A CDDP molecular marker primer set for identifying Polygonatum germplasm resources and its application

Abstract

The present invention belongs to the technical field of plant germplasm resource identification, and specifically relates to a CDDP molecular marker primer set for identifying Polygonatum germplasm resources and its application. The CDDP molecular marker primer set is designed for WRKY, MYB, ERF, KNOX and MADS. The present invention designs primers using the conserved gene sequences corresponding to the five conserved amino acid sequences of WRKY, MYB, ERF, KNOX and MADS as reference sequences. These short conserved gene sequences exist at multiple locations in the plant genome and provide multiple primer binding sites. This type of primer design focuses on gene regions and has advantages over random markers. At the same time, the designed primers are used to amplify genomic DNA of Polygonatum germplasm, which can be used for the identification, differentiation and genetic diversity analysis of Polygonatum germplasm resources.

Description

Technical Field

[0001] The invention belongs to the technical field of plant germplasm resource identification, and particularly relates to a CDDP molecular marker primer set for identifying Polygonatum germplasm resources and an application thereof. Background Art

[0002] Polygonatum sibiricum ( Polygonatum ) is a genus of rhizomatous plants in the Liliaceae family of the angiosperm phylum. All species in this genus are rhizomatous herbs. It comprises approximately 40 species, distributed throughout the northern temperate zone, with 31 found in China. The type species is Polygonatum sibiricum (P. sibiricum. Delar. ex Redout6). The rhizomes of most Polygonatum species are used medicinally, including the Chinese medicinal herbs "Yuzhu" (Yuzhu) and "Huangjing" (Huangjing). The former nourishes yin, moistens dryness, and promotes fluid production to quench thirst, while the latter nourishes the heart and lungs, promotes fluid production to nourish the stomach, and replenishes essence.

[0003] Although Polygonatum sibiricum is widely recognized in the field as a plant of medicinal importance, Polygonatum sibiricum plants from different regions and varieties are believed to have different therapeutic properties. While the diversity of Polygonatum sibiricum germplasm resources in my country is relatively high, the distinction between species within the genus is not high, with no clear boundaries. Currently, there are few studies on its genetic diversity and population structure, and insufficient data are available for its identification. While RAPD, RFLP, ISSR, SCoT, and SSR molecular markers have been applied to Polygonatum sibiricum germplasm identification, genetic diversity analysis, and germplasm resource conservation research, studies using CDDP (conserved DNA-derived polymorphism) molecular markers for Polygonatum sibiricum germplasm identification and genetic diversity analysis have not yet been conducted.

[0004] CDDP molecular marker technology is a molecular marker technology that uses primers designed based on conserved gene sequences of functional genes and gene families in plants to rapidly identify different species. Currently, CDDP molecular marker technology has been applied to the identification of some plant germplasm resources and genetic diversity research, but CDDP molecular markers for identifying and differentiating different species of Polygonatum sibiricum are still lacking. Therefore, there is an urgent need to develop CDDP molecular markers that can distinguish and identify different species of Polygonatum sibiricum. Summary of the Invention

[0005] The object of the present invention is to provide a CDDP molecular marker primer set for identifying Polygonatum germplasm resources and its application. The CDDP molecular marker primer set can be used for the identification, differentiation and genetic diversity analysis of Polygonatum germplasm resources.

[0006] The present invention provides a CDDP molecular marker primer set for identifying Polygonatum germplasm resources. The CDDP molecular marker primer set is designed for WRKY, MYB, ERF, KNOX and MADS.

[0007] Preferably, the CDDP molecular marker primer set includes WRKY-R1, WRKY-R2, WRKY-R3, WRKY-R3B, Myb2, ERF2, KNOX-2, KNOX-3, MADS-1, MADS-2 and MADS-4;

[0008] The nucleotide sequences of WRKY-R1, WRKY-R2, WRKY-R3, WRKY-R3B, Myb2, ERF2, KNOX-2, KNOX-3, MADS-1, MADS-2 and MADS-4 are shown in SEQ ID NO.1 to SEQ ID NO.11.

