AU2020101755A4 - A method for constructing a high-throughput cotton variety DNA fingerprint library based on a capillary four-color fluorescence electrophoresis detection system and multiplex fluorescence PCR amplification - Google Patents
A method for constructing a high-throughput cotton variety DNA fingerprint library based on a capillary four-color fluorescence electrophoresis detection system and multiplex fluorescence PCR amplification Download PDFInfo
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- 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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Abstract
The invention discloses a method for constructing a high-throughput cotton variety DNA
fingerprint library based on a capillary four-color fluorescence electrophoresis detection
system and multiplex fluorescence PCR amplification, which comprises the following steps:
carrying out multiplex fluorescence PCR combination on 52 pairs of core primers distributed
on 26 chromosomes of the whole genome of cotton subgroup A or D, extracting cotton DNA
as a sample to be detected. Five combinations obtained by combining 52 pairs of core primers
according to multiple fluorescent PCR combinations were used to carry out pSSR-PCR
amplification reaction on the sample to be tested to obtain PCR amplification products, and
the PCR amplification products were detected by capillary four-color fluorescent
electrophoresis and data analysis. The method can greatly improve the detection flux and has
high detection efficiency, and is especially suitable for the construction of DNA fingerprint
database of a large number of cotton varieties.
Description
PATENTS ACT 1990
A method for constructing a high-throughput cotton variety DNA fingerprint
library based on a capillary four-color fluorescence electrophoresis detection system and
multiplex fluorescence PCR amplification
The invention is described in the following statement:-
A method for constructing a high-throughput cotton variety DNA fingerprint
library based on a capillary four-color fluorescence electrophoresis detection system and
multiplex fluorescence PCR amplification
The invention belongs to the field of molecular biology, and particularly relates to a
construction method of a high-throughput cotton variety DNA fingerprint library based on
a capillary four-color fluorescence electrophoresis detection system and multiple
fluorescence PCR amplification.
Cotton is the main cash crop in China, and the cultivation and popularization of fine
new varieties is of great significance to the development of national economy and the
improvement of people's living standards. According to statistics, from 2000 to 2009, there
were more than 700 cotton varieties approved by the state and provincial level in China.
As of February 29th, 2012, 335 cotton varieties have applied for protection of new plant
varieties. However, due to the centralized use of backbone parents and the large-scale
application of transgenic technology in cotton breeding, the genetic differences among
cotton varieties are getting smaller and smaller, which makes it increasingly difficult to
identify varieties completely depending on traditional morphological traits. At the same time, due to the poor timeliness and heavy workload of morphological identification, it is easy to be affected by environmental and subjective factors, and it is difficult to effectively control the phenomenon of many varieties.
The development of DNA molecular marker technology promotes the progress of
variety identification technology. Among them, SSR marker technology is widely used in
variety identification of rice, corn, cotton, wheat and other crops because of its stable
amplification, rich polymorphism and co-dominant inheritance. Multicolor fluorescence
detection technology uses fluorescent dyes of different colors to mark the 5' end of SSR
primers. The fluorescence detector collects the signals of the amplified products marked
with fluorescent dyes, and compares them with the molecular weight internal standard of
each lane, which can automatically read the size of product fragments, which can not only
improve the detection efficiency, but also ensure the accuracy of data.
However, the conventional method of constructing DNA fingerprint database of
cotton varieties in China based on cotton SSR multicolor fluorescent marker detection
technology is to carry out SSR-PCR amplification reaction on each pair of core primers to
the sample to be detected, that is, to carry out single SSR-PCR amplification reaction,
which requires a large number of SSR-PCR amplification reactions, which will greatly
reduce the detection efficiency, especially when constructing a large number of cotton
variety DNA fingerprint databases. The detection efficiency is very low, and when SSR
PCR amplification reaction is carried out, it extends for 30 minutes at 60°C after 32 cycles,
and when capillary fluorescence electrophoresis is used to detect the amplified products,
the detection time is longer each time, which will also reduce the detection efficiency.
The invention provides a method for constructing a high-throughput cotton variety
DNA fingerprint library based on a capillary four-color fluorescence electrophoresis
detection system 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 above technical problems, the invention adopts the following
technical scheme:
A construction method of a high-throughput cotton variety DNA fingerprint library
based on a capillary four-color fluorescence electrophoresis detection system and multiple
fluorescence PCR amplification is characterized by comprising the following steps:
(1) Carrying out multiplex fluorescent PCR combination on 52 pairs of core primers
distributed on 26 chromosomes of the whole genome of cotton subgroup Aor subgroup D,
wherein each pair of core primers is marked with fluorescent dye,
The nucleotide sequence of the core primer is shown in Table 1 below.
