Molecular marker primer for molecular identification of avocado Zijin variety and application of molecular marker primer

Abstract

The invention provides a molecular marker primer for molecular identification of avocado Zijin varieties and application, and belongs to the field of molecular identification of avocado Zijin varieties, materials are prepared, DNA of a sample to be detected is extracted, a PCR amplification product of the sample is obtained, fluorescence capillary electrophoresis detection is carried out, and the sample is identified through peak value comparison. According to the method, PCR amplification is carried out through specific primers, the amplified product is subjected to fluorescent capillary electrophoresis detection, and then peak values of sample amplimers are respectively compared with standard peak values of the specific primers, so that whether a test sample is the avocado Zijin variety or not is quickly and effectively judged, and the work of avocado cross breeding, variety protection and production technology popularization is facilitated.

Description

Technical Field

[0001] The invention relates to the field of molecular identification of avocado purple gold varieties, and in particular to a molecular marker primer for molecular identification of avocado purple gold varieties and an application thereof. Background Art

[0002] Avocado, also known as avocado, avocado, camphor pear, and happy fruit, belongs to the evergreen tree of the genus Avocado P.Americana, belonging to the dicotyledon class of the angiosperm, the subclass of the Magnoliaceae, the order of the Camphoraceae, the family Lauraceae, and is a famous tropical fruit and one of the woody oil-bearing tree species. Avocado contains a variety of vitamins, rich fats and proteins, as well as mineral elements such as sodium, potassium, magnesium, and calcium. The kernel contains fatty oil, and its content is 8% to 29%. Due to its rich nutrition, it has considerable health benefits for the human body. With the increasing demand for avocados, a wide variety of avocados have appeared in the market, and it is necessary to identify the varieties of avocados. There are more and more avocado resources, but the lack of accurate variety determination restricts the efficient use of these avocado germplasms. Therefore, it is necessary to determine the collected avocado resources to provide a basis for the subsequent hybridization, grafting and other new variety creation technologies. Summary of the invention

[0003] The purpose of the present invention is to provide a molecular marker primer for molecular identification of avocado purple gold variety and its application, so as to solve the technical problem of inaccurate molecular identification of avocado purple gold variety. The avocado variety is identified to facilitate hybrid breeding and processing for consumption.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0005] A molecular marker primer for molecular identification of avocado purple gold variety, consisting of five primer pairs: Pac025, Pac98, Pac113, Pac142 and Pac170.

[0006] They are: primers for molecular marker Pac025, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2;

[0007] Primers for molecular marker Pac98, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4;

[0008] Primers for molecular marker Pac113, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.6;

[0009] Primers for molecular marker Pac142, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.7, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.8;

[0010] The primers for the molecular marker Pac170, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.9, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.10.

[0011] A molecular marker primer for molecular identification of avocado purple gold variety and its application in identification of avocado purple gold variety.

[0012] A method for identifying an avocado variety Zijin, the method comprising the following steps:

[0013] Step 1: Preparation of samples to be tested: Select fresh avocado leaves to be tested, wash, dry, grind under liquid nitrogen, grind into powder and store in a -80℃ refrigerator for testing;

[0014] Step 2: Extract DNA from the sample to be tested;

[0015] Step 3: amplification product of sample PCR;

[0016] Step 4: fluorescence capillary electrophoresis detection;

[0017] Step 5: Compare the peaks to identify the samples.

[0018] (1) PCR amplification is performed according to the five pairs of specific primers provided, including a forward primer and a reverse primer, to obtain five amplification products, which are then detected by fluorescence capillary electrophoresis, and the peak values ​​of the five amplification products are obtained as standard peak values; (2) genomic DNA of the sample to be tested is extracted; (3) PCR amplification is performed using the genomic DNA as a template and the five pairs of designed primers; (4) the amplification products are detected by fluorescence capillary electrophoresis to obtain the peak values ​​of the amplification products of each sample to be tested; (5) the peak values ​​of the five amplification products of the sample are compared with the standard peak values ​​of the corresponding primers, so as to determine whether the sample to be tested is the avocado variety Zijin.

