CN114480711A - SSR-PCR reaction system of flame orchid in Hainan province and application thereof - Google Patents
SSR-PCR reaction system of flame orchid in Hainan province and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular biology, and particularly discloses a simple sequence repeat-polymerase chain reaction (SSR-PCR) system for flame orchid in Hainan province, which comprises Primer-F, Primer-R, DNA polymerase and a DNA template. The invention also discloses application of the Hainan province flame Simple Sequence Repeat (SSR) -PCR reaction system in SSR molecular markers and assisted breeding of Hainan province flame blue and application in preparation of a detection kit. The SSR-PCR reaction system of the flame orchid in Hainan province provided by the invention has good stability, good resolution and high repeatability, can amplify clear, bright and specific electrophoresis strips, and lays an important experimental foundation for the development of later-stage primers.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a SSR-PCR reaction system for flame orchid in Hainan province and application thereof.
Background
The flame orchid (Renanthera coccinea) is a plant of the Ranunculaceae Vandae (Vandeae) Lauraria subfamily (Aeridiae) Fimbristylis (Renanthera), is a tropical rare orchid with characteristics and ornamental value, and is famous and beautiful like the flower color of fire. The flame orchid has the characteristics of large conical inflorescence, small amount of flowers, long flowering period, long pedicel, high temperature resistance, high humidity resistance, pleased sun and the like, and can be widely used for aspects of cut flowers, garden scenery, hedges, pot culture and the like. There are about 20 species of fire orchid, distributed mainly in tropical regions of asia and islands of the pacific ocean. China has 3 species of Yunnan fire blue (R.imschooliana), fire blue and Chinese fire blue (R.sinica), which are mainly distributed in Yunnan, Guizhou, Hainan and the like.
At present, researches on the cymbidium sinense mainly focus on aspects such as in-vitro rapid propagation of the cymbidium sinense (lindani et al, 2006), sterile germination and test tube seedling culture of seeds (lindani et al, 2008), crossbreeding (canadum et al, 2014; chengheming et al, 2019), and analysis of the population situation of the cymbidium sinense (li juan et al, 2018) by a method combining literature reference, specimen record, on-site community and population situation investigation. Researchers may research the resource of the fire orchid in Hainan province later, and only reports in 2015, for example, the genetic diversity analysis (Yao Jing, 2015) of 8 parts of the fire orchid cultivation resource by the sequence-related amplified polymorphism (SRAP) technology of the fire Jing is performed, but the research has less sample amount, the sample is not the wild resource in Hainan province, and the species specificity of the SRAP marker has no superiority, so the situation of the whole germplasm resource of the fire orchid in Hainan province cannot be reflected.
SSR (simple sequence repeat) molecular markers have the advantages of high polymorphism, large marker quantity, good repeatability, wide coverage and the like, and are widely applied to the fields of molecular marker-assisted selection, genetic diversity analysis, genetic map construction and the like. The current commonly used SSR marker development strategies comprise a library building method, a microsatellite enrichment method, a closely related species primer screening method and a bioinformatics analysis method. The library construction method is complex in construction, low in development efficiency, high in requirements on laboratory equipment and instruments, long in time consumption and only suitable for marking and developing species with little knowledge on genome information; compared with a library method, the microsatellite enrichment method omits the step of constructing the library, but the method also needs to obtain the flanking sequence of the SSR molecular marker by using a PCR method, so that the operation is more complicated; the related species primer screening method needs SSR information of related species and has poor universality on the primer of the species with far relativity, so that the application of the method is limited to a certain extent; the bioinformatics analysis method comprises the steps of searching SSR molecular markers in EST sequences in NCBI and EMBL databases by software or searching species which have completed genome sequencing and transcriptome sequencing to find out the SSR molecular markers, designing primers according to flanking sequences of the SSR molecular markers, and screening the designed primers by using samples to screen out primers with polymorphism and containing the SSR molecular markers. Due to the large amount of EST sequences and transcriptome sequencing sequences, this approach still has a high development efficiency compared to other SSR development approaches (summer plum, 2016). In addition, the SSR developed by the method is positioned in the coding region of the gene, and the obtained SSR molecular marker is often closely linked with the gene, so that the gene positioning and the association analysis of the character can be effectively carried out, and the functional SSR molecular marker can be obtained. Therefore, the SSR marker developed based on transcriptome data is not only beneficial to genetic diversity evaluation of germplasm resources, but also more beneficial to correlation analysis of important traits at a later stage, and time and cost are saved for a parent breeding process in molecular marker-assisted breeding work.
