CN110846430A - Soybean SSR (simple sequence repeat) marker high-throughput multiplex PCR (polymerase chain reaction) method - Google Patents
Soybean SSR (simple sequence repeat) marker high-throughput multiplex PCR (polymerase chain reaction) method Download PDFInfo
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Abstract
The invention discloses a soybean SSR marker high-throughput multiplex PCR method, and relates to the technical field of molecular biology DNA markers. Comprises a soybean SSR marker high-throughput multiplex PCR method, a primer group, a kit and application. The soybean SSR marker multiplex PCR multiplex amplification system based on the capillary four-color fluorescence detection system has important significance for constructing a high-flux automatic detection platform, improving the SSR marker detection efficiency, saving the detection cost, and constructing a fingerprint and identifying varieties.
Description
Technical Field
The invention relates to the technical field of molecular biology DNA marking, in particular to a soybean SSR marking high-throughput multiplex PCR method.
Background
At present, with the development of molecular biology technology, SSR markers have been widely applied to germplasm resource identification, genetic diversity analysis, variety purity and authenticity detection, QTL/gene mining, variety DNA fingerprint database construction and the like due to the advantages of rich variation, simple operation, reliable result, good repeatability and the like. Only genotype information of a single locus can be obtained by analyzing one SSR primer, and the data analysis throughput is low. The establishment of a multiplex PCR composite amplification system is an important way for improving the information content of each SSR analysis.
The multiplex PCR refers to the amplification of multiple pairs of primers in one PCR system, and has the advantages of short time, less required reagents and the like. On soybeans, the research of multiplex PCR mostly focuses on the aspect of transgenic component detection, and the research on an SSR marker high-throughput multiplex PCR system is less.
Therefore, the construction of the multiplex PCR composite amplification system based on the capillary four-color fluorescence detection system has important significance for establishing a high-flux automatic detection platform and improving the SSR marker detection efficiency.
Disclosure of Invention
In view of the above, the invention provides a soybean SSR marker high-throughput multiplex PCR method
In order to achieve the purpose, the invention adopts the following technical scheme:
a soybean SSR marker high-throughput multiplex PCR primer group comprises a primer pair 1-10;
primer pair 1:
5' -AGTTAGGGTGATCAGAAAACTGTAA as shown in SEQ ID NO. 1;
r is 5' -GATTGCTCCAACGTATTAAGCACTT, shown as SEQ ID NO. 2;
and (3) primer pair 2:
5' -TGAAACCTGGAAAAAGATACGTGTG as shown in SEQ ID NO. 3;
r is 5' -GTTGAAGGAAATGCTTCAAACCAAC, shown as SEQ ID NO. 4;
and (3) primer pair:
5' -AGTTGGTGTTTTCTTGTGGTTTTCT as shown in SEQ ID NO. 5;
r is 5' -CGTACTCTGTACTGGGAGCTAATTA, shown as SEQ ID NO. 6;
and (3) primer pair 4:
5' -TTTATGACTCTTTGCAGCAAGAAGG as shown in SEQ ID NO. 7;
r is 5' -AATGATCTAATGTGTGACCATGCAC, shown as SEQ ID NO. 8;
and (3) primer pair 5:
5' -TGCTTCCCTATGCAAAAAGATTGAG as shown in SEQ ID NO. 9;
r is 5' -TCAAGTATGGTGGAAAACTTGAAGC, shown as SEQ ID NO. 10;
and (3) primer pair 6:
5' -TCACCTATTTTCAAATTATGCGCTCA as shown in SEQ ID NO. 11;
r is 5' -ATGGAAATACTGAATGAAAGCATATTG, shown as SEQ ID NO. 12;
and (3) primer pair 7:
5' -TCACGAAAGTTTTTCTGTAACATCT as shown in SEQ ID NO. 13;
r is 5' -GCCTGTATATAAAGGTGCACAAAGT, shown as SEQ ID NO. 14;
and (3) primer pair 8:
5' -GCTTACATGCAACCTATAAGGCATT as shown in SEQ ID NO. 15;
r is 5' -AGGTTGTCAAATGATGAGAAGTCTT, shown as SEQ ID NO. 16;
and (3) primer pair 9:
5' -TGGTGACACTGTATACCAAACTCTT as shown in SEQ ID NO. 17;
r is 5' -GGACTCTGCCTCGTAATATTACCAT, shown as SEQ ID NO. 18;
and primer pair 10:
5' -TTCTTGAGAATTACTGTGTCCCTGT as shown in SEQ ID NO. 19;
r is 5' -CCTGATGGGATGCACTTTGTATATT, shown in SEQ ID NO. 20.
