Macrobrachium rosenbergii transcriptome microsatellite multiple PCR system construction method

Abstract

The invention discloses a construction method of a macrobrachium rosenbergii transcriptome microsatellite multiple PCR (Polymerase Chain Reaction) system. The construction method comprises the following steps: obtaining macrobrachium rosenbergii transcriptome information, obtaining macrobrachium rosenbergii microsatellite site information, designing a microsatellite site primer sequence, grouping sites and verifying polymorphism, screening multiple PCR sites and constructing the multiple PCR system. The macrobrachium rosenbergii SSR molecular marker is developed, a multiple PCR system is constructed, a macrobrachium rosenbergii marker resource library is enriched, and a necessary tool is provided for macrobrachium rosenbergii population genetic diversity analysis and germplasm resource protection; according to the invention, the existing transcriptome public database resource is fully utilized, the constructed multiplex PCR system is high in marker flux, short in detection time, simple and convenient to operate, more accurate in genotyping result, and more economical and efficient.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic animal genetic breeding, and particularly relates to a method for constructing a microsatellite multiplex PCR system of Macrobrachium rosenbergii transcriptome. Background Art

[0002] Macrobrachium rosenbergii was introduced to my country from Japan by the Chinese Academy of Agricultural Sciences in 1976. However, with years of self-propagation, my country's farmed Macrobrachium rosenbergii has shown reduced population diversity and variety degeneration, which has hindered the sustainable development of the species and seriously affected the production and economic benefits of Macrobrachium rosenbergii. Understanding the genetic diversity of existing Macrobrachium rosenbergii germplasm resources is crucial for the effective protection and efficient utilization of germplasm resources.

[0003] At present, there are many molecular marker methods used for the study of shrimp genetic diversity. Simple sequence repeat markers (SSR, also known as microsatellite DNA), ISSR, EST-SSR, related sequence polymorphism (SRAP) and amplified fragment length polymorphism (AFLP) and other marker technologies have been adopted in shrimp diversity research. SSR is a DNA sequence composed of 2 to 6 nucleotides as repeating units, which is widely distributed in the genome of eukaryotic organisms. This sequence has the advantages of co-dominance, relatively rich polymorphism, and high genome coverage. Nowadays, the application of microsatellite markers has made rapid progress in kinship identification, genetic diversity analysis, genetic map construction, assisted selection breeding, and commercially important variety identification. However, to date, the microsatellite molecular marker resources of Macrobrachium rosenbergii are still insufficient, and currently only 66 pairs of microsatellite markers have been used to analyze the genetic diversity of this species. Therefore, the present invention provides a method for constructing a microsatellite multiplex PCR system for the transcriptome of Macrobrachium rosenbergii, which provides a necessary tool for the genetic diversity analysis of Macrobrachium rosenbergii population and the protection of germplasm resources. Summary of the invention

[0004] Technical problems solved: In response to the above technical problems, the present invention provides a method for constructing a microsatellite multiplex PCR system for the transcriptome of Macrobrachium rosenbergii, develops SSR molecular markers for Macrobrachium rosenbergii, constructs a multiplex PCR system, enriches the marker resource library of Macrobrachium rosenbergii, and provides necessary tools for genetic diversity analysis of Macrobrachium rosenbergii populations and protection of germplasm resources.

[0005] Technical solution: A method for constructing a microsatellite multiplex PCR system for the transcriptome of Macrobrachium rosenbergii, comprising the following steps:

[0006] S1. Use the public transcriptome database to obtain the transcriptome sequence of Macrobrachium rosenbergii;

[0007] S2. Using MISA software to analyze the transcriptome sequence of Macrobrachium rosenbergii in the above database to obtain the microsatellite locus information of Macrobrachium rosenbergii;

[0008] S3. According to the estimated size distribution of the amplified products, all sites are grouped into interval ranges according to the size of the amplified products, with one interval set for every 100 bp, for a total of three intervals;

[0009] S4. Randomly select 8 marker sites from each interval and use Primer Premier 6 software to design batch primers for 24 microsatellite loci;

[0010] S5. Randomly select 10 individual transcriptomes of Macrobrachium rosenbergii to test the polymorphism and primer amplification of the above microsatellite loci, and eliminate the loci that cannot amplify products or lack polymorphism;

