InDel molecular marker primer group for identifying variety of orchis polyphylla and application of InDel molecular marker primer group

Abstract

The invention belongs to the technical field of molecular markers, and in particular relates to an InDel molecular marker primer group for identifying varieties of Taiwan ivy mosses and application of the InDel molecular marker primer group for identifying the varieties of Taiwan ivy mosses. The InDel molecular marker primer group comprises 40 pairs of primers, and the nucleotide sequences of the primers are as shown in SEQ ID NO. 1 to SEQ ID NO. 80. The primer group is developed on the basis of a high-throughput sequencing technology, InDel fragment length data is obtained by performing PCR amplification and high-throughput sequencing on genome DNA of a sample to be detected, and authenticity identification and purity identification of the variety of the Taiwan drymoglossum are realized by comparing a difference marker number with a standard sample. The method overcomes the defects that a traditional morphological identification method is time-consuming and labor-consuming and is influenced by the environment, has the advantages of being rapid in detection, high in accuracy, high in flux and the like, and is suitable for germplasm resource protection and new variety registration and identification of the Taiwan dryopteris.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker technology, specifically relating to an InDel molecular marker primer set for the identification of dried varieties of *Moss woyangensis* and its application. Background Technology

[0002] Woyang dried moss is beloved by consumers for its unique flavor and nutritional value. Woyang dried moss has a long history of cultivation, and through long-term domestication and genetic improvement, a rich variety of local varieties with relatively stable genetics have been developed. Currently, the Woyang dried moss varieties promoted in production are diverse due to the influence of traditional planting and consumption habits, mainly including early-maturing, mid-maturing, and late-maturing varieties.

[0003] In the evaluation of seed resources, taxonomic classification, uniformity identification, and purity determination of *Moss woyangensis*, traditional methods mainly rely on morphological characteristics. However, these methods are not only time-consuming and labor-intensive, requiring significant land resources, but also often lead to discrepancies in trait descriptions due to differing standards among observers, thus affecting the accuracy of the results. In recent years, with the rapid development of molecular marker technology, different types of molecular markers have been gradually used for vegetable variety identification due to their advantages such as rapid detection, simple operation, and stable and reliable results.

[0004] InDel (insertion-deletion) markers refer to the differences between two parents in their whole genome. Relative to one parent, the other parent's genome contains a certain number of nucleotide insertions or deletions. Based on these insertion / deletion sites in the genome, PCR primers are designed to amplify these sites; these are called InDel markers. As high-throughput molecular markers, InDel markers have advantages such as rich variation, suitability for genomic molecular marker development, high accuracy, stable variation, and strong intra- and inter-species universality. Furthermore, compared to SSR markers, InDel marker amplification products have clearer and simpler band patterns, and their stability and product separation are significantly better than SSR markers. SSR marker amplification products are generally 100bp~450bp, and their amplification results are directly affected by DNA quality, making genotyping of highly degraded DNA samples difficult. In contrast, some InDel markers with shorter amplification products have lower requirements for DNA quality and can effectively genotype mixed DNA samples and highly degraded trace DNA samples.

[0005] Currently, there are very few reports on core InDel markers covering the entire genome of *Echinops vortex* stem based on InDel marker technology. Therefore, developing a primer set of core InDel molecular markers for *Echinops vortex* stem to achieve rapid detection and identification of *Echinops vortex* stem resource materials is a technical problem that urgently needs to be solved by technicians engaged in the genetic breeding, biotechnology, and DUS testing fields of *Echinops vortex* stem. Summary of the Invention

[0006] To address the above problems, this invention provides an InDel molecular marker primer set for the identification of dried varieties of *Moss vortex* and its application.

[0007] This invention is achieved through the following technical solution: An InDel molecular marker primer set for the identification of dried 'Woyang moss' varieties, the sequences of which are shown in SEQ ID NO.1~SEQ ID NO.80.

[0008] The application of the InDel molecular marker primer set in constructing the dry DNA fingerprint database of *Echinochloa vorticata*.

[0009] Application of the InDel molecular marker primer set in verifying the authenticity of dried 'Woyang moss' varieties.

[0010] The preferred and specific application methods are as follows: Genomic DNA was extracted from the dried moss of the test moss and the standard dried moss of the moss; PCR amplification was performed using the InDel molecular marker primer set as described in claim 1 to obtain the amplification product.

