CN110029191B - Primer group for identifying mating types of monokaryons of shiitake mushroom Shenxiang 215 strain and substantive derivative variety thereof, identification method and application - Google Patents
Primer group for identifying mating types of monokaryons of shiitake mushroom Shenxiang 215 strain and substantive derivative variety thereof, identification method and application Download PDFInfo
- Publication number
- CN110029191B CN110029191B CN201910475026.4A CN201910475026A CN110029191B CN 110029191 B CN110029191 B CN 110029191B CN 201910475026 A CN201910475026 A CN 201910475026A CN 110029191 B CN110029191 B CN 110029191B
- Authority
- CN
- China
- Prior art keywords
- primer pair
- primer
- amplification products
- pair
- mating type
- 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.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a primer group for identifying the mating type of a monokaryon of mushroom Shenxiang 215 strain and a substantive derivative variety thereof, an identification method and application, relates to the technical field of molecular marker assisted breeding of edible mushrooms, and particularly comprises four pairs of primers, wherein the identification method specifically comprises the following steps: hypha culture, rapid preparation of genome DNA, PCR detection of mating types and electrophoresis detection to determine the result. Compared with the conventional method for identifying the mating type of the monokaryon, the method has the advantages of short detection time and high accuracy, and by utilizing the identification method, the identification time can be greatly shortened, the false detection rate can be reduced, and the bikaryon can be eliminated.
Description
Technical Field
The invention relates to the technical field of edible fungus molecular marker assisted breeding, in particular to a primer group for identifying the mating type of monokaryons of shiitake mushroom Shenxiang 215 strains and substantive derived varieties thereof, an identification method and application.
Background
The acquisition of monokaryons and the identification of mating types are one of the basic works of shiitake inheritance and breeding research. Mating type determines the compatibility between the two monokaryon parents undergoing crossing, and incompatible monokaryons cannot be crossed to form a bipkaryon progeny.
At present, the mating type identification of the mushrooms is generally carried out by adopting a mode of observing lock-shaped combination through microscopic examination after pairwise opposite cultivation of monokaryons. The cross breeding work usually involves a large number of single karyons, and the combination of the single karyons in opposite culture causes large workload of inoculation and microscopic examination and takes long time. In addition, in the microscopic examination process, due to the early and late formation time of the lock-shaped combination, the time and the position of picking the bacterial colony are different, and the like, the artificial false examination is easy to generate, and the subsequent test is influenced.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a primer group for identifying the mating type of monokaryons of mushroom Shenxiang 215 strains. The primer can quickly identify the mating type of monokaryons of the shiitake mushroom Shenxiang 215 strain through amplification.
The second purpose of the invention is to provide a method for identifying the mating type of monokaryons of mushroom-flavor 215 strain by using primers. Compared with the existing identification method, the method has the advantages of convenient operation, high identification accuracy and short identification period.
The third purpose of the invention is to provide a primer for identifying the mating type of the monokaryon of the substantive derivative variety of the shiitake mushroom Shenxiang 215 strain. The primer can realize the identification of the mating type of the monokaryon of the substantive derived variety of the mushroom Shenxiang 215 strain.
The fourth purpose of the invention is to provide application of the primer group for identifying the mating type of the monokaryon of the mushroom Shenxiang 215 strain in molecular markers, kits and test strips.
The invention is realized by the following steps:
a primer set for identifying the mating type of a monokaryon of shiitake mushroom Shenxiang 215 strain comprises any one or more of the following first to fourth primer pairs;
wherein the first primer pair comprises: a primer shown as SEQ ID NO. 1-2; the second primer pair includes: primers shown in SEQ ID NO. 3-4; the third primer pair comprises: primers shown in SEQ ID NO. 5-6; the fourth primer pair comprises: primers shown in SEQ ID NO. 7-8.
Furthermore, the 4 pairs of primers in the invention are AS-PCR primers developed based on SNP sites of the A and B regions of the 215 mating factors of champignon.
