Molecular marker primer group, kit, and method for identifying oil tree variety tenglong, and application thereof

Abstract

The application discloses a molecular marker primer group for identifying oil olive variety Tenglong, a kit, a marking method and application, and belongs to the technical field of biology. Main identification methods comprise the following steps: (1) extracting genomic DNA of a sample to be detected; (2) taking the genomic DNA as a template, and performing PCR amplification by using five pairs of primers; (3) performing electrophoresis detection on PCR amplification products, and obtaining peak values of the amplification products of the sample to be detected; and (4) comparing the peak values of the five amplification products of the sample to be detected with corresponding standard peak values, so as to determine whether the sample to be detected is the oil olive variety Tenglong. The application can quickly and effectively determine whether a test sample is the oil olive variety Tenglong by comparing the peak values of amplification products with standard peaks by using five pairs of specific primers to perform PCR amplification on genomic DNA of the sample to be detected, the operation is simple and convenient, and the application is favorable for protection and popularization and application of the variety.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomolecule identification, and particularly relates to a molecular marker for identifying an avocado variety Tenglong and a method for the application. BACKGROUND

[0002] Avocado is also known as alligator pear, butter fruit, cheese pear, camphor pear and happiness fruit, and is a kind of famous tropical fruit and one of woody oil tree species. Avocado is originally from the humid areas of Central America and Mexico, and belongs to the dicotyledonous plant class Magnoliidae, Lauraceae and Persea Americana. Avocado is a kind of evergreen tree, and contains various vitamins, rich fat and protein, and mineral elements such as sodium, potassium, magnesium and calcium. The kernel of avocado contains fatty oil with a content of 8% to 29%, and has considerable health care effects on human body.

[0003] With the increasing demand for avocado, the resources of avocado in Guangxi, Yunnan, Guizhou and Taiwan of China are increasing, and various kinds of avocado appear on the market. However, different avocados have different prices. Some avocado varieties have similar leaves and fruits, but have different DNA sequences and belong to different varieties, and are often mistaken for high-priced varieties for seedling and fruit sales. Due to the lack of accurate variety determination, the efficient use of these avocado germplasm is restricted.

[0004] Avocado variety Tenglong is the first avocado variety with plant new variety right in China, and is the main cultivated variety in Chongzuo region of Guangxi. The variety has high yield and stable yield. Some varieties without variety authorization carry out asexual propagation and cultivation, and it is difficult to distinguish the authenticity of the variety from external morphological characteristics, and even other varieties are used to counterfeit the sales of the variety, so that the purity of the variety is difficult to guarantee. Therefore, it is particularly important to find a kind of technology for quickly and effectively identifying Tenglong variety. SUMMARY

[0005] The application provides a molecular marker primer group, a kit, a method and an application for identifying avocado variety Tenglong.

[0006] The application is achieved by the following technical solutions:

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

[0008] The forward primers of the 5 pairs of primers in the application are all provided with a fluorescent group; 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] In the application, the standard peak values of the Tenglong variety in the primers Pac066 are 203 and 206, the standard peak values in the primers Pac122 are 174 and 177, the standard peak values in the primers Pac131 are 261 and 269, the standard peak values in the primers Pac145 are 277 and 283, and the standard peak values in the primers Pac146 are 252 and 270.

[0010] A rapid detection kit for identifying the oil tree variety Tenglong, comprising the primer set and the fluorescent group described above.

[0011] A method for identifying the oil tree variety Tenglong by using a molecular marker primer set, mainly comprising the following steps,

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

[0013] (2) using the genomic DNA as a template, performing PCR amplification by using the 5 pairs of primers respectively to obtain 5 amplification products; and then performing fluorescent capillary electrophoresis detection on the amplification products to obtain the peak values of the amplification products;

[0014] (3) comparing the peak values of the amplification products in step (2) with the corresponding standard peak values, if the comparison is matched, the sample to be tested is the oil tree variety Tenglong; and if the comparison is not matched, the sample to be tested is not the oil tree variety Tenglong.

[0015] The composition and content of the PCR reaction system used in the application are as follows: 1 μL of genomic DNA, 7.5 μL of 2x 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 program in the application is as follows: 96℃ pre-denaturation for 3 min; 30 cycles; 72℃ extension for 10 min, and 4℃ storage; 30 cycles including 96℃ denaturation for 30 s, 62℃-52℃ gradient annealing for 30 s, and 72℃ extension for 1 min.

[0017] The primer set, the rapid detection kit or the molecular marker method in the application is used in identifying the oil tree variety Tenglong.

[0018] The application has the following beneficial effects:

[0019] (1) The present application adopts 5 pairs of specific primers to identify the oil tree variety Tenglong, and whether it is the oil tree variety Tenglong is judged by comparing the peak value of the amplification product of the test variety with the corresponding standard peak value, the operation method is simple, the detection efficiency is high, the result is reliable, and it is conducive to the protection and popularization and application of the variety.

[0020] (2) When it is difficult to distinguish the variety by simply observing the leaves, flowers and tree trunks, etc., the present application can be used to identify whether it is the oil tree variety Tenglong before hybridization breeding, which can help to determine the parents in the hybridization breeding process.

[0021] (3) Different oil trees have great differences in quality, but due to the similar appearance of oil tree fruits, there are cases of selling low-value oil trees as high-value oil trees on the market, the present application can distinguish the fruit varieties and determine the appropriate price, and maintain the trade order of the oil tree market. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a fruit photo of the oil tree variety Tenglong.

[0023] Figure 2 is a leaf photo of the oil tree variety Tenglong.

[0024] Figure 3 is a standard peak value diagram of the oil tree variety Tenglong under the first pair of primers Pac066.

[0025] Figure 4 is a standard peak value diagram of the oil tree variety Tenglong under the second pair of primers Pac122.

[0026] Figure 5 is a standard peak value diagram of the oil tree variety Tenglong under the third pair of primers Pac131.

[0027] Figure 6 is a standard peak value diagram of the oil tree variety Tenglong under the fourth pair of primers Pac145.

[0028] Figure 7 is a standard peak value diagram of the oil tree variety Tenglong under the fifth pair of primers Pac146. DETAILED DESCRIPTION

[0029] The present application is illustrated by the following examples, but is not limited to the scope of use of the present application.

[0030] Example

[0031] I. Primer set selection

[0032] The application adopts primer groups for identifying oil olive variety Tenglong, and the primer groups include: 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 SEQ ID NO: 1 to SEQ ID NO: 10 in the sequence listing.

[0033] The 5' end of the forward primer is provided with a fluorescent group. The sequence of the primer and the corresponding fluorescent group are specifically shown in Table 1.

[0034] Table 1: Sequence of primer pairs and corresponding fluorescent labels

[0035]

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

[0037] II. Molecular marker method

[0038] 1. The molecular marker method for identifying oil olive variety Tenglong, and the specific operation includes:

[0039] (1) Preparation of the sample to be tested: select fresh oil olive leaves to be tested, wash, dry, grind the sample under liquid nitrogen, grind into powder and store in a-80℃ refrigerator for testing;

[0040] (2) Extracting the genomic DNA of the sample to be tested; in the implementation, the genomic DNA of the oil olive leaves is extracted by using the magnetic bead method genomic DNA extraction kit of Tiangeng Biochemical Technology (Beijing) Co., Ltd., and the specific operation is performed according to the instruction manual of the magnetic bead method genomic DNA extraction kit. 2 μL of the DNA stock solution is added with 2 μL of bromophenol blue for agarose gel electrophoresis to detect the integrity of the DNA.

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

[0042] The 15 μL PCR amplification system includes: (50-200) ng / μL genomic DNA 1 μL, 2x Taq PCR Master Mix 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 procedure 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 amplification. Five PCR amplification products of the samples were obtained.

[0044] (4) Fluorescent capillary electrophoresis detection: the PCR products were subjected to fluorescent capillary electrophoresis detection to obtain the peak values of the amplification products. The sample PCR amplification products diluted to a uniform concentration were added to the on-machine plate, and the on-machine detection reagents were added in a total system of 11.5 μL, including 1 μL of fluorescent PCR product, 0.5 μL of 500LIZ, 10 μL of Hi-Di Formamide, and 9 μL of ROX 400 size marker. After centrifugation, the detection plate was placed on the PCR instrument to run the denaturation program (95 °C, 3 min), and immediately cooled after denaturation. Referring to the ABI 3730xL on-machine operation process, the detection file corresponding to the name of the detection plate was selected, and the SSR sample analysis detection program was run. The results were analyzed using the GeneMarker software to obtain the allele number, peak graph, peak value and genotype of each sample. TM 500LIZ 0.5 μL, Hi-Di TM Formamide 10 μL; the sample and reagents to be detected were added to the detection plate, centrifuged, and placed on the PCR instrument to run the denaturation program (95 °C, 3 min). After denaturation, immediate cooling was performed. Referring to the ABI 3730xL on-machine operation process, the detection file corresponding to the name of the detection plate was selected, and the SSR sample analysis detection program was run. The results were analyzed using the GeneMarker software to obtain the allele number, peak graph, peak value and genotype of each sample.

[0045] (5) Comparing the peak values to identify the sample: the peak values of the five amplification products of the sample to be tested were compared with the corresponding standard peak values, so as to determine whether the sample to be tested was the oil olive variety Tenglong. If the peak values of the five amplification products of the sample to be tested were consistent with the corresponding standard peak values, then the sample to be tested was the oil olive variety Tenglong. If the peak values of the five amplification products of the sample to be tested were inconsistent with at least one of the corresponding standard peak values, then the sample to be tested was not the oil olive variety Tenglong.

[0046] III. Identification and verification

[0047] The 79 oil palm germplasms include GL1, autumn red, 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, Guikengda 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, Guikengda 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 to-be-tested samples are derived from the oil palm germplasm resource garden of Guangxi South Subtropical Agricultural Science Institute.

[0049] Firstly, the leaf genomic DNA of the oil palm is extracted by using the magnetic bead method genomic DNA extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd., and the specific operation is carried out according to the instruction manual of the magnetic bead method genomic DNA extraction kit. Then, the PCR amplification is carried out according to the above-mentioned molecular marker method, and the electrophoresis detection is carried out, so as to obtain the amplification products of the 79 to-be-tested samples.

[0050] Comparative identification

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

[0052] The peak values of the amplification products of the 79 germplasms are compared with the corresponding standard peak values, so as to judge whether the to-be-tested sample is the oil palm variety Tenglong. The specific results are shown in Table 2.

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

[0054]

[0055]

[0056]

[0057] As can be seen from Table 2, the peak values of the sample to be tested Tenglong are consistent with the 10 standard peak values of the 5 primer pairs, and therefore it is judged to be the oil tree variety Tenglong, which is consistent with the actual result. It is shown that the primer set provided in the application can accurately identify the oil tree variety Tenglong.

Claims

1. A molecular marker primer set for identifying the oil tree variety Tenlong, characterized in that, The primer set comprises five pairs of primers, namely Pac066, Pac122, Pac131, Pac145 and Pac146, and the nucleotide sequences are shown as SEQ ID NO. 1-10.

2. The molecular marker primer set for identifying the oil tree variety Tenlong according to claim 1, characterized in that, The forward primers of the five pairs of primers are all provided with a fluorescent group at the 5' end; wherein 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 olive cultivar Tenraion of claim 1, characterized by, The standard peak values of the olive variety Tenglong are 203 and 206 for the primer Pac066, 174 and 177 for the primer Pac122, 261 and 269 for the primer Pac131, 277 and 283 for the primer Pac145, and 252 and 270 for the primer Pac146.

4. A rapid detection kit for identifying the oil tree variety Tenlong, characterized in that The primer set of claim 1 or 2.

5. A method for identifying the molecular marker of the oil tree variety Tenglong by using the molecular marker primer set of claim 1, characterized in that: The method mainly comprises the following steps, (1) extracting the genomic DNA of the sample to be tested; (2) using the genomic DNA as a template, the five pairs of primers are used for PCR amplification respectively to obtain five amplification products; then the amplification products are detected by fluorescence capillary electrophoresis to obtain the peak values of the amplification products; (3) comparing the peak values of the amplification products of step (2) with the corresponding standard peak values, if the comparison is matched, the sample to be tested is the olive variety Tenglong; if the comparison is not matched, it is not the olive variety Tenglong.

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

7. The method of claim 5, wherein the molecular marker is selected from the group consisting of SEQ ID NOs: 1- 12. The PCR reaction program is as follows: 96℃ pre-denaturation for 3 min; 30 cycles; 72℃ extension for 10 min, 4℃ storage; 30 cycles including 96℃ denaturation for 30 s, 62℃-52℃ gradient annealing for 30 s, 72℃ extension for 1 min.

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

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