An SSR molecular marker primer set related to the identification of Polygonatum germplasm resources and its application

Abstract

The present invention provides an SSR molecular marker primer set related to the identification of Polygonatum germplasm resources, and the molecular marker primer set includes: Primer pair 1 of SSR1: the nucleotide sequences are as shown in SEQ ID NO.1-2; Primer pair 2 of SSR2: the nucleotide sequences are as shown in SEQ ID NO.3-4. The SSR polymorphic molecular marker of the present application is beneficial to the identification, evaluation, sharing and utilization of Polygonatum germplasm resources, and provides a technical basis for the digital and intelligent management of Polygonatum germplasm resources. In addition, the SSR molecular marker provided by the present invention is simple to operate, has good reproducibility and high result reliability, and can distinguish different germplasms of Polygonatum plants. It provides a basis for the germplasm identification and innovative breeding of Polygonatum.

Description

Technical Field

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

[0002] Polygonatum is a perennial monocotyledonous plant of the genus Polygonatum in the family Liliaceae, and it is widely distributed. In traditional Chinese medicine theory, Polygonatum plants have the effects of increasing coronary blood flow, enhancing immunity, regulating blood lipid, reducing blood sugar, anti-inflammatory and antibacterial, and delaying aging. Modern pharmacological research believes that it has a variety of medicinal functions such as improving learning and memory ability, relieving fatigue, protecting heart and liver functions, antioxidation, delaying aging, reducing blood sugar concentration, treating osteoporosis, and anti-cancer. There are many types of Polygonatum, and common ones include Polygonatum cyrtonema, Polygonatum kingianum, Polygonatum sibiricum, 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-excavation, resulting in the increasing exhaustion of resources. At the same time, due to the relatively backward cultivation technology and limited planting scale, it is difficult to meet the market demand, further exacerbating the contradiction between resource protection and utilization. In addition, there are many intermediate types among Polygonatum plants, and it is difficult to distinguish them by morphological methods. With the continuous development of molecular biology technology, using molecular markers to identify Polygonatum germplasm resources will be a feasible method.

[0003] SSR, also known as microsatellite DNA, is a kind of DNA sequence formed by tandem repetition of several nucleotides (mostly 1-6), and it is widely distributed at different positions in the whole genome. Among different species of eukaryotes or different varieties of the same species, the number of repetitions at each locus may not be exactly the same, thus resulting in polymorphism at each locus. Usually, it is based on specific PCR products for marking and is widely used in molecular assisted breeding, construction of genetic maps, variety identification, etc.

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

[0005] The object of the present invention is to overcome the defects of the prior art, and provide SSR molecular markers, primer sets thereof and applications related to the identification of Polygonatum germplasm resources. The present invention discovers a primer set of SSR molecular markers related to the identification of Polygonatum germplasm resources, aiming to provide a primer combination that can effectively, quickly and accurately identify Polygonatum germplasm resources by using SSR locus combinations to make up for such deficiencies. At the same time, this method is different from traditional complex means such as genome sequencing of various germplasm plant materials, EST, and construction and analysis of BAC libraries to screen SSR polymorphisms existing in plants. For most medicinal plants whose genomes have not been sequenced, transcriptome sequencing and its assembly series analysis are used to identify the existing SSR polymorphic molecular markers, which is beneficial to the identification, evaluation, sharing and utilization of Polygonatum germplasm resources, and provides a technical basis for the digital 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 result reliability, and can distinguish different germplasms of Polygonatum plants. It provides a basis for the germplasm identification and innovative breeding of Polygonatum.

[0006] To achieve the object of the present invention, the following technical solutions are adopted in the present invention:

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

[0008] Primer pair 1 of SSR1: The nucleotide sequences are as shown in SEQ ID NO.1-2;

[0009] Primer pair 2 of SSR2: The nucleotide sequences are as shown in SEQ ID NO.3-4.

[0010] In the 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 the primer set.

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

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

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

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

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

[0016] Perform PCR amplification on the genomic DNA using the primer sets described above to obtain PCR amplification products;

[0017] Perform PAGE gel electrophoresis on the PCR amplification products and analyze the results to identify the germplasm resources of Polygonatum.

