CN115094153A - Identification method of short and evergreen tape grass strain - Google Patents

Abstract

The invention belongs to the field of plant molecular markers, and particularly relates to an identification method of tape grass. The bitter herb identification related SNP marker fills up the technical blank of bitter herb SNP molecular marker development, can realize high-throughput operation, has very high accuracy, is simple in data reading, saves cost, improves efficiency, provides scientific basis for rapidly identifying and evaluating the genetic relationship of various bitter herb subspecies, provides theory and data support for establishing a bitter herb molecular auxiliary breeding technical platform, and provides a high-efficiency detection method for breeding high-quality bitter herb varieties; provides theoretical basis and technical means for evaluating the genetic characteristics of Chinese bitter herb varieties, identifying specific bitter herb varieties and the like, and has important theoretical value and application significance.

Description

Identification method of short and evergreen tape grass strain

Technical Field

This application is filed on 17.11.2020, under the name 2020112899748: the division application of Chinese invention patent application of 'bitter grass identification related SNP markers and application thereof'. The invention relates to the field of plant molecular markers, in particular to a method for identifying tape grass.

Background

Bitter herbs (Vallisneria), commonly known as "shouli grass" and "Shuihuai leeks", belong to Angiospermae (Angiospermae), monocotyledonous plants (Monocots), Marianales (Helobiae), Hydroxydae (Hydrocharitaceae), perennial stem-free submerged herbaceous plants, have stolons, leaves are linear or ribbon-shaped, grow in creeks, rivers and other environments, are widely distributed all over the world, and are colonizing species and important components of fresh water ecosystems. The eel grass has strong capacity of adsorbing suspended pollutants, has nitrogen and phosphorus removal capacity, can effectively relieve the eutrophication degree of water bodies, effectively inhibits the propagation and growth of algae, can provide guarantee for the growth of other subsequent aquatic plants, enables the aquatic plants to become pioneer species for water purification and ecological restoration of water areas, is dwarf eel grass bred by Taihe water company, has developed root, stem and leaf and strong photosynthesis due to the characteristics of dwarfing, evergreen in four seasons, pollution resistance, weak light resistance and the like, can generate a large amount of primary oxygen, can efficiently absorb and convert nitrogen, phosphorus and other nutritive salts, and is suitable for various water bodies in China. Meanwhile, various underwater lawns and underwater forests with different heights, shapes and colors are configured, so that beautiful underwater landscapes are constructed while the diversity of underwater ecological species is met, and the underwater ecological restoration method is widely applied to the ecological restoration engineering of the water area. In recent years, the variety of the tape grass applied in engineering is various, the naming and the classification are disordered, the morphological identification result has low reliability because the tape grass morphology is greatly influenced by environmental and subjective factors, and the morphological identification method has long period, so that the development of the molecular marker for distinguishing different tape grass subspecies is necessary.

The SNP marker (single nucleotide polymorphism marker) is the most studied and hottest molecular marker in the current genetic marker development in the molecular marker, and is also the molecular marker with the widest application prospect, different sowthistle subspecies can be quickly and accurately identified by utilizing the SNP marker, and no report is found in the domestic method for developing the sowthistle SNP marker.

Disclosure of Invention

In view of the above-mentioned disadvantages of the prior art, it is an object of the present invention to provide a method for identifying tape grass, which solves the problems of the prior art.

To achieve the above and other related objects, the present invention provides a set of eel grass to identify related SNP markers including one or more of a first SNP marker through a second SNP marker.

A set of primers for identification of tape grass, said set of primers comprising primer pairs for detection of a first SNP marker to a twenty-second SNP marker.

A kit for identifying tape grass comprises the primer group.

The SNP marker, the primer group or the kit can be used for identifying the bitter herb strains, establishing a bitter herb molecular assistant breeding technical platform, breeding high-quality bitter herb strains and evaluating the genetic characteristics of the bitter herb strains.

An identification method of tape grass, which comprises detecting SNP markers of tape grass with a primer set or a kit capable of specifically recognizing the SNP markers of tape grass.

Preferably, the kit comprises the primer group capable of specifically recognizing the eel grass SNP marker.

Preferably, the primer group comprises primer pairs shown as SEQ ID NO. 23-SEQ ID NO. 66.

More preferably, the kit further comprises a reagent for extracting genomic DNA from a sample, and a reagent for performing a PCR reaction using the primer set.

Preferably, the SNP markers of the tape grass comprise one or more of a first SNP marker to a second twelve SNP marker.

Preferably, the identification method comprises the steps of:

1) carrying out PCR amplification by using the primer group and using the genomic DNA of the tape grass to be detected as a template;

2) sequencing the PCR amplification product;

3) constructing a phylogenetic tree according to the sequencing result of the step 2).

Preferably, the identification method specifically comprises the following steps:

5) carrying out PCR amplification by using the primer group and using the genomic DNA of the tape grass to be detected as a template;

6) extending the PCR amplification product;

7) carrying out capillary electrophoresis detection on the extension product;

8) the data is read and analyzed.

Preferably, the identification method specifically comprises the following steps:

5) purifying and extending an amplification product obtained by PCR, and extending by adopting a mixed hole;

6) purifying the product obtained by extension and then carrying out capillary electrophoresis;

7) the primers used for extension are shown as SEQ ID NO. 81-SEQ ID NO. 102;

8) the higher the consistency obtained by data analysis is, the closer the consistency is to 100%, the closer the population relationship between the tape grass to be tested and the dwarf tape grass bred by Taihe water company Limited is; when the consistency is 100%, the tape grass to be detected and dwarf tape grass bred by Taihe Shui limited company are the same strain.

Preferably, the identification method of the tape grass is an identification method of a tape grass strain.

As described above, the identification method of tape grass of the present invention has the following advantageous effects:

1) fills the technical blank of the bitter herb SNP molecular marker development.

2) Can identify all the types of the eel grass, can realize high-throughput operation, has very high accuracy, saves the cost and improves the efficiency.

3) Providing scientific basis for rapidly identifying and evaluating the genetic relationship of various eel grass subspecies, providing theoretical and data support for establishing an eel grass molecular auxiliary breeding technical platform, and providing an efficient detection method for breeding high-quality eel grass varieties; provides theoretical basis and technical means for evaluating the genetic characteristics of Chinese bitter herb varieties, identifying specific bitter herb varieties and the like, and has important theoretical value and application significance.

4) The data reading is simple, and whether the tape grass to be detected is the dwarf evergreen tape grass bred by Taihe water company can be directly judged through the fluorescence signal peak.

Drawings

FIG. 1 shows a diagram of a 3730xl parameter set-up for a capillary electrophoresis sequencer according to the present invention.

FIG. 2 shows a phylogenetic tree constructed for 15 populations (18 samples) using the SNP markers of the present invention.

