SNP site combination for cotton DNA fingerprinting, KASP primer and application thereof

Abstract

The application discloses a SNP site combination for cotton DNA fingerprint construction, a KASP primer and application thereof, relates to the technical field of plant genetic breeding, and comprises 26 core SNP sites; the 26 core SNP sites are uniformly distributed on 7 chromosomes of a cotton A subgroup and 7 chromosomes of a D subgroup, and cover 14 chromosomes in total; the SNP site combination for cotton DNA fingerprint construction, the KASP primer and application thereof are characterized in that a standardized KASP primer combination is designed for the 26 core SNP sites, VIC / FAM fluorescent label sequences are added to the tail of the allele-specific primer, PAGE purification is combined to guarantee the quality of the primer, and allele-specific recognition and accurate distinction of the fluorescent signal are ensured, so that the typing result is clear and the misjudgment rate is low.

Description

Technical Field

[0001] This invention relates to the field of plant genetics and breeding technology, specifically to SNP site combinations, KASP primers, and their applications for constructing cotton DNA fingerprinting. Background Technology

[0002] Cotton (Gossypium spp.) is one of my country's important economic crops. Currently, cotton variety identification mainly relies on field phenotypic identification and simple sequence repeat (SSR) molecular marker technology. However, traditional SSR marker detection usually relies on gel electrophoresis or capillary electrophoresis for fragment analysis, which involves complex experimental procedures, limited detection efficiency, and difficulties in data integration between different laboratories, thus significantly limiting its application in large-scale, high-throughput variety identification. In recent years, single nucleotide polymorphisms (SNPs) have gradually become important molecular markers for plant variety identification due to their wide distribution across the genome, abundant quantity, high genetic stability, and suitability for automated high-throughput detection. Gene chips developed based on SNPs have been widely used in cotton genetic research and molecular breeding, but their detection cost is high, they rely on large-scale detection platforms, and are difficult to promote and apply in grassroots seed companies and routine testing.

[0003] However, the following technical problems still exist in the existing technology: (1) Traditional SNP site screening methods mostly rely on a single data source or a single quality indicator, with limited coverage area and easy omission of key variation areas, resulting in the selected marker set failing to fully reflect the genetic diversity of the sample and low variety identification efficiency; (2) The determination of the number of core SNP sites is mostly based on experience, lacking a scientific method for determining the number. Too many markers lead to increased detection costs, while too few markers result in insufficient variety differentiation ability; (3) There is a lack of standardized core SNP site combinations and supporting KASP detection systems suitable for constructing DNA fingerprint maps of cotton varieties, which restricts the standardized development and large-scale application of cotton variety identification work. Therefore, developing a cotton KASP fingerprint map construction method based on core SNP sites is of great significance for cotton variety authenticity identification, seed purity detection, and germplasm resource management. Summary of the Invention

[0004] The purpose of this invention is to provide SNP site combinations, KASP primers and their applications for constructing DNA fingerprinting profiles of cotton, in order to solve the problems of insufficient genetic representativeness of marker sets due to the single SNP site screening method in the prior art, the lack of scientific basis for determining the number of core sites and the difficulty in balancing identification efficiency and detection cost, as well as the lack of standardized core SNP site combinations and KASP detection systems suitable for cotton.

[0005] To achieve the above objectives, the present invention provides the following technical solution: an SNP locus combination for constructing a cotton DNA fingerprint, wherein the SNP locus combination consists of the following 26 core SNP loci: A02_2198127, A02_6551077, A03_103423966, A04_79410894, A04_75510070, A05_6526611, A06_43274017, A08_119076232, A09_14286848, D02_5778414, D0 2_1231245, D04_2753129, D05_54798658, D05_13296669, D05_15195826, D05_860461, D05_859033, D05_12748666, D05 _56872688, D07_21485004, D07_12158735, D09_8614926, D10_4758937, D10_59804947, D10_62797734, D12_12899667;

[0006] The 26 core SNP loci are evenly distributed across 7 chromosomes in subgroup A and 7 chromosomes in subgroup D of cotton, covering a total of 14 chromosomes. The 7 chromosomes in subgroup A are ChrA02, ChrA03, ChrA04, ChrA05, ChrA06, ChrA08, and ChrA09, and the 7 chromosomes in subgroup D are ChrD02, ChrD04, ChrD05, ChrD07, ChrD09, ChrD10, and ChrD12.

[0007] Furthermore, the KASP primer combination is designed for the above 26 core SNP sites, with 3 primers designed for each SNP site: a first allele-specific primer F1 with a VIC fluorescent tag sequence, a second allele-specific primer F2 with a FAM fluorescent tag sequence, and a universal reverse primer R;

[0008] The VIC fluorescent tag sequence is 5'-GAAGGTCGGAGTCAACGGATT-3', and the FAM fluorescent tag sequence is 5'-GAAGGTGACCAAGTTCATGCT-3';

[0009] The primer sequences at each point in the KASP primer combination are shown in SEQ ID NO:1 to SEQ ID NO:78.

[0010] This invention also discloses the application of SNP site combinations in the construction of cotton DNA fingerprinting.

[0011] This invention also discloses the application of KASP primer combinations in the construction of cotton DNA fingerprinting, including the following steps:

[0012] S1. Extract genomic DNA from the cotton sample to be tested and adjust the DNA concentration to 10 ng / μL;

[0013] S2. Using the genomic DNA obtained in S1 as a template, perform PCR amplification using the KASP primer combination to obtain the amplification product;

[0014] S3. Read the VIC fluorescence signal and FAM fluorescence signal of the amplification product, perform genotyping based on the fluorescence signal, and obtain the genotype information of the cotton sample to be tested at 26 core SNP loci.

[0015] S4. Construct the DNA fingerprint of the cotton sample to be tested based on the genotype information obtained in S3.

