CN110453005B - ISSR-SCAR marker for identifying low-temperature stimulated pleurotus geesteranus and application method thereof - Google Patents
ISSR-SCAR marker for identifying low-temperature stimulated pleurotus geesteranus and application method thereof Download PDFInfo
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Abstract
The invention discloses an ISSR-SCAR marker for identifying low-temperature stimulated pleurotus geesteranus and an application method thereof. The invention develops a pair of specific molecular marker primers Xiu1 (5 '-end primer sequence is shown as SEQ ID NO: 1; 3' -end primer sequence is shown as SEQ ID NO: 2), the primers are adopted to carry out PCR amplification on the genome DNA of a target material pleurotus geesteranus strain, if the target material pleurotus geesteranus strain belongs to a low-temperature stimulation type pleurotus geesteranus variety, a specific DNA fragment (the nucleotide sequence of the fragment is shown as SEQ ID NO: 4) with the size of 205bp can be amplified, and thus the low-temperature stimulation type pleurotus geesteranus strain and the non-low-temperature stimulation type pleurotus geesteranus strain can be effectively identified. The method disclosed by the invention is simple to operate, the accuracy rate reaches 100%, and the identification of pleurotus geesteranus varieties and the directional pleurotus geesteranus breeding progress can be accelerated.
Description
Technical Field
The invention relates to the technical field of biomolecular markers, in particular to an ISSR-SCAR marker for identifying low-temperature stimulation type pleurotus geesteranus and application thereof.
Background
The pleurotus geesteranus is delicious in taste and rich in nutrition, and is a kind of edible fungus deeply loved by consumers. The domestic cultivated pleurotus geesteranus strains are various and can be divided into high-temperature type varieties and low-temperature type varieties according to fruiting temperature; according to the need of low-temperature stimulation, the fruiting can be divided into natural fruiting type Pleurotus geesteranus (commonly called fake Pleurotus geesteranus) and low-temperature stimulation type Pleurotus geesteranus (also called out-of-season Pleurotus geesteranus or true Pleurotus geesteranus). The natural fruiting type pleurotus geesteranus has the characteristic that hyphae overgrow with fungus sticks and can grow without low-temperature stimulation after the hyphae are physiologically mature. The low-temperature stimulation type pleurotus geesteranus has the characteristics that hyphae grow full of bags, and the hyphae are required to be stimulated at a low temperature of below 15 ℃ after being physiologically mature to grow regularly. The requirements of the true pleurotus geesteranus and the false pleurotus geesteranus on low-temperature difference are different during fruiting, so that the cultivation management methods are different greatly. If the temperature characteristics required for the fruiting of pleurotus geesteranus are not known in the production process and improper cultivation management methods are adopted for blind planting, the problems of poor fruiting, no fruiting, low yield and the like are caused. Whether the pleurotus geesteranus fruiting needs low-temperature stimulation cannot be judged in the hypha growth stage, and only after the pleurotus geesteranus is cultivated and fruiting is judged. However, the cultivation period is long, the time and the labor are consumed, the fruiting is influenced by various climatic factors, and a judgment result has a large error. Therefore, a simple and rapid method for identifying whether pleurotus geesteranus belongs to a low-temperature-stimulated variety is urgently needed.
In recent years, many reports have been made on classification of genetic diversity and population of edible fungi and identification of strains by using molecular techniques. Such as von Willebrand, Luna, Linyuan, and bear fragrant, the SSR technology is used to classify the collected Pleurotus geesteranus strains into groups, i.e., identify the intimacy between the strains in the species. Although some learners find that the pleurotus geesteranus fruiting temperature is related to the molecular marker in pleurotus geesteranus population classification, the invention does not find the molecular marker related to the temperature, and does not have reports of identifying the molecular marker development of the low-temperature stimulated pleurotus geesteranus, thereby filling the blank in the field.
Disclosure of Invention
The invention aims to: aiming at the existing problems, the ISSR-SCAR marker for identifying the low-temperature stimulated pleurotus geesteranus and the application method thereof are developed, the molecular marker is used for detection, so that the low-temperature stimulated pleurotus geesteranus strain and the non-low-temperature stimulated pleurotus geesteranus strain can be effectively identified, the operation is simple, the accuracy rate reaches 100%, the identification of pleurotus geesteranus varieties and the directional breeding progress of pleurotus geesteranus can be accelerated, and technical support is provided for variety identification and breeding of the low-temperature stimulated pleurotus geesteranus strains in China.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
an ISSR-SCAR molecular marker primer for identifying low-temperature stimulated pleurotus geesteranus, wherein the molecular marker primer is Xiu1, and the sequence of the molecular marker primer is as follows:
the 5' -end primer sequence is shown as SEQ ID NO: 1, the preparation method comprises the following steps of;
the 3' -end primer sequence is shown as SEQ ID NO: 2, the preparation method is as follows.
