Artemisia argyi ssr marker primer and application thereof in construction of artemisia argyi fingerprint

Abstract

The application discloses a folium artemisiae SSR marker primer and application thereof in construction of a folium artemisiae fingerprint, and belongs to the field of molecular biology. 28 pairs of SSR primers are screened through experiments, and primer sequences are shown as SEQ ID NO:1-56. Genetic diversity analysis of 139 folium artemisiae germplasms is carried out by using the 28 pairs of SSR molecular markers, which lays a foundation for accurate identification of folium artemisiae germplasm resources and molecular marker assisted breeding. The SSR marker developed by folium artemisiae transcriptome data has high polymorphism potential, and has high practicability in germplasm identification. The 139 pairs of folium artemisiae germplasms are used as test materials by using the SSR molecular marker, capillary electrophoresis detection is carried out, the use amount of the primer is further simplified according to the polymorphism information content of the primer, 6 pairs of core primers are screened, and the first DNA fingerprint containing the 139 pairs of folium artemisiae germplasms is established, which provides theoretical and technical support for identification and classification of folium artemisiae germplasm resources, germplasm innovation and new variety breeding.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically relating to an SSR marker primer for Artemisia argyi and its application in constructing Artemisia argyi fingerprints. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Artemisia argyi, also known as wormwood, is the dried leaf of Artemisia argyi Lévl. et Van, a plant belonging to the genus Artemisia in the Asteraceae family. It is a traditional Chinese medicine with a long history of application. It possesses the effects of warming the meridians and stopping bleeding, dispelling cold and relieving pain, and externally dispelling dampness and relieving itching. Clinically, it can also be used for hematemesis, metrorrhagia, menorrhagia, threatened abortion, and spermatorrhea. Furthermore, it is a major raw material for the internationally renowned moxibustion therapy, as well as a raw material for numerous pharmaceuticals and health products. Wild Artemisia argyi resources are abundant and distributed throughout most parts of China.

[0004] Research on Artemisia argyi has primarily focused on its chemical composition, pharmacological effects, quality standards, and clinical and daily applications, with limited research on its genetic diversity in molecular biology. In recent years, with the expanding market demand for Artemisia argyi, wild resources have fallen short of supply, leading to large-scale cultivation in many regions. However, due to the morphological similarities among Artemisia species and the numerous adulterants and complex genetic variations in Artemisia argyi germplasm, problems such as introduction confusion, unclear germplasm, and varietal degeneration have arisen during artificial cultivation, severely impacting yield and quality and hindering the protection and utilization of Artemisia argyi germplasm resources. Therefore, developing a set of molecular markers to protect the genetic diversity of Artemisia argyi germplasm is urgently needed.

[0005] Simple sequence repeats (MSLs), also known as microsatellite DNA, are a class of tandem repeat sequences consisting of several nucleotides (usually 1-6) as repeating units. They possess advantages such as high polymorphism, good stability, multiple alleles, codominance, abundant quantity, good genome coverage, and ease of manipulation. In recent years, they have been widely used in germplasm resource research, hybrid identification, genetic map construction, target gene localization, and genetic diversity research.

[0006] Compared to morphological identification methods, DNA fingerprinting possesses high individual specificity and environmental stability. It is an effective technique for strain identification, based on nucleotide site variations within an individual and differentiating strains according to genomic differences. Developing molecular markers is a crucial step in constructing fingerprints. However, large-scale DNA fingerprinting of Artemisia argyi germplasm resources has not yet been constructed using markers, despite its vital role in germplasm resource identification. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide an SRR marker primer for Artemisia argyi, a primer combination, and a fingerprint map. Genetic diversity analysis of 139 Artemisia argyi germplasm resources was conducted using 28 pairs of SSR primers. The amount of primers used was further simplified by utilizing polymorphic information content, resulting in the selection of 6 core SSR primer pairs. This led to the construction of DNA fingerprint maps for the 139 Artemisia argyi germplasm resources, laying the foundation for accurate identification of Artemisia argyi germplasm resources and the breeding of superior varieties.

[0008] The 28 pairs of SSR primers provided by this invention enable the genetic diversity analysis of 139 Artemisia argyi germplasm resources using SSR technology, laying the foundation for the construction of core Artemisia argyi germplasm and the breeding and utilization of germplasm resources.