[0016] Furthermore, the PCR amplification employs a Touchdown gradient cooling annealing strategy, and the PCR amplification program is as follows:

[0017] Pre-denaturation: 94℃, 10 min, 1 cycle;

[0018] Denaturation: 94℃, 20 sec, 10 cycles;

[0019] Refolding / Extension: Temperature was reduced from 61°C to 55°C at -0.6°C / cycle, 45 sec, for 10 cycles;

[0020] Second denaturation: 94℃, 20 sec, 35 cycles;

[0021] Re-annealing / extension: 55°C, 45 sec, 35 cycles.

[0022] Furthermore, the total reaction volume of the PCR amplification reaction system is 5 μL, and the components are: 2×Taq DNAPolymerase Mix 2 μL, 10×SNP Primer Mix 1 μL, and DNA sample 2 μL; each reaction plate is equipped with two template-free controls in which ddH2O is used instead of DNA template.

[0023] Furthermore, the criteria for interpreting genotypes in S3 are as follows: in a scatter plot with VIC fluorescence intensity as the X-axis and FAM fluorescence intensity as the Y-axis, strong VIC / weak FAM indicates AA homozygous genotype, strong FAM / weak VIC indicates BB homozygous genotype, and VIC / FAM signals centered indicate AB heterozygous genotype.

[0024] This invention also discloses the application of the SNP site combination or the KASP primer combination in at least one of the following scenarios: cotton variety authenticity identification, cotton germplasm resource management, cotton plant variety rights protection, cotton breeding material identification, and cotton seed quality testing.

[0025] Compared with existing technologies, the SNP site combinations, KASP primers, and their applications provided by this invention for constructing cotton DNA fingerprinting have the following beneficial effects:

[0026] (1) This invention constructs a multi-dimensional comprehensive quality scoring system, integrates four indicators: missing rate, MAF, Hardy-Weinberg equilibrium test value and Shannon entropy, and adopts a dual-standard collaborative filtering strategy to comprehensively and objectively evaluate large-scale resequencing SNP sites, which improves the limitations of traditional methods that rely on a single indicator and significantly improves the overall data reliability and variety identification efficiency of the marker set.

[0027] (2) By introducing the marker recognition efficiency curve analysis method, this invention accurately determines the number of core sites based on the marginal gain inflection point of the cumulative differentiation ability of varieties with the number of markers, avoiding the arbitrariness of selection based on experience, achieving the optimal variety differentiation effect with a minimum of 26 markers, and significantly reducing the detection cost while ensuring the efficiency of variety identification.

[0028] (3) This invention designs a standardized KASP primer combination for 26 core SNP sites. By adding VIC / FAM fluorescent tag sequences to the tail of allele-specific primers and combining PAGE purification to ensure primer quality, it ensures that allele-specific recognition and fluorescence signals are accurately distinguished, resulting in clear typing results and low misjudgment rate.

[0029] (4) The present invention optimizes the Touchdown PCR amplification program, adopts a pre-denaturation 94℃×10 min activation hot-start enzyme and gradient cooling annealing strategy, which effectively improves primer binding specificity, reduces non-specific amplification, and ensures balanced amplification of 26 sites in the multiplex system.

[0030] (5) This invention establishes a standardized detection process. By unifying the initial DNA concentration (10 ng / μL), setting up dual NTC controls and clear fluorescence signal interpretation standards (RFU difference ≥2000), the interference of template concentration differences is eliminated, a pollution monitoring system is constructed, and the repeatability and reliability of the results are significantly improved.

[0031] (6) The 26 core SNP loci screened in this invention are evenly distributed on 14 chromosomes in cotton subgroups A and D, with strong genetic representativeness. Only 26 loci are needed to distinguish 2880 out of 2882 samples (discrimination rate 99.93%). The genotyping success rate in 87 cotton samples is ≥99%, and the sample discrimination rate reaches 95.4%. It can effectively support the identification of cotton variety authenticity, kinship analysis and genetic diversity research, and can be widely used in cotton germplasm resource management, variety identification and plant variety rights protection. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0033] Figure 1 This is a schematic diagram of a phylogenetic tree constructed based on 26 core SNP sites, provided in an embodiment of the present invention.

[0034] Figure 2 This is a schematic diagram of a cotton variety DNA fingerprint based on 26 core SNP loci, provided in an embodiment of the present invention. Detailed Implementation

[0035] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0036] Example 1:

[0037] The SNP locus combination used for constructing a cotton DNA fingerprint consists of the following 26 core SNP loci: A02_2198127, A02_6551077, A03_103423966, A04_79410894, A04_75510070, A05_6526611, A06_43274017, A08_119076232, A09_14286848, D02_5778414, D02_1231245, D 04_2753129, D05_54798658, D05_13296669, D05_15195826, D05_860461, D05_859033, D05_12748666, D05_5687 2688, D07_21485004, D07_12158735, D09_8614926, D10_4758937, D10_59804947, D10_62797734, D12_12899667;

[0038] The 26 core SNP loci are evenly distributed across 7 chromosomes in subgroup A and 7 chromosomes in subgroup D of cotton, covering a total of 14 chromosomes. The 7 chromosomes in subgroup A are ChrA02, ChrA03, ChrA04, ChrA05, ChrA06, ChrA08, and ChrA09, and the 7 chromosomes in subgroup D are ChrD02, ChrD04, ChrD05, ChrD07, ChrD09, ChrD10, and ChrD12. Information on the 26 core SNP loci is shown in Table 1.

[0039] The specific implementation method is as follows: (1) Data source: The whole genome resequencing data of 2882 cotton samples are used as the original data source. The 2882 cotton samples cover cotton materials from different sources and with different genetic backgrounds, and have rich genetic diversity.

[0040] (2) Filtering and screening candidate sites without missing information: All SNP sites obtained by resequencing were filtered by the F_MISSING=0 index, and finally 2273 candidate SNP sites were obtained.

