CN112980991B - Identification method of SSR (simple sequence repeat) marker fingerprint of Shanghai Zhen 30 strain of hypsizigus marmoreus, construction method and application thereof - Google Patents

Abstract

The invention discloses an identification method of SSR (simple sequence repeat) marker fingerprint of a Shanghai No. 30 strain of hypsizigus marmoreus, and a construction method and application thereof, wherein the fingerprint consists of 6 pairs of SSR markers. The construction method comprises the following steps: (1) hypha culture; (2) extraction of genomic DNA; (3) detection of SSR molecular markers; (4) capillary electrophoresis detection. The application comprises the following steps: SSR marker amplification is carried out on hypsizigus marmoreus strains, the obtained banding pattern is compared with the fingerprint pattern, and the banding pattern is consistent with the fingerprint pattern, so that hypsizigus marmoreus Shanghai No. 30 strains are obtained. Compared with the conventional morphological detection, antagonism test and fruiting test, the invention has the advantages of short detection time, high accuracy and good repeatability, and has the specificity of the Shanghai No. 30 strain of the hypsizigus marmoreus in 59 hypsizigus marmoreus cultivation main cultivated strains collected at home and abroad.

Description

Identification method of SSR (simple sequence repeat) marker fingerprint of Shanghai Zhen 30 strain of hypsizigus marmoreus, construction method and application thereof

Technical Field

The invention belongs to the technical field of detection of hypsizigus marmoreus strains, and particularly relates to an identification method of SSR (simple sequence repeat) marker fingerprint of hypsizigus marmoreus Shanghai No. 30 strain, a construction method and application thereof.

Background

Hypsizygus marmoreus (Latin name Hypsizygus marmoreus (Peck) H.E.Bigelow) belongs to Basidiomycota, agaricales, lyophyllum, and is named by marble speckle on the surface of its cap. The seafood has attractive appearance, crisp and tender texture, unique seafood flavor and economic price, and is popular with consumers. The hypsizigus marmoreus not only contains abundant vitamins and 18 amino acids, but also has the functions of hemolysis, antioxidation, leukemia and lymphoma cell inhibition, blood fat reduction, anti-inflammation, antioxidation and the like due to the components such as small molecular compounds, polysaccharide, polypeptide and the like contained in the hypsizigus marmoreus, and is a food and medicine dual-purpose bacterium with high economic value. Since the establishment of industrial cultivation enterprises of the first hypsizigus marmoreus in China in 2001, the hypsizigus marmoreus becomes one of novel edible fungi with faster market development in China. The industrial total yield of the hypsizigus marmoreus in 2019 is 32.8 ten thousand tons, is increased by 58.63 percent compared with the industrial total yield of the edible fungi in 2018, accounts for 9.6 percent of the industrial total yield of the edible fungi, and is ranked third in the industrial edible fungi production total amount in China.

The high-quality strain plays a significant role in the industrial cultivation of hypsizigus marmoreus. At present, hypsizigus marmoreus strains used by industrial production enterprises in China are mainly prepared by introducing strains in Japan or further hybridizing and breeding on the basis of the strains, wherein fruiting bodies after cultivation are brown and are called as hypsizigus marmoreus, and the fruiting bodies are white and are also called as white hypsizigus marmoreus or hypsizigus marmoreus. Although the yield of industrial production is improved year by year, the problems of long cultivation period, low yield per unit, poor uniformity of the appearance of the single fruiting body, easy occurrence of tumor covers and the like still exist in part of strains, and the defects can cause the increase of production cost and reduce the market competitiveness of the products. On the other hand, the variety of the industrial production strains of the hypsizigus marmoreus is not high, the appearance of the product is similar, and the diversified demands of market consumers cannot be met. The Chinese has rich wild and natural cultivation resources of hypsizigus marmoreus, efficiently utilizes the high-quality resources, expands the genetic foundation of the strain, and is favorable for breeding high-yield, high-quality and special hypsizigus marmoreus strains in China.

Along with the promulgation and implementation of International plant New variety protection laws, the establishment of a mature, quick and accurate molecular biological identification technology system becomes a powerful means for protecting the intellectual property rights of edible fungus varieties.