The development of SSR molecular markers distributed in the Hippocampus flamethrows by adopting a transcriptome sequencing strategy is an effective means for evaluating genetic diversity of the Hippocampus flamethrows, reports about genetic diversity of the Hippocampus flamethrows are not yet found at present, the primary condition of molecular marker detection is to establish a reaction system with clear amplification bands, a foundation is laid for screening of polymorphism of primer amplification and later-stage population polymorphism analysis, and reports are not found in the aspects at present.
Disclosure of Invention
The invention aims to provide a SSR-PCR reaction system of flame orchid in Hainan province and application thereof, so as to solve at least one of the technical problems. An object of the present invention is to provide: the SSR-PCR reaction system of flame orchid in Hainan province comprises 0.4-0.5 mu L, DNA template 1-1.5 mu L of Primer-F, Primer-R, DNA polymerase (5U/mu L);
the sequence of Primer-F and the sequence of Primer-R comprise any one or more than two groups of SSR1-SSR 11;
the sequence of SSR1-11 is as follows:
SSR1, Primer-F: TGGCATTTGACATGCAGGTG, respectively;
Primer-R：ACACCTCGGGCAAAAATCAA；
SSR2, Primer-F: TGGTGGTGTATATGCAGAATAGT, respectively;
Primer-R：CGCCAATATTAGATCCGGGCT；
SSR3, Primer-F: GAGTTGACACACACACGCAC, respectively;
Primer-R：CCAATTGGGCCGTTAAAGTGG；
SSR4, Primer-F: TTGAATGGGAGGAGTTCGGC, respectively;
Primer-R：ACCGATGCAACCTGGATGTC；
SSR5, Primer-F: CGAGTGGTCAGAGTGCCAAT;
Primer-R：TTCTCCATGAGGCGAAACCC；
SSR6, Primer-F: GGCTTTCACTGGGCTAGACA;
Primer-R：GGAGGGGCTTTTGTTGACCT；
SSR7, Primer-F: TCCCTTCTTCTCTCTCTCGCT, respectively;
Primer-R：AGGAAGAGTGTTTGAGTTAATACCT；
SSR8, Primer-F: AGTGGTCATCCAAACAAGGGA, respectively;
Primer-R：TGTGTACAAGAAAGCCAGCA；
SSR9, Primer-F: CCCAGCCTATGTGACTGACA, respectively;
Primer-R：ACTCTCATAAGTTGCTGATAGCCT；
SSR10, Primer-F: GGCGTCTTGGATCTCAGGTC, respectively;
Primer-R：TGGCTATGTGGTTTTCCTTCA；
SSR11, Primer-F: TGTGACGGAAATTTTGGTGCA;
Primer-R：GCCCAATAATCGCACAACGT。
preferably, the amplification procedure is: 3min at 94 ℃; 30s at 94 ℃, 30s at 56 ℃, 30s at 72 ℃ and 35 cycles; 30min at 72 ℃.
The second object of the present invention is to provide: the SSR-PCR reaction system of the Hippocampus Turkey is applied to SSR molecular markers and assisted breeding of the Hippocampus Turkey.
The third object of the present invention is to provide: the SSR-PCR reaction system of the Hippocampus japonicas is applied to the preparation of a detection kit.
The invention has the beneficial effects that:
the invention is named as flame orchid in Hainan province according to the name of flame orchid distributed in Hainan province, and the flame orchid in Hainan province is not a generic concept but the flame orchid growing in Hainan province, wherein the Hainan province is the first province of China's territorial area (land area plus sea area).
