AU2020103387A4 - Method for Constructing DNA Fingerprint Map of Cotton Variety "Zhongmiansuo 96A" and the Application in Variety Identification - Google Patents

Method for Constructing DNA Fingerprint Map of Cotton Variety "Zhongmiansuo 96A" and the Application in Variety Identification Download PDF

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AU2020103387A4
AU2020103387A4 AU2020103387A AU2020103387A AU2020103387A4 AU 2020103387 A4 AU2020103387 A4 AU 2020103387A4 AU 2020103387 A AU2020103387 A AU 2020103387A AU 2020103387 A AU2020103387 A AU 2020103387A AU 2020103387 A4 AU2020103387 A4 AU 2020103387A4
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zhongmiansuo
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Diancheng Huang
Longyu Huang
Meng KUANG
Jun Peng
Kai PENG
Junling SUN
Yanqin Wang
Yuzhen WU
Pengfei Zhang
Xiling Zhang
Dayun ZHOU
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Institute of Cotton Research of Chinese Academy of Agricultural Sciences
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Abstract

The invention provides a method for constructing DNA fingerprint map of cotton variety "Zhongmiansuo 96A" and the application in variety identification, and it belongs to the field of molecular biology. According to the invention, the extracted genomic DNA of cotton variety "Zhongmiansuo 96A"is used as a template, and 60 pairs of excellent SSR marker combination developed independently are used for PCR detection and construction of DNA fingerprint map. The method of the invention could be applied in "Zhongmiansuo 96A" variety genuineness testing with advantages such as simple, rapid, accurate and stable, and the testing procedure of one sample can be completed in just one day.

Description

Method for Constructing DNA Fingerprint Map of Cotton Variety "Zhongmiansuo 96A"
and the Application in Variety Identification
TECHNICAL FIELD
The invention belongs to the field of molecular biology, and particularly relates to the method for constructing DNA fingerprint map of cotton variety "Zhongmiansuo 96A" and the application in variety identification.
BACKGROUND
In recent years, with the acceleration of the cultivation and verification of new cotton varieties in China, the speed of pushing to production is also accelerating. However, due to the concentrated use of the backbone parents and the large-scale application of the transgenic technology in cotton breeding, the genetic difference among cotton varieties is smaller and smaller, so that the variety identification completely dependent on the traditional morphological characters is more and more difficult. At the same time, because of the bad timeliness of morphological identification, it is easy to be influenced by environment and subjective factors, the phenomena of variety, disorder and heterogeneity are difficult to be effectively controlled, and the infringement acts occur frequently, which seriously damage the rights and interests of breeders and farmers. The DNA fingerprint identification technology based on molecular markers has the advantages of accuracy, reliability, simplicity, rapidness and easiness in automation. The rapid identification of varieties by constructing DNA fingerprint map is the premise of realizing industrialization of the variety identification technology, and is also the development trend of the variety identification technology. It is of great significance to protect the intellectual property rights of cotton breeds and improve the seed quality of the seed market.
The "Zhongmiansuo 96A" is a new variety of early and middle maturing conventional cotton, which is obtained through directional selection, resistance screening and ecological selection after cross-breeding by using "Zhongmiansuo 49" as female parent and using self-bred line 221 as male parent, which is the first in comprehensive performance in Xinjiang regional experiment from 2016 to 2017, and is approved by Xinjiang Crop Variety Certification Committee in 2019. The "Zhongmiansuo 96A" is a high-yield new variety, which has the advantages of big boll, high ginning outturn, good disease resistance, quick boll feeding, centralized boll spitting, early ripening, disease resistance, high quality and suitable mechanical harvesting, etc. The product is expected to become the leading variety in Xinjiang cotton market.
However, at present, the market lacks a technology capable of quickly identifying the genuineness of the "Zhongmiansuo 96A" variety, so that the genuineness identification of the "Zhongmiansuo 96A" is difficult, and the large-scale popularization and application of the "Zhongmiansuo 96A" variety are influenced.
SUMMARY
The invention aims to provide a primer combination for identifying cotton variety "Zhongmiansuo 96A" and method for constructing DNA fingerprint map of cotton variety "Zhongmiansuo 96A" and its application in identifying the cotton variety "Zhongmiansuo 96A", so as to solve the problems existing in the prior art and enable the genuineness judgment of the "Zhongmiansuo 96A" to be simply, rapidly, accurately and stably finished.
