Method for rapidly screening high-purity gastrodia and primer composition used in the method

Abstract

The application discloses a method for rapidly screening high-purity Gastrodia elata and a primer composition used in the method. The application aims to solve the technical problem of how to screen or assist in screening high-purity Gastrodia elata varieties. The application provides a reagent for identifying a RFLP molecular marker site of Gastrodia elata, and identifies or assists in identifying the purity of Gastrodia elata varieties by detecting the genotypes of the RFLP molecular marker site of the Gastrodia elata to be tested. After the PCR products of the RFLP sites of the genomic DNA fragments of different Gastrodia elata varieties are cut and subjected to gel electrophoresis, different numbers of bands can be obtained, and the purity of the RFLP sites of the genomic DNA fragments of the Gastrodia elata varieties is selected to be equal to or greater than 95%, thereby obtaining a high-purity Gastrodia elata variety. The composition for detecting the RFLP molecular marker sites in the genomes of the Gastrodia elata and other substances (such as substances for detecting other single nucleotide polymorphisms or genotypes of molecular markers related to the purity of Gastrodia elata) are combined to prepare a product for identifying high-purity Gastrodia elata varieties.

Description

Technical Field

[0001] This invention relates to the field of molecular genetic breeding, specifically to a method for rapidly screening highly homozygous Gastrodia elata and the primer composition used therein. Background Technology

[0002] Gastrodia elata Bl., the dried tuber of the orchid Gastrodia elata Bl., is sweet and neutral in nature, and enters the liver meridian. It has the effects of calming wind and stopping spasms, suppressing liver yang, and dispelling wind and unblocking the meridians. It is used for infantile convulsions, epilepsy, tetanus, headaches, dizziness, hemiplegia, numbness of limbs, and rheumatic pain. Modern research shows that Gastrodia elata can not only be used as a medicine for sedation, hypnosis, analgesia, anti-inflammation, anti-vertigo, anticonvulsant, and anti-aging, but also can be made into health foods for direct consumption, such as Gastrodia elata wine, fresh Gastrodia elata noodles, Gastrodia elata bean curd cake, and Gastrodia elata slices. At the end of 2019, Gastrodia elata was included as a pilot variety for management as "a substance that is traditionally both food and Chinese medicine," becoming a legally recognized "dual-use" variety. Currently, the artificial cultivation of Gastrodia elata is mainly carried out through asexual reproduction. Its asexual reproduction method involves directly cultivating underground tubers or protocorms with Armillaria mellea and its mycelial material, which has the characteristics of short growth cycle and rapid reproduction. However, asexual reproduction easily leads to varietal degeneration. Currently, there are two main solutions to this problem: tissue culture and hybridization breeding. Tissue culture uses virus-free treatment to avoid varietal degeneration. However, tissue culture techniques for Gastrodia elata suffer from slow growth, low survival rate, and susceptibility to contamination and death by the symbiotic Armillaria mellea. Hybridization breeding avoids varietal degeneration by crossing parents to obtain offspring with stable genetic characteristics. However, obtaining offspring with stable genetic characteristics requires highly homozygous pure-line varieties as parents, and currently, pure-line varieties of Gastrodia elata are lacking.

[0003] Currently, the main methods for obtaining pure-line varieties of crops are haploid breeding and pure-line selection. Haploid breeding utilizes plant tissue culture technology to induce haploid plants, and then uses some means to double the chromosome set (such as treatment with colchicine), thereby restoring the plant to a normal chromosome number. Methods for obtaining haploids include in vitro culture and parthenogenesis. In vitro culture includes anther culture and pollen culture; parthenogenesis mainly includes pollen radiation, chemical induction, delayed pollination with pollen from different species, cytoplasmic-nuclear substitution between different species, and distant hybridization. Pure-line selection involves selecting individuals with superior traits from an existing population, identifying their offspring, maintaining purity, and breeding new varieties. Since *Gastrodia elata* does not yet possess the tissue culture technology required for haploid breeding, but has a population with high genetic diversity, pure-line selection is more feasible for obtaining highly homozygous pure-line varieties. The primary challenge in pure-line selection is how to quickly screen highly homozygous individuals from a large number of individuals as initial breeding materials. Currently, genome resequencing technology can accurately determine the homozygosity of samples, but it is costly in both economy and time. How to quickly screen individuals with high homozygosity from a large number of individuals is a primary problem to be solved in Gastrodia elata breeding. Therefore, there is an urgent need to find a low-cost, rapid method that can effectively assess the homozygosity of Gastrodia elata. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to identify or assist in the identification of high-purity Gastrodia elata varieties.

[0005] To address the aforementioned technical problems, this invention first provides a molecular marker for identifying or assisting in the identification of high-purity Gastrodia elata varieties, and verifies the practicality of the marker, providing a reliable molecular marker for identifying or assisting in the cultivation of high-purity Gastrodia elata varieties.

[0006] This invention first provides a reagent for identifying the RFLP molecular marker sites of Gastrodia elata.

[0007] The present invention provides a reagent for identifying RFLP molecular marker sites in Gastrodia elata. The RFLP molecular marker sites are 20 SNP sites, which can be composed of E1, E2, E3, E4, E5, E6, E7, E8, E9, E10, E11, H1, H2, H3, S1, X1, X2, X3, X4, and X5 marker sites.

[0008] The E1 marker site is a site on chromosome 3 of *Gastrodia elata*, with nucleotide type T or G, and is the 435th nucleotide of SEQ ID No. 1 in the *Gastrodia elata* genome. The E2 marker site is a site on chromosome 7 of *Gastrodia elata*, with nucleotide type A or C, and is the 392nd nucleotide of SEQ ID No. 2 in the *Gastrodia elata* genome. The E3 marker site is a site on chromosome 1 of *Gastrodia elata*, with nucleotide type G or C, and is the 360th nucleotide of SEQ ID No. 3 in the *Gastrodia elata* genome. The E4 marker site is a site on chromosome 2 of *Gastrodia elata*, with nucleotide type C or T, and is the 409th nucleotide of SEQ ID No. 4 in the *Gastrodia elata* genome. The E5 marker site is a site on chromosome 2 of *Gastrodia elata*, with nucleotide type C or G, and is the 171st nucleotide of SEQ ID No. 5 in the *Gastrodia elata* genome. The E6 marker site is a site on chromosome 8 of *Gastrodia elata*, with nucleotide type T or C, and is the SEQ ID No. 1 in the *Gastrodia elata* genome. The E7 marker site is a site on chromosome 17 of *Gastrodia elata*, with nucleotide type A or G, and is the 280th nucleotide of SEQ ID No. 7 in the *Gastrodia elata* genome. The E8 marker site is a site on chromosome 2 of *Gastrodia elata*, with nucleotide type C or A, and is the 347th nucleotide of SEQ ID No. 8 in the *Gastrodia elata* genome. The E9 marker site is a site on chromosome 3 of *Gastrodia elata*, with nucleotide type A or G, and is the 140th nucleotide of SEQ ID No. 9 in the *Gastrodia elata* genome. The E10 marker site is a site on chromosome 4 of *Gastrodia elata*, with nucleotide type C or A, and is the 333rd nucleotide of SEQ ID No. 10 in the *Gastrodia elata* genome. The E11 marker site is a site on chromosome 5 of *Gastrodia elata*, with nucleotide type G or A, and is the 330th nucleotide of SEQ ID No. 11 in the *Gastrodia elata* genome. The H1 marker site is a site on chromosome 5 of *Gastrodia elata*, with nucleotide type C or T, and is the SEQ ID No. 7 in the *Gastrodia elata* genome. The 194th nucleotide of No. 12, the H2 marker site is a site on chromosome 14 of *Gastrodia elata*, its nucleotide type is C or T, and it is the 460th nucleotide of SEQ ID No. 13 in the *Gastrodia elata* genome; the H3 marker site is a site on chromosome 14 of *Gastrodia elata*, its nucleotide type is C or T, and it is the 133rd nucleotide of SEQ ID No. 14 in the *Gastrodia elata* genome; the S1 marker site is a site on chromosome 10 of *Gastrodia elata*, its nucleotide type is G or C, and it is the 387th nucleotide of SEQ ID No. 15 in the *Gastrodia elata* genome; the X1 marker site is a site on chromosome 1 of *Gastrodia elata*, its nucleotide type is A or C, and it is the SEQ ID No. 13 in the *Gastrodia elata* genome.The X2 marker site is located on chromosome 5 of *Gastrodia elata*, and its nucleotide type is A or G. It is the 288th nucleotide of SEQ ID No. 17 in the *Gastrodia elata* genome. The X3 marker site is located on chromosome 8 of *Gastrodia elata*, and its nucleotide type is G or A. It is the 310th nucleotide of SEQ ID No. 18 in the *Gastrodia elata* genome. The X4 marker site is located on chromosome 12 of *Gastrodia elata*, and its nucleotide type is T or C. It is the 311th nucleotide of SEQ ID No. 19 in the *Gastrodia elata* genome. The X5 marker site is located on chromosome 6 of *Gastrodia elata*, and its nucleotide type is A or G. It is the 186th nucleotide of SEQ ID No. 20 in the *Gastrodia elata* genome.

[0009] The reagent may consist of 20 compositions named E1, E2, E3, E4, E5, E6, E7, E8, E9, E10, E11, H1, H2, H3, S1, X1, X2, X3, X4, and X5.

[0010] The E1 group consists of a primer composition named E1 primer pair for amplifying the *Gastrodia elata* genomic DNA fragment including the E1 marker site and the restriction endonuclease EcoRI. The E2 group consists of a primer composition named E2 primer pair for amplifying the *Gastrodia elata* genomic DNA fragment including the E2 marker site and the restriction endonuclease EcoRI. The E3 group consists of a primer composition named E3 primer pair for amplifying the *Gastrodia elata* genomic DNA fragment including the E3 marker site and the restriction endonuclease EcoRI. The E4 group consists of a primer composition named E4 primer pair for amplifying the *Gastrodia elata* genomic DNA fragment including the E4 marker site and the restriction endonuclease EcoRI. The Gastrodia elata genome is composed of the following restriction endonucleases: Group E5 consists of a primer pair (named E5 primer pair) for amplifying the Gastrodia elata genomic DNA fragment including the E5 marker site, and EcoRI; Group E6 consists of a primer pair (named E6 primer pair) for amplifying the Gastrodia elata genomic DNA fragment including the E6 marker site, and EcoRI; Group E7 consists of a primer pair (named E7 primer pair) for amplifying the Gastrodia elata genomic DNA fragment including the E7 marker site, and EcoRI; Group E8 consists of a primer pair (named E8 primer pair) for amplifying the Gastrodia elata genomic DNA fragment including the E8 marker site. The H1 group consists of primer compositions for amplifying the Gastrodia elata genomic DNA fragment including the E9 marker site, and the restriction endonuclease EcoRI. The E9 group consists of primer compositions for amplifying the Gastrodia elata genomic DNA fragment including the E9 marker site (named E9 primer pair) and the restriction endonuclease EcoRI. The E10 group consists of primer compositions for amplifying the Gastrodia elata genomic DNA fragment including the E10 marker site (named E10 primer pair) and the restriction endonuclease EcoRI. The E11 group consists of primer compositions for amplifying the Gastrodia elata genomic DNA fragment including the E11 marker site (named E11 primer pair) and the restriction endonuclease EcoRI. The H1 group consists of primer compositions for amplifying the Gastrodia elata genomic DNA fragment including the E11 marker site (named E11 primer pair) and the restriction endonuclease EcoRI. The primer sets for amplifying the *Gastrodia elata* genomic DNA fragment including the H1 marker site and the restriction endonuclease HindIII are as follows: H2 group consists of primer sets for amplifying the *Gastrodia elata* genomic DNA fragment including the H2 marker site and the restriction endonuclease HindIII; H3 group consists of primer sets for amplifying the *Gastrodia elata* genomic DNA fragment including the H3 marker site and the restriction endonuclease HindIII; and S1 group consists of primer sets for amplifying the *Gastrodia elata* genomic DNA fragment including the S1 marker site and the restriction endonuclease SalⅠ.The X1 group consists of a primer combination (named X1 primer pair) for amplifying the *Gastrodia elata* genomic DNA fragment including the X1 marker site, and the restriction endonuclease XbaI. The X2 group consists of a primer combination (named X2 primer pair) for amplifying the *Gastrodia elata* genomic DNA fragment including the X2 marker site, and the restriction endonuclease XbaI. The X3 group consists of a primer combination (named X3 primer pair) for amplifying the *Gastrodia elata* genomic DNA fragment including the X3 marker site, and the restriction endonuclease XbaI. The X4 group consists of a primer combination (named X4 primer pair) for amplifying the *Gastrodia elata* genomic DNA fragment including the X4 marker site, and the restriction endonuclease XbaI. The X5 group consists of a primer combination (named X5 primer pair) for amplifying the *Gastrodia elata* genomic DNA fragment including the X5 marker site, and the restriction endonuclease XhoI.

