SSR molecular marker primer group related to identification of polygonatum germplasm resources and application of SSR molecular marker primer group

Abstract

The invention provides an SSR (Simple Sequence Repeat) molecular marker primer group related to identification of germplasm resources of polygonatum and application of the SSR molecular marker primer group. The SSR molecular marker primer group comprises a primer pair 1 of SSR1, a primer pair 2 of SSR1 and a primer pair 3 of SSR1, wherein the nucleotide sequence of the primer pair 1 is shown as SEQ ID NO.1-2; and a primer pair 2 of the SSR2: the nucleotide sequence is shown as SEQ ID NO.3-4. The SSR polymorphic molecular marker provided by the invention is beneficial to identification, evaluation and shared utilization of the polygonatum sibiricum germplasm resources, and provides a technical basis for data and intelligent management of the polygonatum sibiricum germplasm resources. In addition, the SSR molecular marker provided by the invention is simple to operate, good in reproducibility and high in result reliability, and different germplasms of the polygonatum plants can be distinguished. And a basis is provided for germplasm identification and innovative breeding of polygonatum kingianum.

Description

Technical Field

[0001] The invention relates to the technical field of molecular marker screening, and in particular to an SSR molecular marker primer set related to identification of Polygonatum germplasm resources and an application thereof. Background Art

[0002] Polygonatum is a perennial monocotyledonous plant of the genus Polygonatum in the Liliaceae family, which is widely distributed. In traditional Chinese medicine theory, Polygonatum plants have the effects of increasing coronary flow, enhancing immunity, regulating blood lipids, lowering blood sugar, anti-inflammatory and antibacterial, and delaying aging. Modern pharmacological research believes that it has multiple medicinal functions such as improving learning and memory, relieving fatigue, protecting heart and liver function, anti-oxidation, delaying aging, lowering blood sugar concentration, treating loose bones, and anti-cancer. There are many types of Polygonatum, the most common ones are Polygonatum multiflorum, Polygonatum yunnanensis, Polygonatum acanthus, etc. With the wide recognition of the medicinal value of Polygonatum and the continuous increase in market demand, wild Polygonatum resources are facing the risk of over-exploitation, resulting in increasing resource depletion. At the same time, due to the relatively backward cultivation technology and limited planting scale, it is difficult to meet market demand, further exacerbating the contradiction between resource protection and utilization. In addition, there are many intermediate types of Polygonatum plants, which are difficult to distinguish by morphological methods. With the continuous development of molecular biology technology, it will be a feasible method to use molecular markers to identify the resources of Polygonatum plants.

[0003] SSR, also known as microsatellite DNA, is a type of DNA sequence composed of several nucleotides (mostly 1-6) as repeating units, which are widely distributed in different positions throughout the genome. The number of repetitions at each seat may not be exactly the same between different species of eukaryotes or different varieties of the same species, resulting in polymorphism at each seat. It is usually marked based on specific PCR products and is widely used in molecular assisted breeding, construction of genetic maps and variety identification.

[0004] At present, the identification technology of Polygonatum plant resources is relatively backward, and there is still a lack of SSR marker development and application of Polygonatum germplasm resources. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects of the prior art, and to provide SSR molecular markers and primer sets and applications related to the identification of Polygonatum germplasm resources. The present invention explores SSR molecular marker primer sets related to the identification of Polygonatum germplasm resources, aiming to use SSR site combinations to provide a primer combination that can effectively, quickly and accurately identify Polygonatum germplasm resources, so as to make up for such deficiencies. At the same time, this method is different from the traditional use of various plant material genome sequencing or EST, and BAC library construction analysis sequence and other complex means to screen SSR polymorphisms in plants. For most medicinal plants that have not completed genome sequencing, transcriptome sequencing and its assembly series are used to analyze and identify the SSR polymorphic molecular markers present therein, which is conducive to the identification, evaluation and shared utilization of Polygonatum germplasm resources, and provides a technical basis for the data-based and intelligent management of Polygonatum germplasm resources. In addition, the SSR molecular markers provided by the present invention are simple to operate, have good reproducibility, and high reliability of results, and can distinguish different germplasms of Polygonatum plants. Provide a basis for the identification of Polygonatum germplasm and innovative breeding.

[0006] In order to achieve the purpose of the present invention, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, there is provided an SSR molecular marker primer set related to the identification of Polygonatum germplasm resources, the molecular marker primer set comprising one of the following primer pairs:

[0008] Primer pair 1 of SSR1: the nucleotide sequence is shown in SEQ ID NO. 1-2;

[0009] Primer pair 2 of SSR2: the nucleotide sequence is shown in SEQ ID NO.3-4.