In order to achieve the above purpose, the invention provides a primer combination, which comprises 60 pairs of primers listed in the following table, for detecting cotton variety "Zhongmiansuo 96A":
Name of Chromosome Annealing temperatu Primer sequence (5'-3') primer (Position) re(°C)
Forward:ccaccagccttaccttatacgc CCRI001 1A 60 Reverse:gtgcttggcctcgctaagtg
Forward: agtcctcccactattcgaagct CCRIO02 2A 60 Reverse: atgcctcgtaccctgttccg
Forward: cacgacgactcttcctcatcac CCRI003 3A 60 Reverse: attgcagccacgaaattgtcac
Forward: aggctcgcattgttgacactagg CCRIO04 4A 60 Reverse: cgagcttgaacgaacgaacctct
Forward: tcgattcaccgatctgacaagc CCRI005 5A 60 Reverse: tgacccagaccgaccgttg
Forward: accaggtcggtaacgataggc CCRI006 6A 60 Reverse: gcccaaagttgaagcggaaaac
Forward: gtatgtccaatcgccaccctag CCRI007 6A 60 Reverse: gatgacggtgttaggcggttc
Forward: ggcctcccatctttatccaatgt CCRIO08 7A 60 Reverse: agcaaagcaaactcgacaatgct
Forward:ttgggcggtttgggtcaagg CCRI009 8A 60 Reverse:gggattcggtcggacactcaag
Forward:gccggtaagtccaacataaggg CCRIO10 9A 60 Reverse:cggggtagtccacctcctattg
Forward:actacgcaactgaagggtttcg CCRI011 10A 60 Reverse:gctgcaaggttcgatggaagtg
Forward:ctgcctcgacctacagtttgac CCRIO12 11A 60 Reverse:agttttaggcggtttgggtttg
Forward:accatccttgccgagtaacctg CCRIO13 12A 60 Reverse:gttgatgcctgggtctcaatgc
Forward:agacgtgagttcgaggactttg CCRIO14 12A 60 Reverse:gcctaccctctcaacagtttgg
Forward:tttcacttgtccgaccttaccc CCRIO15 13A 60 Reverse:ggatatgtgtttgagcgtgctg
Forward:cgaaagagtgtcgagcaatccg CCRIO16 1D 60 Reverse:tcctgtcaacttggaggctgag
Forward:cgccacatgaccccaccatc CCRIO17 2D 60 Reverse:cccaccgagtatacaggacacg
Forward:ccgctcggttcaacaggttt CCRIO18 3D 60 Reverse:agagtttgcggttttggtgcc
Forward:ccagccactcggagatccttg CCRIO19 3D 60 Reverse:gcgtggagaaaccagagggtag
Forward:accactttgtccacctgtccac CCR1020 4D 60 Reverse: atcatcgccatctgcctggaag
Forward: aattgaaccaagggcatcatgc CCRI021 5D 60 Reverse: gaacctgattgacgggtagcac
Forward: tgatgttggcgggactatgtag CCR1022 6D 60 Reverse: cgacttcaccctcgtggtaatc
Forward: cttccccacgccactactatcg CCR1023 7D 60 Reverse: ctcagctttcctcctcattggc
Forward: acgagggaacatgtgatctcct CCR1024 7D 60 Reverse: tcactccaaatcacacgtgcca
Forward: accagcatcctttgtgttaggc CCRIO25 8D 60 Reverse: ggatcgattttggaaccgtgtg
Forward: ttccatgaggctatccacaagc CCR1026 10D 60 Reverse: aatgcaccgcaccccatcac
Forward:gcgggccacctaatgatgattg CCR1027 11D 60 Reverse:ttgtttgacgagggagacgatc
Forward: acaagcgcgtctgccatatcc CCR1028 11D 60 Reverse:cgtggtgggtagtcacagtcag
Forward:acactgatgtggcatcgactag CCR1029 12D 60 Reverse:gaatctgggcaaactgttgtcc
Forward:gccgaggcccctataaccc CCR1030 13D 60 Reverse:ctcatatcacgcaccacaccac
Forward:ccggttcaagccgactattcg CCRI031 1A 60 Reverse:actcgtaacaccgtgctgattg
Forward:ctgaggagaaagacaggacgac CCR1032 1A 60 Reverse: tggcggggtaaatgtgaatgc
Forward: tgggtgagtgtgaggactgaag CCR1033 3A 60 Reverse: tgggtgttgcacaaagtttctg
Forward: gtgggcagcgatgaatatgatg CCR1034 3A 60 Reverse: atgagggtcattgcttgggttg
Forward: gccgtttctgccaacccctt CCR1035 4A 60 Reverse: cgggattccacgtgcccaaa
Forward: cgtctcgtcccacctgtaatgc CCR1036 5A 60 Reverse: ggacttcggcaaggcggttc
Forward: ttcctgcaaaattgccttcacc CCRIO37 7A 60 Reverse: tgctttgatatccccgtgatgg
Forward: agggacaagaatggaccgacag CCR1038 7A 60 Reverse: ttaaccgtcgcagcctcctaac
Forward: tgcccttcttgcccctgtg CCR1039 8A 60 Reverse: gcttgcctaatttggtgggaag
Forward: ttactctgggcgtgtggcatag CCR1040 9A 60 Reverse: atggaaggaacagcagcaaacg
Forward: tgtggctccatggcacaatatg CCRI041 10A 60 Reverse: aggctctgttgcaccaattcac
Forward:gttggaggctgcttttgatggg CCR1042 11A 60 Reverse: tgccattgccatgttggtcaag
Forward:ggacaaacatggccccaact CCR1043 11A 60 Reverse:tgtccaaactcttgccaacttgt
Forward: tcttagggcacaatgaggcaaga CCR1044 12A 60 Reverse:ctaagcagcacctcatccagaaa
Forward:aactcaatgggtgtcggttacg CCR1045 13A 60 Reverse:ggaggtgagctatcttcgcaac
Forward:agcacggaagaacatgatgagg CCR1046 13A 60 Reverse:cgtcttcggctcaaatgtgtgc
Forward:tgcccaacctacatgtgacaca CCR1047 1D 60 Reverse:tcaaatttggttgtcacacccca
Forward:ccacatgccacgccgtattatg CCR1048 1D 60 Reverse:atgatggggtgggctgtaaagg
Forward:ttgggccgaaaagggttgaaac CCRIO49 2D 60 Reverse:ggtcggattctgggcacttttc
Forward:agttcggtggacatcaataggc CCR1050 2D 60 Reverse:tccccagggctttgagaatacc
Forward:cgccacatccaagggtgaatg CCRI051 4D 60 Reverse:ggaattgcggtcccaataccac
Forward:attcatggtcaagtcgggtcac CCR1052 5D 60 Reverse:gcttgctttgggtggagtagac
Forward:gttgctgtggagtggagtggag CCR1053 5D 60 Reverse:ttcgagggaggttggtattggc
Forward:tggctttgctttgcttatggtga CCR1054 6D 60 Reverse:tgcactcaactggacacacttt
Forward:aattgtggaggggcactgtcag CCR1055 7D 60 Reverse:gtccccgccatccaagcac
Forward:gactcatggcgacagcgattag CCR1056 8D 60 Reverse:tgatcactcaaacggtgtcacg
Forward:gtttccaccgtcgaaccactg CCR1057 10D 60 Reverse:ggcaggattaggagatcgaagc
