Primers for identification of mating type of Lentinula edodes 0912 and its application

Abstract

The invention discloses primers for identifying the mating type of shiitake mushroom 0912 variety and applications thereof. The invention provides a set of primer pairs, which are composed of primer pair A1 (SEQ ID No. 1 and SEQ ID No. 2), primer pair A2 (SEQ ID No. 3 and SEQ ID No. 4), primer pair B1 (SEQ ID No. 5 and SEQ ID No. 6) and primer pair B2 (SEQ ID No. 7 and SEQ ID No. 8). The invention uses sequence homology comparison to screen the variation sites on the A mating type site HD1 gene and the B mating type site RCB2 gene on a pair of protoplast monokaryotes SP3 and SP30 of shiitake mushroom 0912, designs specific primers, and establishes a shiitake mushroom mating type identification method based on the variation sites of the HD1 and RCB2 genes, which can be used for the identification of the mating type of the monokaryotes of the 0912 variety and the identification of the relationship between other shiitake mushroom varieties and the 0912 variety.

Description

Technical Field

[0001] The invention relates to the field of edible fungi, and in particular to primers for identifying the mating type of Lentinus edodes 0912 variety and applications thereof. Background Art

[0002] Hybrid breeding refers to the process of mating gametes of different genotypes to produce hybrids. In the field of edible fungi breeding, hybrid breeding is the most important breeding method, and its biological basis is the sexual reproduction process under the mating type system. Mating type genes are important genetic information that controls the hybridization, sexual reproduction and fruiting body formation of edible fungi. Monokaryons can grow vegetatively, but are usually sterile. Monokaryons carrying the same mating type gene cannot form stable heterokaryons when mating with each other. For tetrapolar heterothallic basidiomycetes such as shiitake mushrooms, sexual reproduction depends on two mating loci that do not have a linked genetic relationship, namely: the A mating type loci encoding homeodomain transcription factors (HD) control biological processes such as lock union formation and heterokaryon pairing; the B mating type loci encoding lipopeptide pheromone (PHB) and pheromone receptor (RCB) control nuclear migration, cell membrane melting and other functions. The different alleles of the A and B sites are different between the two haploids, forming a heterologous complex, activating the lock-like union, and allowing the compatible monokaryon hyphae to form heterokaryon hyphae that can produce fruiting bodies. Among them, the A mating type site contains a pair of mating type genes HD1, HD2, mip (mitochondrial intermediate peptidase) gene and β-fg (beta-flanking) gene. The B mating type site is divided into multiple subsites, including the Bα subsite (RCB1), the Bβ mating type subsite (RCB2), the RCB3 and RCB4 subsites, the RCB5 subsite and the free RCB gene.

[0003] Shiitake mushroom is the edible fungus variety with the largest annual production and consumption in my country. In recent years, the shiitake mushroom industry has developed rapidly and the cultivation area has continued to expand. The demand for high-yield and high-quality shiitake mushroom varieties and regionally specific varieties has also increased. Therefore, the selection and breeding of new shiitake mushroom varieties has become a necessary path to ensure the high-quality development of my country's shiitake mushroom industry. Reasonable and appropriate identification of shiitake mushroom homokaryons will help shorten the shiitake mushroom breeding cycle and improve the efficiency of targeted breeding. Shiitake mushroom is a typical heterothallic tetrapolar mating edible fungus, and the mating type gene sequence is rich in polymorphism. Previously, our team has completed the genome mapping of a pair of hybridizable protoplast monokaryotic strains SP3 and SP30 of Lentinula edodes 0912 variety (Liaofu No. 4) (Gao Q, et al. 2022. Haplotype-resolved genome analyses reveal genetically distinct nuclei within a commercial cultivar of Lentinula edodes. Journal of Fungi, 8(2): 167. Gao Q, et al. 2023. Insights into the Global Transcriptome Response of Lentinula edodes Mycelia during Aging. J. Fungi 2023, 9(3), 379; https: / / doi.org / 10.3390 / jof9030379). The study found that there were 17 insertion / deletion fragments between HD1 of SP3 and SP30 strains. Deletion, InDel), HD2 gene has 28 InDels; HD gene of SP3 strain has high homology with W1-26 strain, and HD gene of SP30 strain is relatively homologous with L808. Therefore, the mating type gene of Lentinula edodes is also of great application value in genotype identification. Summary of the invention

[0004] The invention aims to provide primers for identifying the mating type of Lentinus edodes 0912 variety and application thereof.

[0005] In a first aspect, the present invention claims a set of primer pairs or a reagent or a kit containing the set of primer pairs.

[0006] The set of primer pairs claimed in the present invention consists of primer pair A1, primer pair A2, primer pair B1 and primer pair B2;

[0007] The primer pair A1 is composed of two single-stranded DNAs shown in SEQ ID No.1 and SEQ ID No.2, or two single-stranded DNAs having more than 80% identity with SEQ ID No.1 and SEQ ID No.2;

[0008] The primer pair A2 is composed of two single-stranded DNAs shown in SEQ ID No.3 and SEQ ID No.4, or two single-stranded DNAs having more than 80% identity with SEQ ID No.3 and SEQ ID No.4;

[0009] The primer pair B1 consists of two single-stranded DNAs shown in SEQ ID No.5 and SEQ ID No.6, or consists of two single-stranded DNAs having more than 80% identity with SEQ ID No.5 and SEQ ID No.6;

[0010] The primer pair B2 consists of two single-stranded DNAs shown in SEQ ID No.7 and SEQ ID No.8, or consists of two single-stranded DNAs having more than 80% identity with SEQ ID No.7 and SEQ ID No.8.

[0011] Among them, 80% or more identity is preferably 90% or more or 85% or more identity.

[0012] In a second aspect, the present invention claims a primer pair set or a reagent or a kit containing the primer pair set.

[0013] The primer pair set claimed in the present invention is composed of any three or any two of the primer pair A1, the primer pair A2, the primer pair B1 and the primer pair B2 in the first aspect described above.

[0014] Furthermore, the primer pair group is any one of the following:

[0015] (a1) primer pair set 1, consisting of the primer pair A1 and the primer pair B1;

[0016] (a2) primer pair set 2, consisting of the primer pair A2 and the primer pair B2;

[0017] (a3) primer pair group 3, consisting of the primer pair A1 and the primer pair B2;

[0018] (a4) primer pair group 4, consisting of the primer pair A2 and the primer pair B1;

[0019] (a5) primer pair set 5, consisting of the primer pair A1 and the primer pair A2;

[0020] (a6) Primer pair set 6, consisting of the primer pair B1 and the primer pair B2.

[0021] In a third aspect, the present invention claims a primer pair or a reagent or kit containing the primer pair.

[0022] The primer pair claimed in the present invention is any of the following:

[0023] (b1) Primer pair 1, which is the primer pair A1 described in the first aspect above;

[0024] (b2) Primer pair 2, which is the primer pair A2 described in the first aspect above;

[0025] (b3) primer pair 3, which is the primer pair B1 in the first aspect above;

[0026] (b4) Primer pair 4 is the primer pair B2 described in the first aspect above.

[0027] In a fourth aspect, the present invention claims the use of the set of primers or the reagent or kit containing the set of primers in the first aspect above in any of the following:

[0028] (c1) Identify or assist in identifying the mating type of monokaryons of Lentinula edodes 0912 variety;

[0029] (c2) identifying or assisting in identifying whether two monokaryons of the tested Lentinus edodes 0912 variety can hybridize and pair to produce a lock-like union;

[0030] (c3) Identify or assist in identifying the genetic relationship between the strain to be tested and Lentinus edodes 0912 variety.

[0031] In a fifth aspect, the present invention claims protection for the use of the primer pair set or the reagent or kit containing the primer pair set in the second aspect above in any of the following:

[0032] (d1) Use of the primer pair group 1 described in the second aspect above in identifying or assisting in identifying whether the mating type of the monokaryon of Lentinus edodes 0912 variety to be tested is the A1B1 mating type;

[0033] (d2) Use of the primer pair group 2 described in the second aspect above in identifying or assisting in identifying whether the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested is the A2B2 mating type;

[0034] (d3) Use of the primer pair group 3 described in the second aspect above in identifying or assisting in identifying whether the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested is the A1B2 mating type;

[0035] (d4) Use of the primer pair group 4 described in the second aspect above in identifying or assisting in identifying whether the mating type of the monokaryon of Lentinus edodes 0912 variety to be tested is the A2B1 mating type;

[0036] (d5) Use of the primer pair group 5 described in the second aspect above in identifying or assisting in identifying the mating type of Lentinus edodes 0912 variety or a strain of a variety related thereto at the A mating type locus;

[0037] (d6) Use of the primer pair group 6 described in the second aspect above in identifying or assisting in identifying the mating type of Lentinus edodes 0912 variety or strains of varieties related thereto at the B mating type locus.

