Molecular marker primer for molecular identification of olea europaea cv. kalamata and application thereof

Abstract

The application provides a molecular marker primer for molecular identification of an oil olive purple gold variety and application, and belongs to the field of molecular identification of the oil olive purple gold variety. Preparing materials, extracting DNA of a to-be-tested sample, amplifying a product of sample PCR, detecting fluorescence capillary electrophoresis, and identifying the sample by comparing peak values. The specific primer is used for PCR amplification, the amplified product is detected by fluorescence capillary electrophoresis, and the peak values of the sample amplification product are compared with the standard peak values of the specific primer, so that whether the test sample is the oil olive purple gold variety is quickly and effectively judged, and the work of hybrid breeding, variety protection and production technology popularization of the oil olive is facilitated.

Description

Technical Field

[0001] This invention relates to the field of molecular identification of the Purple Gold variety of Avocado, and particularly to a molecular marker primer for molecular identification of the Purple Gold variety of Avocado and its application. Background Technology

[0002] Avocado, also known as alligator pear, butter pear, or lucky pear, is an evergreen tree belonging to the genus *P. Americana* of the family Lauraceae in the order Lauraceae of the class Magnoliopsida in the phylum Angiosperms. It is a famous tropical fruit and one of the woody oilseed trees. Avocados contain various vitamins, abundant fats and proteins, as well as minerals such as sodium, potassium, magnesium, and calcium. The kernel contains fatty oil, ranging from 8% to 29%. Due to its rich nutritional content, it has considerable health benefits. With the increasing demand for avocados, a wide variety of avocados have appeared on the market, necessitating varietal identification. While avocado resources are increasing, the lack of precise varietal identification restricts the efficient utilization of these avocado germplasm. Therefore, it is necessary to identify these collected avocado resources to provide a basis for subsequent hybridization, grafting, and other new variety creation techniques. Summary of the Invention

[0003] The purpose of this invention is to provide a molecular marker primer for the molecular identification of the Purple Gold avocado variety and its application, solving the technical problem of inaccurate molecular identification of the Purple Gold avocado variety in existing methods. This facilitates the identification of avocado varieties, making hybridization breeding and processing for consumption easier.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0005] A molecular marker primer for molecular identification of the Zijin variety of avocado consists of five primer pairs: Pac025, Pac98, Pac113, Pac142, and Pac170.

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

[0007] The primers for the molecular marker Pac98 have the following nucleotide sequences: upstream primer as shown in SEQ ID NO.3 and downstream primer as shown in SEQ ID NO.4.

[0008] The primers for the molecular marker Pac113 are shown in SEQ ID NO.5 for the upstream primer and SEQ ID NO.6 for the downstream primer.

[0009] The primers for the molecular marker Pac142 have the following nucleotide sequences: upstream primer as shown in SEQ ID NO.7 and downstream primer as shown in SEQ ID NO.8.

[0010] The primers for the molecular marker Pac170 have the following nucleotide sequences: upstream primer as shown in SEQ ID NO.9 and downstream primer as shown in SEQ ID NO.10.

[0011] Application of a molecular marker primer for molecular identification of the Zijin avocado variety.

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

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

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

[0015] Step 3: Amplification products of sample PCR;

[0016] Step 4: Fluorescent capillary electrophoresis detection;

[0017] Step 5: Compare peak values ​​to identify the sample.

[0018] (1) PCR amplification was performed using the 5 pairs of specific primers provided, including forward and reverse primers, to obtain 5 amplification products. The peak values ​​of the 5 amplification products were then detected by fluorescent capillary electrophoresis and used as standard peak values. (2) Genomic DNA was extracted from the sample to be tested. (3) PCR amplification was performed using the genomic DNA as a template and the 5 pairs of primers designed. (4) The amplified products were detected by fluorescent capillary electrophoresis and the peak values ​​of the amplification products of each sample to be tested were obtained. (5) The peak values ​​of the 5 amplification products of the sample were compared with the standard peak values ​​of the corresponding primers to determine whether the sample to be tested was the Zijin variety of avocado.

