阅读说明：本技术 一种黑牛肝菌菌株与应用 (Hemicus fuliginosus strain and application thereof ) 是由 罗顺珍 纪开萍 曹旸 陈燕 纪光燕 纪光玉 刀明晶 李恒 陶玲 晏汐蓓 王秋兰 于 2021-06-03 设计创作，主要内容包括：本发明公开一种黑牛肝菌菌株与应用,该菌株是由YL1701-2自体杂交获得,已于2021年3月3日保藏在中国微生物菌种保藏管理委员会普通微生物中心CGMCC,保藏编号CGMCC NO.21921。本发明还公开了黑牛肝菌菌株在生产黑牛肝菌的应用。该菌株具有遗传稳定,活力强,后期出菇速度快且出菇整齐,生长速度快,耐保藏等优点,与亲本相比子实体形态上具有明显差异,产量更高,具有很高的商业价值。属于YL1701-2的纯合菌株。(The invention discloses a black boletus strain and application, wherein the strain is obtained by YL1701-2 self-hybridization and has been preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO.21921 at 3 months and 3 days in 2021. The invention also discloses application of the boletus nigricans strain in production of boletus nigricans. The strain has the advantages of stable heredity, strong activity, high fruiting speed in the later period, regular fruiting, high growth speed, preservation resistance and the like, and compared with parents, the strain has obvious difference in the form of fruiting bodies, higher yield and very high commercial value. A homozygous strain belonging to YL 1701-2.)

1. The black cattle liver bacterial strain is obtained by self-hybridization of black cattle liver bacterial strain YL1701-2, and is classified and named as: bolete nigrum (Phlebopus portentosus) FC0580 was preserved in China general microbiological culture Collection center (CGMCC) at 3 months and 3 days 2021, with the preservation number of CGMCC NO. 21921.

2. Use of the strain of boletus nigricans of claim 1 for the production of boletus nigricans.

Technical Field

The invention belongs to the technical field of artificial breeding of edible fungi, and particularly relates to a novel strain of boletus aereus which is obtained by self-hybridization breeding and selection and has excellent biological characteristics and is suitable for industrial cultivation conditions.

Background

Boletus aereus (Phlebopus portentosus) is a large fungus of Boletales (Boletales) Boletaceae (Boletinellaceae) Phlebopus (Phlebopus), and is commonly known as Phlebopus portentosus. At present, the bolete nigricans are bolete varieties which can leave a host tree, culture mature sporocarp and realize artificial cultivation, and the market prospect is wide.

The excellent strain is the key for the success of artificially cultivating the boletus aereus. At present, the method for obtaining excellent strains mainly comprises wild domestication and crossbreeding. Among them, cross breeding is the most effective way to obtain superior strains, and the specific methods include: self-hybridization, proximal hybridization, heterologous hybridization, distal hybridization, and the like. The process of crossbreeding includes the steps of matching, identifying, screening, expanding and verifying, etc., and one strain with commercial value can be obtained by screening thousands of pairs. The boletus edulis belongs to a quadrupole mating system, in the mating system, gametes generated by an individual have 75% of probability of incompatibility and only 25% of probability of compatibility when the gametes are subjected to inbreeding, so that an autohybridization means for hybridizing inbreeding gametes is selected, and the workload is far greater than that of hybridization of the inbreeding gametes. Compared with other hybridization modes, the self-hybridization has the advantages of aggregation and homozygous excellent genes, and is selected by many breeding workers.

Disclosure of Invention

In view of this, the embodiment of the present invention provides a boletus niger strain FC0580 with excellent cultivation characteristics, which can be used for industrial production, and provides an application of the FC0580 strain in producing boletus niger.

In order to achieve the purpose, the invention mainly provides the following technical scheme:

in one aspect, the present invention provides a strain of hepacius niger, obtained by autohybridization of the strain of hepacius niger YL1701-2, classified under the names: bolete nigrum (Phlebopus portentosus) FC0580, has been preserved in China general microbiological culture Collection center (CGMCC) 3.3.3.2021, with the preservation number of CGMCC NO.21921, and the preservation unit address is: xilu No. 1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.

