SNP site related to growth traits of macrobrachium rosenbergii and application of SNP site

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) site related to growth traits of macrobrachium rosenbergii and application of the SNP site, the SNP site comprises bases at the 5263 site, the 5302 site and the 5743 site from the 3'end of a gene sequence (Gene ID: 136838326) of a macrobrachium rosenbergii IGFBP gene NW027100732.1, the bases are respectively named as IGFBP-5263-G / A, IGFBP-5302-G / T and IGFBP-5743-G / C, the base at the IGFBP-5263-G / A site is G or A, the base at the IGFBP-5302-G / T site is G or T, and the base at the The method can be used for molecular marker-assisted selective breeding, improves the selection accuracy, shortens the breeding period, reduces the breeding cost, and can be widely applied to the breeding process of aquatic animals.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular markers for aquatic animals, and particularly relates to SNP loci related to the growth traits of Macrobrachium rosenbergii and their applications. Background Art

[0002] With years of inbred reproduction, the population diversity of farmed Macrobrachium rosenbergii in China has decreased, the variety has degenerated, and the growth rate has slowed down, which has hindered the sustainable development of this species and seriously affected the yield and economic benefits of Macrobrachium rosenbergii. Breeding new varieties of fast-growing Macrobrachium rosenbergii will greatly promote the development of the Macrobrachium rosenbergii industry. In the early stage, the breeding of improved varieties of aquatic animals mainly relied on the selection of individuals with specific phenotypic traits or better production performance. However, the progress of traditional breeding methods is relatively slow. The discovery of genes or molecular markers associated with economic traits has provided a new approach for traditional aquatic breeding technologies. Through molecular markers, the target traits can be accurately and quickly selected, accelerating the breeding process and shortening the breeding cycle. Currently, there are few studies on the growth-related genes and molecular markers of Macrobrachium rosenbergii. Therefore, it is crucial to use aquatic animal omics technology to screen growth trait-related genes and develop single nucleotide polymorphism (SNP) aquatic animal markers through association analysis with apparent traits for the cultivation of new varieties of fast-growing Macrobrachium rosenbergii. Summary of the Invention

[0003] Technical problems to be solved: Aiming at the above technical problems, the present invention provides SNP loci related to the growth traits of Macrobrachium rosenbergii and their applications, which make full use of the existing genomic public database resources, have a short detection time, are easy to operate, have more accurate genotyping results, and are more economical and efficient; provide important tools for molecular marker-assisted breeding of Macrobrachium rosenbergii, etc., and play an important role in the research of Macrobrachium rosenbergii breeding.

[0004] Technical solution: In the first aspect, the present invention provides SNP loci related to the growth traits of Macrobrachium rosenbergii. The SNP loci include the 5263rd, 5302nd, and 5743rd bases from the 3' end of the NW_027100732.1 gene sequence (GeneID: 136838326) of the IGFBP gene of Macrobrachium rosenbergii, which are named IGFBP-5263-G / A, IGFBP-5302-G / T, and IGFBP-5743-G / C respectively. The base at the IGFBP-5263-G / A locus is G or A, the base at the IGFBP-5302-G / T locus is G or T, and the base at the IGFBP-5743-G / C locus is G or C.

[0005] Preferably, the nucleotide sequences of the primer sets used for amplifying the target fragments are shown in SEQ ID NO.1 and SEQ ID NO.2. The target fragments include the bases at the IGFBP-5263-G / A, IGFBP-5302-G / T, and IGFBP-5743-G / C sites. Specifically:

[0006] SEQ ID NO.1 (IGFBP-X2-F1): 5’-GTTCCGGGGGTGCTTAACTC-3’,

[0007] SEQ ID NO.2 (IGFBP-X2-R1): 5’-AACGGCCTCCGTTTTGGTAA-3’;

[0008] The PCR reaction system is: 10 μL of 2×PCR Mix, 0.5 μL of each upstream and downstream primer, 1 μL of DNA, and 8 μL of ddH2O;

[0009] The PCR reaction conditions are: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 30 sec, annealing at 58 °C for 30 sec, extension at 72 °C for 1.5 min, for 34 cycles; extension at 72 °C for 5 min; detecting the size and quality of the PCR products by 1% agarose gel electrophoresis; sending the PCR products to a sequencing company for sequencing.

