An MNP marker primer combination, kit, and its application for avocado variety identification.
By combining MNP marker primers and high-throughput sequencing technology, the problems of detection throughput and accuracy in avocado variety identification have been solved, achieving efficient and accurate variety identification and improving the standardization and branding of the avocado industry.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- YUNNAN INST OF TROPICAL CROPS
- Filing Date
- 2026-05-27
- Publication Date
- 2026-06-30
Smart Images
Figure CN122303478A_ABST
Abstract
Description
Technical Field
[0001] This disclosure relates to the field of molecular identification technology, and in particular to an MNP marker primer combination, kit, and application for avocado variety identification. Background Technology
[0002] Avocado, a perennial woody plant belonging to the genus *Pyrus* of the Lauraceae family, is a globally renowned economic fruit and oilseed tree. Its flesh is rich in nutrients, containing unsaturated fatty acids, protein, and various minerals, possessing high nutritional and health value. With the increasing demand for healthy consumption, the avocado industry has developed rapidly worldwide, and my country has become an important planting area and consumer market. Currently, avocados are cultivated in many parts of my country. However, current production suffers from widespread problems such as mixed varieties, different varieties with the same name, and inconsistent seedling quality. Due to the lack of varieties with independent intellectual property rights, the sources of seedlings for enterprises and farmers are chaotic, with widespread mixing of seedlings and grafted seedlings, poor orchard uniformity, and low commercialization, severely hindering the standardization and branding development of the industry. Therefore, strengthening research on its genetic background, variety protection, and variety identification is particularly important.
[0003] In recent years, research on avocado provenance identification and genetic diversity analysis based on molecular marker technology has largely employed SSR (Simple Sequence Repeat) techniques. SSRs primarily utilize the highly conserved flanking sequences, designing specific primers for PCR (Polymerase Chain Reaction) amplification and agarose gel electrophoresis, with polymorphism analysis based on the electrophoretic bands. However, this method suffers from limited throughput and insufficient polymorphism, making it difficult to distinguish all varieties and meet the needs of large-scale sample testing. Furthermore, during PCR amplification, DNA polymerase slippage upon template binding leads to non-specific amplification, making it difficult to distinguish the slipped bands from the true genotype bands in the sample, thus affecting typing accuracy. Therefore, developing an accurate and efficient detection technology for avocado variety identification has become an urgent technical problem to be solved. Public content
[0004] To address the problems of existing technologies, this disclosure provides an MNP marker primer combination, a kit, and their applications for identifying avocado varieties. The technical solution is as follows:
[0005] On one hand, embodiments of the present invention provide an MNP marker primer set for avocado variety identification, the primer set comprising: primer pair 1 to primer pair 466, each primer pair comprising a forward primer and a reverse primer, the forward primer of primer pair 1, the reverse primer of primer pair 466, and the reverse primer of primer pair 466 are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 932 in the sequence listing.
[0006] On the other hand, embodiments of the present invention provide a kit for identifying avocado varieties, the kit comprising the above-mentioned MNP marker primer combination.
[0007] In another aspect, embodiments of the present invention provide an application of the above-mentioned MNP marker primer combination in identifying avocado varieties, the application including:
[0008] The DNA of the test sample was amplified by multiplex PCR using the above-mentioned MNP marker primer combination to obtain multiplex PCR amplification products;
[0009] Purify the multiplex PCR amplification product;
[0010] A high-throughput sequencing library was constructed using the purified multiplex PCR amplification products to obtain the high-throughput library of the sample to be tested.
[0011] Purify the high-throughput library of the test sample;
[0012] The purified high-throughput library of the test sample was sequenced to obtain sequencing data.
[0013] Analyze the sequencing data to obtain DNA fingerprint data;
[0014] The DNA fingerprint data is compared with control samples to obtain the genetic similarity coefficient;
[0015] Based on the genetic similarity coefficient, the variety identification conclusions of the test sample and the control sample are obtained.
[0016] Specifically, obtaining the variety identification conclusion of the test sample and the control sample based on the genetic similarity coefficient includes: when the genetic similarity coefficient is equal to or greater than 99%, determining that the test sample and the control sample are extremely similar varieties or the same variety.
