Primer group, kit and method for identifying avocado variety
By using primer sets of 8 primer pairs for PCR amplification and fluorescent capillary electrophoresis detection, the inaccuracy problem of avocado variety identification in the prior art was solved, and the accurate identification and distinction of samples to be tested was achieved.
Patent Information
- Application Number
- CN202411886992.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-20
- Publication Date
- 2025-03-11
AI Technical Summary
The prior art is difficult to accurately identify avocado varieties, especially due to the difficulty of identification caused by similar appearances.
A primer set including 8 primer pairs is provided to accurately identify avocado varieties by PCR amplification and fluorescent capillary electrophoresis detection.
The accurate distinction and identification of 80 samples to be tested was achieved, and the identification inaccuracy based on appearance characteristics was overcome, and the operation was simple, the cost was low and the efficiency was high.
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Abstract
Description
Technical Field
[0001] The present disclosure relates to the field of molecular identification, and in particular to a primer set, a kit and a method for identifying avocado varieties. Background Art
[0002] Avocados are native to Colombia, Ecuador and southern Mexico. They are now mainly distributed in tropical and subtropical regions between 30° south latitude and 30° north latitude. They are cultivated in Yunnan, Guangdong, Guangxi and Taiwan in China.
[0003] Avocado flesh is soft and delicate, rich in unsaturated fatty acid-based oils, a variety of minerals and vitamins, and contains no cholesterol and low sugar content. It is a high-end fruit suitable for both young and old. Its fruit is nutritious and has high nutritional value. According to statistics from the Food and Agriculture Organization of the United Nations, the world's avocado production reached more than 23.36 million tons in 2000, ranking 11th in fruit production. There are more than 30 countries that cultivate it. Due to its rich nutrition, the cultivated area around the world has increased year by year, making it a new fruit with great development prospects in tropical and subtropical areas.
[0004] With the rise of avocado, avocado is widely planted. However, in the process of planting, the diversity of avocado varieties and the richness of genetic resources provide the basis for breeding and improvement. Therefore, accurate and rapid identification of avocado varieties is of great significance for genetic potential mining, germplasm resource protection and anti-counterfeiting.
[0005] At present, the identification of avocado varieties mainly relies on morphological characteristics. However, many avocado varieties have similar appearances, such as fruit size, shape and color, which makes it difficult to distinguish them by naked eye observation, which increases the difficulty of avocado variety identification. Therefore, a real and effective method that can accurately identify avocado varieties is urgently needed.
[0006] Public Content
[0007] In order to solve the problems of the prior art, the present disclosure provides a primer set, a kit and a method for identifying avocado varieties. The technical solution is as follows:
[0008] On the one hand, the present disclosure provides a primer set, a kit and a method for identifying avocado varieties, the primer set comprising: 8 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 eighth primer pair and the reverse primer of the eighth primer pair are respectively as shown in SEQ ID NO: 1 to SEQ ID NO: 16 in the sequence listing.
[0009] Specifically, the avocado varieties identified by the primer set include: SCP1, YL31 Guilong No. 8, DL17, HH1, YL27 GL14, BY2, xx3, K1, GL15, GL10, GL16, GL18, Miguel, K2, GL5, Guilong 7, v11, Donnie, Simmonds, SX1, Qiuyue, Guilong 12, BS2, Lula, YL32( bích)、YL33 KP3, GL1, v5, GL13, Nesbitt, Wilsonseedless, Nanya B, Guilong 2, Guilong 11, Guilong 6, TY1, R, Dupuis, Choquette, Tonnage, Boot, Booth8, NY4, loretta, GL25, S2, TW1-98, Beta, Pollock, Kampong, Qiuhong, Fuerte, S4, BS1, Guiyan 8, RS1, Ettinger, B, Lisa, MM3, Bacon, Walter Hole, Guiyan 1, Duke7, Dusa, HHe1, S5, Pinkerton, Hass, BN3, Brookslate, Nabal, Lamb Hass, BQ1, BLD034, xx1, and v8.