[0010] In a second aspect, the present invention provides a kit for detecting SNP sites related to the growth traits of Macrobrachium rosenbergii, including the primer sets described in the first aspect.

[0011] In a third aspect, the present invention provides the application of the SNP sites related to the growth traits of Macrobrachium rosenbergii described in the first aspect in the breeding of excellent growth trait varieties of Macrobrachium rosenbergii.

[0012] Preferably, during the breeding process of Macrobrachium rosenbergii, after genotyping the IGFBP-5263-G / A site by SNP genotyping method, if the base at the IGFBP-5263-G / A site is A, individuals with the genotype AA are fast-growing individuals; after genotyping the IGFBP-5302-G / T site by SNP genotyping method, if the base at the IGFBP-5302-G / T site is T, individuals with the genotype TT are fast-growing individuals; after genotyping the IGFBP-5743-G / C site by SNP genotyping method, if the base at the IGFBP-5743-G / C site is C, individuals with the genotype CC are fast-growing individuals.

[0013] Preferably, the specific operation method of the application is as follows:

[0014] S1: Extract the genomic DNA of the Macrobrachium rosenbergii sample to be detected;

[0015] S2: Using the genomic DNA obtained in step S1 as a template, perform PCR amplification using a primer set to obtain a PCR product.

[0016] S3: Sequence the PCR amplification product obtained in step S2, determine the base types of the test sample at multiple SNP loci according to the sequencing results, and determine whether the test individual has the potential for rapid growth, that is, whether the individual at the IGFBP-5263-G / A locus is of the AA genotype, whether the individual at the IGFBP-5302-G / T locus is of the TT genotype, or whether the individual at the IGFBP-5743-G / C locus is of the CC genotype.

[0017] Beneficial effects: The SNP loci related to the growth traits of Macrobrachium rosenbergii in the present invention, the SNP loci include the 5263rd, 5302nd, and 5743rd bases from the 3' end of the NW_027100732.1 gene sequence (Gene ID: 136838326) of the IGFBP gene in Macrobrachium rosenbergii, which are respectively named IGFBP-5263-G / A, IGFBP-5302-G / T, and IGFBP-5743-G / C. Among them, the base at the IGFBP-5263-G / A locus is G or A, the base at the IGFBP-5302-G / T locus is G or T, and the base at the IGFBP-5743-G / C locus is G or C. When the base at the IGFBP-5263-G / A locus is A, the individual with the AA genotype is a fast-growing individual; when the base at the IGFBP-5302-G / T locus is T, the individual with the TT genotype is a fast-growing individual; when the base at the IGFBP-5743-G / C locus is C, the individual with the CC genotype is a fast-growing individual. It can be used for molecular marker-assisted selection breeding, improving the accuracy of selection, shortening the breeding period, reducing the breeding cost, and can be widely applied to the breeding process of aquatic animals. Description of the Drawings

[0018] Figure 1 It is a flow chart of the application of the SNP loci related to the growth traits of Macrobrachium rosenbergii in the present invention in the breeding of excellent growth trait varieties of Macrobrachium rosenbergii. Detailed Embodiments

[0019] The present invention will be described in detail below with reference to the drawings and specific embodiments:

[0020] Example 1

[0021] 1. Primer design: Design a primer set for specifically amplifying the 5263rd, 5302nd, and 5743rd bases of the intron from the 3' end of the NW_027100732.1 gene sequence (Gene ID: 136838326) of the IGFBP gene in Macrobrachium rosenbergii. The specific primer sequences are as follows:

[0022] SEQ ID NO.1 (IGFBP-X2-F1): 5’-GTTCCGGGGGTGCTTAACTC-3’,

[0023] SEQ ID NO.2 (IGFBP-X2-R1): 5’-AACGGCCTCCGTTTTGGTAA-3’;

[0024] Synthesized by Shanghai Sangon Biotech Co., Ltd.

[0025] 2. Extraction of Macrobrachium rosenbergii Genomic DNA

[0026] Extract the genomic DNA of Macrobrachium rosenbergii using the Marine Animal Tissue Genomic DNA Extraction Kit (Tiangen, Beijing).

[0027] 3. Amplification and Detection: Use the designed primer pair to perform PCR amplification on the genomic DNA of Macrobrachium rosenbergii, and detect the genotypes of SNP sites of the amplified gene fragments.

