New shiitake mushroom single-double cross breeding method and application
By using the 212 spore mononuclear strain of Qingketa and the father Rongxiang No. 7 in single-double hybrid breeding of shiitake mushrooms, two hybrids with different pilot nuclei were successfully obtained on the same plate, solving the problem that each hybrid combination in the prior art can only obtain one hybrid in it, enriching the methods of shiitake mushroom hybrid breeding and expanding the research direction.
Patent Information
- Application Number
- CN202510433735.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-08
- Publication Date
- 2025-07-01
AI Technical Summary
In the prior art, there are lead nuclei when the single and double hybridization of shiitake mushrooms, and only one hybrid can be obtained per hybrid combination, and there is a lack of hybrid sub-breeding method to obtain different lead nuclei on the same plate.
By using the 212 spore mononuclear strain of Qingke and the father Rongxiang No. 7, hybrid breeding was performed, and hybrids of different pilot nuclei were pre-inoculated on PDA+maple chip juice or PDA+nut peel juice plates, the spacing between inoculation points was controlled, and mycelium blocks were picked after dark culture for purification and identification, so as to obtain hybrids of different pilot nuclei.
Two hybrids with different pilot nuclei were successfully obtained on the same plate, enriching the shiitake hybrid breeding methods, expanding the source of hybrids, and providing new ideas for the study of nuclear migration rules and linkage positioning of genetic traits in shiitake paired hybridization.
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Figure CN120230648A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of Lentinula edodes single and double hybridization breeding, and particularly relates to a new method for Lentinula edodes single and double hybridization breeding and its application. Background Art
[0002] Lentinula edodes is the most widely cultivated and consumed edible mushroom variety in China. The cultivation and popularization of new Lentinula edodes varieties have provided strong support for the development of the Lentinula edodes industry in China. Lentinula edodes hybridization breeding is the most commonly used and effective technical means in the breeding of new varieties, mainly including two methods: single-single hybridization and single-double hybridization.
[0003] Lentinula edodes single-double hybridization is an asymmetric hybridization phenomenon in which one nucleus in the donor dikaryotic parent migrates into the recipient mononuclear parent cell to complete pairing, also known as the Büller phenomenon. The research of the prior art believes that: there is a leading nucleus during Lentinula edodes single-double hybridization. Each hybridization combination can only obtain one hybrid offspring. That is, after the homokaryotic heterokaryon composed of the recipient cytoplasm and the recipient and donor nuclei formed by single-double hybridization is lysed by protoplast mononucleation, without exception, the nuclei with the same mating type as the recipient are in the majority, indicating that the recipient cytoplasmic environment plays an important role in the survival and quantity of the nuclei after the lysis of the heterokaryon obtained by single-double hybridization; in single-double hybridization, only the nucleus with strong affinity among the two donor nuclei preferentially migrates into the recipient to form a new heterokaryon, that is, there is a leading nucleus. The affinity specificity of the leading nucleus is related to the B factor. The higher the specificity, the faster the migration speed of the leading nucleus. In the prior art, when different mononuclear female parents of Lentinula edodes are hybridized with the same dikaryotic male parent combination, the male parent nuclei migrated into different hybrid offspring may be the same kind of nucleus, or may be two different nuclei respectively; while when the same mononuclear female parent of Lentinula edodes is hybridized with the same dikaryotic male parent combination, the male parent nuclei migrated into different hybrid offspring can only be the same kind of nucleus, that is, only one kind of hybrid offspring can be obtained. At present, there is no phenomenon that two kinds of hybrid offspring with different leading nuclei appear on the same plate in the combination of the same mononuclear female parent and the same dikaryotic male parent.
