CN109182482B

CN109182482B - Construction method of Chinese softshell turtle genome microsatellite multiple PCR system

Info

Publication number: CN109182482B
Application number: CN201811111994.9A
Authority: CN
Inventors: 陈辰; 朱新平; 李伟
Original assignee: Pearl River Fisheries Research Institute CAFS
Current assignee: Pearl River Fisheries Research Institute CAFS
Priority date: 2018-09-18
Filing date: 2018-09-18
Publication date: 2022-03-01
Anticipated expiration: 2038-09-18
Also published as: CN109182482A

Abstract

The invention discloses a technical method for quickly constructing a multiple PCR system of more than 10 multiplied by Chinese softshell turtle by using public data, which is mainly characterized by comprising the following steps: 1) using a public database to obtain a genome sequence of the Chinese softshell turtle; 2) analyzing the genome sequence of the Chinese softshell turtle by using MISA software, designing batch primers by using Primer 3, and calculating the compatibility of the Primer sequence of the polymorphic site by using AutoDimer software; 3) reserving 10-12 sites with the strongest primer sequence compatibility to construct a multiplex PCR system, preparing primer premix according to the concentration of 0.4 mu M of an upstream primer and 10 mu M of a downstream primer of each site, and then mixing the primer premix of all the sites in equal volume to prepare a multiplex PCR primer working solution; 4) the multiplex PCR system is amplified using conventional PCR methods and the amplified products are genotyped using capillary electrophoresis. The multiple PCR system constructed by the invention has high marker flux and accurate and rapid genotyping result, and can provide a molecular tool for research works such as Chinese softshell turtle genetic breeding and the like.

Description

Construction method of Chinese softshell turtle genome microsatellite multiple PCR system

Technical Field

The invention relates to a construction method of a microsatellite marker multiplex PCR system, in particular to a method for constructing the microsatellite marker multiplex PCR system based on the genome information of Chinese softshell turtles.

Background

The microsatellite marker is a codominant molecular marker and consists of a repeated unit (Motif, usually 1-6 bases) which is repeated for many times in animal and plant genomes and flanking sequences on two sides. The microsatellite marker has the characteristics of high variation degree, wide distribution in a genome, conformity with Mendelian heredity, easiness in PCR amplification and the like, and is the most commonly used molecular marker in group heredity, paternity test and genetic management.

The traditional gene typing method of microsatellite markers is to separate alleles with different fragment sizes in PCR products by utilizing polyacrylamide gel electrophoresis and develop target bands by silver staining. Theoretically, the polyacrylamide gel electrophoresis method can distinguish alleles with base differences of 1bp-2bp, but the technology has the technical defect of low processing flux, each lane can only process one locus or a group of multiple PCR systems, and the requirements on the amplification performance of the marker and the distribution interval of the alleles are strict. In recent years, with the popularization of capillary electrophoresis technology, the construction of a microsatellite multiplex PCR system by using a fluorescent linker has become a mainstream technology for microsatellite marker application. Capillary electrophoresis uses liquid matrix, has the technical advantages of high electrophoresis speed, high analysis precision reading and automatic sample loading, and greatly increases the processing flux of the microsatellite marker and the genotyping precision. Meanwhile, fluorescent joints with different colors are used for distinguishing, so that the typing of multiple groups of PCR products can be realized simultaneously in one sample loading hole.

At present, the technical difficulty for restricting the application of a microsatellite marker multiplex PCR system is that the development item of a microsatellite locus is time-consuming and labor-consuming. In recent years, with the development of next generation sequencing technologies (NGS), public databases have become important resources for obtaining sequence information of microsatellite loci. By utilizing the resources of a public database and by means of bioinformatics analysis, a large number of microsatellite sequences can be screened in a short time and used for later development and verification work.

Microsatellite markers have wide application in the research of the genetic breeding of aquatic animals. Unlike other species, most aquatic animals are extremely fertile, and a pair of parents can produce tens of thousands of offspring individuals, which undoubtedly adds considerable technical difficulty to the genetic management of aquatic animals. How to improve the application efficiency of the microsatellite marker and reduce the cost of genotyping is an important guarantee for the smooth implementation of molecular marker-assisted breeding (MAS) of aquatic animals. The construction of a multiplex PCR system, particularly the construction of a PCR system with the size of more than 10 x, provides a technical idea for solving the problem.