[0010] In a second aspect of the present invention, an SSR molecular marker related to the identification of Polygonatum germplasm resources is provided, and the molecular marker includes SSR1-SSR2 amplified by using the primer set.

[0011] The sequences amplified by the primer pair include:

[0012] The nucleic acid molecule amplified by primer pair 1 of SSR1 is molecular marker SSR1, and the nucleic acid molecule amplified by primer pair 2 of SSR2 is molecular marker SSR1.

[0013] In the third aspect of the present invention, a kit for identifying Polygonatum germplasm resources is provided, the kit comprising the SSR molecular marker primer set related to the identification of Polygonatum germplasm resources.

[0014] In a fourth aspect of the present invention, a method for identifying Polygonatum germplasm resources is provided, the method comprising:

[0015] Extract genomic DNA from Polygonatum cyrtonema from different germplasm sources;

[0016] Performing PCR amplification on the genomic DNA using the primer set to obtain a PCR amplification product;

[0017] The PCR amplification products were subjected to PAGE gel electrophoresis, and the results were analyzed to identify the germplasm resources of the genus Polygonatum.

[0018] In the above technical solution, the specific method for analyzing the results to identify the germplasm resources of the genus Polygonatum includes:

[0019] The genomic DNA of leaves of Polygonatum cyrtonema plants from different sources was extracted by CTAB method, and the DNA quality was detected by 1% agarose electrophoresis. Then, the extracted Polygonatum cyrtonema genomic DNA was used as a template and the primer pairs shown in SEQ ID NO.1-2 and SEQ ID NO.3-4 were used for PCR amplification.

[0020] The PCR product was tested by DNA-PAGE electrophoresis to obtain the electrophoresis pattern of the tested Polygonatum germplasm, which was compared with the Marker. The electrophoresis bands between different germplasms showed polymorphism, which can be used to identify the Polygonatum germplasm resources.

[0021] The specific judgment rules are as follows:

[0022] 1. If the primer pair shown in SEQ ID NO.1-2 is used for PCR amplification,

[0023] (1) If two bands of 290 bp and 220 bp appear, it is Yunnan Polygonatum cyrtonema;

[0024] (2) If two bands of 255 bp and 480 bp appear, it is Polygonatum cyrtonema;

[0025] (3) If there is only one band of 235 bp, it is Polygonatum cyrtonema;

[0026] (4) If two bands of 230 bp and 290 bp appear, it is Polygonatum yunnanensis;

[0027] (5) If two bands of 232 bp and 290 bp appear, it is Polygonatum cyrtonema;

[0028] (6) If three bands of 590 bp, 480 bp, and 400 bp appear, it is Polygonatum cyrtonema;

[0029] (7) If two bands of 240 bp and 238 bp appear, it is Polygonatum cyrtonema;

[0030] (8) If two bands of 250 bp and 300 bp appear, it is Polygonatum cyrtonema;

[0031] (9) If there is no obvious band, it is Sichuan Polygonatum cyrtonema;

[0032] (10) If a band of 250 bp appears, it is Polygonatum hunanense;

[0033] (11) If three bands of 250 bp, 200 bp, and 140 bp appear, it is Polygonatum cyrtonema;

[0034] (12) If a lighter band of 298 bp appears, it is Polygonatum cyrtonema.

[0035] 2. If the primer pair shown in SEQ ID NO.3-4 is used for PCR amplification,

[0036] (1) If two bands of 280 bp and 150 bp appear, it is Yunnan Polygonatum cyrtonema;

[0037] (2) If four bands of 250 bp, 200 bp, 145 bp, and 100 bp appear, it is Polygonatum cyrtonema;

[0038] (3) If a band of 210 bp appears, it is Polygonatum cyrtonema;

[0039] (4) If there is no obvious band, it is Yunnan Polygonatum cyrtonema;

[0040] (5) If three bands of 500 bp, 180 bp, and 150 bp appear, it is Polygonatum cyrtonema;

[0041] (6) If two bands of 152 bp and 150 bp appear, it is Polygonatum cyrtonema;

[0042] (7) If two bands of 250 bp and 200 bp appear, it is Polygonatum cyrtonema;

[0043] (8) If four bands of 240 bp, 238 bp, 200 bp, and 150 bp appear, it is Polygonatum cyrtonema;

[0044] (9) If four bands of 240 bp, 238 bp, 200 bp, and 150 bp appear, it is Sichuan Polygonatum cyrtonema;

[0045] (10) If two bands of 198 bp and 150 bp appear, it is Polygonatum hunanense;

[0046] (11) If there are four bands with sizes of 270 bp, 250 bp, 200 bp, and 160 bp, it is Polygonatum cyrtonema;

[0047] (12) If two DNA fragments with sizes of 215 bp and 150 bp appear, it is Polygonatum cyrtonema.

