SNP marker primer group for identifying yuanling carya and detection method

Abstract

This invention provides a set of SNP marker primers and a detection method for identifying Yuanling walnuts, belonging to the field of molecular marker technology. This method provides 17 pairs of SNP primers (SNP1, SNP3, SNP4, SNP12, SNP13, SNP14, SNP19, SNP22, SNP25, SNP27, SNP28, SNP31, SNP33, SNP38, SNP39, SNP40, and SNP41) as PCR amplification primers. DNA extracted from fresh walnut leaves or silica gel-dried leaf samples is used as amplification template. Sequence analysis of the amplification products is performed. Each primer set can specifically label differential bases, thereby effectively distinguishing Yuanling walnuts from common Hunan walnuts. This enables rapid and accurate identification of Yuanling walnut seedlings, which is of great significance for protecting seedling intellectual property rights and promoting the high-quality development of the Hunan walnut industry in my country.

Description

Technical Field

[0001] This invention relates to the field of molecular marker technology, specifically to an SNP marker primer set and detection method for identifying Yuanling walnut. Background Technology

[0002] Hunan hickory is an important grain and oil crop with significant practical value, serving as a major source of income for people in the mountainous areas of southwestern my country. To address the issue of late fruiting and low yield among young and middle-aged Hunan hickory forests in western Hunan Province, the Hunan Provincial Botanical Garden conducted a survey, collection, and evaluation of wild Hunan hickory resources throughout the province. From the germplasm resources in Yuanling County, Huaihua City, a high-yielding and superior single plant was identified and named "Yuanling Hickory," whose yield is twice that of ordinary Hunan hickory.

[0003] SNP (Single Nucleotide Polymorphism) refers to DNA sequence polymorphism caused by a single base variation at the gene level. It belongs to the third generation of genetic marker technology and has advantages such as strong genetic stability, wide distribution, and ease of automation. It is currently widely used in germplasm identification. However, there are no reports on SNP markers for identifying Hunan walnut varieties or superior individual plants. Promoting "Yuanling walnut" can fundamentally solve the problem of low yield and value in the Hunan walnut industry. A basic prerequisite is the need to propagate a large number of seedlings through asexual reproduction methods such as grafting. "Yuanling walnut" and ordinary Hunan walnut have no significant differences in the botanical characteristics of their vegetative organs, especially leaf characteristics. Currently, they can only be distinguished by their fruit morphology and yield. However, this process is too time-consuming and cannot meet the need for rapid and accurate identification of seedling origin. Therefore, there is an urgent need to establish an accurate and efficient identification technology system for "Yuanling walnut". Summary of the Invention

[0004] To address the aforementioned technical challenges, a single nucleotide polymorphism (SNP) marker specific to "Yuanling walnut" was developed. This marker was then validated using common Hunan walnuts collected from different provenance areas. This specific SNP molecular marker enables rapid and accurate identification of "Yuanling walnut" seedlings, which is of great significance for protecting seedling intellectual property rights and promoting the high-quality development of the Hunan walnut industry in my country. This solution provides a primer set and detection method for identifying SNP markers in Yuanling walnuts.

[0005] To achieve the above objectives, this solution first provides a set of SNP marker primers for identifying Yuanling walnuts, wherein the set of SNP marker primers includes SNP1, SNP3, SNP4, SNP12, SNP13, SNP14, SNP19, SNP22, SNP25, SNP27, SNP28, SNP31, SNP33, SNP38, SNP39, SNP40, and SNP41.

[0006] The primer sequence of SNP1 is shown in SEQ ID NO.1 to SEQ ID NO.2, and the nucleotide base at position 246 of the amplified fragment of SNP1 is C;

[0007] The primer sequence of SNP3 is shown in SEQ ID NO.3 to SEQ ID NO.4, and the nucleotide base at position 283 of the amplified fragment of SNP3 is C;

[0008] The primer sequence of SNP4 is shown in SEQ ID NO.5 to SEQ ID NO.6, and the nucleotide bases at positions 198 and 235 of the amplified fragment of SNP4 are C and C, respectively.

[0009] The primer sequence of SNP12 is shown in SEQ ID NO.7 to SEQ ID NO.8. The nucleotide base at position 237 of the amplified fragment of SNP12 is base C.