Detailed Description

Unless defined otherwise below, 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 belongs.

The terms: "Single nucleotide polymorphism" or "SNP" refers to a DNA sequence change or genetic variation that occurs when one nucleotide [ e.g., adenine (A), thymine (T), cytosine (C), or guanine (G)) ] is changed to another nucleotide in a genomic sequence.

The terms: the SNAPSHOT method is a micro sequencing technology for SNP typing based on the single base extension principle of a fluorescence labeling ddNTP. If the same reaction system contains allele-specific primers aiming at different SNPs and the lengths of the 5' ends of the primers are different, the synchronous analysis of a plurality of SNPs can be realized. The parting method comprises the following steps: PCR amplification, primer extension reaction and electrophoretic typing.

The invention provides a group of SNP markers related to identification of tape grass, which comprise one or more of the following:

1) a first SNP marker, which is the 40 th base A or G from the 5' end of the nucleotide sequence shown in SEQ ID NO. 1;

2) a second SNP marker, which is the 68 th base G or A from the 5' end of the nucleotide sequence shown in SEQ ID NO. 2;

3) a third SNP marker, which is the 53 rd base G or T from the 5' end of the nucleotide sequence shown in SEQ ID NO. 3;

4) a fourth SNP marker, which is the 71 th base G or A from the 5' end of the nucleotide sequence shown in SEQ ID NO. 4;

5) a fifth SNP marker, which is the 62 nd base G or A from the 5' end of the nucleotide sequence shown in SEQ ID NO. 5;

6) a sixth SNP marker, which is the 65 th base C or G from the 5' end of the nucleotide sequence shown in SEQ ID NO. 6;

7) a seventh SNP marker, which is the 64 th base A or G from the 5' end of the nucleotide sequence shown in SEQ ID NO. 7;

8) an eighth SNP marker, which is the 44 th base T or C from the 5' end of the nucleotide sequence shown in SEQ ID NO. 8;

9) a ninth SNP marker, which is the 69 th base A or T from the 5' end of the nucleotide sequence shown in SEQ ID NO. 9;

10) a tenth SNP marker, which is the 56 th base G or A from the 5' end of the nucleotide sequence shown in SEQ ID No. 10;

11) an eleventh SNP marker, which is the 61 st base C or T from the 5' end of the nucleotide sequence shown in SEQ ID NO. 11;

12) a twelfth SNP marker, which is the 61 st base C or T from the 5' end of the nucleotide sequence shown in SEQ ID NO. 12;

13) a thirteenth SNP marker, which is the 49 th base C or T from the 5' end of the nucleotide sequence shown in SEQ ID NO. 13;

14) a fourteenth SNP marker, which is the 53 rd base A or G from the 5' end of the nucleotide sequence shown in SEQ ID NO. 14;

15) a fifteenth SNP marker, which is the 38 th base T or C from the 5' end of the nucleotide sequence shown in SEQ ID NO. 15;

16) a sixteenth SNP marker, which is the 55 th base C or T from the 5' end of the nucleotide sequence shown in SEQ ID No. 16;

17) a seventeenth SNP marker, wherein the tenth SNP marker is the 60 th base T or G from the 5' end of the nucleotide sequence shown in SEQ ID NO. 17;

18) an eighteenth SNP marker, which is the 63 rd base G or A from the 5' end of the nucleotide sequence shown in SEQ ID NO. 18;

19) a nineteenth SNP marker, which is the 46 th base A or G from the 5' end of the nucleotide sequence shown in SEQ ID NO. 19;

20) a twentieth SNP marker, wherein the tenth SNP marker is a 49 th base T or C from the 5' end of the nucleotide sequence shown in SEQ ID NO. 20;

21) a twenty-first SNP marker, wherein the tenth SNP marker is the 56 th base C or T from the 5' end of the nucleotide sequence shown in SEQ ID NO. 21;

22) a twelfth SNP marker, wherein the tenth SNP marker is the 55 th base T or C from the 5' end of the nucleotide sequence shown in SEQ ID NO. 22.

In the present invention, each SNP marker is considered to be a SNP associated with the identification of Sophora alopecuroides. The herba Swertiae Dilutae in herba Swertiae Dilutae identification refers to genus herba Swertiae (Vallisneria), belonging to family Amyda sinensis (Hydrocharitaceae), is submerged herbaceous plant, and is unisexual, hermaphrodite and heterotrophic. The plants of the genus tape grass are widely distributed all over the world, and 10 to 14 kinds are provided. The Sophora includes Sophora alopecuroides L strain, Asian Sophora alopecuroides L strain, and Sophora alopecuroides L strain. The identification of the tape grass in the invention comprises the identification of a tape grass strain. Preferably, the method comprises the identification of the dwarf type eel grass line bred by Taihe Shuihu limited, namely, the identification of unknown eel grass or eel grass line as the dwarf type eel grass line bred by Taihe Shuihu limited. The dwarf type eel grass strain bred by Taihe water company Limited belongs to a evergreen dwarf type eel grass strain.

In one embodiment, the eel identification-related SNP markers include a first SNP marker through a SNP marker twenty-two SNP marker.

In a preferred embodiment, the first SNP marker to the second twelve SNP markers are SNP markers related to identification of the eel grass strain. The SNP marker can be used for identifying all the eel grass, but the SNP marker of the invention plays a much higher value in identifying different eel grass strains than between different species because different species of eel grass can be generally judged by other simple methods such as morphology, first-generation sequencing and the like.

The alleles of the first SNP marker include an AA genotype, an AG genotype and a GG genotype.

The allelic type of the second SNP marker includes a GG genotype, a GA genotype, and an AA genotype.

The third SNP marker has an allele type including a GG genotype, a GT genotype, and a TT genotype.

The allelic type of the fourth SNP marker includes a GG genotype, a GA genotype, and an AA genotype.

The allelic type of the fifth SNP marker includes a GG genotype, a GA genotype, and an AA genotype.

The alleles of the sixth SNP marker include a CC genotype, a CG genotype and a GG genotype.

Alleles of the seventh SNP marker include an AA genotype, an AG genotype and a GG genotype.

The allelic type of the eighth SNP marker includes TT genotype, TC genotype and CC genotype.

The alleles of the ninth SNP marker comprise an AA genotype, an AT genotype and a TT genotype.

The allele type of the tenth SNP marker includes GG genotype, GA genotype and AA genotype.

The allelic type of the eleventh SNP marker includes CC genotype, CT genotype and TT genotype.

The allele type of the twelfth SNP marker comprises a CC genotype, a CT genotype and a TT genotype.

The allelic type of the thirteenth SNP marker includes a CC genotype, a CT genotype and a TT genotype.

The allele type of the fourteenth SNP marker includes an AA genotype, an AG genotype and a GG genotype.

