SNP molecular marker related to grass carp weight trait and application thereof

Abstract

The application belongs to the technical field of aquatic animal molecular markers and aquatic genetic breeding, and specifically discloses SNP molecular markers significantly related to the body weight of grass carp and application thereof. The SNP molecular markers are as follows: 1) SLG11_23629389, the polymorphic base is T / C, 2) SLG11_27776299, the polymorphic base is A / G, 3) SLG11_27776356, the polymorphic base is G / A, and 4) SLG12_35362696, the polymorphic base is T / C. The application also discloses application of the SNP molecular markers, amplification primers or kits in breeding of grass carp with excellent growth traits. The SNP molecular markers can be used for early breeding of grass carp with excellent growth traits, greatly reducing the breeding workload, significantly shortening the breeding time, accelerating the breeding process, improving the breeding efficiency and reducing the breeding cost. The application has important guiding significance for improving the production performance of grass carp, reducing the breeding cost of grass carp and increasing the breeding income, and is suitable for popularization and application.

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 SNP molecular markers related to grass carp weight traits and applications 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, and directly reflect genetic polymorphisms at the DNA level. Among them, single nucleotide polymorphisms (SNPs), as the third-generation molecular markers, are the most numerous and widely distributed in the genome, and are therefore widely used in molecular breeding of animals and plants. SNPs refer to DNA sequence polymorphisms caused by variations in a single nucleotide at the genomic level, which can be the replacement, insertion, or deletion of a single base. Molecular marker-assisted breeding is a breeding technology that utilizes the characteristics of these molecular markers that are closely linked to the genes that control the target traits, and selects the 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 restricts the improvement of grass carp growth traits. Summary of the Invention

[0004] The purpose of the present invention is to provide a SNP molecular marker related to the weight trait of grass carp and its application to solve the above problems.

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

[0006] In a first aspect, the present invention provides SNP molecular markers associated with grass carp weight traits, wherein the SNP molecular markers are one or more of the following 1), 2), 3), and 4):

[0007] 1) Located at position 23629389 of chromosome 11 of grass carp, the polymorphic base is T / C, referred to as molecular marker SNP SLG11_23629389 in this article;

[0008] 2) located at 27776299 of grass carp chromosome 11, the polymorphic site base is A / G, referred to as molecular marker SNP SLG11_27776299 in this paper;

[0009] 3) located at 27776356 of grass carp chromosome 11, the polymorphic site base is G / A, referred to as molecular marker SNP SLG11_27776356 in this paper;

[0010] 4) located at 35362696 of grass carp chromosome 12, the polymorphic site base is T / C, referred to as molecular marker SNP SLG12_35362696 in this paper.

[0011] In a second aspect, the present application provides amplification primers for detecting the SNP molecular markers, including primer pairs capable of detecting one or several of the above-mentioned 1)-4) four SNP molecular markers.

[0012] Preferably, the amplification primers include one or more of the following pairs:

[0013] A pair of primers as shown in SEQ ID NO: 1 and SEQ ID NO: 2, capable of amplifying molecular marker SNP SLG11_23629389;

[0014] A pair of primers as shown in SEQ ID NO: 3 and SEQ ID NO: 4, capable of amplifying molecular marker SNP SLG11_27776299;

[0015] A pair of primers as shown in SEQ ID NO: 5 and SEQ ID NO: 6, capable of amplifying molecular marker SNP SLG11_27776356;

[0016] A pair of primers as shown in SEQ ID NO: 7 and SEQ ID NO: 8, capable of amplifying molecular marker SNP SLG12_35362696.

[0017] In a third aspect, the present application provides a method for selecting grass carp with excellent growth traits, comprising the following steps:

[0018] Step one, extracting fin DNA of the grass carp individual to be detected;

[0019] Step two, using the genomic DNA in step one as a template, PCR amplification is performed using the amplification primers;

[0020] Step three, sequencing analysis is performed on the amplification product obtained in step two to determine the genotype of the SNP molecular marker of the grass carp individual to be detected, and genotype analysis is performed to determine whether the grass carp has the potential of excellent growth traits.

[0021] Preferably, in step three, when PCR amplification is performed with a pair of primers as shown in SEQ ID NO: 1 and ID NO: 2, the polymorphic site at position 23629389 of chromosome 11 of grass carp is detected, and grass carp with genotype CC (the base sequence at the position is shown as SEQ ID NO: 9) has the potential of excellent growth traits.

[0022] Preferably, in step three, when PCR amplification is performed with a pair of primers as shown in SEQ ID NO: 3 and ID NO: 4, the polymorphic site at position 27776299 of chromosome 11 is detected, and grass carp with genotype AG (the base sequence at the position is shown as SEQ ID NO: 10) has the potential of excellent growth traits.

[0023] Preferably, in step three, when PCR amplification is performed with a pair of primers as shown in SEQ ID NO: 5 and ID NO: 6, the polymorphic site at position 27776356 of chromosome 11 of grass carp is detected, and grass carp with genotype GA (the base sequence at the position is shown as SEQ ID NO: 11) has the potential of excellent growth traits.

[0024] Preferably, in step three, when PCR amplification is performed with a pair of primers as shown in SEQ ID NO: 7 and ID NO: 8, the polymorphic site at position 35362696 of chromosome 12 of grass carp is detected, and grass carp with genotype CC (the base sequence at the position is shown as SEQ ID NO: 12) has the potential of excellent growth traits.

[0025] When the molecular marker is SNP SLG11_23629389, grass carp with genotype CC has faster growth rate, and grass carp with genotypes TC and TT has slower growth rate; when the molecular marker is SNP SLG11_27776299, grass carp with genotype AG has better growth traits than grass carp with genotype AA; when the molecular marker is SNP SLG11_27776356, grass carp with genotype GA has better growth traits than grass carp with genotype GG; when the molecular marker is SNP SLG12_35362696, grass carp with genotype CC has better growth traits than grass carp with genotypes TC and TT.

[0026] In a fourth aspect, the present application provides a kit containing the amplification primers, wherein the amplification primers include one or more of the following pairs: SEQ ID NO: 1-2, SEQ ID NO: 3-4, SEQ ID NO: 5-6, SEQ ID NO: 7-8.

[0027] In a fifth aspect, the present application provides application of the SNP molecular marker, the amplification primer or the kit in selecting grass carp with excellent growth traits.

[0028] The present application has the following advantages:

[0029] (1) The present application analyzes the gene locus controlling the growth traits of grass carp through whole genome association analysis, and screens four candidate SNP loci significantly associated with growth traits (body weight), which are SNP SLG11_23629389, SNP SLG11_27776299, SNP SLG11_27776356 and SNP SLG12_35362696. It is found that in SNP SLG11_23629389, the CC genotype is the preferred genotype; in SNP SLG11_27776299, the AG genotype is the preferred genotype; in SNP SLG11_27776356, the GA genotype is the preferred genotype; and in SNP SLG12_35362696, the CC genotype is the preferred genotype. The individual carrying the above-mentioned preferred genotypes has more outstanding growth traits. The four SNPs can be used as molecular markers for predicting the growth rate of grass carp, and the accuracy is high. The SNP locus of the present application has application prospect in grass carp molecular marker assisted breeding.

[0030] (2) The SNP marker of the present application can be determined by primers, which has the advantages of simple and fast operation, accurate and reliable results, and low cost.

[0031] (3) The SNP molecular marker of the present application is not affected by factors such as individual age and gender. The SNP molecular marker can be used for early selection of grass carp with excellent growth traits, greatly reducing the breeding workload, significantly shortening the breeding time, accelerating the breeding process, improving the breeding efficiency, and reducing the breeding cost. It has important guiding significance for improving the production performance of grass carp, reducing the breeding cost of grass carp and increasing the breeding income, and is suitable for popularization and application. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 The Manhattan plot and QQ plot in the GWAS analysis of the body weight traits of grass carp provided by the embodiments of the present application, wherein 1A is the Manhattan plot and 1B is the QQ plot.

[0033] Figure 2 The figure of the body weight statistics of grass carp carrying different genotypes of SNP SLG11_23629389 in the embodiments of the present application, wherein 2A is the screening result of whole genome high-throughput sequencing, and 2B is the verification result of first generation sequencing.

[0034] Figure 3Figure 3A is a graph of the body weight statistics of grass carp carrying different genotypes of SNP SLG11_27776299 in the embodiments of the present application, wherein 3A is the whole genome high-throughput sequencing screening result, and 3B is the first-generation sequencing verification result.

[0035] Figure 4 Figure 4A is a graph of the body weight statistics of grass carp carrying different genotypes of SNP SLG11_27776356 in the embodiments of the present application, wherein 4A is the whole genome high-throughput sequencing screening result, and 4B is the first-generation sequencing verification result.

[0036] Figure 5 Figure 5A is a graph of the body weight statistics of grass carp carrying different genotypes of SNP SLG12_35362696 in the embodiments of the present application, wherein 5A is the whole genome high-throughput sequencing screening result, and 5B is the first-generation sequencing verification result. DETAILED DESCRIPTION

[0037] The present application focuses on the genetic improvement of grass carp growth traits, applies SNP molecular marker information to the molecular marker breeding of grass carp growth traits, screens molecular markers related to the growth traits of grass carp, and has the advantages of simple and fast detection operation, accurate and reliable results, low cost, and suitability for popularization and use.

[0038] In order to enable those skilled in the art to better understand the technical solutions of the present application, the following examples are provided for description.

[0039] Example 1: Screening of SNP molecular markers related to the body weight traits of grass carp individuals

[0040] 1. Sample collection

[0041] The grass carps used in the present study were collected from the National Four Major Carp Breeding Farms in Wujiang, Jiangsu Province, China. 584 healthy individuals of grass carps were selected, and the body weight data of each fish was measured and recorded. The tail fin tissues of the experimental fish were collected for extraction of genomic DNA.

[0042] 2. Extraction of genomic DNA

[0043] The magnetic bead method was used for DNA extraction. The concentration of the DNA sample was detected by Qubit fluorescence quantification instrument; the integrity of the DNA sample was detected by 1% agarose gel electrophoresis. The extracted DNA was subjected to high-throughput sequencing.

[0044] 3. SNP quality control and population structure analysis

[0045] The original high-throughput sequencing data was processed using quality control and filtering, and the PLINK software was used to analyze the population structure. 49Thresholds were set, including missing rate > 0.1, minor allele frequency (MAF) < 0.05, call rate < 0.1 and Hardy-Weinberg exactness (HWE) < 1e-6, to remove low-quality SNPs from the original data, and finally 62736 high-quality SNP sites were obtained for subsequent analysis.

[0046] Before starting the whole genome association analysis, to avoid the influence of population structure, the principal component analysis (PCA) was performed on the quality-controlled data using PLINK software, and visualized using R package ggplot2. The phylogenetic tree was constructed using raxml-ng software and visualized using the website (https: / / itol.embl.de).

[0047] 4. Whole genome association analysis of body weight traits

[0048] The whole genome association analysis was performed on the body weight traits of 584 individuals using Tassel software based on the general linear model (GLM) method, while fully considering the population structure and the kinship between individuals. The statistical model was as follows:

[0049] y = Xa + Zb + e

[0050] In the formula, y represents the phenotypic trait; Xa is the population structure as a fixed effect; Zb is the marker effect, and e is the residual error.

[0051] Bonferroni correction (Bonferroni 1936) was used to determine the whole genome significant threshold calculation formula p value = 0.05 / N, where N represents the total markers used for association analysis. The QQ plot and Manhattan plot were drawn using R language CMplot package, and the phenotypic variance explanation (PVE) and F value were calculated using Tassel software.

[0052] 5. Results analysis

[0053] After quality control and cleaning by PLINK, 62736 SNP sites of 584 individuals of grass carp were obtained for further GWAS analysis. The whole genome significant SNP marker threshold after Bonferroni correction was set to P = 0.05 / 62736 = 7.97 x 10 -7 The GWAS results of grass carp body weight traits are shown in Figure 1As shown: 8 SNPs associated with grass carp weight traits at the genome level were detected on chromosomes 11, 12, 16 and 24. Among them, 3 SNPs with the strongest association with grass carp weight traits were detected on chromosome 11, 1 on chromosome 12, respectively SNP SLG11_23629389T>C, SNP SLG11_27776299A>G, SNP SLG11_27776356G>A and SNP SLG12_35362696T>C (wherein SNP SLG11_27776299 and SNP SLG11_27776356 are too close in distance and marked overlapping in Figure 1 Table A-5A.

[0054] The statistical results of the weight data of 584 grass carp individuals with different genotypes at four SNP loci are shown in Table A-5A. Figure 2 As shown in Table A-5A, the average weight of grass carp with different genotypes at SNP SLG11_23629389T>C locus was significantly different. The average weight of grass carp with CC genotype (20.8g) was significantly higher than that of individuals with TC genotype (17.6g) and TT genotype (17.3g) (P<0.05). Figure 2 As shown in Table A-5A, the average weight of grass carp with different genotypes at SNP SLG11_27776299A>G locus was significantly different. The average weight of grass carp with AG genotype (21.7g) was significantly higher than that of individuals with AA genotype (17.8g) (P<0.05). Figure 3 As shown in Table A-5A, the average weight of grass carp with different genotypes at SNP SLG11_27776356G>A locus was significantly different. The average weight of grass carp with GA genotype (21.7g) was significantly higher than that of individuals with GG genotype (17.8g) (P<0.05). Figure 4 As shown in Table A-5A, the average weight of grass carp with different genotypes at SNP SLG11_27776356G>A locus was significantly different. The average weight of grass carp with GA genotype (21.7g) was significantly higher than that of individuals with GG genotype (17.8g) (P<0.05). Figure 5 As shown in Table A-5A, the average weight of grass carp with different genotypes at SNP SLG12_35362696T>C locus was significantly different. The average weight of grass carp with CC genotype (33.1g) was significantly higher than that of individuals with TC genotype (21.8g) and TT genotype (18g); there was also a significant difference in average weight between individuals with TC genotype and TT genotype (P<0.05).

[0055] Example 2: Verification of SNP molecular marker related to individual body weight trait of grass carp

[0056] Verification test used different grass carp populations, 50 experimental fish were randomly selected, and the body weight trait indicators of each fish were measured and recorded, and the tail fin samples of each fish were collected and preserved for DNA extraction. The specific process of DNA extraction was the same as in Example 1. Subsequently, the above extracted DNA was used as a template for PCR amplification with SEQ ID NO: 1 and SEQ ID NO: 2 as primers. The amplification system used in the PCR reaction was as follows: 20 μl, 100 ng / μl template DNA 1 μl, 10 pmol / μl forward primer and reverse primer each 0.5 μl, Taq Mix 10 μl, and the rest was double distilled water.

[0057] 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 for 7 min. The gene fragment containing SNP SLG11_23629389T>C (SEQ ID NO: 9) was obtained, as shown in SEQ ID NO: 9, the mutation site was at position 201, and the mutation type was T / C.

[0058] The sequences of the upstream and downstream primer pairs of the SNP SLG11_23629389 are as follows:

[0059] SNP SLG11_23629389-F: 5'-GTAGGAGTGTTGAACTGTCT-3' (SEQ ID NO: 1);

[0060] SNP SLG11_23629389-R: 5'-GCATGTACGCACAATCTT-3' (SEQ ID NO: 2).

[0061] The molecular marker sequence is shown in SEQ ID NO: 9, and SNP SLG11_23629389T>C is located at position 201 of the gene fragment SEQ ID NO: 9, and the mutation type is T / C.

[0062] SEQ ID NO: 9 (wherein the double underlined indicates the mutation site, and the single underlined is the position of the upstream and downstream primers):

[0063]

[0064] The genotypes of each individual were obtained by one-generation sequencing of the PCR amplification product, and the body weight data of individuals with different genotypes were counted. Figure 2B shows the results of the test verification of SNP SLG11_23629389. From Figure 2 B shows that the body weight of grass carp SNP SLG11_23629389 individuals with different genotypes is significantly different; among them, the body weight of individuals carrying CC genotype (22.1 g) is higher than that of individuals carrying genotype TC (15.6 g) and TT (15.8 g), which is consistent with the results of Example 1 Figure 2 A results are consistent.

[0065] The verification method of SNP SLG11_27776299A>G is the same as above, and the upstream and downstream primers used are:

[0066] SNP SLG11_27776299-F:5'-ACAGCAACAGAGTCATCAA-3'(SEQ ID NO:3);

[0067] SNP SLG11_27776299-R:5'-AGGATTGACAACGGCATT-3'(SEQ ID NO:4).

[0068] The gene fragment containing SNP SLG11_27776299 is shown in SEQ ID NO:10, the mutation site is at position 201, and the mutation type is A / G.

[0069] SEQ ID NO:10 (wherein the double underlined indicates the mutation site, and the single underlined is the position of the upstream and downstream primers):

[0070]

[0071]

[0072] Figure 3 B shows the results of the test verification of SNP SLG11_27776299. From Figure 3 B shows that the body weight of grass carp SNP SLG11_27776299 individuals with different genotypes is significantly different; among them, the body weight of individuals carrying AG genotype (21.9 g) is higher than that of individuals carrying genotype AA (17 g), which is consistent with the results of Example 1 Figure 3 A results are consistent.

[0073] The verification method of SNP SLG11_27776356G>A is the same as above, and the upstream and downstream primers used are:

[0074] SNP SLG11_27776356G>A-F:5'-ATGACTCCTCTGTTGACTG-3'(SEQ ID NO:5);

[0075] SNP SLG11_27776356 G>A - R: 5'- AGGATTGACAACGGCATT-3' (SEQ ID NO: 6).

[0076] The gene fragment containing SNP SLG11_27776356 is shown as SEQ ID NO: 11, the mutation site is at position 201, and the mutation type is G / A.

[0077] SEQ ID NO: 11 (wherein, double underlined indicates the mutation site, and single underlined is the position of the upstream and downstream primers):

[0078]

[0079] Figure 4 B shows the results of the test verification of SNP SLG11_27776356. From the results of B, it can be seen that the body weight of grass carp SNP SLG11_27776356 different genotypes is significantly different; among them, the body weight of individuals carrying GA genotype (21.9g) is higher than that of individuals carrying genotype GG (17g), which is consistent with the results of Example 1 Figure 4 Figure 4 A.

[0080] The verification method of SNP SLG12_35362696 T>C is the same as above, and the upstream and downstream primers used are as follows:

[0081] SNP SLG12_35362696 T>C - F: 5'- GAGCAACTGCCATTGAGA-3' (SEQ ID NO: 7);

[0082] SNP SLG12_35362696 T>C - R: 5'- TCATAGTGCTGCTTACTCAA-3' (SEQ ID NO: 8).

[0083] The gene fragment containing SNP SLG12_35362696 is shown as SEQ ID NO: 12, the mutation site is at position 201, and the mutation type is T / C.

[0084] SEQ ID NO: 12 (wherein, double underlined indicates the mutation site, and single underlined is the position of the upstream and downstream primers):

[0085]

[0086] Figure 5 B shows the results of the test verification of SNP SLG12_35362696. From the results of B, it can be seen that the body weight of grass carp SNP SLG12_35362696 different genotypes is significantly different; among them, the body weight of individuals carrying GA genotype (21.9g) is higher than that of individuals carrying genotype GG (17g), which is consistent with the results of Example 1 Figure 5 ​It was found that the body weight of grass carp with different genotypes of SNP SLG12_35362696 was significantly different, and the body weight of grass carp with CC genotype (33.1 g) was higher than that of grass carp with TC (21.6 g) and TT (17.5 g) genotypes. Figure 5 A results are consistent.

[0087] In conclusion, four SNP markers (SNP SLG11_23629389, SNP SLG11_27776299, SNP SLG11_27776356 and SNP SLG12_35362696) are significantly associated with the growth traits of grass carp, and the genotypes of the four SNP markers can be determined by a pair of primers, which is simple and reliable. The four SNP markers have application prospects in the molecular marker assisted breeding and genome selection breeding of grass carp.

[0088] Although the present application has been described in detail with general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application.

Claims

1. Application of a primer pair for detecting a SNP site in breeding grass carp with excellent growth traits, characterized in that: The SNP site is a polymorphic site with T / C at position 201 of the sequence shown in SEQ ID NO:

12. Grass carp with CC genotype at the polymorphic site has the potential for excellent growth traits, and the growth trait refers to grass carp weight.

2. The use according to claim 1, characterized in that The primer pair consists of an upstream primer whose sequence is shown in SEQ ID NO: 7 and a downstream primer whose sequence is shown in SEQ ID NO:

8.

3. A method for breeding grass carp with excellent growth traits, characterized in that: The following steps are involved: Step 1: extracting DNA from the fin rays of individual grass carp to be tested; Step 2: Using the genomic DNA in step 1 as a template, perform PCR amplification using the primer pair described in claim 2; Step 3: Perform sequencing analysis on the amplified product obtained in step 2 to determine the genotype of the grass carp individual to be tested at the SNP site, where the SNP site is a polymorphic site with a T / C at position 201 of the sequence shown in SEQ ID NO:

12. Genotyping analysis is used to determine whether the grass carp has the potential for excellent growth traits. Grass carp with a CC genotype at the polymorphic site has the potential for excellent growth traits, where the growth trait refers to grass carp weight.

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