[0048] Furthermore, in the PCR amplification, the annealing temperature range of the primer set is 53-63°C.

[0049] Furthermore, the reaction procedure of the PCR amplification is: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, 35 cycles, and extension at 72°C for 10 min.

[0050] Furthermore, the PCR amplification system, taking 10 μL as an example, includes: 5 μL of Taq PCR MasterMix, 0.2 μL, 10 μmol / L upstream and downstream primers, 1 μL, 25 ng / μL genomic DNA, and deionized water is used to complete the reaction system.

[0051] In the fifth aspect of the present invention, provided is the use of the SSR molecular marker primer set related to the identification of Polygonatum germplasm resources, the SSR molecular marker related to the identification of Polygonatum germplasm resources, or the kit for identifying Polygonatum germplasm resources in the identification of Polygonatum germplasm resources.

[0052] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0053] 1. The two SSR molecular markers of the genus Polygonatum of the present invention have strong cross-species transfer ability in closely related species such as Polygonatum cyrtonema, Polygonatum multiflorum, Polygonatum verticillata, Polygonatum purpurogenum, and Polygonatum cyrtonema. Therefore, the SSR molecular markers provided by the present invention can be applied to the genetic diversity analysis, genetic structure analysis, comparative transcriptomics, genetic linkage map construction, localization and cloning of key genes of medicinal ingredients, molecular marker-assisted breeding and other studies of other species of the genus Polygonatum.

[0054] 2. The SSR molecular marker provided by the present invention is simple to operate, has good reproducibility, and has high reliability of results. It can distinguish different germplasms of Polygonatum plants, providing a basis for the germplasm identification and innovative breeding of Polygonatum. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1The results of primer S10 amplification and electrophoresis of 12 polygonatum germplasm DNA. Among them, M is a marker, 1, Yunnan polygonatum (Guizhou); 2, Polygonatum multiflorum (Guizhou); 3: Polygonatum cyrtonema (Northeast China); 4, Yunnan polygonatum (Yunnan); 5, Yunnan polygonatum (Guangxi); 6: Purple spot Yunnan polygonatum (Guangxi); 7: Polygonatum cyrtonema (Shaanxi); 8, Polygonatum multiflorum (Hunan); 9, Polygonatum multiflorum (Sichuan); 10: Polygonatum odoratum (Hunan); 11: Purple-leaved Polygonatum (Fujian); 12, Polygonatum multiflorum (Anhui).

[0056] Figure 2 The results of primer S18 amplification and electrophoresis of 12 polygonatum germplasm DNA. M is a marker, 1, polygonatum cyrtonema (Guizhou); 2, polygonatum cyrtonema (Guizhou); 3: polygonatum cyrtonema (Northeast China); 4, polygonatum cyrtonema (Yunnan); 5, polygonatum cyrtonema (Guangxi); 6: polygonatum cyrtonema purpurogenum (Guangxi); 7: polygonatum cyrtonema (Shaanxi); 8, polygonatum cyrtonema (Hunan); 9, polygonatum cyrtonema (Sichuan); 10: Polygonatum odoratum (Hunan); 11: polygonatum cyrtonema purpurogenum (Fujian); 12, polygonatum cyrtonema (Anhui). DETAILED DESCRIPTION

[0057] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0058] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0059] Unless otherwise specified, the drugs, reagents or instruments used in the present invention can be purchased from the market.

[0060] The present application will be described in detail below with reference to embodiments and experimental data.

[0061] Example 1 Development of SSR molecular marker primers related to identification of Polygonatum germplasm resources

[0062] S1: PCR primer design and synthesis:

[0063] The transcriptome sequencing assembly results of different accessions of Polygonatum cyrtonema were analyzed to find SSR tandem repeat sequences, and primers were designed using Primer 5.0 software.

