CN113355444B - Method for identifying highland barley variety by SSR molecular marker method and application thereof - Google Patents
Method for identifying highland barley variety by SSR molecular marker method and application thereof Download PDFInfo
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Abstract
The invention discloses a method for identifying highland barley varieties by using an SSR molecular marker method and application thereof, wherein the primer sequence in the SSR molecular marker method is one or more of primers corresponding to Bmag0745, Bmag0496, Bmag0353, Scssr03381, AWBMS0080, GBM1215, Bmag0919, GBM1008, Scssr03907, HVPLASCIB, HVCH126A, GBM1516, Scssr03906, GBM1440, Scssr04163, GBM1413, HVM33 and EBmac 5, and the highland barley varieties can be better distinguished by performing 2 detection methods of denaturing polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis (SSR 041) on 18 screened primers; wherein the primers Bmag0496, Scssr03381, GBM1215, Scssr03906, Scssr04163 and HVM33 can be used for the variety clustering chart of the SSR analysis of the amplification of 24 highland barley main cultivars. The SSR primer provided by the invention has the characteristics of strong specificity, stable amplification result, high polymorphism and the like, and the molecular markers can be applied to related fields of variety identification, gene positioning, molecular identification and the like.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a method for identifying highland barley varieties by using an SSR molecular marker method and application thereof.
Background
The highland barley (Hordeum vulgare L.var.nudum hook.f.) belongs to the barley subfamily of the wheat family of the Gramineae, belongs to the variety of cultivated barley in the botany, is one of the primary crops in China, is the most distinctive crop in Qinghai-Tibet plateau, is a typical representative of plant adaptive evolution under the extreme environmental conditions of the Qinghai-Tibet plateau, has the excellent properties of drought resistance, barren resistance, short growth period, strong adaptability, stable yield, easy cultivation and the like, and is rich in germplasm resources.
The highland barley has the characteristics of high protein, high soluble fiber, high vitamin, low sugar and low fat, contains zinc, calcium, iron, selenium and other trace elements necessary for human bodies, has comprehensive nutrient components and reasonable structure, contains a large amount of dietary fiber with good effects of clearing intestines, relaxing bowels and removing vivotoxin, and is a good product in cereal crops. The Zanba and the butter fried flour which are eaten by Tibetan siblings in the Tibet plateau all take highland barley as main raw materials. The beta-glucan (the average content is 6.57-8.6 percent) contained in the highland barley grains is a special physiological functional element of the highland barley, and has special curative effect on treating various diseases of human beings. Because the highland barley is important in food, health care, feeding and brewing industries, and has rich germplasm resources and various varieties, how to comprehensively and delicately know the genetic diversity of the highland barley germplasm resources and utilize the highland barley germplasm resources with higher efficiency is very key for breeding excellent and special highland barley new varieties.
The molecular marker can reflect the difference of plant genetic bases on the DNA level, and is a new technology in the field of plant genetic breeding. Simple repeat sequences (SSRs) are widely distributed throughout the genome, and microsatellite sequences of the same type produce polymorphisms at each locus due to differences in the number or degree of repetition. The SSR molecular marker has high polymorphic information content and is in co-dominant inheritance, so the SSR molecular marker is widely applied to the research of variety identification of various barley (highland barley) in recent years.
Disclosure of Invention
The invention mainly solves the technical problem of providing a method for identifying highland barley varieties by using an SSR molecular marker method and application thereof, wherein SSR primers provided in the molecular markers have the characteristics of strong specificity, stable amplification result, high polymorphism and the like, and the molecular markers can be applied to the related fields of variety identification, gene positioning, molecular identification and the like.
SSRs are distributed on different positions of the highland barley genome, and the number and the sequence of the repetitive units on each site of different varieties can be different. Because the sequences on both sides of each simple repetitive sequence are highly conserved and single-copy, a pair of specific primers can be designed according to the sequences on both sides, and the DNA sequence between the two primers is amplified by utilizing the PCR technology. In the electrophoresis process, the obtained PCR products are separated under the action of an electric field due to different molecular weights and are distinguished by silver nitrate dyeing or fluorescent dye marking. Therefore, the highland barley variety can be identified by utilizing PCR amplification and electrophoresis technology according to polymorphism of SSR locus.
