Method for rapidly extracting genome DNA of single rape seed for KASP typing and application

Abstract

The invention relates to the technical field of genome DNA extraction, in particular to a method for rapidly extracting genome DNA of a single rape seed for KASP typing and application. The method comprises the following steps: sampling by adopting a seed sampling plate matched with a deep hole plate; soaking the rape seeds with salicylic acid; grinding the soaked seeds to obtain a grinding material; a TPS extracting solution is added into the grinding material, and a mixed solution is obtained after vibration and uniform mixing; carrying out warm bath treatment on the mixed liquid; centrifuging for the first time, extracting supernate, adding isopropanol into the supernate, oscillating, and freezing; after unfreezing, carrying out second-time centrifugal treatment, pouring out supernate, retaining precipitates, and carrying out drying treatment; and adding ultrapure water into the dried precipitate, and carrying out oscillation dissolution to obtain the genome DNA extracting solution of the rape seeds. According to the application, the method is applied to purity identification of hybrid rape seeds. According to the invention, rapid extraction and rapid typing of genome DNA can be realized.

Description

Technical Field

[0001] The invention relates to the technical field of genomic DNA extraction, and in particular to a method and application of rapid extraction of genomic DNA of single-seed oilseed seeds for KASP typing. Background Art

[0002] Rapeseed is one of the important oil crops in my country, with a planting area of ​​about 100 million mu. Hybrid rapeseed has obvious hybrid vigor and is widely used in rapeseed production in my country. The hybrid vigor of rapeseed is closely related to its seed purity, and the purity of seeds directly affects the yield performance of hybrid rapeseed. The latest "Seed Quality and Inspection Standards" stipulates that the purity of hybrid rapeseed seeds should not be less than 86%. Therefore, a fast, accurate and applicable hybrid rapeseed seed purity detection method is very important and urgent to prevent unqualified seeds from entering the market and causing losses to production.

[0003] Establishing a seed purity identification technology system based on molecular markers is an important way to improve the efficiency of seed purity identification. So far, predecessors have used molecular markers such as SRAP (Sequence Related Amplified Polymorphism), RAPD (Random Amplified Polymorphic DNA), SSR (Simple Sequence Repeat) and ISSR (Inter-Simple Sequence Repeat) to identify the purity of Brassica napus hybrids and achieved good results. However, the above molecular marker technology cannot meet the needs of high throughput and low cost. In recent years, competitive allele-specific PCR (Kompetitive Allele Specific PCR, KASP) has been widely used in a variety of crops due to its simplicity, repeatability, stability, accuracy, rapidity and low cost.

[0004] At present, in the application process of KASP technology, the main speed-limiting link is the DNA extraction process. The Chinese patent application with publication number CN103060311A discloses that rapeseed seeds peeled after germination are used as materials, and the alkali cracking method is used to extract DNA; the Chinese patent application with publication number CN104195133A discloses that young and tender leaves of rapeseed are used as materials, and the high salt method is used to extract DNA. These two methods require germination, cultivation and removal of seed coats of rapeseed, and the extraction of rapeseed DNA is time-consuming, costly and cumbersome. In order to meet the timeliness requirements of seed production for genotyping and purity identification, it is necessary to develop a method and application for rapid extraction of genomic DNA of single-grain rapeseed seeds for KASP typing, and realize the full automation of single-grain rapeseed seed genotyping, so as to solve the problems of time-consuming, high cost and cumbersome operation in the existing technology for extracting rapeseed DNA. Summary of the invention

[0005] The present invention aims to provide a method and application for rapid extraction of genomic DNA from single-seed oilseed seeds for KASP typing. The specific technical scheme is as follows:

[0006] In a first aspect, the present invention provides a method for rapid extraction of genomic DNA from single-seed oilseed seeds for KASP typing, comprising:

[0007] Step S1, sampling using a seed sampling plate matched with a deep-well plate, ensuring that there is one rapeseed seed in each well of the deep-well plate; soaking the rapeseed sample with salicylic acid;

[0008] Step S2, grinding the soaked rapeseed sample to obtain abrasive;

[0009] Step S3, adding TPS extract to the abrasive, and mixing by oscillation to obtain a mixed solution; and subjecting the mixed solution to a warm bath treatment;

[0010] Step S4, after the mixed solution after the warm bath treatment is subjected to the first centrifugation treatment, the supernatant is extracted, isopropanol is added to the supernatant, and after shaking, freezing treatment is performed;

[0011] Step S5: After thawing, centrifuge for the second time, discard the supernatant, retain the precipitate and dry it;

[0012] Step S6, adding ultrapure water to the dried precipitate, and after shaking and dissolving, obtaining a DNA extract of rapeseed seeds, which is used to complete rapid genotyping of single rapeseed seeds.