[0038] In a sixth aspect, the present invention claims the use of the primer pair or the reagent or kit containing the primer pair described in the third aspect above in any of the following:

[0039] (e1) Use of the primer pair 1 described in the third aspect above in identifying or assisting in identifying whether the mating type of the strain of Lentinus edodes 0912 variety or a variety related thereto at the A mating type locus is or contains the A1 mating type;

[0040] (e2) Use of the primer pair 2 described in the third aspect above in identifying or assisting in identifying whether the mating type of the strain of Lentinus edodes 0912 variety or a variety related thereto at the A mating type locus is or contains the A2 mating type;

[0041] (e3) Use of the primer pair 3 described in the third aspect above in identifying or assisting in identifying whether the mating type of the strain of Lentinus edodes 0912 variety or a variety related thereto at the B mating type locus is or contains the B1 mating type;

[0042] (e4) Use of the primer pair 4 described in the third aspect above in identifying or assisting in identifying whether the mating type of the tested Lentinus edodes 0912 variety or a strain of a variety related thereto at the B mating type locus is or contains the B2 mating type.

[0043] In a seventh aspect, the present invention claims a method for identifying or assisting in identifying the mating type of monokaryons of Lentinus edodes 0912 variety.

[0044] The method for identifying or assisting in identifying the mating type of the monokaryon of Lentinus edodes 0912 variety claimed in the present invention may include the following steps: using the genome of the monokaryon of Lentinus edodes 0912 variety to be tested as a template, respectively using each primer pair in the set of primer pairs described in the first aspect above to perform PCR amplification, and determining the mating type of the monokaryon of Lentinus edodes 0912 variety to be tested according to the amplification results as follows:

[0045] If the following condition P1 is met and condition P2 is not met and condition P3 is met and condition P4 is not met, then the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested is or is a candidate for the mating type A1B1;

[0046] If the following condition P1 is not satisfied and condition P2 is satisfied and condition P3 is not satisfied and condition P4 is satisfied, then the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested is or the candidate is the A2B2 mating type;

[0047] If the following condition P1 is met and condition P2 is not met and condition P3 is not met and condition P4 is met, then the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested is or the candidate is the A1B2 mating type;

[0048] If the following condition P1 is not satisfied, condition P2 is satisfied, condition P3 is satisfied, and condition P4 is not satisfied, then the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested is or is a candidate for the mating type A2B1;

[0049] P1, using the primer pair A1 to perform PCR amplification to obtain an amplified product containing a 1784±100 bp target fragment (e.g., a 1784 bp target fragment);

[0050] P2, using the primer pair A2 to perform PCR amplification to obtain an amplified product containing a 1433±100 bp target fragment (e.g., a 1433 bp target fragment);

[0051] P3 uses the primer pair B1 to perform PCR amplification to obtain an amplified product containing a 594±50 bp target fragment (e.g., a 594 bp target fragment);

[0052] P4. The primer pair B2 is used to perform PCR amplification to obtain an amplified product containing a target fragment of 819±50 bp (eg, a target fragment of 819 bp).

[0053] In an eighth aspect, the present invention claims protection for any of the following methods:

[0054] (f1) A method for identifying or assisting in identifying whether the mating type of a monokaryon of a Lentinus edodes 0912 variety to be tested is the A1B1 mating type, comprising the following steps: using the genome of the monokaryon of the Lentinus edodes 0912 variety to be tested as a template, respectively using each primer pair in the primer pair group 1 described in the second aspect above to perform PCR amplification, and determining whether the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested is the A1B1 mating type according to the amplification results as follows: if the amplification product obtained by PCR amplification using the primer pair A1 contains a target fragment of 1784±100 bp (such as a target fragment of 1784 bp), and the amplification product obtained by PCR amplification using the primer pair B1 contains a target fragment of 594±50 bp (such as a target fragment of 594 bp), then the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested is or is a candidate for being the A1B1 mating type; otherwise, the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested is not or is not a candidate for being the A1B1 mating type.

[0055] (f2) A method for identifying or assisting in identifying whether the mating type of a monokaryon of a Lentinus edodes 0912 variety to be tested is the A2B2 mating type, comprising the following steps: using the genome of the monokaryon of the Lentinus edodes 0912 variety to be tested as a template, respectively using each primer pair in the primer pair group 2 described in the second aspect above to perform PCR amplification, and determining whether the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested is the A2B2 mating type according to the amplification results as follows: if the amplification product obtained by PCR amplification using the primer pair A2 contains a target fragment of 1433±100 bp (such as a 1433 bp target fragment), and the amplification product obtained by PCR amplification using the primer pair B2 contains a target fragment of 819±50 bp (such as an 819 bp target fragment), then the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested is or has a candidate to be the A2B2 mating type; otherwise, the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested is not or has a candidate to be not the A2B2 mating type.

[0056] (f3) A method for identifying or assisting in identifying whether the mating type of a monokaryon of a Lentinus edodes 0912 variety to be tested is the A1B2 mating type, comprising the following steps: using the genome of the monokaryon of the Lentinus edodes 0912 variety to be tested as a template, respectively using each primer pair in the primer pair group 3 described in the second aspect above to perform PCR amplification, and determining whether the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested is the A1B2 mating type according to the amplification results as follows: if the amplification product obtained by PCR amplification using the primer pair A1 contains a target fragment of 1784±100 bp (such as a target fragment of 1784 bp), and the amplification product obtained by PCR amplification using the primer pair B2 contains a target fragment of 819±50 bp (such as a target fragment of 819 bp), then the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested is or has a candidate of being the A1B2 mating type; otherwise, the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested is not or has a candidate of not being the A1B2 mating type.

[0057] (f4) A method for identifying or assisting in identifying whether the mating type of a monokaryon of a Lentinus edodes 0912 variety to be tested is the A2B1 mating type, comprising the following steps: using the genome of the monokaryon of the Lentinus edodes 0912 variety to be tested as a template, and using each primer pair in the primer pair group 4 described in the second aspect above to perform PCR amplification, and determining whether the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested is the A2B1 mating type according to the amplification results as follows: if the amplification product obtained by PCR amplification using the primer pair A2 contains a target fragment of 1433±100 bp (such as a 1433 bp target fragment), and the amplification product obtained by PCR amplification using the primer pair B1 contains a target fragment of 594±50 bp (such as a 594 bp target fragment), then the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested is or has a candidate to be the A2B1 mating type; otherwise, the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested is not or has a candidate to be not the A2B1 mating type.

[0058] (f5) A method for identifying or assisting in identifying the mating type of a monokaryon of Lentinus edodes 0912 variety or a variety related thereto at the A mating type locus, comprising the following steps: using the genome of the monokaryon of the Lentinus edodes 0912 variety or a variety related thereto as a template, performing PCR amplification using each primer pair in the primer pair group 5 described in the second aspect above, and determining the mating type of the monokaryon of the Lentinus edodes 0912 variety or a variety related thereto at the A mating type locus according to the amplification result as follows: if the amplified product obtained by PCR amplification using the primer pair A1 contains a target fragment of 1784±100 bp (such as a target fragment of 1784 bp), and the PCR amplification using the primer pair A2 If the obtained amplification product does not contain the target fragment of 1433±100bp (such as the target fragment of 1433bp), then the mating type of the monokaryon of the tested Lentinus edodes 0912 variety or a variety related thereto at the A mating type site is or is a candidate for the A1 mating type; if the amplification product obtained by PCR amplification using the primer pair A1 does not contain the target fragment of 1784±100bp (such as the target fragment of 1784bp), and the amplification product obtained by PCR amplification using the primer pair A2 contains the target fragment of 1433±100bp (such as the target fragment of 1433bp), then the mating type of the monokaryon of the tested Lentinus edodes 0912 variety or a variety related thereto at the A mating type site is or is a candidate for the A2 mating type.

[0059] (f6) A method for identifying or assisting in identifying the mating type of a monokaryon of Lentinus edodes 0912 variety or a variety related thereto at a B mating type locus, comprising the following steps: using the genome of a monokaryon of a Lentinus edodes 0912 variety or a variety related thereto as a template, performing PCR amplification using each primer pair in the primer pair group 6 described in the second aspect above, and determining the mating type of the monokaryon of the Lentinus edodes 0912 variety or a variety related thereto at the B mating type locus according to the amplification result as follows: if the amplified product obtained by PCR amplification using the primer pair B1 contains a 594±50 bp target fragment (e.g., a 594 bp target fragment), and if the PCR amplification using the primer pair B2 contains a 594±50 bp target fragment (e.g., a 594 bp target fragment), and the PCR amplification using the primer pair B3 contains a 594±50 bp target fragment (e.g., a 594 bp target fragment), and the PCR amplification using the primer pair B4 contains a 594±50 bp target fragment (e.g., a 594 bp target fragment), and the PCR amplification using the primer pair B5 contains a 594±50 bp target fragment (e.g., a 594 bp target fragment), and the PCR amplification using the primer pair B6 contains a 594±50 bp target fragment (e.g., a 594 bp target fragment). If the amplification product obtained by PCR amplification with the primer pair B1 does not contain the target fragment of 819±50bp (such as the target fragment of 819bp), then the mating type of the monokaryon of the tested Lentinus edodes 0912 variety or a variety related thereto at the B mating type site is or is a candidate for the B1 mating type; if the amplification product obtained by PCR amplification with the primer pair B1 does not contain the target fragment of 594±50bp (such as the target fragment of 594bp), and the amplification product obtained by PCR amplification with the primer pair B2 contains the target fragment of 819±50bp (such as the target fragment of 819bp), then the mating type of the monokaryon of the tested Lentinus edodes 0912 variety or a variety related thereto at the B mating type site is or is a candidate for the B2 mating type.