In the invention, 14 pairs of primers are designed totally, but sequences designed by software have various problems in practical application, such as finding that the amplification condition of one SSR (GM1155) primer does not meet the experimental requirements, the peak value is low, the amplification efficiency is low and is abandoned, finding that two marked (GM020 and GM003) primers amplify two strips, the standard fingerprint cannot be read, redesigning and optimizing the two primers again, and designing twice, wherein the two primers still do not meet the requirements and do not meet the software prediction, so the primers are not abandoned; the other primer was discarded as a marker (GM006) product affecting the data read of the other primer. And finally, obtaining the 10 pairs of primers by taking the amplification efficiency not lower than 90%, the sizes of different primer amplification products with the same fluorescent label not overlapped and the amplification results of the different fluorescent label products not mutually influenced as selection bases.
Preferably: the 5' end of the primer is marked with a fluorescent reporter group, 1-3 fluorescent reporter groups of the primer pair are FAM, 4-6 fluorescent reporter groups of the primer pair are HEX, 7 fluorescent reporter groups of the primer pair are TAMRA, and 8-10 fluorescent reporter groups of the primer pair are ROX.
Depending on the fluorescent reporter group, the 10 primer sets can still be divided into 4 subgroups: the fluorescent group is recommended to use a fluorescent group, if a fluorescent reporter group is required to be replaced, a group of primers needs to be replaced uniformly, otherwise, the reading abnormality problem is caused by overlapping molecular weight ranges of different primers.
Further: a soybean SSR marker high-throughput multiplex PCR method is characterized in that a fluorescent multiplex PCR system is established, 48 parts of soybean DNA are subjected to multiplex PCR amplification by utilizing the primer group, and electrophoresis is capillary electrophoresis.
Preferably: the amplification system of the multiplex PCR in the step (4) is as follows: total volume 25. mu.L, 0.4. mu.M of each primer, 1.5. mu.L of mixed primers, 20 ng/. mu.L of DNA 5. mu.L, 12.5. mu.L of Premix Ex Taq Hot Start enzyme, and 6. mu.L of water.
Preferably: the amplification procedure of the multiplex PCR in the step (4) is as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 4min, 32 cycles; extension at 72 ℃ for 5 min.
Further: a detection kit containing the primer group of the soybean SSR marker high-throughput multiplex PCR also comprises Premix Ex Taq Hot Start enzyme and water.
Further: the application of the primer group of the soybean SSR marker high-throughput multiplex PCR in detection kits, soybean variety identification, genetic diversity analysis or colony structure.
According to the technical scheme, compared with the prior art, the invention discloses and provides the soybean SSR marker high-throughput multiplex PCR method, and the obtained technical effects are that compared with the traditional SSR marker detection, 10 pairs of primers can complete amplification in one PCR reaction, so that the experiment cost is greatly reduced, and the detection efficiency is improved. Different primers are marked by different fluorescent groups, and the molecular weight of each soybean material at each SSR locus can be obtained by capillary electrophoresis.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a schematic diagram of the electronic glue detection results of Mix 1-Mix 4 for Jidou No.7 in the research and development process of the present invention.
FIG. 2 is a schematic diagram comparing the results of a part of single primer amplification mixed capillary electrophoresis and GmMix-1 direct amplification capillary electrophoresis provided by the present invention.
FIG. 3 is a schematic diagram of SSR clustering of 48 materials provided by the present invention (the variety numbers are as in Table 3).
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the following examples, 48 soybean varieties (lines) were obtained from the national germplasm resources pool of the Chinese academy of agricultural sciences.
Example 1
According to the previous experimental result, 14 SSR markers are selected, a reference sequence is downloaded in a soybean reference genome (Glycine _ max.V1.0), specific Primer 14 pairs meeting multiple PCR are designed by utilizing GenoPlexs Primer Designer software (Shijiazhuang Bordi Biotechnology Co., Ltd.), each pair of primers consists of an upstream Primer and a downstream Primer, and the predicted length of an amplified fragment is 50-350 bp.
The principle of primer combination is as follows: in each group, 3-4 primer pairs with different amplification fragment lengths are selected firstly, 3-4 primers are mixed in equal proportion to form 4 groups of primers Mix 1-4, DNA of Jidou No.7 and Yudou No. 25 of a reference soybean sample is extracted, amplification is carried out by using different primers Mix (PCR program is 95 ℃ for 3 min; 30 cycles: 95 ℃ for 30s and 60 ℃ for 4 min; extension: 72 ℃ for 5min), and an amplification product is detected by using a full-automatic nucleic acid analysis system (the detection result of Mix 1-Mix 4 on Jidou No.7 is shown in figure 1). The primer pair for amplifying GM1155 has a lower peak value and low amplification efficiency, and is discarded.
