CN108456734B - SNP marker related to high alkalinity resistance of litopenaeus vannamei, detection primer and application of SNP marker - Google Patents
SNP marker related to high alkalinity resistance of litopenaeus vannamei, detection primer and application of SNP marker Download PDFInfo
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- CN108456734B CN108456734B CN201810174718.0A CN201810174718A CN108456734B CN 108456734 B CN108456734 B CN 108456734B CN 201810174718 A CN201810174718 A CN 201810174718A CN 108456734 B CN108456734 B CN 108456734B
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Abstract
The invention discloses an SNP marker related to high alkalinity resistance of litopenaeus vannamei, a detection primer and application thereof. The SNP marker disclosed by the invention is positioned at the 237 th base position of the SEQ ID-POLR2A sequence, the base of the variation site is A or C, and the survival rate of prawns in a high alkalinity environment is obviously lower when the genotype of the variation site is AC. 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 the litopenaeus vannamei individuals sensitive to the high-alkalinity environmental pressure can be eliminated as soon as possible, so that the high-alkalinity stress-resistant breeding of the litopenaeus vannamei is facilitated.
Description
Technical Field
The invention belongs to the field of aquatic biotechnology and molecular breeding, and particularly relates to an SNP marker related to high alkalinity resistance of litopenaeus vannamei, a detection primer and application of the SNP marker and the detection primer.
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 an SNP marker related to the high alkalinity resistance of the litopenaeus vannamei, a detection primer and application thereof.
In a DNA fragment sequence (SEQID-POLR2A, the nucleotide sequence of which is shown in SEQ ID NO. 1) of a POLR2A gene of the litopenaeus vannamei, a mutation site exists at the 237 th base position, and the mutation site is obviously related to the survival rate of the litopenaeus vannamei under the high alkalinity pressure environment; when the genotype of the mutation site is AC, the survival rate of the prawns in a high alkalinity environment is obviously lower. The base sequence of SEQ ID-POLR2A is: 5 '-AGCACCTCAAGCAGCACATCACCAAGGTCATCATCAAGGGCCTGCCATCCGTCAATCGAGCTGTCATCAATCAGAATGACACGAGTGGGGTTTCCCAGTACGAGCTGCTTGTGGAAGGGGACAACTTGCAGGAAGTCATTGCAACCTATGGTGTTGATGGCACACGTTGCACCTCAAACAACACGTATGAAGTCTTCACCTCATTAGGGATTGAGGCAGCAAGAGCAACAATCATC (A/C) AAGAGATCACAGTGACTATGGAGACTCACGGCCTGAGTGTGGACCACAGGCATGTAATGCTACTGGCTGAC-3'.
Therefore, the first object of the present invention is to provide a SNP marker associated with high alkalinity resistance of Litopenaeus vannamei, wherein the SNP marker is located at the 237 th base from the 5' end of the sequence shown in SEQ ID NO.1, and the base is A or C.
The second purpose of the invention is to provide a primer for detecting SNP markers related to the high alkalinity resistance of litopenaeus vannamei, which comprises the following primers:
POLR 2A-F: 5'-AGCACCTCAAGCAGCACAT-3' (shown in SEQ ID NO. 2);
POLR 2A-R: 5'-GTCAGCCAGTAGCATTACATGCC-3' (shown in SEQ ID NO. 3).
The third purpose of the invention is to provide the application of the SNP marker related to the high alkalinity resistance 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 POLR2A-F and POLR 2A-R;
c. sequencing the amplified product, determining the genotype of the SNP marker, and selecting an AA genotype individual as a backup parent to perform high-alkalinity stress-resistant variety breeding on the litopenaeus vannamei.
The SNP marker disclosed by the invention is positioned at the 237 th base position of the SEQID-POLR2A sequence, the base of a variation site is A or C, and the survival rate of prawns in a high alkalinity environment is obviously lower when the genotype of the variation site is AC. 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 the litopenaeus vannamei individuals sensitive to the high-alkalinity environmental pressure can be eliminated as soon as possible, so that the high-alkalinity stress-resistant breeding of the litopenaeus vannamei is facilitated.
