A molecular marker primer group, kit, method and application for identifying hevea brasiliensis triploid variety yunyan 77-4

Abstract

The disclosure provides a molecular marker primer group for identifying Hevea brasiliensis triploid variety Yunyan 77-4, a kit, a method and an application, belonging to the field of molecular biology and genetic breeding. The molecular marker primer group is composed of two pairs of primers, each pair of primers is composed of an upstream primer and a downstream primer, the upstream primer of the first pair of primers is shown as SEQ ID NO:1 in the sequence table, the downstream primer of the first pair of primers is shown as SEQ ID NO:2 in the sequence table, the upstream primer of the second pair of primers is shown as SEQ ID NO:3 in the sequence table, and the downstream primer of the second pair of primers is shown as SEQ ID NO:4 in the sequence table. The MNP marker primer group is used for amplifying the sample to be tested, the obtained amplification product is sequenced, and whether the variety to be tested is Yunyan 77-4 is identified according to the sequencing result, and the identification is accurate.

Description

Technical Field

[0001] This disclosure relates to the fields of molecular biology and genetic breeding, and in particular to a molecular marker primer set, kit, method and application for identifying the triploid rubber tree variety Yunyan 77-4. Background Technology

[0002] Rubber tree (Hevea brasiliensis) is a perennial tropical crop, traditionally believed to be unsuitable for planting above 17°N latitude, only suitable for low-latitude regions. China has successfully extended its commercial cultivation northward to around 25°N latitude, becoming the northernmost point of global rubber plantations, which places higher demands on the stress resistance of varieties. Polyploid plants generally have stronger ecological adaptability than their diploid (2n) parents, especially performing better under stressful conditions such as cold. The rubber tree variety Yunyan 77-4 is a triploid (3n) cold-resistant, high-yielding variety bred by the Yunnan Provincial Tropical Crops Research Institute, and is also the main cultivated variety in Yunnan's rubber-growing areas. Its resistance and yield are superior to conventional diploid varieties, making its promotion and cultivation in China of great significance.

[0003] Accurate variety identification is fundamental to breeding and production applications. While SSR molecular markers are relatively stable, DNA polymerase slippage occurs during SSR locus amplification, especially with long SSR sequences exhibiting high polymorphism. This can easily lead to false slipped genotypes that are indistinguishable from the dominant genotype in the sample, making SSR markers unsuitable for identifying polyploid plants. SSR markers can only detect the length of amplified products and identify length differences between amplified fragments, but they cannot detect the specific base sequence of the amplified products or identify base variations across different amplified products. Furthermore, 6% of SSR loci do not exhibit length differences but do show base variations. SNP markers have high throughput, but low polymorphism at individual loci makes it difficult to distinguish polyploids.

[0004] Therefore, an accurate identification method is urgently needed for important polyploid varieties such as Yunyan 77-4. Summary of the Invention

[0005] To address the issue of inaccurate identification of the triploid variety Yunyan 77-4, this disclosure provides a molecular marker primer set, kit, method, and application for identifying Yunyan 77-4. The technical solution is as follows:

[0006] On the one hand, this disclosure provides a molecular marker primer set for identifying the triploid rubber tree variety Yunyan 77-4. The molecular marker primer set consists of two pairs of primers, each pair consisting of an upstream primer and a downstream primer. The upstream primer of the first pair of primers is shown in SEQ ID NO:1 in the sequence listing, and the downstream primer of the first pair of primers is shown in SEQ ID NO:2 in the sequence listing. The upstream primer of the second pair of primers is shown in SEQ ID NO:3 in the sequence listing, and the downstream primer of the second pair of primers is shown in SEQ ID NO:4 in the sequence listing.

[0007] On the other hand, this disclosure provides a kit for identifying the triploid rubber tree variety Yunyan 77-4, the kit comprising the aforementioned molecular marker primer set.

[0008] On another aspect, this disclosure provides a method for identifying the triploid rubber tree variety Yunyan 77-4, the method comprising: amplifying the sample to be tested using the above-mentioned molecular marker primer set to obtain the amplification product;

[0009] The amplified products were subjected to next-generation high-throughput sequencing to obtain sequencing data;

[0010] The sequencing data is aligned to the rubber tree reference genome to obtain the sequencing results of the sample to be tested;

[0011] The sequencing results were analyzed to obtain different sequence types;

[0012] When the first pair of primers is used for amplification, and the sequence type obtained is a combination of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 in the sequence listing, and when the second pair of primers is used for amplification, and the sequence type obtained is a combination of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 17 in the sequence listing, then the sample to be tested is identified as Rubber Tree Yunyan 77-4.

