Artemisia argyi SSR (Simple Sequence Repeat) marker primer and application thereof in construction of Artemisia argyi fingerprint spectrum

Abstract

The invention discloses a folium artemisiae argyi SSR (Simple Sequence Repeat) marker primer and application thereof in constructing a folium artemisiae argyi fingerprint spectrum, and belongs to the field of molecular biology. 28 pairs of SSR primers are screened through tests, the sequences of the primers are as shown in SEQ ID NO: 1-56, the 28 pairs of SSR molecular markers are used for carrying out genetic diversity analysis on 139 parts of folium artemisiae argyi germplasm, and a foundation is laid for accurate identification of folium artemisiae argyi germplasm resources and molecular marker-assisted breeding. The SSR marker developed through folium artemisiae argyi transcriptome data has relatively high polymorphism potential, and has relatively high practicability in the aspect of germplasm identification; according to the method, SSR molecular markers are utilized, germplasm collected in a folium artemisiae argyi germplasm resource garden is taken as a test material, capillary electrophoresis detection is carried out, the usage amount of primers is further simplified according to the polymorphic information content of the primers, 6 pairs of core primers are screened out, and the first DNA fingerprint spectrum containing 139 parts of folium artemisiae argyi germplasm is established; theoretical and technical support is provided for identification and classification of argy wormwood leaf germplasm resources, germplasm innovation and new variety breeding.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and in particular relates to an Artemisia argyi SSR marker primer and an application thereof in constructing an Artemisia argyi fingerprint map. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Artemisia argyi Lévl.et Van is the dried leaf of Artemisia argyi Lévl.et Van, also known as ice platform, medicinal grass, moxibustion grass, etc. It is a traditional Chinese medicine with a long history of application. It has the effects of warming the meridians to stop bleeding, dispelling cold and relieving pain, and externally removing dampness and relieving itching; it can also be used clinically for vomiting blood, metrorrhagia, menorrhagia, fetal bleeding, and cold semen. At the same time, it is the main raw material of the internationally famous moxibustion therapy, as well as the raw material of a large number of medicines and health products. Wild mugwort resources are abundant and distributed in most parts of the country.

[0004] Research on mugwort leaves mainly focuses on chemical composition, pharmacological effects, quality standards, clinical and daily applications, and there are few studies on the genetic diversity of mugwort leaves in molecular biology. In recent years, with the continuous expansion of the market demand for mugwort leaves, the wild resources of mugwort are in short supply, and many areas have begun to plant mugwort on a large scale. Due to the similar morphology of Artemisia species, there are many fake mugwort products, and there are complex genetic variations in mugwort germplasm resources, which leads to confusion in the introduction of mugwort, unclear germplasm, and variety degeneration during the artificial cultivation of mugwort, which seriously affects the yield and quality of mugwort and restricts the protection and utilization of mugwort germplasm resources. Therefore, it is urgent to develop a set of molecular markers to protect the genetic diversity of mugwort germplasm.

[0005] Simple Sequence Repeats, also known as microsatellite DNA, is a type of tandem repeat sequence consisting of several nucleotides (generally 1-6) as repeating units. It has the advantages of high polymorphism, good stability, multiple alleles, co-dominance, abundant quantity, good genome coverage and simple operation. In recent years, it has been widely used in germplasm resource research, hybrid identification, genetic map construction, target gene positioning, genetic diversity research, etc.

[0006] Compared with morphological identification methods, DNA fingerprinting has a high degree of individual specificity and environmental stability. DNA fingerprinting is one of the effective technical means for strain identification. DNA fingerprinting is based on nucleotide site variation within individuals and identifies and distinguishes strains according to the difference in genomic characteristics between different strains. The development of molecular markers is a key link in the construction of fingerprints. However, markers have not yet been used to construct a large-scale DNA fingerprint of mugwort germplasm resources, and DNA fingerprints of mugwort germplasm resources play a very important role in the identification of germplasm resources. Summary of the invention

[0007] In order to address the deficiencies in the prior art, the purpose of the present invention is to provide an SRR marker primer, a marker primer combination and a fingerprint map of Artemisia argyi, and to carry out genetic diversity analysis of 139 Artemisia argyi germplasm resources using 28 pairs of SSR primers. The usage of primers is further simplified by using the polymorphic information content, 6 pairs of core SSR primers are screened out, and a DNA fingerprint map of 139 Artemisia argyi germplasm resources is constructed, thereby laying a foundation for the accurate identification of Artemisia argyi germplasm resources and the breeding of excellent varieties.