[0009] The 28 pairs of SSR marker primers include SSR40, SSR99, SSR109, SSR14, SSR03, SSR15, SSR79, SSR01, SSR191, SSR10, SSR95, SSR65, SSR33, SSR31, SSR172, SSR09, SSR22, SSR55, SSR182, SSR92, SSR70, SSR34, SSR46, SSR162, SSR114, SSR06, SSR118, and SSR175, and the primer sequences are shown in SEQ ID NO:1-56.

[0010] The fingerprinting method of this invention enables the differentiation of 139 Artemisia argyi germplasm resources into different strains. Each variety has a unique fingerprint, exhibiting high accuracy and specificity. For example, the fingerprint of Qi Ai (HBQQ), one of the "Four Famous Artemisia argyi", is: 00000000100000 100100000000000 0000000000000100000 000110000000010010000100; the fingerprint of Hai Ai (ZJNB2) is: 00000100010000 000000000000000 0000000000010000000 000010010000000001000100; The fingerprint spectrum of Qi Ai (HeBBD2) is: 0000001000000000000000000000000000000000 00011010000 1000001000000 00000001000; The fingerprint spectrum of Bei Ai (HeNAY) is: 00010000010000 000000000000010 0000100000 000100001000000100000000000000010.

[0011] It is worth noting that the six pairs of core markers provided by this invention establish the first DNA fingerprint information database containing 139 Artemisia argyi germplasm resources, providing an effective way for the accurate identification of Artemisia argyi germplasm resources; and removing any pair of the six core markers will cause the fingerprint pattern to become invalid and make it impossible to accurately identify Artemisia argyi germplasm resources, and adding any pair of the six core markers will not have any beneficial effect on the identification.

[0012] In a first aspect, the present invention provides an Artemisia argyi SSR marker primer, wherein the Artemisia argyi SSR marker primer comprises a pair of primers selected from the following:

[0013] SSR40 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO.1-2;

[0014] SSR99 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO.3-4;

[0015] SSR109 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO.5-6;

[0016] SSR14 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO.7-8;

[0017] SSR03 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO. 9-10;

[0018] SSR15 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO.11-12;

[0019] SSR79 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO.13-14;

[0020] SSR01 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO.15-16;

[0021] SSR191 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO.17-18;

[0022] SSR10 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO.19–20;

[0023] SSR95 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO.21-22;

[0024] SSR65 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO.23-24;

[0025] SSR33 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO. 25–26;

[0026] SSR31 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO.27-28;

[0027] SSR172 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO.29–30;

[0028] SSR09 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO.31-32;

[0029] SSR22 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO.33-34;

[0030] SSR55 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO.35-36;

[0031] SSR182 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO.37-38;

[0032] SSR92 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO.39–40;

[0033] SSR70 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO.41-42;

[0034] SSR34 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO.43-44;

[0035] SSR46 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO.45–46;

[0036] SSR162 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO.47-48;

[0037] SSR114 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO.49–50;

[0038] SSR06 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO.51-52;

[0039] SSR118 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO. 53-54;

[0040] SSR175 includes forward and reverse primers with nucleotide sequences as shown in SEQ ID NO. 55–56.

[0041] Secondly, the present invention provides a combination of Artemisia argyi SSR marker primers, comprising two or more pairs of the Artemisia argyi SSR marker primers described above.

[0042] (1) Primers for amplifying the SSR molecular marker SSR40:

[0043] SEQ ID NO:1:SSR40-F:5'-ACAACCTCTTACCACCCA-3';

[0044] SEQ ID NO:2:SSR40-R:5'-CCCTCACTTTATCCCAAT-3';

[0045] (2) Primers for amplifying the SSR molecular marker SSR99:

[0046] SEQ ID NO:3:SSR99-F:5'-AATCGTTGACGCATTCCTT-3';

[0047] SEQ ID NO:4:SSR99-R:5'-TTGTTGTAGCAGCAGATGAGTTA-3';

[0048] (3) Primers for amplifying the SSR molecular marker SSR109:

[0049] SEQ ID NO:5:SSR109-F:5'-TACGCAATACACAATAGTCAAAGTC-3';

[0050] SEQ ID NO:6:SSR109-R:5'-CATCAGATCCATCGTTCCAAC-3';

[0051] (4) Primers for amplifying the SSR molecular marker SSR14:

[0052] SEQ ID NO:7:SSR14-F:5'-AGAGCCTAGAATCCAACG-3';

[0053] SEQ ID NO:8:SSR14-R:5'-ATGCGATACCGAATGTGA-3';