Forward:tcaggggctccgtcgttctc CCR1058 10D 60 Reverse:ctcggctctttctccggttgc
Forward:agtacccctattttcccgtgaga CCR1059 12D 60 Reverse:tgggtctcacatgtggattgttg
Forward:ggaaatggcccatctgagagtc CCRIO60 13D 60 Reverse:gccaggagatcggagcgtttg
The invention also provides a method for constructing DNA fingerprint map of cotton variety "Zhongmiansuo 96A", which specifically comprises the following steps:
1) Extracting genomic DNA of cotton variety "Zhongmiansuo 96A";
2) Taking the genomic DNA extracted in the step 1) as a template, and carrying out PCR amplification by using the PCR primer combination;
3) Detecting PCR amplification products.
Preferably, the PCR reaction system is 20 pL reaction system, wherein particularly comprises ddH2013.0pL, 10xbuffer solution 2.OpL of 25 mmol/L MgCl2, dNTPs1.6pL, Taqenzyme 0.2pL, forward primer 0.1pL, reverse primer 0.1pL, DNA3.OpL.
Preferably, the PCR amplification procedure is as follows: a) pre-denaturation: 94 °C for min; b) amplification: denaturation at 94 °C for 45 s, annealing at 60 °C for 45 s, extension at 72 °C for 45 s; the cycle should be run 32 times; c) final extension: 72 °C for 10 min, and d) preservation of the amplified product at 40 C. Preferably, the PCR product is detected by capillary electrophoresis method.
The invention also provides an application of the method for constructing DNA fingerprint map of cotton variety "Zhongmiansuo 96A" in identifying the genuineness of the cotton variety "Zhongmiansuo 96A".
The invention has the following technical effects:
According to the invention, through the development of high-quality SSR marker combination of cotton and the optimization of the molecular detection test system, a rapid, accurate, economic, simple, convenient, stable and reliable cotton DNA fingerprint detection test system is established, and a DNA detection method suitable for cotton variety "Zhongmiansuo 96A" is developed on the basis; and by adopting the method, the detection result is stable and reliable, the operation is simple and convenient, the cost is low, and the standardization of the technology is facilitated. The method is used for detecting the cotton variety "Zhongmiansuo 96A", and one single day is enough for a sample from sending sample to receiving result. The detection result is accurate, stable and reliable, and the method has great significance for guaranteeing long-term large-area popularization and application of "Zhongmiansuo 96A" in production.
DESCRIPTION OF THE INVENTION
Various exemplary embodiments of the invention will now be described in detail, which is not to be construed as limiting the invention, but rather is to be construed as a more detailed description of certain aspects, features and embodiments of the invention.
The terminology described herein should be understood for the purpose of describing particular embodiments only and is not used for limiting the invention. In addition, the numerical values in the invention are all the average values of 6 repeated samples. The numerical range in the invention should be understood that each intermediate value between the upper and lower limits of the range is disclosed. Each smaller range between intermediate values within any stated value or stated range and any other stated value or intermediate values within the stated range is also included within the invention. The upper and lower limits of these smaller ranges may be independently included or excluded in the range.
Unless otherwise indicated, all technical and scientific terminologies used herein have the same meaning as commonly understood by one of regular technicians in the art to which this invention relates. Although the invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing methods and/or materials related to said documents. The contents of this specification prevail in conflict with any incorporated document.
Various modifications and variations can be made in the specific embodiments of the specification without departing from the scope or spirit of the invention, which is obvious to those skilled in the art. Other embodiments derived from the specification of the invention are obvious to those skilled in the art from the description of the invention. The specification and embodiments of the invention are exemplary only.
If not specifically indicated, the embodiments are all follow conventional experimental conditions, such as the molecular cloning experimental manual by Sambrook and other people (Sambrook J&Russell DW, Molecular cloning: a laboratory utility, 2001), or according to the conditions recommended by the manufacturer's instructions.