[0060] (f7) A method for identifying or assisting in identifying whether the mating type of a monokaryon or heterokaryon of Lentinus edodes 0912 variety or a variety that is related thereto at an A mating type locus includes an A1 mating type, comprising the following steps: using the genome of the monokaryon or heterokaryon of the Lentinus edodes 0912 variety or a variety that is related thereto as a template, performing PCR amplification using the primer pair 1 described in the third aspect above, and determining the A mating type locus of the monokaryon or heterokaryon of the Lentinus edodes 0912 variety or a variety that is related thereto according to the amplification result as follows: Whether the mating type includes the A1 mating type: If the amplified product obtained by PCR amplification using the primer pair 1 contains a 1784±100bp target fragment (such as a 1784bp target fragment), the mating type of the monokaryon or heterokaryon of the tested Lentinus edodes 0912 variety or a variety related thereto at the A mating type site includes or is a candidate for including the A1 mating type; otherwise, the mating type of the monokaryon or heterokaryon of the tested Lentinus edodes 0912 variety or a variety related thereto at the A mating type site does not include or is a candidate for not including the A1 mating type.

[0061] (f8) A method for identifying or assisting in identifying whether the mating type of the monokaryon or heterokaryon of Lentinus edodes 0912 variety or a variety that is related thereto at the A mating type site includes the A2 mating type, comprising the following steps: using the genome of the monokaryon or heterokaryon of the Lentinus edodes 0912 variety or a variety that is related thereto as a template, performing PCR amplification using the primer pair 2 described in the third aspect above, and determining whether the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested at the A mating type site includes the A2 mating type according to the amplification result as follows: 2 Mating type: If the amplified product obtained by PCR amplification using the primer pair 2 contains a 1433±100bp target fragment (such as a 1433bp target fragment), the mating type of the monokaryon or heterokaryon of the Lentinus edodes 0912 variety to be tested or a variety related thereto at the A mating type site includes or may include the A2 mating type; otherwise, the mating type of the monokaryon or heterokaryon of the Lentinus edodes 0912 variety to be tested or a variety related thereto at the A mating type site does not include or may not include the A2 mating type.

[0062] (f9) A method for identifying or assisting in identifying whether the mating type of a monokaryon or heterokaryon of Lentinus edodes 0912 variety or a variety that is related thereto at a B mating type locus includes a B1 mating type, comprising the following steps: using the genome of the monokaryon or heterokaryon of the Lentinus edodes 0912 variety or a variety that is related thereto as a template, performing PCR amplification using the primer pair 3 described in the third aspect above, and determining the B mating type locus of the monokaryon or heterokaryon of the Lentinus edodes 0912 variety or a variety that is related thereto according to the amplification result as follows: Whether the mating type at the B mating type site includes the B1 mating type: if the amplification product obtained by PCR amplification using the primer pair 3 contains a 594±50bp target fragment (such as a 594bp target fragment), then the mating type of the monokaryon or heterokaryon of the Lentinus edodes 0912 variety to be tested or a variety that is related to it at the B mating type site includes or is a candidate for including the B1 mating type; otherwise, the mating type of the monokaryon or heterokaryon of the Lentinus edodes 0912 variety to be tested or a variety that is related to it at the B mating type site does not include or is a candidate for not including the B1 mating type.

[0063] (f10) A method for identifying or assisting in identifying whether the mating type of a monokaryon or heterokaryon of Lentinus edodes 0912 variety or a variety that is related thereto at a B mating type locus includes a B2 mating type, comprising the following steps: using the genome of the monokaryon or heterokaryon of the Lentinus edodes 0912 variety or a variety that is related thereto as a template, performing PCR amplification using the primer pair 4 described in the third aspect above, and determining the B mating type locus of the monokaryon or heterokaryon of the Lentinus edodes 0912 variety or a variety that is related thereto according to the amplification result as follows: Whether the mating type at the B mating type site includes the B2 mating type: if the amplification product obtained by PCR amplification using the primer pair 4 contains an 819±50bp target fragment (such as an 819bp target fragment), then the mating type of the monokaryon or heterokaryon of the Lentinus edodes 0912 variety to be tested or a variety that is related to it at the B mating type site includes or is a candidate for including the B2 mating type; otherwise, the mating type of the monokaryon or heterokaryon of the Lentinus edodes 0912 variety to be tested or a variety that is related to it at the B mating type site does not include or is a candidate for not including the B2 mating type.

[0064] (f11) A method for identifying or assisting in identifying whether two monokaryons of the Lentinus edodes 0912 variety to be tested can hybridize and produce a lock-like union, comprising the following steps: detecting the mating types of the two monokaryons of the Lentinus edodes 0912 variety to be tested respectively according to the method described in the seventh aspect above; if the mating types of the two monokaryons of the Lentinus edodes 0912 variety to be tested at the A mating type site and the mating types at the B mating type site are different, then the two monokaryons of the Lentinus edodes 0912 variety to be tested can hybridize and pair and produce a lock-like union; otherwise, the two monokaryons of the Lentinus edodes 0912 variety to be tested cannot hybridize and pair and produce a lock-like union.

[0065] That is, among the four mating types, A1B1 mating type, A2B2 mating type, A1B2 mating type and A2B1 mating type, only "A1B1 mating type and A2B2 mating type" and "A2B1 mating type and A1B2 mating type" can hybridize and produce a locked union.

[0066] (f12) A method for identifying or assisting in identifying the closeness of the relationship between the tested shiitake mushroom and the 0912 variety of shiitake mushroom, comprising the following steps: detecting the mating types contained in the tested shiitake mushroom and the 0912 variety of shiitake mushroom respectively according to the method described in the seventh aspect above, the higher the consistency of the mating types contained in the two, the closer the relationship between the two, and the lower the consistency of the mating types contained in the two, the more distant the relationship between the two.

[0067] If the mating type at the A mating type site and the mating type at the B mating type site are the same, the shiitake mushroom to be tested is most closely related to the shiitake 0912 variety; if the mating type at the A mating type site and the mating type at the B mating type site are different, the shiitake mushroom to be tested is the farthest related to the shiitake 0912 variety; if the mating type at the A mating type site and the mating type at the B mating type site are partially the same, the shiitake mushroom to be tested is moderately closely related to the shiitake 0912 variety.

[0068] In the above aspects, the mating types of the monokaryons of the shiitake 0912 variety include (or are divided into) four types: A1B1 mating type, A2B2 mating type, A1B2 mating type and A2B1 mating type. The mating types of the monokaryons of the shiitake 0912 variety at the A mating type site are divided into two types: A1 mating type and A2 mating type. The mating types of the monokaryons of the shiitake 0912 variety at the B mating type site are divided into two types: B1 mating type and B2 mating type.

[0069] The A1B1 mating type satisfies the following condition (g1):

[0070] (g1) Using the genome of the monokaryon of Lentinus edodes 0912 variety as a template, PCR amplification was performed using the primer pair A1 described in the first aspect of the previous text, and the resulting amplification product contained a 1784±100 bp target fragment (e.g., a 1784 bp target fragment), and PCR amplification was performed using the primer pair B1, and the resulting amplification product contained a 594±50 bp target fragment (e.g., a 594 bp target fragment).

[0071] Furthermore, the A1B1 mating type also satisfies the following condition (g2):

[0072] (g2) Using the genome of the monokaryon of Lentinus edodes 0912 variety as a template, the primer pair A2 described in the first aspect of the previous text is used to perform PCR amplification, and the amplification product obtained does not contain the 1433±100 bp target fragment (such as the 1433 bp target fragment), and the primer pair B2 is used to perform PCR amplification, and the amplification product does not contain the 819±50 bp target fragment (such as the 819 bp target fragment).

[0073] The A2B2 mating type satisfies the following condition (h1):

[0074] (h1) Using the genome of the monokaryon of Lentinus edodes 0912 variety as a template, PCR amplification was performed using the primer pair A2 described in the first aspect of the previous text, and the resulting amplification product contained a 1433±100 bp target fragment (e.g., a 1433 bp target fragment), and PCR amplification was performed using the primer pair B2, and the resulting amplification product contained a 819±50 bp target fragment (e.g., an 819 bp target fragment).

[0075] Furthermore, the A2B2 mating type also satisfies the following condition (h2):

[0076] (h2) Using the genome of the monokaryon of Lentinus edodes 0912 variety as a template, the amplification product obtained by PCR amplification using the primer pair A1 described in the first aspect of the previous text does not contain the 1784±100 bp target fragment (such as the 1784 bp target fragment), and the amplification product obtained by PCR amplification using the primer pair B1 does not contain the 594±50 bp target fragment (such as the 594 bp target fragment).

[0077] The A1B2 mating type satisfies the following condition (i1):

[0078] (i1) Using the genome of the monokaryon of Lentinus edodes 0912 variety as a template, PCR amplification was performed using the primer pair A1 described in the first aspect of the previous text, and the resulting amplification product contained a 1784±100 bp target fragment (e.g., a 1784 bp target fragment), and PCR amplification was performed using the primer pair B2, and the resulting amplification product contained a 819±50 bp target fragment (e.g., an 819 bp target fragment).