[0013] Optionally, in step S1, the concentration of salicylic acid used is 10-20 mg / L.

[0014] Optionally, in step S2, the salicylic acid soaking treatment is performed at a temperature of 25-28°C and a time of 2-6 hours.

[0015] Optionally, in step S3, the temperature of the warm bath treatment is 65-75° C. and the time is 30-40 min; during the warm bath treatment, the mixed solution is shaken intermittently.

[0016] Optionally, in step S4, the freezing treatment temperature is -20--18°C, and the freezing treatment time is more than 1 hour.

[0017] Optionally, in step S4, the first centrifugation treatment adopts a centrifugation temperature of 4°C, a centrifugation time of 3500-4500 rpm, and a centrifugation time of 10-20 min.

[0018] Optionally, in step S5, the drying treatment temperature is 60-70° C. and the time is 20-40 min.

[0019] Optionally, in step S5, the second centrifugation treatment adopts a centrifugation temperature of 4°C, a centrifugation time of 3500-4500 rpm, and a centrifugation time of 10-20 min.

[0020] In a second aspect, the present invention provides an application of the method for rapid extraction of genomic DNA from single-seed rapeseed seeds for KASP typing in the purity identification of hybrid rapeseed seeds.

[0021] Optionally, primers used in KASP genotyping include BN900004_K01.

[0022] The application of the technical solution of the present invention has at least the following beneficial effects:

[0023] The present invention provides a method for rapid extraction of genomic DNA of single rapeseed seeds for KASP typing, which realizes full automation of genomic DNA extraction and genotyping of single rapeseed seeds. Specifically, the present invention adopts a seed sampling plate matched with a deep-well plate in step S1 for sampling, which realizes high-throughput seed sampling and greatly improves the sampling efficiency of seeds; soaking rapeseed seeds in salicylic acid can promote rapeseed seed cell division, increase the DNA inside the seeds, and after combined with steps S2-S6, high-throughput extraction of single rapeseed seed DNA can be achieved to meet the KASP genotyping requirements; in addition, the present invention does not require the removal of seed coats or seed germination, which can shorten the sampling time by 3-5 days, and the use of the TPS method can realize rapid extraction of genomic DNA of single rapeseed seeds. The extracted DNA meets the requirements of the LGCA384 system and can achieve high-throughput rapid typing of rapeseed genotyping. DETAILED DESCRIPTION

[0024] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0025] The seed sampling plate matched with the 96 deep-well plate was used for sampling, and the rapeseed seeds were soaked in salicylic acid with concentrations of 5 mg / L, 10 mg / L, 20 mg / L and 40 mg / L respectively; the temperature of the salicylic acid soaking treatment was 28°C and the time was 2h. DNA extraction of single seeds was carried out according to the TPS method in the invention content, and 1 μl DNA was taken from the extracted DNA extract as a template, and KASP detection typing was performed in the 384 system of LGC (Laboratory of the Government Chemist) Co., Ltd. in the UK, the primer was BN900004_K01, and the KASP marker detection results were shown in Table 1. In the actual operation process, the sample detection rate can reach more than 90%, which can meet the production needs, so the concentration of salicylic acid is determined to be 10-20 mg / L.

[0026] Table 1 KASP marker detection results of rapeseed seeds treated with different concentrations of salicylic acid

[0027] Salicylic acid concentration Number of samples tested (grains) Number of samples detected (grains) Detection rate (%) 5mg / L 96 86 89.58 10mg / L 96 91 94.79 20mg / L 96 94 97.92 40mg / L 96 94 97.92

[0028] The seed sampling plate matched with the 96 deep-well plate was used for sampling, and the rapeseed samples were soaked in salicylic acid with a concentration of 20 mg / L at 28°C for 0 h, 2 h, 4 h, 6 h, and 12 h, respectively. The genomic DNA of a single seed was extracted according to the TPS method in the invention content, and 1 μl of DNA was taken from the extracted DNA extract as a template, and the KASP detection typing was performed in the 384 system of LGC (Laboratory of the Government Chemist) Co., Ltd. in the UK, and the primer was BN900004_K01. The KASP marker detection results are shown in Table 2. In the actual operation process, the sample detection rate can reach more than 90%, which can meet the production demand. Considering the time cost, the soaking time of salicylic acid is determined to be 2-6 h.