[0019] This invention uses specific primers for PCR amplification, detects the amplified products by fluorescent capillary electrophoresis, and compares the peak values ​​of the sample amplified products with the standard peak values ​​of the specific primers to quickly and effectively determine whether the test sample is the Purple Gold variety of avocado, which facilitates avocado hybridization breeding and grafting work.

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

[0021] Take the ground avocado sample and place it in a pre-cooled centrifuge tube. Quickly add 500 μL of lysis buffer, then add 100 μL of antioxidant and 10 μL of Rnase A. Mix thoroughly and incubate at 65°C for 10 min, shaking 2-3 times during incubation. Next, add 150 μL of polysaccharide scavenger and gently mix 15-20 times. Let stand at room temperature for 3-5 min. Centrifuge the tube at 12000 rpm for 5 min at 4°C to obtain the supernatant. Transfer 500 μL of the supernatant to a new centrifuge tube. Add 300 μL of... Mix the chloroform and isoamyl alcohol solutions in a ratio of 24:1, shake vigorously for 15 seconds, and centrifuge at 1200 rpm for 30 seconds to obtain the upper aqueous phase. Transfer 500 μL of the upper aqueous phase to an extraction plate, add an equal volume of isopropanol to the extraction plate, and place it at station 1 of the extraction instrument. In the new extraction plate, aliquot 100 μL of magnetic beads into each well and place them at station 2. Aliquot 500 μL / well of 75% ethanol into three separate plates and place them at stations 3, 4, and 5, respectively. Take the elution plate and aliquot 100 μL, 80 μL, and 60 μL of elution buffer into each well and place them at station 6. Verify the instrument status and extraction plate information, and then run the program. After the program completes, remove the elution plate and perform DNA analysis on the eluted DNA. The remaining eluted DNA can be stored at 4°C. Separately, take 2 μL of the DNA stock solution, add 2 μL of bromophenol blue, and perform agarose gel electrophoresis to check DNA integrity.

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

[0023] Using extracted DNA from the test sample as a template, PCR amplification was performed using the provided 5 pairs of primers. 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℃ pre-denaturation for 3 min; 30 cycles of denaturation at 96℃ for 30 s, gradient annealing at 62℃~52℃ for 30 s, extension at 72℃ for 1 min, and a final extension at 72℃ for 10 min. The sample was then stored at 4℃ to obtain the PCR amplification product.

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

[0025] The PCR products were detected by fluorescent capillary electrophoresis to obtain the peak values ​​of the amplified products. The PCR amplified products of the samples diluted to a uniform concentration were added to the test plate. The test reagents were added separately according to the total system of 11.5 μL: 1 μL of fluorescent PCR product, 0.5 μL of GeneScan™ 500 LIZ, and 10 μL of Hi-Di™ Formamide. After centrifuging the test plate with the added samples and reagents, it was placed in the PCR instrument and the denaturation program was run at 95°C for 3 min. After denaturation, it was cooled immediately. Following the ABI 3730xL test plate operation procedure, the test file corresponding to the name of the test plate was selected, and the SSR sample analysis test program was run. The results were analyzed using GeneMarker software to obtain the number of alleles, peak diagram, peak value, and genotype of each sample.

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

[0027] The peak values ​​of the five amplification products of the sample are compared with the standard peak values ​​of the corresponding primer pairs to determine whether the sample is a Zijin avocado variety. If the peak values ​​of the five amplification products of the sample are consistent with the standard peak values ​​of the corresponding primer pairs, the sample is a Zijin avocado variety. If the peak values ​​of the five amplification products of the sample are inconsistent with at least one of the standard peak values ​​of the corresponding primer pairs, the sample is not a Zijin avocado variety.

[0028] Furthermore, in step 3, the provided 5 pairs of primers are used as the designed specific primers. The 5' ends 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 as follows: the standard peak values ​​of the first primer pair are 238 and 244, the standard peak values ​​of the second primer pair are 270 and 276, the standard peak value of the third primer pair is 218, the standard peak values ​​of the fourth primer pair are 278 and 281, and the standard peak values ​​of the fifth primer pair are 187 and 191.

