A key enzyme and transcription factor gene of rehmannia terpenoid synthesis and a molecular marker primer composition and a method for identifying the same

Abstract

The present application relates to a kind of rehmannia terpenoid synthesis key enzyme and transcription factor gene SSR molecular marker primer composition and its identification method, the sequence of the key enzyme gene is by SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 is composed of, the primer composition of the key enzyme gene of the rehmannia terpenoid synthesis is by primer pair SSR-1, primer pair SSR-10, primer pair SSR-16, primer pair SSR-18 is composed of.The advantage of the present application is based on rehmannia transcriptome data, provides the key enzyme gene SSR molecular marker primer of the synthesis of rehmannia, to facilitate further study the synthesis pathway of the terpenoid active ingredient of rehmannia and the germplasm resource identification of rehmannia.

Description

Technical Field

[0001] This invention relates to the field of molecular markers, and more particularly to a molecular marker primer composition for SSR genes of key enzymes and transcription factor genes involved in the synthesis of rehmannia terpenes and a method for their identification. Background Technology

[0002] Rehmannia ( Rehmannia glutinosa Rehmannia glutinosa is one of my country's traditional medicinal and edible herbs, with its tuberous root being the main part used. The plant's medicinal and edible value primarily stems from its rich content of terpenoids. Significant differences exist in the content of active ingredients among different varieties of Rehmannia glutinosa, and even within the same variety, the content varies among individuals from different populations or under different cultivation environments. Among these, terpenoids are recognized as the main effective components of Rehmannia glutinosa, and differences in their content and activity can directly reflect varietal characteristics, thus possessing development value as a core indicator for varietal identification.

[0003] Rehmannia glutinosa has a long history of cultivation in my country and is an important crop with ecological, economic, and medicinal value. my country not only possesses abundant cultivated varieties but also a large number of wild germplasm resources, resulting in a substantial reserve of germplasm. However, in the current utilization of Rehmannia glutinosa germplasm resources, there are widespread problems such as ambiguous species definition (homologous species, synonyms, mixed varieties, and confusion between medicinal and edible types), leading to low efficiency in germplasm resource screening, hindering the promotion of superior varieties, and seriously affecting the standardized cultivation, quality control, and industrial utilization of this plant. Therefore, establishing efficient and accurate methods for identifying Rehmannia glutinosa varieties is of significant theoretical and practical importance for standardizing its germplasm resource management, promoting the breeding of superior varieties, and improving the quality of industrial development.

[0004] Simple repeating sequences ( 简单序列重复 SSR (Stereotyped Rehmannia glutinosa) molecular markers, as a highly efficient molecular identification technique, possess outstanding advantages such as high polymorphism, abundant quantity, good reproducibility, convenient PCR detection, co-dominant inheritance, and broad genome coverage. They have been widely applied in research fields such as genetic diversity analysis, variety breeding, germplasm resource identification, and phylogenetic analysis in various plants, becoming one of the core technical means to solve the problem of germplasm resource identification. However, research on SSR molecular markers in Rehmannia glutinosa started relatively late, the number of developed SSR markers is limited, and there is a lack of a comprehensive SSR marker database, which severely restricts their application in genetic diversity analysis, precise germplasm identification, and molecular-assisted breeding.

[0005] With the rapid development of DNA sequencing technology, the development of SSR molecular markers based on transcriptome data has become a key approach to solving the problem of insufficient genetic information in non-model plants. This approach can efficiently discover molecular markers related to functional traits, providing technical support for germplasm identification and breeding. However, current SSR marker development for Rehmannia glutinosa mainly focuses on random sites in the whole transcriptome. There are no reports of specific SSR markers and primers based on Rehmannia glutinosa transcriptome data, specifically targeting key enzyme genes and transcription factor genes related to terpene synthesis. Consequently, there is a lack of functional molecular markers that can directly link to the synthesis of effective components. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a primer composition for SSR molecular markers of key enzymes and transcription factor genes involved in Rehmannia glutinosa terpene synthesis, along with their identification method. Based on annotation analysis of Rehmannia glutinosa transcriptome data, SSR molecular markers in key enzyme genes and transcription factor genes related to Rehmannia glutinosa terpene synthesis were identified and screened. Corresponding specific primer compositions were designed, providing an effective molecular tool for the precise screening and identification of Rehmannia glutinosa germplasm resources. This primer composition and identification method can efficiently and accurately distinguish different Rehmannia glutinosa varieties and germplasm resources, solving the problem of ambiguous germplasm delineation such as homonymous species and varietal mixing. Simultaneously, it provides functional molecular markers that can be associated with effective component traits for the breeding of superior Rehmannia glutinosa varieties, helping to improve breeding efficiency.

