CN108707650B - High-throughput cotton variety fingerprint database construction method based on capillary four-color fluorescence electrophoresis detection and multiple fluorescence PCR amplification - Google Patents
High-throughput cotton variety fingerprint database construction method based on capillary four-color fluorescence electrophoresis detection and multiple fluorescence PCR amplification Download PDFInfo
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Abstract
The invention discloses a method for constructing a high-throughput cotton variety fingerprint library based on capillary four-color fluorescence electrophoresis detection and multiple fluorescence PCR amplification, which comprises the steps of carrying out multiple fluorescence PCR combination on 52 pairs of core primers distributed on 26 chromosomes of a cotton A subgroup or a cotton D subgroup whole genome, extracting cotton DNA as a sample to be detected, respectively carrying out pSSR-PCR amplification reaction on the sample to be detected by using 5 combinations obtained by combining the 52 pairs of core primers according to a multiple fluorescence PCR combination mode to obtain a PCR amplification product, and carrying out capillary four-color fluorescence electrophoresis detection and data analysis on the PCR amplification product. The method can greatly improve the detection flux, has high detection efficiency, and is particularly suitable for constructing a mass cotton variety DNA fingerprint database.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a method for constructing a high-throughput cotton variety fingerprint database based on capillary four-color fluorescence electrophoresis detection and multiple fluorescence PCR amplification.
Background
The cotton is a main economic crop in China, and the cultivation and popularization of the excellent new variety have important significance for the development of national economy and the improvement of the living standard of people. According to statistics, in 2000-2009, the number of cotton varieties passing through national approval and provincial approval in China is more than 700. By day 29/2 of 2012, up to 335 cotton varieties have been applied for new plant variety protection. However, due to the centralized use of backbone parents and the large-scale application of transgenic technology in cotton breeding, the genetic difference among cotton varieties is smaller and smaller, so that the variety identification completely depending on the traditional morphological characters is increasingly difficult. Meanwhile, the phenomenon of multiple varieties and impurities is difficult to effectively control due to poor timeliness and large workload of morphological identification, and is easily influenced by environment and subjective factors.
The development of DNA molecular marker technology has promoted the progress of variety identification technology. The SSR marker technology is widely applied to variety identification of crops such as rice, corn, cotton, wheat and the like due to the characteristics of stable amplification, rich polymorphism, co-dominant inheritance and the like. The multicolor fluorescence detection technology is characterized in that fluorescent dyes with different colors are marked at the 5' end of an SSR primer, a fluorescence detector is used for carrying out signal acquisition on an amplification product marked with the fluorescent dyes, and the amplification product is compared with molecular weight internal standards of all lanes, so that the size of a product fragment can be automatically read, the detection efficiency can be improved, and the accuracy of data can be ensured.
However, in the conventional method for constructing the cotton variety DNA fingerprint database in China based on the cotton SSR multicolor fluorescence labeling detection technology, each pair of core primers respectively carries out SSR-PCR amplification reaction on a sample to be detected, namely single SSR-PCR amplification reaction, so that a large amount of SSR-PCR amplification reaction is carried out, further the detection efficiency is greatly reduced, particularly, when a large amount of cotton variety DNA fingerprint databases are constructed, the detection efficiency is very low, and when the amplified products are subjected to capillary fluorescence electrophoresis detection, the time for each detection is long, so the detection efficiency is also reduced.
Disclosure of Invention
The invention provides a method for constructing a high-throughput cotton variety fingerprint database based on capillary four-color fluorescence electrophoresis detection and multiple fluorescence PCR amplification, which can greatly improve the detection efficiency and overcome the defects in the prior art.
