A primer set, kit and method for identifying Yunmang 4

Abstract

The present disclosure provides a primer set, a kit and a method for identifying Yunmang No. 4, which belongs to the field of germplasm resource identification. The primer set includes: 6 primer pairs, each of which includes 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 sixth primer pair and the reverse primer of the sixth primer pair are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 12 in the sequence table. The primer set and the corresponding method provided in this embodiment can overcome the uncertainty of variety identification based on the external morphological characteristics of mangoes, accurately identify the new mango variety Yunmang No. 4, and are simple to operate, low in cost, high in efficiency, and the results are intuitive and reliable. The present disclosure provides scientific and technical guarantees for the protection of the new mango variety Yunmang No. 4, which is conducive to the promotion and application of this mango variety.

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 Yunmang 4. Background Art

[0002] Mango is one of the representative tropical fruits, and is favored by consumers around the world for its unique flavor and nutritional value. In order to meet the needs of domestic and foreign markets, my country's mango industry has grown significantly in both area and output, and has become one of the important mango producers in the world. However, the current mango varieties in my country need to be improved in terms of output and quality. If we want to improve the economic benefits of the mango industry, we must speed up the selection and breeding of mango varieties and promote new mango varieties.

[0003] Yunmang 4 is a new mango variety that was developed through cross breeding and targeted cultivation with Mangosteen as the female parent and Red Cloud as the male parent. Yunmang 4 has the characteristics of high yield and excellent quality. In the future, other varieties may be sold as Yunmang 4 in the market. However, due to the external morphological characteristics of the asexual reproduction seedlings, it is difficult to distinguish the authenticity of the variety, and it is difficult to effectively supervise and arbitrate, which will have a negative impact on the promotion, utilization and protection of the variety.

[0004] At present, there are many challenges in identifying new mango varieties, which are mainly reflected in the following four aspects: first, variety diversity. There are many mango varieties, and there are subtle differences in appearance, flavor, maturity, etc. between different varieties, which increases the difficulty of accurate identification; second, similar morphological characteristics. Many mango varieties have similar appearances, such as fruit size, traits and color, which makes it difficult to distinguish them by naked eye observation; third, the genetic background is complex. The genetic background of mango is complex. There may be hybridization and outcrossing between different varieties, resulting in blurred boundaries between varieties; fourth, environmental influences. Environmental factors such as climate and soil conditions have a great impact on the growth and fruit quality of mangoes, 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, fast and effective identification technology that is real and effective, not affected by the environment, and can accurately identify new mango varieties.

[0005] Public Content

[0006] In order to solve the problem of rapid and effective identification and variety protection, the present disclosure provides a primer set, a kit and a method for identifying Yunmang No. 4. The technical solution is as follows:

[0007] On the one hand, the present disclosure provides a primer set for identifying Yunmang No. 4, the primer set comprising: 6 primer pairs, each of the primer pairs 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 sixth primer pair and the reverse primer of the sixth primer pair are respectively as shown in SEQ ID NO: 1 to SEQ ID NO: 12 in the sequence listing.

[0008] On the other hand, the present disclosure provides a kit for identifying Yunmang 4, the kit comprising the above-mentioned primer set.

[0009] In another aspect, the present disclosure provides a method for identifying Yunmang No. 4, the method comprising:

[0010] Extracting genomic DNA from the sample to be tested;

[0011] Using the genomic DNA as a template, PCR amplification is performed using the above six primer pairs to obtain amplified products;

[0012] The six amplification products are respectively detected by fluorescent capillary electrophoresis to obtain six peaks of the amplification products;

[0013] The peak values ​​of the six amplified products are compared with the corresponding standard peak values ​​of the primer pairs to determine whether the sample to be tested is Yunmang No. 4.

[0014] Specifically, the standard peak values ​​of the first primer pair are 206 and 210, the standard peak values ​​of the second primer pair are 172 and 175, the standard peak value of the third primer pair is 175, the standard peak value of the fourth primer pair is 150, the standard peak value of the fifth primer pair is 159, and the standard peak value of the sixth primer pair is 113. If the peak values ​​of the six amplification products of the sample to be tested are consistent with the corresponding standard peak values ​​of the primer pairs, then the sample to be tested is Yunmang No. 4. If the peak values ​​of the six amplification products of the sample to be tested are inconsistent with at least one of the corresponding standard peak values ​​of the primer pairs, then the sample to be tested is not Yunmang No. 4.