[0054] (5) Primers for amplifying the SSR molecular marker SSR03:

[0055] SEQ ID NO:9:SSR03-F:5'-CAACCAAACCACAGGGAC-3';

[0056] SEQ ID NO:10:SSR03-R:5'-CACAGCTCAAGGCAACAC-3';

[0057] (6) Primers for amplifying the SSR molecular marker SSR15:

[0058] SEQ ID NO:11:SSR15-F:5'-CTCCTCCACCTCCACTCA-3';

[0059] SEQ ID NO:12:SSR15-R:5'-GAATCAGGCTCAAGCAAA-3';

[0060] (7) Primers for amplifying the SSR molecular marker SSR79:

[0061] SEQ ID NO:13:SSR79-F:5'-TAATGATTGAGACACCCTTTTG-3';

[0062] SEQ ID NO:14:SSR79-R:5'-AGCAGCCACTTCACCCAG-3';

[0063] (8) Primers for amplifying the SSR molecular marker SSR01:

[0064] SEQ ID NO:15:SSR01-F:5'-CTATGCCTGGTTCGGTGGTC-3';

[0065] SEQ ID NO:16:SSR01-R:5'-TGTCGTCGTCGCCTTCCT-3';

[0066] (9) Primers for amplifying the SSR molecular marker SSR191:

[0067] SEQ ID NO:17:SSR191-F:5'-TTTCACACTCTCCCTCGTCTTC-3';

[0068] SEQ ID NO:18:SSR191-R:5'-AACTGTTGCGGTTTTGGTACTC-3';

[0069] (10) Primers for amplifying the SSR molecular marker SSR10:

[0070] SEQ ID NO:19:SSR10-F:5'-ATGGATCTCCGTCGTCTA-3';

[0071] SEQ ID NO:20:SSR10-R:5'-TCTGAACCCTAACCAACTC-3';

[0072] (11) Primers for amplifying the SSR molecular marker SSR95:

[0073] SEQ ID NO:21:SSR95-F:5'-CAGGTGTATGGATGGATGTGC-3';

[0074] SEQ ID NO:22:SSR95-R:5'-CTTGTTGGAACCCAGCATAAAT-3';

[0075] (12) Primers for amplifying the SSR molecular marker SSR65:

[0076] SEQ ID NO:23:SSR65-F:5'-TTATCTCACCTGGCACTTGG-3';

[0077] SEQ ID NO:24:SSR65-R:5'-TGTGGAATAGGGACCTCATG-3';

[0078] (13) Primers for amplifying the SSR molecular marker SSR33:

[0079] SEQ ID NO:25:SSR33-F:5'-ATTAAATTCCACGCCATCA-3';

[0080] SEQ ID NO:26:SSR33-R:5'-GGAGGTGTCCCAACAAAC-3';

[0081] (14) Primers for amplifying the SSR molecular marker SSR31:

[0082] SEQ ID NO:27:SSR31-F:5'-TCGCTGTTGGATGAAGTG-3';

[0083] SEQ ID NO:28:SSR31-R:5'-CTAAGCATGGTTTGTGGG-3';

[0084] (15) Primers for amplifying the SSR molecular marker SSR172:

[0085] SEQ ID NO:29:SSR172-F:5'-CCTTCATTCAACTCACTCCAA-3';

[0086] SEQ ID NO:30:SSR172-R:5'-AAAAACCCCATAAATCCCTA-3';

[0087] (16) Primers for amplifying the SSR molecular marker SSR09:

[0088] SEQ ID NO:31:SSR09-F:5'-CCCTCCAGTGAAATACGA-3';

[0089] SEQ ID NO:32:SSR09-R:5'-GCATCTGTCCCACTTCTG-3';

[0090] (17) Primers for amplifying the SSR molecular marker SSR22:

[0091] SEQ ID NO:33:SSR22-F:5'-TGGAAATAAGGACGAATG-3';

[0092] SEQ ID NO:34:SSR22-R:5'-AACACGGCAAATAACTC-3';

[0093] (18) Primers for amplifying the SSR molecular marker SSR55:

[0094] SEQ ID NO:35:SSR55-F:5'-ACGGCGTCGTTTATTCC-3';

[0095] SEQ ID NO:36:SSR55-R:5'-GGTCCTTCCCATCCTGTG-3';

[0096] (19) Primers for amplifying the SSR molecular marker SSR182:

[0097] SEQ ID NO:37:SSR182-F:5'-TAAAGAAGCAGTGAGAACAAGCATC-3'; SEQ ID NO:38:SSR182-R:5'-GGTATCAATAAAAGTCAAGCGAAGT-3';

[0098] (20) Primers for amplifying the SSR molecular marker SSR92:

[0099] SEQ ID NO:39:SSR92-F:5'-CCCAACCACCAACGGCAC-3';

[0100] SEQ ID NO:40:SSR92-R:5'-TGAGAAAAAGGTCGTAGAGGAGG-3';

[0101] (21) Primers for amplifying the SSR molecular marker SSR70:

[0102] SEQ ID NO:41:SSR70-F:5'-GATGGGTATGGAAAGACGA-3';

[0103] SEQ ID NO:42:SSR70-R:5'-ATAAACTTGCCAGCTCAA-3';

[0104] (22) Primers for amplifying the SSR molecular marker SSR34:

[0105] SEQ ID NO:43:SSR34-F:5'-TTGTGGGATGCTGTTTGC-3';

[0106] SEQ ID NO:44:SSR34-R:5'-TTGGTGGTACTGGAAGAAGT-3';

[0107] (23) Primers for amplifying the SSR molecular marker SSR46:

[0108] SEQ ID NO:45:SSR46-F:5'-CGCCAATGGGTCCTTCGT-3';

[0109] SEQ ID NO:46:SSR46-R:5'-TGGGCACTTTCTGGAGTGTATT-3';

[0110] (24) Primers for amplifying the SSR molecular marker SSR162:

[0111] SEQ ID NO:47:SSR162-F:5'-CACAAAAGTCACTATCCCTCTCG-3';

[0112] SEQ ID NO:48:SSR162-R:5'-TTCAAGGTTTAGGCACCACAATA-3';

[0113] (25) Primers for amplifying the SSR molecular marker SSR114:

[0114] SEQ ID NO:49:SSR114-F:5'-CACTCGTGTTTCTCCTTGGCTT-3';

[0115] SEQ ID NO:50:SSR114-R:5'-GAATCGGTCAGGTACTTGGCTA-3';

[0116] (26) Primers for amplifying the SSR molecular marker SSR06:

[0117] SEQ ID NO:51:SSR06-F:5'-ACCTGGTTCCGACTTGA-3';

[0118] SEQ ID NO:52:SSR06-R:5'-ACACGCACATCATTCCCT-3';

[0119] (27) Primers for amplifying the SSR molecular marker SSR118:

[0120] SEQ ID NO:53:SSR118-F:5'-CAAACCACAATACAGGTCGCA-3';

[0121] SEQ ID NO:54:SSR118-R:5'-TGTTTTGTATTTTCGTCCTTTGG-3';

[0122] (28) Primers for amplifying the SSR molecular marker SSR175:

[0123] SEQ ID NO:55:SSR175-F:5'-AGAACAAATATCCAAAACCACAA-3';

[0124] SEQ ID NO:56:SSR175-R:5'-CAGAAGGTATAACAAAGTAGCCG-3'.

[0125] Furthermore, the combination is a combination of SSR40, SSR99, SSR109, SSR14, SSR03, and SSR15.

[0126] Thirdly, the present invention provides a kit containing at least the above-mentioned Artemisia argyi SSR marker primers or Artemisia argyi SSR marker primer combinations.

[0127] Fourthly, the present invention provides the application of the above-mentioned Artemisia argyi SSR marker primers, Artemisia argyi SSR marker primer combinations, or kits in any one or more of the following:

[0128] 1) Analysis of genetic diversity in Artemisia argyi;

[0129] 2) Construct a genetic map of Artemisia argyi;

[0130] 3) Germplasm identification of Artemisia argyi;

[0131] 4) Phylogenetic analysis of Artemisia argyi;

[0132] 5) Discovery of functional genes in Artemisia argyi;

[0133] 6) Artemisia argyi molecular marker-assisted breeding;

[0134] Preferably, in application 2), constructing the genetic map of Artemisia argyi specifically involves constructing the fingerprint map of Artemisia argyi.

[0135] Fifthly, the present invention provides a method for identifying the genetic diversity or germplasm of Artemisia argyi or constructing an Artemisia argyi fingerprint, comprising the following steps:

[0136] (1) Extract total DNA from the Artemisia argyi sample to be tested;

[0137] (2) Using the above Artemisia argyi SSR marker primers, Artemisia argyi SSR marker primer combinations or kits, add the universal M13 adapter sequence to the forward primer of each pair of primers to obtain the M13 adapter forward primers.