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

[0019] Extract the genomic DNA of the leaves of Polygonatum plants from different sources using the CTAB method, detect the DNA quality by 1% agarose electrophoresis, and then use the extracted genomic DNA of Polygonatum as a template to perform PCR amplification with the primer pairs shown in SEQ ID NO.1-2 and SEQ ID NO.3-4;

[0020] Perform DNA-PAGE electrophoresis detection on the PCR products to obtain the electrophoresis pattern of the Polygonatum germplasm to be tested. Compare it with the Marker. The electrophoresis bands among different germplasms show polymorphism, and thus it can be used to identify the germplasm resources of Polygonatum.

[0021] The specific judgment rules are as follows:

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

[0023] (1) If two bands with sizes of 290bp and 220bp appear, it is Polygonatum kingianum Coll. et Hemsl. var. wangianum (Sun) Tsai et Tseng from Guizhou;

[0024] (2) If two bands with sizes of 255bp and 480bp appear, it is Polygonatum cyrtonema Hua var. wangianum (Sun) Tsai et Tseng from Guizhou;

[0025] (3) If only one band with a size of 235bp appears, it is Polygonatum verticillatum (L.) All. var. ussuriense (Regel) Y. C. Tang et K. Y. Lang from Northeast China;

[0026] (4) If two bands with sizes of 230bp and 290bp appear, it is Polygonatum kingianum Coll. et Hemsl. from Yunnan;

[0027] (5) If two bands with sizes of 232bp and 290bp appear, it is Polygonatum kingianum Coll. et Hemsl. var. wangianum (Sun) Tsai et Tseng from Guangxi;

[0028] (6) If three bands with sizes of 590bp, 480bp, and 400bp appear, it is Polygonatum verticillatum (L.) All. var. ussuriense (Regel) Y. C. Tang et K. Y. Lang from Northeast China;

[0029] (7) If two bands with sizes of 240bp and 238bp appear, it is Polygonatum sibiricum Delar. ex Redoute var. giraldii (Loes.) Y. C. Tang et Wang from Shaanxi;

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

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

[0032] (10) If a band with a size of 250bp appears, it is Polygonatum odoratum (Mill.) Druce from Hunan;

[0033] (11) If three bands with sizes of 250bp, 200bp, and 140bp appear, it is Polygonatum cyrtonema Hua var. purpureum S. C. Chen from Fujian;

[0034] (12) If a relatively faint band with a size of 298bp appears, it is Polygonatum cyrtonema Hua from Anhui.

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

[0036] (1) If two bands with sizes of 280bp and 150bp appear, it is Polygonatum kingianum Coll. et Hemsl. from Guizhou;

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

[0038] (3) If a band with a size of 210bp appears, it is Polygonatum verticillatum (L.) All. from Northeast China;

[0039] (4) If there is no obvious band, it is Polygonatum kingianum Coll. et Hemsl. from Yunnan;

[0040] (5) If three bands with sizes of 500bp, 180bp, and 150bp appear, it is Polygonatum kingianum Coll. et Hemsl. from Guangxi;

[0041] (6) If two bands with sizes of 152bp and 150bp appear, it is Polygonatum verticillatum (L.) All. from Northeast China;

[0042] (7) If two bands with sizes of 250bp and 200bp appear, it is Polygonatum cirrhifolium (Wall.) Royle var. wangii (Sun) Hoo & Tsai from Shaanxi;

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

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

[0045] (10) If two bands with sizes of 198bp and 150bp appear, it is Polygonatum odoratum (Mill.) Druce from Hunan;

[0046] (11) If four bands of sizes 270bp, 250bp, 200bp, and 160bp appear, it is Polygonatum cyrtonema Hua var. purpureum S. C. Chen from Fujian.

[0047] (12) If two bands of sizes 215bp and 150bp appear, it is Polygonatum cyrtonema Hua from Anhui.

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

[0049] Furthermore, the reaction program 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, taking 10 μL as an example, the PCR amplification system includes: 5 μL of Taq PCR MasterMix, 0.2 μL of 10 μmol / L upstream and downstream primers, 1 μL of 25 ng / μL genomic DNA, and the reaction system is made up to volume with deionized water.

[0051] In the fifth aspect of the present invention, there is provided the use of the SSR molecular marker primer set related to the identification of Polygonatum germplasm resources, or 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 Polygonatum genus in the present invention have strong cross-species transfer ability within related species such as Polygonatum kingianum Coll. et Hemsl., Polygonatum cyrtonema Hua, Polygonatum verticillatum (L.) All., Polygonatum cirrhifolium (Wall.) Royle, and Polygonatum sibiricum Delar. ex Redoute. Therefore, the SSR molecular markers provided by the present invention can be applied to research such as genetic diversity analysis, genetic structure analysis, comparative transcriptomics, genetic linkage map construction, localization and cloning of key genes for medicinal components, and molecular marker-assisted breeding of other species in the Polygonatum genus.