[0079] Furthermore, the A1B2 mating type also satisfies the following condition (i2):

[0080] (i2) Using the genome of the monokaryon of Lentinus edodes 0912 variety as a template, the primer pair A2 described in the first aspect of the previous text is used to perform PCR amplification, and the amplification product obtained does not contain the 1433±100 bp target fragment (such as the 1433 bp target fragment), and the primer pair B1 is used to perform PCR amplification, and the amplification product does not contain the 594±50 bp target fragment (such as the 594 bp target fragment).

[0081] The A2B1 mating type satisfies the following condition (j1):

[0082] (j1) Using the genome of the monokaryon of Lentinus edodes 0912 variety as a template, PCR amplification was performed using the primer pair A2 described in the first aspect of the previous text, and the resulting amplification product contained a 1433±100 bp target fragment (e.g., a 1433 bp target fragment), and PCR amplification was performed using the primer pair B1, and the resulting amplification product contained a 594±50 bp target fragment (e.g., a 594 bp target fragment).

[0083] Furthermore, the A2B1 mating type also satisfies the following condition (j2):

[0084] (j2) Using the genome of the monokaryon of Lentinus edodes 0912 variety as a template, the primer pair A1 described in the first aspect of the previous text is used to perform PCR amplification, and the amplification product obtained does not contain the 1784±100 bp target fragment (such as the 1784 bp target fragment), and the primer pair B2 is used to perform PCR amplification, and the amplification product does not contain the 819±50 bp target fragment (such as the 819 bp target fragment).

[0085] In the above aspects, the monokaryon or heterokaryon of the Lentinus edodes 0912 variety or a variety related thereto comprises an A1 mating type or an A2 mating type at the A mating type locus.

[0086] The A1 mating type satisfies the following condition (k1):

[0087] (k1) Using the genome of the monokaryon or heterokaryon of Lentinus edodes 0912 variety or a variety related thereto as a template, PCR amplification is performed using the primer pair A1 described in the first aspect above, and the resulting amplification product contains a 1784±100 bp target fragment (e.g., a 1784 bp target fragment).

[0088] Furthermore, when the genome of the monokaryon of Lentinus edodes 0912 variety or a variety related thereto is used as a template, the A1 mating type also satisfies the following condition (k2):

[0089] (k2) Using the genome of the monokaryon of Lentinus edodes 0912 variety or a variety related thereto as a template, PCR amplification is performed using the primer pair A2 described in the first aspect above, and the resulting amplification product does not contain the 1433±100 bp target fragment (such as the 1433 bp target fragment).

[0090] The A2 mating type satisfies the following condition (l1):

[0091] (l1) Using the genome of the monokaryon or heterokaryon of Lentinus edodes 0912 variety or a variety related thereto as a template, PCR amplification is performed using the primer pair A2 described in the first aspect above, and the resulting amplification product contains a 1433±100 bp target fragment (e.g., a 1433 bp target fragment).

[0092] Furthermore, when the genome of the monokaryon of Lentinus edodes 0912 variety or a variety related thereto is used as a template, the A2 mating type also satisfies the following condition (l2):

[0093] (l2) Using the genome of the monokaryon of Lentinus edodes 0912 variety or a variety related thereto as a template, PCR amplification is performed using the primer pair A1 described in the first aspect of the previous text, and the resulting amplification product does not contain the 1784±100 bp target fragment (such as the 1784 bp target fragment).

[0094] In the above aspects, the mating types of the monokaryon or heterokaryon of the Lentinus edodes 0912 variety or its related varieties at the B mating type locus are divided into two types: B1 mating type and B2 mating type.

[0095] The B1 mating type satisfies the following conditions (m1):

[0096] (m1) Using the genome of the monokaryon or heterokaryon of Lentinus edodes 0912 variety or a variety related thereto as a template, PCR amplification is performed using the primer pair B1 described in the first aspect above, and the resulting amplification product contains a 594±50 bp target fragment (e.g., a 594 bp target fragment).

[0097] Furthermore, when the genome of the monokaryon of Lentinus edodes 0912 variety or a variety related thereto is used as a template, the B1 mating type also satisfies the following condition (m2):

[0098] (m2) Using the genome of the monokaryon of Lentinus edodes 0912 variety or a variety related thereto as a template, PCR amplification is performed using the primer pair B2 described in the first aspect above, and the resulting amplification product does not contain the 819±50 bp target fragment (such as the 819 bp target fragment).

[0099] The B2 mating type satisfies the following condition (n1):

[0100] (n1) Using the genome of the monokaryon or heterokaryon of Lentinus edodes 0912 variety or a variety related thereto as a template, PCR amplification is performed using the primer pair B2 described in the first aspect of the previous text, and the resulting amplification product contains a target fragment of 819±50 bp (e.g., a target fragment of 819 bp).

[0101] Furthermore, when the genome of the monokaryon of Lentinus edodes 0912 variety or a variety related thereto is used as a template, the B2 mating type also satisfies the following condition (n2):

[0102] (n2) Using the genome of the monokaryon of Lentinus edodes 0912 variety or a variety related thereto as a template, PCR amplification is performed using the primer pair B1 described in the first aspect of the previous text, and the resulting amplification product does not contain the 594±50 bp target fragment (such as the 594 bp target fragment).

[0103] In the above aspects, the nucleotide sequence of the 1784bp target fragment is as shown in SEQ ID No.9 or has more than 80% identity with SEQ ID No.9; the nucleotide sequence of the 1433bp target fragment is as shown in SEQ ID No.10 or has more than 80% identity with SEQ ID No.10; the nucleotide sequence of the 594bp target fragment is as shown in SEQ ID No.11 or has more than 80% identity with SEQ ID No.11; the nucleotide sequence of the 819bp target fragment is as shown in SEQ ID No.12 or has more than 80% identity with SEQ ID No.12.

[0104] In the above aspects, the monokaryon of Lentinus edodes 0912 variety can be a protoplast monokaryon or a monospore strain. The mating type of the protoplast monokaryon is A1B1 mating type or A2B2 mating type. The mating type of the monospore strain is A1B1 mating type or A2B2 mating type or A1B2 mating type or A2B1 mating type.

[0105] In the ninth aspect, the present invention claims protection for the use of the set of primer pairs or the reagent or kit containing the set of primers described in the first aspect above, or the primer pair group or the reagent or kit containing the primer pair group described in the second aspect above, or the primer pair or the reagent or kit containing the primer pair described in the third aspect above, or the method described in the seventh aspect above or the eighth aspect above in shiitake mushroom breeding and / or strain identification.

[0106] The present invention utilizes sequence homology comparison to screen the variation sites on the A mating type site HD1 gene and the B mating type site RCB2 gene on a pair of protoplast monokaryons SP3 and SP30 of Lentinus edodes 0912, designs specific primers, and establishes a Lentinus edodes mating type identification method based on the variation sites of the HD1 and RCB2 genes. The method can be used for the identification of the monokaryon mating type of the 0912 variety and the identification of Lentinus edodes varieties that are genetically related to the 0912 variety. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] Figure 1 Design regions for the forward primer (A) and reverse primer (B) for the A2 mating type HD1 molecular marker.

[0108] Figure 2 Design regions for the forward primer (A) and reverse primer (B) for the A1 mating type HD1 molecular marker.

[0109] Figure 3 Design regions for the forward primer (A) and reverse primer (B) for the B2 mating type RCB2 molecular marker.

[0110] Figure 4Design regions for the forward primer (A) and reverse primer (B) for the B1 mating type RCB2 molecular marker.

[0111] Figure 5 A1-HD1-F / R primers and B1-RCB2-F / R primers were used to amplify 20 A1B1 mating type monosporic strains. In the figure, the arrows indicate the positions of the target bands.

[0112] Figure 6 The A1-HD1-F / R primer and the B1-RCB2-F / R primer were used to amplify 19 A2B2 mating type monosporic strains. In the figure, the arrows indicate the positions of the target bands.

[0113] Figure 7 The A2-HD1-F / R primer and the B2-RCB2-F / R primer were used to verify 19 A2B2 mating type monosporic strains. In the figure, the arrows indicate the positions of the target bands.

[0114] Figure 8 A2-HD1-F / R primers and B2-RCB2-F / R primers were used to amplify 20 A1B1 mating type monosporic strains. In the figure, the arrows indicate the positions of the target bands.

[0115] Fig. 9 To verify 20 A1B2 type monosporic strains using primers A1-HD1-F / R and B2-RCB2-F / R. In the figure, the arrows indicate the positions of the target bands.

[0116] Fig.10 A2-HD1-F / R primers and B1-RCB2-F / R primers were used to amplify 20 A1B2 type monosporic strains. In the figure, the arrows indicate the positions of the target bands.

[0117] Fig.11 To verify 20 A2B1 type monosporic strains using primers A2-HD1-F / R and B1-RCB2-F / R. In the figure, the arrows indicate the positions of the target bands.

[0118] Fig.12 A1-HD1-F / R primers and B2-RCB2-F / R primers were used to amplify 20 A2B1 type monosporic strains. In the figure, the arrows indicate the positions of the target bands.