In order to solve the technical problems, the invention adopts the following technical scheme:
a construction method of a high-throughput cotton variety DNA fingerprint database based on a capillary four-color fluorescence electrophoresis detection system and multiple fluorescence PCR amplification is characterized by comprising the following steps:
(1) performing multiplex fluorescence PCR combination on 52 pairs of core primers distributed on 26 chromosomes of a cotton A subgroup or a cotton D subgroup whole genome, wherein each pair of core primers is labeled with a fluorescent dye,
the nucleotide sequence of the core primer is shown in the following table 1:
TABLE 1
The invention is based on the following basic principle of multiplex fluorescence PCR combination: firstly, all primers with the same fluorescence are marked in the combination, and the ranges of amplified product fragments cannot be crossed; avoiding the mutual influence of non-specific amplification product peaks; thirdly, adopting a four-color fluorescence labeling system: wherein ROX is red fluorescence, FAM is blue fluorescence, HEX is green fluorescence, and Liz-500 as an internal molecular weight standard is orange.
The multiplex fluorescent PCR combination mode of the 52 pairs of core primers is shown in the following table 2:
TABLE 2
(2) Extracting cotton DNA as a sample to be detected;
(3) respectively carrying out pSSR-PCR amplification reaction on a sample to be detected by using 5 combinations obtained by combining the 52 pairs of core primers in the step (1) according to a multiple fluorescence PCR combination mode to obtain PCR amplification products;
(4) and (4) carrying out capillary four-color fluorescence electrophoresis detection and data analysis on the PCR amplification product in the step (3).
Further, the 52 core primers were obtained from 26 pairs each of cotton A subgroup and cotton D subgroup.
Further, the step (2) of extracting cotton DNA comprises the following steps: crushing the hulled cotton seeds, adding SDS extracting solution, fully whirling, and carrying out water bath at 65 ℃; the volume ratio of the added materials is 25: 24: 1, mixing the mixture of phenol, chloroform and isoamylol uniformly, and centrifuging; taking the supernatant, adding 10mg/mLRNase into the supernatant, and carrying out water bath; and centrifuging after extraction to take supernatant, adding isopropanol, washing DNA precipitate by using ethanol after DNA is clustered and separated out, and adding TE or ddH2O to fully dissolve the DNA for later use.
Further, the step (2) of extracting cotton DNA comprises the following steps: pulverizing the hulled cotton seed, adding 800 μ L SDS extractive solution, whirling, water-bathing at 65 deg.C for 30min,shaking gently once at intervals of 10 min; adding equal volume of 800 mu L, and sequentially adding 25: 24: 1, uniformly mixing the mixture of phenol, chloroform and isoamylol until no layering, and centrifuging at 10000rpm for 10 min; taking the supernatant, adding 1 mu LRNase with the concentration of 10mg/mL, and carrying out water bath at 37 ℃ for 30 min; repeatedly extracting once, centrifuging to obtain supernatant, adding 0.7 times volume of isopropanol, washing DNA precipitate with 70% ethanol for 2 times after DNA conglobation, adding 200 μ L TE or ddH2O fully dissolves the DNA for later use.
