An in-del molecular marker primer set of dictyophora rubrozonata, an in-del-PCR reaction system and application thereof and a method for evaluating population genetic diversity

Abstract

The application discloses an InDel molecular marker primer group of Dictyophora rubrocyannis, an InDel-PCR reaction system and application and a method for evaluating population genetic diversity, and belongs to the technical field of molecular biology. TM 10 μL, and the rest is enzyme-free ddH2O. After amplification, electrophoresis detection of bands, genotype coding, conversion of InDel co-dominant allele data and genetic diversity parameter calculation, the system is used for evaluation of the population genetic diversity of the Dictyophora rubrocyannis and molecular marker assisted breeding. The evolutionary relationship tree of strains can be constructed to clearly determine the genetic relationship, and the identification result is comprehensive, accurate and objective, and can efficiently reveal the genetic relationship and variation level of the population of the Dictyophora rubrocyannis, and solves the problems of poor accuracy and low efficiency of traditional identification methods.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to an InDel molecular marker primer set for *Dictyophora indicum*, an InDel-PCR reaction system, their applications, and a method for evaluating population genetic diversity. Background Technology

[0002] Dictyophora rubrovolvata, a fungus belonging to the family Phallaceae and the genus Phallus, is known for its unique flavor, rich nutritional value, and high nutritional content. It is rich in various polysaccharides, possesses strong reducing power and the ability to inhibit hydroxyl free radicals, and exhibits significant in vitro antioxidant activity. Wild Dictyophora rubrovolvata is mainly distributed in the humus soil of bamboo forests in southwestern my country, including Guizhou and Yunnan provinces, and is a local specialty edible fungus resource. With the continuous advancement of industrialized cultivation technology, the cultivation range of Dictyophora rubrovolvata has gradually expanded nationwide, becoming an important cultivated variety in my country's edible fungus industry. However, the current development of the Dictyophora rubrovolvata industry faces the critical problem of lagging germplasm resource research. The collection, identification, evaluation, and breeding of its germplasm resources lag far behind the expansion of cultivation scale. The lack of efficient and precise molecular biology techniques for genetic analysis of Dictyophora rubrovolvata germplasm greatly restricts the healthy and sustainable development of the industry.

[0003] Currently, the main identification methods for *Dictyophora indicum* strains are morphological identification and antagonistic reaction identification. Morphological identification relies on phenotypic traits such as fruiting body morphology and hyphal characteristics, which are easily affected by external factors such as culture conditions and growth stages, resulting in poor accuracy and stability. Antagonistic reaction identification can only distinguish between different strains and cannot achieve specific identification. Furthermore, the objectivity of the identification results is insufficient, and neither of these methods can meet the needs of genetic diversity analysis of *Dictyophora indicum* populations.

[0004] InDel molecular markers are a type of molecular marker developed based on whole-genome high-throughput sequencing technology. They have advantages such as abundant quantity, high amplification accuracy, good experimental stability, and simple and fast operation. They have been widely used in germplasm identification, genetic diversity analysis, and molecular marker-assisted breeding of various edible fungi. A dedicated InDel molecular marker primer set has been developed for *Dictyophora indicum*, a standardized PCR reaction system and amplification procedure have been constructed, and a supporting genetic diversity evaluation method has been established. This can fill the technical gap in molecular germplasm research of *Dictyophora indicum* and provide core technical support for its germplasm resource protection and variety innovation. Summary of the Invention

[0005] The purpose of this invention is to provide an InDel molecular marker primer set for *Dictyophora rubrum*, an InDel-PCR reaction system, its application, and a method for evaluating population genetic diversity, thereby addressing the lack of efficient molecular marker tools in existing *Dictyophora rubrum* germplasm research. Simultaneously, this invention provides the application of this primer set and reaction system, as well as a method for evaluating the genetic diversity of *Dictyophora rubrum* populations, enabling precise and efficient analysis of the genetic relationships and variation levels of *Dictyophora rubrum* populations, and providing technical support for the identification, protection, and marker-assisted breeding of *Dictyophora rubrum* germplasm resources.

[0006] To address the problems mentioned in the background section, the present invention adopts the following technical solution.

[0007] A primer set of InDel molecular markers for *Dictyophora indicum* consists of 10 pairs of specific InDel primers, each consisting of a forward primer and a reverse primer. The sequences of the 10 pairs of specific InDel primers are shown in SEQ ID NO.1 to SEQ ID NO.20.

