A snp molecular marker related to grass carp weight on grass carp chromosome 19 and application thereof

Abstract

The application discloses a SNP molecular marker related to grass carp weight on a 19th chromosome of the grass carp and an application thereof, and belongs to the technical field of biology. The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO. 1, and the polymorphism of two allelomorphic genes A and G at the 501st base. The application further provides an amplification primer of the SNP molecular marker, and the nucleotide sequences of the amplification primer are shown in SEQ ID NO. 2 and SEQ ID NO. 3, which are used for grass carp genotyping or breeding, and the method comprises the following steps: detecting DNA of a to-be-tested grass carp by using the SNP molecular marker and the amplification primer, selecting the grass carp with the genotype AG for breeding, and the average weight of the grass carp with the genotype AG is large. The application introduces molecular marker assisted selection in the process of breeding the grass carp, overcomes the problems of long breeding cycle, low efficiency and large environmental influence of the traditional breeding method, and realizes early screening and directional breeding of excellent traits of the grass carp.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a SNP molecular marker located on chromosome 19 of grass carp that is related to the weight of grass carp and its application. Background Technology

[0002] Grass carp (Ctenopharyngodonidella) is one of the "four major freshwater fish" in my country and the most important freshwater aquaculture species. It has advantages such as fast growth, strong adaptability and good meat quality, and has the highest output in freshwater aquaculture in my country.

[0003] With the expansion of aquaculture scale and the popularization of high-density farming models, the genetic degradation of grass carp has gradually become apparent, manifested in decreased growth rate, reduced disease resistance, and decreased reproductive performance. Traditional phenotype-based breeding methods are greatly affected by the environment, have long cycles, and low efficiency, making it difficult to meet the needs of modern aquaculture. In recent years, the development of molecular biology and genomics has made marker-assisted selection (MAS) an important tool for grass carp breeding. Researchers have identified genes related to growth and immunity, such as GH and IGF-1, laying the foundation for molecular breeding. Among them, SNP-based molecular markers are widely used due to their wide distribution, strong stability, and high detection efficiency. SNP screening around lipid metabolism genes (such as FABP3 and PPARγ) has yielded markers that are significantly associated with traits such as abdominal fat index and liver fat content, promoting the targeted breeding of new low-fat strains. However, currently disclosed SNP loci are mainly concentrated in a few candidate genes, failing to cover core gene regions that affect key economic traits such as growth rate, and most markers only target a single trait, lacking the ability to integrate and synergistically improve multiple traits such as growth, feed utilization, and disease resistance.

[0004] Although traditional breeding methods have improved the growth performance of grass carp to some extent, they still have the following drawbacks: The breeding cycle is long and inefficient. Grass carp typically reach sexual maturity in 4-5 years, and traditional family breeding requires four consecutive generations of screening, with the entire breeding cycle usually taking 16-20 years, resulting in low efficiency. They are significantly affected by the environment; phenotypic traits are easily influenced by external environmental factors (such as water quality, temperature, and feed formulation), leading to large errors in genetic assessment and insufficient stability of breeding results. The genetic basis of breeding is vague; this method fails to identify genes or molecular markers directly related to target traits (such as rapid growth and disease resistance), and the breeding process lacks precise genetic control. Summary of the Invention

[0005] To address the aforementioned shortcomings of existing technologies, the main objective of this invention is to provide a single nucleotide polymorphism (SNP) molecular marker located on chromosome 19 of grass carp that is associated with grass carp body weight. Through whole-genome resequencing and phenotypic data association analysis, a single nucleotide polymorphism (SNP) site significantly associated with body weight was identified on chromosome 19 of grass carp. This site is located on chromosome 19 of the reference genome GCF019924925.1. Its allele variation is highly positively correlated with daily weight gain, specific growth rate, and final body weight of grass carp, and can be used as a molecular marker for early screening and targeted breeding of fast-growing grass carp strains.

[0006] Another object of the present invention is to provide the application of the SNP molecular markers located on chromosome 19 of grass carp that are related to grass carp body weight in grass carp genotyping or breeding.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides an SNP molecular marker located on chromosome 19 of grass carp that is related to the weight of grass carp, the nucleotide sequence of which is shown in SEQ ID NO.1, wherein polymorphism of two alleles, A and G, is observed at the 501st base. That is, the SNP site is located at the 3400300th base of chromosome 19 of grass carp, and polymorphism of two alleles, A and G, is observed at this site.

[0009] Specifically, the sequence of this SNP molecular marker is as follows:

[0010]

[0011] In a second aspect, the present invention provides amplification primers for an SNP molecular marker located on chromosome 19 of grass carp that is related to the body weight of grass carp, the nucleotide sequences of which are shown in SEQ ID NO.2 and SEQ ID NO.3, as follows:

[0012] Upstream primer: 5'-ACAAAACAGAAGTAAAGCAG-3' (SEQ ID NO.2);

[0013] Downstream primer: 5'-AGGTCATGTTACCACCAG-3' (SEQ ID NO.3).

