-F and PC19-R, PC20-F and PC20-R, PC26-F and PC26-R, PC28-F and PC28-R, PC08-F and PC08-R, PC17-F and PC17-R, and PC27-F and PC27-R. The nucleotide sequence of PC01-F is 5'-NED-CCACCAGCCTTACCTTATACGC-3'; the nucleotide sequence of PC01-R is shown in SEQ ID NO:1; the nucleotide sequence of PC02-F is 5'-FAM-AGTCCTCCCACTATTCGAAGCT-3'; the nucleotide sequence of PC02-R is shown in SEQ ID NO:2; the nucleotide sequence of PC13-F is 5'-NED-ACCATCCTTGCCGAGTAACCTG-3'; the nucleotide sequence of PC13-R is shown in SEQ ID NO:3; the nucleotide sequence of PC19-F is 5'-VIC-CCAGCCACTCGGAGATCCTTG-3'; the nucleotide sequence of PC19-R is shown in SEQ ID NO:4; the nucleotide sequence of PC20-F is 5'-PET-ACCACTTTGTCCACCTGTCCAC-3'; the nucleotide sequence of PC20-R is shown in SEQ ID NO:3. The nucleotide sequence of PC26-F is 5'-FAM-TTCCATGAGGCTATCCACAAGC-3'; the nucleotide sequence of PC26-R is shown in SEQ ID NO:6; the nucleotide sequence of PC28-F is 5'-PET-ACAAGCGCGTCTGCCATATCC-3'; the nucleotide sequence of PC28-R is shown in SEQ ID NO:7; the nucleotide sequence of PC08-F is 5'-VIC-GGCCTCCCATCTTTATCCAATGT-3'; the nucleotide sequence of PC08-R is shown in SEQ ID NO:8; the nucleotide sequence of PC17-F is 5'-PET-CGCCACATGACCCCACCATC-3'; the nucleotide sequence of PC17-R is shown in SEQ ID NO:9; the nucleotide sequence of PC27-F is 5'-FAM-GCGGGCCACCTAATGATGATTG-3'; the nucleotide sequence of PC27-R is shown in SEQ ID NO:10. S2. Randomly select 70 seeds from each sample, grind them, and put 200mg of powder into a 2.0mL centrifuge tube. Use the Tiangen Plant Kit (product number: DP320-03) to extract genomic DNA from the cotton sample to be tested as a template. Use an Implen N60 Touch ultra-micro spectrophotometer to detect the DNA mass concentration. After multiplex PCR amplification using the multiplex PCR amplification primers synthesized in S1, the amplification products are obtained. The reaction system for multiplex PCR amplification was as follows: 3 μL DNA, 10 μL 2×Taq Plus Master Mix, 1 μL primer working solution, and ddH2O to a final volume of 20 μL. The primer working solution comprises: 40 μmol / L PC01-F 0.05μL, 40μmol / L PC01-R 0.05μL, 40μmol / L PC02-F 0.05μL, 40μmol / L PC02-R 0.05μL, 40μmol / L PC13-F 0.05μL, 40μmol / LPC13-R 0.05μL, 40μmol / L PC19-F 0.05μL, 40μmol / L PC19-R 0.05μL, 40μmol / L PC20-F0.05μL, 40μmol / L PC20-R 0.05μL, 40μmol / L PC26-F 0.05μL, 40μmol / L PC26-R 0.05μL, 40μmol / L PC28-F 0.05μL, 40μmol / L PC28-R 0.05μL, 40μmol / L PC08-F 0.05μL, 40μmol / LPC08-R 0.05μL, 40μmol / L PC17-F 0.05μL, 40μmol / L PC17-R 0.05μL, 40μmol / L PC27-F0.05μL and 40μmol / L PC27-R 0.05μL; The reaction program for multiplex PCR amplification was as follows: 94℃ for 5 min; 94℃ for 45 s, 60℃ for 45 s, 72℃ for 45 s, 32 cycles; 72℃ for 7 min; store at 4℃. S3. Take 1 μL of the amplification product obtained in S2 and add it to a 96-well plate for the DNA analyzer. Dilute 40-fold with ddH2O to obtain a diluent. Mix 1 μL of the diluent with 0.15 μL of Liz-500 molecular weight internal standard and 8.85 μL of deionized formamide. Centrifuge for 10 s, then denature at 95°C for 5 min, cool at 4°C for 10 min, and briefly centrifuge for 10 s. Perform electrophoresis using a Thermo Fisher Scientific 3500 sequencer. The fluorescence capillary electrophoresis program is: pre-electrophoresis 15 kV 180 s; 1.6 kV injection 8 s; electrophoresis 19.5 kV 1330 s, using GeneMapper. TM The software reads the detection data; S4. Analyze the detection data read in S3, such as... Figure 2As shown, a) is single amplification with 10 primer pairs, b) is deca-amplification with 10 primer pairs, and c) amplification with 10 primer pairs divided into two groups, with single amplification of 10 primer pairs as a control. Seven primer pairs (PC01, PC02, PC13, PC19, PC20, PC26, and PC28) amplified the results, while three primer pairs (PC08, PC17, and PC27) showed disordered peak shapes and poor detection results. The primers that detected the results were grouped together, and the primers that did not detect the results were grouped together. A second PCR amplification was performed according to the groups. The amplification products of the two groups were mixed in equal volumes and the results were detected. All 10 primer pairs amplified the results.

