Composite amplification kit for authenticity identification based on cotton SSR (Simple Sequence Repeat) marker and application of composite amplification kit

Abstract

The invention discloses a composite amplification kit for authenticity identification based on a cotton SSR (Simple Sequence Repeat) marker and application of the composite amplification kit, and belongs to the field of agricultural molecular detection. According to the invention, 60 cotton SSR loci and amplification primers are divided into 6 groups and are connected with different fluorophores, so that the composite amplification kit for detecting the cotton SSR marker is constructed. According to the invention, multiple PCR amplification products of six groups of primers are respectively subjected to capillary four-color fluorescent electrophoresis detection and data analysis, and 60 cotton SSR markers can be detected only through six reactions. According to the multiplex amplification kit and the detection method provided by the invention, the time and the cost for detecting the authenticity of cotton can be remarkably saved, pollution in an experiment process is reduced, the stability of an experiment result is ensured, and the multiplex amplification kit and the detection method have obvious advantages in the aspects of large-scale sample and high-throughput detection; and a technical means is provided for cotton variety authenticity identification and DNA fingerprint database construction.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural molecular detection, in particular to a realness identification composite amplification kit based on cotton SSR markers and application thereof. BACKGROUND

[0002] Cotton is an important economic crop, not only the main natural fiber raw material crop of the textile industry, but also an important resource of animal feed protein and human edible oil. At present, the number of cotton varieties approved in China reaches 2342. Due to the centralized use of backbone parents and the large-scale application of transgenic technology in cotton breeding, the genetic difference between cotton varieties is becoming smaller and smaller, making it more difficult to rely solely on traditional field trait differences to identify varieties. At the same time, due to the poor timeliness of identification, large workload, high labor cost, and easy influence of environmental and subjective factors, the phenomenon of mixed varieties is difficult to effectively control.

[0003] Simple sequence repeats (SSR) are composed of 2-7 bases as core sequences in tandem repeats, with a tandem repeat number of about 5-60 and a length generally less than 400 bp. There are differences between different individuals, and it follows the Mendelian inheritance law, showing dominant inheritance between parents. Due to the difference in base composition and its length polymorphism, SSR has genetic polymorphism. As an ideal molecular marker, SSR markers have been widely used in cotton germplasm diversity evaluation and genetic research.

[0004] In practical application, only one target can be detected in one reaction, so 60 pairs of primers need to be amplified for a single sample to obtain the site information of the sample. When facing a large number of samples, multiple personnel and multiple devices are needed, which is time-consuming and labor-intensive. Although the cost of a single reaction is relatively low, the total cost will increase with the increase in the number of detection targets due to the need for multiple reactions to detect multiple sites. Therefore, for cotton SSR marker detection, it is urgent to provide an amplification method that reduces detection cost and improves detection efficiency. SUMMARY

[0005] The purpose of the present application is to provide a realness identification composite amplification kit based on cotton SSR markers and application thereof, to solve the problems existing in the prior art. The present application only needs 6 reactions to realize the detection of 60 cotton SSR markers, which has obvious advantages in large-scale sample and high-throughput detection, and provides a technical means for cotton variety realness identification and DNA fingerprint library construction.

[0006] To achieve the above purpose, the present application provides the following scheme:

[0007] The application provides a composite amplification kit based on a cotton SSR marker, comprising 60 primer pairs for amplifying 60 cotton SSR sites, an internal standard and a reaction buffer;

[0008] The 60 primer pairs are divided into groups A, B, C, D, E and F according to 10 primer pairs in each group.

[0009] The primer pair comprises a forward primer and a reverse primer, and the 5' end of the forward primer is connected with a fluorescent group; the color of the fluorescent group is blue, green, yellow or red.

[0010] The sequence, concentration and fluorescent group color of the primer are as follows:

[0011]

[0012]

[0013] Further, the blue fluorescent group is FAM; the green fluorescent group is VIC; the yellow fluorescent group is NED; and the red fluorescent group is PET.