[0019] The invention performs PCR amplification by using specific primers, performs fluorescence capillary electrophoresis detection on the amplified products, and then compares the peak values ​​of the sample amplification products with the standard peak values ​​of the specific primers, so as to quickly and effectively judge whether the test sample is the purple gold variety of avocado, thereby facilitating the cross breeding and grafting of avocado.

[0020] Furthermore, the specific process of step 2 is:

[0021] Take the ground avocado sample and put it into a pre-cooled centrifuge tube, then quickly add 500 μL of lysis solution to the centrifuge tube, then add 100 μL of antioxidant and 10 μL of RNaseA, after fully mixing, incubate at 65°C for 10 minutes, and shake it 2 to 3 times during the period; then add 150 μL of polysaccharide scavenger to the centrifuge tube, gently mix it 15 to 20 times, and let it stand at room temperature for 3 to 5 minutes; centrifuge the centrifuge tube at 4°C and 12000 rpm for 5 minutes to obtain the supernatant, take 500 μL of the supernatant and put it into a new centrifuge tube; add 300 μL of L (chloroform) : V (isoamyl alcohol) = 24:1 mixed solution to the new centrifuge tube, mix it, shake it up and down vigorously for 15 seconds, centrifuge it at 1200 rpm for 30 seconds to obtain the upper aqueous phase; take 500 μL of the upper aqueous phase and put it into the extraction plate, and then add it to the extraction plate Add an equal volume of isopropanol and place it in station 1 of the extractor. In a new extraction plate, dispense 100 μL of magnetic beads into each well and place it in station 2 of the extractor. Dispense three plates with 500 μL / well of 75% ethanol and place them in stations 3, 4 and 5 respectively. Take the elution plate and dispense 100 μL, 80 μL and 60 μL of elution solution into each well and place it in station 6. Run the program after checking the instrument status and extraction plate information. After the run is completed, take out the elution plate and perform DNA detection on the eluted DNA. The remaining eluted DNA can be stored at 4°C. Take another 2 μL of DNA stock solution and add 2 μL of bromophenol blue for agarose gel electrophoresis to detect DNA integrity.

[0022] Furthermore, the specific process of step 3 is:

[0023] The DNA extracted from the sample to be tested was used as a template, and PCR amplification was performed using the 5 pairs of primers provided. The 15 μL PCR amplification system included: 1 μL of 50-200 ng / μL genomic DNA, 7.5 μL of 2×Taq PCR Master Mix, 1.0 μL of 10 pmol / μL forward primer, 1.0 μL of 10 pmol / μL reverse primer, and 4.5 μL of ddH2O. The PCR amplification program included: 96°C pre-denaturation for 3 min; 30 cycles included: 96°C denaturation for 30 s, 62°C-52°C gradient annealing for 30 s, and 72°C extension for 1 min. 72°C extension for 10 min, and after completion, the sample was stored at 4°C to obtain the PCR amplification product of the sample.

[0024] Furthermore, the specific process of step 4 is:

[0025] The PCR products were detected by fluorescent capillary electrophoresis to obtain the peak of the amplified products. The sample PCR amplified products diluted to a uniform concentration were added to the upper plate. The upper detection reagents, 1 μL of fluorescent PCR products, and 1 μL of GeneScan TM 500LIZ 0.5μL, Hi-DiTM Formamide 10μL, centrifuge the test plate with samples and reagents, put it on the PCR instrument and run the denaturation program at 95℃ for 3min. 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 test plate, 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.

[0026] Furthermore, the specific process of step 4 is:

[0027] The peak values ​​of the five amplification products of the sample are compared with the standard peak values ​​of the corresponding primer pairs respectively to determine whether the sample to be tested is the avocado variety Zijin. If the peak values ​​of the five 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 avocado variety Zijin. If the peak values ​​of the five 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 avocado variety Zijin.

[0028] Furthermore, in step 3, the 5 pairs of primers provided are used as the designed specific primers, and the 5' end of the forward primers all have fluorescent groups, the fluorescent group of Pac025 is FAM, the fluorescent group of Pac98 is HEX, the fluorescent group of Pac113 is FAM, the fluorescent group of Pac142 is FAM, and the fluorescent group of Pac170 is FAM.

