Molecular markers, primers, kits and applications for identifying the rubber tree variety 'Yunyan 314'

Abstract

The present disclosure relates to the technical field of variety cultivation and identification in molecular biology, specifically to a molecular marker, primer, kit and application for identifying the rubber tree variety 'Yunyan 314'. The molecular marker is located at positions 17,296,046 to 17,296,214 of chromosome 1, and the nucleotide sequence is as shown in SEQ ID No.1. The present invention provides a molecular marker primer and kit for the rubber tree variety 'Yunyan 314'. This technology can specifically identify and amplify the DNA sequence of the 'Yunyan 314' variety, achieve accurate identification of it, help improve the accuracy of variety identification, optimize variety selection and regional suitability assessment, thereby providing a scientific basis for the cultivation 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 differentiation and identification of rubber tree varieties: Yunyan 314, PB86, Yunyan 277-5, PR107, RRIM600, RRIM515, RRIM523 and IAN873, and is easy to operate and suitable for large-scale application.

Description

Technical Field

[0001] The present disclosure relates to the technical field of variety breeding and identification in molecular biology, and specifically 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 globally, the cultivation and yield of rubber trees (Hevea brasiliensis) have a significant impact on economic and industrial development. Yunnan Province, ranking first in China in terms of rubber tree planting area, yield per unit area, and total output, is of irreplaceable importance in selecting and cultivating rubber tree varieties with strong adaptability to promote the sustainable development of China's rubber industry. Especially in the cultivation and planting strategies of key traits such as high yield, cold resistance, and powdery mildew resistance, it is particularly crucial.

[0003] The rubber tree variety 'Yunyan 314' was selected by the Tropical Crops Science Research Institute of Yunnan Province starting in 2010 and obtained a new variety authorization from the Ministry of Agriculture and Rural Affairs in April 2024. Its authorization number is: CNA20191004741, which is a triploid (2n = 3x = 54) clone with excellent traits of fast growth and cold resistance. The average stem girth at 1 m above the ground is over 60 cm after 8 years of planting in Jinghong. The fast growth and cold resistance of 'Yunyan 314' have been verified through trial plantings 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 State Farms and is suitable for planting in severely cold and moderately cold regions, showing the best performance in environments with sufficient water, fertilizer and cool climate.

[0004] However, due to the narrow genetic basis of currently popular rubber tree varieties and the high similarity of phenotypic traits among varieties, 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 instability of identification results. During the popularization and planting of the excellent rubber tree variety 'Yunyan 314', due to its high similarity in phenotype to other rubber tree varieties, it is difficult to distinguish from the appearance, and misidentification often occurs, leading to variety mixing. The patent with the authorization publication number CN 118064428 B discloses an MNP molecular marker for constructing a DNA fingerprint map of rubber trees, which can amplify the DNA fingerprint map of rubber tree varieties using 600 pairs of MNP primers, but the identification markers for individual varieties are not clearly pointed out, and this method requires high-throughput sequencing to analyze the differences among varieties, with relatively high costs.

[0005] Therefore, developing a new method with a short development cycle, low cost, simple operation and targeted at individual rubber tree variety identification is of great significance for solving rubber tree variety mixing, increasing rubber tree yield, and ensuring the sustainable development of natural rubber. Summary of the Invention

[0006] The object 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 base positions 17296046 to 17296214 on 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 base at position 17296081 is A or C;

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

[0011] The base at position 17296103 is A or G;

[0012] The base at position 17296114 is G or A;

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

[0014] The base at position 17296142 is G or A;

[0015] The base at position 17296149 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 base at position 17296174 is G or A;

[0019] The base at position 17296184 is C or G;

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

[0021] The base at position 17296194 is A or G;

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

[0023] Furthermore, the nucleotide sequence of the molecular marker is as shown in 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', and the sequences of the primer pair are 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', and the kit contains 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', and the steps include:

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

[0028] (2) Using the extracted genomic DNA as a template, perform PCR amplification respectively with the primer pairs shown in SEQ ID No. 2-3;

[0029] (3) Sequence the amplification products by Sanger sequencing to obtain sequencing data, determine the genotypes of 16 loci in each individual, and if the genotypes corresponding to the 16 loci are CC, AA, TT, AA, GG, GG, GG, AA, TT, GG, TT, GG, CC, CC, AA, TT, and the sequence is shown in SEQ ID No. 1, then it is the 'Yunyan 314' variety; otherwise, it indicates that the rubber tree sample to be tested is 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 is: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 61°C for 30 s, decreasing 1°C for 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; finally, extension at 72°C for 7 min.