And (3) screening 13 pairs of primers meeting experimental requirements, and adding a fluorescent reporter group to the 5' end of each primer, wherein the fluorescent reporter groups are FAM, HEX, TAMRA or ROX respectively. Then 13 pairs of primers were mixed and recorded as GmMix0, and universal multiplex PCR by the two-step method, amplification program: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 4min, 32 cycles; extending for 5min at 72 ℃, amplifying 8 soybean varieties by the new primer group under the PCR program, and analyzing the amplification banding pattern and the amplification effect. Two bands are amplified by two markers (GM020 and GM003) and standard fingerprints cannot be read, the two pairs of primers are redesigned and optimized again, and the requirements cannot be met after the two pairs of primers are designed twice, so that the primers are abandoned; the other primer was discarded as a marker (GM006) product affecting the data read of the other primer.
And finally, determining 10 pairs of primers for the multiplex PCR combination according to the selection basis that the amplification efficiency is not lower than 90%, the sizes of different primer amplification products with the same fluorescent label are not overlapped, and the primer amplification results of the different fluorescent label products are not mutually influenced. 10 pairs of primers were grouped into 1 set of mixed primers, named GmMix-1, see Table 1.
TABLE 110 nucleotide information for primers
In order to determine whether the amplification of the primers in a PCR reaction after mixing affects the size of an amplified fragment, the peak value of capillary electrophoresis and the like, 8 soybean varieties are selected, single primer amplification is adopted respectively, amplification products are mixed in equal proportion to carry out capillary electrophoresis, 10 pairs of SSR primers are directly amplified by using GmMix-1 and directly subjected to capillary electrophoresis, the experiment is repeated for 3 times, and the amplification system is as follows: total volume 25. mu.L, 0.4. mu.M of each primer, 1.5. mu.L of mixed primers, 20 ng/. mu.L of DNA 5. mu.L, 12.5. mu.L of Premix Ex Taq HotStart enzyme, and 6. mu.L of water.
The detection results of 8 amplified samples are shown in table 2, wherein the results of partial single primer amplification mixed capillary electrophoresis and GmMix-1 direct amplification capillary electrophoresis are shown in fig. 2.
The result shows that compared with the traditional SSR marker detection, the 10 pairs of primers included in the GmMix-1 can complete amplification in one PCR reaction, thereby greatly reducing the experiment cost and improving the detection efficiency. Different primers are marked by different fluorescent groups, and the molecular weight of each soybean material at each SSR locus can be obtained by capillary electrophoresis.
TABLE 2 GmMix-1 results of 8 soybean varieties amplification
Example 2
And (3) establishing a fluorescent multiplex PCR system, performing multiplex PCR amplification on a part of 270 samples, namely 48 soybean DNA (including 1 material repetition) by using a determined primer group (GmMix-1), and performing electrophoretic analysis.
(1) Extraction of genomic DNA of soybean variety
Selecting 48 parts of soybean variety (series), and taking 0.1g of young leaves of each part of soybean. The soybean genomic DNA was extracted using a DNA extraction Kit (genomic DNA Purification Kit, # K0512, Thermo Fisher Scientific), diluted to 20 ng/. mu.L, and stored in a refrigerator at-20 ℃ for further use.
(2) PCR amplification and capillary electrophoresis detection
The PCR amplification system was 25. mu.L, containing 1.5. mu.L of mixed primers (0.4uM), 5. mu.L of DNA (20 ng/. mu.L), 12.5. mu.L of Premix Ex Taq Hot Start enzyme (RR030A, Takara) and 6. mu.L of water. The PCR amplification procedure was: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 4min, 32 cycles; extension at 72 ℃ for 5 min. After the amplification reaction is finished, the product is subjected to capillary electrophoresis by using an ABI3730 high-throughput gene analyzer, and the fragment length (bp) is detected.
(3) Multiplex PCR detection result analysis of 48 parts of soybean variety (line)
Data normalization of capillary electrophoresis results is shown in table 3, and only 1 band amplified at each site is recorded as homozygous, e.g. 180/180; individual amplified 2 bands scored as heterozygous, e.g., 164/193. 48 parts of materials are clustered, and it is found that 10 SSR markers can clearly distinguish all the materials, and the No.6 and the No. 42 in the 48 parts of materials are the repetition of the materials with the same name, so that the two parts of materials are gathered together (see figure 3), which indicates that the SSR markers obtained in the research can be constructed by using the fingerprint of soybeans and identified by varieties.