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 POLR2A-F (5'-AGCACCTCAAGCAGCACAT-3' shown in SEQ ID NO. 2) and POLR2A-R (5'-GTCAGCCAGTAGCATTACATGCC-3' shown in SEQ ID NO. 3) disclosed by the invention, PCR detection is carried out on 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 genotype statistics for 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 pH tolerance of SNP sites
As can be seen from Table 2, the AC genotype mostly appears in the high pH sensitive group, which indicates that the appearance of the AC genotype reduces the tolerance of the prawns in the high pH environment and increases the individual mortality. Therefore, in the process of breeding with high alkalinity resistance, the genotype of the base site disclosed by the invention can be used as a marker sensitive to a high pH environment, and individuals containing an AC genotype are excluded when high pH resistant backup parent screening is carried out.
Sequence listing
<110> Nanhai ocean institute of Chinese academy of sciences
<120> SNP marker related to high alkalinity resistance of litopenaeus vannamei, detection primer and application thereof
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>308
<212>DNA
<213> Litopenaeus vannamei (Litopenaeus vannamei)
<400>1
agcacctcaa gcagcacatc accaaggtca tcatcaaggg cctgccatcc gtcaatcgag 60
ctgtcatcaa tcagaatgac acgagtgggg tttcccagta cgagctgctt gtggaagggg 120
acaacttgca ggaagtcatt gcaacctatg gtgttgatgg cacacgttgc acctcaaaca 180
acacgtatga agtcttcacc tcattaggga ttgaggcagc aagagcaaca atcatcmaag 240
agatcacagt gactatggag actcacggcc tgagtgtgga ccacaggcat gtaatgctac 300
tggctgac 308
<210>2
<211>19
<212>DNA
<213> Litopenaeus vannamei (Litopenaeus vannamei)
<400>2
agcacctcaa gcagcacat 19
<210>3
<211>23
<212>DNA
<213> Litopenaeus vannamei (Litopenaeus vannamei)
<400>3
gtcagccagt agcattacat gcc 23
Claims (3)
1. A detection primer of an SNP marker related to high alkalinity resistance of Litopenaeus vannamei is characterized by comprising the following primers:
POLR2A-F：5’-AGCACCTCAAGCAGCACAT-3’；
POLR2A-R：5’-GTCAGCCAGTAGCATTACATGCC-3’。
2. the application of SNP markers related to the high alkalinity resistance of the litopenaeus vannamei in the molecular marker-assisted selective breeding of the litopenaeus vannamei; the SNP marker is positioned at the 237 th base position from the 5' end of the sequence shown in SEQ ID NO.1, and the base is A or C.
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 genomic DNA of the litopenaeus vannamei to be detected by using the detection primers POLR2A-F and POLR2A-R in the claim 1;
c. sequencing the amplified product, determining the genotype of the SNP marker according to claim 1, and selecting an AA genotype individual as a backup parent to perform breeding of the litopenaeus vannamei high-alkalinity stress-resistant variety.
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| CN111041012B (en) * | 2019-12-13 | 2022-04-26 | 中国科学院南海海洋研究所 | Litopenaeus vannamei endoplasmic reticulum Ca-ATP enzyme LvSERCA gene and encoding protein and application thereof |
| CN111850133B (en) * | 2020-05-28 | 2021-10-19 | 广东省农业科学院动物科学研究所 | Low-temperature-resistant associated SNP molecular marker of litopenaeus vannamei, detection primer and application of detection primer |
| CN116240308A (en) * | 2023-03-13 | 2023-06-09 | 中国科学院南海海洋研究所 | Combined molecular marker for liver and intestine cytozoon resistance character of litopenaeus vannamei, KASP detection primer and application |
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| CN106048016A (en) * | 2016-06-06 | 2016-10-26 | 中国科学院海洋研究所 | Multi-combination molecular markers related to resistance of litopenaeus vannamei and application |
| CN106834439A (en) * | 2016-12-26 | 2017-06-13 | 中国科学院海洋研究所 | A kind of related molecular labeling of Growth of Litopenaeus vannamei and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106048016A (en) * | 2016-06-06 | 2016-10-26 | 中国科学院海洋研究所 | Multi-combination molecular markers related to resistance of litopenaeus vannamei and application |
| CN106834439A (en) * | 2016-12-26 | 2017-06-13 | 中国科学院海洋研究所 | A kind of related molecular labeling of Growth of Litopenaeus vannamei and its application |
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