Molecular markers, primers, reagent kits and applications for identifying rubber tree variety 'Yunyan 621'

Abstract

This invention relates to the field of variety breeding and identification technology in molecular biology, specifically a molecular marker, primers, kit, and application for identifying the rubber tree variety 'Yunyan 621'. The molecular marker is located at bases 66751525–66751724 on chromosome 10, and its nucleotide sequence is shown in SEQ ID No. 1. This invention provides a molecular marker primer and kit for the rubber tree variety 'Yunyan 621'. This technology can specifically identify and amplify the DNA sequence of the 'Yunyan 621' variety, enabling accurate identification. This helps improve the accuracy of variety identification, optimize variety selection and regional suitability assessment, thereby providing a scientific basis for rubber tree planting and breeding, and promoting the healthy development of the rubber industry. The molecular marker, primer pair, or kit of this invention can be used to differentiate and identify rubber tree varieties 'Yunyan 621', 'Yunyan 651', and 'Yunyan 98-305'. It is simple to operate and suitable for large-scale application.

Description

Technical Field

[0001] This disclosure relates to the field of variety breeding and identification technology in molecular biology, specifically a molecular marker, primer, reagent kit, and application for identifying the rubber tree variety 'Yunyan 621'. Background Technology

[0002] As a crucial source of natural rubber globally, the cultivation and yield of the rubber tree (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, plays an irreplaceable role in promoting the sustainable development of my country's rubber industry by selecting and cultivating adaptable rubber tree varieties. This is particularly true in the breeding and cultivation strategies for key traits such as high yield, cold resistance, and powdery mildew resistance.

[0003] The rubber tree variety 'Yunyan 621' is a new rubber tree variety independently bred by the Yunnan Provincial Tropical Crops Research Institute, and its plant variety rights were applied for in October 2022. This variety has excellent traits such as high yield and cold resistance. The high yield and cold resistance of 'Yunyan 621' have been verified through trial plantings in Dehong, Lincang, Xishuangbanna, and Honghe.

[0004] Currently promoted rubber tree varieties have narrow genetic bases, and their phenotypic traits are highly similar. Traditional rubber tree variety identification methods mainly rely on morphological characteristics and physiological and biochemical indicators. These phenotypic-based identification methods are easily affected by environmental factors, leading to instability in the identification results. The phenotype of rubber tree 'Yunyan 621' is very similar to other rubber tree varieties, making it difficult to distinguish from them visually, resulting in frequent misidentification and causing variety mixing. Patent CN 118064428 B discloses an MNP molecular marker for constructing a DNA fingerprint of rubber trees, which can amplify the DNA fingerprint of rubber tree varieties using 600 pairs of MNP primers. However, it does not clearly specify the identification markers for individual varieties, and the method requires high-throughput sequencing to analyze the differences between varieties, making it relatively costly.

[0005] Therefore, developing new methods that are short-cycle, low-cost, simple to operate, and targeted at the identification of individual rubber tree varieties is of great significance for solving the problem of mixed rubber tree varieties, increasing rubber tree yield, and ensuring the sustainable development of natural rubber. Summary of the Invention

[0006] The purpose of this invention is to provide a molecular marker, primer, reagent kit, and application for identifying the rubber tree variety 'Yunyan 621'.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is: a molecular marker for identifying the rubber tree variety 'Yunyan 621', wherein the physical location of the molecular marker is located at bases 66751525 to 66751724 on chromosome CM021235_1, and its nucleotide sequence is shown in SEQ ID No.1.

[0008] The second objective of this invention is to provide a primer pair for amplifying molecular markers of the rubber tree variety 'Yunyan 621', the sequences of which are shown in SEQ ID No. 2-3.

[0009] A third objective of this invention is to provide a kit for identifying the rubber tree variety 'Yunyan 621', the kit comprising primer pairs for amplifying molecular markers of the rubber tree variety 'Yunyan 621'.