[0011] The amplified products were subjected to high-throughput sequencing to obtain the InDel molecular marker fragment length data of the sample to be tested.

[0012] The InDel molecular marker fragment length data of the test sample and the standard sample are compared to calculate the number of InDel molecular markers with differences.

[0013] If the number of different InDel molecular markers is ≥2, it is interpreted as a different variety; if the number of different InDel molecular markers is 1, it is interpreted as a similar variety; if the number of different InDel molecular markers is 0, it is interpreted as the same or very similar variety.

[0014] Preferably, the PCR amplification reaction program includes: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 60℃ for 40 s, extension at 72℃ for 40 s, repeated 35 times; extension at 72℃ for 10 min; and storage at 4℃.

[0015] Application of the InDel molecular marker primer set in the identification of the purity of dried moss.

[0016] The preferred and specific application methods are as follows: Genomic DNA was extracted from the dried moss of *Moss vortex* to be tested and the dried moss of *Moss vortex* as a standard.

[0017] PCR amplification was performed using the InDel molecular marker primer set as described above to obtain the amplification product.

[0018] High-throughput sequencing was performed on the amplified products to obtain the InDel molecular marker fragment length data of the test sample and the standard sample.

[0019] By comparing the InDel molecular marker fragment length data of the test sample and the standard sample, individuals with spectral bands that are not characteristic of this variety can be identified.

[0020] The purity of a variety is calculated using the formula P = (1 - A / B) × 100%, where A is the number of grains with characteristic bands not belonging to this variety, and B is the total number of grains in the sample to be tested.

[0021] Preferably, the PCR amplification reaction program includes: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 60℃ for 40 s, extension at 72℃ for 40 s, repeated 35 times; extension at 72℃ for 10 min; and storage at 4℃.

[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention provides for the first time an InDel molecular marker system containing 40 pairs of core primers, with nucleotide sequences shown in SEQ ID NO: 1~SEQ ID NO: 80, establishing a complete molecular technical solution for the identification of *Vallisneria vortex* varieties. Based on this primer set, a *Vallisneria vortex* DNA fingerprint database can be efficiently constructed, providing core technical support for germplasm resource management. At the application level, this invention establishes a scientific method for identifying the authenticity of varieties. By setting a clear threshold for the number of differential markers, it achieves accurate differentiation between different varieties, similar varieties, and identical varieties. It also provides a reliable purity identification scheme, enabling rapid and accurate assessment of seed purity through single-plant detection and statistical calculation. Compared with traditional identification methods relying on field phenotypes, this invention allows for direct molecular-level detection during the seedling stage, completely eliminating interference from environmental factors, significantly shortening the identification cycle, and saving substantial land and human resources. Furthermore, the molecular marker set of this invention can effectively distinguish *Vallisneria vortex* from lettuce and different *Vallisneria vortex* varieties, providing key technical guarantees for variety rights protection and new variety breeding. The PCR amplification and high-throughput sequencing technologies used are mature and stable, and the detection process is standardized, making it suitable for large-scale sample testing. It has broad prospects for promotion and application in fields such as seed testing, variety registration, and market supervision. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is the result of the purity test for this invention. Detailed Implementation

[0025] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. 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] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0027] The beneficial effects of the present invention will be illustrated below through specific embodiments: Dried moss from Woyang belongs to the Asteraceae family ( Compositae ), Lettuce ( Lettuce L.), lettuce ( Green lettuce L.), stem lettuce ( Green lettuce L. var. angustata Irish ex Bremer This is a cultivated variety of stem lettuce, also known as Gongcai, Taicai, Xiangcai, etc. It is a local variety of stem lettuce that originated in Yimen Town, Woyang County, Anhui Province. Its cultivation history can be traced back to the Ming and Qing Dynasties. Currently, it is widely cultivated in Woyang, Anhui; Pizhou, Jiangsu; Dali and Chuxiong, Yunnan.

[0028] This invention is based on high-throughput sequencing and multiplex PCR technology. Six varieties of Woyang moss (Gongcai), namely Yimiqing, Laolaiqing, Zhuyeqing, Daqingbang, Wantai No. 1, and Wantai No. 2, underwent 20-fold genome resequencing. The sequencing data was then compared with the lettuce reference genome (https: / / lettucegdb.com / genome) to screen for differentially expressed InDel sites. InDel sites with length differences of 10-20 bp, occurring between Woyang moss and lettuce, were selected. Molecular markers were developed, and after screening, 40 pairs of markers that could be amplified in the same PCR reaction without interference were chosen. The PCR products were then subjected to high-throughput sequencing to analyze fragment size and base arrangement, obtaining a nucleotide sequence length matrix. Finally, the Woyang moss DNA fingerprint database was constructed.