[0011] In the above reagents, the E1 primer pair can be composed of two single-stranded DNAs named E1-F and E1-R, where the nucleotide sequence of E1-F is positions 1-20 of sequence 1 in the sequence listing, and the nucleotide sequence of E1-R is anticomplementary to positions 691-710 of sequence 1 in the sequence listing. The E2 primer pair can be composed of two single-stranded DNAs named E2-F and E2-R, where the nucleotide sequence of E2-F is positions 1-20 of sequence 2 in the sequence listing, and the nucleotide sequence of E2-R is anticomplementary to positions 565-584 of sequence 2 in the sequence listing. The E3 primer pair can be composed of two single-stranded DNAs named E3-F and E3-R, where the nucleotide sequence of E3-F is... The E3-R primer pair consists of positions 1-20 of sequence 3 in the sequence listing. The nucleotide sequence of E3-R is anticomplementary to positions 571-590 of sequence 3 in the sequence listing. The E4 primer pair can be composed of two single-stranded DNAs named E4-F and E4-R. The nucleotide sequence of E4-F is positions 1-20 of sequence 4 in the sequence listing, and the nucleotide sequence of E4-R is anticomplementary to positions 554-573 of sequence 4 in the sequence listing. The E5 primer pair can be composed of two single-stranded DNAs named E5-F and E5-R. The nucleotide sequence of E5-F is positions 1-20 of sequence 5 in the sequence listing, and the nucleotide sequence of E5-R is anticomplementary to positions 395-416 of sequence 5 in the sequence listing. The E6 primer pair can be composed of two single-stranded DNAs named E6-F and E6-R. The nucleotide sequence of E6-F is positions 1-20 of sequence 6 in the sequence listing, and the nucleotide sequence of E6-R is anticomplementary to positions 474-494 of sequence 6 in the sequence listing. The E7 primer pair can be composed of two single-stranded DNAs named E7-F and E7-R. The nucleotide sequence of E7-F is positions 1-22 of sequence 7 in the sequence listing, and the nucleotide sequence of E7-R is anticomplementary to positions 439-458 of sequence 7 in the sequence listing. The E8 primer pair can be composed of two single-stranded DNAs named E8-F and E8-R. The nucleotide sequence of E8-F is... The E8-R nucleotide sequence is reverse complementary to positions 446-466 of sequence 8 in the sequence listing. The E9 primer pair can be composed of two single-stranded DNAs named E9-F and E9-R. The nucleotide sequence of E9-F is positions 1-20 of sequence 9 in the sequence listing, and the nucleotide sequence of E9-R is reverse complementary to positions 381-400 of sequence 9 in the sequence listing. The E10 primer pair can be composed of two single-stranded DNAs named E10-F and E10-R. The nucleotide sequence of E10-F is positions 1-22 of sequence 10 in the sequence listing, and the nucleotide sequence of E10-R is reverse complementary to positions 495-514 of sequence 10 in the sequence listing.The E11 primer pair can be composed of two single-stranded DNAs named E11-F and E11-R. The nucleotide sequence of E11-F is positions 1-21 of sequence 11 in the sequence listing, and the nucleotide sequence of E11-R is anticomplementary to positions 501-522 of sequence 11 in the sequence listing. The H1 primer pair can be composed of two single-stranded DNAs named H1-F and H1-R. The nucleotide sequence of H1-F is positions 1-20 of sequence 12 in the sequence listing, and the nucleotide sequence of H1-R is anticomplementary to positions 482-501 of sequence 12 in the sequence listing. The H2 primer pair can be composed of two single-stranded DNAs named H2-F and H2-R. The nucleotide sequence of H2-F is... The H2-R nucleotide sequence is positions 1-20 of sequence 13 in the sequence listing. Positions 634-653 of the H2-R nucleotide sequence are anticomplementary to positions 634-653 of sequence 13 in the sequence listing. The H3 primer pair can be composed of two single-stranded DNAs named H3-F and H3-R. The H3-F nucleotide sequence is positions 1-20 of sequence 14 in the sequence listing. The H3-R nucleotide sequence is anticomplementary to positions 517-536 of sequence 14 in the sequence listing. The S1 primer pair can be composed of two single-stranded DNAs named S1-F and S1-R. The S1-F nucleotide sequence is positions 1-20 of sequence 15 in the sequence listing. The S1-R nucleotide sequence is anticomplementary to positions 543-564 of sequence 15 in the sequence listing. The X1 primer pair can be composed of two single-stranded DNAs named X1-F and X1-R, where the nucleotide sequence of X1-F is positions 1-20 of sequence 16 in the sequence listing, and the nucleotide sequence of X1-R is anticomplementary to positions 630-649 of sequence 16 in the sequence listing. The X2 primer pair can be composed of two single-stranded DNAs named X2-F and X2-R, where the nucleotide sequence of X2-F is positions 1-22 of sequence 17 in the sequence listing, and the nucleotide sequence of X2-R is anticomplementary to positions 564-585 of sequence 17 in the sequence listing. The X3 primer pair can be composed of two single-stranded DNAs named X3-F and X3-R, where the nucleotide sequence of X3-F is... In the sequence 18 listed, positions 1-20 of the X3-R primer pair are reverse complementary to positions 512-531 of sequence 18 in the sequence listing. The X4 primer pair can be composed of two single-stranded DNAs named X4-F and X4-R. The nucleotide sequence of X4-F is positions 1-21 of sequence 19 in the sequence listing, and the nucleotide sequence of X4-R is reverse complementary to positions 526-547 of sequence 19 in the sequence listing. The X5 primer pair can be composed of two single-stranded DNAs named X5-F and X5-R. The nucleotide sequence of X5-F is positions 1-20 of sequence 20 in the sequence listing, and the nucleotide sequence of X5-R is reverse complementary to positions 511-530 of sequence 20 in the sequence listing.

[0012] The application of the above-mentioned reagents also falls within the scope of protection of this invention.

[0013] The above-mentioned reagents can be used in any of the following applications: (1) to identify or assist in identifying the homozygosity of Gastrodia elata; (2) to screen or breed Gastrodia elata single plants, lines, strains or varieties with high homozygosity; (4) Gastrodia elata breeding; (5) to prepare products for identifying or assisting in identifying the homozygosity of Gastrodia elata; (6) to prepare products for screening or breeding Gastrodia elata single plants, lines, strains or varieties with high homozygosity; (7) to prepare products for screening or breeding Gastrodia elata single plants, lines, strains or varieties with high homozygosity; (8) to prepare products for Gastrodia elata breeding.

[0014] The present invention also provides primer compositions for identifying or assisting in the identification of RFLP molecular marker sites in Gastrodia elata.

[0015] The RFLP molecular marker sites are the 20 SNP sites mentioned above, and the primer composition consists of the following 20 primer pairs: the E1 primer pair, the E2 primer pair, the E3 primer pair, the E4 primer pair, the E5 primer pair, the E6 primer pair, the E7 primer pair, the E8 primer pair, the E9 primer pair, the E10 primer pair, the E11 primer pair, the H1 primer pair, the H2 primer pair, the H3 primer pair, the S1 primer pair, the X1 primer pair, the X2 primer pair, the X3 primer pair, the X4 primer pair, and the X5 primer pair.

[0016] This invention also provides applications of the primer compositions described above, specifically, any of the following applications:

[0017] (1) Identify or assist in identifying the homozygosity of Gastrodia elata varieties; (2) Screen or breed Gastrodia elata single plants, lines, strains or varieties with high homozygosity; (4) Gastrodia elata breeding; (5) Prepare products for identification or assistance in identifying high homozygosity of Gastrodia elata; (6) Prepare products for screening or breeding high homozygosity of Gastrodia elata single plants, lines, strains or varieties; (7) Prepare products for screening or breeding high homozygosity of Gastrodia elata single plants, lines, strains or varieties; (8) Prepare products for Gastrodia elata breeding.

[0018] This invention also provides a method for identifying or assisting in the identification of the homozygosity of Gastrodia elata.

[0019] The method for identifying or assisting in the identification of the homozygosity of Gastrodia elata provided by this invention includes the following steps:

[0020] 1) Using the genomic DNA of the *Gastrodia elata* to be tested as a template, PCR amplification was performed using the 20 primer pairs respectively to obtain the PCR products of the *Gastrodia elata* to be tested using the 20 primer pairs. The PCR product of the *Gastrodia elata* to be tested obtained by PCR amplification using the E1 primer pair was named E1-PCR product, the PCR product of the *Gastrodia elata* to be tested obtained by PCR amplification using the E2 primer pair was named E2-PCR product, the PCR product of the *Gastrodia elata* to be tested obtained by PCR amplification using the E3 primer pair was named E3-PCR product, and the PCR product of the *Gastrodia elata* to be tested obtained by PCR amplification using the E4 primer pair was named E4-PCR product. The PCR products obtained by PCR amplification using the E5 primer pair are named E5-PCR product, the PCR products obtained by PCR amplification using the E6 primer pair are named E6-PCR product, the PCR products obtained by PCR amplification using the E7 primer pair are named E7-PCR product, the PCR products obtained by PCR amplification using the E8 primer pair are named E8-PCR product, the PCR products obtained by PCR amplification using the E9 primer pair are named E9-PCR product, and the PCR products obtained by PCR amplification using the E10 primer pair are named E9-PCR product. The PCR products of *Gastrodia elata* obtained by PCR amplification are named E10-PCR products. The PCR products of *Gastrodia elata* obtained by PCR amplification using the E11 primer pair are named E11-PCR products. The PCR products of *Gastrodia elata* obtained by PCR amplification using the H1 primer pair are named H1-PCR products. The PCR products of *Gastrodia elata* obtained by PCR amplification using the H2 primer pair are named H2-PCR products. The PCR products of *Gastrodia elata* obtained by PCR amplification using the H3 primer pair are named H3-PCR products. The PCR products of *Gastrodia elata* obtained by PCR amplification using the S1 primer pair are named H3-PCR products. The PCR products are named S1-PCR products. The Gastrodia elata PCR products obtained by PCR amplification using the X1 primer pair are named X1-PCR products. The Gastrodia elata PCR products obtained by PCR amplification using the X2 primer pair are named X2-PCR products. The Gastrodia elata PCR products obtained by PCR amplification using the X3 primer pair are named X3-PCR products. The Gastrodia elata PCR products obtained by PCR amplification using the X4 primer pair are named X4-PCR products. The Gastrodia elata PCR products obtained by PCR amplification using the X5 primer pair are named X5-PCR products.

[0021] 2) The PCR products of *Gastrodia elata* using the 20 primer pairs were subjected to RFLP analysis. The RFLP analysis included digesting the PCR products of the 20 primer pairs with restriction endonucleases to obtain 20 digested products, performing electrophoresis on the 20 digested products, and determining the number of bands for each of the 20 digested products based on the electrophoresis results. In the RFLP analysis, the bands of the E1-PCR product, E2-PCR product, E3-PCR product, E4-PCR product, E5-PCR product, E6-PCR product, E7-PCR product, E8-PCR product, and E9-PCR product were analyzed. The PCR products, the E10-PCR product, and the E11-PCR product were all digested with the restriction endonuclease EcoRI. The H1-PCR product, the H2-PCR product, and the H3-PCR product were all digested with the restriction endonuclease HindIII. The S1-PCR product was digested with the restriction endonuclease SalI. The X1-PCR product, the X2-PCR product, the X3-PCR product, and the X4-PCR product were all digested with the restriction endonuclease XbaI. The X5-PCR product was digested with the restriction endonuclease XhoI.