[0008] In another aspect, the present invention provides an InDel-PCR reaction system for *Dictyophora indica*, the total volume of which is 20 μL, and consists of the following components: 2×Es Premix Taq. TM 10 μL, 2 μL of *Dictyophora indicum* genomic DNA template, and 1 μL of any one pair of primers from the *Dictyophora indicum* InDel molecular marker primer set as described in claim 1, with the remainder being enzyme-free ddH2O.

[0009] Preferably, the concentration of the *Dictyophora indicum* genomic DNA template is 50 ng / μL.

[0010] In another aspect, the present invention provides a method for amplifying the above-mentioned reaction system, specifically employing the following technical solution.

[0011] An amplification method for the InDel-PCR reaction system of *Dictyophora indicum* includes the following steps: Step 1: Preheat the InDel-PCR reaction system of *Dictyophora indicum* at 94℃ for 5 min; Step 2: Perform 35 cycles of amplification on the preheated Red-topped Bamboo Fungus InDel-PCR reaction system. Each cycle includes denaturation at 94℃ for 30s, annealing at 55~58℃ for 45s, and extension at 72℃ for 30s. Step 3: After the cycle amplification is completed, perform epitaxy at 72℃ for 7 minutes, and store the amplification product at 4℃.

[0012] In addition, the present invention also provides the application of the above-mentioned InDel molecular marker primer set of *Dictyophora indicum* in the evaluation of genetic diversity of *Dictyophora indicum* populations or in molecular marker-assisted breeding.

[0013] Furthermore, the present invention also provides a method for evaluating population genetic diversity using the above-mentioned molecular marker primer set, specifically employing the following technical solution.

[0014] A method for evaluating the genetic diversity of *Dictyophora indicum* populations using an InDel molecular marker primer set includes the following steps: Step 1: Extract the whole genome DNA of *Dictyophora indicum*. Step 2: Amplify the whole genome DNA of *Dictyophora indicum* from Step 1 using the amplification method of the *Dictyophora indicum* InDel-PCR reaction system as described in claim 4. After amplification, perform electrophoretic separation and detect the bands. Step 3: Perform genetic cluster analysis on the red-topped bamboo fungus from different sources and calculate the genetic diversity parameters of red-topped bamboo fungus.

[0015] Preferably, the steps of the genetic clustering analysis in step three are as follows: Read the band information obtained after electrophoresis separation, and determine the genotype of each pair of InDel primer amplification products according to their relative migration position in the gel within the range of 100–250 bp of the electrophoresis pattern. Based on the genotype determination results, the genotype information of each locus is converted into the corresponding allele data format, so that each locus is represented by two alleles. The converted InDel codominant allele data is imported into NTSYS numerical classification analysis software. The similarity matrix between samples is calculated based on the simple matching coefficient, and the similarity matrix is ​​converted into a distance matrix. The unweighted group average method is used to perform hierarchical cluster analysis on the genetic relationship between samples to construct an evolutionary relationship tree between samples.

[0016] Preferably, the genotype determination steps are as follows: A value of 1 is recorded when only the upper band is detected, a value of 2 is recorded when both the upper and lower bands are detected, and a value of -1 is recorded when only the lower band is detected.

[0017] Preferably, the steps for calculating the genetic diversity parameters of *Dictyophora indicum* in step three are as follows: InDel codominance data converted from allele data format were used to perform genetic diversity analysis on all experimental materials using POPGene32 software. The number of alleles, effective number of alleles, Shannon information index, and gene diversity index were calculated to comprehensively assess the genetic variation level of the *Dictyophora indica* materials.

[0018] Preferably, in step three, electrophoresis is performed using 2.5% agarose gel electrophoresis with electrophoresis parameters of 120V constant voltage for 30 min, and DL5000 DNA Marker is used as the electrophoresis control.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The 10 pairs of InDel primers screened in this invention are primers specifically for *Dictyophora indicum*. They are stable in amplification reactions in the genome of *Dictyophora indicum*, with clear amplified fragments without impurities and significant polymorphism. They can effectively distinguish *Dictyophora indicum* strains from different geographical origins and are suitable for genetic diversity analysis of *Dictyophora indicum* populations.

[0020] (2) The 20μL standardized PCR reaction system and the adapted amplification program constructed in this invention have clear parameters, are easy to operate, do not require complex reagent optimization, and have specific requirements for DNA template concentration. The amplification results have good repeatability and are suitable for routine laboratory testing and batch analysis of large-scale samples.

