A molecular marker associated with lentinus edodes number and application

Abstract

The present application belongs to the technical field of fungal genetic breeding, and particularly relates to a molecular marker associated with the number of Lentinula edodes and application. The molecular marker is located at the 3865993th locus of chromosome 7 of the genome of Lentinula edodes, and is a 15bp insertion mutation. By using the molecular marker and detection method provided by the present application, the number of fruiting bodies (number of mushrooms) of Lentinula edodes at the mushroom stage can be efficiently determined at the mycelium stage, so as to accelerate the breeding process and improve the breeding efficiency. The molecular marker is a universal molecular marker, is less limited by genetic materials, and is widely applied.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of fungal genetic breeding technology, and particularly relates to a molecular marker associated with the number of Lentinula edodes fruiting bodies and application thereof. BACKGROUND

[0002] Lentinula edodes is the most produced edible mushroom in the world (Royse, D. J. et al. Current Overview of Mushroom Production in the World. In: Diego CZ, Pardo-Giménez A eds., Edible and Medicinal Mushrooms: Technology and Applications. Hoboken: Wiley, 2017. 5-13.), and plays an extremely important role in the edible mushroom industry in China. China is also the largest producer, exporter and consumer of Lentinula edodes in the world (Li Y. Lentinula edodes Industry has a good development prospect. Food Science. 2005. 07: 261-266.). Lentinula edodes has a unique aroma and delicious taste, and is known as "Queen of Mushrooms" and "Mountain Delicacy" (Chang, S. T. 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, vitamins and minerals, and has the functions of preventing tumors, increasing immunity, reducing blood lipids, resisting thrombosis, and protecting the stomach and liver (Bian Y. Edible Fungus Cultivation (3rd Edition). Beijing: Higher Education Press, 2017. 138-140). Due to its high nutritional and medicinal value, Lentinula edodes is deeply loved by people.

[0003] Important economic traits of Lentinula edodes are mostly quantitative traits, such as yield, number of fruiting bodies, single fruit weight, early maturity, and mycelial growth rate (Xiao Y et al. Association analysis and its application in fungal genetics research. Mycosystema. 2016. 35: 782-790). Unlike qualitative traits, quantitative traits are mostly continuous variation, regulated by multiple genes, with complex genetic basis, and easily affected by environmental factors. There is no clear correspondence between phenotype and genotype, making it difficult to improve genetically (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 excellent varieties seriously restricts the development of edible mushroom industry. Breeding 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, which has certain effect, but has the disadvantages of low efficiency, low stability, long cycle and large workload. In recent years, with the development of molecular biology and genomics, molecular breeding technology has emerged, including molecular marker-assisted breeding, whole genome selection breeding, transgenic breeding, and molecular design breeding.

[0005] InDel (Insertion / Deletion) molecular marker method, i.e. insertion / deletion polymorphism marker, is a genetic marker based on insertion or deletion (i.e. increase or decrease of sequence) at known positions in the genome. 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 prolyl 4-hydroxylase-like 2-oxoglutarate-Fe(II) dioxygenase (Ofd1) gene has a function of responding to changes in oxygen concentration (Hammarlund, E. U. Harnessing hypoxia as an evolutionary driver of complex multicellularity. Interface Focus, 2020, 10(4): 20190101.). The research group of the inventors previously found that the Lentinula edodes homolog LeOfd1 is a candidate gene for regulating the number of mushrooms through multi-omics analysis and genome-wide association analysis (Zhang, J. C. 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 the number of mushrooms (the number of fruiting bodies) and the single mushroom weight. Both the number of mushrooms and the single mushroom weight are positively correlated with yield, while the number of mushrooms and the single mushroom weight are negatively correlated; the contribution rate of the number of mushrooms to yield is higher than that of the single mushroom weight, and compared with the single mushroom weight, genetic improvement of the number of mushrooms has a higher yield-increasing effect (Gong, W. B., et al. Phenotypic evaluation and analysis of important agronomic traits in the hybrid and natural populations of Lentinula edodes. Scientia Horticulturae, 2014, 179: 271-276). Therefore, development of molecular markers associated with the number of mushrooms of Lentinula edodes is one of the effective means to accelerate the breeding process of new strains of Lentinula edodes and has an important guiding role for breeding new varieties with high yield. SUMMARY

[0008] To solve the above technical problems, the present application provides an InDel marker for molecular-assisted breeding of the number of mushrooms of Lentinula edodes and application thereof.

[0009] The inventors find that an InDel sequence of 15 bp only exists in the strains with more mushrooms by studying the Lentinula edodes LeOfd1 gene and comparing the differences between the strains with more mushrooms and the strains with less mushrooms, the InDel sequence (SEQ ID NO. 1) is located in the exon region of the LeOfd1 gene, specifically at the 3865993th site of the seventh chromosome of the Lentinula edodes genome.

[0010] The application of the above-mentioned InDel marker in the selection or assisted breeding of the mushroom number trait of Lentinula edodes: DNA in the mycelium stage of Lentinula edodes is extracted, and whether the InDel marker exists is detected by a PCR detection method or a sequencing method, and the Lentinula edodes strains containing the InDel marker have more mushroom numbers.

[0011] In specific embodiments of the application, a pair of primers for detecting the InDel marker is provided, and the sequences thereof are shown as SEQ ID NO. 2 and 3, and the mushroom number trait phenotype of Lentinula edodes is judged 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 Lentinula edodes is of the more mushroom number type; if the PCR amplification product has two characteristic bands of 133 bp and 118 bp, the Lentinula edodes is of the more mushroom number type; and if the PCR amplification product has only one characteristic band of 118 bp (SEQ ID NO. 5), the Lentinula edodes is of the less mushroom number type.

[0012] The application has the following advantages:

[0013] 1. The inventors screen out a molecular marker GS related to the mushroom number of Lentinula edodes by studying the LeOfd1 gene of Lentinula edodes and comparing the gene sequence differences between the strains with more mushrooms and the strains with less mushrooms, and the mushroom number trait phenotype of Lentinula edodes can be quickly identified by using the molecular marker of the application, without having to confirm the mushroom number by cultivation test.

[0014] 2. The application belongs to the molecular marker assisted breeding of edible fungi, and the method is simple and feasible, which is beneficial to improving the efficiency and saving the cost.

[0015] 3. The molecular marker of the application has the characteristics of convenient detection, stable amplification product and high specificity, and can be simply, quickly and high-throughput applied to the high-yield breeding practice and strain identification of Lentinula edodes. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 LeOfd1 is detected as a candidate gene for regulating the mushroom number trait by association analysis. The genes in the ±2 kb range of the significant association sites detected by association analysis are trait-associated candidate genes. The LeOfd1 gene is located in the ±2 kb range of the arrow-indicated association site.

[0017] Figure 2To analyze the effect of InDel sequence on the structure of LeOfd1 gene coding protein. According to the model prediction, the site is located in the loop of protein structure, and the insertion of sequence may affect the protein properties, thereby leading to the difference in the number of Lentinula edodes.

[0018] Figure 3 The box plot of the distribution of the number of fruiting bodies of strains with different genotypes of GS marker sites. 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 is no difference)

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

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

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

[0022] 1. The present application uses 133 Lentinula edodes strains (including 99 wild strains and 34 cultivated strains), most of which are from China, except for four strains from South Korea, Brazil, Australia and Finland, and a wild strain from Vietnam. Genome resequencing is performed on these strains for variant site analysis. After artificial cultivation, the number of fruiting strains is 118, and the number of fruiting bodies is systematically investigated.

[0023] 2. Genome-wide association study (GWAS) was performed using linear mixed model in FaST-LMM (Lippert, C., Listgarten, J., Liu, Y. et al. FaST linear mixed models for genome-wide association studies. Nat Methods 8, 833-835, 2011) and LeOfd1 gene was found to be significantly associated with the number of fruiting bodies (Fig. 1). Figure 1 Within this gene, we aligned the re-sequencing data of YS1553, YS1518, YS234, YS115 and YS107, five strains with the most number of fruiting bodies, and ZP28, ZP67, ZP10, YS37 and YS67, five strains with the least number of fruiting bodies, to the reference genome of L. japonicus Wpm-1 by sequencing, and found an InDel sequence of 15 bp in length at the 3865993th site on chromosome 7 of L. japonicus, named GS. The allelic genotypes were 0 / 0 (lack of GS marker), 0 / 1 (heterozygous) and 1 / 1 (containing GS marker). The results of the analysis showed that the number of fruiting bodies of strains with genotype 1 / 1 was significantly higher than that of strains with genotype 0 / 0. Analysis of this fragment showed that the InDel was located in the exon region of LeOfd1, resulting in the insertion of five amino acids. According to the model prediction, the site is located in the loop of the protein structure, and the insertion of the sequence may affect the protein properties, thereby leading to the difference in the number of fruiting bodies of L. japonicus (Fig. 2). Figure 2

[0024] Example 2 Detection of GS molecular marker

[0025] 1. According to the re-sequencing results, we used the software GATK (Genome Analysis Toolkit) to analyze the high-throughput sequencing data, extracted the InDel typing data, found the location of GS, and genotyped 133 strains. By analyzing the number of fruiting bodies corresponding to the genotype, we found that the insertion / deletion fragment ACGTGGCCATCGCTG (SEQ ID NO. 1) on chromosome 7 of L. japonicus had an effect on the number of fruiting bodies of L. japonicus. The L. japonicus strains with the insertion of the fragment shown in SEQ ID NO. 1 were strains with more fruiting bodies, and the L. japonicus strains with the deletion of the fragment shown in SEQ ID NO. 1 were strains with fewer fruiting bodies (Table 2).

