Primer group, kit and method for identifying mango variety Jinhuang
Through primer set PCR amplification and fluorescence capillary electrophoresis detection, the identification problem of the mango variety Jinhuang was solved, and accurate identification was achieved with high efficiency and low cost, ensuring the purity of the variety and the authenticity of the transaction.
Patent Information
- Application Number
- CN202510969034.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-15
- Publication Date
- 2025-10-10
AI Technical Summary
Existing technologies make it difficult to quickly and accurately identify mango varieties, especially the Jinhuang mango variety, which has problems such as similar appearance, complex genetic background and large environmental influences, making identification more difficult.
Specific primer sets were used for PCR amplification and fluorescence capillary electrophoresis detection. Five primer pairs (MGSSR050, MGSSR139, MGSSR167, MGSSR195 and MGSSR275 marker loci) were used to identify the mango variety Jinhuang. The peak of the amplified product was compared with the standard peak for identification.
The accurate identification of the mango variety Jinhuang has been achieved. The operation is simple, low-cost, and efficient. The results are intuitive and reliable, ensuring the purity of the variety and the authenticity of the transaction.
Smart Images
Figure CN120758659A_ABST
Abstract
Description
Technical Field
[0001] The present disclosure relates to the field of germplasm resource identification, and in particular to a primer set, a kit, and a method for identifying the mango variety Jinhuang. Background Art
[0002] Mango is a representative tropical fruit, favored by consumers worldwide for its unique flavor and nutritional value. To meet the demands of both domestic and international markets, my country's mango industry has seen significant growth in both area and output, making it a major global producer of mangoes.
[0003] Currently, mango variety identification faces many challenges, mainly reflected in the following four aspects: First, there are many mango varieties, and there are subtle differences in appearance, flavor, and maturity between different varieties, which increases the difficulty of accurate identification; second, morphological characteristics are similar. Many mango varieties have similar appearances, such as fruit size, traits, and color, which makes it difficult to distinguish them by naked eye observation alone; third, the genetic background is complex. Mango has a complex genetic background, and hybridization and outcrossing may occur between different varieties, resulting in blurred boundaries between varieties; fourth, environmental influences. Environmental factors such as climate and soil conditions have a great influence on mango growth and fruit quality, which may cause the same variety to show differences in different environments, thereby increasing the difficulty of identification. Therefore, there is an urgent need for a simple, rapid, and effective identification technology that is truly effective, unaffected by the environment, and can accurately identify new mango varieties to ensure the purity of mango varieties and avoid confusion.
[0004] Public content
[0005] To solve the problem of rapid and effective identification of mango varieties, the present disclosure provides a primer set, kit, and method for identifying the mango variety Jinhuang. The technical solution is as follows:
[0006] On the one hand, the present disclosure provides a primer set for identifying the mango variety Jinhuang, the primer set consisting of 5 primer pairs, each of the primer pairs consisting of a forward primer and a reverse primer, the forward primer of the first primer pair, the reverse primer of the first primer pair, the forward primer of the fifth primer pair, and the reverse primer of the fifth primer pair are shown in SEQ ID NO: 1 to SEQ ID NO: 10 in the sequence listing, respectively.
[0007] In another aspect, the present disclosure provides a kit for identifying the mango variety Jinhuang, comprising the above-mentioned primer set.
[0008] In another aspect, the present disclosure provides a method for identifying the mango variety Jinhuang, the method comprising:
[0009] Extracting genomic DNA from the sample to be tested;
[0010] Using the genomic DNA as a template, PCR amplification is performed using the primer set of claim 1 to obtain 5 amplification products;
[0011] The five amplified products are detected by fluorescence capillary electrophoresis to obtain peak values of the amplified products;
[0012] The peak values of the amplified products were compared with the standard peak values of the corresponding primer pairs, thereby determining whether the sample to be tested is the mango variety Jinhuang; the standard peak values of the first primer pair are 137 and 148, the standard peak value of the second primer pair is 94, the standard peak value of the third primer pair is 101 and 109, the standard peak value of the fourth primer pair is 167 and 170, and the standard peak value of the fifth primer pair is 183;
[0013] If the peak value of the amplified product of the sample to be tested is consistent with the standard peak value of the corresponding primer pair, then the sample to be tested is the mango variety Jinhuang; if the peak value of the amplified product of the sample to be tested is inconsistent with at least one of the standard peak values of the corresponding primer pair, then the sample to be tested is not the mango variety Jinhuang.