[0022] 3) Calculate the homozygosity based on the number of enzyme digestion bands in the PCR product of the gastrodia elata to be tested.

[0023] This invention also provides a method for breeding Gastrodia elata.

[0024] In one embodiment, the Gastrodia elata breeding method may include using the above-mentioned method for identifying or assisting in identifying the homozygosity of Gastrodia elata, selecting Gastrodia elata with a homozygosity greater than or equal to 95% as parents (candidate parents) for breeding. The formula for calculating the homozygosity is: H = (1-n / 20) × 100%, where H represents the homozygosity and n represents the number of enzyme digestion products with 3 bands in the electrophoresis results after enzyme digestion of the 20 PCR products.

[0025] This study developed 20 RFLP markers for assessing the homozygosity of *Gastrodia elata* based on previously obtained SNP markers. By comprehensively evaluating accuracy and screening efficiency, a rapid method for screening highly homozygous *Gastrodia elata* individuals was established. This marker method demonstrates high precision and accuracy, providing technical support for screening high-homozygous pure-line varieties in *Gastrodia elata* pure-line breeding and hybridization. It is of great significance for obtaining genetically stable *Gastrodia elata* germplasm resources from populations with unclear genetic backgrounds. Currently, other medicinal plants also face serious problems such as varietal mixing, degeneration, and low yields; therefore, this method also has important reference value for the development of germplasm resources of other traditional Chinese medicines. Attached Figure Description

[0026] Figure 1 Gel electrophoresis images showing the enzyme digestion results of PCR amplification products with different primer pairs. Detailed Implementation

[0027] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0029] The following examples include: Hi-DNAsecure Plant Kit (Tiangen Biotech (Beijing) Co., Ltd., batch number: W9907); 2×M5 Super FastTaq PCR MasterMix (Beijing Polymer Biotechnology Co., Ltd., batch number: 21KB0808); 6×loading buffer (Dalian TaKaRa Co., Ltd., batch number: P30925); 2000bp DNA Marker (Dalian TaKaRa Co., Ltd., batch number: P51018); XhoI enzyme (New England Biolabs, batch number: 10020678); EcoRI enzyme (New England Biolabs, batch number: 10107912); HindIII enzyme (New England Biolabs, batch number: 10079084); XbaI enzyme (New England Biolabs, batch number: 10116146); rCutSmart Buffer (New England Biolabs, batch number: 10118973); and nucleic acid dye (Thermomix). Fisher Scientific (batch number: 2399638A); Agar powder (BIOWESTE, batch number: 111860)

[0030] The samples of Gastrodia elata were collected from four major producing areas: Dafang in Guizhou, Zhaotong in Yunnan, Yingshan in Hubei, and Yichang in Hubei. They were identified by Associate Researcher Jin Yan and the materials are preserved at the Center for Chinese Materia Medica Resources, China Academy of Chinese Medical Sciences (see Table 2).

[0031] Example 1: RFLP labeling screening for high-purity Gastrodia elata

[0032] 1. Site selection for RFLP markers

[0033] The previously obtained *Gastrodia elata* reference genome and resequencing data from 151 *Gastrodia elata* individuals were used to perform SNP detection using GATK, and SNP sites with any of the following conditions were removed: (1) the SNP is located within 5 bp upstream or downstream of the insertion / deletion variant; (2) the deletion rate is greater than 30%; (3) the minimum allele frequency is less than 5%. A total of 14,267,316 SNP markers were obtained. Based on the above SNP markers, the homozygosity of the 151 *Gastrodia elata* individuals and the homozygosity of each SNP marker were calculated. With a step size of 1% and an interval length of 5%, SNP markers with different heterozygosities were grouped (e.g., 1%–6%, 2%–7%, 3%–8%). A self-written script was used to randomly select 20 SNP markers from each group each time to evaluate the homozygosity of each individual and compare it with the homozygosity calculated using all SNP markers of each individual. The proportion of samples with consistent identification results in each 5% interval was calculated. The above process was performed using the bootstrap method for 1000 self-sampling. Using a self-written script, the Restriction module in the Biopython package was used to detect whether a restriction endonuclease recognition site existed in the SNP marker and its flanking sequences (800 bp upstream and downstream). If the SNP site was located at a restriction endonuclease recognition site and its flanking sequences did not contain the corresponding restriction endonuclease recognition site, then the SNP marker could be used as an RFLP marker.

[0034] This embodiment selects recognition sites for restriction endonucleases such as EcoRI, HindIII, SalI, XbaI, and XhoI. Using 20 sites with heterozygosity between 7% and 12%, the homozygosity of *Gastrodia elata* individuals can be correctly identified within the 5% range. Among these, 37 SNPs located at restriction enzyme sites can be developed into RFLP markers. Twenty of these were selected for RFLP marker development, including 11 EcoRI markers, 3 HindIII markers, 1 SalI marker, 4 XbaI markers, and 1 XhoI marker. These 20 RFLP markers are the following 20 SNPs:

[0035] 1) The SNP named E1 marker site is a site on chromosome 3 of Gastrodia elata (located at chr3:7756023 in the Gastrodia elata genome, (Genbank Accession number GWHBHOU00000003, position 7756023)). Its nucleotide type is T or G, and it is the 435th nucleotide of SEQ ID No. 1 in the Gastrodia elata genome.

[0036] 2) The SNP named E2 marker site, which is a site on chromosome 7 of Gastrodia elata (located at chr7:2724136 in the Gastrodia elata genome, (Genbank Accession number GWHBHOU00000007, nucleotide 2724136)), has nucleotide type A or C, and is the 392nd nucleotide of SEQ ID No. 2 in the Gastrodia elata genome.

[0037] 3) The SNP named E3 marker site, which is a site on chromosome 1 of Gastrodia elata (located at position chr1:90614251 of the Gastrodia elata genome, (position 90614251 of Genbank Accession number GWHBHOU00000001)), and its nucleotide type is G or C, which is the 360th nucleotide of SEQ ID No. 3 in the Gastrodia elata genome;

[0038] 4) The SNP named E4 marker site, which is a site on chromosome 2 of Gastrodia elata (located at position chr2:106091319 of the Gastrodia elata genome, (position 106091319 of Genbank Accession number GWHBHOU00000002)), and its nucleotide type is C or T, which is the 409th nucleotide of SEQ ID No. 4 in the Gastrodia elata genome;

[0039] 5) The SNP named E5 marker site, which is a site on chromosome 2 of Gastrodia elata (located at position chr2:39372534 of the Gastrodia elata genome, (position 39372534 of Genbank Accession number GWHBHOU00000002)), and its nucleotide type is C or G, which is the nucleotide at position 171 of SEQ ID No. 5 in the Gastrodia elata genome;

[0040] 6) The SNP named E6 marker site, which is a site on chromosome 8 of Gastrodia elata (located at position chr8:33125591 in the Gastrodia elata genome (position 33125591 in Genbank Accession GWHBHOU00000007)), has a nucleotide type of T or C, and is the 313th nucleotide of SEQ ID No. 6 in the Gastrodia elata genome;

[0041] 7) The SNP named E7 marker site, which is a site on chromosome 16 of Gastrodia elata (located at position chr16:9163271 of the Gastrodia elata genome, (position 9163271 of Genbank Accession number GWHBHOU000000016)), and its nucleotide type is A or G, and is the 280th nucleotide of SEQ ID No. 7 in the Gastrodia elata genome;

[0042] 8) The SNP named E8 marker site, which is a site on chromosome 2 of Gastrodia elata (located at position chr2:526568 of the Gastrodia elata genome, (position 526568 of Genbank Accession number GWHBHOU00000002)), and its nucleotide type is C or A, and is the 347th nucleotide of SEQ ID No. 8 in the Gastrodia elata genome;

[0043] 9) The SNP named E9 marker site, which is a site on chromosome 3 of Gastrodia elata (located at position chr3:65507245 of the Gastrodia elata genome, (position 65507245 of Genbank Accession number GWHBHOU00000003)), and its nucleotide type is A or G, and is the 140th nucleotide of SEQ ID No. 9 in the Gastrodia elata genome;

[0044] 10) An SNP named E10 marker site, wherein the E10 marker site is a site on chromosome 4 of Gastrodia elata (located at position chr4:57186102 in the Gastrodia elata genome, (position 57186102 in Genbank Accession number GWHBHOU00000004)), and its nucleotide type is C or A, and is the 333rd nucleotide of SEQ ID No. 10 in the Gastrodia elata genome;

[0045] 11) The SNP named E11 marker site, which is a site on chromosome 5 of Gastrodia elata (located at position chr5:23007754 in the Gastrodia elata genome, (position 23007754 in Genbank Accession number GWHBHOU00000005)), whose nucleotide type is G or A, is the 330th nucleotide of SEQ ID No. 11 in the Gastrodia elata genome;

[0046] 12) The SNP named H1 marker site, which is a site on chromosome 5 of Gastrodia elata (located at position chr5:20038961 in the Gastrodia elata genome, (position 20038961 in Genbank Accession number GWHBHOU00000005)), and its nucleotide type is C or T, and is the 194th nucleotide of SEQ ID No. 12 in the Gastrodia elata genome;

[0047] 13) The SNP named H2 marker site, wherein the H3 marker site is a site on chromosome 13 of Gastrodia elata (located at position chr13:21721655 of the Gastrodia elata genome, (position 21721655 of Genbank Accession number GWHBHOU00000013)), and its nucleotide type is C or T, and is the 460th nucleotide of SEQ ID No. 13 in the Gastrodia elata genome;

[0048] 14) The SNP named H3 marker site, wherein the H2 marker site is a site on chromosome 1 of Gastrodia elata (located at position chr1:16461737 of the Gastrodia elata genome, (position 16461737 of Genbank Accession number GWHBHOU00000001)), and its nucleotide type is C or T, and is the 133rd nucleotide of SEQ ID No. 14 in the Gastrodia elata genome;

[0049] 15) An SNP named S1 marker site, wherein the S1 marker site is a site on chromosome 11 of Gastrodia elata (located at position chr11:47646083 in the Gastrodia elata genome, (position 47646083 in Genbank Accession number GWHBHOU00000011)), and its nucleotide type is G or C, which is the 387th nucleotide of SEQ ID No. 15 in the Gastrodia elata genome;

[0050] 16) An SNP named X1 marker site, wherein the X1 marker site is a site on chromosome 1 of Gastrodia elata (located at position chr1:82379251 of the Gastrodia elata genome, (position 82379251 of Genbank Accession number GWHBHOU00000001)), and its nucleotide type is A or C, and it is the 350th nucleotide of SEQ ID No. 16 in the Gastrodia elata genome;

[0051] 17) An SNP named X2 marker site, wherein the X2 marker site is a site on chromosome 5 of Gastrodia elata (located at position chr5:52061619 of the Gastrodia elata genome, (position 52061619 of Genbank Accession number GWHBHOU00000005)), and its nucleotide type is A or G, and is the 288th nucleotide of SEQ ID No. 17 in the Gastrodia elata genome;

[0052] 18) An SNP named X3 marker site, wherein the X3 marker site is a site on chromosome 8 of Gastrodia elata (located at position chr8:4676518 in the Gastrodia elata genome, (position 4676518 in Genbank Accession number GWHBHOU00000008)), and its nucleotide type is A or G, and it is the 310th nucleotide of SEQ ID No. 18 in the Gastrodia elata genome;

[0053] 19) The SNP named X4 marker site is a site on chromosome 14 of Gastrodia elata (located at position chr12:4216762 in the Gastrodia elata genome, (position 4216762 in Genbank Accession number GWHBHOU00000012)), and its nucleotide type is C or T, which is the 311th nucleotide of SEQ ID No. 19 in the Gastrodia elata genome;

[0054] 20) An SNP named X5 marker site, wherein the X5 marker site is a site on chromosome 6 of Gastrodia elata (located at position chr6:53849642 of the Gastrodia elata genome, (position 53849642 of Genbank Accession number GWHBHOU00000006)), and its nucleotide type is A or G, and it is the 186th nucleotide of SEQ ID No. 20 in the Gastrodia elata genome;

[0055] Twenty RFLP-labeled PCR forward and reverse primer pairs were artificially synthesized. The lengths of the 20 PCR forward and reverse primer pair compositions (hereinafter also referred to as PCR primer pair compositions for identifying high homozygosity of Gastrodia elata or specific PCR primer pair compositions) and their products are shown in Table 1. The above-mentioned PCR primer pair compositions for identifying high homozygosity of Gastrodia elata are all individually packaged, and the molar ratio of the two single-stranded DNAs in each primer pair is 1:1.