A method for identifying the mating type of monokaryons of shiitake mushroom-shen 215 species, comprising: and respectively carrying out PCR amplification on the DNA templates of the samples to be detected by using the first primer pair to the fourth primer pair.
Further, the identification method also comprises the steps of hypha culture and genome DNA extraction. The hypha culture specifically comprises the steps of transferring the mushroom Shenxiang 215 monokaryon strain into a potato glucose agar culture medium PDA, and standing and culturing at 23-25 ℃ in a dark place.
The rapid preparation of the genome DNA specifically comprises the following steps which are carried out in sequence: picking a small amount of hyphae with a sterilized toothpick or an inoculating needle tip after the hyphae germinate, suspending the hyphae in a 0.2ml centrifuge tube filled with 80-120 mu L TE buffer, preserving the heat for 4-8min at 96-98 ℃ on a PCR instrument, and immediately placing on ice for cooling for later use. If a larger amount of hyphae is prepared, a 96-well plate can be used as a hyphae container.
Furthermore, the amplification length of the first pair of primers and the second pair of primers is 506bp, and the amplification length of the third pair of primers and the fourth pair of primers is 1383 bp.
Further, the first primer pair and the second primer pair are used for detecting the factor A, and the third primer pair and the fourth primer pair are used for detecting the factor B.
In a preferred embodiment of the invention, the DNA template of the sample to be tested is genomic DNA of the sample to be tested.
In a preferred embodiment of the invention, the method further comprises performing electrophoretic identification on the amplified product; preferably, the electrophoretic identification specifically comprises adding a fluorescent dye to the amplification product or the gel to perform identification of the amplification product.
In a preferred embodiment of the invention, the method further comprises analyzing the mating types, wherein the method for analyzing the mating types comprises: judging the mating type to be A1B1 if the first pair of primers and the third pair of primers have amplification products and the second pair of primers and the fourth pair of primers have no amplification products; judging the mating type to be A1B2 if the first pair of primers and the fourth pair of primers have amplification products and the second pair of primers and the third pair of primers have no amplification products; judging the mating type to be A2B1 if the second primer pair and the third primer pair have amplification products and the first primer pair and the fourth primer pair have no amplification products; if the second pair of primers and the fourth pair of primers have amplification products, and the first pair of primers and the third pair of primers have no amplification products, judging the mating type to be A2B 2; when the amplification products were present in all of the first to fourth primers, the mating type was judged to be a karyon of A1A2B1B 2.
In a preferred embodiment of the present invention, the PCR amplification conditions are: pre-denaturing at 94-98 deg.c for 1-6min, denaturing at 94-95 deg.c for 1-1.5min, annealing at 61-61.5 deg.c for the first primer pair and the second primer pair, annealing at 63-64 deg.c for the third primer pair and the fourth primer pair for 20-90s, extending at 70-72 deg.c for 20-90s, 29-42 cycles, and final maintaining at 70-72 deg.c for 0-10 min;
preferably, the conditions for PCR amplification are: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 1min, annealing temperatures of the first and second primers are both 61 ℃, annealing temperatures of the third and fourth primers are both 63 ℃, annealing time is 60s, extension at 72 ℃ is 60s, 35 cycles are performed, and finally heat preservation at 72 ℃ is performed for 5 min.
A primer group for identifying the mating type of a monokaryon of a substantially derived variety of the mushroom Shenxiang 215 strain is a primer group for identifying the mating type of the monokaryon of the mushroom Shenxiang 215 strain, and the primer group is any one or more of a first primer pair, a second primer pair and a third primer pair.
A molecular marker produced using the primer set, the molecular marker comprising any one or more of the first to fourth primer pairs.
A kit prepared by using the primer group comprises a plurality of detection reagents, and a first primer pair, a second primer pair, a third primer pair and a fourth primer pair are added in the plurality of detection reagents respectively.
A test strip prepared by using the primer group is coated with any one of the first to fourth primer pairs.