The naked barley is called naked barley because the inner and outer palea of the barley grains are separated from the caryopsis and the grains are naked, but the naked barley is similar to the barley genome, but the naked barley and the barley have a plurality of sequence differences. Therefore, the development of an SSR primer with high polymorphism and strong specificity has great significance for identifying highland barley varieties.
In order to solve the problems, the invention adopts the technical scheme that:
provides an SSR primer, the sequence of the SSR primer is one or more of primers corresponding to Bmag0745, Bmag0496, Bmag0353, Scssr03381, AWBMS0080, GBM1215, Bmag0919, GBM1008, Scssr03907, HVPLASCIB, HVCH126A, GBM1516, Scssr03906, GBM1440, Scssr04163, GBM1413, HVM33 and EBmac 0415.
Further, the sequences of the SSR primers are Bmag0496, Scssr03381, GBM1215, Scssr03906, Scssr04163 and HVM 33.
The invention also provides a method for identifying highland barley varieties by using an SSR molecular marker method, which comprises the following steps:
(1) extracting DNA of a highland barley variety sample;
(2) carrying out PCR amplification, modified polyacrylamide gel electrophoresis, silver staining and capillary electrophoresis by using the SSR primers;
further, the sample is selected from one or more of highland barley grains, embryos, seedlings and leaves.
Further, the reaction procedure of the PCR amplification is as follows:
pre-denaturation at 94 ℃ for 4.5-5.5 min; denaturation at 94 ℃ for 25-35 s, annealing at 50-65 ℃ for 55-65 s, extension at 72 ℃ for 25-35 s, and 10-15 cycles, wherein the temperature is reduced by 0.5-1.2 ℃ per cycle; denaturation at 94 ℃ for 25-35 s s, annealing at 50 ℃ for 40-50 s, extension at 72 ℃ for 25-35 s, and 20-25 cycles; extending for 9-12 min at 72 ℃;
preferably, the pre-denaturation is carried out for 5min at 94 ℃; denaturation at 94 ℃ for 30s, annealing at 50-60 ℃ for 60s, extension at 72 ℃ for 30s, and 12 cycles of cooling to 0.8 ℃ per cycle; denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 45s, and extension at 72 ℃ for 30s for 23 cycles; extension at 72 ℃ for 10 min.
Further, the specific steps of the native polyacrylamide gel electrophoresis comprise:
(a) taking 6% non-denatured polyacrylamide solution, tetramethyl ethylenediamine and 10% ammonium persulfate, mixing uniformly, and making into gel;
(b) and mixing the PCR amplification product with the sample adding buffer solution uniformly, cooling after denaturation, and carrying out electrophoresis.
Further, the denaturation condition is that the denaturation is carried out for 4.5-5.5 min at the temperature of 92-98 ℃, and preferably for 5min at the temperature of 95 ℃;
the electrophoresis conditions are that the constant power is 60-80W, and the electrophoresis time is 1.5-2.5 h;
during electrophoresis, the temperature of the gel is 45-55 ℃, and preferably 50 ℃.
Further, the silver staining step comprises: fixing the gel, washing with water after silver dyeing for 15-20 min, developing until the strip is clear, and fixing;
further, the fixing condition is that the fixing is carried out for 15-25 min at 20-35 rpm, preferably for 20min at 30 rpm;
the time of washing is less than or equal to 10 s/time; the fixing time is 4.5-5.5 min.
