CN112626254B - InDel marker fingerprint spectrum of shiitake mushroom L9319 strain and construction method thereof - Google Patents

Abstract

The invention relates to an InDel marker fingerprint of mushroom L9319 strain and a construction method thereof, wherein the fingerprint consists of 7 InDel markers developed after sequencing based on the whole genome of mushroom. The InDel marked fingerprint of the mushroom L9319 strain can be used for the specificity identification of the mushroom L9319 strain, has the advantages of short detection time, high accuracy and good repeatability, and has the specificity of the mushroom L9319 strain in the collected 44 mushroom strains.

Description

InDel marker fingerprint spectrum of shiitake mushroom L9319 strain and construction method thereof

Technical Field

The invention belongs to the field of detection of mushroom strains, and particularly relates to an InDel marked fingerprint of a mushroom L9319 strain and a construction method thereof.

Background

Lentinus edodes (Lentinula edodes (Berk.) Pegler) is popular among consumers because of its high nutritional and medicinal value. China is the earliest country for artificially cultivating the mushrooms in the world, the cultivation history has been over 800 years, and the mushrooms are the most widely cultivated and highest-yield mushroom species in China at present. According to statistics of the edible fungus society of China, the total yield of the edible fungi in China in 2018 is 3842.04 ten thousand tons, wherein the total yield of the shiitake mushrooms is 1043.12 ten thousand tons, accounts for 27.12 percent of the total yield of the edible fungi in China, and accounts for more than 90 percent of the total yield of the shiitake mushrooms in the world. In recent years, the shiitake mushroom industry becomes an important hand grip for domestic accurate poverty relief, shiitake mushrooms are selected as an important industry from national poor counties of more than 20% of China according to statistics, the cultivation scale of the shiitake mushrooms in China is further expanded, but with the rapid development of the shiitake mushroom industry in China, the problem that the shiitake mushroom production is similar and different in name and is also similar is increasingly prominent, so that the intellectual property of shiitake mushroom breeders is damaged, producers are greatly risky, and hidden dangers are caused for the further development of the shiitake mushroom industry. In order to guarantee the rights and interests of breeders, reduce the risks of producers and promote the healthy, stable and sustainable development of the shiitake mushroom industry in China, a simple, convenient, rapid and reliable shiitake mushroom strain specificity identification technology is urgently established.

With the rapid development of bioinformatics and the continuous maturation of DNA sequencing technology, the sequencing throughput is increasing and the cost is decreasing. The DNA molecular marker technology can detect the genetic specificity of the mushroom strains from the gene level, and the completion of the sequencing of the whole genome of the mushroom provides convenience for the development of InDel markers. The InDel molecular marker is an application based on a high-throughput sequencing technology, reduces the cost of InDel marker development, and is suitable for the development of lentinus edodes whole genome sequencing molecular markers. And the InDel marker has the advantages of rich quantity, high accuracy, good stability, rapidness, simplicity and convenience and the like, and can be used for molecular marker assisted breeding and also for lentinus edodes strain identification.

Disclosure of Invention

The invention aims to solve the technical problem of providing an InDel marked fingerprint of a mushroom L9319 strain and a construction method and application thereof.

The fingerprint of the InDel marker of the mushroom L9319 strain consists of 7 pairs of InDel markers, is an InDel marker primer developed based on an insertion/deletion fragment of a mushroom genome, has good amplification band type and high repeatability, and the detailed information of the markers is shown in Table 1.

TABLE 1 InDel tagged primer information List

The invention also provides an application of the InDel marked fingerprint of the mushroom L9319 strain, which is to utilize 7 pairs of InDel marked primers developed by the insertion/deletion segments of the mushroom genome to carry out InDel marked amplification on the mushroom strain, and compare the obtained banding pattern with the banding pattern of the L9319 strain, and the banding pattern is the mushroom L9319 strain when the banding pattern is consistent with the banding pattern;

the combination of the band type numbers of the mushroom L9319 strain is as follows: 02 (1+2) 12 (1+2) 1.