[0119] Fig.13 A2-HD1-F / R primers and A1-HD1-F / R primers were used to analyze the mating type of different Lentinula edodes varieties.

[0120] Fig.14 This is the evolutionary tree of Lentinula edodes HD1 gene (nearest neighbor method). DETAILED DESCRIPTION

[0121] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0122] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0123] Example 1, mating type specific primer design

[0124] 1. Design of primers specific to A mating type locus

[0125] The protoplast monokaryonization of Lentinula edodes 0912 strain (heterokaryon) preserved by our team obtained a pair of protoplast monokaryons SP3 and SP30 that can mate and hybridize (Gao Q, et al. 2022. Haplotype-resolved genome analyses reveal genetically distinct nuclei within a commercial cultivar of Lentinula edodes. Journal of Fungi, 8(2): 167. Gao Q, et al. 2023. Insights into the Global Transcriptome Response of Lentinula edodes Mycelia during Aging. J. Fungi 2023, 9(3), 379; https: / / doi.org / 10.3390 / jof9030379). The mating type of monokaryon SP3 was defined as A2B2, and the mating type of monokaryon SP30 was defined as A1B1. The HD1 genes of the two strains SP3 and SP30 were sequenced and the mutation sites of the two HD1 genes were determined. According to the single nucleotide polymorphism (SNP) and insertion / deletion fragment InDel mutation sites, A2-type specific primers ( Figure 1 ) and A1-specific primers ( Figure 2 ).

[0126] A1-HD1-F: 5'-TCAGGTGCTTTGCGATACTTT-3' (SEQ ID No. 1);

[0127] A1-HD1-R: 5'-CGCCGTCGTCGTGTTGT-3' (SEQ ID No. 2);

[0128] A2-HD1-F: 5'-TCCTTAAGTAACTGGAAGCATC-3' (SEQ ID No. 3);

[0129] A2-HD1-R: 5'-TCCATTGGGTTCCCATAGTC-3' (SEQ ID No. 4).

[0130] Among them, the A1-HD1-F / R primers can only PCR amplify the correct band (1784bp, the nucleotide sequence is shown in SEQ ID No.9) in the monokaryon (A1 type) with the same mating type as the SP30 strain A, and there is no correct size band in the A2 type homokaryon. The A2-HD1-F / R primers can only PCR amplify the correct band (1433bp, the nucleotide sequence is shown in SEQ ID No.10) in the monokaryon (A2 type) with the same mating type as the SP3 strain A, and there is no correct size band in the A1 type homokaryon.

[0131] 2. Design of primers specific for B mating type locus.

[0132] Sequence comparison of the RCB2 genes of the two strains SP3 and SP30 was performed to determine the mutation sites of the two RCB2 genes. Based on the single nucleotide polymorphism (SNP) and insertion / deletion fragment InDel mutation sites, B2-type specific primers ( Figure 3 ) and B1-specific primers ( Figure 4 ).

[0133] B1-RCB2-F:5'-TATCGTTCAAGGACATCGCT-3' (SEQ ID No. 5);

[0134] B1-RCB2-R:5'-TAATGCCTGCAAAACGAAGA-3' (SEQ ID No. 6);

[0135] B2-RCB2-F:5'-TTTGAAGACTTGGGTTGC-3' (SEQ ID No. 7);

[0136] B2-RCB2-R: 5'-AGGAGACATCTGAAGAGGAT-3' (SEQ ID No. 8).

[0137] Among them, the B1-RCB2-F / R primers can only PCR amplify the correct band (594bp, the nucleotide sequence is shown in SEQ ID No.11) in the monokaryon (B1 type) with the same mating type as the SP30 strain B, and there is no correct size band in the B2 type monokaryon; the B2-RCB2-F / R primers can only PCR amplify the correct band (819bp, the nucleotide sequence is shown in SEQ ID No.12) in the monokaryon (B2 type) with the same mating type as the SP3 strain B, and there is no correct size band in the B1 type monokaryon.

[0138] Example 2: Application example of monokaryon haplotype identification

[0139] 1. Single spore separation and hybrid pairing to identify mating type

[0140] Single spores were separated from the heterokaryon strains of Lentinus edodes 0912, and strains without lock-like union were screened by microscopic examination to obtain single spore strains. The single spore strains were paired and hybridized with SP3 and SP30 respectively, and microscopic examination was used to confirm whether heterokaryon strains with lock-like union could be formed. Since only when the mating types of the A mating type site and the B mating type site are different between Lentinus edodes monokaryota can hybridization produce lock-like union, nuclear migration occur, and fruiting bodies form, therefore: if the single spore strain can hybridize with SP3 (mating type is A2B2 type), the mating type of the single spore strain is A1B1 type; if the single spore strain can hybridize with SP30 (mating type is A1B1 type), the mating type of the single spore strain is A2B2 type.

[0141] Twenty strains (strain numbers: 611, 138, 337, 324, 449, 627, 303, 102, 605, 689, 178, 240, 468, 529, 633, 228, 462, 248, 634, and 506) were randomly selected from the A1B1 type monosporic strains obtained by hybridization verification with the monokaryon SP3, and 19 strains (strain numbers: 15, 25, 40, 91, 327, 128, 129, 159, 219, 232, 143, 214, 249, 297, 18, 135, 311, 288, and 312) were randomly selected from the A2B2 type monosporic strains verified by hybridization with the monokaryon SP30, totaling 39 monosporic strains for subsequent PCR amplification analysis.

[0142] In addition, mature fruiting bodies of Lentinus edodes can produce basidiospores of four mating types, which germinate into monokaryon hyphae, and the mating types are A1B1, A2B2, A1B2, and A2B1. Only monokaryons with different mating type genes in A and B can hybridize to produce lock-like union, undergo nuclear migration, and form fruiting bodies. Among the monospore strains without lock-like union obtained in the early monospore separation, in addition to A1B1 and A2B2 strains that can hybridize with SP3 or SP30, there are also some A1B2 and A2B1 strains that cannot hybridize with either SP3 or SP30, so A1B2 and A2B1 strains were screened next. The monospore strains that cannot hybridize with SP3 and SP30 were selected, and 10 strains were randomly selected for pairwise hybridization, from which a pair of hybridizable strains 120 and strain 369 were obtained. Through subsequent PCR detection, strain 120 can amplify the correct target band using the A2-HD1-F / R primer pair and the B1-RCB2-F / R primer pair, and the mating type is identified as A2B1; strain 369 can amplify the correct target band using the A1-HD1-F / R primer pair and the B2-RCB2-F / R primer pair, and the mating type is identified as A1B2.

[0143] The monosporic strains that could not hybridize with SP3 and SP30 were hybridized with strain 120 and strain 369 respectively. The mating type of the monosporic strain that could hybridize with strain 120 (mating type was A2B1) was A1B2, and the mating type of the monosporic strain that could hybridize with strain 369 (mating type was A1B2) was A2B1.

[0144] Twenty strains (strain numbers: 369, 29, 37, 47, 69, 76, 101, 103, 108, 223, 225, 231, 237, 241, 242, 246, 250, 460, 465, and 608) were randomly selected from the A1B2 type monosporic strains obtained by hybridization verification with monokaryon 120, and 20 strains (strain numbers: 120, 65, 161, 168, 172, 177, 191, 197, 198, 316, 358, 360, 363, 367, 374, 379, 381, 404, 408, and 683) were randomly selected from the A2B1 type monosporic strains verified by hybridization with monokaryon 369, a total of 40 monosporic strains were used for subsequent PCR amplification analysis.

[0145] 2. Identification of the mating type of monosporic strains using specific primers

[0146] The DNA of 20 A1B1 type, 19 A2B2 type, 20 A1B2 type and 20 A2B1 type monospore strains were used as sample templates for amplification respectively:

[0147] The reaction system was: 10 μL of r-Taq enzyme; 1 μL of upstream primer; 1 μL of downstream primer; 2 μL of template; and 6 μL of ddH2O.

[0148] PCR conditions for amplification of A1-HD1 and A2-HD1: 95°C for 5 min; 95°C for 30 s, 50°C for 30 s, 72°C for 2 min, 40 cycles; 72°C for 10 min; 4°C∞.

[0149] PCR conditions for amplification of B1-RCB2 and B2-RCB2: 95°C for 5 min; 95°C for 30 s, 50°C for 30 s, 72°C for 50 s, 35 cycles; 72°C for 5 min; 4°C∞.

[0150] Agarose gel electrophoresis: (1) Prepare 1% agarose gel. (2) Spotting: Spot 1KBPlus Marker into the first gel well, then spot the PCR amplification product into other gel wells in turn, spotting 6μL into each gel well. (3) Run electrophoresis: Set the conditions to 130V for 25min. (4) Place the run gel under the instrument for analysis and observation. (To prevent contamination, wear PE gloves when operating)

[0151] (1) Identification of A1B1 monosporic strains using specific primers

[0152] When the 20 A1B1 monospore strains were amplified using primers A1-HD1-F / R and primers B1-RCB2-F / R (sequences see above), both primers were able to amplify bands of the correct size, which was again verified to be the A1B1 mating type at the genetic level ( Figure 5 ). However, when amplifying monospore strains of other mating types, such as the 19 selected A2B2 monospore strains, both sets of primers did not have the correct size band, indicating that the mating type was not A1B1 ( Figure 6 ).

