CN110093436B - SNP locus multicolor fluorescence detection primer, kit and detection method for identifying eucalyptus clone and application of SNP locus multicolor fluorescence detection primer - Google Patents
SNP locus multicolor fluorescence detection primer, kit and detection method for identifying eucalyptus clone and application of SNP locus multicolor fluorescence detection primer Download PDFInfo
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Abstract
The invention discloses SNP locus multicolor fluorescence detection primers, a kit, a detection method and application thereof for identifying eucalyptus clones. The SNP marker with high polymorphism is screened from a eucalyptus genome, a multicolor fluorescence multiplex detection system of 21 loci is optimized and established, and the typing result has the characteristics of easy interpretation of the banding pattern, high locus polymorphism and easy distinction of fluorescence. The method has the advantages of high experimental efficiency, accurate detection, convenient operation and the like. Can provide important technical support for fast genetic evaluation of germplasm resources, molecular marker assisted breeding, clone molecular fingerprint construction and identification of eucalyptus in future. Meanwhile, molecular fingerprints of 58 domestic common eucalyptus clones are constructed, and the clones are successfully identified, so that the workload of identifying the eucalyptus clones is greatly reduced. The method can effectively discriminate counterfeit and disordered clone, and can practically ensure the rights and interests of improved variety breeders and forest growers.
Description
Technical Field
The invention belongs to the technical field of molecular markers, and particularly relates to SNP locus multicolor fluorescence detection primers, kit, detection method and application thereof for identifying eucalyptus clones.
Background
Eucalyptus is a generic name of Eucalyptus (Myrtaceae) genus, cupola (Angopora) genus and umbrella house (Corymbia) genus tree species, has characteristics of rich genetic diversity, rapid growth, strong adaptability and the like, is widely planted in tropical and subtropical areas of China, and plays an important role in relieving the situation of shortage of wood supply and demand in China.
Eucalyptus is mainly planted in a clone tissue culture mode in production. However, with the increase of tissue culture propagation algebra, the expansion of popularization planting area and the expansion of circulation market, the problems of error recording and secondary filling of eucalyptus clones are increasingly serious, and the phenomena of renaming clones cultivated by others, being own and the like occur in some units. The identification of the eucalyptus clone is carried out, which is not only beneficial to the maintenance of the interests of forest farmers, but also beneficial to the reinforcement of the protection of the intellectual property rights of eucalyptus varieties.
The identification of eucalyptus clones is usually carried out based on morphological characteristics of leaves, barks, fruits and the like, so that different person judgment standards are difficult to unify, the identification result is inaccurate, and the safe production of the eucalyptus is difficult to ensure. The DNA molecular marker, especially the single nucleotide polymorphism (single nucleotide polymorphism, SNP) marker has the characteristics of wide distribution, good repeatability, low mutation rate, easy high-throughput automatic parting and the like, can provide unique polymorphism information for a certain variety, and can be used for accurately detecting and identifying the plant variety. The SNaPshot technology is a multiple micro-sequencing technology for SNP detection, has the characteristics of high detection speed, high accuracy, relatively low cost and the like, and is widely applied to the fields of forensic SNP identification, human genetic disease detection and the like. At present, although a certain DNA fingerprint analysis work has been carried out on eucalyptus clones, a set of simple and easy SNP detection systems still do not exist.
Disclosure of Invention
The invention aims to provide a SNP primer set for eucalyptus clone identification based on SNaPshot technology, which is used for quick and convenient identification of eucalyptus clone and establishment of molecular fingerprint of common eucalyptus clone aiming at the current situation of eucalyptus clone planting and the defects and shortcomings of the existing identification method. The invention can effectively discriminate counterfeit and disordered clone, effectively ensure the rights and interests of improved variety breeders and forest growers, improve the degree of improved variety development of eucalyptus commodity forests, and effectively detect partial DNA degradation detection materials.
The first object of the present invention is to provide a SNP locus multicolor fluorescence detection primer set for identifying eucalyptus clones, which comprises a PCR amplification primer pair and an extension primer of 21 SNP gene loci, wherein the amplification primer pair and the extension primer have the sequences shown in the following table 1:
TABLE 1 primer set for identifying 21 SNP loci of Eucalyptus clones
The invention also provides a SNP locus multicolor fluorescence detection kit for identifying eucalyptus clones, which comprises the SNP locus multicolor fluorescence detection primer set for identifying eucalyptus clones.