[0004] If a breeding method can be found in nature: in the same plate, in the pairing of the Lentinula edodes hybridization combination of the same mononuclear female parent and the same dikaryotic male parent, two kinds of leading nucleus migrations occur to obtain two different hybrid offspring, it will be able to fill the blank of this research. At the same time, it can also effectively enrich the Lentinula edodes hybridization breeding means, expand the source of hybrid offspring, and provide new ideas for the research on the nuclear migration law in Lentinula edodes pairing hybridization and the genetic trait linkage mapping of Lentinula edodes dikaryotic strains. Summary of the Invention
[0005] In view of the above, if a breeding method can be found in nature where, on the same plate, in the mating combination of Lentinula edodes hybrids with the same monokaryotic female parent and the same dikaryotic male parent, two leading nucleus migrations occur to obtain two different hybrids, it will fill the gap in this research. At the same time, it can effectively enrich the means of Lentinula edodes cross-breeding, expand the source of hybrids, and provide new ideas for research on the law of nucleus migration in Lentinula edodes cross-mating and the genetic trait linkage mapping of Lentinula edodes dikaryotic strains.
[0006] To achieve the above purpose, the spore monokaryotic strains of Qingke 212 are used as the female parent, including: Qingke 212038, Qingke 212150, and Qingke 212088;
[0007] The preservation information of the single-spore isolation strain of Qingke 212038 is: Qingke 212038, classified as Lentinula edodes, preservation number CGMCC NO.41799; this strain is preserved in the China General Microbiological Culture Collection Center, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, preservation date March 17, 2025;
[0008] The preservation information of the single-spore isolation strain of Qingke 212150 is: Qingke 212150, classified as Lentinula edodes, preservation number CGMCC NO.41800; this strain is preserved in the China General Microbiological Culture Collection Center, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, preservation date March 17, 2025;
[0009] The present invention also includes the application of the spore monokaryotic strains of Qingke 212 in a new single-double cross-breeding method for obtaining two hybrids with different paternal leading nuclei.
[0010] Furthermore, the male parent (dikaryotic strain) in the single-double cross-breeding is: Rongxiang No. 7 (abbreviated as YX7); its preservation information is: Rongxiang No. 7, classified as Lentinula edodes, preservation number CGMCC NO.40139; this strain is preserved in the Institute of Microbiology, Chinese Academy of Sciences, address: No. 3, Beichen West Road, Chaoyang District, Beijing, preservation date April 24, 2022.
[0011] The present invention also includes a method for using the Qingke 212 spore monokaryon strain and the male parent to carry out single-double hybrid breeding, the method comprising: pre-activating the Qingke 212 spore monokaryon strain and the male parent strain on a PDA plate; inoculating the activated Qingke 212038, Qingke 212150 or Qingke 212088 and the male parent on the same PDA+maple sawdust juice plate culture medium or PDA+nut skin juice plate culture medium, with the inoculation points of the two strains spaced 1.5-2.0 cm apart, and culturing in the dark at 25°C after inoculation; picking a number of mycelium blocks in three columns on the spore monokaryon strain colony after culturing for 20d-30d, purifying and identifying, and thus obtaining hybrids with different male parent leading nuclei on the same plate; performing karyotype analysis on the male parent leading nuclei of the purified hybrids to complete single-double hybrid breeding.
[0012] Furthermore, the PDA+maple sawdust juice plate culture medium is composed of the following ingredients: 200g potato, 20g glucose, 20g agar powder, 20-200g maple sawdust juice, and the balance is diluted to 1000mL with water, with a natural pH value; the maple sawdust juice preparation method is to boil maple sawdust with 1000mL boiling water for 10 minutes and then filter to obtain the juice.
[0013] Furthermore, the PDA+nut skin juice plate culture medium is composed of the following ingredients: 200g potato, 20g glucose, 20g agar powder, 20-200g nut skin juice, and the balance is diluted to 1000mL with water, and the pH is natural; the nut skin juice preparation method is to boil the nut skin with 1000mL boiling water for 10 minutes and then filter the juice.
[0014] Furthermore, the distance between the two pieces of mycelium in the picked mycelium block is 1.0 cm.