The invention has the following beneficial effects:
the invention provides a primer for identifying the mating type of a monokaryon of shiitake mushroom Shenxiang 215 strain and substantive derivative varieties thereof, an identification method and application. Compared with the conventional method for identifying the mating type of the monokaryon in the prior art, the method has the advantages of short detection time and high accuracy. The identification method provided by the invention can greatly shorten the identification time of the mating type of the strain monokaryon and also can eliminate the mixed binuclear by routine false detection. In addition, the identification method provided by the invention can be used for identifying the mating type of the monokaryon of the substantive derivative variety of the Chinese Styrax 215 in addition to the monokaryon of the Chinese Styrax 215.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a drawing showing the mating type A factor amplification pattern of spore monokaryons numbered 1 to 24 in "Shenxiang 215" in example 1 of the present invention (upper: A1 shows the amplification result using the first pair of primers; lower: A2 shows the amplification result using the second pair of primers);
FIG. 2 is a drawing showing the amplification pattern of the mating type B factor of a spore monokaryon of "Shenxiang 215" Nos. 1 to 24 in example 1 of the present invention (upper: B1 shows the amplification result using the third primer pair; lower: B2 shows the amplification result using the fourth primer pair);
FIG. 3 is a graph showing the results of amplification using four primers of the present invention on a double nucleus (A1 shows the result of amplification using a first primer pair; A2 shows the result of amplification using a second primer pair; B1 shows the result of amplification using a third primer pair; B2 shows the result of amplification using a fourth primer pair);
FIG. 4 is a microscope photograph of a locked union structure characteristic of a binuclear body;
FIG. 5 is a mating type A factor amplification profile of spore monokaryons numbered 1-24 of "L1641" in example 2 of the present invention (upper: A1 shows the amplification result using a first pair of primers; lower: A2 shows the amplification result using a second pair of primers);
FIG. 6 is a mating type B factor amplification map of spore monokaryons numbered 1 to 24 in "L1641" in example 2 of the present invention (upper: B1 shows the amplification result using the third primer pair; lower: B2 shows the amplification result using the fourth primer pair).
The sizes of the bands of the DNA molecular weight markers represented by M in FIGS. 1-3, 5 and 6 are, from bottom to top: 100bp, 250 bp, 500 bp, 750bp, 1000 bp and 2000 bp.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The embodiment provides a method for identifying the mating type of a monokaryon of shiitake mushroom-Shenxiang 215, which specifically comprises the following steps of sequentially carrying out:
(1) hypha culture: 176 spore monokaryon strains of shiitake mushroom Shenxiang 215 are transferred to a plate filled with a potato glucose agar culture medium PDA, and the plate with the diameter of 90cm is subjected to static culture at 25 ℃ in the dark.
(2) Rapid preparation of genomic DNA: and after 5 days of hypha culture, adding 100 mu L TE buffer into each sample tube of a 96-well plate, picking a small amount of hypha from each monokaryon strain by using the tip of an inoculation needle, suspending the hypha in the sample tubes respectively, recording numbers, treating the 96-well plate for 5min at 98 ℃ by using a PCR instrument, and immediately placing the plate on ice for cooling for later use.
In the embodiment of the invention, the method for quickly preparing DNA by hyphae can realize the quick identification of a large batch of hyphae, and in addition, in other embodiments, DNA extraction methods such as CTAB and the like can also be adopted to realize the extraction of hyphae DNA. The extraction of the genome DNA in the embodiment of the invention can reasonably adjust the times of extracting the DNA and the purity according to the requirement.
(3) Detecting the mating factor type of the strain: and (3) carrying out AS-PCR amplification on the DNA extracted in the step (2).