[0008] The 28 pairs of SSR primers provided by the present invention can use SSR technology to perform genetic diversity analysis on 139 Artemisia argyi germplasm resources, laying a foundation for the construction of Artemisia argyi core 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 fingerprint of the present invention can be used to distinguish the strains of 139 mugwort germplasm resources. Each variety has a unique fingerprint with high accuracy and specificity. For example, the fingerprint of Qi mugwort (HBQQ) among the "four famous mugworts" is: 00000000100000 100100000000000 000000000000001000000 000110000000010010000100; the fingerprint of Hai mugwort (ZJNB2) is: 00000100010000 000000000000000 000000000000100000000 The fingerprint of HeBBD2 is: 0000001000000000000000000000000000000000 00011010000 100000100000 00000001000; the fingerprint of HeNAY is: 00010000010000 000000000000010 0000100000 000100001000001000000000000000100000000000010.

[0011] It is worth noting that the 6 pairs of core markers provided by the present invention have established the first DNA fingerprint information database containing 139 mugwort germplasms, providing an effective way to accurately identify mugwort germplasm resources; and any removal of any pair of the 6 pairs of core markers will cause the fingerprint map to become invalid, and it will be impossible to accurately identify mugwort germplasm resources. Adding any pair of the 6 pairs of core markers will not have a beneficial effect on the identification.

[0012] In a first aspect, the present invention provides a wormwood SSR marker primer, wherein the wormwood SSR marker primer comprises a pair of the following primers;

[0013] SSR40, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 1 to 2;

[0014] SSR99, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 3 to 4;

[0015] SSR109, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 5 to 6;

[0016] SSR14, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 7 to 8;

[0017] SSR03, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 9 to 10;

[0018] SSR15, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 11 to 12;

[0019] SSR79, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 13 to 14;

[0020] SSR01, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 15 to 16;

[0021] SSR191, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 17 to 18;

[0022] SSR10, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 19 to 20;

[0023] SSR95, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 21 to 22;

[0024] SSR65, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 23 to 24;

[0025] SSR33, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 25 to 26;

[0026] SSR31, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 27 to 28;

[0027] SSR172, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 29 to 30;

[0028] SSR09, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 31 to 32;

[0029] SSR22, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 33 to 34;

[0030] SSR55, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 35 to 36;

[0031] SSR182, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 37-38;

[0032] SSR92, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 39 to 40;

[0033] SSR70, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 41 to 42;

[0034] SSR34, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 43-44;

[0035] SSR46, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 45 to 46;

[0036] SSR162, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 47-48;

[0037] SSR114, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 49 to 50;

[0038] SSR06, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 51 to 52;

[0039] SSR118, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 53 to 54;

[0040] SSR175, which includes forward and reverse primers whose nucleotide sequences are shown in SEQ ID NOs.55-56.

[0041] In a second aspect, the present invention provides a combination of SSR marker primers for Artemisia argyi, comprising two or more pairs of the above-mentioned SSR marker primers for Artemisia argyi.

[0042] (1) SSR40 primers for amplifying SSR molecular markers:

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

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

[0045] (2) SSR99 primers for amplifying SSR molecular markers:

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

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

[0048] (3) SSR109 primers for amplifying SSR molecular markers:

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

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

[0051] (4) SSR14 primers for amplifying SSR molecular markers:

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

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

[0054] (5) SSR03 primers for amplifying SSR molecular markers:

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

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

[0057] (6) SSR15 primers for amplifying SSR molecular markers:

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

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

[0060] (7) SSR79 primers for amplifying SSR molecular markers:

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

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

[0063] (8) SSR01 primers for amplifying SSR molecular markers:

[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 SSR molecular markers SSR191:

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

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

[0069] (10) SSR10 primers for amplifying SSR molecular markers:

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

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

[0072] (11) SSR95 primers for amplifying SSR molecular markers:

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

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

[0075] (12) SSR65 primers for amplifying SSR molecular markers:

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

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

[0078] (13) SSR33 primers for amplifying SSR molecular markers:

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

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

[0081] (14) SSR31 primers for amplifying SSR molecular markers:

[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 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) SSR09 primers for amplifying SSR molecular markers:

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

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

[0090] (17) SSR22 primers for amplifying SSR molecular markers:

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

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

[0093] (18) SSR55 primers for amplifying SSR molecular markers:

[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 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) SSR92 primers for amplifying SSR molecular markers:

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

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

[0101] (21) SSR70 primers for amplifying SSR molecular markers:

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

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

[0104] (22) SSR34 primers for amplifying SSR molecular markers:

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

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

[0107] (23) SSR46 primers for amplifying SSR molecular markers:

[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 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 SSR marker SSR114:

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

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

[0116] (26) SSR06 primers for amplifying SSR molecular markers:

[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 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 SSR 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] In a third aspect, the present invention provides a kit, which comprises at least the above-mentioned Artemisia argyi SSR marker primer or Artemisia argyi SSR marker primer combination.

[0127] In a fourth aspect, the present invention provides the use of the above-mentioned Artemisia argyi SSR marker primer, Artemisia argyi SSR marker primer combination or kit in any one or more of the following:

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

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

[0130] 3) Germplasm identification of Artemisia argyi;

[0131] 4) Analysis of the relationship between Artemisia argyi and its offspring;

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

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

[0134] Preferably, in the application 2), constructing the mugwort genetic map is specifically constructing a mugwort fingerprint map.

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

[0136] (1) extracting the total DNA of the mugwort leaf sample to be tested;

[0137] (2) using the above-mentioned Artemisia argyi SSR marker primer, Artemisia argyi SSR marker primer combination or kit, adding a universal M13 adapter sequence to the forward primer of each pair of primers to obtain an M13 adapter forward primer;

[0138] (3) using the total DNA extracted in step (1) as a template, performing PCR amplification using the above-mentioned Artemisia argyi SSR marker primers, Artemisia argyi SSR marker primer combination or the reverse primer of each pair of primers described in the kit, and the M13 adapter forward primer to obtain a fluorescent PCR amplification product;

[0139] (4) performing electrophoresis on the fluorescent PCR amplification product to obtain electrophoresis data and statistically analyze band detection results;

[0140] (5) According to the statistical band detection results in step (4), genetic diversity analysis of mugwort leaves, calculation of polymorphic information content (PIC) of cluster analysis and construction of DNA fingerprint map are carried out.

[0141] Wherein, the mugwort leaf samples are young and tender leaves of mugwort leaf.

[0142] Furthermore, in the step (3), the PCR amplification system is 10 μL, including: 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 M13 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 procedure is: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s, for a total of 35 cycles; and finally 72°C end extension for 5 min and storage at 4°C.

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

[0145] A. According to the band detection results in step (4), record the size of the amplified fragment of each leaf wormwood sample corresponding to each pair of primers;

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

[0147] C. According to the sizes of the amplified fragments obtained by the comprehensive arrangement in the step A, the amplified fragments were numbered and read from large to small, the presence of amplified bands was recorded as "1", and the absence of bands was recorded as "0", and the original matrix was established. The MEGA11 software was imported to draw the NJ tree and the DNA fingerprint of 139 Artemisia argyi germplasm resources was constructed using the original matrix.

[0148] In the sixth aspect, the present invention provides an wormwood fingerprint, which is constructed by the method described in any one of claims 6 to 8, wherein all alleles are converted into a 1 / 0 format, with 1 and 0 representing the corresponding fragments detected and not detected, respectively.