[0138] (3) Using the total DNA extracted in step (1) as a template, PCR amplification was performed using the above Artemisia argyi SSR marker primers, Artemisia argyi SSR marker primer combinations or the reverse primers of each pair of primers described in the kit, and the forward primers of the M13 adapter, to obtain fluorescent PCR amplification products.

[0139] (4) The fluorescent PCR amplification products were subjected to electrophoresis to obtain electrophoresis data and the band detection results were statistically analyzed.

[0140] (5) Based on the statistical band detection results in step (4), perform genetic diversity analysis of Artemisia argyi, cluster analysis, polymorphic information content (PIC) calculation, and DNA fingerprinting.

[0141] The mugwort sample is a young, tender leaf of mugwort.

[0142] Furthermore, in step (3), the PCR amplification system is 10 μL, comprising: 2 μL of DNA template with a concentration of 50 ng / μL, 5 μL of 2×Es Taq MsaterMix, 0.04 μL of M13 adapter forward primer with a concentration of 10 pmol / μL, 0.25 μL of reverse primer with a concentration of 10 pmol / μL, 0.15 μL of M13 fluorescent modification group, and 2.56 μL of H2O.

[0143] Furthermore, in step (3), the PCR amplification program is as follows: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; finally, 72℃ end extension for 5 min, and storage at 4℃.

[0144] Furthermore, the specific methods for genetic diversity analysis and germplasm genetic identification in step (5) are as follows:

[0145] A. Based on the band detection results in step (4), record the amplified fragment size of each Artemisia argyi sample corresponding to each pair of primers;

[0146] B. Based on the size of the amplified fragment obtained from the comprehensive analysis, the following genetic diversity parameters of each primer pair were calculated using Popgen32 software, such as the number of alleles (Na), the effective number of alleles (Ne), the Shannon information index (I), and the allele frequency of different primers. After calculating the alleles of different primers using Popgen32 software, the polymorphism information content (PIC) was calculated using PIC_Cale software.

[0147] C. Based on the amplified fragment sizes obtained in step A, the amplified fragments are numbered and read from largest to smallest. The presence of an amplified band is recorded as "1", and the absence of a band is recorded as "0". An original matrix is ​​established, imported into MEGA11 software to draw the NJ tree, and the original matrix is ​​used to construct DNA fingerprint maps of 139 Artemisia argyi germplasm resources.

[0148] In a sixth aspect, the present invention provides an Artemisia argyi fingerprint spectrum, which is constructed by the method described in any one of claims 6-8, wherein all alleles are converted into 1 / 0 format, with 1 and 0 representing the corresponding detected and undetected fragments, respectively.

[0149] Furthermore, the mugwort fingerprint spectrum includes 139 varieties of mugwort;

[0150] Furthermore, the Artemisia fingerprint spectrum is constructed by a combination of Artemisia SSR marker primers SSR40, SSR99, SSR109, SSR14, SSR03, and SSR15.

[0151] In a seventh aspect, the present invention provides the application of the above-mentioned Artemisia argyi fingerprint spectrum in the differentiation of strains of Artemisia argyi germplasm resources.

[0152] To conduct molecular biology SSR marker-assisted identification of Artemisia argyi germplasm resources, promote the breeding of superior varieties of Artemisia argyi, accelerate the development and utilization of germplasm resources, and protect the diversity of Artemisia argyi germplasm resources, this invention develops SSR primers for Artemisia argyi, providing a scientific basis for research such as Artemisia argyi germplasm resource assessment, DNA fingerprinting, and screening of core germplasm.

[0153] Specifically, using 139 Artemisia argyi germplasm resources as materials, genetic diversity analysis and DNA fingerprinting were conducted using SSR marker technology, providing a basis for the molecular identification of Artemisia argyi germplasm resources.

[0154] One or more of the above technical solutions have the following advantages or beneficial effects:

[0155] 1. The SSR molecular markers developed by this invention using Artemisia argyi transcriptome data have high polymorphism potential and are highly practical in germplasm identification. Through experiments, 28 pairs of SSR primers were screened, and genetic diversity analysis of 139 Artemisia argyi germplasm resources was conducted using SSR technology, laying the foundation for the construction of core Artemisia argyi germplasm and the breeding utilization of germplasm resources.