[0029] Based on the InDel detection principle, a fixed number of InDel primers are used to verify or identify the authenticity of a variety by comparing it with standard samples or with an InDel fingerprint data comparison platform. Authenticity verification is determined by the number of differences in InDel molecular markers among the specified number of primers, while variety authenticity identification is determined by screening and identification based on the principle that there are no differences in the number of InDel molecular markers.

[0030] By screening suitable primers that can accurately identify fingerprint data or patterns of abnormal individuals in a variety, the variety purity can be estimated by measuring the number or percentage of abnormal individuals in a certain number of test samples, thereby evaluating the overall typicality of the variety.

[0031] The Woyang moss and lettuce varieties used in this invention are both local stem lettuce varieties collected from Yimen Town, Woyang County, Anhui Province.

[0032] Example 1 The more primers used for detection, the lower the probability of missed detections and false positives, but the detection cost also increases. After comprehensive testing on commercial and local varieties of Woyang moss, as well as some lettuce varieties, this invention selected 40 InDel molecular markers of Woyang moss and their corresponding amplification primers based on screening principles such as high resolution and uniform chromosome distribution. The details are shown in Table 1. The primers in Table 1 can be divided into two groups, I and II. Group I is numbered M01~M20, and Group II is numbered M21~M40. Each group contains 20 pairs.

[0033] Table 1. Group I (20 core primer pairs) and Group II (20 core primer pairs) and related information. Table 1. Continued table of 20 core primer pairs in Group I and 20 core primer pairs in Group II and related information. Table 1. Continued table of 20 core primer pairs in Group I and 20 core primer pairs in Group II and related information. Table 1. Continued table of 20 core primer pairs in Group I and 20 core primer pairs in Group II and related information. Example 2: Constructing an InDel fingerprint data comparison platform for known varieties Using 40 representative varieties (lines) of Woyang moss and lettuce, an InDel fingerprint data comparison platform for known varieties was constructed based on the 40 InDel molecular markers and amplification primers described in Example 1.

[0034] Construction and usage of InDel fingerprinting: Forty pairs of InDel molecular markers evenly distributed on chromosomes were selected. After PCR amplification, differences in length polymorphism were observed among different varieties (in this patent, the differences were less than 20 bp). The length differences of each variety (line) were recorded to obtain a statistical table of InDel fragment lengths for that variety. Combining the length tables of different varieties (lines) yielded an InDel fragment length matrix, as shown in Table 2. Subsequent variety authenticity identification can be performed using this set of molecular markers for PCR amplification and high-throughput sequencing. The results are compared with the InDel fragment length list of the original variety. If they are completely consistent, the variety can be identified. If they are inconsistent, they can be compared with the InDel fragment length matrices of other varieties to determine whether other varieties have been used to impersonate this variety. Purity identification follows the same principle. At least 200 seeds or seedlings of a specific variety are selected, and DNA is extracted from each seed (plant) for PCR amplification and high-throughput sequencing. The existing information is compared to determine whether there are differences in InDel fragment lengths between individuals, and the variety purity is calculated.

[0035] Table 2. DNA fingerprint matrix of 40 representative stem lettuce varieties (lines). Table 2. DNA fingerprint matrix of 40 representative stem lettuce varieties (lines), continued. Table 2. DNA fingerprint matrix of 40 representative stem lettuce varieties (lines), continued. Example 3: Identification of Variety Authenticity and Purity Determination 1. Testing Plan For authenticity verification and purity determination, the accuracy and precision of the test results may vary depending on the primers, detection platform, and sample conditions. The principle of "suitability for the detection purpose" should be followed, taking into account the detection scale and capabilities to select appropriate primers, detection platforms, and sample conditions, and to develop a corresponding detection plan.

[0036] Under strictly controlled experimental conditions, selected primers were synthesized, and the samples were tested according to the established detection platform following the procedure of DNA extraction, PCR amplification, electrophoresis or high-throughput sequencing, and data analysis.