[0015] The technical solution provided by the embodiment of the present disclosure has the following beneficial effects: 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 markers MIL035, MIL053, MIL074, MIL083, MIL117 and MIL173, and accurately identify the new mango variety Yunmang 4 in 116 samples to be tested, and the identification process is simple to operate, low in cost, high in efficiency, and the results are intuitive and reliable. It provides scientific technical guarantees for the protection of the new mango variety Yunmang 4, which is conducive to the promotion and application of the mango variety. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the drawings required for use in the description of the embodiments will be briefly introduced below. 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 paying any creative work.

[0017] Figure 1 is the electrophoresis chromatogram of the standard peak provided by the first primer pair provided in Example 3 of the present disclosure;

[0018] Figure 2 is the electrophoresis chromatogram of the standard peak provided by the second primer pair provided in Example 3 of the present disclosure;

[0019] Figure 3 is the electrophoresis chromatogram of the standard peak provided by the third primer pair provided in Example 3 of the present disclosure;

[0020] Figure 4 is the electrophoresis chromatogram of the standard peak provided by the fourth primer pair provided in Example 3 of the present disclosure;

[0021] Figure 5 is the electrophoresis chromatogram of the standard peak provided by the fifth primer pair provided in Example 3 of the present disclosure;

[0022] Figure 6 It is the electrophoresis chromatogram of the standard peak provided by the sixth primer pair provided in Example 3 of the present disclosure. DETAILED DESCRIPTION

[0023] 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.

[0024] Embodiment 1

[0025] On the one hand, the present disclosure provides a primer set for identifying Yunmang No. 4, the primer set comprising: 6 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 sixth primer pair and the reverse primer of the sixth primer pair are respectively as shown in SEQ ID NO: 1 to SEQ ID NO: 12 in the sequence listing.

[0026] In this embodiment, the 5' end of the forward primer is fluorescently labeled. The sequence of the primer set and the corresponding fluorescent label are shown in Table 1.

[0027] Table 1 shows the sequences of the primer sets and the corresponding fluorescent markers

[0028]

[0029]

[0030] Embodiment 2

[0031] The present disclosure provides a kit for identifying Yunmang No. 4, the kit comprising the primer set provided in Example 1. The kit provided in this embodiment also comprises: 2×Taq PCR Master Mix, ddH2O, GeneScan TM 500LIZ and Hi-Di TM Formamide.

[0032] Embodiment 3

[0033] The present disclosure provides a method for identifying Yunmang 4, the method comprising:

[0034] Extracting genomic DNA from the sample to be tested;

[0035] Using genomic DNA as a template, PCR amplification was performed using the six primer pairs provided in Example 1 to obtain six amplification products;

[0036] The six amplification products were respectively detected by fluorescent capillary electrophoresis to obtain the peak values ​​of the six amplification products;

[0037] The peak values ​​of the six amplified products were compared with the standard peak values ​​of the corresponding primer pairs to determine whether the sample to be tested is Yunmang No. 4.