[0021] (3) The evaluation method constructed in this invention is efficient and the results are comprehensive: The genetic diversity evaluation method combines gel electrophoresis technology with bioinformatics analysis. Through unified genotype coding rules and professional bioinformatics software, it can not only construct the evolutionary relationship tree of strains to clarify the kinship, but also calculate multiple genetic diversity parameters to assess the level of population variation. The identification results are comprehensive, accurate and objective.

[0022] Low cost and wide range of applications: The reagents used in this invention are all commercially available products in molecular biology, and the experimental instruments are general laboratory equipment, resulting in low detection costs. Moreover, the primer set and reaction system can not only be used for the evaluation of genetic diversity of *Dictyophora indicum* populations, but also for the detection of germplasm purity of *Dictyophora indicum*, molecular marker-assisted breeding, and other fields. It has high application value and is of great significance for promoting the standardization and high-quality development of the *Dictyophora indicum* industry. Attached Figure Description

[0023] Figure 1 The diagram shows the amplification effect of the primers for the nucleotide sequences of the present invention, as shown in SEQ ID NO.1 and SEQ ID NO.2. Figure 2 This invention is based on a cluster diagram of 19 samples of *Dictyophora indicum* with InDel labeling. Detailed Implementation

[0024] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0025] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0026] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places throughout this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that mutually excludes other embodiments. The present invention provides the following embodiments.

[0027] Example 1 Test strains: Nineteen strains of wild and cultivated *Dictyophora indicum* were collected from Guizhou, Yunnan, Zhejiang and other regions. The specific strain numbers and their sources are shown in Table 2 below. In this embodiment, the above 19 strains of *Dictyophora indicum* were isolated by tissue culture to obtain pure strains. The pure strain blocks were inoculated into culture dishes lined with cellophane and cultured. After obtaining mycelium, it was used for whole genome DNA extraction.

[0028] Experimental reagents: 2×Es Premix Taq TM The DL5000 DNA Marker is a commercially available reagent for molecular biology; the genomic DNA extraction kit is a commercially available general-purpose fungal DNA extraction kit; and the enzyme-free ddH2O is a routinely prepared laboratory solution.

[0029] Experimental instruments: fully automated sample grinder, high-speed refrigerated centrifuge, PCR amplification instrument, agarose gel electrophoresis instrument, gel imaging system, nucleic acid quantification instrument. All instruments were calibrated and used in accordance with the operating instructions.

[0030] In this embodiment, a primer set of InDel molecular markers for *Dictyophora indicum* was designed. This primer set consists of 10 pairs of InDel molecular marker primers for *Dictyophora indicum*. Each pair of InDel molecular marker primers for *Dictyophora indicum* consists of a forward primer and a reverse primer. The specific process is as follows.

[0031] Through high-throughput sequencing of the whole genome of *Dictyophora indicum*, screening of variant sites, and primer design, 10 pairs of *Dictyophora indicum*-specific InDel primers were obtained. The nucleotide sequences are shown in Table 1. Table 1. Nucleotide sequences of the primer sets .

[0032] In this embodiment, the sequences of the 10 pairs of specific InDel primers are shown in SEQ ID NO.1 to SEQ ID NO.20. Furthermore, this embodiment utilizes these 10 pairs of specific InDel primers to evaluate the genetic diversity of the *Dictyophora indicum* population, specifically employing the following steps.

[0033] Step 1: Extract whole genome DNA from *Dictyophora indicum*. Specifically, whole genome DNA is extracted from the 19 species of *Dictyophora indicum* listed in Table 2 below. The specific steps are as follows.

[0034] 20 mg of dried mycelium of *Dictyophora indicum* pure culture was ground into powder using an automated rapid sample grinder. Whole-genome DNA was extracted according to the instructions of the fungal genomic DNA extraction kit. The extracted DNA was tested for integrity by 1% agarose gel electrophoresis, and the concentration was determined by a nucleic acid quantification instrument. The DNA template was then diluted to 50 ng / μL and stored at -20°C for later use. Table 2. Test Material Numbers and Sources .

[0035] Step 2: InDel-PCR amplification and agarose gel electrophoresis detection This embodiment provides an InDel-PCR reaction system for *Dictyophora indicum*, and also provides an amplification method for the above system to amplify the extracted DNA templates of the above 19 *Dictyophora indicum* species, as detailed below.

[0036] The InDel-PCR reaction system for *Dictyophora indicum* used in this embodiment is: 2×Es Premix Taq. TM 10 μL, any one of the primer sets in Table 1, including 1 μL each of the forward and reverse primers, totaling 21 μL, 2 μL of DNA template from one of the red-topped bamboo fungus in Table 2, 6 μL of enzyme-free ddH2O; the total reaction volume is 20 μL.

