Molecular marker associated with number of shiitake mushrooms and application of molecular marker

Abstract

The invention belongs to the technical field of fungus genetic breeding, and particularly relates to a molecular marker associated with the number of shiitake mushrooms and application. The molecular marker is located at the 3865993th site of a chromosome 7 of a mushroom genome and is a section of insertion mutation of 15bp. By utilizing the molecular marker and the detection method provided by the invention, the quantity (mushroom number) of mushroom sporocarp in the fruiting stage can be efficiently judged in the mushroom hypha stage, so that the breeding process is accelerated, and the breeding efficiency is improved. The molecular marker is a universal molecular marker, is slightly limited by genetic materials and is wide in application.

Description

Technical Field

[0001] The invention belongs to the technical field of fungal genetic breeding, and in particular relates to a molecular marker associated with the number of shiitake mushrooms (the number of fruiting bodies) and an application thereof. Background Art

[0002] Shiitake mushroom is the edible fungus with the largest output in the world (Royse, DJ et al. Current Overview of Mushroom Production in the World. In: Diego CZ, Pardo-Giménez Aeds., Edible and Medicinal Mushrooms: Technology and Applications. Hoboken: Wiley, 2017.5-13.), and plays an extremely important role in my country's edible fungus industry. China is also the world's largest producer, exporter and consumer of shiitake mushrooms (Li Yuemei. The shiitake mushroom industry has good development prospects. Food Science. 2005.07:261-266.). Lentinula edodes has a unique aroma and delicious taste. It is known as the "Queen of Mushrooms" and "Mountain Treasure" (Chang, ST Past and present trends in the production of Lentinula edodes in Asia. Mushroom Biology and Mushroom Products. 2002. 4: 1-8.). Lentinula edodes is rich in protein, amino acids, multiple vitamins and mineral elements, and has the effects of preventing and treating tumors, increasing immunity, lowering blood lipids, resisting thrombosis, and strengthening the stomach and protecting the liver (Bian Yinbing. Edible Fungus Cultivation (3rd Edition). Beijing: Higher Education Press. 2017. 138-140). It is deeply loved by people because of its high nutritional value and medicinal value.

[0003] Most of the important economic traits of shiitake mushrooms are quantitative traits, such as yield, number of mushrooms (number of fruiting bodies), single mushroom weight, early maturity, mycelial growth rate, etc. (Xiao Yang et al. Association analysis and its application in fungal genetics research. Journal of Mycology. 2016. 35: 782-790). Unlike quality traits, quantitative traits mostly show continuous variation, are regulated by multiple genes, have a complex genetic basis, and are easily affected by environmental factors. There is no clear correspondence between phenotype and genotype, making it difficult to carry out genetic improvement (Santoyo, F. et al. Quantitative linkage mapping of lignin degrading enzymatic activities in Pleurotus ostreatus. Enzyme and Microbial Technology. 2008. 43 (2): 137-143.).

[0004] The lack of high-quality varieties has seriously restricted the development of the edible fungi industry. Cultivating new varieties with high yield, high quality and high resistance is the most effective measure to solve this problem. Conventional breeding methods such as systematic breeding and hybrid breeding are aimed at phenotypic selection. Although they have certain effects, they have disadvantages such as low efficiency, low stability, long cycle and large workload. In recent years, with the development of disciplines such as molecular biology and genomics, molecular breeding technology has emerged, mainly including molecular marker-assisted breeding, whole genome selection breeding, transgenic breeding, molecular design breeding, etc.

[0005] InDel (Insertion / Deletion) molecular marker method, that is, insertion / deletion polymorphism marker, is a genetic marker based on the insertion or deletion (i.e., increase or decrease of sequence) at a known position in the genome sequence. This type of molecular marker is widely present in the genome and can be used to study genetic diversity, genetic mapping, population genetics, association analysis and molecular evolution.

[0006] The proline 4-hydroxylase-like 2-oxoglutarate-Fe(II) dioxygenase (Ofd1) gene has the function of responding to changes in oxygen concentration (Hammarlund, EU Harnessing hypoxia as an evolutionary driver of complex multicellularity. Interface Focus, 2020, 10(4): 20190101.). The inventor's research group previously combined multi-omics analysis and genome-wide association analysis to find that its shiitake homologous gene LeOfd1 is a candidate gene for regulating mushroom number (Zhang, JC et al. Population genomics provides insights into the genetic basis of adaptive evolution in the mushroom-forming fungus Lentinula edodes. Journal of Advanced Research, 2022, 38: 91-106.).

[0007] The yield traits of Lentinula edodes are composed of two traits: mushroom number (number of fruiting bodies) and single mushroom weight. Both mushroom number and single mushroom weight are positively correlated with yield, while mushroom number and single mushroom weight are negatively correlated; the contribution rate of mushroom number to yield is higher than that of single mushroom weight. Compared with single mushroom weight, genetic improvement of mushroom number traits has a higher yield-increasing effect (Gong, WB, et al.. Phenotypicevaluation and analysis of important agronomic traits in the hybrid and natural populations of Lentinula edodes. Scientia Horticulturae, 2014, 179: 271-276). Therefore, the development of molecular markers associated with the mushroom number trait is one of the effective means to accelerate the breeding process of new strains of Lentinula edodes, and plays an important guiding role in the breeding of new high-yield varieties. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides an InDel marker for molecule-assisted selection of mushroom number and its application.

[0009] The inventors studied the LeOfd1 gene of Lentinus edodes and compared the differences between strains with more mushrooms and strains with fewer mushrooms. They found that a 15bp InDel sequence only existed in strains with more mushrooms. The InDel sequence (SEQ ID NO.1) was located in the exon region of the LeOfd1 gene, specifically at site 3865993 of chromosome VII of the Lentinus edodes genome.

[0010] The application of the above InDel marker in the selection of mushroom number traits or auxiliary breeding of Lentinus edodes: extract DNA from the mycelium stage of Lentinus edodes, detect the presence of the InDel marker by PCR detection or sequencing, and the Lentinus edodes strain containing the InDel marker has a larger number of mushrooms.

[0011] In a specific embodiment of the present invention, a pair of primers for detecting the InDel marker is provided, and the sequences thereof are shown in SEQID NO.2 and 3. The phenotype of the mushroom number trait of Lentinus edodes is determined according to the DNA amplification band type: if the PCR amplification product has only one characteristic band of 133 bp (SEQ ID NO.4), the mushroom is of a large mushroom number type; if the PCR amplification product has two characteristic bands of 133 bp and 118 bp, the mushroom is of a large mushroom number type; if the PCR amplification product has only one characteristic band of 118 bp (SEQ ID NO.5), the mushroom is of a small mushroom number type.

[0012] The present invention has the following advantages:

[0013] 1. The inventors studied the LeOfd1 gene of Lentinus edodes and screened out a molecular marker GS related to the number of Lentinus edodes by comparing the gene sequence differences between strains with more mushroom numbers and strains with less mushroom numbers. The molecular marker of the present invention can be used to quickly identify the phenotype of the number of Lentinus edodes mushrooms without having to confirm the number of mushrooms after cultivation tests.

[0014] 2. The present invention relates to molecular marker-assisted breeding of edible fungi, and the method is simple and feasible, which is beneficial to improving efficiency and saving costs.

[0015] 3. The molecular marker of the present invention has the characteristics of convenient detection, stable amplification product and high specificity, and can be applied to high-yield breeding practice of shiitake mushrooms and strain identification in a simple, fast and high-throughput manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The association analysis detected LeOfd1 as a candidate gene regulating mushroom number traits. The red horizontal line is the significant association site detected by the association analysis, and the genes within the ±2kb range are the trait-associated candidate genes. The LeOfd1 gene is located within the ±2kb range of the association site indicated by the arrow.

[0017] Figure 2Effect of InDel sequence pair on the structure of the protein encoded by LeOfd1 gene. According to the model prediction, this locus is located on the loop of the protein structure, and the insertion of the sequence may affect the protein properties, resulting in the difference in the number of fruiting bodies of Lentinula edodes.

[0018] Figure 3 Box plot of the phenotypic values of the fruiting body number trait among strains with different GS marker locus genotypes. 0 / 0 is the genotype lacking the GS marker fragment, 1 / 1 is the genotype containing the GS marker fragment, and 0 / 1 is the heterozygous genotype. (**0.01 < p < 0.05, ****p < 0.01, ns means no difference)

[0019] Figure 4 Electrophoretogram of the PCR amplification products of InDel molecular marker GS. The DNA template was extracted from 12 randomly selected Lentinula edodes strains, and the concentration of the PAGE gel was 7%. According to the electrophoresis results, the strains of 0 / 0 type are ZP9, YS72, and YS45, the strains of 0 / 1 type are YS62, YS73, YS27, YS82, YS48, YS99, and YS84, and the strains of 1 / 1 type are YS90 and YS116. Specific implementation mode