The third object of the invention is to provide the application of the SNP locus multicolor fluorescence detection primer set for identifying eucalyptus clones or the SNP locus multicolor fluorescence detection kit for identifying eucalyptus clones in identifying eucalyptus clones.
The fourth object of the present invention is to provide a method for identifying a clone of eucalyptus by using the SNP locus multicolor fluorescence detection primer set for identifying a clone of eucalyptus, comprising the steps of:
a. extracting clone DNA of eucalyptus to be detected;
b. c, taking the eucalyptus clone DNA extracted in the step a as a template, and performing multiplex PCR amplification by using the amplification primers in the SNP locus multicolor fluorescence detection primer set for identifying the eucalyptus clone to obtain an amplification product;
c. removing redundant dNTPs and primers in the amplified product by adopting exonuclease I and shrimp alkaline phosphatase to obtain a purified amplified product;
d. performing an extension reaction on the purified amplification product by using the extension primer in the SNP locus multicolor fluorescence detection primer set for identifying the eucalyptus clone to obtain an extension product;
e. purifying the extension product by using shrimp alkaline phosphatase, and removing redundant extension primers to obtain a purified extension product;
f. diluting the extension product purified in the step e, adding a test reagent, uniformly mixing, carrying out denaturation treatment to obtain a denatured mixed solution, carrying out typing detection on the mixed solution on an ABI 3130xl genetic analyzer, and determining genotypes of 21 SNP loci according to positions and colors of peaks of the extension product by using a GeneMapper 4.1.
Specifically, the multiplex PCR amplification in the step b uses a reaction system of 20. Mu.L, comprising: 2 XType-it Multiplex PCR Master Mix. Mu.L, 4. Mu.L of water, 3. Mu.L of 21 pairs of amplification primers in a SNP locus multicolor fluorescence detection primer set for identifying eucalyptus clone, which are mixed in equal amounts, wherein the final concentration of a single amplification primer in a reaction system is 0.037. Mu.M, 30 ng/. Mu.L of eucalyptus clone DNA to be detected is 3. Mu.L; the reaction procedure is as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 60℃for 90s, elongation at 72℃for 30s,32 cycles; extending at 60deg.C for 30min; preserving heat at 20 ℃.
In the step c, the purification reaction system for removing redundant dNTPs and primers in the amplified product by adopting exonuclease I and shrimp alkaline phosphatase comprises the following steps: to 2. Mu.L of Exo SAP-IT was added 5. Mu.L of amplification product under the following reaction conditions: 37 ℃ for 1h,75 ℃ for 15min, and finally 4 ℃ for preservation.
The extension reaction in the step d described above employs 10. Mu.L of a reaction system comprising: SNaPShot Multiplex Ready Reaction Mix 5. Mu.L, 3. Mu.L of purified amplification product, and 2. Mu.L of 21 extension primers in a SNP locus multicolor fluorescence detection primer set for identifying eucalyptus clone, which are mixed in equal amounts, wherein the final concentration of the SNaPshot04 extension primer in the reaction system is 0.14. Mu.M, and the final concentrations of the SNaPshot01-SNaPshot03 and the SNaPshot05-SNaPshot21 extension primer in the reaction system are both 0.09. Mu.M; the extension reaction conditions are as follows: pre-denaturation at 96 ℃ for 10s; denaturation at 96℃for 10s, annealing at 50℃for 5s, extension at 60℃for 30s,25 cycles; extending at 60deg.C for 30s, and preserving at 4deg.C.
The purification of the extension product by shrimp alkaline phosphatase in step e above uses a 10. Mu.L reaction system comprising: rSAP 1. Mu.L, extension product 5. Mu.L, cutSmart buffer 1. Mu.L, water 3. Mu.L; purification reaction conditions: storing at 37deg.C for 1h, 75deg.C for 15min, and 4deg.C.