[0015] Furthermore, the method for karyotype analysis of the male parent lead nucleus is to name the two cell nuclei of the male parent Rongxiang No. 7 as A nucleus and B nucleus respectively; design a PA primer pair and a PB primer pair for A nucleus and B nucleus, use the genomic DNA of the hybrid to be tested as a template, and use the PA primer pair and the PB primer pair to perform PCR amplification respectively; if the PA primer pair amplifies a band, the male parent lead nucleus of the hybrid is the A nucleus; if the PB primer pair amplifies a band, the male parent lead nucleus of the hybrid is the B nucleus;
[0016] The upstream sequence of the PA primer pair is: 5'-AATCTCCCAAAGGACTGGATCG-3'; the downstream sequence is: 5'-CACCATTTCGGAGCCAAGAGC-3';
[0017] The upstream sequence of the PB primer pair is: 5'-CATTCAGCGCCAATCGAGCAG-3'; the downstream sequence is: 5'-GAGAAGCTTCGTCAGCCACTG-3'.
[0018] Furthermore, the reaction system for PCR amplification is as follows: 10 μL of 2×Taq PCR MasterMix, 1 μL each of forward and reverse primers (10 μmol / L), 1 μL of template DNA (150 ng), and 7 μL of ddH2O;
[0019] The PCR reaction conditions are as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 60 / 70°C for 30 s, extension at 72°C for 60 s, 30 cycles, fill-in at 72°C for 7 min, and storage at 4°C.
[0020] The present invention also includes the application of the method in localizing the paternal genetic traits on the nucleus. The application is as follows: Using the method to perform single and double hybridization breeding on the monokaryotic strain of Qingke 212 spores and the paternal parent, obtaining two different hybrids with the same cytoplasm, maternal nucleus, and different leading nuclei derived from the paternal parent. Then, respectively identify the karyotypes of the leading nuclei of the two different hybrids and analyze their biological characteristics and cultivation agronomic characteristics, find out the phenotypic trait differences between the two hybrids, and then associate the differential traits with the leading nuclei derived from the paternal parent, thereby obtaining the localization of the paternal genetic traits on its nucleus.
[0021] The present invention has the following beneficial effects: In the research process of the parental strains Rongxiang 7 and Qingke 212, it is found that when certain monosporic isolates of Qingke 212, including Qingke 212038, Qingke 212150, or Qingke 212088, etc., are used as the female parent and the dikaryotic strain Rongxiang 7 is used as the male parent for hybridization, two hybrids with different leading nuclei can be obtained on the same plate. The discovery of this parental strain and the overall breeding method effectively enrich the means of Lentinula edodes hybridization breeding, break the assertion in the prior art that there are leading nuclei during single and double hybridization of Lentinula edodes and only one hybrid can be obtained for each hybridization combination, fill the blank in the research of single and double hybridization breeding of Lentinula edodes, expand the source of hybrids, and also provide new ideas for the research on the nuclear migration law in the paired hybridization of Lentinula edodes and the genetic trait linkage mapping of Lentinula edodes dikaryotic strains. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 It is a paired hybridization plate diagram of the monosporic isolate of Qingke 212038 and YX7; in the figure, the left colony is the colony of the monosporic isolate of the female parent Qingke 212038, and the right is the colony of the male parent YX7 dikaryotic strain.
[0023] Figure 2 It is an ISSR identification electrophoresis diagram of the hybrids obtained from the single and double hybridization of the monosporic isolate of Qingke 212038 and YX7.
[0024] Figure 3Electrophoresis map for karyotype identification of the leading nucleus obtained from the hybrid strain picked at 15 d of paired cultivation of the single spore isolate Qingke 212038 and YX7.
[0025] Figure 4 Electrophoresis map for karyotype identification of the leading nucleus obtained from the hybrid strain picked at 20 d of paired cultivation of the single spore isolate Qingke 212038 and YX7.
