A haplotype molecular marker associated with the length of Gastrodia elata tubers and its application

Abstract

This invention discloses a haplotype molecular marker related to the tuber length of Gastrodia elata and its application. One technical solution to be protected by this invention is the application of two SNPs (SNP10 and SNP11) or one SNP in the identification or auxiliary identification of Gastrodia elata quality: SNP10 is a SNP in the Gastrodia elata genome, located at nucleotide 176 of SEQ ID No. 10 in the sequence listing, and its nucleotide value is C or G; SNP11 is a SNP in the Gastrodia elata genome, located at nucleotide 288 of SEQ ID No. 11 in the sequence listing, and its nucleotide value is C or G. Another technical solution to be protected by this invention is the application of the two SNP sites in Gastrodia elata breeding. The embodiments of this invention illustrate that molecular-assisted breeding can be carried out using the above-mentioned SNP sites to select superior Gastrodia elata germplasm with long tuber lengths, thus promoting rapid and efficient Gastrodia elata breeding.

Description

Technical Field

[0001] This application is a divisional application of application number 202111095727.9, filed on September 17, 2021, entitled "A haplotype molecular marker related to the superior shape and quality of Gastrodia elata and its application". Background Technology

[0002] In recent years, with the rapid development of the traditional Chinese medicine (TCM) industry, the demand for "high-quality" medicinal herbs has been increasing. At the same time, the growing scarcity of wild medicinal resources has placed increasingly higher demands on cultivated medicinal herbs. The breeding of medicinal herbs differs from that of crops. First, a crucial indicator of the quality of medicinal herbs is their clinical efficacy; ensuring both yield and quality has become a pressing issue in the breeding process. Second, the long breeding cycle of medicinal plants, often perennial, further complicates the selection of "high-quality" herbs. Currently, the main standard for evaluating "high quality" is the content of indicative components listed in the Chinese Pharmacopoeia. However, with the deepening research on authentic medicinal herbs, increasing experimental evidence shows that their chemical composition and phenotypes are determined by their intrinsic genes and gene regulatory networks, and are influenced by external factors such as growth environment, harvesting stage, and processing methods. The phenotypic characteristics of medicinal materials can be manifested in their "superior form" and "superior quality," which in turn translates into "superior efficacy" in their use. From a biological perspective, the "superior form" and "superior quality" characteristics of medicinal materials are unified. [1] .

[0003] The yield and quality of Chinese medicinal herbs are mainly affected by factors such as germplasm, environment, and harvesting time. Among these, germplasm selection is the source of Chinese medicinal herb breeding and a key factor affecting the quality of Chinese medicinal herbs. Molecular marker-assisted breeding has been widely used in agricultural production, but its application in the cultivation of Chinese medicinal herbs is relatively limited.

[0004] The dried tuber of *Gastrodia elata* Bl., an orchid, has the effects of calming wind and stopping spasms, suppressing liver yang, and dispelling wind and unblocking meridians. It is mainly used to treat internal liver wind, epilepsy, convulsions, dizziness, headache, numbness of limbs, hemiplegia, and rheumatic pain. How to utilize molecular markers to accelerate the breeding of superior-shaped and high-quality *Gastrodia elata* is an urgent technical problem to be solved. Summary of the Invention

[0005] The technical problem to be solved by this invention is to use molecular markers to assist in the breeding of Gastrodia elata varieties with superior shape and quality characteristics.

[0006] To address the aforementioned technical problems, the present invention first provides applications, namely, the application of any one or more of the following A1-A6 in identifying or assisting in the identification of the quality of Gastrodia elata:

[0007] The application of SNP sites related to polysaccharide content in the identification or auxiliary identification of Gastrodia elata quality, wherein the SNP sites related to polysaccharide content are 4 SNPs, 3 SNPs, 2 SNPs, or 1 SNP selected from SNP1, SNP2, SNP3, and SNP4:

[0008] The SNP1 is a SNP in the Gastrodia elata genome, which is the 222nd nucleotide in SEQ ID No. 1 in the sequence listing. It is either C or T. The letter Y in SEQ ID No. 1 represents either C or T.

[0009] The SNP2 is a SNP in the Gastrodia elata genome, which is the 219th nucleotide in SEQ ID No. 2 in the sequence listing. Its nucleotide is either T or A. The letter W in SEQ ID No. 2 represents either T or A.

[0010] The SNP3 is a SNP in the Gastrodia elata genome, which is the 270th nucleotide in SEQ ID No. 3 in the sequence listing. Its nucleotide is either T or A. The letter W in SEQ ID No. 3 represents either T or A.

[0011] The SNP4 is a SNP in the Gastrodia elata genome, which is the 193rd nucleotide in SEQ ID No. 4 in the sequence listing. Its nucleotide is C or T. The letter Y in SEQ ID No. 4 represents either C or T.

[0012] The application of the SNP site named SNP5, which is related to the content of gastrodin in A2, in the identification or auxiliary identification of the quality of Gastrodia elata; SNP5 is an SNP in the Gastrodia elata genome, which is the 228th nucleotide of SEQ ID No. 5 in the sequence listing, and its nucleotide is C or T. The letter Y in SEQ ID No. 5 represents either C or T nucleotide.

[0013] The application of SNP sites related to the content of p-hydroxybenzyl alcohol (SNP3) in the identification or auxiliary identification of the quality of Gastrodia elata, wherein the SNP sites related to p-hydroxybenzyl alcohol are two SNPs or one SNP from SNP6 and SNP7:

[0014] The SNP6 is a SNP in the Gastrodia elata genome, which is the 238th nucleotide in SEQ ID No. 6 in the sequence listing. Its nucleotide is C or G, and the letter S in SEQ ID No. 6 represents either C or G.

[0015] The SNP7 is a SNP in the Gastrodia elata genome, which is the 238th nucleotide in SEQ ID No. 7 in the sequence listing. Its nucleotide is C or T, and the letter Y in SEQ ID No. 7 represents either C or T.