Table 1
Upstream sequence (5'-3') Downstream sequence (5'-3') Core primer name
BD7-7 CTTCCCCACGCCACTACTATCG CTCAGCTTTCCTCCTCATTGGC
BD6-11 TGATGTTGGCGGGACTATGTAG CGACTTCACCCTCGTGGTAATC
Al-2 CCGGTTCAAGCCGACTATTCG ACTCGTAACACCGTGCTGATTG
BD5-8 ATTCATGGTCAAGTCGGGTCAC GCTTGCTTTGGGTGGAGTAGAC
D12-2 CGGGATGGGGTGTGCAGATC GAGAAAGCAACAAGGCGGGATG
A2-3 AGTCCTCCCACTATTCGAAGCT ATGCCTCGTACCCTGTTCCG
BD1-7 CCACATGCCACGCCGTATTATG ATGATGGGGTGGGCTGTAAAGG
BD9-3 TTGACGTGACTTGCAGCAGATC ATCGCCAGAATGCTGAAAGGTG
BA3-4 TGGGTGAGTGTGAGGACTGAAG TGGGTGTTGCACAAAGTTTCTG
BA9-11 GCCGGTAAGTCCAACATAAGGG CGGGGTAGTCCACCTCCTATTG
DA4-16 AGGCTCGCATTGTTGACACTAGG CGAGCTTGAACGAACGAACCTCT
BD10-1 TTCCATGAGGCTATCCACAAGC AATGCACCGCACCCCATCAC
BD13-21 GCCGAGGCCCCTATAACCC CTCATATCACGCACCACACCAC
BA13-1 AACTCAATGGGTGTCGGTTACG GGAGGTGAGCTATCTTCGCAAC
BD8-13 ACCAGCATCCTTTGTGTTAGGC GGATCGATTTTGGAACCGTGTG
BA5-10 CGTCTCGTCCCACCTGTAATGC GGACTTCGGCAAGGCGGTTC
BD9-2 GTCGCCGATGTCGCCAATG ATGGGTGACAACGGTTACAACC
BA10-6 TGTGGCTCCATGGCACAATATG AGGCTCTGTTGCACCAATTCAC
BD2-11 AGTTCGGTGGACATCAATAGGC TCCCCAGGGCTTTGAGAATACC
BA12-15 AGACGTGAGTTCGAGGACTTTG GCCTACCCTCTCAACAGTTTGG
BA10-9 ACTACGCAACTGAAGGGTTTCG GCTGCAAGGTTCGATGGAAGTG
BA13-4 AGCACGGAAGAACATGATGAGG CGTCTTCGGCTCAAATGTGTGC
Dl-10 TGCCCAACCTACATGTGACACA TCAAATTTGGTTGTCACACCCCA
BAl-4 CTGAGGAGAAAGACAGGACGAC TGGCGGGGTAAATGTGAATGC
BD13-3 GGAAATGGCCCATCTGAGAGTC GCCAGGAGATCGGAGCGTTTG
BA5-4 TCGATTCACCGATCTGACAAGC TGACCCAGACCGACCGTTG
BD7-17 ACCATCCCACTGTTTTCCTCTC GGAAGAACGACCACAGGAGTG
BA7-5 AGGGACAAGAATGGACCGACAG TTAACCGTCGCAGCCTCCTAAC
BD6-3 CAGACTTGGGCCTAGAAAGCG CGAGTCGAGACCTAGAAAACGG
BA12-13 TGTTGAAGCTGGAAGCCTGATG TGGCAAGTCTTCACCCAATGTC
A6-4 AGCAAAGCCAACAGAGGTCAAC GCCAACCATGATTTCGATGCAC
WD12-1 TTGAAAGTCCATCTTAACTTGGTGTGA TCCAGGACAGCAAACTTACGCC
BD5-10 GTTGCTGTGGAGTGGAGTGGAG TTCGAGGGAGGTTGGTATTGGC
BD2-2 TTGGGCCGAAAAGGGTTGAAAC GGTCGGATTCTGGGCACTTTTC
BA9-3 TTACTCTGGGCGTGTGGCATAG ATGGAAGGAACAGCAGCAAACG
D3-1 CCAGCCACTCGGAGATCCTTG GCGTGGAGAAACCAGAGGGTAG
BA8-12 TGCCCTTCTTGCCCCTGTG GCTTGCCTAATTTGGTGGGAAG
BD11-5 ACAAGCGCGTCTGCCATATCC CGTGGTGGGTAGTCACAGTCAG
D10-10 GTTTCCACCGTCGAACCACTG GGCAGGATTAGGAGATCGAAGC
BA3-8 GTGGGCAGCGATGAATATGATG ATGAGGGTCATTGCTTGGGTTG
BD4-3 GCCCACCTACCCACCTGTTTC CCCCTTTTGCTCCCCGATGTAC
BA2-12 CGCACTATTTCTAGCAGCTTGC GCGTGTCGTCCGATCCTAAC
BA11-13 GTTGGAGGCTGCTTTTGATGGG TGCCATTGCCATGTTGGTCAAG
BA8-5 TTGGGCGGTTTGGGTCAAGG GGGATTCGGTCGGACACTCAAG
BD3-6 GCAACACCCTTTAGACGCAGTG ACGGAATGCAGCCCATTGACC
BD8-12 GACTCATGGCGACAGCGATTAG TGATCACTCAAACGGTGTCACG
BA7-1 TTCCTGCAAAATTGCCTTCACC TGCTTTGATATCCCCGTGATGG
BD4-8 ACCACTTTGTCCACCTGTCCAC ATCATCGCCATCTGCCTGGAAG
BA6-2 ACCAGGTCGGTAACGATAGGC GCCCAAAGTTGAAGCGGAAAAC
BD13-3 GGAAATGGCCCATCTGAGAGTC GCCAGGAGATCGGAGCGTTTG
DA4-12 GCCGTTTCTGCCAACCCCTT CGGGATTCCACGTGCCCAAA
BA11-8 AACTGCGACATCACCTGTAACG CCGCCACCGCTGAGTCTC
The invention is based on the following basic principles of multiplex fluorescence
PCR combination: (1) The amplification product fragment ranges of primers labelled with
the same fluorescence in the combination cannot be crossed;@ Avoid the mutual influence
of non-specific amplification product peaks;@ Four-color fluorescence labelling system
was adopted: ROX was red fluorescence, FAM was blue fluorescence, HEX was green
fluorescence, and molecular weight internal standard Liz-500 was orange.
The multiplex fluorescence PCR combination mode of the 52 pairs of core primers is
shown in the following table 2:
Table 2
Combination Core primer name Fluorescent labeling Combination Core primer name Fluorescent labeling
BD7-7 ROX BA13-4 FAM
BD6-11 ROX DI-10 ROX
A1-2 FAM BA1-4 FAM
BD5-8 ROX BD13-3 ROX
D12-2 ROX BA5-4 FAM
Combination A2-3 FAM Combination2 BD7-17 ROX
BD1-7 ROX BA7-5 FAM
BD9-3 ROX BD6-3 ROX
BA3-4 FAM BA12-13 FAM
BA9-11 FAM A6-4 FAM
DA4-16 HEX WD12-1 HEX
BD1O-1 ROX BD5-1O ROX
BD13-21 ROX BD2-2 ROX
BA13-1 FAM BA9-3 FAM
BD8-13 ROX D3-1 ROX
BA5-1O FAM BA8-12 FAM Combination3 Combination4 BD9-2 ROX BD11-5 ROX
BA1O-6 FAM DIO-10 ROX
BD2-11 ROX BA3-8 FAM
BA12-15 FAM BD4-3 ROX
BA1O-9 FAM BA2-12 FAM
BAII-13 FAM BD4-8 ROX
BA8-5 FAM BA6-2 FAM
Combination5 BD3-6 ROX Combination6 BD11-3 ROX
BD8-12 ROX DA4-12 HEX
BA7-1 FAM BA1 1-8 FAM
(2) Extracting cotton DNA as a sample to be tested;
(3) Carrying out pSSR-PCR amplification reaction on the sample to be detected by
using five combinations obtained by combining 52 pairs of core primers in the step (1)
according to a multiplex fluorescent PCR combination mode to obtain PCR amplification
products;
(4) Performing capillary four-color fluorescence electrophoresis detection and data
analysis on the PCR amplification product in the step (3).
