CN116356038A - Breeding method for screening Fugu rubripes individuals with rapid growth performance - Google Patents
Breeding method for screening Fugu rubripes individuals with rapid growth performance Download PDFInfo
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- 241001441722 Takifugu rubripes Species 0.000 title claims abstract description 47
- 238000009395 breeding Methods 0.000 title claims abstract description 12
- 238000012216 screening Methods 0.000 title claims description 10
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- 239000002773 nucleotide Substances 0.000 claims abstract description 10
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 10
- 230000001488 breeding effect Effects 0.000 claims abstract description 6
- 238000012163 sequencing technique Methods 0.000 claims description 16
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- 108020004414 DNA Proteins 0.000 description 8
- 239000003147 molecular marker Substances 0.000 description 4
- 102100020948 Growth hormone receptor Human genes 0.000 description 3
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- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
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- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
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Abstract
The invention provides a SNP locus related to the weight of the early growth of the fugu rubripes, the provided SNP locus is related to the weight growth trait of the fugu rubripes, and the SNP locus can be used for breeding the juvenile fish and the selected parent of the fugu rubripes with the rapid fattening trait. The invention firstly provides SNP loci related to the weight growth traits of the fugu rubripes, wherein the SNP loci are positioned in a sequence of SEQ ID NO:1, and the base at position 264 of the nucleotide fragment of the formula 1 is C or T. According to the invention, through analyzing the correlation of locus genotype frequency and the growth trait of the fugu rubripes, the SNP locus related to the weight trait exists at 264 bases of the GHR 2gene of the fugu rubripes, and the genotype is that the weight of a CC homozygous individual is obviously higher than the phenotype value (p < 0.05) of the weight trait of a CT genotype individual. Thus, individuals with CC type locus can be preferentially selected as parents in production or can be cultivated on a large scale.
Description
Technical Field
The invention belongs to the technical field of fish genetic breeding, and particularly relates to a breeding method for screening Fugu rubripes individuals with rapid growth performance.
Background
Fugu rubripes (Takifugu rubripes) belongs to the order of Fugu (Telraodontiforms), and Fugu (Fugu) takes Japanese coast as a main distribution center, and the wild population in coast of China is less and has impurity germplasm; and the wild population is reduced due to the factors of marine pollution and the like, so the annual fishing amount of the wild population is gradually reduced. The Fugu rubripes have fresh and tender meat quality and rich nutrition, and are known as fish king. Although the liver, ovary and other tissues of the wild fugu rubripes are rich in fugu rubripes toxin and are very strong neurotoxic, the toxin preparation can be used for analgesics, anesthetics and the like and is high in price. Therefore, the fish has higher economic value. At present, the natural resource quantity of the fugu rubripes is seriously reduced, the cultivation scale is gradually increased, and the phenomenon of germplasm degeneration exists in the artificial cultivation process. Therefore, the breeding of Fugu rubripes variety with excellent growth characteristics is very necessary.
At present, morphological character selective breeding is commonly used for breeding, but in practice, the genetic power of the growth characters is about 0.2, the effect of character selection in offspring is not ideal, and molecular marker assisted selective breeding enables excellent characters to be inherited to offspring and expressed completely with high probability. However, the biggest problem of molecular marker assisted selective breeding at present is the lack of effective molecular markers.
Disclosure of Invention
The invention aims to provide a breeding method for screening a fugu rubripes individual with rapid growth performance, namely, the fugu rubripes juvenile fish with rapid growth characteristics and a selected parent are bred through SNP loci related to the weight, the body length and the whole length growth characteristics of the fugu rubripes, so that the genetic breeding speed of the fugu rubripes is increased.
The invention provides a breeding method for screening Fugu rubripes individuals with rapid growth performance, which comprises the steps of screening by detecting genotypes of SNP loci related to Fugu rubripes growth traits;
the SNP locus is positioned in a sequence of SEQ ID NO:1, and the base at position 264 of the nucleotide fragment of the formula 1 is C or T.
Gacattgaggaaaccaccagtagattgaaagatctggaccccgactgcctcatgcaaccctcactcttgtccgattgcacaccacccatttttagcttcagagatgatgactcaggtcgagctagctgctgtgaccctgatctttccagtgaaccagaggcctcaactgtccatccagcaattctaaatcaagtcatcaatcaaacattttgctcgacaggatgtgcgggctcaggtttgctgaatcagacccctaatgtgagcgagactgaaaccttggacagagaggcactgtatacccaagtgagtgaagtgaggtcaactggcaaggtgctactatctcccgagttggagaaaatcagcagcagcaaagggatgccattagagaacgagggtaaggacctccacatcttagtggtgaatgcgcatcatggcagtaatatggcaggaaatgtaagtcaaacatttcccagaccagacacgagtgaactttttgacagttctcatgcatccacctcacattcccatgaatcagatgccacctccaaccgccccgctcctgcctacactgtggtagatggtgtaagtgggc(SEQ ID NO:1)；
Furthermore, the genotype of the SNP locus is CC homozygote.