[0029] Table 2 KASP marker detection results of rapeseed seeds treated with salicylic acid at different soaking times

[0030] Soaking time Number of samples tested (grains) Number of samples detected (grains) Detection rate (%) 0h 96 76 79.17 2h 96 92 95.83 4h 96 93 97.92 6h 96 94 97.12 12h 96 93 96.20

[0031] Embodiment 1:

[0032] A method for rapid extraction of genomic DNA from single-seed oilseed seeds suitable for KASP typing, comprising:

[0033] Step S1, sampling using a seed sampling plate matched with a 96-deep-well plate, ensuring that there is one rapeseed seed in each well of the deep-well plate; adding 20 μl of salicylic acid with a concentration of 20 mg / L to soak the rapeseed seed sample; the temperature of the salicylic acid soaking treatment is 28° C. and the time is 2 h;

[0034] Step S2, grinding the soaked rapeseed sample with a steel ball to obtain abrasive;

[0035] Step S3, adding 500 μl of TPS extract (the formula of the TPS extract is as follows: 10 ml of 1M (pH 8.0) Tris-HCl, 2 ml of 0.5M EDTA (pH 8.0), 7.45 g of KCl and adding ddH2O to 100 ml) to the abrasive, and obtaining a mixed solution after oscillation and mixing; subjecting the mixed solution to warm bath treatment; the temperature of the warm bath treatment is 75° C., and the time is 40 min; during the warm bath treatment, the mixed solution is intermittently oscillated;

[0036] Step S4, after the mixed solution after the warm bath treatment is subjected to the first centrifugation treatment, 190 μl of the supernatant is extracted, and 190 μl of isopropanol is added to the supernatant, and after vortexing on a vortex oscillator, a freezing treatment is performed; the freezing treatment temperature is -20°C, and the freezing treatment time is more than 1 hour; the centrifugation temperature used in the first centrifugation treatment is 4°C, the centrifugation time is 4000 rpm, and the centrifugation time is 10 min;

[0037] Step S5, after thawing, perform a second centrifugation, pour out the supernatant, retain the precipitate and dry it; the drying temperature used is 65°C, and the time is 30 minutes; the second centrifugation temperature used is 4°C, the centrifugation time is 4000rpm, and the centrifugation time is 10min;

[0038] Step S6, adding ultrapure water to the dried precipitate, and dissolving it by vortex oscillator to obtain a genomic DNA extract of a single rapeseed seed, which is used to complete rapid genotyping of the single rapeseed seed.

[0039] The rapeseed seeds used in step S1 are seeds of the restorer line SC4R.

[0040] Embodiment 2:

[0041] The difference from Example 1 is that the rapeseed seeds used in step S1 are seeds of maintainer line 20B.

[0042] Embodiment 3:

[0043] The difference from Example 1 is that the rapeseed seeds used in step S1 are seeds of the sterile line 20A.

[0044] Embodiment 4:

[0045] Different from the embodiment 1, the rapeseed seeds used in step S1 are seeds of the hybrid rapeseed variety Fengyou 306 .

[0046] Embodiment 5:

[0047] Different from Example 1, the rapeseed seeds used in step S1 are seeds of the hybrid rapeseed variety Qinyou 30.

[0048] Embodiment 6:

[0049] Different from Example 1, the rapeseed seeds used in step S1 are seeds of the hybrid rapeseed variety Fengyou 737. Take 1 μl of DNA from the DNA extract obtained in Example 1-6 as a template, and perform KASP typing in the 384 system of LGC (Laboratory of the Government Chemist) Co., Ltd. in the UK. The primer used for detection is BN900004_K01. The results of KASP marker detection are shown in Table 3. In addition, 400 rapeseed seeds of Example 1-6 were sown in the identification plot of the planting base of Hunan Crop Research Institute. Each plot has 9 rows, a row length of 2.0 m, and a row spacing of 0.5 m. They are sown evenly and are not thinned out during the entire growth period. In the flowering period, the purity is investigated according to the fertility performance of the plants. The field planting identification purity (%) = (number of plants for inspection - number of sterile plants) × 100 / number of plants for inspection.