[0156] 2. This invention utilizes SSR markers, using germplasm collected from the Artemisia argyi germplasm resource garden as test materials. Through capillary electrophoresis detection, the amount of primers used is further simplified based on the polymorphic information content of the primers, and 6 pairs of core markers are screened out. The first DNA fingerprint map containing 139 Artemisia argyi germplasm accessions is established. This method is rapid, accurate, precise, reproducible and stable, and can identify multiple varieties simultaneously, which is convenient and fast. At the same time, it provides theoretical and technical support for the identification and classification of Artemisia argyi germplasm resources, germplasm innovation and new variety breeding. Attached Figure Description

[0157] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0158] Figure 1 This is a partial amplification capillary fluorescence electrophoresis detection peak diagram using the labeled primer SSR09 in an embodiment of the present invention. The five smaller diagrams from top to bottom represent the detection peak diagrams of SSR09 for a total of five samples: HBQQ, HeNAY, HeBBD2, ZJNB2, and HeNNY2.

[0159] Figure 2 This is a diagram showing the cluster analysis results of Artemisia argyi germplasm in an embodiment of the present invention. Detailed Implementation

[0160] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0161] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0162] Example 1

[0163] 1. Artemisia argyi germplasm resources

[0164]

[0165] 2. SSR primer development

[0166] A. Using existing Artemisia argyi transcriptome sequences, SSR sites were scanned and screened using the MISA script (http: / / pgrc.ipk-gatersleben.de / misa / ) to obtain a large number of SSR sites; primers were designed for these sites using Primer 5.0 software.

[0167] B. In this invention, the screening criteria are as follows: single nucleotides must be repeated at least 10 times, dinucleotides at least 6 times, and trinucleotides, tetranucleotides, pentanucleotides, and hexanucleotides at least 5 times to be identified as SSR sites. Primers for these sites were designed using Primer 5.0 software with the following parameters: primer length 18-27 bp; annealing temperature 55-65℃; PCR product length between 100 bp and 400 bp; GC content between 40% and 60%. A total of 197 pairs of SSR primers were successfully designed. The amplification efficiency and polymorphism of the primers were studied using eight samples. Finally, 28 pairs of primers that could stably amplify clear and polymorphic bands were selected from the 197 pairs. The primer sequences are shown in Table 1. These primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0168] 3. Genomic DNA extraction

[0169] 139 healthy young leaves were selected, and DNA was extracted from them using a modified CTAB method. The concentration and purity of the DNA in the test materials were detected by Nanodrop nucleic acid analyzer and 1% agarose gel electrophoresis. Qualified DNA samples were stored at -20℃ for later use.

[0170] 4. Capillary electrophoresis detection

[0171] A. Add the universal M13 adapter sequence “GTAAAACGACGGCCAGT” to the forward primers of 28 pairs of SSR primers to obtain the M13 adapter forward primers;

[0172] B. Using the genomic DNA extracted in step 3 as a template, PCR amplification was performed using a reverse primer and an M13 adapter forward primer to obtain fluorescent PCR amplification products. The PCR amplification system was as follows: 2 μL of 50 ng / μL DNA template, 5 μL of 2×Es Taq MsaterMix, 0.04 μL of 10 pmol / μL M13 adapter forward primer, 0.25 μL of 10 pmol / μL reverse primer, 0.15 μL of M13 fluorescent modification group (ROX, HEX, FAM, or TAMRA), and 3.65 μL of H2O. The PCR amplification reaction program was as follows: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; final extension at 72℃ for 5 min, and storage at 4℃.

[0173] C. The fluorescent PCR amplification products obtained in step B were detected by capillary fluorescence electrophoresis using an ABI3730XL DNA sequencer. GeneMarker 2.6.3 was used to read the sample fragment size and the number of alleles, and the band detection results were statistically analyzed.

[0174] 5. Data processing

[0175] The obtained amplified fragment sizes were compiled, and the following genetic diversity parameters for each primer pair were calculated using Popgen32 software, such as the number of alleles (Na), the effective number of alleles (Ne), the Shannon information index (I), and the allele frequency of different primers. After calculating the allele count of different primers using Popgen32 software, the polymorphism information content (PIC) was calculated using PIC_CALC software. Based on the compiled amplified fragment sizes, the fragments were numbered and read from largest to smallest, with the presence of an amplified band marked as "1" and the absence of a band marked as "0". An original matrix was established and imported into MEGA11 software to draw an NJ tree. Cluster analysis was performed on 139 Artemisia argyi samples. Based on the polymorphism information content (PIC) of the markers, the markers were further simplified, and a standard fingerprint map for each variety was constructed.

