CN114292938A - SSR fingerprint identification method and construction method of stropharia rugoso-annulata strain - Google Patents
SSR fingerprint identification method and construction method of stropharia rugoso-annulata strain Download PDFInfo
- Publication number
- CN114292938A CN114292938A CN202111486742.6A CN202111486742A CN114292938A CN 114292938 A CN114292938 A CN 114292938A CN 202111486742 A CN202111486742 A CN 202111486742A CN 114292938 A CN114292938 A CN 114292938A
- Authority
- CN
- China
- Prior art keywords
- ssr
- strain
- fingerprint
- annulata
- stropharia rugoso
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of strain quality identification, and particularly relates to a fingerprint identification method and a construction method of a golden stropharia rugoso-annulata strain, which are characterized in that 6 pairs of SSR markers are constructed to form a fingerprint, wherein the fingerprint comprises the following primers: DQGGSSR007, DQGGSSR031, DQGGSSR034, DQGGSSR037, DQGGSSR065, DQGGSSR 088. The construction method comprises the following steps: s1, hypha culture: inoculating the golden stropharia rugoso-annulata strain to a potato glucose agar solid medium (PDA), culturing at 25 ℃ for 15 days, and collecting hypha; s2, extracting genome DNA: extracting the genome DNA of the hyphae by using a TSINGKE plant DNA extraction kit (TSP101-200), and detecting the concentration and the quality of the total genome DNA by using an ultraviolet spectrophotometry: compared with conventional morphological detection, antagonism test and fruiting test, the method has the advantages of short detection time, high accuracy and good repeatability. The operation time required for detection is within 24h, while the time required for a conventional antagonism test is at least two weeks, and the time required for a fruiting test is at least 3 months.
Description
Technical Field
The invention belongs to the technical field of strain quality identification, and particularly relates to an SSR fingerprint identification method and a construction method of a stropharia rugoso-annulata strain.
Background
Stropharia rugosoannulata Farl. ex Murrill, also known as Stropharia rugosoannulata, Gliocladium viniferum, etc., and its trade name is Tricholoma rubrum. Numerous studies show that the stropharia rugoso-annulata fruiting body contains rich nutritional ingredients such as protein, vitamins, minerals and polysaccharide, is one of the famous edible fungi artificially cultivated in countries in Europe and America, and is also one of the edible fungi recommended to be cultivated to developing countries by the Food and Agriculture Organization (FAO) of United nations. Secondly, the stropharia rugoso-annulata polysaccharide has the effects of preventing coronary heart disease, helping digestion, relieving mental fatigue and the like, and the inhibition rate of the stropharia rugoso-annulata polysaccharide on S-180 sarcoma of mice and Ehrlich carcinoma is more than 70%.
The contribution rate of the high-quality strains in the yield and the quality of the golden stropharia rugoso-annulata is very important. After the excellent strains which are verified by tests are obtained, the strains of the excellent strains need to be accurately defined so as to conveniently distinguish different strains of the strains, thereby avoiding the phenomenon that the bred excellent strains are mixed with other strains in the production, operation and market circulation processes to cause synonyms and economic loss brought to production enterprises and influence the rapid development of the stropharia rugoso-annulata industry. Therefore, the development of a precise and effective stropharia rugoso-annulata variety identification system by utilizing modern molecular biology technology is very important work.
The contribution rate of the high-quality strains in the yield and the quality of the golden stropharia rugoso-annulata is very important. After the excellent strains which are verified by tests are obtained, the strains of the excellent strains need to be accurately defined so as to conveniently distinguish different strains of the strains, thereby avoiding the phenomenon that the bred excellent strains are mixed with other strains in the production, operation and market circulation processes to cause synonyms and economic loss brought to production enterprises and influence the rapid development of the stropharia rugoso-annulata industry. Therefore, the development of a precise and effective stropharia rugoso-annulata variety identification system by utilizing modern molecular biology technology is very important work.
The SSR marker technology is widely used for the researches of genetic map construction, target gene calibration, fingerprint map drawing, variety identification, pedigree analysis, inter-population genetic distance analysis, evolution, genetic diversity and the like. The SSR molecular marker has the advantages of rich quantity, wide distribution, site specificity, high polymorphism, good repeatability and the like, and can be used for identifying the variation among different varieties of the same species with high precision.