Embodiment 1 Construction of characteristic fingerprint map of cotton variety "Zhongmiansuo 96A"
The method for constructing the "Zhongmiansuo 96A" fingerprint map comprises the following steps:
1. DNA extraction
After the sample seeds are fully grounded, 100-200mg of seed powder is taken and placed into a 2.OmL centrifuge tube; 700pL of SDS extracting solution is added into each tube to be evenly mixed; the mixture is washed in a water bath at 650 C for 30min, and the mixture is gently reversed and evenly mixed for a plurality of times during the period. Add equal volume of phenol to each tube: chloroform: isoamyl alcohol (25:24:1) mixed liquor, mixed upside down until no stratification occurred, and centrifuged at 12,000 rpm for 10 min. Sucking supernatant, adding equal volume of precooled isopropanol, gently reversing and mixing until flocculent DNA is agglomerated and precipitated. The DNA is absorbed by a shearing gun head, transferred to a centrifuge tube filled with 70% ethanol for washing once, absolute ethyl alcohol for washing once again, dried under natural conditions. Add 200pL of ultrapure water or TE buffer solution, dissolve it fully and reserve at 4 °C.2. SSR-PCR amplification
Using the primer combinations CCRI 001-060 of Table 1 (as shown in SEQ ID NO. 1-SEQ ID NO. 120), the PCR reaction system is shown in Table 2.
Reaction procedure: a) pre-denaturation: 94 °C for 5 min; b) amplification: denaturation at 94 °C for 45 s, annealing at 60 °C for 45 s, extension at 72 °C for 45 s; the cycle should be run 32 times ; c) final extension: 72 °C for 10 min, and d) preservation of the amplified product at 4 °C.
Table 1 PCR primer information
Annealing Chromosom Name of temperatur e Primer sequence (5'-3') primer (Position) (°C)
Forward:CCACCAGCCTTACCTTATACGC CCRI001 1A 60 Reverse:GTGCTTGGCCTCGCTAAGTG
Forward: AGTCCTCCCACTATTCGAAGCT CCRIO02 2A 60 Reverse: ATGCCTCGTACCCTGTTCCG
Forward:CACGACGACTCTTCCTCATCAC CCRI003 3A 60 Reverse: ATTGCAGCCACGAAATTGTCAC
Forward: AGGCTCGCATTGTTGACACTAGG CCRIO04 4A 60 Reverse:CGAGCTTGAACGAACGAACCTCT
Forward:TCGATTCACCGATCTGACAAGC CCRI005 5A 60 Reverse:TGACCCAGACCGACCGTTG
Forward: ACCAGGTCGGTAACGATAGGC CCRI006 6A 60 Reverse:GCCCAAAGTTGAAGCGGAAAAC
Forward:GTATGTCCAATCGCCACCCTAG CCRI007 6A 60 Reverse:GATGACGGTGTTAGGCGGTTC
Forward:GGCCTCCCATCTTTATCCAATGT CCRIO08 7A 60 Reverse: AGCAAAGCAAACTCGACAATGCT
Forward:TTGGGCGGTTTGGGTCAAGG CCRI009 8A 60 Reverse:GGGATTCGGTCGGACACTCAAG
Forward:GCCGGTAAGTCCAACATAAGGG CCRIO10 9A 60 Reverse:CGGGGTAGTCCACCTCCTATTG
Forward: ACTACGCAACTGAAGGGTTTCG CCRI011 10A 60 Reverse:GCTGCAAGGTTCGATGGAAGTG
Forward:CTGCCTCGACCTACAGTTTGAC CCRIO12 11A 60 Reverse: AGTTTTAGGCGGTTTGGGTTTG
Forward: ACCATCCTTGCCGAGTAACCTG CCRIO13 12A 60 Reverse:GTTGATGCCTGGGTCTCAATGC
Forward: AGACGTGAGTTCGAGGACTTTG CCRIO14 12A 60 Reverse:GCCTACCCTCTCAACAGTTTGG
Forward:TTTCACTTGTCCGACCTTACCC CCRIO15 13A 60 Reverse:GGATATGTGTTTGAGCGTGCTG
Forward:CGAAAGAGTGTCGAGCAATCCG CCRIO16 1D 60 Reverse:TCCTGTCAACTTGGAGGCTGAG
Forward:CGCCACATGACCCCACCATC CCRIO17 2D 60 Reverse:CCCACCGAGTATACAGGACACG
Forward:CCGCTCGGTTCAACAGGTTT CCRIO18 3D 60 Reverse: AGAGTTTGCGGTTTTGGTGCC
Forward:CCAGCCACTCGGAGATCCTTG CCRIO19 3D 60 Reverse:GCGTGGAGAAACCAGAGGGTAG
Forward: ACCACTTTGTCCACCTGTCCAC CCR1020 4D 60 Reverse: ATCATCGCCATCTGCCTGGAAG
Forward: AATTGAACCAAGGGCATCATGC CCRI021 5D 60 Reverse:GAACCTGATTGACGGGTAGCAC
Forward:TGATGTTGGCGGGACTATGTAG CCR1022 6D 60 Reverse:CGACTTCACCCTCGTGGTAATC
Forward:CTTCCCCACGCCACTACTATCG CCR1023 7D 60 Reverse:CTCAGCTTTCCTCCTCATTGGC
Forward: ACGAGGGAACATGTGATCTCCT CCR1024 7D 60 Reverse:TCACTCCAAATCACACGTGCCA
Forward: ACCAGCATCCTTTGTGTTAGGC CCR1025 8D 60 Reverse:GGATCGATTTTGGAACCGTGTG
Forward:TTCCATGAGGCTATCCACAAGC CCR1026 10D 60 Reverse: AATGCACCGCACCCCATCAC
Forward:GCGGGCCACCTAATGATGATTG CCR1027 11D 60 Reverse:TTGTTTGACGAGGGAGACGATC
Forward: ACAAGCGCGTCTGCCATATCC CCR1028 11D 60 Reverse:CGTGGTGGGTAGTCACAGTCAG
