Lecsl2-2 gene for improving fragrance of lentinus edodes and application of Lecsl2-2 gene

Abstract

The invention relates to the technical field of edible mushroom gene breeding, in particular to a Lecsl2-2 gene for improving the fragrance of shiitake mushrooms and application of the Lecsl2-2 gene. According to expression analysis of fragrance genes of Mu-Xiang No.7 and commercial shiitake mushrooms L808, it is found that the expression quantity of the fragrance gene Lecsl2 of the Mu-Xiang No.7 variety with rich fragrance is remarkably higher than that of the commercial shiitake mushrooms L808; in-depth analysis finds that only Meixiang-7 contains a high-expression Lecsl2-2 gene, and a filial generation containing the Lecsl2-2 gene has higher lentinine, an improved breeding method is adopted, namely PCR amplification identification is performed on haploid strains of shiitake mushrooms, the haploid strains containing the Lecsl2-2 gene are directionally selected for hybridization, and the hybridized haploid strains containing the Lecsl2-2 gene are obtained. Therefore, filial generations with higher lentinus edodes content can be bred, a new breeding thought is provided for further improving the fragrance of commercial shiitake mushrooms, and the breeding time is effectively shortened.

Description

Technical Field

[0001] The invention relates to the technical field of edible fungus gene breeding, and in particular to a Lecsl2-2 gene for improving the flavor of shiitake mushrooms and an application thereof. Background Art

[0002] Lentinula edodes is an edible fungus belonging to the phylum Basidiomycota, class Agaricomycetes, order Agaricales, family Omphalotaceae, genus Lentinula. Lentinula is fleshy, delicious, nutritious, and has health benefits such as improving immunity, lowering blood pressure, lowering cholesterol, and anti-tumor. In particular, it has a unique and rich aroma and is deeply loved by consumers. Due to the artificial directional breeding that focuses on the appearance of commodity and the degeneration of varieties, the aroma quality of commercial cultivated shiitake mushroom varieties is far inferior to that of wild shiitake mushrooms. With the improvement of living standards, people are increasingly pursuing health, nutrition and sensory quality in food.

[0003] Therefore, breeding high-quality varieties of strong-flavored shiitake mushrooms is of great significance to improving the quality of shiitake mushrooms, meeting market demand, and promoting the high-quality development of the shiitake mushroom industry.

[0004] In the prior art, we usually improve the aroma of commercial shiitake mushrooms by selecting wild shiitake mushrooms and hybridizing them with commercial shiitake mushrooms. However, we found in actual hybridization that due to the randomness of hybridization, we often need to cultivate to the fruiting stage to detect the aroma of shiitake mushrooms. The breeding process is very slow, and the large amount of screening of hybrid offspring also further increases our workload. In order to improve the breeding efficiency, the research on the aroma gene of shiitake mushrooms using gene-assisted technology for breeding is the focus of current research by technicians. The research related to the aroma gene of shiitake mushrooms in the prior art is reported as follows:

[0005] The unique and rich aroma of dried shiitake mushrooms mainly comes from volatile sulfur-containing compounds, such as 1,2,4-trithiolane, 1,2,4,6-tetrathiolane and 1,2,3,5,6-pentathiolane (lentinan), among which lentinan is considered to be the main aroma substance. 2. In the main metabolic pathways of volatile sulfur-containing compounds in shiitake mushrooms, γ-glutamine transpeptidase (GGT) and L-cysteine ​​sulfoxide lyase (CS lyase) are the key rate-limiting enzymes for the synthesis of sulfur-containing compounds such as lentinan. 3. The shiitake mushroom GGT gene family consists of 7 genes, namely Leggt1, Leggt2, Leggt3, Leggt4, Leggt5, Leggt6 and Leggt7. Among them, Leggt1 and Leggt5 have always maintained a high expression level during the heat drying process, and are believed to play an important role in the formation of shiitake mushroom aroma during heat drying. The expression of the remaining GGT genes is low or down-regulated during the heat drying process. 4. The CS lyase gene family of Lentinus edodes consists of five genes, namely Lecsl1, Lecsl2, Lecsl3, Lecsl4 and Lecsl5. Among them, Lecsl2 and Lecsl3 are considered to be the two CS lyase genes that play a key role in the formation of volatile sulfur-containing aroma compounds during the drying process of Lentinus edodes.

