Molecular marker primers, kit and application for identifying rubber tree Yunyan 7346

Abstract

The invention discloses a molecular marker primer, a kit and an application for identifying a rubber tree Yunyan 7346. The molecular marker primer comprises an upstream primer and a downstream primer, wherein the upstream primer is shown in SEQ ID NO: 1 in the sequence table, and the downstream primer is shown in SEQ ID NO: 2 in the sequence table. The kit comprises the above molecular marker primer. The application comprises: using the molecular marker primer to identify the rubber tree Yunyan 7346. The sequencing results of the marker are used to distinguish the varieties, such as the difference of one or more bases between different sequences, or substitution, insertion, deletion, or duplication, etc., so as to realize the identification of Yunyan 7346, and the rubber trees Yunyan 3117, GT1, Yunyan 772, Reken 524, Yunyan 3089, Reken 502, Reyan 73397 and Reyan 917 can be distinguished, and the rubber tree varieties can be accurately identified.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology and genetic breeding, and in particular to a molecular marker primer, a kit and an application for identifying rubber tree Yunyan 7346. Background Art

[0002] Rubber tree is a typical perennial tropical rainforest tree species, native to Brazil, Venezuela and Guyana in the Amazon River Basin of South America. It was later introduced to my country and is now mainly planted in Hainan, Yunnan and Guangdong. Natural rubber is made from the latex that flows out when rubber trees are tapped, which is solidified and dried. In recent years, my country's demand for natural rubber has continued to grow, and the gap between supply and demand has continued to widen.

[0003] Natural rubber has unique and excellent properties in the fields of weapons and equipment, aircraft tires, high-elastic shock absorption and high-altitude detection. The natural rubber tree industry has broad development prospects in the future.

[0004] At present, the direct object of rubber tree variety identification, authorization and breeding is traits, and trait testing is still the industry standard for many plant variety-specific tests. However, the expression of traits is easily affected by environmental and human factors, and it takes several years to accurately identify them. Rubber tree germplasm resources, important breeding materials, and varieties promoted and applied in production have the phenomenon of "same name different things" and "same things different names", which is not conducive to rubber tree variety breeding, authorization, rapid promotion and intellectual property protection. In particular, the uncertainty of rubber tree varieties in actual production will seriously restrict the healthy and sustainable development of the rubber industry. Impure or even wrong varieties of rubber tree seedlings will seriously affect the economic interests of rubber farmers.

[0005] Public Content

[0006] In order to solve the problem of inaccurate identification of rubber tree Yunyan 7346 variety, the present disclosure provides a molecular marker primer, a kit and an application for identifying rubber tree Yunyan 7346. The technical solution is as follows:

[0007] On the one hand, the present disclosure provides molecular marker primers for identifying rubber tree Yunyan 7346, the molecular marker primers comprising an upstream primer and a downstream primer, the upstream primer is shown as SEQ ID NO: 1 in the sequence listing, and the downstream primer is shown as SEQ ID NO: 2 in the sequence listing.

[0008] On the other hand, the present disclosure provides a kit for identifying the rubber tree Yunyan 7346, which includes the above-mentioned molecular marker primers.

[0009] On the other hand, the present disclosure provides an application of molecular marker primers for identifying the rubber tree Yunyan 7346 variety, the application comprising: using the molecular marker primers to identify the rubber tree Yunyan 7346, the molecular marker primers comprising an upstream primer and a downstream primer, the upstream primer being shown as SEQ ID NO: 1 in the sequence listing, and the downstream primer being shown as SEQ ID NO: 2 in the sequence listing.

[0010] Furthermore, the application also includes: the molecular marker primers can distinguish rubber trees Yunyan 3117, GT1, Yunyan 772, Reken 524, Yunyan 3089, Reken 502, Reyan 73397 and Reyan 917.

[0011] The technical solution provided by the embodiments of the present disclosure has the following beneficial effects: the embodiments of the present invention provide molecular marker primers, kits and applications for identifying rubber tree Yunyan 7346. The molecular marker primers distinguish varieties through sequencing results of amplified products, such as the presence of one or more base differences, or substitutions, or insertions, or deletions, or duplications, etc. between different sequences, thereby achieving the identification of rubber tree Yunyan 7346, and can distinguish rubber trees Yunyan 3117, GT1, Yunyan 772, Reken 524, Yunyan 3089, Reken 502, Reyan 73397 and Reyan 917, and the identification results are not affected by the environment and other human factors, thereby accurately identifying rubber tree varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0013] Figure 1 It is a sequence comparison diagram named a to f provided in Example 3 of the present disclosure. DETAILED DESCRIPTION

[0014] In order to make the objectives, technical solutions and advantages of the present disclosure more clear, the embodiments of the present disclosure will be further described in detail below with reference to the accompanying drawings.

[0015] Embodiment 1

[0016] The present disclosure provides molecular marker primers for identifying rubber tree Yunyan 7346, which include an upstream primer and a downstream primer, the upstream primer is shown in SEQ ID NO: 1 in the sequence list, and the downstream primer is shown in SEQ ID NO: 2 in the sequence list. Specifically, the sequence of the upstream primer is: TCTGAGGTCTTCATCAT TCATTGCT, and the sequence of the downstream primer is: AGGTGATGTTCGGTAGTTCATTAGG.

[0017] Embodiment 2

[0018] The present disclosure provides a kit for identifying the rubber tree Yunyan 7346, and the kit includes the molecular marker primers provided in Example 1.

[0019] Embodiment 3

[0020] The present disclosure provides an application of molecular marker primers for identifying the rubber tree Yunyan 7346, and the application includes: using the molecular marker primers provided in Example 1 to identify the rubber tree Yunyan 7346.

[0021] Furthermore, the application also includes: this molecular marker primer can distinguish rubber trees Yunyan 3117, GT1, Yunyan 772, Reken 524, Yunyan 3089, Reken 502, Reyan 73397 and Reyan 917.

[0022] Specifically, 42 rubber tree varieties were selected as samples to be tested, and the variety names are shown in Table 1.

[0023] Table 1 shows the names of the samples to be tested.

[0024]

[0025]

[0026] Extract the DNA of the 42 samples to be tested. In practice, a new plant genomic DNA extraction kit produced by Tiangen Biochemical Technology (Beijing) Co., Ltd. was used to extract the DNA of the samples to be tested. The specific operation was carried out according to the instructions of the new plant genomic DNA extraction kit.

[0027] 42 samples to be tested were amplified using the labeled primers provided in Example 1 of the present invention. Specifically, the amplification system includes: 50 ng of DNA fragments of the sample to be tested, 0.5 μL of 10 mM dNTP (deoxy / ribonucleoside triphosphate), 0.5 μL of upstream primer, 0.5 μL of downstream primer, 2.5 μL of Tap Buffer, 0.2 μL of Taq enzyme, and ddH 2O The amplification system was supplemented to 20 μL, and the amplification program included: 95°C for 3 min; (95°C for 30 sec, 60°C for 30 sec) × 30 cycles; 72°C for 6 min. The amplified products were sequenced by second-generation high-throughput sequencing, and the sequencing data were aligned to the rubber tree reference genome (NCBI: PRJNA587314) using Bowtie2 software to obtain the sequencing results of each sample to be tested.

[0028] The sequencing results were analyzed using Microsoft Office Excel software, and there were 6 different sequence types, which were named a, b, c, d, e and f, as shown in Table 2.

[0029] Table 2 shows the different sequences obtained by sequencing after amplification of 42 samples by molecular marker primers

[0030]

[0031]

[0032] The software DNASTAR lasergene 11 was used to align the six different sequence types. The alignment results are shown in the figure. Figure 1 As shown, in Figure 1 There are differences in 6 base positions among the 6 different sequences, and the specific sequence differences include: G / T at the 7th base position, G / A at the 26th base position, C / T at the 54th base position, C / T at the 105th base position, A / G at the 136th base position, and C / A at the 144th base position.

[0033] Among the 42 samples to be tested provided in this embodiment, 9 samples to be tested can be effectively distinguished using the primer pairs provided in this embodiment. The different sequence combinations of the 9 samples to be tested are shown in Table 3.

[0034] Table 3 shows the different sequence combinations of the 9 samples to be tested.

[0035] Serial number Name of the sample to be tested Sequence combination 1 Cloud Research 3117 a 2 GT1 ac 3 Cloud Research 772 acf 4 Yunyan7346 ae 5 Reken 524 c 6 Yunyan 3089 cef 7 Reken 502 cf 8 Reyan 73397 de 9 Hot Research 917 ed

[0036] It can be seen from Table 3 that the above 9 samples to be tested have different sequence combinations. It can be seen that the primers provided in Example 1 of the present invention can amplify the sequence of Yunyan 7346, and the sequence combination is ae. According to different sequence combinations, Yunyan 3117, GT1, Yunyan 772, Reken 524, Yunyan 3089, Reken 502, Reyan 73397 and Reyan 917 can also be distinguished.

[0037] Analysis on the accuracy of molecular marker primer identification in rubber tree

[0038] Three reproducibility experiments were used to perform accuracy analysis. In this embodiment, three independent experiments were performed using different personnel, different batches of reagents, and different laboratories, thereby simulating the identification of different batches of rubber trees. A high reproducibility means that the identification results of different laboratories can be accurately compared with each other.

[0039] Reproducibility experiments were conducted on 9 rubber tree samples: Yunyan 7346, Yunyan 3117, GT1, Yunyan 772, Reken 524, Yunyan 3089, Reken 502, Reyan 73397 and Reyan 917. Each sample was repeated three times and the sequence combination of each result was recorded. The specific results are shown in Table 4.

[0040] Table 4 shows the results of three repeated experiments.

[0041]

[0042] As shown in Table 4, the sequence combinations of 3 repeated experiments are all the same. It can be seen from this that the accuracy of the molecular marker primer identification provided by the embodiment of the present invention is 100%. As can be seen, the embodiment of the present invention adopts the variety identification conclusion between different batches of different laboratories or the same laboratory to have higher consistency, thereby there is no need to adopt parallel experiments to reduce experimental error, which provides great convenience for rubber tree variety identification.

[0043] The embodiment of the present invention provides molecular marker primers, a kit and an application for identifying rubber tree Yunyan 7346, and utilizes the sequencing results of the markers to distinguish varieties, such as the presence of one or more base differences, or substitutions, or insertions, or deletions, or duplications, etc. between different sequences, thereby achieving the identification of the Yunyan 7346 variety. The rubber tree Yunyan 7346 is an important variety in Yunnan Province. At the same time, the rubber trees Yunyan 3117, GT1, Yunyan 772, Reken 524, Yunyan 3089, Reken 502, Reyan 73397 and Reyan 917 can be distinguished, and the identification results are not affected by the environment and other human factors, thereby accurately identifying the rubber tree varieties.

[0044] 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 molecular marker primer for identifying rubber tree Yunyan 7346, It is characterized in that The molecular marker primers include an upstream primer and a downstream primer, the upstream primer is shown as SEQ ID NO: 1 in the sequence list, and the downstream primer is shown as SEQ ID NO: 2 in the sequence list.

2. A kit for identifying rubber tree Yunyan 7346, It is characterized in that The kit comprises the molecular marker primer as claimed in claim 1.

3. Application of molecular marker primers for identification of rubber tree Yunyan 7346, It is characterized in that The application includes: using molecular marker primers to identify rubber tree Yunyan 7346, the molecular marker primers include an upstream primer and a downstream primer, the upstream primer is shown as SEQ ID NO: 1 in the sequence list, and the downstream primer is shown as SEQ ID NO: 2 in the sequence list.

4. The use according to claim 3, It is characterized in that The application also includes: the molecular marker primers can also distinguish: rubber trees Yunyan 3117, GT1, Yunyan 772, Reken 524, Yunyan 3089, Reken 502, Reyan 73397 and Reyan 917.

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