A red-crowned bamboo fungus issr molecular marker primer set, a reaction system and application thereof in population genetic diversity

Abstract

The application provides a Dictyophora rubrovolvata ISSR molecular marker primer group, a reaction system and application thereof in population genetic diversity, and belongs to the technical field of molecular biology. The nucleotide sequences of the Dictyophora rubrovolvata ISSR molecular marker primer group provided by the application are shown in SEQ ID NO. 1-SEQ ID NO. 34, and the ISSR-PCR reaction system for genetic diversity analysis of the Dictyophora rubrovolvata contains 1 ul of template DNA, 1 ul of the primer, 12.5 ul of 2x Taq PCR StarMix with Loading Dye and 10.5 ul of ddH2O in every 25 ul. The ISSR molecular marker primer group and the reaction system are stable in the PCR amplification reaction of the Dictyophora rubrovolvata, the amplified fragments are clear, and the polymorphism is high. The primer group and the reaction system can accelerate the identification speed of the Dictyophora rubrovolvata resources, and the result is stable and reliable.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biology, and particularly relates to a Dictyophora rubrovolvata ISSR molecular marker primer group, a reaction system and application thereof in population genetic diversity. BACKGROUND

[0002] Dictyophora rubrovolvata belongs to Phallaceae and Phallus, is one of the most characteristic edible fungi in Guizhou, and is an important cultivated variety in the province. Wild resources are mainly distributed in humus soil under bamboo in Guizhou, Yunnan and other places, and are mainly produced in Guizhou. Dictyophora rubrovolvata has unique flavor, rich nutrition and high value, is rich in various polysaccharides, has strong reducing capacity and hydroxyl radical inhibition capacity, and has obvious in-vitro antioxidant activity.

[0003] Molecular marker technology is a breeding-related genetic marker different from morphological markers, cell markers and biochemical markers, has co-dominance and is more convenient for selection of recessive traits, and can directly reflect differences in a genome between biological individuals or populations. The detection means of molecular markers are widely used in identification of edible fungus germplasm resource systems and breeding research, and the ISSR (inter-simple sequence repeat) molecular marker technology has the characteristics of simple operation, rapidness and high efficiency, and is widely applied to identification of edible fungus germplasm resources, analysis of genetic relationship and genetic diversity. However, at present, the method for applying the ISSR molecular marker to analyze the identification of germplasm resources and genetic diversity of Dictyophora rubrovolvata has not been reported. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a Dictyophora rubrovolvata ISSR molecular marker primer group and a reaction system, and to provide good technical guidance and theoretical support for scientific research such as Dictyophora rubrovolvata resource identification and genetic diversity analysis.

[0005] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions.

[0006] The present application provides a Dictyophora rubrovolvata ISSR molecular marker primer group, and the nucleotide sequence of the primer group is shown in SEQ ID NO. 1-SEQ ID NO. 34.

[0007] The present application also provides a Dictyophora rubrovolvata ISSR-PCR reaction system, which contains 1ul of template DNA, 1ul of any one of the above primers, 12.5ul of 2xTaq PCR StarMix with Loading Dye and 10.5ul of ddH2O in each 25ul reaction system.

[0008] Preferably, the PCR amplification procedure is: 94℃, 2min; 94℃, 30s, 40-60℃ annealing 1min, 72℃, 45s, 35 cycles; 72℃, 10min, 4℃ preservation.

[0009] Preferably, the annealing temperature is determined according to the selected primer, specifically as follows:

[0010]

[0011] The application also provides application of the primer set or the reaction system in evaluation of genetic diversity of Dictyophora rubrovolvata populations or molecular marker assisted breeding.

[0012] The application also provides a method for evaluating genetic diversity of Dictyophora rubrovolvata populations, comprising the following steps:

[0013] Genomic DNA of Dictyophora rubrovolvata is extracted, and the extracted DNA is amplified by using the reaction system, subjected to electrophoretic separation, detected for bands, and subjected to genetic cluster analysis by using the molecular marker primer set, so as to calculate genetic diversity parameters of the Dictyophora rubrovolvata materials.

[0014] Preferably, the electrophoretic separation is electrophoretic separation on a 2% agarose gel.

[0015] Preferably, the cluster analysis is specifically as follows: band information detected after the electrophoretic separation is read, bands in a range of 100-2000bp on the electrophoretogram that are clear and repeatedly appear are recorded as 1, and no band in the same position is recorded as 0, so as to generate a 0 and 1 original matrix; the total number of bands amplified by each primer and the number of polymorphic bands are counted; cluster analysis is performed by using UPGMA method in cluster analysis software NTSYS-pc, a cluster diagram is generated by using a Tree plot module, and a molecular evolution tree is constructed.

[0016] Preferably, the calculation of the genetic diversity parameters of the Dictyophora rubrovolvata materials is specifically as follows: according to the 0 and 1 binary data matrix, the total number of bands and the number of polymorphic bands of the ISSR amplification products are counted, and the ratio of the polymorphic bands is calculated; POPGene32 software is used to calculate genetic diversity parameters of all test materials: allele number, effective allele number, Shannon's information index and genetic diversity index.

[0017] The application has the following beneficial effects:

[0018] The application optimizes the ISSR reaction condition of the genomic DNA of Dictyophora rubrocyannis, and 34 ISSR primers screened by using the optimized PCR reaction system are stable in the PCR amplification reaction of the Dictyophora rubrocyannis, clear in the amplified fragments, and high in polymorphism. The primer group and the reaction system can accelerate the identification speed of the Dictyophora rubrocyannis resources, shorten the experimental time, and the result is stable and reliable, and the deficiency of the traditional morphological identification method is made up. The application has low cost, and can complete the identification of a large number of experimental materials in a short time. The method can well reveal the genetic diversity among the populations of the Dictyophora rubrocyannis, and distinguish the genetic relationship among the germplasm resources of the Dictyophora rubrocyannis, and has important significance for the protection and utilization of the Dictyophora rubrocyannis resources. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The figure is a primer amplification effect diagram of the nucleotide sequence shown as SEQ ID NO. 1, wherein M is Marker DL2000, 1-29 respectively correspond to the Dictyophora rubrocyannis materials numbered as Z1-Z29;

[0020] Figure 2 The application provides a sample clustering diagram of 29 Dictyophora rubrocyannis based on the ISSR marker. DETAILED DESCRIPTION

[0021] The application provides a Dictyophora rubrocyannis ISSR molecular marker primer group, and the nucleotide sequence of the primer group is shown as SEQ ID NO. 1-SEQ ID NO. 34, and specifically shown as Table 1:

[0022] Table 1 nucleotide sequence of the primer group

[0023]

[0024]

[0025] The application further provides a Dictyophora rubrocyannis ISSR-PCR reaction system, and each 25ul of the reaction system contains 1ul of template DNA, 1ul of any one of the primers, 12.5ul of 2xTaq PCR StarMix with Loading Dye, and 10.5ul of ddH2O.

[0026] The specific source of the 2xTaq PCR StarMix with Loading Dye and ddH2O is not particularly limited in the present application, and the conventional commercially available products in the art can be used. The extraction method of the template DNA is not particularly limited in the present application, and in the specific embodiment, the 2xCTAB method is used to extract the DNA, and the concentration of the template DNA is preferably 50 ng / μL. In the reaction system of the present application, the PCR amplification procedure is preferably as follows: 94℃, 2 min; 94℃, 30 s, 40-60℃ annealing for 1 min, 72℃, 45 s, 35 cycles; 72℃, 10 min, 4℃ storage. In the PCR amplification procedure of the present application, the annealing temperature is preferably determined according to the selected primer, and is specifically shown in Table 2:

[0027] Table 2 Annealing temperature of different primers

[0028]

[0029] The present application also provides an application of the primer set or the reaction system in the evaluation of the genetic diversity of Dictyophora rubrocyannis population or the molecular marker assisted breeding.

[0030] The present application also provides a method for evaluating the genetic diversity of Dictyophora rubrocyannis population, comprising the following steps:

[0031] The whole genome DNA of Dictyophora rubrocyannis is extracted, and the extracted DNA is amplified by using the above reaction system, electrophoretically separated, detected for the band, and the different sources of Dictyophora rubrocyannis are subjected to genetic cluster analysis by using the above molecular marker primer set, and the genetic diversity parameters of the Dictyophora rubrocyannis material are calculated.

[0032] The extraction method of the whole genome DNA of Dictyophora rubrocyannis is not particularly limited in the present application, and in the specific embodiment, the 2xCTAB method is used to extract the DNA, and the extracted DNA sample is dissolved in TE buffer and stored at -20℃ for standby, and before amplification, the DNA sample is diluted into a working solution of 50 ng / μL with double distilled water, which is used as a template for PCR amplification reaction. In the present application, the electrophoretic separation is preferably performed on a 2% agarose gel, and after electrophoresis, the G:BOX gel imaging system is used to take pictures to detect the band, and the pictures are saved. In the present application, the 2% agarose gel is used to separate the ISSR-PCR amplification product, and the resolution can reach the effect of non-denaturing polyacrylamide gel, and the operation is simpler and safer.

[0033] In the present application, the cluster analysis is preferably reading the band information detected after electrophoretic separation, taking the bands clearly and repeatedly appearing in the range of 100-2000 bp on the electrophoretogram as 1, and taking no band at the same position as 0, thereby generating a 0 and 1 original matrix; the total band number and the polymorphic band number amplified by each primer are counted; the cluster analysis is carried out by UPGMA method in the cluster analysis software NTSYS-pc (2.10e), and the cluster diagram is generated by the Tree plot module to construct the molecular phylogenetic tree. The calculation of the genetic diversity parameters of the Dictyophora rubrovolvata material is specifically preferably as follows: according to the 0 and 1 binary data matrix, the total band number and the polymorphic band number of the ISSR amplification product are counted, and the ratio of the polymorphic bands is calculated; the POPGene32 software is used to calculate the genetic diversity parameters of all test materials: the allele number, the effective allele number, Shannon's information index and the genetic diversity index.

[0034] The technical solutions provided by the present application are described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0035] Example 1

[0036] 29 Dictyophora rubrovolvata materials were collected from the main cultivation areas of Bijie, Zunyi and Qianxinan in Guizhou, and the details are shown in Table 3.

[0037] Table 3 Test material number and sampling point

[0038]

[0039] The germplasm resources collected were subjected to tissue separation to obtain pure strains, and the pure strain blocks were picked into a shake flask for mycelium culture to obtain mycelium balls for whole genome DNA extraction. The 2xCTAB method was used to extract DNA, and the extracted DNA sample was dissolved in TE buffer and stored at -20℃ for standby, and before amplification, the DNA sample was diluted with double distilled water to 50 ng / μL of working solution as the template for PCR amplification reaction.

[0040] PCR amplification of the template was performed using the primer sequences shown in Table 1. The PCR reaction system (25 μL) consisted of: 1 μL template DNA (50 ng / μL), 1 μL primer, 12.5 μL 2×TaqPCR StarMix with Loading Dye, and 10.5 μL ddH2O. The PCR amplification program was as follows: 94℃ for 2 min; 94℃ for 30 s, 40-60℃ (annealing temperature varies depending on the primers, see Table 2) for 1 min, 72℃ for 45 s, 35 cycles; 72℃ for 10 min, and storage at 4℃. After PCR amplification, the PCR products were separated by electrophoresis on a 2% agarose gel. After electrophoresis, the bands were photographed using a G:BOX gel imaging system and the images were saved. Figure 1 This is a diagram showing the amplification effect using primers with the nucleotide sequence shown in SEQ ID NO.1 of this invention. Figure 1 It can be seen that the bands amplified by the ISSR-PCR reaction system of *Dictyophora indicum* established in this invention have the advantages of high polymorphism, strong specificity, clear background and strong stability, and are suitable for genetic diversity analysis of *Dictyophora indicum*.

[0041] Example 2

[0042] Clear and repetitive bands within the 100-2000 bp range of the electrophoresis pattern obtained in Example 1 were marked as 1, and no band at the same position was marked as 0, thus generating an original matrix of 0 and 1. The total number of bands amplified by each primer and the number of polymorphic bands were counted. Cluster analysis of 29 *Dictyophora indicum* accessions was performed using NTSYS-pc (2.10e) software. The results are as follows: Figure 2 As shown. By Figure 2 It can be seen that the genetic similarity coefficients among the 29 *Dictyophora indicum* strains collected in Example 1 ranged from 0.56 to 0.89. At a genetic similarity coefficient of 0.672, the 29 *Dictyophora indicum* strains could be divided into three categories: Category I included strains 5, 6, 7, 8, 9, and 10; Category II included strains 11, 12, 13, 15, 21, and 22; and Category III included strains 1, 2, 4, and 17. These results indicate that there are significant genetic differences among the tested *Dictyophora indicum* strains.

[0043] Example 3

[0044] The 29 Ph. rubrocaeruleum strains of example 2 were analyzed by using software PopGen32. The results of genetic property analysis showed that the average allele (Na) of Ph. rubrocaeruleum germplasm was 2.000, the average effective allele number (Ne) was 1.5137, the average Nei's genetic diversity index (He) was 0.3114, and the average Shannon information index (I) was 0.4769. The results of genetic distance and genetic similarity analysis are shown in Table 4. The Nei's genetic distance value of the 29 Ph. rubrocaeruleum strains varied between 0.1312-0.9974, and the genetic similarity coefficient varied between 0.4016-0.8770. Thus, the 29 Ph. rubrocaeruleum germplasms collected in example 1 had a high level of genetic diversity, which provided a good resource reserve for subsequent germplasm innovation.

[0045] Table 4 Genetic distance and genetic similarity between 29 Ph. rubrocaeruleum strains

[0046]

[0047] Table 4 Genetic distance and genetic similarity between 29 Ph. rubrocaeruleum strains

[0048]

[0049]

[0050] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application. SEQUENCE LISTING <110> Guizhou Crop Variety Resource Institute <120> A Ph. rubrocaeruleum ISSR molecular marker primer set, reaction system and application thereof in population genetic diversity <160> 34 <170> SIPOSequenceListing 1.0 <210> 1 <211> 17 <212> DNA <213> Artificial Sequence <400> 1 agagagagag agagagt 17 <210> 2 <211> 17 <212> DNA <213> Artificial Sequence <400> 2 agagagagag agagagc 17 <210> 3 <211> 17 <212> DNA <213> Artificial Sequence <400> 3 agagagagag agagagg 17 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <400> 4 gagagagaga gagagac 17 <210> 5 <211> 17 <212> DNA <213> Artificial Sequence <400> 5 gagagagaga gagagaa 17 <210> 6 <211> 17 <212> DNA <213> Artificial Sequence <400> 6 cacacacaca cacacag 17 <210> 7 <211> 17 <212> DNA <213> Artificial Sequence <400> 7 gtgtgtgtgt gtgtgta 17 <210> 8 <211> 17 <212> DNA <213> Artificial Sequence <400> 8 tctctctctc tctctcc 17 <210> 9 <211> 17 <212> DNA <213> Artificial Sequence <400> 9 acacacacac acacact 17 <210> 10 <211> 17 <212> DNA <213> Artificial Sequence <400> 10 acacacacac acacacc 17 <210> 11 <211> 17 <212> DNA <213> Artificial Sequence <400> 11 acacacacac acacacg 17 <210> 12 <211> 17 <212> DNA <213> Artificial Sequence <400> 12 tgtgtgtgtg tgtgtga 17 <210> 13 <211> 17 <212> DNA <213> Artificial Sequence <400> 13 tgtgtgtgtg tgtgtgc 17 <210> 14 <211> 17 <212> DNA <213> Artificial Sequence <400> 14 tgtgtgtgtg tgtgtgg 17 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <400> 15 agagagagag agagagyt 18 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <400> 16 gagagagaga gagagayt 18 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <400> 17 gagagagaga gagagayg 18 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <400> 18 gtgtgtgtgt gtgtgtyc 18 <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <400> 19 acacacacac acacacyt 18 <210> 20 <211> 18 <212> DNA <213> Artificial Sequence <400> 20 tgtgtgtgtg tgtgtgrc 18 <210> 21 <211> 18 <212> DNA <213> Artificial Sequence <400> 21 accaccacca ccaccacc 18 <210> 22 <211> 18 <212> DNA <213> Artificial Sequence <400> 22 atgatgatga tgatgatg 18 <210> 23 <211> 18 <212> DNA <213> Artificial Sequence <400> 23 ccgccgccgc cgccgccg 18 <210> 24 <211> 18 <212> DNA <213> Artificial Sequence <400> 24 ctcctcctcc tcctcctc 18 <210> 25 <211> 18 <212> DNA <213> Artificial Sequence <400> 25 ggcggcggcg gcggcggc 18 <210> 26 <211> 18 <212> DNA <213> Artificial Sequence <400> 26 gttgttgttg ttgttgtt 18 <210> 27 <211> 16 <212> DNA <213> Artificial Sequence <400> 27 gacagacaga cagaca 16 <210> 28 <211> 16 <212> DNA <213> Artificial Sequence <400> 28 gatagataga cagaca 16 <210> 29 <211> 16 <212> DNA <213> Artificial Sequence <400> 29 ggatggatgg atggat 16 <210> 30 <211> 15 <212> DNA <213> Artificial Sequence <400> 30 cttcacttca cttca 15 <210> 31 <211> 15 <212> DNA <213> Artificial Sequence <400> 31 ggagaggaga ggaga 15 <210> 32 <211> 15 <212> DNA <213> Artificial Sequence <400> 32 gggtggggtg gggtg 15 <210> 33 <211> 17 <212> DNA <213> Artificial Sequence <400> 33 bdbcacacac acacaca 17 <210> 34 <211> 17 <212> DNA <213> Artificial Sequence <400> 34 vhvgtgtgtg tgtgtgt 17

Claims

1. A Dictyophora rubrovolvata ISSR molecular marker primer set, characterized in that, The nucleotide sequences of the primer set are shown as SEQ ID NO. 1-SEQ ID NO.

34.

2. The primer set of claim 1 is applied in evaluation of genetic diversity of Dictyophora rubrocyannis population or molecular marker assisted breeding.

3. A method for evaluating the genetic diversity of Dictyophora rubrovolvata population, characterized in that, The method comprises the following steps: Extracting the whole genome DNA of Dictyophora rubrocyannis, amplifying the DNA obtained by using the reaction system, performing electrophoretic separation, detecting the bands, performing genetic cluster analysis of Dictyophora rubrocyannis from different sources by using the molecular marker primer set of claim 1, and calculating the genetic diversity parameters of the Dictyophora rubrocyannis materials.

4. The method of claim 3, wherein, The electrophoretic separation is performed on a 2% agarose gel.

5. The method of claim 3, wherein, The cluster analysis is specifically as follows: reading the band information detected after the electrophoretic separation, marking the bands in the range of 100-2000 bp on the electrophoretogram which are clear and repeatedly appeared as 1, and marking the same position without bands as 0, thereby generating a 0 and 1 original matrix; counting the total band number and the polymorphic band number amplified by each primer; performing cluster analysis by using the UPGMA method in the cluster analysis software NTSYS-pc software, generating a cluster diagram by using the Tree plot module, and constructing a molecular evolution tree.

6. The method of claim 3, wherein, The calculation of the genetic diversity parameters of the Dictyophora rubrocyannis materials is specifically as follows: according to the 0 and 1 binary data matrix, counting the total band number and the polymorphic band number of the ISSR amplification products, and calculating the ratio of the polymorphic bands; using POPGene32 software to calculate the genetic diversity parameters of all test materials: the allele number, the effective allele number, Shannon's information index, and the genetic diversity index.

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