Diluting the extension product purified in the step f, adding a test reagent, uniformly mixing, and carrying out denaturation treatment, wherein the specific steps are as follows: diluting the extension product purified in the step e by 6 times, taking 1 mu L of the diluent, adding 9.25 mu L of Hi-Di formamide and 0.3 mu L GeneScan Size Standards-120LIZ, uniformly mixing, denaturing for 5min at 95 ℃, and treating for 4min at 4 ℃.
The typing detection conditions in the step f are as follows: 15000V, 36cm capillary, POP4 gel, electrophoresis for 30min.
Compared with the prior art, the invention has the advantages that:
(1) The SNP marker with high polymorphism is screened from a eucalyptus genome, a multicolor fluorescence multiplex detection system of 21 loci is optimized and established, and the typing result has the characteristics of easy interpretation of the banding pattern, high locus polymorphism and easy distinction of fluorescence. The method has the advantages of high experimental efficiency, accurate detection, convenient operation and the like. Can provide important technical support for fast genetic evaluation of germplasm resources, molecular marker assisted breeding, clone molecular fingerprint construction and identification of eucalyptus in future.
(2) By using the method, molecular fingerprints of 58 domestic common eucalyptus clones are constructed, and the clones are successfully identified, so that the workload of identifying the eucalyptus clones is greatly reduced. The method can effectively discriminate counterfeit and disordered clone, and can practically ensure the rights and interests of improved variety breeders and forest growers.
Drawings
FIG. 1 shows the detection of eucalyptus clones DH32-29 and A13 by a multicolor fluorescence multiplex detection system of 21 SNP loci.
FIG. 2 is a genetic cluster map of 58 eucalyptus clones based on 21 SNP locus multicolor fluorescence detection primer sets, wherein eucalyptus clone Guangzhou1 and LH1 are the same clone.
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
Example 1
(1) Eucalyptus clone DNA extraction
Leaves of eucalyptus clone (Table 2) were collected, genomic DNA was extracted by CTAB method, and the extract was frozen and stored for later use after detection.
TABLE 2 clone of Eucalyptus 58 parts
58 parts of eucalyptus clone (Table 2) She Yang 0.3.3 g are weighed, ground in liquid nitrogen, transferred into a 2mL centrifuge tube, added with 1mL CTAB extract, kept at 60-65 ℃ for 45-60 min and shaken once for 10 min. The sample tube was taken out, centrifuged at 12000rpm at 4℃for 10min, and the supernatant was collected. Adding an equal volume of chloroform and isoamyl alcohol mixed solution (chloroform: isoamyl alcohol=24:1, volume ratio), sealing, and shaking uniformly; centrifuging at 12000rpm at 4deg.C for 10min, and collecting supernatant. Equal volumes of a mixture of chloroform and isoamyl alcohol (chloroform: isoamyl alcohol=24:1, volume ratio) were added and centrifuged at 12000rpm for 10min, and the supernatant was taken. Adding refrigerated 2/3 volume isopropanol into the supernatant, gently shaking and mixing, and standing at-20deg.C for more than 1 hr. Centrifuge at 12000rpm for 10min, pour out supernatant, add 1mL of 70% and 95% ethanol solution to wash once each, invert the tube on toilet paper after washing, suck dry, and stand at room temperature for 30min. 110. Mu.L of 1 XTE was added, and the mixture was flicked or shaken to dissolve the DNA sufficiently. Slightly centrifuging, transferring the solution into a 1.5mL centrifuge tube, centrifuging at 10000rpm for 5min, and taking supernatant into another 1.5mL centrifuge tube. Storing at-80deg.C for a long period of time.
The CTAB extract comprises the following components: 100mmol/L Tris-HCl (pH 8.0), 20mmol/L EDTA (disodium ethylenediamine tetraacetate), l.4mol/L NaCl,2% CTAB (w/v), 2% PVP (polyvinylpyrrolidone), 1% beta-mercaptoethanol (used after mixing well before each extraction).
The 1×TE comprises the following components: 10mmol/L Tris-HCl, 1mmol/L EDTA, pH=8.0.