[0026] Figure 5 Electrophoresis map for karyotype identification of the leading nucleus obtained from the hybrid strain picked at 30 d of paired cultivation of the single spore isolate Qingke 212038 and YX7.
[0027] Figure 6 Results graph of the antagonism experiment between different karyotype hybrids and Qingke 212 and YX7.
[0028] Figure 7 Electrophoresis map for karyotype identification of the leading nucleus obtained from the hybrid strain picked at 20 d of paired cultivation of the single spore isolate Qingke 212150 and YX7.
[0029] Figure 8 Electrophoresis map for karyotype identification of the leading nucleus obtained from the hybrid strain picked at 20 d of paired cultivation of the single spore isolate Qingke 212088 and YX7.
[0030] Biological material preservation information
[0031] The strain information preserved in this application is as follows: The Lentinula edodes strain Rongxiang No. 7, its taxonomic name is: Lentinula edodes, and the preservation number is CGMCC NO. 40139; This strain is preserved in the Institute of Microbiology, Chinese Academy of Sciences, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation date is April 24, 2022.
[0032] The preservation information of the single spore isolate Qingke 212038 is as follows: Qingke 212038, taxonomic name: Lentinula edodes, preservation number is CGMCC NO. 41799; This strain is preserved in the China Center for Type Culture Collection, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation date is March 17, 2025;
[0033] The preservation information of the single spore isolate Qingke 212150 is as follows: Qingke 212150, taxonomic name: Lentinula edodes., preservation number is CGMCC NO. 41800; This strain is preserved in the China Center for Type Culture Collection, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation date is March 17, 2025. Detailed implementation manners
[0034] All the features disclosed in this specification, or the steps in all the methods or processes disclosed, except for mutually exclusive features and / or steps, can be combined in any manner.
[0035] Any feature disclosed in this specification (including any additional claims, abstract), unless otherwise specifically stated, each feature is only an example of a series of equivalent or similar features.
[0036] Example 1
[0037] This example is a method for hybrid breeding of Lentinula edodes:
[0038] 1. Strains: Rongxiang No. 7 (the accession number of the strain preservation is CGMCC NO. 40139) is a wild-domesticated Lentinula edodes strain, also known as Yexiang No. 7 (abbreviated as YX7); Qingke 212 (Zhe (non)-examined fungus: 2015001) is a domestic commercial cultivated Lentinula edodes variety; Qingke 212038 is a single-spore isolate of the Qingke 212 strain.
[0039] 2. The mating type of each of the above single-spore isolates is A1B1.
[0040] 3. Media: PDA (200 g of potato, 20 g of glucose, 20 g of agar powder, made up to 1000 mL with water, pH natural); PDA + maple sawdust juice (this medium consists of the following components: 200 g of potato, 20 g of glucose, 20 g of agar powder, 20 - 200 g of maple sawdust juice, the balance made up to 1000 mL with water, pH natural; the preparation method of the maple sawdust juice is to boil maple sawdust with 1000 mL of boiling water for 10 minutes and then filter to obtain the juice); PDA + nut skin juice (this medium consists of the following components: 200 g of potato, 20 g of glucose, 20 g of agar powder, 20 - 200 g of nut skin juice, the balance made up to 1000 mL with water, pH natural; the preparation method of the nut skin juice is to boil nut skins with 1000 mL of boiling water for 10 minutes and then filter to obtain the juice).
[0041] 4. Pairing hybridization: First, separately activate the Lentinula edodes single-spore strain Qingke 212038 and the male parent strain YX7 on PDA plates. Inoculate the activated Qingke 212038 and YX7 strains on the same PDA + maple sawdust juice or PDA + nut skin juice plate medium, with a distance of 1.5 - 2.0 cm between the inoculation points of the two strains. After inoculation, place them in the dark at 25°C for cultivation. Take out one plate after culturing for 15 d, 20 d, 25 d, and 30 d respectively. As Figure 1 shown, pick mycelium blocks in three columns L1, L2, and L3 on the single-spore strain colonies, and number them as L1-① to ⑥, L2-① to ⑤, and L3-① to ④ respectively. The picked mycelium blocks are transferred to new PDA plates for purification culture 3 times and stored as hybrid strains for standby.
