Method for constructing dominant mononuclear body library, mushroom breeding method and application thereof

Abstract

The invention discloses a method for constructing a dominant monokaryon library, a mushroom breeding method and application thereof, and the method comprises the following steps: carrying out selfing and phenotype evaluation on spore monokaryons from the same parent according to a mating type matching principle, and determining monokaryons carrying recessive bad characters; hybridizing the spore monokaryons with all other copulable monokaryons in a spore monokaryon group to be screened, classifying the monokaryons of which offspring have bad characters into a first monokaryon material library, and classifying the monokaryons of which offspring do not have any bad characters into a second monokaryon material library; homozygous pairing of the monokaryons with recessive bad characters is avoided, the monokaryons in the first monokaryon material library and the monokaryons in the second monokaryon material library with different parent sources are hybridized, and the monokaryons with different parent sources in the second monokaryon material library are hybridized; and counting the probability that strains with excellent characters appear in a plurality of filial generations formed by each monokaryon, and screening the monokaryons with the probability greater than or equal to 60% to obtain a dominant monokaryon library.

Description

Technical Field

[0001] The invention belongs to the technical field of edible fungus breeding, and in particular relates to a method for constructing a dominant monokaryon library, a shiitake mushroom breeding method and application thereof. Background Art

[0002] Edible fungus breeding technology is the use of modern biotechnology to genetically improve and select edible fungi to obtain new varieties with excellent traits. With the rapid development of the edible fungus industry, the demand for excellent varieties of edible fungi is also increasing, so edible fungus breeding technology plays an important role in the edible fungus industry. With the continuous development of technology, edible fungus breeding technology is also constantly improving and upgrading. At present, edible fungus breeding technology mainly includes domestication breeding, mutagenesis breeding, single-single hybridization, single-double hybridization, multi-spore hybridization, multi-spore selfing and gene editing breeding. Among them, hybridization and selfing based on monokaryon pairing are the most convenient and common ways to obtain new genotype combinations, and are also the main technologies for current edible fungus breeding.

[0003] As a high-yield edible fungus, the normal fruiting rate of the breeding population of shiitake mushrooms is very low. In 2022, Yu Hailong et al. counted 14,797 hybrid offspring of 16 F1 generations of different strains of shiitake mushrooms, and the normal fruiting rate was 31.35%, which was inversely proportional to the genetic distance between the parents. If both the fruiting rate of offspring and the rate of high-quality can be improved, the probability of breeding new varieties will be increased, and innovation in the theory of edible fungus breeding will be achieved.

[0004] The reason for the low fruiting rate of parents with close genetic distance is speculated to be that the close parents carry recessive loci that are difficult to fruit or sterile. When the recessive loci become homozygous, the offspring will not produce mushrooms. However, between parents with long genetic distance, the probability of such recessive loci becoming homozygous is greatly reduced. However, there are often few options for excellent parents that are beneficial to production. Unless a long time and a lot of work are spent on parental improvement, hybridization with current excellent parents is still the best way to obtain excellent offspring. However, if the fruiting rate and excellent rate can be improved, the disadvantages of using close parents can be avoided, and the advantages can be magnified to achieve a significant increase in breeding efficiency.

[0005] Edible fungi form basidiospores or ascospores produced by meiosis on the fruiting body. Except for a few species such as Agaricus bisporus, most of these spores are haploid, that is, gametophytes. Spores can germinate to form monokaryon hyphae that can grow independently. Two compatible monokaryon hyphae pair up to form new binucleate (diploid) hyphae, achieve genetic recombination, and produce new strains. By taking advantage of the independent growth characteristics of gametophytes, it is possible to trace and evaluate the gametophyte materials through pre-preservation of gametophyte materials and carry out hybrid breeding of edible fungi. Summary of the invention

[0006] One object of the present invention is to provide a method for constructing a dominant monocyte library.

[0007] Another object of the present invention is to provide a method for breeding shiitake mushrooms.

[0008] Another object of the present invention is to provide the method for constructing a dominant mononuclear body library and related applications of the mushroom breeding method.