[0038] In this embodiment, 116 mango germplasms including Yunmang 4 were used as samples to be tested for identification. Specifically, the 116 mango germplasms included: Yunmang 3, 905, Aiwen, X2, Tom, 7003, Pink Mango, Dwarf Acid, Black Bens, Myanmar 5, Thailand C, Ivory Coast, Autumn Mango, Luzon, Tainong 1, Machisu, Dabaiyu, Thai Mango, Liuzhou Luzon, Yuexi 1, Jinlong, 90- Menghai, Little Ivory, Thai Seedling Tree, Sickle Mango, X1, Cangjiang 393, Guire No. 10, Guire No. 3, Yunmang No. 2, 2, Shuoshuai Mango, Thailand A, Anning Red Mango, Thailand D, Longjing, New Red River Mango, Four Seasons Honey Mango, Banana Mango, Siamese Mango, Xiamao Mango, Kuichong No. 1, 906D-7, 90-5, Money Mango, 903, CC Asia, White Jade, Longan Xiang Mango, White Wuming, Gaozhou Luzon, Mengbanghong, Ma Li mango, Yunmang No. 4, dwarf mango, 90-9, string mango, 7102, forest mango, small winter mango, big three years, golden spike, cream mango, 90-15, yellow mango, Guinea No. 2, chest ditch mango, Cuba No. 3, raw mango, similar to 905, 635, Long mango, Machesu variant, Mengtai, four-season mango, ivory No. 22, coconut fragrance, 91-Dongfeng, Guifei, Guangnong Research No. 2, 907, Renong No. 1, golden white flower , 8, Jinhuang, Cuba No. 4, 906C-6, 90-12, milk mango, Thailand E, coral mango, ball mango variant No. 1, white ivory, cloud mango No. 1, ball mango variant No. 2, B, 90-11, wild red, 90-7, 811, tiger and leopard teeth, red eagle mango, black, red ivory, big fruit autumn mango, ball mango, red cloud No. 5, Myanmar No. 7, Kent, Kate, sacred heart mango, winter mango, Jingsan, red cloud, Fujian mango and Yuantuan. Among them, the female parent of cloud mango No. 4 is ball mango, and the male parent is red cloud. The samples to be tested provided in this embodiment are all derived from the mango germplasm resource garden of Yunnan Tropical Crop Science Research Institute in Xishuangbanna Tropical Flower Garden.

[0039] Specifically, extract the genomic DNA of the sample to be tested. Extracting the genomic DNA of the above 116 samples to be tested includes: taking an appropriate amount of leaves of the sample to be tested, grinding them into powder, and putting them into a pre-cooled centrifuge tube, and then quickly adding 500 μL of lysis solution to the centrifuge tube, and then adding 100 μL of antioxidant and 10 μL of RnaseA, and after sufficient mixing, incubating at 65°C for 10 minutes, and shaking 2 to 3 times during the period; then adding 150 μL of polysaccharide scavenger to the centrifuge tube, gently mixing 15 to 20 times, and leaving it at room temperature for 3 to 5 minutes; centrifuging the centrifuge tube at 4°C and 12000 rpm for 5 minutes to obtain the supernatant, taking 500 μL of the supernatant and putting it into a new centrifuge tube; adding 300 μL of the new centrifuge tube. The mixture of V (chloroform) and V (isopropanol) = 24:1 was mixed, shaken up and down vigorously for 15 seconds, and centrifuged at 1200rpm for 30 seconds to obtain the upper aqueous phase; 500μL of the upper aqueous phase was loaded into an extraction plate, and then an equal volume of isopropanol was added to the extraction plate and placed in the extraction instrument station 1, 100μL of magnetic beads were dispensed into each well of the new extraction plate and placed in the extraction instrument station 2, three plates of 500μL / well of 75% ethanol were dispensed and placed in stations 3, 4 and 5 respectively, an elution plate was taken, 100μL, 80μL and 60μL of elution solution were dispensed into each well (the corresponding volume was dispensed according to the state of the sample to be tested) and placed in station 6, and the program was run after checking the instrument state and extraction plate information; after the operation was completed, the elution plate was taken out, and the eluted DNA was subjected to DNA detection, and the remaining eluted DNA could be stored at 4°C. Another 2 μL of DNA stock solution was added with 2 μL of bromophenol blue for agarose gel electrophoresis to detect DNA integrity.

[0040] Specifically, genomic DNA was used as a template, and the primer set provided in Example 1 was used for PCR amplification to obtain an amplified product. The 10 μL PCR amplification system includes: 1 μL 20 ng / μL genomic DNA, 5.0 μL 2×Taq PCR Master Mix, 0.5 μL 10 pmol / μL forward primer, 0.5 μL 10 pmol / μL reverse primer, and 3.0 μL ddH2O. The PCR amplification program includes: 95°C pre-denaturation for 5 min; the first cycle includes: 95°C denaturation for 30 s, 62°C to 52°C gradient annealing for 30 s, 72°C extension for 30 s, a total of 10 first cycles; the second cycle includes: 95°C denaturation for 30 s, 52°C annealing for 30 s, 72°C extension for 30 s, a total of 25 second cycles; 72°C extension for 20 min, and storage at 4°C after the amplification is completed.