[0010] The primer sequence of SNP13 is shown in SEQ ID NO.9 to SEQ ID NO.10, and the nucleotide base at position 185 of the amplified fragment of SNP13 is C;

[0011] The primer sequence of SNP14 is shown in SEQ ID NO.11 to SEQ ID NO.12, and the nucleotide base at position 113 of the amplified fragment of SNP14 is A;

[0012] The primer sequence for SNP19 is shown in SEQ ID NO.13 to SEQ ID NO.14, and the nucleotide base at position 173 of the amplified fragment of SNP19 is A;

[0013] The primer set sequence of SNP22 is shown in SEQ ID NO.15~SEQ ID NO.16, and the nucleotide base at position 239 of the SNP22-labeled amplified fragment is C.

[0014] The primer sequence for SNP25 is shown in SEQ ID NO.17 to SEQ ID NO.18, and the nucleotide bases at positions 241 and 262 of the amplified fragment of SNP25 are C and C, respectively.

[0015] The primer sequence for SNP27 is shown in SEQ ID NO.19 to SEQ ID NO.20, and the nucleotide base at position 194 of the amplified fragment of SNP27 is G;

[0016] The primer sequence for SNP28 is shown in SEQ ID NO.21 to SEQ ID NO.22, and the nucleotide base at position 196 of the amplified fragment of SNP28 is G;

[0017] The primer sequence of SNP31 is shown in SEQ ID NO.23~SEQ ID NO.24, and the nucleotide base at position 173 of the amplified fragment of SNP31 is C;

[0018] The primer sequence of SNP33 is shown in SEQ ID NO.25~SEQ ID NO.26, and the nucleotide base at position 196 of the amplified fragment of SNP33 is T;

[0019] The primer sequence of SNP38 is shown in SEQ ID NO.27~SEQ ID NO.28, and the nucleotide base at position 198 of the amplified fragment of SNP38 is G;

[0020] The primer sequence of SNP39 is shown in SEQ ID NO.29~SEQ ID NO.30, and the nucleotide base at position 210 of the amplified fragment of SNP39 is A;

[0021] The primer sequence of SNP40 is shown in SEQ ID NO.31 to SEQ ID NO.32, and the nucleotide base at position 174 of the amplified fragment of SNP40 is T;

[0022] The primer sequence of SNP41 is shown in SEQ ID NO.33 to SEQ ID NO.34, and the 69th nucleotide base of the amplified fragment of SNP41 is T.

[0023] Based on a general inventive concept, this solution also provides a detection method for identifying SNP marker primer sets of Yuanling walnuts, characterized by the following steps:

[0024] S1. Collect fresh leaves or silica gel-dried leaves of the Hunan walnut to be tested as samples and extract DNA;

[0025] S2. Using the DNA extracted in step S1 as an amplification template, PCR amplification is performed using the 17 SNP marker primer sets in claim 1 as amplification primers.

[0026] S3. Perform sequence analysis on the products amplified in step S2, detect the base differences of each group of SNP markers, and determine whether the Hunan walnut to be tested is Yuanling walnut.

[0027] Preferably, the fresh tissue in step S1 includes young leaves.

[0028] Preferably, the PCR amplification system in step S2 is as follows: 25 μL of 2×PhantaMax buffer, 1 μL of 10 μmol / L dNTP mixture, 1 μL each of 10 μmol / L upstream and downstream primers, 1 μL of 1 U / μL PhantaMax Super-Fidelity high-fidelity DNA polymerase, 19 μL of ddH2O, and 2 μL of DNA from the test sample at 50 ng / μL, for a total system volume of 50 μL.

[0029] Preferably, the PCR amplification program in step S2 is as follows: pre-denaturation at 4℃ for 3 min; denaturation at 94℃ for 0.5 min, annealing at 52-58℃ for 0.5 min, extension at 72℃ for 0.5 min, for 35 cycles; and finally extension at 72℃ for 5 min.

[0030] Preferably, the method for determining whether the Hunan walnut to be tested is Yuanling walnut in step S3 is as follows: if at least one SNP marker site in the amplification product of the Hunan walnut to be tested has a base that matches the base of the 17 SNP marker sites described in claim 1, then the Hunan walnut to be tested is Yuanling walnut.