[0011] S6. According to the grouping in step S3, the sequence compatibility of primers within and between polymorphic site groups in step S5 is calculated using AutoDimer software, and the six sites with the strongest sequence compatibility are retained;

[0012] S7, labeling the upstream primer of the site retained in step S6 with a fluorescent group, and resynthesizing the upstream primer;

[0013] S8, preparing a primer premix with the same concentration and a volume ratio of 1:25 for each upstream primer and downstream primer of each site retained in step S7, and then mixing equal volumes of the primer premixes of all sites to prepare a multiplex PCR primer working solution to construct a microsatellite multiplex PCR reaction system;

[0014] S9. Use conventional PCR methods to amplify the multiplex PCR system, use capillary electrophoresis to perform genotyping on the amplified products, and adjust the primer mixing ratio between each site according to the experimental results to achieve the best amplification effect.

[0015] Preferably, the nucleotide sequences of the primers for the 24 microsatellite loci in step S4 are shown in SEQ ID No. 1 to 48, as shown in Table 1 below:

[0016] Table 1 SSR primer sequences in the transcriptome of Macrobrachium rosenbergii

[0017]

[0018]

[0019]

[0020] Preferably, the PCR amplification program is shown in Table 2 below:

[0021] Table 2 PCR amplification program

[0022]

[0023] Preferably, the detailed information of the six sites with the strongest sequence compatibility retained in step S6 is shown in Table 3 below:

[0024] Table 3 Detailed information of the six sites with the strongest sequence compatibility

[0025]

[0026]

[0027] Preferably, in step S7, the fluorescent group is FAM.

[0028] Beneficial effects: The present invention provides a method for constructing a microsatellite multiplex PCR system for the transcriptome of Macrobrachium rosenbergii, develops SSR molecular markers for Macrobrachium rosenbergii, constructs a multiplex PCR system, enriches the marker resource library of Macrobrachium rosenbergii, and provides necessary tools for genetic diversity analysis of Macrobrachium rosenbergii populations and protection of germplasm resources; the present invention makes full use of existing transcriptome public database resources, and the constructed multiplex PCR system has high labeling flux, short detection time, simple operation, more accurate genotyping results, and is more economical and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The present invention is a schematic flow chart of a method for constructing a microsatellite multiplex PCR system for the transcriptome of Macrobrachium rosenbergii. DETAILED DESCRIPTION

[0030] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments:

[0031] Example 1

[0032] like Figure 1 As shown, a method for constructing a microsatellite multiplex PCR system for the transcriptome of Macrobrachium rosenbergii comprises the following steps:

[0033] S1. Use the public transcriptome database to obtain the transcriptome sequence of Macrobrachium rosenbergii;

[0034] S2. The transcriptome sequences of Macrobrachium rosenbergii in the above database were analyzed using MISA software to obtain the microsatellite loci information of Macrobrachium rosenbergii. The SSR search results in the transcriptome of Macrobrachium rosenbergii are shown in Table 4 below:

[0035] Table 4 Search results for SSRs in the transcriptome of Macrobrachium rosenbergii

[0036]

[0037]

[0038] The distribution of different repeat motifs of Macrobrachium rosenbergii SSR is shown in Table 5:

[0039] Table 5 Distribution of different repeat motifs in Macrobrachium rosenbergii SSR

[0040]

[0041] The types of SSR repeat motifs of Macrobrachium rosenbergii are shown in Table 6 below:

[0042] Table 6 Types of SSR repeat motifs in Macrobrachium rosenbergii

[0043]

[0044]

[0045] S3. According to the estimated size distribution of the amplified products, all sites are grouped into interval ranges according to the size of the amplified products, with one interval set for every 100 bp, for a total of three intervals;

[0046] S4. Randomly select 8 marker sites from each interval, use Primer Premier 6 software to design batch primers for 24 microsatellite sites. The SSR primer sequences in the transcriptome of Macrobrachium rosenbergii are shown in Table 1. The polymorphism information of the 24 polymorphic microsatellite marker sites of Macrobrachium rosenbergii is shown in Table 7 below:

[0047] Table 7 Polymorphism information of 24 polymorphic microsatellite marker loci in Macrobrachium rosenbergii

[0048]

[0049]

[0050] S5. Randomly select 10 individual transcriptomes of Macrobrachium rosenbergii to test the polymorphism and primer amplification of the above microsatellite loci, and eliminate the loci that cannot amplify products and lack polymorphism. The PCR amplification program for polymorphic marker detection is shown in Table 2, and the PCR amplification system is shown in Table 8 below:

[0051] Table 8 PCR amplification system

[0052]

[0053] The concentration of the template DNA was 100 ng / μL, the concentration of the upstream and downstream primers was 0.15 μM, and the two were mixed at a volume ratio of 1:25 to obtain an upstream and downstream primer mixture;

[0054] S6. According to the grouping in step S3, the sequence compatibility of primers within and between polymorphic site groups in step S5 was calculated using AutoDimer software, and the six sites with the strongest sequence compatibility were retained. The detailed information is shown in Table 3;

[0055] S7, labeling the upstream primer of the site retained in step S6 with a fluorescent group, and resynthesizing the upstream primer;

[0056] S8. Prepare the upstream primer and downstream primer of each site retained in step S7 into a primer premix at a ratio of 1:25, and then mix the primer premixes of all sites in equal volumes to prepare a multiplex PCR primer working solution to construct a microsatellite multiplex PCR reaction system, as shown in Table 9 below:

[0057] Table 9 Microsatellite multiplex PCR reaction system

[0058]

[0059] The concentration of the template DNA was 100 ng / μL, the concentration of the upstream and downstream primers was 0.15 μM, and the two were mixed at a volume ratio of 1:25 to obtain an upstream and downstream primer mixture;

[0060] S9. The multiplex PCR system was amplified using conventional PCR methods, and the amplified products were genotyped using capillary electrophoresis. The primer mixing ratio between each site was adjusted according to the experimental results to achieve the best amplification effect. Finally, the multiplex PCR system was obtained as shown in Table 3, and the genotyping results of the sites in the multiplex PCR were consistent with the genotyping results of each site amplified separately.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for constructing a microsatellite multiplex PCR system for Macrobrachium rosenbergii transcriptome, characterized in that: The following steps are involved: S1. Use the public transcriptome database to obtain the transcriptome sequence of Macrobrachium rosenbergii; S2. Using MISA software to analyze the transcriptome sequence of Macrobrachium rosenbergii in the above database to obtain the microsatellite locus information of Macrobrachium rosenbergii; S3. According to the estimated size distribution of the amplified products, all sites are grouped into interval ranges according to the size of the amplified products, with one interval set for every 100 bp, for a total of three intervals; S4. Randomly select 8 marker sites from each interval and use Primer Premier 6 software to design batch primers for 24 microsatellite loci; S5. Randomly select 10 individual transcriptomes of Macrobrachium rosenbergii to test the polymorphism and primer amplification of the above microsatellite loci, and eliminate the loci that cannot amplify products or lack polymorphism; S6. According to the grouping in step S3, the sequence compatibility of primers within and between polymorphic site groups in step S5 is calculated using AutoDimer software, and the six sites with the strongest sequence compatibility are retained; S7, labeling the upstream primer of the site retained in step S6 with a fluorescent group, and resynthesizing the upstream primer; S8, preparing a primer premix with the same concentration and a volume ratio of 1:25 for each upstream primer and downstream primer of each site retained in step S7, and then mixing equal volumes of the primer premixes of all sites to prepare a multiplex PCR primer working solution to construct a microsatellite multiplex PCR reaction system; S9. Use conventional PCR methods to amplify the multiplex PCR system, use capillary electrophoresis to perform genotyping on the amplified products, and adjust the primer mixing ratio between each site according to the experimental results to achieve the best amplification effect.

2. The construction method according to claim 1, characterized in that: The nucleotide sequences of the primers for the 24 microsatellite loci in step S4 are shown in SEQ ID Nos. 1 to 48.

3. The construction method according to claim 1, characterized in that: The PCR amplification procedure is as follows:

4. The construction method according to claim 1, characterized in that: The detailed information of the six sites with the strongest sequence compatibility retained in step S6 is as follows:

5. The construction method according to claim 1, characterized in that: In step S7, the fluorescent group is FAM.

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