A set of ssr molecular marker primers for identifying yuanling carya and a detection method thereof

Abstract

This invention provides an SSR molecular marker primer set and detection method for identifying Yuanling walnut, belonging to the field of molecular marker technology. It employs eight pairs of SSR molecular marker primers, using leaves as material, extracting DNA as an amplification template, and using the primer set as amplification primers. The size and SSR sites of the SSR marker amplification fragments in Yuanling walnut differ from those in common Hunan walnut. Therefore, the eight pairs of SSR molecular marker primers designed in this scheme can specifically identify Yuanling walnut. The SSR molecular markers exhibit good stability and are easy to operate, unaffected by plant development stage, growth stage, or environment. Only a small amount of fresh leaves or rapidly dried leaf samples using silica gel are needed for accurate and rapid identification of the sample. This achieves rapid and accurate identification of Yuanling walnut seedlings, which is of great significance for 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 SSR marker primer set for identifying Yuanling walnuts and its detection method. Background Technology

[0002] Hunan hickory (Carya hunanensis Cheng.) is one of the five species of the genus Carya Nutt. native to my country. It has a large wild distribution in western and southwestern Hunan, southeastern and southern Guizhou, and northwestern Guangxi, and is one of the later-developed wild fruit tree resources of the genus Carya in my country. Hunan hickory is an important grain and oil crop with significant practical value, and is a major source of income for people in the mountainous areas of southwestern my country. To address the problem of late fruiting and low yield in young 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 wild germplasm resources in Yuanling County, Huaihua City, a high-yielding superior single plant was identified and named "Yuanling Hickory," whose yield is twice that of ordinary Hunan hickory.

[0003] Molecular markers are powerful tools for studying plant genetic diversity, offering advantages such as high accuracy, good repeatability, and immunity to environmental interference. Simple sequence repeats (SSRs), also known as microsatellites, are co-dominant molecular markers characterized by ease of operation, high accuracy, and good stability. They are widely used in the analysis of plant genetic diversity and phylogenetic relationships. Therefore, it is essential to develop SSR markers for identifying Hunan pecan germplasm resources or superior individual plants. Promoting "Yuanling pecan" can fundamentally solve the problem of low yield and value in the Hunan pecan industry. However, a basic prerequisite is the need to propagate a large number of seedlings through asexual reproduction methods such as grafting. "Yuanling pecan" and common Hunan pecan show no significant differences in the botanical characteristics of their vegetative organs, especially leaf features. Currently, they can only be distinguished by fruit morphology and yield, which is too time-consuming and susceptible to the influence of plant growth and development and the environment, failing to meet the need for rapid and accurate identification of seedling origin. Therefore, establishing an accurate and efficient identification technology system for "Yuanling Mountain Walnut" is of great significance for protecting the intellectual property rights of seedlings and promoting the high-quality development of the Hunan mountain walnut industry in my country. Summary of the Invention

[0004] To address the aforementioned technical challenges and achieve rapid and accurate identification of Yuanling walnut seedlings, this invention provides an SSR molecular marker primer set and its detection method for identifying Yuanling walnuts. By analyzing the chloroplast genome sequences of Yuanling walnuts and common Hunan walnuts from different provenances, a Yuanling walnut-specific simple sequence repeat (SSR) length polymorphism genetic marker is developed. Simultaneously, the developed marker is validated using common Hunan walnut materials collected from different provenances. This specific SSR 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.

[0005] To achieve the above objectives, the present invention first provides an SSR molecular marker primer set for identifying Yuanling walnut, the SSR molecular marker primer set including ChSSR2, ChSSR6, ChSSR8, ChSSR10, ChSSR15, ChSSR30, ChSSR32, and ChSSR34.

[0006] The primer sequence of ChSSR2 is shown in SEQ ID NO.1 to SEQ ID NO.2;

[0007] The primer sequence of ChSSR6 is shown in SEQ ID NO.3 to SEQ ID NO.4;

[0008] The primer sequence of ChSSR8 is shown in SEQ ID NO.5 to SEQ ID NO.6;

[0009] The primer sequence of ChSSR10 is shown in SEQ ID NO.7 to SEQ ID NO.8;

[0010] The primer sequence of ChSSR15 is shown in SEQ ID NO.9 to SEQ ID NO.10;

[0011] The primer sequence of ChSSR30 is shown in SEQ ID NO.11 to SEQ ID NO.12;

[0012] The primer sequence of ChSSR32 is shown in SEQ ID NO.13~SEQ ID NO.14;

[0013] The primer sequence of ChSSR34 is shown in SEQ ID NO.15 to SEQ ID NO.16.