[0031] The principle behind this SNP-marked primer set for identifying Yuanling walnuts is as follows:

[0032] This scheme designs a set of SNP-labeled primers to amplify the DNA of Yuanling walnut samples, specifically labeling differentially expressed bases. For example, SNP1 labels the 246th base of the amplified fragment, which is C in Yuanling walnuts; SNP3 labels the 283rd base of the amplified fragment, which is C in Yuanling walnuts; SNP4 labels the 198th and 235th bases of the amplified fragment, which are C and C in Yuanling walnuts, respectively; SNP12 labels the amplified fragment... The 237th base of the amplified fragment is C (from Yuanling walnut); the 185th base of the amplified fragment labeled by SNP13 is C (from Yuanling walnut); the 113th base of the amplified fragment labeled by SNP14 is A (from Yuanling walnut); the 173rd base of the amplified fragment labeled by SNP19 is A (from Yuanling walnut); the 239th base of the amplified fragment labeled by SNP22 is C (from Yuanling walnut); the 239th base of the amplified fragment labeled by SNP25 is C (from Yuanling walnut); the 237th base of the amplified fragment labeled by SNP25 is C (from Yuanling walnut). The 241st and 262nd bases of the amplified fragment correspond to C and C in Yuanling walnuts; SNP27 labels the 194th base of the amplified fragment, which corresponds to G in Yuanling walnuts; SNP28 labels the 196th base of the amplified fragment, which corresponds to G in Yuanling walnuts; SNP31 labels the 173rd base of the amplified fragment, which corresponds to C in Yuanling walnuts; SNP33 labels the 196th base of the amplified fragment, which corresponds to T in Yuanling walnuts. SNP38 marks the 198th base of the amplified fragment, which corresponds to base G in Yuanling walnut; SNP39 marks the 210th base of the amplified fragment, which corresponds to base A in Yuanling walnut; SNP40 marks the 174th base of the amplified fragment, which corresponds to base T in Yuanling walnut; SNP41 marks the 69th base of the amplified fragment, which corresponds to base T in Yuanling walnut. Therefore, based on the differential bases of these SNP markers, Yuanling walnut can be specifically identified.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) The SNP molecular markers of the present invention are not affected by the plant development period, growth stage and environment. Only a small amount of fresh leaves or silica gel-dried leaf samples are needed to accurately and quickly identify the sample to be tested. Under the premise of complete instruments and equipment, the test results can be obtained in only 4 to 6 hours.

[0035] (2) This invention establishes SNP markers for the first time to identify Yuanling walnut germplasm resources in early tissues such as young leaves, effectively protecting the rights and interests of breeders and providing technical support for the efficient protection of Yuanling walnut germplasm resources and rapid identification of seedlings. Attached Figure Description

[0036] 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 some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 Comparison of SNP1 marker amplification sequence and sequencing peak diagram in Experiment Example 1;

[0038] Figure 2 Comparison of SNP3 marker amplification sequence and sequencing peak diagram in Experiment Example 1;

[0039] Figure 3 Comparison of SNP4 marker amplification sequence and sequencing peak diagram in Experiment Example 1;

[0040] Figure 4 Comparison of SNP12 marker amplification sequence and sequencing peak diagram in Experiment Example 1;

[0041] Figure 5 Comparison of SNP13 marker amplification sequence and sequencing peak diagram in Experiment Example 1;

[0042] Figure 6 Comparison of SNP14 marker amplification sequence and sequencing peak diagram in Experiment Example 1;

[0043] Figure 7 Comparison of SNP19 marker amplification sequence and sequencing peak diagram in Experiment Example 1;

[0044] Figure 8 Comparison of SNP22 marker amplification sequence and sequencing peak diagram in Experiment Example 1;

[0045] Figure 9 Comparison of SNP25 marker amplification sequence and sequencing peak diagram in Experiment Example 1;

[0046] Figure 10 Comparison of SNP27 marker amplification sequence and sequencing peak diagram in Experiment Example 1;