[0007] This invention is achieved through the following technical solution: On one hand, a primer composition for SSR molecular markers of key enzymes and transcription factor genes for the synthesis of rehmannia terpenoids is provided, characterized in that the sequence of the key enzyme gene is composed of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, and the primer composition for the key enzyme gene for the synthesis of rehmannia terpenoids is composed of primer pair SSR-1, primer pair SSR-10, primer pair SSR-16, and primer pair SSR-18.

[0008] Through the above technical solutions, this invention, based on the annotation analysis of Rehmannia glutinosa transcriptome data, mines and screens SSR molecular markers in key enzyme genes and transcription factor genes related to terpene synthesis in Rehmannia glutinosa, and designs corresponding specific primer compositions, providing an effective molecular tool for the precise screening and identification of Rehmannia glutinosa germplasm resources. Using this primer composition and identification method, different Rehmannia glutinosa varieties and germplasm resources can be efficiently and accurately distinguished, solving the problem of ambiguous germplasm delineation such as homonymous species and varietal mixing. Simultaneously, it provides functional molecular markers that can be associated with the traits of effective components for the breeding of superior Rehmannia glutinosa varieties, helping to improve breeding efficiency. Furthermore, it lays an important molecular marker resource and theoretical basis for the mining, identification, and regulatory mechanism research of functional genes related to the synthesis of effective components in Rehmannia glutinosa, which is of positive significance for promoting the standardization and high-quality development of the Rehmannia glutinosa industry.

[0009] As shown in c52655.graph_c0 (SEQ ID NO.1):

[0010]

[0011] As shown in c48814.graph_c0 (SEQ ID NO.2):

[0012] As shown in c52230.graph_c0 (SEQ ID NO.3):

[0013] As shown in c53110.graph_c0 (SEQ ID NO.4):

[0014] The primer composition for the key enzyme gene for the synthesis of Rehmannia glutinosa terpenoids consists of primer pair SSR-1, primer pair SSR-10, primer pair SSR-16, and primer pair SSR-18.

[0015] The DNA sequence information for SSR-1 and SSR-10 is given below:

[0016] With the rapid advancement of DNA sequencing technology, the development of SSR molecular markers based on transcriptome data has played a crucial role in addressing the lack of genetic information in medicinal plants. This invention provides SSR molecular marker primers for key enzyme genes involved in Rehmannia glutinosa synthesis, based on Rehmannia glutinosa transcriptome data, to facilitate further research on the synthetic pathways of terpenoid active components in Rehmannia glutinosa and the identification of Rehmannia glutinosa germplasm resources.

[0017] This invention is based on existing Rehmannia glutinosa transcriptome data in the laboratory, according to the Rehmannia glutinosa genome (… 生物项目 登录号:PRJNA631301 ) Parameterized annotation of transcripts was completed, key enzyme genes for rehmannia terpene synthesis were screened, SSR molecular markers for key enzyme genes for rehmannia terpene synthesis were developed, and the above two pairs of ESTs with good polymorphism were obtained. SSR markers. These two new ESTs The SSR marker differs from previously reported Rehmannia glutinosa SSR markers. Using this marker, not only was an EST-SSR-based fingerprint successfully constructed, laying the foundation for the identification of Rehmannia glutinosa varieties, but it also allows for further screening and analysis of the synthetic pathways of active substances such as terpenoids, promoting the exploration and utilization of Rehmannia glutinosa resources.