[0010] In another aspect, the present disclosure provides a kit for identifying avocado varieties, the kit comprising the above primer set.
[0011] Specifically, the kit also includes: 2×TaqPCRMasterMix, ddH2O, GeneScan TM 500LIZ and Hi-Di TM Formamide.
[0012] In another aspect, the present disclosure provides a method for identifying an avocado variety, the method comprising:
[0013] Extracting genomic DNA from the sample to be tested;
[0014] Using the genomic DNA as a template, PCR amplification was performed using the above 8 primer pairs to obtain 8 amplification products;
[0015] The eight amplification products are respectively detected by fluorescent capillary electrophoresis to obtain eight peaks of the amplification products;
[0016] The peak values of the eight amplified products are compared with the peak values corresponding to each pair of the primer pairs. If the peak values of the eight amplified products are consistent with the peak values corresponding to each pair of the primer pairs, the avocado variety is the corresponding variety.
[0017] Specifically, the identified avocado varieties include: SCP1, YL31 Guilong No. 8, DL17, HH1, YL27 GL14, BY2, xx3, K1, GL15, GL10, GL16, GL18, Miguel, K2, GL5, Guilong 7, v11, Donnie, Simmonds, SX1, Qiuyue, Guilong 12, BS2, Lula, YL32( bích)、YL33 KP3, GL1, v5, GL13, Nesbitt, Wilsonseedless, Nanya B, Guilong 2, Guilong 11, Guilong 6, TY1, R, Dupuis, Choquette, Tonnage, Boot, Booth8, NY4, loretta, GL25, S2, TW1-98, Beta, Pollock, Kampong, Qiuhong, Fuerte, S4, BS1, Guiyan 8, RS1, Ettinger, B, Lisa, MM3, Bacon, Walter Hole, Guiyan 1, Duke7, Dusa, HHe1, S5, Pinkerton, Hass, BN3, Brookslate, Nabal, Lamb Hass, BQ1, BLD034, xx1, and v8.
[0018] Specifically, the peak values of the avocado varieties corresponding to each primer pair are as follows:
[0019]
[0020]
[0021]
[0022] The technical solution provided by the disclosed embodiment has the following beneficial effects: the embodiment provides a primer set, a kit and a method for identifying avocado varieties, which can overcome the inaccuracy of variety identification based on external morphological characteristics of avocado by detecting markers such as Pac001, Pac082, Pac085, Pac091, Pac115, Pac134, Pac154 and Pac186, and accurately distinguish and identify 80 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 is of great significance for the exploration of the genetic potential of avocado varieties, the protection of germplasm resources and the fight against counterfeiting, and is conducive to the promotion and application of avocado varieties. 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 described in further detail below.
[0024] Embodiment 1
[0025] The disclosed embodiments provide a primer set, a kit, and a method for identifying avocado varieties. The primer set includes: 8 primer pairs, each primer pair includes 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 eighth primer pair, and the reverse primer of the eighth primer pair are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 16 in the sequence listing.
[0026] 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.
[0027] Table 1 shows the sequences of the primer sets and the corresponding fluorescent groups
[0028]
[0029]
[0030] Specifically, the avocado varieties identified by the primer set include: SCP1, YL31, Guilong 8, DL17, HH1, YL27, GL14, BY2, xx3, K1, GL15, GL10, GL16, GL18, Miguel, K2, GL5, Guilong 7, v11, Donnie, Simmonds, SX1, Qiuyue, Guilong 12, BS2, Lula, YL32, YL33, KP3, GL1, v5, GL13, Nesbitt, Wil sonseedless, Nanya B, Guilong 2, Guilong 11, Guilong 6, TY1, R, Dupuis, Choquette, Tonnage, Boot, Booth8, NY4, loretta, GL25, S2, TW1-98, Beta, Pollock, Kampong, Qiuhong, Fuerte, S4, BS1, Guiyan 8, RS1, Ettinger, B, Lisa, MM3, Bacon, Walter Hole, Guiyan 1, Duke7, Dusa, HHe1, S5, Pinkerton, Hass, BN3, Brooks late, Nabal, Lamb Hass, BQ1, BLD034, xx1, and v8.