[0153] (2) Identification of A2B2 monosporic strains using specific primers

[0154] When the 19 A2B2-type monospore strains were amplified using primers A2-HD1-F / R and B2-RCB2-F / R, both primers were able to amplify bands of the correct size, which was again verified to be the A2B2 mating type at the genetic level ( Figure 7 ). However, when amplifying monospore strains of other mating types, such as the 20 selected A1B1 monospore strains, both sets of primers did not have the correct size band, indicating that the mating type was not A2B2 ( Figure 8 ).

[0155] (3) Identification of A1B2 monosporic strains using specific primers

[0156] When the 20 selected A1B2 monospore strains were amplified, the A1-HD1-F / R primers and B2-RCB2-F / R primers could amplify the correct bands ( Fig. 9 ), A2-HD1-F / R primers and B1-RCB2-F / R primers have no correct bands ( Fig.10 ), indicating that this type of strain has A1 type A mating type locus and B2 type B mating type locus, but does not have A2 type A mating type locus and B1 type B mating type locus. This result is consistent with the mating type identified by hybridization pairing.

[0157] (4) Identification of A2B1 monospore strains using specific primers

[0158] When the 20 selected A2B1 monosporic strains were amplified, the A2-HD1-F / R primers and the B1-RCB2-F / R primers could amplify the correct bands ( Fig.11 ), A1-HD1-F / R primers and B2-HD1-F / R primers have no correct bands ( Fig.12 ), indicating that this type of strain has A2 type A mating type locus and B1 type B mating type locus, but does not have A1 type A mating type locus and B2 type B mating type locus. This result is consistent with the mating type identified by hybridization pairing.

[0159] The above results show that primers A1-HD1-F / R and A2-HD1-F / R designed based on the SNP and InDel mutation sites of the HD1 gene at the A mating type locus, and primers B1-RCB2-F / R and B2-RCB2-F / R designed based on the SNP and InDel mutation sites of the RCB2 gene at the B mating type locus, can be used as molecular markers for different mating type monokaryons (including protoplast monokaryonized strains and monospore isolated strains, etc.) of Lentinula edodes 0912, and accurately distinguish the four mating types. The mating type identification method has accurate detection results, and at the same time saves the time of hybridization and mating mycelial growth, which can realize rapid mating type identification.

[0160] Example 3: Identification of mating type relationships of Lentinus edodes varieties

[0161] The information of the Lentinus edodes strains involved in this example is shown in Table 1.

[0162] Table 1. Information on Lentinula edodes strains in this example

[0163]

[0164]

[0165] A2-HD1-F / R primers and A1-HD1-F / R primers were used to perform PCR amplification on different varieties of Lentinus edodes (for specific methods, see the relevant steps in Example 2). The hybrid strain 0912SP3×SP30 obtained by the Lentinus edodes 0912 variety collected from Fuping, Hebei, Fengning, Hebei, Shilou, Fangshan, Beijing, etc. and SP3×SP30 had two HD1 genotypes, A2 and A1 ( Fig.13 ). Two L808 strains: 808SP45×SP29 and 808 Guopin JZB2102198 only have the A1 type HD1 genotype consistent with SP30, and do not contain the A2 type HD1 genotype, indicating that the A1 mating type HD1 genes of 0912 and L808 have similar parental sources. This is consistent with the results of phylogenetic tree analysis that the HD1 gene of SP30 is highly homologous to L808 ( Fig.14 ). Similarly, the results showed that the T2 variety of shiitake mushrooms, which are widely cultivated in northern China, have two mating type HD1 genes, A1 and A2. Therefore, A2-HD1-F / R and A1-HD1-F / R can be used to identify the relationship between other shiitake mushroom varieties and the A mating type of 0912 variety.

[0166] PCR amplification was performed on different varieties of Lentinus edodes using primers B2-RCB2-F / R and primers B1-RCB2-F / R (for specific methods, see the relevant steps in Example 2). The hybrid strain 0912SP3×SP30 obtained by Lentinus edodes 0912 collected from Fuping, Hebei, Fengning, Hebei, Shilou, Fangshan, Beijing, etc. and SP3×SP30 had two RCB2 genotypes, B2 and B1 ( Fig.13 ). Similarly, the results showed that the T2 variety of Lentinus edodes, which is widely cultivated in the northern region, has two mating type RCB2 genes, B1 and B2. The two L808 strains (808SP45×SP29 and 808 Guopin JZB2102198) only have the B1 type RCB2 genotype consistent with SP30, and do not have the B2 type RCB2 genotype, indicating that the B1 mating type RCB2 genes of 0912 and L808 have similar parental sources. In addition, 808SP45×0912SP3 is a heterokaryon strain obtained by hybridizing the protoplast monokaryotic strain SP45 of Lentinus edodes 808 variety with 0912SP3. Because SP3 has the B2 type RCB2 gene, the correct band can be amplified using the B2-RCB2-F / R primers, but the band cannot be amplified using the B1-RCB2-F / R primers, indicating that 808SP45 does not have the B1 type RCB2 gene. Based on the experimental results of 808SP45×SP29 and 808SP45×0912SP3, it can be shown that the B mating type locus of the protoplast mononuclear strain SP29 of Lentinula edodes 808 is B1 type RCB2. Therefore, primers B2-RCB2-F / R and B1-RCB2-F / R can be used to identify the relationship between other Lentinula edodes varieties and the B mating type of 0912 variety.

[0167] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A set of primer pairs or a reagent or a kit containing the set of primer pairs, characterized in that: The set of primer pairs consists of primer pair A1, primer pair A2, primer pair B1 and primer pair B2; The primer pair A1 consists of two single-stranded DNAs shown in SEQ ID No.1 and SEQ ID No.2; The primer pair A2 consists of two single-stranded DNAs shown in SEQ ID No.3 and SEQ ID No.4; The primer pair B1 consists of two single-stranded DNAs shown in SEQ ID No.5 and SEQ ID No.6; The primer pair B2 consists of two single-stranded DNAs shown in SEQ ID No.7 and SEQ ID No.

8.

2. A primer pair set or a reagent or kit containing the primer pair set, characterized in that: The primer pair group is any one of the following: (a1) Primer pair set 1, consisting of primer pair A1 described in claim 1 and primer pair B1 described in claim 1; (a2) Primer pair set 2, consisting of primer pair A2 described in claim 1 and primer pair B2 described in claim 1; (a3) Primer pair set 3, consisting of primer pair A1 described in claim 1 and primer pair B2 described in claim 1; (a4) Primer pair set 4, consisting of primer pair A2 described in claim 1 and primer pair B1 described in claim 1; (a5) Primer pair set 5, consisting of primer pair A1 described in claim 1 and primer pair A2 described in claim 1; (a6) Primer pair set 6, consisting of the primer pair B1 described in claim 1 and the primer pair B2 described in claim 1.

3. A primer pair or a reagent or a kit containing the primer pair, characterized in that: The primer pair is any of the following: (b1) Primer pair 1, which is the primer pair A1 in claim 1; (b2) Primer pair 2, which is the primer pair A2 in claim 1; (b3) Primer pair 3, which is the primer pair B1 in claim 1; (b4) Primer pair 4 is the primer pair B2 in claim 1.

4. Use of the primer set according to claim 1 or a reagent or kit containing the primer set in any of the following: (c1) Identify or assist in identifying the mating type of monokaryons of Lentinula edodes cultivar 0912; (c2) Identify or assist in identifying whether two monokaryons of the tested Lentinula edodes 0912 variety can hybridize and pair to produce a lock-like union; (c3) Identify or assist in identifying the relationship between the strain to be tested and Lentinus edodes 0912 variety.

5. Use of the primer pair set according to claim 2 or a reagent or kit containing the primer pair set in any of the following: (d1) Use of the primer pair group 1 described in claim 2 in identifying or assisting in identifying whether the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested is the A1B1 mating type; (d2) Use of the primer pair group 2 described in claim 2 in identifying or assisting in identifying whether the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested is the A2B2 mating type; (d3) Use of the primer pair group 3 described in claim 2 in identifying or assisting in identifying whether the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested is the A1B2 mating type; (d4) Use of the primer pair group 4 described in claim 2 in identifying or assisting in identifying whether the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested is the A2B1 mating type; (d5) Use of the primer pair group 5 described in claim 2 in identifying or assisting in identifying the mating type of Lentinus edodes 0912 variety or strains of varieties related thereto at the A mating type locus; (d6) Use of primer pair group 6 described in claim 2 in identifying or assisting in identifying the mating type of Lentinus edodes 0912 variety or strains of varieties related thereto at the B mating type locus.

6. Use of the primer pair according to claim 3 or a reagent or kit containing the primer pair in any of the following: (e1) Use of the primer pair 1 described in claim 3 in identifying or assisting in identifying whether the mating type of the strain of Lentinus edodes 0912 variety or a variety related thereto at the A mating type locus is or contains the A1 mating type; (e2) Use of the primer pair 2 described in claim 3 in identifying or assisting in identifying whether the mating type of the strain of Lentinus edodes 0912 variety or a variety related thereto at the A mating type locus is or contains the A2 mating type; (e3) Use of the primer pair 3 described in claim 3 in identifying or assisting in identifying whether the mating type of the strain of Lentinus edodes 0912 variety or a variety related thereto at the B mating type locus is or contains the B1 mating type; (e4) Use of the primer pair 4 described in claim 3 in identifying or assisting in identifying whether the mating type of the strain of the Lentinus edodes 0912 variety or a variety related thereto at the B mating type locus is or contains the B2 mating type.

