Four- to six-base repeat SSR microsatellite markers, primers and their applications in two strong parasitic Armillaria species

Abstract

The present invention discloses two four- to six-base repeat SSR microsatellite markers of strongly parasitic Armillaria and primer sets thereof. The microsatellite markers are SSR7, SSR109, SSR129, SSR221, SSR228, SSR262, SSR263, SSR354, SSR387, SSR462, and SSR540, and their nucleotide sequences are shown in SEQ ID No.1 to 11. The nucleotide sequences of the primer pairs are shown in SEQ ID No.12 to 33, respectively. Based on the genome assembly data of different strains of two strongly parasitic Armillaria, the present invention develops a set of four- to six-base repeat SSR microsatellite markers with strong polymorphism and high stability, and uses them in population genetic diversity analysis, germplasm resource identification, and molecular assisted breeding of strongly parasitic Armillaria. Provide technical support for population genetic diversity analysis, germplasm resource identification, and molecular assisted breeding of Armillaria.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and specifically relates to two strong parasitic honey fungus four to six base repeat SSR microsatellite markers, primers and applications thereof. Background Art

[0002] Armillaria fungus ( Armillaria spp.) belongs to the phylum Basidiomycetes, class Agaricales, order Agaricales, family Aquilegiaceae, genus Armillaria, and is an indispensable symbiotic fungus in the cultivation of the precious Chinese medicinal material Gastrodia elata. However, according to previous studies, not all Armillaria species found in China are suitable for Gastrodia elata cultivation, especially the highly parasitic Armillaria can devour Gastrodia elata, causing problems such as reduced Gastrodia elata yield or empty nests. Guo et al., a study found that the Armillaria that can be used for Gastrodia elata cultivation is divided into 7 lineages, including biological species CBSK, CBSG, etc. Armillaria mellea The species complex is highly parasitic (invasive) to Gastrodia elata. Although these Armillaria species are not suitable for Gastrodia elata cultivation, they are still well-known edible fungi. Developing molecular markers with strong specificity, high stability and high polymorphism for highly parasitic Armillaria will not only facilitate the identification and protection of Armillaria germplasm resources, but also accelerate the genetic breeding of Armillaria and promote the sustainable development and technological progress of the edible fungi industry.

[0003] Simple sequence repeats (SSRs), also known as microsatellites or short tandem repeats (STRs), are non-coding repetitive DNA regions composed of tandem repetitions of small motifs of 1 to 6 nucleotides. Due to its co-dominance, high polymorphism (the same core repeating unit sequence shows different repeat times in different species or even different individuals), and genetic stability, SSR has been widely used as a molecular marker in the construction of genetic maps of eukaryotic organisms, fingerprint identification, germplasm identification, population genetic structure analysis and other studies.

[0004] In the detection of SSR microsatellite markers, when shorter core repeat unit sequences (such as dinucleotide repeats or trinucleotide repeats) are used, more SSR loci can be detected in one experiment. However, the widely used dinucleotide repeat SSRs are prone to produce multiple products of slightly different lengths during PCR amplification, affecting the accuracy of genotyping and, in turn, the subsequent determination of alleles, calculation of heterozygosity, etc.

[0005] Therefore, it is necessary to provide a more specific molecular marker to improve the accuracy of genotyping and provide technical support for the identification of Armillaria germplasm resources. Summary of the invention

[0006] The present invention aims to provide a strongly parasitic Armillaria mellea SSR microsatellite marker with stable primer amplification and high polymorphism, and provides two strongly parasitic Armillaria mellea four- to six-base repeat SSR microsatellite markers, primers and applications thereof.

[0007] To achieve the above object, the present invention is implemented through the following technical solutions:

[0008] In the first aspect of the present application, two four- to six-base repeat SSR microsatellite markers of strongly parasitic Armillaria mellea are provided, the SSR microsatellite markers are SSR7, SSR109, SSR129, SSR221, SSR228, SSR262, SSR263, SSR354, SSR387, SSR462, and SSR540; their nucleotide sequences are shown in SEQ ID No. 1~11.

[0009] Further, the core repeating unit sequence of the SSR7 is: AATG;

[0010] The core repeat unit sequence of SSR109 is: ATTGGT;

[0011] The core repeat unit sequence of SSR129 is: CAAA;

[0012] The core repeat unit sequence of SSR221 is: CTGCTC;

[0013] The core repeat unit sequence of SSR228 is: CTTC;

[0014] The core repeat unit sequence of SSR262 is: GAAG;

[0015] The core repeat unit sequence of SSR263 is: GAAG;

[0016] The core repeat unit sequence of SSR354 is: GGAA;

[0017] The core repeat unit sequence of SSR387 is: GTAG;

[0018] The core repeat unit sequence of SSR462 is: TCAC;

[0019] The core repeating unit sequence of SSR540 is: TTCC.