Disclosure of Invention
Compared with conventional morphological detection, antagonism test and fruiting test, the SSR fingerprint spectrum identification method and the construction method of the stropharia rugoso-annulata strain have the advantages of short detection time, high accuracy and good repeatability.
The SSR marker fingerprint spectrum of the stropharia rugoso-annulata ZJJQG001 strain consists of 6 pairs of SSR markers, is an SSR primer developed based on simple repetitive sequence fragments of a genome of the stropharia rugoso-annulata, and has the advantages of good amplification band pattern and high repeatability. Detailed information of SSR marker primers is shown in FIG. 8.
The technical scheme adopted by the invention for solving the technical problems is as follows: the identification method of the SSR fingerprint of the stropharia rugoso-annulata strain is characterized in that the fingerprint constructed by 6 pairs of SSR markers is composed of the following primers: DQGGSSR007, DQGGSSR031, DQGGSSR034, DQGGSSR037, DQGGSSR065, DQGGSSR 088: the construction method comprises the following steps:
s1, hypha culture: inoculating the golden stropharia rugoso-annulata strain to a potato glucose agar solid medium (PDA), culturing at 25 ℃ for 15 days, and collecting hypha;
s2, extracting genome DNA: extracting the genome DNA of the hyphae by using a TSINGKE plant DNA extraction kit (TSP101-200), and detecting the concentration and the quality of the total genome DNA by using an ultraviolet spectrophotometry;
s3, detection of SSR molecular markers: carrying out PCR amplification of SSR markers on the extracted DNA;
s4, electrophoresis detection: carrying out agarose gel electrophoresis on the amplified PCR product to obtain an identification gel picture, determining the concentration of the template through the gel picture, and adding water to dilute to the concentration required by capillary electrophoresis;
s5, carrying out capillary electrophoresis detection;
s6, GeneMapper data analysis.
Preferably, the specific method for extracting the genome DNA of the hyphae by the kit method in S2 comprises the following steps:
s1, placing Spin Colu2 in a Collection Tube, adding 250 mu l of Buffer BL, and centrifuging at 12000rpm/min for 1min to activate the silica gel membrane;
and S2, taking a hypha sample, adding liquid nitrogen, and fully grinding. Grinding, placing in a 1.5ml centrifuge tube, adding 400 μ l Buffer gP1, vortex oscillating for 1min, and water bath at 65 ℃ for 10-30 min, wherein the mixture can be taken out, inverted and mixed uniformly for full cracking;
s3, adding 150 mu l of Buffer gP2, carrying out vortex oscillation for 1min, and carrying out ice bath for 5 min;
s4, centrifuging at 12000rpm for 5min, and transferring the supernatant into a new centrifuge tube;
s5, adding absolute ethyl alcohol with the same volume as the supernatant, immediately and fully oscillating and uniformly mixing, transferring all liquid into Spin Colu2, centrifuging at 12,000rpm/min for 30S, and discarding waste liquid;
s6, adding 500 mu l of Buffer Pw (absolute ethyl alcohol is added before use) into Spin Colu2, centrifuging at 12000rpm/min for 30S, and discarding the waste liquid;
s7, adding 500 mu l of WasN006# Buffer (absolute ethyl alcohol is added before use) into Spin Colu2, centrifuging at 12000rpm/min for 30S, and discarding the waste liquid;
s8, repeating the operation step S7;
s9, putting Spin Colu2 back into the Collection Tube, centrifuging at 12,000rpm/min for 2min, uncovering and airing for 1 min;
s10, taking out Spin Colu2, putting the Spin Colu2 into a clean centrifugal tube, adding 50-100 mu l of TE Buffer (preheating the TE Buffer at 65 ℃) at the center of an adsorption film, standing for 2min at 20-25 ℃, and centrifuging for 2min at 12,000 rpm/min;
s11, taking 2ul of DNA stock solution for 1.2% agarose gel electrophoresis detection, taking 2ul of DNA stock solution for NanoDrop spectrophotometer to detect concentration and quality, and storing the residual DNA at-20 ℃.
Preferably, the PCR amplification system in S3 is: total volume 20ul, including: pomaceae TSE 101-gold Mix (green)16.45uL, 10uM Tag DNase 1.2uL, 10umol/L SSR marker forward primer and reverse primer 1.2uL respectively, and template DNA 1 uL.