[0018] Furthermore, the primer sequences for SSR-1 are as follows: Forward primer SSR-1F: 5'-ACTCGTGAGCCATGTGTATAT-3'; Reverse primer SSR-1R: 5'-CCCCACAGGCCCATACATTTA-3'; Primer sequences for SSR-10: Forward primer SSR-10F: 5'-CGGAGAGCCACCCTTTCACTAA-3'; Reverse primer SSR-10R: 5'-GCGCCACGTCACCATTGTTTAG-3'; Primer sequences for SSR-16: Forward primer SSR-16F: 5'-GGCAGCCAATCTTCCAGAAAAC-3'; Reverse primer SSR-16R: 5'-GGGCGTGGTTCTTGCAGAGGAA-3'; Primer sequences for SSR-18: Forward primer SSR-18F: 5'-CATTGGCAAGTTTCGAGCATGA-3'; Reverse primer SSR-18R: 5'-GAGCCTCGGATTCACTGTAGCA-3'.

[0019] The above-described technical solution and the aforementioned SSR molecular marker primers have the following advantages: ① It can be used for research on the analysis of Rehmannia glutinosa genotypes, identification of germplasm resources, analysis and evaluation of genetic diversity, mapping of functional genes and QTLs, and molecular marker-assisted breeding, which helps to solve problems such as unclear sources of Rehmannia glutinosa varieties and blind hybridization breeding; ②This lays a foundation for further research on the functional and active components of Rehmannia glutinosa, especially the synthetic pathways of active substances such as terpenoids, which is conducive to the further in-depth exploration and utilization of Rehmannia glutinosa resources.

[0020] A kit for identifying Rehmannia glutinosa varieties is provided, comprising the primer composition described above.

[0021] The above technical solutions and reagent kits can provide theoretical support for the screening and identification of Rehmannia glutinosa germplasm resources and molecular marker-assisted breeding, and provide guarantees for the identification of Rehmannia glutinosa varieties and genetic improvement.

[0022] Another method for identifying the species of Rehmannia glutinosa is provided, comprising the following steps: Step 1) Genomic DNA extraction and quality testing; Step 2) SSR-PCR reaction; using the Rehmannia glutinosa genome extracted in Step 1) as a template, amplify the Rehmannia glutinosa sample to be tested based on the above primer combination; Step 3) Denaturing polyacrylamide gel electrophoresis and silver staining; the PCR amplification fragments from Step 2) were analyzed by electrophoresis using a 12% denaturing PAGE gel to obtain the SSR fingerprint polymorphism map of Rehmannia glutinosa.

[0023] Furthermore, two steps are included before step 1): the first step is the screening of key enzyme genes for the synthesis of rehmannia terpenes; the second step is SSR analysis, primer design and screening.

[0024] Furthermore, in the first step, using existing Rehmannia transcriptome data in the laboratory, based on the Rehmannia genome ( 生物项目登录号:PRJNA631301 ) Complete the reference annotation of the transcripts and screen out the key enzyme genes for the synthesis of rehmannia terpenoids.

[0025] Furthermore, in the second step, tools such as SSRminer are used to perform functional SSR analysis and primer design on the key enzyme sequences for rehmannia terpenoid synthesis obtained above. Primers are randomly selected for verification, resulting in primers with clear amplification bands and polymorphism.

[0026] Through the above technical solutions, this invention, based on the annotation analysis of transcriptome data of Rehmannia glutinosa from Hebei and Rehmannia glutinosa from Henan, has identified and screened key enzyme genes and SSR molecular markers for Rehmannia glutinosa synthesis, and designed relevant sequence primers. This not only provides theoretical support for the screening and identification of Rehmannia glutinosa germplasm resources and molecular marker-assisted breeding, but also lays a foundation for the identification of functional genes related to the synthesis of Rehmannia glutinosa's effective components.

[0027] Furthermore, step 1) includes the following operational steps: a. Sample preparation: The experimental sample to be tested is stored in liquid nitrogen, and a certain amount of the liquid nitrogen-stored sample is thoroughly ground into powder. The powder is then transferred to a centrifuge tube and centrifuged. b. Extraction of genomic DNA: Following the instructions of the Plant Genome Extraction Kit from Weizan Biotechnology Co., Ltd. ( 快速纯植物DNA提取迷你试剂盒 DNA was extracted from Rehmannia glutinosa. c. Preparation of gel solution: Prepare an appropriate amount of 1% agarose gel as needed. Heat the gel until the agarose is completely dissolved and uniformly transparent. After cooling to about 60°C, add an appropriate amount of gel red, shake well, and pour the gel onto the gel plate. After the gel solution has fully solidified, pull out the comb vertically, place the gel plate into the electrophoresis tank, and pour in a sufficient amount of 1xTAE buffer into the electrophoresis tank. d. Sample loading; e. Electrophoresis; f. Detection: The main band of the extracted genomic DNA was bright and showed no obvious tailing, while the sample wells did not show obvious luminescence, indicating that the DNA sample was suitable.