[0030] The present invention, by adopting the above-described technical solution, has the following beneficial effects:

[0031] (1) When the leaves, flowers and trunk of the Avocado Purple Gold variety cannot be distinguished by the naked eye, this invention can be used to identify whether it is the Avocado Purple Gold variety before hybridization breeding, thus clarifying the parent plants for hybridization breeding.

[0032] (2) Nowadays, avocados are becoming more and more popular, and there are more and more varieties on the market. However, avocados look similar but have different qualities and different prices. This method can help people quickly distinguish the fruits, determine the price, and maintain the trade order of the avocado market. Attached Figure Description

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

[0034] Figure 2 These are the standard peak diagram and peak value diagram of the second pair of primers of this invention;

[0035] Figure 3 These are the standard peak diagram and peak value diagram of the third primer pair of this invention;

[0036] Figure 4 These are the standard peak diagram and peak value diagram of the fourth primer pair of this invention;

[0037] Figure 5 These are the standard peak diagram and peak value diagram of the fifth primer pair of this invention. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be 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 merely to provide the reader with a thorough understanding of one or more aspects of the present invention, and these aspects of the invention can be implemented even without these specific details.

[0039] Example 1:

[0040] A primer set for identifying the avocado variety Zijin includes 5 primer pairs, each primer pair including 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.

[0041] In this embodiment, the 5' ends of the forward primers all contain fluorescent groups. See Table 1 for the specific sequences of the primer sets and their corresponding fluorescent groups.

[0042] Table 1 shows the primer sequences and corresponding fluorescent labels.

[0043]

[0044] Example 2:

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

[0046] Example 3:

[0047] This disclosure provides a method for identifying the Zijin variety of avocado, the method comprising:

[0048] Extract 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] Five amplification products were detected by fluorescence capillary electrophoresis to obtain the peak values ​​of the five amplification products.

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

[0052] In this embodiment, 73 avocado germplasm accessions, including Zijin, were used as test samples for identification. Specifically, the 73 avocado germplasm accessions included: BN3, MM3, Fuerte, Tenglong, GL13, SCP1, Guilong 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, and Lamb. Hass, Qiuhong, Guilong 2, Boot, v11, v8, Guiyan 8, Guiyan 1, Booth8, YL27, Zijin, Nanya B, HH1, Nesbitt, Qiuyue, YL33, xx3, HN2, xx1, Guikenda 2, HHe1, Guikenda 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 provided in this example were obtained from the Guangxi South Subtropical Agricultural Research Institute.

[0053] Specifically, genomic DNA was extracted from the samples to be tested. The extraction of genomic DNA from the 73 samples included: grinding an appropriate amount of leaf material from the sample into powder, placing it in a pre-cooled centrifuge tube, then quickly adding 500 μL of lysis buffer, followed by 100 μL of antioxidant and 10 μL of RNase A. After thorough mixing, incubate at 65°C for 10 min, shaking 2–3 times during this period; then add 150 μL of polysaccharide scavenger to the centrifuge tube, gently mixing 15–20 times, and incubating at room temperature for 3–5 min; centrifuging the centrifuge tube at 4°C and 12000 rpm for 5 min to obtain the supernatant, transferring 500 μL of the supernatant to a new centrifuge tube; adding 300 μL of lysate to the new centrifuge tube... Mix the chloroform and isoamyl alcohol solutions in a ratio of 24:1, shake vigorously for 15 seconds, and centrifuge at 1200 rpm for 30 seconds to obtain the upper aqueous phase. Take 500 μL of the upper aqueous phase and transfer it to an extraction plate. Add an equal volume of isopropanol to the extraction plate and place it at station 1 of the extraction instrument. Dispense 100 μL of magnetic beads into each well of the new extraction plate and place it at station 2. Dispense 500 μL / well of 75% ethanol into three separate plates and place them at stations 3, 4, and 5, respectively. Take the elution plate and dispense 100 μL, 80 μL, and 60 μL of elution buffer into each well (the appropriate volume depends on the state of the sample) and place it at station 6. Verify the instrument status and extraction plate information, and then run the program. After the program finishes, remove 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, add 2 μL of bromophenol blue, and perform agarose gel electrophoresis to detect DNA integrity.

