Molecular marker, primer and kit for identifying rubber tree variety Yunnan research 314 and application

Abstract

The invention relates to the technical field of variety cultivation and identification in molecular biology, in particular to a molecular marker, a primer and a kit for identifying a rubber tree variety 'Yunnan research 314' and application of the molecular marker, the primer and the kit, the molecular marker is positioned on a No. 1 chromosome 17296046-17296214 base, and the nucleotide sequence is shown as SEQ ID No. 1. The invention provides a molecular marker primer and a kit aiming at a rubber tree variety Yunnan research 314, the technology can specifically identify and amplify a DNA sequence of the Yunnan research 314, realizes accurate identification of the Yunnan research 314, is helpful for improving the accuracy of variety identification, optimizes variety selection and regional suitability evaluation, and has the advantages of high specificity, high sensitivity, high sensitivity and the like. Therefore, a scientific basis is provided for planting and breeding of rubber trees, and healthy development of the rubber industry is promoted. The molecular marker, the primer pair or the kit disclosed by the invention can be used for distinguishing and identifying rubber tree varieties: Yunnan research 314, PB86, Yunnan research 277-5, PR107, RRIM600, RRIM515, RRIM523 and IAN873, and is simple and convenient to operate and 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 314'. 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 314' was selected and bred by the Yunnan Tropical Crops Science Research Institute in 2010 and obtained the new variety authorization from the Ministry of Agriculture and Rural Affairs in April 2024. Its authorization number is: CNA20191004741. It is a triploid (2n=3x=54) asexual line with excellent traits of fast growth and cold resistance. After being planted in Jinghong for 8 years, the average stem circumference is more than 60 cm at 1m above the ground. The fast growth and cold resistance of 'Yunyan 314' have been verified by trial planting in Dehong, Lincang, Xishuangbanna, Honghe and other places, and good yield levels have been obtained. 'Yunyan 314' has been included in the recommended varieties of Yunnan Agricultural Reclamation, 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] However, due to the narrow genetic basis of the rubber tree varieties currently promoted for planting, and the high similarity of phenotypic traits between varieties, the traditional rubber tree variety identification method mainly relies on morphological characteristics and physiological and biochemical indicators. These phenotypic-based identification methods are easily affected by environmental factors, resulting in the instability of identification results. In the promotion and planting process of the rubber tree fine variety 'Yunyan 314', due to its phenotype and other rubber tree varieties and their similarity, it is difficult to distinguish 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 of CN 118064428 B discloses a MNP molecular marker for constructing a DNA fingerprint of a rubber tree, which can utilize 600 pairs of MNP primers to amplify the DNA fingerprint of a rubber tree variety, but the identification marker for a single variety is 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 314'.

[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 314', the physical position of the molecular marker is located at bases 17296046 to 17296214 of chromosome CM021234_1; the molecular marker includes the following 16 base polymorphisms, specifically:

[0008] The base at position 17296073 is C or T;

[0009] The 17296081st base is A or C;

[0010] The base at position 17296082 is T or C;

[0011] The 17296103rd base is A or G;

[0012] The 17296114th base is G or A;

[0013] The base at position 17296126 is G or A;

[0014] The 17296142nd base is G or A;

[0015] The 17296149th base is A or G;

[0016] The base at position 17296167 is T or C;

[0017] The base at position 17296169 is G or G;

[0018] The 17296174th base is G or A;

[0019] The 17296184th base is C or G;

[0020] The base at position 17296188 is C or T;

[0021] The 17296194th base is A or G;

[0022] The base at position 17296208 is T or C.

[0023] Furthermore, the nucleotide sequence of the molecular marker is shown as SEQ ID No.1.

[0024] The second object of the present invention is to provide a primer pair for amplifying molecular markers of the rubber tree variety 'Yunyan 314', wherein the primer pair sequence is shown in SEQ ID No. 2-3.

[0025] The third object of the present invention is to provide a kit for identifying the rubber tree variety 'Yunyan 314', the kit comprising a primer pair for amplifying molecular markers of the rubber tree variety 'Yunyan 314'.

[0026] The fourth object of the present invention is a method for identifying the rubber tree variety 'Yunyan 314', the steps comprising:

[0027] (1) Use a plant total DNA extraction kit to extract the total DNA of all rubber tree samples to be tested;

[0028] (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;

[0029] (3) The amplified products were sequenced by Sanger sequencing to obtain sequencing data, and the genotypes of 16 loci in each individual were determined. The genotypes corresponding to the 16 loci were CC, AA, TT, AA, GG, GG, GG, AA, TT, GG, TT, GG, CC, CC, AA, TT. If the sequence was as shown in SEQ ID No. 1, it was the 'Yunyan 314' variety; otherwise, it indicated that the rubber tree sample to be tested was not the 'Yunyan 314' variety.

