An SNP molecular marker for identifying the species of Fritillaria unibracteata Hsiao et K. C. Hsia and its application
By developing SNP molecular markers and primers for PCR amplification reaction, combined with Sanger sequencing analysis, the problem of identification of fritillaria wab was solved, and the accurate distinction of confusing species was achieved, which improved the identification efficiency and reduced the cost.
Patent Information
- Application Number
- CN202510302705.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-14
- Publication Date
- 2025-08-01
- Estimated Expiration
- 2045-03-14
AI Technical Summary
The prior art lacks effective methods to distinguish species that are easily confused with the same genus of Fritillaria, resulting in serious confounding phenomena in cultivation and lack of simple and reliable identification methods.
A SNP molecular marker was developed, using a nucleotide sequence with a polymorphism of C/G at the 5' end of the nucleotide sequence, and a specific primer was designed for PCR amplification reaction, and the genotype of the PCR amplification product was analyzed by Sanger sequencing to achieve accurate identification of fritillaria wab.
The accurate distinction between fritillaria wab and its easily confused species is achieved, the identification efficiency is improved, and the cultivation and research costs are reduced.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of plant molecular biology, in particular to a SNP molecular marker for identifying Fritillaria var. balboa species and an application thereof. Background Art
[0002] Wabu Fritillaria ( Fritillaria unibracteata var. wabuensis Fritillaria wabuensis belongs to the genus Fritillaria in the Liliaceae family and is one of the original plants of the traditional Chinese medicine "Chuan Fritillaria cirrhosa." The dried bulbs of Fritillaria wabuensis have the effects of clearing heat and moistening the lungs, resolving phlegm and relieving coughs, and dispersing stagnation and eliminating carbuncles. They are used to treat lung heat and dry coughs, dry coughs with little sputum, yin deficiency and fatigue-induced coughs, blood in sputum, scrofula, mastitis, and lung abscesses. There is a high demand for these dried bulbs in the market.
[0003] Wabu Fritillaria is a Class II national protected plant, and its wild resources are scarce. Currently, Wabu Fritillaria is one of the most suitable species for cultivation among the original species of Sichuan Fritillaria. However, mixed plantings still exist in Wabu Fritillaria cultivation sites, and there is a lack of effective means to distinguish Wabu Fritillaria from other easily confused species of the same genus, such as the thick honey Fritillaria, dark purple Fritillaria, Taibai Fritillaria, Huaxi Fritillaria, Gansu Fritillaria, curled leaf Fritillaria, thunbergia, Yi Fritillaria, flat Fritillaria, and Zhejiang Fritillaria.
[0004] With the development of high-throughput sequencing technology, it has become possible to develop molecular markers for sex identification through SNP analysis. However, no SNP molecular markers for species identification of Fritillaria var.
[0005] In summary, how to develop an effective, reliable and easy-to-operate identification method or molecular marker to distinguish Fritillaria wabuensis has become a technical problem that needs to be solved urgently. Summary of the Invention
[0006] The present invention aims to provide a SNP molecular marker for identifying Fritillaria var. wabuensis species and application thereof.
[0007] To achieve the purpose of the present invention, in a first aspect, the present invention provides a SNP molecular marker for identifying the species of Fritillaria var. balnoffii, wherein the SNP molecular marker comprises a nucleotide sequence with a C / G polymorphism at position 192 of the 5' end of the sequence shown in SEQ ID NO: 1.
[0008] Furthermore, the genotype of the site with the polymorphism is CC, corresponding to the species of Fritillaria wabuensis, and the genotype of the site with the polymorphism is CG or GG, corresponding to the species of non-Fritillaria wabuensis.
[0009] In a second aspect, the present invention provides primers for amplifying the SNP molecular marker.
[0010] Preferably, it comprises an upstream primer as shown in SEQ ID NO: 2 and a downstream primer as shown in SEQ ID NO: 3.
[0011] In a third aspect, the present invention provides a detection reagent or kit containing the primer.
[0012] In a fourth aspect, the present invention provides the application of the SNP molecular marker, the primer, or the detection reagent or kit in the identification of Fritillaria unibracteata.
[0013] In a fifth aspect, the present invention provides a method for identifying Fritillaria unibracteata, including: using the genomic DNA of the to-be-detected Fritillaria plant as a template, performing a PCR amplification reaction with the primer, analyzing the PCR amplification product, determining the genotype of the to-be-detected Fritillaria plant, and identifying Fritillaria unibracteata according to the genotype.
[0014] Further, if the genotype of the site with the polymorphism in the SNP molecular marker contained in the amplification product is CC, it corresponds to the Fritillaria unibracteata species, and if the genotype of the site with the polymorphism is CG or GG, it corresponds to non-Fritillaria unibracteata species.
[0015] Preferably, the reaction conditions of the PCR amplification reaction are: 95°C for 3 min; 94°C for 25 s - 30 s, 60°C - 64°C for 25 s - 30 s, 72°C for 10 s, 30 - 40 cycles; 72°C for 5 min.
[0016] By the above technical solutions, the present invention has at least the following advantages and beneficial effects:
[0017] The present invention can distinguish Fritillaria unibracteata from its easily confused species, such as Fritillaria delavayi, Fritillaria unibracteata var. wabuensis, Fritillaria taipaiensis, Fritillaria cirrhosa D. Don var. ecirrhosa Franch., Fritillaria przewalskii Maxim., Fritillaria cirrhosa D. Don var. cirrhosa, Fritillaria delavayi Franch., Fritillaria pallidiflora Schrenk, Fritillaria thunbergii Miq. var. chekiangensis Hsiao & K. C. Hsia, Fritillaria thunbergii Miq.. The SNP molecular marker of the present invention is beneficial to be applied to the cultivation, identification, and research process of Fritillaria unibracteata, so as to quickly distinguish Fritillaria unibracteata species, improve the identification efficiency, help purify the cultivation area, and reduce the cultivation cost and research cost. Description of the Drawings
[0018] Figure 1 It is the representative Sanger sequencing diagram of Example 1 of the present invention, where the upper figure is Fritillaria unibracteata (genotype is CC), and the lower figure is Fritillaria delavayi, Fritillaria unibracteata var. wabuensis, Fritillaria taipaiensis, Fritillaria cirrhosa D. Don var. ecirrhosa Franch., Fritillaria przewalskii Maxim., Fritillaria cirrhosa D. Don var. cirrhosa, Fritillaria delavayi Franch., Fritillaria pallidiflora Schrenk, Fritillaria thunbergii Miq. var. chekiangensis Hsiao & K. C. Hsia, Fritillaria thunbergii Miq. (genotypes are CG or GG respectively).
[0019] Figure 2 It is the Sanger sequencing diagram of the Fritillaria unibracteata sample at position 192 in Example 2 of the present invention.
[0020] Figure 3This is the Sanger sequencing map at position 192 of samples of Fritillaria cirrhosa D. Don var. ecirrhosa Hsiao et K. C. Hsia, Fritillaria unibracteata Hsiao et K. C. Hsia, Fritillaria taipaiensis P. Y. Li, Fritillaria sichuanica P. K. Hsiao & K. C. Hsia, Fritillaria przewalskii Maxim., Fritillaria cirrhosa D. Don var. cirrhosa, Fritillaria delavayi Franch., Fritillaria pallidiflora Schrenk, Fritillaria thunbergii Miq. var. chekiangensis Hsiao & K. C. Hsia in Example 2 of the present invention. Detailed implementation manners
[0021] The present invention provides an SNP molecular marker related to the identification of Fritillaria wabuensis, and the SNP molecular marker is located at the 192nd base at the 5' end of the nucleotide sequence shown in SEQ ID NO: 1.
[0022] The polymorphism of the 192nd base is C / G.
[0023] Further, when the 192nd base is C / C homozygous, the sample is Fritillaria wabuensis; when the 192nd base is C / G heterozygous or G / G homozygous, the sample is not Fritillaria wabuensis.
[0024] The present invention also provides primers for amplifying the SNP molecular marker.
[0025] Preferably, the primers are as shown in SEQ ID NO: 2 and SEQ ID NO: 3.
[0026] The present invention also provides a reagent or kit for identifying the species of Fritillaria wabuensis, which contains the primers described in any of the above solutions.
[0027] The present invention also provides the application of the SNP molecular marker, the primers, or the reagent or kit in the identification of the species of Fritillaria wabuensis.
[0028] The present invention further provides a method for identifying the species of Fritillaria wabuensis, including: using the genomic DNA of the to-be-detected Fritillaria plant as a template, performing a PCR amplification reaction with the primers, analyzing the sequence information of the PCR amplification product, determining the genotype of the to-be-detected Fritillaria plant, and identifying whether it is Fritillaria wabuensis.
[0029] Preferably, the analysis of the sequence information of the PCR amplification product includes: sequencing the PCR amplification product and comparing the sequencing result with a reference sequence.
[0030] Preferably, the reference sequence is as shown in SEQ ID NO: 1.
[0031] Preferably, the sequencing is first-generation Sanger sequencing.
[0032] Preferably, the reaction conditions of the PCR amplification reaction are: 95°C for 3 min, and then 30 - 40 cycles are carried out. The cycle includes 94°C for 25 s - 30 s, 60°C - 64°C for 25 s - 30 s, 72°C for 10 s; finally, it ends with 72°C for 5 min.
[0033] Most preferably, the reaction conditions for the PCR amplification reaction are as follows: 95°C for 3 min; then 34 cycles are carried out, including 94°C for 25 s, 62°C for 25 s, 72°C for 10 s; and finally, it ends with 72°C for 5 min.
[0034] Preferably, the system for the PCR amplification reaction includes: 2× M5 Taq HiFi PCR mix, upstream primer, downstream primer, water, and genomic DNA.
[0035] Most preferably, the system for the PCR amplification reaction is a 20-μL system, which includes: 10 μL of 2× M5 Taq HiFi PCR mix, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, 8 μL of ddH2O, and 1 μL of genomic DNA.
[0036] Preferably, when the 192nd base is C / C homozygous, the sample species is Fritillaria unibracteata Hsiao et K. C. Hsia; when the 192nd base is C / G heterozygous or G / G homozygous, the sample is non-Fritillaria unibracteata Hsiao et K. C. Hsia.
[0037] The following examples are used to illustrate the present invention, but not to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.
[0038] Example 1
[0039] In this example, genomic high-throughput sequencing comparison was carried out to obtain single nucleotide polymorphism sites (SNPs) that are different and characterize genders between Fritillaria unibracteata Hsiao et K. C. Hsia and Fritillaria delavayi Franch., Fritillaria cirrhosa D. Don, Fritillaria taipaiensis P. Y. Li, Fritillaria sichuanica P. K. Hsiao & K. C. Hsia, Fritillaria przewalskii Maxim., Fritillaria cirrhosa D. Don var. cirrhosa, Fritillaria delavayi Franch. var. ecirrhosa Franch., Fritillaria pallidiflora Schrenk, Fritillaria thunbergii Miq. var. chekiangensis Hsiao & K. C. Hsia. The specific steps are as follows:
[0040] Ten samples of Fritillaria unibracteata, four samples of Fritillaria unibracteata var. wabuensis, two samples of Fritillaria cirrhosa var. ecirrhosa, and one sample each of Fritillaria taipaiensis, Fritillaria sichuanica, Fritillaria przewalskii, Fritillaria cirrhosa, Fritillaria delavayi, Fritillaria pallidiflora, Fritillaria thunbergii, and Fritillaria ussuriensis were selected for RNA extraction. According to the requirements of the Illumina library construction process, a transcriptome library was constructed. Then, the Illumina NovaSeq 6000 sequencing platform was used to perform high-throughput sequencing on each sample. The sequencing volume for each sample was 6 Gb, and the sequencing strategy was Pair-End 150 bp. The sequencing data of all samples were integrated and assembled into reference transcripts using Trinity. Further, the transcripts of each sample were assembled using Trinity separately. Sentieon was used to retrieve SNPs in the transcripts of each sample. The SNP retrieval results were screened, and the SNP sites that were exactly the same among the Fritillaria unibracteata samples and different between Fritillaria unibracteata and Fritillaria cirrhosa var. ecirrhosa, Fritillaria unibracteata var. wabuensis, Fritillaria taipaiensis, Fritillaria sichuanica, Fritillaria przewalskii, Fritillaria cirrhosa, Fritillaria delavayi, Fritillaria pallidiflora, Fritillaria thunbergii, and Fritillaria ussuriensis samples were selected as candidate molecular markers for the identification of Fritillaria unibracteata.
[0041] After analyzing the candidate molecular markers, it was found that the SEQ ID NO:1 sequence might contain SNP molecular markers that distinguish Fritillaria unibracteata from other samples. Primers were designed according to the sequence shown in SEQ ID NO:1 for PCR amplification. The upstream and downstream primers are shown in SEQ ID NO:2 and SEQ ID NO:3, respectively. The system for the PCR amplification reaction was 20 μL, including: 10 μL of 2× M5 Taq HiFi PCR mix, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, 8 μL of ddH2O, and 1 μL of genomic DNA. The reaction conditions for the PCR amplification reaction were: 95°C for 3 min; then 35 cycles were carried out, including 94°C for 25 s, 52°C for 25 s, and 72°C for 10 s; finally, it ended with 72°C for 5 min.
[0042] The results showed that a sequence of the target length could be amplified. By comparing the sequences through Sanger sequencing, it was found that among the above 10 samples of Fritillaria unibracteata, 4 samples of Fritillaria unibracteata var. wabuensis, 2 samples of Fritillaria cirrhosa var. ecirrhosa, and one sample each of Fritillaria taipaiensis, Fritillaria sichuanica, Fritillaria przewalskii, Fritillaria cirrhosa, Fritillaria delavayi, Fritillaria pallidiflora, Fritillaria thunbergii, and Fritillaria ussuriensis, the 192nd base of all Fritillaria unibracteata samples was C / C homozygous; the 192nd base of all Fritillaria cirrhosa var. ecirrhosa, Fritillaria unibracteata var. wabuensis, Fritillaria taipaiensis, Fritillaria sichuanica, Fritillaria przewalskii, Fritillaria cirrhosa, Fritillaria delavayi, Fritillaria pallidiflora, Fritillaria thunbergii, and Fritillaria ussuriensis samples was C / G heterozygous or G / G homozygous. The representative diagram of Sanger sequencing is as Figure 1 shown.
[0043] Example 2
[0044] To verify the accuracy of the SNP loci in Example 1 for identifying Fritillaria unibracteata, in this example, during the flowering period, extensive sampling was carried out in the distribution areas of Fritillaria unibracteata and its easily confused species. Genomic DNA was extracted from the samples to be tested. The specificity of the primers was verified by PCR and Sanger sequencing, and it was compared with the morphological and molecular characteristics of Fritillaria unibracteata to confirm the accuracy of the SNP molecular markers and primers in Example 1 for the species identification of Fritillaria unibracteata.
[0045] 1. Sample Preparation
[0046] Twenty-eight samples of Fritillaria from multiple regions such as Sichuan, Chongqing, Gansu, Yunnan, and Zhejiang were selected for verification, including 10 samples of Fritillaria unibracteata, 4 samples of Fritillaria delavayi var. euprepes, 4 samples of Fritillaria cirrhosa, 2 samples of Fritillaria taipaiensis, 2 samples of Fritillaria sichuanica, 1 sample of Fritillaria przewalskii, 1 sample of Fritillaria cirrhosa D. Don var. cirrhosa, 1 sample of Fritillaria thunbergii Miq. var. chekiangensis Hsiao et K. C. Hsia, 1 sample of Fritillaria pallidiflora Schrenk, 1 sample of Fritillaria thunbergii Miq., and 1 sample of Fritillaria thunbergii Miq. var. chekiangensis Hsiao et K. C. Hsia.
[0047] 2. Genomic DNA Extraction
[0048] The leaves of the collected Fritillaria plants were selected, and the genomic DNA of the Fritillaria plants to be tested was extracted using a DNA extraction kit.
[0049] 3. PCR Amplification
[0050] Using the genomic DNA of the Fritillaria plants to be tested as a template, PCR amplification reactions were carried out using the upstream primer SEQ ID NO:2 and the downstream primer SEQ ID NO:3.
[0051] The system of the PCR amplification reaction was 20 μL, including: 10 μL of 2× M5 Taq HiFi PCR mix, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, 8 μL of ddH2O, and 1 μL of genomic DNA.
[0052] The reaction conditions of the PCR amplification reaction were: 95°C for 3 min; then 35 cycles were carried out, including 94°C for 25 s, 62°C for 25 s, and 72°C for 10 s; finally, it ended at 72°C for 5 min.
[0053] 4. Analyze by combining Sanger sequencing to determine the genotype of the Fritillaria plants to be tested and identify whether it is Fritillaria unibracteata. The identification criteria are as follows: when the base at position 192 is C / C homozygous, the sample to be tested is Fritillaria unibracteata; when the base at position 192 is C / G heterozygous or G / G homozygous, the sample to be tested is not Fritillaria unibracteata.
[0054] The Sanger sequencing diagrams of 28 verification samples are as Figures 2 - 3 shown, and the identification results are shown in Table 1.
[0055] Table 1
[0056]
[0057] The results show that the Fritillaria unibracteata species information reflected by the SNP locus according to Example 1 is consistent with the species information of the collected samples, that is, the primers and SNP loci designed by the present invention can achieve accurate identification of Fritillaria unibracteata species.
[0058] Although the present invention has been described in detail with general descriptions and specific embodiments above, some modifications or improvements can be made based on the present invention, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.
Claims
1. A method for identifying the species of Fritillaria unibracteata, characterized in that, Comprising: Using the genomic DNA of the Fritillaria plant to be tested as a template, performing a PCR amplification reaction with primers shown in SEQ ID NO: 2-3, analyzing the PCR amplification product, determining the genotype of the Fritillaria plant to be tested, and identifying Fritillaria unibracteata according to the genotype; If the genotype of the polymorphic site in the SNP molecular marker of the amplification product is CC, it corresponds to the Fritillaria unibracteata species, and if the genotype of the polymorphic site is CG or GG, it corresponds to non-Fritillaria unibracteata species; Wherein, the SNP molecular marker is a nucleotide sequence with a polymorphism of C / G at the 192nd position at the 5' end of the sequence shown in SEQ ID NO:
1.
2. The authentication method according to claim 1, wherein The reaction conditions of the PCR amplification reaction are: 95°C for 3 min; 94°C for 25 s - 30 s, 60°C - 64°C for 25 s - 30 s, 72°C for 10 s, 30 - 40 cycles; 72°C for 5 min.