Chloroplast SSR (simple sequence repeat) labeled primers for identifying butterfly orchid protospecies and variant thereof and application of chloroplast SSR labeled primers
By developing primer combinations for chloroplast SSR markers of Phalaenopsis orchid native species and their variants, and using TP-M13-SSR technology, the molecular identification problem of Phalaenopsis orchid native species and variants has been solved, enabling rapid and accurate identification of germplasm resources and analysis of genetic diversity, thus promoting the breeding process.
Patent Information
- Application Number
- CN202511868982.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-11
- Publication Date
- 2026-01-27
AI Technical Summary
In the current technology, the development of molecular identification techniques for Phalaenopsis orchid native species and variants is limited, and there is a lack of effective chloroplast SSR markers, which restricts classification, identification and utilization.
A set of chloroplast SSR marker primer combinations for Phalaenopsis orchid native species and their variants was developed and detected using TP-M13-SSR technology. The combination includes 4 pairs of SSR marker primers, and DNA fingerprinting was constructed by PCR amplification and capillary electrophoresis for identification.
It enables rapid and accurate identification of Phalaenopsis orchid native species and their variants, improves the efficiency and reliability of germplasm resource identification, promotes genetic diversity analysis and molecular marker-assisted breeding, shortens the breeding cycle and reduces costs.
Smart Images
Figure CN121406820A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular biology technology, specifically relating to a set of chloroplast SSR marker primers for identifying Phalaenopsis orchid native species and their variants, and their applications. Background Technology
[0003] Chloroplasts are plant-specific organelles with independent DNA and widely distributed microsatellite sequences, making them ideal for developing chloroplast SSR molecular markers. These markers not only possess the advantages of widespread distribution, high polymorphism, and ease of operation associated with SSR markers, but also maintain the relative conservatism and maternal inheritance of the chloroplast genome. They hold significant application value in molecular phylogenetic analysis, genetic diversity analysis, kinship identification, and species molecular identification. The TP-M13-SSR technology combines SSR molecular marker technology with fluorescence sequencing technology, solving a series of problems associated with traditional gel electrophoresis analysis, such as low throughput, cumbersome amplification product detection procedures, and heavy data recording workload. It has been successfully applied in orchids, including the development and utilization of universal primers for the genera *Phalaenopsis* and *Aster*, the identification of mutants in *Phalaenopsis*, and the construction of core germplasm for *Cymbidium*. Currently, the development and application of chloroplast SSR markers for *Phalaenopsis* native species and varieties have not been reported, limiting the development of molecular identification techniques for *Phalaenopsis* native species and varieties. Therefore, based on the published chloroplast genome data of Phalaenopsis xiaolanyu, we identified its chloroplast SSR loci and developed SSR markers. We screened out polymorphic chloroplast SSR markers that can be used for molecular identification of multiple Phalaenopsis native species and varieties, and used TP-M13-SSR technology for detection. This is of great significance for the classification, identification and development of Phalaenopsis native species and varieties. Summary of the Invention
[0004] This invention aims to address the technical deficiencies of existing technologies by providing a set of chloroplast SSR marker primers for identifying Phalaenopsis orchid native species and their variants, and their applications.
[0005] The first objective of this invention is to provide a primer set for identifying Phalaenopsis orchid native species and their variants, the primer set comprising the following four pairs of SSR marker primers:
[0006] A second objective of this invention is to provide a kit for identifying Phalaenopsis orchid native species and their variants, the kit comprising the above-described SSR marker primer combination.
[0007] A third objective of this invention is to provide the application of the above-described SSR marker primer combination or kit in the identification of Phalaenopsis orchid native species and their variants.
[0008] Preferably, the original species and variants of Phalaenopsis orchids include white-flowered Phalaenopsis simonii (variant), blue-lipped Phalaenopsis simonii (variant), Phalaenopsis simonii, deep red Phalaenopsis staghorn (variant), yellow-flowered Phalaenopsis staghorn (variant), Phalaenopsis staghorn, fluorescent Phalaenopsis, white-flowered fluorescent Phalaenopsis (variant), and purple-flowered fluorescent Phalaenopsis (variant).
[0009] The fourth objective of this invention is to provide a method for identifying Phalaenopsis orchid native species and their variants, comprising the following steps:
[0010] (1) Using the total DNA of the Phalaenopsis orchid sample to be tested as a template, PCR amplification was performed using the above 4 pairs of SSR marker primers;
[0011] (2) Perform capillary electrophoresis on the obtained PCR products and read the length of the allele fragment and the number of peaks corresponding to each pair of primers;
[0012] (3) Based on the fragment length-peak fingerprint generated by the four primer pairs, compare it with the known original species / variant standard fingerprint library to determine the original species or variant to which the sample belongs.