The PCR amplification system is as follows: total volume 20 μ L, including: 2 XPCR mix 10 uL, 10 umol/L forward primer and reverse primer each 1 uL, extracted strain template DNA 1 uL, ddH 2 O 7μL；
And (3) PCR reaction conditions: 5min at 94 ℃; 94 ℃ for 1min, the annealing temperature of the first pair of primers and the second pair of primers is 61 ℃, the annealing temperature of the third pair of primers and the fourth pair of primers is 63 ℃, the annealing time of the 4 pairs of primers is 1min, the extension is 1min at 72 ℃, and 35 cycles are carried out; keeping the temperature at 72 ℃ for 5 min. Specific information on the AS-PCR primer set is shown in Table 1. In Table 1, matAF1 and matAF2 represent the forward primers of the first and second primer pairs, respectively, matAR represents the reverse primers of the first and second primer pairs, and the reverse primers for detecting A1 and A2 factors are the same. matBF1 and matBF2 indicate the forward primers of the third primer pair and the fourth primer pair, respectively, matBR indicates the reverse primers of the third primer pair and the fourth primer pair, and the reverse primers for detecting factors B1 and B2 are the same.
Table 1: detailed information List of AS-PCR primers
(4) And (3) electrophoresis detection: and (3) carrying out electrophoresis on the product obtained by the PCR amplification in the step (3) through a 1.5% agarose gel (1/10000 SYBR Green I stain is added into the agarose gel in advance), carrying out electrophoresis for 40min at a voltage of 120V, and observing and recording the result by using a photographic system.
And 4 pairs of primers are adopted to carry out PCR amplification on all the monokaryon strains, and the mating type of each monokaryon is judged by recording the molecular weight of an amplified band of each pair of primers and the existence of the band. Judging the mating type to be A1B1 if the first pair of primers and the third pair of primers have amplification products and the second pair of primers and the fourth pair of primers have no amplification products; judging the mating type to be A1B2 if the first primer pair and the fourth primer pair have amplification products and the second primer pair and the third primer pair have no amplification products; judging the mating type to be A2B1 if the second primer pair and the third primer pair have amplification products and the first primer pair and the fourth primer pair have no amplification products; if the second pair of primers and the fourth pair of primers have amplification products, and the first pair of primers and the third pair of primers have no amplification products, judging the mating type to be A2B 2; when the amplification products were present in all of the first to fourth primers, the mating type was judged to be a karyon of A1A2B1B 2.
Referring to FIGS. 1 and 2, the top view of FIG. 1 shows an electrophoretogram after amplification using a first pair of primers, the bottom view of FIG. 1 shows an electrophoretogram after amplification using a second pair of primers, the top view of FIG. 2 shows an electrophoretogram after amplification using a third pair of primers, and the bottom view of FIG. 2 shows an electrophoretogram after amplification using a fourth pair of primers. The electrophoresis results show that: the ratio of the four mating types A1B1, A2B2, A1B2, A2B1 in 176 spore monokaryon strains was 33: 47: 53: 41.
four pairs of primers are used for detecting the genotypes A1, A2, B1 and B2 of the two strains of 2 strains respectively, and the electrophoresis patterns of the amplification products are shown in figure 3, wherein the electrophoresis pattern of the amplification products of one strain is shown in figure 3, and the electrophoresis patterns of the amplification results of the two strains are the same. From FIG. 3, it can be seen that these two strains contained the A1, A2, B1 and B2 genotypes.
The mycelia of the double nucleosomes were further confirmed by microscopic examination, and the microscopic examination results are shown in FIG. 4. In FIG. 4, the arrows indicate the unique phenomenon of the locklike association of the twin nuclei, whereas the single nuclei do not have this structure. Thus, both strains were judged to be binuclear strains.
In this embodiment, DNA prepared by hypha in a batch and rapidly is directly subjected to PCR amplification, so that the quality of a small amount of prepared DNA is unstable, which affects the amplification result, and in order to ensure that all detection results are obtained, some DNA with poor amplification effect needs to be repeatedly prepared and amplified. We randomly selected 10 strains from the monokaryon population of each mating type to form compatible combinations for one-to-one hybridization to verify the reliability of the molecular marker identification result. The test microscopic examination result shows that 20 pairs of hybridization combinations have bikaryon offspring, namely the mating types of the two matched monokaryons can be compatible, and the PCR identification result is completely accurate.
