CN111057771B - SNP molecular marker for distinguishing 'Zhongyang No. 1' from common fugu obscurus and application thereof - Google Patents
SNP molecular marker for distinguishing 'Zhongyang No. 1' from common fugu obscurus and application thereof Download PDFInfo
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Abstract
The invention discloses an SNP molecular marker for distinguishing a 'Zhongyang No. 1' and common fugu obscurus and application thereof, wherein the sequence of the SNP molecular marker is shown as SEQ ID NO:1 (derived from CIRP gene sequence of the fugu obscurus), and the SNP molecular marker is positioned in the sequence shown as SEQ ID NO:1, the SNP site is the base A or T at position 66 of the sequence shown in the figure. The SNP locus disclosed by the invention is obviously related to low temperature resistance, and can be used for distinguishing a Fugu obscurus new variety 'Zhongyang No. 1' population from a common Fugu obscurus population. The SNP locus can also be effectively applied to the early identification of the Fugu obscurus new species 'Zhongyang No. 1' and the common Fugu obscurus seedlings. The method is accurate, simple and practical in operation.
Description
Technical Field
The invention relates to an SNP molecular marker for distinguishing 'Zhongyang No. 1' from common fugu obscurus and application thereof, belonging to the field of genetic breeding of fugu obscurus.
Background
Takifugu obscurus (Takifugu fasciatus) is commonly called puffer, belongs to Osteichthyes, Actinopterygii, Fugu morphes (Letrodontiforms), Takifudae (Tetraodontidae) and Takifugu eastern (Takifugu), and is mainly distributed in yellow sea, Bohai sea, offshore sea area and rivers and lakes in China. The fugu obscurus meat-quality fresh and tender food is good, has high nutritional value, is deeply popular with consumers, and is an aquaculture fish with higher economic, nutritional and culture values. Takifugu obscurus is one of a few species of Takifugu obscurus having the habit of migration of fresh and sea water, and cannot normally survive in the middle and lower reaches of Yangtze river when the temperature is reduced to about 18 ℃ every 10 months, and needs to overwinter in warm ocean current. In the large-scale breeding and production process of the fugu obscurus, the water temperature in winter is far lower than the optimal growth temperature, which causes the fugu obscurus to die greatly and causes huge economic loss. The method comprises the following steps of continuously breeding 5 generations by adopting a group breeding technology in three breeding units of Jiangsu Zhongyang group member Limited company, China institute of aquatic science, fresh water fishery research center and Nanjing Master university, gradually reducing the temperature from 15 ℃ to 7 ℃ after 3 times of low-temperature stress of each generation, eliminating Fugu obscurus individuals without low-temperature resistance until the 5 th generation retains a parent group with low-temperature resistance, and breeding a new variety 'Zhongyang No. 1' (the variety registration number is GS-01-003-.
Studies have shown that common Fugu obscurus stops feeding at 16 ℃ and starts to die below 13 ℃. Compared with the common Fugu obscurus population, the population of the novel Fugu obscurus variety 'Zhongyang No. 1' has normal ingestion at the temperature of more than 12 ℃ and the lowest ingestion temperature is reduced by 4 ℃ (the low temperature resistance is improved by 4 ℃) under the same culture condition; the overwintering survival rate is averagely improved by 11.8 percent. So far, no method for effectively distinguishing the population of the new variety 'Zhongyang No. 1' of the Fugu obscurus from the population of the common Fugu obscurus exists, and the truth of the new variety 'Zhongyang No. 1' of the Fugu obscurus in the market is difficult to distinguish, so that the popularization and the application of the new variety 'Zhongyang No. 1' of the Fugu obscurus are limited to a certain extent. One of the most effective methods is to develop DNA molecular markers and find out the markers related to the low temperature tolerance of the 'Zhongyang No. 1' population and the ordinary Fugu obscurus population of the new variety of Fugu obscurus, so that the 'Zhongyang No. 1' population and the ordinary Fugu obscurus population of the new variety of Fugu obscurus can be effectively distinguished. Single Nucleotide Polymorphism (SNPs) markers as third-generation molecular markers have the advantages of high polymorphism, genetic stability, convenience in detection and the like, and are widely applied to the field of animal and plant molecular breeding research. CIRP, a cold-inducible RNA-binding protein, was found to be closely linked to low temperature stimulation in early studies. Under the low-temperature environment, the CIRP reduces the requirement of cells on nutrient substances by inhibiting cell division, thereby slowing down the apoptosis rate, and is a functional gene with wide distribution and strong action. However, at present, there is no report on the application of CIRP combined with SNP sites in population identification and molecular assisted breeding.
