Molecular marker primer group for identifying avocado variety Tenglong, kit, marking method and application

Abstract

The invention discloses a molecular marker primer group for identifying an avocado variety Tenglong, a kit, a marking method and application, and belongs to the technical field of biology. The main identification method comprises the following steps: (1) extracting genome DNA of a sample to be detected; (2) taking the genome DNA as a template, and carrying out PCR (Polymerase Chain Reaction) amplification by utilizing five pairs of primers; (3) performing electrophoresis detection on the PCR amplification product to obtain a peak value of the amplification product of the sample to be detected; and (4) respectively comparing the peak values of the five amplification products of the sample to be detected with corresponding standard peak values so as to judge whether the sample to be detected is the avocado variety Tenglong or not. The five pairs of specific primers are utilized to perform PCR amplification on the genome DNA of the sample to be tested, and the peak value of the amplification product is compared with the standard peak, so that whether the test sample is the avocado variety Tenglong or not can be quickly and effectively judged, the operation is simple and convenient, and the protection, popularization and application of the variety are facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of biological molecular identification, and specifically relates to molecular markers for molecular identification of an avocado variety, Tenglong, and an application method. Background Art

[0002] Avocado, also known as alligator, avocado, camphor pear, and happy fruit, is native to the humid areas of Central America and Mexico. It belongs to the angiosperms, dicots, magnolias, camphors, lauraceae, alligator genus, alligator P.Americana, an evergreen tree, a famous tropical fruit and one of the woody oil-bearing tree species. Avocado contains a variety of vitamins, rich fat and protein, as well as mineral elements such as sodium, potassium, magnesium, and calcium. The kernel contains fatty oil, the content of which is 8% to 29%. Due to its rich nutrition, it has considerable health benefits for the human body.

[0003] As people's demand for avocados increases, the avocado resources in Guangxi, Yunnan, Guizhou, Taiwan and other places in China are increasing, and a wide variety of avocados have appeared on the market, but different avocados have different prices. However, some avocado varieties have similar leaves and fruits, but their DNA sequences are different, belonging to different varieties. They are often mistaken for high-priced varieties and sold as seedlings and fruits. The lack of accurate variety identification restricts the efficient use of these avocado germplasms.

[0004] The avocado variety Tenglong is the first avocado variety in my country with a new plant variety right. It is currently the main variety in Chongzuo, Guangxi. It has the excellent characteristics of high and stable yield. Some asexual reproduction and cultivation without obtaining variety authorization on the market makes it difficult to distinguish the authenticity of the variety from the external morphological characteristics. There is even a phenomenon of selling other varieties as this variety, making it difficult to ensure the homozygosity of the variety. Therefore, it is particularly important to find a technology that can quickly and effectively identify the Tenglong variety. Summary of the invention

[0005] The present invention provides a molecular marker primer set, a kit, a method and an application thereof for identifying an avocado variety Tenglong. The operation is simple and convenient, and molecular marker identification can be performed by five pairs of specific primers to determine whether the variety to be tested belongs to the avocado variety Tenglong, which has a high guiding significance.

[0006] The present invention is achieved through the following technical solutions:

[0007] A molecular marker primer set for identifying an avocado variety Tenglong, the primer set comprising 5 pairs of primers, namely Pac066, Pac122, Pac131, Pac145 and Pac146, and the nucleotide sequences thereof are shown in SEQ ID NO.1-SEQ ID NO.10.

[0008] The 5' ends of the forward primers of the five pairs of primers of the present invention all carry fluorescent groups; the fluorescent group of Pac066 is HEX, the fluorescent group of Pac122 is HEX, the fluorescent group of Pac131 is HEX, the fluorescent group of Pac145 is HEX, and the fluorescent group of Pac146 is FAM.

[0009] The standard peak values ​​of Tenglong in primer Pac066 are 203 and 206, the standard peak values ​​in Pac122 are 174 and 177, the standard peak values ​​in Pac131 are 261 and 269, the standard peak values ​​in Pac145 are 277 and 283, and the standard peak values ​​in Pac146 are 252 and 270.