[0014] Based on a general inventive concept, this solution also provides a detection method for identifying Yuanling walnut using SSR molecular marker primer sets, including the following steps:

[0015] S1. Extract DNA from fresh leaves or silica gel-dried leaves of the Hunan walnut samples to be tested.

[0016] S2. Using the genomic DNA obtained in step S1 as a template, PCR amplification was performed using the primer set ChSSR2, ChSSR6, ChSSR8, ChSSR10, ChSSR15, ChSSR30, ChSSR32, and ChSSR348 as amplification primers.

[0017] S3. Perform sequence analysis on the amplification products of step S2, detect and analyze the size of the amplified fragments of the above 8 SSR markers and SSR sites, and determine whether the Hunan walnut to be tested is Yuanling walnut.

[0018] Preferably, the PCR amplification system in step S2 is as follows: 2 μL of 50 ng / μL DNA template to be tested, 1 μL each of 10 μmol / L upstream and downstream primers, 25 μL of 2×Phanta Max buffer, 19 μL of ddH2O, 1 μL of 1 U / μL Phanta Max Super-Fidelity high-fidelity DNA polymerase, and 1 μL of 10 μmol / L dNTP mixture, with a total system volume of 50 μL.

[0019] Preferably, the PCR amplification program in step S2 is as follows: pre-denaturation at 94℃ for 3 min; denaturation at 94℃ for 0.5 min, annealing at 48.2–57.0℃ for 0.5 min, extension at 72℃ for 0.5 min, for 35 cycles; and finally extension at 72℃ for 5 min.

[0020] Preferably, the method for determining whether the Hunan walnut to be tested is a Yuanling walnut in step S3 is as follows:

[0021] If the ChSSR2 marker amplification product size is 398 bp, and the SSR site is A (11) That is, the number of A repeats in the sequence of the amplified product is 11;

[0022] Alternatively, the ChSSR6-labeled amplification product is 385 bp in size, with the SSR site being T. (11) That is, the number of T repeats in the sequence of the amplified product is 11;

[0023] Alternatively, the ChSSR8-labeled amplified fragment is 445 bp in size, with the SSR site being T. (11) That is, the number of T repeats in the sequence of the amplified product is 11;

[0024] Alternatively, the ChSSR10-labeled amplified fragment is 484 bp in size, with the SSR site being A. (8) That is, the number of A repeats in the sequence of the amplified product is 8;

[0025] Or the SSR sites labeled with ChSSR15 are A (11) , and T (10) That is, the number of A repeats in the sequence of the amplified product is 11, and the number of T repeats in the sequence of the amplified product is 10.

[0026] Alternatively, the ChSSR30-labeled amplified fragment is 292 bp in size, with the SSR site being T. (10) That is, the number of T repeats in the sequence of the amplified product is 10;

[0027] Alternatively, the ChSSR32-labeled amplified fragment is 291 bp in size, with the SSR site being A. (11) That is, the number of A repeats in the sequence of the amplified product is 11;

[0028] Or the ChSSR34-labeled amplified fragment is 367 bp in size, and the SSR site is A( 10 If the number of A repeats in the sequence of the amplified product is 10, then the Hunan walnut to be tested is Yuanling walnut.

[0029] The principle behind this SSR molecular marker primer set for identifying Yuanling walnut is as follows:

[0030] The SSR molecular marker primer set designed in this scheme can specifically label *Walnut yuanlingensis*, and the size of the amplified marker fragment and the SSR site differ from those of common *Walnut yuanlingensis*. Using young leaves of *Walnut yuanlingensis* as material, DNA was first extracted, and then amplified using the designed SSR molecular marker primer set. Sequencing of the PCR products revealed that the 8 primer pairs designed in this scheme can specifically identify *Walnut yuanlingensis*, with the ChSSR2 marker amplification product being 398 bp in size and the SSR site being A. (11) The amplified product sequence has 11 A repeats; the ChSSR6-labeled amplified product is 385 bp in size, and the SSR site is T. (11) The amplified product sequence has 11 T repeats; the ChSSR8-labeled amplified fragment is 445 bp in size, and the SSR site is T. (11) The amplified product sequence has 11 T repeats; the ChSSR10 labeled amplified fragment is 484 bp in size, and the SSR site is A. (8) The amplified product sequence contains 8 repeats of the base A; the SSR sites labeled with ChSSR15 are A... (11) , and T (10)The amplified product sequence contains 11 A repeats and 10 T repeats; the ChSSR30 labeled amplified fragment is 292 bp in size, and the SSR site is T. (10) The amplified product sequence has 10 T repeats; the ChSSR32-labeled amplified fragment is 291 bp in size, and the SSR site is A. (11) The amplified product sequence has 11 A repeats; the ChSSR34-labeled amplified fragment is 367 bp in size, and the SSR site is A. (10) If the amplified product has 10 repeats of base A in its sequence, and the amplified product of the sample meets the above requirements for the size of any of the SSR molecular marker amplified fragments and the SSR site, then the sample can be identified as Yuanling walnut.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) This scheme is the first to develop a specific SSR length polymorphism genetic marker for Yuanling walnut. At the same time, the developed marker is verified by using common Hunan walnut materials collected from different seed sources. The specific SSR marker can realize the 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 Hunan walnut industry in my country.

