Abelmoschus esculentus SNP primer and variety identification method

Abstract

The invention discloses an abelmoschus esculentus SNP primer and a variety identification method, and belongs to the technical field of variety identification. The invention discloses an abelmoschus esculentus SNP (Single Nucleotide Polymorphism) primer and a method for identifying the variety of abelmoschus esculentus based on the SNP primer. The method comprises the following steps: (1) extracting genome DNA (Deoxyribose Nucleic Acid) of the abelmoschus esculentus; (2) respectively mixing the 14 groups of primers with the genome DNA of the abelmoschus esculentus and a KASP reaction solution, carrying out KASP amplification, and reading a fluorescence signal; (3) performing SNP typing on the abelmoschus esculentus according to the fluorescence signal; and (4) judging a variety identification result. According to the invention, a technical method for performing okra typing analysis and variety identification by adopting a third-generation molecular marker is established for the first time, and the method has the advantages of good stability, high detection efficiency, multiple detection sites, good result accuracy and the like; and a useful research tool is provided for genetic diversity research, breeding material screening and variety authenticity inspection of the crops.

Description

Technical Field

[0001] The invention belongs to the technical field of variety identification, and particularly relates to a coffee sunflower SNP primer and a variety identification method. Background Art

[0002] Crop variety identification, also known as seed authenticity testing, is the analysis of the blood relationship between two crop samples (seeds or seedlings) through biological or molecular biological methods to determine whether they are two different varieties or the same variety. Variety identification can help breeders screen breeding materials and is very useful for genetic diversity and molecular marker-assisted breeding research of crops. Strengthen intellectual property protection in the breeding field to safeguard the interests of farmers, breeders and seed companies.

[0003] Single nucleotide polymorphism (SNP) is a type of molecular marker widely present in biological genomes, usually referred to as the third-generation molecular marker. SNP sequence variation is common in biological genomes, and different crop varieties have rich SNP polymorphisms, that is, different varieties of the same crop have different SNP sequences at specific genomic sites. By detecting the similarities and differences between a series of SNP markers in two different biological samples, it can be determined whether they belong to the same variety. Compared with the second-generation molecular markers (simple sequence repeats, SSR, i.e. microsatellite markers), SNP markers have more advantages in detection stability, detection efficiency, automation, and the number of detection sites. Variety identification technology based on SNP markers is the mainstream detection method internationally.

[0004] Coffee sunflower ( Abelmoschus esculentus ) commonly known as okra, is a special vegetable widely welcomed by people in recent years, with unique flavor and nutritional value. my country has rich germplasm resources of coffee sunflower, which is a good breeding material. Although there are technical standards for variety identification of crops such as rice and corn based on SNP markers, there is no technical method for coffee sunflower variety identification using SNP markers. Due to the great differences in gene sequences of different species, the SNP method used for identification of other crop varieties cannot be used for coffee sunflower variety identification. The present invention intends to fill this gap. Summary of the invention