[0029] Furthermore, in step 5, the standard peak values ​​of the primer pairs are specifically 238 and 244 for the first primer pair, 270 and 276 for the second primer pair, 218 for the third primer pair, 278 and 281 for the fourth primer pair, and 187 and 191 for the fifth primer pair.

[0030] The present invention has the following beneficial effects due to the adoption of the above technical solution:

[0031] (1) When the leaves, flowers and trunks of Avocado Zijin cannot be distinguished by naked eye observation, the method can be used to identify whether it is Avocado Zijin variety before hybrid breeding, so as to identify the parents for hybrid breeding.

[0032] (2) Nowadays, avocados are becoming more and more popular among people, and there are more and more varieties on the market. However, although the appearance of avocado fruits is similar, their quality varies greatly, and different varieties have different prices. This method can help people quickly distinguish the fruits, determine the prices, and maintain the trade order in the avocado market. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1is the standard peak diagram of the first primer pair of the present invention;

[0034] Figure 2 It is the standard peak diagram and peak diagram of the second pair of primers of the present invention;

[0035] Figure 3 is the standard peak graph and peak graph of the third primer pair of the present invention;

[0036] Figure 4 is the standard peak graph and peak graph of the fourth primer pair of the present invention;

[0037] Figure 5 It is the standard peak diagram and peak diagram of the fifth primer pair of the present invention. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and preferred embodiments. However, it should be noted that many details listed in the specification are only for the purpose of enabling the reader to have a thorough understanding of one or more aspects of the present invention, and these aspects of the present invention can be implemented even without these specific details.

[0039] Embodiment 1:

[0040] A primer set for identifying an avocado variety Zijin comprises five primer pairs, each primer pair comprises 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 sequence as SEQ ID NO: 1 to SEQ ID NO: 10 in the sequence table.

[0041] In this embodiment, the 5' end of the forward primers all carry a fluorescent group. The sequences of the primer sets and the corresponding fluorescent groups are shown in Table 1.

[0042] Table 1 shows the sequences of the primer sets and the corresponding fluorescent markers

[0043]

[0044] Embodiment 2:

[0045] A method for identifying the avocado variety Zijin, the method includes the primer set provided in Example 1. The kit provided in this example also includes: 2×Taq PCR Master Mix, ddH2O, GeneScan TM 500LIZ and Hi-Di TM Formamide.

[0046] Embodiment 3:

[0047] The present disclosure provides a method for identifying an avocado variety Zijin, the method comprising:

[0048] Extracting genomic DNA from the sample to be tested;

[0049] Using genomic DNA as a template, PCR amplification was performed using the five primer pairs provided in Example 1 to obtain five amplification products;

[0050] The five amplification products were respectively detected by fluorescent capillary electrophoresis to obtain the peak values ​​of the five amplification products;

[0051] The peak values ​​of the five amplified products were compared with the standard peak values ​​of the corresponding primer pairs to determine whether the sample to be tested was the avocado variety Zijin.

[0052] In this embodiment, 73 avocado germplasms including Zijin were used as samples to be tested for identification. The specific 73 avocado germplasms included: BN3, MM3, Fuerte, Tenglong, GL13, SCP1, Guilong No. 8, Wilson seedless, BY2, S5, Beta, K1, Simmonds, GL1, TW1-98, K2, Pinkerton, loretta, Choquette, GL9, DL17, Nabal, Kampong, Pollock, YL31, GL25, TW1-86, Hass, GL18, Lamb Hass, Qiuhong, Guilong 2, Boot, v11, v8, Guiyan 8, Guiyan 1, Booth8, YL27, Zijin, Nanya B, HH1, Nesbitt, Qiuyue, YL33, xx3, HN2, xx1, Guiken 2, HHe1, Guiken 3, TY1, Guilong 12, Lula, S2, BS1, S4, Brooks late, S6, Dusa, Ettinger, Walter Hole, RS1, YL32, NY4, GL14, BS2, GL10, Guilong 11, v5, GL16, Duke7 and Bacon. The samples to be tested provided in this embodiment are from the South Asian Tropical Agricultural Science Research Institute of Guangxi Zhuang Autonomous Region.