[0032] The molecular markers, primer pairs or kits provided by the present invention can be used for the differentiation and identification of rubber tree varieties: Yunyan 314, PB86, Yunyan 277-5, PR107, RRIM600, RRIM515, RRIM523, and IAN873.

[0033] The beneficial technical effects of the present invention are as follows: The present invention provides a molecular marker primer and kit for the rubber tree variety 'Yunyan 314'. This technology can specifically identify and amplify the DNA sequence of the 'Yunyan 314' variety, achieving accurate identification of it, which helps to 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 markers, primer pairs or kits of the present invention can be used for the differentiation and identification of rubber tree varieties: Yunyan 314, PB86, Yunyan 277-5, PR107, RRIM600, RRIM515, RRIM523 and IAN873, and the operation is simple, suitable for large-scale application. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0035] Figure 1 It is a sequence feature diagram of 9 types in Example 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0036] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0037] Example 1

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

[0039] (1) From the Wickham germplasm resources planted and preserved in the Jinghong Rubber Tree Germplasm Resource Nursery of the Ministry of Agriculture and Rural Affairs, including 123 Yunyan series germplasms including 'Yunyan 314' and 24 domestic and foreign varieties; collect fresh leaves without pest and disease infestation of 147 germplasms, quickly freeze them in liquid nitrogen, and store them at -80 °C for later use.

[0040] (2) Use a plant total DNA extraction kit to extract the whole genome DNA of 147 samples in step (1), and then send the extracted whole genome DNA samples to Beijing Biomarker Technologies Co., Ltd. for resequencing.

[0041] (3) Perform quality control on the original re-sequencing data to obtain clean data for each sample.

[0042] (4) Use the MEM algorithm of the BWA software to align the clean data to the rubber tree reference genome (the reference genome version is GCA_010458925.1) to obtain an intermediate result file in sam format; use the samtools software to convert the sam file to a bam file and perform result merging, duplicate sequence removal, and sorting.

[0043] (5) Use the GATK software to detect and genotype polymorphic sites in the bam file to generate a vcf file; use the vcftools software to perform quality control on the sites in the vcf file to obtain high-quality polymorphic sites that meet the screening conditions. The quality control conditions are: minor allele frequency MAF > 0.05; P-value > 0.01 in the Hardy-Weinberg test; site deletion rate < 0.1; polymorphism information content PIC > 0.2; population site heterozygosity < 0.2. After quality control, core polymorphic sites are obtained.

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

[0045] After screening, 1 molecular marker with significant differences in the 'Yunyan 314' variety was obtained. This molecular marker has polymorphisms at 16 base positions.

[0046] (7) Use the BioEdit software to perform sequence alignment on all sample sequences, and a total of 9 different sequence types were statistically obtained (as shown in Figure 1 ), and the 9 different sequence types were respectively 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), i (SEQ ID No.11). The nucleotide sequences of the 9 sequence types are shown in Table 1.

[0047] Table 1 Different sequences obtained by amplifying and sequencing 147 rubber tree germplasm resources

[0048]

[0049] Analyze the genotype of the 'Yunyan 314' variety. The genotypes corresponding to 16 loci are CC, AA, TT, AA, GG, GG, GG, AA, TT, GG, TT, GG, CC, CC, AA, TT, and only 'Yunyan 314' has this homozygous genotype among all the samples to be tested. The sequence type is a, and the nucleotide sequence is as shown in SEQ ID No.1.

[0050] Example 2

[0051] This example is for the reliability verification of using the screened specific molecular marker to identify the rubber tree variety 'Yunyan 314'.

[0052] Design specific primers for the screened specific molecular marker (SEQ ID No.1), and the designed sequences are as follows:

[0053] F: 5’-ATTGCATTTTCTTTCCACTTGAGGT-3’ (SEQ ID No.2)

[0054] R: 5’- GCAAGCCCCAAATGAGCAATATAAT-3’ (SEQ ID No.3).

