SNP (Single Nucleotide Polymorphism) marker related to growth rate of penaeus vannamei and application thereof

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) marker related to the growth rate of penaeus vannamei boone and application of the SNP marker, the nucleotide sequence of the SNP marker is shown as SEQ ID NO: 1, the polymorphism of the SNP marker is reflected at the 501st site of the SEQ ID NO: 1, and the polymorphic site is G or T. The invention further discloses an application of the SNP marker related to the growth rate of the penaeus vannamei boone. According to the SNP marker related to the growth speed of the penaeus vannamei boone and the application of the SNP marker, the growth speed can be selected according to actual breeding requirements to perform early selection on breeding materials of the penaeus vannamei boone, the breeding efficiency and accuracy can be further effectively improved, the genetic level of breeding groups of the penaeus vannamei boone can be improved, and the breeding cost can be reduced. Therefore, the excellent variety of the penaeus vannamei boone can be accurately and efficiently bred.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a SNP marker related to the growth rate of Penaeus vannamei and an application thereof. Background Art

[0002] Whiteleg shrimp (Litopenaeus vannamei) is an important aquaculture animal. In the past thirty years, the artificial breeding technology of whiteleg shrimp has been very mature, and the scale of whiteleg shrimp farming has gradually expanded. The rapid development of the whiteleg shrimp industry is of great significance for increasing fishermen's income, meeting national aquatic product needs, and optimizing the structure of aquaculture. However, germplasm degradation and lack of excellent varieties are the main bottlenecks for the sustainable and healthy development of the whiteleg shrimp industry. Therefore, cultivating new excellent whiteleg shrimp varieties has become the key to the development of my country's whiteleg shrimp industry.

[0003] Traditional fish breeding methods rely entirely on phenotypes, and there are insurmountable obstacles such as long cycles and low efficiency. Molecular breeding, also known as molecular marker-assisted selection breeding, refers to the use of DNA molecular markers to select breeding materials and comprehensively improve the important economic traits of selected species. It is a breeding method that organically combines traditional genetic breeding with modern molecular biology. Molecular breeding has opened up a new path for fish breeding. With the development of modern biotechnology, the role of molecular markers in fish breeding will become increasingly prominent. In the breeding of white shrimp, people hope to achieve the goal of early selection and improving breeding accuracy by selecting DNA markers that are closely related to growth traits and closely linked to quantitative traits, thereby achieving greater genetic progress.

[0004] However, at present, molecular markers related to growth traits that can be effectively used in the breeding of whiteleg shrimp have yet to be discovered. The present invention provides a SNP marker related to the growth rate of whiteleg shrimp. By detecting the SNP polymorphism of whiteleg shrimp, its growth rate can be effectively determined, which is beneficial to the early screening of whiteleg shrimp, and has the advantages of saving time, low cost and high accuracy. Summary of the invention

[0005] In order to solve the above technical problems, one object of the present invention is to propose a SNP marker related to the growth traits of Penaeus vannamei and can be effectively used for the breeding of Penaeus vannamei.

[0006] It should be noted that SNP (single nucleotide polymorphism, SNP) is a type of molecular genetic marker proposed by Lander, a scholar at the Human Genome Research Center of the Massachusetts Institute of Technology in 1996. It mainly refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level. The polymorphism exhibited by SNP only involves the variation of a single base, which is manifested by conversion, transversion, insertion and deletion, etc.

[0007] According to one aspect of the present invention, the present invention provides a SNP marker related to the growth rate of white shrimp, the nucleotide sequence of the SNP marker is shown in SEQ ID NO: 1, the polymorphism of the SNP marker is reflected in the 501st position of the SEQ ID NO.1 sequence, and the polymorphic site is G or T.

[0008] The inventors found that the weight of the white shrimp with a homozygous TT genotype at this site is significantly higher than that of the white shrimp with a heterozygous GT genotype here. Furthermore, according to an embodiment of the present invention, by detecting the above-mentioned SNP of white shrimp, its growth rate can be effectively determined. Specifically, as mentioned above, the weight of the white shrimp with a homozygous TT SNP site is significantly higher than that of the white shrimp with a heterozygous GT genotype here. For example, when the genotype of the SNP site is TT, it can be determined that the white shrimp to be tested belongs to an individual with a fast growth rate. Therefore, the inventors determined that the SNP marker of the present invention is closely related to the growth rate of white shrimp, and can be effectively used for molecular marker-assisted breeding of white shrimp. Furthermore, the growth rate can be selected according to actual breeding needs to select white shrimp breeding materials for early selection, which can further effectively improve the efficiency and accuracy of breeding, and improve the genetic level of the white shrimp breeding population, so that the white shrimp can be accurately and efficiently bred. In addition, according to some embodiments of the present invention, the SNP markers of the present invention are used for molecular marker-assisted breeding of white shrimp, which has the advantages of early screening, time saving, low cost and high accuracy.