[0037] This embodiment also provides a method for amplifying the above reaction system, the specific steps of which are as follows.

[0038] 1) Preheat the InDel-PCR reaction system of *Dictyophora indicum* at 94℃ for 5 min; 2) The preheated Red-topped Bamboo Fungus InDel-PCR reaction system was subjected to 35 cycles of amplification, each cycle including denaturation at 94℃ for 30s, annealing at 55~58℃ for 45s, and extension at 72℃ for 30s; 3) After the cycle amplification is completed, perform extrapolation at 72℃ for 7 min, and store the amplification product at 4℃.

[0039] Then, the amplification products were subjected to agarose gel electrophoresis. Specifically, 5 μL of PCR amplification product was mixed with 1 μL of loading buffer and subjected to 2.5% agarose gel electrophoresis. The electrophoresis parameters were 120V constant voltage electrophoresis for 30 min, with DL5000 DNA Marker as the electrophoresis control. After electrophoresis, the amplification band information was recorded by taking pictures and detecting the amplification band information using a gel imaging system.

[0040] The results showed that the primer set of the present invention could amplify clear bands in all 19 samples of *Dictyophora indicum*, with no obvious non-specific bands. The amplified bands showed significant polymorphism, making it suitable for genetic diversity analysis of *Dictyophora indicum* populations.

[0041] Step 3: Genotype coding and InDel codominant allele data conversion: See Figure 1 Within the 100~250bp range of the gel imaging pattern, a unified genotype determination was made based on the relative migration position of the amplified bands. Specifically, only the upper band was detected as 1, both upper and lower bands were detected as 2, and only the lower band was detected as -1. Based on the above genotype coding results, data transformation was performed according to the requirements of codominant markers. The genotype information of each InDel marker site was converted into the form of two alleles, resulting in a standardized InDel codominant allele dataset, which is used to prepare for subsequent bioinformatics analysis.

[0042] Step 4: Genetic cluster analysis of *Dictyophora indicum* strains: See Figure 2 The obtained standardized InDel codominant allele dataset was imported into NTSYS numerical classification analysis software. The genetic similarity matrix among 19 *Dictyophora indicum* strains was calculated based on simple matching coefficients, and the similarity matrix was converted into a genetic distance matrix. Hierarchical clustering analysis of *Dictyophora indicum* strains was performed using the unweighted group average method (UPGMA) to construct an evolutionary relationship tree.

[0043] The results showed that the genetic similarity coefficients of the 19 *Dictyophora indicum* strains ranged from 0.585 to 0.90. When the genetic similarity coefficient was 0.69, the 19 *Dictyophora indicum* strains could be divided into four groups: Group I included strains 1, 5, 10, and 12; Group II included strains 2, 3, 8, 9, 13, 16, 17, and 18; Group III included strains 4, 7, and 11; and Group IV included strains 6, 14, 15, and 19. The clustering results of strains from different production areas were correlated with their geographical origin and could clearly reflect the kinship among the strains.

[0044] Step 5: Calculation of genetic diversity parameters of the *Dictyophora indicum* population: The obtained standardized InDel codominant allele dataset was imported into POPGene32 software for genetic diversity analysis of 19 *Dictyophora indica* strains, and core parameters of population genetic diversity were calculated. Simultaneously, Nei's genetic distance and genetic similarity coefficient among strains were analyzed. The results are shown in Table 3. Table 3. Genetic distances and genetic similarities among 19 strains of *Dictyophora indicum*. .

[0045] Genetic characteristic analysis results showed that the average number of alleles (Na) of the *Dictyophora indicum* germplasm in this embodiment was 2.000, the average effective number of alleles (Ne) was 1.7855, the average Nei's gene diversity index (He) was 0.4245, and the average Shannon information index (I) was 0.6099. The Nei's genetic distance between strains varied between 0.00 and 0.70, and the genetic similarity coefficient varied between 0.30 and 1.00, indicating that the tested *Dictyophora indicum* population had a high level of genetic diversity, providing a good resource foundation for the innovation of *Dictyophora indicum* germplasm resources and variety breeding.

[0046] The InDel molecular marker primer set, PCR reaction system, and genetic diversity evaluation method of *Dictyophora indicum* of this invention are simple to operate, highly efficient, and accurate. The reagents and instruments used are all commercially available products in the field of molecular biology, requiring no special equipment. They are suitable for large-scale industrial sample testing and can be widely used in the identification of *Dictyophora indicum* germplasm resources, genetic diversity analysis, molecular marker-assisted breeding, and other fields, with significant industrial application value.