[0009] The present invention provides a kit for identifying Polygonatum germplasm resources, comprising the CDDP molecular marker primer set described in the above technical solution.

[0010] The present invention also provides the use of the CDDP molecular marker primer set or kit described in the above technical solution in the identification of Polygonatum germplasm.

[0011] The present invention also provides the use of the CDDP molecular marker primer set or kit described in the above technical solution in the genetic diversity analysis of Polygonatum.

[0012] The present invention also provides the use of the CDDP molecular marker primer set or kit described in the above technical solution in molecular breeding of Polygonatum.

[0013] Preferably, the genus Polygonatum includes Polygonatum sibiricum, Polygonatum multiflorum, Polygonatum yunnanensis, Polygonatum sibiricum and Polygonatum odoratum.

[0014] The present invention also provides a method for identifying Polygonatum germplasm resources, comprising the following steps:

[0015] Using the CDDP molecular marker primer set described in the above technical solution, PCR amplification is performed on the genomic DNA of the Polygonatum germplasm to obtain a PCR amplification product;

[0016] Detecting the PCR amplification product by gel electrophoresis to obtain band information;

[0017] Statistical analysis is performed on the spectral band information, and the Polygonatum germplasm is determined based on the statistical results.

[0018] Preferably, the statistical analysis includes matrix analysis, cluster analysis, principal coordinate analysis and genetic diversity parameter statistics.

[0019] Preferably, the PCR amplification reaction system includes 10 μL of 10×PCR buffer, 50 ng of genomic DNA, 1.5 μL of 10 μmol / L primer set, and ddH2O is added to 20 μL;

[0020] The PCR amplification procedure was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 2 min, for a total of 35 cycles; and extension at 72°C for 7 min.

[0021] Beneficial effects:

[0022] The present invention provides a CDDP molecular marker primer set for identifying Polygonatum germplasm resources. The CDDP molecular marker primer set is designed for WRKY, MYB, ERF, KNOX, and MADS. The present invention uses conserved gene sequences corresponding to the five conserved amino acid sequences of WRKY, MYB, ERF, KNOX, and MADS as reference sequences to design primers. These short conserved gene sequences are present in multiple locations in plant genomes and provide multiple primer binding sites. This type of primer design focuses on gene functional regions and has advantages over random markers. Furthermore, the designed primers can be used to amplify genomic DNA from Polygonatum germplasm resources for identification, differentiation, and genetic diversity analysis of Polygonatum germplasm resources.

[0023] In addition, the bands obtained by amplifying the Polygonatum germplasm using the CDDP molecular marker primer set are clear, polymorphic, specific, repeatable, and stable, which makes it easy to identify the differences at the genetic level among different plants. The identification results are stable and reliable, filling the shortcomings of traditional morphological identification methods and providing a technical basis for the identification of Polygonatum germplasm resources and the molecular breeding of Polygonatum plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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 are briefly introduced below.

[0025] Figures 1 to 11 The electrophoresis results of 23 Polygonatum sibiricum accessions amplified using WRKY-R1, WRKY-R2, WRKY-R3, WRKY-R3B, Myb2, ERF2, KNOX-2, KNOX-3, MADS-1, MADS-2, and MADS-4, respectively, where M represents DL2000 DNA Marker, and lanes 1 to 23 represent the Polygonatum sibiricum samples numbered 1 to 23 in Table 2, respectively;

[0026] Figure 12 This is the cluster analysis result of Polygonatum sibiricum germplasm samples from 23 regions;

[0027] Figure 13This is the principal coordinate analysis result of Polygonatum sibiricum germplasm samples from 23 regions, where the horizontal axis is the first principal component axis and the vertical axis is the second principal component axis. P1~P23 in the figure represent Polygonatum sibiricum samples from 23 regions. DETAILED DESCRIPTION

[0028] The present invention provides a CDDP molecular marker primer set for identifying Polygonatum germplasm resources. The CDDP molecular marker primer set is designed for WRKY, MYB, ERF, KNOX and MADS.

