CN109652515B - Method for identifying new variety Jinjian of Chinese wax by using capillary electrophoresis fluorescence SSR fingerprint - Google Patents
Method for identifying new variety Jinjian of Chinese wax by using capillary electrophoresis fluorescence SSR fingerprint Download PDFInfo
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention discloses a method for identifying a new variety 'Jinjian' of white wax by utilizing a capillary electrophoresis fluorescence SSR fingerprint, which is characterized in that white wax genome DNA is extracted from young and tender leaves of white wax, the extracted DNA is taken as a template, PCR amplification is carried out by utilizing a screened SSR primer pair, a PCR amplification product is subjected to capillary electrophoresis, and the SSR fingerprint of the new variety 'Jinjian' of white wax is formed by combination according to the peak value of each pair of SSR primers in an electrophoresis result, so that the identification of the new variety 'Jinjian' of white wax is realized. According to the method, 6 pairs of SSR specific primers with stable specificity and good polymorphism are screened out, the fingerprint of the new white wax variety 'Jinjian' is constructed, and the identification of the new white wax variety 'Jinjian' sample can be simply, conveniently, quickly and accurately completed.
Description
Technical Field
The invention belongs to the technical field of plant variety molecular identification, and particularly relates to a method for identifying a new variety 'Jinjian' of white wax by utilizing a capillary electrophoresis fluorescence SSR fingerprint.
Background
The fraxinus plants are anemophilous flowers, natural pollination and hybridization can easily occur during mixed cultivation, particularly, the fraxinus velutina (F.velutina), the fraxinus (F.pennsylvanica) and the fraxinus (F.americica) are equivalent to subgenus-like groups, the morphological characteristics are very similar, the genetic relationship is very close, and the traditional methods for filial generations cannot be distinguished. In the past, macroscopic genetic analysis biological characteristics and morphological characteristic indexes are adopted when seedlings are distinguished, but seedlings with extremely similar shapes or extremely close genetic relations are difficult to distinguish, and particularly improved varieties at different levels in the seeds cannot be identified, so that mixed varieties, mixed lines and mixed plants appear in production so as to falsely and truly, and great loss is brought to production as a result. The germplasm identification on the molecular level is not influenced by the environment and the growth stage, and has stable result and strong reliability. Therefore, it is of great practical significance to seek to truly and accurately identify the improved variety of the white wax from the molecular level.
Simple 5-sequence repeat (SSR) molecular markers have the advantages of high information content, co-dominant inheritance, abundant quantity, Simple analysis method, good result repeatability, time saving, labor saving, money saving and the like, and are the most sensitive and reliable molecular markers in the aspect of germplasm identification at present. The method for identifying certain plant varieties based on capillary electrophoresis fluorescent SSR fingerprints by utilizing the capillary electrophoresis fluorescent SSR molecular marker technology is reported to be applied to crops such as corn, rice, soybean, wheat, cotton and the like, but the related reports of the method applied to forest trees, particularly white wax, are rare.
The new variety of Chinese ash, Jinjian, is an male strain of Fraxinus velutina 'jinjian', is cultivated by a subject group of scientific research institute of forestry in Shandong province, and passes through national approval in 2016. The variety has the advantages of straight dry shape, salt and alkali resistance, perennial, yellow branches, greenish yellow branches in the same year, leathery and glossy leaves and wide application range. However, the 'gold arrow' at the seedling stage is difficult to identify through phenotypic characteristics, which brings trouble to operators or causes loss of production. Therefore, in order to rapidly identify the new white wax variety 'Jinjian', the development of a method for identifying the new white wax variety 'Jinjian' by using the capillary electrophoresis fluorescence SSR fingerprint has important significance.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for identifying a new variety 'Jinjian' of white wax by utilizing a capillary electrophoresis fluorescence SSR fingerprint.