The allele type of the fifteenth SNP marker includes TT genotype, TC genotype and CC genotype.

The allele type of the sixteenth SNP marker comprises a CC genotype, a CT genotype and a TT genotype.

The alleles of the seventeenth SNP marker include TT genotype, TG genotype and GG genotype.

The allele type of the eighteenth SNP marker includes a GG genotype, a GA genotype and an AA genotype.

The alleles of the nineteenth SNP marker include an AA genotype, an AG genotype and a GG genotype.

The allele type of the twentieth SNP marker includes TT genotype, TC genotype and CC genotype.

The alleles of the twenty-first SNP marker include a CC genotype, a CT genotype and a TT genotype.

The allelic type of the twelfth SNP marker includes TT genotype, TC genotype and CC genotype.

The correspondence between the SNP markers and the SNP site sequence numbers thereof is shown in the following table:

SNP markers	SNP site number
		First SNP marker	Re344168_1
Second SNP marker	Re462581_1
		Third SNP marker	Re547001_1
Fourth SNP marker	Re567049_1
		Fifth SNP marker	Re568567_1
Sixth SNP marker	Re569904_1
		Seventh SNP marker	Re572019_1
Eighth SNP marker	Re588584_1
		Ninth SNP marker	Re598414_1
Tenth SNP marker	Re601118_1
		Eleventh SNP marker	Re623292_1
Twelfth SNP marker	Re625719_1
		Thirteenth SNP marker	Re632532_1
Fourteenth SNP marker	Re642863_1
		Fifteenth SNP marker	Re656094_1
Sixteenth SNP marker	Re656788_1
		Seventeenth SNP marker	Re657798_1
Eighteenth SNP marker	Re660420_1
		Nineteenth SNP marker	Re667567_1
Twentieth SNP marker	Re683984_1
		Twenty-first SNP marker	Re687111_1
The second twelve SNP marker	Re689563_1

The invention provides a group of primer groups for identifying tape grass, which comprises primer pairs for detecting any one or more SNP markers of a first SNP marker, a second SNP marker, a third SNP marker, a fourth SNP marker, a fifth SNP marker, a sixth SNP marker, a seventh SNP marker, an eighth SNP marker, a ninth SNP marker, a tenth SNP marker, an eleventh SNP marker, a twelfth SNP marker, a thirteenth SNP marker, a fourteenth SNP marker, a fifteenth SNP marker, a sixteenth SNP marker, a seventeenth SNP marker, an eighteenth SNP marker, a nineteenth SNP marker, a twentieth SNP marker, a twenty-first SNP marker and a twenty-second SNP marker. The forward and reverse nucleotide sequences of each primer pair are selected from one or more of SEQ ID NO. 23-SEQ ID NO. 66.

Specifically, the primer pair comprises a first primer pair, a second primer pair and up to a twenty-second primer pair. The first primer pair is used for detecting the first SNP marker, the second primer pair is used for detecting the second SNP marker, the third primer pair is used for detecting the third SNP marker, and the rest can be done until the twenty-second primer pair and the twenty-second SNP marker.

The specific base sequence of the primer pair exemplified above may be replaced with 1 or more bases or 1 or more bases may be added to the 3 'end or 5' end, as long as the specific recognition region can be specifically recognized under the conditions for carrying out PCR (preferably, annealing and self-annealing do not occur between primers used in a single reaction vessel). Here, the number of the plurality is, for example, 2 to 3. When 1 or more bases are added to the primer, it is preferable to add the base to the 5' end of the primer.

The identity of a nucleotide sequence obtained by substituting 1 or more bases in a specific nucleotide sequence of the primer exemplified above with other bases is preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, and more preferably 95% or more, with the nucleotide sequence before substitution (i.e., the nucleotide sequence represented by the sequence number).

The length of each primer is not particularly limited as long as it can specifically recognize the corresponding specific recognition region and hybridization does not occur between the primers, and is preferably 15 bases or more and 40 bases or less. The lower limit of the length of the primer is more preferably 16 bases or more, still more preferably 17 bases or more, and still more preferably 18 bases or more. More preferably, the upper limit of the length of the primer is 39 bases or less, still more preferably 38 bases or less, and still more preferably 37 bases or less.

According to the embodiment of the invention, the primers have very good specificity, and the primers can be used for accurately and effectively carrying out PCR amplification on the fragment where the related SNP marker of the tape grass to be detected is located.

The invention provides a kit for identifying tape grass, which comprises the primer group.

In one embodiment, the kit further comprises a reagent for extracting genomic DNA from a sample, a reagent for performing a PCR reaction using the primer.

In one embodiment, the reagent for extracting genomic DNA from a sample may be an existing kit.

In one embodiment, the reagent for performing PCR reaction using the primer set may be selected from DNA polymerase, PCR buffer, dNTP mixture and aqueous medium, or a premixed solution of the above reagents. For example, the premix is selected from the group consisting of extaq (hs): premix Ex Taq ^TM Hot Start Version,takara(RR030Q)。

The PCR buffer can generally provide the most suitable conditions for the enzymatic reaction in the PCR system. The buffer solution may be any buffer solution that can exert the above-described effects.

The dNTP mixture is usually used as a raw material for DNA synthesis, and specifically includes dATP, dGTP, dTTP, dCTP, and the like.

The aqueous medium may be used to adjust the concentration of the components of the PCR system, and may generally act as a dilution solvent.

The invention provides the application of the SNP marker related to the identification of the bitter herbs, a primer group for detecting the SNP marker and a kit for detecting the SNP marker in the identification of bitter herb strains, the establishment of a bitter herb molecular assisted breeding technical platform, the breeding of high-quality bitter herb strains and the evaluation of the genetic characteristics of the bitter herb strains.

The method for detecting the SNP marker of the present invention is not particularly limited. The SNP detection can be realized by the technologies of sequencing, single-strand conformation polymorphism polymerase chain reaction (PCR-SSCP), restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP), time-of-flight mass spectrometry and the like. The sequencing is a detection technology with high accuracy, strong flexibility, high flux and short detection period. Only a pair of primers are designed on two sides of the SNP locus, a product of 400-and 700-bp is amplified, and the genotype of the SNP locus can be directly detected through sequencing.

The invention provides an identification method of tape grass, which comprises the step of detecting SNP markers of the tape grass by using the primers or the kit.

In one embodiment, the method of identification comprises the steps of:

1) carrying out PCR amplification by using the primer group and using the genomic DNA of the tape grass to be detected as a template;

2) sequencing the PCR amplification product;

3) constructing a phylogenetic tree according to the sequencing result of the step 2).

The sequencing in step 2) is carried out by using the existing sequencing method, such as Sanger sequencing.

The phylogenetic tree can be constructed using existing software and methods. The ML cluster map is constructed, for example, using Phylilp.695, according to the Maximum likelihood Method (Maximum Likelyhood Method).