The invention also provides a specific DNA fragment obtained by amplification in the low-temperature stimulation type pleurotus geesteranus genome DNA, wherein the nucleotide sequence of the fragment is shown as SEQ ID NO: 4, the method is described in the specification.
A method for identifying low-temperature stimulation type pleurotus geesteranus by using the molecular marker primers comprises the following specific steps: the molecular marker primer Xiu1 is adopted to carry out PCR amplification on the genome DNA of the target material pleurotus geesteranus strain, and the amplification product is detected, if the amplified product can be shown as SEQ ID NO: 4, the DNA fragment with the size of 205bp shows that the target material pleurotus geesteranus strain belongs to a low-temperature stimulation type pleurotus geesteranus variety.
Further, the low-temperature stimulation type pleurotus geesteranus is a pleurotus geesteranus variety which can be normally grown in a high-temperature season after being stimulated at a low temperature of below 15 ℃ for 12-14 hours, and is different from a pleurotus geesteranus variety which can be grown in a high-temperature season without low-temperature stimulation;
in the method, a 25 μ l system is adopted for PCR amplification; the 25 mu l reaction system is l mu l template DNA (30-50 ng), 12 mu l2 XEs Taq Master mix (Dye), 1 mu l Forward Primer (10 mu M), 1 mu l Reverse Primer (10 mu M), 10 mu l ddH2O, the total volume is 25 mu l;
the reaction conditions of the PCR amplification are as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30sec, annealing at 62 ℃ for 30sec, extension at 72 ℃ for l min, 35 cycles, and final extension at 72 ℃ for 10 min;
the PCR amplified product is a DNA band which shows amplification by detecting 1% agarose gel.
The application of the molecular marker primer is the application of the molecular marker primer in identifying Pleurotus geesteranus varieties and breeding anti-season Pleurotus geesteranus germplasm gene resources.
The invention also provides a method for screening the molecular marker primer, which comprises the following steps:
(1) strain classification and mycelium acquisition: carrying out antagonism test and fruiting test on 18 strains collected and cultivated pleurotus geesteranus germplasm resources, preliminarily dividing the 18 strains into two categories of low-temperature stimulated pleurotus geesteranus and non-low-temperature stimulated pleurotus geesteranus according to fruiting performance, and separating fruiting body tissues after fruiting to obtain pure mycelia;
(2) DNA extraction: extracting genome DNA of 18 bacterial strain mycelia by using a plant DNA extraction kit, and performing DNA quality detection by using 1% agarose gel;
(3) screening of universal primers: screening an ISSR universal primer 8 (shown in SEQ ID NO: 3) which has polymorphism for the two strains and has a specific band for low-temperature stimulated pleurotus geesteranus from 100 pairs of ISSR primers;
(4) recovering Taixiu 57-mesh fragment glue: selecting 4 strains (low-temperature stimulating strains: Taixiu 57 and Jinxiu; non-low-temperature stimulating strains: P54 and Xiuzhen 18) from 18 strains, carrying out PCR amplification on the DNA of the 4 strains by using the selected ISSR universal primer 8 (shown in SEQ ID NO: 3), carrying out agarose gel electrophoresis detection on the product, checking the specific DNA fragment of the low-temperature stimulating strain, and carrying out gel recovery on the specific fragment of the Taixiu 57 strain;
(5) cloning and sequencing the gel recovered DNA fragment, and designing and screening an SCAR primer according to a sequencing sequence to obtain a low-temperature stimulation type pleurotus geesteranus specific molecular marker primer Xiu 1;
(6) and carrying out SCAR molecular marker verification on the pleurotus geesteranus strain by using the screened primer Xiu 1.
Compared with the prior art, the invention has the beneficial effects that:
1. the research of the applicant finds out the molecular marker closely related to the pleurotus geesteranus strain and low-temperature stimulation fruiting.
2. The molecular marker primer developed by the invention has definite gene fragment position of the pleurotus geesteranus strain and convenient identification. The molecular marker is used for amplifying the genome DNA of the Pleurotus geesteranus strain, if 205bp of amplified fragment can be amplified, the strain is the low-temperature stimulation Pleurotus geesteranus strain.
3. The breeding selection target of the auxiliary low-temperature stimulation type pleurotus geesteranus (out-of-season pleurotus geesteranus) is definite, the efficiency is improved, and the cost is saved.