[0016] Comparative Example 1 This comparative example shows a comparison between the results of singleton PCR amplification and multipleton PCR amplification.

[0017] The materials used in this comparative example are four cotton varieties collected from the market: Xinta Cotton No. 4, Xinta Cotton No. 13, Anonglu No. 23, and Xinmian No. 92.

[0018] The 10 primer pairs from Example 1 were used to perform singleton and multiplex amplification of DNA from four cotton varieties, as shown in Table 1. The fragment sizes of the amplified products were consistent, and the peak diagrams of singleton and multiplex amplification of Xinta Cotton 4 were basically the same. Compared with singleton amplification, the multiplex amplification system of this invention reduces the number of amplification steps from 10 primers to 2, while also reducing the amount of primers, mixes, and other reagents used, saving costs and improving sample detection efficiency.

[0019] Table 1. Primer electrophoresis detection results of different PCR amplification methods

[0020] In summary, this invention employs a stepwise elimination method, dividing 10 primer pairs into two multiplex amplification combinations through two amplification detections. This reduces the amount of primers, mixes, and other reagents used, saves costs, and improves sample detection efficiency.

[0021] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for optimizing a multiplex PCR amplification primer system for SSR marker detection in cotton, characterized in that, Includes the following steps: S1. Synthesize multiplex PCR amplification primer pairs for cotton SSR marker detection; The multiplex PCR amplification primer pairs are PC01-F and PC01-R, PC02-F and PC02-R, PC13-F and PC13-R, PC19-F and PC19-R, PC20-F and PC20-R, PC26-F and PC26-R, PC28-F and PC28-R, PC08-F and PC08-R, PC17-F and PC17-R, and PC27-F and PC27-R. S2. Extract genomic DNA from the cotton sample to be tested as a template, and perform multiplex PCR amplification using the multiplex PCR amplification primers synthesized in S1 to obtain the amplification product. S3. Take 1 μL of the amplification product obtained in S2, dilute it 40 times with ddH2O to obtain a diluent, mix the diluent with Liz-500 molecular weight internal standard and deionized formamide, centrifuge for 10s, and then perform capillary electrophoresis detection and read the detection data after denaturation, cooling and centrifugation in sequence. S4. Analyze the detection data read in S3 and divide the multiplex PCR amplification primer pairs into two groups of primer pairs. The first group of primer pairs includes PC01, PC02, PC13, PC19, PC20, PC26, and PC28, and the second group of primer pairs includes PC08, PC17, and PC27. S5. Perform a second multiplex PCR amplification using the two primer pairs described in S4. Mix the two amplification products in equal volumes, perform capillary electrophoresis detection, and read the detection data. Both primer pairs can amplify the results. The nucleotide sequence of PC01-F in S1 is 5'-NED-CCACCAGCCTTACCTTATACGC-3'; the nucleotide sequence of PC01-R is shown in SEQ ID NO:1; the nucleotide sequence of PC02-F is 5'-FAM-AGTCCTCCCACTATTCGAAGCT-3'; the nucleotide sequence of PC02-R is shown in SEQ ID NO:2; the nucleotide sequence of PC13-F is 5'-NED-ACCATCCTTGCCGAGTAACCTG-3'; the nucleotide sequence of PC13-R is shown in SEQ ID NO:3; the nucleotide sequence of PC19-F is 5'-VIC-CCAGCCACTCGGAGATCCTTG-3'; the nucleotide sequence of PC19-R is shown in SEQ ID NO:4; the nucleotide sequence of PC20-F is 5'-PET-ACCACTTTGTCCACCTGTCCAC-3'; the nucleotide sequence of PC20-R is shown in SEQ ID NO:

3. The nucleotide sequence of PC26-F is 5'-FAM-TTCCATGAGGCTATCCACAAGC-3'; the nucleotide sequence of PC26-R is shown in SEQ ID NO:6; the nucleotide sequence of PC28-F is 5'-PET-ACAAGCGCGTCTGCCATATCC-3'; the nucleotide sequence of PC28-R is shown in SEQ ID NO:7; the nucleotide sequence of PC08-F is 5'-VIC-GGCCTCCCATCTTTATCCAATGT-3'; the nucleotide sequence of PC08-R is shown in SEQ ID NO:8; the nucleotide sequence of PC17-F is 5'-PET-CGCCACATGACCCCACCATC-3'; the nucleotide sequence of PC17-R is shown in SEQ ID NO:9; the nucleotide sequence of PC27-F is 5'-FAM-GCGGGCCACCTAATGATGATTG-3'; the nucleotide sequence of PC27-R is shown in SEQ ID NO:

10.

2. The method for optimizing a multiplex PCR amplification primer system for cotton SSR marker detection according to claim 1, characterized in that, The reaction system for multiplex PCR amplification described in S2 is as follows: 3 μL DNA, 10 μL 2×Taq Plus Master Mix, 1 μL primer working solution, and ddH2O to a final volume of 20 μL. The primer working solution comprises: 0.05 μL of 40 μmol / L PC01-F, 0.05 μL of 40 μmol / L PC01-R, 0.05 μL of 40 μmol / L PC02-F, 0.05 μL of 40 μmol / L PC02-R, 0.05 μL of 40 μmol / L PC13-F, 0.05 μL of 40 μmol / L PC13-R, 0.05 μL of 40 μmol / L PC19-F, 0.05 μL of 40 μmol / L PC19-R, 0.05 μL of 40 μmol / L PC20-F, and 0.05 μL of 40 μmol / L PC20-R. 0.05μL, 40μmol / L PC26-F 0.05μL, 40μmol / L PC26-R 0.05μL, 40μmol / L PC28-F 0.05μL, 40μmol / L PC28-R 0.05μL, 40μmol / LPC08-F 0.05μL, 40μmol / L PC08-R 0.05μL, 40μmol / L PC17-F 0.05μL, 40μmol / L PC17-R0.05μL, 40μmol / L PC27-F 0.05μL and 40μmol / L PC27-R 0.05μL; The reaction program for the multiplex PCR amplification was as follows: 94℃ for 5 min; 94℃ for 45 s, 60℃ for 45 s, 72℃ for 45 s, 32 cycles; 72℃ for 7 min; and stored at 4℃.

3. The method for optimizing a multiplex PCR amplification primer system for cotton SSR marker detection according to claim 1, characterized in that, The denaturation conditions described in S3 are: denaturation at 95°C for 5 minutes; and the cooling conditions are: cooling at 4°C for 10 minutes.

4. The method for optimizing a multiplex PCR amplification primer system for cotton SSR marker detection according to claim 1, characterized in that, The volume ratio of the diluent, Liz-500 molecular weight internal standard, and deionized formamide in S3 is 1 μL: 0.15 μL: 8.85 μL.

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