[0028] Furthermore, in step 2), the PCR experimental procedure is as follows: First, preheat at 94°C for 2 minutes; Then, perform 10 cycles: hold at 94°C for 30 seconds, then cool down to 60°C and hold for 30 seconds, then extend at 72°C for 40 seconds, with the 72°C temperature gradually decreasing by 1°C in each cycle; Next, perform 20 cycles: 94°C for 30 seconds, 50°C for 30 seconds, and 72°C for 40 seconds. Finally, extend at 72°C for 8 minutes.

[0029] Further, in step 3), the silver staining includes the following steps: ① Fixation: After electrophoresis, turn off the power and separate the glass plates. Take out the gel and rinse both sides with deionized water 3 times each, each time for 10 seconds. Then place the gel in the fixative (10% glacial acetic acid solution) and place it on a shaker with a speed of 45-50 r / min. ② Staining: After fixing for 20 minutes, rinse both sides of the gel with deionized water 3 times each, each time for 10 seconds; then place the gel in the staining solution (0.1% silver nitrate solution), place it on a shaker, set the speed to 45 r / min, and stain for 20 minutes. ③Staining: After staining, immediately rinse both sides of the gel three times with deionized water, each time for 10 seconds; then place the gel in the staining solution (a mixture of 1.5% NaOH and 0.5% HCHO) until complete and clear bands appear, which takes about 6 minutes; after staining, rinse both sides of the gel three times with deionized water again, each time for 10 seconds; finally, fix the gel in the fixative for 5 minutes; after fixing, clean the gel and take a picture to obtain the polymorphic spectral characteristics of the Rehmannia glutinosa variety.

[0030] Beneficial effects Based on NCBI Rehmannia glutinosa transcriptome data and Rehmannia glutinosa var. chinensis, this invention obtained 41,976 and 38,047 transcripts of Rehmannia glutinosa var. chinensis and Rehmannia glutinosa var. chinensis respectively through reference transcriptome annotation. The SSR sequences of the transcriptomes of the two Rehmannia glutinosa were analyzed, laying the foundation for the development of transcriptome-based Rehmannia glutinosa SSR molecular markers.

[0031] This invention also utilizes existing Rehmannia transcriptome data from the laboratory, based on the Rehmannia genome ( 生物项目 登录号:PRJNA631301 The transcripts were annotated with references, and key enzyme genes for the synthesis of rehmannia terpenoids were screened. The SSR sequences of these genes were analyzed, and compared with the transcriptome, the proportion of dinucleotide and trinucleotide SSR sites in these genes was higher.

[0032] This invention developed SSR molecular markers for key enzyme genes involved in the synthesis of terpenoids in Rehmannia glutinosa, and screened two pairs of EST-SSR markers with good polymorphism. Validation was performed on different Rehmannia glutinosa varieties, and the polymorphism of the terpenoid SSR molecular markers in the four varieties was found to be as follows: D. alata, D. persimilis, D. fordii, and D. polystachya. These two new EST-SSR markers differ from previously reported Rehmannia glutinosa SSR markers. Using these markers, not only was an EST-SSR-based fingerprint successfully constructed, laying the foundation for the identification of Rehmannia glutinosa varieties, but it also allows for further screening and analysis of the synthetic pathways of terpenoids and other active substances, promoting the exploration and utilization of Rehmannia glutinosa resources. Attached Figure Description