Disclosure of Invention

Hypsizygus marmoreus Shanghai No. 30, 20 months 2021, was deposited with the Guangdong province microbiological strain collection center at the following addresses: guangzhou city, first, middle road 100, institute of technology, building 59, 5, guangdong province microbiological institute, accession No. GDMCC No:61536.

the invention aims to solve the technical problem of providing an SSR (simple sequence repeat) marker fingerprint of a hypsizigus marmoreus 'Huzhen No. 30' strain, and a construction method and application thereof.

The invention relates to an SSR marker fingerprint of hypsizigus marmoreus 'Shanghai No. 30' strain, which consists of 6 pairs of SSR markers, wherein SSR primers are developed based on simple repeated sequence segments of hypsizigus marmoreus genome, the amplified bands are good, the repeatability is high, and the marker detailed information is shown in table 1:

TABLE 1 SSR marker detailed information List

The invention relates to a construction method of SSR mark fingerprint of hypsizigus marmoreus 'Shanghai No. 30' strain, which comprises the following steps:

(1) Hypha culture: transferring hypsizigus marmoreus strain to potato glucose agar solid medium, culturing at 22 ℃ for 8d, and collecting mycelium;

(2) Extraction of genomic DNA: genomic DNA was extracted using the Meimei fungus DNA extraction kit according to the kit experimental procedure, and 2. Mu.L of DNA was taken for detection by 1.2% agarose gel electrophoresis. Detecting the concentration and purity of the total genome DNA by an ultraviolet spectrophotometry, and adjusting the concentration of the sample DNA to be consistent;

(3) SSR molecular marker primer development: according to the genome re-sequencing result of the hypsizigus marmoreus strain, 20 pairs of SSR primers are selected and designed and synthesized;

(4) Detection of SSR molecular markers: carrying out PCR amplification of gene SSR markers on the extracted DNA;

(5) And (3) electrophoresis detection: mixing the PCR amplified product with formamide sampling buffer, denaturing and detecting;

(6) GeneMapper data analysis.

The construction method of SSR marker finger print of the Shanghai No. 30 strain of hypsizigus marmoreus comprises the following steps of:

(1) Adding the fungus sample into liquid nitrogen for fully grinding;

(2) Adding 360 mu LBufferste and 40 mu L Buffer SDS into the ground powder rapidly, mixing rapidly and uniformly by vortex, placing the centrifuge tube in a water bath at 65 ℃ for 15min, and inverting the centrifuge tube in the water bath process to mix the samples for several times;

(3) Adding 5 mu L of RNase Solution into the lysate, mixing by vortex, and standing at room temperature for 15-30min;

(4) Adding 140 mu L Buffer PS, vortex shaking for 30s, and standing on ice for 10min;

(5) At room temperature, 13000g was centrifuged for 5min and 400 μl of supernatant was carefully transferred to a new centrifuge tube;

(6) Adding 600 mu L Buffer PBD into the sample, and vortex mixing for 30s;

(7) Loading the DNA binding column in a collecting pipe, transferring half of the mixed solution into the column, centrifuging at 8000g for 1min;

(8) Pouring out the filtrate, filling the column into a recovery header, transferring the residual mixed solution into the column, and centrifuging for 1min at 8000 g;

(9) Pouring out the filtrate, filling the column into a recovery header, adding 600 mu L Buffer GW2 into the column, centrifuging 8000g for 1min;

(10) Repeating the step 9;

(11) Pouring out the filtrate, filling the column into a recovery header, centrifuging 10000g for 2min, and removing residual ethanol in the column;

(12) Transferring the column to a new 1.5ml centrifuge tube, adding 30. Mu.L Buffer AE preheated to 65 ℃ to the center of the membrane of the column, standing for 2min at room temperature, and centrifuging for 1min with 10000 g;

(13) mu.L of DNA was used for detection by 1.2% agarose gel electrophoresis, 2. Mu.L of DNA was used for concentration measurement by a NanoDrop spectrophotometer, and the remaining DNA was stored at-20 ℃.