[0041] (3) Multidimensional Comprehensive Quality Score: For the above 2273 SNP loci, the missing rate, MAF, Hardy-Weinberg equilibrium test value, and Shannon entropy of each SNP were calculated. These four quality indicators were then weighted and summed according to preset weights to obtain the comprehensive quality score for each SNP locus. The formula for calculating the comprehensive quality score is as follows:

[0042] Overall quality score = Missing rate × 0.3 + MAF × 0.25 + HW balance value × 0.2 + Shannon entropy × 0.25

[0043] The overall quality score ranged from 0 to 1. Loci with an overall quality score of 0.7 or higher were selected, resulting in 220 high-quality candidate SNP loci.

[0044] (4) Marker recognition efficiency curve analysis: Marker recognition efficiency curve analysis was performed on the above 220 candidate loci. As the number of loci increases, the marginal gain of the cumulative variety differentiation ability was tracked to determine the inflection point where the efficiency gain tends to stabilize, so as to achieve the best variety differentiation effect with the minimum number of markers. Finally, 26 core SNP loci were determined, as shown in Table 1. The comprehensive quality score of each locus is shown in Table 2.

[0045] Table 1. 26 core SNP sites used for constructing cotton DNA fingerprinting

[0046]

[0047] The chromosomal location information of the above 26 core SNP loci was determined based on the CRI_v1 version (CRI-TM1_v1) of the Gossypium hirsutum (AD1) 'TM-1' reference genome, which can be obtained from the CottonGen database (https: / / www.cottongen.org / species / Gossypium_hirsutum / CRI-TM1).

[0048] Table 2. Overall quality score of 26 core SNP loci

[0049]

[0050] After the above screening, 26 core SNP loci were able to distinguish 2880 out of 2882 cotton samples, with a discrimination rate of 99.93%.

[0051] Example 2:

[0052] The KASP primer combination used for constructing cotton DNA fingerprinting was designed for the 26 core SNP sites in Example 1. Three primers were designed for each SNP site: a first allele-specific primer F1 with a VIC fluorescent tag sequence, a second allele-specific primer F2 with a FAM fluorescent tag sequence, and a universal reverse primer R.

[0053] The VIC fluorescent tag sequence is 5'-GAAGGTCGGAGTCAACGGATT-3', and the FAM fluorescent tag sequence is 5'-GAAGGTGACCAAGTTCATGCT-3';

[0054] The primer sequences at each point in the KASP primer combinatorial are shown in SEQ ID NO:1 to SEQ ID NO:78.

[0055] The specific implementation method is as follows: (1) Primer design: According to the KASP primer design principle, for each of the 26 core SNP sites, 300 bp sequences upstream and downstream of each site are selected for primer design, and it is noted whether there are other non-target SNP sites in the sequence to avoid primer amplification failure. Three primers are designed for each SNP site: two allele-specific primers (F1 and F2) and one universal primer (R).

[0056] Allele-specific primers F1 were modified by adding a specific sequence 5'-GAAGGTCGGAGTCAACGGATT-3' that binds to VIC fluorescence to the tail, and primers F2 were modified by adding a specific sequence 5'-GAAGGTGACCAAGTTCATGCT-3' that binds to FAM fluorescence to the tail.

[0057] Primer design parameters: The average GC content of the specific primers was 42.8%, the average annealing temperature (Tm) was 59℃, covering 14 chromosomes in cotton subgroups A and D, and the length of the amplified products ranged from 39 to 100 bp.

[0058] (2) Primer purification and synthesis: All primers were purified by PAGE to remove short oligonucleotides and synthetic byproducts, ensuring primer sequence integrity and reducing non-specific amplification interference. The designed primers were synthesized and purified by Beijing Qingke Biotechnology Co., Ltd.

[0059] The KASP primer sequences corresponding to the 26 core SNP sites are shown in Table 3.

[0060] Table 3. KASP primer sequences corresponding to the 26 core SNP sites

[0061]

[0062] Table 3 shows that the 5' end of primer F1 carries the VIC fluorescent tag sequence GAAGGTCGGAGTCAACGGATT, and the 5' end of primer F2 carries the FAM fluorescent tag sequence GAAGGTGACCAAGTTCATGCT.

[0063] Example 3:

[0064] KASP genotyping was performed on 87 cotton samples based on 26 core SNP loci.

[0065] Using the 26 core SNP loci identified by this invention and their corresponding KASP primers, KASP genotyping was performed on 87 cotton samples to verify the genotyping effect of the marker system.

[0066] (1) Experimental materials:

[0067] Sample source: 87 cotton samples were provided by the Cotton Research Institute of the Chinese Academy of Agricultural Sciences and were grown at the Yacheng Base of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences in Sanya, Hainan. The samples cover cotton materials with different genetic backgrounds and have rich genetic diversity.

[0068] Main reagents: Plant genomic DNA extraction kit (Tiangen Biochemical); 2×Taq DNA Polymerase Mix (Gude Biotechnology); primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. (PAGE purification grade).

[0069] Main instruments: Real-time quantitative PCR instrument (Thermo Fisher Scientific); Micro-volume UV spectrophotometer (Ausun Instruments); 96-well PCR instrument (Bori Technology).

[0070] (2) Genomic DNA extraction and quality testing:

[0071] Fresh leaves from 87 cotton samples were collected, and genomic DNA was extracted using a plant genomic DNA extraction kit. The specific procedure was performed according to the kit's instructions. After extraction, the DNA concentration and purity were determined using a micro-ultraviolet spectrophotometer, requiring a specific OD value. 260 / OD 280 Values ​​between 1.8 and 2.0, OD 260 / OD 230 Value greater than 1.8. Dilute all sample DNA concentrations to 10 ng / μL and store at -20℃ for later use.

[0072] By standardizing the DNA concentration to 10 ng / μL, Taq enzyme activity inhibition (too high concentration) or insufficient fluorescence signal (too low concentration) caused by differences in template concentration can be effectively avoided, ensuring the consistency of amplification efficiency of the 26 SNP sites across different samples (Ct value standard deviation ≤ 0.5).