[0020] The second aspect of the present application provides primers for the above two strong parasitic Armillaria four- to six-base repeat SSR microsatellite markers, the forward sequence and reverse sequence of the primer SSR7 are shown in SED ID No. 12 and SED ID No. 13;

[0021] The forward and reverse sequences of the primers for SSR109 are shown in SED ID No. 14 and SED ID No. 15;

[0022] The forward and reverse sequences of the primers for SSR129 are shown in SED ID No. 16 and SED ID No. 17;

[0023] The forward and reverse sequences of the primers for SSR221 are shown in SED ID No. 18 and SED ID No. 19;

[0024] The forward and reverse sequences of primers for SSR228 are shown in SED ID No. 20 and SED ID No. 21;

[0025] The forward and reverse sequences of the primers for SSR262 are shown in SED ID No. 22 and SED ID No. 23;

[0026] The forward and reverse sequences of the primers for SSR263 are shown in SED ID No. 24 and SED ID No. 25;

[0027] The forward and reverse sequences of primers for SSR354 are shown in SED ID No. 26 and SED ID No. 27;

[0028] The forward and reverse sequences of the primers for SSR387 are shown in SED ID No. 28 and SED ID No. 29;

[0029] The forward and reverse sequences of the primers for SSR462 are shown in SED ID No. 30 and SED ID No. 31;

[0030] The forward and reverse sequences of the primers for SSR540 are shown as SED ID No. 32 and SED ID No. 33.

[0031] Furthermore, the 5' end of the forward primer of the SSR microsatellite marker carries a fluorescent marker.

[0032] Furthermore, the fluorescent marker of SSR7, SSR109, and SSR221 is FAM; the fluorescent marker of SSR129, SSR228, SSR262, SSR263, and SSR354 is HEX; the fluorescent marker of SSR387 and SSR462 is ROX; and the fluorescent marker of SSR540 is TAMRA.

[0033] The third aspect of the present application provides the application of the above-mentioned two highly parasitic Armillaria four- to six-base repeat SSR microsatellite markers in the population genetic diversity analysis, germplasm resource identification, and molecular-assisted breeding of highly parasitic Armillaria.

[0034] Beneficial effects:

[0035] The present invention is based on two strong parasitic honey fungi Armillaria mellea The genome assembly data of 12 different strains were used to predict four to six base repeat SSR microsatellite markers by comparative genomic technology. 42 loci with higher scores and corresponding primer sequences were selected. The results were used to isolate the two strong parasitic Armillaria mellea The SSR microsatellite markers and primers provided by the present invention can provide powerful tools for the identification of germplasm resources, population evolution and genetic diversity analysis of strong parasitic Armillaria mellea. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is the BUSCO evaluation result of the genome assembly quality of the population samples used for SSR locus prediction and primer development in the present invention;

[0037] Figure 2 It is the alleles and allele frequency distribution diagram of 11 SSR microsatellite markers amplified in 12 strains of two strong parasitic Armillaria species in the present invention;

[0038] Figure 3 It is Nei's (1983) standard genetic distance based on the neighbor-joining method among 12 strains based on 11 SSR loci in the present invention. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with examples. It should be understood that the examples are only for illustrative purposes and are not intended to limit the present invention. Reagents not described in detail in the examples are all conventional reagents and can be obtained from commercial sources; experimental methods and equipment not specifically described in detail are all conventional experimental methods and equipment in the art.

[0040] In the detection of SSR microsatellite markers, when a longer core repeating unit sequence (such as four to six base repeats) can be used, the frequency of frameshift phenomenon can be reduced and the amplification specificity can be improved. The detected SSR sites have higher conservation and specificity while maintaining a certain polymorphism, which is suitable for a wider range of species and population studies, as well as long-term genetic research and evolutionary analysis. Therefore, based on the two to three base repeat SSR microsatellite markers previously developed in this laboratory (Liu Jianwei, Yang Shimei, Yang Zhuliang, et al. Two universal polymorphic microsatellite molecular markers for two strongly parasitic Armillaria mellea and their primers and applications [P]. Yunnan Province: CN202310299313.0, 2023-12-15.), the present invention is based on two strongly parasitic Armillaria mellea universal polymorphic microsatellite molecular markers and their primers and applications [P]. Yunnan Province: CN202310299313.0, 2023-12-15.). Armillaria mellea The genome assembly data of the population samples were used to predict the polymorphic SSR microsatellite markers, and the four- to six-base repeat microsatellite loci with relatively ideal scores were selected and verified in the samples of the highly parasitic Armillaria population to obtain more specific molecular markers.