Disclosure of Invention
The purpose of the invention is as follows: in view of the above technical problems, the first objective of the present invention is to provide a SNP molecular marker for distinguishing "fugu Zhongyang No. 1" from ordinary fugu obscurus, i.e. a group identification of the "fugu Zhongyang No. 1" population and the ordinary fugu obscurus population is performed by using a SNP site significantly related to low temperature resistance on the CIRP gene coding region of the fugu obscurus.
The second objective of the invention is to provide a pair of primers for detecting the SNP molecular marker.
The third purpose of the invention is to provide the application of the SNP molecular marker.
The technical scheme is as follows: in order to achieve the purpose, the invention adopts the following technical scheme:
the invention discloses an SNP molecular marker for distinguishing a 'Zhongyang No. 1' and common fugu obscurus, the sequence of the SNP molecular marker is shown as SEQ ID NO:1 (derived from the CIRP gene of the fugu obscurus), and the SNP molecular marker is positioned in the sequence shown as SEQ ID NO:1, the SNP site is the base A or T at position 66 of the sequence shown in the figure.
AT the SNP site, the AT genotype frequency of the Fugu obscurus 'Zhongyang No. 1' population is obviously higher than that of the common Fugu obscurus population.
AT the SNP locus, the AT genotype frequency of the Fugu obscurus population Zhongyang No. 1 is more than 50%, and the AT genotype frequency of the common Fugu obscurus population is less than 50%.
Specific primers for detecting the SNP molecular markers comprise a forward primer and a reverse primer, and the nucleotide sequences of the specific primers are respectively shown as follows:
a forward primer: 5'-AGCGCCTACCTGTTGTCT-3', as shown in SEQ ID NO: 2;
reverse primer: 5'-AAGCGAGCGTTTCTACCC-3', as shown in SEQ ID NO. 3.
The kit for detecting the SNP molecular marker is characterized by comprising the specific primer.
The SNP molecular marker, the specific primer or the kit are applied to distinguishing the Fugu obscurus population of the Zhongyang No. 1 population and the common Fugu obscurus population.
A method for distinguishing the "Zhongyang No. 1" population of Fugu obscurus from the ordinary population of Fugu obscurus includes detecting the SNP molecular marker of said Fugu obscurus to be detected, and analyzing the difference in the genotype frequency of SNP locus.
Further, the method comprises the steps of extracting the genomic DNA of two takifugu obscurus populations, carrying out PCR amplification by using the specific primer or the kit, counting the genotype frequency of the SNP locus in the populations to be detected, and analyzing the difference of the genotype frequencies of the SNP locus of the two populations.
AT the SNP site, the AT genotype frequency of the Fugu obscurus 'Zhongyang No. 1' population is obviously higher than that of the common Fugu obscurus population.
AT the SNP locus, the AT genotype frequency of the Fugu obscurus population Zhongyang No. 1 is more than 50%, and the AT genotype frequency of the common Fugu obscurus population is less than 50%.