The SSR-PCR reaction system of the flame orchid in Hainan province provided by the invention has good stability, good resolution and high repeatability, can amplify clear, bright and specific electrophoresis strips, and lays an important experimental foundation for the development of later-stage primers.
Drawings
FIG. 1 is a gel electrophoresis chart of examples 1 to 4 of the present invention;
FIG. 2 is a diagram of gel electrophoresis comparing comparative example and example 3 of the present invention.
Detailed Description
The following is further detailed by way of specific embodiments:
the SSR-PCR reaction system of flame orchid in Hainan province comprises the following reagents, and is specifically shown in the following table 1:
TABLE 1 DNA polymerase reaction System
The sequences of Primer-F and Primer-R comprise any one or more than two groups of SSR1-SSR 11;
TABLE 2 SSR1-SSR11
The reaction procedure was as follows:
s1 PCR amplification
(1) Extracting DNA of a Plant to be detected according to an operation instruction of the Kit, specifically, extracting the DNA Isolation Kit of Plant of Foregene. The sample collection plot of the plant to be detected is as follows: sand in Hainan Peak Ridge; the species are as follows: renanthera coccine (flame blue).
(2) By ddH2O the extracted DNA was diluted 10-fold and used as a DNA template for PCR reaction.
(3) After mixing and centrifugation of the above solutions, amplification was performed according to the procedure of table 2 below:
TABLE 3 DNA polymerase reaction procedure
S2, detecting PCR amplification products by adopting vertical gel (polyacrylamide gel)
(1) Preparing a glass plate (cleaned), a binder (used for fixing the edge sealing strip and the glass plate), and an edge sealing strip (the glass plate, a comb and an adhesive tape are cleaned and dried, cleaned by absolute ethyl alcohol, and filled with the glass plate and clamped by a clamp after the glass is dried in the air);
(2) the glass plate was sealed at both the bottom and sides with 1.5% agar powder. The preparation method of 1.5% agar powder is shown in the following table 3:
TABLE 4 preparation method of 1.5% agar powder
(3) The following reagents were prepared:
a 30% polyacrylamide solution was prepared according to table 4 below:
table 5: preparation method of 30% polyacrylamide solution
② a 10 percent ammonium persulfate solution is prepared according to the following table 5:
table 6: preparation method of 10% ammonium persulfate solution
③ 0.1 percent of AgNO is prepared according to the following table 63：
Table 7: preparation method of 0.1% AgNO3
(4) In a designated beaker, 21.28mL of 30% polyacrylamide, ddH2O30.16 mL, 5 XTBE 8mL, 10% ammonium persulfate 0.56 mL.
Stirring the four reagents uniformly by a glass rod, and then adding 60 mu l of TEMEP (tetramethylethylenediamine);
pouring the glue, namely quickly pouring the liquid in the beaker into the plate along the glass rod, and inserting a comb (a comb with 40 holes can be formed);
waiting for 0.5-1h, adding 0.5 xTBE into an electrophoresis tank to submerge a sample application hole after glue is solidified, taking 3 ul of PCR product to perform sample application (ensuring that 6 xloading buffer is added into the PCR product to enable the final concentration to be 6 x), and simultaneously adding 3 ul of 2k Marker into an empty glue hole;
adjusting the electrophoresis apparatus to a constant current of 60mA, and performing electrophoresis for about 2h10min (if 2 electrophoresis tanks need to use about 3 h);
after electrophoresis, putting the glass plate into water, taking out the glue (aiming at preventing the glue from cracking), and washing the glue for 1 time by using tap water; 0.1% AgNO for3Silver staining for 10min while shaking;
washing with water for 2 times, 10s each time;
after staining with 0.4% formaldehyde in NaOH (100 mL of 1.5% NaOH was added 400. mu.l of formaldehyde) for about 1-2min (bands clearly shown are acceptable).