Further, the reaction solution of the pSSR-PCR amplification reaction corresponding to each combination in the step (3) comprises all primers, PCR Buffer solution, dNTPs, DNA template, Taq HS polymerase and ddH corresponding to the combination2O。
Furthermore, the reaction solution of the pSSR-PCR amplification reaction corresponding to each combination in the step (3) is 20 μ L,
in combination 1, 20. mu.L of the reaction solution included: 0.03 muL of upstream and downstream of core primer BA11-13, 0.025 muL of upstream and downstream of core primer BD7-7, 0.3 muL of upstream and downstream of core primer BD6-11, 0.07 muL of upstream and downstream of core primer A1-2, 0.025 muL of upstream and downstream of core primer BD5-8, 0.3 muL of upstream and downstream of core primer D12-2, 0.1 muL of upstream and downstream of core primer BA7-1, 0.1 muL of upstream and downstream of core primer 4-8, 0.1 muL of upstream and downstream of core primer BA6-2, 0.3 muL of upstream and downstream of core primer BD9-3, 0.1 muL of upstream and downstream of core primer BA9-11, the concentration of each core primer is 40 mumol/L, 2 muL of 10 Xbuffer solution, concentration of 10mmol/L of PCR, concentration of 0.4. mu.4 ng/L of DNA template, taq HS polymerase 0.25. mu.L at a concentration of 5U/. mu.L, 12.45. mu.L ddH 2O;
in combination 2, 20. mu.L of the reaction solution included: 0.05 muL of upstream and downstream of core primer BA12-13, 0.025 muL of upstream and downstream of core primer BA13-4, 0.07 muL of upstream and downstream of core primer BA1-4, 0.08 muL of upstream and downstream of core primer BD13-3, 0.2 muL of upstream and downstream of core primer BA5-4, 0.15 muL of upstream and downstream of core primer BA 7-17, 0.05 muL of upstream and downstream of core primer BA7-5, 0.3 muL of upstream and downstream of core primer BD6-3, 0.3 muL of upstream and downstream of core primer A6-4, 0.3 muL of upstream and downstream of BD template, 40 mumol/L of core primer, 2 muL of 10 Xbuffer, 10mmol/L of PCR Buffer, 0.05 muL of dNTPs/S/L of PCR, 0.60 muL of dNTPs/L, taq HS polymerase 0.25. mu.L at a concentration of 5U/. mu.L, 12.4. mu.L ddH 2O;
in combination 3, 20. mu.L of the reaction solution included: 0.04 mul of upstream and downstream of core primer BD10-1, 0.08 mul of upstream and downstream of core primer BD13-21, 0.05 mul of upstream and downstream of core primer BA13-1, 0.03 mul of upstream and downstream of core primer BD8-13, 0.2 mul of upstream and downstream of core primer BA5-10, 0.05 mul of upstream and downstream of core primer BD9-2, 0.1 mul of upstream and downstream of core primer BA10-6, 0.35 mul of upstream and downstream of core primer 2-11, 0.35 mul of upstream and downstream of core primer BA12-15, 0.35 mul of upstream and downstream of core primer BA10-9, 40 mul mol/L of each core primer, 2 mul of 10 XPCR Buffer, 0.4 mul of dNTPs with concentration of 10mmol/L, 2 ng/mul of DNA template concentration of 60 ng/mul, 0.5 mul of Taq polymerase U/L of HS 25 mul, 12.19 μ L of ddH 2O;
in combination 4, 20. mu.L of the reaction solution included: 0.025 muL of each upstream and downstream of a core primer BD5-10, 0.3 muL of each upstream and downstream of a core primer BD2-2, 0.05 muL of each upstream and downstream of a core primer BA9-3, 0.05 muL of each upstream and downstream of a core primer D3-1, 0.1 muL of each upstream and downstream of a core primer BA8-12, 0.06 muL of each upstream and downstream of a core primer BD11-5, 0.15 muL of each upstream and downstream of a core primer D10-10, 0.3 muL of each upstream and downstream of a core primer BA3-8, 0.2 muL of each upstream and downstream of a core primer BD4-3, 0.3 muL of each upstream and downstream of a core primer BA2-12, the concentration of each core primer is 40 mumol/L, 2 muL of 10 XPCR Buffer, 0.4 muL of dNTPs with a concentration of 10mmol/L, 2 ng/muL of DNA template with a concentration of 60 ng/muL, and 0.5 muL of Taq 5 muL of DNA polymerase, 12.28 μ L of ddH 2O;
combination 5 20. mu.L of the reaction solution included: 0.03 muL of upstream and downstream of core primer EA8-12, 0.03 muL of upstream and downstream of core primer ED3-5, 0.2 muL of upstream and downstream of core primer BD8-12, 0.3 muL of upstream and downstream of core primer A2-3, 0.35 muL of upstream and downstream of core primer BD1-7, 0.25 muL of upstream and downstream of core primer BD11-3, 0.3 muL of upstream and downstream of core primer BA3-4, 0.3 muL of upstream and downstream of core primer DA4-12, 0.3 muL of upstream and downstream of core primer DA4-16, 0.35 muL of upstream and downstream of core primer BA11-8, 40 mumol/L of each core primer, 2 muL of 10 XPCR Buffer, 0.4 muL of dNTPs with concentration of 10mmol/L, 2 ng/muL of DNA template concentration of 60 ng/muL, 0.5 muL of Taq polymerase of 5 muL, 11.05 μ L of ddH 2O.