[0056] The PCR primer pair composition for identifying high homozygosity of Gastrodia elata is a primer composition for identifying RFLP molecular marker sites in Gastrodia elata. The primer composition consists of the following 20 primer pairs: primer pair E1 for amplifying the Gastrodia elata genomic DNA fragment including the E1 marker site; primer pair E2 for amplifying the Gastrodia elata genomic DNA fragment including the E2 marker site; primer pair E3 for amplifying the Gastrodia elata genomic DNA fragment including the E3 marker site; and primer pair E4 for amplifying the Gastrodia elata genomic DNA fragment including the E4 marker site. The primer compositions include: primer E5 (for amplifying the Gastrodia elata genomic DNA fragment including the E5 marker site), primer E6 (for amplifying the Gastrodia elata genomic DNA fragment including the E6 marker site), primer E7 (for amplifying the Gastrodia elata genomic DNA fragment including the E7 marker site), primer E8 (for amplifying the Gastrodia elata genomic DNA fragment including the E8 marker site), primer E9 (for amplifying the Gastrodia elata genomic DNA fragment including the E9 marker site), and primer E10 (for amplifying the Gastrodia elata genomic DNA fragment including the E9 marker site). Primer compositions including the E10 marker site of *Gastrodia elata* genomic DNA fragment; primer composition named E11 for amplifying the *Gastrodia elata* genomic DNA fragment including the E11 marker site; primer composition named H1 for amplifying the *Gastrodia elata* genomic DNA fragment including the H1 marker site; primer composition named H2 for amplifying the *Gastrodia elata* genomic DNA fragment including the H2 marker site; primer composition named H3 for amplifying the *Gastrodia elata* genomic DNA fragment including the H3 marker site; and primer composition named S1 for amplifying the S1 marker site... Primer compositions for amplifying Gastrodia elata genomic DNA fragments, primer composition named X1 for amplifying Gastrodia elata genomic DNA fragments including the X1 marker site, primer composition named X2 for amplifying Gastrodia elata genomic DNA fragments including the X2 marker site, primer composition named X3 for amplifying Gastrodia elata genomic DNA fragments including the X3 marker site, primer composition named X4 for amplifying Gastrodia elata genomic DNA fragments including the X4 marker site, and primer composition named X5 for amplifying Gastrodia elata genomic DNA fragments including the X5 marker site.

[0057] E1 primers are PCR primer pairs consisting of single-stranded DNA whose nucleotide sequence is the first 20th position of Sequence 1 in the sequence listing and single-stranded DNA whose nucleotide sequence is the reverse complementary position of Sequence 1 from the 691st to the 710th position of Sequence 1 in the sequence listing.

[0058] The E2 primer pair consists of a single-stranded DNA nucleotide sequence from positions 1-20 of sequence 2 in the sequence listing and a single-stranded DNA nucleotide sequence that is reverse complementary to positions 565-584 of sequence 2 in the sequence listing.

[0059] E3 primers are PCR primer pairs consisting of single-stranded DNA whose nucleotide sequence is the first 20th position of sequence 3 in the sequence listing and single-stranded DNA whose nucleotide sequence is the reverse complementary position of sequence 3 from position 571 to 590 in the sequence listing.

[0060] The E4 primer is a PCR primer pair consisting of single-stranded DNA whose nucleotide sequence is the first 20th position of sequence 4 in the sequence listing and a single-stranded DNA whose nucleotide sequence is the reverse complementary single-stranded DNA from the 554th to the 573rd position of sequence 4 in the sequence listing.

[0061] The E5 primer is a PCR primer pair consisting of single-stranded DNA whose nucleotide sequence is the first 20th position of sequence 5 in the sequence listing and a single-stranded DNA whose nucleotide sequence is the reverse complementary single-stranded DNA from the third 395th to the fourth 416th position of sequence 5 in the sequence listing.

[0062] The E6 primer is a PCR primer pair consisting of single-stranded DNA whose nucleotide sequence is the first 20th position of sequence 6 in the sequence listing and a single-stranded DNA whose nucleotide sequence is the reverse complementary single-stranded DNA from the 474th to the 494th position of sequence 6 in the sequence listing.

[0063] The E7 primer is a PCR primer pair consisting of single-stranded DNA whose nucleotide sequence is the first 22nd position of sequence 7 in the sequence listing and a single-stranded DNA whose nucleotide sequence is the reverse complementary position of sequence 7 from position 439 to 458 in the sequence listing.

[0064] The E8 primer is a PCR primer pair consisting of single-stranded DNA whose nucleotide sequence is the first 20th position of sequence 8 in the sequence listing and a single-stranded DNA whose nucleotide sequence is the reverse complementary single-stranded DNA from the fourth 446th to the fourth 466th position of sequence 8 in the sequence listing.

[0065] The E9 primer is a PCR primer pair consisting of single-stranded DNA whose nucleotide sequence is the first 20th position of sequence 9 in the sequence listing and a single-stranded DNA whose nucleotide sequence is the reverse complementary position of sequence 9 from the 381st to the 400th position in the sequence listing.

[0066] The E10 primer is a PCR primer pair consisting of single-stranded DNA whose nucleotide sequence is the first 22nd position of sequence 10 in the sequence listing and a single-stranded DNA whose nucleotide sequence is the reverse complementary position of sequence 10 from position 495 to 514 in the sequence listing.

[0067] The E11 primer is a PCR primer pair consisting of single-stranded DNA whose nucleotide sequence is the first 21st position of sequence 11 in the sequence listing and single-stranded DNA whose nucleotide sequence is the reverse complementary position of sequence 11 from position 501 to 522 in the sequence listing.

[0068] The H1 primer is a PCR primer pair consisting of single-stranded DNA whose nucleotide sequence is the first 20th position of sequence 12 in the sequence listing and single-stranded DNA whose nucleotide sequence is the reverse complementary position of sequence 12 from the 482nd to the 501st position in the sequence listing.

[0069] H2 primers are PCR primer pairs consisting of single-stranded DNA whose nucleotide sequence is the first 20th position of sequence 13 in the sequence listing and single-stranded DNA whose nucleotide sequence is the reverse complementary position of sequence 13 from position 634 to 653 in the sequence listing.

[0070] The H3 primer is a PCR primer pair consisting of single-stranded DNA whose nucleotide sequence is the first 20th position of sequence 14 in the sequence listing and single-stranded DNA whose nucleotide sequence is the reverse complementary position of sequence 14 from the 517th to the 536th position in the sequence listing.

[0071] The S1 primer is a PCR primer pair consisting of single-stranded DNA whose nucleotide sequence is the first 20th position of sequence 15 in the sequence listing and a single-stranded DNA whose nucleotide sequence is the reverse complementary position of sequence 15 from position 543 to 564 in the sequence listing.

[0072] The X1 primer is a PCR primer pair consisting of single-stranded DNA whose nucleotide sequence is the first 20th position of sequence 16 in the sequence listing and a single-stranded DNA whose nucleotide sequence is the reverse complementary position of sequence 16 from position 630 to 649 in the sequence listing.

[0073] The X2 primer is a PCR primer pair consisting of single-stranded DNA whose nucleotide sequence is the first 20th position of sequence 17 in the sequence listing and a single-stranded DNA whose nucleotide sequence is the reverse complementary single-stranded DNA from the 564th to the 585th position of sequence 16 in the sequence listing.

[0074] The X3 primer is a PCR primer pair consisting of a single-stranded DNA whose nucleotide sequence is the first 20th position of sequence 18 in the sequence listing and a single-stranded DNA whose nucleotide sequence is the reverse complementary position of sequence 18 from position 512 to 531 in the sequence listing.

[0075] The X4 primer is a PCR primer pair consisting of single-stranded DNA whose nucleotide sequence is the first 21st position of sequence 19 in the sequence listing and a single-stranded DNA whose nucleotide sequence is the reverse complementary single-stranded DNA from the fifth 26th to the fifth 47th position of sequence 19 in the sequence listing.

[0076] The X5 primer is a PCR primer pair consisting of single-stranded DNA whose nucleotide sequence is the first 20th position of sequence 20 in the sequence listing and a single-stranded DNA whose nucleotide sequence is the reverse complementary single-stranded DNA from the 511th to the 530th position of sequence 20 in the sequence listing.

[0077] Table 1. Forward and reverse primers and product lengths for 20 RFLP markers.

[0078]

[0079]

[0080] 2. Genomic DNA extraction

[0081] Fresh tubers of Gastrodia elata Bl. were collected, pulverized, and 10 mg of the powder was extracted using the Hi-DNAsecure Plant Kit for high-efficiency plant genomic DNA extraction. The DNA concentration was determined using a Nanodrop 2000 micro-volume nucleic acid quantification analyzer, and DNA quality was judged based on absorbance at A260 nm / 280 nm and A260 nm / 230 nm. Genomic DNA samples from 15 Gastrodia elata samples were obtained.

[0082] 3. PCR amplification

[0083] The genomic DNA of the above 15 Gastrodia elata samples was amplified by PCR using specific PCR primer pairs. The PCR reaction volume was 25 μL, containing 13 μL of 2×M5 Super FastTaq PCR MasterMix, 1 μL of forward primer (sequences shown in Table 1), 1 μL of reverse primer (sequences shown in Table 1), 1 μL of DNA template, and sterile water to a final volume of 25 μL. The reaction conditions were as follows: 94℃ for 2 min, 35 cycles (94℃ for 30 s, 58℃ for 15 s, 72℃ for 1 min), 72℃ for 5 min. Fifteen samples of Gastrodia elata were obtained as E1-PCR products (using the E1 primer pair, which consists of single-stranded DNA with nucleotide sequences from positions 1 to 20 of sequence 1 in the sequence listing and single-stranded DNA with reverse complementary nucleotide sequences from positions 691 to 710 of sequence 1 in the sequence listing) and E2-PCR products (using the E2 primer pair, which consists of single-stranded DNA with nucleotide sequences from positions 1 to 20 of sequence 2 in the sequence listing and single-stranded DNA with reverse complementary nucleotide sequences from positions 565 to 584 of sequence 2 in the sequence listing). The PCR primer pairs are: E3-PCR product (the primer pair used is the E3 primer, which is a PCR primer pair composed of single-stranded DNA with nucleotide sequences of positions 1-20 of sequence 3 in the sequence listing and single-stranded DNA with reverse complementary nucleotide sequences of positions 571-590 of sequence 3 in the sequence listing); and E4-PCR product (the primer pair used is the E4 primer, which is a PCR primer pair composed of single-stranded DNA with nucleotide sequences of positions 1-20 of sequence 4 in the sequence listing and single-stranded DNA with reverse complementary nucleotide sequences of positions 554-573 of sequence 4 in the sequence listing). CR primer pair), E5-PCR product (the primer pair used is the E5 primer, which is a PCR primer pair composed of single-stranded DNA with nucleotide sequences of positions 1-20 of sequence 5 in the sequence listing and single-stranded DNA with nucleotide sequences of positions 395-416 of sequence 5 in the sequence listing), E6-PCR product (the primer pair used is the E6 primer, which is a PCR primer pair composed of single-stranded DNA with nucleotide sequences of positions 1-20 of sequence 6 in the sequence listing and single-stranded DNA with reverse complementary nucleotide sequences of positions 474-494 of sequence 6 in the sequence listing). The E7-PCR product (using the E7 primer pair, which consists of a single-stranded DNA nucleotide sequence of positions 1-22 of sequence 7 in the sequence listing and a single-stranded DNA nucleotide sequence of positions 439-458 of sequence 7 in the sequence listing, respectively), and the E8-PCR product (using the E8 primer pair, which consists of a single-stranded DNA nucleotide sequence of positions 1-20 of sequence 8 in the sequence listing and a single-stranded DNA nucleotide sequence of positions 446-466 of sequence 8 in the sequence listing, respectively).E9-PCR product (using the E9 primer pair, which consists of single-stranded DNA with nucleotide sequences from positions 1-20 of sequence 9 in the sequence listing and single-stranded DNA with reverse complementary nucleotide sequences from positions 381-400 of sequence 9 in the sequence listing); E10-PCR product (using the E10 primer pair, which consists of single-stranded DNA with nucleotide sequences from positions 1-22 of sequence 10 in the sequence listing and single-stranded DNA with reverse complementary nucleotide sequences from positions 495-514 of sequence 10 in the sequence listing); E11-PCR product (using the E11 primer pair, which consists of single-stranded DNA with nucleotide sequences from positions 1-21 of sequence 11 in the sequence listing). The H1-PCR product (using primers consisting of single-stranded DNA and nucleotide sequences from positions 501-522 of sequence 11 in the sequence listing), and the H2-PCR product (using primers consisting of single-stranded DNA from positions 1-20 of sequence 12 in the sequence listing and nucleotide sequences from positions 482-501 of sequence 12 in the sequence listing), are both primer pairs. The primer pairs used are H3 primers, which consist of single-stranded DNA with nucleotide sequences from positions 1-20 of sequence 14 in the sequence listing and single-stranded DNA with reverse complementary nucleotide sequences from positions 517-536 of sequence 14 in the sequence listing; S1 primers, which consist of single-stranded DNA with nucleotide sequences from positions 1-20 of sequence 15 in the sequence listing and single-stranded DNA with reverse complementary nucleotide sequences from positions 543-564 of sequence 15 in the sequence listing; and X1 primers, which consist of single-stranded DNA with nucleotide sequences from positions 1-20 of sequence 16 in the sequence listing. The primer pairs used are: X2-PCR product (using the X2 primer pair, which consists of single-stranded DNA with reverse complementary positions 630-649 of sequence 16 in the sequence listing), and X3-PCR product (using the X3 primer pair, which consists of single-stranded DNA with reverse complementary positions 512-531 of sequence 18 in the sequence listing).X4-PCR products (using X4 primers, which consist of single-stranded DNA with nucleotide sequences from positions 1-21 of sequence 19 in the sequence listing and single-stranded DNA with nucleotide sequences from positions 526-547 of sequence 19 in the sequence listing), and X5-PCR products (using X5 primers, which consist of single-stranded DNA with nucleotide sequences from positions 1-20 of sequence 20 in the sequence listing and single-stranded DNA with reverse complementary nucleotide sequences from positions 511-530 of sequence 20 in the sequence listing), for a total of 20 PCR products.