[0009] To achieve the above object, the present invention provides a method for constructing a dominant monocyte library, the method comprising:

[0010] Providing spore monokaryons from a plurality of parents as a spore monokaryon population to be screened;

[0011] The spore monokaryons from the same parent in the spore monokaryon population to be screened are self-pollinated according to the mating type combination principle to obtain a self-pollination offspring population;

[0012] Phenotypic evaluation of the self-pollinated progeny population was performed to identify the monokaryons carrying recessive undesirable traits;

[0013] Hybridizing the monokaryon carrying the recessive undesirable trait with all other matable monokaryons in the spore monokaryon population to be screened, and forming all the progeny obtained from the hybridization into a first hybrid progeny population;

[0014] Performing phenotypic evaluation on the first hybrid offspring population; wherein the monokaryons that have undesirable traits in the first hybrid offspring are marked as monokaryons carrying recessive undesirable traits and are included in the first monokaryon material pool, and the monokaryons that have not developed any undesirable traits in the first hybrid offspring are marked as monokaryons that do not carry recessive undesirable traits and are included in the second monokaryon material pool;

[0015] Avoiding homozygous pairing of monokaryons carrying recessive adverse traits, hybridizing monokaryons in the first monokaryon material library with monokaryons in the second monokaryon material library with different parental sources, and hybridizing monokaryons in the second monokaryon material library with different parental sources to obtain a second hybrid offspring population;

[0016] The second hybrid offspring population is phenotypically evaluated, the probability of the emergence of strains with excellent traits in multiple second hybrid offspring formed by each monokaryon is counted, and monokaryons with a probability of ≥60% of the emergence of excellent traits are screened to obtain a superior monokaryon library.

[0017] The method for constructing a dominant mononuclear body library and the method for breeding shiitake mushrooms provided by the present invention can significantly improve the fruiting rate and high-quality rate of offspring.

[0018] In the above method, preferably, when spore monokaryons from the same parent are selfed according to the mating type combination principle, each monokaryon is used at least 4 times.

[0019] In the above method, preferably, the recessive adverse traits include no mushroom production and / or abnormal fruiting body development.

[0020] In the above method, preferably, the abnormal development of the fruiting body includes the fruiting body being "popcorn" shaped and / or the fruiting body having "sterile folds".

[0021] In the above method, preferably, the excellent traits include one or a combination of two or more of high yield, high quality and stress resistance.

[0022] In the above method, preferably, the number of the parents is 2-8, more preferably 3-5.

[0023] In the above method, preferably, when the mononuclear bodies in the first mononuclear body material library are hybridized with the mononuclear bodies in the second mononuclear body material library with different parental origins, and when the mononuclear bodies in the second mononuclear body material library with different parental origins are hybridized with each other, each mononuclear body is used 6-10 times, more preferably 6-8 times.

[0024] According to a specific embodiment of the present invention, there are mononuclear materials from 4 parents A, B, C, and D in the mononuclear library. According to the principle of mathematical combination, C (4, 2), 6 combinations are obtained, namely AB, AC, AD, BC, BD, and CD, so as to cover all types of genetic variations as much as possible. The present invention sets the number of mononuclear uses in this step to 6-10 times. In theory, the more mononuclears are used, the better, but in actual operation, the more mononuclears are used, the larger the population size. Taking into account operability, statistical analysis, and mating type restrictions, the present invention reasonably sets the number of mononuclear uses in this step.

[0025] In the above method, when the mononuclear bodies in the first mononuclear body material library are hybridized with the mononuclear bodies in the second mononuclear body material library with different parental sources, the hybridization combinations are 1000-5000, preferably 1000-4000, and more preferably 1500-3500.

[0026] In the above method, mononuclear bodies with different parental origins in the second mononuclear body material library are hybridized with each other, and the number of hybridization combinations is 1000-5000, preferably 1000-4000, and more preferably 1500-3500.

[0027] In the above method, preferably, the monokaryon includes one or more of a spore monokaryon and a protoplast monokaryon.

[0028] According to a specific embodiment of the present invention, the high yield is defined as the first-tide yield of ≥300g when the subsequent generations are fruiting.

[0029] According to a specific embodiment of the present invention, the high quality is defined as the high quality of the appearance of the offspring fruiting bodies, such as round fruiting bodies, thick flesh, short stipes, and no obvious deformities.

[0030] According to a specific embodiment of the present invention, the stress resistance is defined as the ability of the offspring strain to resist foreign bacteria and adverse conditions during the cultivation process.

[0031] In the above method, preferably, the method for constructing a dominant monocyte library further comprises the following steps:

[0032] Spore prints from each parent are collected, spore monokaryons are obtained by single spore separation technology, and mating type identification is performed to obtain the mating type of the spore monokaryons of each parent. Mating type identification is performed by hybridization pairing, which is a mating type identification method commonly used in the art.