[0010] The fourth objective of this invention is a method for identifying the rubber tree variety 'Yunyan 621', comprising the following steps:

[0011] (1) Total DNA was extracted from all rubber tree samples to be tested using a plant total DNA extraction kit;

[0012] (2) Using the extracted genomic DNA as a template, PCR amplification was performed using the primer pairs shown in SEQ ID No. 2-3 respectively;

[0013] (3) The amplified products were sequenced by Sanger sequencing to obtain sequencing data. If the amplified products contained a sequence as shown in SEQ ID No. 1, it was the 'Yunyan 621' variety; otherwise, it indicated that the rubber tree sample to be tested was not the 'Yunyan 621' variety.

[0014] Furthermore, the PCR amplification system is 20 μL, comprising: 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;

[0015] The reaction procedure was as follows: pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 30 s, repeated 30 times; and finally extension at 72℃ for 7 min.

[0016] For those skilled in the art, the application of reagents containing the molecular markers described in this invention in the identification of the rubber tree variety 'Yunyan 621' also falls within the scope of protection of this invention.

[0017] The beneficial technical effects of this invention are as follows: This invention provides a molecular marker primer and kit for the rubber tree variety 'Yunyan 621'. This technology can specifically identify and amplify the DNA sequence of the 'Yunyan 621' variety, achieving accurate identification. This helps improve the accuracy of variety identification, optimize variety selection and regional suitability assessment, thereby providing a scientific basis for rubber tree planting and breeding, and promoting the healthy development of the rubber industry. The molecular markers, primer pairs, or kits of this invention can be used to differentiate and identify rubber tree varieties / lines 'Yunyan 621', 'Yunyan 651', and 'Yunyan 98-305'. The operation is simple and suitable for large-scale application. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 These are the six types of sequence feature maps in Embodiment 1 of the present invention. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Example 1

[0022] This embodiment describes a method for screening and identifying molecular markers specific to the rubber tree 'Yunyan 621', including the following steps:

[0023] (1) The germplasm resources of Wei Kehan ​​planted and preserved in the Jinghong Rubber Tree Germplasm Resource Nursery include 123 germplasm accessions of the Yunyan series, including 'Yunyan 621', and 24 domestic and foreign varieties; fresh leaves of 147 germplasm accessions without disease or pest infection were collected, quick-frozen with liquid nitrogen, and stored at -80℃ for later use.

[0024] (2) The whole genome DNA of 147 samples in step (1) was extracted using a plant total DNA extraction kit, and then the extracted whole genome DNA samples were sent to Beijing Biomarker Biotechnology Co., Ltd. for resequencing.

[0025] (3) Perform quality control on the raw resequencing data to obtain clean data for each sample.

[0026] (4) The clean data was aligned to the rubber tree reference genome (the reference genome version is GCA_010458925.1) using the MEM algorithm of BWA software to obtain intermediate result files in sam format; the sam files were converted to bam files using samtools software and the results were merged, duplicate sequences were removed and sorted.

[0027] (5) Using GATK software, polymorphic sites were detected and genotyped in the bam file to generate a vcf file; the sites in the vcf file were quality controlled using vcftools software to obtain high-quality polymorphic sites that meet the screening criteria. The quality control criteria were: minor allele frequency MAF>0.05; P-value in Hardy-Weinberg test>0.01; site deletion rate<0.1; polymorphic information content PIC>0.2; population site heterozygosity rate<0.2. After quality control, core polymorphic sites were obtained.

[0028] (6) Using the vcftools software, we further screened the specific molecular markers of the rubber tree variety 'Yunyan 621' 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 621' was 1, and the homozygous mutation rate of the polymorphic sites in other rubber tree germplasms was <0.1.

[0029] After screening, a molecular marker showing significant differences in the 'Yunyan 621' variety was obtained. This molecular marker has polymorphisms at 5 base positions.

[0030] (7) Using BioEdit software, sequence alignment was performed on all sample sequences, and a total of 6 different sequence types were obtained (e.g., Figure 1 As shown in Table 1, the six different sequence types are 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), and f (SEQ ID No. 8).