Forward: ACACTGATGTGGCATCGACTAG CCR1029 12D 60 Reverse:GAATCTGGGCAAACTGTTGTCC
Forward:GCCGAGGCCCCTATAACCC CCR1030 13D 60 Reverse:CTCATATCACGCACCACACCAC
Forward:CCGGTTCAAGCCGACTATTCG CCRIO31 1A 60 Reverse: ACTCGTAACACCGTGCTGATTG
Forward:CTGAGGAGAAAGACAGGACGAC CCR1032 1A 60 Reverse:TGGCGGGGTAAATGTGAATGC
Forward:TGGGTGAGTGTGAGGACTGAAG CCR1033 3A 60 Reverse:TGGGTGTTGCACAAAGTTTCTG
Forward:GTGGGCAGCGATGAATATGATG CCR1034 3A 60 Reverse: ATGAGGGTCATTGCTTGGGTTG
Forward:GCCGTTTCTGCCAACCCCTT CCR1035 4A 60 Reverse:CGGGATTCCACGTGCCCAAA
Forward:CGTCTCGTCCCACCTGTAATGC CCR1036 5A 60 Reverse:GGACTTCGGCAAGGCGGTTC
Forward: TTCCTGCAAAATTGCCTTCACC CCR1037 7A 60 Reverse:TGCTTTGATATCCCCGTGATGG
Forward: AGGGACAAGAATGGACCGACAG CCR1038 7A 60 Reverse:TTAACCGTCGCAGCCTCCTAAC
Forward:TGCCCTTCTTGCCCCTGTG CCR1039 8A 60 Reverse:GCTTGCCTAATTTGGTGGGAAG
Forward:TTACTCTGGGCGTGTGGCATAG CCR1040 9A 60 Reverse: ATGGAAGGAACAGCAGCAAACG
Forward:TGTGGCTCCATGGCACAATATG CCRI041 10A 60 Reverse: AGGCTCTGTTGCACCAATTCAC
Forward:GTTGGAGGCTGCTTTTGATGGG CCR1042 11A 60 Reverse:TGCCATTGCCATGTTGGTCAAG
Forward:GGACAAACATGGCCCCAACT CCR1043 11A 60 Reverse:TGTCCAAACTCTTGCCAACTTGT
Forward:TCTTAGGGCACAATGAGGCAAGA CCR1044 12A 60 Reverse:CTAAGCAGCACCTCATCCAGAAA
Forward: AACTCAATGGGTGTCGGTTACG CCR1045 13A 60 Reverse:GGAGGTGAGCTATCTTCGCAAC
Forward: AGCACGGAAGAACATGATGAGG CCR1046 13A 60 Reverse:CGTCTTCGGCTCAAATGTGTGC
Forward:TGCCCAACCTACATGTGACACA CCR1047 1D 60 Reverse:TCAAATTTGGTTGTCACACCCCA
Forward:CCACATGCCACGCCGTATTATG CCR1048 1D 60 Reverse:ATGATGGGGTGGGCTGTAAAGG
Forward:TTGGGCCGAAAAGGGTTGAAAC CCR1049 2D 60 Reverse:GGTCGGATTCTGGGCACTTTTC
Forward: AGTTCGGTGGACATCAATAGGC CCR1050 2D 60 Reverse:TCCCCAGGGCTTTGAGAATACC
Forward:CGCCACATCCAAGGGTGAATG CCRI051 4D 60 Reverse:GGAATTGCGGTCCCAATACCAC
Forward: ATTCATGGTCAAGTCGGGTCAC CCR1052 5D 60 Reverse:GCTTGCTTTGGGTGGAGTAGAC
Forward:GTTGCTGTGGAGTGGAGTGGAG CCR1053 5D 60 Reverse:TTCGAGGGAGGTTGGTATTGGC
Forward:TGGCTTTGCTTTGCTTATGGTGA CCR1054 6D 60 Reverse:TGCACTCAACTGGACACACTTT
Forward: AATTGTGGAGGGGCACTGTCAG CCR1055 7D 60 Reverse:GTCCCCGCCATCCAAGCAC
Forward:GACTCATGGCGACAGCGATTAG CCR1056 8D 60 Reverse:TGATCACTCAAACGGTGTCACG
Forward:GTTTCCACCGTCGAACCACTG CCR1057 10D 60 Reverse:GGCAGGATTAGGAGATCGAAGC
Forward:TCAGGGGCTCCGTCGTTCTC CCR1058 10D 60 Reverse:CTCGGCTCTTTCTCCGGTTGC
Forward: AGTACCCCTATTTTCCCGTGAGA CCR1059 12D 60 Reverse:TGGGTCTCACATGTGGATTGTTG
Forward:GGAAATGGCCCATCTGAGAGTC CCR1060 13D 60 Reverse:GCCAGGAGATCGGAGCGTTTG
Table 2 PCR amplification reaction system
Reaction Original Recommended reaction Final concentration component concentration volume (20pL)
ddH20 13.0
xbuffer solutionr
(contains 25 lox 1x 2.0
mmol/L MgCl2)
dNTPs 2.5 mmol/L 0.2 mmol/L each 1.6 each
Taqenzyme 5 U/pL 1 U 0.2
Forward primer 20pmol/L 0.1pmol/L 0.1
Reverse primer 20pmol/L 0.1pmol/L 0.1
DNA 40ng/pL 6.Ong/pL 3.0
3. Capillary electrophoresis detection
The amplification products of different fluorescence labels of the same combination primer are taken in equal volume according to the preset combination primer, and are fully
and uniformly mixed. 1pL mixed liquor is extracted and added to the 96 hole template dedicated to the gene sequencer. 0.15 pL molecular weight internal standard and 8.85 PL deionized formamide are added in each hole; denaturation at 95 °C for 5 min, cooling for more than 10 min, and instantaneous centrifugation for 10 s for later use. The gene sequencer is turned on to check the working state and reagent state of the instrument. A sample plate with 96-hole is placed on a sample rack base; A buffer plate containing electrode buffer solution is placed on the buffer plate rack base; data collection software is opened; and the operation is carried out according to a manual of a gene sequencer. The gene sequencer will automatically run the parameters and store the electrophoresis original data.