7. A method for identifying or assisting in identifying the mating type of a monokaryon of Lentinus edodes 0912 variety, comprising the following steps: using the genome of the monokaryon of Lentinus edodes 0912 variety to be tested as a template, respectively using each primer pair in the set of primer pairs of claim 1 to perform PCR amplification, and determining the mating type of the monokaryon of Lentinus edodes 0912 variety to be tested according to the amplification results as follows: If the following condition P1 is met and condition P2 is not met and condition P3 is met and condition P4 is not met, then the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested is or is a candidate for the mating type A1B1; If the following condition P1 is not satisfied and condition P2 is satisfied and condition P3 is not satisfied and condition P4 is satisfied, then the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested is or the candidate is the A2B2 mating type; If the following condition P1 is met and condition P2 is not met and condition P3 is not met and condition P4 is met, then the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested is or is a candidate for the mating type A1B2; If the following condition P1 is not satisfied, condition P2 is satisfied, condition P3 is satisfied, and condition P4 is not satisfied, then the mating type of the monokaryon of the Lentinus edodes 0912 variety to be tested is or is a candidate for the mating type A2B1; P1, using the primers to perform PCR amplification on A1, the amplified product obtained contains a 1784 bp target fragment; P2, using the primers to perform PCR amplification on A2, the resulting amplification product contains a 1433 bp target fragment; P3, using the primers to perform PCR amplification on B1, the amplified product obtained contains a 594 bp target fragment; P4. The amplified product obtained by PCR amplification of B2 using the primer pair contains a target fragment of 819 bp.

8. Either of the following methods: (f1) A method for identifying or assisting in identifying whether the mating type of a monokaryon of Lentinus edodes 0912 variety is the A1B1 mating type, comprising the following steps: using the genome of the monokaryon of Lentinus edodes 0912 variety to be tested as a template, respectively using each primer pair in the primer pair group 1 of claim 2 to perform PCR amplification, and determining whether the mating type of the monokaryon of Lentinus edodes 0912 variety to be tested is the A1B1 mating type according to the amplification results as follows: if the amplification product obtained by PCR amplification using the primer pair A1 contains a 1784 bp target fragment, and the amplification product obtained by PCR amplification using the primer pair B1 contains a 594 bp target fragment, then the mating type of the monokaryon of Lentinus edodes 0912 variety to be tested is or is a candidate for being the A1B1 mating type; otherwise, the mating type of the monokaryon of Lentinus edodes 0912 variety to be tested is not or is not a candidate for being the A1B1 mating type; (f2) A method for identifying or assisting in identifying whether the mating type of a monokaryon of Lentinus edodes 0912 variety is the A2B2 mating type, comprising the following steps: using the genome of the monokaryon of Lentinus edodes 0912 variety to be tested as a template, respectively using each primer pair in the primer pair group 2 described in claim 2 to perform PCR amplification, and determining whether the mating type of the monokaryon of Lentinus edodes 0912 variety to be tested is the A2B2 mating type according to the amplification results as follows: if the amplification product obtained by PCR amplification using the primer pair A2 contains a 1433 bp target fragment, and the amplification product obtained by PCR amplification using the primer pair B2 contains an 819 bp target fragment, then the mating type of the monokaryon of Lentinus edodes 0912 variety to be tested is or is a candidate for being the A2B2 mating type; otherwise, the mating type of the monokaryon of Lentinus edodes 0912 variety to be tested is not or is not a candidate for being the A2B2 mating type; (f3) A method for identifying or assisting in identifying whether the mating type of a monokaryon of Lentinus edodes 0912 variety is the A1B2 mating type, comprising the following steps: using the genome of the monokaryon of Lentinus edodes 0912 variety to be tested as a template, respectively using each primer pair in the primer pair group 3 described in claim 2 to perform PCR amplification, and determining whether the mating type of the monokaryon of Lentinus edodes 0912 variety to be tested is the A1B2 mating type according to the amplification results as follows: if the amplification product obtained by PCR amplification using the primer pair A1 contains a 1784 bp target fragment, and the amplification product obtained by PCR amplification using the primer pair B2 contains an 819 bp target fragment, then the mating type of the monokaryon of Lentinus edodes 0912 variety to be tested is or is a candidate for being the A1B2 mating type; otherwise, the mating type of the monokaryon of Lentinus edodes 0912 variety to be tested is not or is not a candidate for being the A1B2 mating type; (f4) A method for identifying or assisting in identifying whether the mating type of a monokaryon of Lentinus edodes 0912 variety is the A2B1 mating type, comprising the following steps: using the genome of the monokaryon of Lentinus edodes 0912 variety to be tested as a template, respectively using each primer pair in the primer pair group 4 of claim 2 to perform PCR amplification, and determining whether the mating type of the monokaryon of Lentinus edodes 0912 variety to be tested is the A2B1 mating type according to the amplification results as follows: if the amplification product obtained by PCR amplification using the primer pair A2 contains a 1433 bp target fragment, and the amplification product obtained by PCR amplification using the primer pair B1 contains a 594 bp target fragment, then the mating type of the monokaryon of Lentinus edodes 0912 variety to be tested is or is a candidate for being the A2B1 mating type; otherwise, the mating type of the monokaryon of Lentinus edodes 0912 variety to be tested is not or is not a candidate for being the A2B1 mating type; (f5) A method for identifying or assisting in identifying the mating type of the monokaryon of Lentinus edodes 0912 variety or a variety related thereto at the A mating type locus, comprising the following steps: using the genome of the monokaryon of the Lentinus edodes 0912 variety or a variety related thereto as a template, performing PCR amplification using each primer pair in the primer pair group 5 of claim 2, and determining the mating type of the monokaryon of the Lentinus edodes 0912 variety or a variety related thereto at the A mating type locus according to the amplification result as follows: if the amplified product obtained by PCR amplification using the primer pair A1 contains a 1784 bp target fragment, and If the amplified product obtained by PCR amplification using the primer pair A2 does not contain the 1433 bp target fragment, then the mating type of the monokaryon of the tested Lentinus edodes 0912 variety or a variety related thereto at the A mating type site is or is a candidate for the A1 mating type; if the amplified product obtained by PCR amplification using the primer pair A1 does not contain the 1784 bp target fragment, and the amplified product obtained by PCR amplification using the primer pair A2 contains the 1433 bp target fragment, then the mating type of the monokaryon of the tested Lentinus edodes 0912 variety or a variety related thereto at the A mating type site is or is a candidate for the A2 mating type; (f6) A method for identifying or assisting in identifying the mating type of the monokaryon of Lentinus edodes 0912 variety or a variety related thereto at the B mating type locus, comprising the following steps: using the genome of the monokaryon of the Lentinus edodes 0912 variety or a variety related thereto as a template, performing PCR amplification using each primer pair in the primer pair group 6 of claim 2, and determining the mating type of the monokaryon of the Lentinus edodes 0912 variety or a variety related thereto at the B mating type locus according to the amplification result as follows: if the amplified product obtained by PCR amplification using the primer pair B1 contains a 594 bp target fragment, If the amplification product obtained by PCR amplification using the primer pair B2 does not contain the 819 bp target fragment, then the mating type of the monokaryon of the tested Lentinus edodes 0912 variety or a variety related thereto at the B mating type site is or is a candidate for the B1 mating type; if the amplification product obtained by PCR amplification using the primer pair B1 does not contain the 594 bp target fragment, and the amplification product obtained by PCR amplification using the primer pair B2 contains the 819 bp target fragment, then the mating type of the monokaryon of the tested Lentinus edodes 0912 variety or a variety related thereto at the B mating type site is or is a candidate for the B2 mating type; (f7) A method for identifying or assisting in identifying whether the mating type of a monokaryon or heterokaryon of Lentinus edodes 0912 variety or a variety that is related to it contains an A1 mating type at the A mating type locus, comprising the following steps: using the genome of the monokaryon or heterokaryon of the Lentinus edodes 0912 variety or a variety that is related to it as a template, performing PCR amplification using the primer pair 1 described in claim 3, and determining the monokaryon or heterokaryon of the Lentinus edodes 0912 variety or a variety that is related to it according to the amplification result as follows: Whether the mating type of the monokaryon at the A mating type site includes the A1 mating type: if the amplified product obtained by PCR amplification using the primer pair 1 contains a 1784 bp target fragment, the monokaryon or heterokaryon of the tested Lentinus edodes 0912 variety or a variety with a kinship relationship therewith includes or is a candidate to include the A1 mating type at the A mating type site; otherwise, the monokaryon or heterokaryon of the tested Lentinus edodes 0912 variety or a variety with a kinship relationship therewith does not include or is a candidate to include the A1 mating type at the A mating type site; (f8) A method for identifying or assisting in identifying whether the monokaryon or heterokaryon of Lentinus edodes 0912 variety or a variety that is related to it contains the A2 mating type at the A mating type locus, comprising the following steps: using the genome of the monokaryon or heterokaryon of the Lentinus edodes 0912 variety or a variety that is related to it as a template, performing PCR amplification using the primer pair 2 described in claim 3, and determining the monokaryon or heterokaryon of the Lentinus edodes 0912 variety or a variety that is related to it according to the amplification result as follows: Whether the mating type of the monokaryon at the A mating type site includes the A2 mating type: if the amplified product obtained by PCR amplification using the primer pair 2 contains a 1433 bp target fragment, the monokaryon or heterokaryon of the Lentinus edodes 0912 variety to be tested or a variety with a kinship relationship therewith includes or is a candidate to include the A2 mating type at the A mating type site; otherwise, the monokaryon or heterokaryon of the Lentinus edodes 0912 variety to be tested or a variety with a kinship relationship therewith does not include or is a candidate to not include the A2 mating type at the A mating type site; (f9) A method for identifying or assisting in identifying whether the mating type of the monokaryon or heterokaryon of the tested Lentinus edodes 0912 variety or a variety that is related thereto at the B mating type locus includes the B1 mating type, comprising the following steps: using the genome of the monokaryon or heterokaryon of the tested Lentinus edodes 0912 variety or a variety that is related thereto as a template, performing PCR amplification using the primer pair 3 described in claim 3, and determining the mating type of the tested Lentinus edodes 0912 variety or a variety that is related thereto according to the amplification result as follows: Whether the monokaryon or heterokaryon contains the B1 mating type at the B mating type site: if the amplified product obtained by PCR amplification using the primer pair 3 contains a 594 bp target fragment, the monokaryon or heterokaryon of the Lentinus edodes 0912 variety to be tested or a variety with a genetic relationship therewith contains or is a candidate to contain the B1 mating type at the B mating type site; otherwise, the monokaryon or heterokaryon of the Lentinus edodes 0912 variety to be tested or a variety with a genetic relationship therewith does not contain or is a candidate to not contain the B1 mating type at the B mating type site; (f10) A method for identifying or assisting in identifying whether a monokaryon or heterokaryon of Lentinus edodes 0912 variety or a variety that is related thereto contains a B2 mating type at the B mating type locus, comprising the following steps: using the genome of the monokaryon or heterokaryon of the Lentinus edodes 0912 variety or a variety that is related thereto as a template, performing PCR amplification using the primer pair 4 described in claim 3, and determining the monokaryon or heterokaryon of the Lentinus edodes 0912 variety or a variety that is related thereto according to the amplification result as follows: Whether the mating type of the karyon at the B mating type site includes the B2 mating type: if the amplified product obtained by PCR amplification using the primer pair 4 contains the 819 bp target fragment, then the monokaryon or heterokaryon of the tested Lentinus edodes 0912 variety or a variety with a kinship relationship therewith includes or is a candidate to include the B2 mating type at the B mating type site; otherwise, the monokaryon or heterokaryon of the tested Lentinus edodes 0912 variety or a variety with a kinship relationship therewith does not include or is a candidate to not include the B2 mating type at the B mating type site; (f11) A method for identifying or assisting in identifying whether two monokaryons of the tested shiitake 0912 variety can hybridize, pair, and produce a lock-like union, comprising the following steps: detecting the mating types of the two monokaryons of the tested shiitake 0912 variety respectively according to the method of claim 7; if the mating types of the two monokaryons of the tested shiitake 0912 variety at the mating type locus A and the mating types at the mating type locus B are different, then the two monokaryons of the tested shiitake 0912 variety can hybridize, pair, and produce a lock-like union; otherwise, the two monokaryons of the tested shiitake 0912 variety cannot hybridize, pair, and produce a lock-like union; (f12) A method for identifying or assisting in identifying the closeness of the relationship between the tested shiitake mushroom and the shiitake 0912 variety, comprising the following steps: detecting the mating types contained in the tested shiitake mushroom and the shiitake 0912 variety respectively according to the method described in claim 7, the higher the consistency of the mating types contained in the two, the closer the relationship between the two, and the lower the consistency of the mating types contained in the two, the more distant the relationship between the two.