[0054] Specifically, using genomic DNA as a template, PCR amplification was performed using the primer set provided in Example 1 to obtain the amplification product. The 10 μL PCR amplification system included: 1 μL of 20 ng / μL genomic DNA, 5.0 μL of 2×Taq PCR Master Mix, 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: 95℃ pre-denaturation for 5 min; the first cycle consisted of: 95℃ denaturation for 30 s, gradient annealing from 62℃ to 52℃ for 30 s, and extension at 72℃ for 30 s, for a total of 10 first cycles; the second cycle consisted of: 95℃ denaturation for 30 s, 52℃ annealing for 30 s, and extension at 72℃ for 30 s, for a total of 25 second cycles; the extension at 72℃ for 20 min, and the product was stored at 4℃ after amplification.

[0055] Specifically, the PCR products were detected by fluorescent capillary electrophoresis to obtain the peak values ​​of the amplified products. Fluorescent PCR products diluted to a uniform concentration were added to the PCR plate, and the following reagents were added in a total volume of 10 μL: 1 μL of fluorescent PCR product, 0.5 μL of GeneScan™ 500 LIZ, and 8.5 μL of Hi-Di™ Formamide. The plate containing the samples and reagents was centrifuged and then placed on the PCR instrument to run the denaturation program (95℃, 3 min). After denaturation, the plate was immediately cooled. Following the ABI 3730xL PCR procedure, the corresponding detection file for the plate name was selected, and the SSR sample analysis detection program was run. The results were analyzed using GeneMarker software to obtain the allele count, peak plot, peak value, and genotype for each sample.

[0056] This embodiment provides the standard peak values ​​for primer pairs 1 through 5 (Pac025, Pac98, Pac113, Pac142, and Pac170). Specifically, the standard peak values ​​for primer pair 1 are 238 and 244, for primer pair 2 are 270 and 276, for primer pair 3 is 218, for primer pair 4 are 278 and 281, and for primer pair 5 are 187 and 191. Figures 1 to 5 As shown.

[0057] Specifically, the peak values ​​of the five amplification products were compared with the standard peak values ​​of their corresponding primer pairs to determine whether the sample was *Avocado Purple Gold*. If the peak values ​​of the five amplification products of the sample matched the standard peak values ​​of their corresponding primer pairs, the sample was *Avocado Purple Gold*; if the peak values ​​of the five amplification products of the sample did not match at least one of the standard peak values ​​of their corresponding primer pairs, the sample was not *Avocado Purple Gold*. The specific results are shown in Table 2.

[0058] Table 2 compares the peak values ​​of the five primer pairs in 92 test samples with the provided standard peak values.