[0014] Further, the components of the reaction buffer include 10 mM ammonium sulfate, 10 mM potassium chloride, 55 mM Tris-HCl, 2 mM magnesium ion, 0.8 μg / μL BSA, 5% DMSO, 8% ethylene glycol, 1 mM Na4P2O7 and 0.2 mM dNTP and 2 U Taq enzyme.

[0015] Further, the internal standard is Liz-500.

[0016] The application also provides application of the composite amplification kit in real identification of cotton varieties.

[0017] The application also provides application of the composite amplification kit in construction of a cotton DNA fingerprint library.

[0018] The application also provides a method for detecting the authenticity of a cotton variety, comprising the following steps:

[0019] Extracting genomic DNA of the cotton to be detected;

[0020] Performing multiplex PCR amplification on the genomic DNA by using the composite amplification kit to obtain an amplification product;

[0021] Performing capillary electrophoresis detection on the amplification product, analyzing the detection result and judging the authenticity of the cotton variety.

[0022] Further, the multiplex PCR amplification is: mixing 10 primer pairs of group A, group B, group C, group D, group E or group F respectively to form a primer mixture, and taking the genomic DNA as a template to perform a multiplex PCR reaction respectively.

[0023] Further, the reaction system of the multiplex PCR amplification is:

[0024] The primer mixture is 2 μL, the reaction buffer is 4 μL, the genomic DNA is 1 μL and the ddH2O is 3 μL.

[0025] Further, the reaction procedure of the multiplex PCR amplification is:

[0026] 95 ℃ pre-denaturation for 2 min; 95 ℃ denaturation for 5 s, 58 ℃ annealing for 90 s, 28 cycles; 60 ℃ final extension for 30 min.

[0027] The present application discloses the following technical effects:

[0028] The present application divides 60 cotton SSR sites and amplification primers into 6 groups, connects different fluorescent groups, and constructs a composite amplification kit for detecting cotton SSR markers. The present application respectively performs capillary four-color fluorescence electrophoresis detection and data analysis on the multiplex PCR amplification products of the 6 groups of primers, and only 6 reactions are needed to realize the detection of 60 cotton SSR markers. The composite amplification kit and the detection method provided by the present application can significantly save the time and cost of cotton authenticity detection, reduce the pollution in the experimental process, ensure the stability of the experimental results, have obvious advantages in large-scale sample and high-throughput detection, and provide a technical means for cotton variety authenticity identification and DNA fingerprint library construction. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0030] Figure 1 The amplification spectrum obtained by using the amplification system of the present application for the first group of amplification sites;

[0031] Figure 2 The amplification spectrum obtained by using the amplification system of the present application for the second group of amplification sites;

[0032] Figure 3 The amplification spectrum obtained by using the amplification system of the present application for the third group of amplification sites;

[0033] Figure 4 An amplification profile obtained using the amplification system of the present application for the fourth group of amplification sites;

[0034] Figure 5 An amplification profile obtained using the amplification system of the present application for the fifth group of amplification sites;

[0035] Figure 6 An amplification profile obtained using the amplification system of the present application for the sixth group of amplification sites. DETAILED DESCRIPTION

[0036] Various illustrative embodiments of the present application are now described in detail. Such description, however, should not be taken to limit the scope of the present application, but rather, should be taken as merely illustrative of some aspects, features and embodiments thereof.

[0037] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, where particular ranges of values are given, understand that each intervening value, to the upper or lower limit of the ranges is also specifically included. Each smaller range that falls within the broader ranges is also specifically included. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and each range is inclusive of its end points.

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the patents, patent applications, publications, and descriptions are cited.

[0039] Many modifications and variations of this application can be made without departing from its spirit or scope, which will be apparent to those skilled in the art. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. For example, the specification can be used to design equivalent processes and equivalent structures to those described herein. The specification and examples provide no indication that their disclosure is limited to those embodiments. Constituent steps of the processes can be rearranged or performed in parallel.

[0040] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed material or step.

