Coffee Amur Sunflower SNP Primers and Variety Identification Method

Abstract

The present invention discloses SNP primers for coffee sunflower and a method for variety identification, belonging to the technical field of variety identification. The present invention discloses SNP primers for coffee sunflower and proposes a method for variety identification of coffee sunflower based on the SNP primers, comprising the following steps: (1) extracting genomic DNA of coffee sunflower; (2) mixing 14 sets of primers with the genomic DNA of coffee sunflower and a KASP reaction solution, performing KASP amplification, and reading the fluorescence signal; (3) performing SNP typing of coffee sunflower based on the fluorescence signal; and (4) determining the variety identification result. The present invention establishes for the first time a technical method for typing analysis and variety identification of coffee sunflower using third-generation molecular markers, 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.

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, involves analyzing the genetic relationship of two crop samples (seeds or seedlings) using biological or molecular methods to determine whether they are truly different varieties or the same. Variety identification helps breeders screen breeding materials and is extremely useful for research on crop genetic diversity and molecular marker-assisted breeding. Strengthening intellectual property protection in the breeding sector safeguards the interests of farmers, breeders, and seed companies.

[0003] Single nucleotide polymorphisms (SNPs) are a type of molecular marker widely found in biological genomes and are often referred to as third-generation molecular markers. SNP sequence variation is ubiquitous in biological genomes, and SNP polymorphism is abundant among different crop varieties. This means that different varieties of the same crop exhibit different SNP sequences at specific genomic loci. By detecting similarities and differences in a series of SNP markers between two different biological samples, it is possible to determine whether they belong to the same variety. Compared with second-generation molecular markers (simple sequence repeats, SSRs, or microsatellite markers), SNP markers offer advantages in detection stability, 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 Okra, commonly known as yellow okra or autumn okra, has become a popular specialty vegetable in recent years, boasting a unique flavor and nutritional value. my country possesses a rich germplasm resource of yellow okra, providing excellent breeding material. While SNP-based cultivar identification standards exist for crops such as rice and corn, there are no established methods for identifying yellow okra varieties using SNP markers. Due to the significant differences in gene sequences between species, SNP-based methods used for identifying other crop varieties are not applicable to identifying yellow okra varieties. This present invention aims to address this gap. Summary of the Invention

[0005] In response to the above-mentioned existing technologies, the present invention provides SNP primers and a method for variety identification in C. coffea. This is the first method to utilize third-generation molecular markers for typing analysis and variety identification in C. coffea. The method boasts excellent stability, high detection efficiency, multiple detection sites, and high accuracy, providing a useful research tool for studying the genetic diversity of this crop, screening breeding materials, and verifying variety authenticity.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is to provide a method for SNP primers and variety identification of coffee sunflower, including 14 sets of primers, whose sequence information is as follows:

[0007] The forward primer AES01A is shown as SEQ ID No. 1 in the sequence listing;

[0008] The forward primer AES01B is shown as SEQ ID No. 2 in the sequence listing;

[0009] The reverse primer AER01 is shown as SEQ ID No. 3 in the sequence listing;

[0010] The forward primer AES02A is shown as SEQ ID No. 4 in the sequence listing;

[0011] The forward primer AES02B is shown as SEQ ID No. 5 in the sequence listing;

[0012] Reverse primer AER02 is shown as SEQ ID No. 6 in the sequence listing;

[0013] The forward primer AES03A is shown as SEQ ID No. 7 in the sequence listing;

[0014] The forward primer AES03B is shown as SEQ ID No. 8 in the sequence listing;

[0015] Reverse primer AER03 is shown as SEQ ID No. 9 in the sequence listing;

[0016] The forward primer AES04A is shown as SEQ ID No. 10 in the sequence listing;

[0017] Forward primer AES04B is shown as SEQ ID No. 11 in the sequence listing;

[0018] Reverse primer AER04 is shown as SEQ ID No. 12 in the sequence listing;

[0019] The forward primer AES05A is shown as SEQ ID No. 13 in the sequence listing;

[0020] Forward primer AES05B is shown as SEQ ID No. 14 in the sequence listing;

[0021] Reverse primer AER05 is shown as SEQ ID No. 15 in the sequence listing;

[0022] The forward primer AES06A is shown as SEQ ID No. 16 in the sequence listing;

[0023] Forward primer AES06B is shown as SEQ ID No. 17 in the sequence listing;

[0024] Reverse primer AER06 is shown as SEQ ID No. 18 in the sequence listing;

[0025] The forward primer AES07A is shown as SEQ ID No. 19 in the sequence listing;

[0026] Forward primer AES07B is shown as SEQ ID No. 20 in the sequence listing;

[0027] Reverse primer AER07 is shown as SEQ ID No. 21 in the sequence listing;