[0084] After the PCR reaction was completed, 5 μL of the reaction product was taken, 1 μL of 6× loading buffer was added, and after mixing, it was detected by electrophoresis on a 2.0% agarose gel stained with nucleic acid dye. The gel imaging system was used for observation and imaging.

[0085] 4. RFLP analysis of PCR products

[0086] The PCR product obtained in step 3 above was used for RFLP analysis. The enzyme digestion reaction system was 25 μL, containing 2.5 μL of 10×Buffer, 15 μL of PCR product, and restriction endonuclease (10 U / μL). －1 0.5 μL of dd H2O and 7 μL of dd H2O were added. The reaction was carried out in a water bath at 37℃ for 120 min. Among them, the 12 PCR products from E1-PCR to E11-PCR were digested with restriction endonuclease EcoRI, the 3 PCR products from H1-PCR to H3-PCR were digested with restriction endonuclease HindIII, the S1-PCR product was digested with restriction endonuclease SalI, the 4 PCR products from X1-PCR to X4-PCR were digested with restriction endonuclease XbaI, and the X5-PCR product was digested with restriction endonuclease XhoI.

[0087] Take 5 μL of the enzyme digestion product, add 1 μL of 6× Loading buffer, mix well, and then perform electrophoresis on a 2.0% agarose gel stained with nucleic acid dye. Observe and image using a gel imaging system. The results for the restriction enzyme sites of different samples may show 1 band, 2 bands, or 3 bands. 1 band and 2 bands indicate that the sample is homozygous at that labeling site; 3 bands indicate that the sample is heterozygous at that labeling site.

[0088] Using the 20 RFLP marker sites selected above, DNA extraction, specific primer amplification, and restriction endonuclease experiments were performed on 15 Gastrodia elata samples. The results are as follows: Figure 1As shown, A: Marker site E1; B: Marker site E2; C: Marker site E3; D: Marker site E4; E: Marker site E5; F: Marker site E6; G: Marker site E7; H: Marker site E8; I: Marker site E9; J: Marker site E10; K: Marker site E11; L: Marker site H1; M: Marker site H2; N: Marker site H3; O: Marker site S1; P: Marker site X1; Q: Marker site X2; R: Marker site X3; S: Marker site X4; T: Marker site X5; 1-15 are 15 different Gastrodia elata samples; 16 is the blank control, using ddH2O as a template; M.DL 2000 Marker. Each row represents one type of marker site, and each column of 1-15 represents a different Gastrodia elata sample.

[0089] Depend on Figure 1 It can be seen that samples 1, 3, 4, 5, 7, 8, and 12 all showed only one or two bands at the 20 marker sites. Sample 2 showed three bands at marker site H1; Sample 6 showed three bands at four marker sites: E2, E6, E10, and X5; Sample 9 showed three bands at marker site X5; Sample 10 showed three bands at three marker sites: E2, H1, and X5; Sample 11 showed three bands at marker site H1; Samples 13 and 14 showed three bands at three marker sites: E3, E6, and E7; Sample 15 showed three bands at three marker sites: E2, E3, and X5. Samples showed only one or two bands at all other sites except those mentioned above.

[0090] Homozygosity based on RFLP marker evaluation is calculated using the following formula: Figure 1 Homozygosity was calculated by counting the number of sites with three bands at different sites in each sample. The formula for calculating homozygosity is: H = (1 - n / m) × 100%, where H represents homozygosity, n represents the number of sites with three bands (i.e., the number of enzyme digestion products with three bands in the electrophoresis results after enzyme digestion of 20 PCR products), and m represents the total number of sites, which is 20. The calculation results are shown in Table 2.

[0091] 5. Genome resequencing was used to verify the accuracy of the above RFLP marker identification results.

[0092] Following step 2, 15 Gastrodia elata genomic DNA samples were obtained. These samples were randomly fragmented into 350–500 bp DNA fragments using Covaris. Fragments of approximately 500 bp were recovered for library construction. After successful library construction, the inserted fragments were sequenced using the DNBSEQ-T7 sequencing platform. The raw sequencing data were quality controlled and filtered using FASTP software. Alignment was performed using the Gastrodia elata genome (accession number GWHBHOU00000000, stored at the National Genome Data Center https: / / ngdc.cncb.ac.cn / gwh) as a reference genome using BWA software. The results were further compared using samtools software to identify and sort repetitive sequences. Then, the SNPs and genotypes were detected using the HaplotypeCaller and GenotypeGVCFs programs in GATK software, respectively. Finally, the obtained SNP sites were quality controlled using bcftools (QD<4.0||FS>60.0||MQ<40.0||MAF[0]<0.05||F_MISSING>0.3). Using the obtained SNP and genotype information, the total number of sites (N(NM)) and the number of homozygous sites (O(HOM)) in the results file) of each individual were calculated using the "--het" function of plink software. The homozygosity of the sample was calculated as the number of homozygous sites / total sites × 100%.

[0093] Table 2 shows the homozygosity of Gastrodia elata as assessed by genome resequencing.

[0094] Table 2 Homozygosity of 15 Gastrodia elata samples

[0095]

[0096]

[0097] RFLP marker evaluation showed that the homozygosity of 15 Gastrodia elata samples was in the range of 80%–100%. Among them, 10 samples had a homozygosity of ≥95% (samples 1, 2, 3, 4, 5, 7, 8, 9, 11, and 12); 4 samples had a homozygosity of 85% (samples 10, 13, 14, and 15); and sample 6 had a homozygosity of 80%.

[0098] Genome resequencing technology assessed the homozygosity of 15 Gastrodia elata samples to be between 68% and 99%. Among them, 9 samples had a homozygosity of ≥95% (samples 1, 2, 3, 4, 6, 7, 8, 9, and 12); 5 samples had a homozygosity between 70% and 80% (samples 10, 11, 13, 14, and 15); and sample 5 had a homozygosity of 68%.

[0099] Eight samples showed consistent results from both methods. The homozygosity of these eight Gastrodia elata samples, assessed by RFLP markers and genome resequencing, was greater than 95%. These samples were samples 1, 2, 3, 4, 7, 8, 9, and 12. This indicates that the RFLP marker method has an accuracy of 80% and a precision of 88.9%, demonstrating high reliability. In practical applications, RFLP markers can be combined with genome resequencing. First, low-cost RFLP markers are used to initially screen individuals with homozygosity higher than 95% from a large number of samples. Then, resequencing is performed on the initially screened individuals for precise evaluation, ultimately obtaining genetically homozygous superior parents suitable for pure-line breeding.

