A snp molecular marker related to grass carp growth traits and application thereof

Abstract

The application belongs to the technical field of aquatic animal molecular marker and aquatic genetic breeding, and particularly relates to a SNP molecular marker significantly related to growth traits of grass carp and application thereof. The SNP molecular marker is located at 23608308 bp of the 11th chromosome of the grass carp (SLG11_23608308), and is a T>A base mutation. Experiments show that the SNP molecular marker developed in the application has a significant influence on four growth traits of the grass carp, including body length, body height, body width and body weight (P<0.05), and the allele A has more excellent growth traits. The application discloses application of the SNP molecular marker, amplification primers or kits in breeding of the grass carp with excellent growth traits. The SNP molecular marker provided in the application is not limited by the age and gender of the grass carp, and can be used for breeding of the grass carp with excellent growth traits, and even accurate screening can be performed just after hatching, so that the breeding process of the grass carp with excellent growth traits is greatly accelerated.

Description

Technical Field

[0001] The present invention relates to the technical field of aquatic animal molecular markers and aquatic genetic breeding, and in particular to a SNP molecular marker significantly correlated with grass carp growth traits and an application thereof. Background Art

[0002] Grass carp (Ctenopharyngodon idella) is the dominant commercial fish species in freshwater aquaculture in my country. Genetic improvement of its growth traits is crucial for improving aquaculture profitability. However, the long 4-5 year period of sexual maturity for grass carp results in an extremely long breeding cycle and low breeding efficiency, severely restricting the development of the grass carp breeding industry. Therefore, there is an urgent need to develop molecular markers associated with growth traits for use in marker-assisted breeding of grass carp, thereby identifying individuals with superior growth traits for future breeding.

[0003] Molecular markers are genetic markers based on nucleotide sequence variations in genetic material between individuals, which directly reflect the genetic polymorphism at the DNA level. Among them, single nucleotide polymorphism (SNP) is the third generation molecular marker, and its number is the largest and the most widely distributed in the genome, so it is widely used in the molecular breeding of plants and animals. SNP refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genomic level, and this variation can be the replacement, insertion or deletion of a single base. Molecular marker-assisted breeding is a breeding technique that utilizes these molecular markers to closely link with the control target trait genes, and selects target traits by detecting molecular markers. However, there is currently a lack of molecular markers for molecular marker-assisted breeding of grass carp growth traits, which has restricted the improvement of grass carp growth traits. Summary of the Invention

[0004] The present invention aims to provide a SNP molecular marker (SLG11_23608308) associated with grass carp growth traits and applications thereof.

[0005] In order to achieve the above object, the specific technical solutions adopted by the present invention are as follows:

[0006] In the first aspect, the present invention provides a SNP molecular marker related to the growth traits of grass carp, which is located at 23608308bp of chromosome 11 of grass carp (SLG11_23608308), specifically the T / A polymorphic site at position 201 of the sequence shown in SEQ ID NO: 3; when the genotype of the polymorphic site is AA, it corresponds to high growth performance, and when the genotype of the polymorphic site is TT, it corresponds to low growth performance.

[0007] Furthermore, the growth traits include grass carp body length, body height, body width and weight, and these four traits are better than other genotypes when the genotype of the SNP molecular marker is AA.

[0008] In a second aspect, the present invention provides primers for detecting the SNP molecular markers.

[0009] Furthermore, the sequences of the primers are shown in SEQ ID NO: 1-2.

[0010] In a third aspect, the present invention provides a reagent or kit containing the primer.

[0011] In a fourth aspect, the present invention provides any of the following uses of the SNP molecular marker, the primer for detecting the SNP molecular marker, the reagent or kit containing the primer:

[0012] (1) Used for the breeding of grass carp with excellent growth traits;

[0013] (2) Used to screen fry with excellent growth traits;

[0014] (3) Used for the breeding of grass carp strains with excellent growth traits;

[0015] (4) Used for genetic improvement of grass carp varieties.