4. Statistics and analysis of data
The electrophoresis original data file is derived, the data is discriminated by data analysis software, and the DNA molecular fingerprint map of the electrophoresis original data file is established. Table 3 shows the characteristic fingerprint map of the CCRI 001-060 primer combination in the cotton variety "Zhongmiansuo 96A".
Table 3 Characteristic fingerprints of cotton variety "Zhongmiansuo 96A"
Name of primer Clip size Primer name Clip size Primer name Clip size
CCRI001 313 CCRI021 189 CCRI041 307
CCRIO02 290 CCR1022 223 CCR1042 166
CCRI003 178 CCR1023 178 CCR1043 319
CCRIO04 367 CCR1024 201 CCR1044 384
CCRI005 276 CCR1025 240 CCR1045 232
CCRI006 348 CCR1026 187 CCR1046 192
CCRI007 270 CCR1027 334 CCR1047 219
CCRIO08 153 CCR1028 258 CCR1048 324
CCRI009 216 CCR1029 175 CCR1049 221
CCRIO10 365 CCR1030 220 CCR1050 331
CCRI011 392 CCRI031 220 CCRI051 319
CCRIO12 305 CCR1032 223 CCR1052 249
CCRIO13 215 CCR1033 335 CCR1053 178
CCRIO14 352 CCR1034 307 CCR1054 240
CCRIO15 239 CCR1035 349 CCR1055 270
CCRIO16 188 CCR1036 271 CCR1056 266
CCRIO17 196 CCR1037 305 CCR1057 304
CCRIO18 238 CCR1038 297 CCR1058 223
CCRIO19 235 CCR1039 255 CCR1059 308
CCRIO20 311 CCRIO40 226 CCRIO60 230
Embodiment 2 Genuineness identification of cotton variety "Zhongmiansuo 96A"
Identify whether A cotton variety "X" in the market is "Zhongmiansuo 96A". According to the procedure of embodiment 1, the seed sample of the test variety "X" and the control variety "Zhongmiansuo 96A" randomly selected for DNA preparation, and PCR amplification and capillary electrophoresis detection are performed using the CCRI 001-060 primer combination. Table 4 shows the characteristic fingerprint map of the variety "X" to be tested.
Table 4 Fingerprint of "X" characteristic of cotton variety to be tested
Name of primer Clip size Name of primer Clip size Name of primer Clip size
CCRI001 313 CCRI021 189 CCRI041 301
CCRIO02 290 CCR1022 218 CCR1042 172
CCRI003 178 CCR1023 184 CCR1043 323
CCRIO04 373 CCR1024 199 CCR1044 384
CCRI005 268 CCR1025 235 CCR1045 232
CCRI006 348 CCR1026 172 CCR1046 192
CCRI007 260 CCR1027 340 CCR1047 219
CCRIO08 153 CCR1028 256 CCR1048 324
CCRI009 216 CCR1029 177 CCR1049 214
CCRIO10 369 CCR1030 224 CCR1050 331
CCRI011 392 CCRI031 220 CCRI051 312
CCRIO12 307 CCR1032 218 CCR1052 255
CCRIO13 217 CCR1033 335 CCR1053 173
CCRIO14 343 CCR1034 307 CCR1054 240
CCRIO15 239 CCR1035 342 CCR1055 267
CCRIO16 190 CCR1036 271 CCR1056 266
CCRIO17 192 CCR1037 305 CCRIO57 298
CCRIO18 238 CCRIO38 297 CCRIO58 223
CCRIO19 225 CCRIO39 255 CCRIO59 308
CCR1020 306 CCR1040 226 CCR1060 230
Result analysis: the characteristic DNA fingerprint map of the two varieties at 60 primer sites are obtained by the above tests. Through comparative analysis, 31 primer sites such as CCRI 004, CCR 005, CCRI 007 and the like of the sample "X" to be tested show different gene types with "Zhongmiansuo 96A", which indicated that the variety "X" to be tested is not "Zhongmiansuo 96A".
The above-described embodiments are merely description of the preferred methods of the invention and are not intended to limit the scope of the invention. Various modifications and improvements thereof made by those of regular technicians in the art without departing from the spirit of the design of the invention shall fall within the scope of protection defined by the claims.