[0041] The amplification reaction of the application is performed on an ABI 9700 thermal cycler, electrophoresis and detection are performed on an ABI 3130xL genetic analyzer, and data analysis is performed using GeneMapper ID-X software. The capillary electrophoresis detection method of the application has been disclosed in the literature "Kuang Meng, Wang Yanqin, Zhou Dayun, et al. Construction of SSR fingerprint database of cotton DUS test standard varieties [J]. Cotton Science, 2015, 27(01): 46-52."

[0042] Example 1

[0043] 1. Grouping of SSR sites

[0044] The 60 sites and the corresponding SSR primers are divided into 6 groups (named as group A to group F in turn) for multiplex PCR amplification, and the site information of a single sample obtained by the original 60 reactions is reduced to 6 reactions.

[0045] Each group of sites is labeled with four colors of fluorescence, i.e. blue, green, red and yellow, which is realized by the fluorescent groups connected to the 5' end of the forward primer; the blue fluorescence label is FAM, the green fluorescence label is VIC, the yellow fluorescence label is NED, and the red fluorescence label is PET. The six groups of combinations and fluorescence labels are as follows:

[0046] A group blue: CCRI030, CCRI012;

[0047] A group green: CCRI004, CCRI015, CCRI003;

[0048] A group yellow: CCRI025, CCRI016;

[0049] A group red: CCRI005, CCRI006, CCRI024;

[0050] B group blue: CCRI011, CCRI018, CCRI023;

[0051] B group green: CCRI014, CCRI021;

[0052] B group yellow: CCRI007, CCRI009;

[0053] B group red: CCRI010, CCRI022, CCRI029;

[0054] C group blue: CCRI002, CCRI026, CCRI027;

[0055] C group green: CCRI008, CCRI019;

[0056] Group C yellow: CCRI001, CCRI013;

[0057] Group C red: CCRI020, CCRI028, CCRI017;

[0058] Group D blue: CCRI039, CCRI041, CCRI042;

[0059] Group D green: CCRI043, CCRI045;

[0060] Group D yellow: CCRI031, CCRI035;

[0061] Group D red: CCRI036, CCRI049, CCRI059;

[0062] Group E blue: CCRI032, CCRI050, CCRI055;

[0063] Group E green: CCRI038, CCRI047;

[0064] Group E yellow: CCRI057, CCRI060;

[0065] Group E red: CCRI051, CCRI052, CCRI053;

[0066] Group F blue: CCRI034, CCRI044, CCRI058;

[0067] Group F green: CCRI037, CCRI046;

[0068] Group F yellow: CCRI033, CCRI040, CCRI056;

[0069] Group F red: CCRI048, CCRI054.

[0070] 2. Experimental steps

[0071] 2.1 DNA collection

[0072] After the shelled cotton seeds are crushed, the SDS extraction solution is added, and after vortexing, it is placed in a 65°C water bath; a mixture of phenol, chloroform and isoamyl alcohol with a volume ratio of 25:24:1 is added, mixed well, and centrifuged; the supernatant is taken, 10 mg / mL RNase is added, and the water bath is used; after extraction, centrifugation is performed to obtain the supernatant, isopropanol is added, and after the DNA is precipitated, the DNA precipitate is washed with ethanol, TE or ddH2O is added to dissolve the DNA, and it is ready for use.

[0073] 2.2 Reaction system

[0074] After mixing the reagents (buffer, primer composition, genomic DNA, etc.) by shaking, the PCR reaction mixture was prepared according to the following amplification system.

[0075] The total volume of the amplification system was 10 μL, including primer mixture (5x Primer mix) 2 μL, reaction buffer (PCR Master mix) 4 μL, genomic DNA 1 μL (extracted template DNA), ddH2O 3 μL.

[0076] The components of the primer mix included primer mix of groups A\B\C\D\E\F\, and the primer concentrations are shown in Table 1.

[0077] The components of the reaction buffer included 10 mM ammonium sulfate, 10 mM potassium chloride, 55 mM Tris-HCl, 2 mM magnesium ion, 0.8 μg / μL BSA, 5% DMSO, 8% ethylene glycol, 1 mM Na4P2O7, and 0.2 mM dNTP, and 2 U Taq enzyme.