Preferably, the PCR reaction conditions: 2min at 98 ℃; 10second at 98 ℃, 10second at 60 ℃, 10second at 72 ℃, 35 cycles; 5min at 72 ℃.
Preferably, the electrophoresis in S4 is carried out by adding 2ul of sample and 6ul of bromophenol blue, the voltage is 300V, and the time is 12 minutes.
Preferably, the capillary electrophoresis in S5 is specifically:
s1, mixing ABI HiDi Formamide buffer solution with GeneScan 500LIZ Size Standard internal Standard reagent according to the proportion of 130: 1, mixing to prepare mix;
s2, loading mix in a 96-well reaction plate, and adding 10ul mix in each well;
s3, correspondingly adding 0.5ul of sample template into a 96-well plate, and stopping when the sample template is centrifuged to 4000 rpm;
s4, heating the mixing plate by a metal bath heater at 95 ℃ for pre-denaturation for 5 minutes, taking out and immediately placing the mixing plate at-20 ℃;
s5, cooling, taking out, centrifuging at 4000rpm, thawing, and mixing uniformly;
s6, capillary electrophoresis using 3730 sequencer.
Preferably, the data analysis in S6 specifically includes: importing the obtained original data file into analysis software genemapperID 4.1, and performing group structure analysis, clustering and heterozygosity analysis and genetic diversity analysis by using software such as Popgen32 and posts-1 _2_ 30; the analysis includes allele (Na), effective allele (Ne), shannon index (I), polymorphism information index (PIC), observed heterozygosity (Ho), expected heterozygosity (He), major allele frequency (MP), and inbreeding coefficient (Fis), and the like.
An SSR fingerprint identification method of a stropharia rugoso-annulata strain is suitable for the construction method of the stropharia rugoso-annulata strain, and comprises the following steps: screening a large number of SSR primers, determining the number and numbering of allelic fragments amplified by 6 pairs of SSR primers in each stropharia rugosoannulata cultivar by performing banding amplification on the SSR primers of 13 collected stropharia rugosoannulata cultivars (figure 9), and effectively identifying the ZJJQG001 strain in 13 collected cultivars by the combination of the numbering of different SSR allelic sites; the relative molecular weight of the amplified allelic locus of each SSR primer can be determined by analyzing capillary electrophoresis combined software, the strain with the specific SSR allelic fragment of the strain ZJJQG001 is Stropharia rugosoannulata ZJQG 001, and the serial number combination of the strain is 2/(3+ 5)/1/1/1/1.
The invention has the beneficial effects that:
1. the invention provides a fingerprint identification method and a construction method of a golden stropharia rugoso-annulata strain. The operation time required for detection is within 24h (including genome DNA extraction, PCR amplification, electrophoresis analysis and data analysis), while the time required for conventional antagonism test is at least two weeks, and the time required for fruiting test is at least 3 months.
2. The invention provides a fingerprint identification method and a construction method of golden stropharia rugoso-annulata strains, wherein 13 stropharia rugoso-annulata cultivation strains (comprising DQG1-12 and ZJJQG001) collected by the method have the specificity of golden stropharia rugoso-annulata ZJQG 001 strains, and have good application prospect.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the invention without limiting the invention. In the drawings:
FIG. 1 is a peak diagram of the relative molecular weights of the allelic sites obtained by sequentially detecting the primer DQGGSSR007 in the selected Stropharia rugosoannulata ZJJQG001 and several Stropharia rugosoannulata cultivars;
FIG. 2 is a peak diagram of the relative molecular weights of the allelic sites obtained by sequentially detecting the primers DQGGSSR031 in the selected stropharia rugoso-annulata ZJQG 001 and several stropharia rugoso-annulata cultivars;
FIG. 3 is a peak diagram of the relative molecular weights of the allelic sites obtained by sequentially detecting the primer DQGGSSR034 in the selected Stropharia rugoso-annulata ZJJQG001 and several Stropharia rugoso-annulata cultivars;
FIG. 4 is a peak diagram of the relative molecular weights of the allelic sites obtained by sequentially detecting the primer DQGGSSR037 in the selected Stropharia rugoso-annulata ZJJQG001 and several Stropharia rugoso-annulata cultivars;
FIG. 5 is a peak diagram of the relative molecular weights of the allelic sites obtained by sequentially detecting the primer DQGGSSR065 in the selected stropharia rugoso-annulata ZJJQG001 and several stropharia rugoso-annulata cultivars;
FIG. 6 is a peak diagram of the relative molecular weights of the allelic sites obtained by the sequential detection of the primers DQGGSSR088 in the selected Stropharia rugoso-annulata ZJJQG001 and several Stropharia rugoso-annulata cultivars respectively;
FIG. 7 is a UPGMA clustering tree of 13 stropharia rugoso-annulata constructed based on SSR molecular markers;
FIG. 8 is a list of SSR marker primer information;
FIG. 9 is a summary table of the allele fraction information of primer amplifications;
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.