[0054] 2. The SSR molecular markers provided by the present invention are simple to operate, have good reproducibility, and high result reliability, and can distinguish different germplasms of Polygonatum plants, providing a basis for germplasm identification and innovative breeding of Polygonatum. Description of the Drawings

[0055] Figure 1Amplification results and electrophoresis results of 12 Polygonatum germplasm DNAs with primer S10. Among them, M is the marker, 1, Polygonatum kingianum (Guizhou); 2, Polygonatum cyrtonema (Guizhou); 3, Polygonatum verticillatum (Northeast); 4, Polygonatum kingianum (Yunnan); 5, Polygonatum kingianum (Guangxi); 6, Polygonatum kingianum var. purpureopunctatum (Guangxi); 7, Polygonatum sibiricum (Shaanxi); 8, Polygonatum cyrtonema (Hunan); 9, Polygonatum cyrtonema (Sichuan); 10, Polygonatum odoratum (Hunan); 11, Polygonatum cyrtonema var. thomsonii (Fujian); 12, Polygonatum cyrtonema (Anhui).

[0056] Figure 2 Amplification results and electrophoresis results of 12 Polygonatum germplasm DNAs with primer S18. Among them, M is the marker, 1, Polygonatum kingianum (Guizhou); 2, Polygonatum cyrtonema (Guizhou); 3, Polygonatum verticillatum (Northeast); 4, Polygonatum kingianum (Yunnan); 5, Polygonatum kingianum (Guangxi); 6, Polygonatum kingianum var. purpureopunctatum (Guangxi); 7, Polygonatum sibiricum (Shaanxi); 8, Polygonatum cyrtonema (Hunan); 9, Polygonatum cyrtonema (Sichuan); 10, Polygonatum odoratum (Hunan); 11, Polygonatum cyrtonema var. thomsonii (Fujian); 12, Polygonatum cyrtonema (Anhui). Specific embodiments

[0057] The present invention will be specifically described below in combination with specific embodiments and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present invention, rather than limiting the present invention.

[0058] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention belongs. In case of conflict, this specification shall prevail.

[0059] Unless otherwise specifically stated, 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 in combination with examples and experimental data.

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

[0062] S1: PCR primer design and synthesis:

[0063] Analyze the sequence of the transcriptome sequencing and assembly results of different germplasm Polygonatum, search for SSR tandem repeat sequences, and design primers using Primer 5.0 software.

[0064] S2: Extract genomic DNA of Polygonatum germplasm by CTAB method:

[0065] (1) Select the fresh leaves of 12 Polygonatum sibiricum germplasms shown in Table 2 into a mortar, add 2 mL of CTAB extraction solution and an appropriate amount of quartz sand, thoroughly grind the leaves, and use a pipette to aspirate about 1 mL of the ground solution into a sterilized 1.5 mL centrifuge tube. Place it in a water bath at 65 °C for 10 min, centrifuge at 8000 xg at room temperature for 5 min, aspirate about 600 μL of the supernatant into a newly numbered 1.5 mL centrifuge tube, in a fume hood, add 600 μL of chloroform, mix well for 5 min, centrifuge at 8000 xg at room temperature for 5 min, in the fume hood, aspirate about 500 μL of the supernatant into a new 1.5 mL centrifuge tube, add 700 μL of pre-cooled absolute ethanol, mix well, let it stand at -20 °C for 30 min, centrifuge at 8000 xg at room temperature for 5 min. At this time, a white precipitate can be seen, which is DNA. Pour out the supernatant, add 1 mL of 75% ethanol, gently flick the centrifuge tube wall to wash the DNA, centrifuge at room temperature for 2 min, pour out the supernatant, open the lid for 5 min. After the 75% ethanol has completely evaporated, add 200 μL of TE solution. After the DNA has completely dissolved, store it at -20 °C;

