CN113789392B

CN113789392B - SNP (Single nucleotide polymorphism) marker related to growth of channel catfish and application thereof

Info

Publication number: CN113789392B
Application number: CN202111176337.4A
Authority: CN
Inventors: 钟立强; 王明华; 张世勇; 姜虎成; 陈校辉
Original assignee: Freshwater Fisheries Research Institute of Jiangsu Province
Current assignee: Freshwater Fisheries Research Institute of Jiangsu Province
Priority date: 2021-10-09
Filing date: 2021-10-09
Publication date: 2024-02-02
Anticipated expiration: 2041-10-09
Also published as: CN113789392A

Abstract

The invention is thatDiscloses an SNP marker related to growth of channel catfish and application thereof, wherein the SNP marker is positioned on a channel catfish flesh-engendering regulatory factorMyoGThe gene sequence SEQ ID NO:2 at position 391. By utilizing the SNP marker, the channel catfish parent of the 391 st genotype AA is reserved in production, individuals with other genotypes are removed, the channel catfish parent with high growth speed and stable inheritance can be quickly selected, the breeding period is shortened, and the breeding process is accelerated.

Description

SNP (Single nucleotide polymorphism) marker related to growth of channel catfish and application thereof

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to an SNP marker related to growth of channel catfish and application thereof.

Background

The channel catfish is also called as a channel catfish, is native to North America, has fresh meat quality, strong disease resistance, rapid growth, easy capture and wider temperature adaptation, and has rapid industrial development and annual output of breaking through 30 ten thousand tons after being introduced into China in the 1980 s. However, in the rapid development process of the channel catfish breeding industry in China, production units do not pay attention to parent seed reserving operation, and even the parent seed reserving operation is large and small, so that the genetic diversity of the breeding channel catfish is reduced, the growth is slowed down, the specification is uneven, and the disease resistance is reduced. And the growth characters directly affect the culture yield and benefit, so that the development of the breeding work of the channel catfish with fast growth and good economic characters is very important. The traditional main method for breeding the grown fish is mainly group breeding and family breeding, and the fish with larger individuals and strong bodies is selected as a seed-reserving parent according to quantitative genetic analysis of phenotypes. The method is time-consuming, labor-consuming and high in workload, and meanwhile, the quality of the channel catfish parents is often evaluated inaccurately in phenotype judgment, so that the breeding effect is poor.

With the development of molecular biology and genetics, a plurality of genetic markers, such as AFLP, RAPD, SSR, SNP and the like, have emerged, wherein SNP markers are suitable for high-throughput automatic analysis due to wide distribution, and are genetically stable, so that the SNP markers are increasingly becoming the first genetic markers in genetic breeding research. SNP markers refer to polymorphisms in genomic DNA sequences that result from single nucleotide changes, mainly caused by single base transitions or transversions. SNPs in the coding region of a gene are relatively few, and are attracting attention because the mutation rate in an exon is only 1/5 of that of the surrounding sequence, but it is of great importance in the study of genetic breeding.

The Myostatin gene (MSTN), insulin-like growth factor-1 gene (Insulin-like growth factor I, IGF-1) and myogenic regulatory factor gene (Myogenin, myoG) are important for the regulation of growth in animals. Therefore, the genes are taken as candidate genes of growth traits, and correlation analysis of SNP and the growth traits is widely reported in livestock, poultry and aquatic animals. SNP markers of the gene coding regions related to growth are screened out from the channel catfish, so that channel catfish parents with high growth speed and stable inheritance can be quickly selected, the breeding period is shortened, and the breeding process is accelerated.

Disclosure of Invention

The invention aims to provide an SNP marker related to growth of channel catfish and application thereof, and the SNP marker related to growth characteristics of a channel catfish MyoG gene coding region is detected to perform early selection and rapid screening, so that generation intervals are shortened, selection intensity is improved, and breeding efficiency and accuracy are improved.

Another object of the invention is to provide a screening method for rapid growth channel catfish parents.

The invention adopts the following technical scheme:

an application of channel catfish myogenic regulatory factor MyoG gene in parent screening.

As another aspect of the invention, the invention provides a SNP marker affecting the growth of channel catfish, wherein: the DNA sequence of the SNP marker is shown as SEQ ID NO:2, wherein W at 391 is a base T or A.