Through the banding amplification of the collected InDel marker primers of 44 mushroom varieties, the invention determines the number and the serial number of the allelic fragments of 7 pairs of InDel marker primers amplified in 44 mushroom varieties (table 2), and the L9319 strain can be effectively identified through the serial number combination of different InDel allelic sites (figures 2-8). The relative molecular weight of the amplified allelic locus of each InDel marker primer can be determined by DNA molecular weight control D2000bp DNA ladder, and the species with L9319 species specific InDel allelic fragment combination exists, namely the shiitake mushroom L9319 species, and the number combination of the species is as follows: 02 (1+2) 12 (1+2) 1.

TABLE 2 summary of allelic fragment information for InDel primer amplification

The invention also provides a construction method of the InDel marked fingerprint of the mushroom L9319 strain, which comprises the following steps:

(1) Hypha culture: transferring the mushroom hyphae into a potato glucose culture medium PDA, culturing at 23-22 ℃ in the dark, and collecting the hyphae after 10 days;

(2) Extraction of genomic DNA: extracting genome DNA of the hypha by using a CTAB method, detecting the concentration and purity of the total genome DNA by using an ultraviolet spectrophotometry, and adjusting the concentration of the sample DNA to be 20-30 ng/uL;

the CTAB method for extracting genome DNA of hyphae comprises the following steps:

(1) putting the freeze-dried mushroom mycelia into a 2mL centrifuge tube, and putting 2-3 steel balls;

(2) putting the centrifuge tube into a multi-sample tissue grinder, setting the frequency to be 60Hz, and grinding for 30s until the powder is uniform;

(3) adding 2 × CTAB extractive solution preheated at 62 deg.C for 1 hr, maintaining at 62 deg.C for 60min, and shaking gently once every 20 min;

(4) centrifuging at 12000rpm and 4 deg.C for 20min, subpackaging the supernatant, and subpackaging two tubes each with 800uL;

(5) adding a volume ratio of 22:24:1, gently mixing the mixed solution of phenol, chloroform and isoamylol uniformly for 10min, centrifuging at 12000rpm and 4 ℃ for 10min, and taking supernate and transferring the supernate into a new centrifugal tube;

(6) adding the centrifuge tube into a reactor with the volume ratio of 24:1, gently mixing the chloroform and isoamylol mixture for 10min, centrifuging the mixture for 10min at 12000rpm and 4 ℃, and transferring supernatant (recording volume) into another centrifugal tube;

(7) adding pre-cooled isopropanol of-20 ℃ into the centrifuge tube with volume being 2/3 of that of the supernatant in the step (6), uniformly mixing, standing and precipitating for 1h at-20 ℃, centrifuging for 10min at 8000rpm and 4 ℃, and discarding the supernatant;

(8) adding 1mL of 70% ethanol into the obtained precipitate, washing for 1 time at 8000rpm, and centrifuging at room temperature for 2min;

(9) discarding the ethanol, and drying in an ultra-clean workbench; adding 100. Mu.L of 10 XTE buffer solution, gently tapping to dissolve the precipitate, adding 1. Mu.L of 10mg/mL RNaseA in water bath at 37 ℃ for 1h, and removing RNA;

storing the obtained DNA extract in refrigerator at-20 deg.C for use;

detection of InDel molecular marker: carrying out PCR amplification of a whole genome InDel marker on the extracted DNA;

the PCR amplification system is as follows: total volume 20 μ L, including: premix Taq ^TM (1.22U/22 uL Taq DNase, 0.4mM/L dNTP,0.3mM/L PCR buffer) 10 uL InDel marker forward primer and reverse primer each 1 uL in total volume, 2 uL template DNA extracted at the concentration of 20-30 ng/uL, ddH ₂ O 6μL；