(2) Amplification of
Performing multiplex PCR amplification on an amplification instrument by using the DNA obtained in the step (1) as a template and using the SNP primer set to obtain an amplification product; the multiplex PCR amplification was performed using a 20. Mu.L system comprising: 2 XType-it Multiplex PCR Master Mix. Mu.L, 4. Mu.L of water, 3. Mu.L of 21 pairs of amplification primers (SNaPshot 01-SNaPshot21 amplification primers in Table 1, forward and reverse primers shown as SEQ ID NOS.1-42) mixed in equal amounts (the final concentration of the single amplification primer in the reaction system is 0.037. Mu.M), and 3. Mu.L of 30 ng/. Mu.L of DNA of the eucalyptus clone to be tested; the reaction procedure is as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 60℃for 90s, elongation at 72℃for 30s,32 cycles; extending at 60deg.C for 30min; preserving heat at 20 ℃.
The multiplex PCR amplification was performed using the Type-it Microsatellite PCR Kit kit (QIAGEN, cat. No. 206241).
(3) Amplification product purification
And (3) purifying the amplified product by adopting exonuclease I and shrimp alkaline phosphatase, and removing redundant dNTPs and primers in the amplified product to obtain the purified amplified product. The purification reaction system is as follows: mu.L of Exo SAP-IT (Applied Biosystems company, catalog number 78201.1. ML) was added to 5. Mu.L of amplification product. Purification reaction conditions: 37 ℃ for 1h,75 ℃ for 15min, and finally 4 ℃ for preservation.
(4) Extension reaction
Adding the purified amplification product into a reaction reagent for extension reaction to obtain an extension product. The extension reaction used a 10. Mu.L system comprising: SNaPShot Multiplex Ready Reaction Mix (Applied Biosystems, cat# 4323163) 5. Mu.L of purified amplification product, 3. Mu.L of 21 extension primers (SNaPshot 01-SNaPshot21 extension primers in Table 1, SEQ ID NOS.43-63) mixed in equal amounts, 2. Mu.L (wherein the final concentration of the SNaPshot04 extension primer in the reaction system is 0.14. Mu.M, and the final concentrations of the SNaPshot01-SNaPshot03 and the SNaPshot05-SNaPshot21 extension primers in the reaction system are 0.09. Mu.M); extension reaction conditions: pre-denaturation at 96 ℃ for 10s; denaturation at 96℃for 10s, annealing at 50℃for 5s, extension at 60℃for 30s (25 cycles); extending at 60deg.C for 30s, and preserving at 4deg.C.
(5) Purification of the extension product
Purifying the extension product by using shrimp alkaline phosphatase, and removing redundant extension primers to obtain the purified extension product. Extension product purification uses a 10 μl system comprising: rSAP (NEB Co., catalog number: M0371S) 1. Mu.L, extension product 5. Mu.L, cutSmart buffer (NEB Co., catalog number: M0371S) 1. Mu.L, water 3. Mu.L. Purification reaction conditions: storing at 37deg.C for 1h, 75deg.C for 15min, and 4deg.C.
(6) Detection of extension products
Diluting the purified extension product by 6 times, taking 1 mu L of diluent, adding 9.25 mu L of Hi-Di formamide and 0.3 mu LGeneScan Size Standards-120LIZ (Applied Biosystems company, catalog number: 4324287), uniformly mixing, denaturing for 5min at 95 ℃, and treating for 4min at 4 ℃ to obtain denatured mixed solution. The denatured mixed solution is subjected to electrophoresis detection on an ABI 3130xl genetic analyzer under the detection conditions: 15000V voltage, 36cm capillary, POP4 gel, electrophoresis for 30min; results were analyzed using GeneMapper 4.1 software and the partial typing results are shown in FIG. 1.
(7) Molecular fingerprint of eucalyptus clone
Characteristic bands of different clones were analyzed by GeneMapper4.0 software to construct molecular fingerprints of eucalyptus clones (Table 3), genetic distances between eucalyptus clones were calculated by using PowerMarker 3.25, and NTSYS-pc version2.1 to construct a cluster map between clones (FIG. 2). In FIG. 2, the eucalyptus clone Guangzhou1 and LH1 are the same clone, and the detection result is the same as the actual result. When the unknown eucalyptus clone characteristic band to be detected is consistent with the corresponding eucalyptus clone fingerprint, the name of the clone to be detected can be determined.