[0073] (3) Preparation of KASP primers:

[0074] Mix each primer pair (F1, F2, R) at a ratio of 12 μmol / L (F1): 12 μmol / L (F2): 30 μmol / L (R), bring the volume to 1×TAE buffer, and prepare a 10×SNP Primer Mix. Store at -20℃ protected from light.

[0075] (4) PCR amplification:

[0076] A 5 μL reaction system was used in a 96-well plate. The amounts of each component were as follows: 2 μL of 2×Taq DNA Polymerase Mix, 1 μL of 10×SNP Primer Mix, and 2 μL of DNA sample (10 ng / μL), for a total volume of 5 μL. Two template-free controls (NTCs) were set up in each 96-well plate, with an equal volume of ddH2O replacing the DNA template, to eliminate false positive signals caused by contamination.

[0077] PCR amplification uses a Touchdown gradient cooling annealing strategy, as follows:

[0078] Pre-denaturation: 94℃, 10 min, 1 cycle;

[0079] Denaturation: 94℃, 20 sec, 10 cycles; Renaturation / Extension: Temperature decreased from 61℃ to 55℃ at -0.6℃ / cycle, 45 sec, 10 cycles;

[0080] Second denaturation: 94℃, 20 sec, 35 cycles; Second annealing / extension: 55℃, 45 sec, 35 cycles.

[0081] The Touchdown strategy starts with a high initial annealing temperature (61°C), which allows primers to bind to perfectly matched target sequences preferentially in the early stages of amplification. As the temperature gradually decreases, it transitions to a stable, specific amplification stage, ensuring both amplification specificity and efficiency.

[0082] (5) Fluorescence signal reading and genotyping:

[0083] After PCR amplification, real-time quantitative PCR was used at 25℃ to read the final fluorescence signal, and the fluorescence signal intensity data of the two fluorescence channels, VIC and FAM, were collected. The VIC / FAM scatter plot cluster analysis was performed using analysis software.

[0084] The criteria for interpreting genotypes are as follows: In a scatter plot with VIC fluorescence intensity on the X-axis and FAM fluorescence intensity on the Y-axis, a strong VIC / weak FAM (lower right region) indicates a homozygous AA genotype; a strong FAM / weak VIC (upper left region) indicates a homozygous BB genotype; and a centered VIC / FAM signal (middle region) indicates a heterozygous AB genotype.

[0085] In the scatter plot of the 26 SNP loci, the boundaries of each genotype cluster were clear, the samples within the clusters were concentrated, and the difference in fluorescence signal intensity between VIC and FAM was ≥2000 RFU, ensuring the accuracy of genotype interpretation (genotyping error rate ≤0.5%). The NTC control wells showed no obvious fluorescence signal, indicating that there was no contamination in this batch of experiments and the genotype interpretation results were reliable.

[0086] (6) Genotyping results:

[0087] The fluorescence typing results of the 26 loci were converted into dibasic genotype codes. Homozygous reference alleles, homozygous variant alleles, and heterozygous types were represented by corresponding dibasic genotypes (such as CC, TT, CT, etc.). Missing data were denoted as NN. The results were integrated into a genotype matrix of 26 core SNP loci corresponding to 87 cotton samples, as shown in Table 4.

[0088] Table 4 Genotyping results of 26 core SNP loci corresponding to 87 cotton samples