[0005] In view of the above-mentioned prior art, the present invention provides a method for SNP primers and variety identification of coffee sunflower. For the first time, a technical method for typing analysis and variety identification of coffee sunflower using third-generation molecular markers has been established, which has the advantages of good stability, high detection efficiency, multiple detection sites, and good result accuracy; it provides a useful research tool for genetic diversity research, breeding material screening and variety authenticity testing of this crop.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is: to provide a method for coffee sunflower seed SNP primer and variety identification, including 14 sets of primers, whose sequence information is as follows: The forward primer AES01A is shown as SEQ ID No. 1 in the sequence listing; The forward primer AES01B is shown as SEQ ID No. 2 in the sequence listing; The reverse primer AER01 is shown as SEQ ID No. 3 in the sequence listing; The forward primer AES02A is shown as SEQ ID No. 4 in the sequence listing; The forward primer AES02B is shown as SEQ ID No. 5 in the sequence listing; Reverse primer AER02 is shown as SEQ ID No.6 in the sequence listing; The forward primer AES03A is shown as SEQ ID No. 7 in the sequence listing; The forward primer AES03B is shown as SEQ ID No. 8 in the sequence listing; Reverse primer AER03 is shown as SEQ ID No. 9 in the sequence listing; The forward primer AES04A is shown as SEQ ID No. 10 in the sequence listing; The forward primer AES04B is shown as SEQ ID No. 11 in the sequence listing; Reverse primer AER04 is shown as SEQ ID No. 12 in the sequence listing; The forward primer AES05A is shown as SEQ ID No. 13 in the sequence listing; The forward primer AES05B is shown as SEQ ID No. 14 in the sequence listing; Reverse primer AER05 is shown as SEQ ID No. 15 in the sequence listing; The forward primer AES06A is shown as SEQ ID No. 16 in the sequence listing; The forward primer AES06B is shown as SEQ ID No. 17 in the sequence listing; Reverse primer AER06 is shown as SEQ ID No. 18 in the sequence listing; The forward primer AES07A is shown as SEQ ID No. 19 in the sequence listing; The forward primer AES07B is shown as SEQ ID No. 20 in the sequence listing; Reverse primer AER07 is shown as SEQ ID No. 21 in the sequence listing; The forward primer AES08A is shown as SEQ ID No. 22 in the sequence listing; The forward primer AES08B is shown as SEQ ID No. 23 in the sequence listing; Reverse primer AER08 is shown as SEQ ID No. 24 in the sequence listing; The forward primer AES09A is shown as SEQ ID No. 25 in the sequence listing; Forward primer AES09B is shown as SEQ ID No. 26 in the sequence listing; Reverse primer AER09 is shown as SEQ ID No. 27 in the sequence listing; The forward primer AES10A is shown as SEQ ID No. 28 in the sequence listing; The forward primer AES10B is shown as SEQ ID No. 29 in the sequence listing; The reverse primer AER10 is shown as SEQ ID No. 30 in the sequence listing; The forward primer AES11A is shown as SEQ ID No. 31 in the sequence listing; The forward primer AES11B is shown as SEQ ID No. 32 in the sequence listing; The reverse primer AER11 is shown as SEQ ID No. 33 in the sequence listing; The forward primer AES12A is shown as SEQ ID No. 34 in the sequence listing; The forward primer AES12B is shown as SEQ ID No. 35 in the sequence listing; The reverse primer AER12 is shown as SEQ ID No. 36 in the sequence listing; The forward primer AES13A is shown as SEQ ID No. 37 in the sequence listing; The forward primer AES13B is shown as SEQ ID No. 38 in the sequence listing; Reverse primer AER13 is shown as SEQ ID No. 39 in the sequence listing; The forward primer AES14A is shown as SEQ ID No. 40 in the sequence listing; The forward primer AES14B is shown as SEQ ID No. 41 in the sequence listing; The reverse primer AER14 is shown as SEQ ID No. 42 in the sequence listing.

[0007] Based on the above technical solution, the present invention can also be improved as follows.

[0008] Furthermore, the 5' end of the forward primer is connected to a fluorescent marker, and two forward primers in the same group are respectively connected to fluorescent markers of different colors.

[0009] Furthermore, the above 14 groups of coffee sunflower SNP primers were used to identify coffee sunflower varieties.

[0010] Further, the method for identifying the coffee sunflower variety comprises the following steps: (1) Extracting genomic DNA from Amur cordata; (2) The 14 sets of primers were mixed with the genomic DNA of C. cerana and the KASP reaction solution, respectively, and KASP amplification was performed, and the fluorescence signal was read; (3) SNP typing of coffee sunflower based on fluorescence signals; (4) Determination of variety identification results: Comparison of coffee sunflower SNP typing, the determination criteria are: at the 14 SNP sites, if the number of difference sites ≥ 2, it is determined to be a different variety; if the number of difference sites = 1, it is determined to be a similar variety; if the number of difference sites = 0, it is determined to be very similar or the same variety.

[0011] Further, the conditions for KASP amplification are: First round: 95℃ for 10min; 95℃ for 20s, 61-55℃ for 1min, 10 touchdown PCR cycles, decreasing 0.6℃ each cycle; Second round: 95°C for 20 s, 55°C for 60 s, 35 cycles; finally, read the fluorescence signal data at 25°C.