The invention relates to a method for identifying a new variety 'Jinjian' of white wax by utilizing a capillary electrophoresis fluorescence SSR fingerprint, which comprises the following steps:
(1) extracting genome DNA of the fraxinus chinensis from young and tender leaves of the fraxinus chinensis;
(2) carrying out PCR amplification by using the extracted DNA as a template and the screened SSR primer pair;
(3) performing capillary electrophoresis on the PCR amplification product;
(4) according to the peak value of each pair of SSR primers in the electrophoresis result, the SSR fingerprint of the new white wax variety 'Jinjian' is formed by combination, so that the identification of the new white wax variety 'Jinjian' is realized;
the method is characterized in that:
extracting DNA in the step (1) by adopting a CTAB method, and adding 2% of beta-mercaptoethanol and 2% of PVP into an extraction buffer solution; measuring DNA concentration and purity by ultraviolet spectrophotometry, and diluting to 30 ng. mu.l-1Storing at-20 deg.C for use;
the SSR primer pairs screened in the step (2) are 6 pairs, namely F186 and R186, F187 and R187, F202 and R202, F203 and R203, F208 and R208, F213 and 213, wherein the 5' end of each pair of primers is added with any one fluorescent label selected from FAM, TAMRA, HEX and ROX, and the forward primer consists of TP-M13 primer carrying the fluorescent label; the nucleotide sequence of F186 in the F186 and R186 primer pair is the forward primer sequence shown in SEQ ID No.1, the nucleotide sequence of R186 is the forward primer sequence shown in SEQ ID No.2, the nucleotide sequence of F187 in the F187 and R187 primer pair is the forward primer sequence shown in SEQ ID No.3, the nucleotide sequence of R187 is the forward primer sequence shown in SEQ ID No.4, the nucleotide sequence of F202 in the F202 and R202 primer pair is the forward primer sequence shown in SEQ ID No.5, the nucleotide sequence of R202 is the forward primer sequence shown in SEQ ID No.6, the nucleotide sequence of F203 in the F203 and R203 primer pair is the forward primer sequence shown in SEQ ID No.7, the nucleotide sequence of R203 in the SEQ ID No.8, the nucleotide sequence of F202 in the F208 and R208 primer pair is the forward primer sequence shown in SEQ ID No.9, the nucleotide sequence of R208 is a forward primer sequence shown as SEQ ID No.10, the nucleotide sequence of F213 in the F213 and R213 primer pair is a forward primer sequence shown as SEQ ID No.11, and the nucleotide sequence of R213 is a forward primer sequence shown as SEQ ID No. 12;
the PCR reaction system is 10 μ L: 10ng. uL-1DNA template of (1. mu.L), 2 XTaq plus PCR Master Mix 5. mu.L, 10. mu. mol. L-10.1. mu.L and ddH of each of the forward and reverse primers of (1)2O accounts for 10 mu L;
the PCR amplification program adopts a Touchdown mode: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, and annealing at 56 ℃ for 30 s; 0.5 ℃ reduction per cycle; extension at 72 ℃ for 30s for 15 cycles; denaturation at 95 ℃ for 30s, annealing at 54 ℃ for 30s, and extension at 72 ℃ for 30s, for 20 cycles; extending for 10min at 72 ℃, and storing at 4 ℃;
the method for performing capillary electrophoresis on the PCR amplification product in the step (3) comprises the following steps: mixing 0.3 mu L, GS-500 mu L of LIZ molecular weight internal standard of PCR product with 9.5 mu L of deionized formamide, adding into a PCR plate, denaturing at 95 ℃ for 5min, cooling at 4 ℃ and centrifuging, and detecting on a machine by 1 Xbuffer Buffer solution; capillary electrophoresis was performed using a 3730xl DNA sequencer: pre-electrophoresis for 3min at 15 kV; injecting sample for 15s at 1.6 kV; electrophoresis is carried out for 20min under 15 kV;
the identification method in the step (4) comprises the following steps: performing data sorting and image analysis by using GenemarkerZ.2.0 software, and combining to form an SSR fingerprint of a new variety 'Jinjian' of white wax according to the peak value of each pair of SSR primers; wherein the peak value of the primer pair F186 and R186 is 93/99, the peak value of the primer pair F187 and R187 is 134/140, the peak value of the primer pair F202 and R202 is 109/112, the peak value of the primer pair F203 and R203 is 157, the peak value of the primer pair F208 and R208 is 139/145, and the peak value of the primer pair F213 and R213 is 81/90/96.