Drawings
FIG. 1-PCR amplification profiles of 18 Pleurotus geesteranus strains with ISSR primer 8 (shown in SEQ ID NO: 3); wherein M is DNA Marker DL2000, and the bands are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom in sequence; numbers 1-18 respectively represent 18 pleurotus geesteranus strains to be tested in sequence; the arrows show the specific DNA bands of the cold-stimulated strains.
FIG. 2-PCR amplification profiles of ISSR primer 8 (shown in SEQ ID NO: 3) against 4 representative Pleurotus geesteranus strains; wherein M is DNA Marker DL2000, and the bands are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom in sequence; from left to right, non-low temperature stimulating strains Nongxiu 18 and Xiuzhen P54, and low temperature stimulating strains Jinxiu and Taixiu 57, respectively; the arrow indicates the specific DNA band recovered from the mock gel.
FIG. 3-PCR product of ISSR primer 8 from Taixiu 57, DNA fragment obtained by gel recovery, and PCR product of recovered fragment clone; wherein M is DNA Marker DL2000, and the bands are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom in sequence; the arrow shows the specific DNA band obtained by amplification of the primer 8, the recovered DNA fragment and the cloned DNA fragment, which are consistent in size, indicating that the cloned DNA fragment is accurate.
FIG. 4-PCR amplification profiles of SCAR primer Xiu1 for 4 Pleurotus geesteranus strains; wherein M is DNA Marker DL2000, and the bands are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom in sequence; the numbering is from left to right respectively target material strain Taixiu 57, Jinxiu, Xiuzhen P54 and nongxiu 18; the DNA fragment with the size of 333bp shown by an arrow is a nonspecific fragment, and the DNA fragment with the size of 205bp shown by the arrow is a specific fragment of the low-temperature stimulation type pleurotus geesteranus variety.
FIG. 5-PCR amplification map of SCAR primer Xiu1 for 18 Pleurotus geesteranus strains; wherein M is DNA Marker DL2000, and the bands are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom in sequence; the numbers 1 to 18 sequentially represent the 18 strains tested, respectively. The DNA fragment with the size of 333bp shown by an arrow is a nonspecific fragment, and the DNA fragment with the size of 205bp shown by the arrow is a specific fragment of the low-temperature stimulation type pleurotus geesteranus variety.
Detailed Description
All of the features disclosed in this specification, or all of the steps in any method or process so disclosed, may be combined in any combination, except combinations of features and/or steps that are mutually exclusive.
Any feature disclosed in this specification (including any accompanying claims, abstract) may be replaced by alternative features serving equivalent or similar purposes, unless expressly stated otherwise. That is, unless expressly stated otherwise, each feature is only an example of a generic series of equivalent or similar features.
Example (b):
1. test strains
The collected 18 pleurotus geesteranus germplasm resources can be divided into two categories of low-temperature stimulated pleurotus geesteranus and non-low-temperature stimulated pleurotus geesteranus through an antagonistic test and a fruiting test, and see the table above. And (4) separating fruiting body tissues after fruiting to obtain pure mycelium.
1.1 procedure steps for molecular marker acquisition:
1.1.1 extraction of genomic DNA of Pleurotus geesteranus strains and quality detection
The DNA of 18 tested Pleurotus geesteranus strains was extracted according to the instructions of the plant-type DNA extraction kit of Kangji century Co. The OD260/OD280 of 18 strain DNAs measured by an ultraviolet spectrophotometer ranged from 1.8 to 1.9. Electrophoresis was performed on a 1% agarose gel (containing the DNA dye from Goldview) at 120v for 30min, and the image was taken on a gel imaging system with clear and complete bands and qualified DNA samples.
1.1.2 screening of ISSR primers
ISSR primer 8 (shown in SEQ ID NO: 3) which has polymorphism for the two strains and has specific band for low-temperature stimulated Pleurotus geesteranus was selected from 100 ISSR universal primers. The map is shown in figure 1.
ISSR-PCR amplification reaction system (2 XEs Taq Master Mix: l μ l Template DNA (30 ng), 12 μ l2 XEs Taq Master Mix (Dye), 1 μ l Primer (10 μ M), finally with ddH2And O, supplementing the total volume to 25 mu l.
ISSR amplification reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s; annealing at 52 ℃ for 30 s; lmin extension at 72 ℃, 35 cycles, and finally 10 min extension at 72 ℃.