[0012] Furthermore, after the second round, 95°C for 20 s and 57°C for 60 s were added for 3 cycles; and the fluorescence signal data were read at 25°C.

[0013] Furthermore, KASP amplification was performed on a high-throughput SNP typing detection platform, and the system was: Add 0.5 μL of 10 μM forward and reverse primer mixture to each well. After drying, add 0.8 μL of genomic DNA and 0.8 μL of 2×KASP reaction solution to form a 1.6 μL KASP total reaction system; 0.8 μL of genomic DNA contains 10-20 ng DNA.

[0014] Further, KASP amplification was performed on a real-time fluorescence quantitative PCR instrument, and the system was: Add 1 μL of 10 μM forward and reverse primer mixture, 1 μL of genomic DNA, 5 μL of 2×KASP reaction solution, and 3 μL of H 2 O, constitutes 10μL of KASP total reaction system; 1μL of genomic DNA contains 10~20ng DNA.

[0015] The beneficial effects of the present invention are as follows: SNP sequence variation is common in biological genomes, and different crop varieties have rich SNP polymorphisms, that is, different varieties of the same crop have different SNP sequences at specific genomic loci; by detecting the similarities and differences between a series of SNP markers in two different biological samples, it is possible to identify whether they belong to the same variety, and SNP markers have advantages in detection stability, detection efficiency, automation, and the number of detection sites. Competitive allele-specific PCR (Kompetitive Allele Specific PCR) is a SNP marker detection method based on PCR technology, which is used to detect whether SNP polymorphism (sequence difference) exists at the same SNP site. The present invention compares and analyzes the genome sequences of multiple coffee sunflower varieties / materials, selects SNP sites with sequence differences between different varieties / materials, screens 14 SNP markers that can be used to distinguish these coffee sunflower materials, and then designs fluorescent detection primers based on the sequences of these SNP markers. The 14 groups of coffee sunflower SNP primers and the method for variety identification in the present invention have the advantages of good stability, high detection efficiency, many detection sites, and good result accuracy. The present invention is the first to establish a technical method for typing analysis and variety identification of coffee sunflower using third-generation molecular markers (SNP markers), providing a useful research tool for genetic diversity research, breeding material screening and variety authenticity testing of this crop. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 These are the results of variety identification for 55 samples of C. coffee ambrette. DETAILED DESCRIPTION

[0017] The specific implementation modes of the present invention are described in detail below with reference to the embodiments.

[0018] Example 1 The specific steps for screening SNP loci for coffee sunflower variety identification and designing coffee sunflower SNP primers are as follows: (1) Simplified genome sequencing was performed on 50 samples of coffee sunflower, and sequence comparison analysis was performed to find the sites where SNP differences existed between the two samples; (2) There are 64 pairs of chromosomes in the coffee sunflower. For each pair of chromosomes, 10 SNP sites were selected according to the distribution of SNP sites on them and the principle of roughly uniform intervals (the distance between two adjacent SNP sites is roughly equal), thus obtaining a total of 640 candidate SNP markers; (3) Use primer design software (Primer Premier 5) to evaluate the DNA sequences near the candidate SNP markers, screen SNP sites suitable for primer design, and further screen out 48 candidate SNP markers; design forward primers and reverse primers (the forward primer is fluorescently labeled) for these SNP markers, and order primers from a biotechnology company; (4) DNA of the tested coffee sunflower material was extracted, KASP amplified with SNP marker primers, and typing analysis was performed on the instrument supporting software; SNP markers with poor amplification effect and SNP markers that could not effectively distinguish different varieties were eliminated, and finally 14 SNP markers that can be used to distinguish different coffee sunflower varieties were obtained. The SNP site number, chromosome, specific physical location and nucleotide typing information are as follows: AeSNP01 is located at position 29,276 on chromosome 1, and its nucleotide typing is G / T; AeSNP02 is located at position 5,733,858 on chromosome 3, and its nucleotide typing is C / T; AeSNP03 is located at position 184,591 on chromosome 5, and its nucleotide typing is C / T; AeSNP04 is located at position 97,320 on chromosome 6, and its nucleotide typing is A / G; AeSNP05 is located at position 1,008,375 on chromosome 9, and its nucleotide typing is A / T; AeSNP06 is located at position 813,241 on chromosome 11, and its nucleotide typing is A / G; AeSNP07 is located at position 64,592 on chromosome 25, and its nucleotide typing is A / C; AeSNP08 is located at position 338,911 on chromosome 28, and its nucleotide typing is C / G; AeSNP09 is located at position 39,517 on chromosome 36, and its nucleotide typing is A / C; AeSNP10 is located at position 2,216,790 on chromosome 42, and its nucleotide typing is A / T; AeSNP11 is located at position 468,135 on chromosome 48, and its nucleotide typing is A / C; AeSNP12 is located at position 75,286 on chromosome 55, and its nucleotide typing is C / T; AeSNP13 is located at position 8,271,696 on chromosome 59, and its nucleotide typing is G / T; AeSNP14 is located at position 581,117 on chromosome 62, and its nucleotide typing is G / T.