[0029] In the present invention, the CDDP molecular marker primer set preferably includes WRKY-R1, WRKY-R2, WRKY-R3, WRKY-R3B, Myb2, ERF2, KNOX-2, KNOX-3, MADS-1, MADS-2 and MADS-4; the nucleotide sequences of WRKY-R1, WRKY-R2, WRKY-R3, WRKY-R3B, Myb2, ERF2, KNOX-2, KNOX-3, MADS-1, MADS-2 and MADS-4 are preferably as shown in SEQ ID NO.1 to SEQ ID NO.11; the nucleotide sequences shown in SEQ ID NO.1 to SEQ ID NO.11 are specifically shown in Table 1:

[0030] Table 1 Primer sequences of CDDP molecular marker primer set

[0031]

[0032] Note: The base "S" in the nucleotide sequence in Table 1 represents a degenerate base.

[0033] The present invention provides a kit for identifying Polygonatum germplasm resources, comprising the CDDP molecular marker primer set described in the above technical solution. The kit preferably also includes a PCR amplification reagent. The present invention does not specifically limit the PCR amplification reagent; conventional PCR amplification reagents in the art can be used. In a specific embodiment of the present invention, the PCR amplification reagent includes 10 μL of 10× PCR buffer, 50 ng of genomic DNA, 1.5 μL of a 10 μmol / L primer set, and ddH2O to 20 μL.

[0034] The present invention also provides the use of the CDDP molecular marker primer set or kit described in the above technical solution for any one or more of the identification of Polygonatum germplasm, analysis of Polygonatum genetic diversity, and molecular breeding of Polygonatum. The present invention uses the conserved gene sequences corresponding to the five conserved amino acid sequences of WRKY, MYB, ERF, KNOX, and MADS as reference sequences to design primers. These short conserved gene sequences exist at multiple locations in the plant genome and provide multiple primer binding sites. This type of primer design focuses on gene regions and has advantages over random labeling. The designed CDDP molecular marker primer set is used to amplify genomic DNA of Polygonatum germplasm, which can be used for the identification, differentiation, and genetic diversity analysis of Polygonatum germplasm resources. The Polygonatum genus described in the present invention preferably includes Polygonatum sibiricum, Polygonatum multiflorum, Polygonatum yunnanensis, Polygonatum hupehensis, and Polygonatum odoratum.

[0035] The present invention also provides a method for identifying Polygonatum germplasm resources, comprising the following steps: performing PCR amplification on Polygonatum germplasm genomic DNA using the CDDP molecular marker primer set described in the above technical solution to obtain PCR amplification products; detecting the PCR amplification products using gel electrophoresis to obtain spectral band information; performing statistical analysis on the spectral band information, and judging the Polygonatum germplasm based on the statistical results.

[0036] The present invention utilizes a CDDP molecular marker primer set to perform PCR amplification on genomic DNA from Polygonatum sibiricum germplasm to obtain a PCR amplification product. The present invention preferably includes extracting genomic DNA from Polygonatum sibiricum germplasm before performing the PCR amplification. The present invention does not specifically limit the extraction step for the genomic DNA from Polygonatum sibiricum germplasm; conventional DNA extraction methods in the art, such as the CTAB method, can be used. The PCR amplification reaction system preferably comprises 10 μL of 10× PCR buffer, 50 ng of genomic DNA, 1.5 μL of a 10 μmol / L primer set, and ddH2O to 20 μL. The PCR amplification procedure preferably includes: 94°C pre-denaturation for 3 minutes; 94°C denaturation for 1 minute, 50°C annealing for 1 minute, and 72°C extension for 2 minutes, for a total of 35 cycles; and 72°C extension for 7 minutes. The PCR reaction and reaction procedure of the present invention for CDDP molecular marker amplification yield clear bands, good polymorphism, high reproducibility, strong specificity, and strong stability, making it easy to identify genetic differences between different plants and laying a solid foundation for genetic research in Polygonatum sibiricum.