And (3) PCR reaction conditions: 94 ℃ for 2min; 42second at 94 ℃, 42second at 22-60 ℃, 30second at 72 ℃ for 32 cycles; 7min at 72 ℃; storing at 10 deg.C;

and (3) electrophoresis detection: spotting 7 mu L of the product obtained by the PCR amplification on agarose gel added with nucleic acid dye for electrophoresis, wherein the volume percentage concentration of the agarose gel is 2.2 percent, the electrophoresis buffer solution is 1 XTAE, the voltage is 90v, the current is 300mA, the power is 100W, the electrophoresis is performed for 2h, and the result is photographed and analyzed;

performing PCR amplification on the mushroom strain by adopting 7 pairs of InDel marker primers, determining the number and relative molecular weight of allelic fragments amplified by each InDel marker primer by using a DNA molecular weight control D2000bp DNA ladder, and finding a combination meeting the number: 02 (1 + 2) 12 (1 + 2) 1, namely the strain is determined to be the mushroom L9319 strain.

The invention has the advantages of

The InDel marked fingerprint of the mushroom L9319 strain can be used for identifying the L9319 strain, and has the advantages of short detection time, high accuracy and good repeatability compared with conventional morphological detection, antagonistic test and fruiting test. The detection time only needs 3-4 days, while the conventional antagonism test needs at least two weeks, and the fruiting test needs at least 3 months; the method has the specificity of the L9319 strain in the collected 44 mushroom strains in China, and has good application prospect.

Drawings

FIG. 1 is an InDel marker fingerprint of L9319 strain of Lentinus edodes, wherein M is D2000bp DNA ladder, numerals 1-7 represent 7 pairs of InDel marker primers used, and the arrow indicates the specific InDel allelic fragment combination of L9319 strain of Lentinus edodes;

FIG. 2 is an amplification map of a primer Lefp _ id001 in 44 selected mushroom materials;

FIG. 3 is an amplification map of primer Lefp _ id002 in 44 selected shiitake mushroom materials;

FIG. 4 is an amplification map of primer Lefp _ id003 in 44 selected mushroom materials;

FIG. 5 is an amplification map of primer Lefp _ id004 in selected 44 shiitake mushroom materials;

FIG. 6 shows the amplification pattern of primer Lefp _ id002 in 44 selected shiitake mushroom materials;

FIG. 7 shows the amplification pattern of primer Lefp _ id006 in 44 selected mushroom materials;

FIG. 8 shows the amplification pattern of primer Lefp _ id007 in 44 selected mushroom materials.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the teaching of the present invention, and these equivalents also fall within the scope of the claims appended to the present application.

Marker、Premix Taq ^TM All purchased from Takala Bio physician technology Co., ltd

The rest materials and reagents are all common commercial products.

44 strains were derived:

1. CV108 strain, sourced from edible fungus institute of academy of agricultural sciences of Shanghai city; 2. xiangjiu strain, available from institute of microorganisms of Guangdong province; 3. the L7402 strain is originated from edible and medicinal fungi research institute in Songyang county; 4. 8404 strain, from edible fungus of Yichangsen origin, hubei province; 2. huaxiang No. 2 strain, sourced from university of agriculture in Huazhong; 6. CV442 strain, available from institute of domestic fungus, academy of agricultural sciences, shanghai city; 7. guangxi incense strain, sourced from Guangdong institute for microbiology; 8. l132 strain, available from Sanming fungus institute of Fujian province; 9. the L9319 strain is from the research and development company Limited of the large mountain mushroom industry of Lishui city, zhejiang province; 10. minfeng No. 1 strain, san Ming fungus institute of Fujian province; 11. senyuan No. 1 strain, available from edible fungus of Yichangsen source, limited liability company, hubei province; 12. the strain Shenxiang No. 18 is obtained from edible fungus institute of academy of agricultural sciences in Shanghai city; 13. incense such as strain, sourced from edible fungus institute of academy of agricultural sciences of Shanghai city; 14. shenxiang No. 12 strain, originated from edible fungus institute of academy of agricultural sciences, shanghai city; 12. CV102 strain, sourced from edible fungus institute of academy of agricultural sciences of Shanghai city; 16. the L241 strain is obtained from edible fungus institute of academy of agricultural sciences of Shanghai city; 17. 26 fragrant impurities, sourced from Guangdong institute for microorganisms; 18. l9608 strain, research center of edible fungi of Xixia county, henan; 19. the Z3244 strain is from edible fungus institute of academy of agricultural sciences of Shanghai city; 20. shenxiang No. 16 strain, sourced from edible fungus institute of academy of agricultural sciences in Shanghai city; 21. huaxiang No. 8 strain, sourced from university of agriculture in Huazhong; 22. the N7 strain is sourced from edible fungus institute of academy of agricultural sciences of Shanghai city; 23. the RBL1 strain is sourced from edible fungus institute of academy of agricultural sciences of Shanghai city; 24. the Z3210 strain originated from edible fungus institute of academy of agricultural sciences of Shanghai city; 22. the Shanghai fragrant F2 strain is sourced from edible fungus institute of academy of agricultural sciences of Shanghai city; 26. l241-4 strain, which is sourced from technical research center of edible fungi science in Qingyuan county, zhejiang province; 27. l26 strain, available from Sanming fungus institute of Fujian province; 28. the Junxing No. 8 strain is sourced from research and development centers of edible fungi in Lishui city, zhejiang province; 29. CV201 strain, sourced from edible fungus institute of academy of agricultural sciences of Shanghai city; 30. the source of the suxiang strain is edible fungus institute of academy of agricultural sciences of Shanghai city; 31. l922 strain, sourced from university of agriculture in china; 32. the Z3239 strain is from edible fungus institute of academy of agricultural sciences of Shanghai city; 33. the Z3243 strain is from edible fungus institute of academy of agricultural sciences of Shanghai city; 34. HK3161 strain, sourced from edible fungus institute of academy of agricultural sciences of Shanghai city; 32. the L212 strain is obtained from edible fungus institute of academy of agricultural sciences of Shanghai city; 36. l868 strain, sourced from edible fungus institute of academy of agricultural sciences of Shanghai city; 37. the strain TW1121 was derived from edible fungus institute 38, L9102 strain of academy of agricultural sciences of Shanghai city, and was derived from technical research center of edible fungus science of Qingyuan county, zhejiang province; 39. shenxiang No. 12 strain, originated from edible fungus institute of academy of agricultural sciences in Shanghai city; 40. j868 strain, sourced from edible fungus institute of academy of agricultural sciences, shanghai; 41. senyuan No. 10 strain, available from edible fungus of Yichangsen source, limited liability company, hubei province; 42. WD4204 strain, available from institute of edible fungi of academy of agricultural sciences, shanghai; 43. WD2140 strain from edible fungus institute of academy of agricultural sciences, shanghai; 44. 931 Strain, sourced from edible fungus institute of academy of agricultural sciences of Shanghai city.

Wherein 7 pairs of InDel marked primer sequences (2 '-3') are as follows:

lefp _ id001 forward primer: ACGGATGGAGAGACACAGGA

Reverse primer: TGCCCCCACTTTACTCTCAACC

Lefp _ id002 forward primer: CCTTTCTCAAAAGCGGACTG

Reverse primer: GGGAGTGGGGTTGTTGGATA

Lefp _ id003 forward primer: CGGATGTTATGCACTGGAAG

Reverse primer: CGTACGGTTGGACCATTTGAA

Lefp _ id004 forward primer: AGCCTTTCAAGTCAGCTCG

Reverse primer: GTCAACGGAGGGAAACAGAG

Lefp _ id002 forward primer: GTAGCACTCCTCACAACC

Reverse primer: ACCAAATGTCACACAGCACAGG

Lefp _ id006 forward primer: ACATTGGGCGAAGGCTGTACG

Reverse primer: CCCTTTTGCCTATAAGGCG

Lefp _ id007 forward primer: AACAGTAACCTGTGTGCGCTGC

Reverse primer: CATGATCAGATCACACAGCG.