In one embodiment, the identification method specifically comprises the following steps:

1) carrying out PCR amplification by using the primer group and taking the genome DNA of the tape grass to be detected as a template;

2) extending the PCR amplification product;

3) carrying out capillary electrophoresis detection on the extension product;

4) the data is read and analyzed.

In one embodiment, step 2) is extended using a single well or a mixed well. The single well can only detect one SNP site per well. The mixed wells can simultaneously detect a plurality of SNP sites per well, and simultaneously perform multiplex analysis on up to 10 SNPs. The mixed hole detection can realize that one sample can detect all SNP sites through one-time PCR. The mixed well assay can be carried out by prior art techniques, for example using the SNaPshot Multiplex Kit. The mixed hole detection can save cost and improve efficiency.

In one embodiment, the amplification product obtained from PCR is purified and then extended.

In one embodiment, the product obtained from the extension is purified and then subjected to capillary electrophoresis.

In one embodiment, the primers used for extension are as set forth in SEQ ID NO.81 to SEQ ID NO. 102.

In particular, data reading and analysis may be performed with different internal standards for different experimental purposes.

In one embodiment, the internal standard is commercially available GS120-LIZ and the internal standard fragment sizes are 15, 20, 25, 35, 50, 62, 80, 110, 120, respectively.

Specifically, during data analysis, whether the tape grass to be detected and the dwarf tape grass bred by Taihe Shui limited company are of the same line or not and the distance of the group relationship between the tape grass to be detected and the dwarf tape grass bred by the Taihe Shui limited company are judged according to the consistency. When the consistency is higher and is closer to 100%, the population relationship between the tape grass to be detected and dwarf tape grass bred by Taihe Shui limited company is closer. When the consistency is 100%, the tape grass to be detected and the dwarf tape grass bred by Taihe Shui limited company are the same line.

The degree of agreement is calculated by the following method: p ═ (m-n)/(m-u) 100%; wherein, P is the consistency, n is the number of differential sites, m is the total number of sites (22), and u is the number of Null-value sites.

Because the tape grass strains to be detected and the tape grass strains bred by Taihe Shuihe limited company are all of the tape grass genus, and simultaneously, the hybridization condition possibly exists among the strains, the phylogenetic tree can not distinguish the strains with close genetic relationship, so that the judgment of the genetic relationship according to the consistency is more accurate than that of the phylogenetic tree.

The embodiments of the present invention are described below with specific examples, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and its several details are capable of modifications and variations in various obvious respects, all without departing from the spirit of the invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention is otherwise indicated. Unless defined otherwise, 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 belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention. The starting materials and reagents used in the invention are either commercially available or are conventionally selected, except where specifically indicated.

EXAMPLE 1 screening of polymorphic SNP site markers

(1) Primer design

SNP primers were developed using Primer 3 software for the first 100 SNP sites of the dwarf type eel grass of Taihe Shui Co., Ltd. and the Primer sequences are shown in Table 3. The sequencing verification of SNP obtained by RAD-seq sequencing is carried out by using Sanger sequencing, 5 samples of GS1a, GS4a, TH7, SH14 and HH23 are selected for carrying out sequencing verification on 100 sites, a tailed primer is adopted for amplification, a Common-F (AGTCACGACGTTGTAAAACGAC) is used as a sequencing primer, and an amplification system and a program are as follows:

TABLE 1 amplification procedure

TABLE 2 amplification System

(2) Development test

29 sites with preliminary polymorphisms were obtained by Sanger sequencing screening, followed by population validation using SNAPSHOT. The experimental procedure was as follows:

taking 1 mu l of DNA sample to carry out gel electrophoresis to identify bands, then using a Nano-100 ultraviolet spectrophotometer to determine the DNA concentration, and diluting the sample to 5-10 ng/mu l. The primers were diluted to the required concentration of 50. mu.M, 1. mu.l of each primer was added to 100. mu.l of the amplification primer Premix system, and 1. mu.l of each primer was added to 25. mu.l of the extension primer Premix system.

1) Multiplex PCR reaction (20. mu.l):

mu.l amplification primer Premix, 10. mu.L ExTaq (HS), 2. mu.L DNA, 4. mu. L H ₂ O

Procedure:

95℃2min+(95℃20s+65°(TD-0.5/C)40s+72℃1min)*12+(95℃20s+59°30s+72℃1min)*24+72℃10min+4℃∞

2) purification of PCR amplification product

mu.L of SAP enzyme and 2. mu.L of Exonuclease I enzyme were added to 15. mu.L of PCR product, and water-washed at 37 ℃ for 60 minutes, and then inactivated at 75 ℃ for 15 minutes.

3) SNAPSHOT multiplex single base extension reaction

mu.L of the SNaPshot Multiplex Kit (ABI), 2. mu.L of the purified Multiplex PCR product, 1. mu.L of the extension primer Premix, 2. mu. L H ₂ O

The procedure is as follows: 95 ℃ 10s + (95 ℃ 10s +50 ℃ 5s +60 ℃ 30s) 35+60 ℃ 30s +4 ℃ infinity

4) Purification of extension products

mu.L of SAP enzyme was added to 10. mu.L of extension product, and then inactivated in a water bath at 37 ℃ for 60 minutes and 85 ℃ for 15 minutes.

5) ABI3730XL sequencer sequencing on extension products

mu.L of the purified extension product was mixed with 0.5. mu.L of Liz120 SIZE STANDARD and 8.5. mu.L of Hi-Di, denatured at 95 ℃ for 5 minutes, and sequenced using ABI3730XL sequencer

6) Raw data detected on the ABI3730XL sequencing instrument was imported into GeneMapper 4.0(Applied Biosystems co., ltd., USA) for analysis.

The pre-developed primer sequence information (Table 3) and the split-well case (Table 4) are shown in the following table:

TABLE 3 Pre-development primer sequence information

TABLE 4 Split case

After 2 development experiments, the primer sequence, the primer proportion and the experimental system are optimized, 7 sites (Re654330_1, Re659448_1, Re687832_1, Re592992_1, Re689819_1, Re675055_1 and Re677724_1) in the original 29 sites can not be detected through SNAPSHOT, and after the sites are cancelled, the rest 22 sites can be detected through SNAPSHOT according to the original scheme.

Example 2

Examples 2 and 3 identification processes and phylogenetic trees were constructed using 22 SNP polymorphic sites selected in example 1, for example, dwarf type eel (GS) and 14 wild eel populations (Taihu TH, Honghu HH, Shanghai SH, Huaian HA, Huangshan HS, Hangzhou HZ, Guangzhou GZ, Dali DL, Kunming KM, Guiyang GY, Liuzhou LZ, Changsha CS, Yingtan YT, and Jingdezhen JDZ) selected from Taihe Water corporation.