Referring to fig. 1 to 9, a method for constructing stropharia rugosoannulata strain includes the following steps:
s1, hypha culture, namely transferring the stropharia rugoso-annulata strain to a potato dextrose agar solid medium (PDA), and collecting hypha after culturing for 15d at 25 ℃;
extraction of S2 genomic DNA: extracting the genome DNA of the hyphae by using a TSINGKE plant DNA extraction kit (TSP101-200), and detecting the concentration and the quality of the total genome DNA by using an ultraviolet spectrophotometry.
The specific method for extracting the genome DNA of the hyphae by the kit method comprises the following steps:
s1, placing Spin Colu2 in a Collection Tube, adding 250 mu l of Buffer BL, and centrifuging at 12000rpm/min for 1min to activate the silica gel membrane;
and S2, taking a hypha sample, adding liquid nitrogen, and fully grinding. Grinding, placing in a 1.5ml centrifuge tube, adding 400 μ l Buffer gP1, vortex oscillating for 1min, and water bath at 65 ℃ for 10-30 min, wherein the mixture can be taken out, inverted and mixed uniformly for full cracking;
s3, adding 150 mu l of Buffer gP2, carrying out vortex oscillation for 1min, and carrying out ice bath for 5 min;
s4, centrifuging at 12000rpm for 5min, and transferring the supernatant into a new centrifuge tube;
s5, adding absolute ethyl alcohol with the same volume as the supernatant, immediately and fully oscillating and uniformly mixing, transferring all liquid into Spin Colu2, centrifuging at 12,000rpm/min for 30S, and discarding waste liquid;
s6, adding 500 mu l of Buffer Pw (absolute ethyl alcohol is added before use) into Spin Colu2, centrifuging at 12000rpm/min for 30S, and discarding the waste liquid;
s7, adding 500 mu l of WasN006# Buffer (absolute ethyl alcohol is added before use) into Spin Colu2, centrifuging at 12000rpm/min for 30S, and discarding the waste liquid;
s8, repeating the operation step S7;
s9, putting Spin Colu2 back into the Collection Tube, centrifuging at 12,000rpm/min for 2min, uncovering and airing for 1 min;
s10, taking out Spin Colu2, putting the Spin Colu2 into a clean centrifugal tube, adding 50-100 mu l of TE Buffer (preheating the TE Buffer at 65 ℃) at the center of an adsorption film, standing for 2min at 20-25 ℃, and centrifuging for 2min at 12,000 rpm/min;
s11, taking 2ul of DNA stock solution for 1.2% agarose gel electrophoresis detection, taking 2ul of DNA stock solution for detecting the concentration and quality by a NanoDrop spectrophotometer, and storing the residual DNA at-20 ℃;
and detecting the SSR molecular marker, namely performing PCR amplification of the SSR marker on the extracted DNA.
The PCR amplification system is as follows: total volume 20ul, including: pomaceae TSE 101-gold Mix (green)16.45uL, 10uM Tag DNase 1.2uL, 10umol/L SSR marker forward primer and reverse primer 1.2uL respectively, and template DNA 1 uL.
The PCR reaction conditions are as follows: 2min at 98 ℃; 10second at 98 ℃, 10second at 60 ℃, 10second at 72 ℃, 35 cycles; 5min at 72 ℃.
And (3) performing agarose gel electrophoresis (2ul of sample +6ul of bromophenol blue) on the amplified PCR product, obtaining an identification gel picture under the voltage of 300V for 12 minutes, determining the concentration of the template through the gel picture, and adding water to dilute to the concentration required by capillary electrophoresis. .