1.1.3 gel recovery of ISSR specific target fragments
4 representative strains (low temperature stimulated strains Taixiu 57 and Jinxiu; non-low temperature stimulated strains P54 and Xiuzhen 18) were selected from 18 test strains, and genomic DNAs of the 4 strains were PCR-amplified using ISSR primer 8 (shown in SEQ ID NO: 3) and subjected to agarose gel electrophoresis. And (3) checking specific fragments of the same strain on the agarose gel under ultraviolet light, and recovering the gel from the specific fragments.
The band spectrum of the recovered glue is as follows: the position of the DNA fragment with the size of 1312bp of the Taixiu 57 is shown in the map of FIG. 2.
Gel recovery was performed according to the instructions of a general-purpose DNA purification recovery cassette (Tiangen Biochemical technology Co., Ltd.).
The specific steps of gum recovery are as follows (description step):
(1) add 500. mu.l of equilibration fluid BL to the adsorption column (adsorption column into collection tube), centrifuge at 12,000 rpm for 1min, remove waste from the collection tube, and replace the adsorption column back into the collection tube.
(2) A single band of the target DNA was cut from the agarose gel and placed in a clean centrifuge tube and weighed.
(3) Add equal volume of solution PC (100. mu.l of PC solution if the gel weight is 0.1 g and the volume can be considered as 100. mu.l) to the gel mass, and place in a 50 ℃ water bath for about 10 min while gently turning the centrifuge tube up and down to ensure that the gel mass is sufficiently dissolved.
(4) Adding the solution obtained in the previous step into an adsorption column CB2 (the adsorption column is placed into a collection tube), centrifuging at 12,000 rpm for 1min, pouring off the waste liquid in the collection tube, and placing the adsorption column CB2 into the collection tube.
(5) Adding 600 μ l rinsing solution PW into adsorption column CB2, centrifuging at 12,000 rpm for 1min, pouring off waste liquid in the collection tube, and placing adsorption column CB2 into the collection tube.
(6) And (5) repeating the operation step.
(7) The adsorption column CB2 was placed in a collection tube and centrifuged at 12,000 rpm (-13, 400 Xg) for 2 min to remove the rinse as much as possible. The column was left at room temperature for several minutes and completely air dried.
(8) Placing adsorption column CB2 into a clean centrifuge tube, adding a proper amount of elution buffer EB into the middle position of the adsorption film, and standing at room temperature for 2 min. Centrifugation is carried out at 12,000 rpm (-13, 400 Xg) for 2 min, and DNA solution is collected and stored at-20 ℃ for later use.
And (3) detecting a glue recovery product: electrophoresis detection is carried out on the Taixiu 57 ISSR-PCR product and the glue recovery product by using 1% agarose gel, and the bands to be recovered in the ISSR-PCR product are consistent with the sites of the glue recovery product, which indicates that the glue recovery product is correct.
1.1.4 cloning and characterization of recovered fragment DNA
(1) The purified DNA fragment was recovered and ligated to the Vector pMD18-T Vector according to the instructions of the ligation kit (TaKaRa kit). Connecting a reaction system: mu.l of pMD19-T Vector, 4. mu.l of recovered DNA solution, 5. mu.l of solution I. The system is evenly mixed after being prepared, and can be directly used for transformation after being connected for 12 hours at 16 ℃.
(2) And (3) transformation:
a) taking out the competent cells (DH 5 a) preserved at-80 deg.C, and thawing on ice;
b) adding the connecting product, slightly rotating the centrifuge tube to uniformly mix the contents, and carrying out ice bath for 30 min;
c) placing the centrifuge tube at 42 deg.C for 60-90 s, and rapidly ice-cooling for 2-3 min;
d) adding 500 mul LB culture medium (without antibiotics) into each centrifuge tube, mixing uniformly, and placing in a shaking table at 37 ℃ for shaking culture for 45 min (150 revolutions per minute);
e) mixing the contents of the centrifuge tube uniformly, sucking 100-400 microlitres, adding into IPTG, X-Gal and Amp LB flat plates, and coating uniformly. The plate was left at room temperature until the liquid was completely absorbed, inverted and incubated overnight at 37 ℃.
2.1 detection of Positive clones (colony PCR)
The white colonies were picked from the plate medium with sterilized toothpicks and dipped in 10. mu.l of sterilized ddH2And 0, uniformly mixing, and taking 2 mu l of the mixture for a template for PCR amplification.
System of PCR reaction:
| Reaction Component | Concentration | Volume （μl） | |
| 2×Es Taq | 2X | 10 | |
| M13（47） | 10μM | 0.5 | |
| M13（48） | 10μM | 0.5 | |
| white single colony | Small amount of | ||
| ddH2O | 9 | ||
| Total | 20 |
PCR amplification reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s; annealing at 56 ℃ for 30 s; extension at 72 ℃ for 90 s, 25 cycles, and finally extension at 72 ℃ for 5 min.