The invention also discloses an SSR primer pair used for the method, which is characterized in that: the SSR primer pairs are 6 pairs, namely F186 and R186, F187 and R187, F202 and R202, F203 and R203, F208 and R208, and F213 and 213, wherein the 5' end of each pair of primers is added with a FAM fluorescent label, and the forward primer consists of a TP-M13 primer carrying the fluorescent label; the nucleotide sequence of F186 in the F186 and R186 primer pair is the forward primer sequence shown in SEQ ID No.1, the nucleotide sequence of R186 is the forward primer sequence shown in SEQ ID No.2, the nucleotide sequence of F187 in the F187 and R187 primer pair is the forward primer sequence shown in SEQ ID No.3, the nucleotide sequence of R187 is the forward primer sequence shown in SEQ ID No.4, the nucleotide sequence of F202 in the F202 and R202 primer pair is the forward primer sequence shown in SEQ ID No.5, the nucleotide sequence of R202 is the forward primer sequence shown in SEQ ID No.6, the nucleotide sequence of F203 in the F203 and R203 primer pair is the forward primer sequence shown in SEQ ID No.7, the nucleotide sequence of R203 in the SEQ ID No.8, the nucleotide sequence of F202 in the F208 and R208 primer pair is the forward primer sequence shown in SEQ ID No.9, the nucleotide sequence of R208 is a forward primer sequence shown as SEQ ID No.10, the nucleotide sequence of F213 in the F213 and R213 primer pair is a forward primer sequence shown as SEQ ID No.11, and the nucleotide sequence of R213 is a forward primer sequence shown as SEQ ID No. 12. Specifically, the information of the 6 pairs of SSR primer pairs is shown in Table 1.
Table 1: 6 pairs of SSR primer pair information
The SSR primer pair is applied to the construction of the SSR fingerprint of a new variety 'Jinjian' of white wax.
According to sequence data design obtained by transcriptome sequencing and a large amount of screening, 6 pairs of SSR specific primers with stable specificity and good polymorphism are selected and used for constructing an SSR fingerprint of a new variety 'Jinjian' of white wax; the method has the characteristics of high resolution, simplicity and convenience in operation, rapidness, accurate result and the like, and can be used for rapidly identifying the samples of the new white wax variety 'Jinjian'.
The invention provides an SSR fingerprint of a new variety 'Jinjian' of white wax, which is characterized in that: the chromatogram is constructed by the related steps in the method for identifying the new variety 'Jinjian' of white wax by utilizing the capillary electrophoresis fluorescence SSR fingerprint.
In the SSR fingerprint of the new white wax variety 'Jinjian', the peak value of an SSR primer pair F186 and R186 is 93/99, the peak value of a primer pair F187 and R187 is 134/140, the peak value of a primer pair F202 and R202 is 109/112, the peak value of a primer pair F203 and R203 is 157, the peak value of a primer pair F208 and R208 is 139/145, and the peak value of a primer pair F213 and R213 is 81/90/96.
Compared with the prior art, the invention has the advantages that:
1) the method provided by the invention is a method for quickly and efficiently identifying the new variety of the white wax on a molecular level, has the advantages of quickness, accuracy, convenience in operation and the like, and the identification result is not easily influenced by the environment.
2) The invention utilizes the capillary electrophoresis fluorescence labeling electrophoresis technology, overcomes the defects of the polyacrylamide gel electrophoresis, can accurately read the size of the product fragment, has the accuracy within l bp, can accurately identify the variety, and has lower cost and great application value.
3) The SSR primer pair and the SSR fingerprint of the new variety 'Jinjian' of white wax provided by the invention provide solid theoretical support for further application research of the DNA fingerprint on white wax.
Drawings
FIG. 1: and (3) a capillary electrophoresis picture of PCR amplification products of the SSR primer pair 186 in 'gold arrow'.
FIG. 2: capillary electrophoresis of PCR amplification product of SSR primer pair 187 in 'gold arrow'.
FIG. 3: capillary electrophoresis of PCR amplification product of SSR primer pair 202 in 'gold arrow'.
FIG. 4: and (3) a capillary electrophoresis picture of PCR amplification products of the SSR primer pair 203 in 'gold arrow'.