[0033] In the above method, preferably, the protoplast monokaryon is obtained by removing the cell wall using a lytic enzyme, and the mating type identification is performed by hybridization pairing.

[0034] According to a specific embodiment of the present invention, the method for constructing a dominant monocyte library of the present invention comprises the following steps:

[0035] (1) Monokaryons carrying recessive trait loci such as failure to produce mushrooms or abnormal fruiting body development are obtained by screening the self-pollinated progeny of 2-4 main cultivated varieties of Lentinula edodes;

[0036] (2) pairing the monokaryons from the same parent and other parents to be used with the recessive monokaryons carrying the undesirable trait obtained in step (1) to construct a test cross population for the recessive undesirable trait, and marking the monokaryons carrying the recessive undesirable trait;

[0037] (3) The monokaryons carrying recessive undesirable traits are hybridized and paired with the monokaryons not carrying recessive undesirable traits, and the monokaryons not carrying recessive undesirable traits are hybridized and paired with each other, avoiding recessive homozygous pairing of undesirable traits to improve the mushroom yield of the population. Each monokaryon is used 6-10 times, and the probability of occurrence of high-yield, high-quality, stress-resistant and other traits in the offspring obtained from each monokaryon is counted. The monokaryons with an occurrence probability of ≥60% are screened to obtain a dominant monokaryon library.

[0038] The present invention also provides a method for breeding shiitake mushrooms, which comprises the following steps:

[0039] Using the monokaryons in the superior monokaryon library constructed by the above method as breeding parents for breeding to obtain a superior monokaryon hybrid breeding population;

[0040] The dominant mononuclear hybrid breeding population was evaluated by phenotype, and shiitake mushroom strains with excellent traits were screened out.

[0041] In the above-mentioned shiitake mushroom breeding method, preferably, the breeding method utilized includes one or a combination of two or more of single-single hybrid breeding, single-double hybrid breeding, self-pollination breeding, backcross breeding and mutagenesis breeding.

[0042] The present invention also provides a method for constructing a dominant mononuclear body library and an application of the mushroom breeding method in mushroom breeding.

[0043] The present invention also provides an application of the above method for constructing a dominant mononuclear body library and the mushroom breeding method in improving one or a combination of two or more of the offspring fruiting rate, high yield rate and high quality rate in mushroom breeding.

[0044] In the above application, the fruiting rate is the ratio of the remaining strains capable of normal fruiting to the total number of the constructed population after excluding undesirable traits such as no fruiting and abnormal fruiting body development.

[0045] In the above application, the high yield is the ratio of the number of strains with a single yield of ≥300g in the first flush to the total number of the constructed population when the next generation produces mushrooms.

[0046] In the above application, the high-quality rate is the ratio of the number of strains with high-quality appearance of offspring fruiting bodies, such as round fruiting bodies, thick flesh, short stipes, and no obvious deformities to the total number of the constructed population.

[0047] The method for constructing a superior monokaryon library of the present invention can eliminate spore monokaryon materials carrying recessive undesirable trait genes through evaluation of spore monokaryon materials, and construct a superior monokaryon library. The shiitake mushroom breeding method of the present invention marks monokaryons carrying recessive sites of undesirable traits such as failure to produce mushrooms or abnormal fruiting body development, and avoids recessive homozygous combinations when pairing offspring groups, thereby improving the fruiting rate of breeding offspring. By randomly hybridizing monokaryons multiple times to screen superior monokaryons carrying excellent traits such as high yield, high quality, and stress resistance, these superior monokaryons are used to pair to improve the excellent rate of breeding offspring, thereby achieving the purpose of improving shiitake mushroom breeding efficiency.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] 1. The present invention provides a new breeding technology for edible fungi breeding.

[0050] 2. The present invention can continuously accumulate a library of superior mononuclear bodies, and using superior mononuclear bodies for breeding can significantly improve the fruiting rate, high yield, high quality rate, etc. of the breeding offspring, thereby improving the efficiency of breeding.

[0051] 3. The present invention provides a method for accumulating excellent monokaryotic strains, which is more conducive to accumulating new gene resources by gradually eliminating bad genes in gametes. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The present invention provides a flow chart of the method for constructing a dominant monokaryon library, the method for breeding shiitake mushrooms and their application.

[0053] Figure 2 This is the performance of a normal mushroom log.