[0016] The application of the SNP site named SNP8, which is related to the weight of tubers in A4 tubers, in the identification or auxiliary identification of the quality of Gastrodia elata; SNP8 is an SNP in the Gastrodia elata genome, which is the 218th nucleotide of SEQ ID No. 8 in the sequence listing, and its nucleotide is any one of G, A or T. The letter D in SEQ ID No. 8 represents any one of G, A and T nucleotides.

[0017] The application of the SNP site named SNP9, located 1 cm from the navel, in identifying or assisting in the identification of the quality of Gastrodia elata; SNP9 is an SNP in the Gastrodia elata genome, which is the 193rd nucleotide of SEQ ID No. 9 in the sequence listing, and its nucleotide is C or T. The letter Y in SEQ ID No. 9 represents either C or T nucleotide.

[0018] The application of A6 tuber length-related SNP sites in identifying or assisting in the identification of Gastrodia elata quality, wherein the tuber length-related SNP sites are two SNPs or one SNP from SNP10 and SNP11:

[0019] The SNP10 is a SNP in the Gastrodia elata genome, which is the 176th nucleotide of SEQ ID No. 10 in the sequence listing. Its nucleotide is C or G, and the letter S in SEQ ID No. 10 represents either C or G.

[0020] SNP11 is a SNP in the Gastrodia elata genome, which is the 288th nucleotide in SEQ ID No. 11 in the sequence listing. Its nucleotide is C or G, and the letter S in SEQ ID No. 11 represents either C or G.

[0021] In the above applications, the quality of Gastrodia elata includes one or more of the following B1-B6:

[0022] B1. Total polysaccharide content;

[0023] B2. Gastrodin content;

[0024] B3. Content of p-hydroxybenzyl alcohol;

[0025] B4. Tuber weight;

[0026] B5. Width 1cm from the navel;

[0027] B6. Tuber length.

[0028] The present invention also provides the application of substances for detecting haplotypes in the identification or auxiliary identification of the quality of Gastrodia elata: the haplotype is a polymorphic combination of one or more SNPs from SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, SNP10 and / or SNP11 on the Gastrodia elata chromosome.

[0029] In the above applications, the quality of Gastrodia elata includes one or more of the following B1-B6:

[0030] B1. Total polysaccharide content;

[0031] B2. Gastrodin content;

[0032] B3. Content of p-hydroxybenzyl alcohol;

[0033] B4. Tuber weight;

[0034] B5. Width 1cm from the navel;

[0035] B6. Tuber length.

[0036] To solve the above-mentioned technical problems, the present invention also provides a product containing the substance for detecting haplotypes, and the product may be any one of the following G1)-G7):

[0037] G1) Products that test for single nucleotide polymorphisms or genotypes related to the quality of Gastrodia elata;

[0038] G2) Products for identifying or assisting in the identification of the quality of Gastrodia elata;

[0039] G3) Products used in Gastrodia elata breeding;

[0040] G4) Products for identification or auxiliary identification of Gastrodia elata polysaccharide content;

[0041] G5) Products for identification or auxiliary identification of gastrodin content;

[0042] G6) Products used for identification or auxiliary identification of Gastrodia elata yield;

[0043] G7) Products that are identified or used to assist in the identification of the shape of Gastrodia elata.

[0044] This invention also provides a method for breeding Gastrodia elata, comprising: detecting the genotype of one or more SNPs from the eleven SNPs (SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, SNP10, SNP11) in the Gastrodia elata genome, and selecting Gastrodia elata that satisfy one or more of the following C1-C11 for breeding:

[0045] The genotypes of C1 and SNP1 are CC or TC; CC is the homozygous type of SNP1 being C; TC is the heterozygous type of SNP1 being T and C.

[0046] The genotypes of C2 and SNP2 are TT or TA; TT is the homozygous type of SNP2 being T; TA is the heterozygous type of SNP2 being T and A.

[0047] The genotypes of C3 and SNP3 are TT or TA; TT is the homozygous type of SNP3 being T; TA is the heterozygous type of SNP3 being T and A.

[0048] The genotypes of C4 and SNP4 are CC or TC; CC is the homozygous type of SNP4 being C; TC is the heterozygous type of SNP4 being T and C.

[0049] The genotypes of C5 and SNP5 are CC or CT; CC is the homozygous type of SNP5 being C; CT is the heterozygous type of SNP5 being T and C.

[0050] The genotypes of C6 and SNP6 are CC or CG; CC is the homozygous type of SNP6 being C; CG is the heterozygous type of SNP6 being both C and G.

[0051] The genotypes of C7 and SNP7 are CC or TC; CC is the homozygous type of SNP7 being C; TC is the heterozygous type of SNP7 being T and C.

[0052] The genotypes of C8 and SNP8 are GG or AG; GG is the homozygous type of SNP8 being G; AG is the heterozygous type of SNP8 being A and G.

[0053] The genotypes of C9 and SNP9 are CC or CT; CC is the homozygous type of SNP9 being C; CT is the heterozygous type of SNP9 being C and T.

[0054] The genotypes of C10 and SNP10 are GG or CG; GG is the homozygous type of SNP10 being G; CG is the heterozygous type of SNP10 being C and G.

[0055] The genotypes of C11 and SNP11 are CC or CG; CC is the homozygous type of SNP11 being C; CG is the heterozygous type of SNP11 being C and G.

[0056] In the above method, the detection of the genotype of one or more SNPs among the eleven SNPs (SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, SNP10, and SNP11) in the Gastrodia elata genome can be achieved by determining the nucleotide types of the eleven sites (SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, SNP10, and / or SNP11) in the Gastrodia elata genome using at least one of the following methods: DNA sequencing, restriction fragment length polymorphism, single-strand conformation polymorphism, denaturing high-performance liquid chromatography, and SNP microarray. Among them, SNP chips include chips based on nucleic acid hybridization reactions, chips based on single base extension reactions, chips based on allele-specific primer extension reactions, chips based on "one-step" reactions, chips based on primer ligation reactions, chips based on restriction endonuclease reactions, chips based on protein DNA binding reactions, and chips based on fluorescent molecule DNA binding reactions.