[0042] 5. Identification of the authenticity of hybrid offspring: Genomic DNA of the hybrid strains was extracted separately, and PCR amplification was performed using the ISSR1 primer (sequence: AGAGAGAGAGAGAGAGT). The ISSR-PCR reaction system was as follows: 10 μL of 2×Taq PCR MasterMix, 1 μL of primer (10 μmol / L), 1 μL of template DNA (100 ng), and 8 μL of ddH2O. PCR reaction conditions: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 54°C for 45 s, extension at 72°C for 90 s, 35 cycles, filling at 72°C for 7 min, and storage at 4°C. According to the electrophoresis pattern of the amplification products, analyze whether each hybrid strain is a true hybrid offspring, that is, whether the hybridization is successful.
[0043] 6. Karyotype analysis of the leading nucleus of hybrid offspring: Two pairs of primers, PA and PB, were used to specifically label the A nucleus and B nucleus of the male parent YX7 strain, respectively. The upstream primer sequence of PA was: 5’-AATCTCCCAAAGGACTGGATCG-3’, and the downstream primer sequence was: 5’-CACCATTTCGGAGCCAAGAGC-3’. The primer annealing temperature was 60°C; the upstream primer sequence of PB was: 5’-CATTCAGCGCCAATCGAGCAG-3’, and the downstream primer sequence was: 5’-GAGAAGCTTCGTCAGC CACTG-3’. The primer annealing temperature was 70°C. Using the genomic DNA of the hybrid offspring to be tested as a template, PCR amplification was performed with the two pairs of primers PA and PB respectively. The PCR reaction system was: 10 μL of 2×Taq PCR MasterMix, 1 μL of each upstream and downstream primer (10 μmol / L), 1 μL of template DNA (150 ng), and 7 μL of ddH2O. PCR reaction conditions: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 60 / 70°C for 30 s, extension at 72°C for 60 s, 30 cycles, filling at 72°C for 7 min, and storage at 4°C.
[0044] II. Results and Analysis
[0045] 1. Identification of the authenticity of hybrid offspring: As Figure 2 shown, m and n are the characteristic bands of the monosporic strain Qingke 212038 and the male parent YX7, respectively. The strain that simultaneously has the two characteristic bands m and n is the hybrid offspring with successful hybridization, as Figure 2Strains such as 15dL1-①, 20dL1-⑤, 20dL1-⑥, 20dL2-④, 20dL3-④, 25dL1-①, 25dL2-①, 25dL3-①, 30dL1-①, 30dL1-②, 30dL2-①, 30dL2-⑤; while strain 20dL3-③ only has m bands and lacks n bands, indicating that its hybridization was not successful and it is not a true hybrid.
[0046] 2. Karyotype analysis of the leading nucleus of hybrids
[0047] As Figure 3 shown, the leading nuclei of the hybrids paired and cultured for 15 days are of only one type, all being A nuclei.
[0048] As Figure 4 shown, the leading nuclei of the hybrids paired and cultured for 20 days are of two types: the leading nuclei of 6 strains such as L1-②, L1-③, L1-④, L2-①, L2-③, L2-④ are the A nuclei of the male parent, the leading nuclei of 3 strains such as L1-⑤, L1-⑥, L3-④ are the B nuclei of the male parent, and 4 strains such as L2-②, L3-①, L3-②, L3-③ did not obtain any nuclei of the male parent (non-hybrids). It can be seen that two different hybrids, namely s7d2-38-A and s7d2-38-B, were obtained from the same pairing combination of Qingke 212038 and YX7, and were obtained from different parts of the same plate.