[0100] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. SEQUENCE LISTING <110> Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences <120> A method for rapid screening of high-purity Gastrodia elata and the primer composition used therein <160> 20 <170> PatentIn version 3.5 <210> 1 <211> 710 <212> DNA <213> Gastrodia elata <400> 1 ctgccagcta ctaagtgcca attaacacta aactaactaa ccgataccta tatatacgtg 60 tgtatatata tatacaaaat ggatcatgac ttaaagtttc tgtttaattg aatgcatata 120 tatatatata acctattcat agagtttcaa ttaagttaat atgtctcaga atttttattt 180 acatcatgtt tttatttttt ttttggctac aaacatatac aatggcaact tttactgaac 240 atacgtagat tcacaatgag gaataatcca tggtattaac tgcttgctta aaaactaagg 300 ttaaactagc tacattgaac actagatata atccaattgt ttgtaaaatc taaaatacag 360 caaggaaact aattcataag atcaaagcac gcactttcaa ttatgcttga aggataatgg 420 ggtactcaag gagctaattc taactaagag agtgataaat tttgaagcat tgtgtaaaac 480 atcagacaga aaaaaaagag aaaaagattg cagcatataa catggatagt gaacagcaag 540 ttggccaatt tatgaataat ctttagtcaa aatctgtgtg aaattaggaa gtgttatgca 600 aagcatacaa cgatttaaag attggaggag aggtgtatca tcaaattgta ggaagggagg 660 tataggggta cctagatgcg ttgaagggaa gatcagttac acgcgacgga 710 <210> 2 <211> 584 <212> DNA <213> Gastrodia elata <400> 2 atcccgtcgc caaaagagtt atctttgcat gagcggcggg gtggggcact acctttcaac 6 ctgaataaca tctttaaaca tattattagg gagattgatc agattagtaa tgcattccct 120 tttaactctt tcagctagaa gctaattaac gaaactaaat ttacaatctt aatcaattat 180 tatggtgctg aaggataatg ttgttggttt gtggattaat tggctcatta ttgttggttt 240 gtggtttcat ttgagctttg ttgtctttgt tgtgttttgg cttcttatga tgttgttttg 300 tggcagcatt tgcatattat tgtttttgtt gttgtttggt tggttatggt gacggttaac 360 agtgctaaga tttccgtctc tgtggagaat tcttggtcaa tcattatctt atgttggcag 420 cgatgtgagt tatgtttgtt ataaacttgc atttatagat tctaagttga aagatttata 480 cggtgctggc ggcaggattg cttgtaggtc ggagggaact gttgcgtttg cgttgttgtt 540 caaattactc ttgcatgagt tgaatttaga cctggtgctg ctgg 584 <210> 3 <211> 590 <212> DNA <213> Gastrodia elata <400> 3 gaaccttctg cgccaaaagg ctgtcgaggc ggtttcggag agtgggggaa taaatagagg 60 aaatgcaatt ttctttaaaa ggaaaattta aaataatact caaagttaaa ttattcacat 120 ttttctgccg tttgaagttc aaaaataacg aaacttggca ccgatgtgtg ttatgatata 180 acaaacacca ttgcgccaaa aattttcgat tcggacaaat attttatttt ttcattaatt 240 tttgaagttt aaaacttcga aaattaatca cgatcaaacc gactctcaga atttgacgaa 300 actttttgag cagcctttaa ataatattct taacacaacg acactaaaat agatgaattc 360 tgacatgtat gccaatattc ttgatttatt ggatttaatt aattcaaaaa attaaaatga 420 gagttttcgt tatccaaatt ctcccaaact agggcatttt gatctaaaaa ttatttctat 480 ctaacgtaaa ttttgtttgc cataatttga aattttcgta ggtcttgatg tctgagacgg 540 gttttgatct aatttgaaaa gtaacgatat tgggtcgtta caaactcccc 590<​​​​​​​​​​​​​ actttgattc ttttcattag aatgatatta ttttaagggt cttaatgttg actttaagat 180 atatatttcc aaattgaaat ttctctattt ctatgggttg cttaatgtct aaggagtaaa 240 tcaacaccta atttggacaa gtcttgatct taatattcat taattaagtt ttaacctaat 300 taaggggaga ttaacaaaat tacctacaga aagtggaatt atatttcata attagattcc 360 ttactctttc tttatcataa tggtatataa tattgcaata attgaattta aattgattga 420 attatatcaa aaatacattc tgaattaatt tcctaaaatt ttaatttaat tattcaacga 480 cattgttatt aatatgtgaa tattccggga atattctcat tccgtcccgt gtttaaccgc 540 gcgggctgac ggaacccaac gggcctgaga ata 573 <210> 5 <211> 416 <212> DNA <213> Gastrodia elata <400> 5 aagagctctg attgcccagt aaaccctaga gaataatttt agacccttaa aaccaatttt 60 ggacatttaa tttttttaac cactccttaa ttaccttaat taaggattag taaattaaac 120 cctaaattag tctttgattt tgggtaggtt agttcctaat gccatcttaa gtctattaga 180 acctaagtgt aaggttggat tatcctagaa tgccctaaat tcactaatca ctctaattgg 240 cctaaaaata tgattagtgt taattagctt aattaggctg tcaaaaccta agataagtgt 300 gtttcccact tcattaacca atatgaaaca cattagaaca gattagattt aaattggaga 360 actaaaccct aaaatcccaa ttttagcctt atgaggggta ttttggtcat tttggc 416 <210> 6 <211> 494 <212> DNA <213> Gastrodia elata <400> 6 atgctggtta ggaggcaacc caacaaatta ttttatgttt ttgtttgttt cagattccga 60 gtgatacatg tgtgcgtcta taaaggagca ggaggaatct cgaacatgat tgcagtaacg 120 acaatttgtc attatttaga tcaggttatt ttattcttta ctactccgag taaagtatgt 180 tcattttgcc ttcaatattg tgctttttgt ttttgtttaa ataaatctta gtgaattact 240 atcacccaat ttttgtgcca acaaaatgtc attatctcta caatttttct gaaaatctta 300 atgagctgaa ttcaattgta atggaacata tgaagatttt atttattcta atttacttaa 360 caagaggtga tcaagcaaaa tgtacagttc taaaattttt catgctaaat cattttacac 420 tcattttact ttaagtctcc ataataaaaa aatgtatgat gctttaaacc aaaaggtggg 480 gataaggtca agga 494 <210> 7 <211> 458 <212> DNA <213> Gastrodia elata <400> 7 cctagtcatc caaacatagg caatatgggt cttaaaatac tcaggaaaat aaccccatta 60 ctatagtagt attagttttt tttattttga catcaagata aaaagtcaac tctttgaccc 120 cataattgac cttagagaaa aataaccaaa atacccttag aatttaattg accataactt 180 gatcaattaa actccaaatt cagaaattca aagtgctaac tagcttaaaa tttcaatgtc 240 atcaataaat tgaaataata tttaggatct gaaccttaag aaattagaga atgaccgaaa 300 tacctctaac tttagttagg tcataactta acaatttgat gtccaaattg ggtctactcc 360 ctgtcaacac actcactata ttataaggaa gctatagaaa ctgagtttgt tgaaattgga 420 cttcaaacga aaaagttcgg ttttgtcacc tgttggcc 458 <210> 8 <211> 466 <212> DNA <213> Gastrodia elata <400> 8 tgagagaacc agtgatgtca tttatttcat tatgaatcaa atgctatcat gtgatcaaat 60 gctgccatct ttagtattat aagatcaaga gtgatcaaat gttattagtc atatccatgt 120 gatcaaatgc ttccatattt agtattataa gatcaagagt gatcaaatgc tattagttat 180 atccatgtga tcaaatgctg ccatctgtag tattataaga tcaagagtga tcaaaatgct 240 attagtcata tccatgtgaa caaatgatgt tattcattta aggtagatca aatgatgcca 300 ttttattttt atgagaatga atgctatcat ttctttcatt agaattacat attgccatta 360 tattcatgag atctaaatgc tgccatgttt catataaaaa ttcaatgctg ccatttatat 420 tatgaaccaa atgctgccat ttatattgag aatcaaattg ctgcca 466 <210> 9 <211> 400 <212> DNA <213> Gastrodia elata <400> 9 ttgcttggtg cttggtggta tcaaatttat tacctatctt tggcgagtta gattattgtc 60 ttatttatat tttattttaa ctatactctt attctcataa cctcaaacca tcaatacaaa 120 atcttcttca gcatcttaag tattctttaa aattattaaa tcattatcta atctctcaaa 180 cactctccaa actcccccac aaccaaatct gaaaatagcc cgacctagtt ttactccttg 240 cccctcggca cccatctaaa cacaatctaa cctatccatc tgctccagat ccaaactaca 300 catcttaggc tacatatatt aatttattta cagcccttta acccactaaa acttcacata 360 caaaagggat attttgaatc atcccaagct aggctcgatc 400 <210> 10 <211> 514 <212> DNA <213> Gastrodia elata <400> 10 accatgtgat gaagcaaatg caaaactaca tcaagattta atcataataa tcaccacacc 60 atcacttaga ggagagctga ttacaaacta atgttcagaa tatccaaaaa tatcttaatg 120 gtgattataa catgagctca aaatatgtat acttgtacaa caagatgatc accacttaga 180 gaattttcta acactatctt tattaatatc caaaaaaacc acaataatct tataaacgga 240 acaaatggac aacatcgata tacaaacaac aaattgccta atcaagctca atatagcatg 300 atcaattaaa acaaaatttc aaatatagaa ttataattac aatatgtata ttaatagaca 360 agggaataca aagtttttga ttggctagat ttatcattag gatcatgtat tcaaatgttg 420 gccatcacga ttgattatga cttttgactt cgattaaaat ttaaattatt ttttaggata 480 ttattatttt gcttaaaagg tgtggcaatg ctcg 514 <210> 11 <211> 522 <212> DNA <213> Gastrodia elata <400> 11 ggattgcctc ccaactaatg cttagtttaa tgtcctcaac ctgaccaaaa agagtttata 60 tccctttata atttctttcc aaaattttta ttaaggcttt aggatgagta tgagcttgat 120 taataatgta atcgttttga ataacagcta cactatcttt ttccttccat gtccaccttg 180 tatcttctaa cttacaatag tttttctttt tattcttctt ttcattcttc tcatttcttg 240 ccttatgttt gtcataatac tcatcaaatt catcatcaat atcattctca atttcatttt 300 gcaaaaaggc gtaaaattca attttcttaa gagtaagatc aaagcatttc tccacaaatt 360 cacattgcac acaataatca tcaatattac aaaagggttt gttgatgttt gagatacctc 420 aaatggtttt gacaccttta ttattttgaa tcactttacc ttgcttatcc tcaaataatg 480 catcacttat tattaaacat tgaggtagat gcacttgagg tg 522 <210> 12 <211> 501 <212> DNA <213> Gastrodia elata <400> 12 acatgcctcc tcctagtggt atgaattgag ctatgtagct aagagaattt cttatgtatt 60 cccatggctc aagtgatcaa gctatagtcc aaaggttatg tatgagctgc tttagcacaa 120 caaatgttgt cacatttatt tgtgacatta gcactcaagg tgcacaactt tttcataatg 180 cgatcattaa gcttaatgag gttttcctta aagagtttaa tctaaaattt gtctgcaaca 240 ttcatcattc ttcaactcat ctcataaaat aatatattaa tgtaccatta gctctaaatg 300 atatttcatt caaaagaata gtttgtacat gatatcaacc atgacttact gcatgttttt 360 gcctaatcct tcccaaatga ggggtttttg gctaccctta tactagatac aaaaagaaac 420 aacaaaagat gctgagcata tgcatagctc ttacaaggaa atagaaaaaa aaagtgagaa 480 tactaaaagc agcaacagcc c 501 <210> 13 <211> 653 <212> DNA <213> Gastrodia elata <400> 13 atcctaagca tggtgggacc tctgggtaag gttggattgc cctaagaatg cctaaattca 60 ctaatcaccc taattggctt aagggagtga ttagtgctaa ttagcttaat taagttgtca 120 aaacctaaga taagtgtgtt tcccacttca ttaatcaata tgaaacacat taaaataaat 180 tagacttaat ttgtataact aaaccttaaa atcctaattt ttgccttaag aggggcattt 240 tggtcatttt aaccaaaatg tgcttatatg cccctttaac ttcgaaaat tcagtaagct 300 acccttagag agattataaa tgttttactg actcttagat tataatgtt gatcatgtgg 360 aagtttattt aagaaatctt caattagccc taaagaagtc acttagcctt aataattgaa 420 tctaataatt ttagatgtta aatttcagtt tataagctt aactaagtta gcttatgagc 480 ttaaattcta aaaaaaaaca catttaggaa attctaggtt agaaaattag ttaagtttaa 540 agtgatcaaa tcttagatcc taaactttat atcttctact tgaattaaag tatctttaat 600 tgaatttctt aaaatggttt attgtttaac cttttgaagc cttccatgtg ggt 653 <210> 14 <211> 536 <212> DNA <213> Gastrodia elata <400> 14 gcccgagacc aatactcaca agattttgtt ccattatcat acttcaccct atggaggaca 60 ctatagatct gaaagaacag ccataaatgt cttgcaatca agattcaatt ggctaaccat 120 ctttatggaa gcctctgagt atgtaaagag ctgtgttcaa tgctaaataa tgggaaatat 180 tggtaaaata gatgaattcc ccttcaagat aattttaaag taaaactttt tgatgtttgg 240 ggaatagaca ttgtgggtcc ttttcccaca tcaagcatga acaactatat cttggtggtc 300 gtagaatata tatcaatatg ggtggaagct attgtatgct caaataatga ctctaaaata 360 gttatagatt ttctcaagaa gaatgttctc atcagatttg ttacacatag agttcttatc 420 agtgatagag gtaaacattc ttacaataaa caacatgaaa cattgttaag aaaatatggg 480 tttaaacaca aaataagtat tgcttataac ccactaacaa gcggacaagt ggaagt 536 <210> 15 <211> 长564 <212> DNA <213> Gastrodia elata <400> 15 tcaaacgagc tgaagaggca tacaacttag gctacactct actgaatcaa gatcagtaaa 60 gccaaaaaca tcgatgaaat gcacatgctg tttatgtaaa tcaaatttga tatgtaatac 120 tcatcaagta tcctatatcc gacatatatg tctaaggtaa taagctcaat ttcaaggaaa 180 acaaagcatc aaagttcaaa gagagcaata gactatacta agacacaaga gatatccatt 240 attatttaat tattcattct atttattcta agttacacaa gatcagaatc atcaattctt 300 atactatata taacttatat tcatggcctt ataagttgaa aataattaga actgatcagg 360 ttctaatatg atttaatcag tgtcgactac ataatttgaa tagataatca atcgacctat 420 tttgataaat taaataaccc aaacctgttc tgattaagac aaatcaagat ggaatgatta 480 agaattaaac tccaaatatt gaggtttaat tatttaatct aaaggtgatt ctaaaggact 540 aaagcttaca acccaatctc acga 564 <210> 16 <211> 649 <212> DNA <213> Gastrodia elata <400> 16 gattcgagat tccgcgggat ttaatcctag ccatcgggtt gatagggtgg atggtgaata 60 tgggggaagc accgctgtca cgcgacctgg gctccactgc ggatcgcctg cggtccgcac 120 aacaaatcct agatgtttag ttagttttaa gggtatgaat gcaaatttga aatgttggaa 180 atcctaaatg caattagacc attttaattt aaattgaatt atggtctaaa taatctccaa 240 attttaccaa agtttcactc ctagctaatt tgggggtgag gaattcaaaa atgcaatcaa 300 attttaattc taagatcaaa atattttagg tctaaactat ggcatctaga ctaatcaaaa 360 tgccatcttg acttagagaa gtccaattga gttcaaattt tcaggattag cttaacttga 420 tattctaaaa ataatgggat caaaataaat gctcaattag gttaaaaatt aacagagtaa 480 caacctgggt tgttatagac tctcccactt aagaatgatg tcatcctcga cattaactta 540 agagagaaga cacggatcaa aaatcttaag attagaattt caattcagaa aattaatgga 600 tacctagatt ttcaaagaga ctgggatact tccttcgctt atcggcctc 649 <210> 17 <211> 585 <212> DNA <213> Gastrodia elata <400> 17 ggttggatta gtgtttgagt gattaatttt gctaatcact cactaatcac tcttaaccct 60 aattaagtca caacttagtc aaattatgtc caaattgtgc ctaatacatg tcattgtgat 120 cagtggacta tttgtaaacc atagccactg actttactta atttggacac taattgacaa 180 agttaggatt ttgacccata attaaggatt ccttaattac acttataacc ctaaatcttg 240 tattagccat aacttggcca atacttatca gaatttagca aatctagatt ctatggaaag 300 cttatatagt aaacaacaat aatctgaaag aaaatttaac tttcacttag ccaaattact 360 aatttgccct tatgagtcaa attcactaat ttgacttcat atggcttaat aatcacccaa 420 aattaattta gttactgtct aatcttactt tagacaataa gaaatcacct tccaaaagaa 480 aactttagat tttaaagccc tagatcaaaa tttgatctct tgaaccctta agtatgggca 540 aaatggtcat tttgcccaaa tttagggttt agcatcttaa agccc 585 <210> 18 <211> 531 <212> DNA <213> Gastrodia elata <400> 18 caaaccgaga gctctccctc cttcctccat tgttgttctt tggctgaaga agaaaaagaa 60 aaagttcatc ctccaagagt tctgagtctt cttcctcctt ttcttgcaaa gtcaaatgta 120 ccccctttcc ttccattagt taatccaatg gctattctat ttcgttttta tgctcattta 180 tatatttaag attctgtgtt accttagttg tgtatctata aagctatatt taagatgtag 240 gaactcaaaa aagtaaaaat ctgtaaacaa tagggaatga tatgtaaagt tgctaatacg 300 aatttctaga cacccagtca tttaacccta cggtttgcat tgataactta atcttttggt 360 gctgatttgg gtatcaatgg aaaggtttca gaaagagctt tccaacggta tatgacaccg 420 catgtgaagt gctctaggtt aagagtaatt gagcccgaaa gttgaaccat tgcggttttc 480 gcatagacga aatcaccgtc tggcgctaca gcgctcgtag cacccagaaa t 531 <210> 19 <211> 547<00​​​​​​​​​​​​catttaggac ctaaccctaa ttagcctaat ttgacctaga acttcaaagg accataagtt 240 gaccatctta gctccaaatt aggtgatcca aataccaatt tagatcagca aaacctaatc 300 tacagagatc tacaaaaata tttaggatcc aacagttaga ttttctaatc acccctaatt 360 accctaaaac ttgattagtg aatattagta gatttaataa tccaaattgg acaaactaag 420 tttcagagaa ctcctagaac ctattagaac ctaactctag ggttggatta gtgattgggt 480 gattaatttc actaatcact ctaaactcta attaagccat aacttagcca aatggagtcc 540 aaattga 547 <210> 20 <211> 530 <212> DNA <213> Gastrodia elata <400> 20 aatcgaaccc gggtctgtac cgtggcaggg tactattcta ccactagacc actggtgcaa 60 cacgttaatt tttgttaaat ttataaaatt ataaacgtaa ctcatatatc gaagacagat 120 catatatgtg aagtcacccg ttctcattcg agcaaaaccc tacttcttcc catgacgctc 180 ctcgagtcca tcatacaagc agcggaggcc gccgacaccg gcgatctttc tccatttccc 240 aagaatgcga tccccatcat tctaaacact aatgacattt tttcaaactt aaaggaggac 300 tctgaattcc ttacctctcc gcccatcgtc cgcgtctccg gctggtccat ctcaaagttc 360 gattctagca tcatcgaatc catctcctta ttctcccaaa ctctaaaaaga caagctccaa 420 caatcccagt ccctcaaccg ggctgaattc ctcaagctcc tcggtgcctt cctgcaaacc 480 ctcggagcga atctcggcct cggcttcaat tccgaggaac cttctccgta 530