[0030] Further, the PCR amplification system 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;

[0031] The reaction procedure was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 61°C for 30 s, decreasing by 1°C each cycle, extension at 72°C for 30 s, for 10 cycles; denaturation at 94°C for 30 s, annealing at 51°C for 30 s, extension at 72°C for 30 s, for 30 cycles; and finally extension at 72°C for 7 min.

[0032] The molecular marker, primer pair or kit provided by the invention can be used for distinguishing and identifying rubber tree varieties: Yunyan 314, PB86, Yunyan 277-5, PR107, RRIM600, RRIM515, RRIM523 and IAN873.

[0033] 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 314', the technology can specifically identify and amplify the DNA sequence of the 'Yunyan 314' 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 rubber tree varieties: Yunyan 314, PB86, Yunyan 277-5, PR107, RRIM600, RRIM515, RRIM523 and IAN873, is easy to operate, and is suitable for large-scale application. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] 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.

[0035] Figure 1 These are 9 types of sequence feature graphs in Example 1 of the present invention. DETAILED DESCRIPTION

[0036] 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.

[0037] Example 1

[0038] This embodiment is a method for screening and identifying molecular markers specific to rubber tree 'Yunyan 314', comprising the following steps:

[0039] (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 314' 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.

[0040] (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.

[0041] (3) Perform quality control on the raw resequencing data to obtain clean data for each sample.

[0042] (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.

[0043] (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.

[0044] (6) The vcftools software was used to further screen specific molecular markers for the rubber tree variety 'Yunyan 314' based on the genotypic differences of the core polymorphic loci in different rubber tree germplasms. The screening criteria were: the homozygous mutation rate of the polymorphic loci in the rubber tree variety 'Yunyan 314' was 1, and the homozygous mutation rate of the polymorphic loci in other rubber tree germplasms was <0.1.

[0045] After screening, a molecular marker with significant difference in 'Yunyan 314' variety was obtained, and the molecular marker had polymorphisms at 16 base positions.

[0046] (7) All sample sequences were aligned using BioEdit software, and a total of 9 different sequence types were obtained (e.g. Figure 1 As shown in Table 1, the 9 different sequence types were named a (SEQ ID No.1), b (SEQ ID No.4), c (SEQ ID No.5), d (SEQ ID No.6), e (SEQ ID No.7), f (SEQ ID No.8), g (SEQ ID No.9), h (SEQ ID No.10), and i (SEQ ID No.11). The nucleotide sequences of the 9 sequence types are shown in Table 1.

[0047] Table 1 Different sequences obtained after amplification and sequencing of 147 rubber tree germplasm resources

[0048] (8) The genotype of the 'Yunyan 314' variety was analyzed. The genotypes corresponding to the 16 loci were CC, AA, TT, AA, GG, GG, GG, AA, TT, GG, TT, GG, CC, CC, AA, TT. Among all the samples tested, only 'Yunyan 314' had this homozygous genotype, the sequence type was a, and the nucleotide sequence was as shown in SEQ ID No.1.

[0049] Example 2

[0050] This example is a reliability verification of identifying the rubber tree variety 'Yunyan 314' using the screened specific molecular markers.

[0051] Specific primers were designed for the screened specific molecular marker (SEQ ID No. 1), and the designed sequences are as follows:

[0052] F: 5'-ATTGCATTTTCTTTCCACTTGAGGT-3' (SEQ ID No. 2)

[0053] R: 5'-GCAAGCCCCAAATGAGCAATATAAT-3' (SEQ ID No. 3).

[0054] 19 rubber tree Wickham germplasms were selected and genotyped using Sanger sequencing for the developed specific molecular markers; specifically:

[0055] (1) Extract whole genome DNA of test samples;

[0056] (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;

[0057] (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:

[0058] Table 2 Genotyping of 19 rubber tree germplasms after amplification

[0059]

[0060] 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 314' variety in rubber tree germplasm.

[0061] Example 3

[0062] A molecular marker, primer, and kit for identifying the rubber tree variety 'Yunyan 314' and its application in variety identification, comprising the following steps:

[0063] (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.

[0064] Table 3 Germplasm names of the 50 samples to be tested

[0065]

[0066] (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.