Example 2
The embodiment provides a method for identifying the mating type of a monokaryon of a mushroom strain L1641 (a substantive derivative variety of Shenxiang 215), which specifically comprises the following steps in sequence:
(1) hypha culture: 159 spore monokaryon strains of the Lentinus edodes strain L1641 were transferred to a plate containing a potato dextrose agar medium PDA, and the plate with a diameter of 90cm was subjected to static culture at 25 ℃ in the absence of light.
(2) Rapid preparation of genomic DNA: after 5 days of hypha culture, adding 100 mu L TE buffer into each sample tube of a 96-well plate, picking a small amount of hypha from each monocaryon strain by using the tip of an inoculation needle, suspending the hypha in the sample tubes respectively, recording numbers, treating the 96-well plate for 5min at 98 ℃ by using a PCR instrument, and immediately placing the plate on ice for cooling for later use.
(3) Detecting the mating factor type of the strain: and (3) carrying out AS-PCR amplification on the DNA extracted in the step (2).
The PCR amplification system is as follows: total volume 20 μ L, including: 2 XPCR mix 10 uL, 10 umol/L forward primer and reverse primer each 1 uL, extracted strain template DNA 1 uL, ddH 2 O 7μL；
And (3) PCR reaction conditions: 5min at 94 ℃; 94 ℃ for 1min, the annealing temperature of the first pair of primers and the second pair of primers is 61 ℃, the annealing temperature of the third pair of primers and the fourth pair of primers is 63 ℃, the annealing time of the 4 pairs of primers is 1min, the extension is 1min at 72 ℃, and 35 cycles are carried out; keeping the temperature at 72 ℃ for 5 min.
(4) Electrophoresis detection: and (4) carrying out electrophoresis on the product obtained by the PCR amplification in the step (3) through 1.5% agarose gel (adding 1/10000 SYBR Green I staining agent into the agarose gel in advance), carrying out electrophoresis at the voltage of 120V for 40min, and observing and recording the result by using a photographic system.
And 4 pairs of primers are adopted to carry out PCR amplification on all the monokaryon strains, and the mating type of each monokaryon is judged by recording the molecular weight of an amplified band of each pair of primers and the existence of the band. Judging the mating type to be A1B1 if the first pair of primers and the third pair of primers have amplification products and the second pair of primers and the fourth pair of primers have no amplification products; judging the mating type to be A1B2 if the first pair of primers and the fourth pair of primers have amplification products and the second pair of primers and the third pair of primers have no amplification products; judging the mating type to be A2B1 if the second primer pair and the third primer pair have amplification products and the first primer pair and the fourth primer pair have no amplification products; if the second pair of primers and the fourth pair of primers have amplification products, and the first pair of primers and the third pair of primers have no amplification products, judging the mating type to be A2B 2; when the amplification products were present in all of the first to fourth primers, the mating type was judged to be a karyon of A1A2B1B 2.
Electrophorograms referring to fig. 5 and 6, statistically determined the ratio of the four mating types A1B1, A2B2, A1B2, A2B1 in 159 spore monokaryon strains was 35: 42: 34: 48.
in this embodiment, DNA prepared by hyphae in a batch and rapidly is directly subjected to PCR amplification, and thus, the quality of a small amount of prepared DNA is unstable, which affects the amplification result. We randomly selected 10 strains from the monokaryon population of each mating type to form compatible combinations for one-to-one hybridization to verify the reliability of the molecular marker identification result. The microscopic examination result shows that 20 pairs of hybridization combinations have bikaryon offspring, namely the mating types of the two matched monokaryons can be compatible, and the PCR identification result is completely accurate.