[0009] Another aspect of the present invention provides a method for determining the growth rate of Penaeus vannamei, the method comprising the following steps:

[0010] 1) Extracting genomic DNA of Penaeus vannamei to be tested;

[0011] 2) using the genomic DNA of the tested white shrimp as a template, and performing a PCR amplification reaction using primers with nucleotide sequences as shown in SEQ ID NO: 2-3;

[0012] 3) The PCR amplification product was sequenced and analyzed to detect the genotype at the 501 bp position of the amplification product to determine the genotype of the tested white shrimp. The growth rate of the TT genotype individual was significantly higher than that of the GT genotype individual.

[0013] Specifically, the reaction system of the PCR amplification reaction used in step 2) is calculated as 25 μL, including: 1 μL of 50-100 ng / μL of the tested white shrimp DNA, 1 μL of each of the upstream and downstream primers shown in SEQ ID NO: 2-3 at 10 pmol / μL, 2.0 μL of 10 mmol / L dNTP mix, 0.125 μL of 5 U / μL Taq DNA polymerase, 2.5 μL of 10×PCR reaction buffer, and the balance is double distilled water.

[0014] Specifically, the reaction program of the PCR amplification reaction used in step 2) is: 94°C, 5 minutes; 94°C, 30 seconds; 53°C, 30 seconds; 72°C, 30 seconds; 30 cycles in total; and incubation at 72°C for 5 minutes.

[0015] Preferably, the method of sequencing the PCR amplification product of the present invention is not particularly limited, as long as the sequence of the PCR amplification product, i.e., the fragment where the SNP marker is located, can be effectively obtained. According to some specific examples of the present invention, the PCR amplification product can be sequenced by at least one selected from the first generation gene sequencing, the second generation high-throughput gene sequencing, or the third generation high-throughput gene sequencing. Thus, the sequencing results can be obtained in a high-throughput, fast, efficient, and accurate manner.

[0016] Another aspect of the present invention provides a primer pair for detecting SNP markers related to the growth rate of white shrimp, and the nucleotide sequence of the primer pair is shown in SEQ ID NO: 2-3. The primer pair of the present invention can effectively perform PCR amplification on the fragment where the SNP marker related to the growth rate of the white shrimp to be tested is located, and then the SNP marker can be effectively detected by sequencing, and the genotype of the SNP marker site of the white shrimp to be tested can be determined, and then the growth rate of the white shrimp to be tested can be effectively determined. Specifically, the growth rate of white shrimp with a genotype of homozygous TT at the SNP marker site is significantly higher than that of white shrimp with a genotype of heterozygous GT here. For example, when the genotype of the SNP site is TT, it can be determined that the white shrimp to be tested belongs to an individual with a fast growth rate. Therefore, the primer pair for detecting the SNP marker of the present invention described above can be effectively used for molecular marker-assisted breeding of white shrimp, and can assist in the early selection of excellent white shrimp varieties in a short time, low cost and high accuracy.

[0017] In another aspect of the present invention, a kit for detecting SNP markers related to the growth rate of whiteleg shrimp is provided, the kit comprising a primer pair as shown in claim 5. By using the primer pair contained in the kit of the present invention, the polymorphism detection of the above-mentioned SNP markers related to the growth rate of the whiteleg shrimp to be tested can be effectively realized, and the genotype of the SNP marker site of the whiteleg shrimp to be tested can be determined, thereby effectively determining the growth rate of the whiteleg shrimp to be tested. Specifically, the growth rate of the whiteleg shrimp with a homozygous TT genotype at the SNP marker site is significantly higher than that of the whiteleg shrimp with a heterozygous GT genotype here. For example, when the genotype of the SNP site is TT, it can be determined that the whiteleg shrimp to be tested belongs to an individual with a fast growth rate. Therefore, the kit for detecting the SNP marker of the present invention described above can be effectively used for molecular marker-assisted breeding of whiteleg shrimp, thereby assisting in the early selection of excellent whiteleg shrimp varieties in a short time, at low cost and with high accuracy.