The PCR amplification system in the step (3) is as follows: a total volume of 10 μl, comprising: 10 XPCR buffer 1. Mu.L, 2.5mmol/L dNTP 0.8. Mu.L, 5U/mu.L TAKARA HSTaq enzyme 0.1. Mu.L, 5. Mu.mol/LTP-M130.5. Mu.L, 5. Mu.mol/L SSR labeled specific primers total volume of 0.6 eachMu L, concentration 20ng ~ 30 ng/mu L extracted template DNA 1.2 mu L, ddH ₂ O 5.2μL；

PCR reaction conditions: 95 ℃ for 5min;95 ℃ for 30s,60 ℃ for 30s,72 ℃ for 30s,30 cycles; 95 ℃ for 5min;95 ℃ for 30s,53 ℃ for 30s,72 ℃ for 30s,10 cycles; 30min at 60 ℃.

The sample adding buffer solution in the step (5) is 9 mu L of a mixed solution (0.5:8.5) of an internal molecular weight standard and formamide; the PCR amplification product was added in an amount of 1. Mu.L.

The specific process of denaturation in the step (5) is that denaturation is carried out for 3min at 95 ℃, and the mixture is placed in an ice-water mixture for cooling for 3min after the denaturation is finished.

The electrophoresis in the step (5) has the following technological parameters: the modified polyacrylamide gel is commercial POP7 gel, the electrophoresis buffer is 3730buffer EDTA, the injection voltage is 2000V, the operation voltage is 15000V, the sample injection time is 10s, the temperature is 60 ℃, the capillary length is 50cm, the power is 200W, the electrophoresis is 20min, and the current and the power are all dynamic.

The data analysis in the step (6) specifically includes: the detected original data file is imported into analysis software GeneMapper ID3.2, and analysis of group structure analysis, clustering and heterozygosity is carried out by using POPGENE, NTSYS and other software, so that core germplasm resources are calculated and analyzed. Allele factors (Na, ne), nei's genetic diversity index (He), shannon's diversity information index (I), and gene observed heterozygosity (Ho) were analyzed.

TABLE 2 allelic fragment information summary of SSR primer amplifications

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The application of SSR marker finger print of hypsizigus marmoreus 'Shanghai No. 30' strain is that 6 pairs of SSR primers developed by utilizing simple repeated sequence segments of hypsizigus marmoreus genome are utilized, a large number of SSR primers are screened, the number and numbering (Table 2) of allelic segments amplified by 6 pairs of SSR primers in each hypsizigus marmoreus cultivar are determined by carrying out band amplification on the SSR primers of the collected 59 main hypsizigus marmoreus cultivars, and the 'Shanghai No. 30' strain can be effectively identified in the collected 59 main hypsizigus marmoreus cultivars through the numbering combination of different SSR allelic sites. The relative molecular weight of the allele amplified by each SSR primer can be determined by capillary electrophoresis combined software analysis, and the strain with the combination of the specific SSR allele fragments of the strain 'Shanghai No. 30' is the strain 'Shanghai No. 30' of hypsizigus marmoreus, and the serial number combination of the strains is as follows: (8+16)/1/4/5/4/4.

The invention has the beneficial effects that: compared with the conventional morphological detection, antagonism test and fruiting test, the invention has the advantages of short detection time, high accuracy and good repeatability. The operation time required for detection is within 24 hours (including genomic DNA extraction, PCR amplification, electrophoresis analysis and data analysis), while the time required for the conventional antagonism test is at least two weeks, and the fruiting test is at least 3 months; the method has the specificity of 'Shanghai Zhu No. 30' strain among 59 commercial hypsizigus marmoreus main cultivation strains, and has good application prospect.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:

FIG. 1 is a graph showing the relative molecular weight peaks of the allele sites detected by the primer HMSSR5 in sequence in the selected hypsizigus marmoreus cultivation material "Huzhen No. 30" and several main cultivated commercial varieties, respectively;

FIG. 2 is a graph showing the relative molecular weight peaks of the allele sites detected by the primer HMSSR11 in sequence in the selected hypsizigus marmoreus cultivation material "Huzhen No. 30" and several main cultivated commercial varieties, respectively;

FIG. 3 is a graph showing the relative molecular weight peaks of the allele of the primer HMSSR18 detected sequentially in the selected hypsizigus marmoreus cultivation material "Huzhen No. 30" and several main cultivated commercial varieties, respectively;

FIG. 4 is a graph showing the relative molecular weight peaks of the allele of the primer HMSSR19 detected sequentially in the selected hypsizigus marmoreus cultivation material "Huzhen No. 30" and several main cultivated commercial varieties, respectively;

FIG. 5 is a graph showing the relative molecular weight peaks of the allele sites detected by the primer HMSSR31 in sequence in the selected hypsizigus marmoreus cultivation material "Huzhen No. 30" and several main cultivated commercial varieties, respectively;

FIG. 6 is a graph showing the relative molecular weight peaks of the allele sites detected by the primer HMSSR48 in the selected hypsizigus marmoreus cultivation material "Huzhen No. 30" and several main cultivars, respectively.