The invention provides a primer group for identifying the mating types of monokaryons of shiitake mushroom Shenxiang 215 strains and substantive derived varieties thereof, an identification method and application. Compared with the conventional method for identifying the mating type of the monokaryon in the prior art, the method has the advantages of short detection time and high accuracy. The identification method provided by the invention can greatly shorten the identification time of the mating type of the strain monokaryon and also can eliminate the mixed binuclear by routine false detection. In addition, the identification method provided by the invention can be used for identifying the mating type of the monokaryon of the substantive derivative variety of the Chinese Styrax 215 in addition to the monokaryon of the Chinese Styrax 215. With the identification method of the present invention, the time of 1-2 months required for conventional identification can be shortened to 1 week, and a double nucleus that is erroneously detected can be excluded.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Shanghai city academy of agricultural sciences
<120> primer set for identifying the mating types of monokaryons of shiitake mushroom Shenxiang 215 strain and its substantive derivative variety
Method and use
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213> Artificial sequence
<400> 1
actcccaaat ggcaacctca ca 22
<210> 2
<211> 22
<212> DNA
<213> Artificial sequence
<400> 2
tatcccgata aagcatccgc gc 22
<210> 3
<211> 22
<212> DNA
<213> Artificial sequence
<400> 3
actcccaaat ggcaacctca cc 22
<210> 4
<211> 22
<212> DNA
<213> Artificial sequence
<400> 4
tatcccgata aagcatccgc gc 22
<210> 5
<211> 19
<212> DNA
<213> Artificial sequence
<400> 5
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence
<400> 6
ctcgagacta gttgagaatc 20
<210> 7
<211> 19
<212> DNA
<213> Artificial sequence
<400> 7
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence
<400> 8
ctcgagacta gttgagaatc 20
Claims (8)
1. A primer group for identifying the mating types of monokaryons of mushroom Shenxiang 215 strains and substantive derivative varieties thereof is characterized by comprising a first primer pair, a second primer pair, a third primer pair and a fourth primer pair;
wherein the first primer pair has a primer shown as SEQ ID NO. 1-2; the second primer pair has primers shown as SEQ ID NO. 3-4; the third primer pair has primers shown as SEQ ID NO. 5-6; the fourth primer pair has primers shown as SEQ ID NO. 7-8; the method for analyzing the mating type of the monokaryons corresponding to the primer group comprises the following steps: judging the mating type to be A1B1 if the first primer pair and the third primer pair both have amplification products and the second primer pair and the fourth primer pair both have no amplification products; judging the mating type to be A1B2 if the first primer pair and the fourth primer pair both have amplification products and the second primer pair and the third primer pair both have no amplification products; judging the mating type to be A2B1 if the second primer pair and the third primer pair both have amplification products and the first primer pair and the fourth primer pair both have no amplification products; judging the mating type to be A2B2 if the second primer pair and the fourth primer pair both have amplification products and the first primer pair and the third primer pair both have no amplification products; when the amplification products were present in all of the first to fourth primers, the mating type was judged to be a karyon of A1A2B1B 2.
2. A method for identifying the mating type of monokaryons of mushroom-flavor holy 215 strains is characterized by comprising the following steps: performing PCR amplification on the DNA templates of the samples to be detected by using the first to fourth primer pairs according to claim 1;
the method also comprises the analysis of the mating types, and the method for analyzing the mating types comprises the following steps: judging the mating type to be A1B1 if the first primer pair and the third primer pair both have amplification products and the second primer pair and the fourth primer pair both have no amplification products; judging the mating type to be A1B2 if the first primer pair and the fourth primer pair both have amplification products and the second primer pair and the third primer pair both have no amplification products; judging the mating type to be A2B1 if the second primer pair and the third primer pair both have amplification products and the first primer pair and the fourth primer pair both have no amplification products; judging the mating type to be A2B2 if the second primer pair and the fourth primer pair both have amplification products and the first primer pair and the third primer pair both have no amplification products; when the amplification products were present in all of the first to fourth primers, the mating type was judged to be a karyon of A1A2B1B 2.
3. The method of claim 2, wherein the DNA template of the test sample is genomic DNA of the test sample.
4. The method of claim 2, further comprising: carrying out electrophoretic identification on the amplified product; the electrophoretic identification specifically comprises the step of adding a fluorescent dye into an amplification product or gel to identify the amplification product.