[0019] (5) Based on the above 14 SNP loci, SNP primers for coffee sunflower were designed as shown in Table 1.

[0020] Table 1 Sequence information of 14 pairs of SNP primers of coffee sunflower

[0021]

[0022] Example 2 The method for identifying the coffee sunflower variety is as follows: 1. Extraction of Genomic DNA from Coffea chinensis (1) For each seed sample to be tested, randomly select 30 to 50 seeds for germination, and then randomly select 20 seedlings, and cut 0.1 g of seedling leaves from each seedling; (2) Mix the leaves cut from the 20 seedlings as one sample, grind them into powder with liquid nitrogen, add 10 mL of 2×CTAB extraction buffer, mix thoroughly, and incubate at 65°C for 30 min. (3) Extract twice with 10 mL of a mixture of chloroform and isopropanol (the volume ratio of the two is 24:1), centrifuge at 12,000 rpm for 5 min at room temperature, take the supernatant, add an equal volume of isopropanol, and precipitate at room temperature for 30 min; (4) The precipitate was dissolved in 4 mL of double distilled water, washed once with pre-cooled 70% ethanol, dried naturally, dissolved in 2 mL of 0.1×TE buffer, and stored in a -20°C refrigerator for later use.

[0023] 2. KASP Amplification The 14 sets of primers were mixed with the genomic DNA of Amaranthus coffeei and KASP reaction solution respectively and KASP amplification was performed.

[0024] 1. KASP amplification and detection On the high-throughput SNP typing detection platform, amplification was performed using a 384-well reaction plate. 0.5 μL of a 10 μM forward and reverse primer mixture was added to each well. After drying, 0.8 μL (about 15 ng) of template DNA and 0.8 μL of 2×KASP Master Mix reaction solution were added in sequence to form a 1.6 μL KASP total reaction system. After sealing with the Nexar component, the 384-well plate was taken out for PCR amplification.

[0025] The template DNA is the mixed DNA from the 20 seedlings mentioned above.

[0026] The sequence information of 14 sets of coffee sunflower SNP marker detection primers used for KASP amplification is shown in Table 1, wherein the 5' end of the forward primer with the primer name ending in A is connected to red fluorescent protein, and the 5' end of the forward primer with the primer name ending in B is connected to green fluorescent protein.

[0027] 2. Thermal cycling conditions for KASP amplification are: (1) First round: 95°C for 10 min; 95°C for 20 s, 61-55°C for 1 min, 10 touchdown PCR cycles, decreasing 0.6°C each cycle; (2) Second round: 95°C for 20 s, 55°C for 60 s, 35 cycles; finally read the fluorescence signal data at 25°C.

[0028] (3) If the amplification result is not ideal, add 95℃ for 20s and 57℃ for 60s for 3 cycles after the second round; then read the fluorescence signal data at 25℃.