[0066] (2) Quality detection of the genomic DNA of Polygonatum sibiricum germplasm. Weigh 0.25 g of agarose powder and add 25 mL of 1x TAE solution to a conical flask, heat it in a microwave oven until the solution boils about three times (the agarose powder melts), cool it to 60 °C (as long as it is not hot to the touch), add 1 μL of nucleic acid staining solution, 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 let it 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 solution of the electrophoresis tank. Use a pipette to add 5 μL of 1 kb DNA Ladder to the first well of the gel. Take another clean disposable glove, use a pipette to aspirate 1 μL of DNA Loading Buffer, change the pipette tip and aspirate 4 μL of the sample, mix it evenly with the DNA Loading Buffer and then add it to the sample well after the Marker. Electrophoresis at 220 V and 150 mA. When the band of 1 kb DNA Ladder runs to two-thirds of the gel, the gel can be taken out, placed on the gel imaging system's gel plate, and the results are photographed and recorded.

[0067] S3: PCR amplification: The total volume of the amplification reaction is 10 μL: 5 μL of 2×TaqMasterMix, 0.3 μL of the forward primer, 0.3 μL of the reverse primer, 1 μL of the DNA template, 3.4 μL of ddH2O. The PCR amplification reaction program 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, extension at 72 °C for 10 min. The PCR products are subjected to DNA-PAGE electrophoresis and then silver-stained, and the results are photographed and saved after color development.

[0068] S4: DNA-PAGE electrophoresis:

[0069] Preparation of agarose bottom gel: Wash the glass plate repeatedly with distilled water, wipe it with alcohol, dry it, and then assemble the glass plate. Weigh 0.2 g of agarose and 20 ml of TAE water into a beaker, heat it in a microwave oven for 2 min until completely dissolved. Add about 3 mL of agarose bottom gel to each rubber bottom plate, place the glass plate on it while it is hot so that the agarose bottom gel evenly fills its bottom, and 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 to a conical flask, mix well, and then quickly pour it into the gap between the glass plates with a glass rod to prevent the generation of bubbles. After filling the gel, insert a 1 mm×40 gel comb, wipe off the overflowing gel. After about 50 min, wait for the gel to solidify, then pull out the gel comb and assemble the electrophoresis tank.

[0071] Gel electrophoresis: Pour 1×TBE electrophoresis buffer into the electrophoresis tank, place the gel in the electrophoresis tank, pour 1×TBE electrophoresis buffer over the upper part of the gel in the electrophoresis tank. Add 2 μL of Marker to the first or the first two sample wells, and add 3 μL of PCR product to each of the remaining wells. Connect the power supply, with a voltage of 200 V and a current of 200 mA. The electrophoresis time is determined according to 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, take out the gel and put it into distilled water, wash it once with distilled water, pour out the distilled water, and put it into a pre-prepared 250 mL silver nitrate solution (0.35 g of silver nitrate, 250 mL of pure water). Gently shake it on a shaker for 10 min, then 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 gel.

[0073] Color development and recording: Place the washed gel on a shaker, add 250 mL of sodium hydroxide solution (2 g of sodium hydroxide, 250 mL of pure water) and 2.5 mL of formaldehyde solution, gently shake until the bands are clear. After color development is completed, wash away the residual color development solution with distilled water, lay the gel flat under a fluorescent light box, and take a photo for preservation.

[0074] After re-screening, finally obtain 2 pairs of SSR primer combinations with clear, stable bands, good repeatability, and high polymorphism. Then use 12 Polygonatum sibiricum germplasms to verify the SSR primer combinations. The sequences of the 2 pairs of SSR primer combinations are shown in Table 1.

[0075] The sequences of the SSR primer combinations for rapid identification of Polygonatum sibiricum germplasm resources in this application are shown in Table 1.

[0076] Table 1

[0077]

[0078] The polygonatum germplasm is shown in Table 2 as follows.

[0079] Table 2

[0080]

[0081]

[0082] Example 2: A method for identifying polygonatum germplasm

[0083] Extracting genomic DNA of the polygonatum germplasm to be tested;

[0084] Using the genomic DNA of the extracted polygonatum germplasm as a template, performing PCR amplification on it with the primer combination described in Example 1;

[0085] Performing DNA-PAGE electrophoresis detection on the PCR product. The reaction system for PCR amplification includes: 5 μL of 2×Taq Master Mix, 0.3 μL of forward label, 0.3 μL of reverse label, 1 μL of DNA template, 3.4 μL of ddH2O. The PCR amplification reaction program 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, extension at 72°C for 10 min, and storing at -20°C after completion.