[0047] Figure 11 Comparison of SNP28 marker amplification sequence and sequencing peak diagram in Experiment Example 1;

[0048] Figure 12 Comparison of SNP31 marker amplification sequence and sequencing peak diagram in Experiment Example 1;

[0049] Figure 13 Comparison of SNP33 marker amplification sequence and sequencing peak diagram in Experiment Example 1;

[0050] Figure 14Comparison of SNP38 marker amplification sequence and sequencing peak diagram in Experiment Example 1;

[0051] Figure 15 Comparison of SNP39 marker amplification sequence and sequencing peak diagram in Experiment Example 1;

[0052] Figure 16 Comparison of SNP40 marker amplification sequence and sequencing peak diagram in Experiment Example 1;

[0053] Figure 17 Comparison of SNP41 marker amplification sequence and sequencing peak diagram for Experiment Example 1. Detailed Implementation

[0054] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0055] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0056] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the embodiments are all commercially available.

[0057] Example 1

[0058] Obtaining the SNP marker primer set for identifying Yuanling walnut

[0059] 1. Assemble the chloroplast genome of Yuanling walnut

[0060] Tender leaves of Yuanling walnut were collected, and total DNA was extracted using a modified CTAB method. After DNA testing was passed, the DNA was fragmented using mechanical fragmentation (ultrasound). Fragments of 400–600 bp were then purified, end-repaired, 3′-A-added, and sequencing adapters ligated. Fragment size selection was performed using agarose gel electrophoresis, followed by PCR amplification to form sequencing libraries. The constructed libraries underwent quality control. Libraries that passed quality control were sequenced using the Illumina NovaSeq 6000 platform with paired-end (PE) sequencing at a read length of 150 bp, ensuring a sequencing depth of at least 30 × (no less than 10 Gb of data).

[0061] The raw data is filtered using the fastp (version 0.20.0, https: / / github.com / OpenGene / fastp) software. This includes removing sequencing adapters and primer sequences from the reads, filtering out reads with an average quality value less than Q5, and filtering out reads with more than 5 consecutive undetected bases. The high-quality reads obtained after this series of quality control measures are called Clean Data.

[0062] The high-throughput sequencing data were assembled using SPAdes 3.10.1 software with a k-mer parameter set to 95. Using the published walnut chloroplast genome sequence as a reference, fragments belonging to the chloroplast genome were selected from the assembled data using Sequencher v5.4 software for preliminary assembly. The preliminary assembly results were mapped to the original reads using Geneious R 10.2.3 software to complete the splicing and assembly of the chloroplast genome, which was then manually proofread. The chloroplast genome was annotated and proofread using Plann and Sequin software, thus obtaining the complete Yuanling walnut chloroplast genome.

[0063] 2. Discovering specific SNP sites in Yuanling walnut

[0064] The chloroplast genome sequences of four common Hunan walnuts were downloaded from GenBank, with accession numbers MT955359.1, MW298527.1, NC046435.1, and MH188303.1, respectively. Then, multiple alignments of the chloroplast genomes of Yuanling walnut and the four common Hunan walnuts were performed using MEGA 7.0 software, and the alignment results were exported in EXCEL format. Yuanling walnut-specific SNP sites were manually screened.

[0065] 3. Design of PCR primers for specific SNP sites in Yuanling walnut

[0066] Using the chloroplast genome sequence of *Juglans regia* from Yuanling as a template, PCR primers for specific SNP sites were designed using Primer Premier 6.0 software. The upstream and downstream primers were spaced 50 to 200 bases away from the SNP sites to ensure the accuracy of subsequent sequencing.

[0067] Experimental Example 1

[0068] Identifying Yuanling hickory and common Hunan hickory using SNP-marked primer sets

[0069] 1. Experimental Materials

[0070] Tender leaves were collected from Yuanling hickory, common Hunan hickory, Jingzhou County, Huitong County, Tongdao County, and Liping County, Guizhou Province, totaling 32 materials, for SNP marker screening and specificity verification.

[0071] 2. SNP marker detection

[0072] 2.1 DNA Extraction

[0073] Total DNA was extracted from 32 samples of Yuanling walnut and common Hunan walnut using a modified CTAB method.