Further, the procedure of the pSSR-PCR amplification reaction in step (3) is as follows: pre-denaturation at 94 ℃ for 4min for 1 cycle; denaturation at 94 ℃ for 45s, annealing at 60 ℃ for 45s, and extension at 72 ℃ for 45s for 32 cycles; extension at 72 ℃ for 12min for 1 cycle; storing at 4 ℃ for later use.
Further, in step (4), 1. mu.L of PCR amplification product is added with 8.5. mu.L of deionized formamide and 0.5. mu.L of LLIZ-500 molecular weight internal standard, and capillary four-color fluorescence electrophoresis detection is carried out on a DNA analyzer.
Further, the conditions for detecting the capillary four-color fluorescence electrophoresis in the step (4) are as follows: pre-electrophoresis at 15kV for 3 min; 2kV sample introduction for 2 s; electrophoresis at 15kV for 20 min.
Compared with the prior art, the invention has the beneficial effects that:
based on the basic principle of multiplex fluorescence PCR combination, 52 pairs of core primers distributed on 26 chromosomes of a cotton subgroup A or cotton subgroup D whole genome are combined, 3 10-fold and 2-fold PCR combinations are realized, a multiplex SSR-PCR amplification system and a reaction program suitable for a capillary four-color fluorescence electrophoresis detection system are established, and capillary four-color fluorescence electrophoresis detection is performed on an ABI3730xl DNA analyzer. The invention only needs 5 pSSR-PCR amplification reactions, greatly improves the detection flux, further greatly improves the detection efficiency, and is particularly suitable for the detection and analysis of a large amount of materials; in addition, during the pSSR-PCR amplification reaction, because the optimal amplification temperature of Taq HS polymerase is 72 ℃, and the amplification product is fully tailed after 32 cycles and extends for 12min at 72 ℃, the high consistency of the tail end of the amplification product is ensured, the insufficient tailgating caused by the difference between a core primer and a sample is avoided, and the Taq HS polymerase is a hot start enzyme, so that the non-specific amplification in the system can be reduced, and thus, some miscellaneous peaks can not appear during the capillary four-color fluorescence electrophoresis detection; when the amplification product is subjected to capillary four-color fluorescence electrophoresis detection, the sample introduction time is reduced from the original 10s to 2s, and the electrophoresis time is reduced from the original 40min to 20min, so that the time for each detection is shortened, and the detection efficiency is further improved; according to the invention, the detection efficiency is improved by more than 100 times by the construction of a multiple SSR-PCR amplification system and the capillary four-color fluorescence electrophoresis detection, the size of an amplified product fragment is obtained by automatic comparison of molecular weight internal standards, the manual plate reading error is reduced, and the accuracy and reliability of a data result are ensured to the greatest extent.
Drawings
FIG. 1 is a four-color fluorescence electrophoresis image of a cotton variety, flower 547, in multiplex fluorescence PCR set 1;
FIG. 2 is a four-color fluorescence electrophoresis image of cotton variety flower 547 in multiplex fluorescence PCR assembly 2;
FIG. 3 is a four-color fluorescence electrophoresis image of a cotton variety, flower 547 in multiplex fluorescence PCR assembly 3;
FIG. 4 is a four-color fluorescence electrophoresis image of cotton variety flower 547 in multiplex fluorescence PCR assembly 4;
FIG. 5 is a capillary four color fluorescence electrophoresis detection of cotton variety protein 547 in multiplex fluorescence PCR set 5.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
A DNA fingerprint library of cotton variety shen 547 was constructed, wherein cotton variety shen 547 was obtained from the Cotton institute of agricultural sciences, China.
