Primer group, kit and method for identifying mango variety Guifei
Through primer set PCR amplification and fluorescence capillary electrophoresis detection, and using MGSSR marker site comparison peaks, the identification problem of the mango variety Guifei was solved, and accurate identification with high efficiency and low cost was achieved, ensuring the purity and authenticity of the variety.
Patent Information
- Application Number
- CN202510969005.3
- 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 the Guifei mango variety, especially due to their similar appearance, complex genetic background and environmental influences, which makes identification difficult and easy to confuse.
A specific primer set was used for PCR amplification and fluorescence capillary electrophoresis detection. The marker loci MGSSR069, MGSSR126, MGSSR227, MGSSR246 and MGSSR283 were used to identify the mango variety Guifei by comparing the peak of the amplified product with the standard peak.
The accurate identification of the Guifei mango variety among 63 test samples was achieved with simple operation, low cost, high efficiency and reliable results, ensuring the purity and authenticity of the variety.
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Figure CN120758658A_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 Guifei. 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, a kit, and a method for identifying the mango variety Guifei. The technical solution is as follows:
[0006] On the one hand, the present disclosure provides a primer set for identifying the mango variety Guifei, 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] On the other hand, the present disclosure provides a kit for identifying the mango variety Guifei, comprising the above-mentioned primer set.
[0008] In another aspect, the present disclosure provides a method for identifying the mango variety Guifei, the method comprising:
[0009] Extracting genomic DNA from the sample to be tested;
[0010] PCR amplification is performed on the genomic DNA as a template by using the primer set in claim 1 to obtain five amplification products;
[0011] The five amplification products are respectively detected by fluorescence capillary electrophoresis to obtain peak values of the amplification products; the peak values of the amplification products are respectively compared with standard peak values of the corresponding primer pairs to determine whether the sample to be tested is the mango variety Gefei; the standard peak values of the first primer pair are 219 and 222, the standard peak values of the second primer pair are 159 and 192, the standard peak values of the third primer pair are 193 and 199, the standard peak values of the fourth primer pair are 129 and 142, and the standard peak values of the fifth primer pair are 214 and 223;
[0012] If the peak values of the amplification products of the sample to be tested are consistent with the standard peak values of the corresponding primer pairs, the sample to be tested is the mango variety Gefei; if the peak values of the amplification products of the sample to be tested are inconsistent with at least one of the standard peak values of the corresponding primer pairs, the sample to be tested is not the mango variety Gefei.
[0013] The technical solution provided by the embodiment of the present disclosure has the beneficial effects that: the primer set provided by the embodiment can overcome the uncertainty of variety identification based on the external morphological characteristics of mangoes by detecting the MGSSR069, MGSSR126, MGSSR227, MGSSR246 and MGSSR283 marker sites, accurately identifies the mango variety Gefei in 63 samples to be tested, and the identification process is simple, low in cost, high in efficiency, and the result is intuitive and reliable. The scientific technical support is provided for the identification of the mango variety Gefei, which is conducive to the variety purity guarantee of the mango variety during planting, and is also conducive to the variety true and false identification of the variety during listing and trading. BRIEF DESCRIPTION OF DRAWINGS
[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the drawings needed in the embodiment description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present disclosure, and other drawings can also be obtained by those skilled in the art without creative labor.
[0015] Figure 1 is the standard peak value of the fragment corresponding to the detection of the first primer pair at the MGSSR069 marker site provided by the third embodiment of the present disclosure;
[0016] Figure 2 is the standard peak value of the fragment corresponding to the detection of the second primer pair at the MGSSR126 marker site provided by the third embodiment of the present disclosure;
[0017] Figure 3 It is the standard peak corresponding to the detection fragment of the third primer pair provided in Example 3 of the present disclosure at the MGSSR227 marker site;
[0018] Figure 4 It is the standard peak corresponding to the detection fragment of the fourth primer pair provided in Example 3 of the present disclosure at the MGSSR246 marker site;
[0019] Figure 5 It is the standard peak corresponding to the detection fragment of the fifth primer pair provided in Example 3 of the present disclosure at the MGSSR283 marker site. DETAILED DESCRIPTION
[0020] In order to make the objectives, technical solutions and advantages of the present disclosure more clear, the embodiments of the present disclosure will be further described in detail below with reference to the accompanying drawings.
[0021] Example 1
[0022] In one aspect, the present disclosure provides a primer set for identifying the mango variety Guifei. The primer set comprises five primer pairs, each primer pair comprising 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, respectively, as SEQ ID NOs: 1 to 10 in the sequence listing. Detailed information about the primer set is shown in Table 1.
[0023] Table 1 shows the sequence and information of the primer set
[0024]
[0025] Example 2
[0026] The present disclosure provides a kit for identifying the mango variety Guifei, which includes the primer set provided in Example 1. The kit provided in this embodiment also includes: 2×TaqPCRMasterMix, ddH2O, GeneScan TM 500 LIZ and Hi-Di TM Formamide.