[0037] After obtaining the PCR amplification product, the present invention uses gel electrophoresis to detect the PCR amplification product to obtain band information. The present invention has no special limitation on the gel electrophoresis detection step, and conventional gel electrophoresis detection steps in the art can be used.

[0038] After obtaining the band information, the present invention preferably performs statistical analysis on the band information and determines the Polygonatum germplasm based on the statistical results. The statistical analysis of the present invention preferably includes matrix analysis, cluster analysis, principal coordinate analysis and genetic diversity parameter statistics. The present invention preferably uses the SimQual program in the NTSYS2.10 software to perform matrix analysis, and the matrix analysis is preferably a similarity coefficient matrix. The present invention preferably uses Clustering software to perform cluster analysis and principal coordinate analysis. The present invention preferably uses POPGENE1.32 to perform the genetic diversity parameter statistics; the genetic diversity parameters preferably include the effective number of alleles, the number of alleles, Shannon's information index, gene diversity index genetic diversity parameters, intra-population gene diversity Hs, population total gene diversity Ht, inter-population genetic differentiation coefficient and gene flow genetic differentiation index.

[0039] After completing the statistical analysis, the present invention determines the Polygonatum germplasm based on the statistical analysis. The present invention preferably utilizes genetic diversity analysis, cluster analysis, and principal coordinate analysis to differentiate Polygonatum germplasm from different geographic or species sources. The present invention does not particularly limit the specific analysis steps of genetic diversity analysis, cluster analysis, and principal coordinate analysis; conventional analysis steps in the art may be employed.

[0040] The method described in this paper can effectively reveal the genetic diversity among Polygonatum sibiricum populations and distinguish the phylogenetic relationships among Polygonatum sibiricum germplasm resources. Furthermore, this method, using a conserved DNA-derived polymorphism (CDDP) marker analysis method, provides the first insight into the Polygonatum sibiricum genome and assesses genetic diversity, thus improving the molecular database for the genus. This is of great significance for the conservation and utilization of Polygonatum sibiricum resources.

[0041] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Example 1

[0043] 1. Material collection:

[0044] Polygonatum sibiricum samples from 23 regions were collected from 14 cities in 8 provinces across the country, as shown in Table 2.

[0045] Table 2 Polygonatum sibiricum test material number and sampling point information

[0046]

[0047]

[0048] 2. Extraction of genomic DNA from Polygonatum sibiricum germplasm

[0049] Young leaves of Polygonatum sibiricum samples from 23 regions were collected for whole-genome DNA extraction. Eight to 15 samples were collected from each region. Total genomic DNA was extracted from the 23 Polygonatum sibiricum samples listed in Table 2 using the CTAB method (equal amounts of 8 to 15 samples from each region were mixed). The extracted DNA samples were dissolved in TE buffer and stored at -20°C until use. Prior to amplification, the DNA samples were diluted to 50 ng / μL in double-distilled water and used as templates in PCR amplification reactions.

[0050] 3. PCR reaction system and primers for CDDP molecular markers in Polygonatum sibiricum

[0051] Genomic DNA of Polygonatum sibiricum samples from 23 regions was amplified by PCR using 11 primers, including WRKY-R1, WRKY-R2, WRKY-R3, WRKY-R3B, Myb2, ERF2, KNOX-2, KNOX-3, MADS-1, MADS-2, and MADS-4. One amplification tube corresponded to one sample and one primer, and three biological replicates were set for each primer. The specific sequences of the primers are shown in Table 1 and are not repeated here.

[0052] PCR amplification system: 10 μL of 10× PCR buffer (Dongsheng Biotechnology), 50 ng of genomic DNA, 1.5 μL of 10 μmol / L primers, and ddH2O to 20 μL. PCR amplification program: 94°C initial denaturation for 3 min; 35 cycles of 94°C denaturation for 1 min, 50°C annealing for 1 min, and 72°C extension for 2 min; finally, 72°C extension for 7 min. Store at 4°C.