[0006] Based on the above research, we found that among the four genes (Leggt1, Leggt5, Lecsl2 and Lecsl3) that play a key role in the formation of aroma, only the Lecsl2 gene has a higher expression in the wild strong-aroma variety. This gene is composed of two alleles in Rongxiang 7, of which the allele Lecsl2-1 exists in both varieties of shiitake mushrooms, while the other allele Lecsl2-2 only exists in the strong-aroma variety Rongxiang 7 and has a higher transcriptional expression level. This differential expression may cause the aroma of the two varieties of shiitake mushrooms to be very different, which helps us to select single spores containing this specific gene, and then select these single spores for hybrid breeding, thereby effectively improving the aroma of shiitake mushrooms and shortening the breeding time of shiitake mushrooms. Summary of the invention

[0007] In view of the above, it is necessary to study the aroma genes of shiitake mushrooms and develop single spores containing certain specific genes for fragrant wild varieties, so as to select these single spores for hybrid breeding, which can effectively improve the aroma of shiitake mushrooms and shorten the breeding time of shiitake mushrooms.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] The Lecsl2-2 gene for improving the aroma of shiitake mushrooms is characterized in that the Lecsl2-2 gene has an amino acid sequence as shown in SEQ ID NO.4.

[0010] Furthermore, the CDS coding sequence of the Lecsl2-2 gene is shown as SEQ ID NO.3, and the nucleic acid sequence is shown as SEQ ID NO.2.

[0011] The high-lentinan shiitake mushroom containing the Lecsl2-2 gene, the shiitake mushroom is the Rongxiang No. 7 strain preserved in the General Microbiology Center of the China Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCCNO: 40139, and the preservation date is April 24, 2022.

[0012] A primer pair for detecting Lecsl2-2 gene, wherein the upstream primer sequence of the primer pair is 5'-CGAAGCTTGCAATGAGCTGATG-3'; and the downstream primer sequence is 5'-GATCCATGAC CTTCG TACTC GTT-3'.

[0013] Furthermore, the reaction conditions for PCR amplification using the specific molecular marker primer pair are 94°C for 5 min; 94°C for 30 s, 60°C for 45 s, 72°C for 60 s, 30 cycles; 72°C for 5 min; and stored at 4°C.

[0014] The present invention also includes a method for detecting high-lentinin shiitake mushroom varieties using the specific molecular marker primer pair, the method comprising: using the primer pair to perform PCR amplification on shiitake mushroom genomic DNA, if the target fragment can be amplified, it indicates that the shiitake mushroom variety is a high-lentinin variety; if the product cannot be amplified, it indicates that the shiitake mushroom variety is a low-lentinin variety.

[0015] The present invention also includes a haploid strain comprising the Lecsl2-2 gene, wherein the haploid strain is a single spore isolate of Rongxiang No. 7, and the single spore isolate of Rongxiang No. 7 can be amplified by the specific molecular marker primer pair to obtain a target fragment.

[0016] A method for hybrid breeding of shiitake mushrooms for improving lentinan, the method comprising: using the haploid strain to hybridize with other haploid or diploid strains of shiitake mushrooms, and obtaining hybrid offspring that can produce mushrooms normally.

[0017] The present invention also includes a method for identifying high-lentinin hybrid offspring, which comprises: using the specific molecular marker primer pair to detect the hybrid offspring of Rongxiang No. 7 or its haploid strain and other commercial shiitake mushrooms, and if the target fragment can be amplified, it is proved that the hybrid offspring is a high-lentinin variety.

[0018] The present invention has the following beneficial effects:

[0019] 1. The present invention uses the aroma genes Leggt1, Leggt5, Lecsl2 and Lecsl3 of the wild variety with strong aroma, Rongxiang No. 7, and the commercial Lentinus edodes L808, which are screened and domesticated by the applicant, for expression analysis. It is found that the expression level of the aroma gene Lecsl2 of the fragrant Rongxiang No. 7 variety is significantly higher than that of the commercial Lentinus edodes L808. Based on this conclusion, an in-depth analysis is conducted and it is found that among the alleles of the Lecsl2 gene, only Rongxiang No. 7 contains the Lecsl2-2 gene. It is speculated that the hybrid offspring containing this gene will show higher levels of lentinan. After isolating the haploid strains containing this gene, we performed single-double hybridization and measured the lentinan in the hybrid offspring. Finally, it is found that the hybrid offspring containing the Lecsl2-2 gene show higher levels of lentinan. Therefore, we can improve the breeding method: that is, conduct PCR amplification and identification on the haploid strains of shiitake mushrooms, and selectively select the haploid strains containing the Lecsl2-2 gene for hybridization, so as to breed hybrid offspring with higher lentinan content, which provides a new breeding idea for the next step of improving the aroma of commercial shiitake mushrooms and effectively shortens the breeding time. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 These are the transcriptional expression levels of Leggt1, Leggt5, Lecsl2 and Lecsl3 genes in two shiitake mushroom varieties, Rongxiang No. 7 and L808, where A is the relative expression level of the Leggt1 gene, B is the relative expression level of the Leggt5 gene, C is the relative expression level of the Lecsl2 gene, and D is the relative expression level of the Lecsl3 gene.

[0021] Figure 2 This is the phylogenetic tree of pyridoxal phosphate-dependent transferase in Lentinus edodes.

[0022] Figure 3 The figure shows the amplification of different shiitake mushroom samples by primers of two alleles of Lecsl2: Lecsl2-1 and Lecsl2-2; among them, M is: marker-DL2000, from top to bottom: 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp; lanes 1-2 are parents: YX7, L808; 3-7 are haploid strains isolated from single spores of Rongxiang No. 7, namely YX7-16-1, YX7-5-1, YX7-42-1, YX7-49-1 and YX7-71-13A; Figure A shows the amplification result of primer YX7-Lecsl2-1P; Figure B shows the specific amplification result of YX7-Lecsl2-2P for shiitake mushrooms.

[0023] Figure 4The figure shows the amplification of different shiitake mushroom samples by fluorescent quantitative primers of two alleles of Lecsl2: Lecsl2-1 and Lecsl2-2; M is marker-DL2000, from top to bottom: 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp; 1-2 are parents: L808, YX7; 3-6 are haploid strains of Rongxiang No. 7: YX7-5-1, YX7-16-1, YX7-42-1 and YX7-49-1; Figure A shows the amplification results of primer YX7-Lecsl2-1FP, and Figure B shows the amplification results of YX7-Lecsl2-2FP.

[0024] Figure 5 The transcriptional expression levels of two alleles of Lecsl2: Lecsl2-1 and Lecsl2-2 in the fruiting bodies of Rongxiang No. 7.

[0025] Figure 6 Figure 1 is a diagram of the amplification of the Lecsl2 gene in different hybrid offspring; M is marker-DL2000, and from top to bottom they are: 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp; 1 to 15 are haploids of Rongxiang No. 7: YX7-5-2, YX7-35-1, YX7-35-2, YX7-71-1, YX7-8-1, YX7-8-2, YX7-7-1, YX7-7-2, YX7-7-3, YX7-42-2, YX7-42-3, YX7-49-2, YX7-49-3, YX7-71-2 and YX7-71-3; in the figure, A is the amplification product of YX7-Lecsl2-1P, and B is the amplification product of YX7-Lecsl2-2P.

[0026] Biomaterial Deposit Information

[0027] The strain information deposited in this application is: Rongxiang No. 7 strain, its classification name is: Lentinula edodes, and its Chinese classification name is: Lentinula edodes. It is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a preservation number of CGMCC NO: 40139 and a preservation date of April 24, 2022. DETAILED DESCRIPTION

[0028] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0029] Embodiment 1:

[0030] The parents used in the embodiments of the present invention are: Rongxiang No. 7 and L808; Rongxiang No. 7 is a shiitake variety domesticated by our company, and its preservation number is CGMCC No. 40139; L808 is a shiitake variety bred by Dashan Mushroom Research and Development Co., Ltd., Lishui City, Zhejiang Province (National Product Identification No. 2008009). For these two varieties of shiitake, we found in the content and phenotype determination that the fragrance of the dried product of Rongxiang No. 7 is significantly stronger than that of L808. The lentinan content of the dried product of Rongxiang No. 7 is 301 μg / g, which is significantly higher than that of L808 (only 105 μg / g).

[0031] 1. qPCR analysis and screening of key genes of the present invention: Fluorescence quantitative PCR technology was used to compare the transcriptional expression levels of Leggt1, Leggt5, Lecsl2 and Lecsl3 genes of two mushroom varieties, Rongxiang No. 7 and L808, during the drying process. The results are as follows: Figure 1 As shown. In the figure: A is the relative expression of Leggt1 gene, B is the relative expression of Leggt5 gene, C is the relative expression of Lecsl2 gene, and D is the relative expression of Lecsl3 gene; the transcriptional expression level of Lecsl2 gene is significantly higher in Rongxiang No. 7 than in L808, which is consistent with the trend of lentinan content in the two varieties; the transcriptional expression levels of Lecsl3 and Leggt5 genes in L808 are significantly higher than those in Rongxiang No. 7, which is opposite to the trend of lentinan content in the two varieties; and there is no significant difference in the transcriptional expression level of Leggt1 gene between the two varieties. Therefore, it is speculated that the Lecsl2 gene of Rongxiang No. 7 is the key gene for the higher fragrance concentration of this variety than that of L808.

[0032] 2. In step 1, the fluorescent quantitative PCR primers of the gene to be tested and the reference gene are shown in Table 1. The reaction system (10 μL) is as follows: 2.5 μL of 5-fold diluted cDNA template, 5 μL of AceQ qPCR SYBR Green Master Mix, 0.25 μL of each forward and reverse quantitative primer (10 μM), and 2 μL of ddH2O. qRT-PCR program: 95℃3min, 95℃20s, 60℃30s, 72℃30s; 40 cycles; the melting curve temperature is set to 60℃-90℃, and the data is read every 0.5℃ to collect the fluorescent signal. The relative expression of genes is calculated using 2 -ΔΔCT (Pfaffl 2001).

[0033] Table 1 Quantitative primer information of Leggett and Lecsl family protein genes and internal reference genes

[0034]

[0035] 3. Target gene sequence determination and functional analysis:

[0036] The genomic DNA and cDNA of the shiitake mushroom variety Rongxiang No. 7 were amplified by PCR using primers csl-1: 5'-CTAGCTAGCATGTCCAATACACAATCCATCGCGC-3' and csl-2: 5'-CCGCTCGAGTCAAGGCCTAATGGAAGCAAGCTGC-3'. The amplified fragments were cloned and sequenced, and it was found that the Lecsl2 gene of Rongxiang No. 7 had two alleles, named YX7-Lecsl2-1 and YX7-Lecsl2-2, respectively. The sequences are shown in the sequence listing: SEQ ID NO.1 (YX7-Lecsl2-1) and SEQ ID NO.2 (YX7-Lecsl2-2), respectively.

[0037] The YX7-Lecsl2-1 sequence and the YX7-Lecsl2-2 sequence were compared with the genome of L808 in the NCBI gene library. It was found that the YX7-Lecsl2-1 sequence was completely consistent with the sequence of gene ID CM027198.1 on chromosome 2 of L808, which corresponds to an unknown protein (hypothetical protein) with protein ID KAF8830084.1. The YX7-Lecsl2-2 sequence was significantly different from the CM027198.1 sequence of L808. The comparison results of the CM027198.1 sequence of L808 and the YX7-Lecsl2-2 gene are as follows:

[0038]

[0039] YX7:1951-GCTTCCATTAGGCCTTGA-1968

[0040] The comparison results showed that there were 31 base differences in CDS sequence and 35 base differences in intron between L808-CM027198 and YX7-Lecsl2-2 genes.

[0041] Using the amino acid sequence of YX7-Lecsl2-2 (SEQ ID NO. 2) as a template, a BlastP sequence similarity search (E ≤ 0.01) was performed on the NCBI database against the whole genome database of Lentinus edodes, and 45 homologous proteins were obtained (Table 2). The phylogenetic tree was constructed by repeating the neighbor-joining method 500 times using the MEGA5.017 software package. The results are shown in Figure 2 .

[0042] Table 2: Basic biological information of Lentinula edodes CS lyase homologous proteins

[0043]

[0044]

[0045] PLP-dependent transferase, that is, pyridoxal phosphate-dependenttransferase, pyridoxal phosphate-dependent transferase.

[0046] From Table 2 and Figure 2It can be seen that YX7-Lecsl2-2 of Rongxiang No. 7 has the highest homology with XP046091377, LE01Gene02830, KAJ3912023, UHN91801, KAJ3901745 and GAW05867, with an amino acid sequence similarity of 100%, belonging to completely identical enzyme proteins. The next is KAF8830084 (L808), KAJ3890521, KAJ3875707 and KAJ4486755, all of which belong to the CS lyase of the pyridoxal phosphate-dependent transferase class. XP046091377 is a PLP-dependent transferase of Lentinus edodes strain Len (Bin) 0899ss11, which has cysteine ​​sulfoxide lyase (CS lyase) activity; LE01Gene02830 is a new CS lyase encoded by the Lecsl2 gene of Lentinus edodes strain W1-26, which has not only cysteine ​​sulfoxide lyase activity but also cysteine ​​desulfurase activity; YX7-Lecsl2-2 has the same amino acid sequence as them, indicating that it is also a cysteine ​​sulfoxide lyase (CS lyase) with the same enzymatic activity function.

[0047] Although the amino acid sequences of YX7-Lecsl2-2 and XP046091377 are exactly the same, their gene sequences are not exactly the same. There are 11 base differences in the CDS sequences of the two genes, that is, there are 11 SNP sites, but they are all synonymous mutations; there are 7 base differences in the intron sequences of the two genes. The difference in gene sequence may affect gene expression.

[0048] Embodiment 2:

[0049] This example compares the transcription levels of the target genes YX7-Lecsl2-2 and YX7-Lecsl2-1 based on the experimental results of Example 1.

[0050] (1) Design of specific primers for highly expressed genes: Two alleles coexist in the Rongxiang No. 7 strain. Specific primers were designed based on their SNP sites (YX7-Lecsl2-2P: 5'-CGAAGCTTGCAATGAGCTGATG-3' and 5'-GATCCATGACCTTCGTACTCGTT-3'; YX7-Lecsl2-1P: 5'-CGAAGCTTGCAATGAGC TGATG-3' and 5'-GATCCATGACCTTCG TATTCGTC-3'). The third to last base at the 3' end of the above reverse primers was artificially mismatched to improve the specificity of the alleles; the PCR amplification reaction conditions were: 94℃5min; 94℃30s, 60℃45s, 72℃60s, 30cycles; 72℃5min; stored at 4℃. The specific results of the two pairs of primers for amplifying the shiitake mushroom strains are shown in the figure. Figure 3 shown. Figure 3 The M of marker-DL2000 from top to bottom are: 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp; lanes 1-2 are parents: YX7, L808 respectively; lanes 3-7 are haploid strains isolated from single spore of Rongxiang No. 7, namely YX7-16-1, YX7-5-1, YX7-42-1, YX7-49-1 and YX7-71-13A respectively; Figure A is the amplification result of primer YX7-Lecsl2-1P; Figure B is the specific amplification result of YX7-Lecsl2-2P for Lentinus edodes.

[0051] from Figure 3 It can be seen that primer YX7-Lecsl2-1P amplified bands for both L808 and Rongxiang 7, indicating that both strains have the allele YX7-Lecsl2-1; YX7-Lecsl2-2P only amplified bands for Rongxiang 7 (YX7), but not for L808, indicating that Rongxiang 7 has the YX7-Lecsl2-2 allele, while L808 does not have this allele. Among the haploid strains isolated from the single spore of Rongxiang 7 strain, strain YX7-5-1 can only be amplified by primer YX7-Lecsl2-1P, indicating that they have acquired the allele YX7-Lecsl2-1; YX7-16-1, YX7-42-1, YX7-49-1 and YX7-71-1 can only be amplified by primer YX7-Lecsl2-2P, indicating that they have acquired the allele YX7-Lecsl2-2. The above results also show that the two pairs of primers YX7-Lecsl2-1P and YX7-Lecsl2-2P can effectively distinguish the two alleles, and primer YX7-Lecsl2-2P can be used as a specific molecular marker for the highly expressed gene YX7-Lecsl2-2.

[0052] (2) Design of specific fluorescent quantitative PCR primers for highly expressed genes: YX7-Lecsl2-1FP (5'-GTTCATGTTGCTTTGCAATG-3' and 5'-GATCCATGACCTTCG TATTCGTC-3') and YX7-Lecsl2-2FP (5'-GTTCATGTTGCTTTGCAATG-3' and 5'-GATCC ATGACCTTCG TACTCGTT-3'). The genomic DNA of strains L808, Rongxiang No. 7, YX7-5-1, YX7-16-1, YX7-42-1, and YX7-49-1 were amplified using conventional PCR methods. The PCR reaction program was: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, 30 cycles; 72°C for 3 min; stored at 4°C. The amplification results are shown in Figure 2. Figure 4 As shown, Figure 4 M is marker-DL2000, from top to bottom are: 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp; 1-2 are parents: L808, YX7; 3-6 are haploid strains of Rongxiang No. 7: YX7-5-1, YX7-16-1, YX7-42-1 and YX7-49-1; Figure A is the amplification result of primers YX7-Lecsl2-1FP, Figure B is the amplification result of primers YX7-Lecsl2-2FP, from Figure 4 It can be seen that YX7-Lecsl2-1FP only amplified bands for L808, YX7-5-1 (only YX7-Lecsl2-1 gene) and YX7 (with YX7-Lecsl2-1 and YX7-Lecsl2-2 genes), while YX7-Lecsl2-2FP only amplified bands for YX7-16-1, YX7-42-1, YX7-49-1 (only YX7-Lecsl2-2 gene) and YX7. It can be seen that the two pairs of primers specifically amplified only the two alleles of YX7-Lecsl2-1 or YX7-Lecsl2-2, respectively, and the primers had strong specificity.

[0053] This indicates that the above primers can specifically amplify the YX7-Lecsl2-1 or YX7-Lecsl2-2 gene of Lentinus edodes.

[0054] Example 3

[0055] In our study, we found that the allele YX7-Lecsl2-2 has a greater effect on aroma than YX7-Lecsl2-1. The details are as follows:

[0056] (1) qPCR analysis of gene expression levels: qPCR fluorescence quantitative analysis was performed on the transcriptional expression levels of the two alleles in the fruiting bodies of Rongxiang No. 7 Lentinula edodes using specific fluorescence quantitative PCR primers. The fluorescence quantitative PCR reaction system and reaction procedure and the method for calculating the relative expression of genes refer to Example 1. The results of the fluorescence quantitative analysis are shown in Figure 5 As shown, in the samples after hot drying for 0h, 2h, 4h and 6h, the expression level of YX7-Lecsl2-2 gene was much higher than that of YX7-Lecsl2-1 gene, indicating that YX7-Lecsl2-2 is a highly expressed gene compared with YX7-Lecsl2-1, and has a greater contribution to the strong aroma characteristics of Rongxiang No. 7.

[0057] (2) Comparative analysis of the aroma of hybrid offspring with different alleles of Lentinus edodes: Basidiospores of Lentinus edodes Rongxiang No. 7 were collected and single spores were isolated to obtain haploid strains. The primer pair YX7-Lecsl2-1P and YX7-Lecsl2-2P were used to identify the YX7-Lecsl2 genotype of the haploid strain. The haploid strain of Rongxiang No. 7 was hybridized with L808 for single and double hybridization to obtain a batch of hybrids. The amplification results are shown in Figure 2. Figure 6 As shown, Figure 6M is marker-DL2000, and from top to bottom they are: 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp; 1 to 15 are haploids of Rongxiang No. 7: YX7-5-2, YX7-35-1, YX7-35-2, YX7-71-1, YX7-8-1, YX7-8-2, YX7-7-1, YX7-7-2, YX7-7-3, YX7-42-2, YX7-42-3, YX7-49-2, YX7-49-3, YX7-71-2 and YX7-71-3; in the figure, A is the amplification product of YX7-Lecsl2-1P, and B is the amplification product of YX7-Lecsl2-2P; it can be seen from the figure: YX7-5-2, YX7-35-1, YX7-35-2, YX7-8-1 and YX7-8-2 only contain the allele YX7-Lecsl2-1; YX7-71-1, YX7-7-1, YX7-7-2, YX7-7-3, YX7-42-2, YX7-42-3, YX7-49-2, YX7-49-3, YX7-71-2 and YX7-71-3 only contain the allele YX7-Lecsl2-2; Referring to the method of patent ZL202210723974.7, ISSR primer P1: 5'-AGAGAGAGAGAGAGAGT-3' was used to identify two different donor nuclei of L808, and the hybrids receiving the same donor nucleus were grouped together. 18 hybrid strains in the same group were selected. At the same time, the selected 18 hybrids and parent strains Rongxiang No. 7 and L808 were cultivated and grown, and the fruiting bodies were harvested and dried to determine their lentinan content. The results are shown in Table 3:

[0058] Table 3 Comparison of genotypes and lentinan content in the offspring of the single and double hybrids of Rongxiang 7 and L808

[0059]

[0060]