(1) Collecting and storing samples: collecting dwarf type eel Grasses (GS) bred by Taihe water company and 14 wild eel grass groups (Taihu TH, Honghu HH, Shanghai SH, Huaian HA, Huangshan HS, Hangzhou HZ, Guangzhou GZ, Dali DL, Kunming KM, Guiyang GY, Liuzhou LZ, Changsha CS, Yingtan YT and Jingdezhen JDZ), temporarily culturing in clean water in a laboratory for three days, soaking for 15min by using 5 per thousand potassium permanganate diluent, removing microorganisms on the surface of leaves, and avoiding pollution caused by subsequent DNA extraction experiments; after soaking, selecting healthy leaves, cleaning with clear water, wiping, putting into a 10ml centrifuge tube, marking, and putting into a refrigerator at minus 80 ℃ for freezing and storage for later use.

(2) Extracting plant group DNA: TIANGEN DP305 kit is selected to extract leaf genome DNA of the selected tape grass material.

(3) And (3) PCR amplification: PCR amplification was performed using the 22 SNP polymorphism primer pairs (shown in Table 3) selected in example 1. The primers of Table 3 were first mixed in the proportions shown in Table 5 and amplified using the extracted genomic DNA as a template.

TABLE 5 mixing ratio of labeled primers

The concentration of the primer working solution is 50 mu M, the total volume is 100 mu l after forward and reverse primers and deionized water are added respectively, and the amplification system is as follows:

TABLE 6 amplification System

Reagent	Volume (μ l)
		EXTaq(HS)	5
Pmix	1
		H 2 O	3
DNA	1
		Total	10

Wherein EXTTaq (HS) is Premix Ex Taq ^TM Hot Start Version, takara (RR 030Q); pmix is the primer mixture in Table 6; DNA was diluted to a concentration of 30 ng/. mu.l and then 1. mu.l was added.

The amplification procedure was: 2min at 95 ℃; 12 × (94 ℃ for 20 s; 65 ℃ (TD-0.5/C) for 40 s; 72 ℃ for 1 min); 24X (94 ℃ C. for 20 s; 59 ℃ C. for 30 s; 72 ℃ C. for 1 min); 10min at 72 ℃. The annealing temperature was reduced by 0.5 ℃ per cycle for the first 12 cycles.

The experimental procedure was as follows:

1) according to the experimental sample size, the EXTaq, the Pmix and the H are prepared ₂ And (4) O, a premixed liquid.

2) A96-well PCR plate was prepared, and 9. mu.l of the above premix was added to each well.

3) Add 1. mu.l of diluted DNA to the well.

4) After covering the rubber pad, it was centrifuged at 1500rpm for 30 seconds.

5) Put into a PCR instrument and run the PCR program.

Remarking: the whole experiment was conducted on an ice box.

(4) And (3) PCR product purification: the total amplification product of step (3) was purified according to the following system and procedure.

TABLE 7 purification System

Reagent	Volume of(μl)
		H 2 O	1
FASTAP	1
		EXO 1	0.05
PCR product	3
		Total	5

Purification procedure: 50min at 37 ℃; 15min at 85 ℃; 4 ℃ forever

Experimental procedures

1) The H20, FASTAP, EXOI premix was prepared according to the sample size.

2) A new 96-well PCR plate was taken and 3. mu.l of the 2.1 PCR product was added to the new PCR plate.

3) Mu.l of the premix of 1) was added to each well.

4) After covering the rubber pad, it was centrifuged at 1500rpm for 30 seconds.

5) Put into a PCR instrument and run the purification program.

Remarking: the whole experiment was conducted on an ice box.

(5) And (3) extension reaction: mixing the purified products obtained in the step (4) according to the following proportion and extending.

TABLE 8 extended primer mix ratio

TABLE 9 elongation System

Primer working solution 50uM, total volume after primer extension and deionized water 25 ul.

TABLE 10 elongation system

Reagent	Volume (μ l)
		SNP KIT	1
Pmix	1
		Purification of the product	3
Total	5

And (3) extension procedure: 30s at 95 ℃; 35 × (95 ℃ C. for 10 s; 50 ℃ C. for 5 s; 60 ℃ C. for 30 s); 30s at 60 ℃; the 4 ℃ forever experiment was performed as follows

1) And configuring SNP KIT and Pmix premix according to the experimental sample size.

2) A96-well PCR plate was used, and 3. mu.l of the purified product was added thereto.

3) Mu.l of the premix was added to each well.

4) After covering the rubber pad, it was centrifuged at 1500rpm for 30 seconds.

5) Put into a PCR instrument and run the extension program.

Remarking: the whole experiment was conducted on an ice box.

(6) And (3) purification of an extension product: the extension product obtained in the step (4) is operated according to the following purification system and experimental steps

TABLE 11 purification System

Reagent	Volume (μ l)
		H 2 O	1.5
FASTAP	0.5
		Extension product	5
Total	7

And (3) purification procedure: 50min at 37 ℃; 15min at 85 ℃; 4 ℃ forever

Experimental procedures

1) Configure H according to sample size ₂ O, FASTAP premix liquid.

2) Adding the premixed solution of 1) into the 2.3 extension product

3) After covering the rubber pad, the mixture was centrifuged at 1500rpm for 30 seconds.

4) Put into a PCR instrument and run the purification program.

Remarking: the whole experiment was conducted on an ice box.

(7) And (3) capillary electrophoresis detection: detecting the purified product obtained in step (6) according to the following system and operation

TABLE 12 test System

Reagent	Volume of
		Liz 120	0.5μl
HiDi	8.5μl
		Purified product	1μl
total	10μl

Sequencer 3730xl parameter settings are shown in FIG. 1.

Experimental procedures

1) The LIZ120, HIDI premix is preconfigured.

2) Take 96-well plate, add 9. mu.l of 1) premix per well.

3) Add 1. mu.l of purified product to a 96 well plate.

4) After covering the rubber pad, it was centrifuged at 1500rpm for 30 seconds.

5) Placing into a PCR instrument, and running at 95 deg.C for 5 min.

6) Detecting the product treated by 5) by capillary electrophoresis (3730 xl).

7) Data reading: reading data of the qualified sample obtained in the step 6) on a computer, wherein the data is read by using GeneMapper 4.0(applied biosystems Co., Ltd., USA) software; the internal standard is selected to be GS 120-LIZ. The internal standard fragment sizes were 15, 20, 25, 35, 50, 62, 80, 110, 120, respectively, according to product introduction. The relevant panel, bin and Analysis Methods files were analyzed. The allele position of each locus in the bin file can be fine-tuned according to the actual position.