[0037] The test results should be filled out in accordance with the regulations, and the test report should indicate the key information that affects the test results based on the selected testing plan.

[0038] 2. Primers 2.1 Authenticity Verification 2.1.1 The more primers used, the lower the probability of missed detections and false positives, but the higher the detection cost. Therefore, a balance between accuracy and cost is necessary. After comprehensive testing on commercial and local varieties of Woyang moss, as well as some lettuce varieties, this standard selected 40 pairs of primers for variety authenticity identification based on screening principles such as high resolution and uniform chromosome distribution. These are detailed in Table 1, and an InDel fingerprint data comparison platform for known varieties was constructed accordingly. The primers in Table 1 are divided into two groups, I and II. Group I is numbered M01-M20, and Group II is numbered M21-M40, with 20 pairs in each group.

[0039] 2.1.2 Verification of variety authenticity emphasizes the rejection of results, and relatively speaking, the requirement for the number of primers is not high. A stepwise approach is allowed for testing. That is, the test is first performed using primer group I. If no differential sites are detected or the number of differential sites that can be used to determine the discrepancy is reached, the test can be terminated. If differential sites are detected but the number of differential sites that can be used to determine the discrepancy is not reached, the test is continued using primer group II.

[0040] 2.1.3 Variety authenticity identification emphasizes the affirmation of the results. Given the availability of an InDel fingerprint data comparison platform for known varieties, the specific variety can be identified through screening using known detection information. To minimize false positives, a high number of primers is required during testing. A stepwise approach can be used, or the 40 pairs of InDel primers in Table A.1 can be used directly until a match can be made with the InDel fingerprint data comparison platform. If, after comparison, there is still no locus difference with known varieties and a conclusion cannot be drawn, other distinguishable InDel molecular markers are permitted for detection.

[0041] 2.2 Purity Determination 2.2.1 Purity determination requires identifying the type of abnormal individual being tested. Different types of abnormal individuals require different primers and their numbers, and sometimes DNA fingerprint data from the corresponding parents is also needed.

[0042] 2.2.2 The primers selected for variety purity determination should be screened through preliminary tests on the test samples to identify abnormal individuals of the variety. Screening should also consider primer heterozygosity, rapid DNA extraction, and the potential for multiplex electrophoresis. Samples showing genetic instability at individual loci should be discarded, and purity determination should be terminated for samples exhibiting severe genetic instability.

[0043] 3. Testing Platform 3.1 Electrophoresis is a key step in the detection process. For authenticity verification, non-denaturing PAGE electrophoresis or capillary electrophoresis can be used. However, it should be noted that denaturing PAGE electrophoresis data is difficult to compare with the InDel fingerprint data comparison platform, making it difficult to verify authenticity. For purity determination, PAGE electrophoresis or capillary electrophoresis can be used. Agarose gel electrophoresis can also be used if primers with significant differences in allele amplification fragments are selected.

[0044] 3.2 For samples with large volumes, a sample grinder, an automated DNA extraction and pipetting workstation, a high-throughput PCR amplification instrument, and capillary electrophoresis combined with multiplex PCR can be combined to improve the overall efficiency of detection.

[0045] 3.3 High-throughput sequencing platforms, such as Illunima NovaSeq 6000, Hermo Fisher SOLiD, and Roche 454, can perform high-throughput sequencing of PCR products, avoiding the shortcomings of electrophoresis detection. This patent recommends prioritizing the use of high-throughput sequencing, as this method is simple, fast, and yields highly accurate results.

[0046] 3.4 Technical requirements for DNA extraction, PCR amplification, and electrophoresis: Provided that the requirements are suitable for the detection purpose and do not affect the detection quality, appropriate local modifications to the provisions of this standard are permitted in accordance with the requirements of the detection platform.

[0047] 4. Sample 4.1 Authenticity Verification 4.1.1 The sample submitted for testing shall be seeds, and the weight shall be not less than 5g or not less than 2000 seeds.

[0048] 4.1.2 Take representative samples from the submitted samples and test them using either a mixed sample or a single individual sample. The mixed sample should contain at least 10 individuals, and the single individual sample should contain at least 5 individuals.

[0049] 4.2 Purity Determination 4.2.1 The submitted sample shall be seeds. For pure-line seeds, the quantity shall be no less than 5g or no less than 1000 seeds. If the test is carried out simultaneously with the authenticity identification, the same submitted sample may be used.