TABLE 3 SNP molecular fingerprint of 58 Eucalyptus clones
(8) Detection of unknown eucalyptus clones
Extracting genomic DNA of a eucalyptus clone to be detected by the method, carrying out multi-color fluorescent multiplex PCR amplification of SNP loci, carrying out genotyping on the amplified products by using a genetic analyzer to obtain SNP conditions of corresponding loci of the clone, and obtaining the strain name of the unknown eucalyptus clone if the detection result is completely consistent with the SNP types in the SNP molecular fingerprint of the eucalyptus clone. Meanwhile, the detection can be carried out on a plurality of unknown eucalyptus clones, and the relationship among samples can be confirmed according to the typing result.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.
Sequence listing
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tgaagcaact gcgaaacg 18
<210> 41
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
gttgtaagtg aaactgt 17
<210> 42
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
tttatctaat tctgagt 17
<210> 43
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 43
gtctgggtga agatcgatcc 20
<210> 44
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 44
tctcccttac aagagggtca tgga 24
<210> 45
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 45
tctctcctct ttagcttcta caaggc 26
<210> 46
<211> 32
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 46
tctcctaaaa tcaaagatta caaagaacag ac 32
<210> 47
<211> 38
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 47
tctctctctc cagtttttag agtcttcaag tcaatatc 38
<210> 48
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 48
tctctctctc tctctctctc ttagtcggtg ccaccttgga 40
<210> 49
<211> 42
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 49
tctctctctc tctctctctc tcttatccca tagacctgct cg 42
<210> 50
<211> 44
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 50
tctctctctc tctctctctc tctctctggc ggactgatca agac 44
<210> 51
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 51
tctctctctc tctctctctc tctctctcga ttcactgtcg aggtcc 46
<210> 52
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 52
tctctctctc tctctctctc tctctctaga aaggaagttc tcagcttc 48
<210> 53
<211> 50
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 53
tctctctctc tctctctctc tctctctctc gctgattacc ctattcgtag 50
<210> 54
<211> 54
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 54
tctctctctc tctctctctc tctctctctc tctctcagtc ctgacggatc catt 54
<210> 55
<211> 56
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 55
tctctctctc tctctctctc tctctctctc tctctctagg caccaaatct agcttg 56
<210> 56
<211> 58
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 56
tctctctctc tctctctctc tctctctctc tctctctcta caagctcttg gcgaaatt 58
<210> 57
<211> 62
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 57
tctctctctc tctctctctc tctctctctc tctctctctc tctccttcaa ggggaggacc 60
<210> 58
<211> 64
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 58
tctctctctc tctctctctc tctctctctc tctctctctc tccggttcca acaagacatc 60
<210> 59
<211> 68
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 59
tctctctctc tctctctctc tctctctctc tctctctctc tctctctctc gggcagtgga 60
<210> 60
<211> 70
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 60
ctctctctct ctctctctct ctctctctct ctctctctct ctctcttggg aagcaactag 60
<210> 61
<211> 74
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 61
tctctctctc tctctctctc tctctctctc tctctctctc tctctctctc ttgagttgat 60
gattttcttc tgcc 74
<210> 62
<211> 78
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 62
ctctctctct ctctctctct ctctctctct ctctctctct ctctctctct ctctagtatt 60
<210> 63
<211> 81
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 63
tctctctctc tctctctctc tctctctctc tctctctctc tctctctctc tctctcaact 60
gtcataattg tcaatgacaa t 81
Claims (10)
1. The SNP locus multicolor fluorescence detection primer set for identifying eucalyptus clone is characterized by comprising 21 SNP gene loci and extension primers, wherein the sequences of the PCR amplification primers and the extension primers are as follows:
SNaPshot01, PCR amplification primer shown as SEQ ID NO.1-2 and extension primer shown as SEQ ID NO. 43;
SNaPshot02, the PCR amplification primer is shown as SEQ ID NO.3-4, and the extension primer is shown as SEQ ID NO. 44;
SNaPshot03, a PCR amplification primer shown as SEQ ID NO.5-6 and an extension primer shown as SEQ ID NO. 45;
SNaPshot04, a PCR amplification primer is shown as SEQ ID NO.7-8, and an extension primer is shown as SEQ ID NO. 46;
SNaPshot05, the PCR amplification primer is shown as SEQ ID NO.9-10, and the extension primer is shown as SEQ ID NO. 47;
SNaPshot06, the PCR amplification primer is shown as SEQ ID NO.11-12, and the extension primer is shown as SEQ ID NO. 48;
SNaPshot07, the PCR amplification primer is shown as SEQ ID NO.13-14, and the extension primer is shown as SEQ ID NO. 49;
SNaPshot08, PCR amplification primer shown as SEQ ID NO.15-16 and extension primer shown as SEQ ID NO. 50;
SNaPshot09, the PCR amplification primer is shown as SEQ ID NO.17-18, and the extension primer is shown as SEQ ID NO. 51;
SNaPshot10, the PCR amplification primer is shown as SEQ ID NO.19-20, and the extension primer is shown as SEQ ID NO. 52;
SNaPshot11, PCR amplification primers shown as SEQ ID NO.21-22 and extension primers shown as SEQ ID NO. 53;
SNaPshot12, the PCR amplification primer is shown as SEQ ID NO.23-24, and the extension primer is shown as SEQ ID NO. 54;
SNaPshot13, the PCR amplification primer is shown as SEQ ID NO.25-26, and the extension primer is shown as SEQ ID NO. 55;
SNaPshot14, the PCR amplification primer is shown as SEQ ID NO.27-28, and the extension primer is shown as SEQ ID NO. 56;
SNaPshot15, the PCR amplification primer is shown as SEQ ID NO.29-30, and the extension primer is shown as SEQ ID NO. 57;
SNaPshot16, the PCR amplification primer is shown as SEQ ID NO.31-32, and the extension primer is shown as SEQ ID NO. 58;
SNaPshot17, the PCR amplification primer is shown as SEQ ID NO.33-34, and the extension primer is shown as SEQ ID NO. 59;
SNaPshot18, the PCR amplification primer is shown as SEQ ID NO.35-36, and the extension primer is shown as SEQ ID NO. 60;
SNaPshot19, a PCR amplification primer is shown as SEQ ID NO.37-38, and an extension primer is shown as SEQ ID NO. 61;
SNaPshot20, the PCR amplification primer is shown as SEQ ID NO.39-40, and the extension primer is shown as SEQ ID NO. 62;
SNaPshot21, the PCR amplification primers are shown as SEQ ID NO.41-42, and the extension primers are shown as SEQ ID NO. 63.
2. A SNP locus multicolor fluorescence detection kit for identifying eucalyptus clones is characterized by comprising the SNP locus multicolor fluorescence detection primer set for identifying eucalyptus clones as set forth in claim 1.
3. The use of the SNP site multicolor fluorescence detection primer set for identifying eucalyptus clones according to claim 1 or the SNP site multicolor fluorescence detection kit for identifying eucalyptus clones according to claim 2 for identifying eucalyptus clones.
4. A method for identifying a eucalyptus clone using the SNP site multicolor fluorescence detection primer set for identifying a eucalyptus clone according to claim 1, comprising the steps of:
a. extracting clone DNA of eucalyptus to be detected;
b. c, carrying out multiplex PCR (polymerase chain reaction) amplification by using the eucalyptus clone DNA extracted in the step a as a template and using the amplification primers in the SNP locus multicolor fluorescence detection primer set for identifying the eucalyptus clone according to claim 1 to obtain an amplification product;
c. removing redundant dNTPs and primers in the amplified product by adopting exonuclease I and shrimp alkaline phosphatase to obtain a purified amplified product;
d. performing an extension reaction on the purified amplification product by using the extension primer in the SNP locus multicolor fluorescence detection primer set for identifying eucalyptus clone according to claim 1 to obtain an extension product;
e. purifying the extension product by using shrimp alkaline phosphatase, and removing redundant extension primers to obtain a purified extension product;
f. diluting the extension product purified in the step e, adding a test reagent, uniformly mixing, carrying out denaturation treatment to obtain a denatured mixed solution, carrying out typing detection on the mixed solution on an ABI 3130xl genetic analyzer, and determining genotypes of 21 SNP loci according to positions and colors of peaks of the extension product by using a GeneMapper 4.1.
