SNP (Single Nucleotide Polymorphism) molecular marker for identifying species of fritillaria wangsiensis and application thereof

Abstract

The invention relates to the technical field of plant molecular biology, and provides an SNP (Single Nucleotide Polymorphism) molecular marker for identifying species of fritillaria wangsiensis and application of the SNP molecular marker. The SNP molecular marker contains a nucleotide sequence of which the polymorphism is C / G at the 192nd site at the 5'end of a sequence shown as SEQ ID NO: 1. The invention also provides an SNP molecular marker for identifying the species of the fritillaria wussuriensis and an identification method of the species of the fritillaria wussuriensis. According to the method, the fritillaria wabsiensis can be distinguished from easily-confused species such as the fritillaria thunbergii, the fritillaria unibiacteata, the fritillaria taipaiensis, the fritillaria pallidiflora, the fritillaria pallidiflora, the fritillaria cirrhosa, the fritillaria fusiformis, the fritillaria pallidiflora, the fritillaria ussuriensis and the fritillary bulb. The SNP molecular marker disclosed by the invention is favorably applied to the cultivation, identification and research processes of the fritillaria wussuriensis, so that the species of the fritillaria wussuriensis can be quickly distinguished, the identification efficiency is improved, the cultivation land is helped to be purified, and the planting cost and the research cost are reduced.

Description

Technical Field

[0001] The invention relates to the technical field of plant molecular biology, in particular to a SNP molecular marker for identifying species of Fritillaria var. Background Art

[0002] Fritillaria wabuensis Fritillaria unibracteata var. wabuensis ), belongs to the genus Fritillaria of the Liliaceae family, and is one of the original plants of the traditional Chinese medicine "Chuan Fritillaria". The dried bulbs of Wabu Fritillaria have the effects of clearing heat and moistening the lungs, resolving phlegm and relieving cough, dispersing nodules and eliminating carbuncle. It is used to treat lung heat and dry cough, dry cough with little phlegm, yin deficiency and fatigue cough, blood in sputum, scrofula, mastitis, and lung carbuncle. In the market, there is a large demand for its dried bulbs.

[0003] Wabu Fritillaria is a national second-class protected plant, and its wild resources are scarce. At present, Wabu Fritillaria is one of the most suitable species for cultivation among the original species of Sichuan Fritillaria, but there is still a phenomenon of mixed planting in the cultivation area of ​​Wabu Fritillaria, and there is a lack of effective means to distinguish Wabu Fritillaria from easily confused species of the same genus, such as Nongmi Fritillaria, Dark Purple Fritillaria, Taibai Fritillaria, Huaxi Fritillaria, Gansu Fritillaria, Curly Leaf Fritillaria, Suosa Fritillaria, Yi Fritillaria, Ping 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. At present, 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 purpose of the present invention is to provide a SNP molecular marker for identifying the species of Fritillaria var.

[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 balbisiana, wherein the SNP molecular marker contains a nucleotide sequence with a polymorphism of C / G at position 192 at the 5' end of the sequence as shown in SEQ ID NO:1.

[0008] Further, the genotype of the site with the polymorphism is CC, corresponding to the Wabu Fritillaria species, and the genotype of the site with the polymorphism is CG or GG, corresponding to non-Wbu Fritillaria species.

[0009] In a second aspect, the present invention provides primers for amplifying the SNP molecular markers.

[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 a kit containing the primers.

[0012] In a fourth aspect, the present invention provides the use of the SNP molecular marker, the primer or the detection reagent or kit in the identification of Fritillaria var.

[0013] In a fifth aspect, the present invention provides a method for identifying the species of Fritillaria dasyphylla, comprising: using the genomic DNA of the Fritillaria plant to be tested as a template, performing a PCR amplification reaction using the primers, analyzing the PCR amplification products, determining the genotype of the Fritillaria plant to be tested, and identifying the Fritillaria dasyphylla based on 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 species of Fritillaria wabuensis, and the genotype of the site with the polymorphism is CG or GG, it corresponds to the non-Fritillaria wabuensis species.

[0015] Preferably, the reaction conditions of the PCR amplification reaction are: 95°C for 3 min; 94°C for 25s-30s, 60°C-64°C for 25s-30s, 72°C for 10s, 30-40 cycles; 72°C for 5 min.

[0016] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The present invention can distinguish the Wabu Fritillaria from its easily confused species, such as the Nongmi Fritillaria, the Dark Purple Fritillaria, the Taibai Fritillaria, the Huaxi Fritillaria, the Gansu Fritillaria, the Curly-leaved Fritillaria, the Suosa Fritillaria, the Yi Fritillaria, the Ping Fritillaria, and the Zhejiang Fritillaria. The SNP molecular marker of the present invention is advantageously applied to the cultivation, identification and research of Wabu Fritillaria, so as to quickly distinguish the Wabu Fritillaria species, improve the identification efficiency, help purify the cultivation site, and reduce the planting cost and research cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a representative Sanger sequencing diagram of Example 1 of the present invention, wherein the upper diagram is of Fritillaria dasyphylla (genotype is CC), and the lower diagram is of Fritillaria thunbergii, Fritillaria thunbergii, Fritillaria taibai, Fritillaria huaxiensis, Fritillaria gansuensis, Fritillaria volvacea, Fritillaria thunbergii, Fritillaria yi, Fritillaria ping, and Fritillaria thunbergii (genotypes are CG or GG, respectively).

[0018] Figure 2 It is the Sanger sequencing map of the Fritillaria wabuensis sample at position 192 in Example 2 of the present invention.

[0019] Figure 3It is the Sanger sequencing map of the samples of Congmi Fritillaria, Dark Purple Fritillaria, Taibai Fritillaria, Huaxi Fritillaria, Gansu Fritillaria, Curly Leaf Fritillaria, Suosa Fritillaria, Yi Fritillaria, Ping Fritillaria, and Zhejiang Fritillaria at position 192 in Example 2 of the present invention. DETAILED DESCRIPTION

[0020] The invention provides a SNP molecular marker related to identification of Fritillaria var. wabuensis. The SNP molecular marker is located at the 192nd base at the 5' end of the nucleotide sequence shown in SEQ ID NO:1.

[0021] The polymorphism of the 192nd base is C / G.

[0022] Furthermore, when the 192nd base is homozygous for C / C, the sample is Fritillaria wabuensis; when the 192nd base is heterozygous for C / G or homozygous for G / G, the sample is not Fritillaria wabuensis.

[0023] The present invention also provides primers for amplifying the SNP molecular marker.

[0024] Preferably, the primers are shown as SEQ ID NO:2 and SEQ ID NO:3.

[0025] The present invention also provides a reagent or a kit for identifying the species of Fritillaria wabuensis, which contains the primers described in any of the above schemes.

[0026] The present invention also provides the use of the SNP molecular marker, the primer, or the reagent or the kit in the identification of Fritillaria var.

[0027] The present invention further provides a method for identifying the species of Fritillaria wabuensis, comprising: using the genomic DNA of the Fritillaria plant to be tested as a template, using the primers to perform a PCR amplification reaction, analyzing the sequence information of the PCR amplification product, determining the genotype of the Fritillaria plant to be tested, and identifying whether it is Fritillaria wabuensis.

[0028] Preferably, analyzing the sequence information of the PCR amplification product includes: sequencing the PCR amplification product, and comparing the sequencing result with a reference sequence.

[0029] Preferably, the reference sequence is shown as SEQ ID NO:1.

[0030] Preferably, the sequencing is first-generation Sanger sequencing.

[0031] Preferably, the reaction conditions of the PCR amplification reaction are: 95°C for 3 min, followed by 30-40 cycles, wherein the cycles include 94°C for 25s-30s, 60°C-64°C for 25s-30s, and 72°C for 10s; and finally 72°C for 5 min.

[0032] Most preferably, the reaction conditions of the PCR amplification reaction are: 95°C for 3 min; then 34 cycles, including 94°C for 25 s, 62°C for 25 s, and 72°C for 10 s; and finally 72°C for 5 min.

[0033] Preferably, the PCR amplification reaction system includes: 2× M5 Taq HiFi PCR mix, upstream primers, downstream primers, water, and genomic DNA.

[0034] Most preferably, the system for PCR amplification reaction is a 20 μL system, which includes: 2× M5 Taq HiFi PCR mix 10 μL, 10 μM upstream primer 0.5 μL, 10 μM downstream primer 0.5 μL, ddH2O 8 μL, and genomic DNA 1 μL.