[0026] ​2.118 Lentinula edodes strains in Table 2, in which 52 strains have genotype 0 / 0, 9 strains have genotype 0 / 1, and 59 strains have genotype 1 / 1. The average number of fruiting bodies of the 52 strains containing 0 / 0 genotype is 24.95 per stick, and the average number of fruiting bodies of the 9 strains containing 0 / 1 genotype and the 59 strains containing 1 / 1 genotype is 44.58 per stick and 55.64 per stick, respectively. Variance analysis shows that the number of fruiting bodies of strains containing 0 / 1 and 1 / 1 genotype is significantly more than that of 0 / 0 genotype (P<0.01), but there is no significant difference in the number of fruiting bodies between strains containing 0 / 1 and 1 / 1 genotype. Figure 3

[0027] Table 2 Phenotype of fruiting body number of 118 Lentinula edodes strains and genotype at GS locus

[0028]

[0029]

[0030]

[0031]

[0032] Example 3 Verification of molecular markers related to fruiting body number of Lentinula edodes

[0033] Twelve strains were randomly selected from the above table, and genomic DNA was extracted using the CTAB method. The PCR amplification system was as follows: 2x Phanta Max Buffer 25 μL, primers (10 μmol / L, primer sequences are shown in SEQ ID NO. 2 and 3) 2 μL each, dNTPMix (10 mmol / L) 1 μL, Phanta Max Super-Fidelity DNA Polymerase 1 μL, template 200 ng, and ddH2O to 50 μL. Reaction parameters: 95°C pre-denaturation for 3 min, 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 30 s, 34 cycles, and 72°C extension for 10 min.

[0034] Electrophoresis was performed using 7% polyacrylamide gel with a program of 400 V voltage, 130 mA current, and 30 min. The gel was stained with Ultra GelRed (10,000x) dye for 15 min, and the gel was placed in a gel illuminator to obtain a gel picture.

[0035] ​The PCR product is subjected to 7% polyacrylamide gel electrophoresis. If there is a characteristic band of 133 bp (SEQ ID NO. 4), the Lentinula edodes is a multiple number type; if there is a characteristic band of 133 bp (SEQ ID NO. 4) and a characteristic band of 118 bp (SEQ ID NO. 5), the Lentinula edodes is also a multiple number type; if there is only a characteristic band of 118 bp (SEQ ID NO. 5), the Lentinula edodes is a less number type. The multiple number type can be marked as M, and the less number type can be marked as L. We define the number of single-stick mushrooms less than 30 as a less number type, which is marked as L; and the number of single-stick mushrooms greater than 30 as a multiple number type, which is marked as M.

[0036] As Figure 4 As shown in Table 3, the band types of the strains with more mushrooms and the strains with less mushrooms are different after PCR amplification, so that the strains of Lentinula edodes with different mushroom numbers are distinguished. According to the mushroom number phenotype verification of this batch of Lentinula edodes strains, it is found that the accuracy of the marker for judging the multiple mushroom number is 66.7%.

[0037] The above identification results show that the molecular marker and method provided by the present application can accurately and efficiently judge the number of mushrooms at the mycelial stage of Lentinula edodes, effectively reduce the cultivation scale of breeding materials, reduce the workload of phenotype identification in the later cultivation test, speed up the breeding process, and improve the breeding efficiency. The molecular marker is a universal molecular marker, which is less limited by genetic materials and widely applicable. Therefore, the InDel molecular marker of the present application can effectively identify the phenotype of the mushroom number trait, and can be used for molecular marker assisted selection of Lentinula edodes varieties with multiple mushroom numbers.

[0038] Table 3: Molecular marker PCR electrophoresis detection of 12 Lentinula edodes strains and their corresponding mushroom number trait phenotypes

[0039] Lentinula edodes strain name Marker determination result Number of fruiting bodies phenotype Number of fruiting bodies (per 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 above-described embodiments are only preferred embodiments of the present application, merely used to explain the present application, and are not intended to limit the scope of the present application. For those skilled in the art, of course, other embodiments can be easily made by substitution or change based on the technical content disclosed in the present specification, and therefore, any changes and improvements made on the principle of the present application shall be included in the scope of the present application.

Claims

1. The use of molecular markers in the selection or assisted breeding of the number of fruiting bodies of Lentinula edodes, characterized by, The molecular marker is an InDel marker located at the 3865993th site on the chromosome 7 of the Lentinula edodes monokaryon Wpm-1 reference genome, and the sequence is shown as SEQ ID NO.

1. Compared with Lentinula edodes without the molecular marker, the Lentinula edodes strain with the molecular marker has more mushroom numbers.

2. The use of primers in the selection or assisted breeding of Lentinula edodes fruit number traits, characterized in that, The genomic DNA of the Lentinula edodes sample is subjected to PCR amplification by using the primers shown as SEQ ID NO. 2 and 3. If the amplification product has only one characteristic band of 133 bp, and the sequence of the characteristic band is shown as SEQ ID NO. 4, the Lentinula edodes is of the high-mushroom-number type. If the amplification product has two characteristic bands of 133 bp and 118 bp, and the sequences of the characteristic bands are shown as SEQ ID NO. 4 and 5, the Lentinula edodes is of the high-mushroom-number type. If the amplification product has only one characteristic band of 118 bp, and the sequence of the characteristic band is shown as 5, the Lentinula edodes is of the low-mushroom-number type.

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