[0033] (2) The SSR molecular marker proposed in this application has good stability and is easy to operate. It is not affected by the plant development period, growth stage and environment. Only a small amount of fresh leaves or silica gel-dried tissue leaves 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.

[0034] (3) The method provided by the present invention has the advantages of high throughput, accuracy, low cost, simple operation, saving manpower and material resources, and has broad application prospects. Attached Figure Description

[0035] 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.

[0036] Figure 1 A comparison of the ChSSR2 marker amplification sequence and sequencing peak diagram in Experiment Example 1;

[0037] Figure 2 Comparison of ChSSR6 marker amplification sequence and sequencing peak diagram in Experiment Example 1;

[0038] Figure 3 Comparison of ChSSR8 marker amplification sequence and sequencing peak diagram in Experiment Example 1;

[0039] Figure 4 A comparison of the ChSSR10 marker amplification sequence and sequencing peak diagram in Experiment Example 1;

[0040] Figure 5 Comparison of ChSSR15 marker amplification sequence and sequencing peak diagram in Experiment Example 1;

[0041] Figure 6 A comparison of the ChSSR30 marker amplification sequence and sequencing peak diagram in Experiment Example 1;

[0042] Figure 7 A comparison of the ChSSR32 marker amplification sequence and sequencing peak diagram in Experiment Example 1;

[0043] Figure 8 Comparison of the ChSSR34 marker amplification sequence and sequencing peak diagram in Experiment Example 1. Detailed Implementation

[0044] 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.

[0045] 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.

[0046] 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.

[0047] Example 1

[0048] Design of an SSR molecular marker primer set for identifying Yuanling walnut

[0049] 1. Assemble the chloroplast genome of Yuanling walnut

[0050] 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).

[0051] 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.

[0052] The high-throughput sequencing data were assembled using SPAdes 3.10.1 software with a k-mer parameter set to 95. Based on the published walnut chloroplast genome sequence as a reference sequence, 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 with 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.

[0053] 2. Discovering specific SSR loci in Yuanling walnut

[0054] The chloroplast genome sequences of four common hickory species were downloaded from GenBank, with accession numbers MT955359.1, MW298527.1, NC046435.1, and MH188303.1. Then, multiple alignments of the chloroplast genomes of Yuanling hickory and the four common Hunan hickory species were performed using MEGA 7.0 software, and the alignment results were exported in EXCEL format. Specific SSR loci for Yuanling hickory were manually screened.

[0055] 3. Design of PCR primers for specific SSR sites in Yuanling walnut

[0056] 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 positioned 50 to 200 base pairs away from the SSR site to ensure the accuracy of subsequent detection.

[0057] Experimental Example 1

[0058] Using an SSR molecular marker primer set for identifying Yuanling walnuts to distinguish between Yuanling walnuts and common Hunan walnuts.

[0059] 1. Experimental Materials

[0060] 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 screening and specificity verification of SSR markers.

[0061] 2. SSR marker detection

[0062] 2.1 DNA Extraction

[0063] Total DNA was extracted from 32 samples of Yuanling walnut and common Hunan walnut using a modified CTAB method.

[0064] 2.2 PCR amplification

[0065] Using the total DNA extracted from Yuanling walnuts and common Hunan walnuts in step 2.1 as amplification templates, PCR amplification was performed using 8 pairs of specific marker primers, as shown in Table 1 below.

[0066] Table 1 Information on Specific Marker Primers

[0067]

[0068] 2.3 PCR reaction system: The PCR reaction system is 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 total DNA of the test material at 50 ng / μL.

[0069] 2.4 PCR reaction procedure: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 0.5 min, annealing at 48.2–57.0℃ for 0.5 min, extension at 72℃ for 0.5 min, for 35 cycles; final extension at 72℃ for 5 min, storage at 4℃. PCR amplification products were electrophoresed on a 1% agarose gel (5 μL of GelRed 10000× stock solution was added to every 50 mL of agarose solution) at 100V for 40 min in 1×TAE buffer. Electrophoresis results were photographed 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.

