A pleurotus strain Z72 and a breeding method thereof

Abstract

The present application provides a kind of pleurotus strain Z72 and its breeding method, method includes the following steps: (1) parent strain cultivation out mushroom, gather just about to open umbrella pleurotus fruit body and carry out spore collection;(2) to the spore obtained is diluted, the diluted spore suspension is irradiated, culture, respectively screening out single spore strain and binucleate strain;(3) take the mycelium of single spore strain and binucleate strain and cross, purification, screening, obtain hybrid strain, namely this.The breeding method of the application adopts mutagenesis and hybridization breeding technology to obtain new strains, selects new varieties of edible fungi which are rich in nutrition, high yield, storage resistant and suitable for the climate characteristics of Liaoning, enriches the category of edible fungi market, improves the quality and technical level of edible fungi, provides the competitiveness of edible fungi enterprises, and promotes the development of Liaoning edible fungi industry.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microorganisms, and particularly relates to a Pleurotus ostreatus strain Z72 and a breeding method thereof. BACKGROUND

[0002] Pleurotus ostreatus is tender and thick, rich in nutrition, delicious in taste, has important edible and medicinal values, is one of edible fungi with important production in China, and is always in the core position of world edible fungi cultivation. Long-term consumption of Pleurotus ostreatus can enhance the defense ability of human body, regulate metabolism, blood pressure, etc. At present, the cultivated varieties are faced with problems such as loss of genetic ability, degeneration of strains and weakening of stress resistance. At present, the strains used in the factory cultivation of edible fungi in China are dominated by foreign strains, and too few strains are made in China. Some large factory production enterprises of edible fungi need to pay several hundred million to several billion of strain fees to foreign countries every year, so it is urgent to develop new strains of edible fungi with independent intellectual property rights, which has great significance and necessity for the sustainable development of the edible fungi industry. The core of restricting the development of the industry is the good seed source, and it is urgent to breed excellent strains with excellent quality characteristics and productivity and strong adaptability for the problems in the Pleurotus ostreatus industry.

[0003] Mutagenic breeding technology, as a traditional breeding method with a long history, is still an important method to obtain high-yield strains, and is a relatively new technology in edible fungi breeding. The method is simple, easy to operate, and has a short breeding period, and can quickly obtain new varieties. Mutagenic breeding is to make the genetic characteristics of strains vary by using physical and chemical methods on the DNA of strains, so as to obtain excellent strains. The existing hybrid breeding method is to make gene recombination, combine the excellent characteristics of two or more varieties together, and the disadvantages are long breeding period and complex process; only existing gene recombination can be used, new genes cannot be created, and segregation phenomenon will occur in hybrid offspring. The advantages of mutagenic breeding are that the mutation rate can be improved, and more excellent variation types can be obtained in a short time. SUMMARY

[0004] Therefore, the present application aims at overcoming the defects in the prior art, and provides a Pleurotus ostreatus strain Z72 and a breeding method thereof.

[0005] To achieve the above-mentioned purposes, the technical scheme of the present application is as follows:

[0006] A pleurotus strain Z72, the preservation number of the pleurotus strain Z72 in China General Microbiological Culture Collection Center is: CGMCC NO. 41177; the preservation date is March 6, 2024; the preservation unit is China Microbial Culture Collection Management Committee General Microorganism Center. The preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing; the taxonomic name of the pleurotus strain Z72 is Pleurotus ostreatus.

[0007] A mutagenic hybrid breeding method of a pleurotus strain, comprising the following steps:

[0008] (1) After the parent strain is cultivated to grow mushrooms, the spores of the pleurotus fruiting bodies about to open are collected for spore collection;

[0009] (2) The obtained spores are diluted, and the diluted spore suspension is subjected to irradiation treatment and culture, and single spore strains and dikaryon strains are respectively screened out;

[0010] (3) The mycelia of the single spore strains and the dikaryon strains are hybridized, purified and screened to obtain hybrid strains.