Furthermore, the simple sequence repeat in the amplification product of the core
primer is greater than or equal to 3bp.
Furthermore, the 52 pairs of core primers are respectively taken from cotton subgroup
A and cotton subgroup D, with 26 pairs each.
Further, the extraction of cotton DNA in step (2) includes the following steps: after
the dehulled cotton seeds are crushed, SDS extract is added, and after the vortex is
sufficient, water bath is carried out at 65 °C;Adding a mixed solution of phenol, chloroform
and isoamyl alcohol with a volume ratio of 25: 24: 1 in turn, mixing uniformly, and
centrifuging; Collect supernatant, adding RNase with concentration of l0mg/mL RNase
for water bath; After extraction, centrifuge to take supernatant, add isopropanol, wash DNA
precipitate with ethanol after DNA clumps and precipitate, and add TE or ddH20 to fully
dissolve DNA for later use.
Further, the extraction of cotton DNA in step (2) includes the following steps: after
the hulled cotton seeds are fully crushed, 800L SDS extract is added, and after the vortex
is sufficient, the cotton seeds are bathed in water at 65°C for 30min, and gently shaken
once every 10min;Adding an equal volume of 800pL mixed solution of phenol, chloroform
and isoamyl alcohol with a volume ratio of 25: 24: 1 in turn, mixing evenly until no
stratification, and centrifuging at 10000rpm for 10 minutes;1 L RNase with a
concentration of 10mg/mL was added to the supernatant, and the supernatant was bathed
in water at 37°C for 30min ; After repeated extraction, centrifuge to take supernatant, add
0.7 times of isopropanol, wash DNA precipitate twice with 70% ethanol after DNA
clumping and precipitation, and add 200 L TE or ddH20 to fully dissolve DNA for later
use.
Furthermore, the reaction solution of pSSR-PCR amplification reaction corresponding
to each combination in step (3) includes all primers, PCR Buffer solution, dNTPs, DNA
template, Taq HS polymerase and ddH20.
Furthermore, the reaction solution of pSSR-PCR amplification reaction corresponding
to each combination in step (3) is 20L,
The 20 L reaction solution in combination 1 includes 0.024L upstream and
downstream of core primer BD7-7, 0.3tL upstream and downstream of core primer BD6
11, 0.04 L upstream and downstream of core primer Al-2, 0.025tL upstream and
downstream of core primer BD5-8 and 0.3tL upstream and downstream of core primer
D12-20.35tL upstream and downstream of core primer BD1-7, 0.3tL upstream and downstream of core primer BD9-3, 0.25 L upstream and downstream of core primer BA3
4, 0.15 L upstream and downstream of core primer BA9-11, 0.35tL upstream and
downstream of core primer DA4-16, and the concentration of each core primer is
jmol/L. 24L lOx PCR Buffer, 0.4tL dNTPs with a concentration of 10mmol/L, 2L
DNA template with a concentration of 60ng/L, 0.24L Taq HS polymerase with a
concentration of 5U/4L and 11.15 L ddH20;
The 20tL reaction solution in combination 2 includes 0.025tL upstream and
downstream of core primer BA13-4, 0.1 L upstream and downstream of core primer D1
, 0.074L upstream and downstream of core primer BAl-4, 0.08 L upstream and
downstream of core primer BD13-3, and 0.2L upstream and downstream of core primer
BA5-40.05 L upstream and downstream of core primer BA7-5, 0.35tL upstream and
downstream of core primer BD6-3, 0.03 L upstream and downstream of core primer
BA12-13, 0.3tL upstream and downstream of core primer A6-4, 0.15 L upstream and
downstream of core primer WD12-1, and the concentration of each core primer is
jmol/L. 24L lOx PCR Buffer, 0.4tL dNTPs with a concentration of 10mmol/L, 2L
DNA template with a concentration of 60ng/L, 0.24L Taq HS polymerase with a
concentration of 5U/L and 12.29tL ddH20;
The 20 L reaction solution in combination 3 includes 0.04pL upstream and
downstream of core primer BD1O-1, 0.08 L upstream and downstream of core primer
BD13-21, 0.03 L upstream and downstream of core primer BA13-1, 0.03 L upstream and
downstream of core primer BD8-13 and 0 upstream and downstream of core primer BA5
100.1 L upstream and downstream of core primer BA1O-6, 0.35tL upstream and downstream of core primer BD2-11, 0.35tL upstream and downstream of core primer
BA12-15, 0.35tL upstream and downstream of core primer BA1O-9, andthe concentration
of each core primer is 40jmol/L, 2L 1Ox PCR Buffer, 0.4tL dNTPs with a concentration
of 10mmol/L, 2L DNA template with a concentration of 60ng/4L, 0.24L Taq HS
polymerase with a concentration of 5U/L and 12.24tL ddH20;
The 20tL reaction solution in combination 4 includes 0.025tL upstream and
downstream of core primer BD5-10, 0.24L upstream and downstream of core primer BD2
2, 0.05 L upstream and downstream of core primer BA9-3, 0.05 L upstream and
downstream of core primer D3-1 and 0.1 L upstream and downstream of core primer BA8
120.1jL upstream and downstream of core primer D1O-10, 0.3tL upstream and
downstream of core primer BA3-8, 0.24L upstream and downstream of core primer BD4
3, and 0.3tL upstream and downstream of core primer BA2-12, and the concentration of
each core primer is 40jmol/L, 24L 10 x PCR Buffer, 0.4tL dNTPs with a concentration of
1Ommol/L, 24L DNA template with a concentration of60ng/4L, 0.2L Taq HS polymerase
with a concentration of 5U/L and 12.63tL ddH20;
The 20tL reaction solution in combination 5 includes 0.04pL upstream and
downstream of core primer BA1-13, 0.24L upstream and downstream of core primer
BA8-5, 0.24L upstream and downstream of core primer BD3-6, 0.3tL upstream and
downstream of core primer BD4-8-12 and 0.1 L upstream and downstream of core primer
BA7-1. 0.1 L upstream and downstream of core primer BA6-2, 0.3tL upstream and
downstream of core primer BD11-3, 0.3tL upstream and downstream of core primer DA4
12, 0.3tL upstream and downstream of core primer BA11-8, and the concentration of each core primer is 40jmol/L, 24L lOx PCR Buffer, 0.4tL dNTPs with a concentration of mmol/L, 24L DNA template with the concentration of 60ng/L, 0.24L Taq HS polymerase with the concentration of 5U/4L and 11.52L ddH20.