FIG. 5: capillary electrophoresis of PCR amplification product of SSR primer pair 208 in 'gold arrow'.
FIG. 6: capillary electrophoresis chart of PCR amplification product of SSR primer pair 213 in 'gold arrow'.
Detailed Description
Example 1
1. Authentication material
The 'Jinjian' tender leaves are selected to extract genome DNA.
Extraction of DNA
2.1 placing prepared CTAB extraction buffer solution in a water bath kettle at 65 deg.C for preheating for 30min, placing chloroform-isoamyl alcohol (24: 1), anhydrous ethanol, 70% ethanol in a refrigerator at-20 deg.C for precooling, pre-cooling with mortar in advance, and pre-cooling with a small amount of liquid nitrogen before use;
2.2 taking 0.4-0.5g of fraxinus chinensis young leaves, adding sufficient liquid nitrogen, grinding into powder, quickly transferring into a 2mL centrifuge tube, adding 600 mu L of 2% CTAB extract (containing 2% beta-mercaptoethanol), and fully mixing. Water bath at 65 deg.C for 30min, and slightly inverting for 3-4 times;
2.3 after cooling to room temperature 600. mu.L of chloroform: extracting with isoamyl alcohol (24: 1) for 10min while gently shaking, cooling to room temperature, and centrifuging at 12000r/min for 10 min;
2.4 transfer the supernatant (ca. 500. mu.l) to another centrifuge tube, add an equal volume of chloroform: isoamyl alcohol (24: l) repeat step 2.3;
2.5 taking out the supernatant, transferring the supernatant into another clean centrifugal tube with the volume of 1.5ml, adding 1ml of precooled absolute ethyl alcohol to precipitate DNA (placing the DNA for 30min at the temperature of minus 20 ℃), slightly rotating the centrifugal tube to enable flocculent DNA to appear, placing the DNA for 1h at the temperature of minus 20 ℃, and observing the generation of precipitate;
2.68000 rmp, centrifuging at room temperature for 5min, pouring out the supernatant, washing the precipitate with 1mL of 75% alcohol twice, and standing on an ultraclean bench to volatilize ethanol completely.
2.7 after the DNA is air-dried (the DNA is not suitable for over-drying, otherwise is not easy to dissolve), dissolving with appropriate amount of TE (pH8.0, 50 μ l) or ultrapure water, and standing at 4 deg.C for 6-12h to dissolve completely;
2.8 adding 2 mul RNA enzyme 10mg/mL, water bathing at 37 deg.C for 1h, and storing at-80 deg.C for use;
DNA quantification and quality detection
3.1 taking 2 mu L of DNA and detecting the DNA by using a trace nucleic acid protein detector, the concentration of the DNA and the ratio of A260/A280 can be directly read, and the ratio is between 1.8 and 2.0 and meets the requirement.
3.2 taking another 3 mul DNA and adding 1 mul bromophenol blue, electrophoresing on 0.8% agarose gel for 15-20 min, stabilizing at 100V, electrophoresis buffer solution of 1 XTBE, observing under 325nm ultraviolet lamp, taking picture, and estimating the concentration of unknown DNA by comparing with the fluorescence of standard solution.
3.3 Total DNA samples of plants exhibited a clear band with a large relative molecular weight and a small migration rate.
3.4 the samples were diluted to a concentration of 20 ng/. mu.L and stored at-80 ℃ until use.
4. Capillary electrophoresis system detection
4.1 screening in 196 parts of known resources one by one, selecting 6 pairs of SSR primers with good polymorphism (detailed information is shown in Table 1), and adding a fluorescence-labeled FAM (6-methoxy-fluorescein) at the 5' end of each pair of primers. The forward primer is composed of a TP-M13 primer carrying a fluorescent label, and is synthesized by Shandong Huabo genetic engineering Co., Ltd.
Table 1: 6 pairs of SSR primer pair information
4.2 PCR amplification by fluorescence labeling capillary electrophoresis Using 10. mu.L of the reaction: 10ng. uL-1DNA template of (1. mu.L), 2 XTaq plus PCR Master Mix 5. mu.L, forward and reverse primers of 0.1. mu.L each (10. mu. mol. L)-1) And ddH2O amounted to 10. mu.L.
