CN112626255B - InDel marker fingerprint spectrum of mushroom CV108 strain and construction method thereof - Google Patents
InDel marker fingerprint spectrum of mushroom CV108 strain and construction method thereof Download PDFInfo
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Abstract
The invention relates to an InDel mark fingerprint of mushroom CV108 strain and a construction method thereof, wherein the fingerprint consists of 7 InDel marks developed after sequencing based on mushroom whole genome. The InDel marked fingerprint of the mushroom CV108 strain can be used for the specificity identification of the mushroom CV108 strain, has the advantages of short detection time, high accuracy and good repeatability, and has the specificity of the mushroom CV108 strain in the collected 44 mushroom strains.
Description
Technical Field
The invention belongs to the field of detection of mushroom strains, and particularly relates to an InDel marked fingerprint of a mushroom CV108 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 seeds in China at present. According to statistics of the China edible fungus Association, the total yield of Chinese edible fungi in 2018 is 3842.04 ten thousand tons, wherein the total yield of the shiitake mushrooms is 1043.12 ten thousand tons, which accounts for 27.15% of the total yield of domestic edible fungi and accounts for more than 90% 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 50% 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 throughput of sequencing is increasing and the cost is decreasing. The DNA molecular marker technology can detect the genetic specificity of mushroom strains on the gene level, and the completion of mushroom whole genome sequencing 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 developing lentinus edodes whole genome sequencing molecular markers. And the InDel marker has the advantages of abundant quantity, high accuracy, good stability, rapidness, convenience and the like, and can be used for molecular marker assisted breeding and mushroom strain identification.
Disclosure of Invention
The invention aims to solve the technical problem of providing an InDel marked fingerprint of mushroom CV108 strain and a construction method and application thereof, wherein the fingerprint has the advantages of short detection time, high accuracy and good repeatability compared with conventional morphological detection, antagonistic test and fruiting test.
The fingerprint of the InDel marker of the mushroom CV108 strain consists of 7 pairs of InDel markers, is an InDel marker primer developed based on an insertion/deletion fragment of a mushroom genome, and has the advantages of good amplification band type, high repeatability and detailed marking information 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 CV108 strain, which is to utilize 7 pairs of InDel marked primers developed by the insertion/deletion fragment of the mushroom genome to carry out InDel marked amplification on the mushroom strain, compare the obtained banding pattern with the banding pattern of the CV108 strain, and obtain the mushroom CV108 strain if the banding pattern is consistent with the banding pattern;
the combination of the band type numbers of the mushroom CV108 strains is as follows: (1+2) 2 (1+2) 12 (1+2) 1.
Through the banding amplification of the collected InDel marked primers of 44 mushroom varieties, the invention determines the number and the numbering of the allelic fragments of 7 pairs of InDel marked primers amplified in the 44 mushroom varieties (table 2), and CV108 strains can be effectively identified through the combination of the numbering 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 D2000 bp DNA ladder, a CV108 strain specific InDel allelic fragment combination strain exists, namely the mushroom CV108 strain, and the serial number combination of the strain is as follows: (1+2) 2 (1+2) 12 (1+2) 1.
TABLE 2 summary of allelic fragment information from InDel primer amplification
The invention also provides a construction method of the InDel marked fingerprint of the mushroom CV108 strain, which comprises the following steps:
(1) Hypha culture: transferring the mushroom hyphae into a potato glucose culture medium PDA, culturing at 23-25 ℃ in a dark place, 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 process for extracting the genome DNA of hyphae by the CTAB method 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 65 deg.C for 1 hr, keeping the temperature at 65 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 25:24:1, 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 at 12000rpm and 4 ℃ for 10min, and taking supernatant (recording volume) and transferring the supernatant 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 5min;
(9) removing 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 (1.25U/25. 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: 5min at 94 ℃; 45second at 94 ℃, 45second at 55-60 ℃, 30second at 72 ℃,35 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 2.5 percent, the electrophoresis buffer solution is 1 × TAE, the voltage is 90v, the current is 300mA, the power is 100W, the electrophoresis is performed for 5h, and the result is photographed and analyzed;
performing PCR amplification on the mushroom strain by adopting 7 pairs of InDel labeled primers, determining the number and relative molecular weight of allelic fragments amplified by each InDel labeled primer by using a DNA molecular weight control D2000 bp DNA ladder, and finding a combination of No. Fu Gebian: the strain of (1+2) 2 (1+2) 12 (1+2) 1 can be determined as the mushroom CV108 strain.