Furthermore, the procedure of pSSR-PCR amplification reaction in step (3) is: pre
denaturation at 94°C for 4min, one cycle; Denaturing at 94°C for 45s, annealing at 60°C
for 45s, and extending at 72°C for 45s, for 32 cycles.12min at 72°C, 1 cycle; Store at 4°C
for later use.
Furthermore, in step (4), 1 L of PCR amplification product was added with 8.5tL of
deionized formamide and 0.5 L of Liz-500 molecular weight internal standard, and then
detected by capillary four-color fluorescence electrophoresis on DNA analyzer.
Further, the conditions of capillary four-color fluorescence electrophoresis detection
in step (4) are as follows: pre-electrophoresis 15kV, 3 min; Sampling 2kV for 2s;
Electrophoresis 15kV, 20min.
Compared with the prior art, the invention has the beneficial effects that:
According to the invention, based on the basic principle of multiplex fluorescent PCR
combination, 52 pairs of core primers distributed on 26 chromosomes of the whole genome
of cotton subgroup a or subgroup d are combined, three 10-fold and two 11-fold PCR
combinations are realized, a multiplex SSR-PCR amplification system and a reaction
program suitable for a capillary four-color fluorescent electrophoresis detection system are
established, and capillary four-color fluorescent electrophoresis detection is carried out on
an ABI3730xl DNA analyzer. According to the method, only five times of pSSR-PCR amplification reaction are needed, so that the detection flux is greatly improved, and the detection efficiency is further greatly improved, and the method is particularly suitable for detecting and analyzing a large number of materials; In addition, during the pSSR-PCR amplification reaction, the Taq HS polymerase has an optimum amplification temperature of 72°C, and after 32 cycles, it extends at 72°C for 12min, so that the amplified products are fully tailed, which ensures the high consistency of the ends of the amplified products and avoids insufficient tailing due to the difference between the core primers and the samples. In addition, Taq HS polymerase is a hot starter enzyme, which reduces the nonspecific amplification in the system, so some miscellaneous peaks will not appear during capillary four-color fluorescence electrophoresis detection. Moreover, when the amplified products were detected by capillary four-color fluorescence electrophoresis, the injection time was reduced from 10s to 2s, and the electrophoresis time was reduced from min to 20min, which shortened the time for each detection and improved the detection efficiency. The simple sequence repeat in the amplification product of the core primer of the invention is greater than or equal to 3bp, so that the detection result is simple and easy to interpret. According to the invention, the construction of multiple SSR-PCR amplification systems and capillary four-color fluorescence electrophoresis detection improve the detection efficiency by more than 100 times, and the fragment size of amplification products is obtained through automatic comparison of molecular weight internal standards, so that the accuracy and reliability of data results are guaranteed to the greatest extent while the error of manual reading of plates is reduced; and the method is especially suitable for the construction of DNA fingerprint databases of large quantities of cotton varieties.
Fig. 1 is a capillary four-color fluorescence electrophoresis detection chart of cotton
variety Xin 547 in multiplex fluorescence PCR combination 1;
Fig. 2 is a capillary four-color fluorescence electrophoresis detection chart of cotton
variety Xin 547 in multiplex fluorescence PCR combination 2;
Fig. 3 is a capillary four-color fluorescence electrophoresis detection chart of cotton
variety Xin 547 in multiplex fluorescence PCR combination 3;
Fig. 4 is a capillary four-color fluorescence electrophoresis detection chart of cotton
variety Xin 547 in multiplex fluorescence PCR combination 4;
Fig. 5 is a capillary four-color fluorescence electrophoresis detection chart of cotton
variety Xin 547 in multiplex fluorescence PCR combination 5.
The following examples further illustrate the contents of the present invention, but
should not be construed as limiting the present invention. Without departing from the
spirit and essence of the invention, modifications or substitutions made to the methods,
steps or conditions of the invention belong to the scope of the invention.
The DNA fingerprint database of cotton variety Xin 547 was constructed, which
originated from Cotton Research Institute of Chinese Academy of Agricultural Sciences.
1. DNA extraction
(1) Shelling a single cotton seed, fully crushing, and transferring into a 2mL centrifuge
tube;
(2) Adding 800pL 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% - mercaptoethanol), swirl until fully
mixed, and then water bath at 65°C for 30min with gently shaken once every 10min.
(3) After the water bath is finished, add an equal volume of 800pL mixed solution of
phenol, chloroform and isoamyl alcohol (the volume ratio is 25: 24: 1 in turn), mix it upside
down until it is not layered, and centrifuge it for10min at 10000rpm;
(4) Transfer the supernatant to another 2mL centrifuge tube, add l pL RNase enzyme
(1Omg/mL), mix well, and then water bath at 37°C for 30min.
(5) Add equal volume of 800pL phenol: chloroform: isoamyl alcohol (the volume
ratio is 25: 24: 1 in turn), mix it upside down until it does not delaminate, and centrifuge it
for 10min at 10000rpm.
(6) Transfer the supernatant to another 2mL centrifuge tube, add 0.7 times of isopropyl
alcohol and shake it slowly for several times, and let it stand for 30min, and flocculent
DNA will precipitate out.
(7) Absorb DNA with a snip gun, transfer it to a centrifuge tube containing 70%
ethanol, and soak it twice, the first time for about 2 hours, and the second time for
overnight.
(8) Pour out the ethanol, dry the DNA by natural ventilation, add 200pL TE(pH 8.0)
or ddH20, and fully dissolve it for later use.
And (9) detecting the concentration of DNA by ultraviolet rays, diluting the DNA
stock solution to the use concentration of 60ng/pL with ddH20, and storing at 4 °C for later
use to obtain a sample to be detected.