Preferably, the 391 st genotype of the SNP marker is TT, AA and AT.

As another aspect of the invention, the invention provides an application of SNP markers in judging the growth speed of channel catfish and/or in screening fast-growing channel catfish.

As another aspect of the invention, the invention provides a method for judging MyoG genotype of channel catfish, wherein whether the 391 st base of SNP marker of channel catfish is of AA genotype or not is detected, and the DNA sequence of the SNP marker is as shown in SEQ ID NO:2 is shown in the figure; if the strain is AA genotype, the strain can be used as a backup parent of the channel catfish with high growth speed and stable inheritance.

Preferably, the method comprises the following steps of 1) extracting channel catfish genomic DNA; 2) PCR detection of the 391 st genotype of the SNP marker by using the extracted genomic DNA as a template.

Preferably, in step 2), the PCR detection is specifically performed by PCR amplification of channel catfish genomic DNA using primers F1 and R1 to obtain a PCR product,

F1：5'-TCTTCCCTTCCAGGCTTACA-3'(SEQ ID NO：3)；

R1：5'-AGCAGCCGAGGACCTGTAAT-3'(SEQ ID NO：4)。

the PCR products were detected by agarose gel electrophoresis and then subjected to sequencing analysis.

Preferably, the reaction system of the primary PCR amplification is that a PCR amplification uses a ready-to-use UltraTaq enzyme PCR kit, which comprises 2X UltraTaq Master Mix mu l, 2 mu l of genome DNA, 2 mu l of upstream and downstream primers and ddH ₂ O14. Mu.l; the amplification conditions were: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 59℃for 30s, elongation at 72℃for 60s,30 cycles; extending at 72℃for 10min.

The PCR products were detected by 1% agarose gel electrophoresis, and the electrophoresis results were observed by a gel imager. All amplified products were sent to Shanghai Biotechnology Co., ltd and sequenced directly using the ABI3730 XL sequencer (ABI, USA). Sequencing peak patterns were observed using Chromas software, and DNA sequence alignment was performed in combination with ClustalX software to determine the base type of SNP sites. Individuals with genotype AA AT 391 had significantly higher body weight and length than those AT and TT (P < 0.05).

The beneficial effects of the invention are as follows:

the invention greatly improves the screening accuracy of the channel catfish parent, can quickly select the backup parent of the channel catfish with high growth speed and stable inheritance in advance, and reduces the cost by directional cultivation. Compared with the traditional method, the method has the advantages of strong purposefulness, direct action effect, simple operation, rapid detection, low detection cost and convenient wide popularization and application.

Detailed Description

For a better description of the objects, technical solutions and advantages of the present invention, the present application will be further described with reference to the specific examples below.

Example 1: acquisition of SNP markers related to growth traits of Ictalurus punctatus

1. Experimental animal

The channel catfish samples used in the experiment are all from the channel catfish genetic breeding center in the national institute of freshwater aquaculture in Jiangsu province. In order to reduce the influence of the environment on the growth of the channel catfish, the G3 generation family of the channel catfish core breeding population is constructed in 6 months 2019, and the seed culture is carried out according to the same standard, namely uniform water changing rate, feeding amount, culture density, oxygenation amount and water temperature. When the average age of the family is 170d, measuring the weight and the body length data of the individual, randomly selecting 6 300 fish of the family, collecting tail fin tissues of each fish, soaking in 95% alcohol, and preserving at-20 ℃.

2. Primer design

According to the MyoG gene (AY 534329.1), MSTN gene (AF 396747.1) and IGF-1 gene (AH 015082.2) published by GenBank database, a pair of primers F1/R1, F2/R2 and F3/R3 was designed by using PrimerPremier 5 software, and the amplified products were 464bp, 550bp and 1776bp in size, respectively.

