CN108192894B

CN108192894B - SNP (Single nucleotide polymorphism) marker of high-alkalinity stress-resistant character associated gene of litopenaeus vannamei, detection primer and application of SNP marker

Info

Publication number: CN108192894B
Application number: CN201810174970.1A
Authority: CN
Inventors: 黄文�; 程楚杭; 胡超群; 霍达; 任春华
Original assignee: South China Sea Institute of Oceanology of CAS
Current assignee: South China Sea Institute of Oceanology of CAS
Priority date: 2018-03-02
Filing date: 2018-03-02
Publication date: 2020-05-26
Anticipated expiration: 2038-03-02
Also published as: CN108192894A

Abstract

The invention discloses a SNP marker of a high-alkalinity stress-resistance associated gene of litopenaeus vannamei, a detection primer and application thereof. The mutation site disclosed by the invention is positioned in the coding region of the Crustacanin A gene of the litopenaeus vannamei, the base of the mutation site is T or C, and when the genotype of the mutation site is TT, the survival rate of the litopenaeus vannamei in a high alkalinity environment is obviously lower. The invention also discloses a primer combination for detecting the mutation site and a breeding application method thereof. In the high-alkalinity stress-resistant breeding work of the litopenaeus vannamei, the method disclosed by the invention can be used for screening the backup parent population and eliminating the litopenaeus vannamei individuals sensitive to the high-alkalinity environmental pressure as soon as possible, so that the method is favorable for integrally improving the tolerance of the offspring of the litopenaeus vannamei in the high-alkalinity environmental condition and has higher potential application value.

Description

SNP (Single nucleotide polymorphism) marker of high-alkalinity stress-resistant character associated gene of litopenaeus vannamei, detection primer and application of SNP marker

Technical Field

The invention belongs to the field of aquatic biotechnology and molecular breeding, and particularly relates to a SNP marker of a high-alkalinity stress-resistance associated gene of litopenaeus vannamei, a detection primer and application of the SNP marker.

Background

Litopenaeus vannamei (Litopenaeus vannamei, commonly known as Penaeus vannamei) is originally produced in the Pacific coastal waters of Central and south America, and the Litopenaeus vannamei is introduced into China from the United states in 1988, and is cultured in large scale in 1998, and is now the most important prawn culture variety in China. By 2016, the annual output of the culture in China exceeds 160 million tons, the annual output value exceeds 600 hundred million yuan, the annual demand of seedlings exceeds 5000 hundred million tails, and the fine breed breeding has important significance for the litopenaeus vannamei breeding industry.

Because the litopenaeus vannamei has stronger environmental adaptability and higher economic value, the litopenaeus vannamei has been popularized to various water areas for cultivation, wherein the water areas comprise saline-alkali soil sea and fresh water areas such as Shanxi, Shandong and the like. According to reports, a high alkalinity environment reduces the immunocompetence of a prawn (Chang-Che Li and Jiannn-Chu Chen. the immune response of the white shrimp Litopenaeus vannamei and its society of vitality to Vibrio nonlympathobiology under low and high pH stress. Fish & Shellfish Immunology,2008,25, 701-. Therefore, the breeding work of the high alkalinity stress-resistant character can provide scientific support for the breeding industry of the litopenaeus vannamei.

The traditional breeding method completely depends on phenotype, and has the disadvantages of long period, low efficiency, instability and the like. Molecular breeding, i.e., molecular marker-assisted selective breeding, refers to a technique of selecting breeding materials by using DNA molecular markers, and a molecular breeding method carries out backup parent selection according to effective molecular markers to breed offspring with favorable economic properties. SNP is the abbreviation of Single Nucleotide Polymorphism, and refers to the variation of Single Nucleotide on genome, and the SNP site is generally double Polymorphism, and the occurrence frequency in human genome is about 1/300 bases. Compared with RAPD (first generation molecular marker) and SSR (second generation molecular marker), the SNP molecular marker has the advantages of stable heredity, large quantity, wide distribution, high information content, various detection means and the like, and is the most widely and latest molecular marker currently applied.

Disclosure of Invention

The invention aims to provide a SNP marker of a high-alkalinity stress-resistance associated gene of Litopenaeus vannamei, a detection primer and application thereof.

A conservative coding region sequence (SEQID-CA, the nucleotide sequence of which is shown as SEQ ID NO. 1) exists in the DNA fragment sequence of the Litopenaeus vannamei Crustayanin A gene, an SNP variation site exists at the position of the 11 th base from the 5' end of the coding region sequence, the base is T or C, and the genotype of the variation site is obviously related to the high-alkalinity anti-stress property of the Litopenaeus vannamei. The base sequence of SEQID-CA is as follows: 5 '-TACTGCGTGG (T/C) CGCTGGTATCAG-3'.

Therefore, the first purpose of the invention is to provide a SNP marker of a high alkalinity stress resistance associated gene of Litopenaeus vannamei, wherein the SNP marker is positioned at the 11 th base from the 5' end of the sequence shown in SEQ ID NO.1, and the base is T or C.

The second purpose of the invention is to provide a detection primer of the SNP marker of the high alkalinity stress resistance associated gene of the litopenaeus vannamei, which comprises the following primers:

CA-F: 5'-GAAGTCTGGAATGCCGTCG-3' (shown in SEQ ID NO. 2);

CA-R: 5'-CATCCACTCCAACTACGACTAC-3' (shown in SEQ ID NO. 3).