[0057] In the above method, the substance used to detect the polymorphism or genotype of one or more of the eleven SNPs (SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, SNP10, and SNP11) in the Gastrodia elata genome can be D1), D2), or D3).

[0058] D1) Contains PCR primer pairs for amplifying the Gastrodia elata genomic DNA fragment including the SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, SNP10 and / or SNP11 sites.

[0059] D2) PCR reagents containing the PCR primer pairs described in D1);

[0060] D3) A kit containing the PCR primer pair described in D1) or the PCR reagent described in D2).

[0061] The PCR primer pairs mentioned above are primer pairs consisting of F1 and R1, F2 and R2, F3 and R3, F4 and R4, F5 and R5, F6 and R6, F7 and R7, F8 and R8, F9 and R9, F10 and R10, and / or F11 and R11.

[0062] F1, Single-stranded DNA as shown in SEQ ID No. 12 of the sequence listing;

[0063] R1, the single-stranded DNA shown in SEQ ID No. 13 of the sequence listing;

[0064] F2, Single-stranded DNA as shown in SEQ ID No. 14 of the sequence listing;

[0065] R2, the single-stranded DNA shown in SEQ ID No. 15 of the sequence listing;

[0066] F3, Single-stranded DNA as shown in SEQ ID No. 16 of the sequence listing;

[0067] R3, the single-stranded DNA shown in SEQ ID No. 17 of the sequence listing;

[0068] F4, Single-stranded DNA as shown in SEQ ID No. 18 of the sequence listing;

[0069] R4, the single-stranded DNA shown in SEQ ID No. 19 of the sequence listing;

[0070] F5, Single-stranded DNA as shown in SEQ ID No. 20 of the sequence listing;

[0071] R5, the single-stranded DNA shown in SEQ ID No. 21 of the sequence listing;

[0072] F6, Single-stranded DNA as shown in SEQ ID No. 22 of the sequence listing;

[0073] R6, Single-stranded DNA as shown in SEQ ID No. 23 of the sequence listing;

[0074] F7. Single-stranded DNA as shown in SEQ ID No. 24 of the sequence listing;

[0075] R7, Single-stranded DNA as shown in SEQ ID No. 25 of the sequence listing;

[0076] F8. Single-stranded DNA as shown in SEQ ID No. 26 of the sequence listing;

[0077] R8, Single-stranded DNA as shown in SEQ ID No. 27 of the sequence listing;

[0078] F9. Single-stranded DNA as shown in SEQ ID No. 28 of the sequence listing;

[0079] R9, Single-stranded DNA as shown in SEQ ID No. 29 of the sequence listing;

[0080] F10, Single-stranded DNA as shown in SEQ ID No. 30 of the sequence listing;

[0081] R10, Single-stranded DNA as shown in SEQ ID No. 31 of the sequence listing;

[0082] F11, Single-stranded DNA as shown in SEQ ID No. 32 of the sequence listing;

[0083] R11, Single-stranded DNA as shown in SEQ ID No. 33 of the sequence listing.

[0084] In the above method, the PCR primers may or may not be labeled with a marker. The marker refers to any atom or molecule that can be used to provide a detectable effect and can be linked to nucleic acids. Markers include, but are not limited to, dyes; radioactive markers, such as... 32 P; binding moieties, such as biotin; haptens, such as digoxigenin (DIG); luminescent, phosphorescent, or fluorescent moieties; and fluorescent dyes alone or in combination with moieties whose emission spectra can be inhibited or shifted by fluorescence resonance energy transfer (FRET). The label can provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetric determination, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The label can be a charged moieties (positive or negative) or, optionally, charge-neutral. The label can include nucleic acid or protein sequences or combinations thereof, provided that the sequence containing the label is detectable. In some embodiments, nucleic acids are detected directly without labeling (e.g., direct sequence reading).

[0085] In the above methods, the product can be a reagent, kit, or system. The system may include a combination of reagents or kits, instruments, and analysis software, such as a product consisting of PCR primers, PARMS master mix reagents, an ELISA reader, and the online software SNPdecoder (http: / / www.snpway.com / snpdecoder01 / ), or a combination of PCR primers, PARMS mastermix reagents, the online software SNP decoder, and a real-time PCR instrument. The product may include the substances described above for detecting polymorphisms or genotypes at SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, SNP10, and SNP11 loci in the Gastrodia elata genome.

[0086] The application of the above-mentioned products or methods in the breeding of Gastrodia elata also falls within the protection scope of this invention.

[0087] In embodiments of this invention, genetic variation analysis of related populations of *Gastrodia elata* inbred lines revealed 11 SNPs: SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, SNP10, and SNP11. Among these, SNP1, SNP2, SNP3, and SNP4 are correlated with *Gastrodia elata* polysaccharide content; SNP5 is correlated with gastrodin content; SNP6 and SNP7 are correlated with *Gastrodia elata* p-hydroxybenzyl alcohol content; SNP8 is correlated with *Gastrodia elata* tuber weight; SNP9 is correlated with the width of the tuber 1 cm from the navel; and SNP10 and SNP11 are correlated with *Gastrodia elata* tuber length. These SNP loci can be used for molecular-assisted breeding to select superior *Gastrodia elata* germplasm with a "cucumber-shaped" appearance and high levels of active ingredients, thus promoting rapid and efficient *Gastrodia elata* breeding. Attached Figure Description

[0088] Figure 1 The bar chart shown in Example 1 is a screening chart of polymorphic sites. The data in the chart are mean ± standard deviation, with 3 replicates. The significance difference between the two different allele germplasms was analyzed by a two-tailed t-test. ** indicates that the significance analysis result is P<0.01. Detailed Implementation

[0089] 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.

[0090] 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.

[0091] The instruments used in the following examples were a Veriti™ PCR instrument, a Gene Amp 9700 PCR instrument (Applied Biosystems), a Nanodrop 2000 micro-quantitative nucleic acid analyzer (Thermo Scientific), and a SYNGENE gel imaging system (Gene).