[0010] A rapid detection kit for identifying the avocado variety Tamlong comprises the above-mentioned primer set and fluorescent group.

[0011] A method for identifying an avocado variety Tenglong using a molecular marker primer set mainly comprises the following steps:

[0012] (1) extracting genomic DNA from the sample to be tested;

[0013] (2) Using genomic DNA as a template, PCR amplification was performed using five pairs of primers to obtain five amplification products; the amplification products were then detected by fluorescent capillary electrophoresis to obtain the peak value of the amplification products;

[0014] (3) Compare the peak of the amplified product of step (2) with the corresponding standard peak. If the comparison is matched, the sample to be tested is the avocado variety Tenglong; if the comparison is not matched, the sample is not the avocado variety Tenglong.

[0015] The composition and content of the PCR reaction system used in the present invention are as follows: 1 μL of genomic DNA, 7.5 μL of 2×Taq PCR Master Mix, 1.0 μL of forward primer, 1.0 μL of reverse primer and 4.5 μL of ddH2O.

[0016] The PCR reaction procedure in the present invention is: 96°C pre-denaturation for 3 minutes; 30 cycles; 72°C extension for 10 minutes, 4°C storage; 30 cycles include 96°C denaturation for 30 seconds, 62°C-52°C gradient annealing for 30 seconds, and 72°C extension for 1 minute.

[0017] The primer set, rapid detection kit or molecular marker method of the present invention is used in identifying the avocado variety Tenglong.

[0018] Beneficial effects of the present invention:

[0019] (1) The present invention uses 5 pairs of specific primers to identify the avocado variety Tenglong. By comparing the peak value of the amplified product of the variety to be tested with the corresponding standard peak value, it is determined whether it is the avocado variety Tenglong. The operation method is simple, the detection efficiency is high, the result is reliable, and it is beneficial to the protection and promotion and application of the variety.

[0020] (2) When it is difficult to distinguish varieties simply by observing leaves, flowers and trunks with the naked eye, the method of the present invention can be used to identify whether it is an Avocado Tenglong variety before hybrid breeding, which can help identify the parents during the hybrid breeding process.

[0021] (3) The quality of different avocados varies greatly, but since the appearance of avocado fruits is similar, low-priced avocados are sold as high-value avocados in the market. The method of the present invention can distinguish the fruit varieties, determine the appropriate prices, and maintain the trading order of the avocado market. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a photo of the fruit of the avocado variety Tamron.

[0023] Figure 2 This is a photo of the leaves of the avocado variety Tamron.

[0024] Figure 3 This is the standard peak map of the avocado variety Tamron with the first primer pair Pac066.

[0025] Figure 4 This is the standard peak diagram of the avocado variety Tamron using the second pair of primers Pac122.

[0026] Figure 5 This is the standard peak map of the avocado variety Tamlong using the third primer pair Pac131.

[0027] Figure 6 This is the standard peak diagram of the avocado variety Tamron using the fourth primer pair Pac145.

[0028] Figure 7 This is the standard peak map of the avocado variety Tamron using the fifth primer pair Pac146. DETAILED DESCRIPTION

[0029] The present invention is illustrated by the following examples, but is not intended to limit the scope of use of the present invention.

[0030] Example

[0031] 1. Primer set selection

[0032] The present invention adopts a primer set for identifying the avocado variety Tenglong, including: 5 pairs of primers, each pair of primers includes a forward primer and a reverse primer, and the first pair of primers to the fifth pair of primers are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 10 in the sequence table.

[0033] The 5' end of the forward primers all have a fluorescent group. The sequences of the primers and the corresponding fluorescent groups are specifically shown in Table 1.

[0034] Table 1: Primer pairs and corresponding fluorescent markers

[0035]

[0036] The kit used in the identification process of the present invention includes: 5 pairs of primers, 2×Taq PCR Master Mix, ddH2O, GeneScan TM 500LIZ and Hi-Di TM Formamide.

[0037] 2. Molecular marker method

[0038] 1. Molecular marker method for identifying the avocado variety Tenglong, the specific operations include:

[0039] (1) Preparation of samples: Select fresh avocado leaves to be tested, wash, dry, grind under liquid nitrogen, grind into powder and store in a -80°C refrigerator for testing;

[0040] (2) Extracting genomic DNA from the sample to be tested; in this implementation, a magnetic bead-based genomic DNA extraction kit from Tiangen Biochemical Technology (Beijing) Co., Ltd. was used to extract genomic DNA from avocado leaves. The specific operation was performed according to the instructions of the magnetic bead-based genomic DNA extraction kit. Another 2 μL of DNA stock solution was added with 2 μL of bromophenol blue for agarose gel electrophoresis to detect DNA integrity.

[0041] (3) Sample PCR amplification: Using the DNA extracted from the sample to be tested as a template, PCR amplification was performed using the 5 pairs of primers in Table 1.

[0042] The 15 μL PCR amplification system includes: (50-200) ng / μL genomic DNA 1 μL, 2×Taq PCR MasterMix 7.5 μL, 10 pmol / μL forward primer 1.0 μL, 10 pmol / μL reverse primer 1.0 μL and ddH2O 4.5 μL.

[0043] The PCR amplification program was as follows: 96°C pre-denaturation for 3 min; 30 cycles including (96°C denaturation for 30 s, 62°C-52°C gradient annealing for 30 s, 72°C extension for 1 min); 72°C extension for 10 min, and storage at 4°C after the amplification. Five PCR amplification products of the sample were obtained.

[0044] (4) Fluorescence capillary electrophoresis detection: Detect the PCR product by fluorescence capillary electrophoresis to obtain the peak value of the amplified product. Add the sample PCR amplification product diluted to a uniform concentration to the upper plate, and add the upper detection reagent according to the total system of 11.5 μL, 1 μL of fluorescent PCR product, and 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). 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.

[0045] (5) Comparison of peak values ​​to identify samples: The peak values ​​of the five amplified products of the sample to be tested are respectively compared with the corresponding standard peak values ​​to determine whether the sample to be tested is the avocado variety Tenglong. If the peak values ​​of the five amplified products of the sample to be tested are consistent with the corresponding standard peak values, the sample to be tested is the avocado variety Tenglong; if the peak values ​​of the five amplified products of the sample to be tested are inconsistent with at least one of the corresponding standard peak values, the sample to be tested is not the avocado variety Tenglong.

[0046] 3. Identification and Verification

[0047] 79 avocado germplasms were used as samples for identification, including: GL1, Qiuhong, Hass, Nabal, Pinkerton, Lamb Hass, MM3, Choquette, HH1, NY4, Beta, GL18, TY1, Guiyan No. 10, TB, DL17, xx1, K1, Nanya B, Brooks late, Boot, YL31, SCP1, Fuerte, S5, Bacon, Ettinger, Dusa, GL13, BS1, Guiken No. 2, Guiyan No. 8, Guiyan No. 1, BN3, v11, Tonnage, yl33, Tenglong, Guilong No. 8, Duke7, Kampong, Booth8, G, Booth7, KP3, BS2, loretta, Wilson seedless, Simmonds, Zijin, GL25, TW1-86, TW1-98, Guilong No. 11, Guikenda No. 3, HHe1, K2, Donnie, Lula, Miguel, SX1, Guilong No. 7, v5, BY2, GL10, v8, GL15, GL14, Qiuyue, GL5, YL27, Walter Hole, RS1, S6, HN2, GL16, Guilong No. 6 and S4.

[0048] The above-mentioned samples to be tested are from the avocado germplasm resource garden of Guangxi South Asian Tropical Agricultural Science Research Institute.

[0049] First, the genomic DNA of avocado leaves was extracted using a magnetic bead method genomic DNA extraction kit from Tiangen Biochemical Technology (Beijing) Co., Ltd. The specific operation was carried out according to the instructions of the magnetic bead method genomic DNA extraction kit. Then, PCR amplification and electrophoresis detection were performed according to the molecular marker method described above to obtain amplification products of 79 samples to be tested.

