Primer for repeating SSR (simple sequence repeat) microsatellite marker of four to six bases of armillaria auruginosa

Abstract

The invention relates to a primer for repeating SSR (Simple Sequence Repeat) microsatellite markers of four to six basic groups of Armillaria aululifera, and belongs to the technical field of molecular biology. The microsatellite markers are CPSSR-1, CPSSR-91, CPSSR-96, CPSSR-209, CPSSR-334, CPSSR-336, CPSSR-339, CPSSR-364, CPSSR-406, CPSSR-409, CPSSR-481, CPSSR-518, CPSSR-521, CPSSR-531, CPSSR-583 and CPSSR-637, and the nucleotide sequences of the microsatellite markers are as shown in SEQ ID No. 1 to SEQ ID No. 16. The nucleotide sequence of the primer is as shown in SEQ ID No. 33 to 64. Compared with a traditional method, the method has the remarkable advantages of being short in detection time, high in accuracy, good in repeatability and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to primers for Armillaria gallica tetra- to hexa-base repeat SSR microsatellite markers. Background Art

[0002] Armillaria fungus ( Armillaria spp.) belongs to the phylum Basidiomycetes, class Agaricales, order Agaricales, family Aquilegiaceae, genus Armillaria. It is not only a famous edible fungus, but also an indispensable symbiotic fungus in the cultivation of Gastrodia elata. With the in-depth study of the pharmacological effects of Gastrodia elata, the demand for Gastrodia elata has been increasing. Since the 1970s, Gastrodia elata has gradually turned to artificial cultivation to make up for the serious shortage of natural resources. In the process of symbiosis with Gastrodia elata, Armillaria can provide Gastrodia elata with the nutrients needed for growth, which makes it the key to the success of Gastrodia elata cultivation. In the production of Gastrodia elata, Armillaria gallica is the most widely used Armillaria species. However, due to the lack of molecular fingerprint recognition system related to strain identification, strain identification in production is difficult, and it is extremely difficult to identify strains based on morphological characteristics. Therefore, in order to accurately identify the biological species and phylogenetic species of Armillaria and provide a scientific basis for its species classification, further strengthening the research on Armillaria resources, and developing molecular markers with strong specificity, good stability and high polymorphism for Armillaria gallica will be beneficial to the identification and protection of Armillaria germplasm resources, the analysis of population genetic diversity, and provide technical support for improving the quality and yield of Gastrodia elata cultivation.

[0003] Simple sequence repeats (SSRs), also known as microsatellites or short tandem repeats (STRs), are non-coding repetitive DNA regions composed of tandem repeats of small motifs of 1 to 6 nucleotides. Due to its co-dominance, high polymorphism (the same repeat unit shows different numbers of repeats 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, and population genetic structure analysis. With the development of sequencing technology and multiplex PCR technology, it has become possible to identify a large number of SSR loci in non-model species at a low cost, and the use of SSR markers continues to increase.

[0004] In SSR marker detection, the use of shorter repeat units (such as di- or tri-nucleotide repeats) can detect more SSR sites in one experiment; the use of longer repeat units (such as four to six base repeats) can reduce frameshift phenomena and improve amplification specificity. At the same time, while maintaining polymorphism, it increases the conservation and specificity of the site, which is suitable for a wide range of species and population studies, as well as long-term genetic research and evolutionary analysis.

[0005] Therefore, the present invention provides a molecular marker with stronger specificity to improve the accuracy of genotyping and provide technical support for the identification of germplasm resources of Armillaria gallica. Summary of the invention

[0006] The present invention aims to provide an Armillaria Gallicum SSR marker with good amplification stability and high polymorphism, and proposes an Armillaria Gallicum SSR marker based on four to six base repeats, corresponding primers and applications thereof.

[0007] To achieve the above object, the present invention is implemented through the following technical solutions: The four to six base repeat SSR microsatellite markers of Armillaria gallica are CPSSR-1, CPSSR-91, CPSSR-96, CPSSR-209, CPSSR-334, CPSSR-336, CPSSR-339, CPSSR-364, CPSSR-406, CPSSR-409, CPSSR-481, CPSSR-518, CPSSR-521, CPSSR-531, CPSSR-583, and CPSSR-637, and their nucleotide sequences are shown in SEQ ID No. 1~16.