[0092] In the following examples, the 2000bp DNA Marker was a product of Beijing TransGen Biotech Co., Ltd. (batch number BM101); and the rTaq DNA polymerase was a product of Bio-Rad Biotechnology (Beijing) Co., Ltd. (batch number R001A).

[0093] The Gastrodia elata germplasm resources in the following examples were collected from the following Gastrodia elata producing areas: Zhaotong, Yunnan; Dafang, Guizhou; Yichang, Hubei; Yingshan, Hubei; and Ningshan, Shaanxi. [2] The public can obtain the aforementioned parent plants from the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, to replicate the experiments described in this application; however, they may not be used for any other purpose.

[0094] Based on literature review, relevant traits of "superior shape" in Gastrodia elata were screened, specifically including tuber weight, tuber length, total length from the "parrot's beak" to the navel, and width at 1 cm from the navel. The specific measurement methods are referenced in the literature "Quantitative Taxonomic Study of Agronomic Traits of Cultivated Gastrodia elata". [2] "Correlation and Path Analysis of Underground Tuber Yield and Major Agronomic Traits of Gastrodia elata in Zhaotong" [3] And "The Influence of Different Planting Densities and Seedling Grades on the Main Agronomic Traits and Economic Indicators of Gastrodia elata Underground Tubers" [4] The screening for "high-quality" traits in Gastrodia elata was conducted based on the 2020 edition of the Pharmacopoeia and relevant literature. Specifically, these traits included total polysaccharide content, p-hydroxybenzyl alcohol content, and total gastrodin content. The determination methods were based on the Pharmacopoeia of the People's Republic of China. [5] Study on the quality evaluation of Gastrodia elata medicinal materials from different major producing areas based on UPLC-MS [6] Methodological Study and Application of Pre-Column Derivatization UPLC-MS / MS for Determination of 12 Monosaccharide Contents [7] The Tricarboxylic Acid Cycle in Forward and Reverse Directions [8] And "The Discovery and Implications of the Tricarboxylic Acid Cycle" [9] The method described in the text is as follows.

[0095] Example 1

[0096] 1. Correlation analysis between "superior shape and quality" characteristics and single nucleotide polymorphism (SNP) sites.

[0097] Polymorphism validation was performed on 506 loci associated with "superior form and quality" traits identified by genome-wide association studies (GWAS). Loci with polymorphism were further screened, and the detected single nucleotide polymorphisms (SNPs) were validated in a natural population of 200 Gastrodia elata germplasm accessions. The statistical analysis results of the trait content determined by each polymorphic locus are shown in Table 1. Figure 1 As shown:

[0098] Table 1. Selected sites with significant functions

[0099]

[0100] Table 1 and Figure 1The results showed that 11 loci were polymorphic, of which 4 loci were associated with the "superior shape" characteristics of Gastrodia elata (SNP8-10): Scaffold8-5761880 (SNP10) was significantly associated with tuber length, with Gastrodia elata with the GG allele exhibiting longer tuber length; tuber length was also associated with the Scaffold5-8521686 (SNP11) locus, with the CC allele exhibiting longer tuber length; the locus Scaffold3-10602379 (SNP8) was significantly associated with weight, with the average tuber weight of Gastrodia elata with the GG allele being 78.41g, significantly higher than that of Gastrodia elata with the AA allele; the locus Scaffold2-11556326 (SNP9) with the width at 1cm from the navel showed an advantage for the CC allele.

[0101] The seven "high-quality" trait-related loci (SNP1-7) include: four polysaccharide content loci, where the CC allele at Scaffold3-108855 (SNP1) and Scaffold0-8535479 (SNP4) shows higher polysaccharide content, while the TT allele at Scaffold4-5593251 (SNP2) and Scaffold2-10276736 (SNP3) shows higher polysaccharide content; one gastrodin content locus, where the CC allele at Chr8-15360782 (SNP5) shows higher gastrodin content; and two p-hydroxybenzyl alcohol content loci, Chr2-62639910 (SNP6) and Chr4-15523768 (SNP7), where the CC allele shows higher p-hydroxybenzyl alcohol content.

[0102] 2. Validation and screening of SNP sites in superior Gastrodia elata germplasm.

[0103] Samples of *Gastrodia elata* (Gastrodia elata) from Ningxia, Shaanxi; Yingshan, Hubei; Yichang, Hubei; Zhaotong, Yunnan; and Dafang, Guizhou were collected for germplasm resource identification. For 25 samples, SNP primers were used to detect their loci and genotypes, and *Gastrodia elata* germplasm with "superior morphology and superior quality" genotypes were screened. Flowers from the above 25 *Gastrodia elata* resource samples after bolting were harvested, photographed, and genomic DNA was extracted using a modified CTAB method. The operation steps are as follows:

[0104] Take approximately 25-50 mg of Gastrodia elata sample into a 2.0 mL centrifuge tube, add liquid nitrogen to pulverize; add 900 μL of 2×CTAB buffer preheated to 65℃, shake well, and incubate in a 65℃ water bath for 30 min (add a small amount of PVP and 10 μL of β-mercaptoethanol), shaking once every 10 min; remove, cool to below 45℃, add an equal volume of chloroform:isoamyl alcohol (24:1), shake well to form an emulsion, and incubate at 12000 r·min-1 Centrifuge for 10 min; transfer the supernatant to a new centrifuge tube, add an equal volume of chloroform:isoamyl alcohol (24:1), gently shake to mix thoroughly, and centrifuge at 12000 rpm. -1 Centrifuge for 10 min; add 330 μL (approximately 2 / 3 volume of supernatant) to 500 μL of supernatant, mix well, and incubate at -20°C for 30 min; centrifuge at 12000 rpm below 4°C. -1 Centrifuge for 10 min; discard the supernatant, add 500 μL of 70% ethanol, resuspend the DNA, and centrifuge at 12000 rpm. -1 Centrifuge for 5 min, repeat once; discard the supernatant, add 500 μL of anhydrous ethanol, resuspend the DNA, and centrifuge at 12000 rpm. -1 Centrifuge for 5 min; discard the supernatant, evaporate the ethanol, add 75 μL of sterile water, and after the DNA is dissolved, determine its concentration and purity using a Nanodrop 2000 micro-nucleic acid quantification analyzer for PCR reaction, and store at 4℃.