The capillary tube electrophoresis is specifically as follows:
s1, mixing ABI HiDi Formamide buffer solution with GeneScan 500LIZ Size Standard internal Standard reagent according to the proportion of 130: 1, mixing to prepare mix;
s2, loading mix in a 96-well reaction plate, and adding 10ul mix in each well;
s3, correspondingly adding 0.5ul of sample template into a 96-well plate, and stopping when the sample template is centrifuged to 4000 rpm;
s4, heating the mixing plate by a metal bath heater at 95 ℃ for pre-denaturation for 5 minutes, taking out and immediately placing the mixing plate at-20 ℃;
s5, cooling, taking out, centrifuging at 4000rpm, thawing, and mixing uniformly;
s6, performing capillary electrophoresis by using a 3730 sequencer;
the method comprises the steps of carrying out PCR amplification and capillary electrophoresis on a stropharia rugosoannulata ZJJQG001 strain by adopting 6 pairs of SSR primers, and finding out coincidence codes which are DQGGSSR007, DQGGSSR031, DQGGSSR034, DQGGSSR037, DQGGSSR065 and DQGGSSR088 by analyzing allele (Na), effective allele (Ne), shannon index (I), polymorphism information index (PIC), observed heterozygosity (Ho), expected heterozygosity (He), main allele frequency (MP), inbreeding coefficient (Fis) and the like and combining relative molecular weight peak maps of allelic sites, wherein the corresponding band types are as follows: 2/(3+5)/1/1/1/1, namely determining the strain to be stropharia rugoso-annulata ZJJQG 001. To ensure the accuracy of the identification, three replicates were recommended.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed.
Claims (9)
1. The identification method of the SSR marker fingerprint of the stropharia rugoso-annulata strain is characterized in that 6 pairs of SSR markers comprise DQGGSSR007, DQGGSSR031, DQGGSSR034, DQGGSSR037, DQGGSSR065 and DQGGSSR088, and the specific sequences are as follows:
the corresponding combination of the belt type numbers is as follows: 2/(3+5)/1/1/1/1.
2. The method for constructing the SSR fingerprint of the stropharia rugosoannulata strain according to claim 1, which comprises the following steps:
s1, hypha culture: inoculating the golden stropharia rugoso-annulata strain to a potato glucose agar solid medium (PDA), culturing at 25 ℃ for 15 days, and collecting hypha;
s2, extracting genome DNA: extracting the genome DNA of the hyphae by using a TSINGKE plant DNA extraction kit (TSP101-200), and detecting the concentration and the quality of the total genome DNA by using an ultraviolet spectrophotometry;
s3, detection of SSR molecular markers: carrying out PCR amplification of SSR markers on the extracted DNA;
s4, electrophoresis detection: carrying out agarose gel electrophoresis on the amplified PCR product to obtain an identification gel picture, determining the concentration of the template through the gel picture, and adding water to dilute to the concentration required by capillary electrophoresis;
s5, carrying out capillary electrophoresis detection;
s6, GeneMapper data analysis.
3. The method for constructing the SSR fingerprint of the stropharia rugoso-annulata strain according to claim 2, wherein the SSR fingerprint comprises the following steps: the specific method for extracting the genome DNA of the hyphae by the kit method in the S2 comprises the following steps:
s1, placing Spin Colu2 in a Collection Tube, adding 250 mu l of Buffer BL, and centrifuging at 12000rpm/min for 1min to activate the silica gel membrane;
and S2, taking a hypha sample, adding liquid nitrogen, and fully grinding. Grinding, placing in a 1.5ml centrifuge tube, adding 400 μ l Buffer gP1, vortex oscillating for 1min, and water bath at 65 ℃ for 10-30 min, wherein the mixture can be taken out, inverted and mixed uniformly for full cracking;
s3, adding 150 mu l of Buffer gP2, carrying out vortex oscillation for 1min, and carrying out ice bath for 5 min;
s4, centrifuging at 12000rpm for 5min, and transferring the supernatant into a new centrifuge tube;
s5, adding absolute ethyl alcohol with the same volume as the supernatant, immediately and fully oscillating and uniformly mixing, transferring all liquid into Spin Colu2, centrifuging at 12,000rpm/min for 30S, and discarding waste liquid;
s6, adding 500 mu l of Buffer Pw (absolute ethyl alcohol is added before use) into Spin Colu2, centrifuging at 12000rpm/min for 30S, and discarding the waste liquid;
s7, adding 500 mu l of WasN006# Buffer (absolute ethyl alcohol is added before use) into Spin Colu2, centrifuging at 12000rpm/min for 30S, and discarding the waste liquid;
s8, repeating the operation step S7;
s9, putting Spin Colu2 back into the Collection Tube, centrifuging at 12,000rpm/min for 2min, uncovering and airing for 1 min;
s10, taking out Spin Colu2, putting the Spin Colu2 into a clean centrifugal tube, adding 50-100 mu l of TE Buffer (preheating the TE Buffer at 65 ℃) at the center of an adsorption film, standing for 2min at 20-25 ℃, and centrifuging for 2min at 12,000 rpm/min;
s11, taking 2ul of DNA stock solution for 1.2% agarose gel electrophoresis detection, taking 2ul of DNA stock solution for NanoDrop spectrophotometer to detect concentration and quality, and storing the residual DNA at-20 ℃.