[0149] Further, the wormwood fingerprint includes 139 wormwood varieties;

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

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

[0152] In order to carry out molecular biology SSR marker-assisted identification of mugwort germplasm resources, promote the breeding of mugwort varieties, accelerate the development and utilization of germplasm resources, and protect the diversity of mugwort germplasm resources, the present invention develops SSR primers for mugwort, which provide a scientific basis for research such as mugwort germplasm resource evaluation, DNA fingerprint map construction, and screening of core germplasm.

[0153] Specifically, 139 mugwort germplasm resources were used as materials, and genetic diversity analysis and DNA fingerprint map construction were carried out through SSR marker technology, providing a basis for the molecular identification of mugwort germplasm resources.

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

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

[0156] 2. The present invention utilizes SSR markers and takes the germplasm collected in the mugwort germplasm resource garden as test materials. Through capillary electrophoresis detection, the usage of primers is further simplified according to the polymorphic information content of the primers, and 6 pairs of core markers are screened out to establish the first DNA fingerprint map containing 139 mugwort germplasms. This method is fast, accurate, precise, reproducible and stable, and can identify multiple varieties at the same time. It is convenient and fast, and provides theoretical and technical support for the identification and classification of mugwort germplasm resources, germplasm innovation and new variety breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0157] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

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

[0159] Figure 2 This is a diagram showing the cluster analysis results of the mugwort germplasm in the embodiment of the present invention. DETAILED DESCRIPTION

[0160] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

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

[0162] Example 1

[0163] 1. Artemisia argyi germplasm resources

[0164]

[0165] 2.SSR Primer Development

[0166] A. Using the existing mugwort transcriptome sequence, the MISA script (http: / / pgrc.ipk-gatersleben.de / misa / ) was used to scan and screen SSR sites to obtain a large number of SSR sites; and Primer 5.0 software was used to design primers for the sites.

[0167] B. In the present invention, the screening criteria are: mononucleotides are repeated at least 10 times, dinucleotides are repeated at least 6 times, and trinucleotides, tetranucleotides, pentanucleotides and hexanucleotides are repeated at least 5 times to be identified as SSR sites. The present invention uses Primer5.0 software to design primers for these sites, and the parameters are set as follows: primer length 18-27bp; annealing temperature is 55-65°C; PCR product length range is between 100bp and 400bp; GC content is between 40% and 60%, and finally 197 pairs of SSR primers are successfully designed. The amplification efficiency and polymorphism of primers were studied using 8 samples, and finally 28 pairs of primers that can stably amplify clear and polymorphic bands were screened out from 197 pairs of primers. The primer sequences are shown in Table 1 and were commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0168] 3. Genomic DNA Extraction

[0169] Healthy young leaves of 139 materials were selected, and their DNA was extracted using the improved CTAB method. The DNA concentration and purity of the test materials were detected using Nanodrop nucleic acid analyzer and 1% agarose gel electrophoresis. The 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 primer;

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

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

[0174] 5. Data collation

[0175] The size of the amplified fragments obtained by comprehensive sorting was used to calculate the following genetic diversity parameters of each primer pair, such as the number of alleles (Na), the number of effective alleles (Ne), the Shannon information index (I) and the allele frequency of different primers, etc. After calculating the alleles of different primers with Popgen32 software, the polymorphic information content (PIC) was calculated with PIC_CALC software; according to the size of the amplified fragments obtained by comprehensive sorting, the amplified fragments were numbered and read from large to small, and the presence of amplified bands was recorded as "1" and the absence of bands was recorded as "0". The original matrix was established and imported into MEGA11 software to draw the NJ tree, and cluster analysis was performed on the 139 individual samples of mugwort leaves. The markers were further simplified according to the polymorphic information content (PIC) of the markers to construct the standard fingerprint map of each variety.

[0176] Example 2: Design of SSR primers

[0177] According to the above-mentioned SSR primer development method, 197 SSR markers were screened. Using capillary electrophoresis detection, 28 pairs of primers with high polymorphism were screened, as shown in Table 2, corresponding to sequences Seq NO ID.1~56.