Detailed Description

In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.

Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

Example 1:

(1) Hypha culture: transferring hypsizigus marmoreus strain to potato glucose agar solid medium, culturing at 22 ℃ for 8d, and collecting mycelium;

(2) Extraction of genomic DNA: genomic DNA was extracted using the Meimei fungus DNA extraction kit according to the kit experimental procedure, and 2. Mu.L of DNA was taken for detection by 1.2% agarose gel electrophoresis. Detecting the concentration and purity of the total genome DNA by an ultraviolet spectrophotometry, and adjusting the concentration of the sample DNA to be consistent;

the process for extracting the genome DNA of the mycelium by the CTAB method comprises the following steps:

(1) adding the fungus sample into liquid nitrogen for fully grinding;

(2) adding 360 mu L of Buffer STE and 40 mu L of Buffer SDS into the ground powder rapidly, vortex and mix uniformly rapidly, placing the centrifuge tube in a water bath at 65 ℃ for 15min, and inverting the centrifuge tube to mix the samples for several times during the water bath;

(3) adding 5 mu L of RNase Solution into the lysate, mixing by vortex, and standing at room temperature for 15-30min;

(4) adding 140 mu L Buffer PS, vortex shaking for 30s, and standing on ice for 10min;

(5) at room temperature, 13000g was centrifuged for 5min and 400 μl of supernatant was carefully transferred to a new centrifuge tube;

(6) 600 μl Buffer PBD (diluted with absolute ethanol) was added to the sample, and vortexed for 30s;

(7) loading the DNA binding column in a collecting pipe, transferring half of the mixed solution into the column, centrifuging at 8000g for 1min;

(8) pouring out the filtrate, filling the column into a recovery header, transferring the residual mixed solution into the column, and centrifuging for 1min at 8000 g;

(9) pouring out the filtrate, filling the column into a recovery header, adding 600 mu L Buffer GW2 (diluted by absolute ethyl alcohol) into the column, centrifuging 8000g for 1min;

repeating step 9;

pouring out the filtrate, filling the column into a recovery header, centrifuging 10000g for 2min, and removing residual ethanol in the column;

turning the columnTransferring to a new 1.5ml centrifuge tube, adding 30 μl of Buffer AE preheated to 65deg.C to the center of the membrane of the column, standing at room temperature for 2min, centrifuging with 10000g for 1min;

mu.L of DNA was used for detection by 1.2% agarose gel electrophoresis, 2. Mu.L of DNA was used for concentration measurement by a NanoDrop spectrophotometer, and the remaining DNA was stored at-20 ℃.

(3) Detection of SSR molecular markers: carrying out PCR amplification of gene SSR markers on the extracted DNA;

the PCR amplification system is as follows: a total volume of 10 μl, comprising: 10 XPCR buffer 1. Mu.L, 2.5mmol/LdNTP 0.8. Mu.L, 5U/mu.L TAKARA HSTaq enzyme 0.1. Mu.L, 5. Mu.mol/L TP-M130.5. Mu.L, 5. Mu.mol/L SSR labeled specific primers each 0.6. Mu.L total volume, template DNA 1.2. Mu.L extracted at a concentration of 20 ng-30 ng/mu.L, ddH ₂ O 5.2μL；

PCR reaction conditions: 95 ℃ for 5min;95 ℃ for 30s,60 ℃ for 30s,72 ℃ for 30s,30 cycles; 95 ℃ for 5min;95 ℃ for 30s,53 ℃ for 30s,72 ℃ for 30s,10 cycles; 30min at 60 ℃. (4) electrophoresis detection: mixing 1 μl of the PCR amplified product with 9 μl of sample buffer, denaturing at 95deg.C for 3min, and cooling in ice-water mixture for 3min; applying 3 μL sample onto modified polyacrylamide gel for electrophoresis, wherein the modified polyacrylamide gel is commercial POP7 gel, the electrophoresis buffer is 3730buffer EDTA, the injection voltage is 2000V, the operation voltage is 15000V, the sample injection time is 10s, the temperature is 60 ℃, the capillary length is 50cm, the power is 200W, the electrophoresis is 20min, and the current and the power are all dynamic,