1. DNA extraction
(1) Shelling single cotton seeds, fully crushing the cotton seeds, and transferring the crushed cotton seeds into a 2mL centrifuge tube;
(2) adding 800 μ L of DNA extract (1% SDS, 0.01mol/L EDTA 8.0, 0.7mol/L NaCl, 0.05mol/L Tris-HCl, 0.5% sorbitol, 1% PVP, 1% beta-mercaptoethanol), swirling to mix well, water bathing at 65 deg.C for 30min, and shaking gently at intervals of 10 min;
(3) after the water bath is finished, adding 800 mu L of mixed solution of phenol, chloroform and isoamylol (the volume ratio is 25: 24: 1 in sequence), turning upside down and uniformly mixing until no layering occurs, and centrifuging at 10000rpm for 10 min;
(4) the supernatant was pipetted into another 2mL centrifuge tube, 1. mu.L RNase enzyme (10mg/mL) was added and mixed well, followed by a 37 ℃ water bath for 30 min.
(5) An equal volume of 800 μ L phenol was added: chloroform: isoamyl alcohol (the volume ratio is 25: 24: 1 in sequence), turning upside down and mixing evenly until no layering, and centrifuging for 10min at 10000 rpm.
(6) The supernatant was transferred to another 2mL centrifuge tube, 0.7 volume isopropanol was added and the tube was shaken slowly several times and allowed to stand for 30min, whereupon flocculent DNA was precipitated as a pellet.
(7) The DNA is sucked by a shearing gun head and transferred into a centrifugal tube filled with 70% ethanol, and the DNA is soaked twice, about 2 hours for the first time and overnight for the second time.
(8) The ethanol was decanted, the DNA dried by natural ventilation, and 200. mu.L of TE (pH 8.0) or ddH2O was added and dissolved thoroughly for further use.
(9) And (3) detecting the DNA concentration by ultraviolet, diluting the DNA stock solution to the use concentration of 60 ng/mu L by ddH2O, and storing at 4 ℃ for later use to obtain a sample to be detected.
2. pSSR-PCR amplification
The PCR amplification products were obtained by performing pSSR-PCR amplification reactions on samples to be tested using 5 combinations obtained by combining 52 pairs of core primers (the nucleotide sequences of the core primers are shown in Table 1) in accordance with the multiplex fluorescent PCR combination method, the multiplex fluorescent PCR combination method of 52 pairs of core primers is shown in Table 2, and 20. mu.L of reaction solutions for the pSSR-PCR amplification reactions corresponding to combination 1, combination 2, combination 3, combination 4 and combination 5 are shown in tables 3, 4, 5, 6 and 7, respectively,
TABLE 3
TABLE 4
TABLE 5
TABLE 6
TABLE 7
The procedure for the pSSR-PCR amplification reaction was: pre-denaturation at 94 ℃ for 4min for 1 cycle; denaturation at 94 ℃ for 45s, annealing at 60 ℃ for 45s, and extension at 72 ℃ for 45s for 32 cycles; extension at 72 ℃ for 12min for 1 cycle; storing at 4 ℃ for later use. The amplification reaction system extends for 12min at 72 ℃ after 32 cycles, so that the amplification products are fully tailed, the high consistency of the tail ends of the amplification products is ensured, and the insufficient tailgating caused by the difference between the primers and the samples is avoided.