[0047] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. For those skilled in the art, various improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0048] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A set of InDel molecular marker primers for *Dictyophora indicum*, characterized in that, It consists of 10 pairs of specific InDel primers, each consisting of a forward primer and a reverse primer. The sequences of the 10 pairs of specific InDel primers are shown in SEQ ID NO.1 to SEQ ID NO.

20.

2. A red-topped bamboo fungus InDel-PCR reaction system, characterized in that, The total volume of the reaction system is 20 μL, and it consists of the following components: 2×Es Premix Taq TM 10 μL, 2 μL of *Dictyophora indicum* genomic DNA template, and 1 μL of any one pair of primers from the *Dictyophora indicum* InDel molecular marker primer set as described in claim 1, with the remainder being enzyme-free ddH2O.

3. The *Dictyophora indica* InDel-PCR reaction system according to claim 2, characterized in that, The concentration of the *Dictyophora indicum* genomic DNA template was 50 ng / μL.

4. An amplification method using the *Dictyophora indica* InDel-PCR reaction system as described in claim 2, characterized in that, Includes the following steps: Step 1: Preheat the InDel-PCR reaction system of *Dictyophora indicum* at 94℃ for 5 min; Step 2: Perform 35 cycles of amplification on the preheated Red-topped Bamboo Fungus InDel-PCR reaction system. Each cycle includes denaturation at 94℃ for 30s, annealing at 55~58℃ for 45s, and extension at 72℃ for 30s. Step 3: After the cycle amplification is completed, perform epitaxy at 72℃ for 7 minutes, and store the amplification product at 4℃.

5. The application of the InDel molecular marker primer set of *Dictyophora indicum* as described in claim 1 in the evaluation of genetic diversity of *Dictyophora indicum* populations or in marker-assisted breeding.

6. A method for evaluating the genetic diversity of *Dictyophora indicum* populations using an InDel molecular marker primer set, characterized in that... Includes the following steps: Step 1: Extract the whole genome DNA of *Dictyophora indicum*. Step 2: Amplify the whole genome DNA of *Dictyophora indicum* from Step 1 using the amplification method of the *Dictyophora indicum* InDel-PCR reaction system as described in claim 4. After amplification, perform electrophoretic separation and detect the bands. Step 3: Perform genetic cluster analysis on the red-topped bamboo fungus from different sources and calculate the genetic diversity parameters of red-topped bamboo fungus.

7. The method for evaluating the genetic diversity of *Dictyophora indicum* populations using the InDel molecular marker primer set according to claim 6, characterized in that... in, The steps of genetic cluster analysis in step three are as follows: Read the band information obtained after electrophoresis separation, and determine the genotype of each pair of InDel primer amplification products according to their relative migration position in the gel within the range of 100–250 bp of the electrophoresis pattern. Based on the genotype determination results, the genotype information of each locus is converted into the corresponding allele data format, so that each locus is represented by two alleles; The converted InDel codominant allele data were imported into NTSYS numerical classification analysis software. The similarity matrix between samples was calculated based on the simple matching coefficient, and the similarity matrix was converted into a distance matrix. The unweighted group average method was used to perform hierarchical cluster analysis on the genetic relationship between samples, and an evolutionary relationship tree between samples was constructed.

8. The method for evaluating the genetic diversity of *Dictyophora indicum* populations using the InDel molecular marker primer set according to claim 7, characterized in that... The genotype determination steps are as follows: A value of 1 is recorded when only the upper band is detected, a value of 2 is recorded when both the upper and lower bands are detected, and a value of -1 is recorded when only the lower band is detected.

9. The method for evaluating the genetic diversity of *Dictyophora indicum* populations using the InDel molecular marker primer set according to claim 7, characterized in that... in, The steps for calculating the genetic diversity parameters of *Dictyophora indicum* in step three are as follows: InDel codominance data converted from allele data format were used to perform genetic diversity analysis on all experimental materials using POPGene32 software. The number of alleles, effective number of alleles, Shannon information index, and gene diversity index were calculated to comprehensively assess the genetic variation level of the *Dictyophora indica* materials.

10. The method for evaluating the genetic diversity of *Dictyophora indicum* populations using the InDel molecular marker primer set according to claim 6, characterized in that, In step three, electrophoresis was performed using 2.5% agarose gel electrophoresis with a constant voltage of 120V for 30 minutes, using DL5000 DNA Marker as the electrophoresis control.

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