[0031] Table 1. Different sequences obtained from sequencing 147 rubber tree germplasm resources after amplification.

[0032] a TAGCAATGCTGAAGCAAGAGATATAAGCAGAGGAGCTCGGGAAGACACTGATCCGCATCGAGCCAAAGCTAATGAAATGCATTGTAGTAGAAAACTAGTTTTGCCGTCATTTCCTATACCAGAAAAGCCTCCTGTGCTAGCAATCAAATCCTGCGCCACAAATTCTATTCTATTTTCTGCAGAAATTGTTAATCGTGAAA b TAGCAATGCTGAAGCAAGAGATATAAGCAGAGGAGCTCGGGAAGACACTGATCCGCATCGAGCCAAAGCTAATGAAATGCATTGTAGTAGAAAACTAGTTTTGCCGTCATTTCCTATACCAGAAAAGCCTCCTGTGCTAGCAATCAAATCCTGCGCCACAAATTCTATTCTATTTTCTGCAGAAATCCTTAATCGTGAAA c TAGCAATGCTGAAGCAAGAGATATAAGCAGAGGAGCTCGGGAAGACACTGATCCGCATCGAGCCAAAGCTAATGAAATGCATTGTAGTAGAAAACTAGTTTTGCCGTCATTTCCTATACCAGAAAAGTCTCCTGTGCTAGCAATCAAATCCTGCGCCACAAATTCTATTCTATTTTCTGCAGAAATCCTTAATCGTGAAA d TAGCAATGCTGAAGCAAGAGATATAAGCAGAGGAGCTCGGGAAGACACTGATCCGCATCGAGCCAAAGCTAATGAAATGCATTGTAGTAGAAAACTAGTTTTGCCGTCATTTCCTATACCAGAAAAGCCTCCTGTGCTAGCAATCAAATCCTGCGCCACAAATTCTATTCTATTTTCCGCAGAAATCCTTAATCGTGAAA e TAGCAATGCTGAAGCAAGAGATATAAGCAGAGGAGCTCGGGAAGACACTGATCCGCATCGAGCCAAAGCTAATGAAATGCATTGTAGTAGAAAACTAGTTTTGCCGTCATTTCCTATACCAGAAAAGCCTCCTGTGCTAGCAATCAAATCCTGCGCCACAAATTCTATTCTATTTTCTGCAGAAATCGTTAATCGTGAAA f TAGCAATGCTGAAGCAAGAGATATAAGCAGAGGAGCTCGGGAAGACACTGATCCGCATCGAGCCAAAGCTAATGAAATGCATTGTAGTAGAAAACTAGTTTTGCCGTCATTTCCTATACCAGAAAAGCCTCCTGTGCTAGCAATCAAATCCTGCGCCACAAATTCTATTCTATTTTCCGCAGAAATCCTTAATCGTGAAA

[0033] (8) Analyze the genotype of the 'Yunyan 621' variety. The genotypes corresponding to the 5 loci are GG, CC, TT, TT, and GG. Among all the samples to be tested, only 'Yunyan 621' has this homozygous genotype. The sequence type is a, and the nucleotide sequence is as shown in SEQ ID No. 1.

[0034] Example 2

[0035] This embodiment verifies the reliability of identifying the rubber tree variety 'Yunyan 621' using the screened specific molecular markers.

[0036] Specific primers were designed for the screened specific molecular marker (SEQ ID No. 1), and the designed sequences are shown below:

[0037] F: 5'-CTTGCATATAAAACGCTGTAAGGGAA-3' (SEQ ID No. 2)

[0038] R: 5'-ATAGATCAATATCAAGTGGCCACGA-3' (SEQ ID No. 3).

[0039] Thirty-two accessions of the rubber tree Wekehane were selected, and genotyping was performed using Sanger sequencing based on developed specific molecular markers; specifically including:

[0040] (1) Extract whole-genome DNA from the sample to be tested;

[0041] (2) Using the DNA from step (1) as a template and the above nucleotide sequence as a primer, perform PCR amplification on the molecular marker site to obtain the PCR amplification product.