Further, the capillary electrophoresis comprises the following specific steps: taking different fluorescence-labeled amplification products of the same combined primer with the same volume, uniformly mixing to obtain a mixed solution, sucking the mixed solution, a molecular weight internal standard and deionized formamide, adding the mixed solution to a 96-well plate, performing denaturation, cooling, centrifuging and performing electrophoresis;
further, the dosage of the internal standard is 0.05-0.15 muL, preferably 0.1 muL; the dosage of the deionized formamide is 8-9.5 mu L, preferably 8.9 mu L; the preferable denaturation condition is 90-100 ℃ for 3-7 min, and the preferable denaturation condition is 95 ℃ for 5 min.
The difference of 1bp can be distinguished through capillary electrophoresis, and the highland barley varieties can be distinguished more accurately.
The invention also provides the application of the primer in highland barley variety identification.
The invention also provides the application of the primer in the analysis of the genetic relationship of highland barley varieties.
The invention has the beneficial effects that: the SSR primers for highland barley diversity analysis provided by the invention have the characteristics of strong specificity, stable amplification result, high polymorphism and the like, 18 pairs of SSR primers are screened, 2 detection methods of modified polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis are adopted, the acquisition of SSR labeled molecular data of 24 highland barley varieties, variety authenticity verification or identity identification are carried out according to different primer amplification sites, and meanwhile, genetic breeding analysis and genetic relationship research on highland barley can be carried out by combining means such as modern molecular biology and the like, so that the method has very important theoretical significance and practical significance.
Drawings
FIG. 1 is a non-denaturing polyacrylamide gel electrophoresis pattern of 24 highland barley varieties corresponding to 18 pairs of primers;
FIG. 2 is the allele data of the species to be tested at the core primer site;
FIG. 3 is a variety clustering chart of applying 6 pairs of primers to 24 parts of main highland barley cultivar amplification SSR analysis.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
1. Instrument and reagent
An instrument device: PCR instrument, high-pressure electrophoresis instrument (maximum voltage is not less than 2000V, and has constant voltage, constant current and constant power function, vertical electrophoresis tank and matched glue making accessories, common electrophoresis instrument, DNA analyzer (based on capillary electrophoresis, has fragment analysis function and data analysis software, and can distinguish difference of at least 1 nucleotide), high-speed refrigerated centrifuge (maximum eccentricity is not less than 20000r), horizontal shaking table, film observing lamp, electronic balance, micropipettor, magnetic stirrer, nucleic acid concentration tester or ultraviolet spectrophotometer, microwave oven, high-pressure sterilization pot, acidimeter, water bath (temperature control precision is plus or minus 1 ℃), refrigerator, ice maker, tissue crusher, gel imaging system or ultraviolet projector or camera;
reagent: sodium dodecyl benzene sulfonate, chloroform, isoamylol, disodium ethylene diamine tetraacetate, trihydroxymethyl amino, methane, concentrated hydrochloric acid, sodium hydroxide, hydrochloric acid, 10 Xbuffer solution, magnesium chloride, sodium chloride, 4 kinds of deoxyribonucleotides and Taq DNA polymerase.
2. The primer, highland barley varieties and reference samples are shown in the table 1 and the table 2:
by analysis, 18 pairs of SSR primers were screened as shown in table 1:
1. TABLE 1 allele data information for known varieties at core primer sites
TABLE 2 list of reference samples
| Serial number | Name of breed | Serial number | Name of breed | Serial number | Name of breed | Serial number | Name of |
| 1 | Kunlun No. 14 | 2 | Kunlun No. 15 | 3 | Himalayan No. 19 | 4 | Yellow blue No.2 |
| 5 | Yunqing No.2 | 6 | Yellow green No.1 | 7 | Diqing No.3 | 8 | Aqing No. 5 |
| 9 | Highland barley No. 5 | 10 | Highland barley No.4 | 11 | Aqing No. 6 | 12 | Highland barley No.4 |
3. Identification method
(1) Sample preparation
The sample division and preservation of the seed sample meet the GB/T3543.2 regulations. 40 individuals (seeds, leaves or other equivalent) were tested per sample and mixed for analysis. Samples with poor consistency were analyzed individually for each individual.
