Primer design method for detecting the cytoplasmic origin of hybrid strains of Pleurotus eryngii, primers and their applications

Abstract

The present invention discloses a primer design method for detecting the cytoplasm source of a hybrid strain of Pleurotus eryngii, primers and applications thereof, and relates to the field of biotechnology. The primers include, the nucleotide sequence of the upstream primer MT‑S5‑F is: 5'‑AGCGTTAACACACCCCC‑3'; the nucleotide sequence of the downstream primer MT‑S5‑R is: 5'‑CTGAAAATGCAGACCCG‑3'. The present invention designs a specific identification primer MT‑S5, which can identify which parent the cytoplasm of the hybrid strain of Pleurotus eryngii comes from by comparing and referencing the results of the electrophoresis bands observed after DNA in vitro amplification, and provides great convenience for the rapid screening and identification of excellent hybrid offspring. By clarifying the cytoplasm source of the hybrid strain, the method of parent identification is introduced into the hybrid breeding of Pleurotus eryngii, which can not only clarify the parental composition and cytoplasmic inheritance rules in the hybridization process of Pleurotus eryngii, but also provide certain reference ideas for the genetic breeding of other edible fungi, which is conducive to analyzing the genetic composition of the hybrid strain, and then conducive to the screening and confirmation of excellent parents.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a primer design method for detecting the cytoplasmic origin of a hybrid strain of Pleurotus eryngii, primers and applications thereof. Background Art

[0002] Pleurotus citrinopileatus Sing., commonly known as elm yellow mushroom, belongs to the genus Pleurotus, class Hymenomycetes, order Agaricales, family Pleurotus, and is a famous edible fungus with medicinal and edible properties in Northeast China. ... is a famous edible fungus with medicinal and edible properties in Northeast China. Pleurotus citrinopileatus Sing., commonly known as elm yellow mushroom, belongs to the genus Pleurotus, class Basidiomycetes, class Agaricales, family Pleurotus, and is a famous edible

[0003] The hybrid breeding operation of Pleurotus eryngii is not very difficult. The real difficulty lies in the screening of excellent hybrid offspring from a large number of hybrid strains and the analysis of the genetic composition of the hybrid strains. Like all edible fungi, the gametes of Pleurotus eryngii are not divided into male and female. Whether the gametes are protoplast monokaryons that have not undergone meiosis or spores that have undergone meiosis, there is no clear distinction between the maternal and paternal parent gametes in the hybridization. Since the monokaryon hybridization of Pleurotus eryngii is a process of bidirectional nuclear migration, it is necessary to identify the cytoplasmic source of the hybrid strain to determine the parental composition in the hybridization process and clarify the cytoplasmic genetic rules.

[0004] Although there are relatively abundant studies on variety breeding, cluster analysis, component determination, etc. of Pleurotus eryngii, there are relatively few studies on the genetic laws, and there are no reports on the development of molecular markers for the cytoplasmic genome and the exploration of genetic laws. Summary of the invention

[0005] The present invention provides a primer design method for detecting the cytoplasmic origin of a hybrid strain of Pleurotus eryngii, primers and applications thereof, aiming to solve the problems existing in the above-mentioned background technology.

[0006] In order to achieve the above technical objectives, the present invention mainly adopts the following technical solutions:

[0007] In a first aspect, the present invention discloses a method for designing primers for detecting the cytoplasmic origin of a hybrid strain of Pleurotus eryngii, comprising the following steps:

[0008] S1 Pleurotus eryngii mycelium culture and DNA extraction: After culturing the Pleurotus eryngii monokaryotic hybrid parent and hybrid strain to full plate, the mycelium was scraped and genomic DNA was extracted;

[0009] S2 Resequencing of the mitochondrial genome of Pleurotus eryngii: Resequence the mitochondrial genome of hyphae genomic DNA to detect the variation compared with the mitochondrial reference genome;

[0010] S3 Analysis of differential loci in Pleurotus eryngii: Check all the variation detection information in the mitochondrial genome, select the common variation types applicable to the two parents, and locate the variation types based on this;

[0011] S4 Primer design for differential sites of Pleurotus eryngii: Based on the information of variable sites in the mitochondrial genome resequencing data, by comparing the variable sites between the single-nuclear hybrid parents, 200bp sequences upstream and downstream of the differential sites were selected in the mitochondrial reference genome to design primers to be screened;

[0012] S5 Primers for identifying the cytoplasm of hybrid strains of Pleurotus eryngii: All the primers to be screened designed in S4 are amplified using the DNA of the monokaryotic parent strains as templates, and primers that can clearly observe the different molecular weights of the amplification products of the two monokaryotic parents are selected, so as to obtain identification primers that can distinguish which monokaryotic parent the cytoplasm of the hybrid strain of Pleurotus eryngii comes from.