[0059] Name of the sample to be tested Pac025 (standard peak values ​​are 238 and 244). Pac98 (standard peak values ​​are 270 and 276) Pac113 (standard peak value is 218) Pac142 (standard peak values ​​are 278 and 281). Pac170 (standard peak values ​​are 187 and 191). BN3 235 / 235 276 / 276 218 / 221 269 / 278 191 / 191 MM3 235 / 244 276 / 276 218 / 218 278 / 278 191 / 191 Fuerte 238 / 238 0 / 0 218 / 224 269 / 278 191 / 191 Tenglong 238 / 238 270 / 276 218 / 218 278 / 281 187 / 191 GL13 238 / 238 270 / 276 218 / 221 278 / 278 191 / 191 SCP1 238 / 238 270 / 276 218 / 221 284 / 284 187 / 187 Guilong No. 8 238 / 238 270 / 276 218 / 227 278 / 281 191 / 191 Wilsonseedless 238 / 238 270 / 276 218 / 227 281 / 284 187 / 191 BY2 238 / 238 270 / 276 221 / 221 284 / 284 191 / 191 S5 238 / 238 273 / 273 218 / 218 278 / 278 191 / 191 Beta 238 / 238 273 / 276 201 / 201 284 / 284 191 / 191 K1 238 / 238 273 / 276 201 / 218 278 / 284 187 / 191 Simmonds 238 / 238 273 / 276 201 / 221 284 / 284 191 / 191 GL1 238 / 238 273 / 276 218 / 218 278 / 281 191 / 191 TW1-98 238 / 238 273 / 276 218 / 218 281 / 284 187 / 191 K2 238 / 238 273 / 276 218 / 218 284 / 284 191 / 191 Pinkerton 238 / 238 273 / 276 218 / 224 269 / 281 191 / 191 loretta 238 / 238 273 / 276 221 / 221 278 / 284 191 / 191 Choquette 238 / 238 273 / 288 201 / 218 284 / 284 191 / 191 GL9 238 / 238 276 / 276 201 / 201 284 / 284 191 / 191 DL17 238 / 238 276 / 276 201 / 221 284 / 284 191 / 191 Nabal 238 / 238 276 / 276 218 / 218 278 / 278 191 / 191 Kampong 238 / 238 276 / 276 218 / 218 278 / 281 191 / 191 Pollock 238 / 238 276 / 276 218 / 218 278 / 284 191 / 191 YL31 238 / 238 276 / 276 218 / 218 281 / 281 187 / 191 GL25 238 / 238 276 / 276 218 / 221 281 / 284 191 / 191 TW1-86 238 / 238 276 / 276 218 / 221 284 / 284 187 / 191 Hass 238 / 238 276 / 276 218 / 224 278 / 281 191 / 191 GL18 238 / 238 276 / 276 221 / 221 269 / 278 191 / 191 Lamb Hass 238 / 238 276 / 280 218 / 218 278 / 278 191 / 191 Autumn Red 238 / 238 276 / 298 218 / 227 281 / 284 191 / 191 Guilong No. 2 238 / 244 0 / 0 218 / 221 278 / 284 191 / 191 Boot 238 / 244 264 / 264 218 / 221 278 / 284 191 / 191 v11 238 / 244 270 / 273 201 / 218 281 / 284 187 / 191 v8 238 / 244 270 / 276 218 / 218 281 / 284 187 / 191 Guiyan No. 8 238 / 244 270 / 276 218 / 221 278 / 281 187 / 191 Guiyan No. 1 238 / 244 270 / 276 218 / 221 278 / 284 187 / 187 Booth8 238 / 244 273 / 276 201 / 218 278 / 281 191 / 191 YL27 238 / 244 273 / 276 201 / 218 281 / 284 187 / 191 Zijin 238 / 244 273 / 276 218 / 218 278 / 281 187 / 191 South Asia B 238 / 244 273 / 276 218 / 218 278 / 281 191 / 191 HH1 238 / 244 273 / 276 218 / 221 278 / 281 191 / 191 Nesbitt 238 / 244 273 / 276 218 / 221 281 / 284 191 / 191 Autumn Moon 238 / 244 273 / 276 221 / 221 269 / 284 191 / 191 YL33 238 / 244 276 / 276 201 / 218 284 / 284 187 / 191 xx3 238 / 244 276 / 276 201 / 218 284 / 284 191 / 191 HN2 238 / 244 276 / 276 201 / 221 284 / 284 187 / 191 xx1 238 / 244 276 / 276 201 / 221 284 / 284 191 / 191 Guiken No. 2 238 / 244 276 / 276 218 / 218 278 / 284 191 / 191 HHe1 238 / 244 276 / 276 218 / 218 281 / 284 187 / 191 Guiken No. 3 238 / 244 276 / 276 218 / 218 281 / 284 191 / 191 TY1 238 / 244 276 / 276 218 / 221 281 / 284 191 / 191 Guilong No. 12 238 / 244 276 / 276 221 / 221 269 / 284 191 / 191 Lula 238 / 244 276 / 276 221 / 221 284 / 284 191 / 191 S2 238 / 244 276 / 276 221 / 224 281 / 284 191 / 191 BS1 238 / 250 270 / 276 218 / 218 278 / 284 187 / 191 S4 238 / 250 273 / 276 218 / 218 284 / 284 191 / 191 Brooks late 238 / 250 276 / 276 201 / 218 278 / 278 191 / 191 S6 238 / 250 276 / 276 201 / 218 278 / 284 191 / 191 Dusa 238 / 250 276 / 276 215 / 218 269 / 278 191 / 191 Ettinger 238 / 250 276 / 280 218 / 218 269 / 278 191 / 191 Walter Hole 238 / 250 276 / 280 218 / 221 269 / 269 191 / 191 RS1 238 / 250 280 / 280 218 / 224 269 / 269 191 / 191 TB 244 / 244 270 / 273 218 / 218 284 / 284 187 / 191 NY4 244 / 244 273 / 276 201 / 218 278 / 281 191 / 191 GL14 244 / 244 273 / 276 218 / 218 278 / 281 191 / 191 BS2 244 / 244 273 / 276 218 / 218 284 / 284 191 / 191 GL10 244 / 244 273 / 276 218 / 221 278 / 284 187 / 191 Guilong No. 11 244 / 244 273 / 276 221 / 227 281 / 284 191 / 191 v5 244 / 244 276 / 276 218 / 227 281 / 281 187 / 191 GL16 244 / 244 276 / 276 221 / 221 281 / 284 187 / 191 Duke7 250 / 250 276 / 280 215 / 221 269 / 269 191 / 191 Bacon 250 / 250 276 / 280 218 / 224 269 / 278 191 / 191

[0060] Table 2 provides the peak combinations of 73 samples to be tested. The standard peak combinations for the avocado variety Zijin (Pac025, Pac98, Pac113, Pac142, and Pac170) are, respectively: 238 and 244, 270 and 276, 218, 278 and 281, and 187 and 191. In this embodiment, only the peak value of the tested sample Zijin matches the 9 standard peak values, so it is identified as the avocado variety Zijin, which is consistent with the actual results. Therefore, the primer set provided in this embodiment can accurately identify the avocado variety Zijin.

[0061] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A molecular marker primer for molecular identification of the Purple Gold variety of avocado, characterized in that: It consists of five primer pairs: Pac025, Pac98, Pac113, Pac142, and Pac170. The primers are: the primers for the molecular marker Pac025, the upstream primer nucleotide sequence of which is shown in SEQ ID NO.1, and the downstream primer nucleotide sequence of which is shown in SEQ ID NO.2; The primers for the molecular marker Pac98 have the following nucleotide sequences: upstream primer as shown in SEQ ID NO.3 and downstream primer as shown in SEQ ID NO.

4. The primers for the molecular marker Pac113 are shown in SEQ ID NO.5 for the upstream primer and SEQ ID NO.6 for the downstream primer. The primers for the molecular marker Pac142 have the following nucleotide sequences: upstream primer as shown in SEQ ID NO.7 and downstream primer as shown in SEQ ID NO.

8. The primers for the molecular marker Pac170 have the following nucleotide sequences: upstream primer as shown in SEQ ID NO.9 and downstream primer as shown in SEQ ID NO.

10.

2. The application of the molecular marker primer for molecular identification of the Purple Gold variety of Avocado according to claim 1 in the identification of the Purple Gold variety of Avocado.

3. A method for identifying the Zijin avocado variety using the molecular marker primers for molecular identification of the Zijin avocado variety as described in claim 1, characterized in that: The method includes the following steps: Step 1: Preparation of the sample to be tested: Select fresh avocado leaves to be tested, wash them, dry them, grind them under liquid nitrogen conditions, grind them into powder, and store them in a -80℃ refrigerator for testing. Step 2: Extract DNA from the sample to be tested; Step 3: Amplification products of sample PCR; Step 4: Fluorescent capillary electrophoresis detection; Step 5: Compare peak values ​​to identify the sample.