(2) DNA extraction
Taking 40 mixed tissues capable of extracting DNA, such as 200-300 mg highland barley seeds, embryos, seedlings or leaves, and the like, putting the mixed tissues into a 2.0mL centrifuge tube, quickly grinding the mixed tissues under liquid nitrogen, adding 700 mu L CTAB extracting solution preheated at 65 ℃, and shaking up; putting the centrifugal tube into a water bath kettle with the constant temperature of 65 ℃ for 45-60 min, and reversing and uniformly mixing the centrifugal tube for 1 time every 10 min; taking out the centrifuge tube, and cooling to room temperature; adding equal volume of chloroform isoamyl alcohol (24:1, v/v), shaking up and down slightly, reversing for multiple times, and mixing until the liquid is milky white; centrifuging at 12000rpm for 10min, transferring the supernatant to a new 2.0mL centrifuge tube, adding precooled anhydrous ethanol with the volume about 1.5 times of that of the supernatant, and gently mixing the mixture; centrifuging at 12000rpm for 3min to separate phases, and discarding the supernatant; washing with 70% ethanol solution for 2 times, and naturally drying; the precipitate was dissolved by adding 50. mu.L of 1 XTE buffer (pH 8.0); detecting the quality of the DNA stock solution by 0.8% agarose gel electrophoresis, detecting the concentration of the DNA stock solution by a nucleic acid concentration determinator or an ultraviolet spectrophotometer, and diluting to 50-150 ng/mu L; storing at-20 deg.C for use.
Note: other methods that yield DNA of a quality that meets the requirements of PCR amplification are suitable for use in this standard.
(3) PCR amplification reaction system
The final concentrations of the total system and components of the 20. mu.L PCR amplification reaction system are shown in Table 3, and can be adjusted according to different experimental conditions. The buffers in Table 3 contain MgCl2MgCl is not added any more2The solution was replaced with an equal volume of sterile water. The volume of the reaction system can be adjusted according to specific conditions.
TABLE 3 PCR amplification reaction System
(4) Reaction procedure
The reaction parameters in the reaction program can be properly adjusted according to the type of the PCR amplification instrument, Taq enzyme, primers and the like. The following reaction sequence is generally employed: pre-denaturation at 94 deg.C for 5 min; denaturation at 94 ℃ for 30s, annealing at 50-60 ℃ for 60s, and extension at 72 ℃ for 30s (each cycle is reduced by 0.8 ℃, and 12 cycles are total); denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 45s, and extension at 72 ℃ for 30s for 23 cycles; extension at 72 ℃ for 10 min. The amplification product was stored at 4 ℃.
(5) Denaturing polyacrylamide gel electrophoresis (PAGE) detection
S1 glass plate treatment
And cleaning the glass plate, washing with double distilled water and drying. Scrubbing with absolute ethyl alcohol for 2 times, and wiping with absorbent paper. 2-3 mL of affinity silane working solution is coated on the long plate, and 2-3 mL of stripping silane working solution is coated on the short plate with the groove. The two glass plates are prevented from being contaminated with each other during the operation. After the glass plate is completely dried, the plastic strips are placed on the two sides of the flat plate in order, the concave plate is covered, and after the plastic strips are fixed by the frame, the plastic strips are leveled by a level meter.
S2, preparing glue
Adding 70 mu L of tetramethyl ethylenediamine and 210 mu L of newly configured 10% ammonium persulfate solution into 100 mu L of 6.0% modified polyacrylamide gel solution respectively, shaking up gently, quickly filling the glass plate interlayer with the gel, and inserting the flat end of the shark tooth comb into the gel solution by about 5mm at the groove. Prevent the bubble in the encapsulating process. And (3) after the glue is polymerized for 1 hour, cleaning the glue solution on the surface of the rubber plate, slightly pulling out the comb, and cleaning the comb by using clear water for later use.
S3 preliminary electrophoresis
The rubber plate is arranged on an electrophoresis tank, about 800mL of 1 xTBE buffer solution preheated to 60-70 ℃ is added into an upper tank, about 1000mL of 1 xTBE buffer solution preheated to 60-70 ℃ is added into a lower tank, and 80W constant power electrophoresis is carried out for 40-60 min.