2. pSSR-PCR amplification
Five combinations obtained by combining 52 pairs of core primers (see Table 1 for
nucleotide sequences of core primers) according to multiplex fluorescence PCR were used
to perform pSSR-PCR amplification reaction on the sample to be tested to obtain PCR
amplification products. The multiplex fluorescence PCR combination of 52 pairs of core
primers is shown in Table 2, and the 20pL reaction solutions of 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 below, respectively:
Table 3
Reagent Concentration Volume
BD7-7 (upstream/downstream) 40tmol/L 0.02pL
BD6-11 (upstream/downstream) 40ptmol/L 0.3pL
A1-2 (upstream/downstream) 40ptmol/L 0.04pL
BD5-8 (upstream/downstream) 40ptmol/L 0.025piL
D12-2 (upstream/downstream) 40ptmol/L 0.3tL A2-3 (upstream/downstream) 40pmol/L 0.04pL
BD1-7 (upstream/downstream) 40pmol/L 0.35pL
BD9-3 (upstream/downstream) 40pmol/L 0.3pL
BA3-4 (upstream/downstream) 40pmol/L 0.25pL
BA9-11 (upstream/downstream) 40pmol/L 0.15pL
DA4-16 (upstream/downstream) 40pmol/L 0.35pL
PCR Buffer lox 2pL
dNTPs 10mmol/L 0.4pL
DNA 60ng/pL 2pL
Taq HS polymerase 5U/pL 0.2pL ddH20 11.15pL
Total volume 20pL
Table 4
Reagent Concentration Volume
BA13-4 (upstream/downstream) 40pmol/L 0.025pL
Dl-10 (upstream/downstream) 40pmol/L 0.1pL
BAl-4 (upstream/downstream) 40pmol/L 0.07pL
BD13-3 (upstream/downstream) 40pmol/L 0.08pL
BA5-4 (upstream/downstream) 40pmol/L 0.2pL
BD7-17 (upstream/downstream) 40pmol/L 0.2pL
BA7-5 (upstream/downstream) 40pmol/L 0.05pL
BD6-3 (upstream/downstream) 40pmol/L 0.35pL
BA12 13 (upstream/downstream) 40pmol/L 0.03pL
A6-4 (upstream/downstream) 40pmol/L 0.3pL
WD12-1 WD12-140pmol/L 0. 15pgL (upstream/downstream)
PCR Buffer lox 2pL
dNTPs l0mmol/L 0.4pL
DNA 60ng/pL 2pL
Taq HS polymerase 5U/pL 0.2pL
ddH20 12.29pL
Total volume 20pL
Table 5
Reagent Concentration Volume
BD10-1 (upstream/downstream) 40pmol/L 0.04pL
BD13-21 (upstream/downstream) 40pmol/L 0.08pL
BA13-1 (upstream/downstream) 40pmol/L 0.03pL
BD8-13 (upstream/downstream) 40pmol/L 0.03pL
BA5-10 (upstream/downstream) 40pmol/L 0.2pL
BD9-2 (upstream/downstream) 40pmol/L 0.05pL
BA10-6 (upstream/downstream) 40pmol/L 0.1pL
BD2-11 (upstream/downstream) 40pmol/L 0.35pL
BA12 15 (upstream/downstream) 40pmol/L 0.35pL
BA10-9 (upstream/downstream) 40pmol/L 0.35pL
PCR Buffer lox 2pL
dNTPs l0mmol/L 0.4pL
DNA 60ng/pL 2pL
Taq HS polymerase 5U/pL 0.2pL
ddH20 12.24pL
Total volume 20pL
Table 6
Reagent Concentration Volume
BD5-10 (upstream/downstream) 40pmol/L 0.025pL
BD2-2 (upstream/downstream) 40pmol/L 0.2pL
BA9-3 (upstream/downstream) 40pmol/L 0.05pL
D3-1 (upstream/downstream) 40pmol/L 0.05pL
BA8-12 (upstream/downstream) 40pmol/L 0.1pL
BD11-5 (upstream/downstream) 40pmol/L 0.06pL
D10-10 (upstream/downstream) 40pmol/L 0.1pL
BA3-8 (upstream/downstream) 40pmol/L 0.3pL
BD4-3 (upstream/downstream) 40pmol/L 0.2pL
BA2-12 (upstream/downstream) 40pmol/L 0.3pL
PCR Buffer lox 2pL
dNTPs l0mmol/L 0.4pL
DNA 60ng/pL 2pL
Taq HS polymerase 5U/pL 0.2pL
ddH20 12.63pL
Total volume 20pL
Table 7
Reagent Concentration Volume
BAl1-13 (upstream/downstream) 40pmol/L 0.04pL
BA8-5 (upstream/downstream) 40pmol/L 0.2pL
BD3-6 (upstream/downstream) 40pmol/L 0.2pL
BD8-12 (upstream/downstream) 40pmol/L 0.3pL
BA7-1 (upstream/downstream) 40pmol/L 0.1pL
BD4-8 (upstream/downstream) 40pmol/L 0.1pL
BA6-2 (upstream/downstream) 40pmol/L 0.lpL
BD11-3 (upstream/downstream) 40pmol/L 0.3pL
DA4-12 (upstream/downstream) 40pmol/L 0.3pL
BA11-8 (upstream/downstream) 40pmol/L 0.3pL
PCR Buffer lox 2pL
dNTPs l0mmol/L 0.4pL
DNA 60ng/pL 2pL
Taq HS polymerase 5U/pL 0.2pL
ddH20 11.52pL
Total volume 20pL
The procedure of pSSR-PCR amplification reaction is: pre-denaturation at 94°C for
4min, 1 cycle; Denaturing at 94°C for 45s, annealing at 60°C for 45s, and extending at
72°C for 45s, for 32 cycles.12min at 72°C, 1 cycle; Store at 4°C for later use. The
amplification reaction system was extended at 72°C for 12min after 32 cycles, so that the
amplification products were fully tailed, ensuring the high consistency of the ends of the amplification products and avoiding insufficient tailing due to the difference between primers and samples.
3. Capillary four-color fluorescence detection
1 L PCR amplification product was added with 8.5 L deionized formamide and
0.5tL Liz-500 molecular weight internal standard, and was detected by capillary four
color fluorescence electrophoresis on ABI3730xl DNA analyzer. The conditions of
capillary four-color fluorescence electrophoresis detection were as follows: pre
electrophoresis 15kV, 3 min;2 kV injection for 2 s; electrophoresis at 15 kV for 20 min,
and then the detection results were collected and analyzed by GeneMapper software.
Sample injection time is reduced from 10s to 2s, and electrophoresis time is reduced from
min to 20min, thus shortening the time for each detection and improving the detection
efficiency.
The fingerprint information of cotton variety Xin 547 was obtained by the above
method, namely, the fingerprint database of cotton variety Xin 547 was constructed. The
fingerprint information of cotton variety Xin 547 was shown in Table 8 below. The
capillary four color fluorescence electrophoresis detection maps of cotton variety Xin 547
in combination 1, combination 2, combination 3, combination 4 and combination 5 were
shown in Fig. 1, Fig. 2, Fig. 3, Fig. 4and Fig. 5, respectively in which the abscissa is the
fragment size (BP) of the amplified product, and the ordinate is the peak height
(representing the fluorescence signal intensity).