TABLE 3 result of amplification of 48 soybean varieties (lines) by GmMix-1
A summary of primer amplification range information obtained by further amplifying 270 samples with the GmMix-1 mixed primer is shown in Table 4.
TABLE 4 information of GmMix-1 Mixed primers
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
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Claims (7)
1. A soybean SSR marker high-throughput multiplex PCR primer group is characterized by comprising a primer pair 1-10;
primer pair 1:
5' -AGTTAGGGTGATCAGAAAACTGTAA as shown in SEQ ID NO. 1;
r is 5' -GATTGCTCCAACGTATTAAGCACTT, shown as SEQ ID NO. 2;
and (3) primer pair 2:
5' -TGAAACCTGGAAAAAGATACGTGTG as shown in SEQ ID NO. 3;
r is 5' -GTTGAAGGAAATGCTTCAAACCAAC, shown as SEQ ID NO. 4;
and (3) primer pair:
5' -AGTTGGTGTTTTCTTGTGGTTTTCT as shown in SEQ ID NO. 5;
r is 5' -CGTACTCTGTACTGGGAGCTAATTA, shown as SEQ ID NO. 6;
and (3) primer pair 4:
5' -TTTATGACTCTTTGCAGCAAGAAGG as shown in SEQ ID NO. 7;
r is 5' -AATGATCTAATGTGTGACCATGCAC, shown as SEQ ID NO. 8; (ii) a
And (3) primer pair 5:
5' -TGCTTCCCTATGCAAAAAGATTGAG as shown in SEQ ID NO. 9;
r is 5' -TCAAGTATGGTGGAAAACTTGAAGC, shown as SEQ ID NO. 10;
and (3) primer pair 6:
5' -TCACCTATTTTCAAATTATGCGCTCA as shown in SEQ ID NO. 11;
r is 5' -ATGGAAATACTGAATGAAAGCATATTG, shown as SEQ ID NO. 12;
and (3) primer pair 7:
5' -TCACGAAAGTTTTTCTGTAACATCT as shown in SEQ ID NO. 13;
r is 5' -GCCTGTATATAAAGGTGCACAAAGT, shown as SEQ ID NO. 14;
and (3) primer pair 8:
5' -GCTTACATGCAACCTATAAGGCATT as shown in SEQ ID NO. 15;
r is 5' -AGGTTGTCAAATGATGAGAAGTCTT, shown as SEQ ID NO. 16;
and (3) primer pair 9:
5' -TGGTGACACTGTATACCAAACTCTT as shown in SEQ ID NO. 17;
r is 5' -GGACTCTGCCTCGTAATATTACCAT, shown as SEQ ID NO. 18;
and primer pair 10:
5' -TTCTTGAGAATTACTGTGTCCCTGT as shown in SEQ ID NO. 19;
r is 5' -CCTGATGGGATGCACTTTGTATATT, shown in SEQ ID NO. 20.
2. The primer group for soybean SSR-labeled high-throughput multiplex PCR according to claim 1, wherein a fluorescent reporter group is labeled at the 5' end of the primer, the fluorescent reporter groups of 1-3 of the primer pairs are FAM, the fluorescent reporter groups of 4-6 of the primer pairs are HEX, the fluorescent reporter group of 7 of the primer pairs is TAMRA, and the fluorescent reporter groups of 8-10 of the primer pairs are ROX.
3. A soybean SSR marker high-throughput multiplex PCR method is characterized in that a fluorescent multiplex PCR system is established, and the primer group in claim 1 is utilized to perform multiplex PCR amplification and electrophoretic analysis on soybean DNA.
4. The method for soybean SSR marker high-throughput multiplex PCR according to claim 3, wherein the amplification system of the multiplex PCR is: total volume 25. mu.L, 0.4. mu.M of each primer, 1.5. mu.L of mixed primers, 20 ng/. mu.L of DNA 5. mu.L, 12.5. mu.L of Premix Ex Taq Hot Start enzyme, and 6. mu.L of water.
5. The method for soybean SSR marker high-throughput multiplex PCR according to claim 3, wherein the amplification procedure of the multiplex PCR is as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 4min, 32 cycles; extension at 72 ℃ for 5 min.
6. A detection kit comprising the primer set for soybean SSR marker high-throughput multiplex PCR of claim 1, further comprising Premix Ex Taq Hot Start enzyme and water.
7. The use of the soybean SSR marker high-throughput multiplex PCR primer set according to claim 1 in detection kits, construction of finger prints and variety identification.
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