8) And judging the parting result, wherein the preset judging scheme is as follows:

a) the target region of the typing map has a normal peak pattern: alleles were read normally.

b) There is a hetero-peak interference in the typing pattern: processing according to Null value Null.

c) And (3) no parting result of a target area in the parting map: processing according to Null value Null.

d) All the site typing results were completely consistent: the consistency P is 100 percent, and belongs to Taihe water breeding Group (GS). Calculating the formula: p ═ (m-n)/(m-u) 100%; p: consistency, n: number of ectopic sites, m: total number of sites (22), u: number of Null value sites.

e) There are sites of inconsistent typing results: the higher the degree of agreement (the greater the number of identical sites), the closer the relationship to the GS population.

Null value Null: and (4) carrying out rejection processing, namely rejecting Null loci when calculating the P value.

9) The 300 sample identity results are shown in tables 13-1 to 13-5. The origin of the samples is shown in tables 13-6.

TABLE 13-1 sample conformity

TABLE 13-2 sample conformity

TABLE 13-3 sample conformity

TABLE 13-4 sample conformity

TABLE 13-5 sample conformity

TABLE 13-6 sample sources

Sampling point	Longitude (G)	Latitude	Sample numbering
				Gold mountain area of Shanghai city	121°21'34.7"E	30°50'50.9"N	GS1～GS30
High and new district of Jiangsu Suzhou city	120°29'00.2"E	31°12'40.5"N	TH1～TH30
				Pudong New Area, Shanghai	121°50'09.1"E	30°51'39.5"N	SH1～SH30
Huai Yin district of Huai' an city of Jiangsu province	118°55'52.1"E	33°26'30.0"N	HA1～HA30
				Hubei Jingzhou City Authentic City	113°15'10.2"E	29°49'12.1"N	HH1～HH30
Tunxi district of Huangshan city, Anhui	118°20'55.4"E	29°43'07.5"N	HS1～HS30
				City of Hangzhou Zhejiang	120°11'0.36"E	30°16'48.77"N	HZ1～HZ30
Huangpu district of Guangdong Guangzhou City	113°27'8.18"E	23°5'40.7"N	GZ1～GZ30
				Yunan Dali Bai autonomous State Dali City	100°13'44.86"E	25°37'0.07"N	DL1～DL30
Yunan Kunming City Yongzong	102°46'47.1"E	24°51'01.5"N	KM1～KM30
				Mountain lake area of Guizhou Guiyang city	106°35'33.49"N	26°38'16.03"N	GY1～GY30
Middle region of Liuzhou city	109°27'47.9"E	24°21'01.7"N	LZ1～LZ30
				Wangcheng city of Changsha in Hunan province	112°56'08.4"E	28°18'59.3"N	CS1～CS30
Jiangxi Yingtan city moon and lake region	117°04'03.6"E	28°16'29.1"N	YT1～YT30
				Changjiang district of Jingdezhen city of Jiangxi	117°10'12.7"E	29°15'07.3"N	JDZ1～JDZ30

Example 3 construction of phylogenetic Tree

Sample collection, genomic DNA extraction, PCR amplification procedures were the same as in example 2. Sequencing the PCR amplification product. According to the sequencing results, a phylogenetic tree was constructed using the 22 polymorphic SNP loci described in this application for 15 populations (18 samples), and an ML cluster map was constructed using phylip.695 according to the Maximum likelihood Method (Maximum Likelyhood Method) (fig. 2). According to the established phylogenetic tree, the group genetic relationship between the tape grass to be detected and the dwarf tape grass strain bred by Taihe Shui Limited company can be visually seen.

The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods set forth herein, as well as variations of the methods of the invention, will be apparent to persons skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

Sequence listing

<110> Shanghai Taihe water science and technology development Co., Ltd

Shanghai Ocean University

<120> an identification method of short and evergreen tape grass strain

<160> 102

<170> SIPOSequenceListing 1.0

<210> 1

<211> 110

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

aattctttca aaccctcaga gcaacgtggt ctttttatar ccgtaggatc ctagatccaa 60

cgggtaagaa tccttgtacg cattacaagg aggctttgca acactaaagt 110

<210> 2

<211> 105

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tcaggatggt ccatcatgag ttccatcttg gagtattagt agcagtagta gggatgcaaa 60

cggatacrga tatatccgtt tccgtccgct tatatctact accat 105

<210> 3

<211> 105

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aaaattttat tttttgaatc tgacaatcat caattccttc ataaaagaaa gtkaatttga 60

caagggtcct tcattgaaaa gaaaaaaaca gtgatcttgt gccac 105

<210> 4

<211> 105

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

taaccaatgg gattgtcttc ccccaaagtt ttctcttgaa tctatcaatg attttgatgg 60

gaatctctcg raaagtcaag tcctcctgga catctaccac tggct 105

<210> 5

<211> 105

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gttatggttt tgagcatgaa acaaccttgg aaggttgtag gagcgtggat tcgtgtttct 60

crtcgtgttg aggtagggat catacctatt ctctataacg tcccg 105

<210> 6

<211> 105

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cccattaagt tgtttgttca tcgcattact tttaacctca actgtcgtct catattcgat 60

aaaasttctt gtattatgca ggatttggca gcaaggaaga ggatt 105

<210> 7

<211> 110

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

aattcgggaa gaggattcag gttatgatta gataagtcta gcctccctta agagggactg 60

ctaragggct tgaagccttg ataattgcct aatagaagtt aggtgaaggc 110

<210> 8

<211> 105

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tctgcatgcc caatacatat ccgtccgccg tggtcacctg agtygcggct ctatttctca 60

tgtttgtttg cagccaattt tgcaaaaact agtgtgagct tgtgt 105

<210> 9

<211> 105

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

taacatttgg cttaatgtct tcggagatgc ctcgggtttg atggttaata aaaataaatt 60

tggtttttwa ctatttagtt tccttgatga tgagaaccat tagtt 105

<210> 10

<211> 105

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ctacaaagct tcagtgattg taataaaaca cgatctattc actgaaataa catacratat 60

aattgcttct catccaatta ttagctctac caactcttct tatcc 105

<210> 11

<211> 105

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

acttggaagt gggaatggca gcaagagcaa caatacctgt agcgctcaac ccaagggaag 60

yagtagcatg gacatcctgg gtagagttgg agtgcaacat attca 105

<210> 12

<211> 105

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gctgtggttt tgagcgtggc ttgaccaaag gagtcttgta gaacgtggat tcgtgtttcg 60

ygacgttcta aggtaggtat cctacccatt ttcatagtca tttat 105

<210> 13

<211> 105

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tgcctaattc tcaactcata tccctaaggg aagaagattt tgatgatcyc tcagaattgt 60

ctgccatact tcctctcatg gaccctccat ttgagaagat cactc 105

<210> 14

<211> 105

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gttgtggttt cgagtgtaga atgaccaagg aaggctagag gaacgtggat tcrtgtttcc 60