S4 denaturation of PCR amplification product
mu.L of 6 Xloading buffer (loading buffer composed of 0.25% bromophenol blue, 0.25% xylene green FF, and 30% glycerol aqueous solution) was added to 20. mu.L of the amplification product of LPCR, and mixed well. Denaturing at 95 deg.c for 5min in water bath or PCR instrument, taking out, setting on ice and cooling for over 10 min.
S5 electrophoresis
And (4) blowing and sucking the sample adding groove by using a liquid transfer device to remove bubbles and impurities. Inserting the shark teeth end into the gel with the thickness of 1 mm-2 mm. 3-5 mul of amplification sample to be detected is spotted in each sample adding hole, and DNAarkers are spotted on two sides of the gel plate. In addition to the sample to be tested, the amplification product of the reference variety should be added. Electrophoresis is carried out at a constant power of 60W to 80W, and the temperature of the gel is kept at about 50 ℃. Electrophoresis is carried out for 1.5h to 2.5 h.
S6 silver staining
After electrophoresis is finished, carefully separating the two glass plates, taking down the long glass plate attached to the gel, immersing the long glass plate into a stationary liquid, and enabling the stationary liquid to submerge the gel and be fixed for 20min at 30rpm on a shaking table; taking out the rubber plate, and rinsing the rubber plate for 1-2 times by using distilled water for no more than 10 s; taking out the gel plate and putting the gel plate into a dyeing solution to ensure that the dyeing solution is submerged in the gel, and shaking gently at 30rpm for 15-20 min; taking out the rubber plate, and rinsing with distilled water for 1 time, wherein the time is not more than 10 s; and (3) placing the rubber plate into a developing solution, shaking gently at 30rpm until the strips are clear, then placing the rubber plate into a fixing solution for fixing for 5min, and rinsing with distilled water for 1 min. And (4) draining after taking out, and scanning or shooting for imaging. The electrophoresis pattern of 18 pairs of primers corresponding to 24 highland barley varieties is shown in figure 1.
S7 capillary electrophoresis fluorescence detection
The primer grouping scheme of labeling four-color fluorescence by 18 SSR primers and 18 pairs of SSR primers of varieties which are respectively distinguished is shown in a table 4:
TABLE 4
② sample preparation
Respectively taking different fluorescence-labeled amplification products of the same combined primer with the same volume according to the predetermined combined primer, and fully and uniformly mixing. mu.L of the mixture was pipetted and applied to a 96-well plate dedicated to a genetic analyzer. And respectively adding 0.1 mu L of molecular weight internal standard and 8.9 mu L of deionized formamide into each hole, placing the sample on a PCR instrument for denaturation at 95 ℃ for 5min, taking out, immediately placing on ice, cooling for more than 10min, and centrifuging for 10s for later use.
(iii) electrophoretic detection
And opening the DNA analyzer, checking the working state of the instrument, replacing the buffer solution and filling the gel. The 96-well loading plate with the sample is placed on the sample holder base. The data collection software was turned on and the operation was performed according to the manual of the genetic analyzer. And editing the sample table, executing the operation program and storing data.
S8, collecting allelic variation data
Allelic variation for each SSR site in a sample is expressed in terms of amplified fragment size.
For the method for detecting the denaturing polyacrylamide gel electrophoresis, comparing the allelic variation of each amplification site with the size of an allelic compiling fragment of a corresponding reference variety, and determining the allelic variation of a sample to be detected at the site. For the method of capillary electrophoresis fluorescence detection, the equipotential variation data of the variety to be detected and the reference variety are read out by using fragment analysis software of a DNA analyzer. Comparing the data of the allelic variation of the reference variety with the data of the allelic variation of the reference variety in the table 2, wherein the difference value between the two data is the system error. And removing the system error from the allelic variation data of the sample to be detected, wherein the obtained data is the allelic variation size of the sample to be detected at the position point.
