Primers for four to six-base repeat SSR microsatellite markers of Armillaria gallica

Abstract

The present invention relates to primers for four- to six-base repeat SSR microsatellite markers of Armillaria gallica, belonging 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, CPSSR-637, and their nucleotide sequences are as shown in SEQ ID No. 1 to 16. The nucleotide sequences of the primers are as shown in SEQ ID No. 33 to 64. Compared with traditional methods, the present invention has significant advantages such as short detection time, high accuracy, and good repeatability.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and particularly relates to primers for four- to six-base repeat SSR microsatellite markers of Armillaria gallica. Background Art

[0002] Armillaria ( Armillaria spp.) belongs to Basidiomycota, Agaricomycetes, Agaricales, Physalacriaceae, Armillaria, and is not only a well-known edible mushroom but also an indispensable symbiotic fungus in Gastrodia elata cultivation. With the in-depth study of the pharmacological effects of Gastrodia elata, the demand for Gastrodia elata has been increasing continuously. Since the 1970s, Gastrodia elata has gradually been converted to artificial cultivation to make up for the serious shortage of natural resources. During the symbiotic process with Gastrodia elata, Armillaria can provide the nutrients required for the growth of Gastrodia elata, which makes it the key to the success of Gastrodia elata cultivation. In Gastrodia elata production, Armillaria gallica is the most widely used Armillaria species. However, due to the lack of a molecular fingerprint recognition system related to strain identification, it is difficult to identify strains in production, and it is extremely difficult to identify species based on morphological characteristics. Therefore, to accurately identify the biological species and phylogenetic species of Armillaria and provide a scientific basis for its species classification, further strengthen the research on Armillaria resources, and develop 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, population genetic diversity analysis, and provide technical support for improving the cultivation quality and yield of Gastrodia elata.

[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. Because of its characteristics of codominance, high polymorphism (the number of repeats of the same repeat unit shows differences in different species or even different individuals), and genetic stability, SSR has been widely used as a molecular marker in the research of eukaryotic genetic map construction, fingerprint recognition, germplasm identification, population genetic structure analysis, etc. With the development of sequencing technology and multiplex PCR technology, it has become possible to identify a large number of SSR loci at a lower cost in non-model species, and the use of SSR markers continues to increase.

[0004] In SSR marker detection, using shorter repeat units (such as dinucleotide or trinucleotide repeats) can detect more SSR loci in one experiment; using longer repeat units (such as four- to six-base repeats) can reduce frameshift phenomena, improve amplification specificity, and at the same time, on the basis of maintaining polymorphism, increase the conservation and specificity of loci, which is suitable for extensive species and population research, and is also applicable to 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*, etc. Summary of the Invention

[0006] The object of the present invention is to provide an *Armillaria gallica* SSR marker with good amplification stability and high polymorphism, and to propose an *Armillaria gallica* SSR marker based on four- to six-base repeats, corresponding primers and their applications.

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

[0008] 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, CPSSR-637, and their nucleotide sequences are as shown in SEQ ID No. 1~16.

[0009] Further, the nucleotide sequence of the microsatellite marker CPSSR-406 is as shown in SED ID No. 1;

[0010] The nucleotide sequence of the microsatellite marker CPSSR-481 is as shown in SED ID No. 2;

[0011] The nucleotide sequence of the microsatellite marker CPSSR-1 is as shown in SED ID No. 3;

[0012] The nucleotide sequence of the microsatellite marker CPSSR-209 is as shown in SED ID No. 4;

[0013] The nucleotide sequence of the microsatellite marker CPSSR-91 is as shown in SED ID No. 5;

[0014] The nucleotide sequence of the microsatellite marker CPSSR-96 is as shown in SED ID No. 6;

[0015] The nucleotide sequence of the microsatellite marker CPSSR-334 is as shown in SED ID No. 7;

[0016] The nucleotide sequence of the microsatellite marker CPSSR-336 is as shown in SED ID No. 8;

[0017] The nucleotide sequence of the microsatellite marker CPSSR-339 is shown in SED ID No. 9;

[0018] The nucleotide sequence of the microsatellite marker CPSSR-364 is shown in SED ID No. 10;

[0019] The nucleotide sequence of the microsatellite marker CPSSR-409 is shown in SED ID No. 11;

[0020] The nucleotide sequence of the microsatellite marker CPSSR-518 is shown in SED ID No. 12;

[0021] The nucleotide sequence of the microsatellite marker CPSSR-521 is shown in SED ID No. 13;

[0022] The nucleotide sequence of the microsatellite marker CPSSR-531 is shown in SED ID No. 14;

[0023] The nucleotide sequence of the microsatellite marker CPSSR-583 is shown in SED ID No. 15;

[0024] The nucleotide sequence of the microsatellite marker CPSSR-637 is shown in SED ID No. 16.

[0025] Furthermore, the present invention provides the nucleotide core repeat units of the above-mentioned Armillaria gallica four to six-base repeat SSR microsatellite markers;

[0026] The repeat unit of CPSSR-406 is: GAAAGG;

[0027] The repeat unit of CPSSR-481 is: GCTCTG;

[0028] The repeat unit of CPSSR-1 is: AAATA;

[0029] The repeat unit of CPSSR-209 is: CACAC;

[0030] The repeat unit of CPSSR-91 is: AGAT;

[0031] The repeat unit of CPSSR-96 is: AGAT;

[0032] The repeat unit of CPSSR-334 is: CTAC;

[0033] The repeat unit of CPSSR-336 is: CTAC;

[0034] The repeating unit of CPSSR-339 is: CTAC;

[0035] The repeating unit of CPSSR-364 is: CTTC;

[0036] The repeating unit of CPSSR-409 is: GAAG;

[0037] The repeating unit of CPSSR-518 is: GGTA;

[0038] The repeating unit of CPSSR-521 is: GGTA;

[0039] The repeating unit of CPSSR-531 is: GTAT;

[0040] The repeating unit of CPSSR-583 is: TATC;

[0041] The repeating unit of CPSSR-637 is: TCCT;

[0042] The present invention also provides a primer for the four- to six-base repeating SSR marker of Armillaria gallica. The forward and reverse sequences of the primer of CPSSR-406 are as shown in SED ID No. 17 and SED ID No. 18;

[0043] The forward and reverse sequences of the primer of CPSSR-481 are as shown in SED ID No. 19 and SED ID No. 20;

[0044] The forward and reverse sequences of the primer of CPSSR-1 are as shown in SED ID No. 21 and SED ID No. 22;

[0045] The forward and reverse sequences of the primer of CPSSR-209 are as shown in SED ID No. 23 and SED ID No. 24;

[0046] The forward and reverse sequences of the primer of CPSSR-91 are as shown in SED ID No. 25 and SED ID No. 26;

[0047] The forward and reverse sequences of the primer of CPSSR-96 are as shown in SED ID No. 27 and SED ID No. 28;

[0048] The forward and reverse sequences of the primer of CPSSR-334 are as shown in SED ID No. 29 and SED ID No. 30;

[0049] The forward and reverse sequences of the primers of CPSSR-336 are shown in SED ID No. 31 and SED ID No. 32;

[0050] The forward and reverse sequences of the primers of CPSSR-339 are shown in SED ID No. 33 and SED ID No. 34;

[0051] The forward and reverse sequences of the primers of CPSSR-364 are shown in SED ID No. 35 and SED ID No. 36;

[0052] The forward and reverse sequences of the primers of CPSSR-409 are shown in SED ID No. 37 and SED ID No. 38;

[0053] The forward and reverse sequences of the primers of CPSSR-518 are shown in SED ID No. 39 and SED ID No. 40;

[0054] The forward and reverse sequences of the primers of CPSSR-521 are shown in SED ID No.41 and SED ID No. 42;

[0055] The forward and reverse sequences of the primers of CPSSR-531 are shown in SED ID No. 43 and SED ID No. 44;

[0056] The forward and reverse sequences of the primers of CPSSR-583 are shown in SED ID No. 45 and SED ID No. 46;

[0057] The forward and reverse sequences of the primers of CPSSR-637 are shown in SED ID No. 47 and SED ID No. 48.

[0058] Furthermore, the 5'-ends of the forward primers in the corresponding primer pairs of the SSR markers carry different fluorescent markers, and the characteristics are as follows: the fluorescent markers of the primer pairs of CPSSR-406, CPSSR-481, CPSSR-1, CPSSR-209, and CPSSR-91 are FAM; the fluorescent markers of the primer pairs of CPSSR-96, CPSSR-334, CPSSR-336, CPSSR-339, and CPSSR-364 are HEX; the fluorescent markers of the primer pairs of CPSSR-409, CPSSR-518, CPSSR-521, CPSSR-531, and CPSSR-583 are ROX; the fluorescent marker of the primer pair of CPSSR-637 is TAMRA.

[0059] The beneficial effects of the present invention are as follows:

[0060] Based on the genomic assembly data of 18 different strains of Armillaria gallica, the present invention predicts tetra- to hexa-base repeat SSR markers through comparative genomics technology, selects 42 loci with higher scores and corresponding primer sequences among them, and verifies them in 12 population samples of Armillaria gallica collected from Heilongjiang Province, Liaoning Province, and Jilin Province. Finally, 16 SSR molecular markers with high polymorphism are developed. 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. Description of the Drawings

[0061] Figure 1 is the BUSCO assessment result of the genomic assembly quality of the population samples used for SSR locus prediction and primer development in the present invention;

[0062] Figure 2 is the distribution map of alleles and allele frequencies amplified by 16 SSR microsatellite markers in 12 strains of Armillaria gallica in the present invention;

[0063] Figure 3 is the Nei's (1983) standard genetic distance among 12 strains based on 16 SSR loci in the present invention. Detailed Embodiments

[0064] The present invention will be further described below in conjunction with embodiments. It should be understood that the embodiments are only for illustrative purposes and are not used to limit the present invention. The reagents not specifically described in detail in the embodiments are all conventional reagents and can be obtained through commercial channels; the experimental methods and equipment not specifically described in detail are all conventional experimental methods and equipment in the art.

[0065] Embodiment

[0066] I. Genome Extraction and Detection

[0067] Total DNA was extracted using a magnetic bead method plant DNA extraction kit with the help of a KingFisher fully automatic nucleic acid extractor - Auto-Pure 96. The specific steps are as follows:

[0068] (1) Take the dry weight tissue of the sample (not more than 20 mg) into a 2 ml centrifuge tube, add steel beads, and place it in a high-throughput tissue grinder SCIENTZ-48, grind it into powder at 60 Hz, 60 s, 2 times;

[0069] (2) Add 500 μL of Buffer GT, vortex for 1 min to fully disperse the sample, and let it stand at room temperature for 15 minutes;

[0070] (3) Centrifuge at 13,000×g for 3 min, and set aside the supernatant.

[0071] (4) Collect and aliquot according to Table 1 as follows:

[0072] Table 1

[0073]

[0074] (5) Set the program according to Table 2 as follows:

[0075] Table 2

[0076]

[0077] (6) After the program is completed, aspirate the DNA at well position 8 into a 1.5-ml centrifuge tube.

[0078] (7) Detect the DNA concentration with a spectrophotometer, adjust the concentration to 100 ng / µL, and store it in a -20°C refrigerator.

[0079] II. Development of SSR loci and primers for the Armillaria gallica genome:

[0080] Development was carried out using the genomic assembly data of 18 different strains of Armillaria gallica completed in the laboratory. The genomic data of the 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, with all parameters set to default values. Primers with a deletion rate of 0 were screened out from the obtained results, and then sorted in descending order according to the standard deviation. The top 42 corresponding primer pairs with the highest standard deviation were selected for subsequent population verification.

[0081] Figure 1 It is the BUSCO assessment result of the genomic assembly quality of the population samples used for SSR locus prediction and primer development in the present invention.

[0082] III. PCR amplification of SSR loci

[0083] Synthesize the 42 primer pairs screened above. The forward primer of each primer pair is labeled with a fluorescent dye at the 5' end. Subsequently, PCR amplification is performed in 12 Armillaria gallica samples (SJ73, SJ180, SJ154, SJ22, SJ105, SJ96, SJ76, SJ40, SJ175, SJ172, SJ182, SJ35) to detect the presence or absence of bands. The PCR amplification system is as follows: 17 μL of GoldMix (green), 1 μL of 10 μM Primer F (with fluorescent label), 1 μL of 10 μM Primer R, and 1 μL of 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, for 35 cycles; 72 °C for 5 min; among which the amplification annealing temperature of the primers is: the first-round annealing temperature is 65 °C, and the second-round annealing temperature is 56 °C.

[0084] IV. Detection of PCR Products and Genotyping of SSR Loci

[0085] Perform agarose gel electrophoresis on the amplified PCR products (2 μL of sample + 6 μL of bromophenol blue) to obtain an identification gel image to determine the template concentration. Subsequently, perform capillary electrophoresis to genotype the PCR amplification results of all samples. Among them, the genotyping of all samples is carried out on an ABI 3730 DNA Analyzer, and GeneMapper 4.1 is used to detect the number of alleles of each sample, and the size of the alleles is determined relative to the internal standard GS500LIZ in the molecule.

[0086] V. Locus Analysis and Screening

[0087] Use the software GeneMapper 4.1 to analyze the accurate loci of the data, and determine the accurate size of the loci according to the core base repeat number of the SSR loci corresponding to the primers. Subsequently, judge whether the detection primers have locus polymorphism based on the obtained peak images and data, and select loci with high specificity and good polymorphism for subsequent analysis.

[0088] After the above experiments, the microsatellite molecular markers obtained were: 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. Their nucleotide sequences are shown in SEQ ID No. 1-16, and the nucleotide sequences corresponding to the SSR molecular marker primers are shown in SED ID No. 17-48.

[0089] Table 3 Core repeat unit sequences of 16 SSR microsatellite loci in the present invention

[0090]

[0091] Table 4 16 SSR microsatellite loci in the present invention and their corresponding primer information

[0092]

[0093] 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

[0094] VI. Genetic diversity analysis of SSR loci

[0095] Use GenAlEx version 6.501 and PowerMarker V3.25 software to calculate genetic parameters such as the number of alleles (Na), effective number of alleles (Ne), and Shannon's information index (I) of the developed SSRs in 12 Armillaria gallica populations for genetic diversity analysis. Figure 2 Shown are the allele and allele frequency distribution maps amplified by 16 SSR microsatellite markers in the present invention in 12 strains of Armillaria gallica.

[0096] The results are shown in Table 5. The observed number of alleles (Na) of 16 SSR microsatellite loci in 12 samples was 4-9, the average value of the Shannon diversity index (I) was 1.549, the average value of the observed heterozygosity (Ho) was 0.393, the average value of the expected heterozygosity (He) was 0.716, and the average value of the polymorphism information content Pic was 0.689.

[0097] The above results indicate that the 16 SSR loci in the present invention have high polymorphism in the samples and can distinguish all those from the wild environment (such as Figure 3 ).

[0098] Table 5 Genetic diversity information of 16 SSR microsatellite loci

[0099]

[0100] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not restrictive. For those skilled in the art, without departing from the principle of the present invention, several improvements made are regarded as the protection scope of the present invention.

Claims

1. Primers for 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; 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 and reverse primer sequences of CPSSR-406 are shown as SED ID No. 17 and SED ID No. 18; The forward and reverse primer sequences of CPSSR-481 are shown as SED ID No. 19 and SED ID No. 20; The forward and reverse primer sequences of CPSSR-1 are shown as SED ID No. 21 and SED ID No. 22; The forward and reverse primer sequences of CPSSR-209 are shown as SED ID No. 23 and SED ID No. 24; The forward and reverse primer sequences of CPSSR-91 are shown as SED ID No. 25 and SED ID No. 26; The forward and reverse primer sequences of CPSSR-96 are shown as SED ID No. 27 and SED ID No. 28; The forward and reverse primer sequences of CPSSR-334 are shown in SEQ ID No. 29 and SEQ ID No. 30; The forward and reverse primer sequences of CPSSR-336 are shown in SEQ ID No. 31 and SEQ ID No. 32; The forward and reverse primer sequences of CPSSR-339 are shown in SEQ ID No. 33 and SEQ ID No. 34; The forward and reverse primer sequences of CPSSR-364 are shown in SEQ ID No. 35 and SEQ ID No. 36; The forward and reverse primer sequences of CPSSR-409 are shown in SEQ ID No. 37 and SEQ ID No. 38; The forward and reverse primer sequences of CPSSR-518 are shown in SEQ ID No. 39 and SEQ ID No. 40; The forward and reverse primer sequences of CPSSR-521 are shown in SEQ ID No. 41 and SEQ ID No. 42; The forward and reverse primer sequences of CPSSR-531 are shown in SEQ ID No. 43 and SEQ ID No. 44; The forward and reverse primer sequences of CPSSR-583 are shown in SEQ ID No. 45 and SEQ ID No. 46; The forward and reverse primer sequences of CPSSR-637 are shown in SEQ ID No. 47 and SEQ ID No. 48.

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