The invention has the advantages of
The InDel marked fingerprint of the mushroom CV108 strain can be used for identifying the CV108 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 CV108 strains in the collected 44 mushroom strains in China, and has good application prospect.
Drawings
FIG. 1 is fingerprint of mushroom CV108 strain InDel marker, where M is D2000 bp DNA ladder, numerals 1-7 represent 7 pairs of InDel marker primers, and the arrow indicates the specific InDel allelic fragment combination of mushroom CV108 strain;
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 is an amplification map of primer Lefp _ id005 in selected 44 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 the primer Lefp _ id007 in the selected 44 shiitake 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 may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Marker、Premix Taq TM All purchased from: takala Bio doctor Biotech Co., ltd
The remaining materials and reagents were 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 from research institute of edible and medicinal fungi in Songyang county; 4. 8404 strain, from edible fungus of Yichangsen origin, hubei province; 5. huaxiang No. 5 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. l135 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. shenxiang No. 18 strain, sourced from edible fungus institute of academy of agricultural sciences in Shanghai city; 13. incense such as bacterial strain, obtained from edible fungus institute of academy of agricultural sciences of Shanghai city; 14. the Shenxiang No. 15 strain is obtained from edible fungus institute of academy of agricultural sciences in Shanghai city; 15. CV105 strain, sourced from edible fungus institute of academy of agricultural sciences of Shanghai city; 16. the L241 strain is 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. the Shenxiang No. 16 strain is originated from edible fungus institute of academy of agricultural sciences of 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. RBL1 strain, 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; 25. the Shanghai fragrant F2 strain is sourced from edible fungus institute of academy of agricultural sciences of Shanghai city; 26. l241-4 strain, sourced from technical research center of edible fungi 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. CV501 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. l952 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; 35. l212 strain, sourced 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 TW1151 strain is from edible fungi institute 38 and L9105 strain of agricultural academy of sciences of Shanghai city, and is from technical research center of edible fungi of Qingyuan county of 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 of Shanghai city; 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, academy of agricultural sciences, shanghai city; 43. WD5140 strain, sourced from edible fungus institute of academy of agricultural sciences of Shanghai city; 44. 931 Strain, sourced from edible fungus institute of academy of agricultural sciences of Shanghai city.