3. Capillary four-color fluorescence detection
Taking 1 mu L of PCR amplification product, adding 8.5 mu L of deionized formamide and 0.5 mu L of Liz-500 molecular weight internal standard, and carrying out capillary four-color fluorescence electrophoresis detection on an ABI3730xl DNA analyzer, wherein the conditions of the capillary four-color fluorescence electrophoresis detection are as follows: pre-electrophoresis at 15kV for 3 min; 2kV sample introduction for 2 s; electrophoresis is carried out for 20min at 15kV, and then data acquisition and analysis are carried out on the detection result by using GeneMapper software. The sample introduction time is reduced from the original 10s to 2s, and the electrophoresis time is reduced from the original 40min to 20min, so that the time for each detection is shortened, and the detection efficiency is further improved.
The fingerprint information of cotton variety height 547 obtained by the above method is a fingerprint database (abbreviated as fingerprint database) of cotton variety height 547, which is shown in table 8 below, and the capillary four-color fluorescence electrophoresis detection images of cotton variety height 547 in multiplex fluorescence PCR combination 1, combination 2, combination 3, combination 4 and combination 5 are shown in fig. 1, fig. 2, fig. 3, fig. 4 and fig. 5, respectively, wherein the abscissa in each figure is the size (bp) of the amplified product and the ordinate is the peak height (representing the fluorescence signal intensity).
TABLE 8
Note: the amplified fragment size in the table is the fragment size of the PCR amplified product of each pair of core primers.
Claims (9)
1. A method for constructing a high-throughput cotton variety fingerprint database based on capillary four-color fluorescence electrophoresis detection and multiplex fluorescence PCR amplification is characterized by comprising the following steps:
(1) performing multiplex fluorescence PCR combination on 52 pairs of core primers distributed on 26 chromosomes of a cotton A subgroup or a cotton D subgroup whole genome, wherein each pair of core primers is labeled with a fluorescent dye,
the nucleotide sequence of the core primer is shown as follows:
the multiplex fluorescent PCR combination mode of the 52 pairs of core primers is as follows:
(2) extracting cotton DNA as a sample to be detected;
(3) respectively carrying out pSSR-PCR amplification reaction on a sample to be detected by using 5 combinations obtained by combining the 52 pairs of core primers in the step (1) according to a multiple fluorescence PCR combination mode to obtain PCR amplification products;
(4) and (4) carrying out capillary four-color fluorescence electrophoresis detection and data analysis on the PCR amplification product in the step (3).
2. The method for constructing the high-throughput cotton variety fingerprint database based on capillary four-color fluorescence electrophoresis detection and multiplex fluorescence PCR amplification according to claim 1, which is characterized in that: the 52 pairs of core primers are respectively taken from 26 pairs of each cotton A subgroup and cotton D subgroup.
3. The method for constructing the high-throughput cotton variety fingerprint database based on capillary four-color fluorescence electrophoresis detection and multiplex fluorescence PCR amplification according to claim 1, wherein the step (2) of extracting cotton DNA comprises the following steps: crushing the hulled cotton seeds, adding SDS extracting solution, fully whirling, and carrying out water bath at 65 ℃; the volume ratio of the added materials is 25: 24: 1, mixing the mixture of phenol, chloroform and isoamylol uniformly, and centrifuging; taking the supernatant, adding RNase with the concentration of 10mg/mL, and carrying out water bath; and centrifuging after extraction to take supernatant, adding isopropanol, washing DNA precipitate by using ethanol after DNA is clustered and separated out, and adding TE or ddH2O to fully dissolve the DNA for later use.
4. The method for constructing the high-throughput cotton variety fingerprint database based on capillary four-color fluorescence electrophoresis detection and multiplex fluorescence PCR amplification according to claim 3, wherein the step (2) of extracting cotton DNA comprises the following steps: fully crushing the hulled cotton seeds, adding 800 mu L SDS extracting solution, fully whirling, carrying out water bath at 65 ℃ for 30min, and gently shaking once at intervals of 10 min; adding equal volume of 800 mu L, and sequentially adding 25: 24: 1, uniformly mixing the mixture of phenol, chloroform and isoamylol until no layering, and centrifuging at 10000rpm for 10 min; taking the supernatant, adding 1 microliter RNase with the concentration of 10mg/mL, and carrying out water bath at 37 ℃ for 30 min; repeatedly extracting once, centrifuging to obtain supernatant, adding 0.7 times volume of isopropanol, washing DNA precipitate with 70% ethanol for 2 times after DNA conglobation, adding 200 μ L TE or ddH2O fully dissolves the DNA for later use.