F1:5'-TGTTGGATTGGTCTGGAGTGG-3'(SEQ ID NO:3)

R1:5'-CGTCTACTCTCCACCTGCTTC-3'(SEQ ID NO:4)

F2:5'-ACGGTGTTCCTGTTACTGC-3'(SEQ ID NO:5)

R2:5'-CACCAGATGTTGCTATGC-3'(SEQ ID NO:6)

F3:5'-AAGACGCACGAGCCAGGATTATT-3'(SEQ ID NO:7)

R3:5'-TGAGGAGCACATTAGCACTTCTGG-3'(SEQ ID NO:8)

3. PCR amplification

PCR amplification uses a ready-to-use UltraTaq enzyme PCR kit (Shanghai JieRui bioengineering limited)Company), the reaction system is: 2X UltraTaq Master Mix. Mu.l, 2. Mu.l of genomic DNA, 2. Mu.l of upstream and downstream primers, ddH ₂ O 14μl。

The MyoG gene amplification conditions were: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 59℃for 30s, elongation at 72℃for 60s,30 cycles; extending at 72℃for 10min. The MSTN gene amplification conditions were: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 58℃for 30s, elongation at 72℃for 60s,30 cycles; extending at 72℃for 10min. IGF-1 gene amplification conditions were: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 64℃for 30s, elongation at 72℃for 60s,30 cycles; extending at 72℃for 10min.

The PCR products were detected by 1% agarose gel electrophoresis, and the electrophoresis results were observed by a gel imager.

4. Sequencing

The electrophoretically-acceptable samples were sent to Shanghai Biotechnology Co., ltd, and sequenced directly using an ABI3730 XL sequencer (ABI, USA). Sequencing peak patterns were observed using Chromas software, and DNA sequence alignment was performed in combination with ClustalX software to determine the base type of SNP sites.

5. SNP typing

The MyoG gene found two coding region SNPs in total, g.391T > A and g.4819A > G, respectively. Wherein g.489A > G is synonymous mutation, the coded amino acid after mutation is changed, and the coded amino acid is still Gln; and g.391T > A is missense mutation, the coded amino acid is changed, and the mutation is from Ser to Thr.

MSTN gene finds two coding region SNP, g.1859C > G and g.1907C > G/T, which are synonymous mutation, and coding amino acid is unchanged after mutation and is still Leu.

Five coding region SNPs were found in IGF-1 gene, g.1695G > T, g.1744G > C, g.1747G > A, g.1750G > A and g.1826C > G, respectively. Wherein, three mutations of g.1744G > C, g.1747G > A and g.1750G > A are synonymous mutations, and the coded amino acid is unchanged after mutation, and the ratio is Leu, thr and Pro; g.1695G > T and g.1826C > G are missense mutations, the encoded amino acid is changed, and Arg is mutated into Leu and Gly respectively.

6. Frequency statistical analysis of SNP loci

The genotype of the 300-tail channel catfish of the randomly selected 6 families is respectively judged by using the 3 missense mutation SNP detection method, and the frequency of SNP loci is counted.

Genotype frequency refers to the ratio of the number of individuals of a certain genotype for a trait in a population to the total number of individuals:

P _YY ＝N _YY /N

wherein P is _YY Represents the YY genotype frequency of a certain site, N _YY Representing the number of individuals in the population having a YY genotype; n is the total number of individuals tested.

Gene frequency refers to the relative ratio of a certain base factor to the total number of alleles in a population:

P _y ＝(2N _yy +N _yy1 +N _yy2 +…+N _yyi …+N _yyn )/2N

wherein P is _y Indicating allele y frequency, N _yy Representing the number of individuals in the population having yy genotype, N _yyi Representing the number of individuals with the yyi genotype in the population, y1-yn is the n different complex alleles of allele y. The statistical results are shown in Table 1.

TABLE 1 genotype and allele frequency of 3 missense mutation SNP loci in Ictalurus punctatus populations

7. Correlation analysis of Gene Effect

The correlation of individuals with different genotypes of the channel catfish and the growth traits of the individuals with the comprehensive character records is subjected to significance test (see table 2).

1) The measured body ruler data mainly comprise: body weight (170 days old) and length.

2) Correlation analysis is a generally linear model: the SPSS 20.0 software general linear model GLM (General linear models procedure) was invoked to perform a significance test on the effect of each genotype on growth traits. The statistical model is as follows:

Y _ijk ＝μ+G _i +T _j +β _1P +E _ijk

wherein: y is Y _ijk : recording the phenotype of the individual; mu: overall average; g _i : genotype fixing effect; tj: fixing effect of cultivation time; beta _1P : linear covariates of initial body weight, eijk: random errors.

It can be seen from Table 2 that individuals with genotype AA AT the MyoG gene g.399 SNP locus are significantly higher in body weight and length than individuals with genotype TT and AT (P < 0.05). And the IGF-1 gene g.1695 and g.1826SNP loci have no obvious difference (P > 0.05) in weight and body length among channel catfish of different genotypes. Therefore, AA in the single nucleotide polymorphic site (reference sequence AY534329.1, 391 st position of the gene) of the MyoG gene of the channel catfish is a dominant genotype and can be used as a DNA marker. Thus, in the breeding work, individuals with the MyoG gene of the channel catfish with the AA genotype should be selected as channel catfish parents for breeding the growth traits.

TABLE 2 correlation analysis between different genotypes and growth traits of Ictalurus punctatus SNP loci

Note that: the numerical values in the table are average value plus or minus standard error; the same column is marked with a, b as a significant level of difference P < 0.05.

In order to verify the reliability of the excavated 1SNP molecular markers, weight gain measurement and SNP typing based on Sanger sequencing are carried out on the channel catfish breeding population with the market specification, and further prove that the polymorphism of the SNP locus is extremely obviously related to the channel catfish growth trait (P < 0.05), so that the channel catfish growth trait can be selected by using the SNP markers.

Example 2: verification of SNP molecular markers related to growth traits of channel catfish

1. Cultivation experiment and sample collection

After the growth characters such as weight, body length and the like of the channel catfish core breeding population G3 generation family are measured for the first time, PIT electronic chips are injected into the abdominal cavity, and the channel catfish core breeding population G3 generation family is put into a pond with the same mouth of 6 mu for mixed culture for 18 months, so that the channel catfish core breeding population G3 generation family reaches the market specification. And (3) taking off the net for fishing in 11 months in 2020, randomly selecting 500 tail, measuring the weight and the body length, and collecting tail fin tissues.

2. Extraction of genomic DNA and SNP typing

Genomic DNA was extracted from the tail fins of the test fish. The PCR amplification uses a ready-to-use UltraTaq enzyme PCR kit (Shanghai JieRui bioengineering Co., ltd.) and the reaction system is: 2X UltraTaq Master Mix. Mu.l, 2. Mu.l of genomic DNA, 2. Mu.l of upstream and downstream primers, ddH ₂ O14. Mu.l. The amplification primers were F1/R1 (SEQ ID NO:3 and SEQ ID NO: 4). The amplification conditions were: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 59℃for 30s, elongation at 72℃for 60s,30 cycles; extending at 72℃for 10min. The PCR products were detected by 1% agarose gel electrophoresis, and the electrophoresis results were observed by a gel imager. The qualified PCR amplified products were subjected to Sanger sequencing using an ABI3730 sequencer, and the primers used for sequencing were identical to those used for PCR amplification. Sequencing peak patterns were read using Chromas software and each sample was recorded at the SNP site (g.391T>A) Is a genotype of the subject.

3. Growth trait and SNP association analysis

After each sample SNP locus (g.3991T > A) is successfully typed, the body weight and body length characters are associated with each sample genotype for analysis. The results are shown in table 3, indicating that the weight and length of AA type individuals are extremely significantly higher than those of TT and AT type (P < 0.05).

TABLE 3 results of analysis relating growth traits to SNP genotypes of Ictalurus punctatus

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Sequence listing

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Claims

1. A method for screening channel catfish parents is characterized by comprising the following steps:

detecting whether the 391 st base of the SNP marker of the channel catfish is of an AA genotype, wherein the DNA sequence of the SNP marker is shown as SEQ ID NO. 2;

if the channel catfish is of AA genotype, the channel catfish can be used as a backup parent of the channel catfish with high growth speed and stable inheritance.

2. The method according to claim 1, characterized in that: comprises the following steps of the method,

1) Extracting channel catfish genome DNA;

2) PCR detection of the 391 st genotype of the SNP marker by using the extracted genomic DNA as a template.

3. The method according to claim 2, characterized in that: the specific operation of the PCR detection is that,

PCR amplification is carried out on the channel catfish genome DNA by using the primers F1 and R1 to obtain a PCR product,

the sequence of the primer F1 is shown as SEQ ID NO. 3, and the sequence of the primer R1 is shown as SEQ ID NO. 4.