4.3PCR amplification program adopts Touchdown mode: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s (0.5 ℃ per cycle), extension at 72 ℃ for 30s, 15 cycles; denaturation at 95 ℃ for 30s, annealing at 54 ℃ for 30s, and extension at 72 ℃ for 30s, for 20 cycles; extending for 10min at 72 ℃, and storing at 4 ℃.
4.4PCR product denaturation: diluting the PCR product by 10-20 times with double distilled water, uniformly mixing 0.3 mu L of the PCR product, 0.5 mu L of the molecular weight internal standard and 9.5 mu L of deionized formamide, and adding the mixture into a PCR plate; the denaturation program was run on a PCR instrument: denaturation at 95 deg.C for 5min, cooling at 4 deg.C, centrifuging, and detecting with 1 × Buffer solution on machine.
4.5PCR product amplification detection: the PCR product detection adopts a 3730xl DNA analyzer produced by ABI company of America, adopts 50cm of capillary electrophoresis of 96 channels produced by ABI company of America, and carries out pre-electrophoresis for 3min under 15 kV; injecting sample for 15s at 1.6 kV; electrophoresis is carried out for 20min at 15 kV.
And (4) importing the computer result original file into Genemarker 2.2.0 software for data collection and image analysis.
5. Data analysis
And determining the molecular weight according to the molecular weight determined during primer screening and the primer color, and introducing the collected original Data into a Gene Marker 2.2.0 system by adopting Data Collection software for analysis. And comparing the GS-500LIZ molecular weight internal standard in the lane with target peak size system software, accurately calculating the size of the target DNA fragment, and converting the electrophoresis result into a PDF picture format for exporting.
Each fluorescently labeled capillary electrophoresis was examined for 3 replicates, and the average of the 3 replicates was used as data for the experimental material at that locus.
6, SSR fingerprint construction and molecular identification:
according to the peak value of the 6 pairs of SSR primers, an SSR fingerprint of a new variety 'Jinjian' of white wax is constructed, and the SSR fingerprint is shown in a table 2.
TABLE 26 SSR fingerprint of new variety 'Jinjian' of white wax constructed by combining SSR fluorescent primers
In the SSR fingerprint of the new white wax variety 'Jinjian', the peak value of an SSR primer pair F186 and R186 is 93/99, the peak value of a primer pair F187 and R187 is 134/140, the peak value of a primer pair F202 and R202 is 109/112, the peak value of a primer pair F203 and R203 is 157, the peak value of a primer pair F208 and R208 is 139/145, and the peak value of a primer pair F213 and R213 is 81/90/96.
Sequence listing
<110> Shandong province forestry science research institute, Shandong Huabo Gene engineering science and technology Co., Ltd
<120> method for identifying new variety Jinjian of Chinese wax by using capillary electrophoresis fluorescence SSR fingerprint
<141> 2018-12-20
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<170> SIPOSequenceListing 1.0
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tcttcacgtc ttctgtttgt tca 23
<210> 2
<211> 23
<212> DNA
<213> Artificial sequence
<221> primer R186 nucleotide sequence
<400> 2
gaaaacgtgt gaatgagttt ggt 23
<210> 3
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<213> Artificial sequence
<221> primer F187 nucleotide sequence
<400> 3
tcgatctttc catctaaaca agc 23
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<211> 23
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<213> Artificial sequence
<221> primer R187 nucleotide sequence
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aacgtgtgaa tgagtttggt ttt 23
<210> 5
<211> 23
<212> DNA
<213> Artificial sequence
<221> nucleotide sequence of primer F202
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agttttcacc gctttcagtg tta 23
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<221> primer R202 nucleotide sequence
<400> 6
gggaatgaac atgagtttca gta 23
<210> 7
<211> 23
<212> DNA
<213> Artificial sequence
<221> nucleotide sequence of primer F203
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gttatcagta gatgcaaccg cac 23
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<221> primer R203 nucleotide sequence
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aacaccggtt ttcaacattt ct 22
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<213> Artificial sequence
<221> primer F208 nucleotide sequence
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cctcctattg aatcattcgc tta 23
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<213> Artificial sequence
<221> primer R208 nucleotide sequence
<400> 10
attttgattt ccctcctctg aag 23
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<213> Artificial sequence
<221> primer F213 nucleotide sequence
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gacaacatgc ctaaattgga ctc 23
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<212> DNA
<213> Artificial sequence
<221> primer R213 nucleotide sequence
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aattctgaac ttcaaggtgg gat 23
Claims (3)
1. A method for identifying a Fraxinus chinensis variety Jinjian by using a capillary electrophoresis fluorescence SSR fingerprint comprises the following steps:
(1) extracting genome DNA of the fraxinus chinensis from young and tender leaves of the fraxinus chinensis;
(2) carrying out PCR amplification by using the extracted DNA as a template and the screened SSR primer pair;
(3) performing capillary electrophoresis on the PCR amplification product;
(4) combining to form an SSR fingerprint of the white wax variety Jinjian according to the peak value of each pair of SSR primers in the electrophoresis result, and realizing the identification of the white wax variety Jinjian;
the method is characterized in that:
extracting DNA in the step (1) by adopting a CTAB method, and adding 2% of beta-mercaptoethanol and 2% of PVP into an extraction buffer solution; measuring DNA concentration and purity by ultraviolet spectrophotometry, and diluting to 30 ng. mu.l-1Storing at-20 deg.C for use;
the SSR primer pairs screened in the step (2) are 6 pairs, namely F186 and R186, F187 and R187, F202 and R202, F203 and R203, F208 and R208, F213 and R213, wherein the 5' end of each pair of primers is added with any one fluorescent label selected from FAM, TAMRA, HEX and ROX, and the forward primer consists of TP-M13 primer carrying the fluorescent label; the nucleotide sequence of F186 in the F186 and R186 primer pair is shown as SEQ ID No.1, the nucleotide sequence of R186 is shown as SEQ ID No.2, the nucleotide sequence of F187 in the F187 and R187 primer pair is shown as SEQ ID No.3, the nucleotide sequence of R187 is shown as SEQ ID No.4, the nucleotide sequence of F202 in the F202 and R202 primer pair is shown as SEQ ID No.5, the nucleotide sequence of R202 is shown as SEQ ID No.6, the nucleotide sequence of F203 in the F203 and R203 primer pair is shown as SEQ ID No.7, the nucleotide sequence of R203 is shown as SEQ ID No.8, the nucleotide sequence of F208 in the F208 and R208 primer pair is shown as SEQ ID No.9, the nucleotide sequence of R208 is shown as SEQ ID No.10, the nucleotide sequence of F213 in the F213 and R213 primer pair is shown as SEQ ID No.11, and the nucleotide sequence of R213 is shown as SEQ ID No. 12;
the PCR reaction system is 10 μ L: 10ng. uL-1DNA template of (1. mu.L), 2 XTaq plus PCR Master Mix 5. mu.L, 10. mu. mol. L-10.1. mu.L and ddH of each of the forward and reverse primers of (1)2O accounts for 10 mu L;
the PCR amplification program adopts a Touchdown mode: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, and annealing at 56 ℃ for 30 s; 0.5 ℃ reduction per cycle; extension at 72 ℃ for 30s for 15 cycles; denaturation at 95 ℃ for 30s, annealing at 54 ℃ for 30s, and extension at 72 ℃ for 30s, for 20 cycles; extending for 10min at 72 ℃, and storing at 4 ℃;
the method for performing capillary electrophoresis on the PCR amplification product in the step (3) comprises the following steps: mixing 0.3 mu L, GS-500 mu L of LIZ molecular weight internal standard of PCR product with 9.5 mu L of deionized formamide, adding into a PCR plate, denaturing at 95 ℃ for 5min, cooling at 4 ℃ and centrifuging, and detecting on a machine by 1 Xbuffer Buffer solution; capillary electrophoresis was performed using a 3730xl DNA sequencer: pre-electrophoresis for 3min at 15 kV; injecting sample for 15s at 1.6 kV; electrophoresis is carried out for 20min under 15 kV;
the identification method in the step (4) comprises the following steps: performing data sorting and image analysis by using GenemarkerZ.2.0 software, and combining to form an SSR fingerprint of the Fraxinus chinensis breed Jinjian according to the peak value of each pair of SSR primers; wherein the peak value of the primer pair F186 and R186 is 93/99, the peak value of the primer pair F187 and R187 is 134/140, the peak value of the primer pair F202 and R202 is 109/112, the peak value of the primer pair F203 and R203 is 157, the peak value of the primer pair F208 and R208 is 139/145, and the peak value of the primer pair F213 and R213 is 81/90/96.