The strain is obtained by self-hybridization of the monocosporium hyphae of the black boletus strain YL 1701-2. The sporocarp morphological characteristics of the edible fungus comprise dark brown fungus cap, yellow fungus stalk, wine bottle shape, longer fungus hole, yellow fungus, compact and compact meat quality, golden fungus meat, basically no brown stain after cooking and light fishy smell. Identified by SSR marks, has certain genetic difference with a strain YL1701-2, and is homozygous by partial marks.

In another aspect, embodiments of the present invention provide the use of the above-described strain of boletus nigricans FC0580 in the production of boletus nigricans.

The invention has the advantages that:

compared with the strain YL1701-2 used in the conventional industrial culture of boletus nigricans, the FC0580 strain has more stable culture character, stronger growth vigor in the fruiting process, higher yield, stronger pollution resistance and wine bottle-shaped stipe, meets the morphological characteristics of the boletus which is commonly called as the boletus giganteus, is a good new variety for the industrial production of the boletus nigricans, and has very high commercial value.

Drawings

FIG. 1 is a graph of the results of non-weighted pairwise arithmetic mean (UPGMA) clustering as described in example 3.

Detailed Description

The present invention is further described in detail with reference to the following examples, which are not intended to limit the technical scope of the present invention, and all equivalent substitutions and changes based on the teachings of the present invention should fall within the protection scope of the present invention.

The raw materials and the like used in the present invention are all conventional products which can be obtained commercially.

The invention relates to a black cattle liver bacterial strain, which is obtained by self-hybridization of a black cattle liver bacterial strain YL1701-2 and is classified and named as: bolete nigrum (Phlebopus portentosus) FC0580 is preserved in China general microbiological culture Collection center (CGMCC) at 3 months and 3 days 2021, and the preservation number is CGMCC NO. 21921.

The present invention will be described in further detail with reference to specific examples, wherein:

example 1:

culture method of boletus nigricans (Phlebopus portentosus) FC0580 strain: the invention is obtained by adopting a method of self-hybridization breeding selection.

1.1 parent strain YL1701-2 fruiting body culture and spore collection:

fruiting bodies of YL1701-2 strain were cultured and spores were collected.

Specifically, fruiting bodies of YL1701-2 strains with good growth of mushroom types are selected in mushroom houses, and spores are collected when pileus of the fruiting bodies are completely unfolded and pileus edge fungus holes are obvious.

1.2 parent strain YL1701-2 spore coating and single colony transfer culture and identification:

and coating the spores obtained in the steps on a culture medium and performing transfer culture to obtain the mononuclear hyphae. Wherein the culture medium is preferably M1 culture medium comprising potato 200g (boiled water), glucose 20g, yeast extract 2g, and MgSO₄ 1g、KH₂PO₄1g of agar and 16g of agar, and the volume is up to 1000 mL.

Specifically, the collected spores are diluted by sterile water and then coated on an M1 culture medium, the spores are transferred to an M1 culture medium for culture after germination, and the mononuclear hyphae are identified and stored after the hyphae overgrow.

1.3 identifying the mating type of the mononuclear hyphae.

1.4 compatible spore mononuclear hypha pairing, microscopic examination, culture and fruiting verification:

and (3) pairing compatible mononuclear hyphae in the mononuclear hyphae obtained in the step (a), transferring the hyphae successfully paired to a culture medium for culture, and performing fruiting verification, thereby breeding the boletus nigricans FC0580 strain.

Specifically, after pairing, parent spore mononuclear hyphae are cultured until the hyphae contact with each other, microscopic examination is carried out, and strains which do not form locked combination under special conditions are eliminated. Transferring the successfully matched hyphae to a slant culture medium for culture. And (4) fruiting verification can be carried out after the hypha grows full. And (5) fruiting verification and recording the fruiting rate, fruiting uniformity, yield, product characteristics and the like of the selfing strain. After primary screening, secondary screening and production verification, strains with high fruiting rate, low pollution rate, high average yield, complete and non-malformed fruit body shape, hard and preserved meat quality and light fishy smell are screened and finally bred to obtain the FC0580 strain.