Claims (6)

1. A primer combination for detecting a cotton variety "Zhongmiansuo 96A" is
characterized in that the primer combination comprises 60 pairs of primers listed in the following table:
Annealing Chromosom Name of temperatur e e Primer sequence (5'-3') primer (Position) (°C)
Forward:ccaccagccttaccttatacgc CCRI001 1A 60 Reverse:gtgcttggcctcgctaagtg
Forward:agtcctcccactattcgaagct CCRIO02 2A 60 Reverse:atgcctcgtaccctgttccg
Forward:cacgacgactcttcctcatcac CCRI003 3A 60 Reverse: attgcagccacgaaattgtcac
Forward:aggctcgcattgttgacactagg CCRIO04 4A 60 Reverse:cgagcttgaacgaacgaacctct
Forward:tcgattcaccgatctgacaagc CCRI005 5A 60 Reverse:tgacccagaccgaccgttg
Forward:accaggtcggtaacgataggc CCRI006 6A 60 Reverse:gcccaaagttgaagcggaaaac
Forward:gtatgtccaatcgccaccctag CCRI007 6A 60 Reverse:gatgacggtgttaggcggttc
CCRIO08 7A 60 Forward: ggcctcccatctttatccaatgt
Reverse: agcaaagcaaactcgacaatgct
Forward:ttgggcggtttgggtcaagg CCRI009 8A 60 Reverse:gggattcggtcggacactcaag
Forward:gccggtaagtccaacataaggg CCRIO10 9A 60 Reverse:cggggtagtccacctcctattg
Forward:actacgcaactgaagggtttcg CCRI011 10A 60 Reverse:gctgcaaggttcgatggaagtg
Forward:ctgcctcgacctacagtttgac CCRIO12 11A 60 Reverse:agttttaggcggtttgggtttg
Forward:accatccttgccgagtaacctg CCRIO13 12A 60 Reverse:gttgatgcctgggtctcaatgc
Forward:agacgtgagttcgaggactttg CCRIO14 12A 60 Reverse:gcctaccctctcaacagtttgg
Forward:tttcacttgtccgaccttaccc CCRIO15 13A 60 Reverse:ggatatgtgtttgagcgtgctg
Forward:cgaaagagtgtcgagcaatccg CCRIO16 1D 60 Reverse:tcctgtcaacttggaggctgag
Forward:cgccacatgaccccaccatc CCRIO17 2D 60 Reverse:cccaccgagtatacaggacacg
Forward:ccgctcggttcaacaggttt CCRIO18 3D 60 Reverse:agagtttgcggttttggtgcc
CCRIO19 3D 60 Forward: ccagccactcggagatccttg
Reverse:gcgtggagaaaccagagggtag
Forward:accactttgtccacctgtccac CCR1020 4D 60 Reverse: atcatcgccatctgcctggaag
Forward: aattgaaccaagggcatcatgc CCRI021 5D 60 Reverse:gaacctgattgacgggtagcac
Forward:tgatgttggcgggactatgtag CCR1022 6D 60 Reverse:cgacttcaccctcgtggtaatc
Forward:cttccccacgccactactatcg CCR1023 7D 60 Reverse:ctcagctttcctcctcattggc
Forward:acgagggaacatgtgatctcct CCR1024 7D 60 Reverse:tcactccaaatcacacgtgcca
Forward: accagcatcctttgtgttaggc CCR1025 8D 60 Reverse:ggatcgattttggaaccgtgtg
Forward: ttccatgaggctatccacaagc CCR1026 10D 60 Reverse: aatgcaccgcaccccatcac
Forward:gcgggccacctaatgatgattg CCR1027 11D 60 Reverse:ttgtttgacgagggagacgatc
Forward: acaagcgcgtctgccatatcc CCR1028 11D 60 Reverse:cgtggtgggtagtcacagtcag
Forward:acactgatgtggcatcgactag CCR1029 12D 60 Reverse:gaatctgggcaaactgttgtcc
CCRIO30 13D 60 Forward: gccgaggcccctataaccc
Reverse:ctcatatcacgcaccacaccac
Forward:ccggttcaagccgactattcg CCRI031 1A 60 Reverse:actcgtaacaccgtgctgattg
Forward:ctgaggagaaagacaggacgac CCR1032 1A 60 Reverse: tggcggggtaaatgtgaatgc
Forward:tgggtgagtgtgaggactgaag CCR1033 3A 60 Reverse:tgggtgttgcacaaagtttctg
Forward:gtgggcagcgatgaatatgatg CCR1034 3A 60 Reverse:atgagggtcattgcttgggttg
Forward:gccgtttctgccaacccctt CCR1035 4A 60 Reverse:cgggattccacgtgcccaaa
Forward:cgtctcgtcccacctgtaatgc CCR1036 5A 60 Reverse:ggacttcggcaaggcggttc
Forward:ttcctgcaaaattgccttcacc CCR1037 7A 60 Reverse:tgctttgatatccccgtgatgg
Forward:agggacaagaatggaccgacag CCR1038 7A 60 Reverse: ttaaccgtcgcagcctcctaac
Forward: tgcccttcttgcccctgtg CCR1039 8A 60 Reverse:gcttgcctaatttggtgggaag
Forward: ttactctgggcgtgtggcatag CCR1040 9A 60 Reverse: atggaaggaacagcagcaaacg
CCRI041 10A 60 Forward: tgtggctccatggcacaatatg
Reverse: aggctctgttgcaccaattcac
Forward:gttggaggctgcttttgatggg CCR1042 11A 60 Reverse:tgccattgccatgttggtcaag
Forward:ggacaaacatggccccaact CCR1043 11A 60 Reverse:tgtccaaactcttgccaacttgt
Forward:tcttagggcacaatgaggcaaga CCR1044 12A 60 Reverse:ctaagcagcacctcatccagaaa
Forward:aactcaatgggtgtcggttacg CCR1045 13A 60 Reverse:ggaggtgagctatcttcgcaac
Forward:agcacggaagaacatgatgagg CCR1046 13A 60 Reverse:cgtcttcggctcaaatgtgtgc