Table 8
Combination Core primer Fluorescent Amplified Combination Core primer Fluorescent Amplified
name labeling fragment size name labeling fragment
size
BD7-7 ROX 180 BA12-13 FAM 180
BD6-11 ROX 217 BA13-4 FAM 192
Al-2 FAM 220 WD12-1 HEX 217
BD5-8 ROX 254 D1-10 ROX 220
D12-2 ROX 280 BA1-4 FAM 223
Combination A2-3 FAM 290 Combination2 BD13-3 ROX 232 1 BD1-7 ROX 323 BA5-4 FAM 266
BD9-3 ROX 374 BD7-17 ROX 255
BA3-4 FAM 335 BA7-5 FAM 295
BA9-11 FAM 367 BD6-3 ROX 306
DA4-16 HEX 372 A6-4 FAM 393
BD10-1 ROX 174 BD5-10 ROX 172
BD13-21 ROX 225 BD2-2 ROX 213
BA13-1 FAM 231 BA9-3 FAM 226
BD8-13 ROX 236 D3-1 ROX 237
Combination BA5-10 FAM 268 Combination4 BA8-12 FAM 255 3 BD9-2 ROX 300 BD11-5 ROX 255
BA10-6 FAM 301 D10-10 ROX 305
BD2-11 ROX 332 BA3-8 FAM 306
BA12-15 FAM 342 BD4-3 ROX 353
BA10-9 FAM 392 BA2-12 FAM 351
BA11-13 FAM 166/172 BD4-8 ROX 310
BA8-5 FAM 216 BA6-2 FAM 339
Combination BD3-6 ROX 232 Combination6 BD11-3 ROX 340
BD8-12 ROX 265 DA4-12 HEX 342
BA7-1 FAM 305 BA11-8 FAM 392
Note: The amplified fragment size in the table is the fragment size of each pair of core
primer PCR amplification products.
Claims (10)
1. A construction method of high-throughput cotton variety DNA fingerprint database
based on capillary four-color fluorescence electrophoresis detection system and multiplex
fluorescence PCR amplification, which is characterized by comprising the following steps:
(1) Carrying out multiplex fluorescent PCR combination on 52 pairs of core primers
distributed on 26 chromosomes of the whole genome of cotton subgroup a or subgroup d,
wherein each pair of core primers is marked with fluorescent dye,
The nucleotide sequence of the core primer is as follows:
Core primer Upstream sequence (5'-3') Downstream sequence (5'-3')
name
BD7-7 CTTCCCCACGCCACTACTATCG CTCAGCTTTCCTCCTCATTGGC
BD6-11 TGATGTTGGCGGGACTATGTAG CGACTTCACCCTCGTGGTAATC
A1-2 CCGGTTCAAGCCGACTATTCG ACTCGTAACACCGTGCTGATTG
BD5-8 ATTCATGGTCAAGTCGGGTCAC GCTTGCTTTGGGTGGAGTAGAC
D12-2 CGGGATGGGGTGTGCAGATC GAGAAAGCAACAAGGCGGGATG
A2-3 AGTCCTCCCACTATTCGAAGCT ATGCCTCGTACCCTGTTCCG
BD1-7 CCACATGCCACGCCGTATTATG ATGATGGGGTGGGCTGTAAAGG
BD9-3 TTGACGTGACTTGCAGCAGATC ATCGCCAGAATGCTGAAAGGTG
BA3-4 TGGGTGAGTGTGAGGACTGAAG TGGGTGTTGCACAAAGTTTCTG
BA9-11 GCCGGTAAGTCCAACATAAGGG CGGGGTAGTCCACCTCCTATTG
DA4-16 AGGCTCGCATTGTTGACACTAGG CGAGCTTGAACGAACGAACCTCT
BD10-1 TTCCATGAGGCTATCCACAAGC AATGCACCGCACCCCATCAC
BD13-21 GCCGAGGCCCCTATAACCC CTCATATCACGCACCACACCAC
BA13-1 AACTCAATGGGTGTCGGTTACG GGAGGTGAGCTATCTTCGCAAC
BD8-13 ACCAGCATCCTTTGTGTTAGGC GGATCGATTTTGGAACCGTGTG
BA5-10 CGTCTCGTCCCACCTGTAATGC GGACTTCGGCAAGGCGGTTC
BD9-2 GTCGCCGATGTCGCCAATG ATGGGTGACAACGGTTACAACC
BA1O-6 TGTGGCTCCATGGCACAATATG AGGCTCTGTTGCACCAATTCAC
BD2-11 AGTTCGGTGGACATCAATAGGC TCCCCAGGGCTTTGAGAATACC
BA12-15 AGACGTGAGTTCGAGGACTTTG GCCTACCCTCTCAACAGTTTGG
BA1O-9 ACTACGCAACTGAAGGGTTTCG GCTGCAAGGTTCGATGGAAGTG
BA13-4 AGCACGGAAGAACATGATGAGG CGTCTTCGGCTCAAATGTGTGC
D1-10 TGCCCAACCTACATGTGACACA TCAAATTTGGTTGTCACACCCCA
BA1-4 CTGAGGAGAAAGACAGGACGAC TGGCGGGGTAAATGTGAATGC
BD13-3 GGAAATGGCCCATCTGAGAGTC GCCAGGAGATCGGAGCGTTTG
BA5-4 TCGATTCACCGATCTGACAAGC TGACCCAGACCGACCGTTG
BD7-17 ACCATCCCACTGTTTTCCTCTC GGAAGAACGACCACAGGAGTG
BA7-5 AGGGACAAGAATGGACCGACAG TTAACCGTCGCAGCCTCCTAAC
BD6-3 CAGACTTGGGCCTAGAAAGCG CGAGTCGAGACCTAGAAAACGG
BA12-13 TGTTGAAGCTGGAAGCCTGATG TGGCAAGTCTTCACCCAATGTC
A6-4 AGCAAAGCCAACAGAGGTCAAC GCCAACCATGATTTCGATGCAC
WD12-1 TTGAAAGTCCATCTTAACTTGGTGTGA TCCAGGACAGCAAACTTACGCC
BD5-10 GTTGCTGTGGAGTGGAGTGGAG TTCGAGGGAGGTTGGTATTGGC
BD2-2 TTGGGCCGAAAAGGGTTGAAAC GGTCGGATTCTGGGCACTTTTC
BA9-3 TTACTCTGGGCGTGTGGCATAG ATGGAAGGAACAGCAGCAAACG
D3-1 CCAGCCACTCGGAGATCCTTG GCGTGGAGAAACCAGAGGGTAG
BA8-12 TGCCCTTCTTGCCCCTGTG GCTTGCCTAATTTGGTGGGAAG
BD11-5 ACAAGCGCGTCTGCCATATCC CGTGGTGGGTAGTCACAGTCAG
D1O-10 GTTTCCACCGTCGAACCACTG GGCAGGATTAGGAGATCGAAGC
BA3-8 GTGGGCAGCGATGAATATGATG ATGAGGGTCATTGCTTGGGTTG
BD4-3 GCCCACCTACCCACCTGTTTC CCCCTTTTGCTCCCCGATGTAC
BA2-12 CGCACTATTTCTAGCAGCTTGC GCGTGTCGTCCGATCCTAAC
BAI1-13 GTTGGAGGCTGCTTTTGATGGG TGCCATTGCCATGTTGGTCAAG
BA8-5 TTGGGCGGTTTGGGTCAAGG GGGATTCGGTCGGACACTCAAG
BD3-6 GCAACACCCTTTAGACGCAGTG ACGGAATGCAGCCCATTGACC
BD8-12 GACTCATGGCGACAGCGATTAG TGATCACTCAAACGGTGTCACG
BA7-1 TTCCTGCAAAATTGCCTTCACC TGCTTTGATATCCCCGTGATGG
BD4-8 ACCACTTTGTCCACCTGTCCAC ATCATCGCCATCTGCCTGGAAG
BA6-2 ACCAGGTCGGTAACGATAGGC GCCCAAAGTTGAAGCGGAAAAC
BD13-3 GGAAATGGCCCATCTGAGAGTC GCCAGGAGATCGGAGCGTTTG
DA4-12 GCCGTTTCTGCCAACCCCTT CGGGATTCCACGTGCCCAAA