[0105] The primers in Table 2 were used to perform PCR to detect the genotypes of loci associated with the "superior morphology and quality" characteristics. The PCR reaction system was as follows: 25 μL PCR reaction system, containing 2.5 μL of 10×rtaq buffer, 1.5 μL of dNTPs (10 mmol·L⁻¹), 0.5 μL of upstream and downstream primers, 0.4 μL of rTaq DNA polymerase, 1 μL of DNA template, and sterile water to a final volume of 25 μL. The PCR reaction program was: 94℃ pre-denaturation for 3 min (94℃ denaturation for 30 s, annealing temperatures for each primer are shown in Table 2), 72℃ extension for 30 s, for a total of 35 cycles, and a final extension at 72℃ for 5 min.

[0106] Table 2. Primers for "High-Quality" Loci

[0107]

[0108]

[0109] The genotyping results of the "superior form" related loci of Gastrodia elata are shown in Table 3: H1-2, H2-2, H2-3, H2-5, H3-2, and H3-3 are homozygous for GG at the tuber length locus Scaffold8-5761880 (SNP10) and homozygous for CC at Scaffold5-8521686 (SNP11); at the weight locus Scaffold3-10602379 (SNP8), they are homozygous for GG. W2-2 and W2-3... The Scaffold2-11556326(SNP9) at a width of 1 cm from the navel on tubers W2-4, W3-1, W3-2, W3-3, W3-4, W3-5, W3-6, W3-7, W3-10, W3-11, W4-2, W4-3, W4-6, W4-7, and W4-12 were homozygous for CC, while the Scaffold2-11556326(SNP9) on W4-1 and W4-4 were heterozygous for CT.

[0110] Table 3. Genotyping results of Gastrodia elata "superior form" related loci

[0111]

[0112]

[0113] The genotyping results of the "high-quality" related loci in Gastrodia elata are shown in Table 4: At the Chr4-15523768 (SNP7) and Chr2-62639910 (SNP6) loci of p-hydroxybenzyl alcohol in Gastrodia elata, H1-2, H2-2, H2-3, H2-5, H3-2, H3-3, W2-2, W2-3, W2-4, W3-1, W3-2, W3-3, W3-4, W3-5, W3-6, W3-7, W3-10, W3-11, W4-2, W4-3, W4-6, W4-7, and W4-12 are all homozygous for CC, indicating a higher quality. High p-hydroxybenzyl alcohol content; Gastrodin content: H1-2, H2-2, H2-3, H2-5, H3-2, H3-3, W4-1, and W4-3 at the Chr8-15360782 (SNP5) site are homozygous (CC), possibly indicating higher gastrodin content; At the Gastrodia polysaccharide Scaffold3-108855 (SNP1) site, H2-2, H2-3, H2-5, H3-2, H3-3, W2-2, W2-3, W2-4, W3-1, W3-2, W3-3, W3-4, W3-5, W3-6, W3-7, and W3... -10, W3-11, W4-2, W4-3, W4-6, W4-7, and W4-12 are all homozygous for CC. At Scaffold4-5593251 (SNP2), W2-2, W2-3, W2-4, W3-1, W3-2, W3-3, W3-4, W3-5, W3-6, W3-7, W3-10, W3-11, W4-2, W4-6, W4-7, and W4-12 are all homozygous for TT. At Scaffold12-10275736 (SNP3), H2-2, H2-3, H2-5, and H3- 2. H3-3, W2-2, W2-3, W2-4, W3-1, W3-2, W3-3, W3-4, W3-5, W3-6, W3-7, W3-10, W3-11, W4-2, W4-3, W4-6, W4-7, and W4-12 are all TT homozygous, S W2-2, W2-3, W2-4, W3-1, W3-2, W3-3, W3-4, W3-5, W3-6, W3-7, W3-10, W4-2, W4-7, and W4-12 in the caffold0-18535479 (SNP4) locus are CC homozygous.

[0114] Table 4. Genotyping results of loci related to "high quality" in Gastrodia elata

[0115]

[0116]

[0117] The above verification revealed that among the 11 SNP sites related to "superior shape and quality" of Gastrodia elata selected in step 1, four SNP sites were related to the total polysaccharide content of Gastrodia elata, namely SNP1, SNP2, SNP3 and SNP4:

[0118] SNP1 corresponds to position 108855 of the Gastrodia elata genome sequence Scaffold3, with nucleotide C, corresponding to position 222 of SEQ ID No. 1 in the sequence listing (SEQ ID No. 1 is the nucleotide sequence of the PCR product obtained by amplification using primers F1 (SEQ ID No. 12) and R1 (SEQ ID No. 13)). The determination results show three genotypes for the SNP1 locus (referred to as SNP1 genotypes): CC, TT, or CT. Genotype CC is homozygous for SNP1 with C, indicating a high-glucose genotype; genotype TT is homozygous for SNP1 with T, indicating a low-glucose genotype; and genotype CT is heterozygous for SNP1.

[0119] SNP2 corresponds to position 5593251 of the Gastrodia elata genome sequence Scaffold4, with nucleotides T or A, corresponding to position 219 of SEQ ID No. 2 in the sequence listing (SEQ ID No. 2 is the nucleotide sequence of the PCR product obtained by amplification using primers F2 (SEQ ID No. 14) and R2 (SEQ ID No. 15)). The determination results show two genotypes for SNP2 (referred to as SNP2 genotypes): TT or AA. Genotype TT is homozygous for SNP2 being T, indicating a high-glucose genotype; genotype AA is homozygous for SNP2 being A, indicating a low-glucose genotype.