[0041] Specifically, the PCR product is detected by fluorescent capillary electrophoresis to obtain the peak value of the amplified product. The fluorescent PCR product diluted to a uniform concentration is added to the upper plate, and the upper detection reagents are added to the total 10 μL system: 1 μL of fluorescent PCR product, GeneScanTM 500 LIZ 0.5μL, Hi-Di TM Formamide 8.5μL; put the plate to be tested with samples and reagents on the PCR instrument after centrifugation and run the denaturation program (95℃, 3min), and cool it immediately after the denaturation is completed; refer to the ABI 3730xL machine operation process, select the test file corresponding to the name of the plate to be tested, and run the SSR sample analysis test program. Use GeneMarker software to analyze the results and obtain the number of alleles, peak graph, peak value and genotype of each sample.

[0042] This embodiment provides standard peak values ​​for the first to sixth primer pairs (MIL035, MIL053, MIL074, MIL083, MIL117, and MIL173). Specifically, the standard peak values ​​for the first primer pair are 206 and 210, the standard peak values ​​for the second primer pair are 172 and 175, the standard peak value for the third primer pair is 175, the standard peak value for the fourth primer pair is 150, the standard peak value for the fifth primer pair is 159, and the standard peak value for the sixth primer pair is 113. Figures 1 to 6 shown.

[0043] Specifically, the peak values ​​of the six amplification products are compared with the standard peak values ​​of the corresponding primer pairs, so as to determine whether the sample to be tested is Yunmang No. 4. If the peak values ​​of the six 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 Yunmang No. 4. If the peak values ​​of the six 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 Yunmang No. 4. The specific results are shown in Table 2.

[0044] Table 2 is the comparison of the peak values ​​of 6 primer pairs of 116 samples to be tested with the standard peak values ​​provided

[0045]

[0046]

[0047]

[0048]

[0049] Table 2 provides the peak combinations of 116 samples to be tested, and the standard peak combinations of Yunmang No. 4 (MIL035, MIL053, MIL074, MIL083, MIL117 and MIL173) are: 206 and 210, 172 and 175, 175, 150, 159 and 113. In this embodiment, only the peak of the sample to be tested Yunmang No. 4 is consistent with the 8 standard peaks, and it is judged to be Yunmang No. 4, which is consistent with the actual results. It can be seen that the primer set provided in this embodiment can accurately identify Yunmang No. 4.

[0050] The above description is only an optional embodiment of the present disclosure and is not intended to limit the present disclosure. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the protection scope of the present disclosure.

Claims

1. A primer set for identifying Yunmang 4, characterized in that: The primer set consists of 6 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 to the forward primer of the sixth primer pair and the reverse primer of the sixth primer pair are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 12 in the sequence listing.

2. A kit for identifying Yunmang No. 4, characterized in that: The kit comprises the primer set as claimed in claim 1.

3. A method for identifying Yunmang No. 4, characterized in that: The method comprises: Extracting genomic DNA from the sample to be tested; Using the genomic DNA as a template, PCR amplification is performed using the six primer pairs described in claim 1 to obtain six amplification products; The six amplification products are respectively detected by fluorescent capillary electrophoresis to obtain six peaks of the amplification products; The peak values ​​of the six amplification products are compared with the corresponding standard peak values ​​of the primer pairs respectively to determine whether the sample to be tested is Yunmang No.

4. The standard peak values ​​of the first primer pair are 206 and 210, the standard peak values ​​of the second primer pair are 172 and 175, the standard peak value of the third primer pair is 175, the standard peak value of the fourth primer pair is 150, the standard peak value of the fifth primer pair is 159, and the standard peak value of the sixth primer pair is 113. If the peak values ​​of the six amplification products of the sample to be tested are consistent with the corresponding standard peak values ​​of the primer pairs, the sample to be tested is Yunmang No.

4. If the peak values ​​of the six amplification products of the sample to be tested are inconsistent with at least one of the corresponding standard peak values ​​of the primer pairs, the sample to be tested is not Yunmang No. 4.

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