[0064] S2: Extraction of genomic DNA from Polygonatum cyrtonema germplasm using CTAB method:

[0065] (1) Select fresh leaves of the 12 accessions of Polygonatum cyrtonema shown in Table 2 and put them into a mortar. Add 2 mL of CTA B extract and an appropriate amount of quartz sand. Grind the leaves thoroughly. Use a pipette to draw about 1 mL of grinding solution into a sterilized 1.5 mL centrifuge tube. Place the tube in a water bath at 65°C for 10 min. Centrifuge at 8000 x g for 5 min at room temperature. Draw about 600 μL of the supernatant into a new 1.5 mL centrifuge tube. Add 600 μL of chloroform in a fume hood and mix for 5 min. Centrifuge at 8000 x g for 5 min at room temperature. Draw about 500 μL of the supernatant into a new 1.5 mL centrifuge tube in a fume hood. mL centrifuge tube, add 700μL pre-cooled anhydrous ethanol, mix well, let stand at -20℃ for 30min, centrifuge at 8000xg for 5min at room temperature, then white precipitate can be seen, which is DNA, pour out the supernatant, add 1mL75% ethanol, tap the tube wall to clean the DNA, centrifuge at room temperature for 2min, pour out the supernatant, open the lid for 5min, add 200μLTE solution after 75% ethanol is completely evaporated, and store at -20℃ after the DNA is completely dissolved;

[0066] (2) Quality test of genomic DNA of Polygonatum cyrtonema germplasm: weigh 0.25g of agarose powder and add 25mL of 1xTAE solution to a conical flask, heat in a microwave oven until the solution boils about three times (the agarose powder melts), cool to 60°C (not too hot), add 1μL of nucleic acid dye, and shake well. Pour the prepared 1% agarose gel into the electrophoresis gel plate (the gel plate is placed horizontally and poured to two-thirds of the comb), insert the electrophoresis gel comb, and cool at room temperature until the gel solidifies. After the gel solidifies, pull out the gel comb and place the gel electrophoresis plate in the buffer of the electrophoresis tank. Use a pipette to add 5μL of 1kbDNA Ladder to the first well of the gel, take another clean disposable glove, use a pipette to absorb 1μL of DNA Loading Buffer, change the pipette tip to absorb 4μL of sample, mix it evenly with DNA Loading Buffer, and add it to the spotting well behind the marker. Perform electrophoresis at 220V and 150mA. When the 1kb DNA ladder runs to two-thirds of the gel, take out the gel and place it on the gel imaging plate of the gel imaging system. Take a photo and record the results.

[0067] S3: PCR amplification: The total volume of the amplification reaction was 10 μL: 2×TaqMasterMix 5 μL, forward label 0.3 μL, reverse label 0.3 μL, DNA template 1 μL, ddH2O 3.4 μL. The PCR amplification reaction program was: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 30 s, 35 cycles, 72°C extension for 10 min. The PCR product was silver stained after DNA-PAGE electrophoresis and photographed after color development.

[0068] S4: DNA-PAGE electrophoresis:

[0069] Preparation of agarose sealing glue: Wash the glass plate repeatedly with distilled water, wipe it with alcohol, dry it and assemble the glass plate. Weigh 0.2g of agarose and 20ml of TAE water into a beaker and heat it in a microwave for 2min until it is completely dissolved. Add about 3mL of agarose sealing glue to each rubber bottom plate, put the glass plate on it while it is hot so that the agarose sealing glue evenly fills the bottom. It can be used after cooling and solidifying.

[0070] Gel preparation: Add 17.5 mL of 30% gel stock solution, 5 mL of 5×TBE solution, 500 μL of 10% APS solution, 15 mL of ddH2O, and 40 μL of TEMED into a conical flask. After mixing evenly, use a glass rod to drain and quickly inject into the gap of the glass plate to prevent bubbles. After the gel is poured, insert a 1mm×40 gel comb and wipe the overflowed gel. Let the gel run for about 50 minutes. After the film solidifies, pull out the gel comb and assemble the electrophoresis tank.

[0071] Gel electrophoresis: Inject 1×TBE electrophoresis buffer into the electrophoresis tank, place the film in the electrophoresis tank, inject 1×TBE electrophoresis buffer into the top of the electrophoresis tank to cover the top of the film, add 2μL Marker to the first or first two sample wells, add 3μL PCR product to each of the remaining wells, turn on the power supply, voltage 200V, current 200mA, and the electrophoresis time is determined by the actual situation.

