Polygonatum IRAP marker sequence based on retrotransposon sequence, kit and use thereof

Abstract

The present invention discloses a polygonatum IRAP marker sequence, a kit and a use thereof based on a retrotransposon sequence, including identification of polygonatum germplasm by screening 17 IRAP marker primers. The identification method comprises extracting genomic DNA, designing IRAP primers based on the RT sequences of the polygonatum retrotransposons Ty1‑copia and Ty3‑gypsy, selecting DNA of different germplasms to perform PCR reaction amplification, gel electrophoresis screening and polymorphism analysis on the IRAP primers according to the PCR reaction system and PCR reaction program. The present invention develops an IRAP marker based on the polygonatum retrotransposon sequence, which is easy to operate, fast, has stable results, high efficiency, is not easily affected by the environment, and can be used in the identification of polygonatum germplasm, genetic diversity evaluation, species kinship analysis and other studies in various regions, providing an effective means for excellent germplasm mining, assisted breeding and protection of germplasm property rights.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology, and in particular to a method for identifying polygonatum germplasm at the DNA molecular level, and involves the development of IRAP primers, PCR amplification and the application of IRAP markers. The IRAP primers developed based on the retrotransposon sequence are specifically used for rapid identification and kinship analysis of polygonatum germplasm at the DNA level. Background Art

[0002] Polygonatum sibiricum, also known as pendant, wild ginger, sun grass, etc., is a perennial herb known as the "king of blood and qi supplements". Polygonatum sibiricum contains polysaccharides, alkaloids, saponins and other compounds, which can resist fatty liver, protect kidneys, and resist Alzheimer's disease.

[0003] The identification methods of Polygonatum cyrtonema mainly include morphological markers, cytological markers, biochemical markers and molecular markers. Among them, molecular markers make up for the shortcomings of the other three identification methods with their advantages such as rapidity, no restrictions on seasons and environments, and wide application range. Molecular markers such as ISSR, RAPD, and SCAR have been widely used in the research fields of genetic diversity analysis, germplasm identification, and phylogeny of Polygonatum cyrtonema.

[0004] For example, in Chinese patent literature, the invention patent with publication number CN114164290A discloses a method for ISSR-PCR molecular markers of polygonatum for rapid identification of polygonatum germplasm resources. Ye Bihuan and other scholars proposed the SCAR molecular identification method of long-pedunculated polygonatum, which was based on simple sequence repeat interval amplification polymorphism (ISSR) and related sequence amplification polymorphism (SRAP) molecular markers to perform interspecific polymorphism analysis on the genomic DNA of Polygonatum multiflorum and long-pedunculated polygonatum, obtain ISSR and SRAP-PCR differential fragments, and design species-specific characteristic fragment amplification region (SCAR) primers after sequencing, which were used for accurate and rapid identification of the two species of Polygonatum ("SCAR molecular identification of long-pedunculated polygonatum", Ye Bihuan et al., Chinese Journal of Pharmaceutical Sciences, 2019).

[0005] However, the above molecular markers still have some disadvantages, such as: ISSR markers are Mendelian in inheritance, and it is difficult to distinguish between dominant homozygous and heterozygous genotypes; RAPD markers have dominant inheritance problems, cannot distinguish between homozygous and heterozygous types, and are easily affected by temperature, resulting in poor stability and repeatability of the test; SCAR markers have problems such as a single amplification site, a high annealing temperature, and insensitivity to reaction conditions.

[0006] Retrotransposons are a type of mobile genetic factor that is widely present in the genomes of eukaryotic organisms. They use RNA as an intermediate and adopt a "copy-paste" mechanism for transposition. There are two main types of retrotransposons, one is LTR and the other is non-LTR. Among them, LTR can be divided into two groups, Ty1-copia and Ty3-gypsy. The main difference between them is the different positions of RT in their structural domains.

[0007] LTR retrotransposons have the characteristics of universality, rich copy number, and polymorphic insertion sites, so they can be used to develop molecular markers such as IRAP, REMAP, and SSAP. Among them, IRAP markers have the advantages of high throughput, wide genome coverage, and rich polymorphism, and have been applied to genetic diversity analysis, germplasm identification, and genetic linkage map construction. However, no technical solution for using IRAP markers for Polygonatum cyrtonema germplasm identification has been disclosed. Summary of the invention

[0008] The purpose of the present invention is to provide a polygonatum IRAP marker sequence based on a retrotransposon sequence, a kit and its use, and a method for identifying polygonatum germplasm related thereto. The present invention develops an IRAP marker based on a polygonatum retrotransposon sequence, uses the marker to identify polygonatum germplasm, and provides services for the identification, preservation, and new germplasm mining of excellent polygonatum germplasm.

[0009] Based on the first main aspect of the present invention, an IRAP marker primer for retrotransposon sequence for identification of Polygonatum cyrtonema germplasm is provided, wherein the IRAP marker primer comprises one or more specific primers listed in Table 1 below:

[0010] Table 1 IRAP primer sequence list

[0011]

[0012] According to the second main aspect of the present invention, a method for identifying IRAP-labeled polygonatum germplasm based on retrotransposon sequences is provided, the method comprising at least the following steps:

[0013] S1, extraction of Polygonatum sibiricum genomic DNA;

[0014] S2, IRAP primers were designed based on the RT sequences of Ty1-copia and Ty3-gypsy retrotransposons of Polygonatum cyrtonema;

[0015] S3, selecting DNA from different germplasms and performing PCR amplification with IRAP primers according to the PCR reaction system and PCR reaction procedure;

[0016] S4, preliminarily screening the amplified IRAP primers by agarose gel electrophoresis, and selecting IRAP primers with good polymorphism and clear bands as IRAP marker primers for subsequent steps;

[0017] S5, select the IRAP marker primers that have been initially screened to perform IRAP marker analysis on all test materials, and determine the results based on the presence, number and size of the amplified bands.

[0018] In the above steps, the IRAP marker primers preliminarily screened in step S5 are selected to perform PCR amplification on the test material germplasm according to the same PCR reaction system and PCR reaction procedure as step S3. The experiment is repeated 3 times for each IRAP primer, and 1.5% agarose gel electrophoresis is used to detect the PCR reaction products.

[0019] As a further preferred embodiment, in step S3 and step S5, the PCR reaction system is a 10 μL system: 0.6 μL template DNA, 1.0 μL primer, 6.0 μL PCR Mix and 2.4 μL ddH2O. The PCR reaction program is: 94°C pre-denaturation for 4 min, 94°C denaturation for 30 s, 57°C annealing for 30 s, 72°C extension for 1 min, a total of 40 cycles; 72°C reaction for another 10 min, and storage at 4°C.

[0020] As a preferred solution, the process of designing IRAP primers based on the RT sequences of Ty1-copia and Ty3-gypsy of Polygonatum cyrtonema retrotransposon includes the following steps:

[0021] Firstly, the sequence information of Ty1-copia and Ty3-gypsy of Polygonatum cyrtonema retrotransposon was obtained by cloning and sequencing the RT sequence of LTR retrotransposon of Polygonatum cyrtonema.

[0022] Use sequence alignment software, such as BLAST, to determine the conserved regions of these transposon sequences. Through multiple sequence alignment, identify the conserved regions in the Ty1-copia and Ty3-gypsy sequences, which may serve as candidate sites for designing primers.

[0023] Then, use primer design software such as Primer3 or Oligo to design IRAP primers based on the conserved sequence, taking into account the Tm value of the primers, the probability of dimer formation, the specificity of the primer pair, and the coverage.

[0024] Finally, the specificity and amplification efficiency of the primers were verified by PCR experiments using in vitro synthesized DNA templates or genomic DNA. IRAP-PCR amplification was performed on different Polygonatum germplasm samples to screen out primer combinations with good polymorphism.

[0025] As a possible alternative, high-throughput sequencing technologies (such as Illumina or Nanopore) can be used to sequence the amplified fragments to improve the accuracy and resolution of identification. Alternatively, the CRISPR-Cas system can be used to edit specific transposon sequences to verify their function and role in the Polygonatum cyrtonema genome.

[0026] In some embodiments, it includes developing or using advanced bioinformatics analysis tools, such as machine learning algorithms, to predict and explain the polymorphism patterns of transposon sequences. Experimentally optimize the reaction conditions of IRAP-PCR, such as Mg2+ concentration, annealing temperature, and number of cycles, to improve amplification efficiency and specificity. As a further preferred embodiment, it also includes collecting and analyzing the IRAP-PCR results of different Polygonatum germplasms, establishing a database containing standard band patterns for subsequent germplasm identification and comparison; and integrating IRAP-PCR data with other molecular marker data, using multivariate statistical analysis methods, such as principal component analysis (PCA) or cluster analysis, to reveal the genetic relationship between Polygonatum germplasms.

[0027] As a third main aspect of the present invention, a kit for performing the aforementioned method is provided, the kit comprising at least one or more primers among the IRAP labeled primers, and reagents suitable for IRAP-PCR reaction.

[0028] In some embodiments, the kit comprises one or more of the IRAP labeling primers; and

[0029] PCRMix suitable for IRAP-PCR reactions; and

[0030] ddH2O for DNA amplification.

[0031] As a further preferred embodiment, the PCRMix suitable for IRAP-PCR reaction comprises one or more combinations of the following components: DNA polymerase, dNTPs mixture, PCR buffer, and MgCl2.

[0032] Furthermore, the PCR buffer contains appropriate salts, stabilizers and pH adjusters.

[0033] In some embodiments, as a further preferred embodiment, a method for preparing the kit comprises the following steps:

[0034] First, use a high-quality DNA synthesis service to synthesize all specific IRAP-labeled primers based on the sequence information provided. Prepare PCR buffer containing appropriate salt concentrations, pH adjusters, and stabilizers. Prepare a dNTPs mix containing appropriate amounts of the four deoxynucleotide triphosphates. Select an appropriate thermostable DNA polymerase and determine its concentration in the PCRMix according to the manufacturer's instructions. Determine the optimal concentration of MgCl2 and add it to the PCRMix.

[0035] Then dispense PCRMix and ddH2O into suitable containers, such as PCR reaction tubes or microcentrifuge tubes. Make sure the volume of the aliquot is consistent with the requirements of the identification method, such as a 10 μL system.

[0036] The synthetic IRAP-labeled primers are then diluted to the desired final concentration and dispensed into individual tubes or premixed into PCRMix.

[0037] In the above steps, all synthesized primers and prepared PCRMix need to be quality tested to ensure their purity and functionality. Stability tests are performed on the packaged reagents to ensure stability at room temperature and refrigerated conditions.

[0038] Finally, the aliquoted PCR Mix, ddH2O, primers, and instructions are assembled into a kit that is easy to transport and store. The completed kit is subjected to a final quality inspection to ensure that all components meet the standards.

[0039] As a fourth main aspect of the present invention, the use of the above kit is provided, wherein the kit is used for: identification of polygonatum germplasm resources, or analysis of polygonatum genetic diversity; wherein

[0040] The use of identification of Polygonatum cyrtonema germplasm resources includes marking according to the presence or absence of amplified bands, i.e.

[0041] The bands at the same electrophoresis position were marked as "1", and the bands without bands were marked as "0". Then a 0 / 1 data matrix was established, and the polymorphic information content (PIC) was calculated using PowerMarker v3.25 software, and the core primers were determined. The obtained 0 / 1 data matrix was used to construct the germplasm fingerprint code.

[0042] Finally, the electronic ID card of the test germplasm was constructed through the barcode and QR code generator.

[0043] Advantages and beneficial effects of the present invention:

[0044] The present invention develops IRAP markers based on the sequence of the retrotransposon of Polygonatum cyrtonema. The markers are easy to operate, fast, stable, efficient, and not easily affected by the environment. They can be used in the identification of Polygonatum cyrtonema germplasm in various regions, genetic diversity evaluation, species relationship analysis, and other research, providing effective means for excellent germplasm mining, assisted breeding, and germplasm property protection. The key technical problems that the present invention needs to solve are obtaining efficient IRAP primers and establishing a PCR amplification system.

[0045] Specifically, the present invention has at least the following outstanding substantive features and significant improvements compared to the prior art:

[0046] (1) Simple and rapid operation: The present invention uses IRAP primers developed based on the Polygonatum sibiricum retrotransposon sequence to perform PCR amplification and agarose gel electrophoresis on the sample to obtain the test results.

[0047] (2) Accurate and reliable results: The present invention performs detection based on the retrotransposon sequence at the DNA molecular level and detects dozens of Polygonatum cyrtonema germplasms. The results of repeated detections remain consistent and the results are accurate and reliable;

[0048] (3) High sensitivity of the test results: only 30 ng of template DNA is required to accurately identify the germplasm to which the sample belongs;

[0049] (4) Good polymorphism: After testing 10 Polygonatum germplasms, the polymorphism of IRAP primers is generally high, which can effectively identify different germplasms;

[0050] (5) The optimal system of IRAP-PCR for Polygonatum cyrtonema was determined, and IRAP markers were used to identify Polygonatum cyrtonema germplasm economically and efficiently;

[0051] (6) The present invention develops IRAP markers based on the Polygonatum cyrtonema retrotransposon sequence, which can be effectively used in the research of Polygonatum cyrtonema germplasm identification, genetic diversity evaluation, species kinship analysis, etc. in various regions, providing an effective means for the mining of excellent germplasm, auxiliary breeding and protection of germplasm property rights. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0053] Figure 1The results of the preliminary screening of IRAP primers PsRT-5, PsRT-7, PsRT-14 and PsRT-21 by 1.5% agarose gel electrophoresis after PCR amplification in one embodiment of the present invention are shown, where M is a DNA marker, and 1-4 represent four different Polygonatum cyrtonema germplasms respectively;

[0054] Figure 2 The results of 1.5% agarose gel electrophoresis detection after PCR amplification of 19 polygonatum germplasms using IRAP primers PsRT-5, PsRT-7, PsRT-21, and REPs-34 in one embodiment of the present invention are shown, where M is a DNA marker, and 1-19 represent polygonatum samples No. 1-19, respectively. DETAILED DESCRIPTION

[0055] The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings so that the purpose, features and advantages of the present invention can be more clearly understood. It should be understood that the embodiments shown in the accompanying drawings are not intended to limit the scope of the present invention, but are only intended to illustrate the essential spirit of the technical solution of the present invention.

[0056] In the following description, certain specific details are set forth for the purpose of illustrating the various disclosed embodiments to provide a thorough understanding of the various disclosed embodiments. However, those skilled in the relevant art will recognize that the embodiments may be practiced without one or more of these specific details. In other cases, well-known devices, structures, and techniques associated with the present application may not be shown or described in detail to avoid unnecessarily obscuring the description of the embodiments.

[0057] References throughout the specification to "one embodiment" or "an embodiment" indicate that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearance of "in one embodiment" or "in an embodiment" in various places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any manner in one or more embodiments.

[0058] Embodiment 1:

[0059] 1. Sample Collection

[0060] There were 19 germplasms for this experiment, collected from Huaxi District, Guiyang City and Miaowangcheng, Songtao County, Tongren City. Fresh young leaves of each identification material were randomly selected, packed with tin foil and marked. Two copies of the same sample were collected and placed in an ice box with ice bags. One copy was then brought back to the laboratory for testing and the other was stored in a -80℃ refrigerator for later use. Detailed information on the 19 identification materials is shown in Table 2.

[0061] Table 2 Polygonatum germplasm information

[0062]

[0063] 2. Genomic DNA Extraction

[0064] All sample DNA was extracted using the new plant genomic DNA extraction kit (centrifugal column type) from Tiangen Biochemical Technology (Beijing) Co., Ltd. The operation steps were carried out according to the kit instructions. After DNA extraction, 1% agarose gel electrophoresis was used to detect DNA integrity, and the full-wavelength microplate reader Multiskan GO was used to detect its concentration and purity. After the quality was qualified, it was stored in a -20℃ ultra-low temperature refrigerator for use.

[0065] The 1% agarose gel electrophoresis showed that the genomic DNA of Polygonatum cyrtonema leaves had high integrity, the DNA sample bands were clear, the main band was obvious, and there was no DNA degradation.

[0066] Using the full-wavelength microplate reader Multiskan GO for detection, the OD260 / OD280 values ​​of the 19 sample DNAs were all between 1.7-2.0, and the concentrations were between 38ng / μL-122ng / μL. Both the purity and concentration met the PCR amplification requirements for SSR molecular markers.

[0067] 3. IRAP Primer Design and Synthesis

[0068] The sequences obtained by cloning and sequencing the RT sequences of the LTR-type retrotransposon of Polygonatum were analyzed to remove the shorter and repeated sequences. IRAP marker primers were designed using snapgene, and a total of 87 IRAP unidirectional primers were designed. The primers were synthesized by Bioengineering (Shanghai) Co., Ltd.

[0069] 4. Establishment of IRAP-PCR reaction system

[0070] The PCR reaction system was a 10 μL system: 0.6 μL of 50 ng / L template DNA, 1.0 μL of primers, 6.0 μL of PCR Mix and 2.4 μL of ddH2O, and the number of PCR cycles was 40.

[0071] 5. Establishment of IRAP-PCR Reaction Procedure

[0072] The PCR reaction program was as follows: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 1 min, for a total of 40 cycles; finally, reaction at 72°C for 10 min and storage at 4°C.

[0073] 6. Screening of IRAP Primers

[0074] DNA from four germplasms with obvious morphological differences was selected for PCR amplification with 87 primers. The PCR reaction products were detected by 1.5% agarose gel electrophoresis. IRAP primers with good polymorphism and clear bands were selected. The results of some primer screening are as follows Figure 1 .

[0075] Seventeen IRAP primers with good polymorphism and clear bands were screened out, and the specific primer sequences are shown in Table 1.

[0076] 7. IRAP Marker Detection

[0077] 17 IRAP primers were used to perform PCR amplification on 19 germplasms, and the experiment was repeated 3 times for each IRAP primer. The PCR reaction products were detected by 1.5% agarose gel electrophoresis. Some PCR amplification results are shown in Figure 2 .

[0078] 8. Statistics

[0079] The reproducible and clear bands in the three repeated experiments were counted, and the bands at the same electrophoresis position were marked as "1", and the bands without bands were marked as "0", and a 0 / 1 data matrix of 19 germplasms was established.

[0080] 9. Production of electronic ID card

[0081] The polymorphic information content (PIC) was calculated using PowerMarker v3.25 software, and the core primers were determined based on the principle of using the least primers to identify as many test germplasms as possible, as shown in Table 3. After the core primers were determined, the germplasm fingerprint code was constructed using the obtained 0 / 1 data matrix. Finally, the electronic ID card of the test germplasm was constructed using a barcode and QR code generator (http: / / qr-batch.com).

[0082] Table 3 PIC values ​​of primers and number of identified germplasms

[0083]

[0084] It can be seen from Table 3 that the core primer is PsRT-21, and the constructed fingerprint code is shown in Table 4.

[0085] Table 4 Fingerprint codes of 19 Polygonatum cyrtonema germplasms

[0086]

[0087]

[0088] The matters not described in detail in the present invention are all known technologies to those skilled in the art.

[0089] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. The use of IRAP labeled primers in the preparation of a kit for the identification of Polygonatum cyrtonema germplasm, characterized in that: The IRAP marker primer is selected from any one of the following primers: Primer PsRT-21, sequence 5′-TCTACATGCACCAACCAGTGG-3′; Primer REPs-34, sequence: 5′-GGAGCGAGTGTCTTCTCAAA-3′.

2. A method for identifying Polygonatum cyrtonema germplasm using IRAP markers based on retrotransposon sequences, characterized in that: The method comprises at least the following steps: S1, extraction of Polygonatum sibiricum genomic DNA; S2, based on the Ty1- copia and Ty3- gyps y RT sequence to design IRAP primers; S3, selecting DNA from different germplasms and performing PCR amplification with IRAP primers according to the PCR reaction system and PCR reaction procedure; S4, preliminarily screening the amplified IRAP primers by agarose gel electrophoresis, and selecting IRAP primers with good polymorphism and clear bands as IRAP marker primers for subsequent steps; the IRAP marker primer is selected from any one of the following primers: Primer PsRT-21, sequence 5′-TCTACATGCACCAACCAGTGG-3′; Primer REPs-34, sequence 5′-GGAGCGAGTGTCTTCTCAAA-3′; S5, select the IRAP marker primers that have been initially screened to perform IRAP marker analysis on all test materials, and determine the results based on the presence, number and size of the amplified bands.

3. The method for identifying Polygonatum cyrtonema germplasm using IRAP markers based on retrotransposon sequences according to claim 2, characterized in that: In step S5, the IRAP marker primers preliminarily screened are selected to perform PCR amplification on the test material germplasm according to the same PCR reaction system and PCR reaction procedure as in step S3. The experiment is repeated 3 times for each IRAP primer, and the PCR reaction products are detected by 1.5% agarose gel electrophoresis.

4. The method for identifying polygonatum germplasm using IRAP markers based on retrotransposon sequences according to claim 2 or 3, characterized in that: The PCR reaction system is a 10 μL system: 0.6 μL template DNA, 1.0 μL primer, 6.0 μL PCR Mix and 2.4 μL ddH2O.

5. The method for identifying polygonatum germplasm using IRAP markers based on retrotransposon sequences according to claim 2 or 3, characterized in that: The PCR reaction procedure is: pre-denaturation at 94°C for 4 minutes, denaturation at 94°C for 30 seconds, annealing at 57°C for 30 seconds, extension at 72°C for 1 minute, a total of 40 cycles; reaction at 72°C for another 10 minutes, and storage at 4°C.

6. A use of a kit, characterized in that: The kit is used for: identification of polygonatum germplasm resources, or analysis of polygonatum genetic diversity; wherein in the use of identification of polygonatum germplasm resources, it includes marking according to the presence or absence of amplified bands, that is, marking the presence of bands at the same electrophoresis position as "1", and marking the absence of bands as "0", then establishing a 0 / 1 data matrix, calculating the polymorphic information content PIC using software, determining the core primers, and constructing a germplasm fingerprint code using the obtained 0 / 1 data matrix; Finally, the electronic ID card of the tested Polygonatum cyrtonema germplasm was constructed through the barcode and QR code generator; The kit comprises an IRAP labeled primer, and the IRAP labeled primer is selected from any one of the following primers: Primer PsRT-21, sequence 5′-TCTACATGCACCAACCAGTGG-3′; Primer REPs-34, sequence: 5′-GGAGCGAGTGTCTTCTCAAA-3′.

7. The use of the kit according to claim 6, characterized in that: The kit contains PCR Mix suitable for IRAP-PCR reaction; and ddH2O for DNA amplification.

8. The use of the kit according to claim 7, characterized in that: The PCRMix suitable for IRAP-PCR reaction includes the following components: DNA polymerase, dNTPs mixture, PCR buffer and MgCl2.

9. The use of the kit according to claim 8, characterized in that: The PCR buffer contains appropriate salts, stabilizers and pH adjusters.

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