Molecular markers, primers, kits and applications for identifying the rubber tree variety 'Yunyan 614'

Abstract

The present disclosure relates to the technical field of variety cultivation and identification in molecular biology, specifically a molecular marker, primer, kit and application for identifying the rubber tree variety 'Yunyan 614', wherein the molecular marker is located at bases 11867535 to 11867651 of chromosome 1, and the nucleotide sequence is shown in SEQ ID No.5. The present invention provides a molecular marker primer and kit for the rubber tree variety 'Yunyan 614', which can specifically identify and amplify the DNA sequence of the 'Yunyan 614' variety, realize accurate identification thereof, help improve the accuracy of variety identification, optimize variety selection and regional suitability assessment, thereby providing a scientific basis for the planting and breeding of rubber trees, and promoting the healthy development of the rubber industry. The molecular marker, primer pair or kit of the present invention can be used for the distinction and identification of the rubber tree varieties 'Yunyan 614' and 'IAN873', is easy to operate, and is suitable for large-scale application.

Description

Technical Field

[0001] The present invention relates to the technical field of variety cultivation and identification in molecular biology, and in particular to a molecular marker, primer, kit and application for identifying the rubber tree variety 'Yunyan 614'. Background Art

[0002] As an important source of natural rubber in the world, the planting and production of rubber trees (Hevea brasiliensis) have a significant impact on economic and industrial development. Yunnan Province ranks first in China in terms of rubber tree planting area, yield per unit area and total output. The selection and planting of rubber tree varieties with strong adaptability are of irreplaceable importance in promoting the sustainable development of my country's rubber industry. In particular, the cultivation and planting strategies of varieties with key traits such as high yield, cold resistance and powdery mildew resistance are particularly critical.

[0003] The rubber tree variety 'Yunyan 614' is a new variety of rubber tree independently cultivated by the Yunnan Tropical Crops Science Research Institute. It has applied for a new plant variety right in October 2022, and its application number is: 20221005624. This variety has the excellent traits of fast growth, cold resistance, and drought resistance. After being planted in Jinghong for 8 years, the average stem circumference is more than 62cm at 1m above the ground. The fast growth and cold resistance of 'Yunyan 614' have been verified by trial planting in Dehong, Lincang, Xishuangbanna, Honghe and other places, and good yield levels have been obtained. It is suitable for planting in severe cold areas and moderate cold areas, and performs best in an environment with sufficient water and fertilizer conditions and a warm and cool climate.

[0004] The rubber tree varieties currently being promoted for planting have a narrow genetic basis, and the phenotypic traits between varieties are highly similar. Traditional rubber tree variety identification methods mainly rely on morphological characteristics and physiological and biochemical indicators. These phenotype-based identification methods are easily affected by environmental factors, resulting in the instability of identification results. The phenotype of rubber tree 'Yunyan 614' is very similar to other rubber tree varieties, and it is difficult to distinguish them from the appearance, and identification errors occur from time to time, thereby causing the phenomenon of variety mixing. The patent with the authorization publication number CN 118064428 B discloses a MNP molecular marker for constructing a DNA fingerprint of rubber tree, which can utilize 600 pairs of MNP primers to amplify the DNA fingerprint of rubber tree varieties, but the identification markers for a single variety are not clearly pointed out, and the method needs to be subjected to high-throughput sequencing to analyze the differences of each variety, and the cost is relatively high.

[0005] Therefore, a new method for identifying individual rubber tree varieties with a short development cycle, low cost, and simple operation is of great significance for solving the mixing of rubber tree varieties, increasing rubber tree yields, and ensuring the sustainable development of natural rubber. Summary of the invention

[0006] The purpose of the present invention is to provide a molecular marker, primer, kit and application for identifying the rubber tree variety 'Yunyan 614'.

[0007] To achieve the above object, the technical solution adopted by the present invention is: a molecular marker for identifying the rubber tree variety 'Yunyan 614', the physical position of the molecular marker is located at bases 11867535 to 11867651 of chromosome CM021236_1; the molecular marker includes the following four base polymorphisms, specifically:

[0008] The 11867552nd base is C or A;

[0009] The base at position 11867562 is T or C;

[0010] The 11867604th base is A or missing (denoted as "-");

[0011] The base at position 11867626 is T or C.

[0012] Furthermore, the nucleotide sequence of the molecular marker is shown as SEQ ID No.5.

[0013] The second object of the present invention is to provide a primer pair for amplifying molecular markers of the rubber tree variety 'Yunyan 614', wherein the primer pair sequence is shown in SEQ ID No. 2-3.

[0014] The third object of the present invention is to provide a kit for identifying the rubber tree variety 'Yunyan 614', the kit comprising a primer pair for amplifying molecular markers of the rubber tree variety 'Yunyan 614'.