(5) Analysis of results

Performing PCR amplification and capillary electrophoresis on the hypsizigus marmoreus strain by adopting 6 pairs of SSR primers, and finding out the coincidence number combination by analyzing an allele factor (Na, ne), a Nei's genetic diversity index (He), a shannon's diversity information index (I) and a gene observation heterozygosity (Ho) and combining a relative molecular weight peak diagram of an allele site: HMSSR5, HMSSR11, HMSSR18, HMSSR19, HMSSR31, HMSSR48, the corresponding bands are: the strain of (8+16)/1/4/5/4/4 can be determined to be the Hypsizygus marmoreus No. 30. To ensure accuracy of the identification, three replicates were recommended.

Taking several main cultivated commercial varieties as an example, 6 pairs of allele site relative molecular weight peak diagrams obtained by sequential detection of primers are given as shown in figures 1-6 (in sequence, shanghai No. 1 (2K 3B 2 15-X2 (5) 14-X1 (6) 13-X6 (7) H2 8B 027A 9X 5 DTG)

B4 A kind of electronic device. The fingerprint spectrum of the invention refers to the primer and the band type combination thereof.

It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered in the scope of the claims of the present invention.

Sequence listing

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Claims (2)

1. A fingerprint identification method of SSR mark of Shanghai Zhen 30 strain of hypsizigus marmoreus is characterized in that: the fingerprint consists of 6 SSR marker loci, and the specific sequences of the corresponding 6 pairs of primers are as follows:

HMSSR5 forward primer: CACACCTTACGAGGTGAGCA;

reverse primer: TGTTTGATGTTAGCCGAACG;

HMSSR11 forward primer: GGAGTTTGAGTTGAGGCAGC;

reverse primer: ATGAACCAGACCAAAGACCG;

HMSSR18 forward primer: GAGGATTGAAGGGACTGTCG;

reverse primer: CCTCATCCTCCGACTCTACG;

HMSSR19 forward primer: GCTTCGGCATAACAATGTCC;

reverse primer: TGATTTGGTGTTGATGGTGAG;

HMSSR31 forward primer: ACGACGCACCGTGGTTTAT;

reverse primer: GTAGGGTAGCGGCATCGTT;

HMSSR48 forward primer: GCCGTGTGACACCAAATACA;

reverse primer: ACATCTAACCGACCCACGAC;

the corresponding band number combinations are: (8+16)/1/4/5/4/4;

the band type corresponding to the primer HMSSR5 is 8+16, wherein the band size of the allele fragment corresponding to the number 8 is 206-206.99 bp, and the band size of the allele fragment corresponding to the number 16 is 236-236.99 bp;

the band type number corresponding to the primer HMSSR11 is 1, and the band size of the corresponding allele fragment is 263-263.99 bp;

the band type number corresponding to the primer HMSSR18 is 4, and the band size of the corresponding allele fragment is 262-262.99 bp;

the band type number corresponding to the primer HMSSR19 is 5, and the band size of the corresponding allele fragment is 132-132.99 bp;

the band type number corresponding to the primer HMSSR31 is 4, and the band size of the corresponding allele fragment is 153-153.99 bp;

the band type number corresponding to the primer HMSSR48 is 4, and the band size of the corresponding allele fragment is 118-118.99 bp;

the preservation number of the Shanghai Zhen 30 strain of the hypsizigus marmoreus is GDMCCNo:61536.

2. the application of the SSR marked fingerprint of the Shanghai 30 strain of hypsizigus marmoreus as claimed in claim 1, which is characterized in that: the fingerprint of SSR mark of the Shanghai No. 30 strain of the hypsizigus marmoreus is used for identifying the specific allelic variation of the Shanghai No. 30 strain of the hypsizigus marmoreus and/or identifying the specificity of the Shanghai No. 30 strain of the hypsizigus marmoreus.

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