[0074] 2.2 PCR amplification

[0075] Using the DNA extracted from Yuanling walnuts and common Hunan walnuts in step 2.1 as templates, PCR amplification was performed using 17 pairs of specific marker primers. The primers are shown in Table 1 below:

[0076] Table 1. Specific primer information

[0077]

[0078]

[0079] 2.3 PCR reaction system

[0080] The PCR reaction system consisted of 50 μL, including 25 μL of 2×PhantaMax buffer, 1 μL of dNTP mixture (10 μmol / L), 1 μL each of forward and reverse primers (10 μmol / L), 1 μL of PhantaMax Super-Fidelity high-fidelity DNA polymerase (1 U / μL), 19 μL of ddH2O, and 2 μL of DNA from the test material at a concentration of 50 ng / μL.

[0081] 2.4 PCR reaction procedure

[0082] Pre-denaturation at 94℃ for 3 min; denaturation at 94℃ for 0.5 min, annealing at 52-58℃ for 0.5 min, extension at 72℃ for 0.5 min, for 35 cycles; final extension at 72℃ for 5 min, and storage at 4℃. PCR amplification products were electrophoresed on 1% agarose gel (5 μL of GelRed 10,000× stock solution added to every 50 mL of agarose solution) at 100V for 40 min in 1×TAE buffer. Electrophoresis results were recorded using a gel imaging system. If the size of the PCR product matched the expected value, the target nucleic acid fragment was considered successfully amplified.

[0083] 2.5 Sequencing of PCR products

[0084] The successfully amplified PCR products were subjected to Sanger first-generation sequencing using an ABI 3730XL sequencer.

[0085] 2.6 Sequence Analysis

[0086] The sequencing peak image file was read using the DNA sequencing analysis software Chromas, and multiple sequence alignment was performed using DNAMAN 9.0 software.

[0087] 3. Specific identification and detection analysis of SNP markers in Yuanling walnut

[0088] 3.1 SNP1 marker detection results

[0089] Table 2 and Figure 1 The results showed that the 246th base of the SNP1 marker amplification fragment in the 32 tested materials was base C in Yuanling walnut and base A in common Hunan walnut.

[0090] Table 2 Detection results of SNP1 markers

[0091]

[0092] 3.2 SNP3 marker detection results

[0093] Table 3 and Figure 2 The results showed that the 283rd base of the SNP3 marker amplification fragment in the 32 tested materials was C in Yuanling walnut and T in common Hunan walnut.

[0094] Table 3. Detection results of SNP3 markers

[0095]

[0096] 3.3 SNP4 marker detection results

[0097] Table 4 and Figure 3 The results showed that the 198th and 235th bases of the SNP4 marker amplification fragments in the 32 tested materials were C and C for Yuanling walnuts, and A and A for common Hunan walnuts.

[0098] Table 4 shows the detection results of SNP4 markers.

[0099]

[0100]

[0101] 3.4 SNP12 marker detection results

[0102] Table 5 and Figure 4The results showed that the 237th base of the SNP12 marker amplification fragment in the 32 tested materials was C in Yuanling walnut and T in common Hunan walnut.

[0103] Table 5 Detection results of SNP12 markers

[0104]

[0105] 3.5 SNP13 marker detection results

[0106] Table 6 and Figure 5 The results showed that the 185th base of the SNP13 marker amplification fragment in the 32 tested materials was base C in Yuanling walnut and base A in common Hunan walnut.

[0107] Table 6 Detection results of SNP13 markers

[0108]

[0109]

[0110] 3.6 SNP14 marker detection results

[0111] Table 7 and Figure 6 The results showed that the 113th base of the SNP14 marker amplification fragment in the 32 tested materials was base A in Yuanling walnut and base T in common Hunan walnut.

[0112] Table 7 Detection results of SNP14 markers

[0113]

[0114]

[0115] 3.7 SNP19 marker detection results

[0116] Table 8 and Figure 7 The results showed that the 173rd base of the SNP19 marker amplification fragment in the 32 tested materials was base A in Yuanling walnut and base T in common Hunan walnut.