5. The method for identifying eucalyptus clones according to claim 4, wherein the multiplex PCR amplification in the step b is performed by using a reaction system of 20. Mu.L, comprising: 2 XType-it Multiplex PCR Master Mix. Mu.L, 4. Mu.L of water, 3. Mu.L of 21 pairs of amplification primers in the SNP locus multicolor fluorescence detection primer set for identifying eucalyptus clone according to claim 1, which are mixed in equal amounts, wherein the final concentration of single amplification primer in a reaction system is 0.037. Mu.M, 30 ng/. Mu.L of eucalyptus clone DNA to be detected is 3. Mu.L; the reaction procedure is as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 60℃for 90s, elongation at 72℃for 30s,32 cycles; extending at 60deg.C for 30min; preserving heat at 20 ℃.
6. The eucalyptus clone identification method according to claim 4, wherein the purification reaction system for removing redundant dNTPs and primers in the amplified product by using exonuclease I and shrimp alkaline phosphatase in the step c is as follows: to 2. Mu.L of Exo SAP-IT was added 5. Mu.L of amplification product under the following reaction conditions: 37 ℃ for 1h,75 ℃ for 15min, and finally 4 ℃ for preservation.
7. The method for identifying a clone of eucalyptus according to claim 4, wherein the extension reaction in the step d uses a reaction system of 10 μl, comprising: SNaPShot Multiplex Ready Reaction Mix 5. Mu.L, 3. Mu.L of purified amplification product, 2. Mu.L of 21 extension primers in the SNP locus multicolor fluorescence detection primer set for identifying eucalyptus clone according to claim 1, which are mixed in equal amounts, wherein the final concentration of the SNaPshot04 extension primer in the reaction system is 0.14. Mu.M, and the final concentrations of the SNaPshot01-SNaPshot03 and the SNaPshot05-SNaPshot21 extension primer in the reaction system are each 0.09. Mu.M; the extension reaction conditions are as follows: pre-denaturation at 96 ℃ for 10s; denaturation at 96℃for 10s, annealing at 50℃for 5s, extension at 60℃for 30s,25 cycles; extending at 60deg.C for 30s, and preserving at 4deg.C.
8. The method for identifying eucalyptus clones according to claim 4, wherein said step e of purifying the extension product by using shrimp alkaline phosphatase comprises the steps of: rSAP 1. Mu.L, extension product 5. Mu.L, cutSmart buffer 1. Mu.L, water 3. Mu.L; purification reaction conditions: storing at 37deg.C for 1h, 75deg.C for 15min, and 4deg.C.
9. The method for identifying the clone of eucalyptus according to claim 4, wherein in the step f, the extension product purified in the step e is diluted, and then a test reagent is added, and the mixture is uniformly mixed to carry out denaturation treatment, specifically: diluting the extension product purified in the step e by 6 times, taking 1 mu L of the diluent, adding 9.25 mu L of Hi-Di formamide and 0.3 mu L GeneScan Size Standards-120LIZ, uniformly mixing, denaturing for 5min at 95 ℃, and treating for 4min at 4 ℃.
10. The method for identifying the clone of eucalyptus according to claim 4, wherein the typing detection conditions in the step f are as follows: 15000V, 36cm capillary, POP4 gel, electrophoresis for 30min.
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| JP2007037501A (en) * | 2005-08-05 | 2007-02-15 | Oji Paper Co Ltd | New dna derived from plant of genus eucalyptus and method for individual identification of plant of genus eucalyptus using the dna |
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| CN107385052B (en) * | 2017-08-08 | 2020-10-30 | 中国林业科学研究院热带林业研究所 | STR primer for identifying clone of eucalyptus and application thereof |
| CN107354222B (en) * | 2017-08-30 | 2020-10-30 | 中国林业科学研究院热带林业研究所 | STR primer, PCR kit and method for identifying clone of eucalyptus |
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