[0086] Obtaining the electrophoresis pattern of the polygonatum germplasm to be tested, comparing it with the Marker, and the electrophoresis bands among different germplasms show polymorphism, thereby being used to identify polygonatum germplasm resources.

[0087] Specifically manifested as:

[0088] At Figure 1Among them, the target product size of primer S10 (i.e., primer pair 1 of SSR1: the nucleotide sequences are shown in SEQ ID NO.1-2) is 292bp, and it is the target band (located between 200bp - 300bp) after comparison with the Marker. Different Polygonatum germplasms in the figure showed obvious polymorphisms after amplification with primer S10. 1 and 2 are Polygonatum kingianum and Polygonatum cyrtonema from Guizhou respectively, showing two bands of different heights; 3 is Polygonatum verticillatum from Northeast China with only 1 band; 4 and 5 are Polygonatum kingianum from Yunnan and Polygonatum kingianum from Guangxi, and their bands are similar to those of Polygonatum kingianum from Guizhou; 6 produced 3 relatively faint bands in the range of 400bp - 600bp; 7 is two relatively close bands; 8 and 11 are Polygonatum cyrtonema from different producing areas, and the bands produced by their products after electrophoresis are polymorphic with other species; 9 and 12 are Polygonatum cyrtonema from Sichuan and Anhui without products (bands), and 12 only has a relatively faint stained band, which is significantly different from other germplasms. Different Polygonatum germplasm resources can be distinguished from the number and size of the bands after electrophoresis of PCR products.

[0089] In Figure 2 Among them, the target product size of primer S18 is 114bp, and it is the target band (located between 100bp - 250bp) after comparison with the Marker. Different Polygonatum germplasms in the figure showed obvious polymorphisms after amplification with primer S18. 1 and 2 are Polygonatum kingianum and Polygonatum cyrtonema from Guizhou respectively, showing bands of different heights and numbers; 3 is Polygonatum verticillatum from Northeast China with only 1 band, and it is significantly different from other germplasms; 4 is Polygonatum kingianum from Yunnan without products and bands; 5 and 6 are Polygonatum kingianum from Guangxi and Polygonatum cirrhifolium var. wangianum, and their bands are significantly different; 7 is Polygonatum sibiricum, only producing two bands, and it is completely different from other germplasms; 8 and 9 are both Polygonatum cyrtonema, and the bands produced by their products after electrophoresis are similar, but they are polymorphic compared with other species; 11 and 12 are Polygonatum cyrtonema from Fujian and Anhui, and their products (bands) are significantly different and polymorphic with other germplasms. Different Polygonatum germplasm resources can be distinguished from the number and size of the bands after electrophoresis of PCR products.

[0090] Example 3. Effect Identification

[0091] The method shown in Example 2 was used for the experiment, and the results are as follows. Sequencing was used for verification, and the correct rate of the determination result was 100%.

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

[0093] Material / strip type 1 2 3 4 5 6 7 8 9 10 11 12 Flos Lonicerae (Suining) + + Dian (Wenshan) + + + Jitou (Shangluo) + Verticillate + + Flos Lonicerae (Nanchong) + + Flos Lonicerae (Hunan) + Flos Lonicerae (Hubei) + Dian (Baise) + + Dian (Pu'er) + Dian (Nanning) + + Jitou (Luoyang) + Dian (Guizhou) + +

[0094] Note: + indicates presenting positive

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

[0096] Material / strip type 1 2 3 4 5 6 7 8 9 10 11 12 Flos Lonicerae (Suining) + + Dian (Wenshan) + Jitou (Shangluo) + Verticillate + Flos Lonicerae (Nanchong) + + Flos Lonicerae (Hunan) + + Flos Lonicerae (Hubei) + Dian (Baise) + Dian (Pu'er) + Dian (Nanning) + Jitou (Luoyang) + Dian (Guizhou) +

[0097] Note: + indicates a positive result

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

[0099] Finally, it should also be noted that the terms "comprise", "include" or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements but also other elements not expressly listed, or elements inherent to such process, method, article or device.

[0100] Although the preferred embodiments of the embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling 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 equivalent technologies, the embodiments of the present invention are also intended to include these changes and modifications.

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: The nucleotide sequences are as shown in SEQ ID NO.1-2; Primer pair 2: The nucleotide sequences are as shown in SEQ ID NO.3-4; The rules for identifying the germplasm resources of the Polygonatum genus using the primer pairs are as follows: If PCR amplification is performed using the primer pair shown in SEQ ID NO.1-2, (1) If two bands with sizes of 290bp and 220bp appear, it is Polygonatum kingianum Coll. et Hemsl. var. wangianum (Sun) Hoo & Tsai from Guizhou; (2) If two bands with sizes of 255bp and 480bp appear, it is Polygonatum cyrtonema Hua from Guizhou; (3) If only one band with a size of 235bp appears, it is Polygonatum verticillatum (L.) All. var. ussuriense (Regel) Kitag. from Northeast China; (4) If two bands with sizes of 230bp and 290bp appear, it is Polygonatum kingianum Coll. et Hemsl. from Yunnan; (5) If two bands with sizes of 232bp and 290bp appear, it is Polygonatum kingianum Coll. et Hemsl. from Guangxi; (6) If three bands with sizes of 590bp, 480bp, and 400bp appear, it is Polygonatum verticillatum (L.) All. var. ussuriense (Regel) Kitag. from Northeast China; (7) If two bands with sizes of 240bp and 238bp appear, it is Polygonatum cirrhifolium (Wall.) Royle var. sutchuenense (Franch.) Y. C. Tang & Tsai from Shaanxi; (8) If two bands with sizes of 250bp and 300bp appear, it is Polygonatum cyrtonema Hua from Hunan; (9) If no obvious bands appear, it is Polygonatum cyrtonema Hua from Sichuan; (10) If one band with a size of 250bp appears, it is Polygonatum odoratum (Mill.) Druce var. pluriflorum (Miq.) Ohwi from Hunan; (11) If three bands with sizes of 250bp, 200bp, and 140bp appear, it is Polygonatum cyrtonema Hua var. purpureum S. C. Chen from Fujian; (12) If one relatively faint band with a size of 298bp appears, it is Polygonatum cyrtonema Hua from Anhui; If PCR amplification is performed using the primer pair shown in SEQ ID NO.3-4, (1) If two bands with sizes of 280bp and 150bp appear, it is Polygonatum kingianum Coll. et Hemsl. var. wangianum (Sun) Hoo & Tsai from Guizhou; (2) If four bands with sizes of 250bp, 200bp, 145bp, and 100bp appear, it is Polygonatum cyrtonema Hua from Guizhou; (3) If one band with a size of 210bp appears, it is Polygonatum verticillatum (L.) All. var. ussuriense (Regel) Kitag. from Northeast China; (4) If no obvious bands appear, it is Polygonatum kingianum Coll. et Hemsl. from Yunnan; (5) If three bands with sizes of 500bp, 180bp, and 150bp appear, it is Polygonatum kingianum Coll. et Hemsl. from Guangxi; (6) If two bands with sizes of 152bp and 150bp appear, it is Polygonatum verticillatum (L.) All. var. ussuriense (Regel) Kitag. from Northeast China; (7) If two bands with sizes of 250bp and 200bp appear, it is Polygonatum cirrhifolium (Wall.) Royle var. sutchuenense (Franch.) Y. C. Tang & Tsai from Shaanxi; (8) If four bands with sizes of 240bp, 238bp, 200bp, and 150bp appear, it is Polygonatum cyrtonema Hua from Hunan; (9) If four bands with sizes of 240bp, 238bp, 200bp, and 150bp appear, it is Polygonatum cyrtonema Hua from Sichuan; (10) If two bands with sizes of 198bp and 150bp appear, it is Polygonatum odoratum (Mill.) Druce var. pluriflorum (Miq.) Ohwi from Hunan; (11) If four bands with sizes of 270bp, 250bp, 200bp, and 160bp appear, it is Polygonatum cyrtonema Hua var. purpureum S. C. Chen from Fujian; (12) If two bands with sizes of 215 bp and 150 bp appear, it is Polygonatum cyrtonema Hua from Anhui.

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

3. A method for identifying germplasm resources of the genus Polygonatum, characterized in that, The method includes: Extracting genomic DNA of Polygonatum from different germplasm sources; Performing PCR amplification on the genomic DNA using the primer set described in claim 1 to obtain a PCR amplification product; Performing PAGE gel electrophoresis on the PCR amplification product and analyzing the results to achieve the identification of Polygonatum germplasm resources; The rules for identifying Polygonatum germplasm resources using the primer pair are as follows: If PCR amplification is performed using the primer pair shown in SEQ ID NO.1 - 2, (1) If two bands with sizes of 290 bp and 220 bp appear, it is Polygonatum kingianum Coll. et Hemsl. from Guizhou; (2) If two bands with sizes of 255 bp and 480 bp appear, it is Polygonatum cyrtonema Hua from Guizhou; (3) If only one band with a size of 235 bp appears, it is Polygonatum verticillatum (L.) All. from Northeast China; (4) If two bands with sizes of 230 bp and 290 bp appear, it is Polygonatum kingianum Coll. et Hemsl. from Yunnan; (5) If two bands with sizes of 232 bp and 290 bp appear, it is Polygonatum kingianum Coll. et Hemsl. from Guangxi; (6) If three bands with sizes of 590 bp, 480 bp, and 400 bp appear, it is Polygonatum verticillatum (L.) All. from Northeast China; (7) If two bands with sizes of 240 bp and 238 bp appear, it is Polygonatum cirrhifolium (Wall.) Royle from Shaanxi; (8) If two bands with sizes of 250 bp and 300 bp appear, it is Polygonatum cyrtonema Hua from Hunan; (9) If no obvious band appears, it is Polygonatum cyrtonema Hua from Sichuan; (10) If one band with a size of 250 bp appears, it is Polygonatum odoratum (Mill.) Druce var. pluriflorum (Miq.) Ohwi from Hunan; (11) If three bands with sizes of 250 bp, 200 bp, and 140 bp appear, it is Polygonatum cyrtonema Hua var. purpureum Rendle from Fujian; (12) If one relatively faint band with a size of 298 bp appears, it is Polygonatum cyrtonema Hua from Anhui; If PCR amplification is performed using the primer pair shown in SEQ ID NO.3 - 4, (1) If two bands with sizes of 280 bp and 150 bp appear, it is Polygonatum kingianum Coll. et Hemsl. from Guizhou; (2) If four bands with sizes of 250 bp, 200 bp, 145 bp, and 100 bp appear, it is Polygonatum cyrtonema Hua from Guizhou; (3) If one band with a size of 210 bp appears, it is Polygonatum verticillatum (L.) All. from Northeast China; (4) If no obvious band appears, it is Polygonatum kingianum Coll. et Hemsl. from Yunnan; (5) If three bands with sizes of 500 bp, 180 bp, and 150 bp appear, it is Polygonatum kingianum Coll. et Hemsl. from Guangxi; (6) If two bands with sizes of 152 bp and 150 bp appear, it is Polygonatum verticillatum (L.) All. from Northeast China; (7) If two bands with sizes of 250 bp and 200 bp appear, it is Polygonatum cirrhifolium (Wall.) Royle from Shaanxi; (8) If four bands with sizes of 240 bp, 238 bp, 200 bp, and 150 bp appear, it is Polygonatum cyrtonema Hua from Hunan; (9) If four bands with sizes of 240 bp, 238 bp, 200 bp, and 150 bp appear, it is Polygonatum cyrtonema Hua from Sichuan; (10) If two bands with sizes of 198 bp and 150 bp appear, it is Polygonatum odoratum (Mill.) Druce var. pluriflorum (Miq.) Ohwi from Hunan; (11) If four bands with sizes of 270 bp, 250 bp, 200 bp and 160 bp appear, it is Polygonatum cyrtonema Hua var. purpureum S. C. Chen from Fujian; (12) If two bands with sizes of 215 bp and 150 bp appear, it is Polygonatum cyrtonema Hua from Anhui.

4. The method for identifying Polygonatum germplasm resources according to claim 3, characterized in that, In the said PCR amplification, the annealing temperature range of the primer set is 53 - 63 °C.

5. The method for identifying Polygonatum germplasm resources according to claim 3, characterized in that, The reaction program of the said 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.

6. The method for identifying Polygonatum germplasm resources according to claim 3, characterized in that, Taking 10 μL as an example, the said PCR amplification system includes: 5 μL of 2×Taq Master Mix, 0.3 μL of forward label, 0.3 μL of reverse label, 1 μL of DNA template, and 3.4 μL of ddH2O.

7. Use of the SSR molecular marker primer set related to the identification of Polygonatum germplasm resources according to claim 1 or the kit for identifying Polygonatum germplasm resources according to claim 2 in the identification of Polygonatum germplasm resources.

Images