A black bolete strain PJ1208 and its application

Abstract

The present invention discloses a Boletus aereus strain and its application. The strain PJ1208 is obtained by the method of double-single hybridization, using the protoplast monokaryotic mycelium of YL01 as the receptor and the wild strain HZ18072 as the donor. It was deposited in the China General Microbiological Culture Collection Center (CGMCC) on March 3, 2021, with the deposit number CGMCC NO. 21925. The present invention also discloses the application of the Boletus aereus strain in the production of Boletus aereus. This strain has the characteristics of genetic stability, strong vitality, fast growth rate, neat fruiting, uniform color of the fruiting body (olive brown), good mushroom shape, soft and glutinous flesh, no color change after injury, and no earthy smell. It belongs to an excellent strain and has high commercial value.

Description

Technical Field

[0001] The present invention belongs to the technical field of artificial breeding of edible fungi, and specifically relates to a strain of Boletus aereus and its application. Background Art

[0002] Boletus aereus is a large fungus belonging to the genus Phlebopus in the family Boletinellaceae of the order Boletales, commonly known as Phlebopus portentosus. Currently, Boletus aereus is the only species of bolete that has achieved industrialized artificial cultivation globally, with broad market prospects.

[0003] When mononuclear and binuclear mycelia of edible fungi are co-cultured, the nuclei in the binuclear mycelia migrate to the mononuclear mycelia to form new binuclear mycelia, namely double-single hybridization. Double-single hybridization is used to further genetically improve strains by selecting strains with various excellent traits. This method only requires mononucleation of the parent with improved parental traits and pairing with the strain to be improved, and is a more simple and effective cross-breeding method. Summary of the Invention

[0004] In view of this, an embodiment of the present invention provides a strain of Boletus aereus PJ1208 with excellent biological characteristics, and provides the application of the PJ1208 strain in the production of Boletus aereus.

[0005] To achieve the above object, the present invention mainly provides the following technical solutions:

[0006] On the one hand, an embodiment of the present invention provides a strain of Boletus aereus, classified and named as: Boletus aereus PJ1208, which was deposited in the China General Microbiological Culture Collection Center (CGMCC) on March 3, 2021, with the deposit number CGMCC NO.21925.

[0007] For the PJ1208 strain, Parent 1: protoplast mononuclear strain 01PS-12 isolated from the binuclear strain YL01 of Boletus aereus, Parent 2: wild binuclear strain HZ18072 of Boletus aereus. The present invention is obtained by double-single hybridization of Parent 1 and Parent 2.

[0008] The strain of the present invention uses the protoplast mononuclear strain 01PS-12 derived from the strain YL01 as the receptor and the wild strain HZ18072 as the donor, and is obtained by the method of double-single hybridization. This strain has the characteristics of genetic stability, strong vitality, fast growth rate, neat fruiting, uniform color of the fruiting body, showing olive brown, good mushroom shape, soft and glutinous flesh, no color change after injury, and no earthy smell. It belongs to an excellent strain and has high commercial value.

[0009] On the other hand, an embodiment of the present invention provides the application of the above-mentioned Boletus atkinsonii PJ1208 strain in the production of Boletus atkinsonii.

[0010] The advantages of the present invention are as follows:

[0011] The strain of the present invention is obtained by double single hybridization breeding. SSR molecular detection shows that it is different from parent 1: mononuclear strain 01PS-12 and parent 2: wild strain HZ18072, and it is a new strain.

[0012] The difference between the strain of the present invention and the existing Boletus atkinsonii production strains: The colors of the cap and stipe of the fruiting body are uniform, olive brown, the flesh is soft and does not change color after injury, and there is no earthy smell. Description of the Drawings

[0013] Figure 1 It is a dendrogram of the unweighted pair group method with arithmetic mean (UPGMA) cluster analysis described in Example 3. Detailed Embodiments

[0014] The following embodiments are used to further illustrate the present invention in detail, but the embodiments do not limit the technical solutions of the present invention. All equivalent substitutions or changes made based on the teachings of the present invention should fall within the protection scope of the present invention.

[0015] The raw materials used in the present invention are all conventional products that can be obtained by commercial purchase.

[0016] A Boletus atkinsonii strain PJ1208 of the present invention is obtained by double single hybridization from protoplast mononuclear strain 01PS-12 isolated from Boletus atkinsonii dikaryotic strain YL01, which is parent 1, and wild Boletus atkinsonii dikaryotic strain HZ18072, which is parent 2. It is classified and named as Boletus atkinsonii PJ1208, and has been deposited in the China General Microbiological Culture Collection Center (CGMCC) on March 3, 2021, with the deposit number CGMCC NO.21925.

[0017] The following will further introduce in detail a Boletus atkinsonii strain and its application of the present invention in combination with specific embodiments:

[0018] Example 1:

[0019] Morphological characteristics of the parental strains of the strain PJ1208 of the present invention

[0020] 1.1 Morphological characteristics of the fruiting body of the strain YL01, the source of parent 1

[0021] The cap is grayish brown, the stipe is dark yellow, the cap diameter is 45 - 51 mm, the stipe is cylindrical, the mushroom shape is good, the average mushroom weight is 115 g, the mushroom emergence is neat, the mushroom emergence rate is 92% - 96%, the yield is stable, the flesh is firm, and there is no color change after injury and no earthy smell.

[0022] 1.2 Morphological characteristics of the fruiting body of parental strain 2 HZ18072

[0023] The cap is dark brown, the stipe is dark brown, the cap diameter is 50 - 85 mm, the stipe is cylindrical, the mushroom shape is good, the average mushroom weight: 75 g, the mushroom emergence rate is 70% - 78.5%, the flesh is soft and glutinous, there is color change after injury, and there is an earthy smell.

[0024] 1.3 Morphological characteristics of the fruiting body of strain PJ1208

[0025] Both the cap and the stipe are olive brown, the cap diameter is 45.08 - 88.04 mm, the stipe is cylindrical, the mushroom shape is good, the average mushroom weight: 97.23 g, the mushroom emergence is neat, the mushroom emergence rate is 96% - 98.5%, the yield is stable, the flesh is soft and glutinous, there is no color change after injury, and there is no earthy smell.

[0026] The Boletus aereus PJ1208 strain of the present invention is applied to the production of Boletus aereus. The Boletus aereus produced by the PJ1208 strain has high yield, neat mushroom emergence, uniform color of the fruiting body, showing olive brown, good mushroom shape, soft and glutinous flesh, no color change after injury, no earthy smell, etc., and is suitable for industrialized production of Boletus aereus.

[0027] Example 2

[0028] Breeding and production method of the Boletus aereus PJ1208 strain: The present invention is obtained by the method of double - single hybridization.

[0029] 2.1 Preparation of protoplasts of Boletus aereus YL1701

[0030] The strain YL1701 was cultured in M1 liquid medium to obtain mycelial pellets. After filtration and washing, the mycelial pellets were dissolved with 1.5% lywallzyme at 30 °C and 185 r / min for 3 h, filtered through 8 - layer 10 g non - woven fabric. The filtrate was centrifuged at 3000 r / min for 10 min at 4 °C, washed twice with isotonic solution, then diluted and spread on the regeneration medium, and cultured in the dark in an incubator at 29 °C. Among them, the composition of M1 medium includes 200 g of potato (boiled in water), 20 g of glucose, 2 g of yeast extract, 1 g of MgSO4, 1 g of KH2PO4, 16 g of agar, and made up to 1000 mL.

[0031] 2.2 Identification of mononuclear hyphae of protoplasts of Boletus aereus YL1701

[0032] When the protoplast colonies germinate until they are visible to the naked eye, pick single colonies and transfer them to a plate. After culturing for 15 days, check under a microscope. If there are no clamp connections, it is a protoplast monokaryotic mycelium.

[0033] 2.3 Cultivation of the wild Boletus aereus strain HZ18072

[0034] Transfer the wild Boletus aereus strain HZ18072 to M1 medium and incubate it in the dark at 29 °C for 20 days.

[0035] 2.4 Double-single hybridization

[0036] Pair-culture the mononuclear strain 01PS-12 obtained by protoplast isolation from the strain YL1701 with the strain HZ18072. Pick the mycelium that produces clamp connections on one side of the 01PS-12 colony and transfer it to a test tube of M1 medium for preservation. This is the new strain PJ1208 obtained by double-single hybridization.

[0037] Example 3

[0038] SSR molecular marker identification.

[0039] Collect the mycelia of the strain PJ1208, its parental dikaryotic strain YL1701, mononuclear strain 01PS-12, dikaryotic strain HZ18072, and 6 other Boletus aereus strains. After extracting DNA, perform PCR amplification on the DNA samples with 21 pairs of primers in the text. Add FAM or HEX fluorescent labels to the 5'-end of the forward primers when synthesizing the primers. Genotype the PCR products using an ABI 3730 sequencer. Use the software Gene mapper4.1 (Applied Biosystems Co., Ltd., USA) to analyze the accurate sites of the data with reference to the core base repeat numbers corresponding to the primers. Calculate the number of alleles, effective number of alleles, Shannon's information index, expected heterozygosity, and observed heterozygosity using Popgene 32. Calculate the genetic similarity coefficient using the NTSYSpc-2.10 software and perform cluster analysis using the unweighted pair-group method with arithmetic means (UPGMA). The UPGMA cluster analysis results are as Figure 1 shown.

[0040] The information of the 21 pairs of SSR primers used is shown in the following table.

[0041] Table 1 SSR primer information for strain identification

[0042]

[0043]

[0044] The analysis results show that strain PJ1208 has genetic differences from other strains including its parental strains YL1701, 01PS-12, and HZ18072 at the SSR loci used for identification, and it is a brand-new strain.

[0045] As described above, it is only the preferred embodiment of the present invention, and there is no restriction on the present invention in any form. Any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A strain of Boletus aereus ([ Phlebopus portentosus Phlebopus portentosus ) PJ1208, characterized in that, It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 3, 2021, with the deposit number CGMCC NO. 21925.

2. Use of the Boletus aereus strain PJ1208 as claimed in claim 1 in the production of Boletus aereus.

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