Claims

1. A reagent for identifying RFLP molecular marker sites in Gastrodia elata, characterized in that: The RFLP molecular marker sites consist of 20 SNP sites, which are composed of E1, E2, E3, E4, E5, E6, E7, E8, E9, E10, E11, H1, H2, H3, S1, X1, X2, X3, X4, and X5 marker sites. The E1 marker site is a site on chromosome 3 of Gastrodia elata, and its nucleotide type is T or G. It is the 435th nucleotide of SEQ ID No. 1 in the Gastrodia elata genome. The E2 marker site is a site on chromosome 7 of Gastrodia elata, and its nucleotide type is A or C. It is the 392nd nucleotide of SEQ ID No. 2 in the Gastrodia elata genome. The E3 marker site is a site on chromosome 1 of Gastrodia elata, and its nucleotide type is G or C. It is the 360th nucleotide of SEQ ID No. 3 in the Gastrodia elata genome. The E4 marker site is a site on chromosome 2 of Gastrodia elata, and its nucleotide type is C or T. It is the 409th nucleotide of SEQ ID No. 4 in the Gastrodia elata genome. The E5 marker site is a site on chromosome 2 of Gastrodia elata, and its nucleotide type is C or G. It is the 171st nucleotide of SEQ ID No. 5 in the Gastrodia elata genome. The E6 marker site is a site on chromosome 8 of Gastrodia elata, and its nucleotide type is T or C. It is the 313th nucleotide of SEQ ID No. 6 in the Gastrodia elata genome. The E7 marker site is a location on chromosome 17 of Gastrodia elata, and its nucleotide type is A or G. It is the 280th nucleotide of SEQ ID No. 7 in the Gastrodia elata genome. The E8 marker site is a site on chromosome 2 of Gastrodia elata, and its nucleotide type is C or A. It is the 347th nucleotide of SEQ ID No. 8 in the Gastrodia elata genome. The E9 marker site is a site on chromosome 3 of Gastrodia elata, and its nucleotide type is A or G. It is the 140th nucleotide of SEQ ID No. 9 in the Gastrodia elata genome. The E10 marker site is a site on chromosome 4 of Gastrodia elata, and its nucleotide type is C or A. It is the 333rd nucleotide of SEQ ID No. 10 in the Gastrodia elata genome. The E11 marker site is a site on chromosome 5 of Gastrodia elata, and its nucleotide type is G or A. It is the 330th nucleotide of SEQ ID No. 11 in the Gastrodia elata genome. The H1 marker site is a site on chromosome 5 of Gastrodia elata, and its nucleotide type is C or T. It is the 194th nucleotide of SEQ ID No. 12 in the Gastrodia elata genome. The H2 marker site is a site on chromosome 14 of Gastrodia elata, and its nucleotide type is C or T. It is the 460th nucleotide of SEQ ID No. 13 in the Gastrodia elata genome. The H3 marker site is a site on chromosome 14 of Gastrodia elata, and its nucleotide type is C or T. It is the 133rd nucleotide of SEQ ID No. 14 in the Gastrodia elata genome. The S1 marker site is a site on chromosome 10 of Gastrodia elata, and its nucleotide type is G or C. It is the 387th nucleotide of SEQ ID No. 15 in the Gastrodia elata genome. The X1 marker site is a site on chromosome 1 of Gastrodia elata, and its nucleotide type is A or C. It is the 350th nucleotide of SEQ ID No. 16 in the Gastrodia elata genome. The X2 marker site is a site on chromosome 5 of Gastrodia elata, and its nucleotide type is A or G, specifically nucleotide 288 of SEQ ID No. 17 in the Gastrodia elata genome. The X3 marker site is a site on chromosome 8 of Gastrodia elata, and its nucleotide type is G or A. It is the 310th nucleotide of SEQ ID No. 18 in the Gastrodia elata genome. The X4 marker site is a site on chromosome 12 of Gastrodia elata, and its nucleotide type is T or C. It is the 311th nucleotide of SEQ ID No. 19 in the Gastrodia elata genome. The X5 marker site is a site on chromosome 6 of Gastrodia elata, and its nucleotide type is A or G, which is the 186th nucleotide of SEQ ID No. 20 in the Gastrodia elata genome; The reagent consists of 20 compositions named E1, E2, E3, E4, E5, E6, E7, E8, E9, E10, E11, H1, H2, H3, S1, X1, X2, X3, X4, and X5. The E1 group consists of a primer composition named E1 primer pair for amplifying the Gastrodia elata genomic DNA fragment including the E1 marker site, and a restriction endonuclease. EcoR I. Composition The E2 group consists of a primer composition named E2 primer pair for amplifying the Gastrodia elata genomic DNA fragment including the E2 marker site, and a restriction endonuclease. EcoR I. Composition The E3 group consists of a primer composition named E3 primer pair for amplifying the Gastrodia elata genomic DNA fragment including the E3 marker site, and a restriction endonuclease. EcoR I. Composition The E4 group consists of a primer composition named E4 primer pair for amplifying the Gastrodia elata genomic DNA fragment including the E4 marker site, and a restriction endonuclease. EcoR I. Composition The E5 group consists of a primer composition named E5 primer pair for amplifying the Gastrodia elata genomic DNA fragment including the E5 marker site, and a restriction endonuclease. EcoR I. Composition The E6 group consists of a primer composition named E6 primer pair for amplifying the Gastrodia elata genomic DNA fragment including the E6 marker site, and a restriction endonuclease. EcoR I. Composition The E7 group consists of a primer composition named E7 primer pair for amplifying the Gastrodia elata genomic DNA fragment including the E7 marker site, and a restriction endonuclease. EcoR I. Composition The E8 group consists of a primer composition named E8 primer pair for amplifying the Gastrodia elata genomic DNA fragment including the E8 marker site, and a restriction endonuclease. EcoR I. Composition The E9 group consists of a primer composition named E9 primer pair for amplifying the Gastrodia elata genomic DNA fragment including the E9 marker site, and a restriction endonuclease. EcoR I. Composition The E10 group consists of a primer composition named E10 primer pair for amplifying the Gastrodia elata genomic DNA fragment including the E10 marker site, and a restriction endonuclease. EcoR I. Composition The E11 group consists of a primer composition named E11 primer pair for amplifying the Gastrodia elata genomic DNA fragment including the E11 marker site, and a restriction endonuclease. EcoR I. Composition The H1 group consists of a primer composition named H1 primer pair for amplifying the Gastrodia elata genomic DNA fragment including the H1 marker site, and the restriction endonuclease HindIII. The H2 group consists of a primer composition named H2 primer pair for amplifying the Gastrodia elata genomic DNA fragment including the H2 marker site, and a restriction endonuclease. Hind Composition III The H3 group consists of a primer composition named H3 primer pair for amplifying the Gastrodia elata genomic DNA fragment including the H3 marker site, and a restriction endonuclease. Hind Composition III The S1 group consists of a primer composition named S1 primer pair for amplifying the Gastrodia elata genomic DNA fragment including the S1 marker site, and a restriction endonuclease. Sal I. Composition The X1 group consists of a primer composition named X1 primer pair for amplifying the Gastrodia elata genomic DNA fragment including the X1 marker site, and the restriction endonuclease XbaI. The X2 group consists of a primer composition named X2 primer pair for amplifying the Gastrodia elata genomic DNA fragment including the X2 marker site, and a restriction endonuclease. Xba I is composed of, The X3 group consists of a primer composition named X3 primer pair for amplifying the Gastrodia elata genomic DNA fragment including the X3 marker site, and the restriction endonuclease XbaI. The X4 group consists of a primer composition named X4 primer pair for amplifying the Gastrodia elata genomic DNA fragment including the X4 marker site, and a restriction endonuclease. Xba I is composed of, The X5 group consists of a primer composition named X5 primer pair for amplifying the Gastrodia elata genomic DNA fragment including the X5 marker site, and a restriction endonuclease. Xho I consists of; The E1 primer pair consists of two single-stranded DNAs named E1-F and E1-R. The nucleotide sequence of E1-F is positions 1-20 of sequence 1 in the sequence listing, and the nucleotide sequence of E1-R is inversely complementary to positions 691-710 of sequence 1 in the sequence listing. The E2 primer pair consists of two single-stranded DNAs named E2-F and E2-R. The nucleotide sequence of E2-F is positions 1-20 of sequence 2 in the sequence listing, and the nucleotide sequence of E2-R is inversely complementary to positions 565-584 of sequence 2 in the sequence listing. The E3 primer pair consists of two single-stranded DNAs named E3-F and E3-R. The nucleotide sequence of E3-F is positions 1-20 of sequence 3 in the sequence listing, and the nucleotide sequence of E3-R is inversely complementary to positions 571-590 of sequence 3 in the sequence listing. The E4 primer pair consists of two single-stranded DNAs named E4-F and E4-R. The nucleotide sequence of E4-F is positions 1-20 of sequence 4 in the sequence listing, and the nucleotide sequence of E4-R is inversely complementary to positions 554-573 of sequence 4 in the sequence listing. The E5 primer pair consists of two single-stranded DNAs named E5-F and E5-R. The nucleotide sequence of E5-F is positions 1-20 of sequence 5 in the sequence listing, and the nucleotide sequence of E5-R is inversely complementary to positions 395-416 of sequence 5 in the sequence listing. The E6 primer pair consists of two single-stranded DNAs named E6-F and E6-R. The nucleotide sequence of E6-F is positions 1-20 of sequence 6 in the sequence listing, and the nucleotide sequence of E6-R is inversely complementary to positions 474-494 of sequence 6 in the sequence listing. The E7 primer pair consists of two single-stranded DNAs named E7-F and E7-R. The nucleotide sequence of E7-F is positions 1-22 of sequence 7 in the sequence listing, and the nucleotide sequence of E7-R is inversely complementary to positions 439-458 of sequence 7 in the sequence listing. The E8 primer pair consists of two single-stranded DNAs named E8-F and E8-R. The nucleotide sequence of E8-F is positions 1-20 of sequence 8 in the sequence listing, and the nucleotide sequence of E8-R is inversely complementary to positions 446-466 of sequence 8 in the sequence listing. The E9 primer pair consists of two single-stranded DNAs named E9-F and E9-R. The nucleotide sequence of E9-F is positions 1-20 of sequence 9 in the sequence listing, and the nucleotide sequence of E9-R is inversely complementary to positions 381-400 of sequence 9 in the sequence listing. The E10 primer pair consists of two single-stranded DNAs named E10-F and E10-R. The nucleotide sequence of E10-F is positions 1-22 of sequence 10 in the sequence listing, and the nucleotide sequence of E10-R is inversely complementary to positions 495-514 of sequence 10 in the sequence listing. The E11 primer pair consists of two single-stranded DNAs named E11-F and E11-R. The nucleotide sequence of E11-F is positions 1-21 of sequence 11 in the sequence listing, and the nucleotide sequence of E11-R is inversely complementary to positions 501-522 of sequence 11 in the sequence listing. The H1 primer pair consists of two single-stranded DNAs named H1-F and H1-R. The nucleotide sequence of H1-F is positions 1-20 of sequence 12 in the sequence listing, and the nucleotide sequence of H1-R is inversely complementary to positions 482-501 of sequence 12 in the sequence listing. The H2 primer pair consists of two single-stranded DNAs named H2-F and H2-R. The nucleotide sequence of H2-F is positions 1-20 of sequence 13 in the sequence listing, and the nucleotide sequence of H2-R is inversely complementary to positions 634-653 of sequence 13 in the sequence listing. The H3 primer pair consists of two single-stranded DNAs named H3-F and H3-R. The nucleotide sequence of H3-F is positions 1-20 of sequence 14 in the sequence listing, and the nucleotide sequence of H3-R is inversely complementary to positions 517-536 of sequence 14 in the sequence listing. The S1 primer pair consists of two single-stranded DNAs named S1-F and S1-R. The nucleotide sequence of S1-F is positions 1-20 of sequence 15 in the sequence listing, and the nucleotide sequence of S1-R is inversely complementary to positions 543-564 of sequence 15 in the sequence listing. The X1 primer pair consists of two single-stranded DNAs named X1-F and X1-R. The nucleotide sequence of X1-F is positions 1-20 of sequence 16 in the sequence listing, and the nucleotide sequence of X1-R is inversely complementary to positions 630-649 of sequence 16 in the sequence listing. The X2 primer pair consists of two single-stranded DNAs named X2-F and X2-R. The nucleotide sequence of X2-F is positions 1-22 of sequence 17 in the sequence listing, and the nucleotide sequence of X2-R is inversely complementary to positions 564-585 of sequence 17 in the sequence listing. The X3 primer pair consists of two single-stranded DNAs named X3-F and X3-R. The nucleotide sequence of X3-F is positions 1-20 of sequence 18 in the sequence listing, and the nucleotide sequence of X3-R is inversely complementary to positions 512-531 of sequence 18 in the sequence listing. The X4 primer pair consists of two single-stranded DNAs named X4-F and X4-R. The nucleotide sequence of X4-F is positions 1-21 of sequence 19 in the sequence listing, and the nucleotide sequence of X4-R is inversely complementary to positions 526-547 of sequence 19 in the sequence listing. The X5 primer pair consists of two single-stranded DNAs named X5-F and X5-R, respectively. The nucleotide sequence of X5-F is positions 1-20 of sequence 20 in the sequence listing, and the nucleotide sequence of X5-R is inversely complementary to positions 511-530 of sequence 20 in the sequence listing.