[0013] Specifically, the rubber tree reference genome is PRJNA587314.

[0014] In another aspect, this disclosure provides an application of the above-mentioned molecular marker primer set, the application of which includes using the molecular marker primer set to distinguish rubber tree varieties: Yunyan 77-4, GT1, PR107, Reken 628, Reken 106, PR228, Reken 501, Yunyan 98296, Yunyan 99229 and RRIM501.

[0015] On the other hand, this disclosure provides an application of the above-mentioned molecular marker primer set, the application of which includes using the molecular marker primer set for parental identification of the triploid rubber tree variety Yunyan 77-4.

[0016] The beneficial effects of the technical solution provided in this disclosure are as follows: This disclosure provides a molecular marker primer set, kit, method, and application for identifying the triploid rubber tree variety Yunyan 77-4. The primer set is labeled with MNP (Multiple Nucleotide Polymorphism) and amplified on the sample to be tested. The amplified products are sequenced, and the differences in different base sequences are analyzed based on the sequencing results to determine whether the variety to be tested is Yunyan 77-4. The identification results are not affected by environmental or other human factors, thereby achieving accurate identification of Yunyan 77-4. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of this disclosure, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a comparison diagram of the a1 sequence provided in Embodiment 3 of this disclosure and the sequences named b1~h1 amplified by the first pair of primers;

[0019] Figure 2 This is a comparison diagram of the a2 sequence provided in Embodiment 3 of this disclosure and the sequences named b2~e2 amplified by the second pair of primers. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this disclosure clearer, the embodiments of this disclosure will be described in further detail below with reference to the accompanying drawings.

[0021] Example 1

[0022] This disclosure provides a molecular marker primer set for identifying the triploid rubber tree variety Yunyan 77-4. The primer set consists of two pairs of primers, each pair comprising an upstream primer and a downstream primer. The upstream primer of the first pair is shown in SEQ ID NO:1 of the sequence listing, specifically: AAGATTAGCCCCAAAAGTTGCTAAC. The downstream primer of the first pair is shown in SEQ ID NO:2 of the sequence listing, specifically: AATCTGTCGGGTTTAGTGGAGGTAT. The upstream primer of the second pair is shown in SEQ ID NO:3 of the sequence listing, specifically: ACTGAGATGCATTGCACAATCAATA. The downstream primer of the second pair is shown in SEQ ID NO:4 of the sequence listing, specifically: TGGAAGCTCCGTTTATAGAAGCTTA. Relevant information for the two primer pairs is shown in Table 1.

[0023] Table 1 shows the relevant information for the two pairs of primers.

[0024]

[0025] Example 2

[0026] This disclosure provides a kit for identifying the triploid rubber tree variety Yunyan 77-4, which includes the molecular marker primer set provided in Example 1.

[0027] Example 3

[0028] This disclosure provides a method for identifying the triploid rubber tree variety Yunyan 77-4, which includes using the molecular marker primers provided in Example 1 to identify Yunyan 77-4.

[0029] In this embodiment, 17 rubber tree varieties were used as the test samples. The specific variety names are shown in Table 2.

[0030] Table 2 lists the variety names of the 17 samples to be tested.

[0031]

[0032] DNA was extracted from the 17 samples to be tested. In practice, the DNA of the samples was extracted using a novel plant genomic DNA extraction kit manufactured by Tiangen Biotech (Beijing) Co., Ltd., to obtain DNA fragments from the samples. Specific procedures were performed according to the instructions for this novel plant genomic DNA extraction kit.

[0033] Seventeen test samples were amplified using the labeled primers provided in Example 1 of this invention to obtain amplification products. Specifically, the amplification system included: 50 ng of DNA fragment from the test sample, 0.5 μL of 10 mM dNTP (Deoxyribonucleoside triphosphate), 0.5 μL of upstream primer, 0.5 μL of downstream primer, 2.5 μL of Tap Buffer, 0.2 μL of Taq enzyme, and ddH2O was added to bring the amplification system to 20 μL. The amplification program included: 95℃ for 3 min; (95℃ for 30 sec, 60℃ for 30 sec) × 30 cycles; 72℃ for 6 min. The amplification products were subjected to next-generation high-throughput sequencing, and the sequencing data were aligned to the rubber tree reference genome PRJNA587314 using Bowtie2 software to obtain the sequencing results for each test sample.