[0072] Silver staining: After electrophoresis, place the glass plate in a tray and add an appropriate amount of distilled water. Carefully separate the two glass plates, remove the film and put it into distilled water. Wash it once with distilled water, pour out the distilled water and put it into a pre-prepared 250mL silver nitrate solution (0.35g silver nitrate, 250mL pure water). After shaking on a shaker for 10 minutes, pour out the staining solution and wash it 3 times with distilled water to wash away the residual silver nitrate solution on the surface of the film.

[0073] Color development and recording: Place the washed film on a shaker, add 250 mL of sodium hydroxide solution (2 g of sodium hydroxide, 250 mL of purified water) and 2.5 mL of formaldehyde solution, and shake gently until the strips are clear. After the color development is completed, wash off the remaining color developing solution with distilled water, lay the film flat under a fluorescent light box, and take pictures for preservation.

[0074] After repeated screening, two pairs of SSR primer combinations with clear and stable bands, good repeatability and high polymorphism were finally obtained. The SSR primer combinations were then verified using 12 Polygonatum cyrtonema germplasms. The sequences of the two pairs of SSR primer combinations are shown in Table 1.

[0075] The sequence of the SSR primer combination for rapid identification of Polygonatum sibiricum germplasm resources in the present application is shown in Table 1.

[0076] Table 1

[0077]

[0078] The Polygonatum sibiricum germplasm is shown in Table 2.

[0079] Table 2

[0080]

[0081]

[0082] Example 2: A method for identifying Polygonatum sibiricum germplasm

[0083] Extraction of genomic DNA from the tested Polygonatum cyrtonema germplasm;

[0084] The extracted genomic DNA of Polygonatum cyrtonema germplasm was used as a template and PCR amplified using the primer combination described in Example 1;

[0085] The PCR products were detected by DNA-PAGE electrophoresis. The reaction system of PCR amplification included: 2×Taq Master Mix 5μL, forward label 0.3μL, reverse label 0.3μL, DNA template 1μL, ddH2O 3.4μL, and the PCR amplification reaction program was: 94℃ pre-denaturation for 5min, 94℃ denaturation for 30s, 56℃ annealing for 30s, 72℃ extension for 30s, 35 cycles, 72℃ extension for 10min, and stored at -20℃ after completion.

[0086] The electrophoresis pattern of the tested Polygonatum germplasm is obtained and compared with the Marker. The electrophoresis bands between different germplasms show polymorphism, which can be used to identify the Polygonatum germplasm resources.

[0087] The specific manifestations are:

[0088] exist Figure 1In the figure, the target product size of primer S10 (i.e., primer pair 1 of SSR1: nucleotide sequence as shown in SEQ ID NO.1-2) is 292bp, and the target band (located between 200bp and 300bp) is obtained after comparison with Marker. In the figure, different Polygonatum germplasms show obvious polymorphism after amplification with S10 primers. 1 and 2 are Polygonatum cyrtonema and Polygonatum cyrtonema from Guizhou, respectively, with two bands of different heights; 3 is Polygonatum cyrtonema from Northeast China with only one band; 4 and 5 are Polygonatum cyrtonema from Yunnan and Polygonatum cyrtonema from Guangxi, and the bands are similar to those of Polygonatum cyrtonema from Guizhou; 6 produces 3 lighter bands located in the range of 400bp-600bp; 7 is two relatively close bands; 8 and 11 are Polygonatum cyrtonema from different origins, and the bands produced by electrophoresis of their products are polymorphic with other species; 9 and 12 are Polygonatum cyrtonema from Sichuan and Anhui without products (bands), and 12 has only one lighter stained band, which is obviously different from other germplasms. Different Polygonatum cyrtonema germplasm resources can be distinguished by the number and size of bands after electrophoresis of PCR products.

[0089] exist Figure 2 In the figure, the target product size of primer S18 is 114bp, and the target band is (located between 100bp-250bp) after comparison with Marker. In the figure, different polygonatum germplasms show obvious polymorphism after amplification with S18 primer. 1 and 2 are Yunnan polygonatum and Polygonatum cyrtonema from Guizhou, respectively, with different heights and numbers of bands; 3 is only one band of Northeast Verticillium cyrtonema, which is obviously different from other germplasms; 4 is Yunnan polygonatum without products and bands; 5 and 6 are Yunnan polygonatum and purple-spotted polygonatum from Guangxi, with obvious differences in bands; 7 is Polygonatum cyrtonema, which only produces two bands, which are completely different from other germplasms; 8 and 9 are both Polygonatum cyrtonema, and the bands produced by their products after electrophoresis are similar, but there are polymorphisms compared with other species; 11 and 12 are Polygonatum cyrtonema from Fujian and Anhui, and their products (bands) are obviously different and polymorphic with other germplasms. Different polygonatum germplasm resources can be distinguished by the number and size of bands after electrophoresis of PCR products.

[0090] Example 3, Effect Evaluation

[0091] The experiment was carried out using the method shown in Example 2, and the results are as follows. The results were verified by sequencing, and the accuracy of the results was 100%.

[0092] Table 3. Band types amplified by primer S10 in different Polygonatum cyrtonema germplasm materials

[0093] Material / Strip Type 1 2 3 4 5 6 7 8 9 10 11 12 Duohua(Suining) + + Yunnan (Wenshan) + + + Chicken head (Shangluo) + Wheel blade + + Duohua(Nanchong) + + Duohua (Hunan) + Duohua(Hubei) + Yunnan (Baise) + + Yunnan (Pu'er) + Yunnan (Nanning) + + Chicken head (Luoyang) + Yunnan (Guizhou) + +

[0094] Note: + indicates positive

[0095] Table 4. Band types amplified by primer S18 in different Polygonatum cyrtonema germplasm materials

[0096] Material / Strip Type 1 2 3 4 5 6 7 8 9 10 11 12 Duohua(Suining) + + Yunnan (Wenshan) + Chicken head (Shangluo) + Wheel blade + Duohua(Nanchong) + + Duohua (Hunan) + + Duohua(Hubei) + Yunnan (Baise) + Yunnan (Pu'er) + Yunnan (Nanning) + Chicken head (Luoyang) + Yunnan (Guizhou) +

[0097] Note: + indicates positive

[0098] It can be seen from the above that the SSR molecular marker provided by the present invention is simple to operate, has good reproducibility, and has high reliability of results, and can distinguish different germplasms of Polygonatum plants.

[0099] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0100] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the embodiments of the present invention.

[0101] Obviously, those skilled in the art can make various changes and modifications to the embodiments of the present invention without departing from the spirit and scope of the embodiments of the present invention. Thus, if these modifications and variations of the embodiments of the present invention fall within the scope of the claims of the embodiments of the present invention and their equivalents, the embodiments of the present invention are also intended to include these modifications and variations.

Claims

1. An SSR molecular marker primer set related to the identification of Polygonatum germplasm resources, characterized in that: The molecular marker primer set includes one of the following primer pairs: Primer pair 1: nucleotide sequence as shown in SEQ ID NO.1-2; Primer pair 2: The nucleotide sequence is shown in SEQ ID NO.3-4.

2. An SSR molecular marker related to the identification of Polygonatum germplasm resources, characterized in that: The molecular marker includes one of the molecular marker SSR1 and the molecular marker SSR2 amplified using the primer set according to claim 1.

3. A kit for identifying Polygonatum germplasm resources, characterized in that: The kit comprises the SSR molecular marker primer set related to the identification of Polygonatum germplasm resources as described in claim 1.

4. A method for identifying Polygonatum germplasm resources, characterized in that: The method comprises: Extract genomic DNA from Polygonatum cyrtonema from different germplasm sources; Performing PCR amplification on the genomic DNA using the primer set of claim 1 to obtain a PCR amplification product; The PCR amplification products were subjected to PAGE gel electrophoresis, and the results were analyzed to identify the germplasm resources of the genus Polygonatum.

5. A method for identifying Polygonatum germplasm resources according to claim 4, characterized in that: In the PCR amplification, the annealing temperature range of the primer set is 53-63°C.

6. A method for identifying Polygonatum germplasm resources according to claim 4, characterized in that: The reaction procedure of the PCR amplification was as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, 35 cycles, and extension at 72°C for 10 min.

7. A method for identifying Polygonatum germplasm resources according to claim 4, characterized in that: The PCR amplification system, taking 10 μL as an example, includes: 5 μL of 2×Taq MasterMix, 0.3 μL of forward marker, 0.3 μL of reverse marker, 1 μL of DNA template, and 3.4 μL of ddH2O.

8. Use of the SSR molecular marker primer set related to the identification of Polygonatum germplasm resources described in claim 1, the SSR molecular marker related to the identification of Polygonatum germplasm resources described in claim 2, or the kit for identifying Polygonatum germplasm resources described in claim 3 in the identification of Polygonatum germplasm resources.

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