[0011] Further, the irradiation dose of the irradiation treatment in the step (2) is 500-900 Gy, the dose rate is 2.5 Gy / min, and the treatment time is 200-360 min.

[0012] Preferably, the irradiation dose of the irradiation treatment in the step (2) is 600-800 Gy, and the treatment time is 240-320 min.

[0013] The application of the breeding method in breeding the pleurotus strain.

[0014] The application of the breeding method in artificially cultivating the pleurotus strain Z72.

[0015] The application of the breeding method in propagating the pleurotus strain Z72.

[0016] Compared with the prior art, the present application has the following advantages:

[0017] The breeding method of the present application adopts mutagenic and hybrid breeding techniques to obtain new strains, and selects new varieties of edible fungi which are rich in nutrition, high in yield, storage-resistant and suitable for the climate characteristics of Liaoning, enriches the categories of the edible fungi market, improves the quality and technical level of edible fungi, provides the competitiveness of edible fungi enterprises, and promotes the development of the edible fungi industry in Liaoning Province. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The cluster analysis diagram of the pleurotus hybrid material based on the SSR marker in embodiment 1 of the present application.

[0019] Figure 2 Physical map of mutagenized strain "9408" as described in Example 1 of the present application;

[0020] Figure 3 Physical map of mutagenized strain "Grey Beauty No. 2" as described in Example 1 of the present application;

[0021] Figure 4 Physical map of the back of the fruiting body of Z72 strain as described in Example 1 of the present application;

[0022] Figure 5 Physical map of the front of the fruiting body of Z72 strain as described in Example 1 of the present application;

[0023] Figure 6 Phenotypic variation map of different Pleurotus ostreatus strains as described in Example 2 of the present application. DETAILED DESCRIPTION

[0024] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which the present application belongs. The experimental reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods described, unless otherwise specified, are all conventional methods.

[0025] The present application will be described in detail below with reference to examples.

[0026] Example 1 Selection of hybridization

[0027] 1.1 Test materials

[0028] 1.1.1 Test strains

[0029] The test strain P1 is 5178, P2 is 9408, P3 is Grey Beauty No. 2, and P4 is Laetiporus sulphureus T2. They are stored in the Institute of Edible Fungi, Liaoning Academy of Agricultural Sciences (Table 1).

[0030] Table 1 Test strains

[0031]

[0032] 1.1.2 Main instruments

[0033] Liaoning Academy of Agricultural Sciences Irradiation Center: 60 Co-γ rays. Analytical balance NewClassic ML, Mettler Toledo Company. Clean bench: VS-1300L-U, Suzhou Antai Air Technology Co., Ltd. of Suzhou Clean Group; biochemical incubator: SPX-250B-Z type, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory.

[0034] 1.1.3 Culture medium

[0035] PDA solid medium: pH natural, potato 200 g, agar 18 g, glucose 20 g, ultrapure water 1000 ml.

[0036] 1.2 Test method

[0037] 1.2.1 Preparation of single spores of mutagenized material

[0038] After the parent strain was cultivated to produce mushrooms, the mushroom fruiting bodies with opening caps were collected for spore collection. The operation was as follows: in the clean bench, the stipes of the fruiting bodies were cut off, the caps were sterilized with 75% ethanol, and then washed with sterile water for 2-3 times. After the water on the surface of the cap and gill was absorbed with filter paper, the cap was placed in a culture dish; both the filter paper and the culture dish were sterilized before use, and the sterilized fruiting body was placed on a sterile fixed collection device. The fixed device was made of copper wire, and the lower end was used to collect spore prints with sterile filter paper.

[0039] 1.2.2 Ray mutagenesis

[0040] The mushroom fruiting bodies with opening caps were collected for spore collection, and the collection device was placed in a well-ventilated laboratory with a temperature of 23°C. After 24 or 48 hours, a circle of light yellow spore prints was visible on the filter paper at the lower end of the device. The filter paper with spores was taken out on the clean bench, cut into small pieces, and dissolved in 0.9% sterile NaCl. The spore concentration was counted using a hemocytometer, and the concentration of the spore suspension was diluted to the order of 1x106 / mL using a microscope. The diluted spore suspension was divided into 2 mL sterile centrifuge tubes. The prepared sample was sent to the cobalt source irradiation center of Liaoning Provincial Academy of Agricultural Sciences for further irradiation treatment. The irradiation dose was set to 0, 100 Gy, 200 Gy, 300 Gy, 400 Gy, 500 Gy, 600 Gy, 700 Gy, 800 Gy, 900 Gy, and 1000 Gy, with a dose rate of 2.5 Gy / min. The specific parameters are shown in Table 2. After mutagenesis, 200 μL of spore suspension was taken on the clean bench and spread on PDA flat plate medium, which was incubated at 23°C. Three replicates were observed and the mortality rate was calculated after 6 days. The mortality rate (%) = (number of untreated regenerated fungi - number of mutagenized regenerated single fungi) / number of untreated regenerated fungi x 100%.

[0041] Table 2 Mutagenesis parameters

[0042] Irradiation dose / Gy Treatment time / min 0 0 100 40 200 80 300 120 400 160 500 200 600 240 700 280 800 320 900 360 1000 400

[0043] 60In Co-γ mutagenic breeding, the use of mutagenic dose is too high, resulting in low survival rate, which is not conducive to the screening of mutant strains and the establishment of mutant population. Similarly, the mutagenic dose is too low, the survival rate of offspring is high, which leads to large workload of phenotype variation strain screening and too few variation population. The lethal rate of different strains under different mutagenic treatment gradients is shown in Table 3. As can be seen from the table, the spore suspension of different varieties of Pleurotus strains is used as the mutagenic material, which has tolerance. They are sensitive to 60 The tolerance of Co-γ mutagenic dose is obviously different. Among the 10 treatment groups, the lethal rate of P1 is the lowest, which is 27.99% under the treatment condition of 100 Gy. Overall, P2 and P3 are more sensitive than P1 and P4 under the mutagenic conditions of 200-400 Gy, and the lethal rate is high. When the irradiation dose is 100-1000 Gy, the lethal rate gradually increases with the increase of irradiation dose, which is 27.58-96.10%. The lethal rate is proportional to the irradiation time, which reaches a significant level. The half lethal dose of irradiation is 500 Gy, which indicates that the spores of Pleurotus are tolerant to Co-γ irradiation. 60 Co-γ irradiation. The irradiation dose is crucial to the mutagenic results. The radiation source, mutagenic dose and mutagenic material are important factors affecting mutation. Selecting appropriate mutation frequency, survival rate and other factors is conducive to the rapid screening of mutant strains. Referring to the research of Song Bing et al., the half lethal dose can be used as the best radiation dose of Pleurotus. The half lethal rate can cause the strain to mutate towards the positive direction, and the probability of positive mutation is high when the lethal rate is 70-80%. In order to obtain a high positive mutation rate, the irradiation dose with a lethal rate of 70-80% is selected as the best mutagenic time. When the irradiation dose is larger, the lethal rate is higher. When the mutagenic dose is the same, the sensitivity of different Pleurotus materials to radiation is different, and the difference in lethal rate is obvious. The dose rate of P1 is 75% under the irradiation dose of 500 Gy, which is 24% higher than the half lethal rate, which is suitable for the dose of spores. The suitable mutagenic dose of P2 is 600 Gy, the suitable mutagenic dose of P3 is 700 Gy, and the suitable mutagenic dose of P4 is 400 Gy.

[0044] Table 3 Lethal rate of different strains under different mutagenic treatment gradients

[0045]

[0046] 1.2.3 Preliminary screening of mutagenic mononuclear mycelium

[0047] The regenerated Pleurotus single colony was picked on PDA plate and numbered. The single spore strain was screened by microscope to identify whether there was lock-like joint structure. The culture was preserved for use. A 5mm diameter puncher was used to take 60 The strains obtained by Co-γ mutagenesis and the mycelial blocks of the starting strains were subjected to antagonistic experiment on PDA plate, and the antagonistic lines were preserved.

[0048] 1.2.4 Construction of hybrid population

[0049] The diluted Pleurotus spore suspension was plated on PDA plate medium, and cultured in a 23°C incubator for 15 days. Single colony was selected under a microscope, and the strain without lock-like joint structure was identified as a single spore strain. The spore monokaryon mycelium blocks of '9408' and 'grey beauty No. 2', and '5178' and '9408' were paired in groups of two, with a distance of 1.5 cm, and inoculated on a common PDA plate medium in a 23°C incubator. Observation and recording were performed every day, and when the mycelium combination contacted and merged, the mycelium at the merging junction was picked and observed under a microscope for lock-like joint structure to identify hybrid strains and construct hybrid population. After transfer and preservation, they were kept in reserve.

[0050] The mutagenized spore monokaryon and dikaryon donors were randomly paired to create hybrid combinations, which were inoculated on PDA plates at a distance of 1.5 cm for pairwise pairing. The mycelium on the monokaryon side was picked and inoculated, purified, and preserved to obtain 168 hybrid offspring. By analyzing and comparing the growth conditions of the strains, such as mycelial solid and liquid growth state, speed, and biomass, strains with poor mycelial vigor, extremely slow growth speed, and contamination were eliminated, and 31 hybrid offspring with good mycelial growth, dense mycelium, and strong vigor were obtained.

[0051] Table 4 Collection of monokaryons

[0052] Parent name Mononuclear number Binuclear number P1 2 6 P2 21 69 P3 2 51 P4 4 2 Total 29 128

[0053] 1.2.5 Preliminary screening of hybrid offspring

[0054] All hybrid offspring were observed under a microscope, and strains with lock-like joint affinity were selected. The colony morphology, growth speed, and liquid shake flask culture mycelial ball growth conditions, biological efficiency, and other traits of the mycelium were compared to select strains with fast mycelial growth speed and strong vigor. A total of 35 strains with relatively stable traits and lock-like joint were finally obtained.

[0055] 1.2.6 Screening of hybrid strains after mutation based on SSR molecular markers

[0056] PCR amplification was performed using the DNA of the strains as template: the amplification system was 10 μL reaction system, which included 5 μL 2×TaqPlus Master MIx II, 0.5 μL forward primer, 0.5 μL reverse primer, 1 μL DNA template, 3 μL ddH2O. BioLab T100 PCR instrument was used for amplification, and the specific amplification procedure was 95°C for 3 min; 95°C for 30 sec, 55°C for 30 sec, 72°C for 1 min, 35 cycles, 72°C for 7 min. The PCR amplification product was subjected to 1.25% agarose gel electrophoresis in 1×TBE buffer, the loading amount was 10 μL, the voltage was 150 V, the current was 80 mA, and the electrophoresis time was 20 min. The polymorphic bands obtained after electrophoresis of 8 pairs of SSR primers were counted, and the bands were recorded as 1 and no bands were recorded as 0, and then the data were arranged into an Excel table, and UPGMA genetic diversity cluster analysis was performed using NTSYS2.10 software to obtain the UPGMA genetic diversity cluster analysis diagram.

[0057] The identification results of the mutagenized hybrids by SSR showed that 2 groups of mutagenized hybrids presented different specific bands under the amplification of 8 pairs of primers (conventional primers), indicating that these strains had undergone mutation. UPGMA cluster analysis was performed according to the genetic similarity coefficient, and a cluster dendrogram was drawn as shown in Figure 1 The hybrid Z168 was clustered together with the starting strain M29, and Z129 was clustered together with the starting strain M5. The 8 pairs of SSR markers could be used to distinguish the 39 pieces of P. beilvii materials in the 2 groups, and the 8 pairs of SSR markers could be used to identify and distinguish the hybrid offspring and mutagenized parent materials of P. beilvii. The band types under different primers were coded in sequence, and each primer was numbered according to the type of band. Different numbers represented the band type under the primer. If the band type exceeded 9, the letter was continued to represent. The molecular ID card of each mutant strain was constructed according to the band type under different primers. The numbers in the molecular ID card represented the band type under the first to eighth primer in sequence. The molecular ID card of the hybrid is shown in Table 5. Different digital codes of the molecular ID card were obtained for each strain, which could be used to distinguish different strains simply and clearly.

[0058] Table 5 SSR molecular ID card of the hybrid

[0059] Serial number Primer 2 Primer 3 Primer 4 Primer 5 Primer 6 Primer 7 Primer 8 Primer 9 M14 1 1 1 1 1 1 1 1 M5 2 1 1 1 1 1 1 2 Z72 6 4 3 2 1 1 2 6

[0060] 60 Co-γ ray dose was used to irradiate the spore suspensions of different P. beilvii, the mortality rate was counted, and the spore monokaryons of the mutagenized strains "9408" and "grey beauty No. 2" were obtained. 60 ​Co-γ mutagenesis and hybrid breeding research, Z72 strain was obtained. Fruiting bodies were clustered or superimposed type, cap was gray or black gray, uniform color, fan-shaped or fan hemispherical; gill arranged regularly; stem was thick, high yield, good consistency, short cycle, obvious tide, the real picture of the mutant strain "9408" as shown in Figure 2 the real picture of the mutant strain "gray beauty 2" as shown in Figure 3 the real picture of the Z72 strain as shown in Figure 4-5

[0061] Example 2 Agronomic traits of hybrid

[0062] 1. Materials and methods

[0063] 1.1 Materials

[0064] The test strain G1 was 9408, G2 was gray beauty 2, G3 was wild pleurotus, and G4 was the hybrid Z72 of G1 and G2. It was stored in Liaoning Academy of Agricultural Sciences (Table 6).

[0065] Table 6 Number and source of test strains

[0066] Number Variety name Origin G1 9408 Liaoning Provincial Academy of Agricultural Sciences G2 Hui Mei 2 Liaoning Provincial Academy of Agricultural Sciences G3 PO2 Liaoning Provincial Academy of Agricultural Sciences G4 Z72 Liaoning Provincial Academy of Agricultural Sciences

[0067] 1.2 Test method

[0068] PDA medium: potato 200g, glucose 20g, water 1L, pH natural, used for plate mycelium culture.

[0069] The formula of the original culture medium: cottonseed hull 98%, gypsum 2%, moisture content 50%-55%.

[0070] The formula of the cultivation species culture medium: cottonseed hull 30%, corn cob 48%, bran 18%, soybean meal 2%, gypsum 1%, lime 1%, moisture content 63%.

[0071] According to the formula, put the culture medium into the blender, stir evenly after adding water, so that the moisture content is 65%, automatic bottle filling machine is used for filling, and the matching plastic cover is used for sealing. The high-pressure polypropylene fungus bag with the specification of 17cmx35cmx0.05cm is used for filling, 900g of culture medium is filled in each bag, and 121℃ high-pressure steam sterilization is performed for 2h. After sterilization, when the temperature decreases to 80℃, the fungus bag is moved to the pre-cooling room, and when the temperature cools to about 25℃, it is transferred to the inoculation room through the conveying belt to be inoculated under sterile conditions, 5ml of strain is inoculated in each bag, and 50 bags are inoculated for each variety. It is cultured in the room at 22℃ in the dark, the relative humidity of air is less than 60%, the light intensity is less than 50lx, and the volume fraction of carbon dioxide is less than 0.3%. After the mycelium grows full of the fungus bag, it is fruiting. The fruiting temperature is 16℃, the relative humidity of air is 95%, the light intensity is 150lx, and the volume fraction of carbon dioxide is less than 0.08%. ​

[0072] 1.3 Project determination

[0073] Agronomic trait index determination: 5 bags of fungus were randomly selected from each treatment, and the indexes of different varieties were recorded (from the beginning of the full bag of mycelium, the first flush of mushrooms). The full bag time (PT), mycelial growth (HG), cap length (PL), stem length (SL), stem diameter (SD), cap thickness (CT), cap color (CC), single bag yield (YP), mycelial growth rate (HGS), and biological efficiency (BC) were measured.