[0050] 4.2.2 A specified number of test specimens shall be taken from the submitted samples, and each specimen shall be tested independently. The extraction and division of the submitted samples or test specimens shall be representative and comply with the provisions of GB / T3543.2. The number of test specimens shall contain at least 96 (including control) × 4 seeds.

[0051] 5. Testing conditions Authenticity verification or purity determination should be performed under controlled conditions that facilitate the correct implementation of the test, including but not limited to the following conditions: Seed inspectors possess the knowledge and skills to be familiar with the testing technologies used.

[0052] All instruments are compatible with the technology used and have been regularly maintained, verified, and calibrated.

[0053] Use appropriate grade reagents and sterilized consumables.

[0054] The appropriate reference sample used in the calibration affects the evaluation of test results.

[0055] 6. Instruments, equipment, reagents, and solution preparation 6.1 Instruments and Equipment High-speed centrifuge; PCR amplification instrument; high-voltage electrophoresis apparatus (3000V, 400mA, 400W); DNA electrophoresis tank; electronic balance (0.01g, 0.001g); mortar and pestle or tissue homogenizer; water bath; fume hood; micropipette (10L, 200μL, 1000μL); magnetic stirrer; film viewing lamp; horizontal shaker; autoclave; pH meter; ultra-low temperature freezer; beakers; forceps; graduated cylinders; volumetric flasks (500ml, 1000ml); centrifuge tubes (1.5ml, 2mL); pipette tips (10μL, 200μL, 1000μL); 96-well PCR plate; sealing film; disposable gloves; centrifuge tube rack; staining box; level; clamps, etc.

[0056] 6.2 Reagents Hexadecyltrimethylammonium bromide (CTAB); sodium chloride; chloroform; isoamyl alcohol; β-mercaptoethanol; tris(hydroxymethyl)aminomethane (Tris-base); 37% hydrochloric acid (v / v); disodium ethylenediaminetetraacetate (Na2EDTA-2H2O); sodium hydroxide; boric acid; 40% non-denaturing polyacrylamide stock solution (19:1) (v / v); tetramethylethylenediamine (TEMED); ammonium persulfate; silver nitrate; formaldehyde; ethanol.

[0057] Note: All reagents are of analytical grade. 6.3 Solution Preparation For the preparation of stock solutions of DNA extraction and electrophoresis reagents, please refer to Appendix B. The water used for reagent preparation should meet the requirements of Grade I water as specified in GB / T 6682, except that Grade III water can be used for the preparation of silver staining and developing solutions.

[0058] 7. Methods and Steps 7.1 DNA Extraction 7.1.1 Take the sample seed, grind it into powder using a mortar and pestle or tissue homogenizer, place it in a 2ml centrifuge tube, add 800L of 2% CTAB DNA extraction solution preheated at 65℃, and vortex for 1min.

[0059] 7.1.2 Place the centrifuge tube in a 65°C water bath for 40 min, inverting and mixing every 10 min. Remove the sample from the water bath, let it stand for a moment, add a mixture of 800 chloroform and isoamyl alcohol (24 chloroform: 1 isoamyl alcohol), mix for 15 min, then let it stand for 10 min, and centrifuge at 10000 r / min at room temperature for 10 min.

[0060] 7.1.3 Transfer 600 μL of the supernatant to a new 2 mL centrifuge tube, add 900 μL of 95% ethanol (v / v), and incubate at -20°C for 30 min. Centrifuge at 10000 rpm for 10 min, discard the supernatant, and retain the precipitate.

[0061] 7.1.4 Add 500 μL of 75% ethanol (v / v), gently tap the precipitate, centrifuge at 10000 r / min for 5 min, and discard the supernatant.

[0062] 7.1.5 Repeat step 7.1.4 once.

[0063] 7.1.6 Place the centrifuge tubes with the opening facing down to allow excess ethanol to drain out, and dry at room temperature for 1 hour or at 50°C for 20 minutes. Add 150 μL of sterile ddH2O (containing 1 μg / mL RNase) and dissolve in a 65°C water bath for 1 hour.

[0064] 7.1.7 Remove the centrifuge tube, place it at room temperature, centrifuge at 10000 r / min for 2 min, and transfer the supernatant to a new 1.5 mL centrifuge tube to remove starch and other precipitates.