9. The method according to claim 7 or 8, characterized in that: The mating types of the monokaryons of the Lentinus edodes 0912 variety include four types: A1B1 mating type, A2B2 mating type, A1B2 mating type and A2B1 mating type; The A1B1 mating type satisfies the following condition (g1): (g1) using the genome of the monokaryon of Lentinus edodes 0912 variety as a template, the primer pair A1 described in claim 1 is used to perform PCR amplification, and the amplified product obtained by PCR amplification contains a 1784 bp target fragment, and the primer pair B1 is used to perform PCR amplification, and the amplified product obtained by PCR amplification contains a 594 bp target fragment; The A2B2 mating type satisfies the following condition (h1): (h1) Using the genome of the monokaryon of Lentinus edodes 0912 variety as a template, the primer pair A2 described in claim 1 is used to perform PCR amplification, and the amplified product obtained by PCR amplification contains a 1433 bp target fragment, and the primer pair B2 is used to perform PCR amplification, and the amplified product obtained by PCR amplification contains an 819 bp target fragment; The A1B2 mating type satisfies the following condition (i1): (i1) using the genome of the monokaryon of Lentinus edodes 0912 variety as a template, the primer pair A1 described in claim 1 is used to perform PCR amplification, and the amplified product obtained by PCR amplification contains a 1784 bp target fragment, and the primer pair B2 is used to perform PCR amplification, and the amplified product obtained by PCR amplification contains an 819 bp target fragment; The A2B1 mating type satisfies the following condition (j1): (j1) Using the genome of the monokaryon of Lentinus edodes 0912 variety as a template, the primer pair A2 described in claim 1 is used to perform PCR amplification, and the amplified product obtained by PCR amplification contains a 1433 bp target fragment, and the primer pair B1 is used to perform PCR amplification, and the amplified product obtained by PCR amplification contains a 594 bp target fragment; and / or The monokaryon or heterokaryon of the Lentinus edodes 0912 variety or a variety related thereto comprises an A1 mating type or an A2 mating type at the A mating type locus; The A1 mating type satisfies the following condition (k1): (k1) using the genome of the monokaryon or heterokaryon of Lentinus edodes 0912 variety or a variety related thereto as a template, PCR amplification was performed using the primer pair A1 described in claim 1, and the resulting amplification product contained a 1784 bp target fragment; The A2 mating type satisfies the following condition (l1): (l1) Using the genome of the monokaryon or heterokaryon of Lentinus edodes 0912 variety or a variety related thereto as a template, PCR amplification was performed using the primer pair A2 described in claim 1, and the resulting amplification product contained a 1433 bp target fragment; and / or The monokaryon or heterokaryon of the Lentinus edodes 0912 variety or a variety related thereto comprises a B1 mating type or a B2 mating type at the B mating type locus; The B1 mating type satisfies the following conditions (m1): (m1) using the genome of the monokaryon or heterokaryon of Lentinus edodes 0912 variety or a variety related thereto as a template, PCR amplification of B1 using the primer pair described in claim 1 to obtain an amplified product containing a 594 bp target fragment; The B2 mating type satisfies the following conditions (n1): (n1) Using the genome of the monokaryon or heterokaryon of Lentinus edodes 0912 variety or a variety related thereto as a template, PCR amplification is performed using the primer pair B2 described in claim 1, and the resulting amplified product contains a target fragment of 819 bp.

10. The method according to claim 9, characterized in that: The A1B1 mating type also satisfies the following condition (g2): (g2) Using the genome of the monokaryon of Lentinus edodes 0912 variety as a template, the amplified product obtained by PCR amplification using the primer pair A2 in claim 1 does not contain the 1433 bp target fragment, and the amplified product obtained by PCR amplification using the primer pair B2 does not contain the 819 bp target fragment; The A2B2 mating type also satisfies the following condition (h2): (h2) Using the genome of the monokaryon of Lentinus edodes 0912 variety as a template, the amplified product obtained by PCR amplification using the primer pair A1 in claim 1 does not contain the 1784 bp target fragment, and the amplified product obtained by PCR amplification using the primer pair B1 does not contain the 594 bp target fragment; The A1B2 mating type also satisfies the following condition (i2): (i2) Using the genome of the monokaryon of Lentinus edodes 0912 variety as a template, the amplified product obtained by PCR amplification using the primer pair A2 in claim 1 does not contain the 1433 bp target fragment, and the amplified product obtained by PCR amplification using the primer pair B1 does not contain the 594 bp target fragment; The A2B1 mating type also satisfies the following condition (j2): (j2) Using the genome of the monokaryon of Lentinus edodes 0912 variety as a template, the amplification product obtained by PCR amplification using the primer pair A1 described in claim 1 does not contain the 1784 bp target fragment, and the amplification product obtained by PCR amplification using the primer pair B2 does not contain the 819 bp target fragment.

11. Use of the primer set of claim 1 or a reagent or kit containing the primer set, the primer set of claim 2 or a reagent or kit containing the primer set, the primer pair of claim 3 or a reagent or kit containing the primer pair, or any method of claims 7-10 in shiitake mushroom breeding and / or strain identification.

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