[0014] The technical solution provided by the disclosed embodiments has the following beneficial effects: the primer set provided in this embodiment, by detecting the marker loci MGSSR050, MGSSR139, MGSSR167, MGSSR195, and MGSSR275, can overcome the uncertainty of variety identification based on the external morphological characteristics of mangoes, accurately identifying the mango variety Jinhuang in 63 test samples, and the identification process is simple to operate, low-cost, highly efficient, and the results are intuitive and reliable. This provides scientific technical support for the identification of the mango variety Jinhuang, which is beneficial for ensuring the purity of the mango variety during planting and for distinguishing the authenticity of the mango variety when it is listed for trading. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.
[0016] Figure 1 It is the standard peak corresponding to the detection fragment of the first primer pair provided in Example 3 of the present disclosure at the MGSSR050 marker site;
[0017] Figure 2 It is the standard peak corresponding to the detection fragment of the second primer pair provided in Example 3 of the present disclosure at the MGSSR139 marker site;
[0018] Figure 3 is a standard peak value corresponding to a fragment detected by the 3rd primer pair at the MGSSR167 marker site provided in Embodiment Three of the present disclosure;
[0019] Figure 4 is a standard peak value corresponding to a fragment detected by the 4th primer pair at the MGSSR195 marker site provided in Embodiment Three of the present disclosure;
[0020] Figure 5 is a standard peak value corresponding to a fragment detected by the 5th primer pair at the MGSSR275 marker site provided in Embodiment Three of the present disclosure. DETAILED DESCRIPTION
[0021] In order to make the objectives, technical solutions and advantages of the present disclosure clearer, the following will further describe the embodiments of the present disclosure in detail with reference to the accompanying drawings.
[0022] Embodiment One
[0023] In one aspect, the present disclosure provides a primer set for identifying mango variety Jinhuang, which comprises five primer pairs, each of which comprises a forward primer and a reverse primer, the forward primer of the 1st primer pair, the reverse primer of the 1st primer pair, the forward primer of the 5th primer pair and the reverse primer of the 5th primer pair are sequentially shown as SEQ ID NO: 1 to SEQ ID NO: 10 in the sequence listing. The specific information of the primer set is shown in Table 1.
[0024] Table 1 shows the sequences and information of the primer set
[0025]
[0026] Embodiment Two
[0027] The present disclosure provides a kit for identifying mango variety Jinhuang, which comprises the primer set provided in Embodiment One. The kit provided in the present embodiment further comprises: 2x Taq PCR Master Mix, ddH2O, GeneScan TM 500 LIZ and Hi-Di TM Formamide.
[0028] Embodiment Three
[0029] The present disclosure provides a method for identifying mango variety Jinhuang, specifically:
[0030] extracting genomic DNA from the sample to be tested;
[0031] using the genomic DNA as a template, performing PCR amplification using the primer set provided in Embodiment One to obtain an amplification product;
[0032] The amplified product is detected by fluorescence capillary electrophoresis to obtain the peak of the amplified product;
[0033] The peak of the amplified product was compared with 8 standard peaks, and the comparison results were used to determine whether the sample to be tested was the mango variety Jinhuang.