The boletus nigricans FC0580 strain is applied to producing boletus nigricans, the boletus nigricans produced by the FC0580 strain have higher yield and stronger pollution resistance, grow stronger in the fruiting process, have wine bottle-shaped stipes, meet the morphological characteristics of boletus which is commonly called as 'big foot mushrooms', and are suitable for industrial production of the boletus nigricans. The following example provides the case of the FC0580 strain in a boletus nigricans producing application.

Example 2

The basic situation and the morphological characteristics of the FC0580 strain of the invention are as follows:

2.1 culture Medium

The most suitable culture medium of FC0580 strain is M1 culture medium, and the formula is as follows: 200g of potato (boiled water), 20g of glucose, 2g of yeast extract and MgSO₄ 1g、KH₂PO₄1g of agar and 16g of agar, and the volume is up to 1000 mL.

2.2 hyphal growth

The strain FC0580 grows for 20 days on the M1 culture medium, the bacterial colony is dark yellow brown, the hypha is dense, the growth is vigorous, the sclerotium is not generated, the water is discharged, and the daily growth amount reaches: 3.01 mm/day, 0.48 mm/day faster than the parent strain YL 1701-2.

2.3 morphological characteristics of fruiting bodies

Strain FC0580 has the following heterologies in the daughter morphology compared to the parent strain YL 1701-2:

the same points are as follows: the mushroom cap is brown, hemispherical, yellow in stipe and yellow in mushroom hole, has no earthy taste and basically has no brown stain after being cooked.

The difference is as follows: FC0580 pileus is small in diameter and thick; FC0580 stipe is longer, becomes wine bottle type, has thin upper part and thick lower part, the diameter difference between the upper part and the lower part is larger (about 25-30mm), YL1701-2 stipe is cylindrical, and the diameter difference between the upper part and the lower part is smaller (about 8-16 mm).

The specific data are shown in Table 1.

TABLE 1 comparison of morphological characteristics of fruiting bodies of Boletus nigricans strain FC0580 with YL1701-2

The comparison of the strain FC0580 and YL1701-2 on cultivation characteristics is shown in the following table after the verification of industrialized mushroom production.

TABLE 2 comparison of the cultivation traits of the Boletus nigricans strain FC0580 with those of YL1701-2

Compared with YL1701-2, the strain FC0580 has the advantages that the time and the properties required by the mushroom growing period are basically the same, the fruiting uniformity is slightly lower, but the time required by covering soil to the fruiting, the pollution rate in the mushroom growing period and the average single bottle yield are better than those of the strain YL 1701-2. From this, it was found that the strain FC0580 was an excellent strain for industrial production of Boletus aereus.

Example 3

And (3) identifying SSR molecular markers.

Collecting mycelia of strains FC0580, YL1701-2 and other 6 strains of Boletus edulis, extracting DNA, carrying out PCR amplification on a DNA sample by using 21 pairs of primers in the strains, and adding FAM or HEX fluorescent markers at the tail ends of the forward primers during primer synthesis. PCR products were genotyped using an ABI 3730 sequencer. The data accuracy site was analyzed by reference to the number of core base repeats in the primer mapping using the software Gene mapper 4.1(Applied Biosystems Co., Ltd., USA). The allele fraction, effective allele fraction, Shannon's informative index, expected heterozygosity, observed heterozygosity were calculated using Popgene 32. Genetic similarity coefficients were calculated using NTSYSpc-2.10 software and cluster analysis was performed using unweighted pairwise arithmetic mean (UPGMA). The results of the UPGMA cluster analysis are shown in FIG. 1.

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

TABLE 3 SSR primer information for strain identification

The analysis results show that the strain FC0580 has genetic differences with other strains including its parent YL1701-2 at the SSR site for identification, and is a brand new strain.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent change and modification made to the above embodiment according to the technical spirit of the present invention are still within the scope of the technical solution of the present invention.