The primers were synthesized by Shanghai Bioengineering Co., ltd.

Example 1

(1) Hypha culture: transferring the mushroom hyphae into a potato glucose culture medium PDA, culturing at 23-22 ℃ in a dark place, and collecting the hyphae after 10 days;

(2) Extraction of genomic DNA: extracting genome DNA of the hyphae by a CTAB method, detecting the concentration and purity of the total genome DNA by an ultraviolet spectrophotometry, and adjusting the concentration of the sample DNA to be 20-30 ng/uL;

the CTAB method for extracting genome DNA of hyphae comprises the following steps:

(1) putting the freeze-dried mushroom mycelia into a 2mL centrifuge tube, and putting 2-3 steel balls;

(2) putting the centrifuge tube into a multi-sample tissue grinder, setting the frequency to be 60Hz, and grinding for 30s until the powder is uniform;

(3) adding 2 × CTAB extractive solution preheated at 62 deg.C for 1 hr, maintaining at 62 deg.C for 60min, and shaking gently once every 20 min;

(4) centrifuging at 12000rpm and 4 deg.C for 20min, subpackaging the supernatant, and subpackaging two tubes each with 800uL;

(5) adding a volume ratio of 22:24:1, gently mixing the mixed solution of phenol, chloroform and isoamylol uniformly for 10min, centrifuging at 12000rpm and 4 ℃ for 10min, and taking supernate and transferring the supernate into a new centrifugal tube;

(6) adding the centrifuge tube with the volume ratio of 24:1, gently mixing the chloroform and isoamylol mixture for 10min, centrifuging the mixture for 10min at 12000rpm and 4 ℃, and transferring supernatant (recording volume) into another centrifugal tube;

(7) adding pre-cooled isopropanol of-20 ℃ into the centrifuge tube in an amount which is 2/3 of the volume of the supernatant in the step (6), uniformly mixing, standing and precipitating at-20 ℃ for 1h, centrifuging at 8000rpm and 4 ℃ for 10min, and removing the supernatant;

(8) adding 1mL of 70% ethanol into the obtained precipitate, washing for 1 time at 8000rpm, and centrifuging at room temperature for 2min;

(9) and (5) discarding the ethanol, and drying in an ultra-clean workbench. Adding 100. Mu.L of 10 XTE buffer solution, gently tapping to dissolve the precipitate, adding 1. Mu.L of 10mg/mL RNaseA in water bath at 37 ℃ for 1h, and removing RNA;

storing the obtained DNA extract in refrigerator at-20 deg.C for use;

(3) Detection of InDel molecular marker: carrying out PCR amplification of a whole genome InDel marker on the extracted DNA;

the PCR amplification system is as follows: total volume 20 μ L, including: premix Taq ^TM (1.22U/22. Mu.L Taq DNase, 0.4mM/L dNTP,0.3mM/L PCR buffer) 10. Mu.L InDel labeled forward primer and reverse primer each 1. Mu.L, 2. Mu.L ddH of template DNA extracted at a concentration of 20-30 ng/. Mu.L ₂ O 6μL；

And (3) PCR reaction conditions: 94 ℃ for 2min; 42second at 94 ℃, 42second at 22-60 ℃, 30second at 72 ℃ for 32 cycles; 7min at 72 ℃; storing at 10 deg.C;