[0015] The fourth object of the present invention is a method for identifying the rubber tree variety 'Yunyan 614', the steps comprising:

[0016] (1) Use a plant total DNA extraction kit to extract the total DNA of all rubber tree samples to be tested;

[0017] (2) using the extracted genomic DNA as a template, performing PCR amplification using the primer pairs shown in SEQ ID No. 2 to 3 respectively;

[0018] (3) The amplified products were sequenced by Sanger sequencing to obtain sequencing data and determine the genotypes of the four loci in each individual. The genotypes corresponding to the four loci were AA, CC, -, and CC. If the sequence was as shown in SEQ ID No. 5, it was the 'Yunyan 614' variety; otherwise, it indicated that the rubber tree sample to be tested was not the 'Yunyan 614' variety.

[0019] Furthermore, the PCR amplification system is 20 μL, including: 0.5 μL of 50 ng / μL DNA template, 2 μL of 10×PCR buffer, 25 mM MgCl 2 2μL, 10mM dNTPs 0.5μL, 5U / μL Taq DNA polymerase 0.2μL, 10μM upstream primer 0.5μL, 10μM downstream primer 0.5μL and ddH 2 O 13.8 μL;

[0020] The reaction procedure was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, 30 cycles; and final extension at 72°C for 7 min.

[0021] The molecular marker, primer pair or kit provided by the invention can be used for distinguishing and identifying rubber tree varieties: Yunyan 614 and IAN873.

[0022] The beneficial technical effect of the present invention is: the present invention provides a molecular marker primer and a kit for the rubber tree variety 'Yunyan 614', the technology can specifically identify and amplify the DNA sequence of the 'Yunyan 614' variety, realize accurate identification thereof, help improve the accuracy of variety identification, optimize variety selection and regional suitability assessment, thereby providing a scientific basis for the planting and breeding of rubber trees, and promoting the healthy development of the rubber industry. The molecular marker, primer pair or kit of the present invention can be used for the distinction and identification of the rubber tree varieties 'Yunyan 614' and 'IAN873', is easy to operate, and is suitable for large-scale application. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0024] Figure 1 These are three types of sequence feature graphs in Example 1 of the present invention. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example 1

[0027] This embodiment is a method for screening and identifying molecular markers specific to rubber tree 'Yunyan 614', comprising the following steps:

[0028] (1) The Wei Ke Han germplasm resources planted and preserved in the Jinghong Rubber Tree Germplasm Resource Nursery of the Ministry of Agriculture and Rural Affairs include 123 Yunyan series germplasms including 'Yunyan 614' and 24 domestic and foreign varieties; fresh leaves free of pests and diseases from 147 germplasms were collected, quickly frozen in liquid nitrogen, and stored at -80℃ for future use.

[0029] (2) The whole genome DNA of the 147 samples in step (1) was extracted using a plant total DNA extraction kit, and the extracted whole genome DNA samples were then sent to Beijing Biomarker Biotechnology Co., Ltd. for resequencing.

[0030] (3) Perform quality control on the raw resequencing data to obtain clean data for each sample.

[0031] (4) The clean data was aligned to the rubber tree reference genome (the reference genome version was GCA_010458925.1) using the MEM algorithm of the BWA software to obtain an intermediate result file in the sam format; the sam file was converted into a bam file using the samtools software and the results were merged, duplicate sequences were removed, and sorting was performed.

[0032] (5) Use GATK software to detect and type polymorphic sites in the bam file and generate a vcf file; use vcftools software to perform quality control on the sites in the vcf file to obtain high-quality polymorphic sites that meet the screening criteria. The quality control conditions are: minor allele frequency MAF>0.05; P-value in the Hardy-Weinberg test>0.01; site missing rate<0.1; polymorphism information content PIC>0.2; population site heterozygosity rate<0.2. After quality control, the core polymorphic sites were obtained.

[0033] (6) The vcftools software was used to further screen specific molecular markers for the rubber tree variety 'Yunyan 614' based on the genotypic differences of the core polymorphic sites in different rubber tree germplasms. The screening criteria were: the homozygous mutation rate of the polymorphic sites in the rubber tree variety 'Yunyan 614' was 1, and the homozygous mutation rate of the polymorphic sites in other rubber tree germplasms was <0.1.

[0034] After screening, a molecular marker with significant difference in 'Yunyan 614' variety was obtained, and the molecular marker had polymorphisms at 4 base positions.

[0035] (7) All sample sequences were aligned using BioEdit software, and three different sequence types were obtained (e.g. Figure 1 The three different sequence types were named a (SEQ ID No. 1), b (SEQ ID No. 4), and c (SEQ ID No. 5), and the nucleotide sequences of the three sequence types are shown in Table 1.

[0036] Table 1 Different sequences obtained after amplification and sequencing of 147 rubber tree germplasm resources

[0037]

[0038] (8) The genotype of the 'Yunyan 614' variety was analyzed. The genotypes corresponding to the four loci were AA, CC, -, and CC. Among all the samples to be tested, only 'Yunyan 614' had this homozygous genotype, the sequence type was c, and the nucleotide sequence was as shown in SEQ ID No. 5.

