SSR molecular marker primer combination for identifying dendrobium germplasm and application

Abstract

The invention discloses an SSR (Simple Sequence Repeat) molecular marker primer combination for identifying dendrobium germplasm, the SSR molecular marker primer combination comprises 20 pairs of primers, and the nucleotide sequences of the SSR molecular marker primer combination are as shown in SEQ ID No. 1 to SEQ ID No. 40. The SSR molecular marker composition is high in polymorphism, strong in specificity, stable in amplification, convenient to use and low in cost, and can be widely applied to multiple important fields such as accurate identification of dendrobium germplasm resources, genetic diversity evaluation, genetic relationship analysis, DUS test and molecular breeding.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker technology, specifically relating to an SSR molecular marker primer combination for identifying Dendrobium species and its application. Background Technology

[0002] Dendrobium is a large genus within the Orchidaceae family, encompassing numerous species of significant ornamental, medicinal, and economic value. my country is one of the main distribution areas for Dendrobium species, possessing abundant wild resources, such as Dendrobium officinale, Dendrobium nobile, Dendrobium chrysotoxum, and Dendrobium huoshanense. However, the genus Dendrobium is highly diverse, with many native species or varieties exhibiting very similar morphological characteristics. Especially before flowering, many native species or varieties are almost indistinguishable, exhibiting complex interspecific and intraspecific variations. Relying solely on traditional morphological identification methods is insufficient for accurate differentiation, easily leading to confusion of germplasm resources and the widespread existence of homonyms or synonyms. This severely restricts the effective protection, rational development and utilization, and breeding of new varieties of Dendrobium germplasm resources.

[0003] Molecular marker technology provides an efficient and accurate means for the identification of plant germplasm resources. Among them, SSR (Simple Sequence Repeats) molecular markers have been widely used in various fields such as plant genetic diversity analysis, variety identification, fingerprinting, genetic mapping, and marker-assisted breeding due to their advantages such as high polymorphism, codominant inheritance, good repeatability, wide distribution, and ease of operation.

[0004] Currently, the development of molecular markers for Dendrobium species is mostly based on transcriptome (EST-SSR) or simplified genome sequencing of specific varieties or native species. This approach suffers from problems such as insufficient polymorphism, weak universality, and high development costs, making it difficult to meet the needs for efficient and accurate identification of the vast and complex native species and varieties of Dendrobium.

[0005] Therefore, it is urgent to utilize the increasingly abundant whole-genome sequencing data of Dendrobium in recent years to develop a set of low-cost, highly polymorphic, stable, and highly specific SSR molecular marker combinations to provide strong technical support for the accurate identification of Dendrobium native species and varieties, germplasm resource management, and genetic breeding research. Summary of the Invention

[0006] The purpose of this invention is to provide a combination of SSR molecular marker primers for identifying Dendrobium germplasm and its application, in order to solve the problems of difficulty in identifying Dendrobium germplasm resources, insufficient number of existing SSR markers, limited polymorphism, and high development and application costs in the prior art.

[0007] A primer composition for identifying Dendrobium species includes 20 pairs of primers. The DNA sequences of the primer composition are shown in SEQ ID No. 1 to SEQ ID No. 40, as detailed in Table 2.

[0008] Application of the above-mentioned SSR molecular marker primer combination in the identification of Dendrobium germplasm.

[0009] Application of the above-mentioned SSR molecular marker primer combination in the analysis of genetic diversity or phylogenetic relationships in Dendrobium germplasm.

[0010] The application of the above-mentioned SSR molecular marker primer combination in molecular marker-assisted breeding of Dendrobium.

[0011] A method for germplasm identification, genetic diversity, or phylogenetic analysis of the genus *Dendrobium* includes the following steps:

[0012] (1) Extract genomic DNA from the Dendrobium germplasm samples;

[0013] (2) PCR amplification was performed using the genomic DNA extracted in step (1) as a template. The SSR molecular marker primers used for PCR amplification were the SSR molecular marker primer combination mentioned above.

[0014] (3) The PCR amplification products obtained in step (2) were detected by agarose gel electrophoresis, and the data were collected by reading the graph;

[0015] (4) Statistical analysis of the data obtained in step (3) is performed to determine the genetic diversity or cluster and polymorphism information content.

[0016] Furthermore, the PCR amplification system described in step (2) is as follows: the total volume of the reaction system is 20 μL, including 1.0 µL DNA template, 10.0 µL 2×Rapid Taq Master Mix, 0.5 µL of each primer, and deionized water to make up to 20 µL; the PCR amplification program is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, annealing for 30 s according to the annealing temperature of the primers, 72℃ extension for 15 s, 30 cycles; 72℃ extension for 5 min.

[0017] The beneficial effects of this invention are:

[0018] 1. Novel source: Developed based on whole genome sequencing data, with broad SSR locus coverage, abundant markers, and a large amount of information.

[0019] 2. High specificity: Primer design is based on the genome sequences of multiple native Dendrobium species, resulting in good amplification specificity, clear background, and accurate and reliable results.

[0020] 3. Good stability: The amplification conditions are optimized, the reproducibility is high, and it is not limited by the plant growth stage (roots, stems, leaves, and even some dried medicinal materials) or season.

[0021] 4. Simple and inexpensive: Since the implementation process of this invention only uses conventional PCR technology combined with conventional agarose gel electrophoresis, the technical operation is simple, the development cost is low, and the versatility is good.

[0022] 5. Wide range of applications: This SSR marker composition can be used in many areas, such as the precise identification and molecular identification of native Dendrobium germplasm resources; the evaluation of genetic diversity of Dendrobium germplasm resources and the construction of core germplasm banks; the analysis of interspecific and intraspecific phylogenetic relationships and phylogenetic studies of Dendrobium; the screening of similar varieties and identification of variety authenticity in DUS testing (specificity, uniformity, stability) of new Dendrobium varieties; the association analysis and molecular marker-assisted selection (MAS) of important horticultural or medicinal traits of Dendrobium (such as flower diameter, flowering period, polysaccharide content, etc.) to accelerate the breeding process; the conservation genetics research of endangered Dendrobium species; the detection process is standardized and can be achieved by conventional PCR combined with ordinary agarose gel electrophoresis, which is easy to promote and apply to scientific research, seedling management, market supervision and breeding practice. Attached Figure Description

[0023] Figure 1 UPGMA clustering dendrites of 48 Dendrobium orchid native species based on 20 SSR markers.

[0024] Figure 2 DNA fingerprinting of Dendrobium officinale native species was constructed based on a combination of four pairs of SSR marker primers. Detailed Implementation

[0025] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0026] The experimental methods used in the examples are all conventional methods, and the reagents or instruments used, unless otherwise specified, are all conventional products that can be purchased on the market.

[0027] Example 1

[0028] Primer design based on whole-genome sequencing data of Dendrobium officinale native species

[0029] 1. SSR Site Search: Search for SSR sites in the assembled contig sequences of Dendrobium whole-genome sequencing data. Parameter settings: ≥6 dinucleotide repeats, or ≥5 trinucleotide repeats, or ≥4 tetra, penta, or hexanucleotide repeats.

[0030] 2. Primer Design: PCR primers were designed from the flanking sequences of the SSR loci using Primer 5.0 software. Design parameters: primer length 18-24 bp, Tm value 55-65℃ (Tm difference between upstream and downstream ≤5℃), GC content 40-60%, and typical product size 100-300 bp.

[0031] 3. Approximately 3,000 candidate SSR primer pairs were initially designed. Using the difference in the number of nucleotide repeats predicted by the same primer pair between two different Dendrobium orchid native species as the standard, 70 primer pairs were further screened for subsequent research.

[0032] Example 2

[0033] Primary and secondary screening of SSR primers

[0034] 1. Materials: 48 native species of the Dendrobium genus, encompassing 13 groups, including Dendrobium officinale, Dendrobium nobile, Dendrobium chrysanthum, Dendrobium nobile, and Dendrobium grandiflorum, were selected. Young leaf tissues from these plants were sampled. The native Dendrobium species are listed in Table 1.

[0035] Table 1: Native species names, Latin names, and groups of 48 Dendrobium orchids

[0036]

[0037] Note: The characteristic markers or alternative names (another group name) of the variety are listed in parentheses after the Chinese name or group name of the native species.

[0038] 2. DNA extraction: Genomic DNA was extracted using a commercially available Simgen plant / fungus DNA extraction kit. After passing the tests by 1% agarose gel electrophoresis and NanoDrop 2000 (OD260 / 280≈1.8, concentration≥50 ng / μL), the DNA was diluted to appropriate proportions for later use.

[0039] 3. Initial screening (PCR and agarose gel electrophoresis):

[0040] DNA from six representative native species among the 48 Dendrobium orchid native species was selected as templates, and PCR amplification was performed using 70 primer pairs designed in Example 1.

[0041] (1) PCR system (20 μL): 1.0µL DNA template, 10.0µL 2×Rapid Taq Master Mix, 0.5µL each of primers, and deionized water to make up to 20µL.

[0042] (2) PCR program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, annealing for 30 s according to the primer annealing temperature, 72℃ extension for 15 s, 30 cycles; 72℃ extension for 5 min.

[0043] (3) Electrophoresis: 5 μL of PCR product was subjected to 3% agarose gel electrophoresis. 29 primer pairs were initially screened out, showing relatively single, clear and bright amplification bands, with the main band size meeting expectations and appearing stably in multiple samples.

[0044] 4. Secondary screening (PCR and agarose gel electrophoresis):

[0045] (1) Using 48 Dendrobium officinale DNA samples as templates, PCR amplification was performed using the 29 primer pairs selected in the initial screening (the reaction system and procedure were the same as in the initial screening).

[0046] (2) Perform 3% agarose gel electrophoresis on the PCR products to separate the bands and detect their genotypes.

[0047] (3) Statistical analysis of electrophoresis results, and screening for primers with relatively single, clear and bright amplification bands, main band size that meets expectations and appears stably in multiple samples.

[0048] Finally, a core primer set containing 20 pairs of highly polymorphic SSR markers (marker numbers D1 to D66) was obtained, as shown in Table 2.

[0049] Table 2: 20 SSR primer combinations

[0050] Example 3

[0051] Genetic Polymorphism Analysis of Core SSR Marker Primer Combinations in Dendrobium Germplasm Identification

[0052] 1. Materials: Same as in Example 2.

[0053] 2. DNA extraction: Same as in Example 2.

[0054] 3. PCR amplification and electrophoresis: The PCR reaction system, reaction procedure and electrophoresis settings are the same as in Example 2.

[0055] 4. Data Analysis and Evaluation:

[0056] The number of DNA bands in the electrophoretic amplification products of each sample was counted, and the number of bands was recorded as '1' for samples with a band and '0' for samples without a band. Electrophoretic typing was performed.

[0057] (1) Genetic diversity analysis of 42 Dendrobium species was performed using Popgene software. Genetic parameters such as total number of bands, number of polymorphic bands, percentage of polymorphic sites (%), number of observed alleles (Na), number of effective alleles (Ne), Nei's gene diversity index (H), and Shannon information index (I) for each primer pair were analyzed, as shown in Tables 3 and 4.

[0058] Table 3: Statistics on polymorphism of screening primer bands

[0059] Table 4: Genetic diversity index of 48 Dendrobium officinale germplasm resources

[0060] (2) Using NTSYS software, the Unweighted Group Pairing Method (UPGMA) was used to perform '1,0' data system clustering, obtaining cluster diagrams for 42 Dendrobium officinale native species, as shown in the figure. Figure 1 .

[0061] As shown in Table 3, genetic diversity analysis of 42 native Dendrobium species yielded a total of 474 bands using 20 primer pairs, all of which were polymorphic, representing a polymorphism rate of 100%. The number of bands amplified by each primer ranged from 13 to 37. The primer with the most amplified sites was D62 (37 sites), while the primer with the fewest was D11 (13 sites), with an average of 23.7 polymorphic bands per primer.

[0062] As shown in Table 4, the average number of observed alleles (Na) was 2.000, the average number of effective alleles (Ne) was 1.0885, the average Nei's gene diversity index (H) was 0.0754, and the average Shannon diversity information index (I) was 0.1532. This indicates that the SSR marker combination used in this study is highly efficient in detecting the genetic diversity of Dendrobium germplasm resources, and there is relatively rich genetic diversity among the native species.

[0063] like Figure 1 As can be seen, the UPGMA clustering dendrite can clearly group samples of different Dendrobium species together according to specific clusters. For example, Dendrobium nobile (4), Dendrobium chrysanthum (7), Dendrobium floribundum (16), and Dendrobium fimbriatum (38) belong to the Lantern Group; Dendrobium officinale (1), Dendrobium chromeensis (2), and Dendrobium sparsely flowered (6) belong to the Type Group; Dendrobium chrysanthum (21) and Dendrobium d'Australis (22) belong to the Australian Pigeon Group; Dendrobium scythe (44) and Dendrobium chrysanthum (45) belong to the Wide Mouth Group; and other Dendrobium species with different kinship can also be well classified according to genetic distance.

[0064] Example 4

[0065] Construction of SSR marker fingerprint of Dendrobium native species

[0066] 1. Materials: Same as in Example 2.

[0067] 2. DNA extraction: Same as in Example 2.

[0068] 3. PCR amplification and electrophoresis: The PCR reaction system, reaction procedure and electrophoresis settings are the same as in Example 2.

[0069] 4. Data Analysis and Evaluation:

[0070] The number of DNA bands generated after amplification and electrophoresis of 48 samples using the above-mentioned label combinations was counted. A band with a band was recorded as '1', and no band was recorded as '0'. Four primer pairs, D6, D16, D60, and D66, were selected and found to simultaneously produce effective amplified bands in all tested native species, exhibiting good band polymorphism. These combinations can be used to construct SSR fingerprints of 48 Dendrobium native species. Figure 2 .

[0071] The above atlas can individually identify the 48 original Dendrobium species resources mentioned above, and can provide a certain scientific basis and data support for the classification and identification of Dendrobium.

[0072] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A primer combination of SSR molecular marker for identifying Dendrobium species germplasm, characterized in that, The nucleotide sequences of the SSR molecular marker primer combination comprising 20 pairs of primers are shown as SEQ ID No. 1-SEQ ID No.

40.

2. The application of the SSR molecular marker primer combination of claim 1 in identification of Dendrobium species germplasm.

3. The application of the SSR molecular marker primer combination of claim 1 in genetic diversity or genetic relationship analysis of Dendrobium species germplasm.

4. The application of the SSR molecular marker primer combination of claim 1 in molecular marker assisted breeding of Dendrobium species.

5. A method for identifying germplasm, analyzing genetic diversity or analyzing genetic relationship of Dendrobium, characterized in that, The method comprises the following steps: (1) extracting genomic DNA of the Dendrobium species germplasm sample; (2) performing PCR amplification using the genomic DNA extracted in step (1) as a template, and the SSR molecular marker primer used for PCR amplification is the SSR molecular marker primer combination of claim 1; (3) performing agarose gel electrophoresis detection on the PCR amplification product obtained in step (2), reading the map and collecting data; (4) statistically analyzing the electrophoresis band distribution data obtained in step (3) to determine genetic diversity, clustering and polymorphism information, and for constructing the SSR marker fingerprint of Dendrobium species germplasm resources.

6. The method of claim 5, wherein, The PCR amplification system in step (2) is as follows: the total volume of the reaction system is 20 µL, including 1.0 µL of DNA template, 10.0 µL of 2×Rapid Taq Master Mix, 0.5 µL of primer, and deionized water to make up to 20 µL; the PCR amplification program is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, annealing for 30 s according to the annealing temperature of the primer, 72℃ extension for 15 s, 30 cycles; 72℃ extension for 5 min.

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