[0176] Example 2: Design of SSR primers

[0177] Based on the above SSR primer development method, a total of 197 SSR markers were screened. Using capillary electrophoresis, 28 pairs of primers with high polymorphism were screened out, as shown in Table 2, with corresponding sequences Seq NO ID.1~56.

[0178] Table 2: Information on 28 SSR primers

[0179]

[0180] Capillary electrophoresis was used to detect the amplification of 28 pairs of SSR primers in 139 germplasm resources. Some results are shown in Table 3. A total of 266 alleles (Na) were detected, with an average of 9.5 alleles, and gene fragments ranging from 150 to 400. The effective number of alleles (Ne) ranged from 1.0758 to 1.5007, with an average of 1.2319 alleles. Nei's genetic diversity index (H) ranged from 0.0650 to 0.3138, with an average of 0.1532. The Shannon information diversity index (I) ranged from 0.1350 to 0.4833, with an average of 0.2582. The polymorphism information content (PIC) ranged from 0.2149 to 0.8831, with an average of 0.7121. These results indicate rich genetic diversity among Artemisia argyi materials and that the SSR primer set developed in this invention can be applied to the analysis of genetic diversity and identification of germplasm resources in Artemisia argyi.

[0181] Table 3: Statistical analysis of amplification information of 28 pairs of SSR primers in 139 Artemisia argyi materials

[0182]

[0183] Cluster analysis revealed that 139 Artemisia argyi germplasm accessions were effectively distinguished, such as... Figure 2As shown, the samples can be roughly divided into four major categories (Group I, Group II, Group III, and Group IV), with germplasm from each province distributed in each group. Group I contains 20 germplasm accessions, with 7 each from Hubei and Henan, and 1 each from Guizhou, Jiangxi, Jiangsu, Hebei, Shaanxi, and Shandong. Group II contains 31 Artemisia argyi accessions, mainly from Hunan and Hubei. Group III includes 24 germplasm accessions, primarily composed of 11 from Hubei, 3 from Jiangxi, 2 each from Shaanxi and Shandong, and 1 each from Hunan, Anhui, Sichuan, Zhejiang, Guizhou, and Hebei. Group IV contains 64 germplasm accessions, mainly from Hubei, Anhui, Henan, Sichuan, and Fujian, and is the group with the richest sample sources.

[0184] Example 3: Fingerprint patterns and identification methods of 139 Artemisia argyi germplasm resources

[0185] Based on the principle of minimizing markers in distinguishing 139 germplasms, and combined with the polymorphism information content of the markers, 6 core markers (SSR40, SSR99, SSR109, SSR14, SSR03, and SSR15) were further screened from 28 pairs of SSR markers to construct a molecular fingerprint library for 139 germplasms. The presence or absence of obvious bands at the migration positions of the amplified bands was statistically analyzed, where 1 indicates the presence of an electrophoretic band and 0 indicates the absence of an electrophoretic band, i.e., all alleles were converted to the 1 / 0 format. The DNA fingerprints of the 139 Artemisia argyi germplasms are shown in Table 4.

[0186] Table 4: DNA fingerprints of 139 Artemisia argyi germplasms

[0187]

[0188]

[0189]

[0190] The method for distinguishing strains of Artemisia argyi germplasm resources using the above fingerprinting is as follows: Statistical analysis is performed based on whether there are obvious bands at the migration positions of the amplified bands of SSR40, SSR99, SSR109, SSR14, SSR03, and SSR15. Here, 1 indicates the presence of an electrophoretic band, and 0 indicates the absence of an electrophoretic band. That is, all alleles are converted into 1 / 0 format, with 1 and 0 representing the detected and undetected fragments, respectively. A 74-bit 1 / 0 format string is obtained, which is then compared with the fingerprinting shown in Table 4.

[0191] The aforementioned fingerprint profiles are sufficient to differentiate the cultivars of 139 Artemisia argyi germplasm resources. Each cultivar has a unique fingerprint profile, exhibiting high accuracy and specificity. For example, the fingerprint profile of Qi Ai (HBQQ), one of the "Four Famous Artemisia argyi" varieties, is: 00000000100000 100100000000000 000000000 00001000000000110000000010010000100; the fingerprint profile of Hai Ai (ZJNB2) is: 000001000100000000000000000000 00000000000010000000 0000100100000 00001000100; The fingerprint spectrum of Qi Ai (HeBBD2) is: 00000010000000000000000000000 000000000000011010000 1000001000000 00000001000; The fingerprint spectrum of Bei Ai (HeNAY) is: 00010000010000 000000000000010 0000100000 000100001000000100000000000000010.