cgacgtgttg agatagggat caatcatatt tccgtagcca tttga 105

<210> 15

<211> 105

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

catccattct aggttgacaa cttgacatct ctttattyag gagatagggg gagtgtacat 60

ataatttgat gtgagttcca tccatgccta gttaaggctt acaaa 105

<210> 16

<211> 105

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

cagctctttc atgcagacaa cggctcaatt cccagcaagg agaatgatca ccacyagttg 60

ctctccgttt gtgatgccct gttcccggaa tacccaatag gtgga 105

<210> 17

<211> 105

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

tttacctcaa tcactctgaa gaggaaatca gatggatgga tttttcaagt tgtcaatack 60

tatggtccat gagccccgga atgcaaacct cactttttaa aggaa 105

<210> 18

<211> 105

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gatattgaat atatccatcc agactttaaa cttccaaatt ccttctttga cagacacctt 60

ctrgagcact atgtctactg ctactgactc tttaggcttg cacaa 105

<210> 19

<211> 105

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

ggattttgtt ggtcgcaagt aattaagtgg aattgtgaat ataccrtgta gtagttggag 60

gcatgaacta tctagttcca gtgagctggt ggttaaatta tttct 105

<210> 20

<211> 105

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

cccattgagg tcatgaaggc ttttagagga gagggaaaaa ctatagacyg tgggtccatt 60

gaagaatgta gaactattca agaattggat gacttcacat ccgtg 105

<210> 21

<211> 105

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

aaaagttgaa ttgataaaac gtggcatttt ttacaaacta aagatactga cctctytgaa 60

cccagggaaa caagatgaat aggaaaggga ccaagaacat gtaat 105

<210> 22

<211> 105

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

acatgcttcc ctccattctc gaccgactct ttctcaggta tctcctttat acggyccaat 60

tcctgtgaga cagagagcac agccgcatca aacgataaca cattg 105

<210> 23

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

ttcaaaccct cagagcaacg 20

<210> 24

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

aatgcgtaca aggattctta ccc 23

<210> 25

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

ttggagtatt agtagcagta gtaggga 27

<210> 26

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

tggtagtaga tataagcgga cgga 24

<210> 27

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

tgaatctgac aatcatcaat tcct 24

<210> 28

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

tttttctttt caatgaagga ccct 24

<210> 29

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

ttcccccaaa gttttctctt ga 22

<210> 30

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

cagtggtaga tgtccaggag ga 22

<210> 31

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

catgaaacaa ccttggaag 19

<210> 32

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

gggacgttat agagaatagg tatgatc 27

<210> 33

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

gttgtttgtt catcgcatta ctttta 26

<210> 34

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

atcctcttcc ttgctgcca 19

<210> 35

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

gggaagagga ttcaggttat gatta 25

<210> 36

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

ttctattagg caattatcaa ggct 24

<210> 37

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

gcccaataca tatccgtccg 20

<210> 38

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

tggctgcaaa caaacatgag 20

<210> 39

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

tgggatagca gaaatgggga 20

<210> 40

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

gcattgtcag ttggttgttg g 21

<210> 41

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

cggagatgcc tcgggttt 18

<210> 42

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

actaatggtt ctcatcatca aggaaa 26

<210> 43

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

gcttcagtga ttgtaataaa acacgat 27

<210> 44

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

gttggtagag ctaataattg gatgaga 27

<210> 45

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

ggcagcaaga gcaacaatac c 21

<210> 46

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

gcactccaac tctacccagg at 22

<210> 47

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

ggcttgacca aaggagtctt gta 23

<210> 48

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

aaatgactat gaaaatgggt aggata 26

<210> 49

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 49

tgcctaattc tcaactcata tccc 24

<210> 50

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 50

agtgatcttc tcaaatggag ggtc 24

<210> 51

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 51

agtgtagaat gaccaaggaa gg 22

<210> 52

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 52

aaatggctac ggaaatatga ttga 24

<210> 53

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 53

tcgtgctttt tatcagtatt aagaag 26

<210> 54

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 54

ttgtttcatt atcagcattt cca 23

<210> 55

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 55

catccattct aggttgacaa cttga 25

<210> 56

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 56

ttgtaagcct taactaggca tgg 23

<210> 57

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 57

gcagacaacg gctcaattcc 20

<210> 58

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 58

aacagggcat cacaaacgg 19

<210> 59

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 59

aatcagatgg atggattttt caa 23

<210> 60

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 60

aaagtgaggt ttgcattccg 20

<210> 61

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 61

gctcgtggtg tcttgctact tg 22

<210> 62

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 62

caactgatat cggggctatg c 21

<210> 63

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 63

ttaaacttcc aaattccttc tttgac 26

<210> 64

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 64

aagcctaaag agtcagtagc agtagac 27

<210> 65

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 65

ggattttgtt ggtcgcaagt aa 22

<210> 66

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 66

cagctcactg gaactagata gttcatg 27

<210> 67

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 67

cacgtagcat gtctttggca a 21

<210> 68

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 68

attgttggga ttatattgga agtg 24

<210> 69

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 69

cccatgaggt cgtgatgaag 20

<210> 70

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 70

tgcttttaag gcaattcctt tct 23

<210> 71

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 71

aaggctttta gaggagaggg aa 22

<210> 72

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 72

gatgtgaagt catccaattc ttgaa 25

<210> 73

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 73

ttgaattgat aaaacgtggc at 22

<210> 74

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 74

atgttcttgg tccctttcct att 23

<210> 75

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 75

acaaccccat gcttgagcc 19

<210> 76

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 76

ccattcgccg tgagattctt 20

<210> 77

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 77

catgcttccc tccattctcg 20

<210> 78

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 78

tgcggctgtg ctctctgtc 19

<210> 79

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 79

tcaatttgag ttctttacat ttcacc 26

<210> 80

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 80

tggttgttca atggaaccta caat 24

<210> 81

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 81

tttttttttt tttttcagag caacgtggtc tttttata 38

<210> 82

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 82

tttttttttt tttttttttt tttttatcaa ggcttcaagc cct 43

<210> 83

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 83

tttttttttt tttttttttt ttttccaaat cctgcataat acaagaa 47

<210> 84

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 84

tttttttttt tttttttttt ttttttttta tgtccaggag gacttgactt t 51