The third purpose of the invention is to provide the application of the SNP marker of the high alkalinity stress-resistance associated gene of the litopenaeus vannamei in the molecular marker-assisted selective breeding of the litopenaeus vannamei.

The fourth purpose of the invention is to provide a breeding method of a litopenaeus vannamei high alkalinity stress-resistant variety, which comprises the following steps:

a. extracting the genomic DNA of the litopenaeus vannamei to be detected;

b. carrying out PCR amplification on the genomic DNA of the litopenaeus vannamei to be detected by using the detection primers CA-F and CA-R;

c. sequencing the amplified product, determining the genotype of the SNP marker, and selecting a TC genotype or CC genotype individual as a backup parent to perform breeding of the litopenaeus vannamei high-alkalinity stress-resistant variety.

The SNP marker disclosed by the invention is positioned in a coding region of a Crustacanin A gene of the litopenaeus vannamei, the base of a variation site is T or C, and when the genotype of the variation site is TT, the survival rate of the litopenaeus vannamei in a high alkalinity environment is obviously lower. The invention also discloses a primer combination for detecting the mutation site and a breeding application method thereof. In the high-alkalinity stress-resistant breeding work of the litopenaeus vannamei, the method disclosed by the invention can be used for screening the backup parent population and eliminating the litopenaeus vannamei individuals sensitive to the high-alkalinity environmental pressure as soon as possible, so that the method is favorable for integrally improving the tolerance of the offspring of the litopenaeus vannamei in the high-alkalinity environmental condition and has higher potential application value.

Detailed Description

The present invention is further illustrated in detail by the following examples, which are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.

Example (b):

carrying out a high alkalinity tolerance experiment on 642 descendants from the group cross of Zhengda and Zhongke No.1 under the stress condition of pH 9.6 +/-0.2, taking the dead Litopenaeus vannamei Boone within 2h as a high pH sensitive group, and taking the Litopenaeus vannamei Boone still alive after 24h as a high pH tolerant group.

During the experiment, 1 observation is carried out every half an hour, and dead individuals are placed in 95% alcohol for storage in time. The extraction method of the genomic DNA of the prawns in the high-pH sensitive group and the high-pH tolerant group is carried out according to a tissue genome extraction kit of the Tiangen marine animal (TIANGEN, Tiangen Biochemical technology Co., Ltd.).

By using the detection primers CA-F (5'-GAAGTCTGGAATGCCGTCG-3', shown as SEQ ID NO. 2) and CA-R (5'-CATCCACTCCAACTACGACTAC-3', shown as SEQ ID NO. 3) disclosed by the invention, the PCR detection is carried out on the shrimp genome DNA of a high pH sensitive group and a high pH tolerant group, and the reaction program is as follows: pre-denaturation at 95 ℃ for 4 min, (denaturation at 95 ℃ for 30 sec, annealing at 52-57 ℃ for 20 sec, extension at 72 ℃ for 1 min) x 35 cycles, and final extension at 72 ℃ for 10 min. The PCR results were sequenced and the genotype results for the individuals are shown in table 1:

TABLE 1 Individual genotypes of different treatment groups

Note: medium sensitivity represents high pH sensitive group, tolerance represents high pH tolerant group

Statistical high alkalinity tolerance correlation analysis was performed on the results in table 1, with the results shown in table 2:

TABLE 2 genotype frequency and correlation analysis with high alkali tolerance of SNP sites

As can be seen from Table 2, the TT genotype appeared in the vast majority of individuals in the high pH sensitive group, indicating that individuals with the TT genotype were less resistant in the high alkaline stress environment. The TC or CC genotypes appear in most high pH tolerant groups, indicating that individuals with the TC/CC genotypes are more resistant under high alkalinity stress. Therefore, in the process of breeding with high alkalinity resistance, the genotype of the basic site disclosed by the invention can be used as a marker for breeding with high alkalinity environment resistance, and when high alkalinity resistant backup parents are screened, individuals containing TT homozygote genotypes are excluded and individuals containing TC or CC genotypes are selected as much as possible.

Sequence listing

<110> Nanhai ocean institute of Chinese academy of sciences

<120> SNP marker of Litopenaeus vannamei high-alkalinity stress-resistance character associated gene, detection primer and application thereof

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<170>SIPOSequenceListing 1.0

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<213> Litopenaeus vannamei (Litopenaeus vannamei)

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Claims

1. A detection primer for a high alkalinity stress resistance associated gene SNP marker of Litopenaeus vannamei is characterized by comprising the following primers:

CA-F：5’-GAAGTCTGGAATGCCGTCG-3’；

CA-R：5’-CATCCACTCCAACTACGACTAC-3’；

the SNP marker is positioned at the 11 th base position from the 5' end of the sequence shown in SEQ ID NO.1, and the base is T or C.

2. The application of the detection primer of the SNP marker of the litopenaeus vannamei high-alkalinity stress-resistance associated gene in the molecular marker-assisted selective breeding of litopenaeus vannamei.

3. A breeding method of a high-alkalinity stress-resistant variety of Litopenaeus vannamei is characterized by comprising the following steps:

a. extracting the genomic DNA of the litopenaeus vannamei to be detected;

b. carrying out PCR amplification on the genomic DNA of the litopenaeus vannamei to be detected by using the detection primers CA-F and CA-R of claim 1;

c. sequencing the amplified product, determining the genotype of the SNP marker according to claim 1, and selecting a TC genotype or CC genotype individual as a backup parent to perform breeding of the litopenaeus vannamei high-alkalinity stress-resistant variety.