[0089] Sample No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 NB190074 CA GG TT AT GT CG AG CT GA TT TC GA AA GG GA TC AT CC TC AA GG AA AG CC AA AC NB190084 CA GG TT AT GT CG AG CT GA TT TC GA AA GG GA TC AT CC TC AA GG AA AG CC AA AC NB190386 CA GG TT AT GT CG AG CT GA TT TC GA GA GG GA CC AT CC TC AA GG AA AG CC AA AC NB190149 CA GG TT AT GT CG AG CT GA TT TC GA GG GG GA CC AT CC NN AA GG GA AG CC AA AC NB190154 CA GG TT AT GT CG AG CT GA TT TC GA GG GG GA TC AT CC TC AG GG AA AG CC AA AC NB190168 CA GG TT AT GT CG AG CT GA TT CC GA GA GA GA TC AT CC TC AA GG AA AG CC AA AC NB190192 CA GG TT AT GT CG AG CT GA TT TC GA GA GG GA TC AT CC TC AA GG AA AG CC AA AC NB190235 CA GG TT AT GT CG AG CT GA TT CC GA GA GA GA TC AT CC TC AG GG AA GG CC AA AC NB190243 CA GG TT AT GG CG AG CT GA TT TC GA GA GG GA TC AT CC TC AG GG AA AG CC AA AC NB190248 CA GG TT AT GG CG AG CT GA TT TC GA GA GG GA TC AT CC TC AG GG AA AG CC AA AC NB190251 CA GG TT AT GG CG AG CC GA TT CC GA GA GA GA TC AT CC TC AA GG AA AG CC AA CC NB190252 CA GG TT AT GG CG AG CT GA TT CC GA GA GA GA TC AT CC NN AA GG GA AG CC AA CC NB190257 CA GG TT AT GT CG AG CC GA TT CC GA AA GG GA [[ID=343 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ GG GG GA NN AT CC TC AG GG GA AG CC AA AC NB190436 CA GG TT AT GT CG AG CC GA TT TC GA AA GG GA CC AT CC TC AG GG GA AG CC AA AC NB190452 CA GG TT AT GT CG AG CT GA TT TC GA GG GG AA TC AT CC TC AA GG AA AG CC AA AC NB190096 CA GG TT AT GT CG AG CT GA TT CC GA GG GG GA CC AT CC TC AA GG AA AG CC AA AC NB190217 CA GG TT AT GT CG AG CT GA TT CC GA GA GG GA TC AT CC TC AG GG GA AG CC AA AC NB190502 CA GG TT AT GT NN AG CT GA TT CC GA GA GA GA CC AT CC TC AG GG GG GG CC AA CC NB191618 CA GG TT AT GT CG AG CT GA TT TC GA AA GG GA TC AT CC TC AA GG GA AG CC AA AC NB190472 CA GG TT AT GT CG AG CC GA TT TC GA AA GG GA TC AT CC TC AA GG GA AG CC AA AC NB190456 CA GG TT AT GT CG AG CT GA TT TC GA AA GG GA TC AT CC NN AA GG GA AG CC AA AC NB190459 CA GG TT AT GT CG AG CT GA TT CC GA AA GG GA CC AT CC TC AA GG GG AG CC AA AC NB190473 CA GG TT AT GT CG AG CC GA TT TC GA GG GA GA CC AT CC TC AG GG GG GG CC AA CC NB190202 CA GG TT AT GT CG AG CT GA TT TC GA AA GG GA CC AT CC TC AA GG GA AG CC AA AC NB190454 CA GG TT AT GT CG AG CC GA TT CC GA GG GG GA TC AT CC TC AA GG GA AG CC AA AC NB190525 CA GG TT AT GT CG AG CT GA TT TC GA GA GG GA TC AT CC TC AG GG AA AG CC AA AC NB190529 CA GG TT AT GT CG AG CT GA TT CC GA GA GG GA CC AT CC TC AG GG AA AG CC AA AC NB190531 CA GG TT AT GT CG AG CT GA TT CC GA GG GG AA CC AT CC TC AA GG AA AG CC AA AC NB190507 CA NN TT AT GT CG AG CT GA TT TC GA GG GA GA CC AT CC TC AG GG GG GG CC AA CC NB190468 CA GG TT AT GT CG AG CC GA TT CC GA GG GA GA CC AT CC TC AG GG GG AG CC AA AC NB190246 CA GG TT AT GT CG AG CC GA TT TC GA GA GG GA CC AT CC TC AA GG GA AG CC AA AC NB190256 CA GG TT AT GT CG AG CC GA TT TC GA GG GA GA TC AT CC TC AA GG AA AG CC AA AC NB190496 CA GG TT AT GT CG AG CT GA TT TC GA GA GG GA TC TT CC TC AA GG GG GG CC AA AC NB190176 CA GG TT AT GG CG AG CT GA TT TC GA AA GG GA TC AT CC TC AA GG GA AG CC AA AC NB190258 CA GG TT AT GT CG AG CC GA TT CC GA AA GG GA TC AT CC TC AA GG GA GG CC AA AC NB190261 CA GG TT AT GT CG AG CC GA TT TC GA GA GG GA TC AT CC TC AA GG AA GG CC AA AC NB190269 CA GG TT AT GT CG AG CT GA TT TC GA GG GG GA CC AT CC TC AA GG GA AG CC AA AC NB190325 CA GG TT AT GT CG AG CT GA TT TC GA AA GG GA CC AT CC TC AG GG GA AG CC AA AC NB190337 CA GG TT AT GG CG AG CC GA TT TC GA AA GG GA CC AT CC TC AA GG GA GG CC AA AC NB190359 CA GG TT AT GT CG AG CC GA TT TC GA GG GG GA CC AT CC TC AA GG AA AG CC AA AC NB190367 CA GG TT AT GT CG AG CT GA TT TC GA GA GG GA CC AT CC TC AG GG GA AG CC AA AC NB190398 CA GG TT AT GT CG AG CC GA TT TC GA GG GG GA CC AT CC TC AA GG GA AG CC AA AC NB190521 CA GG TT AT GT CG AG CT GA TT CC GA AA GA AA CC AT CC TC AA GG AA AG CC AA AC NB190286 CA GG TT AT GT CG AG CT GA TT TC GA GA GG GA TC AT CC TC AA GG GA AG CC AA AC NB190295 CA GG TT AT GT CG AG CC GA TT TC GA AA GA GA CC AT CC TC AG GG AA AG CC AA AC NB190309 CA GG TT AT GT CG AG CT GA TT TC GA GG GG GA CC AT CC TC AA GG AA AG CC AA AC NB191623 CA GG TT AT GT CG AG CT GA TT CC GA GA GG GA CC AT CC TC AG GG AA AG CC AA AC Zhong 12 CA GG TT AT GT CG AG CT GA TT TC GA GA GA GA TC NN NN NN NN AG GA AG TC AA AC 108 Fu CA GG TT AT GT CG AG CT GA TT TC GA GA GG GA CC NN CC TC AA AG GA AG TC AA AC 23D036 CA GG TT AT GT CG AG CT GA TT TC GA GA GG GA CC NN CC TC AG AG AA AG TC AA AC 23D120 CA GG TT AT GT CG AG CT GA TT TC GA GA GA GA CC NN CC TC AA AG AA AG TC AA AC 23D148 CA GG TT AT GT CG AG CC GA TT TC GA GA GA GA CC NN CC NN AA AG NN AG TC AA CC C-3174 CA GG TT AT GT CG AG CT GA TT TC GA GA GA GA TC AT CC TC AA AG AA AG TC AA AC C-4744 CA GG TT AT GT CG AG CT GA TT TC GA AA GA GA CC NN CC TC AA AG GA AG TC AA AC J8031 CA GG TT AT GT CG AG CT GA TT TC GA AA GG GA CC NN NN TC AG AG AA AG TC AA AC KK-1543 CA GG TT AT GT CG AG CC GA TT TC GA AA GG GA TC AT CC TC AA AG GA AG TC AA AC Chuangmian 508 CA GG TT AT GT CG AG CC GA TT TC GA GA GA GA CC AT CC TC AG AG AA AG TC AA AC Ta 2 CA GG TT AT GT CG AG CT GA TT TC GA AA GA GA CC AT CC TC AG AG AA AG TC AA AC Xinluzao 1 CA GG TT AT GT CG AG CT GA TT TC GA GA GA GA CC AT CC TC AA AG GA AG TC AA[[ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ AC Xinluzhong 34 CA GG TT AT GT CG AG CC GA TT TC GA AA GG GA CC AT CC TC AA AG AA AG TC AA AC Xinluzhong 42 CA GG TT AT GT CG AG CC GA TT TC GA GA GA GA CC AT CC TC AA AG AA AG TC AA AC Xinluzhong 87 CA GG TT AT GT CG AG CC GA TT TC GA GA GA GA CC AT CC TC AA AG GA AG TC AA AC Zhongmiansuo 35 CA GG TT AT GT CG AG CT GA TT TC GA GA GG GA CC AT CC TC AA AG GA AG TC AA AC Zhongmiansuo 41 CA GG TT AT GT CG AG CT GA TT TC GA GA GG GA TC AT CC TC AA AG GA AG TC AA AC Zhongmiansuo 49 CA GG TT AT GT CG AG CC GA TT TC GA GA GG GA CC AT CC TC AA AG AA AG TC AA AC Zhong G32917 CA GG TT AT GT CG AG CT GA TT TC GA GA GA GA CC AT CC TC AA AG AA AG TC AA AC Zhong G33103 CA GG TT AT GT CG AG CT GA TT CC GA GA GA GA CC AT CC TC AA AG GA AG TC AA AC Shikang 126 CA GG TT AT GT CG AG CT GA TT TC GA GA GA GA CC AT CC TC AG AG GA AG TC AA AC Ji 2016 CA GG TT AT GT CG AG CT GA TT TC GA GA GG GA CC AT CC TC AG AG GA AG TC AA AC Jimian 298⊕ CA GG TT AT GT CG AG CT GA TT TC GA GA GG GA CC AT CC TC AA AG AA AG TC AA AC DP 161 B2RF CA GG TT AT GT CG AG CT GA TT CC GA AA GA GA CC AT CC TC AG AG GA AG TC AA AC DP 143 B2RF CA GG TT NN GT CG AG CT GA TT CC GA GA GA GA CC AT [[ID ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ AG AG AA AG TC AA AC ST 5458B2RF CA GG TT AT GT CG AG CC GA TT CC GA GA GA GA CC AT CC TC AA AG GA AG TC AA AC Zhongmian 9001 CA GG TT AT GT CG AG CT GA TT TC GA AA GG GA CC AT CC TC AA AG GA AG TC AA AC