[0120] SNP3 corresponds to position 10,275,736 of the Gastrodia elata genome sequence Scaffold12, with nucleotides T or A, corresponding to position 270 of SEQ ID No. 3 in the sequence listing (SEQ ID No. 3 is the nucleotide sequence of the PCR product obtained by amplification using primers F3 (SEQ ID No. 16) and R3 (SEQ ID No. 17)). The determination results show two genotypes for SNP3 (referred to as SNP3 genotypes): TT or AA. Genotype TT is homozygous for SNP3 being T, indicating a high-glucose genotype; genotype AA is homozygous for SNP3 being A, indicating a low-glucose genotype.

[0121] SNP4 corresponds to position 18535479 of the Gastrodia elata genome sequence Scaffold0, with nucleotides C or T, corresponding to position 193 of SEQ ID No. 4 in the sequence listing (SEQ ID No. 4 is the nucleotide sequence of the PCR product obtained by amplification using primers F4 (SEQ ID No. 18) and R4 (SEQ ID No. 19)). The determination results show three genotypes for SNP4 (referred to as SNP4 genotypes): CC, TT, or CT. Genotype CC is homozygous for SNP4 as C, indicating a high-glucose genotype; genotype TT is homozygous for SNP4 as T, indicating a low-glucose genotype; and genotype CT is heterozygous for SNP4.

[0122] A single SNP locus associated with gastrodin content in Gastrodia elata was identified and named SNP5. SNP5 corresponds to position 15,360,782 on the Chr8 sequence of the Gastrodia elata genome, with nucleotides C or T, corresponding to position 228 of SEQ ID No. 5 in the sequence listing (SEQ ID No. 5 is the nucleotide sequence of the PCR product obtained by amplification using primers F5 (SEQ ID No. 20) and R5 (SEQ ID No. 21)). The results showed two genotypes for SNP5 (referred to as SNP5 genotypes): CC or TT. Genotype CC is homozygous for SNP5 with the nucleotide value C, indicating a high gastrodin content; genotype TT is homozygous for SNP5 with the nucleotide value T, indicating a low gastrodin content.

[0123] Two SNP sites associated with the content of p-hydroxybenzyl alcohol in Gastrodia elata were identified and named SNP6 and SNP7:

[0124] SNP6 corresponds to position 62639910 of the Chr2 sequence in the Gastrodia elata genome. Its nucleotide is either C or G, corresponding to position 238 of SEQ ID No. 6 in the sequence listing (SEQ ID No. 6 is the nucleotide sequence of the PCR product obtained by amplification using primers F6 (SEQ ID No. 22) and R6 (SEQ ID No. 23)). The results showed one genotype for SNP6 (referred to as SNP6 genotype), namely CC: Genotype CC is homozygous for SNP6 being C, and is a genotype with high p-hydroxybenzyl alcohol content.

[0125] SNP7 corresponds to position 15523768 of the Chr4 sequence in the Gastrodia elata genome. Its nucleotide is either C or T, corresponding to position 238 of SEQ ID No. 7 in the sequence listing (SEQ ID No. 7 is the nucleotide sequence of the PCR product obtained by amplification using primers F7 (SEQ ID No. 24) and R7 (SEQ ID No. 25)). The determination results show two genotypes for SNP7 (referred to as SNP7 genotypes): CC or TT. Genotype CC is homozygous for SNP7 as C, indicating a high p-hydroxybenzyl alcohol content; genotype TT is homozygous for SNP7 as T, indicating a low p-hydroxybenzyl alcohol content.

[0126] A SNP locus associated with the weight of Gastrodia elata was identified as SNP8. SNP8 corresponds to position 10,602,379 of the Gastrodia elata genome sequence Scaffold3, and its nucleotide is any one of G, A, or T. It corresponds to position 218 of SEQ ID No. 8 in the sequence listing (SEQ ID No. 8 is the nucleotide sequence of the PCR product obtained by amplification using primers F8 (SEQ ID No. 26) and R8 (SEQ ID No. 27)). The results showed three genotypes for SNP8 (referred to as SNP8 genotypes): GG, AA, or AT. Genotype GG is homozygous for SNP8 with the genotype G, indicating a high weight; genotype AA is homozygous for SNP8 with the genotype A, indicating a low weight; and genotype AT is heterozygous for SNP8.

[0127] A single SNP locus associated with the width of the tuber at a distance of 1 cm from the navel was identified and named SNP9. SNP9 corresponds to position 11556326 of the Gastrodia elata genome sequence Scaffold2, and its nucleotide is either C or T, corresponding to position 193 of SEQ ID No. 9 in the sequence listing (SEQ ID No. 9 is the nucleotide sequence of the PCR product obtained by amplification using primers F9 (SEQ ID No. 28) and R9 (SEQ ID No. 29)). The results showed three genotypes for SNP9 (referred to as SNP9 genotypes): CC, TT, or CT. Genotype CC is homozygous for SNP9 with the value C, indicating a wide width at 1 cm from the navel; genotype TT is homozygous for SNP9 with the value T, indicating a narrow width at 1 cm from the navel; and genotype CT is heterozygous for SNP9.

[0128] A combination of SNP loci associated with the length of Gastrodia elata tubers was identified. This combination includes two SNP loci, SNP10 and SNP11.

[0129] SNP10 corresponds to position 5761880 of the Gastrodia elata genome sequence Scaffold8, and its nucleotide is either C or G. It corresponds to position 176 of SEQ ID No. 10 in the sequence listing (SEQ ID No. 10 is the nucleotide sequence of the PCR product obtained by amplification using primers F10 (SEQ ID No. 30) and R10 (SEQ ID No. 31)). The results showed three genotypes for SNP10 (referred to as SNP10 genotypes): CC, GG, or CG. Genotype CC is homozygous for SNP10 as C, indicating a short tuber genotype; genotype GG is homozygous for SNP10 as G, indicating a long tuber genotype; and genotype CT is heterozygous for SNP10.