[0013] In a preferred embodiment of the present invention, in step S3, the variation detection information includes SNP, InDel, CNV, and SV variation site information.

[0014] In a preferred embodiment of the present invention, in step S4, primers are designed based on the InDel variation site information in the mitochondrial genome resequencing data;

[0015] In a second aspect, the present invention discloses a primer for detecting the cytoplasmic origin of a hybrid strain of Pleurotus eryngii, which is designed by the design method described in the first aspect, and the primer includes an upstream primer MT-S5-F having a nucleotide sequence of: 5'-AGCGTTAACACACCCCC-3'; a downstream primer MT-S5-R having a nucleotide sequence of: 5'-CTGAAAATGCAGACCCG-3'.

[0016] In a third aspect, the present invention discloses a kit for detecting the cytoplasmic origin of a hybrid strain of Pleurotus eryngii, wherein the kit comprises the primers described in the second aspect.

[0017] In a fourth aspect, the present invention discloses a use of the primers described in the second aspect in detecting the cytoplasmic origin of a hybrid strain of Pleurotus eryngii.

[0018] In a fifth aspect, the present invention discloses a method for detecting the cytoplasmic origin of a hybrid strain of Pleurotus eryngii, using the primers described in the second aspect to detect the cytoplasmic origin of the hybrid strain of Pleurotus eryngii.

[0019] Further, the following steps are included:

[0020] Using the DNA of the hybrid strain of Pleurotus eryngii as a template and the primers described in the second aspect for PCR amplification, the cytoplasmic origin of the hybrid strain can be determined based on the molecular weight of the amplified product and the molecular weight of the product amplified using the DNA of the mononuclear hybrid parent as a template.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention designs a specific identification primer MT-S5, which can identify which parent the cytoplasm of the Pleurotus eryngii hybrid strain actually comes from by observing the electrophoresis band results after in vitro DNA amplification and comparing them with references, providing great convenience for the rapid screening and identification of excellent hybrid offspring.

[0023] 2. The present invention introduces a method for identifying parents in the hybrid breeding of Pleurotus eryngii by clarifying the cytoplasmic origin of the hybrid strains. This can not only clarify the parental composition and cytoplasmic inheritance rules in the hybridization process of Pleurotus eryngii, but also provide certain reference ideas for the genetic breeding of other edible fungi.

[0024] 3. The present invention solves the problem of determining the parents during the hybridization of Pleurotus eryngii, is beneficial to analyzing the genetic composition of the hybrid strains, and is beneficial to the screening and confirmation of excellent parents by combining the trait performance and genetic composition analysis of the hybrid strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The figures are electrophoretic diagrams of the amplification of the parent binukaryotic strain and the monokaryotic strain in the primer screening process of Example 1 of the present invention; wherein, ad are electrophoretic diagrams of the amplification of primers MT-S1, MT-S2, MT-S3, and MT-S4; e are electrophoretic diagrams of the amplification of primers MT-S5; fg are electrophoretic diagrams of the amplification of primers MT-S6 and MT-S7; h are electrophoretic diagrams of the amplification of primers MT-S8, MT-S9, MT-S10, MT-S11, and MT-S12 from left to right; i are electrophoretic diagrams of the amplification of primers MT-S13, MT-S14, MT-S15, MT-S16, MT-S17, and MT-S18 from left to right; jl are electrophoretic diagrams of the amplification of primers MT-S19, MT-S20, and MT-S21;

[0026] Figure 2 The amplification electrophoresis diagram of the parental dikaryotic strain and the monokaryotic strain provided in Example 2 of the present invention;

[0027] Figure 3 The construction diagram of the homokaryotic heteroplasmic strain provided in Example 2 of the present invention;

[0028] Figure 4 This is the amplified electrophoresis diagram of the parent strain and the isokaryotic heterogeneous hybrid strain provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0029] The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings so that the purpose, features and advantages of the present invention can be more clearly understood. It should be understood that the embodiments shown in the accompanying drawings are not intended to limit the scope of the present invention, but are only intended to illustrate the essential spirit of the technical solution of the present invention.

[0030] In the hybrid breeding process, which is the method of selecting excellent strains of most edible fungi, the influence of mitochondrial genes in the cytoplasm of edible fungi on various characteristics of strains is often easily overlooked. However, the influence of mitochondrial genes in edible fungi on their characteristics cannot be underestimated. The cytoplasmic genetic laws that are closely related to them also have a very high scientific status as an important part of the genetic breeding research of edible fungi. The development of this molecular marker technology can specifically mark the cytoplasm of the hybrid parents of Pleurotus eryngii, so as to quickly determine the cytoplasmic source of the hybrid strain of Pleurotus eryngii, and can also provide reference ideas for other edible fungi-related genetic breeding research.

[0031] The following is an explanation through specific embodiments.

[0032] Example 1 Primer Design Method for Detecting the Cytoplasmic Origin of Hybrid Strains of Pleurotus eryngii

[0033] 1. Mycelial Culture and DNA Extraction of Pleurotus eryngii

[0034] After the monokaryotic hybrid parents and hybrid strains of Pleurotus eryngii were cultured in a culture dish with PDA medium covered with cellophane until the plate was full, the hyphae were scraped and the genomic DNA was extracted by the CTAB method.

[0035] 2. Resequencing of the mitochondrial genome of Pleurotus eryngii

[0036] The genomic DNA samples of the mycelium of the mononuclear hybrid parent of Pleurotus eryngii were delivered to a sequencing company for resequencing of the nuclear genome and mitochondrial genome based on the Illumina high-throughput sequencing platform to detect the variations compared with the mitochondrial reference genome.

[0037] 3. Analysis of differential loci in Pleurotus eryngii

[0038] View all variant detection information in the mitochondrial genome, including SNP, InDel, CNV, SV and other variants, select the common variant types between the two parents, and use this as a basis to locate the variant type.

[0039] 4. Primer design for differential loci of Pleurotus eryngii

[0040] Based on the InDel variation site information in the mitochondrial genome resequencing data, by comparing the InDel variation sites between the monokaryotic parents, 200bp sequences upstream and downstream of the differential sites were selected in the mitochondrial reference genome, and primers were designed using Snapgene. The product length was set to 400-800bp and the annealing temperature was set to 50℃-62℃. Primers with strong specificity, few mismatches, and a single expected amplification product were selected as screenable primers for molecular marker development.

[0041] 5. Primer Development for Cytoplasmic Identification of Hybrid Strains of Pleurotus erythrorhizium

[0042] Utilize all designed primers to be screened (named MT-S1, MT-S2, MT-S3, MT-S4, MT-S5, MT-S6, MT-S7, MT-S8, MT-S9, MT-S10, MT-S11, MT-S12, MT-S13, MT-S14, MT-S15, MT-S16, MT-S17, MT-S18, MT-S19, MT-S20, MT-S21, as shown in Table 1 below), carry out PCR amplification with the DNA of monokaryotic parent strain as template, through screening of designed primers, select primers that can obviously observe the difference in molecular weight of the amplification products of the two monokaryotic parents, can be used as identification primers that can distinguish which monokaryotic parent the cytoplasm of the two hybrid strains comes from.

[0043] The results are as follows Figure 1 As shown, primer MT-S5 can be used to distinguish parental cytoplasm.

[0044] Table 1 Primer information for identification of Pleurotus erythrorhizon cytoplasm

[0045]

[0046]

[0047]

[0048] Example 2 Method for Detecting the Cytoplasmic Origin of Hybrid Strains of Pleurotus eryngii

[0049] The developed primer MT-S5 was used to amplify the hybrid strain DNA as a template. The cytoplasmic origin of the hybrid strain could be determined based on the molecular weight of the amplified product and the molecular weight of the product amplified using the hybrid mononuclear parent DNA as a template.

[0050] The following is an explanation and verification through specific steps.

[0051] 1. Preparation and screening of monokaryotic strains of Pleurotus eryngii

[0052] Using three Pleurotus aurantii strains X18, Y21, and Y24 as parents, the monokaryotic strains X18-a, X18-b, Y21-c, Y21-d, Y24-e, and Y24-f of the two mating types of the parents were prepared by protoplast monokaryotic technology. The monokaryotic strains with clear cytoplasmic origin were preliminarily screened out using the specific primer MT-S5 for distinguishing the cytoplasm of the Pleurotus aurantii parent of the present invention, and PCR amplification was performed on the parent binucleate and monokaryotic strains. The results are as follows: Figure 2 The monokaryotic strain Y21-d and its parent Y21 amplified a band of the same size, which was about 480bp; the monokaryotic strain Y24-f and its parent Y24 amplified a band of the same size, which was about 450bp; the monokaryotic strain X18-b and its parent X18 amplified a band of the same size, which was about 380bp, indicating that the cytoplasm of the monokaryotic strain was the same as that of the parent. The monokaryotic strains X18-b, Y21-d, and Y24-f with the same cytoplasm as the parents were screened out as the parent monokaryotic strains of the hybrid strains.

[0053] 2. Preparation of hybrid strains

[0054] Three monokaryotic strains were mated in pairs, and hybrids were picked from the edges of the colonies on both sides of the hybridization culture dish. Two hybrids were picked from each hybrid combination, which were isokaryotic heteroplasmic strains. Figure 3 As shown, a total of 6 homokaryotic heteroplasmic hybrid strains were obtained, and the strain information is shown in Table 2.

[0055] Table 2 Information on homokaryotic heteroplasmic hybrid strains

[0056]

[0057] 3. Identification of the cytoplasm of Pleurotus eryngii homokaryotic heteroplasmic hybrid strains

[0058] PCR amplification was performed on the parent binuclear strain and the hybrid strain using the specific primer MT-S5 for distinguishing the cytoplasm of the parent Pleurotus eryngii of the present invention. The results are as follows: Figure 4 As shown in the figure, the molecular weight of the amplified product of the homokaryotic heteroplasmic hybrid strain is the same as that of the sampling parent, indicating that the cytoplasm of the homokaryotic heteroplasmic hybrid strain is consistent with that of the sampling parent. Primer MT-S5 can accurately and effectively distinguish the cytoplasm of the hybrid strain of Pleurotus eryngii.

[0059] The preferred embodiments of the present invention have been described in detail above, but it should be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention. These equivalent forms are also defined by the claims attached to this application.

Claims

1. Application of primers in detecting the cytoplasmic origin of a hybrid strain of Pleurotus eryngii, the primers comprising: an upstream primer MT-S5-F having a nucleotide sequence of 5'-AGCGTTAACACACCCCC-3'; a downstream primer MT-S5-R having a nucleotide sequence of 5'-CTGAAAATGCAGACCCG-3'; a primer design method comprising the following steps: S1 Pleurotus eryngii mycelium culture and DNA extraction: After culturing the Pleurotus eryngii monokaryotic hybrid parent and hybrid strain to full plate, the mycelium was scraped and genomic DNA was extracted; S2 Resequencing of the mitochondrial genome of Pleurotus eryngii: Resequence the mitochondrial genome of hyphae genomic DNA to detect the variation compared with the mitochondrial reference genome; S3 Analysis of differential loci in Pleurotus eryngii: Check all the variation detection information in the mitochondrial genome, select the common variation types applicable to the two parents, and locate the variation types based on this; S4 Primer design for differential sites of Pleurotus eryngii: Based on the information of variable sites in the mitochondrial genome resequencing data, by comparing the variable sites between the single-nuclear hybrid parents, 200bp sequences upstream and downstream of the differential sites were selected in the mitochondrial reference genome to design primers to be screened; S5 Primers for identifying the cytoplasm of the hybrid strain of Pleurotus aurantii: All the primers designed in S4 to be screened are respectively amplified using the DNA of the monokaryotic parent strain as a template, and primers that can clearly observe the difference in molecular weight of the amplification products of the two monokaryotic parents are selected, that is, identification primers that can distinguish which monokaryotic parent the cytoplasm of the hybrid strain of Pleurotus aurantii comes from are obtained; In step S4, primers are designed based on the InDel variation site information in the mitochondrial genome resequencing data.

2. A kit for detecting the cytoplasmic origin of a hybrid strain of Pleurotus eryngii, characterized in that: The kit contains an upstream primer MT-S5-F, the nucleotide sequence of which is: 5'-AGCGTTAACACACCCCC-3'; and a downstream primer MT-S5-R, the nucleotide sequence of which is: 5'-CTGAAAATGCAGACCCG-3'.

3. A method for detecting the cytoplasmic origin of a hybrid strain of Pleurotus eryngii, characterized in that: Primers are used to detect the cytoplasmic origin of the Pleurotus aurantii hybrid strain, and the primers include: the nucleotide sequence of the upstream primer MT-S5-F is: 5'-AGCGTTAACACACCCCC-3'; the nucleotide sequence of the downstream primer MT-S5-R is: 5'-CTGAAAATGCAGACCCG-3'; The steps include: Using the DNA of the hybrid strain of Pleurotus eryngii as a template and primers for PCR amplification, the cytoplasmic origin of the hybrid strain can be determined based on the molecular weight of the amplified product and the molecular weight of the product amplified using the DNA of the mononuclear hybrid parent as a template.

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