[0034] In this embodiment, 63 representative mango germplasms including Jinhuang were used as test samples for identification. The specific 63 mango germplasms were: CC Asia, YNMGZY1, 7102, Thailand A, 91-Dongfeng, Ball Mango Variety 2, Mengbanghong, YNMGZY4, Ivory Coast, Fujian Mango, Myanmar System, Mar-90, 90-Menghai, 635, Thailand C, Guifei, Kuichong No. 1, Thailand E, Mengtai, Jinhuang, 90-12, Mali Mango, 90-9, Dwarf Acid, Cuba No. 3, 907, White Unknown, Longtai, Xionggou mango, Myanmar No. 7, Thailand D, Ivory No. 22, Tainong No. 1, YNMGZY9, similar to 905, Kate, Marchesu series, Wild Red, 2, 7003, 8, X10, 906C-6, Cangjiang 393, Jingsan, 90-5, YNMGZY11, YNMGZY13, YNMGZY2, Coconut fragrance, YNMGZY3, Liuxiang mango, YNMGZY15, India series, Money Mango, Ball Mango variant No. 1, 906D-7, B, Guinea No. 2, Yuantuan, White Ivory, New Red River Mango and Dwarf Mango.
[0035] All test germplasms in this example were planted and preserved in the mango germplasm resource garden of the Yunnan Institute of Tropical Crops. This resource garden is located in the Xishuangbanna Tropical Flower Garden. It serves as the Yunnan Innovation Base for Mango Germplasm Conservation of the Ministry of Agriculture and Rural Affairs and the provincial-level tropical crop germplasm resource garden in Xishuangbanna, Yunnan Province. It has complete germplasm preservation conditions and standardized management measures.
[0036] Specifically, the genomic DNA of the 63 samples to be tested was extracted by taking an appropriate amount of leaves of the sample to be tested, grinding the leaves into powder, and placing them into a pre-cooled centrifuge tube, then quickly adding 500 μL of lysis solution to the centrifuge tube, and then adding 100 μL of antioxidant and 10 μL of RnaseA, after being fully mixed, incubate at 65℃ for 10min, and shake it 2-3 times during the period; then add 150μL of polysaccharide scavenger to the centrifuge tube, mix gently for 15-20 times, and let it stand at room temperature for 3-5min; centrifuge the centrifuge tube at 4℃ and 12000rpm for 5min to obtain the supernatant, take 500μL of the supernatant and put it into a new centrifuge tube; add 300μL of L(chloroform): V(isoamyl alcohol) = 24:1 mixed solution to the new centrifuge tube, mix it, shake it up and down vigorously for 15s, centrifuge it at 1200rpm for 30s to obtain the upper aqueous phase; take 500μL of the upper aqueous phase and put it into the extraction plate, then add an equal volume of isopropanol to the extraction plate and place it on the extraction plate. Instrument station 1, in a new extraction plate, dispense 100 μL of magnetic beads into each well and place it in extraction instrument station 2, dispense three plates of 500 μL / well of 75% ethanol and place them in stations 3, 4 and 5 respectively, take the elution plate, dispense 100 μL, 80 μL and 60 μL of eluate into each well (the corresponding volume is dispensed according to the status of the sample to be tested) and place it in station 6, check the instrument status and extraction plate information and run the program after the operation is completed; after the operation is completed, remove the elution plate to obtain the eluted genomic DNA, take another 2 μL of genomic DNA stock solution and add 2 μL of bromophenol blue for agarose gel electrophoresis to detect the integrity of the genomic DNA, and the remaining eluted genomic DNA can be stored at 4°C.
[0037] Specifically, using genomic DNA as a template, PCR amplification was performed in sequence using the five primer pairs provided in Example 1 to obtain amplified products. Each 10 μL PCR amplification system included: 1 μL of 20 ng / μL genomic DNA, 5.0 μL of 2×TaqPCRMasterMix, 0.5 μL of 10 pmol / μL forward primer, 0.5 μL of 10 pmol / μL reverse primer, and 3.0 μL of ddH2O. The PCR amplification program included: pre-denaturation at 95°C for 5 minutes; the first cycle included: denaturation at 95°C for 30 seconds, gradient annealing at 62°C to 52°C for 30 seconds, and extension at 72°C for 30 seconds, for a total of 10 first cycles; the second cycle included: denaturation at 95°C for 30 seconds, annealing at 52°C for 30 seconds, and extension at 72°C for 30 seconds, for a total of 25 second cycles; extension at 72°C for 20 minutes, and storage at 4°C after the amplification was completed.