[0013] Preferably, in step (1), the total volume of the PCR amplification reaction system is 10µL, and the composition is as follows: 1.0µL of 50ng / µL DNA template, 1.0µL of 10×Buffer I buffer, 0.1µL of 5U / µL TAKARA HSTaq enzyme, 0.5µL of 5µM upstream primer, 0.5µL of 5µM downstream primer, 0.8µL of 2.5mM dNTP, and 6.1µL of deionized water.
[0014] Preferably, in step (1), the PCR amplification reaction program is as follows: 95℃, 5 min; 95℃ 30 s, 60℃ 30 s, 72℃ 30 s, 35 cycles; 60℃, 30 min.
[0015] The fifth objective of this invention is to provide the application of the above-mentioned SSR marker primer combinations or kits in the analysis of interspecific genetic diversity, identification of germplasm resources, or molecular marker-assisted breeding within the Phalaenopsis genus.
[0016] The sixth objective of this invention is to provide the application of the above-mentioned SSR marker primer combination or kit in constructing a Phalaenopsis orchid chloroplast DNA fingerprint database, constructing a standard fingerprint library by using the chloroplast SSR amplification fragment size information of different Phalaenopsis orchid native species and their variants for rapid comparison and identification of unknown samples.
[0017] The present invention provides a set of chloroplast SSR marker primers that enable rapid and accurate identification of Phalaenopsis orchid native species and their variants, effectively distinguishing phenotypically similar closely related species and variants, and significantly improving the efficiency and reliability of germplasm resource identification. The DNA fingerprint database constructed using these primers provides a standardized and shared technical platform for genetic diversity analysis among Phalaenopsis species, core germplasm screening, kinship clarification, and molecular marker-assisted breeding. This can significantly shorten the traditional breeding cycle, reduce breeding costs, and promote the targeted breeding of new and superior Phalaenopsis orchid varieties and the effective protection of intellectual property rights. Attached Figure Description
[0018] Figure 1 Images of native species and variants of the Phalaenopsis genus.
[0019] Figure 2 Amplification patterns of nine different Phalaenopsis orchid species and variants on SSR2 primers.
[0020] Figure 3 Amplification patterns of nine different Phalaenopsis orchid species and variants on SSR3 primers.
[0021] Figure 4 Amplification patterns of nine different Phalaenopsis orchid species and variants on SSR4 primers.
[0022] Figure 5 Amplification patterns of nine different Phalaenopsis orchid species and variants on SSR7 primers. Detailed Implementation
[0023] The following embodiments are further illustrations of the present invention, but not limitations thereof. Specific experimental conditions and methods are not specified in the following embodiments, and the techniques used are generally conventional methods well known to those skilled in the art.
[0024] Example 1
[0025] 1. Experimental Methods
[0026] (1) Total DNA extraction
[0027] Young root tip tissues from different samples were collected, flash-frozen in liquid nitrogen, ground into powder, and 0.2 g was taken. The DNA was extracted according to the operating procedure of the novel plant genomic DNA extraction kit of Tiangen Biotech (Beijing) Co., Ltd. The extracted DNA was dissolved in 50 µL ddH2O, and 2 µL was taken for NanoDrop spectrophotometry and 1.2% agarose gel electrophoresis to detect the purity and concentration of the extracted DNA. After passing the test, the DNA was stored at -20℃ for later use.
[0028] (2) Development of SSR markers for chloroplasts in Phalaenopsis orchids
[0029] The chloroplast genome sequence of *Phalaenopsis xiaolanyuensis* was downloaded online (https: / / www.ncbi.nlm.nih.gov / search / all / ?term=NC_017609). SSR loci were predicted online using MISA-web (http: / / misaweb.ipk-gatersleben.de / ). The prediction parameters were set as follows: repeat elements of 1 base ≥ 10 times, repeat elements of 2 bases ≥ 5 times, repeat elements of 3 to 6 bases ≥ 3 times, and the distance between two SSR loci was at least 200 bp. Primers were designed and synthesized using Primer Premier 3.0.
[0030] (3) PCR reaction
[0031] The designed primers were used for singleton PCR amplification in different samples, and the primers were synthesized using FAM fluorescent labeling.
[0032] The PCR amplification system is as follows:
[0033] The reaction procedure is as follows:
[0034] (4) Capillary electrophoresis detection and analysis
[0035] 1.2% agarose gel electrophoresis was used to detect amplification products and screen primers with good amplification effects. 1.0 µL of the amplification product of the selected primers and 9 µL of a mixture of ROX-500 molecular weight internal standard and formamide (0.5:8.5) were added to each well of a 96-well plate. After denaturation at 95℃ for 3 min, the sample was analyzed using an ABI 3730XL detector. The sample was injected at 1 kV for 10 s, followed by electrophoresis at 15 kV for 30 min. The raw data collected by DataCollection software was imported into GeneMapper 3.2 software for analysis. The position of each peak was compared with the molecular weight internal standard in its lane to calculate the accurate size of the target DNA fragment. Three replicates of capillary electrophoresis were performed independently at each fluorescently labeled locus. The average of the three replicates was rounded to the nearest integer and used as the data for that locus.
[0036] 2. Experimental Results
[0037] This invention developed four pairs of polymorphic SSR primers (Table 1), which were used in nine germplasm resources from three different Phalaenopsis orchid native species and their variants (Table 2). Figure 1 The fluorescence peaks were stably amplified in 9 different germplasm resources. Figures 2-5Nine fingerprint maps specific to different germplasm resources can be constructed (Table 3), which can effectively distinguish the original species and its variants, proving that the Phalaenopsis chloroplast SSR marker developed in this invention can be applied to the classification and identification of Phalaenopsis original species and variants.
[0038] Table 1 Primer Information
[0039] Table 2. Information on native species and varieties of the genus Phalaenopsis.
[0040] Table 3 Fingerprints of each sample
[0041] Example 2
[0042] According to the chloroplast SSR molecular marker primer composition and germplasm resource identification method described in this invention, nine samples each from three different Phalaenopsis species and their variants were selected for testing. The test results are shown in Table 4 below. Table 4 shows that the chloroplast SSR molecular marker primer composition and germplasm identification method described in this invention can accurately identify nine Phalaenopsis germplasm resources from three different Phalaenopsis species and their variants, with an accuracy rate of 100%.
[0043] Table 4 Accuracy Test Results
[0044] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A primer set for identifying Phalaenopsis orchid native species and their varieties, characterized in that, The primer combination includes the following four pairs of SSR marker primers:
2. A kit for identifying Phalaenopsis orchid native species and their varieties, characterized in that, The kit contains the SSR-labeled primer combination as described in claim 1.
3. The application of the SSR marker primer combination of claim 1 or the kit of claim 2 in the identification of Phalaenopsis orchid native species and their variants.
4. The application according to claim 3, characterized in that, The original species and variants of Phalaenopsis include white-flowered Phalaenopsis simonii (variant), blue-lipped Phalaenopsis simonii (variant), Phalaenopsis simonii, deep red staghorn Phalaenopsis (variant), yellow-flowered staghorn Phalaenopsis (variant), staghorn Phalaenopsis, fluorescent Phalaenopsis, white-flowered fluorescent Phalaenopsis (variant), and purple-flowered fluorescent Phalaenopsis (variant).
5. A method for identifying Phalaenopsis orchid native species and their varieties, characterized in that, Includes the following steps: (1) Using the total DNA of the Phalaenopsis orchid sample to be tested as a template, PCR amplification was performed using the four pairs of SSR marker primers described in claim 1; (2) Perform capillary electrophoresis on the obtained PCR products and read the length of the allele fragment and the number of peaks corresponding to each pair of primers; (3) Based on the fragment length-peak fingerprint generated by the four primer pairs, compare it with the known original species / variant standard fingerprint library to determine the original species or variant to which the sample belongs.
6. The method according to claim 5, characterized in that, In step (1), the total volume of the PCR amplification reaction system is 10µL, and the composition is as follows: 1.0µL of 50ng / µL DNA template, 1.0µL of 10×Buffer I buffer, 0.1µL of 5U / µL TAKARAHSTaq enzyme, 0.5µL of 5µM upstream primer, 0.5µL of 5µM downstream primer, 0.8µL of 2.5mM dNTP, and 6.1µL of deionized water.
7. The method according to claim 5, characterized in that, In step (1), the PCR amplification reaction program is as follows: 95℃, 5 min; 95℃ 30 s, 60℃ 30 s, 72℃ 30 s, 35 cycles; 60℃, 30 min.
8. The application of the SSR marker primer combination of claim 1 or the kit of claim 2 in the analysis of interspecific genetic diversity, identification of germplasm resources or molecular marker-assisted breeding within the genus Phalaenopsis.
9. The application of the SSR marker primer combination of claim 1 or the kit of claim 2 in constructing a Phalaenopsis orchid chloroplast DNA fingerprint database, characterized in that, A standard fingerprint library was constructed using the size information of SSR amplified fragments from different Phalaenopsis orchid species and their variants for rapid comparison and identification of unknown samples.
10. The application according to claim 9, characterized in that, The original species and variants of Phalaenopsis include white-flowered Phalaenopsis simonii (variant), blue-lipped Phalaenopsis simonii (variant), Phalaenopsis simonii, deep red staghorn Phalaenopsis (variant), yellow-flowered staghorn Phalaenopsis (variant), staghorn Phalaenopsis, fluorescent Phalaenopsis, white-flowered fluorescent Phalaenopsis (variant), and purple-flowered fluorescent Phalaenopsis (variant).