4. The method for constructing the SSR fingerprint of the stropharia rugoso-annulata strain according to claim 2, wherein the SSR fingerprint comprises the following steps: the PCR amplification system in S3 is: total volume 20ul, including: pomaceae TSE 101-gold Mix (green)16.45uL, 10uM Tag DNase 1.2uL, 10umol/L SSR marker forward primer and reverse primer 1.2uL respectively, and template DNA 1 uL.
5. The method for constructing the SSR fingerprint of the stropharia rugoso-annulata strain according to claim 2, wherein the SSR fingerprint comprises the following steps: the PCR reaction conditions are as follows: 2min at 98 ℃; 10second at 98 ℃, 10second at 60 ℃, 10second at 72 ℃, 35 cycles; 5min at 72 ℃.
6. The method for constructing the SSR fingerprint of the stropharia rugoso-annulata strain according to claim 2, wherein the SSR fingerprint comprises the following steps: the sample adding amount of electrophoresis in the S4 is 2ul of sample and 6ul of bromophenol blue, the voltage is 300V, and the time is 12 minutes.
7. The method for constructing the SSR fingerprint of the stropharia rugoso-annulata strain according to claim 2, wherein the SSR fingerprint comprises the following steps: the electrophoresis of the capillary tube in the S5 is specifically as follows:
s1, mixing ABI HiDi Formamide buffer solution with GeneScan 500LIZ Size Standard internal Standard reagent according to the proportion of 130: 1, mixing to prepare mix;
s2, loading mix in a 96-well reaction plate, and adding 10ul mix in each well;
s3, correspondingly adding 0.5ul of sample template into a 96-well plate, and stopping when the sample template is centrifuged to 4000 rpm;
s4, heating the mixing plate by a metal bath heater at 95 ℃ for pre-denaturation for 5 minutes, taking out and immediately placing the mixing plate at-20 ℃;
s5, cooling, taking out, centrifuging at 4000rpm, thawing, and mixing uniformly;
s6, capillary electrophoresis using 3730 sequencer.
8. The method for constructing the SSR fingerprint of the stropharia rugoso-annulata strain according to claim 2, wherein the SSR fingerprint comprises the following steps: the data analysis in S6 specifically includes: the obtained original data file is imported into analysis software genemapper ID 4.1, and group structure analysis, clustering and heterozygosity analysis and genetic diversity analysis are carried out by using software such as Popgen32 and posts-1 _2_ 30. The analysis includes allele (Na), effective allele (Ne), shannon index (I), polymorphism information index (PIC), observed heterozygosity (Ho), expected heterozygosity (He), major allele frequency (MP), and inbreeding coefficient (Fis), and the like.