[0017] The beneficial effects of the technical solution provided in this disclosure are as follows: This invention provides an MNP marker primer combination, kit, and its application for avocado variety identification. Using this MNP marker primer combination, DNA fingerprint data of the sample to be tested can be obtained through multiplex PCR amplification and high-throughput sequencing. The genetic similarity coefficient is then obtained by comparing the DNA fingerprint data with a control sample, and the variety identification conclusion is derived based on the genetic similarity coefficient. The DNA fingerprint data consists of the base sequences of multiple MNP markers obtained after sequencing, with a resolution reaching the single-base level. It also boasts high accuracy and digitization. Sequence analysis software can be used to compare hundreds or thousands of samples at once, quickly obtaining the variety identification conclusion, which significantly improves the accuracy and efficiency of avocado variety identification. The MNP marker primer combination provided by this invention has good application value in avocado variety identification and DNA fingerprint database construction, providing technical support for the intellectual property protection of avocado varieties in my country and promoting the healthy development of the industry. Attached Figure Description
[0018] To more clearly illustrate the technical solutions in the embodiments of this disclosure, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0019] Figure 1 This is a statistical chart of the number of markers on chromosomes provided in Embodiment 1 of this disclosure, where the horizontal axis represents the chromosome number of the sample to be tested;
[0020] Figure 2 This is the total logarithm of the 106 avocado test samples provided in Embodiment 3 of this disclosure (using 10^6 logarithms). 6 (represented by) the proportion of MNP marker detection sites, where the horizontal axis is the sequence number of the sample to be tested;
[0021] Figure 3 This is a differential proportion distribution map of MNP marker sites provided in Embodiment 3 of this disclosure. Detailed Implementation
[0022] To make the objectives, technical solutions, and advantages of this disclosure clearer, the embodiments of this disclosure will be described in further detail below with reference to the accompanying drawings.
[0023] Example 1
[0024] This invention provides an MNP marker primer combination for identifying avocado varieties. The MNP marker primer combination includes primer pairs 1 to 466, each primer pair including a forward primer and a reverse primer. The forward primer of primer pair 1, the reverse primer of primer pair 1 to the forward primer of primer pair 466 and the reverse primer of primer pair 466 are shown in SEQ ID NO: 1 to SEQ ID NO: 932 in the sequence listing, as shown in Table 1.
[0025] Table 1 shows the sequences of 466 primer pairs.
[0026] The primer pairs in the above primer set do not interfere with each other, ensuring that each primer pair can react normally in the same amplification reaction and specifically amplify the target gene sequence. The number of markers on the chromosome is as follows: Figure 1 As shown.
[0027] Example 2
[0028] This invention provides a kit for identifying avocado varieties, which uses the MNP marker primer combination provided in Example 1.
[0029] Example 3
[0030] This invention provides an application for identifying avocado varieties using the MNP marker primer combination provided in Example 1. This application includes:
[0031] Multiplex PCR amplification was performed on the DNA of the test sample using an MNP-labeled primer combination to obtain multiplex PCR amplification products;
[0032] Purify multiplex PCR amplification products;
[0033] High-throughput sequencing libraries were constructed using purified multiplex PCR amplification products to obtain high-throughput libraries of the samples to be tested.
[0034] Purifying high-throughput libraries of test samples;
[0035] The purified high-throughput library of the test sample was sequenced to obtain sequencing data.
[0036] Analyze sequencing data to obtain DNA fingerprint data;
[0037] The genetic similarity coefficient is obtained by comparing DNA fingerprint data with control samples;
[0038] Based on the genetic similarity coefficient, the variety identification conclusions of the test sample and the control sample are obtained.
[0039] Specifically, based on the genetic similarity coefficient, the variety identification conclusions of the test sample and the control sample are obtained, including: when the genetic similarity coefficient is equal to or greater than 99%, the test sample and the control sample are determined to be extremely similar varieties or the same varieties.
[0040] In this embodiment, the samples to be tested consist of 106 avocado varieties collected by the project unit. Information on the 106 avocado samples to be tested provided in this embodiment is shown in Table 2.
[0041] Table 2 contains information on 106 avocado samples to be tested.
[0042] DNA extraction from the test samples: DNA was extracted from the leaves of the above 106 avocado varieties using the Plant Genomic DNA Extraction Kit (catalog number: DP320) manufactured by Tiangen Biotech (Beijing) Co., Ltd. Detailed operating procedures are described in the kit's instruction manual. DNA from 106 test samples was obtained. Then, 1 μL of DNA from each test sample was taken and the DNA concentration of the test sample was determined using a Qubit fluorescence quantitative quantitation instrument. In this embodiment, the DNA concentration of the test samples was measured to be between 20 ng / μL and 50 ng / μL.