[0050] The results are shown in Table 3. The sample detection rates of the seeds of the three parent materials and four hybrid rapeseed varieties tested were 98.40%-100.00%, indicating that DNA can be efficiently extracted under this method and applied to KASP typing.

[0051] Table 3 KASP marker detection results of Brassica napus hybrid samples

[0052]

[0053]

[0054] As shown in Table 4, the results of the KASP marker detection purity of the three parental materials and the field planting identification purity are completely consistent, while the difference between the KASP marker detection purity of the three hybrid rapeseed varieties and the field planting identification purity is 1.7%-2.8%. The results show that the results of KASP genotyping by extracting DNA using this method are very consistent with the results of field planting identification, indicating that this method can be well used in the field purity identification of hybrid rapeseed seeds.

[0055] Table 4 Comparison of KASP marker detection results and field purity detection results of Brassica napus hybrid samples

[0056]

[0057] In summary, the present invention has determined a method for rapid extraction of genomic DNA from single-seed rapeseed seeds suitable for KASP typing, which can efficiently extract DNA and realize rapid and high-throughput genotyping of rapeseed seeds through KASP marker detection. The extraction method uses a seed sampling plate matched with a deep-well plate for sampling, and can be sampled by soaking in salicylic acid. Compared with conventional rapeseed seed germination sampling, the sampling time can be shortened by 3-5 days, and the labor cost can be effectively saved, which greatly improves the experimental efficiency.

[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for rapid extraction of genomic DNA from single-seed oilseed seeds for KASP typing, characterized in that: include: Step S1, sampling using a seed sampling plate matched with a deep-well plate, ensuring that there is one rapeseed seed in each well of the deep-well plate; soaking the rapeseed sample with salicylic acid; Step S2, grinding the soaked rapeseed sample to obtain abrasive; Step S3, adding TPS extract to the abrasive, and mixing by shaking to obtain a mixed solution; Treating the mixed solution in a temperature bath; Step S4, after the mixed solution after the warm bath treatment is subjected to the first centrifugation treatment, the supernatant is extracted, isopropanol is added to the supernatant, and after shaking, freezing treatment is performed; Step S5: After thawing, centrifuge for the second time, discard the supernatant, retain the precipitate and dry it; Step S6, adding ultrapure water to the dried precipitate, and after shaking and dissolving, obtaining a genomic DNA extract of a single rapeseed seed for rapid genotyping of the single rapeseed seed.

2. The method for rapid extraction of genomic DNA from single-seed oilseed seeds for KASP typing according to claim 1, characterized in that: In step S1, the concentration of salicylic acid used is 10-20 mg / L.

3. The method for rapid extraction of genomic DNA from single-seed oilseed seeds for KASP typing according to claim 1, characterized in that: In step S1, the temperature of the salicylic acid soaking treatment is 25-28°C and the time is 2-6 hours.

4. The method for rapid extraction of genomic DNA from single-seed oilseed seeds for KASP typing according to claim 1, characterized in that: In step S3, the temperature of the warm bath treatment is 65-75° C. and the time is 30-40 min. During the warm bath treatment, the mixed solution is shaken intermittently.

5. The method for rapid extraction of genomic DNA from single-seed oilseed seeds for KASP typing according to claim 1, characterized in that: In step S4, the freezing treatment temperature is -20-18°C and the freezing treatment time is more than 1 hour.

6. The method for rapid extraction of genomic DNA from single-seed oilseed seeds for KASP typing according to claim 1, characterized in that: In step S4, the centrifugation temperature used in the first centrifugation treatment is 4°C, the centrifugation time is 3500-4500 rpm, and the centrifugation time is 10-20 min.

7. The method for rapid extraction of genomic DNA from single-seed oilseed seeds for KASP typing according to claim 1, characterized in that: In step S5, the drying process is carried out at a temperature of 60-70°C and a time of 20-40 minutes.

8. The method for rapid extraction of genomic DNA from single-seed oilseed seeds for KASP typing according to claim 1, characterized in that: In step S5, the second centrifugation treatment adopts a centrifugation temperature of 4°C, a centrifugation time of 3500-4500 rpm, and a centrifugation time of 10-20 min.

9. Use of the method for rapid extraction of genomic DNA from single-seed rapeseed seeds for KASP typing according to any one of claims 1 to 8 in purity identification of hybrid rapeseed seeds.

10. The use according to claim 9, characterized in that: Primers used in KASP genotyping included BN900004_K01.

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