[0070] 2.5 Sequencing of PCR products

[0071] The successfully amplified PCR products were subjected to Sanger first-generation sequencing using an ABI 3730XL sequencer.

[0072] 2.6 Sequence Analysis

[0073] 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.

[0074] 3. Specific identification and detection analysis of SSR markers in Yuanling walnut

[0075] 3.1 Detection results of ChSSR2 markers

[0076] Table 2 below and Figure 1 The results showed that there were differences in the size of the ChSSR2 marker amplified fragment and the SSR site among the 32 tested materials. The amplified product of common Hunan pecan was 397 bp in size, and the SSR site was A. (10) The amplified product sequence has 10 A repeats, while the amplified product of Yuanling walnut is 398 bp in size, and the SSR site is A. (11) The amplified product sequence contains 11 repeats of the base A.

[0077] Table 2 Detection results of ChSSR2 markers

[0078]

[0079] 3.2 Detection results of ChSSR6 marker

[0080] Table 3 and Figure 2 The results showed that there were differences in the size of the ChSSR6 marker amplified fragment and the SSR site among the 32 tested materials. The amplified product of common Hunan pecan was 384 bp in size, and the SSR site was T. (10)The amplified product sequence has 10 T repeats, while the amplified product of Yuanling walnut is 385 bp in size, and the SSR site is T. (11) The amplified product sequence contains 11 repeats of the T base.

[0081] Table 3. Detection results of ChSSR6 markers

[0082]

[0083]

[0084] 3.3 Detection results of ChSSR8 marker

[0085] Table 4 and Figure 3 The results showed that there were differences in the size of the ChSSR8 marker amplified fragment and the SSR site among the 32 tested materials. The amplified product of common Hunan pecan was 444 bp in size, and the SSR site was T. (10) The amplified product sequence has 10 T repeats, the size of the Yuanling walnut amplified product is 445 bp, and the SSR site is T. (11) The amplified product sequence contains 11 repeats of the T base.

[0086] Table 4. Detection results of ChSSR8 markers

[0087]

[0088]

[0089] 3.4 Detection results of ChSSR10 marker

[0090] Table 5 and Figure 4 The results showed that there were differences in the size of the ChSSR10 marker amplified fragment and the SSR site among the 32 tested materials. The amplified product of common Hunan walnut was 486 bp in size, and the SSR site was A. (10) The amplified product sequence contains 10 A repeats, the amplified product size of Yuanling walnut is 484 bp, and the SSR site is A. (8) The amplified product sequence contains 8 repeats of base A.

[0091] Table 5 Detection results of ChSSR10 markers

[0092]

[0093] 3.5 Detection results of ChSSR15 marker

[0094] Table 6 and Figure 5The results showed that the ChSSR15 marker amplified fragment size was 390 bp in all 32 tested materials, but the SSR sites differed. The two SSR sites in common Hunan walnut were A... (12) and T (9) The amplified product sequence contained 12 A repeats and 9 T repeats; the SSR sites of Yuanling walnut were A... (11) and T (10) The amplified product sequence has 11 A repeats and 10 T repeats.

[0095] Table 6. Detection results of ChSSR15 markers

[0096]

[0097]

[0098] 3.6 Detection results of ChSSR30 marker

[0099] Table 7 and Figure 6 The results showed that there were differences in the size of the ChSSR30 marker amplified fragment and the SSR site among the 32 tested materials. The amplified product of common Hunan walnut was 294 bp in size, and the SSR site was T. (12) The amplified product sequence has 12 T repeats, the amplified product size of Yuanling walnut is 292 bp, and the SSR site is T. (10) The amplified product sequence contains 10 T repeats.

[0100] Table 7 Detection results of ChSSR30 markers

[0101]

[0102]

[0103] 3.7 Detection results of ChSSR32 marker

[0104] Table 8 and Figure 7 The results showed that there were differences in the size of the ChSSR32 marker amplified fragment and the SSR locus among the 32 tested materials. The amplified product of common Hunan pecan was 290 bp in size, and the SSR locus was A. (10) The amplified product sequence has 10 A repeats, the amplified product size of Yuanling walnut is 291 bp, and the SSR site is A. (11) The amplified product sequence contains 11 repeats of the base A.

