Method for identifying mating types of lepista sordida protoplasted monokaryons and special primer pair IS-879b thereof

A technology of is-879b, A. variegata, which is applied in the field of identification of monokaryon mating type of protoplasts of A. variegata, can solve the problems of narrow temperature range, fragile fruiting bodies, poor insect resistance, etc., and achieve accuracy and The effect of high reliability

Inactive Publication Date: 2013-09-04
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in actual production, there are problems such as low yield, poor insect resistance,

Method used

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  • Method for identifying mating types of lepista sordida protoplasted monokaryons and special primer pair IS-879b thereof
  • Method for identifying mating types of lepista sordida protoplasted monokaryons and special primer pair IS-879b thereof
  • Method for identifying mating types of lepista sordida protoplasted monokaryons and special primer pair IS-879b thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0032] The preparation method of the solid regeneration plate (RM): add sorbitol and agar to the liquid MCM medium so that the concentrations are 1M and 20g / L, respectively.

[0033] 1.2 Protoplast preparation

[0034] 1) Take the hyphae of A. japonica and culture them in liquid MCM medium at 160rpm and 25°C for 4 days (3-5 days are acceptable), filter and collect the mycelium with 3 layers of sterile lens paper, and then use 0.6M manna Alcohol aqueous solution washed 2-3 times.

[0035] 2) Suspend the hyphae (about 1 g) of A. japonica in step 1) in 1.5% lysozyme solution (dissolve 1.5 g lysozyme with 0.6M mannitol aqueous solution and set the volume to 100 mL; lysozyme was purchased from Guangdong Bide Biotechnology Co., Ltd., catalog number: Bd_8110001023), in a water-bath shaker (32°C, 60rpm) for 2 hours, filtered with 3 layers of sterile lens tissue and collected the filtrate.

[0036] 3) Centrifuge the filtrate of step 2) at 3000 rpm for 10 min and collect the precipita...

Embodiment 1

[0050] Example 1. Using the PCR primer pair IS-879b to identify the mating type of the protoplast monokaryon of the mushroom

[0051] 1. PCR reagents for identifying or assisting in identifying the mating type of monokaryon protoplasts

[0052] The reagents for identifying or assisting in identifying the mating type of the protoplast monokaryon of Amanita chinensis in this embodiment consist of PCR primer pair IS-879b, 10×Taq buffer, dNTP mix, Taq DNA polymerase and ddH 2 O composition.

[0053] Among them, the PCR primer pair IS-879b is composed of two single-stranded DNAs, IS-879b-F and IS-879b-R, and its sequence is as follows:

[0054] IS-879b-F:5'-CTTCAACAGCAAACGTCAAAC-3' (SEQ ID No.1),

[0055] IS-879b-R: 5'-GTTGATGCCACTTCATGGTT-3' (SEQ ID No. 2).

[0056] 10×Taq buffer, dNTP mix and Taq DNA polymerase were purchased from Beijing Shengxu Baichuan Company (CNS).

[0057] 2. Identify or assist in the identification of monokaryotic mating types

[0058] Inoculate the 3...

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Abstract

The invention discloses a method for identifying the mating types of lepista sordida protoplasted monokaryons and a special primer pair IS-879b thereof. The method comprises the following steps of: taking genomes DNA of two lepista sordida protoplasted monokaryons to be identified as templates respectively, performing polymerase chain reaction (PCR) amplification by using a PCR primer pair consisting of two single chains DNA shown as SEQ ID No. 1 and SEQ ID No. 2, and detecting the magnitude of the obtained PCR products, wherein if the PCR products of both the two lepista sordida protoplasted monokaryons to be identified contain or do not contain DNA fragments of 1000bp-1500bp, the mating types of the two lepista sordida protoplasted monokaryons to be identified are identical; and if the PCR product of one protoplasted monokaryon contains the DNA fragments of 1000bp-1500bp and the PCR product of the other one does not contain the DNA fragments of 1000bp-1500bp, the mating types of the two lepista sordida protoplasted monokaryons to be identified are different.

Description

technical field [0001] The invention relates to a method for identifying the mating type of the protoplast monokaryon of Amanita chinensis and the pair of special primers IS-879b. Background technique [0002] The nutrient incompatibility system is a genetic system for identifying aliens. In most fungi, this system can cause genetically distinct individuals to produce characteristic somatic incompatibility in the junction zone (Qi Yuancheng et al., Chinese cultivated pellagra Comparison of somatic cell incompatibility test and RAPD analysis results of Pleurotus species. Acta Mycophyta Sinica, 2010, 29 (3)) reaction, the intensity of somatic cell incompatibility reaction varies from individual to individual. In fungi, nutritional incompatibility is also known as somatic incompatibility or heterokaryotic incompatibility. fusion to maintain individual genetic stability. The mating type is the type of combination determined according to whether the individuals of the mating fa...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 许峰王鹏刘宇李登进王守现赵爽王兰青耿小丽
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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