[0053] 4. Gel electrophoresis detection

[0054] The obtained PCR amplification products were subjected to gel electrophoresis detection. The gel electrophoresis detection method was as follows: 8 μL of the above-obtained PCR amplification products were subjected to electrophoresis and color development. The electrophoresis was performed on a 1.5% agarose gel at a voltage of 110 V for 50 min. DL2000 DNA Marker was used as a standard reference. The band information was obtained by taking a photo and saving the result. Figures 1 to 11 As shown in Table 3, Figures 1 to 11 These are the amplification results of WRKY-R1, WRKY-R2, WRKY-R3, WRKY-R3B, Myb2, ERF2, KNOX-2, KNOX-3, MADS-1, MADS-2, and MADS-4 for Polygonatum sibiricum samples from 23 regions, respectively. M represents the DL2000 DNA Marker. Lanes 1 to 23 represent the Polygonatum sibiricum samples from regions numbered 1 to 23 in Table 2, respectively.

[0055] Table 3 Amplification results of 23 Polygonatum samples using CDDP molecular marker primer set

[0056]

[0057] Note: TNP: total number of amplified bands; NPB: number of polymorphic bands; PPB: percentage of polymorphism; PIC: polymorphism information content; MI: labeling index.

[0058] Depend on Figures 1 to 11 As can be seen, the band patterns amplified from 23 Polygonatum germplasm genomic DNA samples using the CDDP molecular marker amplification primers described in this invention were stable, clear, and reproducible. They also exhibited strong specificity, clear background, and robust stability. Table 3 shows that the 11 primer combinations amplified a total of 161 bands, 149 of which were polymorphic, with a polymorphism percentage (PPB) of 92.54%. Different primers amplified different bands, and the same primers amplified different bands from Polygonatum spp. in different regions. This demonstrates the rich polymorphism and complex genetic background among the 23 samples, providing opportunities for analyzing the phylogenetic relationships of Polygonatum germplasm.

[0059] 5. Statistical analysis of spectral information

[0060] Analyze the band information obtained in step 4. Using CDDP as the dominant marker, bands with consistent electrophoretic mobility in products amplified by the same primer are considered to represent the same locus. Identical bands at the same locus on the electropherogram are marked as "1," while their absence is marked as "0," thus generating a "0" and "1" matrix.

[0061] The total number of bands and polymorphic bands amplified by each primer pair were counted. The similarity coefficient matrix was calculated using the SimQual program in NTSYS2.10 software. UPGMA (unweighted pair-group method with arithmetic means) clustering was performed using SHAN in the Clustering program. The cluster diagram was generated using the Threeplot module to construct a molecular evolutionary tree. Decenter data transformation was performed based on the calculated similarity coefficient matrix, and then principal coordinate analysis was performed. The cluster analysis results are shown in Figure 2. Figure 12 The principal coordinate analysis results are shown in Figure 13 shown.

[0062] Genetic diversity parameters such as the effective number of alleles Ne, the number of alleles Na, Shannon's information index, and Nei's gene diversity index were calculated using POPGENE1.32, and the results are shown in Table 4.

[0063] Table 4 Genetic diversity analysis results of 23 samples of Polygonatum

[0064]

[0065] Note: Na: observed number of alleles; Ne: effective number of alleles; H: Nei's gene diversity; I: Shannon's information index.

[0066] Table 4 shows that the genetic diversity of a plant population can be reflected by the number of alleles (Na), the effective number of alleles (e), Nei's genetic diversity (H), and Shannon's information index (I). A larger Ne indicates a more important allele and a greater allele effect. Larger I and H values ​​indicate a higher genetic diversity within the sample. Table 4 shows that the Na value for Polygonatum sibiricum is 2.0000, the Ne value is 1.4829, the H value is 0.3033, and the I value is 0.4702. The total genetic diversity (Ht) of Polygonatum sibiricum germplasm from different regions is 0.3033, indicating a high degree of differentiation among different populations.