Further, sequencing and analyzing the fugu rubripes individuals to be screened through a PCR product, and determining the genotype of SNP loci of the individuals to be detected after sequencing and analyzing the PCR product;
the PCR amplification method, wherein the sequence information of the primers used is as follows:
F:5′-GACATTGAGGAAACCACCAG-3′(SEQ ID NO:2)，
R:5′-GCCCACTTACACCATCTACC-3′(SEQ ID NO:3)。
the invention discovers that the nucleotide sequence SEQ ID NO of the fugu rubripes by analyzing the relativity of locus genotype and the weight character of the fugu rubripes: 1, wherein a base at 264bp has SNP locus related to weight character, and the genotype of the genotype CC homozygous individual is obviously higher than the phenotype value (p < 0.05) of the weight character of the CT genotype individual. And the corresponding characters can be rapidly identified by a PCR product sequencing method. Thus, individuals with the genotype CC type at the locus can be preferentially selected as parents or cultivated on a large scale during production.
Drawings
Fig. 1: sequencing peak diagrams of the CC and CT genotypes at the SNP marker locus;
Detailed Description
The single nucleotide polymorphism (Single nucleotide polymorphism, SNP) refers to DNA sequence polymorphism caused by single nucleotide mutation on genomic DNA, seed selection is carried out by determining SNP and genotyping, and is a mature molecular biotechnology, and SNPs molecular markers are widely applied to research on livestock and aquatic economic animals, including QTL positioning, molecular marker assisted selection and the like. As a new generation of genetic marking technology, SNPs play a great role in the field of aquatic economic animal genetic breeding research.
In SNP analysis, a certain target fragment in genomic DNA is amplified by utilizing a PCR technology at fixed points, amplified products are sent to sequencing, and whether mutation exists in the target fragment is judged after comparison according to sequencing results. The result is determined by comparing peak patterns among a plurality of samples, and observing the base difference and the double peaks of SNP loci among the peak patterns, thereby showing the DNA specificity of different biological individuals.
The invention designs 1 pair of primers for the fugu rubripes growth hormone receptor 2gene (Growth Hormone Receptor gene) to carry out SNP genotyping analysis on the sequencing result of PCR products on the fugu rubripes individuals and parents.
Through Seqman alignment, a SNP locus is screened to be positioned at 264 th base of a fragment of the fugu rubripes growth hormone receptor 2gene (nucleotide sequence SEQ ID NO: 1). Then analyzing the correlation between the point mutation genotype frequency and the growth character of the fugu rubripes for the 100 fugu rubripes individuals, and finding that the weight of the genotype CC homozygous individuals is obviously higher than the phenotype value (p < 0.05) of the weight character corresponding to the CT genotype individuals; SNP locus is found at 264 th position of the gene in Fugu rubripes juvenile fish, and the weight of homozygous CC type individuals in 100 identical cell juvenile fish is obviously higher than that of CT heterozygous individuals (p < 0.05).
Thus, individuals with CC type locus can be preferentially selected as parents in production or can be cultivated on a large scale.
The invention is further illustrated by the following examples and figures.
Example 1 screening for SNP loci
The SNP locus screening steps of the invention are as follows:
a) Extraction of the fugu rubripes genome: the marine animal DNA extraction kit extracts genomic DNA from the tail fin and muscle of the fugu rubripes. 3mg of sample tissue is taken and stored in a 1.5ml centrifuge tube after being extracted according to the instruction of a kit, and the sample tissue is stored at the temperature of minus 20 ℃; agarose gel electrophoresis detects DNA.
b) Primer design and screening: according to the sequence of the GHR 2gene of the fugu rubripes, a plurality of pairs of primers are designed on the DNA sequence of the fugu rubripes by utilizing Primer design software Primer 5.0, and a PCR product sequencing method is carried out on a plurality of fugu rubripes juvenile fish individuals to analyze base differences, and the primers with SNP loci are screened out, wherein the Primer sequence information is as follows:
F:5′-GACATTGAGGAAACCACCAG-3′(SEQ ID NO:2)，
R:5′-GCCCACTTACACCATCTACC-3′(SEQ ID NO:3)。
c) PCR amplification of the target gene: the reaction system was 25. Mu.l: 10 Xbuffer 2.5. Mu.l, dNTP 2. Mu.l, F primer (SEQ ID NO. 2) 1. Mu.l, R primer (SEQ ID NO. 3) 1. Mu.l, genomic DNA 1. Mu.l, taq enzyme 0.3. Mu.l, double distilled water make up to 25. Mu.l. The PCR procedure was: pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing at 62.5℃for 30s, extension at 72℃for 30s,30 cycles, and extension at 72℃for 7min. The resulting PCR amplification product was electrophoretically detected at a concentration of 1% agarose gel.
d) Sequencing the target fragment: the PCR product is sent to the China big genetic engineering Co.Ltd for single-molecule fluorescent marking sequencing, and the sequence of the obtained target fragment is SEQ ID NO. 1.
Example 2: PCR product sequencing method proves the correlation between SNP locus and growth character
To determine the sequence of SEQ ID NO:1, carrying out PCR product sequencing analysis of base differences and genotyping on SNP loci associated with the growth traits of the fugu rubripes fragments, and comprising the following steps:
a) Obtaining a fugu rubripes sample: all the adopted experimental sample fishes are taken from Dalian Zhengzhi practical limited company, and 100 fish of the juvenile fish of the fugu rubripes are randomly selected under the same culture management and nutrition conditions.
b) Data collection and extraction of genomic DNA: phenotypic values of body weight and body length of 100 individuals were measured and recorded while tail fin muscle tissue was taken for genomic DNA extraction.
c) And (5) PCR reaction and sequencing. The reaction system was 25. Mu.l: 10 Xbuffer 2.5. Mu.l, dNTP 2. Mu.l, F primer (SEQ ID NO: 2) 1. Mu.l, R primer (SEQ ID NO: 3) 1. Mu.l, genomic DNA 1. Mu.l, taq enzyme 0.3. Mu.l, double distilled water make up to 25. Mu.l. The PCR procedure was: pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing at 62.5℃for 30s, extension at 72℃for 30s,30 cycles, and extension at 72℃for 7min. The PCR amplified product is detected by electrophoresis under 1% agarose gel, and the target fragment band is clear and can be sent out for sequencing.
SNP genotype and growth trait phenotype value association analysis:
(1) SEQ ID NO:1 and the character phenotype values of the body weight, the body length and the body total length of the juvenile fish of the 100-tail fugu rubripes are respectively subjected to single-factor variance analysis, and the difference significance of the growth characters of each genotype of the SNP locus is calculated, and the result is shown in table 1.
Table 1: fugu rubripes nucleotide SEQ ID NO:1 sequence SNP growth traits (mean.+ -. Standard deviation) of each genotype
Note that: the same letters in the same column are not significant in difference, and adjacent letters are significant in difference (P < 0.05)
As can be seen from Table 1, individuals with genotype CC homozygous had significantly higher body weights than those with CT genotype (p < 0.05).
The weight of the takifugu rubripes CC genotype individuals screened by detecting the SNP locus is obviously higher than that of CT type individuals.
Therefore, the SNP locus for breeding provided by the invention can be used as a molecular marker for breeding of good varieties of fugu rubripes.
Claims (8)
1. A breeding method for screening Fugu rubripes individuals with rapid growth performance is characterized in that the method is to screen through detecting the genotype of SNP locus related to Fugu rubripes growth character; the SNP locus is positioned in a sequence of SEQ ID NO:1, and the base at position 264 of the nucleotide fragment of the formula 1 is C or T.
2. The method of claim 1, wherein the genotype of the SNP site is CC homozygous.
3. The method according to claim 1 or 2, wherein the method is to analyze the individual to be screened by sequencing the PCR product, and the genotype of the SNP locus of the individual to be detected is determined after the sequencing analysis of the PCR product.
4. The method of claim 3, wherein the primers used in the PCR amplification method are SEQ ID NO.2 and SEQ ID NO. 3.
5. A SNP site, characterized in that said SNP site is located in a sequence of SEQ ID NO:1, and the base at position 264 of the nucleotide fragment of the formula 1 is C or T.
6. The use of the SNP locus according to claim 5 for genetic breeding of Takifugu rubripes.
7. A molecular product for breeding fugu rubripes, wherein the product comprises a PCR amplification primer for detecting the SNP site as set forth in claim 5.
8. The molecular product according to claim 7, wherein the PCR amplification primers have the sequences SEQ ID NO.2 and SEQ ID NO. 3.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117417432A (en) * | 2023-12-18 | 2024-01-19 | 海南热带海洋学院崖州湾创新研究院 | Fugu rubripes growth hormone and application thereof |
| CN117925860A (en) * | 2024-03-21 | 2024-04-26 | 深圳大学 | SNP molecular marker related to growth traits of puffer green and application thereof |
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2022
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117417432A (en) * | 2023-12-18 | 2024-01-19 | 海南热带海洋学院崖州湾创新研究院 | Fugu rubripes growth hormone and application thereof |
| CN117417432B (en) * | 2023-12-18 | 2024-03-01 | 海南热带海洋学院崖州湾创新研究院 | Fugu rubripes growth hormone and application thereof |
| CN117925860A (en) * | 2024-03-21 | 2024-04-26 | 深圳大学 | SNP molecular marker related to growth traits of puffer green and application thereof |
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