2. The use of the reagent according to claim 1 in any of the following: (1) To identify or assist in identifying the homozygosity of Gastrodia elata; (2) Screening or breeding single plants, strains, varieties or cultivars of Gastrodia elata with high homozygosity; (3) Gastrodia elata breeding; (4) To prepare products for identification or to assist in the identification of high purity of Gastrodia elata; (5) To prepare or select products of high homozygosity single plants, strains, varieties or cultivars of Gastrodia elata; (6) Preparation of products for Gastrodia elata breeding; The purpose of the breeding is to select highly homozygous single plants, strains, varieties, or cultivars of Gastrodia elata.

3. A primer composition for identifying or assisting in the identification of RFLP molecular marker sites in Gastrodia elata, characterized in that: The RFLP molecular marker sites are the 20 SNP sites described in claim 1, and the primer composition consists of the following 20 primer pairs: the E1 primer pair, the E2 primer pair, the E3 primer pair, the E4 primer pair, the E5 primer pair, the E6 primer pair, the E7 primer pair, the E8 primer pair, the E9 primer pair, the E10 primer pair, the E11 primer pair, the H1 primer pair, the H2 primer pair, the H3 primer pair, the S1 primer pair, the X1 primer pair, the X2 primer pair, the X3 primer pair, the X4 primer pair, and the X5 primer pair as described in claim 1.

4. The use of the primer composition of claim 3 in any of the following: (1) To identify or assist in identifying the homozygosity of Gastrodia elata varieties; (2) Screening or breeding single plants, strains, varieties or cultivars of Gastrodia elata with high homozygosity; (3) Gastrodia elata breeding; (4) To prepare products for identification or to assist in the identification of high purity of Gastrodia elata; (5) To prepare or select products of high homozygosity single plants, strains, varieties or cultivars of Gastrodia elata; (6) Preparation of products for Gastrodia elata breeding; The purpose of the breeding is to select highly homozygous single plants, strains, varieties, or cultivars of Gastrodia elata.

5. Methods for identifying or assisting in the identification of the homozygosity of Gastrodia elata, including the following steps: 1) Using the genomic DNA of the *Gastrodia elata* to be tested as a template, PCR amplification was performed using the 20 primer pairs described in claim 3 to obtain 20 PCR products. The PCR product of *Gastrodia elata* to be tested obtained by PCR amplification using the E1 primer pair was named E1-PCR product, the PCR product of *Gastrodia elata* to be tested obtained by PCR amplification using the E2 primer pair was named E2-PCR product, the PCR product of *Gastrodia elata* to be tested obtained by PCR amplification using the E3 primer pair was named E3-PCR product, and the PCR product of *Gastrodia elata* to be tested obtained by PCR amplification using the E4 primer pair was named E4-PCR product. The PCR products of *Gastrodia elata* obtained by PCR amplification using the E5 primer pair are named E5-PCR products, the PCR products of *Gastrodia elata* obtained by PCR amplification using the E6 primer pair are named E6-PCR products, the PCR products of *Gastrodia elata* obtained by PCR amplification using the E7 primer pair are named E7-PCR products, the PCR products of *Gastrodia elata* obtained by PCR amplification using the E8 primer pair are named E8-PCR products, the PCR products of *Gastrodia elata* obtained by PCR amplification using the E9 primer pair are named E9-PCR products, and the PCR products of *Gastrodia elata* obtained by PCR amplification using the E10 primer pair are named E9-PCR products. The PCR product of *Gastrodia elata* obtained by R-amplification is named E10-PCR product. The PCR product of *Gastrodia elata* obtained by PCR amplification using the E11 primer pair is named E11-PCR product. The PCR product of *Gastrodia elata* obtained by PCR amplification using the H1 primer pair is named H1-PCR product. The PCR product of *Gastrodia elata* obtained by PCR amplification using the H2 primer pair is named H2-PCR product. The PCR product of *Gastrodia elata* obtained by PCR amplification using the H3 primer pair is named H3-PCR product. The PCR product of *Gastrodia elata* obtained by PCR amplification using the S1 primer pair is named E10-PCR product. The R product is named S1-PCR product. The Gastrodia elata PCR product obtained by PCR amplification using the X1 primer pair is named X1-PCR product. The Gastrodia elata PCR product obtained by PCR amplification using the X2 primer pair is named X2-PCR product. The Gastrodia elata PCR product obtained by PCR amplification using the X3 primer pair is named X3-PCR product. The Gastrodia elata PCR product obtained by PCR amplification using the X4 primer pair is named X4-PCR product. The Gastrodia elata PCR product obtained by PCR amplification using the X5 primer pair is named X5-PCR product. 2) Perform RFLP analysis on the Gastrodia elata PCR products of the 20 primer pairs to be tested. The RFLP analysis includes digesting the Gastrodia elata PCR products of the 20 primer pairs to be tested with restriction endonucleases to obtain 20 digested products. Perform electrophoresis on the 20 digested products and determine the number of bands of the 20 digested products based on the electrophoresis results. In the RFLP analysis, the E1-PCR product, E2-PCR product, E3-PCR product, E4-PCR product, E5-PCR product, E6-PCR product, E7-PCR product, E8-PCR product, E9-PCR product, E10-PCR product, and E11-PCR product were each processed with restriction endonucleases. EcoR I. Enzyme digestion was performed, and the H1-PCR product, the H2-PCR product, and the H3-PCR product were each digested with restriction endonucleases. Hind III. The S1-PCR product was digested with restriction endonucleases. Sal I. The X1-PCR product, X2-PCR product, X3-PCR product, and X4-PCR product were digested with restriction endonucleases. Xba I was subjected to enzyme digestion, and the X5-PCR product was digested with restriction endonuclease. Xho I undergoes enzyme digestion; 3) Calculate the homozygosity based on the number of enzyme digestion bands in the PCR product of the gastrodia elata to be tested.

6. A method for breeding Gastrodia elata, characterized by: The method includes identifying or assisting in identifying the homozygosity of Gastrodia elata using the method of claim 5, selecting Gastrodia elata with a homozygosity greater than or equal to 95% as parents for breeding, and the formula for calculating the homozygosity is: H=(1-n / 20)×100%, where H represents the homozygosity and n represents the number of enzyme digestion products with 3 bands in the electrophoresis results after enzyme digestion of the 20 PCR products; The purpose of the breeding is to select highly homozygous single plants, strains, varieties, or cultivars of Gastrodia elata.

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