BA11-8 AACTGCGACATCACCTGTAACG CCGCCACCGCTGAGTCTC
The multiplex fluorescence PCR combination mode of the 52 pairs of core primers
is as follows:
Fluorescent Core primer Fluorescent labeling Combination labeling Combination Core primer name name
BD7-7 ROX BA13-4 FAM
BD6-11 ROX D1-10 ROX
Al-2 FAM BA1-4 FAM
BD5-8 ROX BD13-3 ROX
Combination D12-2 ROX Combination2 BA5-4 FAM
A2-3 FAM BD7-17 ROX
BD1-7 ROX BA7-5 FAM
BD9-3 ROX BD6-3 ROX
BA3-4 FAM BA12-13 FAM
BA9-11 FAM A6-4 FAM
DA4-16 HEX WD12-1 HEX
BD10-1 ROX BD5-10 ROX
BD13-21 ROX BD2-2 ROX
BA13-1 FAM BA9-3 FAM
BD8-13 ROX D3-1 ROX
BA5-10 FAM BA8-12 FAM Combination Combination4 BD9-2 ROX BD11-5 ROX
BA10-6 FAM D10-10 ROX
BD2-11 ROX BA3-8 FAM
BA12-15 FAM BD4-3 ROX
BA10-9 FAM BA2-12 FAM
BA11-13 FAM BD4-8 ROX
BA8-5 FAM BA6-2 FAM
Combination5 BD3-6 ROX Combination6 BD11-3 ROX
BD8-12 ROX DA4-12 HEX
BA7-1 FAM BA11-8 FAM
(2) Extracting cotton DNA as a sample to be tested;
(3) Carrying out pSSR-PCR amplification reaction on the sample to be detected by
using five combinations obtained by combining 52 pairs of core primers in the step (1)
according to a multiplex fluorescent PCR combination mode to obtain PCR amplification
products;
And (4) performing capillary four-color fluorescence electrophoresis detection and
data analysis on the PCR amplification product in the step (3).
2. The construction method of high-throughput cotton variety DNA fingerprint
database based on capillary four-color fluorescence electrophoresis detection system and
multiplex fluorescence PCR amplification according to claim 1, characterized in that the
simple sequence repeat in the amplification product of the core primer is greater than or
equal to 3bp.
3. The construction method of high-throughput cotton variety DNA fingerprint
database based on capillary four-color fluorescence electrophoresis detection system and
multiplex fluorescence PCR amplification according to claim 1, characterized in that the
52 pairs of core primers are respectively taken from cotton subgroup A and cotton subgroup
D with 26 pairs each.
4. The construction method of high-throughput cotton variety DNA fingerprint
database based on capillary four-color fluorescence electrophoresis detection system and
multiplex fluorescence PCR amplification according to claim 1, characterized in that the
extraction of cotton DNA in step (2) comprises the following steps: crushing the dehulled
cotton seeds, adding SDS extract, taking a water bath at 65°C after vortex is sufficient;
Adding a mixed solution of phenol, chloroform and isoamyl alcohol with a volume ratio
of 25: 24: 1 in turn, mixing uniformly, and centrifuging; Collect supernatant, adding RNase
with concentration of 10mg/mL , water bath; After extraction, centrifuge to take
supernatant, add isopropanol, wash DNA precipitate with ethanol after DNA clumps and
precipitate, and add TE or ddH20 to fully dissolve DNA for later use.
5. The construction method of high-throughput cotton variety DNA fingerprint
database based on capillary four-color fluorescence electrophoresis detection system and multiplex fluorescence PCR amplification according to claim 4, characterized in that the extraction of cotton DNA in step (2) comprises the following steps: after the hulled cotton seeds are fully crushed, 800L SDS extract is added, and after the vortex is sufficient, water bath at 65°C is carried out for 30min, and shaking once every 10min;Adding an equal volume of 800pL mixed solution of phenol, chloroform and isoamyl alcohol with a volume ratio of 25: 24: 1 in turn, mixing evenly until no stratification, and centrifuging at10000rpm for 10 minutes;1 L RNase with a concentration of1Omg/mL was added to the supernatant, and the supernatant was bathed in water at 37°C for 30min; After repeated extraction, centrifuge to take supernatant, add 0.7 times of isopropanol, wash DNA precipitate twice with 70% ethanol after DNA clumping and precipitation, and add 200 L TE or ddH20 to fully dissolve DNA for later use.
6. The construction method of high-throughput cotton variety DNA fingerprint
database based on capillary four-color fluorescence electrophoresis detection system and
multiplex fluorescence PCR amplification according to claim 1, characterized in that the
reaction solution of pSSR-PCR amplification reaction corresponding to each combination
in step (3) comprises all primers, PCR Buffer, dNTPs, DNA template, Taq HS polymerase
and ddH20.