[0053] Specifically, the genomic DNA of the sample to be tested is extracted. The genomic DNA of the above 73 samples to be tested is extracted, including: 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, 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 incubating at 65°C for 10 minutes after sufficient mixing, 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 a supernatant, taking 500 μL of the supernatant and putting it into a new centrifuge tube; adding 300 μL of a mixture of L (chloroform) : V (isoamyl alcohol) = 24:1 to the new centrifuge tube and mixing, Violently shake up and down for 15s, centrifuge at 1200rpm for 30s to obtain the upper aqueous phase; take 500μL of the upper aqueous phase and load it into the extraction plate, then add an equal volume of isopropanol to the extraction plate and place it in the extraction instrument station 1, dispense 100μL of magnetic beads per well in the new extraction plate and place it in the extraction instrument station 2, dispense three plates of 500μL / well of 75% ethanol and place them in stations 3, 4 and 5 respectively, take the elution plate, dispense 100μL, 80μL and 60μL of elution per well (dispense the corresponding volume according to the state 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, take out the elution plate, perform DNA detection on the eluted DNA, and the remaining eluted DNA can be stored at 4℃. Take another 2μL of DNA stock solution and add 2μL of bromophenol blue for agarose gel electrophoresis to detect DNA integrity.

[0054] Specifically, genomic DNA was used as a template, and PCR amplification was performed using the primer set provided in Example 1 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.

[0055] 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 500LIZ 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.

[0056] This embodiment provides standard peak values ​​for the first to fifth primer pairs (Pac025, Pac98, Pac113, Pac142, and Pac170), specifically, the standard peak values ​​for the first primer pair are 238 and 244, the standard peak values ​​for the second primer pair are 273 and 276, the standard peak values ​​for the third primer pair are 218, the standard peak values ​​for the fourth primer pair are 278 and 281, and the standard peak values ​​for the fifth primer pair are 187 and 191. Figures 1 to 5 shown.

[0057] Specifically, the peak values ​​of the five amplified products are compared with the standard peak values ​​of the corresponding primer pairs to determine whether the sample to be tested is Aoli Zijin. If the peak values ​​of the five amplified 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 Aoli Zijin. If the peak values ​​of the five amplified 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 Aoli Zijin. The specific results are shown in Table 2.

[0058] Table 2 is the comparison of the peak values ​​of 5 primer pairs of 92 samples to be tested with the standard peak values ​​provided

[0059]

[0060]

[0061]

[0062]

[0063]

[0064] Table 2 provides the peak combinations of 73 samples to be tested. The standard peak combinations of Zijin oil pear (Pac025, Pac98, Pac113, Pac142 and Pac170) are: 238 and 244, 270 and 276, 218, 278 and 281, and 187 and 191. In this example, only the peak of Zijin oil pear sample to be tested is consistent with the 9 standard peaks, so it is judged to be Zijin oil pear variety, which is consistent with the actual result. It can be seen that the primer set provided in this example can accurately identify Zijin oil pear variety.

[0065] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A molecular marker primer for molecular identification of avocado purple gold variety, characterized by: It consists of five primer pairs: Pac025, Pac98, Pac113, Pac142, and Pac170; They are: primers for molecular marker Pac025, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2; Primers for molecular marker Pac98, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4; Primers for molecular marker Pac113, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.6; Primers for molecular marker Pac142, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.7, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.8; The primers for the molecular marker Pac170, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.9, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

10.

2. Application of the molecular marker primer for molecular identification of the Zijin variety of Avocado according to claim 1 in the identification of the Zijin variety of Avocado.

3. A method for identifying the avocado variety Zijin, characterized in that: The method comprises the following steps: Step 1: Preparation of samples to be tested: Select fresh avocado leaves to be tested, wash, dry, grind under liquid nitrogen, grind into powder and store in a -80℃ refrigerator for testing; Step 2: Extract DNA from the sample to be tested; Step 3: amplification product of sample PCR; Step 4: fluorescence capillary electrophoresis detection; Step 5: Compare the peaks to identify the samples.