[0028] The forward primer AES08A is shown as SEQ ID No. 22 in the sequence listing;

[0029] The forward primer AES08B is shown as SEQ ID No. 23 in the sequence listing;

[0030] Reverse primer AER08 is shown as SEQ ID No. 24 in the sequence listing;

[0031] The forward primer AES09A is shown as SEQ ID No. 25 in the sequence listing;

[0032] Forward primer AES09B is shown as SEQ ID No. 26 in the sequence listing;

[0033] Reverse primer AER09 is shown as SEQ ID No. 27 in the sequence listing;

[0034] The forward primer AES10A is shown as SEQ ID No. 28 in the sequence listing;

[0035] Forward primer AES10B is shown as SEQ ID No. 29 in the sequence listing;

[0036] The reverse primer AER10 is shown as SEQ ID No. 30 in the sequence listing;

[0037] The forward primer AES11A is shown as SEQ ID No. 31 in the sequence listing;

[0038] The forward primer AES11B is shown as SEQ ID No. 32 in the sequence listing;

[0039] Reverse primer AER11 is shown as SEQ ID No. 33 in the sequence listing;

[0040] The forward primer AES12A is shown as SEQ ID No. 34 in the sequence listing;

[0041] Forward primer AES12B is shown as SEQ ID No. 35 in the sequence listing;

[0042] Reverse primer AER12 is shown as SEQ ID No. 36 in the sequence listing;

[0043] The forward primer AES13A is shown as SEQ ID No. 37 in the sequence listing;

[0044] The forward primer AES13B is shown as SEQ ID No. 38 in the sequence listing;

[0045] Reverse primer AER13 is shown as SEQ ID No. 39 in the sequence listing;

[0046] The forward primer AES14A is shown as SEQ ID No. 40 in the sequence listing;

[0047] The forward primer AES14B is shown as SEQ ID No. 41 in the sequence listing;

[0048] The reverse primer AER14 is shown as SEQ ID No. 42 in the sequence listing.

[0049] On the basis of the above technical solution, the present invention can also be improved as follows.

[0050] Furthermore, the 5' end of the forward primer is connected to a fluorescent marker, and the two forward primers in the same group are respectively connected to fluorescent markers of different colors.

[0051] Furthermore, the above 14 groups of coffee ambrette SNP primers were used to identify coffee ambrette varieties.

[0052] Furthermore, the method for identifying the coffee sunflower variety comprises the following steps:

[0053] (1) Extracting genomic DNA from C. coffee sunflower;

[0054] (2) 14 sets of primers were mixed with the genomic DNA of C. coffee ambrosii and KASP reaction solution, and KASP amplification was performed, and the fluorescence signal was read;

[0055] (3) SNP typing of coffee sunflower based on fluorescence signals;

[0056] (4) Variety identification result determination: The SNP typing of coffee sunflower was compared, and the determination criteria were as follows: at the 14 SNP sites, if the number of difference sites ≥ 2, it was determined to be a different variety; if the number of difference sites = 1, it was determined to be a similar variety; if the number of difference sites = 0, it was determined to be very similar or the same variety.

[0057] Furthermore, the conditions for KASP amplification are:

[0058] First round: 95℃ for 10 min; 95℃ for 20 s, 61-55℃ for 1 min, 10 touchdown PCR cycles, decreasing by 0.6℃ each cycle;

[0059] Second round: 95°C for 20 s, 55°C for 60 s, 35 cycles; finally, the fluorescence signal data was read at 25°C.

[0060] 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 was read at 25°C.

[0061] Furthermore, KASP amplification was performed on a high-throughput SNP typing detection platform, and the system was:

[0062] 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 of DNA.

[0063] Furthermore, KASP amplification was performed on a real-time fluorescence quantitative PCR instrument using the following system:

[0064] 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 H2O to each well to form a 10 μL KASP total reaction system; 1 μL of genomic DNA contains 10-20 ng of DNA.

[0065] The present invention has the following beneficial effects: SNP sequence variation is ubiquitous in biological genomes, and different crop varieties exhibit abundant SNP polymorphisms. Specifically, different varieties of the same crop exhibit 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. SNP markers offer advantages in detection stability, efficiency, automation, and the number of detection sites. Competitive allele-specific PCR (CMS) is a PCR-based SNP marker detection method used to detect the presence of SNP polymorphisms (sequence differences) at the same SNP locus. The present invention comparatively analyzes the genomic sequences of multiple coffee ambrette varieties / materials, selects SNP sites with sequence differences between different varieties / materials, and screens 14 SNP markers that can be used to distinguish these coffee ambrette materials. Fluorescent detection primers are then designed based on the sequences of these SNP markers. The 14 sets of coffee ambrette SNP primers and the variety identification method of the present invention offer the advantages of good stability, high detection efficiency, a large number of detection sites, and high accuracy. This invention establishes for the first time 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

[0066] Figure 1 The results of variety identification of 55 samples of coffee ambrette materials are shown in the figure. DETAILED DESCRIPTION

[0067] The specific implementation methods of the present invention are described in detail below with reference to the embodiments.