[0178] Table 2: Information of 28 pairs of SSR primers

[0179]

[0180] Capillary electrophoresis was used to detect the amplification of 28 pairs of SSR primers in 139 germplasm resources. Some of the results are shown in Table 3. A total of 266 alleles (Na) were detected, with an average of 9.5, and the gene fragments were between 150 and 400; the effective allele number (Ne) ranged from 1.0758 to 1.5007, with an average of 1.2319 effective alleles; Nei's genetic diversity index (H) was between 0.0650 and 0.3138, with an average of 0.1532; Shannon information diversity index (I) was between 0.1350 and 0.4833, with an average of 0.2582; polymorphic information content (PIC) was between 0.2149 and 0.8831, with an average of 0.7121. This shows that there is rich genetic diversity among mugwort materials, and the SSR primer set developed by the present invention can be used for genetic diversity analysis and germplasm resource identification of mugwort.

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

[0182]

[0183] Through cluster analysis, it was found that 139 Artemisia germplasms were effectively distinguished, such as Figure 2As shown in the figure, it can be roughly divided into four categories (group I, group II, group III, group IV), and germplasms from various provinces are distributed in each group. Group I contains 20 germplasms, 7 from Hubei and Henan, and 1 from Guizhou, Jiangxi, Jiangsu, Hebei, Shaanxi, and Shandong; Group II contains 31 germplasms of mugwort, mainly from Hunan and Hubei; Group III includes 24 germplasms, mainly composed of 11 germplasms from Hubei, 3 from Jiangxi, 2 from Shaanxi and Shandong, and 1 from Hunan, Anhui, Sichuan, Zhejiang, Guizhou, and Hebei; Group IV contains 64 germplasms, mainly from Hubei, Anhui, Henan, Sichuan, Fujian and other places, and is the group with the richest sample sources.

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

[0185] According to the principle of the minimum markers for distinguishing 139 germplasms and the polymorphic information content value of the markers, 6 pairs of core markers (SSR40, SSR99, SSR109, SSR14, SSR03, and SSR15) were further screened from the 28 pairs of SSR markers to construct a molecular fingerprint library of the 139 germplasms. Statistics were performed based on whether there were obvious bands at the migration position of the amplified bands, where 1 indicated the presence of an electrophoresis band and 0 indicated the absence of an electrophoresis band, that is, all alleles were converted into a 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 of using the above-mentioned fingerprint map to distinguish the strains of mugwort germplasm resources is to count whether there are obvious bands at the migration position of the SSR40, SSR99, SSR109, SSR14, SSR03, and SSR15 amplified bands, where 1 indicates the presence of an electrophoresis band and 0 indicates the absence of an electrophoresis band, that is, all alleles are converted into a 1 / 0 format, with 1 and 0 representing the corresponding fragments detected and not detected, respectively, to obtain a 74-bit 1 / 0 format string, which can be compared with the fingerprint map shown in Table 4.

[0191] The above fingerprints can distinguish the strains of 139 mugwort germplasm resources. Each variety has a unique fingerprint with high accuracy and specificity. For example, the fingerprint of Qi Ai (HBQQ) among the “Four Famous Mugworts” is: 00000000100000 100100000000000 00000000000 00001000000000110000000010010000100; the fingerprint of Hai Ai (ZJNB2) is: 0000010001000000000000000000 000000000000100000000 00001001000000 The fingerprint of HeBBD2 is: 0000001000000000000000000000 0000000000000011010000 1000001000000 00000001000; the fingerprint of HeNAY is: 00010000010000 000000000000010 0000100000 000100001000001000000000000001000000000000010.

[0192] It is worth noting that the above 6 pairs of core markers have established the first DNA fingerprint information database containing 139 mugwort germplasms, providing an effective way to accurately identify mugwort germplasm resources; and any removal of any pair of the 6 pairs of core markers will cause the fingerprint map to become invalid, and it will be impossible to accurately identify mugwort germplasm resources. Adding any pair of the 6 pairs of core markers will not have a beneficial effect on the identification.