[0033] Figure 1 This involves the screening and validation of 20 primer pairs (Marker: 700 DNA Marker; 1-20 are 20 pairs of SSR sequence primers related to terpene synthesis). Figure 2 These are the SSR-PCR results for 22 Rehmannia glutinosa varieties (Marker: 700 DNA Marker; 1-22 represent: 1-Beijing No. 1, 2-Xin 2, 3-Shanxi Jinjiu 1, 4-Tehong, 5-Xin 8, 6-Kangyu 831, 7-Tunwang, 8-Huaifeng, 9-Beijing No. 3, 10-TDS2, 11-Beijing No. 2, 12-A22, 13-Wanjin No. 1, 14-Huanghouza, 15-85-5 Bai, 16-Hongshuwang, 17-Zhangsi 961, 18-Xingke No. 3, 19-Xingke No. 2, 20-Qinhuai, 21-85-5, 22-Jinjiu). Detailed Implementation To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, all percentages, ratios, proportions or parts are by weight. Unless otherwise specified, the reagents and raw materials used in the embodiments and comparative examples of the present invention are commercially available.

[0035] The main sources of reagents used in the following examples are as follows:

[0036] Example 1 (1) Screening of key enzyme genes for the synthesis of rehmannia terpenes Using existing Rehmannia transcriptome data from the laboratory, based on the Rehmannia genome ( 生物项目登录 号:PRJNA631301 ) Complete the reference annotation of the transcripts and screen out the key enzyme genes for the synthesis of rehmannia terpenoids.

[0037] (2) SSR analysis, primer design and screening Functional SSR analysis and primer design were performed on the obtained key enzyme sequences for terpene synthesis from Rehmannia glutinosa using the SSRminer tool in TBtools and the Mouse Little Brother online tool, respectively. Twenty primer pairs were randomly selected for validation. Using DNA extracted from the Rehmannia glutinosa cultivar 9 as a template, the PCR amplification products of the 20 pairs of terpene synthesis gene SSR primers were analyzed by polyacrylamide gel electrophoresis (see [link to analysis]). Figure 1 As shown in Table 1), primers with clear amplification bands and polymorphism were finally selected as primers for genetic polymorphism analysis of germplasm resources (numbered 1, 10, 16, and 18, a total of 4 pairs).

[0038] Table 14 SSR molecular marker primers for key enzyme sequences in terpene synthesis

[0039] (3) Genomic DNA extraction and quality testing Young leaves from 22 Rehmannia glutinosa species listed in Table 2 were used as experimental samples for testing and stored in liquid nitrogen. The method was followed according to the Plant Genome Extraction Kit of Aladdin Biotechnology Co., Ltd. 快速纯植物DNA 提取迷你试剂盒 DNA was extracted from Rehmannia glutinosa. Subsequently, the DNA concentration (OD) was determined using an ultra-micro UV-Vis spectrophotometer. 260 / OD 280 The ratio was approximately 1.8, and DNA purity was determined by 1% agarose gel electrophoresis.

[0040] The specific steps are as follows: Sample preparation: Grind the 100 mg sample preserved in liquid nitrogen into powder and transfer the powder into a 1.5 mL centrifuge tube.

[0041] Genomic DNA extraction: Extract DNA according to the kit steps, and extend the water bath time and centrifugation time as appropriate.

[0042] Preparation of the gel solution: Prepare an appropriate amount of 1% agarose gel as needed. Heat until the agarose is completely dissolved and uniformly transparent. After cooling to approximately 60°C, add an appropriate amount of gel red, shake well, and pour the gel onto a gel plate. After the gel solution has fully solidified, vertically remove the comb, place the gel plate into the electrophoresis tank, and pour sufficient 1xTAE buffer into the electrophoresis tank.

[0043] Sample loading: Take 5 μL of stock solution, add 1 μL of 6x Loading buffer, mix well, and then spot the mixture into the sample wells sequentially. Add 15000 DNA Markers to one of the sample wells.

[0044] Electrophoresis: Electrophoresis was performed using a 120V regulated voltage for 30 minutes.

[0045] Detection: The electrophoresis results were observed using a UV imaging analyzer. The extracted genomic DNA main band was bright and showed no obvious tailing, while the sample wells did not show significant luminescence, indicating that the DNA samples were suitable for subsequent experiments.