[0029] 3. Genotyping It is completed on the Araya component (enzyme reader) of the high-throughput SNP typing detection platform or on a fluorescence quantitative PCR instrument.

[0030] After KASP amplification, the instrument reads the fluorescence signal and automatically analyzes the amplification results through the analysis software supporting the instrument. The sample SNP typing is performed according to the principle of clear typing and NTC (no sample negative control) without specific amplification.

[0031] 4. Determination of Variety Identification Results (1) The genotype data of homozygous sites are recorded as X / X and Y / Y, where X and Y are two different alleles at the same site; the genotype data of heterozygous sites are recorded as X / Y; the allele data of deletion sites are recorded as - / -.

[0032] (2) Based on the differences in genotypes of different coffee sunflower samples at 14 SNP loci, the variety identification results were determined according to the following criteria: If the number of differential loci is ≥ 2, they are judged to be different varieties; The number of difference sites = 1, which is determined to be a similar variety; The number of difference sites = 0, which means they are judged to be very similar or the same variety.

[0033] Example 3 Compared with Example 2, the method for identifying coffee sunflower varieties is as follows: KASP amplification is performed on a real-time fluorescence quantitative PCR instrument, and a 96-well reaction plate is used for amplification. To each well, 1 μL of a mixture of forward and reverse primers each having a concentration of 10 μM, 1 μL of template DNA (about 15 ng), 5 μL of 2×KASP Master Mix reaction solution, and 3 μL of double distilled water are added to form a 10 μL KASP total reaction system for amplification; the remaining steps remain unchanged.

[0034] Example 4 The method of Example 2 was used to identify the varieties of 55 samples of coffee sunflower from all over the country. The identification results are as follows: Figure 1 As shown, it is shown that the 14 coffee sunflower SNP sites and their corresponding detection primers in the present invention are sufficient to distinguish coffee sunflower materials well.

[0035] Although the specific implementation of the present invention is described in detail in conjunction with the embodiments, it should not be understood as limiting the scope of protection of this patent. Within the scope described in the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. Coffee sunflower SNP primer, characterized in that, Includes 14 sets of primers, and their sequence information is as follows: The forward primer AES01A is shown as SEQ ID No. 1 in the sequence listing; The forward primer AES01B is shown as SEQ ID No. 2 in the sequence listing; The reverse primer AER01 is shown as SEQ ID No. 3 in the sequence listing; The forward primer AES02A is shown as SEQ ID No. 4 in the sequence listing; The forward primer AES02B is shown as SEQ ID No. 5 in the sequence listing; Reverse primer AER02 is shown as SEQ ID No.6 in the sequence listing; The forward primer AES03A is shown as SEQ ID No. 7 in the sequence listing; The forward primer AES03B is shown as SEQ ID No. 8 in the sequence listing; Reverse primer AER03 is shown as SEQ ID No. 9 in the sequence listing; The forward primer AES04A is shown as SEQ ID No. 10 in the sequence listing; The forward primer AES04B is shown as SEQ ID No. 11 in the sequence listing; Reverse primer AER04 is shown as SEQ ID No. 12 in the sequence listing; The forward primer AES05A is shown as SEQ ID No. 13 in the sequence listing; The forward primer AES05B is shown as SEQ ID No. 14 in the sequence listing; Reverse primer AER05 is shown as SEQ ID No. 15 in the sequence listing; The forward primer AES06A is shown as SEQ ID No. 16 in the sequence listing; The forward primer AES06B is shown as SEQ ID No. 17 in the sequence listing; Reverse primer AER06 is shown as SEQ ID No. 18 in the sequence listing; The forward primer AES07A is shown as SEQ ID No. 19 in the sequence listing; The forward primer AES07B is shown as SEQ ID No. 20 in the sequence listing; Reverse primer AER07 is shown as SEQ ID No. 21 in the sequence listing; The forward primer AES08A is shown as SEQ ID No. 22 in the sequence listing; The forward primer AES08B is shown as SEQ ID No. 23 in the sequence listing; Reverse primer AER08 is shown as SEQ ID No. 24 in the sequence listing; The forward primer AES09A is shown as SEQ ID No. 25 in the sequence listing; Forward primer AES09B is shown as SEQ ID No. 26 in the sequence listing; Reverse primer AER09 is shown as SEQ ID No. 27 in the sequence listing; The forward primer AES10A is shown as SEQ ID No. 28 in the sequence listing; The forward primer AES10B is shown as SEQ ID No. 29 in the sequence listing; The reverse primer AER10 is shown as SEQ ID No. 30 in the sequence listing; The forward primer AES11A is shown as SEQ ID No. 31 in the sequence listing; The forward primer AES11B is shown as SEQ ID No. 32 in the sequence listing; The reverse primer AER11 is shown as SEQ ID No. 33 in the sequence listing; The forward primer AES12A is shown as SEQ ID No. 34 in the sequence listing; The forward primer AES12B is shown as SEQ ID No. 35 in the sequence listing; The reverse primer AER12 is shown as SEQ ID No. 36 in the sequence listing; The forward primer AES13A is shown as SEQ ID No. 37 in the sequence listing; The forward primer AES13B is shown as SEQ ID No. 38 in the sequence listing; Reverse primer AER13 is shown as SEQ ID No. 39 in the sequence listing; The forward primer AES14A is shown as SEQ ID No. 40 in the sequence listing; The forward primer AES14B is shown as SEQ ID No. 41 in the sequence listing; The reverse primer AER14 is shown as SEQ ID No. 42 in the sequence listing.