<210> 85

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 85

tttttttttt tttttttttt tttttttttt ttttaaacat gagaaataga gycgc 55

<210> 86

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 86

tttttttttt tttttttttt tttttttttt tttttttttt gatccctacc tcaacacga 59

<210> 87

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 87

tttttttttt tttttttttt tttttttttt tttttttttt taatgaagga cccttgtcaa 60

att 63

<210> 88

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 88

ttttttcctt ctttgacaga caccttct 28

<210> 89

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 89

ttttttttct aagggaagaa gattttgatg atc 33

<210> 90

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 90

tttttttttt acacgatcta ttcactgaaa taacatac 38

<210> 91

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 91

tttttttttt tttttttttt ttttttttta gggatgcaaa cggatac 47

<210> 92

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 92

tttttttttt tttttttttt ttttttctca tcatcaagga aactaaatag t 51

<210> 93

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 93

tttttttttt tttttttttt tttttttttt gatggatttt tcaagttgtc aatac 55

<210> 94

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 94

tttttttttt tttttttttt tttttttttt tttttttctc aggtatctcc tttatacgg 59

<210> 95

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 95

tttttttttt tttttttttt tttttttttt tttttttttt taggatacct accttagaac 60

gtc 63

<210> 96

<211> 67

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 96

tttttttttt tttttttttt tttttttttt tttttttttt ttttacaaac taaagatact 60

gacctct 67

<210> 97

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 97

ttttttttcc caggatgtcc atgctact 28

<210> 98

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 98

ttttttttgt tgacaacttg acatctcttt att 33

<210> 99

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 99

tttttttttt tttttttcat cacaaacgga gagcaayt 38

<210> 100

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 100

tttttttttt tttttttttt ttttcatgcc tccaactact aca 43

<210> 101

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 101

tttttttttt tttttttttt tttttttttt tagaggagag ggaaaaacta tagac 55

<210> 102

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 102

tttttttttt tttttttttt tttttttttt tttttttttt ttaggctaga ggaacgtgga 60

ttc 63

Claims (9)

1. The identification method of the tape grass is characterized by comprising the step of detecting the SNP marker of the tape grass by using a primer group or a kit which can specifically identify the SNP marker of the tape grass.

2. The identification method according to claim 1, wherein the SNP markers of tape grass comprise one or more of a first SNP marker to a twenty-second SNP marker:

1) a first SNP marker, which is the 40 th base A or G from the 5' end of the nucleotide sequence shown in SEQ ID NO. 1;

2) a second SNP marker, which is the 68 th base G or A from the 5' end of the nucleotide sequence shown in SEQ ID NO. 2;

3) a third SNP marker, which is the 53 rd base G or T from the 5' end of the nucleotide sequence shown in SEQ ID NO. 3;

4) a fourth SNP marker, which is the 71 th base G or A from the 5' end of the nucleotide sequence shown in SEQ ID NO. 4;

5) a fifth SNP marker, which is the 62 nd base G or A from the 5' end of the nucleotide sequence shown in SEQ ID NO. 5;

6) a sixth SNP marker, which is the 65 th base C or G from the 5' end of the nucleotide sequence shown in SEQ ID NO. 6;

7) a seventh SNP marker, which is the 64 th base A or G from the 5' end of the nucleotide sequence shown in SEQ ID NO. 7;

8) an eighth SNP marker, which is the 44 th base T or C from the 5' end of the nucleotide sequence shown in SEQ ID NO. 8;

9) a ninth SNP marker, which is the 69 th base A or T from the 5' end of the nucleotide sequence shown in SEQ ID NO. 9;

10) a tenth SNP marker, which is the 56 th base G or A from the 5' end of the nucleotide sequence shown in SEQ ID NO. 10;

11) an eleventh SNP marker, which is the 61 st base C or T from the 5' end of the nucleotide sequence shown in SEQ ID NO. 11;

12) a twelfth SNP marker, which is the 61 st base C or T from the 5' end of the nucleotide sequence shown in SEQ ID NO. 12;

13) a thirteenth SNP marker, which is the 49 th base C or T from the 5' end of the nucleotide sequence shown in SEQ ID NO. 13;

14) a fourteenth SNP marker, which is the 53 rd base A or G from the 5' end of the nucleotide sequence shown in SEQ ID NO. 14;

15) a fifteenth SNP marker, which is the 38 th base T or C from the 5' end of the nucleotide sequence shown in SEQ ID NO. 15;

16) a sixteenth SNP marker, which is the 55 th base C or T from the 5' end of the nucleotide sequence shown in SEQ ID NO. 16;

17) a seventeenth SNP marker, wherein the tenth SNP marker is the 60 th base T or G from the 5' end of the nucleotide sequence shown in SEQ ID NO. 17;

18) an eighteenth SNP marker, which is the 63 rd base G or A from the 5' end of the nucleotide sequence shown in SEQ ID NO. 18;

19) a nineteenth SNP marker, which is the 46 th base A or G from the 5' end of the nucleotide sequence shown in SEQ ID NO. 19;

20) a twentieth SNP marker, wherein the tenth SNP marker is a 49 th base T or C from the 5' end of the nucleotide sequence shown in SEQ ID NO. 20;

21) a twenty-first SNP marker, wherein the tenth SNP marker is the 56 th base C or T from the 5' end of the nucleotide sequence shown in SEQ ID NO. 21;

22) a twelfth SNP marker, wherein the tenth SNP marker is the 55 th base T or C from the 5' end of the nucleotide sequence shown in SEQ ID NO. 22.

3. The method as claimed in claim 1, wherein the kit comprises a primer set capable of specifically recognizing SNP markers of tape grass.

4. The method according to claim 3, wherein the primer set comprises a primer set represented by SEQ ID No.23 to SEQ ID No. 66.

5. The method of claim 1, wherein the kit further comprises a reagent for extracting genomic DNA from a sample, and a reagent for performing a PCR reaction using the primer set.

6. The method of claim 1, wherein the method of identification comprises the steps of:

1) carrying out PCR amplification by using the primer group and using the genomic DNA of the tape grass to be detected as a template;

2) sequencing the PCR amplification product;

3) and constructing a phylogenetic tree according to the sequencing result of the step 2).

7. The method of claim 1, wherein the method of identification comprises the steps of:

1) carrying out PCR amplification by using the primer group and using the genomic DNA of the tape grass to be detected as a template;

2) extending the PCR amplification product;

3) carrying out capillary electrophoresis detection on the extension product;

4) the data is read and analyzed.

8. The method of claim 7, further comprising one or more of the following features:

1) purifying the PCR amplification product and then extending, wherein mixed hole extension is adopted during extension;

2) purifying the extension product and then performing capillary electrophoresis;

3) the primers used for extension are shown as SEQ ID NO. 81-SEQ ID NO. 102;

4) the higher the consistency obtained by data analysis is, the closer the consistency is to 100%, the closer the population relationship between the tape grass to be tested and the short evergreen tape grass bred by Taihe water company Limited is; when the consistency is 100%, the tape grass to be tested and the dwarf tape grass bred by Taihe Shui Limited are of the same strain.

9. The method according to claim 1, wherein the method for identifying tape grass is a method for identifying a line of tape grass.

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