The method for distinguishing the Fugu obscurus population of 'Zhongyang No. 1' from the common Fugu obscurus population specifically comprises the following steps of:
1) extraction of genomic DNA: respectively extracting the genome of not less than 90 individuals in each population of the Fugu obscurus
DNA；
2) Taking the extracted genome DNA as a template, and carrying out PCR amplification by using the primer pair;
3) SNP locus genotype frequency difference analysis: counting the genotype frequency of the SNP locus in the group to be detected, and analyzing the difference of the genotype frequencies of the two groups;
the frequency of allele AT in the SNP locus is significantly higher in the population of "Zhongyang No. 1" than in the general population (P < 0.05);
the invention takes the single nucleotide polymorphic site of the CRIP gene of the fugu obscurus as a research target, and finds that three types of SNP sites AT, AA and TT positioned on the CRIP gene are obviously related to distinguishing a 'Zhongyang No. 1' population of a new species of the fugu obscurus and a common fugu obscurus population. Specifically embodied in the SNP locus, the AT genotype frequency of the population of the new variety of the Fugu obscurus named ' Zhongyang No. 1 ' is obviously higher than that of the population of the Fugu obscurus named ' ordinary Fugu obscurus, wherein the AT genotype frequency of the population of the new variety of the Fugu obscurus named ' Zhongyang No. 1 ' is more than 50%, and the AT genotype frequency of the population of the ordinary Fugu obscurus is less than 50%. The SNP marker of the invention is used for identifying the population of 'Zhongyang No. 1' of a new variety of the Fugu obscurus and the population of the common Fugu obscurus, so that the identification of breeding parents at the population level is simpler and more convenient.
The technical effects are as follows: compared with the prior art, the invention has the following advantages:
1. the SNP molecular marker and the application method can realize the identification of a new species 'Zhongyang No. 1' population of the Fugu obscurus and a common Fugu obscurus population, are not limited by the age, the sex and the like of the Fugu obscurus, can be used for the early breeding of Fugu obscurus population fries, and help the market to accurately distinguish the population fries. The blindness in parent breeding is solved, and on the basis of not killing the fugu obscurus under low-temperature stress, the group identification can be carried out by only using a small number of tail fins, so that a scientific tool is provided for molecular assisted breeding.
2. By SEQ ID NO:2 and SEQ ID NO:3, detecting the primers shown as SEQ ID NO:1 from the 66 th SNP site of the 5' end, the method is accurate and reliable and is convenient to operate.
Drawings
FIG. 1 is a diagram of the partial sequencing peak of the 66 th site from the 5' end of the SNP molecular marker nucleotide sequence of AA genotype Fugu obscurus;
FIG. 2 is a diagram showing the peak of the 66 th site partial sequencing of the SNP molecular marker nucleotide sequence of Takifugu obscurus of the AT genotype;
FIG. 3 is the partial sequencing peak diagram of the SNP molecular marker nucleotide sequence of Takifugu obscurus of TT genotype from the 66 th site at the 5' end.
Detailed Description
The technical solution of the present invention is further described in detail by the following specific examples.
The invention is based on SEQ ID NO:2 and SEQ ID NO:3, extracting the genome DNA of the takifugu obscurus by the pair of primers, carrying out PCR amplification, sequencing the amplified product and analyzing the sequencing result to obtain the SNP locus related to the low-temperature tolerance capability of the takifugu obscurus. Can be applied to the identification of the 'Zhongyang No. 1' population of the new species of the fugu obscurus and the population of the common fugu obscurus.
Example 1
a) Obtaining of Fugu obscurus population:
the invention carries out low temperature resistant experimental treatment on the new variety 'Zhongyang No. 1' of Fugu obscurus cultured in the research institute of Water science of Nanjing city at the Lukou experimental base. Taking the unbalance time as a low temperature resistance parameter, and then carrying out SPSS correlation analysis on the unbalance time and the SNP locus genotype to obtain a low temperature resistance dominant genotype. The specific experimental process is as follows: after the membrane is removed in 4 months in 2019, the fish is cultivated in a pond (580) in the same environment (temperature, density and the like) for 191 days, fish with 121 tail bodies being 15cm +/-0.5 cm long, strong in physique, free of damage and good in vitality are selected, and the feeding is stopped for 1 day before the experiment is carried out; cooling from room temperature 25 deg.C at 1 deg.C/h rate, and cooling at 5 deg.C/h rate when the temperature is reduced to 13 deg.C. The first fish lost balance at 11.8 ℃ and the last fish lost balance at 6.8 ℃. The whole experimental process lasts for 1 hour and 2 minutes; recording time according to the sequence of losing balance of the fish body, and judging the low-temperature tolerance capability as quantitative character; the tail fin of the fish body is cut and stored at the temperature of 95% ethanol to 20 ℃ and is used for extracting genome DNA for detection (see table 2).