[0054] Figure 3 It is a "popcorn" trait.

[0055] Figure 4 It is the manifestation of "sterile folds".

[0056] Figure 5 It is the manifestation of the "no mushroom" trait. DETAILED DESCRIPTION

[0057] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0058] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the art. The experimental methods in each embodiment where specific conditions are not specified are operated according to conventional conditions in the art. The starting reagents and materials used in the embodiments can be obtained commercially or prepared with reference to the prior art in the art.

[0059] In some specific embodiments of the present invention, the method for constructing a dominant monocyte library of the present invention comprises the following steps:

[0060] Providing spore monokaryons from a plurality of parents as a spore monokaryon population to be screened;

[0061] The spore monokaryons from the same parent in the spore monokaryon population to be screened are self-pollinated according to the mating type combination principle to obtain a self-pollination offspring population;

[0062] Phenotypic evaluation of the self-pollinated progeny population was performed to identify the monokaryons carrying recessive undesirable traits;

[0063] Hybridizing the monokaryon carrying the recessive undesirable trait with all other matable monokaryons in the spore monokaryon population to be screened, and forming all the progeny obtained from the hybridization into a first hybrid progeny population;

[0064] Performing phenotypic evaluation on the first hybrid offspring population; wherein the monokaryons that have undesirable traits in the first hybrid offspring are marked as monokaryons carrying recessive undesirable traits and are included in the first monokaryon material pool, and the monokaryons that have not developed any undesirable traits in the first hybrid offspring are marked as monokaryons that do not carry recessive undesirable traits and are included in the second monokaryon material pool;

[0065] Avoiding homozygous pairing of monokaryons carrying recessive adverse traits, hybridizing monokaryons in the first monokaryon material library with monokaryons in the second monokaryon material library with different parental sources, and hybridizing monokaryons in the second monokaryon material library with different parental sources to obtain a second hybrid offspring population;

[0066] The second hybrid offspring population is phenotypically evaluated, the probability of the emergence of strains with excellent traits in multiple second hybrid offspring formed by each monokaryon is counted, and monokaryons with a probability of ≥60% of the emergence of excellent traits are screened to obtain a superior monokaryon library.

[0067] In some specific embodiments of the present invention, the method for breeding shiitake mushrooms of the present invention comprises the following steps:

[0068] Using the monokaryons in the superior monokaryon library constructed by the above method as breeding parents for breeding to obtain a superior monokaryon hybrid breeding population;

[0069] The dominant mononuclear hybrid breeding population was evaluated by phenotype, and shiitake mushroom strains with excellent traits were screened out.

[0070] The flowchart of the method for constructing a dominant mononuclear library, the method for breeding shiitake mushrooms and their application in the present invention is as follows: Figure 1 shown.

[0071] In a specific embodiment of the present invention, four shiitake mushroom strains are selected as parent strains, and the strain codes are A, B, C, and D. Spore prints of the four parent strains are collected using a spore collector, and spore monokaryons are obtained using a single spore separation technique, which is a spore monokaryon population to be screened. Mating type identification is performed by hybridization and pairing.

[0072] (1) A certain number of monokaryons of different mating types (A1B1, A2B2, A1B2, A2B1, etc.) are selected from a monokaryon population derived from any parent, for example, parent A, and hybridized according to the mating type combination principle to obtain self-pollinated offspring, that is, a self-pollinated offspring population derived from the monokaryon population of parent A. Alternatively, a certain number of monokaryons of different mating types are selected from two or more parents, and the monokaryons derived from the same parent are self-pollinated, and the self-pollinated offspring derived from multiple parents together constitute a self-pollinated offspring population (for example, a population consisting of the self-pollinated offspring of A and the self-pollinated offspring of B).

[0073] (2) Cultivating the self-pollinated progeny obtained in step (1), and statistically analyzing the phenotypes after the mushrooms are produced. Counting the undesirable traits that can be stably inherited in the population, including "no mushrooms", "popcorn-shaped" fruiting bodies, and "sterile folds" on fruiting bodies. Counting the number of monokaryon materials with the traits of "no mushrooms", "popcorn-shaped" fruiting bodies, and "sterile folds" on fruiting bodies, and statistically analyzing their mating types.