[0068] Example 1

[0069] The specific steps for screening SNP sites for coffee sunflower variety identification and designing coffee sunflower SNP primers are as follows:

[0070] (1) Simplified genome sequencing was performed on 50 accessions of C. coffee sunflower, and sequence comparison analysis was performed to identify sites where SNP differences existed between each accession.

[0071] (2) C. coffee sunflower has 64 pairs of chromosomes. 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;

[0072] (3) Use primer design software (Primer Premier 5) to evaluate the DNA sequences near the candidate SNP markers, screen for SNP sites suitable for primer design, and further screen out 48 candidate SNP markers; design forward primers and reverse primers for these SNP markers (the forward primer is fluorescently labeled) and order the primers from a biotechnology company;

[0073] (4) DNA was extracted from the tested coffee sunflower material, and KASP amplified using SNP marker primers. Typing analysis was performed on the instrument software. SNP markers with poor amplification effects and those that could not effectively distinguish different varieties were eliminated. Finally, 14 SNP markers that can be used to distinguish different coffee sunflower varieties were obtained. The SNP site number, chromosome location, specific physical location, and nucleotide typing information are as follows:

[0074] AeSNP01 is located at position 29,276 on chromosome 1, and its nucleotide typing is G / T;

[0075] AeSNP02 is located at position 5,733,858 on chromosome 3, and its nucleotide typing is C / T;

[0076] AeSNP03 is located at position 184,591 on chromosome 5, and its nucleotide typing is C / T;

[0077] AeSNP04 is located at position 97,320 on chromosome 6, and its nucleotide typing is A / G;

[0078] AeSNP05 is located at position 1,008,375 on chromosome 9, and its nucleotide typing is A / T;

[0079] AeSNP06 is located at position 813,241 on chromosome 11, and its nucleotide typing is A / G;

[0080] AeSNP07 is located at position 64,592 on chromosome 25, and its nucleotide typing is A / C;

[0081] AeSNP08 is located at position 338,911 on chromosome 28, and its nucleotide typing is C / G;

[0082] AeSNP09 is located at position 39,517 on chromosome 36, and its nucleotide typing is A / C;

[0083] AeSNP10 is located at position 2,216,790 on chromosome 42, and its nucleotide typing is A / T;

[0084] AeSNP11 is located at position 468,135 on chromosome 48, and its nucleotide typing is A / C;

[0085] AeSNP12 is located at position 75,286 on chromosome 55, and its nucleotide typing is C / T;

[0086] AeSNP13 is located at position 8,271,696 on chromosome 59, and its nucleotide typing is G / T;

[0087] AeSNP14 is located at position 581,117 on chromosome 62, and its nucleotide typing is G / T.

[0088] (5) Based on the above 14 SNP loci, SNP primers for coffee sunflower were designed as shown in Table 1.

[0089] Table 1 Sequence information of 14 pairs of SNP primers for Coffea chinensis

[0090]

[0091]

[0092] Example 2

[0093] The specific steps for identifying coffee sunflower varieties are as follows:

[0094] 1. Extraction of Genomic DNA from Coffea chinensis

[0095] (1) For each seed sample to be tested, 30 to 50 seeds were randomly selected for seedling germination, and then 20 seedlings were randomly selected. 0.1 g of seedling leaves were cut from each seedling.

[0096] (2) Leaves cut from these 20 seedlings were mixed together as one sample, ground into powder using liquid nitrogen, and then mixed thoroughly with 10 mL of 2×CTAB extraction buffer. The mixture was then incubated at 65°C for 30 min.

[0097] (3) Extract twice with 10 mL of a mixture of chloroform and isoamyl alcohol (the volume ratio 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;

[0098] (4) The precipitate was dissolved in 4 mL of double-distilled water, washed once with pre-cooled 70% ethanol, dried naturally, and then dissolved in 2 mL of 0.1×TE buffer and stored in a -20°C refrigerator for later use.

[0099] 2. KASP Amplification

[0100] Fourteen primer sets were mixed with the genomic DNA of C. coffee ambrosii and KASP reaction solution respectively and then KASP amplification was performed.

[0101] 1. KASP Amplification and Detection: Amplification was performed using a 384-well reaction plate on a high-throughput SNP typing platform. 0.5 μL of a 10 μM mixture of forward and reverse primers was added to each well. After drying, 0.8 μL (approximately 15 ng) of template DNA and 0.8 μL of 2× KASP Master Mix were added sequentially to form a 1.6 μL KASP total reaction system. After sealing with the Nexar module, the 384-well plate was removed for PCR amplification.