[0038] Specifically, the amplified product was detected by fluorescence capillary electrophoresis to obtain the peak value of the amplified product. The amplified product diluted to a uniform concentration was added to the upper plate, and the upper detection reagents were added according to the total system of 10 μL: 1 μL of amplified product, GeneScan TM500 LIZ 0.5μL and Hi-Di TM 8.5 μL of Formamide was added. After centrifugation, the plate containing the sample and reagents was placed on a PCR instrument and denaturation cycle was run (95°C, 3 minutes). After denaturation, the plate was immediately cooled. Following the ABI 3730xL instrument operation procedure, the test file corresponding to the plate to be tested was selected and the SSR sample analysis program was run. The results were analyzed using GeneMarker software to obtain the number of alleles, peak plot, peak value, and genotype for each sample.
[0039] This example provides standard peaks for detecting fragments at the marker sites MGSSR050, MGSSR139, MGSSR167, MGSSR195, and MGSSR275 using primer pairs 1 to 5.
[0040] Specifically, the standard peak values of the first primer pair are 137 and 148, the standard peak value of the second primer pair is 94, the standard peak value of the third primer pair is 101 and 109, the standard peak value of the fourth primer pair is 167 and 170, and the standard peak value of the fifth primer pair is 183.
[0041] The peaks obtained by amplifying the 63 test samples provided in this example using 5 primer pairs were compared with the 8 standard peaks. The specific results are shown in Table 2.
[0042] Table 2 shows the comparison results of the peak values of 63 samples to be tested and the standard peak values.
[0043]
[0044]
[0045]
[0046] Table 2 provides the peak combinations for the 63 samples tested. Sample No. 21 has peaks at markers MGSSR050, MGSSR139, MGSSR167, MGSSR195, and MGSSR275, respectively: 137 and 148, 94, 101 and 109, 167 and 170, and 183. These peaks are completely consistent with the eight standard peaks for the mango variety Jinhuang provided in this example, and therefore the sample is identified as the mango variety Jinhuang. These results demonstrate that the SSR primer pair combination used in this example can accurately identify the mango variety Jinhuang.
[0047] The above description is merely an optional embodiment of the present disclosure and is not intended to limit the present disclosure. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the scope of protection of the present disclosure.
Claims
1. A primer set for identifying the mango variety Jinhuang, characterized in that: The primer set consists of 5 primer pairs, each of which consists of a forward primer and a reverse primer. The forward primer of the first primer pair, the reverse primer of the first primer pair, the forward primer of the fifth primer pair, and the reverse primer of the fifth primer pair are shown in SEQ ID NO: 1 to SEQ ID NO: 10 in the sequence listing, respectively.
2. A kit for identifying the mango variety Jinhuang, characterized in that: The kit comprises the primer set according to claim 1.
3. A method for identifying the mango variety Jinhuang, characterized in that: The method is: Extracting genomic DNA from the sample to be tested; Using the genomic DNA as a template, PCR amplification is performed using the primer set of claim 1 to obtain 5 amplification products; The five amplified products are detected by fluorescence capillary electrophoresis to obtain peak values of the amplified products; The peak values of the amplified products were compared with the standard peak values of the corresponding primer pairs, thereby determining whether the sample to be tested is the mango variety Jinhuang; the standard peak values of the first primer pair are 137 and 148, the standard peak value of the second primer pair is 94, the standard peak value of the third primer pair is 101 and 109, the standard peak value of the fourth primer pair is 167 and 170, and the standard peak value of the fifth primer pair is 183; If the peak value of the amplified product of the sample to be tested is consistent with the standard peak value of the corresponding primer pair, then the sample to be tested is the mango variety Jinhuang; if the peak value of the amplified product of the sample to be tested is inconsistent with at least one of the standard peak values of the corresponding primer pair, then the sample to be tested is not the mango variety Jinhuang.