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Method for distinguishing mating types of four varieties in black morchella group and primer pair

A technology for mating type and Morchella, applied in the field of biology, can solve problems such as loss of mating type, and achieve the effects of simple method and stable and reliable results

Active Publication Date: 2017-01-04
云南省农业科学院生物技术与种质资源研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Both the spore isolation and tissue isolation of Morchella may only obtain a strain with a mating type, and a mating type will also be lost during the subculture of the strain

Method used

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  • Method for distinguishing mating types of four varieties in black morchella group and primer pair
  • Method for distinguishing mating types of four varieties in black morchella group and primer pair
  • Method for distinguishing mating types of four varieties in black morchella group and primer pair

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Identification of the mating type of Morchella edulis YPL2 monoascospore strain

[0045] The monoascospore strains YPL2-1—YPL2-24 of Morchella chinensis were inoculated on PDA medium, cultivated at 23°C for 10 days, picked appropriate amount of mycelia, extracted DNA by CTAB method, and took 3 μL in 1% (W / V ) agarose gel electrophoresis to detect DNA quality and concentration.

[0046] Using the DNA of YPL2-1—YPL2-24 as a template, PCR amplification was performed on P8-5F / P8-5R with primers: 25 µL amplification system included 2×PCRmix (TSINGKE Bio Inc) 12.5 µL, 10 µM / L 1 µL each of P8-5F and P8-5R 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 30 sec, annealing at 65°C for 30 sec, extension at 72°C for 90 sec, 30 cycles; extension at 72°C for 10 min.

[0047] Using the DNA of YPL2-1—YPL2-24 as a template, PCR amplification was performed on P10-1F / P10-2R with pri...

Embodiment 2

[0049] Example 2 Mating Type Identification of Morchella HL1 Monoascospore Strain

[0050] The monoascospore strain HL1-1—HL1-12 of Morchella liumei was inoculated on PDA medium, cultured at 23°C for 10 days, picked appropriate amount of mycelium, extracted DNA by CTAB method, and took 3 µL on 1% agarose gel DNA quality and concentration were detected by electrophoresis.

[0051] Using the DNA of HL1-1—HL1-12 as a template, PCR amplification was performed on P7-2F / P7-2R with primers. The 25 µL amplification system included 2×PCRmix (TSINGKE Bio Inc) 12.5 µL, 10 µM / L 1 µL each of P7-2F and P7-2R primers, 1 µL of DNA template, ddH 2 O 9.5 µL. The PCR amplification program was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 sec, annealing at 62°C for 30 sec, extension at 72°C for 40 sec, 30 cycles; extension at 72°C for 10 min.

[0052] Using the DNA of HL1-1—HL1-12 as a template, PCR amplification was performed on P10-2F / P10-3R with primers. The 25...

Embodiment 3

[0054] Example 3 Identification of the mating type of the morel 2688 monoascospore strain

[0055] The monoascospore strains 23-S1—23-S12 of Morchella soliformis were inoculated on PDA medium, cultured at 23°C for 10 days, an appropriate amount of mycelium was picked, DNA was extracted by CTAB method, and 3 µL was electrophoresed on 1% agarose gel Check DNA quality and concentration.

[0056] Use the primer pairs P7-2F / P7-2R and P10-2F / P10-3R to detect the mating type, and the operation steps are the same as in Example 2.

[0057] All amplified products were detected by electrophoresis on 1.2% agarose gel, the results are shown in image 3 , the strains 23-S3, 23-S7, 23-S9, 23-S10, and 23-S11 only amplified a band of about 0.6kb by the primer pair P7-2F / P7-2R, which was the mating type of MAT1-1; the strain 23- For S1, 23-S2, 23-S4, 23-S15, 23-S6, 23-S8, and 23-S12, only the primer pair P10-2F / P10-3R amplifies a band of about 1.0 kb, which is MAT1-2 mating type .

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Abstract

The invention relates to a method for distinguishing mating types of four varieties in black morchella group and a primer pair, and belongs to the field of biotechnology. The method comprises the following steps: extracting the DNA of a strain to be detected as a template, performing PCR amplification through the primer pair P7-2F / P7-2R or P8-5F / P8-5R to obtain a strain of MAT1-1 mating type of an expected band, performing PCR amplification through the primer pair P10-1F / P10-2R or P10-2F / P10-3R to obtain a strain of MAT1-2 mating type of an expected band, wherein the strain which can be detected by the two expected amplification bands is of two mating types. The four primers can be general to the mating type detection of the four varieties of morchella. A detection result of the method is high in specificity, and the method is stable, reliable and quick, and is applicable to researches on selection, cultivation, mating type genes and system development of strains of the morchella.

Description

technical field [0001] The invention belongs to the technical field of biology, and in particular relates to a method for identifying the morel ( Morchella importuna ), Liumei Morchella ( Morchella sextelata ), Qimei Morchella ( Morchella septimelata ) and tall morel ( Morchella elata ) Molecular detection method of mating type and universal specific primer pair. The technology of the invention is applicable to the research on the selection, cultivation, mating type gene and phylogeny of hickory chick species. Background technique [0002] O’Donnell et al. used the polygenic pedigree consensus phylogenetic species recognition method (GCPSR), based on LSU, ef1‐a , rpb1 and rpb2 The nucleotide sequences of these four genes divide the fungi of the genus Morchella into yellow Morchella clade (Esculenta Clade), black Morchella clade (Elata Clade) and red Morchella clade (Rufobrunnea Clade) . Morel, Liumei morel, Qimei morel and Gao morel all belong to the black mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 柴红梅赵永昌陈卫民苏开美张小雷刘萍陈立佼
Owner 云南省农业科学院生物技术与种质资源研究所
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