[0043] The DNA of the test sample was amplified by multiplex PCR using the MNP marker primer combination provided in Example 1 of this invention to obtain multiplex PCR amplification products.
[0044] Specifically, each amplification reaction for a test sample included: 4 μL of the MNP marker primer combination provided in this embodiment of the invention, 4 μL of DNA from the test sample, 10 μL of GenoPlexs 3×T Master Mix (manufacturer: Shijiazhuang Borui Biotechnology Co., Ltd.), and 12 μL of water. The mixture was vortexed and mixed to obtain a compound, which was then used as the multiplex PCR amplification system. The multiplex PCR amplification program was: 95℃ for 3 min; (95℃ for 20 sec, 60℃ for 4 min) × 17 cycles; 72℃ for 4 min to obtain the multiplex PCR amplification product.
[0045] Purify the multiplex PCR amplification products.
[0046] Specifically, DNA purification magnetic beads (manufacturer: Nanjing Novizan Biotechnology Co., Ltd.) were used to purify the multiplex PCR amplification products after the reaction. For detailed operating procedures, please refer to the product's instruction manual to obtain purified multiplex PCR amplification products.
[0047] High-throughput sequencing libraries were constructed using purified multiplex PCR amplification products to obtain high-throughput libraries of the samples to be tested.
[0048] Specifically, 10 μL of GenoPlexs 3×T Master Mix, 2 μL of 5 μM Illumina sequencing adapter primers (purchased from Borui Biotechnology Co., Ltd.), and 16 μL of water were added to the purified multiplex PCR amplification product, and the PCR amplification reaction was performed according to the following program: 95℃ for 3 min; (95℃ for 15 s, 58℃ for 15 s, 70℃ for 30 s) × 8 cycles; a final extension at 72℃ for 5 min, and the reaction was terminated at 16℃. After the reaction, a high-throughput sequencing library of the sample to be tested was obtained.
[0049] Purify the high-throughput library of the sample to be tested. Specifically, use DNA purification magnetic beads to purify the high-throughput sequencing library to obtain the purified high-throughput sequencing library. Refer to the instructions for the DNA purification magnetic bead product for specific purification methods.
[0050] The purified high-throughput library of the test sample was sequenced to obtain sequencing data. Specifically, the purified high-throughput sequencing library was sequenced using an Illumina NextSeq1000 sequencer to obtain sequencing data of the test sample. For detailed sequencing procedures, please refer to the instruction manual of this sequencer.
[0051] Sequencing data was analyzed to obtain DNA fingerprint data. Specifically, sequence analysis was performed using Bowtie2 (version 2.1.0) software to obtain the MNP-tagged DNA sequence for each sample.
[0052] Detection rate of MNP markers
[0053] Multiplex PCR amplification and construction of a second-generation high-throughput library were performed using the MNP marker primer combination provided in this embodiment of the invention. The obtained sequencing data were used to analyze the 106 avocado DNA samples. All 466 MNP marker sites provided in this embodiment were detected in the 106 samples to be tested. For specific detection information, please refer to Table 3.
[0054] Table 3 shows the detection information for 106 avocado varieties.
[0055] Table 3 shows that the highest number of detected markers was 466 for each avocado variety, with an average of 464.65 MNP marker loci detected per variety, resulting in an average detection rate of 99.71%. The total logarithm of reads from 106 avocado samples (using 10^6 MNP markers) was [data missing]. 6 (Indicated) the ratio of the detected sites, for example Figure 2 As shown.
[0056] Accuracy Analysis
[0057] To verify the accuracy of the MNP marker primer combinations, reproducibility experiments were conducted on seven test samples (including two independent experiments performed by different personnel, using different batches of reagents, and different instruments). The data from the two experiments for each test sample were compared and analyzed, and the genotyping accuracy was calculated using the formula: accuracy = 1 - (1 - precision) / 2. Precision refers to the proportion of MNP marker sites whose genotyping results are consistent between the two experiments. The statistical results are shown in Table 4.
[0058] Table 4 shows the accuracy of the MNP marker sites in avocado.
[0059] As shown in Table 4, this embodiment used 7 samples for reproducibility testing, comparing a total of 3257 MNP marker sites, with 0 different sites and an accuracy rate of 100%. High marker accuracy indicates that the identification results are not affected by different personnel, different batches of reagents, or different instruments, providing technical support for the sharing of DNA fingerprint data.
[0060] Distinguishing features of MNP-marked varieties
[0061] Genotypes of all detected MNP marker loci in the 106 test samples were compared pairwise. Based on the principle that a difference of at least one SNP at the same MNP marker locus across different varieties is considered significant, the number of differentially expressed MNP marker loci in each pairwise comparison of the 106 test samples provided in this embodiment was counted, resulting in 5565 pairs of comparisons. The average difference per test sample was 408.84 MNP marker loci, with an average difference rate of 88.26%. The distribution of the difference rate is as follows: Figure 3 As shown. By Figure 3 It can be seen that the selected MNP marker sites have high polymorphism and can significantly distinguish any avocado variety.
[0062] Avocado Variety Identification
[0063] The DNA fingerprint data is compared with a control sample to obtain a genetic similarity coefficient. Based on the genetic similarity coefficient, a variety identification conclusion is obtained for the test sample and the control sample, thereby identifying the variety of the test sample. Specifically, when the genetic similarity coefficient is equal to or greater than 99%, the test sample and the control sample are determined to be of very similar or identical varieties. The control sample can be amplified and sequenced together with the test sample to obtain DNA fingerprint data, or it can be amplified and sequenced beforehand and then compared with the test sample, depending on the actual situation.
[0064] Specifically, a new leaf sample was collected from the test sample PH4 and named PH4-X. The above 106 test samples were used as control samples. The above experimental procedure was followed, and the genetic similarity coefficient was obtained through DNA fingerprint data. Referring to the existing national standard "MNP Marker Method for Plant Variety Identification", the genetic similarity coefficient was used as the basis for determining the variety identification conclusion. When the genetic similarity coefficient (GS) is equal to or greater than 99%, the test sample and the control sample were determined to be "very similar varieties or the same varieties". The results are shown in Table 5.
[0065] Table 5 shows the comparison results of DNA fingerprint data between PH4-X and 106 control samples.
[0066] As shown in Table 5, if the number of differing loci between PH4-X and PH4 from the same test sample is 0 and the genetic similarity (GS value) reaches 100%, they are determined to be the same variety. However, if the number of differing loci between PH4-X and other avocado samples is significant (184-464 loci), they are determined to be different varieties. Therefore, the MNP marker primer combination provided in this embodiment of the invention can be used to compare and analyze the DNA fingerprint data of the collected test samples with existing data for accurate identification of avocado varieties.
[0067] The above description is merely an optional embodiment of this disclosure and is not intended to limit this disclosure. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the protection scope of this disclosure.
Claims
1. An MNP marker primer combination for avocado variety identification, characterized in that, The MNP-labeled primer combination includes primer pairs 1 to 466, each primer pair including a forward primer and a reverse primer. The forward primer of primer pair 1, the reverse primer of primer pair 1 to the forward primer of primer pair 466, and the reverse primer of primer pair 466 are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 932 in the sequence listing.
2. A kit for identifying avocado varieties, characterized in that, The kit includes the MNP-labeled primer combination as described in claim 1.
3. The application of the MNP marker primer combination as described in claim 1 in the identification of avocado varieties, characterized in that, The applications include: Multiplex PCR amplification was performed on the DNA of the test sample using the MNP marker primer combination described in claim 1 to obtain multiplex PCR amplification products; Purify the multiplex PCR amplification product; A high-throughput sequencing library was constructed using the purified multiplex PCR amplification products to obtain the high-throughput library of the sample to be tested. Purify the high-throughput library of the test sample; The purified high-throughput library of the test sample was sequenced to obtain sequencing data. Analyze the sequencing data to obtain DNA fingerprint data; The DNA fingerprint data is compared with control samples to obtain the genetic similarity coefficient; Based on the genetic similarity coefficient, the variety identification conclusions of the test sample and the control sample are obtained.
4. The application according to claim 3, characterized in that, Based on the genetic similarity coefficient, the variety identification conclusion of the test sample and the control sample is obtained, including: when the genetic similarity coefficient is equal to or greater than 99%, the test sample and the control sample are determined to be extremely similar varieties or the same varieties.