[0049] As Figure 5 shown, the leading nuclei of the hybrids paired and cultured for 30 days are of two types: the leading nuclei of 3 strains such as L1-①, L1-⑤, L1-⑥ are the A nuclei of the male parent, and the leading nuclei of 9 strains such as L1-②, L1-③, L1-④, L2-①, L2-②, L2-③, L3-②, L3-③, L3-④ are the B nuclei of the male parent. It is proved again that two different hybrids, namely s7d2-38-A and s7d2-38-B, can be obtained from the same pairing combination of Qingke 212038 and YX7, and were obtained from different parts of the same plate.
[0050] 3. Antagonism test of two different types of hybrids
[0051] As Figure 6 shown, the two hybrids s7d2-38-A and s7d2-38-B have a strong antagonistic reaction with Qingke 212, a weak antagonistic reaction with YX7, an obvious antagonistic reaction between the two hybrids, and no antagonistic reaction within the same hybrid. This shows that s7d2-38-A and s7d2-38-B are different strains.
[0052] Example 2
[0053] This example shows the experimental results of the hybridization of different single spore isolates with dikaryotic parents:
[0054] 1. Strains: Rongxiang No. 7 (the preservation number of the strain is CGMCC NO. 40139) is a wild-domesticated Lentinula edodes strain, also known as Yexiang No. 7 (abbreviated as YX7); Qingke 212 (Zhe (non)-examined strain: 2015001) is a domestic commercial cultivated Lentinula edodes variety; Qingke 212038, Qingke 212150, Qingke 212088, Qingke 212008, Qingke 212036, Qingke 212060, Qingke 212080 and Qingke 212160 are all single-spore isolates of the Qingke 212 strain.
[0055] The mating types of the above single-spore isolates are as follows: Qingke 212038 (A1B1), Qingke 212150 (A2B1), Qingke 212088 (A1B1), Qingke 212008 (A1B1), Qingke 212036 (A2B1), Qingke 212060 (A2B1), Qingke 212080 (A2B2) and Qingke 212160 (A1B2).
[0056] Refer to the method and primers of Example 1 for experimental verification. After 20 days of confrontation hybridization between each spore mononuclear body and Rongxiang No. 7 on a PDA plate, 15 samples were taken from different parts of the colony of the spore mononuclear body, and after purification, they were used as 15 hybrid strains of the spore mononuclear body and Rongxiang No. 7. The leading nuclei of the hybrid strains from different sampling parts were detected respectively, and the leading nucleus ratio was counted. The results are shown in Table 1:
[0057] Table 1 Different leading nucleus ratios in the single and double hybrid offspring of the Qingke 212 spore mononuclear body strain and Rongxiang No. 7
[0058]
[0059]
[0060] As can be seen from Table 1, the three single-spore isolates of Qingke 212038, Qingke 212150 and Qingke 212088 can all form two hybrid offspring with Rongxiang No. 7, and they can obtain different leading nuclei YX7-PA and YX7-PB of Rongxiang No. 7 on the same plate respectively; while the four single-spore isolates of Qingke 212008, Qingke 212036, Qingke 212080 and Qingke 212160 can only obtain the leading nucleus YX7-PA of Rongxiang No. 7, and can only get one kind of hybrid offspring s7d2-38-A when hybridized with Rongxiang No. 7. The single-spore isolate of Qingke 212060 can only obtain the leading nucleus YX7-PB of Rongxiang No. 7 and form one kind of hybrid offspring s7d2-38-B.