[0020] Unless otherwise specified, the methods used in the following examples are all conventional biological experimental methods. The primers used were all synthesized by Wuhan Tianyi Huiyuan Biotechnology Co., Ltd., and the sequencing was completed by Wuhan Tianyi Huiyuan Biotechnology Co., Ltd. Unless otherwise specified, the specific experimental methods involved are all conventional methods or implemented according to the conditions recommended in the manufacturer's instructions. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described herein are for illustrative purposes only. The reference genome data of Lentinula edodes monokaryon Wpm-1 and the population genome data of Lentinula edodes used in the experiment are the sequencing results of the research group where the inventor is located.

[0021] Example 1 Development of molecular markers related to the fruiting body number trait of Lentinula edodes

[0022] 1. In this invention, 133 Lentinula edodes strains (including 99 wild strains and 34 cultivated strains) were used. Most of the strains are from China, except for four strains from South Korea, Brazil, Australia, and Finland, and wild strains from Vietnam. These strains were subjected to genome resequencing for variant site analysis. After artificial cultivation, the number of strains that could produce fruiting bodies was 118, and the fruiting body number was systematically investigated.

[0023] 2. Using the linear mixed model in FaST-LMM to conduct genome-wide association analysis (GWAS) (Lippert, C., Listgarten, J., Liu, Y. et al.FaST linear mixed models for genome-wide association studies. Nat Methods 8, 833–835, 2011), it was found that the LeOfd1 gene is a candidate gene significantly associated with the number of mushrooms ( Figure 1 ). Within this gene, we sequenced and aligned the resequencing data of the five strains with the largest number of mushrooms, YS1553, YS1518, YS234, YS115 and YS107, and the five strains with the smallest number of mushrooms, ZP28, ZP67, ZP10, YS37 and YS67, to the reference genome of Lentinus edodes Wpm-1. We found a 15bp InDel sequence located at the 3865993th site on chromosome 7 of Lentinus edodes, named GS. The allele types are 0 / 0 (lacking GS marker), 0 / 1 (heterozygous) and 1 / 1 (containing GS marker). The analysis results showed that the number of mushrooms in the strain with genotype 1 / 1 was significantly higher than that in the strain with genotype 0 / 0. By analyzing the fragment, we found that the InDel was located in the exon region of LeOfd1, resulting in the insertion of five amino acids. According to the model prediction, this site is located in the loop of the protein structure. The insertion of the sequence may affect the protein properties, thus leading to the difference in the number of mushrooms ( Figure 2 ).

[0024] Example 2 Detection of GS Molecular Markers

[0025] 1. According to the resequencing results, we used the software GATK (Genome Analysis Toolkit) to analyze the high-throughput sequencing data, extract InDel typing data, find the location of GS, and perform genotyping on 133 strains. By analyzing the number of mushrooms corresponding to the genotype, it can be seen that the insertion / deletion fragment ACGTGGCCATCGCTG (SEQ ID NO.1) on the seventh chromosome of Lentinus edodes has an effect on the number of mushrooms. The Lentinus edodes with the fragment shown in SEQ ID NO.1 inserted have a larger number of mushrooms; the Lentinus edodes with the fragment shown in SEQ ID NO.1 missing have a smaller number of mushrooms (Table 2).

[0026] 2. The mushroom number traits of 118 shiitake mushroom strains and their corresponding genotypes at the GS locus are shown in Table 2. Among them, 52 shiitake mushroom strains have a genotype of 0 / 0, 9 shiitake mushroom strains have a genotype of 0 / 1, and 59 shiitake mushroom strains have a genotype of 1 / 1. According to the cultivation test, the average number of mushrooms of the 52 shiitake mushroom strains containing the 0 / 0 allele was 24.95 per stick, and the average number of mushrooms of the 9 shiitake mushroom strains containing the 0 / 1 allele and the 59 shiitake mushroom strains containing the 1 / 1 allele was 44.58 per stick and 55.64 per stick, respectively. The variance analysis showed that the number of mushrooms of the strains containing the 0 / 1 and 1 / 1 alleles was significantly greater than that of the 0 / 0 strains ( Figure 3 ), but there was no significant difference in mushroom number between strains with 0 / 1 and 1 / 1 alleles.