[0102] The template DNA is the mixed DNA from the 20 seedlings mentioned above.

[0103] The sequence information of 14 sets of coffee sunflower SNP marker detection primers used for KASP amplification is shown in Table 1. Among them, the forward primers with the primer name ending in A are connected to the 5' end of red fluorescent protein, and the forward primers with the primer name ending in B are connected to the 5' end of green fluorescent protein.

[0104] 2. Thermal cycling conditions for KASP amplification are:

[0105] (1) First round: 95°C for 10 min; 95°C for 20 s, 61-55°C for 1 min, 10 touchdown PCR cycles, decreasing the temperature by 0.6°C per cycle;

[0106] (2) Second round: 95°C for 20 s, 55°C for 60 s, 35 cycles; finally, read the fluorescence signal data at 25°C.

[0107] (3) If the amplification results are not ideal, add 95°C for 20s and 57°C for 60s for 3 cycles after the second round; then read the fluorescence signal data at 25°C.

[0108] 3. Genotyping

[0109] This is done on the Araya component (microplate reader) of the high-throughput SNP typing detection platform or on a fluorescence quantitative PCR instrument.

[0110] After KASP amplification, the instrument reads the fluorescence signal and automatically analyzes the amplification results using the instrument's accompanying analysis software. The sample SNP typing is performed based on the principle of clear typing and no specific amplification of the NTC (no sample negative control).

[0111] IV. Determination of Variety Identification Results

[0112] (1) The genotype data of homozygous sites are recorded as X / X and Y / Y, where X and Y are two different allelic variants at the same site; the genotype data of heterozygous sites are recorded as X / Y; the allelic variant data of deletion sites are recorded as - / -.

[0113] (2) Based on the genotype differences of different coffee sunflower samples at 14 SNP sites, the variety identification results were determined according to the following criteria:

[0114] If the number of differential sites is ≥ 2, the varieties are judged to be different;

[0115] The number of difference sites = 1, which is determined to be a similar variety;

[0116] If the number of difference sites = 0, the varieties are judged to be very similar or identical.

[0117] Example 3

[0118] 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 (approximately 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.

[0119] Example 4

[0120] The method of Example 2 was used to identify 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 C. coffee ambrette SNP sites and their corresponding detection primers in the present invention are sufficient to well distinguish C. coffee ambrette materials.

[0121] Although the specific embodiments of the present invention have been described in detail in conjunction with the embodiments, this should not be construed as limiting the scope of protection of this patent. Within the scope described by 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 set, characterized in that, It consists of the following 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; 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; 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; 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; 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; 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; 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; Forward primer AES12B is shown as SEQ ID No. 35 in the sequence listing; 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; The 5' end of the forward primer is connected to a fluorescent marker, and the two forward primers in the same group are respectively connected to fluorescent markers of different colors.

2. A method for identifying coffee sunflower varieties, characterized by: Identifying a coffee ambrette variety using the coffee ambrette SNP primer set according to claim 1 comprises the following steps: (1) Extracting genomic DNA from C. coffee sunflower; (2) mixing the 14 sets of primers with the genomic DNA of the coffee sunflower and the KASP reaction solution, performing KASP amplification, and reading the fluorescence signal; (3) SNP typing of coffee sunflower based on fluorescence signals; (4) Variety identification result determination: The SNP typing of coffee sunflower was compared, and the determination criteria were as follows: at the 14 SNP sites, if the number of difference sites ≥ 2, it was determined to be a different variety; if the number of difference sites = 1, it was determined to be a similar variety; if the number of difference sites = 0, it was determined to be very similar or the same variety.

3. The method for identifying coffee sunflower varieties according to claim 2, characterized in that: The conditions for the KASP amplification are: First round: 95℃ for 10 min; 95℃ for 20 s, 61-55℃ for 1 min, 10 touchdown PCR cycles, decreasing by 0.6℃ each cycle; Second round: 95°C for 20 s, 55°C for 60 s, 35 cycles; finally, the fluorescence signal data was read at 25°C.

4. The method for identifying coffee sunflower varieties according to claim 3, wherein: After the second round, the cells were heated at 95°C for 20 s and 57°C for 60 s for 3 cycles; the fluorescence signal data were then read at 25°C.

5. The method for identifying coffee sunflower varieties according to claim 3 or 4, characterized in that: KASP amplification was performed on a high-throughput SNP typing detection platform using the following system: 0.5 μL of 10 μM forward and reverse primer mixture was added to each well. 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.

6. The method for identifying coffee sunflower varieties according to claim 3 or 4, 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 H 2 O were added to each well to form a 10 μL KASP total reaction system; the 1 μL genomic DNA contained 10-20 ng of DNA.

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