5. The method of claim 2, wherein the PCR amplification conditions are: pre-denaturing at 94-98 deg.c for 1-6min, denaturing at 94-95 deg.c for 1-1.5min, annealing at 61-61.5 deg.c for the first primer pair and the second primer pair, annealing at 61-63 deg.c for the third primer pair and the fourth primer pair for 20-90s, extending at 70-72 deg.c for 20-90s, 29-42 cycles, and final maintaining at 70-72 deg.c for 0-10 min.
6. The method of claim 5, wherein the PCR amplification conditions are: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 1min, annealing temperatures of the first primer pair and the second primer pair are both 61 ℃, annealing temperatures of the third primer pair and the fourth primer pair are both 63 ℃, annealing time is 60s, extension at 72 ℃ is 60s, 35 cycles are performed, and finally heat preservation at 72 ℃ is performed for 5 min.
7. A kit prepared by using the primer set according to claim 1, wherein the kit comprises a plurality of detection reagents, and the first to fourth primer pairs are added to the plurality of detection reagents, respectively.
8. A test strip prepared by using the primer set of claim 1, wherein the test strip is coated with first to fourth primer pairs.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910475026.4A CN110029191B (en) | 2019-05-31 | 2019-05-31 | Primer group for identifying mating types of monokaryons of shiitake mushroom Shenxiang 215 strain and substantive derivative variety thereof, identification method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910475026.4A CN110029191B (en) | 2019-05-31 | 2019-05-31 | Primer group for identifying mating types of monokaryons of shiitake mushroom Shenxiang 215 strain and substantive derivative variety thereof, identification method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110029191A CN110029191A (en) | 2019-07-19 |
| CN110029191B true CN110029191B (en) | 2022-09-06 |
Family
ID=67243857
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910475026.4A Active CN110029191B (en) | 2019-05-31 | 2019-05-31 | Primer group for identifying mating types of monokaryons of shiitake mushroom Shenxiang 215 strain and substantive derivative variety thereof, identification method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110029191B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110904262B (en) * | 2019-12-16 | 2023-01-24 | 上海市农业科学院 | Method for creating nucleoplasm hybrid of shiitake mushroom cultivation strain by using cytoplasm and nucleus molecular markers |
| CN113151577B (en) * | 2021-06-08 | 2022-12-30 | 上海市农业科学院 | InDel molecular marker and method for identifying mating types A of different monokaryons of same strain of shiitake mushroom |
| CN116144831B (en) * | 2023-04-18 | 2023-07-21 | 云南菌视界生物科技有限公司 | Primer combination for identifying mating type tremella aurantialba mononucleosome and identification method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101712984A (en) * | 2009-11-30 | 2010-05-26 | 浙江省林业科学研究院 | Molecular specificity mark primer for authenticating mushroom strains 241 and 241-4 and detecting method |
| CN103184280A (en) * | 2013-02-04 | 2013-07-03 | 北京市农林科学院 | Method for identifying or assisting in identifying mating types of Lepista sordid protoplast monokaryons and special primer pairs IS-818 thereof |
| CN103194535A (en) * | 2013-02-04 | 2013-07-10 | 北京市农林科学院 | Method for identification or auxiliary identification of mating type of protoplast monokaryon of Lepista sordid and special primer pair SR-5*16 used therein |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106884044B (en) * | 2017-02-07 | 2020-08-11 | 上海市农业科学院 | SSR marker fingerprint spectrum of mushroom Shenxiang 215 strain as well as construction method and application thereof |
| KR101834565B1 (en) * | 2017-02-23 | 2018-03-06 | 충북대학교 산학협력단 | CAPS Marker for Differentiation of Shiitake Mushroom Cultivar Sanmaru-1 and use thereof |
| KR101906037B1 (en) * | 2017-02-23 | 2018-10-08 | 충북대학교 산학협력단 | CAPS Marker for Differentiation of Shiitake Mushroom Cultivar Chunjang-3 and use thereof |
| CN108070674B (en) * | 2018-02-01 | 2021-08-31 | 福建省农业科学院食用菌研究所（福建省蘑菇菌种研究推广站） | SCAR-PCR identification method for mating types of agaricus bisporus homonuclear sterile single spore strains |