[0027] Table 2 Phenotypes of mushroom number traits and genotypes at GS loci of 118 Lentinula edodes strains

[0028]

[0029]

[0030]

[0031]

[0032] Example 3 Verification of molecular markers associated with mushroom number traits

[0033] Randomly select 12 strains from the above table and extract genomic DNA using the CTAB method. The PCR amplification system is as follows: 2×Phanta Max Buffer 25μL, primers (10μmol / L, primer sequences are shown in SEQ ID NO.2 and 3) 2μL each, dNTPMix (10mmol / L) 1μL, Phanta Max Super-Fidelity DNA Polymerase 1μL, template 200ng, supplemented with ddH2O to 50μL. Reaction parameters: 95℃ pre-denaturation 3min, 95℃ denaturation 30s, 60℃ annealing 30s, 72℃ extension 30s, cycle number 34, 72℃ extension 10min.

[0034] 7% polyacrylamide gel electrophoresis was performed at 400 V, 130 mA, and 30 minutes. Ultra GelRed (10,000×) dye was used to stain the gel for 15 minutes, and the gel was spread on a gel imaging apparatus to obtain a gel image.

[0035] The PCR products were subjected to 7% polyacrylamide gel electrophoresis. If there is a characteristic band of 133bp (SEQ ID NO.4), the mushroom is of the type with many mushrooms; if there is both a characteristic band of 133bp (SEQ ID NO.4) and a characteristic band of 118bp (SEQID NO.5), the mushroom is also of the type with many mushrooms; if there is only a characteristic band of 118bp (SEQ ID NO.5), the mushroom is of the type with few mushrooms. The type with many mushrooms can be marked as M, and the type with few mushrooms can be marked as L. We define the type with less than 30 mushrooms per stick as the type with few mushrooms, marked as L; the type with more than 30 mushrooms per stick as the type with many mushrooms, marked as M.

[0036] like Figure 4 As shown in Table 3, after PCR amplification, there were differences in the banding patterns between strains with more mushroom numbers and strains with fewer mushroom numbers, thereby distinguishing shiitake mushroom strains with different mushroom numbers. Based on the mushroom number phenotype verification of this batch of shiitake mushroom strains, it was found that the accuracy of this marker in judging the number of mushrooms was 66.7%.

[0037] The above identification results show that the molecular marker and method provided by the present invention can accurately and efficiently determine the number of mushrooms in the mycelium stage of shiitake mushrooms, effectively reduce the cultivation scale of breeding materials, reduce the workload of phenotypic identification during later cultivation tests, accelerate the breeding process, and improve breeding efficiency. The molecular marker is a universal molecular marker, which is less restricted by genetic materials and has a wide range of applications. Therefore, the InDel molecular marker of the present invention can effectively identify the phenotype of mushroom number traits and can be used for molecular marker-assisted breeding of high-mushroom-number shiitake mushroom varieties.

[0038] Table 3 Molecular markers detected by PCR electrophoresis of 12 Lentinula edodes strains and their corresponding phenotypes of mushroom number traits

[0039] Shiitake mushroom strain name Mark judgment results Mushroom number phenotype Number of mushrooms (pieces / stick) ZP9 L L 22.43 YS72 L L 21.38 YS62 M L 29.38 YS90 M M 67.13 YS73 M M 71.5 YS116 M L 26.75 YS27 M M 62.88 YS82 M M 55 YS48 M L 23 YS45 L L 19.5 YS99 M M 48.13 YS84 M L 24.25

[0040] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, other implementation methods can certainly be easily made by replacement or modification based on the technical contents disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. A molecular marker associated with the number of Lentinula edodes mushrooms, characterized in that: The molecular marker is an InDel marker located at the 3865993rd site on chromosome 7 of Lentinula edodes, and its sequence is shown in SEQ ID NO.

1.

2. Use of the molecular marker described in claim 1 in selection of mushroom number traits or assisted breeding of Lentinus edodes.

3. The use according to claim 2, characterized in that: Compared with shiitake mushrooms not containing the molecular marker, the number of mushrooms of the shiitake mushroom strain containing the molecular marker is greater.

4. Use of a reagent for detecting the molecular markers of claim 1 in selection of mushroom number traits or assisted breeding.

5. The use according to claim 4, characterized in that: The reagent is a primer for amplifying the molecular marker according to claim 1.

6. The use according to claim 5, characterized in that: The sequences of the primers are shown in SEQ ID NOs. 2 and 3.

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