[0074] (3) Monokaryons with the recessive traits of "no mushroom production", "popcorn", and "sterile folds" (one monokaryon for each trait can be selected, or multiple monokaryons can be selected) are hybridized with the spore populations A, B, C, and D to be screened, respectively, to cover all other monokaryons that can mate in all the populations to be screened. According to the mating type combination principle, for example, one monokaryon with a mating type of A1B1 is selected from the traits of "no mushroom production", "popcorn", and "sterile folds" and combined with some spore monokaryons in A, B, C, and D to obtain offspring; one monokaryon with a mating type of A1B2 is selected from the traits of "no mushroom production", "popcorn", and "sterile folds" and combined with the aforementioned spore monokaryons in A, B, C, and D that have not been combined to obtain offspring; Select one monokaryon with mating type A2B1 from each of the "popcorn" and "sterile gills" traits, and mate it with the aforementioned spore monokaryons that have not been paired in A, B, C, and D to obtain offspring; select one monokaryon with mating type A2B2 from each of the "no mushroom production", "popcorn", and "sterile gills" traits, and mate it with the aforementioned spore monokaryons that have not been paired in A, B, C, and D to obtain offspring, and the accumulated hybrid offspring are the first hybrid offspring population.

[0075] (4) The phenotypes of the first hybrid offspring population were evaluated and counted. The monokaryons carrying one of the three traits of "no mushroom production", "popcorn" and "sterile folds" were included in the first monokaryon library; the monokaryons that did not carry the three recessive undesirable traits of "no mushroom production", "popcorn" and "sterile folds" were included in the second monokaryon library.

[0076] (5) Randomly select monokaryotes with different parental origins from the second monokaryon material library and hybridize them with each other according to the mating type combination principle. The monokaryons are used 6 times, 7 times or 8 times to construct at least 1000 hybrid combinations, usually 1000-5000 hybrid combinations, to obtain "screening hybrid population 1", which is included in the second hybrid offspring population.

[0077] Avoid homozygous pairing of monokaryons carrying recessive undesirable traits, randomly select monokaryons carrying recessive traits in the first monokaryon material library and monokaryons that do not carry undesirable traits in the second monokaryon material library with different parental sources, and cross them with each other according to the mating type combination principle. The number of monokaryons used is between 6 and 8 times, and at least 1,000 hybrid combinations, usually 1,000 to 5,000 hybrid combinations, are constructed, namely "screening hybrid population 2", and are included in the second hybrid offspring population. In the actual operation of the above mutual hybridization, due to the restriction of mating type, some monokaryons are hybridized 6 times, some monokaryons are hybridized 7 times, and the remaining part of monokaryons are hybridized 8 times.

[0078] (6) Perform phenotypic evaluation on the second hybrid offspring population, and count the probability of high-quality strains appearing in multiple offspring formed by each monokaryon. Monokaryons with a probability of more than 60% for fruiting, high yield (first-wave yield ≥300 g), and high-quality traits are screened out to obtain a superior monokaryon library.

[0079] Example 1

[0080] This embodiment provides a method for constructing a dominant mononuclear body library, a shiitake mushroom breeding method and applications thereof.

[0081] The strains used in this example are all from Qihe Biological Culture Collection Center.

[0082] Take 4 shiitake mushroom strains as an example as the parent strains of this embodiment, Qihe No. 2, L808, Dandong 1363, Qihe 17, the strain codes are A, B, C, and D respectively, and the strain details are shown in Table 1.

[0083] The spore prints of the above four strains were collected using a spore collector, and 1674 spore monokaryons were obtained using the single spore separation technology, which was the spore monokaryon population to be screened. After mating type identification was performed by hybridization pairing, the mating type identification results were shown in Table 2.

[0084] Table 1 List of breeding parent strains

[0085] Serial number Strain name Strain code 1 Seven Rivers 2 A 2 L808 B 3 Dandong 1363 C 4 Seven Rivers 17 D

[0086] Table 2 Mating type identification results

[0087]

[0088] (1) The spore monokaryons of Qihe No. 2 (A) were used to construct a spore monokaryon selfing population. 49 monokaryons of different mating types A1B1, A2B2, A1B2, and A2B1 were selected from the monokaryon population derived from parent A. Hybridization was performed according to the mating type combination principle. The spore monokaryons of types A1B1 and A2B2 were combined, and the spore monokaryons of types A1B2 and A2B1 were combined. Each monokaryon was used 4 times. A total of 392 selfing progeny were obtained, namely the selfing progeny population derived from the monokaryon population of parent A.

[0089] (2) The self-pollinated offspring obtained in step (1) were cultured, and the phenotypes were counted after the mushrooms were produced. It was found that the undesirable traits that could be stably inherited in this group included "no mushrooms", "popcorn" fruiting bodies, and "sterile folds" in the fruiting bodies. According to statistics, there were 37 monokaryon materials with the "no mushrooms" trait (including 17 A1B1, 9 A1B2, 8 A2B1, and 3 A2B2), 26 monokaryon materials with the "popcorn" trait (including 3 A1B1, 6 A1B2, 11 A2B1, and 6 A2B2), and 18 monokaryon materials with the "sterile folds" trait (including 9 A1B1, 4 A1B2, 4 A2B1, and 1 A2B2). Figure 2 The following is the performance of the normal mushroom sticks. Figure 3 The "popcorn" trait is expressed. Figure 4 It is the performance of "sterile folds". Figure 5 It is the manifestation of the "no mushroom" trait.

[0090] (3) The monokaryons with the recessive traits of "no mushrooms", "popcorn", and "sterile folds" were hybridized with the spore populations of A, B, C, and D. According to the mating type combination principle, one monokaryon with the mating type of A1B1 was selected from each of the traits of "no mushrooms", "popcorn", and "sterile folds" and mated with some of the spore monokaryons in A, B, C, and D to obtain 1839 offspring; one monokaryon with the mating type of A1B2 was selected from each of the traits of "no mushrooms", "popcorn", and "sterile folds" and mated with some of the spore monokaryons in A, B, C, and D to obtain 873 offspring; One monokaryon with mating type A2B1 was selected from each of the "no mushroom production", "popcorn", and "sterile gills" traits, and was mated with some spore monokaryons in A, B, C, and D to obtain 1143 offspring; one monokaryon with mating type A2B2 was selected from each of the "no mushroom production", "popcorn", and "sterile gills" traits, and was mated with some spore monokaryons in A, B, C, and D to obtain 1167 offspring, a total of 5022 hybrid offspring were obtained, namely the first hybrid offspring, and the hybridization situation is shown in Table 3.

[0091] Table 3

[0092]

[0093]

[0094]

[0095] (4) The phenotypic evaluation and statistics of the first hybrid offspring population showed that 888 monokaryons carrying one of the three traits of "no mushroom production", "popcorn" and "sterile folds" were included in the first monokaryon library; 786 monokaryons that did not carry any of the three recessive adverse traits of "no mushroom production", "popcorn" and "sterile folds" were included in the second monokaryon material library.

[0096] (5) Randomly select monokaryons with different parental origins from the second monokaryon material library and hybridize them with each other according to the mating type combination principle. The monokaryons are used 6 times, 7 times or 8 times to construct 3281 hybrid combinations, and obtain "screening hybrid population 1", which is included in the second hybrid offspring population.

[0097] Avoid homozygous pairing of monokaryons carrying recessive undesirable traits, randomly select monokaryons carrying recessive traits in the first monokaryon material library and monokaryons that do not carry undesirable traits in the second monokaryon material library with different parental sources, and cross them with each other according to the mating type combination principle. The number of monokaryons used is between 6 and 8 times, and 1991 hybrid combinations are constructed, namely "screening hybrid population 2", which are included in the second hybrid offspring population. In the actual operation of the above mutual hybridization, due to the restriction of mating type, some monokaryons are hybridized 6 times, some monokaryons are hybridized 7 times, and the remaining part of monokaryons are hybridized 8 times.

[0098] (6) The second hybrid offspring were phenotypically evaluated, and the probability of the emergence of high-quality strains in the multiple offspring formed by each monokaryon was counted. The monokaryons with a probability of more than 60% for fruiting, high yield (first tide yield ≥ 300 g), and high-quality traits were screened out. 62 dominant monokaryons were obtained from the parental monokaryons of "screening hybrid population 1", and 49 dominant monokaryons were obtained from the parental monokaryons of "screening hybrid population 2", for a total of 111 dominant monokaryons, namely the dominant monokaryon library.