| CN108611435B (en) * | 2018-05-21 | 2022-03-25 | 上海市农业科学院 | Method and primer for identifying same-core sterile single spore strain of agaricus bisporus and mating type of same-core sterile single spore strain |
-
2019
- 2019-05-31 CN CN201910475026.4A patent/CN110029191B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101712984A (en) * | 2009-11-30 | 2010-05-26 | 浙江省林业科学研究院 | Molecular specificity mark primer for authenticating mushroom strains 241 and 241-4 and detecting method |
| CN103184280A (en) * | 2013-02-04 | 2013-07-03 | 北京市农林科学院 | Method for identifying or assisting in identifying mating types of Lepista sordid protoplast monokaryons and special primer pairs IS-818 thereof |
| CN103194535A (en) * | 2013-02-04 | 2013-07-10 | 北京市农林科学院 | Method for identification or auxiliary identification of mating type of protoplast monokaryon of Lepista sordid and special primer pair SR-5*16 used therein |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110029191A (en) | 2019-07-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110029191B (en) | Primer group for identifying mating types of monokaryons of shiitake mushroom Shenxiang 215 strain and substantive derivative variety thereof, identification method and application | |
| KR101860232B1 (en) | Method for discrimination of jujube varieties using SSR marker | |
| CN113355446B (en) | InDel molecular marker related to purple character of Chinese cabbage and application thereof | |
| CN111926098B (en) | InDel molecular marker closely linked with epistatic gene Y of eggplant fruit color and application | |
| US5674993A (en) | Nucleic acid markers for rice blast resistance genes and rice blast resistance genes isolated by the use of these markers | |
| CN106916897A (en) | One kind is used to identify the molecular labeling and its application of giant pumpkin ' silver-colored brightness three ' hybrid seed purity | |
| CN114606332A (en) | SNP (Single nucleotide polymorphism) site and Hf-KASP1 marker for judging pulp hardness of watermelon and application thereof | |
| CN106755519B (en) | Method for identifying homozygous and heterozygous transgenic corn double antibody 12-5 based on digital PCR and application | |
| CN106350601A (en) | High throughput test method for corncob color genotype and kit thereof | |
| CN110453003B (en) | Molecular marker closely linked with spinach leaf type character major QTL (quantitative trait loci) and application thereof | |
| CN110331222B (en) | Molecular marker related to cotton fertility restoration and application thereof | |
| CN108179177A (en) | A kind of kit and detection method of quick detection nucleic acid mutation | |
| CN108411030B (en) | Primer pair, kit containing primer pair, application of primer pair and method for detecting ecological A17 and R108 of medicago truncatula | |
| CN114836558B (en) | InDel marker linked with broccoli low Wen Bianzi trait, primer set and application | |
| CN111733220B (en) | Method for high-specificity identification of transgenic carnation | |
| KR102335806B1 (en) | Molecular marker based on chloroplast genome sequence for discriminating Zizyphus jujuba 'SanJo' cultivar and uses thereof | |
| KR20160086177A (en) | Random Amplified Polymorphic DNA primer for discrimination of tobacco cultivars | |
| CN114480666A (en) | Method for distinguishing black porgy and two hybrid offspring thereof by using ISSR molecular marker | |
| Ahmedand et al. | Genetic diversity of mango (Mangifera indica L.) cultivars in Shendi Area | |
| CN104894125B (en) | A kind of RT LAMP kits and its primer special for detecting grape A viruses | |
| CN109536633A (en) | The SNP marker and application isolated with the anti-graywall main effect QTL-qRgls2 of corn | |
| CN113736908B (en) | SNP locus combination for detecting tomato leaf mold resistance and application thereof | |
| CN111850159B (en) | SSR molecular marker identification primer and application thereof | |
| CN117737287A (en) | Primer group for identifying mating type of agrocybe cylindracea AS-5 mononucleosome strain, identification method and application | |
| CN114703322B (en) | Primer pair, kit and detection method for RT-RAA fluorescence detection of avian leukosis virus P12 gene |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |