Application of InDel molecular marker associated with non-fruiting / popcorn abnormal character of lentinus edodes in lentinus edodes breeding

Abstract

The invention provides an application of an InDel molecular marker associated with the unusual character of mushroom fruiting / popcorn in mushroom breeding, and belongs to the technical field of molecular genetic breeding, the InDel molecular marker associated with the unusual character of mushroom fruiting / popcorn comprises BCG5 and / or BCHP7; the BCG5 is a 11bp segment which is inserted or deleted on a chromosome 6 of the shiitake mushroom, the nucleotide sequence of the 11bp segment is shown as SEQ ID NO.1, and the 11bp segment corresponds to the 1163900th to 1163910th sites of the physical position of the chromosome 6 in a reference genome GCA015476405.1 of the shiitake mushroom; the BCHP7 is a 31bp segment inserted or deleted on a chromosome 6 of the shiitake mushroom, the nucleotide sequence of the 31bp segment is shown as SEQ ID NO.2, and the 31bp segment corresponds to the 1247636 to 1247666 of the physical position of the chromosome 6 in a reference genome GCA015476405.1 of the shiitake mushroom. The InDel molecular marker associated with the abnormal character of no fruiting / popcorn of the shiitake mushroom can assist in early screening of the abnormal character of no fruiting / popcorn of the shiitake mushroom, so that the breeding efficiency of the shiitake mushroom is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular genetic breeding, and particularly relates to the application of InDel molecular markers associated with abnormal mushroom production / popcorn production traits of shiitake mushrooms in shiitake mushroom breeding. Background Art

[0002] Lentinula edodes (Berk.) Pegler is popular among consumers for its high nutritional and medicinal value. It is the most widely cultivated and highest-yielding mushroom species in my country. China is also the first country in the world to carry out artificial cultivation of shiitake mushrooms. Excellent shiitake mushroom strains are the foundation of industrial development and the prerequisite for stable cultivation.

[0003] In the breeding of edible fungi such as shiitake mushrooms, monokaryons are usually prepared by protoplast monokaryon preparation or spore monokaryon separation to obtain monokaryons, and then traditional breeding methods such as single-single hybridization or single-double hybridization are used to aggregate the ideal traits of the parents into new varieties through hybridization combinations. Generally, there are four stages: 1) The compatible monokaryon strains are paired to form new binuclear strains, and these binuclear strains are purified to form mother strains (primary strains), which is usually called the pairing stage; 2) The mother strain is inoculated into the culture medium to grow into the original seed stage of the cultivated species (secondary species); 3) The original seed is inoculated into the mushroom stick and the culture stage of the spawn is completed; 4) The mushroom stick enters the mushroom shed for the phenotypic screening stage of mushroom production. The entire breeding cycle usually takes 8-10 months, and strains are eliminated in the constructed population in the four stages, especially in the mushroom production stage, some strains are eliminated because of the fruiting body with abnormal mushroom / popcorn characteristics.

[0004] Due to the long breeding cycle and high elimination rate of shiitake mushrooms, especially the eliminated strains in the final fruiting stage, a lot of ineffective work has been caused, a lot of manpower, material resources and time costs have been wasted, the difficulty of shiitake mushroom breeding has been increased, and the breeding efficiency has been restricted. If the abnormal traits of not producing mushrooms / popcorn can be located and molecular markers can be developed for early screening in the mother strain mycelium stage, the strains with abnormal traits of not producing mushrooms / popcorn that can only be found in the fruiting stage can be identified, which will significantly improve the breeding efficiency of shiitake mushrooms. The method of using molecular markers to assist in the targeted screening of parents with specific traits and the selection of offspring has been widely used in crop breeding, but this method has not been widely used in edible fungi. With the maturity of molecular marker technology and the completion of shiitake mushroom genome sequencing, the application of molecular markers for targeted breeding in edible fungi such as shiitake mushrooms is inevitable. Among them, Indel molecular markers are an application based on high-throughput sequencing technology, which has the advantages of abundant quantity, high accuracy, good stability, and quick and easy typing. Summary of the invention

[0005] In order to solve the problem of early screening of abnormal traits of shiitake mushrooms not producing mushrooms / popcorn, the present invention provides the application of InDel molecular markers associated with abnormal traits of shiitake mushrooms not producing mushrooms / popcorn in shiitake mushroom breeding. The InDel molecular markers associated with abnormal traits of shiitake mushrooms not producing mushrooms / popcorn can assist in the early screening of abnormal traits of shiitake mushrooms not producing mushrooms / popcorn, thereby improving the efficiency of shiitake mushroom breeding.

[0006] The present invention is achieved through the following technical solutions:

[0007] The present invention provides an application of an InDel molecular marker associated with the abnormal trait of no mushroom production / popcorn formation of shiitake mushrooms in shiitake mushroom breeding, wherein the InDel molecular marker associated with the abnormal trait of no mushroom production / popcorn formation of shiitake mushrooms comprises BCG5 and / or BCHP7;

[0008] The BCG5 is an 11 bp fragment inserted or deleted on chromosome 6 of Lentinus edodes, the nucleotide sequence of the 11 bp fragment is shown in SEQ ID NO.1, and the 11 bp fragment corresponds to the physical position 1163900 to 1163910 of chromosome 6 in the Lentinus edodes reference genome GCA_015476405.1;

[0009] The BCHP7 is a 31 bp fragment inserted or deleted on chromosome 6 of Lentinus edodes. The nucleotide sequence of the 31 bp fragment is shown in SEQ ID NO.2. The 31 bp fragment corresponds to the physical position 1247636 to 1247666 of chromosome 6 in the Lentinus edodes reference genome GCA_015476405.1.

[0010] Specifically, the nucleotide sequence of the 11 bp fragment is: 5'-AAATCGTGACA-3';

[0011] The nucleotide sequence of the 31 bp fragment is: 5'-TTCTTCAAATTCTATAGACCATTCGCACACA-3'.

[0012] Based on the same inventive concept, the present invention provides an early selection method for shiitake mushrooms with abnormal traits of not producing mushrooms / popcorn, the early selection method comprising: early selection of shiitake mushrooms with abnormal traits of not producing mushrooms / popcorn based on the genotype of InDel molecular markers BCG5 and / or BCHP7;

[0013] The BCG5 is an 11 bp fragment inserted or deleted on chromosome 6 of Lentinus edodes, the nucleotide sequence of the 11 bp fragment is shown in SEQ ID NO.1, and the 11 bp fragment corresponds to the physical position 1163900 to 1163910 of chromosome 6 in the Lentinus edodes reference genome GCA_015476405.1;

[0014] The BCHP7 is a 31 bp fragment inserted or deleted on chromosome 6 of Lentinus edodes, the nucleotide sequence of the 31 bp fragment is shown in SEQ ID NO.2, and the 31 bp fragment corresponds to the physical position 1247636 to 1247666 of chromosome 6 in the Lentinus edodes reference genome GCA_015476405.1;

[0015] The BCG5 includes an insertion type and a deletion type, the insertion type is named A, and the deletion type is named a; the BCHP7 includes an insertion type and a deletion type, the insertion type is named B, and the deletion type is named b;

[0016] When the genotype of BCG5 in shiitake mushrooms is aa, shiitake mushrooms show abnormal characteristics of no mushrooms / popcorn, and when the genotype of BCG5 is Aa or AA, shiitake mushrooms show normal fruiting body characteristics; when the genotype of BCHP7 in shiitake mushrooms is bb, shiitake mushrooms show abnormal characteristics of no mushrooms / popcorn, and when the genotype of BCHP7 is Bb or BB, shiitake mushrooms show normal fruiting body characteristics.

[0017] Furthermore, the early selection method specifically includes:

[0018] Detecting and determining the genotype of the BCG5 and / or the BCHP7 in the genome of the tested shiitake mushroom;

[0019] Based on the genotype of the BCG5 and / or BCHP7, early selection is performed on the abnormal trait of no mushroom production / popcorn formation of the shiitake mushroom to be tested.

[0020] Furthermore, the detecting and determining the genotype of the BCG5 and / or the BCHP7 in the tested shiitake mushroom genome specifically includes:

[0021] Detect and determine the genotype of BCG5 in the tested mushroom genome:

[0022] Using BCG5-f and BCG5-r as primers, PCR amplification was performed on the genomic DNA of the tested Lentinus edodes;

[0023] Detect the length of the PCR amplification product or sequence the PCR amplification product. When the PCR amplification product does not contain the 11 bp fragment, it is recorded as aa; when the PCR amplification product contains the 11 bp fragment, it is recorded as AA; when the PCR amplification product partially contains the 11 bp fragment, it is recorded as Aa;

[0024] Wherein, the nucleotide sequence of the BCG5-f is shown in SEQ ID NO.3, and the nucleotide sequence of the BCG5-r is shown in SEQ ID NO.4;

[0025] Detect and determine the genotype of the BCHP7 in the tested mushroom genome:

[0026] BCHP7-f and BCHP7-r were used as primers to amplify the genomic DNA of Lentinus edodes.

[0027] Detect the length of the PCR amplification product or sequence the PCR amplification product. When the shiitake mushroom genome to be tested does not contain the 31 bp fragment, the target fragment cannot be amplified, which is recorded as bb. When the shiitake mushroom genome to be tested contains the 31 bp fragment, a target fragment with a length of 215 bp is amplified, which is recorded as BB or Bb.

[0028] Among them, the nucleotide sequence of the BCHP7-f is shown in SEQ ID NO.5, and the nucleotide sequence of the BCHP7-r is shown in SEQ ID NO.6.

[0029] Furthermore, the early selection method is carried out at any stage of the pairing stage, the original seed stage and the cultivation stage of shiitake mushroom breeding.

[0030] Preferably, the early selection method is carried out in the pairing stage of shiitake mushroom breeding.

[0031] Based on the same inventive concept, the present invention provides the use of primers for detecting InDel molecular markers associated with the abnormal trait of no mushroom production / popcorn in shiitake mushroom breeding, wherein the InDel molecular markers associated with the abnormal trait of no mushroom production / popcorn in shiitake mushroom include BCG5 and / or BCHP7;

[0032] The BCG5 is an 11 bp fragment inserted or deleted on chromosome 6 of Lentinus edodes, the nucleotide sequence of the 11 bp fragment is shown in SEQ ID NO.1, and the 11 bp fragment corresponds to the physical position 1163900 to 1163910 of chromosome 6 in the Lentinus edodes reference genome GCA_015476405.1;

[0033] The BCHP7 is a 31 bp fragment inserted or deleted on chromosome 6 of Lentinus edodes, the nucleotide sequence of the 31 bp fragment is shown in SEQ ID NO.2, and the 31 bp fragment corresponds to the physical position 1247636 to 1247666 of chromosome 6 in the Lentinus edodes reference genome GCA_015476405.1;

[0034] The primers for detecting the InDel molecular marker associated with the abnormal trait of no mushroom production / popcorn production of Lentinus edodes include primers for detecting the BCG5 and primers for detecting the BCHP7, the primers for detecting the BCG5 include BCG5-f and BCG5-r, and the primers for detecting the BCHP7 include BCHP7-f and BCHP7-r;

[0035] The nucleotide sequence of the BCG5-f is shown in SEQ ID NO.3, the nucleotide sequence of the BCG5-r is shown in SEQ ID NO.4, the nucleotide sequence of the BCHP7-f is shown in SEQ ID NO.5, and the nucleotide sequence of the BCHP7-r is shown in SEQ ID NO.6.

[0036] Based on the same inventive concept, the present invention provides a kit for early screening of abnormal mushroom / popcorn traits in shiitake mushrooms, the kit comprising primers for detecting the InDel molecular marker BCG5 and / or primers for detecting the InDel molecular marker BCHP7, the primers for detecting the InDel molecular marker BCG5 include BCG5-f and BCG5-r, and the primers for detecting the InDel molecular marker BCHP7 include BCHP7-f and BCHP7-r;

[0037] The nucleotide sequence of the BCG5-f is shown in SEQ ID NO.3, the nucleotide sequence of the BCG5-r is shown in SEQ ID NO.4, the nucleotide sequence of the BCHP7-f is shown in SEQ ID NO.5, and the nucleotide sequence of the BCHP7-r is shown in SEQ ID NO.6.

[0038] Based on the same inventive concept, the present invention also provides a kit for early screening of abnormal mushroom production / popcorn-like traits of shiitake mushrooms for use in shiitake mushroom breeding.

[0039] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0040] The present invention discloses an application of InDel molecular markers associated with abnormal mushroom production / popcorn production in shiitake mushroom breeding. The InDel molecular markers associated with abnormal mushroom production / popcorn production include BCG5 and BCHP7, both of which are located on chromosome 6 of the shiitake mushroom reference genome. A homozygous strain lacking BCG5 or BCHP7 will have fruiting bodies with abnormal mushroom production / popcorn production. Fruiting bodies with abnormal mushroom production and popcorn production are different phenotypes of the lack of BCG5 or BCHP7. Therefore, the InDel molecular markers BCG5 and BCHP7 are applied to shiitake mushroom breeding, so that early screening of abnormal mushroom production / popcorn production of shiitake mushrooms can be performed at the mother strain mycelium stage, and strains with abnormal mushroom production / popcorn production that could only be found in the mushroom production stage can be identified in advance, thereby significantly improving the efficiency of shiitake mushroom breeding and effectively saving manpower and material resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0042] Figure 1 This is a comparison chart of normal fruiting bodies of shiitake mushrooms and fruiting bodies with abnormal popcorn characteristics: A is the morphology of normal fruiting bodies at different developmental stages; B is the morphology of fruiting bodies with abnormal popcorn characteristics at different developmental stages.

[0043] Figure 2 Electropherogram of BCG5 molecular marker screening for NF / PF abnormalities.

[0044] Figure 3 Electropherogram of BCHP7 molecular marker screening for NF / PF abnormality traits. DETAILED DESCRIPTION

[0045] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0046] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0047] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0049] The overall idea of ​​the present invention is as follows:

[0050] The F2 generation population of the "Huxiang F2" strain will experience "No fruiting" (NF) and "popcorn fruiting" (PF) with abnormal fruiting bodies during the fruiting stage. The no fruiting trait has no adverse manifestations in the original species and spawning stages, and the appearance and morphology are normal in the mushroom stick cultivation stage. In the fruiting stage, there is no primordium formation on the surface of the mushroom stick. The mushroom stick has no response to the artificially controlled temperature difference, humidity difference, vibration and other conditions that stimulate mushroom production, and cannot generate primordia, which is an abnormal trait of fruiting body production; the popcorn trait has no adverse manifestations in the original species and spawning stages, and the appearance and morphology are normal in the mushroom stick cultivation stage, but in the fruiting stage, abnormal primordia similar to popcorn are formed on the surface of the mushroom stick, and the primordia have no differentiation of sterile cap and stipe. After the primordium grows to 2-3cm in size, it begins to crack and looks like popcorn, so it is called popcorn mushroom, which is an abnormal trait of fruiting body formation ( Figure 1 B).

[0051] Based on a large number of previous related experiments, the inventors found that the NF abnormal trait and the PF abnormal trait are different phenotypes of a harmful recessive trait, and are controlled by a region located on chromosome 6 of the reference genome.

[0052] Therefore, when the inventors used the shiitake mushroom strain "Huxiang F2" as a parent for breeding operations, they developed InDel molecular markers BCG5 and BCHP7 that are easy to detect by genotyping for the abnormal NF / PF traits that appeared in the mushroom stage, so that the abnormal NF / PF traits can be detected in the mycelium stage. The present invention applies it to the early screening of abnormal NF / PF traits in the breeding population, and at the same time provides a detection method for InDel molecular markers for early screening of abnormal NF / PF traits in the mycelium growth period.

[0053] The application of the InDel molecular marker associated with the abnormal trait of no mushroom production / popcorn production of Lentinus edodes in Lentinus edodes breeding of the present invention will be described in detail below in combination with the embodiments and experimental data.

[0054] Example 1

[0055] The method of this embodiment using BCG5 and BCHP7 molecular markers to detect abnormal mushroom / popcorn characteristics at an early stage includes the following steps:

[0056] 1) Extracting DNA from the mycelial stage of the strain to be tested;

[0057] 2) using the primers of the two InDel molecular markers BCG5 and BCHP7 in Table 2, PCR amplification was performed using the mycelial DNA of the edodes strain to be detected as a template;

[0058] 3) Determine the genotype of the strain to be tested based on the DNA electrophoresis results after PCR amplification and judge the mushroom phenotype.

[0059] Step 1) Specific method:

[0060] 1. Inoculate the mycelium of Lentinus edodes to be tested into PDA culture medium, culture at 23-25℃ in the dark, and collect the mycelium after 10-15 days;

[0061] 2. CTAB method to extract genomic DNA from mycelium: ① Place freeze-dried Lentinus edodes mycelium in a 2 mL centrifuge tube and add 2-3 steel balls; ② Place the centrifuge tube in a multi-sample tissue grinder, set the frequency to 60 Hz, and grind for 30 seconds until it becomes a uniform powder; ③ Add CTAB extract (CTAB 2 g, 1 mol / L Tris-HCl 10 mL, 0.2 mol / L ETA 10 mL, 5 mol / L NaCl) preheated at 65 °C for 1 h ④ Centrifuge at 12000r / min and 4°C for 20min, and dispense the supernatant into two tubes, 800μL each; ⑤ Add a mixture of phenol, chloroform and isoamyl alcohol in a volume ratio of 25:24:1 to the above supernatant, gently mix for 10min, centrifuge at 12000r / min and 4°C for 10min, and transfer the supernatant to a new centrifuge tube; ⑥ Add a mixture of chloroform and isoamyl alcohol in a volume ratio of 24:1 to the above centrifuge tube, and gently Mix for 10 minutes, centrifuge at 12000r / min and 4℃ for 10 minutes, take the supernatant (record the volume) and transfer it to another centrifuge tube; ⑦ Add 2 / 3 volume of -20℃ pre-cooled isopropanol relative to the supernatant in step ⑥ to the above centrifuge tube, mix, let it stand at -20℃ for 1h, then centrifuge at 8000r / min and 4℃ for 10min, and discard the supernatant; ⑧ Add 1mL of 70% ethanol to the obtained precipitate and wash it once, centrifuge at 8000r / min at room temperature for 5min; ⑨ Discard the ethanol and blow dry in the clean bench; add 100μL 10×TE buffer, tap gently to dissolve the precipitate, add 1μL 10mg / mL RNaseA and bathe at 37℃ for 1h to remove RNA; ⑩ Store the obtained DNA extract in a -20℃ refrigerator for later use.

[0062] Step 2) Specific method:

[0063] 1. The PCR amplification reaction system has a total volume of 20 μL, consisting of 2×PCR Premix TaqTM (1.25U / 25μL TaqDNA enzyme, 0.4mM / L dNTP, 0.3mM / L PCR buffer) 10μL, 10μmol / L BCG5 or BCHP7 labeled forward primer and reverse primer 1μL each, 20-30ng / μL template DNA extracted in step 1, 2μL, ddH2O 6μL;

[0064] 2. PCR reaction conditions: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 1 min, annealing at 56°C for 45 s, extension at 72°C for 1 min, 35 cycles; maintain at 72°C for 10 min, and store at 4°C.

[0065] Step 3) Specific method:

[0066] 5 μL of the product obtained by the above PCR amplification was spotted on an agarose gel containing nucleic acid dye for electrophoresis. The volume percentage concentration of the agarose gel was 2.5%, the electrophoresis buffer was 1×TAE, and the electrophoresis was carried out at a voltage of 90 V for 5 hours. The bands were photographed and statistically analyzed: a single 178 bp band was amplified using the BCG5 molecular marker primer, or / simultaneously, no band could be amplified using the BCHP7 molecular marker primer (BCHP7-f was located on the missing 31 bp InDel), which was a homozygous deletion genotype of the NF / PF abnormal trait, and the mushroom production showed the NF / PF abnormal trait strain; two bands of 178 bp and 189 bp or only one band of 189 bp were amplified using the BCG5 molecular marker primer, or / simultaneously, a 215 bp band was amplified using the BCHP7 molecular marker primer, which was a heterozygous genotype of the Norm trait or a homozygous non-deletion genotype, and the mushroom production showed the Norm trait strain.

[0067] Example 2

[0068] This example verifies the association between abnormal NF and PF traits and molecular markers BCG5 and BCHP7.

[0069] Based on the previous phenotypic stability fruiting test of NF abnormal traits and PF abnormal traits, the inventors speculated that NF and PF abnormal traits are different phenotypes of the same trait. By constructing verification experiments with different dominant and recessive combinations, the inventors speculated that NF and PF abnormal traits are recessive traits controlled by a single locus. Then, the BSA mixed pool positioning method was used to determine that NF and PF abnormal traits were co-located on the reference genome chr6, the same region with a length of 0-1.4Mb. According to the insertion / deletion (InDel) variation sites in this interval, InDel molecular markers BCG5 and BCHP7 were developed. The chromosomes where they are located, the specific physical locations and insertion and deletion information are shown in Table 1. At the same time, specific detection primers were designed for the genotypes of these two InDel sites, and the detailed information is shown in Table 2.

[0070] Table 1 InDel molecular markers of NF / PF strains with abnormal traits

[0071]

[0072] Table 2 Allele-specific primers for genotyping of InDel loci

[0073]

[0074] 1. In this example, 6 strains with abnormal NF / PF traits and 6 strains with normal fruiting (Norm) traits were selected from 4 hybrid populations (A2B, BD, GA, and DF) of the F1 population of the "Huxiang F2" strain, that is, 24 strains with abnormal NF / PF traits and 24 strains with Norm traits. The detection method of steps 1) to 3) of Example 1 was used to test the InDel molecular marker BCG5. The results are as follows: Figure 2 As shown. In the BCG5 marker, the NF / PF abnormal trait is a homozygous deletion genotype, and the corresponding amplified fragment of the electrophoresis result is a band of 178bp, which is defined as a negative result; the corresponding Norm trait is a heterozygous genotype or a homozygous non-deleted genotype, and the corresponding amplified fragment is two bands of 178bp and 189bp or only a homozygous genomic band of 189bp, which is defined as a positive result. Among the 24 strains of NF / PF abnormal traits and Norm traits screened and tested, two strains did not amplify bands (marked by orange circles), which is a PCR failure with a low probability of occurrence and does not affect the overall detection; there are 5 Norm strains whose genotypes do not correspond to their phenotypes (marked by red boxes). The genotype test results of these Norm strains are NF / PF abnormal traits, but the actual phenotype is a Norm strain that can produce mushrooms. The test value is negative but the phenotype is normal. Therefore, this type of test error is called a false negative. If the test value is a positive result of a normal strain, but the phenotype is NF / PF abnormal traits, the test error at this time is called a false positive. It can be seen that the false positive rate of BCG5 molecular marker in screening NF / PF abnormal traits is 0, the false negative rate is 20.83%, and the comprehensive error rate is 10.42%.

[0075] In order to reduce the false negative rate of BCG5, BCHP7 molecular marker was used to detect NF / PF abnormal trait strains and Norm trait strains. In BCHP7 marker, NF / PF abnormal trait is a homozygous deletion genotype, and its electrophoresis results (such as Figure 3 ) There should be no amplified fragment band, which is defined as a negative result; the corresponding Norm trait is a heterozygous genotype or a homozygous non-deleted genotype, and the corresponding amplified fragment is only a 215bp amplified band, which is defined as a positive result. The false positive rate of BCHP7 molecular marker in screening NF / PF abnormal traits is 4.17%, the false negative rate is 8.33%, and the comprehensive error rate is 6.25%.

[0076] In summary, when conducting early genotypic screening of NF / PF abnormal trait strains in the mycelium stage for population expansion and identification applications, BCG5 and BCHP7 can be used in combination to improve accuracy.

[0077] 2. The F1 generation segregation population that was rescreened and verified by molecular markers in step 1, each strain has accurately recorded phenotypic traits. The population strain traits are divided into strain rejection, mushroom stick rejection, popcorn mushroom (PF), no mushroom production (NF), normal mushroom production (such as Figure 1 A) Traits. Strains with species rejection and stick rejection were eliminated before entering the fruiting stage because their phenotypic traits in the fruiting stage were unclear, and these strains were not subject to population verification. Table 3 lists 8 complete populations used to expand the verification of NF / PF abnormal traits and Norm trait rescreening. The total number of strains in the hybrid population ranged from 196 to 312. The mycelium stage of NF / PF abnormal trait and Norm trait strains in the population was verified using a combination of two molecular markers, BCG5 and BCHP7. The comprehensive error rate is shown in Table 3. The comprehensive error rates of BCG5 and BCHP7 in the 8 populations were all less than 6%. In actual production, BCG5 and BCHP7 can be used for testing at the same time, and the intersection of the test results is taken, which has higher accuracy.

[0078] Table 3. Results of expanded population verification of NF / PF abnormal traits by BCG5 and BCHP7

[0079]

[0080]

[0081] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0082] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0083] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of InDel molecular markers associated with the abnormal trait of no mushroom production / popcorn in shiitake mushroom breeding, characterized in that: The InDel molecular markers associated with the abnormal trait of no mushroom production / popcorn production of Lentinus edodes include BCG5 and / or BCHP7; The BCG5 is an 11 bp fragment inserted or deleted on chromosome 6 of Lentinus edodes, the nucleotide sequence of the 11 bp fragment is shown in SEQ ID NO.1, and the 11 bp fragment corresponds to the physical position 1163900 to 1163910 of chromosome 6 in the Lentinus edodes reference genome GCA_015476405.1; The BCHP7 is a 31 bp fragment inserted or deleted on chromosome 6 of Lentinus edodes. The nucleotide sequence of the 31 bp fragment is shown in SEQ ID NO.

2. The 31 bp fragment corresponds to the physical position 1247636 to 1247666 of chromosome 6 in the Lentinus edodes reference genome GCA_015476405.

1.

2. An early selection method for mushrooms with abnormal characteristics of no mushroom production / popcorn, characterized in that: The early selection method comprises: performing early selection on the abnormal traits of no mushroom production / popcorn production of shiitake mushrooms based on the genotype of InDel molecular markers BCG5 and / or BCHP7; The BCG5 is an 11 bp fragment inserted or deleted on chromosome 6 of Lentinus edodes, the nucleotide sequence of the 11 bp fragment is shown in SEQ ID NO.1, and the 11 bp fragment corresponds to the physical position 1163900 to 1163910 of chromosome 6 in the Lentinus edodes reference genome GCA_015476405.1; The BCHP7 is a 31 bp fragment inserted or deleted on chromosome 6 of Lentinus edodes, the nucleotide sequence of the 31 bp fragment is shown in SEQ ID NO.2, and the 31 bp fragment corresponds to the physical position 1247636 to 1247666 of chromosome 6 in the Lentinus edodes reference genome GCA_015476405.1; The BCG5 includes an insertion type and a deletion type, the insertion type is named A, and the deletion type is named a; the BCHP7 includes an insertion type and a deletion type, the insertion type is named B, and the deletion type is named b; When the genotype of BCG5 in shiitake mushrooms is aa, shiitake mushrooms show abnormal characteristics of no mushrooms / popcorn, and when the genotype of BCG5 is Aa or AA, shiitake mushrooms show normal fruiting body characteristics; when the genotype of BCHP7 in shiitake mushrooms is bb, shiitake mushrooms show abnormal characteristics of no mushrooms / popcorn, and when the genotype of BCHP7 is Bb or BB, shiitake mushrooms show normal fruiting body characteristics.

3. The early selection method for mushrooms with abnormal characteristics of no mushroom production / popcorn formation according to claim 2, characterized in that: The early selection method specifically includes: Detecting and determining the genotype of the BCG5 and / or the BCHP7 in the genome of the tested shiitake mushroom; Based on the genotype of the BCG5 and / or BCHP7, early selection is performed on the abnormal trait of no mushroom production / popcorn formation of the shiitake mushroom to be tested.