[0074] Mycelial full bag days (d): the number of days required for mycelium to grow throughout the bag after inoculation.

[0075] Mycelial growth: mycelial growth. 6 ideal growth, 5 vigorous, 4 relatively vigorous, 3 average growth, 2 poor growth, 1 worst growth.

[0076] Cap length: the maximum diameter of the cap was measured with a vernier caliper; stem length: the straight-line distance from the top of the cap to the base was measured with a vernier caliper; stem diameter: the diameter of the middle part of the stem was measured with a vernier caliper. Cap thickness: the distance from the top of the cap to the thickest part of the cap where the cap meets the stem was measured.

[0077] Cap color: observe the color of the fruiting body. 6 black, 5 black-brown, 4 gray-brown, 3 gray, 2 gray-white, 1 white.

[0078] Single bag yield and biological efficiency determination: when the first flush of mushrooms grows to 80% cooked, harvest, weigh the fresh weight of each bag, and take the average value. Calculate the biological efficiency.

[0079] Biological efficiency (%) = fresh weight of fruiting body / dry weight of culture medium x 100.

[0080] Determination of moisture content of fruiting body: kill at 105°C for 10 min, and dry at 55°C to constant weight.

[0081] 1.4 Data analysis

[0082] Microsoft excel 2016 software and SPSS17.0 were used for data processing and analysis. R software package pheatmap was used to draw cluster heat map.

[0083] 2 Results and analysis

[0084] 2.1 Statistical analysis of main agronomic traits

[0085] For the strain of Pleurotus ostreatus, the comparison was made based on the full bag time (PT), mycelium growth (HG), pileus length (PL), stipe length (SL), stipe diameter (SD), pileus thickness (CT), pileus color (CC), single bag yield (YP), mycelium growth rate (HGS) and biological efficiency (BC) indexes, as shown in Table 7.

[0086] Table 7 Agronomic traits of strains

[0087]

[0088]

[0089] The coefficient of variation represents the variation degree of each trait of different individuals affected by different conditions, and can reflect the basic dynamics of individual trait changes. According to the analysis of the agronomic traits of different Pleurotus ostreatus strains, it can be seen from Table 7 that there is genetic variation of different degrees among the agronomic traits of the tested Pleurotus ostreatus strains, and the variation coefficient ranges from 4% to 41%. The maximum value of the variation coefficient is 41%, which is the stipe diameter, indicating that different varieties do not have consistent fruiting under the same environment in the factory. The minimum value of the variation coefficient among different varieties is 4%, which is the pileus length, stipe length and mycelium growth rate. The traits from high to low variation coefficient are stipe diameter (41%) > mycelium growth (33%) > pileus color (31%) > full bag time (7%) = single bag yield (7%) = biological efficiency (7%) > pileus length (4%) = stipe length (4%) = mycelium growth rate (4%). The variation coefficients of stipe diameter and mycelium growth of different varieties are relatively large.

[0090] The comprehensive characteristics of high-quality Pleurotus ostreatus mainly include moderate pileus thickness and size, short stipe, fast mycelium growth rate and high yield. The yield factor is an important factor affecting the selection of Pleurotus ostreatus cultivation, and the yield is directly related to the quality of the variety, so the yield is taken as one of the evaluation factors. Figure 6 It can be seen from the trait clustering heat map that the correlation of mycelium growth, single bag yield and biological efficiency is relatively high. From the strain clustering heat map, Z72 is a very potential variety for factory cultivation in terms of mycelium growth, single bag yield and biological efficiency.

[0091] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A Pleurotus strain ( Pleurotus ostreatus ) Z72, characterized by: The Pleurotus ostreatus strain Z72 has a preservation number of CGMCC NO.41177 in China General Microbiological Culture Collection Center, and the preservation date is March 6, 2024, and the preservation unit is China General Microbiological Culture Collection Center. ​

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