[0193] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A SSR marker primer for Artemisia argyi, characterized in that: The wormwood SSR marker primers include a pair of the following primers: SSR40, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 1 to 2; SSR99, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 3 to 4; SSR109, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 5 to 6; SSR14, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 7 to 8; SSR03, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 9 to 10; SSR15, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 11 to 12; SSR79, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 13 to 14; SSR01, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 15 to 16; SSR191, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 17 to 18; SSR10, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 19 to 20; SSR95, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 21 to 22; SSR65, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 23 to 24; SSR33, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 25 to 26; SSR31, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 27 to 28; SSR172, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 29 to 30; SSR09, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 31 to 32; SSR22, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 33 to 34; SSR55, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 35 to 36; SSR182, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 37-38; SSR92, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 39 to 40; SSR70, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 41 to 42; SSR34, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 43-44; SSR46, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 45 to 46; SSR162, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 47-48; SSR114, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 49 to 50; SSR06, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 51 to 52; SSR118, which includes forward and reverse primers having nucleotide sequences as shown in SEQ ID NOs. 53 to 54; SSR175, which includes forward and reverse primers whose nucleotide sequences are shown in SEQ ID NOs.55-56.

2. A combination of SSR marker primers for Artemisia argyi, characterized in that: Including two or more pairs of the mugwort SSR marker primers described in claim 1.

3. The Artemisia argyi SSR marker primer combination according to claim 2, characterized in that: The combination is a combination of SSR40, SSR99, SSR109, SSR14, SSR03, and SSR15.

4. A kit, characterized in that: The kit contains at least the mugwort SSR marker primer described in claim 1 or the mugwort SSR marker primer combination described in claim 2 or 3.

5. Use of the Artemisia argyi SSR marker primer according to claim 1, the Artemisia argyi SSR marker primer combination according to claim 2 or 3, or the kit according to claim 4 in any one or more of the following: 1) Genetic diversity analysis of Artemisia argyi; 2) construct a genetic map of Artemisia argyi; 3) Germplasm identification of Artemisia argyi; 4) Analysis of the relationship between Artemisia argyi and its offspring; 5) Mining of functional genes in Artemisia argyi; 6) Molecular marker-assisted breeding of Artemisia argyi; Preferably, in the application 2), constructing the mugwort genetic map is specifically constructing a mugwort fingerprint map.

6. A method for identifying genetic diversity or germplasm of Artemisia argyi or constructing a fingerprint of Artemisia argyi, characterized in that: The following steps are involved: (1) extracting the total DNA of the mugwort leaf sample to be tested; (2) using the mugwort SSR marker primers described in claim 1, the mugwort SSR marker primer combination described in claim 2 or 3, or the kit described in claim 4, adding a universal M13 adapter sequence to the forward primer of each pair of primers to obtain an M13 adapter forward primer; (3) using the total DNA extracted in step (1) as a template, performing PCR amplification using the mugwort SSR marker primers described in claim 1, the mugwort SSR marker primer combination described in claim 2 or 3, or the reverse primer of each pair of primers described in the kit described in claim 4, and the M13 adapter forward primer, respectively, to obtain a fluorescent PCR amplification product; (4) performing electrophoresis on the fluorescent PCR amplification product to obtain electrophoresis data and statistically analyze band detection results; (5) According to the statistical band detection results in step (4), genetic diversity analysis of mugwort leaves, calculation of polymorphic information content (PIC) of cluster analysis and construction of DNA fingerprint map are carried out.

7. The method according to claim 6, characterized in that In the step (3), the PCR amplification system is 10 μL, including: 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 according to claim 6, characterized in that In the step (3), the PCR amplification procedure is: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s, a total of 35 cycles; final 72°C end extension for 5 min, and storage at 4°C.

9. A fingerprint spectrum of Artemisia argyi, characterized in that: The wormwood fingerprint is constructed by the method according to any one of claims 6 to 8, wherein all the alleles are converted into a 1 / 0 format, with 1 and 0 representing the corresponding fragments detected and not detected, respectively. Further, the wormwood fingerprint includes 139 wormwood varieties; Furthermore, the mugwort fingerprint is constructed by a combination of the mugwort SSR marker primers SSR40, SSR99, SSR109, SSR14, SSR03, and SSR15.

10. Application of the Artemisia argyi fingerprint map as described in claim 9 in distinguishing strains of Artemisia argyi germplasm resources.

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