[0016] Furthermore, the method for breeding grass carp with excellent growth traits is as follows:

[0017] 1) Detecting SNP molecular markers related to grass carp growth traits in reserve broodstock;

[0018] 2) Select the individuals with genotype AA in step 1) as broodstock, and eliminate the broodstock with genotype TA or TT.

[0019] Furthermore, the method for screening fry with excellent growth traits is as follows:

[0020] 1) Detecting SNP molecular markers associated with grass carp growth traits in the fry to be screened;

[0021] 2) When the genotype of the SNP molecular marker detected in step 1) is AA, the fry to be screened has high growth genetic performance and is retained; when the genotype detected is TA or TT, the fry to be screened has low growth performance and is eliminated.

[0022] Furthermore, the breeding method of the grass carp strain with excellent growth traits is as follows:

[0023] 1) Detecting SNP molecular markers related to grass carp growth traits in reserve broodstock;

[0024] 2) selecting the individuals with genotype AA in step 1) as breeding broodstock, and breeding the selected female and male fish;

[0025] 3) Detecting the SNP molecular markers on the grass carp born in step 2), retaining individuals with genotype AA, eliminating individuals with genotype TA or TT, and then breeding to obtain grass carp strains with excellent growth traits.

[0026] Furthermore, the genetic improvement method of the grass carp variety is as follows:

[0027] 1) Detection of SNP molecular markers associated with grass carp growth traits in reserve broodstock;

[0028] 2) Retain individuals with genotype AA in step 1) and eliminate individuals with genotype TA or TT;

[0029] 3) using the individuals with genotype AA in step 2) as broodstock for breeding, continuing to select grass carp individuals with genotype AA from the offspring, and eliminating individuals with genotype TA or TT; thereby increasing the frequency of allele A in the offspring grass carp population generation by generation, thereby reducing the proportion of low growth performance of the offspring grass carp.

[0030] The present invention has the following beneficial effects:

[0031] (1) The present invention screened and identified a molecular marker SNP SLG11_23608308 that is related to the growth traits of grass carp. The body length of grass carp with different genotypes of this marker is extremely significantly different. The average body length of the genotype AA of the SNP SLG11_23608308 molecular marker is 10.4 cm, the average body width is 1.4 cm, the average body height is 2.3 cm, and the average weight is 22.4 g. The average body length of the genotype TT of the molecular marker is 9.6 cm, the average body width is 1.2 cm, the average body height is 2.0 cm, and the average weight is 16.9 g. The A allele significantly improves the growth performance of grass carp, thereby affecting its meat production performance. The AA genotype is the dominant genotype. Therefore, by applying this molecular marker, the growth performance of grass carp can be improved, thereby accelerating the genetic improvement progress and breeding process of grass carp.

[0032] (2) By detecting the SNP SLG11_23608308 molecular marker genotype to assist in the breeding of grass carp, the AA genotype of grass carp was selected, which can effectively improve the growth performance of grass carp and facilitate subsequent comprehensive breeding.

[0033] (3) The SNP SLG11_23608308 molecular marker was applied to the breeding of grass carp growth traits. The grass carp bred can significantly improve the growth performance, thereby improving the production performance of the broodstock.

[0034] (4) The SNP SLG11_23608308 molecular marker is applied to screen fry with excellent growth performance genetic traits. Grass carp with high growth performance traits can be screened and retained at the fry stage, thereby selecting and breeding grass carp with high growth performance traits at the grass carp fry stage, greatly improving breeding efficiency and saving feeding costs.

[0035] (5) Applying the SNP SLG11_23608308 molecular marker to cultivate grass carp strains with excellent growth traits can obtain grass carp strains with significantly improved growth performance, thereby increasing carcass weight and net meat weight, thereby improving the production performance of this strain of grass carp and accelerating the genetic improvement and breeding process of grass carp.