[0046] Table 2 Information on the Rehmannia glutinosa varieties used in the experiment:

[0047] (4) SSR-PCR reaction Using the obtained genome of the sample to be tested as a template, the four pairs of SSR primers obtained through screening were mixed for PCR experiments. The steps were as follows: First, preheating was performed at 95 °C for 5 minutes, followed by 10 cycles: incubation at 94 °C for 30 seconds, then cooling from 65 °C to 60 °C and holding for 45 seconds (each cycle decreasing by 0.5 °C), and finally extension at 72 °C for 60 seconds; then 23 cycles were performed: incubation at 94 °C for 30 seconds, holding at 58 °C for 45 seconds, extension at 72 °C for 60 seconds; finally, extension at 72 °C for 10 minutes. The PCR reaction system is shown in Table 3.

[0048] Table 3 SSR-PCR reaction system

[0049] (5) Denaturing polyacrylamide gel electrophoresis and silver staining The obtained PCR products were analyzed by denaturing polyacrylamide gel electrophoresis. The steps are as follows: ① Cleaning the glass plates: Thoroughly clean the glass plates with dish soap, avoiding touching them with your fingers. Rinse them with double-distilled water and dry them in an oven. After cooling, place them horizontally and secure them with clips, ready for gel casting. ② Gel casting: Prepare a 12% denaturing PAGE gel, the gel formula is shown in Table 4. Place the silane-coated sides of the two glass plates facing each other, and place sealing strips on both sides of the middle of the two glass plates to prevent the gel from flowing out. After mixing all the reagents in the gel formula, slowly pour the gel between the two glass plates, avoiding air bubbles as much as possible. After pouring the gel, secure the bottom and sides of the glass plates with clips, insert the comb vertically, and let it stand for about 1.5 hours to allow the gel to solidify. ③ Sample loading: After the gel solidifies, vertically remove the comb, install the electrophoresis apparatus and glass plates. Add 1×TBE buffer to the electrophoresis tank, covering the glass plates. To minimize the impact of urea on nucleic acid electrophoresis bands, after adding 1×TBE, use a pipette to agitate the sample wells to remove residual urea. Add 4 μL of PCR product to each well using a pipette, inserting the pipette tip to the bottom of the well and slowly dispensing to avoid uneven distribution of the sample, such as stringing or floating. Check the TBE buffer between the two gel plates; it should not be too low. ④ Electrophoresis: Use a two-stage electrophoresis method (pre-electrophoresis followed by electrophoresis). Electrophoresis conditions are constant voltage 100 V, 50 mA, 90 minutes, stopping when the indicator reaches 2 / 3 of the glass plate.

[0050] Table 4. Gel Formulation for Denaturing Polyacrylamide Gel Electrophoresis

[0051] After electrophoresis, the polyacrylamide gel was silver-stained using the following steps: ① Fixation: After electrophoresis, the power was turned off and the glass plates were separated. The gel was removed and rinsed with deionized water on both sides three times each, for 10 seconds each time. Then, the gel was placed in the fixative (10% glacial acetic acid solution) on a shaker at 45-50 r / min. ② Staining: After 20 minutes of fixation, the gel was rinsed again with deionized water on both sides three times each, for 10 seconds each time. Then, the gel was placed in the staining solution (0.1% silver nitrate solution) on a shaker at 45 r / min for 20 minutes. ③ Development: After staining, the gel was immediately rinsed again with deionized water on both sides three times each, for 10 seconds each time. Then, the gel was placed in the developing solution (a mixture of 1.5% NaOH and 0.5% HCHO solution) until clear and complete bands appeared, which took approximately 6 minutes. After development, the gel was rinsed again with deionized water on both sides three times each, for 10 seconds each time. Finally, the gel was fixed in the fixative for 5 minutes. After fixing, the gel was cleaned and photographed to obtain the polymorphic spectral characteristics of 22 Rehmannia glutinosa varieties (see...). Figure 2 (As shown).

[0052] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A primer composition for SSR molecular markers of key enzymes and transcription factor genes involved in the synthesis of rehmannia terpenes, characterized in that, The sequence of the key enzyme gene is composed of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, and the primer composition for the key enzyme gene for the synthesis of rehmannia terpenoids is composed of primer pair SSR-1, primer pair SSR-10, primer pair SSR-16, and primer pair SSR-18.