The Chinese soft-shelled turtles are important famous and excellent aquatic product aquaculture varieties in China, and the annual output of commercial soft-shelled turtles in China is basically stabilized at 33 ten thousand tons and accounts for about 1.2% of the total amount of fresh water aquaculture. At present, the artificial breeding work of the Chinese soft-shelled turtles is carried out for many years, but the development and research of molecular markers have a certain degree of lag, which undoubtedly hinders the research process of the genetic breeding work of the Chinese soft-shelled turtles. Aiming at the problems, the invention provides a technical method for quickly constructing multiple PCR (polymerase chain reaction) of more than 10 multiplied by the number of the Chinese soft-shelled turtles, and can provide research tools for family identification, genetic breeding and population genetic research of the Chinese soft-shelled turtles.

Disclosure of Invention

The invention provides a technical method for quickly constructing a multiple PCR system of more than 10 multiplied by Chinese softshell turtle by using public data, and the specific technical scheme of the invention is as follows:

1. using a public database, obtaining the genome sequence of the Chinese softshell turtle.

2. And analyzing the genome sequence of the Chinese softshell turtle by using MISA software, and searching and positioning the microsatellite loci in the sequence.

3. The above microsatellite sites were subjected to batch Primer design using Primer 3.

4. According to the size distribution of the product, all the sites are grouped according to the size of the product, and each 80bp-100bp is provided with a grouping interval; randomly selecting 10-15 mark/set multiple PCR systems from each group, and synthesizing the upstream and downstream primers of all selected sites.

5. And (3) using 8 or more individual genomes of the Chinese softshell turtles to test the polymorphism of the microsatellite loci and the amplification condition of the primers, and rejecting loci of which the products cannot be amplified or PCR products have no polymorphism.

6. According to the grouping of the step 4, the compatibility of upstream and downstream sequences in the conservation site primer group and between groups in the step 5 is calculated by using AutoDimer software, and 10-12 sites with the strongest compatibility of primer sequences in the group and between groups are reserved.

7. Grouping the sites reserved in the step 6 according to the step 4, respectively marking the 5' end of the upstream primer of each site in each group with fluorescence of one color, and re-synthesizing the upstream primers; the upstream primer is labeled with fluorescence, a linker sequence which contains M13(-21) universal primer and has been experimentally verified to have amplification universality can be selected, PCR amplification is carried out by using M13-SSR method, and a fluorescent group can be directly labeled on the 5' end of the upstream primer of each site to carry out conventional PCR reaction.

8. And (3) preparing primer premix solution for each site in the step (7) according to the concentration of 0.4 mu M of the upstream primer and 10 mu M of the downstream primer, and then mixing the primer premix solution for all the sites in equal volume to prepare multiplex PCR primer working solution to construct a microsatellite multiplex PCR reaction system.

9. And (5) carrying out genotyping on the individual Chinese softshell turtle in the step 5 by using a conventional PCR method and capillary electrophoresis, and checking the amplification efficiency of a multiplex PCR system. According to the experimental result, the concentration ratio of the primers among all the sites in the system is adjusted to achieve the optimal amplification effect.

The invention has the following technical advantages:

1. the existing public database resources are fully utilized, a large amount of microsatellite locus information can be obtained in a short time, and the cost for developing the marker is reduced.

2. The constructed multiple PCR system has high labeling flux, can realize more than 10 locus types in one reaction system, saves the investment of reagents and consumables, and improves the efficiency of genetic analysis experiments.

3. The genotyping result is accurate, and the genotyping result is rapid and accurate by using the technical means of fluorescence capillary electrophoresis as the genotyping method of the microsatellite loci.

Drawings

Fig. 1 is a flow chart showing the main technical features of the present invention.

FIG. 2 shows the genotyping results of the multiplex PCR system constructed in the examples of the present invention.

Detailed Description

The present invention is further illustrated by the following specific examples. The examples are intended to illustrate the invention, but not to limit the scope of the invention. Unless otherwise indicated, the examples were run under conventional experimental conditions or in accordance with the manufacturer's instructions. Reagents and instruments used in the examples were purchased from conventional bioproduct agencies unless otherwise specified.

1. Acquisition of genome information of Chinese softshell turtle

Using a public database to obtain the genome sequence of the Chinese softshell turtle, wherein the download address of the genome sequence of the Chinese softshell turtle isftp：//ftp.ncbi.nlm.nih.gov/genomes/Pelodiscus sinensis/CHR Un/。

2. Acquisition of microsatellite locus sequence information

And analyzing the genome sequence of the Chinese softshell turtle by using MISA software, and searching and positioning the microsatellite loci in the sequence. To simplify the amount of calculation in the data analysis, only one Scaffold (NW _005855045.1) of the genome of Chinese softshell turtle was used in this example. The parameters of the MISA software are set to definition (unit _ size, min _ repeats): 2-63-54-5, intersections (max _ difference _ for _2_ SSRs): 100. the distribution of the base repeat units of the microsatellite loci of the trionyx sinensis is shown in table 1.

TABLE 1 distribution of microsatellite base repeat units in the genome of Trionyx sinensis

3. Microsatellite locus primer sequence design

Using Primer 3 to perform batch Primer design on the microsatellite loci, wherein the Primer _ product _ size _ range is set as 400 according to the parameters of Primer 3 software; primer _ max _ end _ stability 250.

4. Site grouping and polymorphism validation

All sites are divided into 3 groups according to the sizes of products of 90bp-180bp, 180bp-260bp and 260bp-400bp, 12 markers/sets of multiple PCR systems are randomly selected from each group, and the upstream and downstream primers of all selected sites are synthesized. And (3) randomly selecting 30 individual genome DNAs of the Chinese softshell turtles to test the amplification efficiency of the microsatellite locus primers and the polymorphism of the loci, and removing the loci of which the products cannot be amplified or the PCR products have no polymorphism. The PCR reaction program was set up according to the instructions for Taq enzyme, and capillary electrophoresis was performed using an ABI 3130 genetic analyzer.

5. Screening for multiplex PCR sites

And (3) calculating the compatibility of upstream and downstream sequences in the group and between the reserved site primer in the step 4 by using AutoDimer software, and reserving 12 sites with strongest compatibility of primer sequences in the group and between the groups. And (3) respectively labeling the 5' end of the upstream primer of each site in each group with fluorescence of one color, and synthesizing the upstream primer of each site again. The AtuoDimer software parameters were set to minimum score requirement 7, Na⁺(Molar) ═ 0.085. The primer information for the 12 sites is shown in Table 2.

TABLE 2 Trionyx sinensis Wiegmann 12 × microsatellite multiplex PCR System site information

6. Construction of multiplex PCR reaction System

And (3) preparing primer premix solution from the sites according to the concentrations of 0.4 mu M of the upstream primer and 10 mu M of the downstream primer, mixing the primer premix solution of all the sites in equal volume to prepare multiplex PCR primer working solution, and carrying out multiplex PCR amplification according to a reaction system shown in the table 3.

TABLE 3 multiple PCR reaction System for Trionyx sinensis Wiegmann 12 Xmicrosatellite

7. Multiplex PCR system amplification efficiency test

The 12 x Chinese soft-shelled turtle microsatellite multiplex PCR system was tested using 30 individual Chinese soft-shelled turtles from step 4. And calculating the amplification efficiency of the multiplex PCR according to the number of successfully amplified samples of the multiplex PCR/the number of successfully amplified samples of all the sites. The result shows that the amplification efficiency of the 12 XChina soft-shelled turtle microsatellite multiple PCR system is 99.89%, and the genotyping result of the sites in the multiple PCR is consistent with the genotyping result of the single amplification of each site.

The foregoing detailed description has set forth specific details of the invention. Those skilled in the art will appreciate that modifications, additions and substitutions are possible, without departing from the scope of the invention as disclosed in the accompanying claims.

Claims

1. A method for quickly constructing a 10 multiplied Chinese soft-shelled turtle microsatellite marker multiplex PCR system for genotyping by using public data is characterized by comprising the following steps:

1) obtaining a genome sequence of the Chinese softshell turtle by using a public database;

2) analyzing a genome sequence of the Chinese softshell turtle to obtain microsatellite locus information of the Chinese softshell turtle;

3) designing primers for all the microsatellite loci;

4) all sites are subjected to interval grouping according to the product sizes of 90bp-180bp, 180bp-260bp and 260bp-400bp, and 12 markers/sets of multiple PCR system synthesis primers are randomly selected from each group;

5) verifying the polymorphism of each site and the amplification condition of the product by using 8 or more individual genomes of the Chinese softshell turtles, and removing sites of which the product cannot be amplified and the PCR product has no polymorphism;

6) according to the grouping in the step 4), using AutoDimer software to calculate the sequence compatibility of primers in the polymorphic site group and between groups, and reserving 12 sites with strongest sequence compatibility;

7) labeling the upstream primer of each site in each group with a different color fluorescence; the site information of the 12 sites and the primer sequences are shown in the following table:

8) preparing primer premix according to the concentration of 0.4 mu M of the upstream primer and 10 mu M of the downstream primer of each site, mixing the primer premix of all the sites in equal volume to prepare multiplex PCR primer working solution, and constructing a microsatellite multiplex PCR reaction system;

9) the multiplex PCR system is amplified using conventional PCR methods and the amplified products are genotyped using capillary electrophoresis.