4. The early selection method for mushrooms with abnormal characteristics of no mushroom production / popcorn formation according to claim 3, characterized in that: The detecting and determining the genotype of the BCG5 and / or the BCHP7 in the tested shiitake mushroom genome specifically includes: Detect and determine the genotype of BCG5 in the tested mushroom genome: Using BCG5-f and BCG5-r as primers, PCR amplification was performed on the genomic DNA of the tested Lentinus edodes; Detect the length of the PCR amplification product or sequence the PCR amplification product. When the PCR amplification product does not contain the 11 bp fragment, it is recorded as aa; when the PCR amplification product contains the 11 bp fragment, it is recorded as AA; when the PCR amplification product partially contains the 11 bp fragment, it is recorded as Aa; Wherein, the nucleotide sequence of BCG5-f is shown in SEQ ID NO.3, and the nucleotide sequence of BCG5-r is shown in SEQ ID NO.4; Detect and determine the genotype of the BCHP7 in the tested mushroom genome: BCHP7-f and BCHP7-r were used as primers to amplify the genomic DNA of Lentinus edodes. Detect the length of the PCR amplification product or sequence the PCR amplification product. When the shiitake mushroom genome to be tested does not contain the 31 bp fragment, the target fragment cannot be amplified, which is recorded as bb. When the shiitake mushroom genome to be tested contains the 31 bp fragment, a target fragment with a length of 215 bp is amplified, which is recorded as BB or Bb. Among them, the nucleotide sequence of the BCHP7-f is shown in SEQ ID NO.5, and the nucleotide sequence of the BCHP7-r is shown in SEQ ID NO.

6.

5. The early selection method for mushrooms with abnormal characteristics of not producing mushrooms / popcorn-like appearance according to any one of claims 2 to 4, characterized in that: The early selection method is carried out at any stage of the pairing stage, the original seed stage and the cultivation stage of shiitake mushroom breeding.

6. A method for early selection of mushrooms with abnormal characteristics of non-mushroom production / popcorn production according to any one of claims 2 to 4, characterized in that: The early selection method is carried out in the pairing stage of shiitake mushroom breeding.

7. Use of primers for detecting InDel molecular markers associated with the abnormal trait of no mushroom production / popcorn production in shiitake mushroom breeding, characterized in that: The InDel molecular markers associated with the abnormal trait of no mushroom production / popcorn production of Lentinus edodes include BCG5 and / or BCHP7; The BCG5 is an 11 bp fragment inserted or deleted on chromosome 6 of Lentinus edodes, the nucleotide sequence of the 11 bp fragment is shown in SEQ ID NO.1, and the 11 bp fragment corresponds to the physical position 1163900 to 1163910 of chromosome 6 in the Lentinus edodes reference genome GCA_015476405.1; The BCHP7 is a 31 bp fragment inserted or deleted on chromosome 6 of Lentinus edodes, the nucleotide sequence of the 31 bp fragment is shown in SEQ ID NO.2, and the 31 bp fragment corresponds to the physical position 1247636 to 1247666 of chromosome 6 in the Lentinus edodes reference genome GCA_015476405.1; The primers for detecting the InDel molecular marker associated with the abnormal trait of no mushroom production / popcorn production of Lentinus edodes include primers for detecting the BCG5 and primers for detecting the BCHP7, the primers for detecting the BCG5 include BCG5-f and BCG5-r, and the primers for detecting the BCHP7 include BCHP7-f and BCHP7-r; The nucleotide sequence of the BCG5-f is shown in SEQ ID NO.3, the nucleotide sequence of the BCG5-r is shown in SEQ ID NO.4, the nucleotide sequence of the BCHP7-f is shown in SEQ ID NO.5, and the nucleotide sequence of the BCHP7-r is shown in SEQ ID NO.

6.

8. A kit for early screening of abnormal mushroom production / popcorn characteristics of shiitake mushrooms, characterized in that: The kit comprises primers for detecting the InDel molecular marker BCG5 and / or primers for detecting the InDel molecular marker BCHP7, wherein the primers for detecting the InDel molecular marker BCG5 include BCG5-f and BCG5-r, and the primers for detecting the InDel molecular marker BCHP7 include BCHP7-f and BCHP7-r; The nucleotide sequence of the BCG5-f is shown in SEQ ID NO.3, the nucleotide sequence of the BCG5-r is shown in SEQ ID NO.4, the nucleotide sequence of the BCHP7-f is shown in SEQ ID NO.5, and the nucleotide sequence of the BCHP7-r is shown in SEQ ID NO.

6.

9. Use of the kit for early screening of abnormal mushroom production / popcorn-like characteristics of shiitake mushrooms as claimed in claim 8 in shiitake mushroom breeding.

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