[0065] 7.1.8 DNA concentration and quality were determined using an ultra-micro nucleic acid and protein detector, and its OD... 260 With OD 280 The ratio was 2.0, and it was diluted to 20 ng / μL for later use.

[0066] 7.2 PCR Amplification 7.2.1 Reaction System The DNA from the test samples and standard samples was amplified by PCR using 40 pairs of dried InDel core primers from *Echinochloa vorticata* according to this standard. The PCR reaction system was prepared by adding the components and amounts listed in Table 1 to the PCR tubes in an ice tray or at 4°C, as shown in Table 3.

[0067] Table 3 PCR reaction system 7.2.2 PCR reaction procedure The PCR reaction procedure is set up according to the reaction system in Table 3, as shown in Table 4.

[0068] Table 4 PCR reaction procedure Note: " / " indicates that this item is not present.

[0069] 7.3 PCR Product Analysis 7.3.1 Electrophoretic detection of PCR products 7.3.1.1 Preparation of electrophoresis glass plates Electrophoresis was performed using a 340mm×110mm electrophoresis glass plate. The plate was carefully cleaned with detergent, rinsed thoroughly with distilled water, then rinsed with distilled water, and finally wiped with 75% ethanol (by volume) and left to air dry.

[0070] 7.3.1.2 Gel Plate Preparation An 8% (w / w) non-denaturing polyacrylamide gel was used. 40 μL of 8% (w / w) non-denaturing polyacrylamide gel was taken, and 40 μL of TEMED and 400 μL of 10% ammonium persulfate were added. The mixture was thoroughly mixed and poured into the gel. Immediately after pouring, a 40 or 100-tooth high-throughput gel casting comb was inserted, and the mixture was allowed to stand for at least 20 minutes to allow the gel to completely solidify.

[0071] 7.3.1.3 Electrophoresis Assemble the gel plates into the electrophoresis tank and add 0.5×TBE buffer. Add 3 μL of PCR product to each sample, and simultaneously spot a DNA marker (minimum fragment 100 bp) as an indicator of the amplified fragment size. Set the electrophoresis voltage to 220V and the rated power per plate to 25W. Adjust the electrophoresis time according to the size of the amplified product; generally, stop electrophoresis when the xylene cyanide indicator band just appears on the gel plate.

[0072] 7.3.1.4 Silver staining After removing the gel from the glass plate, rinse it twice with distilled water to remove any residual electrophoresis buffer. Stain with AgNO3 staining solution at a final concentration of 0.1% (w / w) for 10 min.

[0073] 7.3.1.5 Development Rinse the gel twice with distilled water, then develop it with 1.5% (w / w) NaOH (containing 0.5% formaldehyde by volume) solution for 10 min. Finally, rinse the gel thoroughly with distilled water to terminate the color development reaction.

[0074] 7.3.1.6 Recording and Analysis of Electrophoresis Results 7.3.1.6.1 Spread the gel evenly on a glass plate, seal it with plastic wrap, and take a photograph. Referring to the DNA Marker electrophoresis results, record the PCR amplification results for each pair of InDel primers based on the number of bands amplified from the standard and test samples and their molecular weight, recording "1" for the presence of a corresponding band and "0" for the absence of a corresponding band. Within the allelic variant fragment size range, specific bands may appear as a single band or two bands. Bands outside the allelic variant fragment size range need to be identified as non-specific amplification or newly added rare allelic variants. Products amplified using a single individual showing three or more bands indicate non-specific amplification; products extracted from mixed samples showing three or more bands at certain sites require individual identification.

[0075] 7.3.1.6.2 The authenticity of the variety is determined by comparison with the standard sample. For the identified specific bands, the test sample and the standard sample are compared site by site on the same electrophoresis plate to determine the difference sites.

[0076] 7.3.2 High-throughput sequencing detection of PCR products 7.3.2.1 Introduction to Raw Data Raw image data files obtained from high-throughput sequencing (such as those from Illumina NovaSeq 6000 sequencing platforms) are converted into raw sequencing reads (Raw Data or Raw Reads) through base calling analysis. These reads are stored in FASTQ (fq) file format, which contains the sequence information of the reads and their corresponding sequencing quality information. Each read in the FASTQ file consists of four lines: the first line begins with "@", followed by Illumina sequence identifiers and descriptive text (optional); the second line is the base sequence; the third line begins with "+", followed by Illumina sequence identifiers (optional); and the fourth line indicates the sequencing quality of the corresponding sequence.