4. The method for identifying the avocado variety Zijin according to claim 3, characterized in that: The specific process of step 2 is: Take the ground avocado sample and put it into a pre-cooled centrifuge tube, then quickly add 500 μL of lysis solution to the centrifuge tube, then add 100 μL of antioxidant and 10 μL of RNaseA, after fully mixing, incubate at 65°C for 10 minutes, and shake it 2 to 3 times during the period; then add 150 μL of polysaccharide scavenger to the centrifuge tube, gently mix it 15 to 20 times, and let it stand at room temperature for 3 to 5 minutes; centrifuge the centrifuge tube at 4°C and 12000 rpm for 5 minutes to obtain the supernatant, take 500 μL of the supernatant and put it into a new centrifuge tube; add 300 μL of V (chloroform) : V (isoamyl alcohol) = 24:1 mixed solution to the new centrifuge tube, mix it, shake it up and down vigorously for 15 seconds, centrifuge it at 1200 rpm for 30 seconds to obtain the upper aqueous phase; take the upper aqueous phase 5 00μL is loaded into the extraction plate, and then an equal volume of isopropanol is added to the extraction plate and placed in station 1 of the extractor. In the new extraction plate, 100μL of magnetic beads are dispensed into each well and placed in station 2 of the extractor. Three plates of 500μL / well of 75% ethanol are dispensed and placed in stations 3, 4 and 5 respectively. Take the elution plate, dispense 100μL, 80μL and 60μL of elution solution into each well and place it in station 6. Run the program after checking the instrument status and extraction plate information. After the run is completed, take out the elution plate, perform DNA detection on the eluted DNA, and the remaining eluted DNA can be stored at 4°C. Take another 2μL of DNA stock solution and add 2μL of bromophenol blue for agarose gel electrophoresis to detect DNA integrity.

5. The method for identifying the avocado variety Zijin according to claim 3, characterized in that: The specific process of step 3 is: The DNA extracted from the sample to be tested was used as a template, and PCR amplification was performed using the 5 pairs of primers provided. The 15 μL PCR amplification system included: 1 μL of 50-200 ng / μL genomic DNA, 7.5 μL of 2×Taq PCR Master Mix, 1.0 μL of 10 pmol / μL forward primer, 1.0 μL of 10 pmol / μL reverse primer, and 4.5 μL of ddH2O. The PCR amplification program included: 96°C pre-denaturation for 3 min; 30 cycles included: 96°C denaturation for 30 s, 62°C-52°C gradient annealing for 30 s, and 72°C extension for 1 min. 72°C extension for 10 min, and after completion, the sample was stored at 4°C to obtain the PCR amplification product of the sample.

6. The method for identifying the avocado variety Zijin according to claim 3, characterized in that: The specific process of step 4 is: The PCR products were detected by fluorescent capillary electrophoresis to obtain the peak of the amplified products. The sample PCR amplified products diluted to a uniform concentration were added to the upper plate. The upper detection reagents, 1 μL of fluorescent PCR products, and 1 μL of GeneScan were added to the system in a total of 11.5 μL. TM 500LIZ 0.5μL, Hi-Di TM Formamide 10μL, centrifuge the test plate with samples and reagents, put it on the PCR instrument and run the denaturation program at 95℃ for 3min. 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 test plate, 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.

7. The method for identifying the avocado variety Zijin according to claim 3, characterized in that: The specific process of step 4 is: The peak values ​​of the five amplification products of the sample are compared with the standard peak values ​​of the corresponding primer pairs respectively to determine whether the sample to be tested is the avocado variety Zijin. If the peak values ​​of the five 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 avocado variety Zijin. If the peak values ​​of the five 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 avocado variety Zijin.

8. The method for identifying the avocado variety Zijin according to claim 3, characterized in that: In step 3, the 5 pairs of primers provided are used as the designed specific primers. The 5' end of the forward primers all have fluorescent groups, the fluorescent group of Pac025 is FAM, the fluorescent group of Pac98 is HEX, the fluorescent group of Pac113 is FAM, the fluorescent group of Pac142 is FAM, and the fluorescent group of Pac170 is FAM.

9. The method for identifying the avocado variety Zijin according to claim 3, characterized in that: In step 5, the standard peak values ​​of the primer pairs are specifically 238 and 244 for the first primer pair, 270 and 276 for the second primer pair, 218 for the third primer pair, 278 and 281 for the fourth primer pair, and 187 and 191 for the fifth primer pair.

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