[0078] Table 1 Primer information and concentration

[0079]

[0080]

[0081]

[0082]

[0083] 2.3 PCR reaction program

[0084] The PCR amplification program used in the composite amplification kit according to the present application is shown in Table 2.

[0085] Table 2 Amplification program of the composite amplification kit according to the present application

[0086]

[0087] 2.4 Capillary electrophoresis detection

[0088] Mix Liz-500 internal standard and formamide at a ratio of 2.5:100, take 10 μL of the mixture and add it to a 96-well plate, then add 1 μL of amplified product sample or allele standard, mix and stand for 5 min, denature at 95°C for 3 min, immediately ice bath for 3 min, centrifuge and then put on an ABI3130xL sequencer for detection.

[0089] 2.5 Data analysis

[0090] Import the raw data, select Add sample to project in the File menu on the main page, find the sample file, select the folder, click add to list, click add, and the sample file is displayed in the Project window; select the analysis parameters. Define analysis method, panel, and size standard. Browse the raw data of the sample electrophoresis, select the file name of a certain sample, and select "Raw data" under the "sample" menu. Move the tracking line and stop the cursor on the right side of the primer peak (in front of the first orange internal standard peak). Take the value displayed on the X-axis in the lower left corner of the window at this time as the starting point in the analysis parameters of the analysis method. Click the green analysis button, and a save project dialog box appears. After naming and saving, the software starts processing the data. After the analysis is complete, analysis completed is displayed in the lower left corner. The data obtained by using the GeneMapper ID-X software for analysis and generating a map, as shown in Figures 1-6 .

[0091] The above-described embodiments are only descriptions of the preferred modes of the present application and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A composite amplification kit based on cotton SSR markers, characterized in that, 60 primer pairs for amplifying 60 SSR loci of cotton, an internal standard and a reaction buffer; The 60 primer pairs are divided into groups A, B, C, D, E and F according to 10 primer pairs in each group. The primer pair comprises a forward primer and a reverse primer, and the 5' end of the forward primer is connected with a fluorescent group; the color of the fluorescent group is blue, green, yellow or red. The sequences, concentrations and fluorescent group colors of the primers are as follows:

2. The multiplex amplification kit of claim 1, wherein The blue fluorescent group is FAM; the green fluorescent group is VIC; the yellow fluorescent group is NED; and the red fluorescent group is PET.

3. The multiplex amplification kit of claim 1, wherein The components of the reaction buffer include 10 mM ammonium sulfate, 10 mM potassium chloride, 55 mM Tris-HCl, 2 mM magnesium ion, 0.8 μg / μL BSA, 5% DMSO, 8% ethylene glycol, 1 mM Na4P2O7 and 0.2 mM dNTP and 2 U Taq enzyme.

4. The multiplex amplification kit of claim 1, wherein The internal standard is Liz-500.

5. Use of the composite amplification kit according to any one of claims 1-4 in the identification of the authenticity of a cotton variety.

6. Use of the composite amplification kit according to any one of claims 1-4 in the construction of a cotton DNA fingerprint library.

7. A method of detecting the authenticity of a cotton variety, characterized by, The method comprises the following steps: extracting genomic DNA of the cotton to be tested; performing multiplex PCR amplification on the genomic DNA using the composite amplification kit according to any one of claims 1-4 to obtain an amplification product; performing capillary electrophoresis detection on the amplification product, analyzing the detection result and determining the authenticity of the cotton variety.

8. The method of claim 7, wherein, The multiplex PCR amplification is: mixing 10 primer pairs of group A, B, C, D, E or F respectively to form a primer mixture, and performing multiplex PCR reaction respectively with the genomic DNA as a template.

9. The method of claim 8, wherein, The reaction system of the multiplex PCR amplification is: The primer mixture is 2 μL, the reaction buffer is 4 μL, the genomic DNA is 1 μL and ddH2O is 3 μL.

10. The method of claim 7, wherein, The reaction program of the multiplex PCR amplification is: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 5 s, 58℃ annealing for 90 s, 28 cycles; 60℃ final extension for 30 min.

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