S9, data recording
The allelic variation data of the pure and the locus are recorded as X/X, wherein X is the size of the allelic variation of the locus; allelic variation data for the heterozygous locus is recorded as X/Y, wherein X, Y is two different allelic variations at the locus, small before and large after; the null allelic variation was recorded as 0/0 (Table 1).
S10, determination method
(ii) direct comparison
A direct comparison method is adopted for simultaneously comparing 2 or more than 2 varieties. The core primers in Table 1 were used for detection to obtain the allele data (FIG. 2) of the variety to be tested at these primer sites, and these data were used for comparison between varieties. When the difference among the varieties is that the number of points is more than or equal to 2, judging the varieties to be different; judging as an approximate variety when the number of the ectopic points of the inter-variety difference is equal to 1; when the number of ectopic sites is 0, the same variety is judged to be suspected. The field identification is carried out according to the regulation of GB/T19557.31 for different varieties and similar varieties.
Database comparison
And comparing the detected variety with the variety data in the database by using a database comparison method after detecting the variety to be detected. 18 pairs of primers in the table 1 are used for detection, allele data of the variety to be detected at the primer sites are obtained, the data are compared with the varieties in the database, and the comparison method and the judgment method are directly compared with the first step.
The operating conditions are as follows: the storage was carried out at room temperature except for the sample storage.
S11 SSR result analysis
FIG. 1 shows the result of PAGE, 18 pairs of primers have good and stable polymorphism, and the primers can be used as alternative core primers for capillary electrophoresis; as shown in the capillary electrophoresis result of FIG. 2, the diversity index of the primers Bmag0496, HVM33, GBM1215, Scssr03381, Scssr04163 and Scssr03906 is high, and 24 highland barley main cultivars can be distinguished by using the 6 pairs of primers, as shown in Table 5 and FIG. 3.
TABLE 5
| Serial number | Primer and method for producing the same | Number of sites | PIC value of |
| 1 | |
4 | 0.54 |
| 2 | |
3 | 0.29 |
| 3 | Scssr04163 | 5 | 0.55 |
| 4 | |
3 | 0.45 |
| 5 | |
3 | 0.43 |
| 6 | |
3 | 0.43 |
| 7 | |
3 | 0.46 |
| 8 | |
3 | 0.51 |
| 9 | |
4 | 0.46 |
| 10 | GBM1008 | 3 | 0.53 |
| 11 | |
4 | 0.66 |
| 12 | |
4 | 0.51 |
| 13 | |
4 | 0.63 |
| 14 | |
3 | 0.36 |
| 15 | |
4 | 0.58 |
| 16 | |
4 | 0.24 |
| 17 | Bmag0496 | 8 | 0.75 |
| 18 | Bmag0745 | 5 | 0.51 |
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
<110> Qinghai university
<120> method for identifying highland barley varieties by SSR molecular marker method and application thereof
<130> 3.30
<160> 36
<170> PatentIn version 3.5
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<213> Artificial Synthesis
<400> 23
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<212> DNA
<213> Artificial Synthesis
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<212> DNA
<213> Artificial Synthesis
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<212> DNA
<213> Artificial Synthesis
<400> 26
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<212> DNA
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<212> DNA
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<400> 33
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Claims (9)
1. An SSR primer consisting of the sequences shown in Seq ID No.3 and Seq ID No.4, Seq ID No.7 and Seq ID No.8, Seq ID No.11 and Seq ID No.12, Seq ID No.25 and Seq ID No.26, Seq ID No.29 and Seq ID No.30, and Seq ID No.33 and Seq ID No. 34.