[0130] SNP11 corresponds to position 8521686 of the Gastrodia elata genome sequence Scaffold5, and its nucleotide is either C or G. It corresponds to position 288 of SEQ ID No. 11 in the sequence listing (SEQ ID No. 11 is the nucleotide sequence of the PCR product obtained by amplification using primers F11 (SEQ ID No. 32) and R11 (SEQ ID No. 33)). The determination results show three genotypes for SNP11 (referred to as SNP11 genotypes): CC, GG, or CG. Genotype CC is homozygous for SNP11 with the nucleotide C trait, indicating a long tuber genotype; genotype GG is homozygous for SNP11 with the nucleotide G trait, indicating a short tuber genotype; and genotype CT is heterozygous for SNP11.

[0131] Based on the above results, materials W4-3 and W4-4 have a high sugar genotype, and W4-3 has a high gastrodin genotype. However, many loci in other traits of these two parents are heterozygous. Therefore, multiple generations of breeding can be carried out on the offspring of these two materials to obtain gastrodia elata that meets the characteristics of "superior shape and high quality".

[0132] 3. Verification of different genotype traits at SNP loci in superior Gastrodia elata germplasm.

[0133] To verify the correspondence between different genotypes and traits at SNP loci related to "superior morphology," six lines were randomly selected from the self-crossed progeny of the aforementioned 25 parents and numbered 1, 2, 3, 4, 5, and 6, respectively, to form a small population for verification. Three replicates were taken from each line.

[0134] The genotypic verification results of the "superior form" related loci of Gastrodia elata are shown in Table 5: 1, 2, and 3 are homozygous for GG at the tuber length locus Scaffold8-5761880 (SNP10) and homozygous for CC at Scaffold5-8521686 (SNP11), which is longer than 4, 5, and 6 (SNP10 is homozygous for CC and SNP11 is homozygous for GG); 1, 2, and 3 are homozygous for GG at the tuber weight locus Scaffold3-10602379 (SNP8), which is significantly heavier than 4, 5, and 6 (SNP8 is homozygous for AA); 4, 5, and 6 are homozygous for CC at the tuber width locus Scaffold2-11556326 (SNP9) at 1 cm from the navel, which is wider than 1, 2, and 3 (SNP9 is homozygous for TT), which is wider at 1 cm from the navel.

[0135] Table 5. Verification of the correspondence between genotypes and traits at loci associated with "superior form" in Gastrodia elata.

[0136]

[0137] To verify the correspondence between different genotypes and traits at SNP loci related to "high quality," five lines were randomly selected from the self-crossed progeny of the aforementioned 25 parents and numbered 1, 2, 3, 4, and 5, forming a small population for verification. Three replicates were taken from each line.

[0138] The genotypic verification results of the "high-quality" related loci in Gastrodia elata are shown in Table 6: At the p-hydroxybenzyl alcohol loci Chr4-15523768 (SNP7) and Chr2-62639910 (SNP6) in Gastrodia elata, loci 2, 3, 4, and 5 are all homozygous (CC), indicating higher p-hydroxybenzyl alcohol content; at the gastrodin content loci Chr8-15360782 (SNP5), loci 1, 2, 3, and 4 are homozygous (CC), potentially indicating higher gastrodin content. In the Scaffold3-108855 (SNP1) site of Gastrodia elata polysaccharide, 2, 3, 4, and 5 are all homozygous for CC; in the Scaffold4-5593251 (SNP2) site, 5 is homozygous for TT; in the Scaffold12-10275736 (SNP3) site, 2, 3, 4, and 5 are homozygous for TT; and in the Scaffold0-18535479 (SNP4) site, 5 is homozygous for CC.

[0139] Table 6. Verification of the correspondence between genotypes and traits at loci associated with "high quality" in Gastrodia elata.

[0140]

[0141]

[0142] The above verification results demonstrate that the genotypes of the 11 SNP loci obtained through screening can be used for molecular marker replication screening of the corresponding traits.

[0143] In summary, this invention, based on GWAS screening to identify SNP loci related to "superior shape and quality," validated the associated "superior shape and quality" loci. Furthermore, primers were designed for the DNA fragments containing these loci to identify relevant loci in Gastrodia elata grown at the germplasm base. Eleven SNP loci were screened and validated, providing a theoretical basis for the breeding of "superior shape and quality" Gastrodia elata varieties. This invention lays the foundation for future cultivation of high-yield, high-activity-content "high-quality" Gastrodia elata germplasm.

[0144] 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.

[0145] References

[0146] [1] Yuan Yuan, Huang Luqi. Research progress and development trend of molecular pharmacognosy of traditional Chinese medicinal materials [J]. Chinese Science Bulletin, 2020, 65(12):1093.

[0147] [2] Qian Run, Li Hui, Hua Zhongyi, et al. Quantitative taxonomic study on agronomic traits of cultivated Gastrodia elata [J]. Chinese Journal of Traditional Chinese Medicine, 2020, 45(13):3085.

[0148] [3] Wang Li, Ma Congji, Liu Dahui, et al. Correlation and path analysis of underground tuber yield and main agronomic traits of Gastrodia elata in Zhaotong[J]. Chinese Journal of Traditional Chinese Medicine, 2017, 42(4):644.

[0149] [4] Wang Li, Ma Congji, Zhang Zhihui, et al. Effects of different planting densities and seedling grades on the main agronomic traits and economic indicators of underground tubers of Gastrodia elata [J]. Southwest China Journal of Agricultural Sciences, 2017, 30(1):62.

[0150] [5] National Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China [S]. 2020 edition. Part I. Beijing: China Medical Science and Technology Press, 2020: 59-60.

[0151] [6] Kang Chuanzhi, Lü Chaogeng, Yang Jian, et al. Study on quality evaluation of Gastrodia elata from different major producing areas based on UPLC-MS [J]. Chinese Journal of Traditional Chinese Medicine, 2017, 32(5):2010-2015.

[0152] [7] Liang Yuting, Zhou Junhui, Nan Tiegui, et al. Methodological study and application of pre-column derivatization UPLC-MS / MS determination of 12 monosaccharides[J]. Chinese Journal of Traditional Chinese Medicine, 2018, 43(22):4469-4473.