Forward: tgcccaacctacatgtgacaca CCR1047 1D 60 Reverse:tcaaatttggttgtcacacccca
Forward:ccacatgccacgccgtattatg CCR1048 1D 60 Reverse: atgatggggtgggctgtaaagg
Forward:ttgggccgaaaagggttgaaac CCR1049 2D 60 Reverse:ggtcggattctgggcacttttc
Forward:agttcggtggacatcaataggc CCR1050 2D 60 Reverse:tccccagggctttgagaatacc
Forward:cgccacatccaagggtgaatg CCRI051 4D 60 Reverse:ggaattgcggtcccaataccac
CCR1052 5D 60 Forward: attcatggtcaagtcgggtcac
Reverse:gcttgctttgggtggagtagac
Forward:gttgctgtggagtggagtggag CCR1053 5D 60 Reverse:ttcgagggaggttggtattggc
Forward: tggctttgctttgcttatggtga CCR1054 6D 60 Reverse:tgcactcaactggacacacttt
Forward:aattgtggaggggcactgtcag CCR1055 7D 60 Reverse:gtccccgccatccaagcac
Forward:gactcatggcgacagcgattag CCR1056 8D 60 Reverse:tgatcactcaaacggtgtcacg
Forward:gtttccaccgtcgaaccactg CCR1057 10D 60 Reverse:ggcaggattaggagatcgaagc
Forward:tcaggggctccgtcgttctc CCR1058 10D 60 Reverse:ctcggctctttctccggttgc
Forward:agtacccctattttcccgtgaga CCR1059 12D 60 Reverse:tgggtctcacatgtggattgttg
Forward:ggaaatggcccatctgagagtc CCR1060 13D 60 Reverse:gccaggagatcggagcgtttg
2. A method for constructing DNA fingerprint map of cotton variety "Zhongmiansuo 96A" is characterized by comprising the following steps:
1) Extracting genomic DNA of cotton variety "Zhongmiansuo 96A";
2) Taking the genomic DNA extracted in the step 1) as a template, and carrying out PCR amplification by using the PCR primer combination according to claim 1;
3) Detecting PCR amplification products.
3. According to claim 2, the method for constructing DNA fingerprint map of cotton variety "Zhongmiansuo 96A" is characterized in that PCR reaction system is 20 pL reaction system, wherein particularly comprises ddH2013.OpL, 10xbuffer solution 2.OpL of 25 mmol/L MgCl2, dNTPs1.6pL, Taqenzyme 0.2pL, forward primer 0.1pL, reverse primer 0.1pL, DNA3.OpL.
4. According to claim 2, the method for constructing DNA fingerprint map of cotton variety "Zhongmiansuo 96A" is characterized by PCR amplification procedure, which is as follows: a) pre-denaturation: 94 °C for 5 min; b) amplification: denaturation at 94 °C for 45 s, annealing at 60 °C for 45 s, extension at 72 °C for 45 s; the cycle should be run 32 times; c) final extension: 72 °C for 10 min, and d) preservation of the amplified product at 40 C.
5. According to claim 2, the method for constructing DNA fingerprint map of cotton variety "Zhongmiansuo 96A" is characterized in that the method for detecting a PCR product is capillary electrophoresis method.
6. The application of the method for constructing DNA fingerprint map of cotton variety "Zhongmiansuo 96A" in claim 2-5 in "Zhongmiansuo 96A" variety genuineness testing.
AU2020103387A 2020-11-11 2020-11-11 Method for Constructing DNA Fingerprint Map of Cotton Variety "Zhongmiansuo 96A" and the Application in Variety Identification Ceased AU2020103387A4 (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
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CN113234847A (en) * 2021-05-11 2021-08-10 安徽省农业科学院水稻研究所 Hybrid plant removing method for molecular identification and fingerprint spectrum construction of normal and cross pollinated crop varieties
CN114328959A (en) * 2021-12-27 2022-04-12 北京百度网讯科技有限公司 Knowledge graph construction and use method, device, equipment and medium

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113234847A (en) * 2021-05-11 2021-08-10 安徽省农业科学院水稻研究所 Hybrid plant removing method for molecular identification and fingerprint spectrum construction of normal and cross pollinated crop varieties
CN114328959A (en) * 2021-12-27 2022-04-12 北京百度网讯科技有限公司 Knowledge graph construction and use method, device, equipment and medium
CN114328959B (en) * 2021-12-27 2023-03-10 北京百度网讯科技有限公司 Knowledge graph construction and use method, device, equipment and medium

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