b) Extracting DNA of the takifugu obscurus:
(1) taking 15mg tail fins, adding 400 mu L ACL Solution, shearing, adding 10 mu L Proteinase K, shaking and mixing uniformly for 1 minute, and standing at 55 ℃ for about 2 hours until the lysate is clear.
(2) Then 300uL of Ext solution and 300. mu.L of AB solution were added in this order, shaken vigorously, and centrifuged at 12,000rpm for 5 minutes.
(3) The tip was passed through the upper solution deep into the lower solution and the solution was carefully aspirated into the GenClean Column, trying to avoid aspiration into the upper solution and precipitation in the middle layer.
(4) Centrifuging at 8000rpm for 1 min, taking down GenClean Column, and pouring off waste liquid in the collecting tube.
(5) GenClean Column was returned to the collection tube, 500. mu.L of land Solution was added, and centrifuged at 8,000rpm for 1 minute at room temperature.
(6) Repeating the step (5) once.
(7) The GenClean column was removed and the waste stream from the collection tube was discarded. The column was placed back into the collection tube and centrifuged at 12,000rpm for 1 minute at room temperature to remove residual Wash Solution.
(8) The column was placed in a fresh, clean 1.5mL centrifuge tube, 60. mu.L of Elution Buffer was added to the center of the column, and the column was left at room temperature for 2 minutes. Then centrifuged at 12,000rpm for 1 minute at room temperature. The liquid in the centrifuge tube is the extracted DNA, and the DNA is preserved at the temperature of minus 20 ℃. Detecting DNA sample by 1% agarose gel electrophoresis, and detecting concentration and purity by ultraviolet spectrophotometer.
c) Carrying out PCR amplification on the genomic DNA of the takifugu obscurus based on the SNP primer
The amplification length of the PCR product is about 123bp, and the PCR primers are as follows:
upstream primer (SEQ ID NO: 2): 5'-AGCGCCTACCTGTTGTCT-3'
Downstream primer (SEQ ID NO: 3): 5'-AAGCGAGCGTTTCTACCC-3'
The PCR reaction system was 20. mu.L of 2 XTaq Master Max 10. mu.L, forward and reverse primers 0.8. mu.L each, DNA template 1. mu.L, and sterilized water 7.4. mu.L. The PCR reaction is carried out for 35 cycles in total, the pre-denaturation is carried out for 5min at the temperature of 95 ℃ before the cycle, and each cycle comprises the denaturation at the temperature of 95 ℃ for 30s, the annealing at the temperature of 53 ℃ for 30s and the extension at the temperature of 72 ℃ for 30 s; after the circulation was completed, the extension was carried out at 72 ℃ for 5 min. And (3) carrying out electrophoresis detection on the amplification product by using 1% agarose gel, and storing the qualified PCR product at-20 ℃ for subsequent sequencing reaction.
d) Sequencing the PCR amplification product, and determining the SNP locus genotype based on the sequencing result. Based on Hiseq2000 high-throughput sequencing platform, the PCR amplification products of the total 423 individuals of the four takifugu obscurus populations are subjected to bidirectional sequencing and splicing on an ABI3730 sequencer. Based on the sequencing results, the takifugu obscurus SNP sites were typed, as shown in fig. 1.
e) Correlation analysis of SNP locus genotype and low temperature resistance of Fugu obscurus
The SNP locus genotype and low-temperature tolerance of 121 individuals of a new variety of Fugu obscurus, Zhongyang No. 1, cultivated in the research institute of Water science in Nanjing City, the Lukou laboratory base are shown in Table 1: from the data in Table 1, a linear analysis model was constructed using the SPSS (25.0) GLM program based on the characteristics of the trait and the test population to analyze the association between the gene polymorphism and the trait. The correlation between different nucleotide types of SNPs and low temperature resistance is shown in Table 2.