2. An SSR primer pair for use in the method of claim 1 characterized by: the SSR primer pairs are 6 pairs, namely F186 and R186, F187 and R187, F202 and R202, F203 and R203, F208 and R208, and F213 and R213, wherein the 5' end of each pair of primers is added with a FAM fluorescent label, and the forward primer consists of a TP-M13 primer carrying the fluorescent label; the nucleotide sequence of F186 in the F186 and R186 primer pair is shown as SEQ ID No.1, the nucleotide sequence of R186 is shown as SEQ ID No.2, the nucleotide sequence of F187 in the F187 and R187 primer pair is shown as SEQ ID No.3, the nucleotide sequence of R187 is shown as SEQ ID No.4, the nucleotide sequence of F202 in the F202 and R202 primer pair is shown as SEQ ID No.5, the nucleotide sequence of R202 is shown as SEQ ID No.6, the nucleotide sequence of F203 in the F203 and R203 primer pair is shown as SEQ ID No.7, the nucleotide sequence of R203 is shown as SEQ ID No.8, the nucleotide sequence of F208 in the F208 and R208 primer pair is shown as SEQ ID No.9, the nucleotide sequence of R208 is shown as SEQ ID No.10, the nucleotide sequence of F213 in the F213 and R213 primer pair is shown as SEQ ID No.11, and the nucleotide sequence of R213 is shown as SEQ ID No. 12.
3. The use of the SSR primer pair of claim 2 in the construction of an SSR fingerprint of a Fraxinus chinensis breed Jinjian.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811622251.8A CN109652515B (en) | 2018-12-28 | 2018-12-28 | Method for identifying new variety Jinjian of Chinese wax by using capillary electrophoresis fluorescence SSR fingerprint |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201811622251.8A CN109652515B (en) | 2018-12-28 | 2018-12-28 | Method for identifying new variety Jinjian of Chinese wax by using capillary electrophoresis fluorescence SSR fingerprint |
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Citations (3)
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| CN104164503A (en) * | 2014-08-04 | 2014-11-26 | 山东省林业科学研究院 | Method for identifying fraxinus chinensis species by virtue of SSR (Simple Sequence Repeat) fingerprint spectrum |
| CN105861643A (en) * | 2015-12-03 | 2016-08-17 | 山东农业工程学院 | Construction method for fraxinus velutina SSR marked molecular genetic linkage map |
| CN106244700A (en) * | 2016-08-24 | 2016-12-21 | 山东省林业科学研究院 | A kind of method utilizing SSR marker technology that Fraxinus velutina and American Red cenospecies are carried out Rapid identification |
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| CN104164503A (en) * | 2014-08-04 | 2014-11-26 | 山东省林业科学研究院 | Method for identifying fraxinus chinensis species by virtue of SSR (Simple Sequence Repeat) fingerprint spectrum |
| CN105861643A (en) * | 2015-12-03 | 2016-08-17 | 山东农业工程学院 | Construction method for fraxinus velutina SSR marked molecular genetic linkage map |
| CN106244700A (en) * | 2016-08-24 | 2016-12-21 | 山东省林业科学研究院 | A kind of method utilizing SSR marker technology that Fraxinus velutina and American Red cenospecies are carried out Rapid identification |
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| MOLECULAR AND MORPHOLOGICAL CHARACTERIZATION OF RECIPROCAL F1 HYBRID ASH (FRAXINUS EXCELSIOR×FRAXINUS ANGUSTIFOLIA,OLEACEAE) AND PARENTAL SPECIES REVEALS ASYMMETRIC CHARACTER INHERITANCE;Muriel Thomasset等;《Int.J.Plant Sci.》;20111231;第172卷(第3期);第423-433页 * |
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