Method for authenticating mating type of eleven varieties in black morchella group

A kind of technology of mating type and Morchella, applied in the field of molecular biology, can solve problems such as being unable to identify whether Morchella strain is reliable, and achieve the effects of stable and reliable results and simple and convenient method.

Active Publication Date: 2017-09-12
KUNMING INST OF BOTANY - CHINESE ACAD OF SCI
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Problems solved by technology

[0005] The purpose of the present invention is to provide a method for identifying the mating types of eleven species in the Black Morchella group and the related general specific primer pairs in order to solve the problem that it is currently impossible to identify whether the morel strains are reliable. The primer pairs are used to identify the mating types of the strains of these eleven species for the presence or absence of bands after PCR amplification, so as to determine whether they are effective strains

Method used

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  • Method for authenticating mating type of eleven varieties in black morchella group
  • Method for authenticating mating type of eleven varieties in black morchella group
  • Method for authenticating mating type of eleven varieties in black morchella group

Examples

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Embodiment 1

[0055] Neutral Morchella (M.tridentina) X255 monoascospore strain mating type identification Neutral Morchella monoascospore strain X255-1—X255-11 was inoculated on PDA medium, cultured at 24°C for 7 days, and an appropriate amount of mycelium was picked , DNA was extracted by the CTAB method, and 4 μL was electrophoresed on a 1% (W / V) agarose gel to detect the quality and concentration of the DNA.

[0056] Using the DNA of X255-1—X255-11 as a template, PCR amplification was performed on MAT1-1L / MAT1-1R or 1L / 1R with primers: 25 μL amplification system included 12.5 μL of 2×PCRmix (TIANGENBioInc), 5 μM MAT1 -1L, 1 μL of MAT1-1R primers, 1 μL of DNA template, ddH 2O 9.5 μL. The PCR amplification program was: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 1 min, annealing at 50°C for 30 sec, extension at 72°C for 1 min, 35 cycles; extension at 72°C for 10 min.

[0057] Using the DNA of X255-1—X255-11 as a template, PCR amplification was performed on MAT1-2L / MAT1-...

Embodiment 2

[0060] Identification of mating type of M. semilibera monoascospore strain D907-1—D907-11 monoascospore strains of M. semilibera were inoculated on PDA medium, cultured at 24°C for 7 days, and picked Take an appropriate amount of mycelium, extract DNA by CTAB method, and take 4 μL to detect DNA quality and concentration by electrophoresis on 1% (W / V) agarose gel.

[0061] Using the DNA of D907-1—D907-11 as a template, PCR amplification was performed on MAT1-1L / MAT1-1R or 1L / 1R with primers: 25 μL amplification system included 2×PCRmix (TIANGENBioInc) 12.5 μL, 5 μM MAT1 -1L, 1 μL of MAT1-1R primers, 1 μL of DNA template, ddH 2 O 9.5 μL. The PCR amplification program was: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 1 min, annealing at 50°C for 30 sec, extension at 72°C for 1 min, 35 cycles; extension at 72°C for 10 min.

[0062] Using the DNA of D907-1—D907-11 as a template, PCR amplification was performed on MAT1-2L / MAT1-2R or 2L / 2R with primers. The 25 μL am...

Embodiment 3

[0065] Mating type identification of M. exuberans K63 monoascospore strain M. exuberans K63-1—K63-11 monoascospore strains were inoculated on PDA medium, cultured at 24°C for 7 days, and appropriate amount of bacteria were picked. Silk, DNA was extracted by CTAB method, and 4 μL was electrophoresed on 1% (W / V) agarose gel to detect the quality and concentration of DNA.

[0066] Using the DNA of K63-1—K63-11 as a template, PCR amplification was performed on MAT1-1L / MAT1-1R or 1L / 1R with primers: 25 μL amplification system included 2×PCRmix (TIANGENBioInc) 12.5 μL, 5 μM MAT1 -1L, 1 μL of MAT1-1R primers, 1 μL of DNA template, ddH 2 O 9.5 μL. The PCR amplification program was: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 1 min, annealing at 50°C for 30 sec, extension at 72°C for 1 min, 35 cycles; extension at 72°C for 10 min.

[0067] Using the DNA of K63-1—K63-11 as a template, PCR amplification was performed on MAT1-2L / MAT1-2R or 2L / 2R with primers. The 25 μL ...

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Abstract

The invention provides a method for authenticating the mating type of eleven varieties in a black morchella group. The method includes the steps of firstly, the strain total DNA of a to-be-authenticated strain is extracted to serve as the PCR amplification template; secondly, a primer pair MAT1-1L / MAT1-1R is used to perform PCR amplification to obtain an expected-band strain which indicates that the strain contains the MAT1-1 mating type, a primer pair MAT1-2L / MAT1-2R to perform PCR amplification to obtain an expected-band strain which indicates that the strain contains the MAT1-2 mating type, and the fact that the strain only contains a male parent or a female parent is indicated if only the strain of one mating type is detected, and the strain is poor in fruiting ability or incapable of fruiting; the strains with two expected amplification bands capable being detected have the male parent mating type and the female parent mating type and have fruiting ability. By the primers and detection method for detection the mating type genes of morchella, the high yield and stable yield reliability during morchella cultivation can be increased greatly. The molecular marker of the mating type genes is simple, convenient, easy to operate and applicable to the morchella mating type gene authentication and cultivation, strain breeding, species identification, sexual reproduction evolution and phylogenic studies.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to the identification of eleven kinds of morels, which are respectively: (M. M.exuberans, Mel-13, Mel-14, M.eximioides, M.eohespera, M.purpurascens, Mel-21 , Molecular detection method and specific primer pair of Morchella (M.dunalii) and Puche Morchella (M.pulchella) mating type gene. The technology of the present invention is applicable to Morchella strain breeding, cultivation, mating Research and development on genotypes, species identification, sexual reproductive evolution and phylogeny. Background technique [0002] O'Donnell, Taskin, and Du Xihui used the polygenic lineage consensus phylogenetic species recognition method (GCPSR), based on the nucleotide sequences of five genes, LSU, ITS, EF1-a, RPB1, and RPB2, to identify Morchella The genus of fungi is divided into yellow morel clade (Esculenta Clade), black morel clade (Elata Clade) and reddish more...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/6895
Inventor 杜习慧杨祝良
Owner KUNMING INST OF BOTANY - CHINESE ACAD OF SCI
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