TABLE 1 relationship between SNP site genotype of 121-tailed individual of Fugu obscurus new variety "Zhongyang No. 1" and low-temperature imbalance time
TABLE 2 correlation of SNP site of CRIP gene of Fugu obscurus with time to loss of balance at low temperature
Note: the expression mode is mean value plus or minus standard deviation, and the difference is significant when P <0.05
f) The gene type identification of the population dominant SNP locus of the new variety of Fugu obscurus, Zhongyang No. 1, is as described above, and the low-temperature unbalance time of the AT type of the SNP locus is obviously longer than that of the AA type and the TT type (P < 0.05). The SNP site AT type is proved to be the dominant genotype of a new variety 'Zhongyang No. 1' population, and is also obviously related to the low temperature resistance of the Fugu obscurus.
Example 2
a) Obtaining different Fugu obscurus populations:
the fish used in the experiments of the invention were taken from four groups: the variety of the Fugu obscurus ' Zhongyang No. 1 ' is taken from the Takifugu obscurus ' new variety of 5-month old, namely Zhongyang group Limited company (Haian) in 2018 for 9 months; taking 99 tails of common unseamed fugu obscurus groups of about 4 months old from fishery science and technology limited of Zhenjiang river source in 2018 months; taking 96 tails of common unseamed fugu obscurus groups of about 5 months old from Sanxian culture Limited company (Jiangyin) Shenhong Ganghong in 2018 in 10 months; the 121 tails of the new variety 'Zhongyang No. 1' of Fugu obscurus bred in the laboratory base of Lukou of the research institute of Water science of Nanjing City. The tail fin was stored at 95% ethanol-20 ℃ for genomic DNA extraction.
b) Extracting genome DNA of different fugu obscurus populations;
the method for extracting the genome DNA of different takifugu obscurus populations is the same as the step of the embodiment 1 b).
c) Carrying out PCR amplification on the takifugu obscurus genome DNA based on the SNP primer;
the method for PCR amplification of genomic DNA of different Fugu obscurus populations was the same as in example 1 c).
d) Sequencing the PCR amplification product, and determining the genotype of the SNP locus based on the sequencing result;
the method for determining SNP site genotype was the same as in example 1 d). The SNP site genotype results are shown in FIG. 1.
e) Comparing the frequency of different genotypes of the SNP locus in different groups;
and (3) counting the allele frequencies of the SNP loci in 4 groups of the Fugu obscurus respectively, and analyzing the difference of the allele frequencies of the SNP loci of the 4 groups by utilizing an SPSS (25.0) GLM program. The analytical procedure is as described in example 1 e). The results are shown in Table 3:
TABLE 3 SNP locus genotype distribution in the Fugu obscurus New breed "Zhongyang No. 1" population and Fugu obscurus general population
f) The SNP locus is applied to the identification of a new species of Fugu obscurus, namely a population of 'Zhongyang No. 1' and a population of common Fugu obscurus.
As described above, in the SNP sites, the frequency of the allele AT was significantly higher in the 2 populations of the new species of fugu obscurus "Zhongyang No. 1" than in the 2 populations of takifugu obscurus derived from jiangyin and Yangzhong.
The invention discovers that the SNP locus AT genotype is the dominant genotype of the 'Zhongyang No. 1' population of the new species of the Fugu obscurus and is also obviously related to the low temperature resistance of the Fugu obscurus by selecting samples of different groups of the Fugu obscurus 423 and carrying out genome DNA extraction, PCR amplification and SNP locus analysis on each individual.