[0031] Embodiment 2
[0032] The disclosed embodiments provide a kit for identifying avocado varieties, the kit comprising the primer set provided in Embodiment 1.
[0033] Specifically, the kit also includes: 2×TaqPCR MasterMix, ddH2O, GeneScan TM 500LIZ and Hi-Di TM Formamide.
[0034] Embodiment 3
[0035] The present disclosure provides a method for identifying avocado varieties, the method comprising:
[0036] Extracting genomic DNA from the sample to be tested;
[0037] Specifically, the identified avocado varieties include: SCP1, YL31, Guilong No. 8, DL17, HH1, YL27, GL14, BY2, xx3, K1, GL15, GL10, GL16, GL18, Miguel, K2, GL5, Guilong No. 7, v11, Donnie, Simmonds, SX1, Qiuyue, Guilong No. 12, BS2, Lula, YL32, YL33, KP3, GL1, v5, GL13, Nesbitt, Wilson nseedless, Nanya B, Guilong No. 2, Guilong No. 11, Guilong No. 6, TY1, R, Dupuis, Choquette, Tonnage, Boot, Booth8, NY4, loretta, GL25, S2, TW1-98, Beta, Pollock, Kampong, Qiuhong, Fuerte, S4, BS1, Guiyan No. 8, RS1, Ettinger, B, Lisa, MM3, Bacon, Walter Hole, Guiyan No. 1, Duke7, Dusa, HHe1, S5, Pinkerton, Hass, BN3, Brooks late, Nabal, Lamb Hass, BQ1, BLD034, xx1 and v8, a total of 80 avocado varieties. In this implementation, the above 80 avocado varieties are selected as samples to be tested. The samples to be tested provided in this embodiment are derived from the avocado germplasm resource garden of Guangxi South Asian Tropical Agricultural Science Research Institute.
[0038] Extract the genomic DNA of the above 80 avocado varieties. In the implementation, the magnetic bead genomic DNA extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. was used to extract the genomic DNA of avocado leaves. The specific operation was carried out according to the instructions of the magnetic bead genomic DNA extraction kit. Take another 2 μL of DNA stock solution and add 2 μL of bromophenol blue for agarose gel electrophoresis to detect DNA integrity.
[0039] Using genomic DNA as a template, PCR amplification was performed using the eight primer pairs provided in Example 1 to obtain eight amplification products.
[0040] Specifically, the 15 μL PCR amplification system includes: 1 μL (50-200) ng / μL genomic DNA, 7.5 μL 2×Taq PCR Master Mix, 1.0 μL 10 pmol / μL forward primer, 1.0 μL 10 pmol / μL reverse primer and 4.5 μL ddH2O.
[0041] Specifically, the PCR amplification program was: pre-denaturation at 96°C for 3 min; 30 cycles including (denaturation at 96°C for 30 s, gradient annealing at 62°C to 52°C for 30 s, extension at 72°C for 1 min); extension at 72°C for 10 min, and storage at 4°C after completion of amplification.
[0042] The eight amplified products were respectively detected by fluorescent capillary electrophoresis to obtain the peak values of the eight amplified products.
[0043] Specifically, the fluorescent PCR product diluted to a uniform concentration was added to the upper plate, and the upper detection reagents were added to the total 11.5 μL system: 1 μL of fluorescent PCR product, GeneScan TM 500LIZ 0.5μL, Hi-Di TM Formamide 10 μL; centrifuge the plate to be tested with samples and reagents and place it on the PCR instrument to run the denaturation program (95°C, 3 min), 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 to be tested.
[0044] The peak values of the eight amplified products were compared with the peak values corresponding to each primer pair. If the peak values of the eight amplified products were consistent with the peak values corresponding to each primer pair, the avocado variety was the corresponding variety.
[0045] Specifically, the peak values of avocado varieties corresponding to each primer pair are shown in Table 2 .
[0046] Table 2 shows the peak values amplified by each primer pair for avocado varieties
[0047]
[0048]
[0049]
[0050] In this embodiment, the peak values of 640 amplified products of 80 samples to be tested are consistent with the peak values amplified by the avocado varieties corresponding to each primer pair provided in Table 2. It can be seen that all 80 samples to be tested can be accurately identified.
[0051] 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 avocado varieties, characterized in that: The primer set includes: 8 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 eighth primer pair and the reverse primer of the eighth primer pair are respectively shown as SEQ ID NO: 1 to SEQ ID NO: 16 in the sequence listing.
2. The primer set according to claim 1, characterized in that The avocado varieties identified by the primer set include: SCP1, YL31, Guilong 8, DL17, HH1, YL27, GL14, BY2, xx3, K1, GL15, GL10, GL16, GL18, Miguel, K2, GL5, Guilong 7, v11, Donnie, Simmonds, SX1, Qiuyue, Guilong 12, BS2, Lula, YL32, YL33, KP3, GL1, v5, GL13, Nesbitt, and Wils onseedless, Nanya B, Guilong 2, Guilong 11, Guilong 6, TY1, R, Dupuis, Choquette, Tonnage, Boot, Booth8, NY4, loretta, GL25, S2, TW1-98, Beta, Pollock, Kampong, Qiuhong, Fuerte, S4, BS1, Guiyan 8, RS1, Ettinger, B, Lisa, MM3, Bacon, Walter Hole, Guiyan 1, Duke7, Dusa, HHe1, S5, Pinkerton, Hass, BN3, Brooks late, Nabal, Lamb Hass, BQ1, BLD034, xx1, and v8.
3. A kit for identifying avocado varieties, characterized in that: The kit comprises the primer set according to claim 1 or 2.
4. The kit according to claim 3, characterized in that The kit also includes: 2×Taq PCRMasterMix, ddH2O, GeneScan TM 500LIZ and Hi-Di TM Formamide.
5. A method for identifying avocado varieties, 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 eight primer pairs described in claim 1 to obtain eight amplification products; The eight amplification products are respectively detected by fluorescent capillary electrophoresis to obtain eight peaks of the amplification products; The peak values of the eight amplified products are compared with the peak values corresponding to each pair of the primer pairs. If the peak values of the eight amplified products are consistent with the peak values corresponding to each pair of the primer pairs, the avocado variety is the corresponding variety.
6. The method according to claim 5, characterized in that The identified avocado varieties include: SCP1, YL31, Guilong No. 8, DL17, HH1, YL27, GL14, BY2, xx3, K1, GL15, GL10, GL16, GL18, Miguel, K2, GL5, Guilong No. 7, v11, Donnie, Simmonds, SX1, Qiuyue, Guilong No. 12, BS2, Lula, YL32, YL33, KP3, GL1, v5, GL13, Nesbitt, Wilson seedless, Nanya B, Guilong 2, Guilong 11, Guilong 6, TY1, R, Dupuis, Choquette, Tonnage, Boot, Booth8, NY4, loretta, GL25, S2, TW1-98, Beta, Pollock, Kampong, Qiuhong, Fuerte, S4, BS1, Guiyan 8, RS1, Ettinger, B, Lisa, MM3, Bacon, Walter Hole, Guiyan 1, Duke7, Dusa, HHe1, S5, Pinkerton, Hass, BN3, Brooks late, Nabal, Lamb Hass, BQ1, BLD034, xx1, and v8.
7. The method according to claim 5, characterized in that The peak values of the avocado varieties corresponding to each primer pair are as follows: