Molecular marker primers, kits and applications for the identification of Hevea brasiliensis Yunyan 3117

Abstract

The present invention discloses a molecular marker primer, kit and application for the identification of Hevea brasiliensis Yunyan 3117. The molecular marker primer comprises an upstream primer and a downstream primer. The upstream primer is as shown in SEQ ID NO: 1 in the sequence listing, and the downstream primer is as shown in SEQ ID NO: 2 in the sequence listing. The kit comprises the above-mentioned molecular marker primer. The application includes: using the molecular marker primer for the identification of Hevea brasiliensis varieties. The sequencing results of the markers are used to distinguish varieties. For example, if there are differences, substitutions, insertions, deletions, duplications, etc. of one or more bases between different sequences, Hevea brasiliensis Yunyan 2775, RRIM600, Reyan 106, Rekan 516, Rekan 525, RRIC52, Rekan 523, Rekan 628 and Rekan 524 can be distinguished, and the Hevea brasiliensis varieties can be accurately identified.

Description

Technical Field

[0001] The present disclosure relates to the fields of molecular biology and genetic breeding, and particularly relates to a molecular marker primer, a kit and an application for identifying Hevea brasiliensis Yunyan 3117. Background Art

[0002] Currently, the direct object of variety identification, authorization and breeding of Hevea brasiliensis is traits, and trait testing is still the industry standard for the specific testing of many plant varieties. However, the performance of traits is easily affected by environmental and human factors, and it takes several years to accurately identify. There are phenomena of "same name but different species" and "same species but different names" in Hevea brasiliensis germplasm resources, important breeding materials and varieties popularized and applied in production, which is not conducive to the variety breeding, authorization, rapid promotion of Hevea brasiliensis and the protection of intellectual property rights. Especially the uncertainty of Hevea brasiliensis varieties in actual production will seriously limit the healthy and sustainable development of the rubber industry, and the impurity or even error of Hevea brasiliensis seedling varieties will seriously affect the economic interests of rubber farmers. Summary of the Invention

[0003] In order to solve the problem of inaccurate identification of Hevea brasiliensis varieties, the embodiments of the present disclosure provide a molecular marker primer, a kit and an application for identifying Hevea brasiliensis Yunyan 3117. The technical solutions are as follows:

[0004] On the one hand, the present disclosure provides a molecular marker primer for identifying Hevea brasiliensis Yunyan 3117. The molecular marker primer includes an upstream primer and a downstream primer. The upstream primer is as shown in SEQ ID NO: 1 in the sequence listing, and the downstream primer is as shown in SEQ ID NO: 2 in the sequence listing.

[0005] On the other hand, the present disclosure provides a kit for identifying Hevea brasiliensis Yunyan 3117. The kit includes the above-mentioned molecular marker primer.

[0006] On the other hand, the present disclosure provides an application of the molecular marker primer for identifying Hevea brasiliensis Yunyan 3117. The application includes: using the molecular marker primer to identify or distinguish Hevea brasiliensis varieties, and the Hevea brasiliensis varieties are: Yunyan 2775, Reyan 106, Rekan 516, Rekan 525, RRIC52, Rekan 523, Rekan 628 and Rekan 524. The molecular marker primer consists of an upstream primer and a downstream primer. The upstream primer is as shown in SEQ ID NO: 1 in the sequence listing, and the downstream primer is as shown in SEQ ID NO: 2 in the sequence listing.

[0007] The beneficial effects brought by the technical solutions provided in the embodiments of the present disclosure are as follows: The embodiments of the present invention provide molecular marker primers, kits and applications for the identification of Hevea brasiliensis Yunyan 3117. These molecular marker primers distinguish varieties based on the sequencing results of amplification products. For example, if there are differences, substitutions, insertions, deletions, duplications, etc. of one or more bases between different sequences, Hevea brasiliensis Yunyan 2775, Reyan 106, Rekan 516, Rekan 525, RRIC52, Rekan 523, Rekan 628 and Rekan 524 can be distinguished and identified, and the identification results are not affected by the environment and other human factors, thus accurately identifying the varieties of Hevea brasiliensis. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present disclosure. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0009] Figure 1 It is a sequence comparison diagram named a - h provided in Embodiment III of the present disclosure. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0010] To make the objectives, technical solutions and advantages of the present disclosure clearer, the following will further describe the embodiments of the present disclosure in detail.

[0011] Embodiment I

[0012] The present disclosure provides molecular marker primers for the identification of Hevea brasiliensis Yunyan 3117. The primers include an upstream primer and a downstream primer. The upstream primer is as shown in SEQ ID NO: 1 in the sequence listing, and the downstream primer is as shown in SEQ ID NO: 2 in the sequence listing. Specifically, the sequence of the upstream primer is: TTCTTACCCTTAAAGTC GATAGCCC, and the sequence of the downstream primer is: TCCCTTAGCTAATGGATGAGAGGTA.

[0013] Embodiment II

[0014] The present disclosure provides a kit for the identification of Hevea brasiliensis Yunyan 3117, and the kit includes the molecular marker primers provided in Embodiment I.

[0015] Embodiment III

[0016] The present disclosure provides an application of the molecular marker primers for the identification of Hevea brasiliensis Yunyan 3117, and the application includes: using the molecular marker primers provided in Embodiment I to identify the varieties of Hevea brasiliensis.

[0017] Further, the application further includes: This molecular marker primer can distinguish Hevea brasiliensis Yunyan 2775, Reyan 106, Rekeng 516, Rekeng 525, RRIC52, Rekeng 523, Rekeng 628, and Rekeng 524.

[0018] Specifically, 42 Hevea brasiliensis varieties were used as test samples, and the variety names are shown in Table 1.

[0019] Table 1 shows the variety names of the test samples

[0020]

[0021]

[0022] Extract the DNA of the above 42 test samples. In implementation, a new plant genomic DNA extraction kit produced by Tiangen Biochemical Technology (Beijing) Co., Ltd. was used to extract the DNA of the test samples. The specific operation was carried out according to the instructions of this new plant genomic DNA extraction kit.

[0023] Use the marker primer provided in Example 1 of the present invention to amplify 42 test samples. Specifically, the amplification system includes: 50 ng DNA fragment of the test sample, 0.5 μL of 10 mM dNTP (deoxy-ribonucleoside triphosphate), 0.5 μL of the upstream primer, 0.5 μL of the downstream primer, 2.5 μL of Tap Buffer, 0.2 μL of Taq enzyme, and ddH2O was added to make up the amplification system to 20 μL. The amplification program includes: 95°C for 3 min; (95°C for 30 sec, 60°C for 30 sec) × 30 cycles; 72°C for 6 min. The amplified product was subjected to second-generation high-throughput sequencing, and the sequencing data was aligned to the Hevea brasiliensis reference genome (NCBI: PRJNA587314) using the Bowtie2 software to obtain the sequencing results of each test sample.

[0024] Use Microsoft Office Excel software to analyze the sequencing results, and there are 8 different sequence types in total. The 8 different sequence types were respectively named a, b, c, d, e, f, g, and h, as shown in Table 2.

[0025] Table 2 shows the different sequences obtained by sequencing after the molecular marker primer amplified 42 test samples

[0026]

[0027]

[0028] The software DNASTAR lasergene 11 was used to align 8 different sequence types, and the alignment results are as Figure 1 shown. Among Figure 1 them, there are 20 base sites with differences among the 8 different sequences. Specifically, the sequence differences include: at the 5th base position A / T, at the 8th base position T / A, at the 15th base position G / A, at the 22nd base position C / T, at the 35th base position G / A, at the 49th base position T / A, at the 51st base position C / T, at the 63rd base position G / A, at the 79th base position C / T, at the 82nd base position C / G, at the 84th base position A / G, at the 90th base position G / A, at the 93rd base position C / T, at the 95th base position G / A, at the 96th base position C / T, at the 98th base position G / A, at the 99th base position C / T, at the 117th base position C / T, at the 122nd base position A / G, and at the 129th base position C / T.

[0029] Among the 42 test samples provided in this embodiment, the primer pairs provided in this embodiment can effectively distinguish 11 test samples. The different sequence combinations of the 11 test samples are shown in Table 3.

[0030] Table 3 shows the different sequence combinations of the 11 distinguished test samples

[0031] Serial number Name of the sample to be tested Sequence combination 1 Yunyan 2775 ab 2 Zhan Shi 32113 ac 3 Yunyan 3089 acd 4 Reyan 106 bc 5 Reken 516 bd 6 Reken 525 be 7 RRIC 52 bg 8 Yunyan 3117 dg 9 Reken 523 ef 10 Reken 628 eg 11 Reken 524 g

[0032] As can be seen from Table 3, the above 11 test samples have different sequence combination ways. It can be seen that the primers provided in Embodiment 1 of the present invention can amplify the sequence of Yunyan 3117, and the sequence combination way is dg. Also, according to the different sequence combinations, the distinction of Hevea brasiliensis Yunyan 2775, Zhanshi 32113, Yunyan 3089, Reyan 106, Rekeng 516, Rekeng 525, RRIC52, Rekeng 523, Rekeng 628, and Rekeng 524 can be achieved.

[0033] Accuracy analysis of molecular marker primers for Hevea brasiliensis

[0034] Three reproducibility experiments were used for accuracy analysis. In this embodiment, three independent experiments were conducted by different personnel, reagents from different batches, and different laboratories, so as to simulate the identification of Hevea brasiliensis in different batches. A high reproducibility rate means that the identification results of different laboratories can be accurately compared with each other.

[0035] A reproducibility experiment was conducted on 11 rubber tree samples to be tested. The 11 samples to be tested were specifically: Yunyan 3117, Yunyan 2775, Zhanshi 32113, Yunyan 3089, Reyan 106, Rekeng 516, Rekeng 525, RRIC52, Rekeng 523, Rekeng 628, and Rekeng 524. Each sample to be tested was subjected to three repeated experiments, and the sequence combinations of each result were recorded. The specific results are shown in Table 4.

[0036] Table 4 shows the results of the three repeated experiments

[0037]

[0038] As can be seen from Table 4, the sequence combinations of the three repeated experiments are the same. Thus, it can be seen that the accuracy rate of the molecular marker primer identification provided in the embodiments of the present invention is 100%. It can be seen that the variety identification conclusions among different laboratories or different batches in the same laboratory in the embodiments of the present invention have high consistency, so there is no need to adopt parallel experiments to reduce experimental errors, which provides great convenience for the variety identification of rubber trees.

[0039] The embodiments of the present invention provide molecular marker primers, kits and applications for the identification of Yunyan 3117 rubber trees. The sequencing results of the markers are used to distinguish varieties. If there are differences, substitutions, insertions, deletions, duplications, etc. of one or more bases between different sequences, Yunyan 2775, Reyan 106, Rekeng 516, Rekeng 525, RRIC52, Rekeng 523, Rekeng 628, and Rekeng 524 rubber trees can be distinguished and identified, and the identification results are not affected by the environment and other human factors, and thus the rubber tree varieties can be accurately identified.

[0040] The above are only optional embodiments of the present disclosure, and are not intended to limit the present disclosure. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present disclosure shall be included within the protection scope of the present disclosure.

Claims

1. Application of molecular marker primers for rubber tree variety identification, characterized in that, The application is to use molecular marker primers for identifying or differentiating rubber tree varieties, and the rubber tree varieties are: Yunyan 2775, Reyanshu 106, Rekeng 516, Rekeng 525, RRIC52, Rekeng 523, Rekeng 628 and Rekeng 524. The molecular marker primers consist of an upstream primer and a downstream primer. The upstream primer is as shown in SEQ ID NO: 1 in the sequence listing, and the downstream primer is as shown in SEQ ID NO: 2 in the sequence listing.

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