2. The primer composition for SSR molecular markers of key enzymes and transcription factor genes involved in the synthesis of rehmannia terpenoids according to claim 1, characterized in that, Primer sequence for SSR-1: Forward primer SSR-1F: 5'-ACTCGTGAGCCATGTGTATAT-3'; Reverse primer SSR-1R: 5'-CCCCACAGGCCCATACATTTA-3'; Primer sequences for SSR-10: Forward primer SSR-10F: 5'-CGGAGAGCCACCCTTTCACTAA-3'; Reverse primer SSR-10R: 5'-GCGCCACGTCACCATTGTTTAG-3'; Primer sequences for SSR-16: Forward primer SSR-16F: 5'-GGCAGCCAATCTTCCAGAAAAC-3'; Reverse primer SSR-16R: 5'-GGGCGTGGTTCTTGCAGAGGAA-3'; Primer sequences for SSR-18: Forward primer SSR-18F: 5'-CATTGGCAAGTTTCGAGCATGA-3'; Reverse primer SSR-18R: 5'-GAGCCTCGGATTCACTGTAGCA-3'.

3. A reagent kit for identifying varieties of Rehmannia glutinosa, characterized in that, It contains the primer composition as described in claim 1 or 2.

4. A method for identifying varieties of Rehmannia glutinosa, characterized in that, Includes the following steps: Step 1) Genomic DNA extraction and quality testing; Step 2) SSR-PCR reaction; using the Rehmannia glutinosa genome extracted in step 1) as a template, amplify the Rehmannia glutinosa sample to be tested based on the primer composition described in claim 1 or 2; Step 3) Denaturing polyacrylamide gel electrophoresis and silver staining; the PCR amplification fragment from step 2) was analyzed by electrophoresis using a 12% denaturing PAGE gel to obtain the fingerprint of Rehmannia glutinosa.

5. The method for identifying Rehmannia glutinosa varieties according to claim 4, characterized in that, Before step 1), there are two additional steps: the first step is the screening of key enzyme genes for the synthesis of rehmannia terpenes; the second step is SSR analysis, primer design and screening.

6. The method for identifying Rehmannia glutinosa varieties according to claim 5, characterized in that, In the first step, using Rehmannia transcriptome data, based on the Rehmannia genome ( BioProject accession number: PRJNA631301 ) Complete the reference annotation of the transcripts and screen out the key enzyme genes for the synthesis of rehmannia terpenoids.

7. The method for identifying Rehmannia glutinosa varieties according to claim 5, characterized in that, In the second step, SSRminer was used to perform functional SSR analysis and primer design on the obtained key enzyme sequences for the synthesis of rehmannia terpenes. Primers were randomly selected for verification, and primers with clear amplification bands and polymorphism were obtained.

8. The method for identifying Rehmannia glutinosa varieties according to claim 4, characterized in that, In step 2), the PCR experiment procedure is as follows: First, preheat at 94°C for 2 minutes; Then, perform 10 cycles: hold at 94°C for 30 seconds, then cool down to 60°C and hold for 30 seconds, then extend at 72°C for 40 seconds, with the 72°C temperature gradually decreasing by 1°C in each cycle; Next, perform 20 cycles: 94°C for 30 seconds, 50°C for 30 seconds, and 72°C for 40 seconds. Finally, extend at 72°C for 8 minutes.

9. The method for identifying Rehmannia glutinosa varieties according to claim 4, characterized in that, In step 3), the silver staining includes the following steps: ① Fixation: After electrophoresis, turn off the power and separate the glass plates. Take out the gel and rinse both sides with deionized water 3 times each, each time for 10 seconds. Then place the gel in the fixative and place it on a shaker with a speed of 45-50 r / min. ② Staining: After fixing for 20 minutes, rinse both sides of the gel with deionized water 3 times each, each time for 10 seconds; then place the gel in the staining solution, place it on a shaker, set the speed to 45 r / min, and stain for 20 minutes. ③Staining: After staining, immediately rinse both sides of the gel with deionized water three times each, for 10 seconds each time; then place the gel in the developing solution until complete and clear bands appear, which takes about 6 minutes; after staining, rinse both sides of the gel with deionized water three times each, for 10 seconds each time; finally, place the gel in the fixative for 5 minutes; after fixing, clean the gel and take a picture to obtain the polymorphic fingerprint characteristics of the Rehmannia glutinosa variety.

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