[0105] Table 8 Detection results of ChSSR32 markers

[0106]

[0107] 3.8 Detection results of ChSSR34 marker

[0108] Table 9 and Figure 8 The results showed that there were differences in the size of the ChSSR34 marker amplified fragment and the SSR locus among the 32 tested materials. The amplified product of common Hunan pecan was 368 bp in size, and the SSR locus was A. (11) The amplified product sequence contains 11 A repeats, the amplified product size of Yuanling walnut is 367 bp, and the SSR site is A. (10) The amplified product has 10 repeats of base A in its sequence.

[0109] Table 9 Detection results of ChSSR34 markers

[0110]

[0111] Therefore, it can be seen that any one of the eight primer pairs ChSSR2, ChSSR6, ChSSR8, ChSSR10, ChSSR15, ChSSR30, ChSSR32, and ChSSR34 can identify Yuanling walnuts and distinguish them from common Hunan walnuts. Through the differences in the size of the amplified product and the SSR site, the primer pairs have a wide range of selectable ranges and high specificity, enabling rapid and accurate identification of Yuanling walnut seedlings.

[0112] 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 SSR molecular marker primer sets for identifying Yuanling walnuts, characterized in that, Includes the following steps: S1. Extract total DNA from fresh leaves or silica gel-dried leaves of the Hunan walnut samples to be tested. S2. Using the total DNA obtained in step S1 as a template, PCR amplification is performed using the SSR molecular marker primer set as amplification primers; the SSR molecular marker primer set is ChSSR2, ChSSR6, ChSSR8, ChSSR10, ChSSR15, ChSSR30, ChSSR32, and ChSSR34. The primer sequence of ChSSR2 is shown in SEQ ID NO. 1 to SEQ ID NO. 2; The primer sequence of ChSSR6 is shown in SEQ ID NO. 3 to SEQ ID NO. 4; The primer sequence of ChSSR8 is shown in SEQ ID NO. 5 to SEQ ID NO. 6; The primer sequence of ChSSR10 is shown in SEQ ID NO. 7 to SEQ ID NO. 8; The primer sequence of ChSSR15 is shown in SEQ ID NO. 9 to SEQ ID NO. 10; The primer sequence of ChSSR30 is shown in SEQ ID NO. 11 to SEQ ID NO. 12; The primer sequence of ChSSR32 is shown in SEQ ID NO. 13 to SEQ ID NO. 14; The primer sequence of ChSSR34 is shown in SEQ ID NO. 15 to SEQ ID NO. 16; S3. Perform sequence analysis on the amplification products from step S2 to detect and analyze the size of the amplified fragments of the above 8 SSR markers and the SSR sites. The method for determining whether the Hunan mountain walnut to be tested is a Yuanling mountain walnut in step S3 is as follows: If the size of the ChSSR2 marker amplification product is 398 bp, the SSR site is A (11) i.e. the number of base A repeats in the sequence of the amplification product is 11; Alternatively, the ChSSR6-labeled amplification product is 385 bp in size, with the SSR site being T. (11) That is, the number of T repeats in the sequence of the amplified product is 11; Alternatively, the ChSSR8-labeled amplified fragment is 445 bp in size, with the SSR site being T. (11) That is, the number of T repeats in the sequence of the amplified product is 11; Alternatively, the ChSSR10-labeled amplified fragment is 484 bp in size, with the SSR site being A. (8) That is, the number of A repeats in the sequence of the amplified product is 8; Alternatively, the ChSSR15-labeled amplified fragment is 390 bp in size, with SSR sites A. (11) and T (10) That is, the number of A repeats in the sequence of the amplified product is 11, and the number of T repeats in the sequence of the amplified product is 10. Alternatively, the ChSSR30-labeled amplified fragment is 292 bp in size, with the SSR site being T( 10 This means that the number of T repeats in the sequence of the amplified product is 10; Alternatively, the ChSSR32-labeled amplified fragment is 291 bp in size, with the SSR site being A( 11 This means that the number of A repeats in the sequence of the amplified product is 11. Alternatively, the ChSSR34-labeled amplified fragment is 367 bp in size, with the SSR site being A( 10 If the number of A repeats in the sequence of the amplified product is 10, 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: 2 μL of 50 ng / μL DNA template to be tested, 1 μL each of 10 μmol / L upstream and downstream primers, 25 μL of 2×Phanta Max buffer, 19 μL of ddH2O, 1 μL of 1 U / μL Phanta Max Super-Fidelity high-fidelity DNA polymerase, and 1 μL of 10 μmol / L dNTP mixture, with a total system volume of 50 μL.

3. The detection method according to claim 1, characterized in that, The PCR amplification program in step S2 is as follows: pre-denaturation at 94℃ for 3 min; denaturation at 94℃ for 0.5 min, annealing at 48.2~57.0℃ 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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