3.1 clone identification and detection.
The Taixiu 57 ISSR-PCR product, the gel recovery product and the clone-PCR product were subjected to electrophoresis detection with 1.0% agarose gel at 120V for 30 min. The sites of the three product amplification bands are consistent, which indicates that the cloning of the positive clone is correct. The map is shown in FIG. 3.
4.1 clonal sequencing analysis
Sequencing the detected positive clones, wherein the sequencing company is Guangzhou Ongke Biotechnology limited, successfully sequencing the sequenced sequences, and the size of the obtained sequence fragment is 1312bp (as shown in SEQ ID NO: 6).
5.1 design of SCAR primers
The sequence of the clone sequencing of the specific DNA fragment amplified according to ISSR primer 8 is SEQ ID NO: 6, designing 10 pairs of SCAR primers, and screening out SCAR primers Xiu1 with specificity (as shown in SEQ ID NO: 1 and SEQ ID NO: 2. after the SCAR primers are designed, carrying out primer synthesis.
2. The flow steps of the verification of the molecular marker are as follows:
(1) the screened SCAR primer Xiu1 is used for carrying out PCR amplification verification on the genomic DNA of 4 strains such as Taixiu 57, Jinxiu, P54, Xiuzhen 18 and the like. All 4 strains amplified a 333bp fragment (as shown in SEQ ID NO: 5), and both the low-temperature-stimulated strains Taixiu 57 and Jinxiu amplified a 205bp fragment (as shown in SEQ ID NO: 4), while the non-low-temperature-stimulated strains P54 and Xiuzhen 18 failed to amplify a 205bp fragment. The amplification profile is shown in FIG. 4.
(2) PCR amplification verification was performed on 18 Pleurotus geesteranus strain genomic DNAs using Xiu1 primers. 18 strains can amplify fragments with the size of 333bp (shown as SEQ ID NO: 5), 8 strains such as low-temperature stimulated strain Taixiu 57 and Jinxiu can also amplify fragments with the size of 205bp (shown as SEQ ID NO: 4), and 10 strains such as non-low-temperature stimulated strain P54 and Xiuzhen 18 cannot amplify fragments with the size of 205 bp. The amplification pattern is shown in FIG. 5.
The above examples are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but not to be construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.
Sequence listing
<110> institute of microbiology of Guangxi Zhuang autonomous region academy of agricultural sciences
<120> ISSR-SCAR marker for identifying low-temperature stimulation type pleurotus geesteranus and application method thereof
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Claims (5)
1. An ISSR-SCAR molecular marker primer for identifying low-temperature stimulated pleurotus geesteranus is characterized in that the molecular marker primer is Xiu1, and the sequence of the molecular marker primer is as follows:
the 5' -end primer sequence is shown as SEQ ID NO: 1, the preparation method comprises the following steps of;
the 3' -end primer sequence is shown as SEQ ID NO: 2, the preparation method is as follows.
2. A specific DNA fragment capable of being amplified in low-temperature stimulation type Pleurotus geesteranus genome DNA is characterized in that the size of the fragment is 205bp, and the nucleotide sequence is shown as SEQ ID NO: 4, the method is described in the specification.
3. The method for identifying low-temperature-stimulated pleurotus geesteranus by using the molecular marker primer as claimed in claim 1, which comprises the following specific steps: performing PCR amplification on the genomic DNA of the target material pleurotus geesteranus strain by using a molecular marker primer Xiu1, detecting an amplification product, and if the DNA fragment of claim 2 can be amplified, indicating that the target material pleurotus geesteranus strain belongs to a low-temperature stimulation type pleurotus geesteranus variety.
4. The method of claim 3, wherein the PCR amplification is performed using a 25 μ l system; the 25 mu l reaction system is 1 mu l of Template DNA containing 30-50ng, 12 mu l of 2 XEs Taq Master mix (Dye), Forward Primer with the 1 mu l concentration of 10 mu M, Reverse Primer with the 1 mu l concentration of 10 mu M, and 10 mu l ddH2O, the total volume is 25 mu l;
the reaction conditions of the PCR amplification are as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30sec, annealing at 62 ℃ for 30sec, extension at 72 ℃ for l min, 35 cycles, and final extension at 72 ℃ for 10 min;
the PCR amplified product is a DNA band which shows amplification by detecting 1% agarose gel.
5. The use of the molecular marker primer of claim 1, wherein said molecular marker primer is used for breeding of Pleurotus geesteranus germplasm gene resources cultivated in out-of-season.
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