[0061] The identification electrophoresis diagrams of the pairing of Qingke 212150 and Qingke 212088 with Rongxiang No. 7 are as Figures 7 - 8 shown, among which, Figure 7The experimental results of the paired culture of the single-spore isolate of Qingke 212150 and YX7 are shown in the figure: On the 20th day after paired culture, a total of 6 strains, namely L1-③, L2-①, L2-②, L2-⑤, L3-①, and L3-②, obtained the nucleus of male parent A, and a total of 8 strains, namely L1-②, L1-④, L1-⑤, L1-⑥, L2-③, L2-④, L3-③, and L3-④, obtained the nucleus of male parent B; The L1-① strain has two male parent cell nuclei, which should be the mycelium of YX7 taken by mistake and is not a true hybrid. It can be seen that two different hybrids, namely s7d2-150-A and s7d2-150-B, were obtained from the same paired combination of Qingke 212150 and YX7, and they were obtained from different parts of the same plate.
[0062] Figure 8 The experimental results of the paired culture of the single-spore isolate of Qingke 212088 and YX7 are shown in the figure: On the 20th day after paired culture, a total of 4 strains, namely L1-②, L2-①, L2-②, and L2-③, obtained the nucleus of male parent A, and a total of 10 strains, namely L1-①, L1-③, L1-④, L1-⑤, L1-⑥, L2-④, L2-⑤, L3-①, L3-②, L3-③, and L3-④, obtained the nucleus of male parent B; The L2-① strain has two male parent cell nuclei, which should be the mycelium of YX7 taken by mistake and is not a true hybrid. It can be seen that two different hybrids, namely s7d2-88-A and s7d2-88-B, were obtained from the same paired combination of Qingke 212088 and YX7, and they were obtained from different parts of the same plate.
[0063] Example 3
[0064] This example is about the application of the hybridization method of the present application to analyze the genetic traits of the male parent in nuclear localization. Specifically: Single and double hybridization breeding was carried out on the single-spore strains of Qingke 212 and the male parent to obtain two hybrids with the same cytoplasm and female parent nucleus but different leading nuclei derived from the male parent. Then, the method of Example 2 was used to analyze the karyotypes of the leading nuclei of the two hybrids, and at the same time, the biological characteristics and cultivation agronomic characteristics of the two hybrids were analyzed to find out the phenotypic differences between them. Then, the differential traits were associated with the leading nuclei derived from the male parent, and the localization of the genetic traits of the male parent on its cell nucleus could be obtained.
[0065] In summary, in the research process of the present invention on the parental strains Rongxiang No. 7 and Qingke 212, it was found that: Some single-spore isolates of Qingke 212, namely Qingke 212038, Qingke 212150, and Qingke 212088, can all obtain two hybrids with different leading nuclei from the male parent Rongxiang No. 7 on the same plate at the same time. This method enriches the means of Lentinula edodes hybridization breeding, expands the source of hybrids, and can also provide new ideas for the research on the nuclear migration law in Lentinula edodes paired hybridization and the genetic trait linkage mapping of Lentinula edodes dikaryotic strains.
[0066] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the appended claims.
Claims
1. Qingke 212 spore monokaryon strain, characterized in that, The spore monokaryon strains of Qingke 212 include: Qingke 212038, Qingke 212150 and / or Qingke 212088; The spore monokaryon strain of Qingke 212038 has a preservation number of CGMCC NO.41799; the spore monokaryon strain of Qingke 212150 has a preservation number of CGMCC NO.41800. The classification and naming of the above two spore monokaryon strains are: Lentinula edodes, both of which are preserved in the China Center for Type Culture Collection, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation date is March 17, 2025.
2. Application of the Qingke 212 spore monokaryon strain as claimed in claim 1 as a female parent in a new method for single-double hybrid breeding of Lentinus edodes.
3. The use according to claim 2, characterized in that: The male parent in the new single-double hybrid breeding method is the Rongxiang No. 7 binuclear strain, whose preservation number is CGMCC NO.40139, and its classification name is: Lentinula edodes, with a preservation number of CGMCC NO.40139; the strain is preserved in the Institute of Microbiology, Chinese Academy of Sciences, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the preservation date is April 24, 2022.