[0034] Based on the upstream and downstream primer sequences of the first primer pair, e-PCR was performed on the reference genome using TBtools software to obtain the chromosomal location, start point, and end point of the target sequence. The target sequence of the first primer pair on the reference genome was then extracted using TBtools software and named a1 (reference genome sequence).

[0035] Using Microsoft Office Excel, the sequencing results of 17 samples from the first primer pair were analyzed, revealing seven different sequence types. These seven sequence types were named b1, c1, d1, e1, f1, g1, and h1, as shown in Table 3.

[0036] Table 3 shows the reference genome sequence a1 and the different sequences obtained by amplification with the first pair of primers.

[0037]

[0038]

[0039] The sequence types in Table 3 were compared using the software DANMAN. The comparison results are as follows: Figure 1 As shown. By Figure 1 It can be seen that there are 7 base positions that differ.

[0040] Using TBtools software, the second pair of primers was used for e-PCR on the reference genome to obtain the chromosomal location, start point, and end point of the target sequence. The corresponding base sequence was then extracted using this software and named a2 (reference genome sequence).

[0041] The sequencing results of 17 samples using the second primer pair were analyzed using Microsoft Office Excel software, revealing four different sequence types. These four sequence types were named b2, c2, d2, and e2, as shown in Table 4.

[0042] Table 4 shows the different sequences obtained after amplification of the reference genome sequence a2 and the second primer pair from 17 test samples.

[0043]

[0044] The sequence types in Table 4 were compared using the software DANMAN. The comparison results are as follows: Figure 2 As shown. By Figure 2 It can be seen that sequence b2 is consistent with the reference genome a2, with 5 base positions differing.

[0045] Table 5 shows the genotypes obtained after amplification of 17 samples using two primer pairs.

[0046]

[0047] In Table 5, the genotype " / " for the first primer pair of Reyan 106 indicates that no sequence was amplified, meaning that the first primer pair did not amplify the target site on the Reyan 106 genome. The sequence at this site differs from other varieties, representing a polymorphism. When amplification is performed using the first primer pair, the sequence types obtained are combinations shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 in the sequence listing. When amplification is performed using the second primer pair, the sequence types obtained are combinations shown in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 17 in the sequence listing. Therefore, the sample to be tested is identified as rubber tree Yunyan 77-4. This method can accurately identify the triploid rubber tree variety Yunyan 77-4.

[0048] Example 4

[0049] Analysis of the genotype combinations obtained after amplification of 17 samples using two primer pairs revealed three repetitive genotype combinations: g1+c2d2 for Zhanshi 32713, Dongfang 7747, and Yunyan 41; h1+b2d2 for IAN873 and Yunyan 590; and f1+c2 for PR300 and IRCI22. In addition, 10 varieties showed unique genotype combinations. This means that the MNP marker primer set disclosed herein can identify the triploid variety Yunyan 77-4, and can also distinguish GT1, PR107, Reken 628, Reken 106, PR228, Reken 501, Yunyan 98296, Yunyan 99229, and RRIM501. Details are shown in Table 6.

[0050] Table 6 Genotype combinations obtained after amplification of 26 samples using two primer pairs.

[0051]

[0052] Example 5

[0053] This disclosure provides an application of molecular marker primers for identifying the triploid rubber tree variety Yunyan 77-4. This application includes using the molecular marker primer set for parental identification of the triploid rubber tree variety Yunyan 77-4. Specifically, referring to Table 5, the genotype of the first primer pair for Yunyan 77-4 is b1c1d1 (triploid variety). Theoretically, its nuclear chromosome 2n has three possible genotypes: b1c1, c1d1, and b1d1. From the 17 samples provided in this embodiment, only the GT1 variety has b1c1; c1d1 and b1d1 are not found. Additionally, only the PR107 genotype contains d1. Therefore, the parents of Yunyan 77-4 are GT1 and PR107, and 2n originates from GT1.