7. The construction method of high-throughput cotton variety DNA fingerprint
database based on capillary four-color fluorescence electrophoresis detection system and
multiplex fluorescence PCR amplification according to claim 6, characterized in that the
reaction solution of pSSR-PCR amplification reaction corresponding to each combination
in step (3) is 20[L,
The 20tL reaction solution in combination 1 includes 0.024L upstream and
downstream of core primer BD7-7, 0.3tL upstream and downstream of core primer BD6
11, 0.04 L upstream and downstream of core primer Al-2, 0.025tL upstream and
downstream of core primer BD5-8 and 0.3tL upstream and downstream of core primer
D12-20.35tL upstream and downstream of core primer BD1-7, 0.3tL upstream and
downstream of core primer BD9-3, 0.25 L upstream and downstream of core primer BA3
4, 0.15 L upstream and downstream of core primer BA9-11, 0.35tL upstream and
downstream of core primer DA4-16, and the concentration of each core primer is
jmol/L, 24L lOx PCR Buffer, 0.4tL dNTPs with a concentration of 10mmol/L, 2L
DNA template with a concentration of 60ng/L, 0.24L Taq HS polymerase with a
concentration of 5U/4L and 11.15 L ddH20;
The 20tL reaction solution in combination 2 includes 0.025tL upstream and
downstream of core primer BA13-4, 0.1 L upstream and downstream of core primer D1
, 0.074L upstream and downstream of core primer BAl-4, 0.08 L upstream and
downstream of core primer BD13-3, and 0.2L upstream and downstream of core primer
BA5-40.05 L upstream and downstream of core primer BA7-5, 0.35tL upstream and
downstream of core primer BD6-3, 0.03 L upstream and downstream of core primer
BA12-13, 0.3tL upstream and downstream of core primer A6-4, 0.15 L upstream and
downstream of core primer WD12-1, and the concentration of each core primer is
jmol/L, 24L lOx PCR Buffer,0.4 L dNTPs with a concentration of 10mmol/L, 2L
DNA template with a concentration of 60ng/L, 0.24L Taq HS polymerase with a
concentration of 5U/L and 12.29tL ddH20;
The 20tL reaction solution in combination 3 includes 0.04pL upstream and
downstream of core primer BD1O-1, 0.08 L upstream and downstream of core primer
BD13-21, 0.03 L upstream and downstream of core primer BA13-1, 0.03 L upstream and
downstream of core primer BD8-13 and 0 upstream and downstream of core primer BA5
100.1 L upstream and downstream of core primer BA1O-6, 0.35tL upstream and
downstream of core primer BD2-11, 0.35tL upstream and downstream of core primer
BA12-15, 0.35tL upstream and downstream of core primer BA1O-9, and the concentration
of each core primer is 40jmol/L, 2L 1Ox PCR Buffer, 0.4tL dNTPs with a concentration
of 10mmol/L, 2L DNA template with a concentration of 60ng/4L, 0.24L Taq HS
polymerase with a concentration of 5U/L and 12.24tL ddH20;
The 20tL reaction solution in combination 4 includes 0.025tL upstream and
downstream of core primer BD5-10, 0.24L upstream and downstream of core primer BD2
2, 0.05 L upstream and downstream of core primer BA9-3, 0.05 L upstream and
downstream of core primer D3-1 and 0.1 L upstream and downstream of core primer BA8
120.1jL upstream and downstream of core primer D1O-10, 0.3tL upstream and
downstream of core primer BA3-8, 0.24L upstream and downstream of core primer BD4
3, and 0.3tL upstream and downstream of core primer BA2-12, and the concentration of
each core primer is 40jmol/L, 24L 1Ox PCR Buffer, 0.4tL dNTPs with a concentration of
1Ommol/L, 24L DNA template with a concentration of60ng/4L, 0.2L Taq HS polymerase
with a concentration of 5U/L and 12.63tL ddH20;
The 20tL reaction solution in combination 5 includes 0.04pL upstream and
downstream of core primer BAl1-13, 0.24L upstream and downstream of core primer
BA8-5, 0.24L upstream and downstream of core primer BD3-6, 0.3tL upstream and
downstream of core primer BD4-8-12 and 0.1 L upstream and downstream of core primer
BA7-1.0.1 L upstream and downstream of core primer BA6-2, 0.3tL upstream and
downstream of core primer BD11-3, 0.3tL upstream and downstream of core primer DA4
12, 0.3tL upstream and downstream of core primer BA11-8, and the concentration of each
core primer is 40jmol/L, 24L lOx PCR Buffer, 0.4tL dNTPs with a concentration of
mmol/L, 24L DNA template with the concentration of 60ng/L, 0.24L Taq HS
polymerase with the concentration of 5U/4L and 11.52L ddH20.
8. The construction method of high-throughput cotton variety DNA fingerprint
database based on capillary four-color fluorescence electrophoresis detection system and
multiplex fluorescence PCR amplification according to claim 1, characterized in that the
procedure of pSSR-PCR amplification reaction in step (3) is: pre-denaturation at 94°C for
4min, one cycle; Denaturing at 94°C for 45s, annealing at 60°C for 45s, and extending at
72°C for 45s, for 32 cycles.12min at 72°C, 1 cycle; Store at 4°C for later use.
9. The construction method of high-throughput cotton variety DNA fingerprint
database based on capillary four-color fluorescence electrophoresis detection system and
multiplex fluorescence PCR amplification according to claim 1, characterized in that, in
step (4), 1 L of PCR amplification product is added with 8.5tL of deionized formamide
and 0.5 L of Liz-500 molecular weight internal standard, and capillary four-color
fluorescence electrophoresis detection is performed on a DNA analyzer.
10. The construction method of high-throughput cotton variety DNA fingerprint
database based on capillary four-color fluorescence electrophoresis detection system and multiplex fluorescence PCR amplification according to claim 1, which is characterized in that the conditions of capillary four-color fluorescence electrophoresis detection in step
(4) are: pre-electrophoresis 15kV, 3min;2 kV injection for 2 s; electrophoresis at 15 kV
for 20 min.
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| CN116200521A (en) * | 2022-12-05 | 2023-06-02 | 东北林业大学 | SSR (simple sequence repeat) marker primer group for identifying Korean pine clone and construction method and application of SSR marker primer group and fingerprint |
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| CN116200521A (en) * | 2022-12-05 | 2023-06-02 | 东北林业大学 | SSR (simple sequence repeat) marker primer group for identifying Korean pine clone and construction method and application of SSR marker primer group and fingerprint |
| CN116200521B (en) * | 2022-12-05 | 2023-08-18 | 东北林业大学 | SSR (simple sequence repeat) marker primer group for identifying Korean pine clone and construction method and application of SSR marker primer group and fingerprint |
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