5. The method for constructing the high-throughput cotton variety fingerprint library based on capillary four-color fluorescence electrophoresis detection and multiple fluorescence PCR amplification according to claim 1, wherein the reaction solution of the pSSR-PCR amplification reaction corresponding to each combination in the step (3) comprises all primers, PCR Buffer solution, dNTPs, DNA template, Taq HS polymerase and ddH of the corresponding combination2O。
6. The method for constructing the high-throughput cotton variety fingerprint database based on capillary four-color fluorescence electrophoresis detection and multiplex fluorescence PCR amplification according to claim 5, wherein the reaction solution of the pSSR-PCR amplification reaction corresponding to each combination in the step (3) is 20 μ L,
in combination 1, 20. mu.L of the reaction solution included: 0.03 muL of upstream and downstream of core primer BA11-13, 0.025 muL of upstream and downstream of core primer BD7-7, 0.3 muL of upstream and downstream of core primer BD6-11, 0.07 muL of upstream and downstream of core primer A1-2, 0.025 muL of upstream and downstream of core primer BD5-8, 0.3 muL of upstream and downstream of core primer D12-2, 0.1 muL of upstream and downstream of core primer BA7-1, 0.1 muL of upstream and downstream of core primer 4-8, 0.1 muL of upstream and downstream of core primer BA6-2, 0.3 muL of upstream and downstream of core primer BD9-3, 0.1 muL of upstream and downstream of core primer BA9-11, the concentration of each core primer is 40 mumol/L, 2 muL of 10 Xbuffer solution, concentration of 10mmol/L of PCR, concentration of 0.4. mu.4 ng/L of DNA template, taq HS polymerase 0.25. mu.L at a concentration of 5U/. mu.L, 12.45. mu.L ddH 2O;
in combination 2, 20. mu.L of the reaction solution included: 0.05 muL of upstream and downstream of core primer BA12-13, 0.025 muL of upstream and downstream of core primer BA13-4, 0.07 muL of upstream and downstream of core primer BA1-4, 0.08 muL of upstream and downstream of core primer BD13-3, 0.2 muL of upstream and downstream of core primer BA5-4, 0.15 muL of upstream and downstream of core primer BA 7-17, 0.05 muL of upstream and downstream of core primer BA7-5, 0.3 muL of upstream and downstream of core primer BD6-3, 0.3 muL of upstream and downstream of core primer A6-4, 0.3 muL of upstream and downstream of BD template, 40 mumol/L of core primer, 2 muL of 10 Xbuffer, 10mmol/L of PCR Buffer, 0.05 muL of dNTPs/S/L of PCR, 0.60 muL of dNTPs/L, taq HS polymerase 0.25. mu.L at a concentration of 5U/. mu.L, 12.4. mu.L ddH 2O;
in combination 3, 20. mu.L of the reaction solution included: 0.04 mul of upstream and downstream of core primer BD10-1, 0.08 mul of upstream and downstream of core primer BD13-21, 0.05 mul of upstream and downstream of core primer BA13-1, 0.03 mul of upstream and downstream of core primer BD8-13, 0.2 mul of upstream and downstream of core primer BA5-10, 0.05 mul of upstream and downstream of core primer BD9-2, 0.1 mul of upstream and downstream of core primer BA10-6, 0.35 mul of upstream and downstream of core primer 2-11, 0.35 mul of upstream and downstream of core primer BA12-15, 0.35 mul of upstream and downstream of core primer BA10-9, 40 mul mol/L of each core primer, 2 mul of 10 XPCR Buffer, 0.4 mul of dNTPs with concentration of 10mmol/L, 2 ng/mul of DNA template concentration of 60 ng/mul, 0.5 mul of Taq polymerase U/L of HS 25 mul, 12.19 μ L of ddH 2O;
