Primer group, kit and method for identifying mango variety dwarf acid

Abstract

The invention provides a primer group, a kit and a method for identifying mango variety dwarf acid, and belongs to the field of germplasm resource identification. The primer group is composed of five primer pairs, each primer pair is composed of a forward primer and a reverse primer, and 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 sequentially shown as SEQ ID NO: 1 to SEQ ID NO: 10 in a sequence table. According to the primer group and the corresponding method provided by the embodiment of the invention, the uncertainty of variety identification based on external morphological characteristics of mangoes can be overcome, the mango variety dwarf acid can be accurately identified, the operation is simple and convenient, the cost is low, the efficiency is high, and the result is visual and reliable. Scientific technical guarantee is provided for protection of the new mango variety dwarf acid, and popularization and application of the mango variety are facilitated.

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 dwarf acid mango varieties. 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 dwarf acid. The technical solution is as follows:

[0006] On the one hand, the present disclosure provides a primer set for identifying dwarf acid mango varieties, the primer set consisting of five 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.

[0007] In another aspect, the present disclosure provides a kit for identifying dwarf acid of mango varieties, the kit comprising the above primer set.

[0008] In another aspect, the present disclosure provides a method for identifying dwarf acid in mango varieties, 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 are respectively compared with the standard peak values ​​of the corresponding primer pairs to determine whether the sample to be tested is a mango variety of dwarf acid; the standard peak values ​​of the first primer pair are 165 and 168, the standard peak value of the second primer pair is 144, the standard peak value of the third primer pair is 129 and 132, the standard peak value of the fourth primer pair is 134 and 143, and the standard peak value of the fifth primer pair is 169 and 185;

[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, the sample to be tested is the mango variety dwarf acid; 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, the sample to be tested is not the mango variety dwarf acid.

[0014] The technical solution provided by the embodiments of the present disclosure has the following beneficial effects: the primer set provided in this embodiment, by detecting the marker loci MGSSR031, MGSSR215, MGSSR224, MGSSR225, and MGSSR249, can overcome the uncertainty of variety identification based on the external morphological characteristics of mangoes, accurately identifying the mango variety dwarf sour in 63 samples to be tested, and the identification process is simple to operate, low in cost, highly efficient, and the results are intuitive and reliable. It provides scientific technical support for the identification of the mango variety dwarf sour, which is beneficial for ensuring the purity of the mango variety during planting and also for distinguishing the authenticity of the mango variety when it is put on the market 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 MGSSR031 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 MGSSR215 marker site;

[0018] 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 MGSSR224 marker site;

[0019] 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 MGSSR225 marker site;

[0020] 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 MGSSR249 marker site. DETAILED DESCRIPTION

[0021] 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.

[0022] Example 1

[0023] In one aspect, the present disclosure provides a primer set for identifying dwarf acid mango varieties. 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.

[0024] Table 1 shows the sequence and information of the primer set

[0025]

[0026] Example 2

[0027] The present disclosure provides a kit for identifying dwarf acid in mango varieties, the kit comprising the primer set provided in Example 1. The kit provided in this embodiment also comprises: 2×TaqPCRMasterMix, ddH2O, GeneScan TM 500 LIZ and Hi-Di TM Formamide.

[0028] Example 3

[0029] The present disclosure provides a method for identifying dwarf acid mango varieties, specifically comprising:

[0030] Extracting genomic DNA from the sample to be tested;

[0031] Using the genomic DNA as a template, PCR amplification was performed using the primer set provided in Example 1 to obtain an amplified 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 the 9 standard peaks, and the comparison results were used to determine whether the sample to be tested was the mango variety dwarf acid.

[0034] In this embodiment, 63 representative mango germplasms including dwarf acid were used as test samples for identification. The specific 63 mango germplasms were: CC sub-, 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 Tropical Crops Science Institute. This resource garden is located in the Xishuangbanna Tropical Flower Garden. It serves as the Yunnan Innovation Base for Mango Germplasm Resource Conservation and the Yunnan Provincial Tropical Crops Xishuangbanna Germplasm Resource Garden, and 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. In station 1 of the instrument, dispense 100 μL of magnetic beads into each well of a new extraction plate and place it in station 2 of the extraction instrument. Dispense three plates of 75% ethanol at 500 μL / well and place them in stations 3, 4, and 5, respectively. Take the elution plate and dispense 100 μL, 80 μL, and 60 μL of eluate into each well (depending on the sample status). Place the elution solution in station 6. Verify the instrument status and extraction plate information before running the program. After the run is complete, remove the elution plate and obtain the eluted genomic DNA. Take another 2 μL of the genomic DNA stock solution and add 2 μL of bromophenol blue for agarose gel electrophoresis to test the integrity of the genomic DNA. 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 MGSSR031, MGSSR215, MGSSR224, MGSSR225, and MGSSR249 using primer pairs 1 to 5.

[0040] Specifically, the standard peak values ​​of the first primer pair are 165 and 168, the standard peak value of the second primer pair is 144, the standard peak value of the third primer pair is 129 and 132, the standard peak value of the fourth primer pair is 134 and 143, and the standard peak value of the fifth primer pair is 169 and 185.

[0041] The peak values ​​obtained by amplifying the 63 test samples provided in this example using the five primer pairs were compared with the nine standard peak values. 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. 25 has peaks at markers MGSSR031, MGSSR215, MGSSR224, MGSSR225, and MGSSR249, respectively: 165 and 168, 144, 129 and 132, 134 and 143, and 169 and 185. These peaks are completely consistent with the nine standard peaks for the mango variety dwarf acid provided in this example, and thus the sample is identified as the mango variety dwarf acid. These results demonstrate that the SSR primer pair combination used in this example can accurately identify the mango variety dwarf acid.

[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 dwarf acid mango varieties, 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 dwarf acid in mango varieties, characterized in that: The kit comprises the primer set according to claim 1.

3. A method for identifying dwarf acid mango varieties, 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 are respectively compared with the standard peak values ​​of the corresponding primer pairs to determine whether the sample to be tested is a mango variety of dwarf acid; the standard peak values ​​of the first primer pair are 165 and 168, the standard peak value of the second primer pair is 144, the standard peak value of the third primer pair is 129 and 132, the standard peak value of the fourth primer pair is 134 and 143, and the standard peak value of the fifth primer pair is 169 and 185; 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, the sample to be tested is the mango variety dwarf acid; 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, the sample to be tested is not the mango variety dwarf acid.

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