Wherein 7 pairs of InDel labeled primer sequences (5 '-3') are as follows:
lefp _ id001 forward primer: ACGGATGGAGAGACACAGGA
Reverse primer: TGCCCCACTTACTCTCAACC
Lefp _ id002 forward primer: CCTTTCTCAAAAGCGGACTG
Reverse primer: GGGAGTGGGTTGTTTGGATA
Lefp _ id003 forward primer: CGGATGTTATGCACTGGAAG
Reverse primer: CGTACGGTTGGACATTTGAA
Lefp _ id004 forward primer: AGCCTTTCACAGTCAGCTCG
Reverse primer: GTCAACGGAGGGAAACAGAG
Lefp _ id005 forward primer: GTAGCACTCCTCATACAACC
Reverse primer: ACCAAATGTCACAGCACAGG
Lefp _ id006 forward primer: ACATTGGCGAAGGCTGTACG
Reverse primer: CCCTTTTGCCCTATAAGGCG
Lefp _ id007 forward primer: AACAGTAACCTGTGCACTGC
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 23-25 ℃ in the dark, 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 65 deg.C for 1 hr, maintaining at 65 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 the supernatant into a mixture with a volume ratio of 25:24:1, 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 at 12000rpm and 4 ℃ for 10min, and taking supernatant (recording volume) and transferring the supernatant 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 5min;
(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 Ι _, comprising: premix Taq TM (1.25U/25. 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: 5min at 94 ℃; 45second at 94 ℃, 45second at 55-60 ℃, 30second at 72 ℃,35 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 2.5 percent, the electrophoresis buffer solution is 1 × TAE, the voltage is 90v, the current is 300mA, the power is 100W, the electrophoresis is performed for 5h, 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 the allelic fragments amplified by each InDel labeled primer can be determined by controlling a DNA ladder by DNA molecular weight D2000 bp, and as shown in figure 1, a combination of No. Fu Gebian is found as follows: (1+2) 2 (1+2) 12 (1+2) 1, that is, the strain is the mushroom CV108 strain, wherein the number 1 in FIGS. 2-8 is CV108 strain, and the band thereof is the same as that in FIG. 1.
Sequence listing
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Claims (1)
1. A method for identifying mushroom CV108 strains is characterized in that the mushroom strains are amplified by utilizing the following 7 InDel labeled primer combinations, and the obtained banding pattern is compared with the banding pattern of the CV108 strains, and the banding pattern is consistent with the banding pattern, namely the mushroom CV108 strains; the combination of the band type numbers of the mushroom CV108 strains is as follows: (1+2)/2/(1+2)/1/2/(1+2)/1; the number corresponding to the primer Lefp _ id001 is a first pair of band types (1+2), wherein 1 corresponds to 203bp, and 2 corresponds to 149bp; the number corresponding to the primer Lefp _ id002 is a second pair of band types 2, wherein 2 corresponds to 214bp; the primer Lefp _ id003 corresponds to 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 primer Lefp _ id006 is the sixth band pair (1+2), where 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 sequences of 7 pairs of InDel labeled primers are 5'-3' as follows:
lefp _ id001 forward primer: ACGGATGGAGAGACACAGGA
Reverse primer: TGCCCCACTTACTCTCAACC
Lefp _ id002 forward primer: CCTTTCTCAAAAGCGGACTG
Reverse primer: GGGAGTGGGTTGTTTGGATA
Lefp _ id003 forward primer: CGGATGTTATGCACTGGAAG
Reverse primer: CGTACGGTTGGACATTTGAA
Lefp _ id004 forward primer: AGCCTTTCACAGTCAGCTCG
Reverse primer: GTCAACGGAGGGAAACAGAG
Lefp _ id005 forward primer: GTAGCACTCCTCATACAACC
Reverse primer: ACCAAATGTCACAGCACAGG
Lefp _ id006 forward primer: ACATTGGCGAAGGCTGTACG
Reverse primer: CCCTTTTGCCCTATAAGGCG
Lefp _ id007 forward primer: AACAGTAACCTGTGCACTGC
Reverse primer: CATGATCAGATCACACAGCG.
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CN106755510A (en) * | 2017-02-07 | 2017-05-31 | 上海市农业科学院 | A kind of SSR marker finger-print of fragrant No. 15 strains in mushroom Shen and its construction method and application |
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CN102876781A (en) * | 2012-09-11 | 2013-01-16 | 上海市农业科学院 | SSR marked fingerprint spectrum of shiitake fungus L808 strain and application thereof |
CN106755510A (en) * | 2017-02-07 | 2017-05-31 | 上海市农业科学院 | A kind of SSR marker finger-print of fragrant No. 15 strains in mushroom Shen and its construction method and application |
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InDel 标记及其在食用菌研究中的应用与展望;沈秀芬等;《上海农业学报》;20200630;第36卷(第3期);第137页第2、6段,第138页第1-2段 * |
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