[0117] Table 8 Detection results of SNP19 markers

[0118]

[0119] 3.8 SNP22 marker detection results

[0120] Table 9 and Figure 8 The results showed that the 239th base of the SNP22 marker amplification fragment in the 32 tested materials was C in Yuanling walnut and T in common Hunan walnut.

[0121] Table 9 Detection results of SNP22 markers

[0122]

[0123]

[0124] 3.9 SNP25 marker detection results

[0125] Table 10 and Figure 9 The results showed that the 241st and 262nd bases of the SNP25-labeled amplified fragments in the 32 tested materials were C and C in Yuanling walnut and A and A in common Hunan walnut.

[0126] Table 10 Detection results of SNP25 markers

[0127]

[0128] 3.10 SNP27 marker detection results

[0129] Table 11 and Figure 10 The results showed that the 194th base of the SNP27 marker amplification fragment in the 32 tested materials was base G in Yuanling walnut and base T in common Hunan walnut.

[0130] Table 1 Detection results of SNP27 markers

[0131]

[0132] 3.11 SNP28 marker detection results

[0133] Table 12 and Figure 11 The results showed that the 196th base of the SNP28-labeled amplified fragment in the 32 tested materials was base G in Yuanling walnut and base T in common Hunan walnut.

[0134] Table 12 Detection results of SNP28 markers

[0135]

[0136]

[0137] 3.12 SNP31 marker detection results

[0138] Table 13 and Figure 12 The results showed that the 173rd base of the SNP31-labeled amplified fragment in the 32 tested materials was C in Yuanling walnut and T in common Hunan walnut.

[0139] Table 13 Detection results of SNP31 markers

[0140]

[0141] 3.13 SNP33 marker detection results

[0142] Table 14 and Figure 13 The results showed that the 196th base of the SNP33-labeled amplified fragment in the 32 tested materials was T in Yuanling walnut and C in common Hunan walnut.

[0143] Table 14 Detection results of SNP33 markers

[0144]

[0145]

[0146] 3.14 SNP38 marker detection results

[0147] Table 15 and Figure 14 The results showed that the 198th base of the SNP38-labeled amplified fragment in the 32 tested materials was base G in Yuanling walnut and base T in common Hunan walnut.

[0148] Table 15 Detection results of SNP38 markers

[0149]

[0150] 3.15 SNP39 marker detection results

[0151] Table 16 and Figure 15 The results showed that the 210th base of the SNP39 marker amplification fragment in the 32 tested materials was base A in Yuanling walnut and base G in common Hunan walnut.

[0152] Table 16 Detection results of SNP39 markers

[0153]

[0154] 3.16 SNP40 marker detection results

[0155] Table 17 and Figure 16 The results showed that the 174th base of the SNP40-labeled amplified fragment in the 32 tested materials was T in Yuanling walnut and G in common Hunan walnut.

[0156] Table 17 Detection results of SNP40 markers

[0157]

[0158]

[0159] 3.17 SNP41 marker detection results

[0160] Table 18 and Figure 17 The results showed that the 69th base of the SNP41-labeled amplified fragment in the 32 tested materials was base T in Yuanling walnut and base A in common Hunan walnut.

[0161] Table 18 Detection results of SNP41 markers

[0162]

[0163] The base differences of the above 17 pairs of SNP primers can achieve specific labeling of Yuanling walnuts, with the advantages of accurate and rapid identification.

[0164] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. For those skilled in the art, any improvements and modifications obtained without departing from the technical concept of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting SNP marker primer sets for identifying Yuanling walnuts, characterized in that, Includes the following steps: S1. Collect fresh leaves or silica gel-dried leaves of the Hunan walnut to be tested as samples and extract DNA; S2. Using the DNA extracted in step S1 as an amplification template, PCR amplification was performed using the SNP-labeled primer set as amplification primers. S3. Perform sequence analysis on the products amplified in step S2, detect the base differences of each group of SNP markers, and determine whether the Hunan walnut to be tested is Yuanling walnut. The SNP marker primer set is SNP1, SNP3, SNP4, SNP12, SNP13, SNP14, SNP19, SNP22, SNP25, SNP27, SNP28, SNP31, SNP33, SNP38, SNP39, SNP40, SNP41; The primer sequence of SNP1 is shown in SEQ ID NO.1 to SEQ ID NO.2, and the nucleotide base at position 246 of the amplified fragment of SNP1 is C; The primer sequence of SNP3 is shown in SEQ ID NO. 3 to SEQ ID NO. 4, and the nucleotide base at position 283 of the amplified fragment of SNP3 is C; The primer sequence of SNP4 is shown in SEQ ID NO. 5 to SEQ ID NO.

6. The nucleotide bases at positions 198 and 235 of the amplified fragment of SNP4 are C and C, respectively. The primer sequence of SNP12 is shown in SEQ ID NO. 7 to SEQ ID NO.

8. The nucleotide base at position 237 of the amplified fragment of SNP12 is base C. The primer sequence of SNP13 is shown in SEQ ID NO. 9 to SEQ ID NO.

10. The nucleotide base at position 185 of the amplified fragment of SNP13 is C. The primer sequence of SNP14 is shown in SEQ ID NO. 11 to SEQ ID NO.

12. The nucleotide base at position 113 of the amplified fragment of SNP14 is A. The primer sequence for SNP19 is shown in SEQ ID NO. 13 to SEQ ID NO. 14, and the nucleotide base at position 173 of the amplified fragment of SNP19 is A; The primer set sequence of SNP22 is shown in SEQ ID NO. 15 to SEQ ID NO.

16. SNP22 marks the nucleotide base at position 239 of the amplified fragment as base C. The primer sequence of SNP25 is shown in SEQ ID NO. 17 to SEQ ID NO.

18. The nucleotide bases at positions 241 and 262 of the amplified fragment of SNP25 are C and C, respectively. The primer sequence for SNP27 is shown in SEQ ID NO. 19 to SEQ ID NO.

20. The nucleotide base at position 194 of the amplified fragment of SNP27 is G. The primer sequence for SNP28 is shown in SEQ ID NO. 21 to SEQ ID NO. 22, and the nucleotide base at position 196 of the amplified fragment of SNP28 is G; The primer sequence of SNP31 is shown in SEQ ID NO. 23 to SEQ ID NO.

24. The nucleotide base at position 173 of the amplified fragment of SNP31 is C. The primer sequence of SNP33 is shown in SEQ ID NO. 25 to SEQ ID NO. 26, and the nucleotide base at position 196 of the amplified fragment of SNP33 is T; The primer sequence of SNP38 is shown in SEQ ID NO. 27 to SEQ ID NO. 28, and the nucleotide base at position 198 of the amplified fragment of SNP38 is G; The primer sequence of SNP39 is shown in SEQ ID NO. 29 to SEQ ID NO. 30, and the nucleotide base at position 210 of the amplified fragment of SNP39 is A; The primer sequence of SNP40 is shown in SEQ ID NO. 31 to SEQ ID NO. 32, and the nucleotide base at position 174 of the amplified fragment of SNP40 is T; The primer sequence of SNP41 is shown in SEQ ID NO. 33 to SEQ ID NO. 34, and the 69th nucleotide base of the amplified fragment of SNP41 is T; The method for determining whether the Hunan walnut to be tested is Yuanling walnut in step S3 is as follows: if at least one SNP marker site in the amplification product of the Hunan walnut to be tested has a base that matches the base of the 17 SNP marker sites, then the Hunan walnut to be tested is Yuanling walnut.

2. The detection method according to claim 1, characterized in that, The PCR amplification system in step S2 is as follows: 25 μL of 2×PhantaMax buffer, 1 μL of 10 μmol / L dNTP mixture, 1 μL each of 10 μmol / L upstream and downstream primers, 1 μL of 1 U / μL PhantaMax Super-Fidelity high-fidelity DNA polymerase, 19 μL of ddH2O, and 2 μL of DNA from the test sample at 50 ng / μL, for a total system volume of 50 μL.

3. The detection method according to claim 1, characterized in that, The PCR amplification procedure in step S2 is as follows: pre-denaturation at 4℃ for 3 min; denaturation at 94℃ for 0.5 min, annealing at 52~58℃ for 0.5 min, extension at 72℃ for 0.5 min, for 35 cycles; and finally extension at 72℃ for 5 min.

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