4. The method for identifying the Zijin variety of avocado according to claim 3, characterized in that: The specific process of step 2 is as follows: Take the ground avocado sample and place it in a pre-cooled centrifuge tube. Quickly add 500 μL of lysis buffer, then add 100 μL of antioxidant and 10 μL of Rnase A. Mix thoroughly and incubate at 65°C for 10 min, shaking 2–3 times during incubation. Next, add 150 μL of polysaccharide scavenger to the centrifuge tube and gently mix 15–20 times. Let it stand at room temperature for 3–5 min. Centrifuge the tube at 12000 rpm for 5 min at 4°C to obtain the supernatant. Transfer 500 μL of the supernatant to a new centrifuge tube. Add 300 μL of [unclear text - possibly a specific ingredient or solution] to the new centrifuge tube. Mix the chloroform and isoamyl alcohol mixture (V:V = 24:1) thoroughly, shake vigorously for 15 seconds, and centrifuge at 1200 rpm for 30 seconds to obtain the upper aqueous phase. Take 500 μL of the upper aqueous phase and transfer it to an extraction plate. Add an equal volume of isopropanol to the extraction plate and place it at station 1 of the extraction instrument. In the new extraction plate, aliquot 100 μL of magnetic beads into each well and place them at station 2. Aliquot 500 μL / well of 75% ethanol into three separate plates and place them at stations 3, 4, and 5, respectively. Take the elution plate and aliquot 100 μL, 80 μL, and 60 μL of elution buffer into each well and place them at station 6. After verifying the instrument status and extraction plate information, run the program. After the program finishes, remove the elution plate and perform DNA analysis on the eluted DNA. Store the remaining eluted DNA at 4°C. Take another 2 μL of the DNA stock solution, add 2 μL of bromophenol blue, and perform agarose gel electrophoresis to check DNA integrity.

5. The method for identifying the Zijin variety of avocado according to claim 3, characterized in that: The specific process of step 3 is as follows: Using DNA extracted from the sample as a template, PCR amplification was performed using the provided 5 pairs of primers. The 15 μL PCR amplification system included: 1 μL of genomic DNA (50-200 ng / μL), 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℃ pre-denaturation for 3 min; 30 cycles including: 96℃ denaturation for 30 s, gradient annealing from 62℃ to 52℃ for 30 s, extension at 72℃ for 1 min, and extension at 72℃ for 10 min. After completion, the sample was stored at 4℃ to obtain the PCR amplification product.

6. The method for identifying the Zijin variety of avocado according to claim 3, characterized in that: The specific process of step 4 is as follows: The PCR products were detected by fluorescent capillary electrophoresis to obtain the peak values ​​of the amplified products. The PCR amplified products of the samples diluted to a uniform concentration were added to the test plate. The test reagents were added separately according to the total system of 11.5 μL: 1 μL of fluorescent PCR product, 0.5 μL of GeneScan™ 500 LIZ, and 10 μL of Hi-Di™ Formamide. After centrifuging the test plate with the added samples and reagents, it was placed in the PCR instrument and the denaturation program was run at 95°C for 3 min. After denaturation, it was cooled immediately. Following the ABI 3730xL test plate operation procedure, the test file corresponding to the name of the test plate was selected, and the SSR sample analysis test program was run. The results were analyzed using GeneMarker software to obtain the number of alleles, peak diagram, peak value, and genotype of each sample.

7. The method for identifying the Zijin variety of avocado according to claim 3, characterized in that: The specific process of step 5 is as follows: The peak values ​​of the five amplification products of the sample are compared with the standard peak values ​​of the corresponding primer pairs to determine whether the sample is an avocado variety, Zijin. If the peak values ​​of the five amplification products of the sample are consistent with the standard peak values ​​of the corresponding primer pairs, the sample is an avocado variety, Zijin. If the peak values ​​of the five amplification products of the sample are inconsistent with at least one of the standard peak values ​​of the corresponding primer pairs, the sample is not an avocado variety, Zijin.

8. The method for identifying the Zijin variety of avocado according to claim 3, characterized in that: In step 3, the provided 5 pairs of primers are used as the designed specific primers. The 5' ends 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 Zijin variety of avocado according to claim 3, characterized in that: In step 5, the standard peak values ​​of the primer pairs are as follows: the standard peak values ​​of the first primer pair are 238 and 244, the standard peak values ​​of the second primer pair are 270 and 276, the standard peak value of the third primer pair is 218, the standard peak values ​​of the fourth primer pair are 278 and 281, and the standard peak values ​​of the fifth primer pair are 187 and 191.

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