2. A method for identifying highland barley varieties by using an SSR molecular marker method is characterized by comprising the following steps:
(1) extracting DNA of a highland barley variety sample;
(2) carrying out PCR amplification, denaturing polyacrylamide gel electrophoresis, silver staining and capillary electrophoresis by using the SSR primers according to claim 1;
the sample is selected from one or more of highland barley seeds, embryos, seedlings and leaves;
the highland barley variety is sweet green No.7, sweet green No. 6, sweet green No. 5, yellow green No.2, sweet green No.4, A green No. 6, A green No. 5, Tibetan No. 13, Kunlun No. 14, rock highland barley, Tibetan 690, Kunlun No. 15, Tibetan green 320, Himalayan No. 19, highland barley No.1, highland barley No.4, kang green No.8, Himalayan No. 22, kang green No. 9, triperyyellow, highland barley No. 5, Tibetan green 2000, sweet green No.2 or highland barley No. 4.
3. The method of claim 2, wherein the reaction procedure of the PCR amplification is:
pre-denaturation at 94 ℃ for 4.5-5.5 min; denaturation at 94 ℃ for 25-35 s, annealing at 50-65 ℃ for 55-65 s, extension at 72 ℃ for 25-35 s, and 10-15 cycles, wherein the temperature is reduced by 0.5-1.2 ℃ per cycle; denaturation at 94 ℃ for 25-35 s, annealing at 50 ℃ for 40-50 s, extension at 72 ℃ for 25-35 s, and 20-25 cycles; extending for 9-12 min at 72 ℃.
4. The method of claim 2, wherein the specific steps of denaturing polyacrylamide gel electrophoresis comprise:
(a) taking 6% modified polyacrylamide solution, tetramethyl ethylenediamine and 10% ammonium persulfate, mixing uniformly, and making into gel;
(b) and (3) uniformly mixing the PCR amplification product with the sample adding buffer solution, cooling after denaturation, and performing electrophoresis.
5. The method according to claim 2, wherein the denaturation condition is 92-98 ℃ for 4.5-5.5 min;
the electrophoresis condition is that the constant power is 60-80W, and the electrophoresis time is 1.5-2.5 h;
and during electrophoresis, the temperature of the gel is 45-55 ℃.
6. The method of claim 2, wherein the silver staining step comprises: fixing the gel, washing with water, carrying out silver dyeing for 15-20 min, washing with water again, developing until the strips are clear, and fixing;
the fixing condition is that the speed is 20-35 rpm for 15-25 min;
the time of washing is less than or equal to 10 s/time; the fixing time is 4.5-5.5 min.
7. The method according to claim 2, wherein the specific steps of capillary electrophoresis comprise: taking different fluorescence-labeled amplification products of the same combined primer with the same volume, uniformly mixing to obtain a mixed solution, sucking the mixed solution, a molecular weight internal standard and deionized formamide, adding the mixed solution to a 96-well plate, performing denaturation, cooling, centrifuging and performing electrophoresis;
the dosage of the internal standard is 0.05-0.15 mu L;
the dosage of the deionized formamide is 8-9.5 mu L;
the denaturation condition is 90-100 ℃ and 3-7 min.
8. The use of the primer of claim 1 in highland barley variety identification;
the highland barley variety is sweet green No.7, sweet green No. 6, sweet green No. 5, yellow green No.2, sweet green No.4, A green No. 6, A green No. 5, Tibetan No. 13, Kunlun No. 14, rock highland barley, Tibetan 690, Kunlun No. 15, Tibetan green 320, Himalayan No. 19, highland barley No.1, highland barley No.4, kang green No.8, Himalayan No. 22, kang green No. 9, triperyyellow, highland barley No. 5, Tibetan green 2000, sweet green No.2 or highland barley No. 4.
9. The use of the primer of claim 1 in the analysis of genetic relationship of highland barley varieties;
the highland barley variety is sweet green No.7, sweet green No. 6, sweet green No. 5, yellow green No.2, sweet green No.4, A green No. 6, A green No. 5, Tibetan No. 13, Kunlun No. 14, rock highland barley, Tibetan 690, Kunlun No. 15, Tibetan green 320, Himalayan No. 19, highland barley No.1, highland barley No.4, kang green No.8, Himalayan No. 22, kang green No. 9, triperyyellow, highland barley No. 5, Tibetan green 2000, sweet green No.2 or highland barley No. 4.
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