[0061] It can be seen from Table 3 that under the same a-nucleus condition of the L808 donor parent, the Lecsl2 genotypes inherited by the hybrids d7s8-5-1, d7s8-5-2, d7s8-35-1, d7s8-35-2, d7s8-8-1, and d7s8-8-2 are YX7-Lecsl2-1 and 808-Lecsl2, and these two genotypes are exactly the same, so they actually have only one Lecsl2 genotype. The lentinan content of these six hybrid strains ranges from 59.3 to 104.8 μg / g, which is comparable to that of the L808 parent. The Lecsl2 genotypes inherited by the hybrids d7s8-7-1, d7s8-7-2, d7s8-7-3, d7s8-42-1, d7s8-42-2, d7s8-42-3, d7s8-49-1, d7s8-49-2, d7s8-49-3, d7s8-71-1, d7s8-71-2, and d7s8-71-3 are YX7-Lecsl2-2 and 808-Lecsl2, which have two Lecsl2 genotypes. The lentinan content of these 12 hybrid strains ranges from 206.6 to 342.0 μg / g, which is comparable to that of the Rongxiang 7 parent and much higher than that of the L808 parent. It can be seen that the YX7-Lecsl2-2 gene plays an important role in improving the lentinan content of hybrid offspring and is an excellent gene related to the aroma quality traits of shiitake mushrooms.

[0062] From the above, it can be seen that the present invention conducted expression analysis of the aroma genes of Rongxiang No. 7 and the commercial shiitake mushroom L808 and found that the Rongxiang No. 7 variety highly expressed the Lecsl2 gene, and only this variety contained the Lecsl2-2 gene. Therefore, we improved the breeding method: PCR amplification and identification of the haploid strains of shiitake mushrooms, and directed selection of haploid strains containing the Lecsl2-2 gene for hybridization, so as to select hybrid offspring with higher lentinan content, which provided a new breeding idea for the next step of improving the aroma of commercial shiitake mushrooms and effectively shortened the breeding time.

[0063] The above-described embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. The Lecsl2-2 gene for improving the aroma of shiitake mushrooms is characterized by: The Lecsl2-2 gene has an amino acid sequence as shown in SEQ ID NO.

4.

2. The Lecsl2-2 gene according to claim 1, characterized in that The CDS coding sequence of the Lecsl2-2 gene is shown in SEQ ID NO.3, and the nucleic acid sequence is shown in SEQ ID NO.

2.

3. A high-lentinin content shiitake mushroom comprising the Lecsl2-2 gene as claimed in claim 1, characterized in that: The shiitake mushroom is Rongxiang No. 7, and its preservation number is: CGMCC No.40139.

4. A specific molecular marker primer pair for detecting Lecsl2-2 gene, characterized in that: The upstream primer sequence of the primer pair is 5'-CGAAGCTTGCAATGAGCTGATG-3'; the downstream primer sequence is 5'-GATCCATGACCTTCG TACTC GTT-3'.

5. The specific molecular marker primer pair according to claim 4, characterized in that: The reaction conditions for the PCR amplification of the specific molecular marker primer pair are 94° C. for 5 min; 94° C. for 30 s, 60° C. for 45 s, 72° C. for 60 s, 30 cycles; 72° C. for 5 min; and storage at 4° C.

6. A method for detecting high lentinan mushroom varieties using the specific molecular marker primer pair as claimed in claim 4, characterized in that: The method comprises the following steps: using the primer pair to perform PCR amplification on genomic DNA of shiitake mushrooms. If a target fragment can be amplified, it indicates that the shiitake mushroom variety is a high-lentinin variety.

7. A haploid strain comprising the Lecsl2-2 gene as claimed in claim 1, characterized in that: The haploid strain is a single spore isolate of Rongxiang No. 7, and the single spore isolate of Rongxiang No. 7 can be amplified by the specific molecular marker primer pair described in claim 4 to obtain the target fragment.

8. A method for crossbreeding shiitake mushrooms to increase lentinan, characterized in that: The method comprises: using the haploid strain as claimed in claim 7 to hybridize with other haploid strains or diploid strains of Lentinus edodes to obtain hybrid offspring that can produce mushrooms normally.

9. A method for identifying high-lentinin hybrid offspring, characterized in that: The method comprises: using the specific molecular marker primer pair as described in claim 4 to detect the hybrid offspring of Rongxiang No. 7 or its haploid strain and other commercial shiitake mushrooms; if the target fragment can be amplified, it is proved that the hybrid offspring is a high-lentinin variety.

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