[0042] (3) The PCR amplification products obtained in step (2) were subjected to Sanger sequencing to determine the genotype of the molecular marker site, as shown in Table 2:

[0043] Table 2. Genotyping of 32 rubber tree germplasm accessions after amplification

[0044] Yunyan 621 GG, CC, TT, TT, GG Yunyan 272 GG, CC, TT, CC, CC Yunyan 314 GG, CC, TT, CC, CC Yunyan 617 GG, CC, TT, CC, CC Yunyan 281 GG, T / C, T / C, CC, CC Yunyan 612 GG, T / C, TT, CC, CC Yunyan 619 GG, CC, TT, CC, CC Yunyan 624 GG, CC, T / C, CC, CC Yunyan 584 GG, T / C, TT, CC, CC Yunyan 588 GG, CC, TT, CC, CC Yunyan 270 GG, T / C, TT, CC, CC Yunyan 1251 GG, T / C, TT, CC, CC Yunyan 190 GG, T / C, T / C, CC, CC Yunyan 77-4 GG, CC, TT, CC, CC Yunyan 73-46 GG, T / C, TT, CC, CC Yunyan 73-477 GG, CC, T / C, CC, CC Yunyan 631 GG, CC, TT, CC, CC RIRIM721 GG, T / C, TT, CC, CC PB86 GG, T / C, TT, CC, CC PR107 GG, CC, T / C, CC, CC IRCI22 GG, CC, TT, CC, CC PB5 / 51 GG, CC, TT ,CC, CC RRIM515 GG, CC, TT, CC, CC GT1 GG, CC, TT, CC, CC Yunyan 1628 GG, CC, TT, CC, CC Yunyan 629 GG, CC, TT, CC, CC Yunyan 13 GG, CC, TT, CC, CC Yunyan 30 GG, T / C, TT, CC, CC Yunyan 341 GG, T / C, TT, CC, CC Yunyan 552 GG, CC, T / C, CC, CC RRIM905 GG, CC, TT, CC, CC RRII105 GG, T / C, TT, CC, CC

[0045] The verification results are consistent with the genotype analysis results obtained in Example 1, indicating that the developed molecular markers can effectively identify the 'Yunyan 621' variety in rubber tree germplasm.

[0046] Example 3

[0047] A molecular marker, primers, and reagent kit for identifying the rubber tree variety 'Yunyan 621', and their application in variety identification, comprising the following steps:

[0048] (1) Fifty rubber tree germplasm samples were randomly selected (sample names are shown in Table 3), and whole genome DNA was extracted from all samples using a plant total DNA extraction kit.

[0049] Table 3. Germplasm names of 50 samples to be tested

[0050] 1 Yunyan 272 18 Yunyan 13 35 Yunyan 636 2 Yunyan 617 19 Yunyan 30 36 Yunyan 644 3 Yunyan 612 20 Yunyan 575 37 RRII105 4 Yunyan 281 21 Yunyan 341 38 Yunyan 646 5 Yunyan 314 22 Yunyan 552 39 Yunyan 648 6 Yunyan 619 23 Yunyan 554 40 Yunyan 98-305 7 Yunyan 621 24 Yunyan 73-46 41 PB5 / 51 8 Yunyan 624 25 Yunyan 73-477 42 RRIM515 9 Yunyan588 26 Yunyan 613 43 Yunyan 277-5 10 Yunyan 584 27 RRIM712 44 Yunyan 80-1983 11 Yunyan 270 28 Yunyan 631 45 PB86 12 Yunyan1251 29 PR107 46 Yunyan667 13 Yunyan 190 30 GT1 47 Yunyan 669 14 Yunyan555 31 IRCI22 48 Yunyan 91-440 15 Yunyan556 32 Yunyan 633 49 Yunyan 672 16 Yunyan558 33 Yunyan 634 50 Yunyan 98-296 17 Yunyan 629 34 Yunyan 635

[0051] (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 respectively. The amplification system is shown in Table 4.

[0052] Table 4 PCR Expansion System

[0053]

[0054] The reaction procedure was as follows: pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 30 s, repeated 30 times; and finally extension at 72℃ for 7 min.

[0055] (3) The PCR amplification products were sequenced by Sanger to determine the genotype of 5 loci in each sample.

[0056] (4) When the genotyping result of the sample is GG, CC, TT, TT, GG at 5 base positions, the sample to be tested is identified as the 'Yunyan 621' variety.

[0057] (5) Among the 50 test samples provided in this embodiment, the primer pairs provided in this embodiment can effectively distinguish 3 test samples. The different sequence combinations of the 3 test samples are shown in Table 5.

[0058] Table 5. Sequence combinations of three rubber tree varieties distinguished using this primer combination.

[0059] 1 Yunyan 621 a 2 Yunyan 651 ae 3 Yunyan 98-305 df

[0060] As shown in Table 5, the three samples to be tested have different sequences. It can be seen that the primers provided in Embodiment 1 of the present invention can amplify the sequence of 'Yunyan 621', and the sequence type is a (SEQ ID No. 1). It can also distinguish between Yunyan 651 and Yunyan 98-305 based on the different sequence types.

[0061] Example 4

[0062] Accuracy analysis of molecular marker primer identification for rubber trees

[0063] Accuracy analysis was performed using three reproducibility experiments. In this embodiment, three independent experiments were conducted using different personnel, different batches of reagents, and different laboratories to simulate the identification of different batches of rubber trees. The high reproducibility rate means that the identification results of different laboratories can be accurately compared with each other.

[0064] Reproducibility experiments were conducted on three rubber tree samples: Yunyan 621, Yunyan 651, and Yunyan 98-305. Each sample was tested three times, and the sequence combination of the results was recorded each time. The specific results are shown in Table 6.

[0065] Table 6 Results of three repeated experiments

[0066] 1 Yunyan 621 a a a 2 Yunyan 651 ae ae ae 3 Yunyan 98-305 df df df

[0067] Table 6 further verifies that the rubber tree molecular markers and amplification primers of the present invention can distinguish and identify Yunyan 621, Yunyan 651 and Yunyan 98-305.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate and not 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 modifications or equivalent substitutions can still be made to the present invention without departing from the spirit and scope of the present invention. Any modifications or partial substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for identifying the rubber tree variety 'Yunyan 621', characterized by the following steps: include: (1) Total DNA was extracted from all rubber tree samples to be tested using a plant total DNA extraction kit; (2) Using the extracted genomic DNA as a template, PCR amplification was performed using the primer pairs shown in SEQ ID No. 2-3 respectively; (3) The amplified products were sequenced by Sanger sequencing to obtain sequencing data. If the amplified products contained a sequence as shown in SEQ ID No. 1, it was the 'Yunyan 621' variety; otherwise, it indicated that the rubber tree sample to be tested was not the 'Yunyan 621' variety.

2. The identification method according to claim 1, characterized in that, The PCR amplification system is 20 μL, comprising: 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℃ for 3 min; denaturation at 95℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 30 s, repeated 30 times; and finally extension at 72℃ for 7 min.

3. The application of a reagent for identifying the molecular marker of the rubber tree variety 'Yunyan 621' in the identification of the rubber tree variety 'Yunyan 621', wherein the molecular marker is physically located at bases 66751525–66751724 on chromosome CM021235_1, and its nucleotide sequence is shown in SEQ ID No. 1, characterized in that… Total DNA was extracted from all rubber tree samples to be tested. PCR amplification was performed using the extracted genomic DNA as a template. The amplification products were sequenced by Sanger sequencing to obtain sequencing data. If the amplification product contained a sequence as shown in SEQ ID No. 1, it was identified as the 'Yunyan 621' variety; otherwise, it indicated that the rubber tree sample to be tested was not the 'Yunyan 621' variety.

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