(4) And (3) electrophoresis detection: spotting 7 mu L of the product obtained by the PCR amplification on agarose gel added with nucleic acid dye for electrophoresis, wherein the volume percentage concentration of the agarose gel is 2.2 percent, the electrophoresis buffer solution is 1 × TAE, the voltage is 90v, the current is 300mA, the power is 100W, the electrophoresis is carried out for 2h, and the result is photographed and analyzed;

the mushroom strains are respectively subjected to PCR amplification by adopting 7 pairs of InDel labeled primers, the number and the relative molecular weight of allelic fragments amplified by each InDel labeled primer can be determined by DNA molecular weight control D2000bp DNA ladder, and as shown in figure 1, the combination meeting the number is found as follows: 02 (1 + 2) 12 (1 + 2) 1, namely the strain is the mushroom L9319 strain, the number 9 in the figures 2-8 is the L9319 strain, and the bands are the same as those in the figure 1.

Sequence listing

<110> Shanghai city academy of agricultural sciences

<120> InDel mark fingerprint spectrum of mushroom L9319 strain and construction method thereof

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

acggatggag agacacagga 20

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tgccccactt actctcaacc 20

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cctttctcaa aagcggactg 20

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gggagtgggt tgtttggata 20

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cggatgttat gcactggaag 20

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cgtacggttg gacatttgaa 20

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

agcctttcac agtcagctcg 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gtcaacggag ggaaacagag 20

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gtagcactcc tcatacaacc 20

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

accaaatgtc acagcacagg 20

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

acattggcga aggctgtacg 20

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cccttttgcc ctataaggcg 20

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

aacagtaacc tgtgcactgc 20

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

catgatcaga tcacacagcg 20

Claims (1)

1. A method for identifying mushroom L9319 bacterial, characterized by that to InDel label primer combination, amplify mushroom bacterial with following 7, the banding pattern got is compared with banding pattern of L9319 bacterial, it is mushroom L9319 bacterial that is identical with the banding pattern; the combination of the band type numbers of the mushroom L9319 strain is as follows: 0/2/(1 + 2)/1; wherein the number corresponding to the primer Lefp _ id001 is a first pair of banding patterns 0, wherein 0 corresponds to no banding; the number corresponding to the primer Lefp _ id002 is a second pair of band types 2, wherein 2 corresponds to 214bp; the number corresponding to the primer Lefp _ id003 is a third pair of band types (1 + 2), wherein 1 corresponds to 265bp, and 2 corresponds to 248bp; the number corresponding to the primer Lefp _ id004 is a fourth pair of band types 1, wherein 1 corresponds to 270bp; the number corresponding to the primer Lefp _ id005 is a fifth pair of band types 2, wherein 2 corresponds to 346bp; the number corresponding to the primer Lefp _ id006 is the sixth pair of band types (1 + 2), wherein 1 corresponds to 277bp, and 2 corresponds to 262bp; the number corresponding to the primer Lefp _ id007 is a seventh pair of band types 1, wherein 1 corresponds to 284bp;

the 7 pairs of InDel labeled primer sequences 5'-3' are as follows:

lefp _ id001 forward primer: ACGGATGGAGAGACACAGGA

Reverse primer: TGCCCCCACTTTACTCTCAACC

Lefp _ id002 forward primer: CCTTTCTCAAAAGCGGACTG

Reverse primer: GGGAGTGGGGTTGTTGGATA

Lefp _ id003 forward primer: CGGATGTTATGCACTGGAAG

Reverse primer: CGTACGGTTGGACCATTTGAA

Lefp _ id004 forward primer: AGCCTTTCAAGTCAGCTCG

Reverse primer: GTCAACGGAGGGAAACAGAG

Lefp _ id005 forward primer: GTAGCACTCCTCACAACC

Reverse primer: ACCAAATGTCACACAGCACAGG

Lefp _ id006 forward primer: ACATTGGGCGAAGGCTGTACG

Reverse primer: CCCTTTTGCCTATAAGGCG

Lefp _ id007 forward primer: AACAGTAACCTGTGTGCGCTGC

Reverse primer: CATGATCAGATCACACAGCG.

Images