[0067] Depend on Figure 12 The dendrogram shows that, at a genetic similarity coefficient of 0.29, the 23 Polygonatum populations were divided into five clusters. Cluster I was divided into two subclusters. Cluster I-1 consisted entirely of Polygonatum accessions from Shandong Province. Cluster I-2 included the vast majority of Polygonatum multiflorum accessions, with accessions from Anhui Province clustered together. Accessions AHQY-3 and AHQY-4, originating from the same region, were closely related. Cluster II consisted of accessions FJYP-1 and FJYP-2 from Fujian Province, SDYT-1 from Shandong Province, JLFY-2 from Jilin Province, and CQ-1 from Chongqing. Cluster III included accessions Hubei Polygonatum, Polygonatum sibiricum, and Polygonatum duniana, all of which were whorled leaf populations. Cluster IV consisted entirely of populations from Anhui Province. Cluster V included the only cultivated accession, AHQY-2, forming a separate cluster. The CDDP primers used in this study were able to fully distinguish all accessions. This result may be partly due to the recent rise in cultivation and cross-pollination. Cross-pollination has facilitated the continuous exchange of accessions during long-term cultivation, resulting in high genetic diversity in accessions. While uneven distribution of accessions may have affected analysis of genetic diversity, the results indicate that most accessions with the same geographic origin or similar genetic background can be clustered into the same group.

[0068] Depend on Figure 13 It can be concluded that principal coordinate analysis showed that homologous accessions mostly clustered together, which may be because they originated from the same ancestor. It can be observed that the results elucidated by principal components corresponded to the results obtained by cluster analysis to a large extent.

[0069] From the results of the above examples, it can be seen that the CDDP molecular marker primer set and method described in the present invention can distinguish Polygonatum germplasm from different sources and obtain genetic diversity information of Polygonatum germplasm, which can be used as one of the methods for identification and genetic diversity research between Polygonatum species.

[0070] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Use of a kit for identifying CDDP molecular markers in genetic diversity analysis of Polygonatum spp.; the kit comprising a CDDP molecular marker primer set; The CDDP molecular marker primer set consists of WRKY-R1, WRKY-R2, WRKY-R3, WRKY-R3B, Myb2, ERF2, KNOX-2, KNOX-3, MADS-1, MADS-2 and MADS-4; The nucleotide sequences of WRKY-R1, WRKY-R2, WRKY-R3, WRKY-R3B, Myb2, ERF2, KNOX-2, KNOX-3, MADS-1, MADS-2 and MADS-4 are shown in SEQ ID NO.1 to SEQ ID NO.11; The Polygonatum genus includes Polygonatum sibiricum, Polygonatum multiflorum, Polygonatum yunnanensis, Polygonatum sibiricum and Polygonatum odoratum.

2. A method for identifying Polygonatum germplasm resources, characterized in that: The steps include: Using the CDDP molecular marker primer set described in claim 1 to perform PCR amplification on the genomic DNA of the Polygonatum germplasm to obtain a PCR amplification product; Detecting the PCR amplification product by gel electrophoresis to obtain band information; Performing statistical analysis on the spectral band information, and determining the Polygonatum germplasm according to the statistical results; The Polygonatum genus includes Polygonatum sibiricum, Polygonatum multiflorum, Polygonatum yunnanensis, Polygonatum sibiricum and Polygonatum odoratum.

3. The method according to claim 2, characterized in that The statistical analysis includes matrix analysis, cluster analysis, principal coordinate analysis and genetic diversity parameter statistics.

4. The method according to claim 2 or 3, characterized in that The PCR amplification reaction system includes 10 μL of 10×PCR buffer, 50 ng of genomic DNA, 1.5 μL of a 10 μmol / L primer set, and ddH2O to 20 μL. The PCR amplification procedure is as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 2 min, for a total of 35 cycles; and extension at 72°C for 7 min.

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