9. An identification method of SSR fingerprint of golden stropharia rugoso-annulata strain is characterized in that: the identification method is suitable for the construction method of the SSR fingerprint of the stropharia rugosoannulata strain as described in the claims 1-8, and comprises the following steps: screening a large number of SSR primers, determining the number and numbering of allelic fragments amplified by 6 pairs of SSR primers in each stropharia rugosoannulata cultivar by performing banding amplification on the SSR primers of 13 collected stropharia rugosoannulata cultivars (figure 9), and effectively identifying the ZJJQG001 strain in 13 collected cultivars by the combination of the numbering of different SSR allelic sites; the relative molecular weight of the amplified allelic locus of each SSR primer can be determined by analyzing capillary electrophoresis combined software, the strain with the specific SSR allelic fragment of the strain ZJJQG001 is Stropharia rugosoannulata ZJQG 001, and the serial number combination of the strain is 2/(3+ 5)/1/1/1/1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111486742.6A CN114292938A (en) | 2021-12-07 | 2021-12-07 | SSR fingerprint identification method and construction method of stropharia rugoso-annulata strain |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111486742.6A CN114292938A (en) | 2021-12-07 | 2021-12-07 | SSR fingerprint identification method and construction method of stropharia rugoso-annulata strain |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114292938A true CN114292938A (en) | 2022-04-08 |
Family
ID=80964841
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111486742.6A Pending CN114292938A (en) | 2021-12-07 | 2021-12-07 | SSR fingerprint identification method and construction method of stropharia rugoso-annulata strain |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114292938A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112410226A (en) * | 2020-09-10 | 2021-02-26 | 云南菌视界生物科技有限公司 | Golden stropharia rugoso-annulata strain |
-
2021
- 2021-12-07 CN CN202111486742.6A patent/CN114292938A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112410226A (en) * | 2020-09-10 | 2021-02-26 | 云南菌视界生物科技有限公司 | Golden stropharia rugoso-annulata strain |
| CN112410226B (en) * | 2020-09-10 | 2023-02-17 | 云南菌视界生物科技有限公司 | Golden stropharia rugoso-annulata strain |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106367496B (en) | kiwi species associated specific single nucleotide molecular marker, detection primer group and application thereof | |
| CN114292944B (en) | SNP molecular marker linked with pepper epidemic disease resistance and application thereof | |
| CN113584216A (en) | KASP marker development of wheat grain weight gene TaCYP78A16 and application thereof | |
| CN114292938A (en) | SSR fingerprint identification method and construction method of stropharia rugoso-annulata strain | |
| CN111206106A (en) | RPA primer, kit and detection method for detecting sweet potato rot stem nematode | |
| CN113151567B (en) | SSR molecular marker and method for identifying Lepista sordida N006# strain | |
| CN104131115A (en) | Gene chip for detecting pathogenic fungi of poplar canker and application thereof | |
| CN112592996B (en) | Molecular marker ZMM1776 closely linked with sesame seed sesamin content major gene locus and application thereof | |
| CN111690759B (en) | Specific primer, kit and method for detecting RPA of citrus canker pathogen | |
| AU2020103706A4 (en) | Ssr molecular marker primer related to walnut black spot disease and application thereof | |
| CN110878376B (en) | SSR molecular marker primer for identifying dendrobium huoshanense and application thereof | |
| CN114517238B (en) | SSR molecular marker and method for identifying tremella aurantialba ZJJJE 001 strain | |
| CN106544438A (en) | A kind of application of genetic marker in purpleapricot identification | |
| WO2023087788A1 (en) | Dna bar code for screening total soluble protein content index of floccularia luteovirens | |
| WO2023087784A1 (en) | Dna barcode for screening floccularia luteovirens using content of total polyphenol as index | |
| CN107557487B (en) | Construction method and application of oat DNA molecular fingerprint | |
| CN112941224A (en) | Identification method of SSR (simple sequence repeat) marker fingerprint of golden 6046 strain of flammulina velutipes, construction method and application thereof | |
| CN110923304B (en) | Molecular marker, primer pair and method for identifying sex of ginkgo biloba | |
| KR102174274B1 (en) | Molecular marker for discriminating Zizyphus jujuba 'Boeun' and 'Chuseok' cultivar and uses thereof | |
| CN107012253B (en) | Method for identifying female parent of cultivated oat | |
| CN111304357A (en) | Fusarium oxysporum bitter gourd specialized LAMP visual detection method and kit thereof | |
| CN110846434B (en) | Primer and kit for identifying tea tree varieties and identification method thereof | |
| CN112980994B (en) | Identification method of SSR marker fingerprint of needle mushroom strain and construction method and application thereof | |
| CN102296124A (en) | Method for quickly differentiating Chinese date varieties by using random amplified polymorphic DNA (RAPD) | |
| CN113186328B (en) | Identification method of microsatellite DNA marker fingerprint of flammulina velutipes xujin 18 strain and construction method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication |