Method for constructing molecular identity card of Yunnan coptis chinensis by utilizing SNP (Single Nucleotide Polymorphism) marker

Abstract

The invention discloses a method for constructing a molecular identity card of Yunnan coptis chinensis by using an SNP (Single Nucleotide Polymorphism) marker, relates to the technical field of molecular identity construction of coptis chinensis, and solves the problems that the basic research of the existing coptis chinensis is weak and a method for developing the SNP marker and molecular identity card construction does not exist. Comprising the following steps: 1, extracting genome DNA of the Yunnan coptis chinensis leaves by using a plant genome DNA extraction kit, and detecting the integrity and purity of the genome DNA by adopting 1% agarose gel electrophoresis and Nanodrop; step 2, simplified genome sequencing: the extracted genome DNA is subjected to simplified genome sequencing, and the simplified genome sequencing comprises DNA sample detection, library construction, library quality control and computer sequencing. A large number of polymorphic SNP variation sites of the Yunnan coptis chinensis are obtained by utilizing a high-throughput simplified genome sequencing technology, and a plurality of molecular identity cards of the Yunnan coptis chinensis are successfully constructed based on a plurality of SNP markers, so that subsequent provenance identification and authenticity identification are facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of molecular identity construction of Coptis chinensis, and specifically to a method for constructing a Yunnan Coptis chinensis molecular identity card by utilizing SNP markers. Background Art

[0002] Yunnan Coptis chinensis, commonly known as Yunlian, is an important medicinal plant produced in Gaoligong Mountain. It contains berberine, jatrorrhizine and other compounds. Yunnan Coptis chinensis is mainly distributed in Tengchong, Fugong, Weixi, Deqin and other places in the northwest of Yunnan. Yunnan Coptis chinensis has both sexual reproduction and asexual reproduction, and is highly heterozygous. Due to natural and artificial propagation, Yunnan Coptis chinensis has accumulated natural evolution and artificial selection variations, forming a diverse Yunnan Coptis chinensis resource. Yunnan Coptis chinensis has rich phenotypic variations both within and between populations.

[0003] The research on Yunnan Coptis chinensis mainly focuses on resource survey, cultivation technology, pollination ecology, component content, pharmacology and other aspects. With the development of molecular marker technology, certain progress has been made in the genetic diversity, kinship and species identification of Coptis chinensis species. Early random amplified polymorphic DNA, simple sequence repeat amplification ISSR, and related sequence amplification polymorphism SRAP markers were developed to study the genetic diversity between and within species of Coptis chinensis. With the development of sequencing technology, simple sequence repeat SSR markers of Coptis chinensis were developed based on transcriptome sequences. Commonly used chloroplast and nuclear barcode DNA gene fragments were developed to identify interspecific kinship and evolutionary relationship analysis of Coptis chinensis. However, these markers have failed to effectively identify interspecific differences in Coptis chinensis, and are even more difficult to use for variety identification, which is not conducive to the protection, development and utilization of Coptis chinensis germplasm resources. DNA molecular ID cards can be used to quickly and accurately identify different individuals within a species, which can provide a scientific basis for the identification and protection of germplasm resources. The molecular markers for constructing DNA molecular ID cards mainly include SSR and single nucleotide polymorphism SNP. SNP markers are the third generation of molecular markers. Due to their large number of polymorphic sites, they can effectively distinguish different germplasm resources. More and more crops are using SNP-based fingerprint maps or molecular identity cards, including rice, corn, soybeans, angelica, Cistanche deserticola, and pecans.

[0004] There are differences between populations of Yunnan Coptis chinensis resources. In the past, due to irregular introduction and frequent exchange and propagation, the phenomenon of homonymous or homonymous products in production has not only brought many difficulties to the collection, protection and utilization of Yunnan Coptis chinensis resources, but also damaged the rights and interests of consumers. The traditional identification basis of Yunnan Coptis chinensis is mainly based on morphological traits such as leaf shape, density of leaflets and length of calyx. There are cross-overlapping of characteristic traits between different populations, making it difficult for morphological identification methods to quickly and accurately identify. The basic research of the genus Coptis chinensis is weak, and there is no method for developing SNP markers and molecular identity cards; therefore, it does not meet the existing needs. In this regard, we propose a method for constructing a molecular identity card of Yunnan Coptis chinensis using SNP markers. Summary of the invention

[0005] The purpose of the present invention is to provide a method for constructing a molecular identity card of Coptis chinensis Yunnanensis using SNP markers, so as to solve the problem mentioned in the above background technology that the basic research of the existing Coptis chinensis genus is weak and no method for constructing SNP markers and molecular identity cards has been developed.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for constructing a molecular ID card of Coptis chinensis using SNP markers, comprising the following steps:

[0007] Step 1: Use a plant genomic DNA extraction kit to extract genomic DNA from leaves of Coptis chinensis var. yunnanensis, and use 1% agarose gel electrophoresis and Nanodrop to detect the integrity and purity of the genomic DNA;

[0008] Step 2: Simplified genome sequencing: the extracted genomic DNA is subjected to simplified genome sequencing, which includes DNA sample detection, library construction, library quality control and on-machine sequencing;

[0009] Step 3: Develop polymorphic markers. For Stacks, tags are generated based on the similarity of the sequencing sequences. Each tag is equivalent to a ddRAD sequencing site. Then, tags between different samples are compared to develop polymorphic markers. The original output results of Stacks are screened to obtain available SNP molecular markers.

[0010] Step 4: Use the neighbor-joining method in MEGA7 software to construct a phylogenetic tree and use ggtree for visualization;

[0011] Step 5: Construct a molecular ID card. Digitally encode the 10 SNP data obtained through screening that can distinguish all samples as the basic information for constructing a molecular ID card. SNP markers have three genotypes, namely 0 / 0, 0 / 1 and 1 / 1, represented by 0, 1 and 2 respectively. The 10 SNPs obtain a 10-digit code. Taking sample 1 as an example, the genotype at V01 is 0 / 1, and the corresponding code is 1. Similarly, all genotypes are converted into 10-digit codes as 1111102212, and the numbers are converted into QR codes.

[0012] Preferably, the library construction comprises the following steps:

[0013] S1: After the sample genomic DNA is qualified, 200 ng genomic DNA is completely digested with restriction endonucleases MseⅠ and SacⅠ;

[0014] S2: The enzyme digestion products were connected with self-designed specific adapters, and then a specific amount of the connection products were pooled and purified with AMPure XP Beads;

[0015] S3: PCR enrichment using high-fidelity polymerase KOD-Plus-Neo;

[0016] S4: Pool all products and perform overnight low-pressure electrophoresis with Bio-Rad Certified Megabase Agarose, select the 300-400 bp range, and then purify with an agarose gel recovery kit.

[0017] Preferably, the library quality control comprises the following steps:

[0018] D1: Preliminary quantification using a fluorescence quantifier;

[0019] D2: Use Agilent 2100 to detect the insert size of the library. The next step of the experiment can be carried out only after the insert size meets expectations and there is no contamination by the adapter;

[0020] D3: Use real-time fluorescence quantitative PCR instrument to accurately quantify the effective concentration of the library, that is, the effective concentration > 2nM is a qualified library.

[0021] Preferably, the sequencing and data quality control comprises the following steps:

[0022] E1: Pool the libraries according to the target data volume, and perform paired-end 150bp sequencing using the Illumina HiSeq platform;

[0023] E2: Convert the obtained raw image data file into the original sequencing sequence through base recognition analysis;

[0024] E3: The mass numbers of bases are represented by different characters;

[0025] E4: The base quality number of Illumina HiSeq is represented by Q, and the sequencing error rate is represented by E. The relationship between Q and E is: Q = -log (E);

[0026] E5: The raw reads obtained by sequencing are filtered to obtain clean reads, and the adapter sequences contained in the clean reads are removed using fastp software;

[0027] E6: Use fastp software to remove low-quality bases in clean reads.

[0028] Preferably, the mass number of the base is the ASCII value corresponding to each character in the original sequencing sequence converted through base recognition analysis minus 33.

[0029] Preferably, the removal of low-quality bases in clean reads is performed by calculating the average mass number with a sliding window of 4 bp, and if the mass number of a base is lower than 15, all subsequent bases are removed.

[0030] Preferably, the original sequencing sequence is stored in a FASTQ file format.

[0031] Preferably, the length of the clean reads is greater than 50 bp.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. The present invention uses high-throughput simplified genome sequencing technology to obtain a large number of polymorphic SNP variation sites of Yunnan Coptis chinensis, and successfully constructs multiple molecular ID cards of Yunnan Coptis chinensis based on multiple SNP markers, which is helpful for subsequent provenance identification and authenticity identification. The phylogenetic tree constructed based on sequence information also successfully shows the different genetic relationships of multiple samples, providing a certain reference for the evolution and geographical distribution characteristics of Yunnan Coptis chinensis;

[0034] 2. The present invention converts genotype data into digital codes and constructs a fingerprint map; the fingerprint map is combined with the commodity code to obtain multiple Yunnan coptis identity cards, which can be used for commercial fast and accurate variety query and identification; after the SNP marker is converted into a KASP marker, it is more convenient to use, and multiple core SNP markers have been successfully developed and directly used to construct the molecular identity card of Yunnan coptis, and the SNP marker can be subsequently converted into a KASP marker for the authenticity detection of the Yunnan coptis variety in production. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The base mass number distribution diagram of sequencing data of five samples of the present invention;

[0036] Figure 2 The base content distribution diagram of sequencing data of five samples of the present invention;

[0037] Figure 3 is a phylogenetic tree diagram of the present invention;

[0038] Figure 4 Schematic diagram of the molecular ID coding of five samples of the present invention. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0040] See also Figures 1 to 4 The present invention provides an embodiment: a method for constructing a molecular ID card of Coptis chinensis using SNP markers, comprising the following steps:

[0041] Step 1: Use a plant genomic DNA extraction kit to extract genomic DNA from leaves of Coptis chinensis var. yunnanensis, and use 1% agarose gel electrophoresis and Nanodrop to detect the integrity and purity of the genomic DNA;

[0042] Step 2: Simplified genome sequencing: the extracted genomic DNA is subjected to simplified genome sequencing, which includes DNA sample detection, library construction, library quality control and on-machine sequencing;

[0043] Step 3: Develop polymorphic markers. For Stacks, tags are generated based on the similarity of the sequencing sequences. Each tag is equivalent to a ddRAD sequencing site. Then, tags between different samples are compared to develop polymorphic markers. The original output results of Stacks are screened to obtain available SNP molecular markers.

[0044] Step 4: Use the neighbor-joining method in MEGA7 software to construct a phylogenetic tree and use ggtree for visualization;

[0045] Step 5: Construct a molecular ID card, digitally encode the multiple SNP data obtained by screening and able to distinguish all samples, as the basic information for constructing a molecular ID card, SNP markers, there are 3 genotypes, namely 0 / 0, 0 / 1 and 1 / 1, represented by 0, 1 and 2 respectively, and multiple SNPs obtain the corresponding number of digital codes;

[0046] The 10 SNP data obtained through screening and capable of distinguishing all samples are now digitally encoded as the basic information for constructing a molecular ID card. The 10 SNPs are encoded with 10 digits. Taking sample 1 as an example, the genotype at V01 is 0 / 1, and the corresponding code is 1. Similarly, all genotypes are converted into 10-digit codes as 1111102212, and the numbers are converted into QR codes.

[0047] Library construction includes the following steps:

[0048] S1: After the sample genomic DNA is qualified, 200 ng genomic DNA is completely digested with restriction endonucleases MseⅠ and SacⅠ;

[0049] S2: The enzyme digestion products were connected with self-designed specific adapters, and then a specific amount of the connection products were pooled and purified with AMPure XP Beads;

[0050] S3: PCR enrichment using high-fidelity polymerase KOD-Plus-Neo;

[0051] S4: Pool all products and perform overnight low-pressure electrophoresis with Bio-Rad Certified Megabase Agarose, select the 300-400 bp range, and then purify with an agarose gel recovery kit.

[0052] Library quality control includes the following steps:

[0053] D1: Preliminary quantification using a fluorescence quantifier;

[0054] D2: Use Agilent 2100 to detect the insert size of the library. The next step of the experiment can be carried out only after the insert size meets expectations and there is no contamination by the adapter;

[0055] D3: Use real-time fluorescence quantitative PCR instrument to accurately quantify the effective concentration of the library, that is, the effective concentration > 2nM is a qualified library.

[0056] Further, sequencing and data quality control include the following steps:

[0057] E1: Pool the libraries according to the target data volume, and perform paired-end 150bp sequencing using the Illumina HiSeq platform;

[0058] E2: The obtained raw image data file is converted into the original sequencing sequence through base recognition analysis, and the original sequencing sequence is stored in the FASTQ file format;

[0059] E3: The mass of the base is represented by different characters. The mass of the base is the ASCII value corresponding to each character in the original sequencing sequence converted by base recognition analysis minus 33;

[0060] E4: The base quality number of Illumina HiSeq is represented by Q, and the sequencing error rate is represented by E. The relationship between Q and E is: Q = -log (E);

[0061] E5: The raw reads obtained by sequencing are filtered to obtain clean reads, and the adapter sequences contained in the clean reads are removed using fastp software;

[0062] E6: Use fastp software to remove low-quality bases in clean reads. The removal of low-quality bases in clean reads is to calculate the average mass number with a 4bp sliding window. If the mass number of the base is lower than 15, all subsequent bases are removed. The length of clean reads is greater than 50bp.

[0063] The test materials are 5 samples of Coptis chinensis collected from the Gaoligong Mountain area of ​​Yunnan. The collection information is shown in Table 1 below:

[0064] Table 1 Collection information of Coptis chinensis population in Yunnan

[0065]

[0066] Results and analysis: Sequencing data quality control and statistics. To ensure the normal progress of subsequent analysis, the quality score of sequencing data should be mainly distributed above 30, such as Figure 1 As shown in the figure, the results show that the quality scores of the sequencing data of the five samples are all above 30. The distribution of bases A, T, C, G, and N in the sequencing data is an important indicator for evaluating data quality. The content of N reflects the quality of sequencing. Figure 2 As shown in Table 2 below, the average GC content of single nucleotides of the five samples is 41.30%, the average contents of Q20 and Q30 are 98.12% and 94.10%, respectively, and the N content is close to 0. Therefore, the base content distribution and quality distribution in the sequencing data meet the expected requirements;

[0067] Table 2 Statistical tables of sequencing data volume and data quality of 5 samples

[0068]

[0069] Note: Reads: the number of clean PE reads measured for each sample; Bases: the number of clean bases measured for each sample; GC: the GC content of each sample; Q20: the percentage of bases with a mass number greater than 20; Q30: the percentage of bases with a mass number greater than 30.

[0070] Development of polymorphic markers: Compare tags between different Yunnan Coptis chinensis samples to develop polymorphic markers, and count the number of tags in each sample and the average sequencing depth, as shown in Table 3 of the following table. The average number of sequencing TAAGs for the five samples was 140908, and the average sequencing depth was 41.42. After screening the original output results of Stacks, a total of 34891 available SNP molecular markers were obtained, as shown in Table 4 of the following table, indicating that Yunnan Coptis chinensis has abundant molecular variation sites, which provides a theoretical reference for the development of SNP molecular markers, genetic diversity analysis, genetic evolution analysis, etc. of Yunnan Coptis chinensis.

[0071] Table 3 Statistics of sample tag number and sequencing depth

[0072] Table3 Statisticaltableoftagnumberandsequencing

[0073] depthofsamples

[0074]

[0075] Table 4 Statistical table of SNP marker type distribution

[0076]

[0077] Phylogenetic tree analysis: In order to study the evolutionary relationship of the five Yunnan Coptis chinensis samples, a phylogenetic tree was constructed using MEGA7 based on all valid SNP sites obtained after sequencing, and visualized using ggtree, as shown in Figure 2. Figure 3 As shown, the results show that sample 2 (Yaping, Fugong County) and sample 3 (Shiyueliang, Fugong County) are clustered together, indicating that the two samples are closely related and may have originated from the same population and then spread to different places; sample 4 (Shangpa Town, Fugong) and sample 5 (Laowo, Lushui) are each clustered into one category, indicating that they are distantly related, and sample 1 (Jiduluo to Dongshaofang, Gongshan County) is relatively closely related to samples 2 and 3, and distantly related to samples 4 and 5.

[0078] Construction of DNA molecular identity cards: Ten SNP markers that can distinguish the five samples were selected at different locations on the chromosome, as shown in Table 5 of the following table. Their numbers on the chromosomes are: Locus7732, Locus8626, Locus11326, Locus14484, Locus23512, Locus25773, Locus37776, Locus47930, Locus79720, Locus139627. Based on the combination of sequence information of the ten SNPs on the five samples, molecular identity cards of the five samples were constructed, as shown in Table 5 . Figure 4 As shown in the figure, a QR code for each sample was generated. These 10 SNP markers have good genotyping results. Samples 2 and 3 show the same genotyping in V01, V02, V05, V06, and V07 markers. Compared with other samples, they have more similarities, which is consistent with the results of the phylogenetic tree. The 10 SNP markers developed can be used for germplasm identification, kinship analysis, parent selection, etc. of Yunnan Coptis chinensis, greatly improving production efficiency and facilitating the viewing of genetic information of various germplasms.

[0079] Table 5 SNP information used to construct molecular identity cards and genotype in each sample

[0080]

[0081]

[0082] Note: 0 corresponds to Ref allele, 1 corresponds to Alt allele, Ref is A, Alt is T, then 0 / 0 corresponds to A / A, 1 / 1 corresponds to T / T, 0 / 1 indicates heterozygous A / T.

[0083] This application uses high-throughput simplified genome sequencing technology to obtain a large number of polymorphic SNP variant sites in Yunnan Coptis chinensis, and successfully constructs molecular ID cards for 5 Yunnan Coptis chinensis based on 10 SNP markers, which is helpful for subsequent provenance identification and authenticity identification. The phylogenetic tree constructed based on sequence information also successfully shows the different genetic relationships of the 5 samples, providing a certain reference for the evolution and geographical distribution characteristics of Yunnan Coptis chinensis.

[0084] In the past, due to the weak genetic basis of the genus Coptis, traditional phenotypic identification methods were mostly used to distinguish different species. However, this method not only requires strong botanical classification capabilities, is not suitable for production use, and is difficult to use to distinguish genotypic differences at the subspecific level. In recent years, the application of molecular marker technology based on sequencing in the identification of species in the genus Coptis has increased. Liu Xiaoguang et al. used PCR and sequencing technology to obtain sequence information of four gene fragments (rbcL4, ITS, trnH-psbA and matK) of 230 individuals of four species and one variant of the genus Coptis (Coptis chinensis, Emei Coptis chinensis, Triangular Leaf Coptis chinensis, Yunnan Coptis chinensis, and Short Calyx Coptis chinensis). However, they could only distinguish Yunnan Coptis chinensis from other species, and the identification ability of ITS was only 20-40%. It can be seen that these four commonly used DNA barcode fragment information is difficult to distinguish differences between species in the genus Coptis chinensis, and it also shows that there are obvious differences between Yunnan Coptis chinensis and other species.

[0085] The existing technology uses psbA-trnH, ycf1, ITS and ETS sequence information to identify 39 samples of the genus Coptis from 16 species and 3 variants from around the world, among which the ITS+ycf1 combination has a resolution of up to 79%. The developed barcode successfully identified Coptis chinensis, Coptis yunnanensis and Coptis triangularis. However, some species still cannot be identified, and the identification of materials at the sub-species level is still difficult. It can be seen that sequencing analysis based on a few gene fragments cannot meet the needs of accurate identification of Coptis chinensis species.

[0086] This application successfully used SNP molecular markers for the first time to effectively identify five genotypes of Coptis chinensis in different geographical regions in the genus Coptis chinensis, which has an important guiding role in the identification of species at the subspecific level of the genus Coptis chinensis. In addition, the results of the phylogenetic tree clustering of the five Coptis chinensis samples constructed based on all SNPs sites are consistent with the results of the molecular ID coding constructed based on the 10 SNP genotyping data, which shows that the 10 SNP markers selected have good representativeness and can be used as a basis for molecular classification of other Coptis chinensis species;

[0087] This application uses simplified genome resequencing technology to obtain a large number of SNP difference sites in Yunnan Coptis chinensis, successfully constructs a phylogenetic tree of 5 Yunnan Coptis chinensis samples based on SNP sequences, and screens out 10 core SNP markers that can be used to construct a molecular ID card and QR code for Yunnan Coptis chinensis, providing technical support for the identification of different Yunnan Coptis chinensis resources in subsequent production.

[0088] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A method for constructing a molecular ID card of Coptis chinensis Yunnanensis using SNP markers, characterized in that: The steps include: Step 1: Use a plant genomic DNA extraction kit to extract genomic DNA from leaves of Coptis chinensis var. yunnanensis, and use 1% agarose gel electrophoresis and Nanodrop to detect the integrity and purity of the genomic DNA; Step 2: Simplified genome sequencing: the extracted genomic DNA is subjected to simplified genome sequencing, which includes DNA sample detection, library construction, library quality control and on-machine sequencing; Step 3: Develop polymorphic markers. For Stacks, tags are generated based on the similarity of the sequencing sequences. Each tag is equivalent to a ddRAD sequencing site. Then, tags between different samples are compared to develop polymorphic markers. The original output results of Stacks are screened to obtain available SNP molecular markers. Step 4: Use the neighbor-joining method in MEGA7 software to construct a phylogenetic tree and use ggtree for visualization; Step 5: Construct a molecular ID card. Digitally encode the multiple SNP data obtained through screening that can distinguish all samples as the basic information for constructing a molecular ID card. There are three genotypes of SNP markers, namely 0 / 0, 0 / 1 and 1 / 1, represented by 0, 1 and 2 respectively. Multiple SNPs obtain the encoding of the corresponding number of digits, and the numbers are converted into QR codes.

2. The method for constructing a molecular ID card of Coptis chinensis using SNP markers according to claim 1, characterized in that: The library construction comprises the following steps: S1: After the sample genomic DNA is qualified, 200 ng genomic DNA is completely digested with restriction endonucleases MseⅠ and SacⅠ; S2: The enzyme digestion products were connected with self-designed specific adapters, and then a specific amount of the connection products were pooled and purified with AMPure XP Beads; S3: PCR enrichment using high-fidelity polymerase KOD-Plus-Neo; S4: Pool all products and perform overnight low-pressure electrophoresis with Bio-Rad Certified Megabase Agarose, select the 300-400 bp range, and then purify with an agarose gel recovery kit.

3. The method for constructing a molecular ID card of Coptis chinensis using SNP markers according to claim 2, characterized in that: The library quality control includes the following steps: D1: Preliminary quantification using a fluorescence quantifier; D2: Use Agilent 2100 to detect the insert size of the library. The next step of the experiment can be carried out only after the insert size meets expectations and there is no contamination by the adapter; D3: Use real-time fluorescence quantitative PCR instrument to accurately quantify the effective concentration of the library, that is, the effective concentration > 2nM is a qualified library.

4. The method for constructing a molecular ID card of Coptis chinensis using SNP markers according to claim 3, characterized in that: The sequencing and data quality control include the following steps: E1: Pool the libraries according to the target data volume, and perform paired-end 150bp sequencing using the Illumina HiSeq platform; E2: Convert the obtained raw image data file into the original sequencing sequence through base recognition analysis; E3: The mass numbers of bases are represented by different characters; E4: The base quality number of Illumina HiSeq is represented by Q, and the sequencing error rate is represented by E. The relationship between Q and E is: Q = -log (E); E5: The raw reads obtained by sequencing are filtered to obtain clean reads, and the adapter sequences contained in the clean reads are removed using fastp software; E6: Use fastp software to remove low-quality bases in clean reads.

5. The method for constructing a molecular ID card of Coptis chinensis using SNP markers according to claim 4, characterized in that: The mass number of the base is the ASCII value corresponding to each character in the original sequencing sequence converted through base recognition analysis minus 33.

6. The method for constructing a molecular ID card of Coptis chinensis using SNP markers according to claim 4, characterized in that: The removal of low-quality bases in clean reads is to calculate the average mass number with a sliding window of 4 bp, and if the mass number of a base is lower than 15, all subsequent bases are removed.

7. The method for constructing a molecular ID card of Coptis chinensis using SNP markers according to claim 4, characterized in that: The raw sequencing sequence is stored in the FASTQ file format.

8. The method for constructing a molecular ID card of Coptis chinensis using SNP markers according to claim 4, characterized in that: The length of the clean reads is greater than 50 bp.

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