Through statistical analysis of SNP locus genotypes of two populations of 2 common unseeded populations of the Fugu rubripes and the Fugu obscurus new variety 'Zhongyang No. 1', the AT genotype frequency of the population of the Fugu obscurus new variety 'Zhongyang No. 1' is found to be remarkably higher than that of the common Fugu obscurus population, wherein the AT genotype frequency of the population of the Fugu obscurus new variety 'Zhongyang No. 1' is higher than 50%, and the AT genotype frequency of the common Fugu obscurus population is lower than 50%. Therefore, the population of the new variety of the fugu obscurus named 'Zhongyang No. 1' and the population of the common fugu obscurus can be effectively distinguished, and the method is effectively applied to the identification of the seedlings of the new variety of the fugu obscurus named 'Zhongyang No. 1' and the population of the common fugu obscurus.
Sequence listing
<110> university of Nanjing university
<120> SNP molecular marker for distinguishing 'Zhongyang No. 1' from common Fugu obscurus and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 123
<212> DNA
<213> CRIP(CRIP)
<400> 1
agggattaac tcccttctgt agcaccgctg cctccaaagc cccggtcatt gttgccatag 60
cttctttcac cataactcct gtcgccatag cttctgtctc caccatagct gcctatttaa 120
ttt 123
<210> 2
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
agcgcctacc tgttgtct 18
<210> 3
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
aagcgagcgt ttctaccc 18
Claims (4)
1. The SNP molecular marker for distinguishing the 'Zhongyang No. 1' and the common fugu obscurus is characterized in that the sequence of the SNP molecular marker is shown as SEQ ID NO:1, and the sequence of the SEQ ID NO:1, the 66 th position of the sequence is SNP site, and the base is A or T.
2. The SNP molecular marker of claim 1, for use in differentiating between a population of Fugu obscurus "Zhongyang No. 1" and a population of Fugu obscurus AT a site where the AT genotype frequency of the population of Fugu obscurus "Zhongyang No. 1" is greater than 50% and the AT genotype frequency of the population of Fugu obscurus is less than 50%.
3. A method for distinguishing the "Zhongyang No. 1" population of Fugu obscurus from the ordinary Fugu obscurus population includes detecting the SNP molecular marker in claim 1 of Fugu obscurus to be detected, analyzing the genotype frequency difference of SNP sites, where the AT genotype frequency of the "Zhongyang No. 1" population of Fugu obscurus is greater than 50% and the AT genotype frequency of the ordinary Fugu obscurus population is less than 50%.
4. The method of claim 3, wherein the method comprises extracting genomic DNA of two Fugu obscurus populations, performing PCR amplification with specific primers, counting the genotype frequency of SNP locus in the population to be detected, analyzing the difference of the genotype frequency of SNP locus of the two populations, the specific primers comprise forward primer and reverse primer, and the nucleotide sequences are respectively as follows:
a forward primer: 5'-AGCGCCTACCTGTTGTCT-3', as shown in SEQ ID NO: 2;
reverse primer: 5'-AAGCGAGCGTTTCTACCC-3', as shown in SEQ ID NO. 3.
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| CN103866004A (en) * | 2014-01-24 | 2014-06-18 | 中国水产科学研究院 | Molecular marker method for identifying fugu rubripes parentage as well as microsatellite and kit used for molecular marker method |
| CN109852703A (en) * | 2019-04-02 | 2019-06-07 | 南京师范大学 | One kind SNP marker relevant to fugu obscurus coefficient of condition and its application |
| CN110241225A (en) * | 2019-04-29 | 2019-09-17 | 中国水产科学研究院 | The SNP marker of species identification for river Puffer combines |
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| CN103866004A (en) * | 2014-01-24 | 2014-06-18 | 中国水产科学研究院 | Molecular marker method for identifying fugu rubripes parentage as well as microsatellite and kit used for molecular marker method |
| CN109852703A (en) * | 2019-04-02 | 2019-06-07 | 南京师范大学 | One kind SNP marker relevant to fugu obscurus coefficient of condition and its application |
| CN110241225A (en) * | 2019-04-29 | 2019-09-17 | 中国水产科学研究院 | The SNP marker of species identification for river Puffer combines |
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