in combination 4, 20. mu.L of the reaction solution included: 0.025 muL of each upstream and downstream of a core primer BD5-10, 0.3 muL of each upstream and downstream of a core primer BD2-2, 0.05 muL of each upstream and downstream of a core primer BA9-3, 0.05 muL of each upstream and downstream of a core primer D3-1, 0.1 muL of each upstream and downstream of a core primer BA8-12, 0.06 muL of each upstream and downstream of a core primer BD11-5, 0.15 muL of each upstream and downstream of a core primer D10-10, 0.3 muL of each upstream and downstream of a core primer BA3-8, 0.2 muL of each upstream and downstream of a core primer BD4-3, 0.3 muL of each upstream and downstream of a core primer BA2-12, the concentration of each core primer is 40 mumol/L, 2 muL of 10 XPCR Buffer, 0.4 muL of dNTPs with a concentration of 10mmol/L, 2 ng/muL of DNA template with a concentration of 60 ng/muL, and 0.5 muL of Taq 5 muL of DNA polymerase, 12.28 μ L of ddH 2O;
combination 5 20. mu.L of the reaction solution included: 0.03 muL of upstream and downstream of core primer EA8-12, 0.03 muL of upstream and downstream of core primer ED3-5, 0.2 muL of upstream and downstream of core primer BD8-12, 0.3 muL of upstream and downstream of core primer A2-3, 0.35 muL of upstream and downstream of core primer BD1-7, 0.25 muL of upstream and downstream of core primer BD11-3, 0.3 muL of upstream and downstream of core primer BA3-4, 0.3 muL of upstream and downstream of core primer DA4-12, 0.3 muL of upstream and downstream of core primer DA4-16, 0.35 muL of upstream and downstream of core primer BA11-8, 40 mumol/L of each core primer, 2 muL of 10 XPCR Buffer, 0.4 muL of dNTPs with concentration of 10mmol/L, 2 ng/muL of DNA template concentration of 60 ng/muL, 0.5 muL of Taq polymerase of 5 muL, 11.05 μ L of ddH 2O.
7. The method for constructing the high-throughput cotton variety fingerprint database based on capillary four-color fluorescence electrophoresis detection and multiplex fluorescence PCR amplification according to claim 1, wherein the procedure of the pSSR-PCR amplification reaction in the step (3) is as follows: pre-denaturation at 94 ℃ for 4min for 1 cycle; denaturation at 94 ℃ for 45s, annealing at 60 ℃ for 45s, and extension at 72 ℃ for 45s for 32 cycles; extension at 72 ℃ for 12min for 1 cycle; storing at 4 ℃ for later use.
8. The method for constructing the high-throughput cotton variety fingerprint database based on the capillary four-color fluorescence electrophoresis detection and the multiplex fluorescence PCR amplification according to claim 1, wherein the step (4) is that 1 μ L of PCR amplification product is taken, 8.5 μ L of deionized formamide and 0.5 μ L of Liz-500 molecular weight internal standard are added, and the capillary four-color fluorescence electrophoresis detection is carried out on a DNA analyzer.
9. The method for constructing the high-throughput cotton variety fingerprint database based on capillary four-color fluorescence electrophoresis detection and multiplex fluorescence PCR amplification according to claim 1, wherein the conditions of the capillary four-color fluorescence electrophoresis detection in the step (4) are as follows: pre-electrophoresis at 15kV for 3 min; 2kV sample introduction for 2 s; electrophoresis at 15kV for 20 min.
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