[0077] 7.3.2.2 Base Sequencing Quality Distribution The sequencing error rate for each base is obtained by converting the sequencing Phred score (Qphred) using Formula 1. The Phred score is calculated during base calling using a model that predicts the probability of errors in base discrimination. In the NovaSeq sequencing system, the library is first prepared into a microarray to immobilize the DNA template onto the chip. During immobilization, each DNA molecule forms a cluster, which represents a sequencing site. A very small number of clusters may physically overlap during immobilization. During sequencing, the sequencing software analyzes and identifies these overlapping points using the first four bases, separating them to ensure that each site represents a single DNA molecule. Therefore, the error rate for the first few bases at the 5′ end of the sequencing sequence is relatively high. Furthermore, the sequencing error rate increases with the length of the sequenced reads due to the consumption of chemical reagents during the sequencing process. Therefore, when performing base sequencing quality distribution analysis, the base quality values ​​of the first 4 bases and the last dozen or so bases in the sample will be lower than those of the middle sequencing bases, but their quality values ​​are all higher than Q30%. The sequencing error rate is related to base quality. According to the characteristics of sequencing technology, the error rate of the first few cycles and the end of the sequencing fragment will be higher.

[0078] 7.3.2.3 Base type distribution Base type distribution checks are used to detect the presence of AT / GC segregation, a phenomenon that can arise from sequencing or library preparation and affect subsequent analysis. High-throughput sequencing involves randomly fragmented DNA segments from the genome. Since the distribution of sites on the genome is approximately uniform, as are the G / C and A / T ratios, according to the law of large numbers, the GC and AT ratios should be equal in each sequencing cycle, and equal to the total GC and AT ratios of the genome. However, due to overlapping clusters, the AT and GC ratios in the first few bases of the sample can fluctuate significantly, exceeding those in other sequencing segments, while the GC and AT ratios in other segments are equal and uniformly distributed without segregation. In simplified sequencing, enzyme cleavage at specific locations fixes the first few bases of the sequence, leading to greater fluctuations in the GC ratio of these first few bases. Furthermore, because enzyme cleavage sites are fixed, the probability of obtaining identical fragments from different species increases, resulting in greater fluctuations in base type content.

[0079] 8. Result Judgment and Calculation 8.1 Judgment of the authenticity of dried moss from Woyang To determine the authenticity of a variety, the number of differentially expressed sites is calculated and verified using the electrophoretic analysis results of 40 InDel markers on the sample and standard samples, or the sequence length matrix obtained from high-throughput sequencing. The test results are expressed as the number of site differences between the sample and the standard sample.

[0080] If all 40 amplified InDel molecular markers in the test sample are completely identical in size and type to fragments of a known variety (line) in the database, they are considered to be from the same variety. If more than one InDel molecular marker amplifies fragments of inconsistent sizes (e.g., fragment size 300 / 300 in the database, but 310 / 310 in the sample), then that site is considered a differential site. The number of such differential sites determines whether they are actually from the same variety. This invention sets a threshold of 2; therefore, if two or more differential sites exist, they can be identified as different varieties.

[0081] If among the 40 InDel markers detected: a) If the number of difference markers between the test sample and the standard sample is ≥2, it is interpreted as a different variety; b) If the number of difference markers between the test sample and the standard sample is 1, it is interpreted as a similar variety; c) If the number of difference markers between the test sample and the standard sample is 0, the sample is identified as the same or very similar variety.

[0082] 8.2 Calculation of Variety Purity In terms of purity testing, the purity of the variety is calculated according to Formula 1 using the percentage table of the number of normal individuals (total number of tested samples minus the number of atypical individuals) to the total number of tested samples.

[0083] P=(1-A / B)×100%(1) In the formula: P is the purity of the sample variety to be tested; A is the number of grains with characteristic bands not of this variety; B is the total number of grains in the sample to be tested.

[0084] For example, randomly select individual plants from 96 varieties to be tested, extract DNA, and perform PCR amplification. Then compare whether there are differences in the length of the amplified fragments among the individuals. If the 96 samples are completely identical in the amplified product fragments of 40 pairs of InDel molecular markers and there are no differences, the purity is considered to be 100%. If there is a significantly different site in one sample, it is considered to be a hybrid (i.e., not a plant of this variety) and should be excluded when calculating the purity. Therefore, the purity = (96-1) / 96×100%≈99.0%, and so on.