[0035] Preferably, when the 192nd base is homozygous for C / C, the sample species is Fritillaria wabuensis; when the 192nd base is heterozygous for C / G or homozygous for G / G, the sample is not Fritillaria wabuensis.

[0036] The following examples are used to illustrate the present invention, but are not intended 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.

[0037] Example 1 This example uses high-throughput genome sequencing to compare and obtain single nucleotide polymorphism sites (SNPs) that are different between Fritillaria dahurica, Fritillaria nigra, Fritillaria taibaiensis, Fritillaria huaxiensis, Fritillaria gansuensis, Fritillaria volvacea, Fritillaria suosa, Fritillaria yi, Fritillaria pingensis, and Fritillaria thunbergii, and that characterize male and female sex. The specific steps are as follows: 10 samples of Fritillaria wabuensis, 4 samples of Fritillaria davidiana, 2 samples of Fritillaria nigra, and 1 sample each of Fritillaria taibaiensis, Fritillaria huaxiensis, Fritillaria gansuensis, Fritillaria truncatum, Fritillaria yi, Fritillaria pingensis, and Fritillaria thunbergii were selected for RNA extraction, and transcriptome libraries were constructed according to the requirements of the Illumina library construction process. Then, the Illumina NovaSeq6000 sequencing platform was used to perform high-throughput sequencing on each sample, with a sequencing volume of 6 Gb per sample and a sequencing strategy of Pair-End 150 bp. The sequencing data of all samples were integrated and assembled as reference transcripts using Trinity. Further, the transcripts of each sample were assembled separately using Trinity. Sentieon was used to perform SNP retrieval on the transcripts of each sample. The SNP retrieval results were screened, and the SNP sites that were exactly the same among the samples of Fritillaria wabui and different from those of Fritillaria wabui, Fritillaria nigra, Fritillaria taibai, Fritillaria huaxi, Fritillaria gansuensis, Fritillaria volvacea, Fritillaria thunbergii, Fritillaria yi, Fritillaria ping, and Fritillaria thunbergii were selected as candidate molecular markers for the identification of Fritillaria wabui.

[0038] After analyzing the candidate molecular markers, it was found that the SEQ ID NO:1 sequence may contain SNP molecular markers that distinguish Fritillaria wabuensis from other samples. Primers were designed according to the sequence shown in SEQ ID NO:1 for PCR amplification reaction, and the upstream and downstream primers are shown in SEQ ID NO:2 and SEQ ID NO:3, respectively. The system of PCR amplification reaction was 20μL, including: 2× M5Taq HiFi PCR mix 10μL, 10μM upstream primer 0.5μL, 10μM downstream primer 0.5μL, ddH2O 8μL, genomic DNA 1μL. The reaction conditions of PCR amplification reaction were: 95℃ 3min; then 35 cycles, including 94℃ 25s, 52℃ 25s, 72℃ 10s; and finally 72℃ 5min.

[0039] The results showed that the sequence of the target length could be amplified. Through Sanger sequencing and sequence alignment, it was found that in the above 10 samples of Fritillaria wabui, 4 samples of Fritillaria davidiana, 2 samples of Fritillaria nigra, and 1 sample each of Fritillaria taibai, Fritillaria huaxi, Fritillaria gansuensis, Fritillaria volvacea, Fritillaria thunbergii, Fritillaria yi, Fritillaria ping, and Fritillaria thunbergii, the 192nd base of all Fritillaria wabui samples was C / C homozygous; the 192nd base of all Fritillaria nigra, Fritillaria davidiana, Fritillaria taibai, Fritillaria huaxi, Fritillaria gansuensis, Fritillaria volvacea, Fritillaria thunbergii, Fritillaria yi, Fritillaria ping, and Fritillaria thunbergii samples was C / G heterozygous or G / G homozygous. The representative Sanger sequencing figures are shown in Figure 1 shown.

[0040] Example 2 In order to verify the accuracy of the SNP sites in Example 1 in identifying Fritillaria wabuensis, this example extensively sampled in the distribution area of ​​Fritillaria wabuensis and its easily confused species during the flowering period, extracted genomic DNA from the samples to be tested, verified the specificity of the primers by PCR and Sanger sequencing, and compared them with the morphological and molecular characteristics of Fritillaria wabuensis to confirm the accuracy of the SNP molecular markers and primers in Example 1 for species identification of Fritillaria wabuensis.

[0041] 1. Sample preparation 28 samples of Fritillaria collected from Sichuan, Chongqing, Gansu, Yunnan, Zhejiang and other regions were selected for verification, including 10 samples of Fritillaria wabui, 4 samples of Fritillaria nigra, 4 samples of Fritillaria purpurogenum, 2 samples of Fritillaria taibai, 2 samples of Fritillaria huaxiensis, 1 sample of Fritillaria gansuensis, 1 sample of Fritillaria curlifolia, 1 sample of Fritillaria thunbergii, 1 sample of Fritillaria yi, 1 sample of Fritillaria pingeba and 1 sample of Fritillaria thunbergii.

[0042] 2. Genomic DNA Extraction Select the leaves of the collected Fritillaria plants, and use a DNA extraction kit to extract the genomic DNA of the Fritillaria plants to be tested.

[0043] 3. PCR amplification A PCR amplification reaction was performed using the genomic DNA of the Fritillaria plant to be tested as a template and using the upstream primer SEQ ID NO: 2 and the downstream primer SEQ ID NO: 3.

[0044] The PCR amplification reaction system was 20 μL, including: 2× M5 Taq HiFi PCR mix 10 μL, 10 μM upstream primer 0.5 μL, 10 μM downstream primer 0.5 μL, ddH2O 8 μL, and genomic DNA 1 μL.

[0045] The reaction conditions of the PCR amplification reaction were: 95°C for 3 min; then 35 cycles, including 94°C for 25 s, 62°C for 25 s, and 72°C for 10 s; and finally 72°C for 5 min.

[0046] 4. Combined with Sanger sequencing analysis, the genotype of the Fritillaria plant to be tested was determined to identify whether it was Fritillaria wabui. The identification criteria were as follows: when the 192nd base was C / C homozygous, the sample to be tested was Fritillaria wabui; when the 192nd base was C / G heterozygous or G / G homozygous, the sample to be tested was not Fritillaria wabui.

[0047] Sanger sequencing of 28 validation samples Figure 2~Figure 3 The identification results are shown in Table 1.

[0048] Table 1

[0049] The results show that the species information of Fritillaria wabui reflected by the SNP sites in Example 1 is consistent with the species information of the collected samples, that is, accurate Fritillaria wabui species identification can be achieved through the primers and SNP sites designed by the present invention.

[0050] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A SNP molecular marker for identifying species of Fritillaria wabuensis, characterized in that: The SNP molecular marker contains a nucleotide sequence with a polymorphism of C / G at position 192 at the 5' end of the sequence shown in SEQ ID NO:

1.

2. The SNP molecular marker according to claim 1, characterized in that: 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 non-Fritillaria wabuensis species.

3. Primers for amplifying the SNP molecular marker according to claim 1 or 2.

4. The primer according to claim 3, characterized in that It includes an upstream primer as shown in SEQ ID NO:2 and a downstream primer as shown in SEQ ID NO:

3.

5. A detection reagent or kit containing the primers according to claim 3 or 4.

6. Use of the SNP molecular marker according to claim 1 or 2, the primer according to claim 3 or 4, or the detection reagent or kit according to claim 5 in the identification of Fritillaria var.

7. A method for identifying the species of Fritillaria wabuensis, characterized in that: include: The genomic DNA of the Fritillaria plant to be tested is used as a template, a PCR amplification reaction is performed using the primers described in claim 3 or 4, the PCR amplification products are analyzed, the genotype of the Fritillaria plant to be tested is determined, and the Wabu Fritillaria is identified according to the genotype.

8. The identification method according to claim 7, characterized in that: If the genotype of the site with the polymorphism in the SNP molecular marker as claimed in claim 1 or 2 contained in the amplification product is CC, it corresponds to the species of Fritillaria wabuensis, and the genotype of the site with the polymorphism is CG or GG, corresponding to the species other than Fritillaria wabuensis.

9. The identification method according to claim 7 or 8, characterized in that: The reaction conditions of PCR amplification reaction are: 95℃ for 3min; 94℃ for 25s~30s, 60℃~64℃ for 25s~30s, 72℃ for 10s, 30~40 cycles; 72℃ for 5min.

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