[0039] Example 2

[0040] This example is a reliability verification of identifying the rubber tree variety 'Yunyan 614' using the screened specific molecular markers.

[0041] Specific primers were designed for the screened specific molecular marker (SEQ ID No. 5), and the designed sequences are as follows:

[0042] F: 5'- TGTTGCTTCAAAGTCTCTCTTCAAC-3' (SEQ ID No. 2)

[0043] R: 5'-AGGTGTAGAGTTTTGAATCATAACATCA-3' (SEQ ID No. 3).

[0044] 28 Wei Ke Han germplasms of rubber tree were selected, and the developed specific molecular markers were genotyped by Sanger sequencing; specifically,

[0045] (1) Extract the whole genome DNA of the sample to be tested;

[0046] (2) using the DNA in step (1) as a template and the above nucleotide sequence as a primer, PCR amplification is performed on the molecular marker site to obtain a PCR amplification product;

[0047] (3) The PCR amplification product obtained in step (2) was subjected to Sanger sequencing to determine the genotype of the molecular marker site, as shown in Table 2:

[0048] Table 2 Genotyping of 28 rubber tree germplasm after amplification

[0049]

[0050] The verification result is consistent with the genotype analysis result obtained in Example 1, indicating that the developed molecular marker can effectively identify the 'Yunyan 614' variety in rubber tree germplasm.

[0051] Example 3

[0052] A molecular marker, primer, and kit for identifying the rubber tree variety 'Yunyan 614' and its application in variety identification, comprising the following steps:

[0053] (1) Fifty rubber tree germplasms were randomly selected (the sample names are shown in Table 3), and the whole genome DNA of all samples was extracted using a plant total DNA extraction kit.

[0054] Table 3 Germplasm names of the 50 samples to be tested

[0055]

[0056] (2) Using the extracted genomic DNA as a template, PCR amplification was performed using the primer pairs described in SEQ ID No. 2 to 3. The amplification system is shown in Table 4.

[0057] Table 4 PCR extension system

[0058]

[0059] The reaction procedure was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, 30 cycles; and final extension at 72°C for 7 min.

[0060] (3) The PCR amplification products were sequenced by Sanger sequencing to determine the genotypes of the four loci of each sample.

[0061] (4) When the genotyping results of the sample are AA, CC, -, CC at the four base positions, the sample for testing is determined to be the 'Yunyan 614' variety.

[0062] (5) Among the 50 samples to be tested provided in this embodiment, the primer pairs provided in this embodiment can effectively distinguish two samples to be tested. The different sequence combinations of the two samples to be tested are shown in Table 5.

[0063] Table 5 Sequence combinations of two rubber tree varieties distinguished by this primer combination

[0064]

[0065] As shown in Table 5, the two samples to be tested have different sequences. It can be seen that the primers provided in Example 1 of the present invention can amplify the sequence of 'Yunyan 614', and the sequence type is c (SEQ ID No. 5). IAN873 can also be distinguished according to the different sequence types.

[0066] Example 4

[0067] Analysis on the accuracy of molecular marker primer identification in rubber tree

[0068] 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.

[0069] Reproducibility experiments were conducted on two rubber tree samples: Yunyan 614 and IAN873. Each sample was repeated three times, and the sequence combination of each result was recorded. The specific results are shown in Table 6.

[0070] Table 6 Results of three repeated experiments

[0071]

[0072] Table 6 further verifies that the rubber tree molecular markers and amplification primers of the present invention can distinguish and identify 'Yunyan 614' and IAN873.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. Identification of molecular markers for the rubber tree variety 'Yunyan 614', characterized in that: The physical position of the molecular marker is located at bases 11867535 to 11867651 of chromosome CM021236_1, and the nucleotide sequence of the molecular marker is shown in SEQ ID No.

5.

2. A method for identifying the rubber tree variety 'Yunyan 614', characterized in that the steps include: (1) Use a plant total DNA extraction kit to extract the total DNA of all rubber tree samples to be tested; (2) using the extracted genomic DNA as a template, and performing PCR amplification using the primer pairs shown in SEQ ID No. 2-3; (3) The amplified products were sequenced by Sanger sequencing to obtain sequencing data and determine the genotypes of the four loci in each individual. The genotypes corresponding to the four loci were AA, CC, -, and CC. When SEQ ID No. 5 was present in the amplified product, it was identified as the 'Yunyan 614' variety; otherwise, it indicated that the rubber tree sample to be tested was not the 'Yunyan 614' variety.

3. The identification method according to claim 2, characterized in that: The amplification system of the PCR amplification is 20 μL, including: 0.5 μL of 50 ng / μL DNA template, 2 μL of 10×PCR buffer, 2 μL of 25 mM MgCl2, 0.5 μL of 10 mM dNTPs, 0.2 μL of 5 U / μL Taq DNA polymerase, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer and 13.8 μL of ddH2O; The reaction procedure was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, 30 cycles; and final extension at 72°C for 7 min.

4. Application of the molecular marker according to claim 1 in identifying the rubber tree variety Yunyan 614.

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