[0090] The quality of the typing results was analyzed: the total genotype data points for the 26 SNP loci in the 87 samples were 2262, with 2241 data points successfully typed, resulting in an overall typing success rate of 99.07% and an average locus deletion rate of 0.93%. These results demonstrate that the KASP detection system established in this invention is stable and reliable, and the SNP fingerprint data from the 87 cotton germplasm resource samples obtained have high integrity. It can be directly used for applications such as cotton variety authenticity identification, phylogenetic analysis, and new variety rights protection.

[0091] Based on genotypic combinations at 26 loci, 83 different DNA fingerprint types were generated from 87 cotton samples. Of these, 83 samples possessed unique DNA fingerprints, achieving a sample discrimination rate of 95.4% and a sample coverage rate of 100%. Further analysis revealed that all 26 core SNP loci could be accurately distinguished in the 87 cotton samples (including 36 mainstream commercially cultivated varieties analyzed), with no duplicate DNA fingerprints found. This indicates that the marker combination has a high ability to identify widely used cultivated varieties in current production, meeting the practical application needs for variety authenticity identification, variety rights protection, and germplasm resource management.

[0092] Example 4:

[0093] Phylogenetic trees of 2882 cotton varieties were constructed based on 26 core SNP loci.

[0094] This embodiment uses VCF data from the resequencing of 2882 cotton varieties as input. Utilizing 26 core SNP loci, it completes the analysis of phylogenetic relationships and population structure display of cotton samples through linkage disequilibrium (LD) pruning, IBS (Identity-By-State) distance matrix calculation, Neighbor-Joining (NJ) tree construction, and online visualization on the iTOL website.

[0095] (1) Data preparation:

[0096] Input data: Resequencing genotype data from 2882 cotton variety samples, stored in VCF compressed format (vcf.gz), containing genotype information for 26 core SNP loci identified through screening.

[0097] Sample grouping information: A file containing the group affiliation information of 2882 samples, separated by tabs, including sample ID (ID column) and group (Group column), covering a total of 3 cotton groups: G1 (upland cotton), G2 (sea island cotton), and G3 (other cotton varieties).

[0098] Software environment: PLINK 1.9 (genotype data processing); Python 3.8, depending on the scikit-bio library (DistanceMatrix and nj modules) and the pandas library (data reading); iTOL (Interactive Tree Of Life, an online phylogenetic tree visualization platform, website: https: / / itol.embl.de).

[0099] (2) LD pruning:

[0100] Using PLINK 1.9 software, with a window size of 50 sites, a step size of 10 sites, and r 2 LD pruning was performed with a threshold of 0.2 to confirm the independence between sites.

[0101] (3) Calculate the IBS distance matrix between samples:

[0102] Based on the sites retained after LD pruning, the IBS distance between all pairs of samples is calculated using PLINK, and the result is output in a square matrix format.

[0103] (4) Constructing a phylogenetic tree based on the NJ method:

[0104] Using the IBS distance matrix as input, a phylogenetic tree is constructed using the adjacency join method. A script written in Python 3.8 calls the nj() function of the scikit-bio library to complete the tree construction.

[0105] The script reads the 2882×2882 IBS distance matrix, calls the scikit-bio nj() function to calculate the adjacency tree topology, and outputs the results in Newick format (.nwk), containing 2882 leaf nodes, with each leaf node corresponding to a cotton sample.

[0106] (5) Generate iTOL group color annotation file:

[0107] An iTOL color bar annotation file is generated based on the sample grouping information file. The color codes corresponding to the three groups are: G1 (#E64B35, upland cotton), G2 (#3C9A3C, Sea Island cotton), and G3 (#4DBBD5, other cotton varieties).

[0108] A script written in Python 3.8 automatically reads grouping information and generates a DATASET_COLORSTRIP annotation file that conforms to the iTOL format specification.