The formulation of 1.5% NaOH is shown in Table 7 below:
table 8: preparation method of 1.5% NaOH
The formulation of 0.4% formaldehyde in NaOH is shown in table 8:
table 9: preparation method of 0.4% formaldehyde NaOH solution
Examples 1 to 4
The main difference between examples 1-4 is the amount of DNA template and the amount of DNA polymerase, as shown in Table 10 below:
TABLE 10 differences between examples 1 to 4
FIG. 1 is a diagram of gel electrophoresis in examples 1 to 4 of the present invention, and it can be seen from FIG. 1 that electrophoresis bands obtained by 1. mu.l and 1.5. mu.l of DNA templates are not very different. The examples 3 and 4, in which the amount of DNA polymerase was 0.5. mu.l, amplified clearer, brighter, and more specific electrophoretic bands than the examples 1 and 2, in which the amount of DNA polymerase was 0.4. mu.l.
Comparative example
The reaction system and reaction procedure of the comparative example differs from example 3 as follows:
mix PCR reaction system is as follows:
table 11: mix reaction system
Table 12: mix PCR reaction procedure
The vertical gel (polyacrylamide gel) is adopted to detect the PCR amplification product, the experimental result is shown in figure 2, and compared with the reaction system and the reaction program (tables 2-9 and tables 11-12) of the comparative example, the reaction system and the reaction program (tables 1-10) of the example 3 are adopted, clear, bright and specific electrophoresis bands can be amplified by the 11 pairs of primers SSR1-SSR11, the resolution is good, the stability is good, the repeatability is high, and an important experimental basis is laid for the development of the later-stage primers. In FIG. 2, the bands are unclear as the amplification results of the comparative example.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (4)
1. The SSR-PCR reaction system of flame orchid in Hainan province is characterized by comprising Primer-F, Primer-R, DNA polymerase and a DNA template;
the sequence of Primer-F and the sequence of Primer-R comprise any one or more than two groups of SSR1-SSR 11;
the sequence of SSR1-11 is as follows:
SSR1, Primer-F: TGGCATTTGACATGCAGGTG, respectively;
Primer-R：ACACCTCGGGCAAAAATCAA；
SSR2, Primer-F: TGGTGGTGTATATGCAGAATAGT, respectively;
Primer-R：CGCCAATATTAGATCCGGGCT；
SSR3, Primer-F: GAGTTGACACACACACGCAC, respectively;
Primer-R：CCAATTGGGCCGTTAAAGTGG；
SSR4, Primer-F: TTGAATGGGAGGAGTTCGGC, respectively;
Primer-R：ACCGATGCAACCTGGATGTC；
SSR5, Primer-F: CGAGTGGTCAGAGTGCCAAT, respectively;
Primer-R：TTCTCCATGAGGCGAAACCC；
SSR6, Primer-F: GGCTTTCACTGGGCTAGACA, respectively;
Primer-R：GGAGGGGCTTTTGTTGACCT；
SSR7, Primer-F: TCCCTTCTTCTCTCTCTCGCT, respectively;
Primer-R：AGGAAGAGTGTTTGAGTTAATACCT；
SSR8, Primer-F: AGTGGTCATCCAAACAAGGGA, respectively;
Primer-R：TGTGTACAAGAAAGCCAGCA；
SSR9, Primer-F: CCCAGCCTATGTGACTGACA;
Primer-R：ACTCTCATAAGTTGCTGATAGCCT；
SSR10, Primer-F: GGCGTCTTGGATCTCAGGTC, respectively;
Primer-R：TGGCTATGTGGTTTTCCTTCA；
SSR11, Primer-F: TGTGACGGAAATTTTGGTGCA, respectively;
Primer-R：GCCCAATAATCGCACAACGT。
2. the SSR-PCR reaction system for flame orchid, Hainan, according to claim 1, wherein the amplification procedure is: 3min at 94 ℃; 30s at 94 ℃, 30s at 56 ℃, 30s at 72 ℃ and 35 cycles; 30min at 72 ℃.
3. The use of a Hainan province Helianthus tuberosus SSR-PCR reaction system according to claim 1 or 2 in the SSR molecular markers and assisted breeding of Hainan province Helianthus tuberosus.
4. Use of a Hainan province flameray SSR-PCR reaction system according to claim 1 or 2 in the preparation of a detection kit.
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