[0085] 8.3 Authenticity Verification Results The sizes of PCR product fragments amplified by 40 pairs of Indel markers from 40 varieties (lines) were determined using the aforementioned molecular markers. For PCR fragments of the same size, M07 had a detection rate of 50% in dried Woyang moss and 7.1% in lettuce; M14 had a detection rate of 83.3% in dried Woyang moss and 7.1% in lettuce; M17 had a detection rate of 75% in dried Woyang moss but only 35.7% in lettuce. M02 had a detection rate of 53.6% in lettuce but none in dried Woyang moss; M03 accounted for 60.7% in lettuce but only 25% in dried Woyang moss. The above-mentioned marker combinations can be used to distinguish between Woyang moss and lettuce varieties. At the same time, the fingerprint database constructed using these 40 pairs of markers for 40 stem lettuce varieties can serve as an important basis for identifying new varieties. If a variety to be identified participating in production trials and regional trials has a fingerprint that is highly similar to or consistent with that of a variety in the database, it can be determined to be a non-new variety.

[0086] 8.4 Purity Test Results The seed production fields of Wantai No. 1 were tested using 20 pairs of Indel molecular markers from Group I. Eighty individual plants were randomly selected for purity testing, and a fingerprint matrix was created. All amplified fragments of consistent length and size were uniformly represented by dark gray, heterozygous bands or bands of inconsistent size were represented by light colors, and no amplified fragments were represented by blank. Figure 1 As shown in the figure, the results indicate that only one strain had an inconsistent amplified fragment size, while the remaining 79 strains showed the same size, thus the purity was 98.8%.

[0087] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0088] 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. An InDel molecular marker primer set for identifying Dryopteris filix-mas, characterized in that, The sequence of the InDel molecular marker primer set is shown as SEQ ID NO. 1-SEQ ID NO.

80.

2. The InDel molecular marker primer set of claim 1 in the construction of a DNA fingerprint database of Guanyang moss.

3. The InDel molecular marker primer set of claim 1 in the authenticity of Guanyang moss varieties.

4. Use according to claim 3, wherein the compound is ###0002### The specific application method is as follows: Genomic DNA of the to-be-tested Guanyang moss and standard Guanyang moss is extracted respectively; PCR amplification is performed using the InDel molecular marker primer set of claim 1 to obtain amplification products; High-throughput sequencing is performed on the amplification products to obtain InDel molecular marker fragment length data of the to-be-tested sample; The InDel molecular marker fragment length data of the to-be-tested sample and the standard sample are compared, and the number of different InDel molecular markers is calculated; If the number of different InDel molecular markers is ≥2, it is judged as different varieties; if the number of different InDel molecular markers is =1, it is judged as similar varieties; and if the number of different InDel molecular markers is =0, it is judged as the same or extremely similar varieties.

5. The use according to claim 4, wherein the compound is ###0002### The reaction program of the PCR amplification includes: 94℃ pre-denaturation for 5min; 94℃ denaturation for 30s, 60℃ annealing for 40s, 72℃ extension for 40s, 35 cycles; 72℃ extension for 10min; 4℃ storage.

6. The InDel molecular marker primer set of claim 1 in the purity identification of Guanyang moss.

7. Use according to claim 6, wherein The specific application method is as follows: Genomic DNA of the to-be-tested Guanyang moss and standard Guanyang moss is extracted respectively; PCR amplification is performed using the InDel molecular marker primer set to obtain amplification products; High-throughput sequencing is performed on the amplification products to obtain InDel molecular marker fragment length data of the to-be-tested sample and the standard sample; The InDel molecular marker fragment length data of the to-be-tested sample and the standard sample are compared, and the number of different InDel molecular markers is calculated; The purity of the variety is calculated according to the formula P=(1-A / B)×100%, wherein A is the number of characteristic bands of non-variety, and B is the total number of to-be-tested samples.

8. Use according to claim 7, wherein the compound is ###0002### The reaction program of the PCR amplification includes: 94℃ pre-denaturation for 5min; 94℃ denaturation for 30s, 60℃ annealing for 40s, 72℃ extension for 40s, 35 cycles; 72℃ extension for 10min; 4℃ storage.

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