[0109] The script reads the grouping information of 2882 samples and generates iTOL leaf node color mapping entries line by line.

[0110] (6) iTOL Online Visualization:

[0111] Log in to the iTOL website (https: / / itol.embl.de), upload the Newick file to the My Tree page, and complete the phylogenetic tree import. Drag and drop iTOL leaf node color mapping entries onto the phylogenetic tree page; the system will automatically recognize the DATASET_COLORSTRIP format and overlay the corresponding population color stripe onto each leaf node. Adjust the tree layout (circle / rectangle), branch length display, and legend position in the visualization interface, and export a high-resolution image (SVG or PDF format).

[0112] The final phylogenetic tree results are shown below. Figure 1 G1 represents upland cotton, G2 represents sea island cotton, and G3 represents other cotton species. The phylogenetic tree shows that the 2882 cotton variety samples were clearly clustered into three genetic populations based on 26 core SNP loci. Samples within each population were genetically close, while inter-population branching was significant. This indicates that the core SNP loci combinations screened in this invention can effectively reflect the population genetic structure of cotton germplasm resources and have good kinship analysis capabilities.

[0113] Example 5:

[0114] DNA fingerprints of 87 cotton varieties were constructed based on 26 core SNP loci.

[0115] This embodiment utilizes the KASP genotyping results of 87 cotton samples × 26 core SNP loci obtained in Example 3 to construct a cotton variety DNA fingerprint heatmap, which visually displays the genotypic distribution characteristics of each sample at the core SNP loci and the genetic differences between samples.

[0116] (1) Data source:

[0117] The KASP genotyping matrix of 87 cotton samples in Example 3 was used as input data. The matrix contains the double base genotype codes (such as AA, AG, GG, etc.) of 87 samples at 26 core SNP sites. Missing data is denoted as NN.

[0118] (2) Fingerprint pattern construction method:

[0119] Genotype classification and coding: Genotypes are classified into four homozygous types (A / A, T / T, C / C, G / G), six heterozygous types (A / C, A / G, A / T, C / G, C / T, G / T), and deletion type (NN), and each is assigned a fixed display color to ensure that the same type is displayed with a consistent color throughout the map, which facilitates intuitive comparison of genotype distribution patterns at different loci and among different samples.

[0120] Matrix layout: The 26 core SNP loci are used as the vertical axis (rows), and the 87 cotton samples are used as the horizontal axis (columns). The color of each cell represents the genotype classification of the sample at the corresponding SNP locus. This layout is suitable for fingerprint visualization scenarios where the number of samples significantly exceeds the number of loci. It can fully present all locus information within a limited map area while maintaining clear and distinguishable genotype differences between samples.

[0121] Sample clustering and sorting: Based on the genotypic differences of 87 samples at 26 loci, Hamming distance was used to calculate the pairwise genetic distance between samples, and hierarchical clustering (average linkage method) was used to rearrange the sample order so that samples with similar genetic backgrounds are arranged adjacently in the graph.

[0122] Graphical presentation: The fingerprint heatmap was plotted using Python (matplotlib library), with a resolution of 300 dpi. The full label name was marked on the vertical axis, and the legend indicated the genotype category represented by each color.

[0123] (3) Results and Analysis:

[0124] Schematic diagram of the constructed DNA fingerprint ( Figure 2 The figure visually presents the genotypic distribution of 87 cotton samples at 26 core SNP loci. As shown in the figure:

[0125] (a) Most core SNP loci showed good polymorphism in 87 samples, with homozygous genotypes (A / A, T / T, C / C, G / G) and heterozygous genotypes (Het) alternating, and no large areas of single color blocks were observed, indicating that the 26 selected core SNP loci can effectively distinguish cotton samples with different genetic backgrounds.

[0126] (b) After hierarchical clustering and sorting, samples with similar genetic backgrounds clustered in the figure to form continuous color blocks, reflecting that there are several closely related sample groups among the 87 samples, further verifying that the DNA fingerprint map constructed in this invention can accurately reflect the genetic similarity and differences between samples.

[0127] (c) The fingerprint spectrum presents genotype information intuitively with color coding, which is more convenient for researchers, variety identification agencies and grassroots seed companies to quickly identify the genetic relationship between samples compared with the traditional digital coding table format.

[0128] This invention utilizes 26 core SNP loci to develop KASP markers for DNA fingerprinting and evaluation of identification capabilities in 87 cotton samples. Based on genotype combinations of the 26 loci, 83 different DNA fingerprint types were formed, of which 83 samples possessed unique DNA fingerprints, achieving a sample discrimination rate of 95.4% and a sample coverage rate of 100%. The results indicate that the 26 selected core markers exhibit high genetic polymorphism and strong variety identification ability.

[0129] Further analysis revealed that all 26 core SNP loci in the 87 cotton samples (including 36 mainstream commercially cultivated varieties analyzed) could be accurately distinguished, with no duplicate DNA fingerprints observed. This indicates that the marker combination has high identification capability for widely used cultivated varieties in current production. Compared to methods that rely on a large number of molecular markers to construct DNA fingerprint maps, this method achieves high sample identification efficiency using a smaller number of core markers, effectively reducing detection costs and improving detection efficiency while maintaining identification capability.

[0130] Example 6:

[0131] Establishment of a standardized testing process: This embodiment details the standardized KASP testing process established by the present invention, including the following steps:

[0132] S1, DNA template standardization:

[0133] Genomic DNA was extracted from cotton samples, supporting both wet (fresh leaves) and dry (dried leaves) samples. The initial DNA concentration was uniformly adjusted to 10 ng / μL to avoid amplification deviations caused by excessively high concentrations (inhibiting Taq enzyme activity) or excessively low concentrations (insufficient signal detection), ensuring consistent amplification efficiency (Ct value standard deviation ≤ 0.5) for the 26 core SNP loci across different samples.