[0014] A third aspect of the present invention provides the SNP molecular marker located on chromosome 19 of grass carp that is related to the weight of grass carp, primers for amplifying the SNP molecular marker, or the application of the amplification primers in grass carp genotyping or breeding.

[0015] Preferably, the nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.1, wherein polymorphism of two alleles, A and G, appears at the 501st base;

[0016] The amplification primer sequences are shown in SEQ ID NO.2 and SEQ ID NO.3;

[0017] The genotyping includes: the average body weight of grass carp with genotype AG;

[0018] The breeding includes: selecting grass carp with genotype AG for breeding, wherein the grass carp with genotype AG have an average body weight;

[0019] The application is for screening grass carp with a large average weight.

[0020] A fourth aspect of the present invention provides a method for breeding grass carp, comprising:

[0021] The SNP molecular marker and the amplification primers were used to detect the DNA of the grass carp to be tested; grass carp with the genotype AG were selected for breeding, and the grass carp with the genotype AG had an average body weight; the SNP molecular marker sequence is shown in SEQ ID NO.1, in which polymorphism of two alleles, A and G, appears at the 501st base; the amplification primer sequences are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0022] This invention proposes a molecularly assisted breeding method for grass carp growth rate based on SNP molecular markers related to grass carp body weight. The implementation process includes sample collection, genomic DNA extraction, specific primer amplification, genotype determination, and breeding population screening, as follows: First, tail fin rays of grass carp individuals to be tested are collected, with approximately 10 cm² of fin rays cut off. The collection time is limited to the juvenile stage to ensure that genetic potential assessment can be completed early and to avoid invalid individuals occupying aquaculture resources. After collection, the samples are immediately stored in a -20°C freezer to prevent DNA degradation. Subsequently, a commercially available column-type genomic DNA extraction kit is used to extract genomic DNA from the samples according to standard operating procedures. After extraction, the DNA concentration and purity are measured using a UV spectrophotometer. The concentration is not less than 50 ng / μL to meet the template quality requirements for subsequent amplification reactions.

[0023] Specific primer pairs were designed and synthesized targeting the SNP site. The upstream primer of this specific primer pair is 5'-ACAAAACAGAAGTAAAGCAG-3', and the downstream primer is 5'-AGGTCATGTTACCACCAG-3'. This primer pair amplifies a target fragment of 600 base pairs, containing the SNP site and approximately 500 base pairs of flanking sequences upstream and downstream, ensuring that the amplified product contains complete restriction enzyme recognition sites and is of sufficient length for sequencing verification. During primer design, BLAST alignment was used to confirm its uniqueness within the grass carp genome, avoiding non-specific amplification.

[0024] Using extracted genomic DNA as a template, target fragments containing SNP sites were amplified by polymerase chain reaction (PCR). The total volume of the amplification reaction system was 20 μL, containing 5 μL of pure water, 1.5 μL each of forward and reverse primers, 2 μL of DNA, and 10 μL of TaqMaster Mix solution. The amplification program was set as follows: 94℃ pre-denaturation for 5 minutes, followed by 35 cycles of 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 30 seconds, and a final extension at 72℃ for 5 minutes. After amplification, 5 μL of the amplification product was loaded onto a 1% agarose gel and electrophoresed at 120 V for 30 minutes. The amplification was confirmed to be successful by observing the appearance of a single, clear band using a gel electrophoresis apparatus.

[0025] Next, the products were sent to a sequencing service provider using direct sequencing. The polymerase chain reaction (PCR) amplification products were purified using a commercially available PCR product purification kit to remove unreacted primers and dNTPs. Sequencing primers included upstream and downstream primers, with the sequencing direction covering the SNP site. After the sequencing results were returned, the sequencing peak diagram was compared with a reference genome using the sequence alignment software SnapGene to determine the nucleotide type at that site. If it was AG, it was identified as a fast-growing dominant genotype; if it was AA or GG, it was identified as a normal-growing genotype. This sequencing method can simultaneously detect heterozygotes and provide complete flanking sequence information, making it suitable for research validation and high-precision genotyping needs.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] This invention overcomes the shortcomings of traditional breeding methods, such as long breeding cycles, low efficiency, and significant environmental impact, by introducing molecular marker-assisted selection and genotype-phenotype association analysis into the grass carp breeding process. It achieves rapid and accurate breeding of superior traits in grass carp. Furthermore, by constructing a molecular marker system related to grass carp growth traits, combined with whole-genome resequencing and population association analysis of target traits, a closed-loop breeding process is established, encompassing genotype identification, trait prediction, and selection of superior parents. This invention can determine individuals with superior genotypes through early molecular marker screening, shortening the breeding cycle by more than 50% and improving breeding efficiency. Attached Figure Description

[0028] Figure 1 The above is a sequencing peak diagram of different genotypes after sequencing and typing of SNP molecular markers on chromosome 19 of grass carp that are related to the weight of grass carp in the example. Detailed Implementation

[0029] To more fully understand and demonstrate the technical solutions, objectives, and advantages of the present invention, the technical effects produced by the present invention will be further described in detail and completely below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. It should be noted that other embodiments obtained by those skilled in the art without departing from the concept of the present invention are all within the protection scope of the present invention.

[0030] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0031] Example 1: Application of growth-related molecular markers

[0032] 1. Measurement of test materials and growth traits

[0033] An experimental population of grass carp was constructed by intraspecies hybridization of female grass carp from the Hanjiang River in the Yangtze River system and male grass carp from the Zhaoqing River system in the Pearl River system. Male and female individuals with superior growth traits were selected as parents for artificial insemination. The fertilized eggs were cultured in incubators until fry were developed, then transferred to ponds for further rearing. After 10 months of rearing, 514 grass carp were randomly selected for body weight and length measurements. Small pieces of caudal fin tissue were then removed and placed in 96-well plates containing anhydrous ethanol and stored at -20°C for later use.

[0034] 2. Sequence Information

[0035] Molecular marker 500 bp sequences were extracted from the genome for primer construction, where S represents forward and A represents reverse, as detailed below:

[0036] S: 5'-ACAAAACAGAAGTAAAGCAG-3'

[0037] A: 5'-AGGTCATGTTACCACCAG-3'

[0038] 3. PCR amplification

[0039] The PCR amplification system consisted of 20 μL of components, including 10 μL Taq Master Mix, 5 μL ddH2O, 2 μL DNA, and 1.5 μL each of forward and reverse primers.

[0040] PCR amplification program: The amplification program was set to 94℃ pre-denaturation for 5 minutes, followed by 35 cycles of 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 30 seconds, and finally 72℃ final extension for 5 minutes.

[0041] After amplification, take 5 μL of the amplification product and load it onto a 1% agarose gel. Electrophoresis is performed at 120 V for 30 minutes. Observe the gel using a gel electrophoresis apparatus to see if a single clear band appears, confirming successful amplification.

[0042] Using the measured genotype data and phenotypic data, the gene frequencies and average body weight of different genotypes were statistically analyzed. The results are expressed as mean ± standard deviation. The dominant genotype of the experimental population was determined by the mean. The statistical results are shown in Tables 1 and 2. First, the allele and gene frequencies of individual SNP loci were statistically analyzed (Table 1). The results show that the A gene frequency of the SNP locus was relatively high. According to Table 2, the effects of SNP loci on body length and body weight were statistically significant (p < 0.05). The body length (20.855±3.203 mm) and weight (224.412±111.122 mm) of individuals with the AG genotype were significantly higher than those with the AA genotype (body length 19.942±3.003 mm, weight 193.626±105.886 mm). However, the GG genotype (body length 20.931±3.862 mm, weight 221.133±102.341 mm) showed no significant difference from either the AG or AA genotypes. This suggests that the AG genotype may have an advantage in promoting growth, while the AA genotype may have an inhibitory effect on growth traits.

[0043] Table 1: Statistics on SNP loci genotypes and allele frequencies

[0044]

[0045] Table 2: Association analysis between SNP sites and growth traits

[0046]

[0047] Note: Different letters in the same column at the same site indicate significant differences (P<0.05).

[0048] Example 2: Development of Molecular Markers

[0049] (1) Construction of experimental population: Normal-growing male and female grass carp without deformities were selected as parents to construct the experimental population. After hatching, they were first cultured in a flowing water pond (4m). 3 The fish are raised using powdered feed as their first food, gradually transitioning to formulated feed. When they are about 15 days old, they are transferred to an outdoor cement pond (5m×3m×2m) for standardized rearing. They are then fed artificial formulated feed three times a day (morning, noon, and evening), and their feeding is observed. The aerator is turned on.

[0050] (2) Growth data measurement: After hatching and raising the experimental population constructed in step (1) for 10 months, the weight, body length, body width, and body height of the offspring individuals were measured. An electronic balance was used to measure the body weight trait (accurate to 0.1g), and a standard ruler was used to measure the body length, body width, and body height (accurate to 0.1cm). Subsequently, a small piece of caudal fin tissue was cut off and placed in a 96-well plate containing anhydrous ethanol and stored in a -20℃ freezer for later use.

[0051] (3) Major SNP screening: Total DNA of grass carp in the caudal fin tissue of step (2) was extracted using a DNA extraction kit. The DNA was subjected to simplified genome sequencing. After obtaining the raw data, it was compared with the reference genome using BWA. SNP development and genotyping were performed using Stacks software. SNPs that did not meet the standard were filtered using VCFtools. Finally, only SNPs with MAF (minor allele frequency) > 10% were retained. Genetic linkage map was constructed using JoinMap4.0 software. QTL location was performed using MapQTL6 software. Combined with the genetic linkage map, molecular markers in the QTL intervals related to growth traits were found. Molecular marker development was completed, and SNP molecular markers related to grass carp body weight were obtained. The nucleotide sequence is shown in SEQ ID NO.1. Polymorphism of two alleles, A and G, appeared at the 501st base. The SNP site is located at the 3400300th base of the 19th chromosome of grass carp. Polymorphism of two alleles, A and G, appeared at this site.

[0052] Specifically, the sequence of this SNP molecular marker is as follows:

[0053]

[0054] (4) Molecular marker fragment primer design: Using the detailed molecular marker information developed in step (3), a grass carp reference genome index was constructed using the BWA software. Then, flanking sequences containing SNPs were extracted using bedtools. Primers were designed using the obtained sequences in the Primer Premier5 software to obtain an amplification primer for a SNP molecular marker related to grass carp body weight. Its nucleotide sequence is shown in SEQ ID NO.2 and SEQ ID NO.3, as follows:

[0055] Upstream primer: 5'-ACAAAACAGAAGTAAAGCAG-3' (SEQ ID NO.2);

[0056] Downstream primer: 5'-AGGTCATGTTACCACCAG-3' (SEQ ID NO.3).

[0057] Example 3: Application of Molecular Markers

[0058] (1) Measurement of test materials and growth traits: The method is the same as step (1) and (2) in the development of molecular markers.

[0059] (2) Sequence information: Based on the flanking sequences of the SNPs obtained in step (4) of the molecular marker development, the sequence information and the annealing temperature required for PCR amplification of each SNP fragment are obtained in Primer 5.

[0060] (3) PCR amplification: The PCR amplification system consisted of 20 μL of 10 μL of Taq Master Mix solution. The amplification program was set to 94℃ for pre-denaturation for 5 minutes, followed by 35 cycles of 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 30 seconds, and a final extension at 72℃ for 5 minutes. After amplification, 5 μL of the amplification product was loaded onto a 1% agarose gel and electrophoresed at 120V for 30 minutes. The amplification was confirmed to be successful by observing whether a single clear band appeared using a gel electrophoresis apparatus.

[0061] (4) SNP genotyping: SNP genotyping is performed using first-generation sequencing. The genotype of each SNP locus is determined based on the shape of the sequencing peak.

[0062] (5) Evaluation of dominant genotypes: The mean and standard deviation of the phenotypic data corresponding to different genotypes in the sequencing data are calculated, and the dominant genotype is determined by comparing the phenotypic values ​​corresponding to different genotypes.

[0063] Table 1: The AG genotype is highly positively correlated with daily weight gain, specific growth rate, and final weight of grass carp.

[0064]

[0065] Note: *p<0.05, **p<0.01

[0066] Table 2: The GG genotype was highly positively correlated with daily weight gain, specific growth rate, and final weight of grass carp.

[0067]

[0068] Note: *p<0.05, **p<0.01

[0069] Through whole-genome resequencing and phenotypic data association analysis, a single nucleotide polymorphism (SNP) site significantly associated with growth rate was identified on chromosome 19 of grass carp. This site is located on chromosome 19 of the reference genome GCF019924925.1. Its allele variation is highly positively correlated with daily weight gain, specific growth rate, and final body weight of grass carp (Tables 1 and 2). It can be used as a molecular marker for early screening and targeted breeding of fast-growing grass carp strains.

[0070] The above are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of primers for amplifying SNP molecular markers in grass carp genotyping or breeding; The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.1, wherein polymorphism of two alleles, A and G, appears at the 501st base; The sequences of the primers are shown in SEQ ID NO:2 and SEQ ID NO:3; The genotyping includes: Grass carp with genotype AG have an average body weight; The breeding includes: selecting grass carp with genotype AG for breeding, wherein the grass carp with genotype AG have an average body weight; The application is for screening grass carp with a large average weight.

2. A method for breeding grass carp, characterized in that, This includes using the SNP molecular marker and primers described in claim 1 to detect the grass carp DNA to be tested; Grass carp with genotype AG were selected for breeding, and the average body weight of grass carp with genotype AG was [not specified]. The breeding program includes selecting grass carp with a large average weight.

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