[0028] The PCR reaction system is: 10 μL of 2×PCR Mix, 0.5 μL of each upstream and downstream primer, 1 μL of DNA, and 8 μL of ddH2O;

[0029] The PCR reaction conditions are: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 sec, annealing at 58°C for 30 sec, extension at 72°C for 1.5 min, for 34 cycles; extension at 72°C for 5 min; use 1% agarose gel electrophoresis to detect the size and quality of the PCR products; send the PCR products to a sequencing company for sequencing. After sequence alignment and screening of sites with polymorphism greater than 3%, 11 SNP candidate sites are obtained, and the gene frequencies and genetic parameters are shown in Table 1 below:

[0030] Table 1 Gene Frequencies and Genetic Parameters of 11 SNP Sites in the IGFBP Gene of Macrobrachium rosenbergii

[0031]

[0032] These 11 loci were associated with the growth traits of 100 individuals of Macrobrachium rosenbergii to identify 3 SNP loci in this gene fragment of the Macrobrachium rosenbergii IGFBP gene, located at the 3' end of the gene sequence, named IGFBP-5263-G / A, IGFBP-5302-G / T, and IGFBP-5743-G / C. The IGFBP-5263-G / A locus was significantly associated with the body weight of Macrobrachium rosenbergii. The IGFBP-5302-G / T locus was significantly associated with the body weight, carapace height, and height of the first abdominal segment of Macrobrachium rosenbergii. The IGFBP-5743-G / C locus was significantly associated with the body weight, carapace length, carapace height, height of the first abdominal segment, and abdominal length of Macrobrachium rosenbergii. When the base at the IGFBP-5263-G / A locus is A, individuals with the AA genotype are fast-growing individuals. When the base at the IGFBP-5302-G / T locus is T, individuals with the TT genotype are fast-growing individuals. When the base at the IGFBP-5743-G / C locus is C, individuals with the CC genotype are fast-growing individuals.

[0033] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. The SNP loci associated with the growth traits of Macrobrachium rosenbergii are characterized by: The SNP sites include bases 5263, 5302 and 5743 from the 3' end of the gene sequence of the IGFBP gene NW_027100732.1 of Macrobrachium rosenbergii, which are named IGFBP-5263-G / A, IGFBP-5302-G / T and IGFBP-5743-G / C respectively.

2. The SNP locus associated with the growth traits of Macrobrachium rosenbergii according to claim 1, characterized in that: The nucleotide sequences of the primer set used to amplify the target fragment are shown in SEQ ID NO.1 and SEQ ID NO.2, and the target fragment includes the bases of IGFBP-5263-G / A, IGFBP-5302-G / T and IGFBP-5743-G / C sites.

3. A kit for detecting SNP sites associated with growth traits of Macrobrachium rosenbergii, characterized in that: Comprising the primer set described in claim 2.

4. Use of the SNP loci associated with the growth traits of Macrobrachium rosenbergii according to claim 1 in the breeding of varieties with excellent growth traits of Macrobrachium rosenbergii.

5. The use according to claim 4, characterized in that: In the breeding process of Macrobrachium rosenbergii, after typing the IGFBP-5263-G / A site marker by the SNP typing method, the base of the IGFBP-5263-G / A site is A, and the individual with the genotype of AA is a fast-growing individual; after typing the IGFBP-5302-G / T site marker by the SNP typing method, the base of the IGFBP-5302-G / T site is T, and the individual with the genotype of TT is a fast-growing individual; after typing the IGFBP-5743-G / C site marker by the SNP typing method, the base of the IGFBP-5743-G / C site is C, and the individual with the genotype of CC is a fast-growing individual.

6. The use according to claim 4, characterized in that: The specific operation method is as follows: S1: Extracting genomic DNA of the sample to be tested from Macrobrachium rosenbergii; S2: using the genomic DNA obtained in step S1 as a template, performing PCR amplification using a primer set to obtain a PCR product; S3: Sequence the PCR amplification products obtained in step S2, and determine the base types of the test samples at multiple SNP sites based on the sequencing results to determine whether the individual to be tested has the potential for rapid growth, that is, whether the IGFBP-5263-G / A site is the AA genotype, whether the IGFBP-5302-G / T site is the TT genotype, or whether the IGFBP-5743-G / C site is the CC genotype.

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