[0055] Select 19 accessions of Wickham germplasm of rubber tree, and use Sanger sequencing to perform genotyping detection on the developed specific molecular marker; specifically including:

[0056] (1) Extract the genomic DNA of the samples to be tested;

[0057] (2) Using the DNA in step (1) as a template and the above nucleotide sequences as primers, perform PCR amplification on this molecular marker locus to obtain a PCR amplification product;

[0058] (3) Perform Sanger sequencing on the PCR amplification product obtained in step (2) to determine the genotype of this molecular marker locus, as shown in Table 2 specifically:

[0059] Table 2 Genotyping after amplification of 19 rubber tree germplasms

[0060]

[0061] The verification results are consistent with the genotype analysis results obtained in Example 1, indicating that the developed molecular marker can effectively identify the 'Yunyan 314' variety in rubber tree germplasm.

[0062] Example 3

[0063] A molecular marker, primer, kit for identifying the rubber tree variety 'Yunyan 314' and its application in variety identification, the steps include:

[0064] (1)Randomly select 50 rubber tree germplasms (sample names are shown in Table 3), and extract the whole genome DNA of all samples using a plant total DNA extraction kit.

[0065] Table 3 Germplasm names of 50 samples to be tested

[0066]

[0067] (2)Using the extracted genomic DNA as a template, perform PCR amplification with the primer pairs described in SEQ ID No.2 - 3 respectively. The amplification system is shown in Table 4.

[0068] Table 4 PCR amplification system

[0069]

[0070] The reaction program is 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 1°C for 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; finally, extension at 72°C for 7 min.

[0071] (3)The PCR amplification products are subjected to Sanger sequencing to determine the genotypes of 16 loci for each sample.

[0072] (4)When the genotype of the 16 base positions of the sample is CC, AA, TT, AA, GG, GG, GG, AA, TT, GG, TT, GG, CC, CC, AA, TT, determine that the sample to be tested is the 'Yunyan 314' variety.

[0073] (5)Among the 50 samples to be tested provided in this example, the primer pairs provided in this example can effectively distinguish 8 samples to be tested. The different sequence combinations of the 8 samples to be tested are shown in Table 5.

[0074] Table 5 Sequence combinations of 8 rubber tree varieties distinguished by this primer combination

[0075]

[0076] As can be seen from Table 5, the above - mentioned 8 samples to be tested have different sequence combination methods. It can be seen that the primer 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.

[0077] Example 4

[0078] Accuracy Analysis of Molecular Marker Primers for Rubber Trees

[0079] Three reproducibility experiments were used for accuracy analysis. In this embodiment, three independent experiments were conducted with different personnel, reagents from different batches, and different laboratories, thus simulating the identification of rubber trees from different batches. A high reproducibility rate means that the identification results from different laboratories can be accurately compared with each other.

[0080] Reproducibility experiments were carried out on 8 rubber tree samples to be tested. The 8 samples to be tested were specifically: Yunyan 314, PB86, Yunyan 277-5, PR107, RRIM600, RRIM515, RRIM523, IAN873. Three repeated experiments were conducted for each sample to be tested, and the sequence combinations of each result were recorded. The specific results are shown in Table 6.

[0081] Table 6 Results of Three Repeated Experiments

[0082]

[0083] Table 6 further verifies that the molecular markers and amplification primers for rubber trees of the present invention can distinguish and identify Yunyan 314, PB86, Yunyan 277-5, PR107, RRIM600, RRIM515, RRIM523, and IAN873.

[0084] 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 of ordinary skill in the art should understand that: modifications or equivalent replacements can still be made to the present invention. Any modification or partial replacement without departing from the spirit and scope of the present invention shall be covered by the scope of the claims of the present invention.

Claims

1. A molecular marker for identifying 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 nucleotide sequence of the molecular marker is shown in SEQ ID No.

1. When the molecular marker is detected in a rubber tree variety, it is 'Yunyan 314'.

2. 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 product is sequenced by Sanger sequencing to obtain sequencing data. If the amplified product contains a sequence as shown in SEQ ID No. 1, it is the variety 'Yunyan 314'; otherwise, it indicates that the rubber tree sample to be tested is not the variety 'Yunyan 314'.

3. The identification method according to claim 2, characterized in that: The PCR amplification: The reaction system was 20 μL, including: 50 ng / μL DNA template 0.5 μL, 10× PCR buffer 2 μL, 25 mM MgCl2 2 μL, 10 mM dNTPs 0.5 μL, 5 U / μL Taq DNA polymerase 0.2 μL, 10 μM upstream primer 0.5 μL, 10 μM downstream primer 0.5 μL and ddH2O 13.8 μL; 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.

4. Application of the reagent for detecting the molecular marker described in claim 1 in identifying the rubber tree variety Yunyan 314.

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