[0027] Example 3
[0028] The present disclosure provides a method for identifying the mango variety Guifei, specifically:
[0029] Extracting genomic DNA from the sample to be tested;
[0030] Using the genomic DNA as a template, PCR amplification was performed using the above primer set to obtain 5 amplification products;
[0031] The peak values of the amplification products are respectively compared with the standard peak values of the corresponding primer pairs, so as to determine whether the sample to be tested is the mango variety Gefei.
[0032] The peak values of the amplification products are respectively compared with the standard peak values of the corresponding primer pairs, so as to determine whether the sample to be tested is the mango variety Gefei.
[0033] In this embodiment, 63 representative mango germplasms including Gefei are used as samples to be tested for identification. The 63 mango germplasms are as follows: CC sub, YNMGZY1, 7102, Thailand A, 91-dongfeng, ball mango variety No. 2, Mengbang red, YNMGZY4, Ivory Coast, Fujian mango, Myanmar system, Mar-90, 90-Menghai, 635, Thailand C, Gefei, Guichong No. 1, Thailand E, Mengtai, Jinhuang, 90-12, Mali mango, 90-9, dwarf acid, Cuba No. 3, 907, white unknown, Longtai, chestnut mango, Myanmar No. 7, Thailand D, Ivory No. 22, Tainong No. 1, YNMGZY9, similar to 905, Kate, Machasu system, wild red, 2, 7003, 8, X10, 906C-6, Cangjiang 393, Jing three, 90-5, YNMGZY11, YNMGZY13, YNMGZY2, coconut flavor, YNMGZY3, Liuxiang mango, YNMGZY15, Indian system, Jinqian mango, ball mango variety No. 1, 906D-7, B, Guinea No. 2, Yuantuan, white ivory, new Honghe mango and dwarf mango.
[0034] All the test germplasms in this embodiment are planted and preserved in the mango germplasm resource garden of the Yunnan Institute of Tropical Crop Sciences, which is located in the Xishuangbanna Tropical Flower Garden and simultaneously serves as the Yunnan Innovation Base for the Protection of Mango Germplasm Resources of the Ministry of Agriculture and Rural Affairs and the Yunnan Provincial Tropical Crop Banna Germplasm Resource Garden, with perfect germplasm preservation conditions and standardized management measures.
[0035] 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.
[0036] 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.
[0037] 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 0.5 μL and Hi-Di TM Formamide 8.5 μL; the sample and reagent added detection plate was centrifuged and then placed on the PCR instrument to run the denaturation program (95℃, 3 min), and immediately cooled after denaturation; according to the ABI 3730xL instrument operation process, the detection file corresponding to the name of the detection plate was selected, and the SSR sample analysis detection program was run. The results were analyzed using GeneMarker software to obtain the allele number, peak graph, peak value and genotype of each sample to be tested.
[0038] The first to fifth primer pairs provided in the embodiment detect the standard peak values of the fragments at the MGSSR069, MGSSR126, MGSSR227, MGSSR246 and MGSSR283 marker sites.
[0039] Specifically, the standard peak values of the first primer pair are 219 and 222, the standard peak values of the second primer pair are 159 and 192, the standard peak values of the third primer pair are 193 and 199, the standard peak values of the fourth primer pair are 129 and 142, and the standard peak values of the fifth primer pair are 214 and 223.
[0040] The peak values obtained by amplifying the 63 samples to be tested using the five primer pairs were compared with the standard peak values, and the specific results are shown in Table 2.
[0041] Table 2 shows the comparison of the peak values of the 63 samples to be tested with the standard peak values
[0042]
[0043]
[0044]
[0045] Table 2 provides the peak value combinations of the 63 samples to be tested. The peak values of sample No. 17 at the MGSSR069, MGSSR126, MGSSR227, MGSSR246 and MGSSR283 marker sites are 219 and 222, 159 and 192, 193 and 199, 129 and 142, and 214 and 223, respectively. After comparison, it is found that the peak values are completely consistent with the 10 standard peak values of the mango variety Gefei provided in the embodiment, and therefore it is determined that the sample is the mango variety Gefei. The results show that the SSR primer pair combination used in the embodiment can accurately identify the mango variety Gefei.
[0046] The above only describes optional embodiments of the disclosure and is not intended to limit the disclosure. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the disclosure shall be included in the protection scope of the disclosure.
Claims
1. A primer set for identifying the mango variety Guifei, 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 Guifei, characterized in that: The kit comprises the primer set according to claim 1.
3. A method for identifying the mango variety Guifei, 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 amplification products are respectively subjected to fluorescence capillary electrophoresis detection to obtain peak values of the amplification products; the peak values of the amplification products are respectively compared with the standard peak values of the corresponding primer pairs, thereby determining whether the test sample is the mango variety Guifei; the standard peak values of the first primer pair are 219 and 222, the standard peak values of the second primer pair are 159 and 192, the standard peak values of the third primer pair are 193 and 199, the standard peak values of the fourth primer pair are 129 and 142, and the standard peak values of the fifth primer pair are 214 and 223; 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 Guifei; 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 Guifei.