[0036] (6) Applying the SNP SLG11_23608308 molecular marker to the genetic improvement of grass carp can greatly shorten the years of genetic breeding, improve the production performance of grass carp, and accelerate the genetic improvement and breeding process of grass carp. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a Manhattan plot of the four traits of body length, body height, body width, and body weight in the grass carp GWAS analysis in an embodiment of the present invention.

[0038] Figure 2 2A is the Manhattan plot of the grass carp body length trait, 2B is the Manhattan plot of the grass carp body height trait, 2C is the Manhattan plot of the grass carp body width trait, and 2D is the Manhattan plot of the grass carp weight trait.

[0039] Figure 3 3A is the QQ graph of the grass carp body length trait, 3B is the QQ graph of the grass carp body height trait, 3C is the QQ graph of the grass carp body width trait, and 3D is the QQ graph of the grass carp weight trait.

[0040] Figure 4 4A is a statistical result diagram of the body length of grass carp carrying different genotypes of SNP SLG11_23608308 in the embodiment of the present invention, wherein 4A is the screening result of whole-genome high-throughput sequencing, and 4B is the verification result of first-generation sequencing.

[0041] Figure 5 5A is a graph showing the statistical results of grass carp with different genotypes of SNP SLG11_23608308 in an embodiment of the present invention, wherein 5A is the screening result of whole-genome high-throughput sequencing, and 5B is the verification result of first-generation sequencing.

[0042] Figure 66A is a statistical result diagram of the body width of grass carp carrying different genotypes of SNP SLG11_23608308 in the embodiment of the present invention, wherein 6A is the screening result of whole-genome high-throughput sequencing, and 6B is the verification result of first-generation sequencing.

[0043] Figure 7 7A is a graph showing the statistical results of the weight of grass carp carrying different genotypes of SNP SLG11_23608308 in an embodiment of the present invention, wherein 7A is the screening result of whole-genome high-throughput sequencing, and 7B is the verification result of first-generation sequencing. DETAILED DESCRIPTION

[0044] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0045] Example 1: Screening of SNP molecular markers related to grass carp growth traits

[0046] 1. Sample Collection

[0047] The grass carp used in this study were collected from the four major national family carp farms in Wujiang, Jiangsu Province, China. A total of 584 healthy grass carp individuals were selected, and the body length, height, width, and weight data of each fish were measured and recorded. At the same time, the tail fin tissue of the experimental fish was collected for genomic DNA extraction.

[0048] 2. Extraction of Genomic DNA

[0049] DNA samples were extracted using a magnetic bead method. DNA concentration was determined using a Qubit fluorescence quantifier, and DNA integrity was assessed using 1% agarose gel electrophoresis. High-throughput sequencing was performed on the extracted DNA.

[0050] 3. SNP quality control and population structure analysis

[0051] The raw high-throughput sequencing data were processed using quality control and filtering, and the PLINK software [ 49 ] set thresholds, including missing rate > 0.1, minor allele frequency (MAF) < 0.05, call rate < 0.1, and Hardy-Weinberg accuracy (HWE) < ​​1e-6, to remove low-quality SNPs from the original data, and finally obtained 62,736 high-quality SNP sites for subsequent analysis.

[0052] Before starting the genome-wide association analysis, to minimize the influence of population structure, principal component analysis (PCA) was performed on the quality-controlled data using PLINK software and visualized using the R package ggplot2. The phylogenetic tree was constructed using raxml-ng software and visualized using the website (https: / / itol.embl.de).

[0053] 4. Genome-wide association analysis of growth traits

[0054] A genome-wide association study (GWAS) was conducted using Tassel software and the general linear model (GLM) approach for body length, height, width, and weight in 584 individuals, taking into account population structure and inter-individual relatedness. The statistical model is as follows:

[0055] y=Xα+Zβ+e

[0056] Where y represents the phenotypic trait; Xα represents population structure as a fixed effect; Zβ represents the marker effect; and e represents the residual. A Bonferroni correction (Bonferroni 1936) was used to determine the genome-wide significance threshold. The formula p-value = 0.05 / N, where N represents the total number of markers used in the association analysis, was used. QQ plots and Manhattan plots were created using the CMplot package in R. Phenotypic variance explained (PVE) and F-scores were calculated using Tassel software.

[0057] 5. Results Analysis

[0058] After PLINK quality control cleaning, 62,736 SNPs were obtained from 584 grass carp individuals for further GWAS analysis. The genome-wide significant SNP marker threshold after Bonferroni correction was set at P = 0.05 / 62,736 = 7.97 × 10 -7 GWAS analysis results are as follows. Figure 1-3 Among them, the GWAS results of grass carp body length traits are as follows Figure 2 A, 3A: Three SNPs were detected on chromosomes 11 and 20 that were significantly associated with grass carp height traits at the genomic level. The GWAS results for grass carp height traits are as follows: Figure 2 B, 3B: A total of 1 SNP was detected on chromosome 11 that was significantly associated with grass carp body height traits at the genomic level. The GWAS results for grass carp body width traits are as follows Figure 2 As shown in C and 3C: A total of 8 SNPs were detected on chromosomes 2, 4, 6, 8, 11 and 19 that were significantly associated with the body width trait of grass carp at the genomic level. The GWAS results for the body weight trait of grass carp are as follows: Figure 2 D, 3D shows: A total of 5 SNPs were detected on chromosomes 11 and 12 that were significantly associated with grass carp weight traits at the genomic level. Figure 1 The Manhattan plot of the four traits shown is the same as Figure 2Comparing the individual Manhattan plots for each trait, we can see that SNP SLG11_23608308 is a significant SNP. This indicates that the single SNP on chromosome 11 with the strongest association with all four growth traits, SNP SLG11_23608308T>A, was identified.

[0059] The statistical results of body length, height, width and weight of grass carp with different genotypes of SNP SLG11_23608308 in this example are as follows Figure 4 As shown in A-7A, the average body length of the SNP SLG11_23608308 molecular marker genotype AA is 10.4 cm, the average body height is 2.3 cm, the average body width is 1.4 cm, and the average weight is 22.4 g; the average body length of the molecular marker genotype TT is 9.6 cm, the average body height is 2.0 cm, the average body width is 1.2 cm, and the average weight is 16.9 g. The A allele significantly improves the growth performance of grass carp and thus affects its meat production performance. The AA genotype is the dominant genotype.

[0060] Example 2: Verification of SNP molecular markers related to grass carp growth traits

[0061] The verification test used different grass carp populations, randomly selected 50 experimental fish, measured and recorded the four growth traits of each fish, namely body length, body height, body width, and weight, and simultaneously collected and preserved the tail fin sample of each fish for DNA extraction. The specific process of DNA extraction was the same as that in Example 1.

[0062] Subsequently, PCR amplification was performed using the extracted DNA as a template and SEQ ID NO: 1 and SEQ ID NO: 2 as primers to obtain a gene fragment containing SNP SLG11_23608308T>A (such as SEQ ID NO: 3).

[0063] The sequences of the upstream and downstream primer pairs of SNP SLG11_23608308 are: SNP SLG11_23608308-F: 5'-TCGGTGATGGCTCATGTT-3' (SEQ ID NO: 1); SNP SLG11_23608308-R: 5'-GCCAATCAGAATCCATCCT-3' (SEQ ID NO: 2).

[0064] The molecular marker sequence is shown in SEQ ID NO: 3, SNP SLG11_23608308T>A is located at position 201 of the gene fragment SEQ ID NO: 3, and the mutation type is T / A.

[0065] SEQ ID NO:3 (wherein, double underlined indicates mutation site, and single underlined is the position of upstream and downstream primers):

[0066]

[0067] The amplification system used in the PCR reaction was as follows: 100 ng / μl template DNA 1 μl, 10 pmol / μl forward primer and reverse primer 0.5 μl each, Taq Mix 10 μl, and the rest was double distilled water.

[0068] The PCR reaction program was as follows: the PCR reaction conditions were as follows: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 55°C or 60°C annealing for 30 s, 72°C extension for 30 s, a total of 30-35 cycles; 72°C incubation for 7 min.

[0069] The PCR amplification products were sequenced, and the genotype of each individual was obtained by first-generation sequencing, and the body length, body height, body width, and body weight data of individuals with different genotypes were counted, and the results are shown in Figure 3 B-6B, and the correlation of the SNP SLG11_23608308 molecular marker with the body length, body height, body width, and body weight four growth traits was successfully verified. Among them:

[0070] Figure 4 B shows that there is a significant difference in body length among individuals with different genotypes of grass carp SNP SLG11_23608308, and the body length of individuals carrying AA genotype (average 11.4 cm) is higher than that of individuals carrying genotype TA (average 9.7 cm) and TT (average 9.7 cm). This is consistent with the statistical results of body length of 584 grass carps with different genotypes in Example 1 (4A).

[0071] Figure 5 B shows that there is a significant difference in body height among individuals with different genotypes of grass carp SNP SLG11_23608308, and the body height of individuals carrying AA genotype (average 2.6 cm) is higher than that of individuals carrying genotype TA (average 2.1 cm) and TT (average 2.1 cm). This is consistent with the statistical results of body height of 584 grass carps with different genotypes in Example 1 (5A).

[0072] Figure 6 B shows that there is a significant difference in body width among individuals with different genotypes of grass carp SNP SLG11_23608308, and the body width of individuals carrying AA genotype (average 1.6 cm) is higher than that of individuals carrying genotype TA (average 1.3 cm) and TT (average 1.3 cm). This is consistent with the statistical results of body width of 584 grass carps with different genotypes in Example 1 (6A).

[0073] Figure 7 Figure B shows significant differences in body weight between individuals with different genotypes of grass carp SLG11_23608308. Individuals with the AA genotype weighed more (average 26.9 g) than those with the TA (average 17.9 g) and TT (average 17.3 g) genotypes. This is consistent with the statistical results (7A) of the 584 grass carp with different genotypes in Example 1.

[0074] Example 3: Application of SNP molecular markers related to grass carp growth traits

[0075] Based on the conclusion drawn in the above embodiment that the SLG11_23608308 molecular marker is significantly correlated with grass carp growth traits, in practical applications, this molecular marker can be used for breeding grass carp with excellent growth traits, screening fry with excellent growth traits, breeding grass carp strains with excellent growth traits, or genetic improvement of grass carp varieties. This embodiment provides methods for these applications, as follows:

[0076] 1. Application of the SLG11_23608308 molecular marker in the selection of grass carp for meat yield during the fattening period

[0077] The genotype of the SLG11_23608308 molecular marker is detected for grass carp; individuals with the AA genotype obtained by the detection are selected as broodstock, and grass carp individuals with TA or TT genotypes are eliminated. Grass carp with high growth performance traits can be selected as broodstock to achieve the selection of grass carp for growth traits.

[0078] 2. Application of the SLG11_23608308 molecular marker in screening for protein content in fattening grass carp fry

[0079] The genotype of the SLG11_23608308 molecular marker in the fry to be screened is detected; when the genotype of the detected molecular marker is AA, the fry to be screened has a genetic trait of high growth performance and is retained; when the genotype is TA or TT, the fry to be screened has a genetic trait of low protein content during the fattening period and is eliminated.

[0080] 3. Application of the SLG11_23608308 molecular marker in developing grass carp strains with muscle protein content during the fattening period

[0081] The genotype of the SLG11_23608308 molecular marker is detected for grass carp; individuals with the AA genotype obtained by the detection are selected as grass carp broodstock, and the male and female broodstock are fertilized; the SLG11_23608308 molecular marker is detected for the grass carp born from breeding, the AA genotype individuals are retained, and the TA or TT genotype individuals are eliminated, and breeding is carried out to cultivate a high-growth performance and high-production performance grass carp strain.

[0082] IV. Application of the LG11_23608308 molecular marker in genetic improvement of grass carp

[0083] Detect the genotype of the SLG11_23608308 molecular marker in grass carp; select grass carp individuals with the genotype AA and eliminate individuals with the genotype TA or TT; use the screened individuals as grass carp broodstock for breeding, and continue to select grass carp individuals with the SLG11_23608308 molecular marker genotype AA in the offspring, and eliminate individuals with the genotype TA or TT; thereby increasing the frequency of allele A in the offspring grass carp population generation by generation, thereby increasing the muscle protein content of the offspring grass carp during the fattening period.

[0084] In the above application, the method in Example 2 is still used to detect the genotype of the SLG11_23608308 molecular marker, including the following steps:

[0085] 1) Extract genomic DNA of the grass carp to be tested.

[0086] 2) Using genomic DNA as a template, PCR amplification was performed using the primers shown in SEQ ID NOs: 1-2;

[0087] The amplification system used in the PCR reaction was as follows in 20 μl: 100 ng / μl template DNA 1 μl, 0.5 μl each of 10 pmol / μl forward primer and reverse primer, 10 μl Taq Mix, and the balance was double-distilled water;

[0088] The PCR reaction program was as follows: PCR reaction conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C or 60°C for 30 s, extension at 72°C for 30 s, for a total of 30-35 cycles; and insulation at 72°C for 7 min.

[0089] 3) Sequencing the PCR amplification product obtained in step 2) to determine the genotype of the grass carp SLG11_23608308 molecular marker to be tested.

[0090] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A SNP molecular marker associated with grass carp growth traits, characterized in that: The polymorphic site is located at 23608308bp of chromosome 11 of grass carp, specifically the T / A polymorphic site at position 201 of the sequence shown in SEQ ID NO: 3; the growth trait is body length, height, width or weight of grass carp, and when the genotype of the polymorphic site is AA, it corresponds to high growth performance, and when the genotype of the polymorphic site is TT, it corresponds to low growth performance.

2. Any of the following uses of the SNP molecular marker according to claim 1: (1) Used for the breeding of grass carp with excellent growth traits; (2) Used to screen fry with excellent growth traits; (3) Used for the selection and breeding of grass carp strains with excellent growth traits; The growth traits described in (1)-(3) are body length, body height, body width or body weight of grass carp.

3. The use according to claim 2, characterized in that The excellent growth trait breeding method of grass carp is as follows: 1) detecting SNP molecular markers related to the growth traits of the grass carp in the reserve broodstock; 2) Select the individuals with genotype AA in step 1) as broodstock, and eliminate the broodstock with genotype TA or TT.

4. The use according to claim 2, characterized in that The method for screening fry with excellent growth traits is as follows: 1) detecting SNP molecular markers associated with the growth traits of the grass carp in the fry to be screened; 2) When the genotype of the SNP molecular marker detected in step 1) is AA, the fry to be screened has high growth genetic performance and is retained; when the genotype detected is TA or TT, the fry to be screened has low growth performance and is eliminated.

5. The use according to claim 2, characterized in that The breeding method of the excellent growth trait grass carp strain is as follows: 1) detecting SNP molecular markers related to the growth traits of the grass carp in the reserve broodstock; 2) selecting the individuals with genotype AA in step 1) as breeding broodstock, and breeding the selected female and male fish; 3) Detecting the SNP molecular markers on the grass carp born in step 2), retaining individuals with genotype AA, eliminating individuals with genotype TA or TT, and then breeding to obtain grass carp strains with excellent growth traits.

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