[0153] [8] Zhu Qinshi. Forward and reverse tricarboxylic acid cycle [J]. Bulletin of Biology, 2015, 50(1):16-19.

[0154] [9] Chen Mu, Liu Rui, Weng Yi. Discovery and inspiration of the tricarboxylic acid cycle [J]. Medicine and Philosophy (A), 2012, 33(1):71-73.

Claims

1. The application of a substance for detecting the genotype of SNP loci related to tuber length in the identification or auxiliary identification of the quality of Gastrodia elata, characterized in that: The tuber length-related SNP sites are either two SNPs or one SNP from SNP10 and SNP11: SNP10 is a SNP in the Gastrodia elata genome, located at nucleotide 176 of SEQ ID No. 10 in the sequence listing, and its nucleotide is C or G. SNP10 has three genotypes: CC, GG, and CG. Genotype CC is homozygous for SNP10 as C, which is a short tuber genotype; genotype GG is homozygous for SNP10 as G, which is a long tuber genotype; and genotype CG is heterozygous for SNP10. SNP11 is a SNP in the Gastrodia elata genome, located at nucleotide 288 of SEQ ID No. 11 in the sequence listing, and its nucleotide is C or G. SNP11 has three genotypes: CC, GG, and CG. Genotype CC is homozygous for SNP11 with the genotype C, and is a long tuber genotype. Genotype GG is homozygous for SNP11 with the genotype G, and is a short tuber genotype. Genotype CG is heterozygous for SNP11. The quality of Gastrodia elata is defined as the length of its tuber.

2. The application of haplotype detection substances in the identification or auxiliary identification of the quality of Gastrodia elata, characterized in that: The haplotype is a polymorphic combination of SNP10 and / or SNP11 as described in claim 1 on the Gastrodia elata chromosome; SNP10 has three genotypes: CC, GG, or CG. Genotype CC is homozygous for SNP10 with the genotype C, indicating a short tuber; genotype GG is homozygous for SNP10 with the genotype G, indicating a long tuber; and genotype CG is heterozygous for SNP10. SNP11 also has three genotypes: CC, GG, or CG. Genotype CC is homozygous for SNP11 with the genotype C, indicating a long tuber; genotype GG is homozygous for SNP11 with the genotype G, indicating a short tuber; and genotype CG is heterozygous for SNP11. The quality of *Gastrodia elata* refers to tuber length. The substance used for detecting haplotypes is either D1), D2), or D3): D1) Contains PCR primer pairs that amplify the Gastrodia elata genomic DNA fragment including the SNP10 and / or SNP11 sites; D2) PCR reagents containing the PCR primer pairs described in D1); D3) A kit containing the PCR primer pair described in D1) or the PCR reagent described in D2); The PCR primer pairs are primer pairs consisting of F10 and R10 and / or primer pairs consisting of F11 and R11: F10, Single-stranded DNA as shown in SEQ ID No. 30 of the sequence listing; R10, Single-stranded DNA as shown in SEQ ID No. 31 of the sequence listing; F11, Single-stranded DNA as shown in SEQ ID No. 32 of the sequence listing; R11, Single-stranded DNA as shown in SEQ ID No. 33 of the sequence listing.

3. The product, characterized in that: The product contains the substance for detecting haplotypes as described in claim 2, and may be any one of the following G1)-G4): G1) Products that test for single nucleotide polymorphisms or genotypes related to the quality of Gastrodia elata; G2) Products for identifying or assisting in the identification of the quality of Gastrodia elata; the quality of Gastrodia elata refers to the tuber length; G3) Products used for Gastrodia elata breeding; the goal of the breeding is to select long-tubered Gastrodia elata. G4) Products for identifying or assisting in the identification of the shape of Gastrodia elata, wherein the shape of Gastrodia elata is the length of the tuber.

4. A method for breeding Gastrodia elata, characterized in that: include: Detect the genotypes of SNP10 and / or SNP11 as described in claim 1 in the Gastrodia elata genome, and select Gastrodia elata that satisfy the following C1 and / or C2 for breeding: The genotypes of C1 and SNP10 are GG or CG; GG is the homozygous type of SNP10 being G; CG is the heterozygous type of SNP10 being C and G. The genotypes of C2 and SNP11 are CC or CG; CC is the homozygous type of SNP11 being C; CG is the heterozygous type of SNP11 being C and G. The substances used to detect the genotypes of SNP10 and / or SNP11 in the Gastrodia elata genome are as follows: D1), D2), or D3): D1) Contains PCR primer pairs that amplify the Gastrodia elata genomic DNA fragment including the SNP10 and / or SNP11 sites; D2) PCR reagents containing the PCR primer pairs described in D1); D3) A kit containing the PCR primer pair described in D1) or the PCR reagent described in D2); The PCR primer pairs are primer pairs consisting of F10 and R10 and / or primer pairs consisting of F11 and R11: F10, Single-stranded DNA as shown in SEQ ID No. 30 of the sequence listing; R10, Single-stranded DNA as shown in SEQ ID No. 31 of the sequence listing; F11, Single-stranded DNA as shown in SEQ ID No. 32 of the sequence listing; R11, Single-stranded DNA as shown in SEQ ID No. 33 of the sequence listing.

5. The application of the product according to claim 3 in the breeding of Gastrodia elata, characterized in that: The breeding objective is to select for long-tubered Gastrodia elata. SNP10 has three genotypes: CC, GG, or CG. Genotype CC is homozygous for SNP10 with the genotype C, indicating a short tuber. Genotype GG is homozygous for SNP10 with the genotype G, indicating a long tuber. Genotype CG is heterozygous for SNP10. SNP11 also has three genotypes: CC, GG, or CG. Genotype CC is homozygous for SNP11 with the genotype C, indicating a long tuber. Genotype GG is homozygous for SNP11 with the genotype G, indicating a short tuber. Genotype CG is heterozygous for SNP11.

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