[0008] Furthermore, the nucleotide sequence of the microsatellite marker CPSSR-406 is shown in SED ID No. 1; The nucleotide sequence of the microsatellite marker CPSSR-481 is shown in SED ID No. 2; The nucleotide sequence of the microsatellite marker CPSSR-1 is shown in SED ID No. 3; The nucleotide sequence of the microsatellite marker CPSSR-209 is shown in SED ID No. 4; The nucleotide sequence of the microsatellite marker CPSSR-91 is shown in SED ID No. 5; The nucleotide sequence of the microsatellite marker CPSSR-96 is shown in SED ID No. 6; The nucleotide sequence of the microsatellite marker CPSSR-334 is shown in SED ID No. 7; The nucleotide sequence of the microsatellite marker CPSSR-336 is shown in SED ID No. 8; The nucleotide sequence of the microsatellite marker CPSSR-339 is shown in SED ID No. 9; The nucleotide sequence of the microsatellite marker CPSSR-364 is shown in SED ID No. 10; The nucleotide sequence of the microsatellite marker CPSSR-409 is shown in SED ID No. 11; The nucleotide sequence of the microsatellite marker CPSSR-518 is shown in SED ID No. 12; The nucleotide sequence of the microsatellite marker CPSSR-521 is shown in SED ID No. 13; The nucleotide sequence of the microsatellite marker CPSSR-531 is shown in SED ID No. 14; The nucleotide sequence of the microsatellite marker CPSSR-583 is shown in SED ID No. 15; The nucleotide sequence of the microsatellite marker CPSSR-637 is shown in SED ID No. 16.

[0009] Furthermore, the present invention provides the nucleotide core repeating unit of the above-mentioned Armillaria gallica four to six base repeating SSR microsatellite marker; The repeating unit of the CPSSR-406 is: GAAAGG; The repeating unit of the CPSSR-481 is: GCTCTG; The repeating unit of the CPSSR-1 is: AAATA; The repeating unit of the CPSSR-209 is: CACAC; The repeating unit of the CPSSR-91 is: AGAT; The repeating unit of the CPSSR-96 is: AGAT; The repeating unit of the CPSSR-334 is: CTAC; The repeating unit of the CPSSR-336 is: CTAC; The repeating unit of the CPSSR-339 is: CTAC; The repeating unit of the CPSSR-364 is: CTTC; The repeating unit of the CPSSR-409 is: GAAG; The repeating unit of the CPSSR-518 is: GGTA; The repeating unit of the CPSSR-521 is: GGTA; The repeating unit of the CPSSR-531 is: GTAT; The repeating unit of the CPSSR-583 is: TATC; The repeating unit of the CPSSR-637 is: TCCT; The present invention also provides a primer for a four- to six-base repeat SSR marker of Armillaria gallica, and the forward sequence and reverse sequence of the primer CPSSR-406 are shown in SED ID No. 33 and SED ID No. 34; The forward and reverse sequences of primers for CPSSR-481 are shown in SED ID No. 35 and SED ID No. 36; The forward and reverse sequences of primers for CPSSR-1 are shown in SED ID No. 37 and SED ID No. 38; The forward and reverse sequences of primers for CPSSR-209 are shown in SED ID No. 39 and SED ID No. 40; The forward and reverse sequences of primers for CPSSR-91 are shown in SED ID No. 41 and SED ID No. 42; The forward and reverse sequences of primers for CPSSR-96 are shown in SED ID No. 43 and SED ID No. 44; The forward and reverse sequences of primers for CPSSR-334 are shown in SED ID No. 45 and SED ID No. 46; The forward and reverse sequences of primers for CPSSR-336 are shown in SED ID No. 47 and SED ID No. 48; The forward and reverse sequences of the primers for CPSSR-339 are shown in SED ID No. 49 and SED ID No. 50; The forward and reverse sequences of primers for CPSSR-364 are shown in SED ID No. 51 and SED ID No. 52; The forward and reverse sequences of primers for CPSSR-409 are shown in SED ID No. 53 and SED ID No. 54; The forward and reverse sequences of primers for CPSSR-518 are shown in SED ID No. 55 and SED ID No. 56; The forward and reverse sequences of primers for CPSSR-521 are shown in SED ID No. 57 and SED ID No. 58; The forward and reverse sequences of primers for CPSSR-531 are shown in SED ID No. 59 and SED ID No. 60; The forward and reverse sequences of primers for CPSSR-583 are shown in SED ID No. 61 and SED ID No. 62; The forward and reverse sequences of the primers for CPSSR-637 are shown in SED ID No. 63 and SED ID No. 64.

[0010] Furthermore, the 5' end of the forward primer in the primer pair corresponding to the SSR marker carries a different fluorescent marker, which is characterized in that: the fluorescent marker of the primer pairs CPSSR-406, CPSSR-481, CPSSR-1, CPSSR-209, and CPSSR-91 is FAM; the fluorescent marker of the primer pairs CPSSR-96, CPSSR-334, CPSSR-336, CPSSR-339, and CPSSR-364 is HEX; the fluorescent marker of the primer pairs CPSSR-409, CPSSR-518, CPSSR-521, CPSSR-531, and CPSSR-583 is ROX; and the fluorescent marker of the primer pair CPSSR-637 is TAMRA.

[0011] The beneficial effects of the present invention are: The present invention is based on the genome assembly data of 18 different strains of Armillaria gallica, predicts four to six base repeat SSR markers by comparative genomic technology, selects 42 sites with higher scores and corresponding primer sequences, and verifies them in 12 Armillaria gallica population samples collected from Heilongjiang Province, Liaoning Province, and Jilin Province, and finally develops 16 SSR molecular markers with high polymorphism. The SSR 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 Armillaria gallica. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] 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; Figure 2 It is the alleles and allele frequency distribution diagram of 16 SSR microsatellite markers amplified in 12 strains of Armillaria gallica; Figure 3 It is Nei's (1983) standard genetic distance based on the neighbor-joining method among 12 strains based on 16 SSR loci in the present invention. DETAILED DESCRIPTION

[0013] 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.

[0014] Example 1. Genome extraction and detection Total DNA was extracted using a magnetic bead plant DNA extraction kit with the help of KingFisher fully automatic nucleic acid extractor - Auto-Pure 96. The specific steps are as follows: (1) Take the dry weight tissue of the sample (no more than 20 mg) and put it into a 2 ml centrifuge tube, add steel beads, and put it into the high-throughput tissue grinder SCIENTZ-48, 60 Hz, 60 s, 2 times, and grind it into powder; (2) Add 500 μL Buffer GT, vortex for 1 min to fully disperse the sample, and leave at room temperature for 15 minutes; (3) Centrifuge at 13,000 × g for 3 min and keep the supernatant for later use; (4) Collect and package as shown in Table 1: Table 1 (5) Set up the program according to the settings described in Table 2: Table 2 (6) After the procedure is completed, transfer the DNA from plate position 8 to a 1.5 ml centrifuge tube; (7) Detect the DNA concentration using a spectrophotometer, adjust the concentration to 100 ng / µL, and store in a -20°C refrigerator.

[0015] 2. SSR loci and primer development of Armillaria gallica genome: The genome assembly data of 18 different strains of Armillaria gallica completed in the laboratory were used for development. The genome data of 18 strains BJ-M1, CBSB03002, CBSB19036, CBSB96003, CBSB96011, CBSB96031, CBSB96032, HS-A25, HS-A9, LD-M8, LZL-17, LZL-19, LZL-1, LZL-4, MF-M5, SS-01, WSB-M1, and Y3 were assembled from Illumina second-generation whole genome sequencing data. The assembly data of CBSB96032 was selected as the reference sequence, and the CandiSSR (Xia et al., 2016) software was used for SSR locus prediction 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.

[0016] 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.

[0017] 3. PCR amplification of SSR loci 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. Then, PCR amplification was performed in 12 samples of Armillaria gallica (SJ73, SJ180, SJ154, SJ22, SJ105, SJ96, SJ76, SJ40, SJ175, SJ172, SJ182, and SJ35) to detect the presence of bands. The PCR amplification system was: 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 was: 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 was: 65°C for the first amplification annealing temperature and 56°C for the second amplification annealing temperature.

[0018] IV. PCR product detection and genotyping of SSR loci 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.

[0019] 5. Site Analysis and Screening 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.

[0020] After the above experiments, the microsatellite molecular markers obtained are: CPSSR-1, CPSSR-91, CPSSR-96, CPSSR-209, CPSSR-334, CPSSR-336, CPSSR-339, CPSSR-364, CPSSR-406, CPSSR-409, CPSSR-481, CPSSR-518, CPSSR-521, CPSSR-531, CPSSR-583, and CPSSR-637, whose nucleotide sequences are shown in SEQ ID No. 1-16, the nucleotide sequences of the core repeating units are shown in SED ID No. 17-32, and the nucleotide sequences corresponding to the SSR molecular marker primers are shown in SED ID No. 33-64.

[0021] Table 3 The core repeating unit sequences of the 16 SSR microsatellite loci in the present invention Table 4 16 SSR microsatellite loci and their corresponding primer information in the present invention 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 VI. Genetic diversity analysis of SSR loci GenAlEx version 6.501 and PowerMarker V3.25 software were used to calculate the genetic parameters of the SSRs developed in 12 Armillaria gallica populations, including the number of alleles (Na), the number of effective alleles (Ne), and the Shannon's information index (I), and genetic diversity analysis was performed. Figure 2 Shown are the alleles and allele frequency distribution diagrams amplified by 16 SSR microsatellite markers in the present invention in 12 strains of Armillaria gallica.

[0022] The results are shown in Table 5. The observed allele numbers (Na) of the 16 SSR microsatellite loci in 12 samples ranged from 4 to 9, the average Shannon diversity index (I) was 1.549, the average observed heterozygosity (Ho) was 0.393, the average expected heterozygosity (He) was 0.716, and the average polymorphism information content Pic was 0.689.

[0023] The above results show that the 16 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 ).

[0024] Table 5 Genetic diversity information of 16 SSR microsatellite loci 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 the four- to six-base repeat SSR microsatellite markers of Armillaria gallica, characterized in that: The microsatellite markers are: CPSSR-1, CPSSR-91, CPSSR-96, CPSSR-209, CPSSR-334, CPSSR-336, CPSSR-339, CPSSR-364, CPSSR-406, CPSSR-409, CPSSR-481, CPSSR-518, CPSSR-521, CPSSR-531, CPSSR-583, CPSSR-637, and their nucleotide sequences are shown in SEQ ID No. 1-16; The repeating unit of the microsatellite marker CPSSR-406 is: GAAAGG; The repeating unit of the microsatellite marker CPSSR-481 is: GCTCTG; The repeating unit of the microsatellite marker CPSSR-1 is: AAATA; The repeating unit of the microsatellite marker CPSSR-209 is: CACAC; The repeating unit of the microsatellite marker CPSSR-91 is: AGAT; The repeating unit of the microsatellite marker CPSSR-96 is: AGAT; The repeating unit of the microsatellite marker CPSSR-334 is: CTAC; The repeating unit of the microsatellite marker CPSSR-336 is: CTAC; The repeating unit of the microsatellite marker CPSSR-339 is: CTAC; The repeating unit of the microsatellite marker CPSSR-364 is: CTTC; The repeating unit of the microsatellite marker CPSSR-409 is: GAAG; The repeating unit of the microsatellite marker CPSSR-518 is: GGTA; The repeating unit of the microsatellite marker CPSSR-521 is: GGTA; The repeating unit of the microsatellite marker CPSSR-531 is: GTAT; The repeating unit of the microsatellite marker CPSSR-583 is: TATC; The repeating unit of the microsatellite marker CPSSR-637 is: TCCT; The forward sequence and reverse sequence of the primer of CPSSR-406 are shown in SED ID No. 33 and SED ID No. 34; The forward sequence and reverse sequence of the primer of CPSSR-481 are shown in SED ID No. 35 and SED ID No. 36; The forward sequence and reverse sequence of the primer of CPSSR-1 are shown in SED ID No. 37 and SED ID No. 38; The forward sequence and reverse sequence of the primer of CPSSR-209 are shown in SED ID No. 39 and SED ID No. 40; The forward sequence and reverse sequence of the primer of CPSSR-91 are shown in SED ID No. 41 and SED ID No. 42; The forward sequence and reverse sequence of the primer of CPSSR-96 are shown in SED ID No. 43 and SED ID No. 44; The forward sequence and reverse sequence of the primer of CPSSR-334 are shown in SED ID No. 45 and SED ID No. 46; The forward sequence and reverse sequence of the primer of CPSSR-336 are shown in SED ID No. 47 and SED ID No. 48; The forward sequence and reverse sequence of the primer of CPSSR-339 are shown in SED ID No. 49 and SED ID No. 50; The forward sequence and reverse sequence of the primer of CPSSR-364 are shown in SED ID No. 51 and SED ID No. 52; The forward sequence and reverse sequence of the primer of CPSSR-409 are shown in SED ID No. 53 and SED ID No. 54; The forward sequence and reverse sequence of the primer of CPSSR-518 are shown in SED ID No. 55 and SED ID No. 56; The forward sequence and reverse sequence of the primer of CPSSR-521 are shown in SED ID No. 57 and SED ID No. 58; The forward sequence and reverse sequence of the primer of CPSSR-531 are shown in SED ID No. 59 and SED ID No. 60; The forward sequence and reverse sequence of the primer of CPSSR-583 are shown in SED ID No. 61 and SED ID No. 62; The forward sequence and reverse sequence of the primer of CPSSR-637 are shown as SED ID No. 63 and SED ID No. 64.

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