[0134] S2, PCR reaction system:

[0135] Using the genomic DNA obtained in S1 as a template, PCR amplification was performed using KASP primer combinations to obtain amplification products; two template-free controls (NTCs) were included for each primer pair. In a 5 μL 96-well plate system: 2 × Taq DNA Polymerase Mix 2 μL, 10 × SNP Primer Mix 1 μL, DNA sample 2 μL, total reaction volume 5 μL. The control group used ddH2O instead of DNA template.

[0136] The PCR amplification procedure is as follows:

[0137] PCR amplification uses a touchdown cooling annealing strategy, and the specific procedure is as follows:

[0138] Pre-denaturation: 94℃, 10 min, 1 cycle;

[0139] Denaturation: 94°C, 20 sec, 10 cycles; Renaturation / Extension: Temperature decreased from 61°C to 55°C at -0.6°C / cycle, 45 sec, 10 cycles;

[0140] Second denaturation: 94℃, 20 sec, 35 cycles; Second annealing / extension: 55℃, 45 sec, 35 cycles.

[0141] S3. Using a real-time quantitative PCR instrument, VIC / FAM fluorescence signals are read to distinguish between the AA, BB, and AB genotypes. The intensity difference between VIC / FAM fluorescence signals must be ≥2000 RFU to ensure accurate genotype interpretation (genotyping error rate ≤0.5%). A characteristic fingerprint map is generated based on the genotyping results from 26 core loci, providing a digital basis for variety identification and kinship analysis.

[0142] S4. Construct the DNA fingerprint of the cotton sample to be tested based on the genotyping information obtained in S3. Genotyping quality control standards:

[0143] The KASP marker had an average genotyping success rate of ≥99% and showed good polymorphism in 2882 cotton variety samples (mean MAF ≥0.2).

[0144] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. The application of SNP site combinations in the construction of cotton DNA fingerprinting, characterized in that, The SNP locus combination consists of the following 26 core SNP loci: A02_2198127, A02_6551077, A03_103423966, A04_79410894, A04_75510070, A05_6526611, A06_43274017, A08_119076232, A09_14286848, D02_5778414, D02_1231245, D04_2753129 , D05_54798658, D05_13296669, D05_15195826, D05_860461, D05_859033, D05_12748666, D05_56872688, D07_21485004, D07_12158735, D09_8614926, D10_4758937, D10_59804947, D10_62797734, D12_12899667; The chromosomal location information of the 26 core SNP loci is based on the upland cotton reference genome CRI-TM1_v1 version; The 26 core SNP loci are evenly distributed across 7 chromosomes in subgroup A and 7 chromosomes in subgroup D of cotton, covering a total of 14 chromosomes. The 7 chromosomes in subgroup A are ChrA02, ChrA03, ChrA04, ChrA05, ChrA06, ChrA08, and ChrA09, and the 7 chromosomes in subgroup D are ChrD02, ChrD04, ChrD05, ChrD07, ChrD09, ChrD10, and ChrD12.

2. A KASP primer combination for constructing cotton DNA fingerprinting, characterized in that, The KASP primer combination is designed for the 26 core SNP sites described in claim 1, with 3 primers designed for each SNP site: a first allele-specific primer F1 with a VIC fluorescent tag sequence, a second allele-specific primer F2 with a FAM fluorescent tag sequence, and a universal reverse primer R; The VIC fluorescent tag sequence is 5'-GAAGGTCGGAGTCAACGGATT-3', and the FAM fluorescent tag sequence is 5'-GAAGGTGACCAAGTTCATGCT-3'; The primer sequences at each point in the KASP primer combination are shown in SEQ ID NO:1 to SEQ ID NO:

78.

3. The application of the KASP primer combination according to claim 2 in the construction of cotton DNA fingerprinting, characterized in that, Includes the following steps: S1. Extract genomic DNA from the cotton sample to be tested and adjust the DNA concentration to 10 ng / μL; S2. Using the genomic DNA obtained in S1 as a template, perform PCR amplification using the KASP primer combination to obtain the amplification product; S3. Read the VIC fluorescence signal and FAM fluorescence signal of the amplification product, perform genotyping based on the fluorescence signal, and obtain the genotype information of the cotton sample to be tested at 26 core SNP loci. S4. Construct the DNA fingerprint of the cotton sample to be tested based on the genotype information obtained in S3.

4. The application of the KASP primer combination according to claim 3 in the construction of cotton DNA fingerprinting, characterized in that, The PCR amplification employed a Touchdown gradient cooling annealing strategy, and the PCR amplification program was as follows: Pre-denaturation: 94℃, 10 min, 1 cycle; Denaturation: 94℃, 20 sec, 10 cycles; Refolding / Extension: Temperature was reduced from 61°C to 55°C at -0.6°C / cycle, 45 sec, for 10 cycles; Second denaturation: 94℃, 20 sec, 35 cycles; Re-annealing / extension: 55°C, 45 sec, 35 cycles.

5. The application of the KASP primer combination according to claim 3 in the construction of cotton DNA fingerprinting, characterized in that, The total reaction volume of the PCR amplification reaction system was 5 μL, and the components were: 2 μL of 2×Taq DNA Polymerase Mix, 1 μL of 10×SNP Primer Mix, and 2 μL of DNA sample; each reaction plate was equipped with two template-free controls in which ddH2O was used instead of DNA template.

6. The application of the KASP primer combination according to claim 3 in the construction of cotton DNA fingerprinting, characterized in that, The criteria for interpreting genotypes in S3 are as follows: in a scatter plot with VIC fluorescence intensity on the X-axis and FAM fluorescence intensity on the Y-axis, strong VIC / weak FAM indicates AA homozygous genotype, strong FAM / weak VIC indicates BB homozygous genotype, and VIC / FAM signals centered indicate AB heterozygous genotype.

7. The application of the KASP primer combination as described in claim 2 in at least one of the following scenarios: cotton variety authenticity identification, cotton germplasm resource management, cotton plant variety rights protection, cotton breeding material identification, and cotton seed quality testing.

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