[0067] Table 4 PCR extension system

[0068]

[0069] The reaction procedure was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 61°C for 30 s, decreasing by 1°C each cycle, extension at 72°C for 30 s, for 10 cycles; denaturation at 94°C for 30 s, annealing at 51°C for 30 s, extension at 72°C for 30 s, for 30 cycles; and finally extension at 72°C for 7 min.

[0070] (3) The PCR amplification products were sequenced by Sanger sequencing to determine the genotype of 16 loci for each sample.

[0071] (4) When the genotyping results of the sample are CC, AA, TT, AA, GG, GG, GG, AA, TT, GG, TT, GG, CC, CC, AA, TT at the 16 base positions, the sample is determined to be the 'Yunyan 314' variety.

[0072] (5) Among the 50 samples to be tested provided in this embodiment, 8 samples to be tested can be effectively distinguished using the primer pairs provided in this embodiment. The different sequence combinations of the 8 samples to be tested are shown in Table 5.

[0073] Table 5 Sequence combinations of 8 rubber tree varieties distinguished by this primer combination

[0074]

[0075] It can be seen from Table 5 that the above 8 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 314', and the sequence type is a (SEQ ID No. 1). It can also distinguish PB86, Yunyan 277-5, PR107, RRIM600, RRIM515, RRIM523, and IAN873 according to different sequence combinations.

[0076] Example 4

[0077] Analysis on the accuracy of molecular marker primer identification in rubber tree

[0078] 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.

[0079] A reproducibility experiment was conducted on 8 rubber tree samples to be tested. The 8 samples to be tested were: Yunyan 314, PB86, Yunyan 277-5, PR107, RRIM600, RRIM515, RRIM523, and IAN873. Each sample to be tested was repeated three times, and the sequence combination of each result was recorded. The specific results are shown in Table 6.

[0080] Table 6 Results of three repeated experiments

[0081]

[0082] Table 6 further verifies that the rubber tree molecular markers and amplification primers of the present invention can distinguish and identify Yunyan 314, PB86, Yunyan 277-5, PR107, RRIM600, RRIM515, RRIM523 and IAN873.

[0083] 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 314', characterized in that: The physical position of the molecular marker is located at bases 17296046 to 17296214 of chromosome CM021234_1; the molecular marker includes the following 16 base polymorphisms, specifically: The base at position 17296073 is C or T; The 17296081st base is A or C; The base at position 17296082 is T or C; The 17296103rd base is A or G; The 17296114th base is G or A; The base at position 17296126 is G or A; The 17296142nd base is G or A; The 17296149th base is A or G; The base at position 17296167 is T or C; The base at position 17296169 is G or G; The 17296174th base is G or A; The 17296184th base is C or G; The base at position 17296188 is C or T; The 17296194th base is A or G; The base at position 17296208 is T or C.

2. The molecular marker according to claim 1, characterized in that The nucleotide sequence of the molecular marker is shown as SEQ ID No.

1.

3. A primer pair for amplifying the molecular marker of rubber tree variety 'Yunyan 314', characterized in that: The primer pair sequences are shown in SEQ ID No. 2-3.

4. A kit for identifying the rubber tree variety 'Yunyan 314', characterized in that: The kit comprises the primer pair according to claim 3.

5. A method for identifying the rubber tree variety 'Yunyan 314', 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 described in SEQ ID No. 2 to 3 respectively; (3) The amplified products were sequenced by Sanger sequencing to obtain sequencing data, and the genotypes of 16 loci in each individual were determined. The genotypes corresponding to the 16 loci were CC, AA, TT, AA, GG, GG, GG, AA, TT, GG, TT, GG, CC, CC, AA, TT. If the sequence was as shown in SEQ ID No. 1, it was the 'Yunyan 314' variety; otherwise, it indicated that the rubber tree sample to be tested was not the 'Yunyan 314' variety.

6. The identification method according to claim 5, characterized in that: The PCR amplification system 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 TaqDNA 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 61°C for 30 s, decreasing by 1°C each cycle, extension at 72°C for 30 s, for 10 cycles; denaturation at 94°C for 30 s, annealing at 51°C for 30 s, extension at 72°C for 30 s, for 30 cycles; and finally extension at 72°C for 7 min.

7. Use of the molecular marker according to claim 1 or 2, the primer pair according to claim 3, or the kit according to claim 4 in identifying rubber tree varieties.

8. According to the application of the molecular marker described in claim 1 or 2 or the primer pair described in claim 3 or the test kit described in claim 4 in identifying the rubber tree variety, it is characterized in that, The rubber tree varieties are: Yunyan 314, PB86, Yunyan 277-5, PR107, RRIM600, RRIM515, RRIM523 and IAN873.

Images