[0099] Using the monokaryons of the dominant monokaryon library as breeding parents, according to the mating type combination principle, 2124 hybrid offspring were constructed, namely the "dominant monokaryon hybrid breeding population". In contrast, the A, B, C, and D monokaryon populations were randomly crossed in pairs to construct a total of 2230 hybrids, namely the "traditional hybrid breeding population". The statistical phenotypic data of mushroom production are shown in Table 4, where the strain rejection rate is the number of strains rejected due to poor germination, slow growth, contamination, etc. during the strain growth stage divided by the total number of strains in the breeding population; the mushroom stick rejection rate is the number of strains rejected due to slow mycelial growth, mycelial degeneration, contamination, etc. during the mushroom stick culture stage divided by the total number of strains in the breeding population. From the statistical results in Table 4, it can be seen that the "dominant monokaryon hybrid breeding population" is significantly superior to the "non-dominant monokaryon hybrid breeding population" in terms of mushroom production rate, first tide yield, and high-quality rate. The difference in the strain elimination rate was not obvious, because the strain elimination process included hybridization, mother strain transfer, and strain preparation, and the intermediate human and environmental factors were relatively large. This result did not affect the improvement of the overall superiority level in the dominant mononuclear hybrid breeding population. According to the phenotypic evaluation results, shiitake mushroom strains with excellent traits can be screened from the "dominant mononuclear hybrid breeding population".

[0100] Table 4 Mushroom production statistics

[0101]

[0102] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for constructing a dominant mononuclear body library, the method comprising: Providing spore monokaryons from a plurality of parents as a spore monokaryon population to be screened; The spore monokaryons from the same parent in the spore monokaryon population to be screened are self-pollinated according to the mating type combination principle to obtain a self-pollination offspring population; Phenotypic evaluation of the self-pollinated progeny population was performed to identify the monokaryons carrying recessive undesirable traits; Hybridizing the monokaryon carrying the recessive undesirable trait with all other matable monokaryons in the spore monokaryon population to be screened, and forming all the progeny obtained from the hybridization into a first hybrid progeny population; Performing phenotypic evaluation on the first hybrid offspring population; wherein the monokaryons that have undesirable traits in the first hybrid offspring are marked as monokaryons carrying recessive undesirable traits and are included in the first monokaryon material pool, and the monokaryons that have not developed any undesirable traits in the first hybrid offspring are marked as monokaryons that do not carry recessive undesirable traits and are included in the second monokaryon material pool; Avoiding homozygous pairing of monokaryons carrying recessive adverse traits, hybridizing monokaryons in the first monokaryon material library with monokaryons in the second monokaryon material library with different parental sources, and hybridizing monokaryons in the second monokaryon material library with different parental sources to obtain a second hybrid offspring population; The second hybrid offspring population is phenotypically evaluated, the probability of the emergence of strains with excellent traits in multiple second hybrid offspring formed by each monokaryon is counted, and monokaryons with a probability of ≥60% of the emergence of excellent traits are screened to obtain a superior monokaryon library.

2. The method according to claim 1, wherein: The recessive undesirable traits include no mushroom production and / or abnormal fruiting body development; the excellent traits include one or a combination of two or more of high yield, high quality and stress resistance.

3. The method according to claim 1, wherein: The number of the parents is 2-8, preferably 3-5.

4. The method according to claim 1, wherein: When the monokaryons in the first monokaryon material library are hybridized with the monokaryons in the second monokaryon material library from different parental sources, and when the monokaryons in the second monokaryon material library from different parental sources are hybridized with each other, each monokaryon is used 6-10 times.

5. The method according to claim 1, wherein: The monokaryon includes one or more of a spore monokaryon and a protoplast monokaryon.

6. The method according to claim 1, wherein: The method for constructing a dominant mononuclear body library also includes the following steps: Spore prints from each parent are collected, spore monokaryons are obtained through single spore separation technology, and mating type identification is performed to obtain the mating type of the spore monokaryons of each parent.

7. A method for breeding shiitake mushrooms, the method comprising the following steps: Using the monokaryons in the dominant monokaryon library constructed by the method described in any one of claims 1 to 6 as breeding parents for breeding to obtain a dominant monokaryon hybrid breeding population; The dominant mononuclear hybrid breeding population was evaluated by phenotype, and shiitake mushroom strains with excellent traits were screened out.

8. The method according to claim 7, wherein: The breeding utilization method includes one or a combination of two or more of single-single hybrid breeding, single-double hybrid breeding, self-pollination breeding, backcross breeding and mutation breeding.

9. Use of the method according to any one of claims 1 to 6, or the method according to claim 7 or 8 in shiitake mushroom breeding.

10. Use of the method according to any one of claims 1 to 6, or the method according to claim 7 or 8, in improving the offspring fruiting rate, high yield rate, and high quality rate, or a combination of two or more thereof in shiitake mushroom breeding.

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