[0192] It is worth noting that the above six pairs of core markers established the first DNA fingerprint information database containing 139 Artemisia argyi germplasm resources, providing an effective way for the accurate identification of Artemisia argyi germplasm resources. Moreover, removing any pair of the six core markers will cause the fingerprint pattern to become invalid and make it impossible to accurately identify Artemisia argyi germplasm resources. Adding any pair of the six core markers will not have any beneficial effect on the identification.

[0193] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An Artemisia leaf SSR marker primer, characterized in that, The Artemisia argyi SSR marker primer comprises the following primers: SSR40, the nucleotide sequences of the forward and reverse primers are shown as SEQ ID NO. 1-2; SSR99, the nucleotide sequences of the forward and reverse primers are shown as SEQ ID NO. 3-4; SSR109, the nucleotide sequences of the forward and reverse primers are shown as SEQ ID NO. 5-6; SSR14, the nucleotide sequences of the forward and reverse primers are shown as SEQ ID NO. 7-8; SSR03, the nucleotide sequences of the forward and reverse primers are shown as SEQ ID NO. 9-10; SSR15, the nucleotide sequences of the forward and reverse primers are shown as SEQ ID NO. 11-12.

2. A primer combination of an Artemisia leaf SSR marker, characterized in that, The Artemisia argyi SSR marker primer of claim 1.

3. A kit characterized in that, The kit at least contains the Artemisia argyi SSR marker primer of claim 1 or contains the Artemisia argyi SSR marker primer combination of claim 2.

4. The application of the Artemisia argyi SSR marker primer of claim 1, the Artemisia argyi SSR marker primer combination of claim 2 or the kit of claim 3 in any one or more of the following: 1) genetic diversity analysis of Artemisia argyi; 2) construction of genetic map of Artemisia argyi; 3) germplasm identification of Artemisia argyi; 4) genetic relationship analysis of Artemisia argyi.

5. Use according to claim 4, characterized in that, In the application 2), the construction of genetic map of Artemisia argyi is specifically the construction of fingerprint map of Artemisia argyi.

6. A method for identifying genetic diversity or germplasm of Artemisia argyi or constructing a fingerprint of Artemisia argyi, characterized in that, The method comprises the following steps: (1) extracting total DNA of the Artemisia argyi sample to be tested; (2) using the Artemisia argyi SSR marker primer of claim 1, the Artemisia argyi SSR marker primer combination of claim 2 or the kit of claim 3, the forward primer of each primer is added with a universal M13 adapter sequence to obtain a M13 adapter forward primer; (3) using the reverse primer of each primer in the Artemisia argyi SSR marker primer of claim 1, the Artemisia argyi SSR marker primer combination of claim 2 or the kit of claim 3, the M13 adapter forward primer and the total DNA extracted in step (1) as a template, performing PCR amplification to obtain a fluorescent PCR amplification product; (4) performing electrophoresis on the fluorescent PCR amplification product to obtain electrophoresis data and count the band detection results; (5) performing genetic diversity analysis of Artemisia argyi, cluster analysis of polymorphic information content (PIC) and construction of DNA fingerprint map according to the counted band detection results in step (4).

7. The method of claim 6, wherein, In step (3), the PCR amplification system is 10 μL, which comprises: 2 μL of DNA template with a concentration of 50 ng / μL, 5 μL of 2×Es Taq MsaterMix, 0.04 μL of M13 adapter forward primer with a concentration of 10 pmol / μL, 0.25 μL of reverse primer with a concentration of 10 pmol / μL, 0.15 μL of M13 fluorescent modification group and 2.56 μL of H2O.

8. The method of claim 6, wherein, In step (3), the PCR amplification procedure is as follows: pre-denaturation at 94℃ for 5 min, denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 30 s, for a total of 35 cycles; final end extension at 72℃ for 5 min, and preservation at 4℃.

9. Application of a fingerprint of folium artemisiae in line differentiation of folium artemisiae germplasm resources, characterized in that, The fingerprint spectrum of the folium artemisiae argyi is constructed by the method of any one of claims 6-8, wherein all alleles are converted into 1 / 0 format, and 1 and 0 represent the detected and undetected corresponding fragments, respectively.

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