[0054] Analyzing the genotypes of the second primer pair in Table 5, the genotype of the second primer pair for Yunyan 77-4 is b2c2e2 (triploid variety). Theoretically, its nuclear chromosome 2n has three possible genotypes: b2c2, c2e2, and b2e2. Searching through the 17 samples provided in this example, only variety GT1 has b2c2; c2e2 and b2e2 are not found. Furthermore, only PR107 has the genotype e2. This also proves that the parents of Yunyan 77-4 are GT1 and PR107, and that 2n originates from GT1.

[0055] Using the primer set provided in Example 1, it is possible to effectively identify that the nuclear chromosome of the triploid rubber tree variety Yunyan 77-4 originates from GT1 and PR107, and confirm that the 2n chromosome of the triploid variety Yunyan 77-4 originates from GT1, and can definitively rule out the possibility that other test samples are its parents.

[0056] Accuracy analysis of molecular marker primer set identification

[0057] Accuracy analysis was performed using two reproducibility experiments. In this embodiment, two independent experiments were conducted using different personnel, different batches of reagents, and different laboratories to simulate the identification of different batches. A high reproducibility rate means that the identification results from different laboratories can be accurately compared with each other.

[0058] A reproducibility experiment was conducted using 17 samples to be tested. The results of each experiment were analyzed, and the genotypes and combinations were recorded. See Table 7 for details.

[0059] Table 7 shows the results of the two repeated experiments.

[0060]

[0061] As shown in Table 7, the genotypes and combinations were identical in both replicate experiments. Therefore, the accuracy of the molecular marker primer set identification provided in the embodiments of this disclosure is 100%.

[0062] The identification conclusions of this invention, obtained by different personnel, batches, and laboratories, show high consistency, thus eliminating the need for parallel experiments to reduce experimental errors. This greatly facilitates the identification of triploid rubber tree varieties.

[0063] This invention provides a molecular marker primer set, kit, method, and application for identifying the triploid rubber tree variety Yunyan 77-4. The identification results are unaffected by environmental or other human factors, with a short identification cycle and high accuracy. The molecular marker primer set is used to amplify the test sample, and the amplified products are sequenced. Analysis of the sequencing results reveals multiple base differences between different varieties, enabling accurate identification of the triploid rubber tree variety Yunyan 77-4. The target sequence is amplified using a highly polymorphic molecular marker primer set, and the base differences between different varieties are compared to achieve the identification of the triploid rubber tree variety Yunyan 77-4. Reproducibility experiments further verify the accuracy and reliability of this technology.

[0064] The above description is merely an optional embodiment of this disclosure and is not intended to limit this disclosure. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the protection scope of this disclosure.

Claims

1. A molecular marker primer set for identifying the triploid rubber tree variety Yunyan 77-4, characterized in that, The molecular marker primer set consists of two pairs of primers, each pair consisting of an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer of the first pair of primers is shown in SEQ ID NO:1, the nucleotide sequence of the downstream primer of the first pair of primers is shown in SEQ ID NO:2, the nucleotide sequence of the upstream primer of the second pair of primers is shown in SEQ ID NO:3, and the nucleotide sequence of the downstream primer of the second pair of primers is shown in SEQ ID NO:

4.

2. A reagent kit for identifying the triploid rubber tree variety Yunyan 77-4, characterized in that, The kit includes the molecular marker primer set as described in claim 1.

3. A method for identifying the triploid rubber tree variety Yunyan 77-4, characterized in that, The method includes: amplifying the sample to be tested using the molecular marker primer set as described in claim 1 to obtain amplification products; The amplified products were subjected to next-generation high-throughput sequencing to obtain sequencing data; The sequencing data is aligned to the rubber tree reference genome to obtain the sequencing results of the sample to be tested; The sequencing results were analyzed to obtain different sequence types; When the first pair of primers is used for amplification, and a combination of nucleotide sequences such as SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 is obtained, and when the second pair of primers is used for amplification, and a combination of nucleotide sequences such as SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 17 is obtained, then the sample to be tested is identified as Yunyan 77-4.

4. The method according to claim 3, characterized in that, The reference genome for the rubber tree is PRJNA587314.

5. An application of the molecular marker primer set as described in claim 1, characterized in that, The application includes using the molecular marker primer set to distinguish rubber tree varieties: Yunyan 77-4, GT1, PR107, Reken 628, Reken 106, PR228, Reken 501, Yunyan 98296, Yunyan 99229 and RRIM501.

6. An application of the molecular marker primer set as described in claim 1, characterized in that, The application includes using the molecular marker primer set for parent identification of the triploid rubber tree variety Yunyan 77-4.

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