[0050] Comparative Identification

[0051] The standard peak values ​​of the Tenglong variety in the present invention for the five pairs of primers (Pac066, Pac122, Pac131, Pac145 and Pac146) are specifically as follows: the standard peak values ​​for the first primer pair are 203 and 206, the standard peak values ​​for the second primer pair are 174 and 177, the standard peak values ​​for the third primer pair are 261 and 269, the standard peak values ​​for the fourth primer pair are 277 and 283, and the standard peak values ​​for the fifth primer pair are 252 and 280. Figures 1 to 5 shown.

[0052] The peak values ​​of the amplified products of the 79 germplasms were compared with the corresponding standard peak values ​​to determine whether the sample to be tested was the avocado variety Tenglong. The specific results are shown in Table 2.

[0053] Table 2 Comparison of the peak values ​​of 5 primer pairs in 92 samples tested with the standard peak values

[0054]

[0055]

[0056]

[0057] By comparing Table 2, it can be seen that the peak value of the sample Tenglong is consistent with the 10 standard peak values ​​of the five pairs of primers, so it is judged to be the avocado variety Tenglong, which is consistent with the actual result. This shows that the primer set provided by the present invention can accurately identify the avocado variety Tenglong.

Claims

1. A molecular marker primer set for identifying the avocado variety Tenglong, characterized in that: The primer set comprises 5 pairs of primers, namely Pac066, Pac122, Pac131, Pac145 and Pac146, and the nucleotide sequences thereof are shown in SEQ ID NO.1-SEQ ID NO.

10.

2. The molecular marker primer set for identifying the avocado variety Tenglong according to claim 1, characterized in that: The 5' ends of the forward primers of the five pairs of primers all carry fluorescent groups; the fluorescent group of Pac066 is HEX, the fluorescent group of Pac122 is HEX, the fluorescent group of Pac131 is HEX, the fluorescent group of Pac145 is HEX, and the fluorescent group of Pac146 is FAM.

3. The molecular marker primer set for identifying the avocado variety Tenglong according to claim 1, characterized in that: The standard peaks of avocado variety Tamlong in primer Pac066 are 203 and 206, the standard peaks in Pac122 are 174 and 177, the standard peaks in Pac131 are 261 and 269, the standard peaks in Pac145 are 277 and 283, and the standard peaks in Pac146 are 252 and 270.

4. A rapid detection kit for identifying the avocado variety Tenglong, characterized in that A primer set comprising claim 1 or 2.

5. A molecular marker method for identifying the avocado variety Tenglong using a molecular marker primer set, characterized in that: The method mainly includes the following steps: (1) extracting genomic DNA from the sample to be tested; (2) Using genomic DNA as a template, PCR amplification was performed using five pairs of primers to obtain five amplification products; the amplification products were then detected by fluorescent capillary electrophoresis to obtain the peak value of the amplification products; (3) Compare the peak value of the amplified product of step (2) with the corresponding standard peak value. If the comparison is matched, the sample to be tested is the avocado variety Tenglong; if the comparison is not matched, the sample to be tested is not the Tenglong avocado variety.

6. The molecular marker method of avocado variety Tenglong according to claim 5, characterized in that: The composition and content of the PCR reaction system are as follows: 1 μL of genomic DNA, 7.5 μL of 2×Taq PCR Master Mix, 1.0 μL of forward primer, 1.0 μL of reverse primer and 4.5 μL of ddH2O.

7. The molecular marker method of avocado variety Tenglong according to claim 5, characterized in that: The PCR reaction program was as follows: pre-denaturation at 96°C for 3 min; 30 cycles; extension at 72°C for 10 min, and storage at 4°C; the 30 cycles included denaturation at 96°C for 30 s, gradient annealing at 62°C to 52°C for 30 s, and extension at 72°C for 1 min.

8. Use of the primer set according to any one of claims 1 to 3, the rapid detection kit according to claim 4, or the molecular marker method according to any one of claims 5 to 7 in identifying the avocado variety Tenglong.

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