Primer group, kit and method for identifying mango variety Katt

Abstract

The invention provides a primer group, a kit and a method for identifying a mango variety Katt, and belongs to the field of germplasm resource identification. The primer group is composed of four 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 fourth primer pair and the reverse primer of the fifth primer pair are sequentially shown as SEQ ID NO: 1 to SEQ ID NO: 8 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 Katt 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.

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 Kate. 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 Kate. The technical solution is as follows:

[0006] On the one hand, the present disclosure provides a primer set for identifying the mango variety Kate, the primer set consisting of four 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 fourth primer pair, and the reverse primer of the fourth primer pair are shown in SEQ ID NO: 1 to SEQ ID NO: 8 in the sequence listing, respectively.

[0007] In another aspect, the present disclosure provides a kit for identifying the mango variety Kate, comprising the above primer set.

[0008] In another aspect, the present disclosure provides a method for identifying mango variety Kate, 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 to obtain four amplification products; the four amplification products are respectively detected by fluorescent capillary electrophoresis to obtain peak values ​​of the amplification products;

[0011] Comparing the peak values ​​of the amplified products with the corresponding standard peak values ​​of the primer pairs, thereby determining whether the sample to be tested is the mango variety Kate;

[0012] Specifically, the standard peak values ​​of the first primer pair are 210 and 214, the standard peak values ​​of the second primer pair are 90 and 97, the standard peak values ​​of the third primer pair are 120 and 129, and the standard peak values ​​of the fourth primer pair are 170 and 179;

[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 Kate; 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 Kate.

[0014] The technical solutions provided by the disclosed embodiments have the following beneficial effects: The primer set provided in this embodiment, by detecting the marker loci MGSSR013, MGSSR114, MGSSR141, and MGSSR266, can overcome the uncertainty of variety identification based on external morphological characteristics of mangoes, accurately identifying the mango variety Kate in 63 test samples. The identification process is simple, low-cost, and highly efficient, and the results are intuitive and reliable. This provides scientific technical support for the identification of the mango variety Kate, which is beneficial for ensuring the purity of this mango variety during planting and for distinguishing the authenticity of this 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 MGSSR013 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 MGSSR114 marker site;

[0018] Figure 3It is the standard peak corresponding to the detection fragment of the third primer pair provided in Example 3 of the present disclosure at the MGSSR141 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 MGSSR266 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 Kate. The primer set comprises four 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 fourth primer pair, and the reverse primer of the fourth primer pair are shown, respectively, as SEQ ID NOs: 1 to 8 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 Kate, which includes the primer set provided in Example 1. The kit provided in this embodiment also includes: 2×TaqPCRMasterMix, ddH2O, GeneScan TM 500LIZ and Hi-Di TM Formamide.

[0027] Example 3

[0028] The present disclosure provides a method for identifying the mango variety Kate, 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 four amplification products;

[0031] The four amplification products are respectively subjected to fluorescence capillary electrophoresis detection to obtain peak values ​​of the amplification products;

[0032] The peak values ​​of the amplified products are compared with the corresponding standard peak values ​​of the primer pairs, so as to determine whether the sample to be tested is the mango variety Kate.

[0033] In this embodiment, 63 representative mango germplasms including Kate 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.

[0034] All the test germplasms in this example were planted and preserved in the mango germplasm resource garden of the Yunnan Institute of Tropical Crops Sciences, which is located in the Xishuangbanna Tropical Flower Garden. It is also the Yunnan Innovation Base for Mango Germplasm Resource Conservation of the Ministry of Agriculture and Rural Affairs and the Yunnan Provincial Tropical Crops Xishuangbanna Germplasm Resource Garden, with complete 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, genomic DNA was used as a template, and PCR amplification was performed in sequence using the four 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 min; the first cycle included: denaturation at 95°C for 30 s, gradient annealing at 62°C to 52°C for 30 s, and extension at 72°C for 30 s, for a total of 10 first cycles; the second cycle included: denaturation at 95°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C for 30 s, for a total of 25 second cycles; extension at 72°C for 20 min, 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 TM500LIZ 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.

[0038] This example provides standard peak values ​​for detecting fragments at the marker sites MGSSR013, MGSSR114, MGSSR141, and MGSSR266 using the first to fourth primer pairs.

[0039] Specifically, the standard peak values ​​of the first primer pair are 210 and 214, the standard peak values ​​of the second primer pair are 90 and 97, the standard peak values ​​of the third primer pair are 120 and 129, and the standard peak values ​​of the fourth primer pair are 170 and 179.

[0040] The peak values ​​obtained by amplifying the 63 test samples provided in this example using the four primer pairs were compared with the standard peak values. The specific results are shown in Table 2.

[0041] Table 2 shows the comparison results of the peak values ​​of 63 samples to be tested and the standard peak values.

[0042]

[0043]

[0044] Table 2 provides the peak combinations for 63 test samples. Sample 36 has peaks at marker loci MGSSR013, MGSSR114, MGSSR141, and MGSSR266, respectively: 210 and 214, 90 and 97, 120 and 129, and 170 and 179. These peaks are completely consistent with the eight standard peaks for the mango variety Kate provided in this example, thus confirming that the sample is the mango variety Kate. This demonstrates that the primer pair combination provided in this example can accurately identify the test sample as the mango variety Kate.

[0045] 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 Kate, characterized in that: The primer set consists of four 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 fourth primer pair, and the reverse primer of the fourth primer pair are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 8 in the sequence listing.

2. A kit for identifying mango variety Kate, characterized in that: The kit comprises the primer set according to claim 1.

3. A method for identifying mango variety Kate, 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 four amplification products; The four amplified products are respectively subjected to fluorescence capillary electrophoresis detection 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, thereby determining whether the sample to be tested is the mango variety Kate; the standard peak values ​​of the first primer pair are 210 and 214, the standard peak values ​​of the second primer pair are 90 and 97, the standard peak values ​​of the third primer pair are 120 and 129, and the standard peak values ​​of the fourth primer pair are 170 and 179; 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 Kate; 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 Kate.

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