[0041] Example

[0042] 1. Genome extraction and detection

[0043] Total DNA was extracted using a plant genomic DNA extraction kit (200), and the specific steps are as follows:

[0044] (1) Place the adsorption column in a collection tube, add 250 μL of Buffer BL, and centrifuge at 12,000 × g for 1 min to activate the silica membrane.

[0045] (2) Take the dry weight tissue of the sample (no more than 20 mg), add liquid nitrogen and grind it thoroughly. After grinding, place it in a 1.5 mL centrifuge tube, add 400 μL Buffer gP1, vortex and oscillate for 1 min, and then place it in a 65°C water bath for 10-30 min;

[0046] (3) Add 150 μL Buffer gP2, vortex for 1 min, and place on ice for 5 min;

[0047] (4) Centrifuge at 12,000 × g for 5 min and transfer the supernatant to a new centrifuge tube;

[0048] (5) Add an equal volume of anhydrous ethanol to the supernatant, vortex and mix thoroughly, transfer all the liquid to the adsorption column, centrifuge at 12,000 × g for 30 s, and discard the waste liquid;

[0049] (6) Add 500 μL of Buffer PW (anhydrous ethanol has been added before use) to the adsorption column, centrifuge at 12,000 × g for 30 s, and discard the waste liquid;

[0050] (7) Add 500 μL Wash Buffer (anhydrous ethanol has been added before use) to the adsorption column, centrifuge at 12,000 × g for 30 s, and discard the waste liquid;

[0051] (8) Repeat step (7);

[0052] (9) Place the adsorption column back into the collection tube, centrifuge at 12,000 × g for 2 min, and open the lid to dry for 1 min.

[0053] (10) Remove the adsorption column and place it in a clean centrifuge tube. Add 50-100 μL TEBuffer (preheated at 65°C) to the center of the adsorption membrane. Incubate at 20-25°C for 2 min and centrifuge at 12,000 × g for 2 min.

[0054] (11) Detect the DNA concentration using a spectrophotometer, adjust the concentration to 100 ng / µL, and store in a -20°C refrigerator.

[0055] 2. Genomic SSR loci and primer development

[0056] The genome assembly data of 12 different strains of two strongly parasitic Armillaria melleae were used for development. The genome data of strains CBSK00111 and CBSG19037 in biological species CBSK and CBSG were assembled from PacBio HiFi sequencing data; the genome data of the remaining 10 strains, including 21LJ03, 21SNJ05, A10, CBSK19083, guo242, guo256, HKAS49004, HKAS81832, LJW849, and XCB003, were assembled from Illumina second-generation whole genome sequencing data. The assembly data of CBSK00111 was selected as the reference sequence, and the CandiSSR (Xia et al., 2016) software was used for the prediction of SSR microsatellite loci and primer design, and all parameters were default parameters. Primers with a missing rate of 0 were selected from the obtained results, and then arranged in descending order according to the standard deviation, and the corresponding primer pairs ranked in the top 42 of the standard deviation were selected for subsequent population verification.

[0057] Figure 1 It is the BUSCO evaluation result of the genome assembly quality of population samples used for SSR site prediction and primer development in the present invention.

[0058] 3. PCR amplification of SSR loci

[0059] The 42 primer pairs screened above were synthesized, and the forward primer of each primer pair was labeled with a fluorescent dye at the 5' end, and then PCR amplification was performed in 12 highly parasitic Armillaria samples (CBSK19083, SJ113, guo237, A10, 21SNJ05, K19015, 21LJ03, guo242, HKAS49004, XCB003, CBSG19037, HKAS81832). The components of the PCR amplification system were: 17 μL Gold Mix (green), 1 μL 10 μM Primer F (with fluorescent marker), 1 μL 10 μM Primer R, and 1 μL Template (gDNA). The amplification program of the PCR amplification system is: 98°C for 2 min; 98°C for 10 s, 65 / 56°C for 10 s, 72°C for 10 s, 35 cycles; 72°C for 5 min; the amplification annealing temperature of the primers is: 65°C for the first amplification annealing temperature and 56°C for the second amplification annealing temperature.

[0060] IV. PCR product detection and genotyping of SSR loci

[0061] The amplified PCR products were subjected to agarose gel electrophoresis (2 μL sample + 6 μL bromophenol blue), and the identification gel image was obtained to determine the template concentration, followed by capillary electrophoresis to genotype the PCR amplification results of all samples. Among them, the genotyping of all samples was performed on the ABI 3730 DNA Analyzer, and the number of alleles in each sample was detected using GeneMapper 4.1, and the size of the allele was determined relative to the molecular internal standard GS500LIZ.

[0062] 5. Site Analysis and Screening

[0063] The software GeneMapper 4.1 was used to analyze the accurate sites of the data, and the exact size of the site was determined based on the core base repeat number of the primer corresponding to the SSR site. Then, the peak graph and data obtained from the analysis were used to determine whether the detection primer had site polymorphism, and sites with high specificity and good polymorphism were selected for subsequent analysis.

[0064] After the above experiments, the microsatellite molecular markers obtained were: SSR7, SSR109, SSR129, SSR221, SSR228, SSR262, SSR263, SSR354, SSR387, SSR462, and SSR540, whose nucleotide sequences correspond to SED ID No. 1-11 (Table 1), and the corresponding primer sequences are shown in SEQ ID No. 12-33 (Table 3).

[0065] Table 1 Nucleotide sequence information of SSR molecular markers

[0066]

[0067] Table 1 Nucleotide sequence information of SSR molecular markers

[0068]

[0069] Table 2 The core repeating unit sequences of the 11 SSR microsatellite loci in the present invention

[0070]

[0071] Table 3 Information of 11 SSR microsatellite loci and their corresponding primers in the present invention

[0072]

[0073] Note: F is the forward primer; R is the reverse primer; FAM is 6-carboxyfluorescein; HEX is hexachloro-fluorescein; ROX is carboxy-X-rhodamine; TAMRA is tetramethylrhodamine.

[0074] VI. Genetic diversity analysis of SSR loci

[0075] GenAlEx version 6.501 and PowerMarker V3.25 software were used to calculate the genetic parameters of the developed SSR in 12 strong parasitic Armillaria populations, such as the number of alleles (Na), the number of effective alleles (Ne), and the Shannon's information index (I), and genetic diversity analysis was performed. The results are shown in Table 4. The observed number of alleles (Na) of the 11 SSR microsatellite loci in the 12 samples was 3-8, the average Shannon's diversity index (I) was 1.366, the average observed heterozygosity (Ho) was 0.416, the average expected heterozygosity (He) was 0.679, and the average polymorphism information content PIC was 0.662.

[0076] The above results show that the 11 SSR loci in the present invention have high polymorphism in the samples and can distinguish all the samples from the wild environment (such as Figure 3 ).

[0077] Table 4 Genetic diversity information of 11 SSR microsatellite loci

[0078]

[0079] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. For those skilled in the art, several improvements made without departing from the principles of the present invention are considered to be within the protection scope of the present invention.

Claims

1. Primers for two strong parasitic Armillaria tetra- to hexa-base repeat SSR microsatellite markers, characterized in that: The SSR microsatellite markers are SSR7, SSR109, SSR129, SSR221, SSR228, SSR262, SSR263, SSR354, SSR387, SSR462, and SSR540; The core repeating unit sequence of the SSR7 is: AATG; The core repeating unit sequence of SSR109 is: ATTGGT; The core repeating unit sequence of SSR129 is: CAAA; The core repeating unit sequence of SSR221 is: CTGCTC; The core repeating unit sequence of SSR228 is: CTTC; The core repeating unit sequence of SSR262 is: GAAG; The core repeating unit sequence of SSR263 is: GAAG; The core repeating unit sequence of SSR354 is: GGAA; The core repeating unit sequence of SSR387 is: GATG; The core repeating unit sequence of SSR462 is: TGAC; The core repeating unit sequence of the SSR540 is: TTCC; The forward sequence and reverse sequence of the SSR7 primer are shown in SED ID No. 12 and SED ID No. 13; The forward sequence and reverse sequence of the primer of SSR109 are shown in SED ID No. 14 and SED ID No. 15; The forward sequence and reverse sequence of the primer of SSR129 are shown in SED ID No. 16 and SED ID No. 17; The forward sequence and reverse sequence of the primer of SSR221 are shown in SED ID No. 18 and SED ID No. 19; The forward sequence and reverse sequence of the primer of SSR228 are shown as SED ID No. 20 and SED ID No. 21; The forward sequence and reverse sequence of the primer of SSR262 are shown in SED ID No. 22 and SED ID No. 23; The forward sequence and reverse sequence of the primer of SSR263 are shown in SED ID No. 24 and SED ID No. 25; The forward sequence and reverse sequence of the primer of SSR354 are shown in SED ID No. 26 and SED ID No. 27; The forward sequence and reverse sequence of the primer of SSR387 are shown in SED ID No. 28 and SED ID No. 29; The forward sequence and reverse sequence of the primer of SSR462 are shown as SED ID No. 30 and SED ID No. 31; The forward sequence and reverse sequence of the primer of SSR540 are shown in SED ID No. 32 and SED ID No. 33; The two strong parasitic Armillaria are CBSK and CBSG.

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