2. The coffee sunflower SNP primer according to claim 1, characterized in that: The 5' end of the forward primer is connected to a fluorescent marker, and two forward primers in the same group are respectively connected to fluorescent markers of different colors.

3. A method for identifying coffee sunflower varieties, characterized in that: The coffee ambrette variety is identified using the coffee ambrette SNP primers described in claim 1 or 2.

4. The method for identifying coffee sunflower varieties according to claim 3, characterized in that: The following steps are involved: (1) Extracting genomic DNA from Amur cordata; (2) mixing the 14 sets of primers with the genomic DNA of the coffee sunflower and the KASP reaction solution respectively, performing KASP amplification, and reading the fluorescence signal; (3) SNP typing of coffee sunflower based on fluorescence signals; (4) Determination of variety identification results: Comparison of coffee sunflower SNP typing, the determination criteria are: at the 14 SNP sites, if the number of difference sites ≥ 2, it is determined to be a different variety; if the number of difference sites = 1, it is determined to be a similar variety; if the number of difference sites = 0, it is determined to be very similar or the same variety.

5. The method for identifying coffee sunflower varieties according to claim 4, characterized in that: The conditions for the KASP amplification are: First round: 95℃ for 10min; 95℃ for 20s, 61-55℃ for 1min, 10 touchdown PCR cycles, decreasing 0.6℃ each cycle; Second round: 95°C for 20 s, 55°C for 60 s, 35 cycles; finally, read the fluorescence signal data at 25°C.

6. The method for identifying coffee sunflower varieties according to claim 5, characterized in that: After the second round, the temperature was increased to 95°C for 20 s and 57°C for 60 s for 3 cycles; the fluorescence signal data was then read at 25°C.

7. The method for identifying coffee sunflower varieties according to claim 5 or 6, characterized in that: KASP amplification was performed on a high-throughput SNP typing detection platform, the system is: 0.5 μL of 10 μM forward and reverse primer mixture was added to each well, and after drying, 0.8 μL of genomic DNA and 0.8 μL of 2×KASP reaction solution were added to form a 1.6 μL KASP total reaction system; the 0.8 μL genomic DNA contained 10-20 ng DNA.

8. The method for identifying coffee sunflower varieties according to claim 5 or 6, characterized in that: KASP amplification was performed on a real-time fluorescence quantitative PCR instrument using the following system: 1 μL of 10 μM forward and reverse primer mixture, 1 μL of genomic DNA, 5 μL of 2×KASP reaction solution, and 3 μL of H2O were added to each well to form a 10 μL KASP total reaction system; the 1 μL genomic DNA contained 10-20 ng DNA.

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