4. A method for single-double hybrid breeding using the Qingke 212 spore monokaryon strain as claimed in claim 1 as the female parent and the binuclear male parent, characterized in that: The method comprises the following steps: respectively activating the Qingke 212 spore monokaryon strain and the father parent strain on a PDA plate in advance; respectively inoculating the activated Qingke 212038, Qingke 212150 or Qingke 212088 and the father parent on the same PDA+maple sawdust juice plate culture medium or PDA+nut skin juice plate culture medium, with the inoculation points of the two strains spaced 1.5-2.0 cm apart, and culturing in the dark at 25°C after inoculation; picking a number of hyphae blocks from the spore monokaryon strain colony in three columns after culturing for 20 days to 30 days, purifying and identifying, and thus obtaining hybrids of different father parent leading nuclei on the same plate; and performing karyotype analysis on the father parent leading nuclei of the purified hybrids to complete the single-double hybrid breeding.
5. The method according to claim 4, characterized in that The PDA+maple sawdust juice plate culture medium is composed of the following ingredients: 200g potato, 20g glucose, 20g agar powder, 20-200g maple sawdust juice, and the balance is diluted to 1000mL with water, with a natural pH value; the maple sawdust juice preparation method is to boil maple sawdust in 1000mL boiling water for 10 minutes and then filter to obtain juice.
6. The method according to claim 4, characterized in that The PDA+nut skin juice plate culture medium is composed of the following ingredients: 200g potato, 20g glucose, 20g agar powder, 20-200g nut skin juice, and the balance is diluted to 1000mL with water, and the pH is natural; the nut skin juice preparation method is to boil nut skins with 1000mL boiling water for 10 minutes and then filter to obtain juice.
7. The method according to claim 4, characterized in that The distance between the two pieces of mycelium in the picked mycelium block is 1.0 cm.
8. The method according to claim 4, characterized in that The method for karyotype analysis of the male parent lead nucleus is as follows: the two cell nuclei of the male parent Rongxiang No. 7 are named A nucleus and B nucleus respectively; a PA primer pair and a PB primer pair are designed for the A nucleus and the B nucleus, and the genomic DNA of the hybrid to be tested is used as a template, and PCR amplification is performed using the PA and PB primer pairs respectively; if the PA primer pair amplifies a band, the male parent lead nucleus of the hybrid is the A nucleus; if the PB primer pair amplifies a band, the male parent lead nucleus of the hybrid is the B nucleus; The upstream sequence of the PA primer pair is: 5'-AATCTCCCAAAGGACTGGATCG-3'; the downstream sequence is: 5'-CACCATTTCGGAGCCAAGAGC-3'; The upstream sequence of the PB primer pair is: 5'-CATTCAGCGCCAATCGAGCAG-3'; the downstream sequence is: 5'-GAGAAGCTTCGTCAGCCACTG-3'.
9. The method according to claim 8, characterized in that The reaction system of the PCR amplification is: 2×Taq PCRMasterMix 10 μL, 1 μL of 10 μmol / L upstream and downstream primers, 1 μL of 150 ng template DNA, and 7 μL of ddH2O; The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 60 / 70°C for 30 s, extension at 72°C for 60 s, 30 cycles, fill-in at 72°C for 7 min, and storage at 4°C.
10. Use of the method according to claim 4 for localizing paternal genetic traits in the cell nucleus, characterized in that: The application is: using the method to carry out single-double hybrid breeding of the Qingke 212 spore monokaryon strain and the binucleate parent, obtaining two hybrids with the same cytoplasm and maternal cell nucleus but different leading nuclei derived from the paternal parent, performing karyotype analysis on the leading nuclei of the two hybrids according to the method of claim 8, and simultaneously analyzing the biological characteristics and cultivation agronomic characteristics of the two hybrids to find out the differences in phenotypic traits of the two hybrids, and then associating the differential traits with the leading nucleus derived from the paternal parent, so as to obtain the location of the genetic traits of the paternal parent on its cell nucleus.