[0018] In another aspect of the present invention, there is provided an application of a SNP marker in detecting growth traits of Penaeus vannamei, such as the application of the aforementioned SNP marker associated with growth rate of Penaeus vannamei in detecting growth traits of Penaeus vannamei.

[0019] Specifically, the growth rate of individuals with TT genotype of the SNP marker is significantly higher than that of individuals with GT genotype.

[0020] The present invention also provides a product containing the aforementioned SNP marker associated with the growth rate of Penaeus vannamei, wherein the product is any one of the following products:

[0021] 1) Products for detecting SNP polymorphism or genotype associated with growth rate of Penaeus vannamei;

[0022] 2) Identify or assist in identifying products for the growth cycle of Penaeus vannamei;

[0023] 3) Products used for breeding of Penaeus vannamei.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention is the first to provide a SNP marker based on the species of Penaeus vannamei by using a genomic method. The marker can be effectively used in molecular marker-assisted breeding of Penaeus vannamei in combination with upstream and downstream primers. By detecting the SNP polymorphism of Penaeus vannamei, its growth rate can be effectively determined, thereby facilitating the early screening of Penaeus vannamei.

[0026] 2. The SNP markers related to the growth rate of Penaeus vannamei provided by the present invention can select the growth rate according to the actual breeding needs, and then perform early selection of Penaeus vannamei breeding materials, further effectively improve the efficiency and accuracy of breeding, and improve the genetic level of the Penaeus vannamei breeding population, so that excellent varieties of Penaeus vannamei can be bred accurately and efficiently;

[0027] 3. The SNP marker provided by the present invention is not limited by the age, gender, etc. of the whiteleg shrimp, and can be used for the early breeding of whiteleg shrimp, which can significantly promote the breeding process of whiteleg shrimp. DETAILED DESCRIPTION

[0028] The technical scheme of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention. The materials, instruments and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources. The technical means used in the embodiments, unless otherwise specified, are conventional means well known to those skilled in the art.

[0029] Unless otherwise specified, the terms used in the present invention have the general meanings in the field to which the present invention belongs.

[0030] The present invention is described below with reference to specific examples. It should be noted that these examples are merely illustrative and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the examples, conventional experimental conditions, such as the Molecular Cloning Laboratory Manual (Green MR & Sambrook J, Molecular cloning: a laboratory manual (Fourth Edition), 2012), or the conditions recommended in the manufacturer's instructions, are used. Where the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0031] Example 1 Obtaining SNP markers associated with growth rate of Penaeus vannamei

[0032] 1.1 Obtaining the white shrimp population

[0033] The group used was white shrimp hatched on March 10, 2020 in a white shrimp farm in Hainan. On April 12, 2020, 20,000 shrimp fry (total length 0.8 cm) were transferred to an effective water body with an area of ​​10m 3Cultivated in indoor cement pools. On August 8, 2020, 300 individuals were randomly selected from the cement pools, and tail limbs with a length of about 0.3 cm were cut and stored in 95% ethanol at -20°C for genomic DNA extraction.

[0034] 1.2 Extraction of genomic DNA from Penaeus vannamei

[0035] This experiment used the conventional phenol-chloroform method to extract genomic DNA from the fin rays of white shrimp. The specific steps are as follows:

[0036] (1) Take 0.3-0.5 g of tail limbs into a 1.5 mL Eppendorf tube, cut into pieces, and dry on a clean bench with the lid open for 20 min;

[0037] (2) After the ethanol has basically evaporated, wash with TE buffer (10 mmol / mL Tris, 1 mmol / mL EDTA, SDS 5%, pH = 8.0) for 1 to 2 times, then add 600 μL DNA extraction solution (0.001 mol / L Tris-Cl, 0.1 mol / L EDTA, SDS 5%, pH = 8.0) and 3 μL proteinase K (200 mg / ml), and digest in a 55°C water bath for about 3 h. During the first 30 min, gently shake the centrifuge tube once every 10 min until the liquid in the tube is clear.

[0038] (3) Add 600 μL of self-prepared phenol-chloroform solution (phenol: chloroform: isoamyl alcohol = 25:24:1), gently invert the centrifuge tube back and forth for 10 minutes, and centrifuge at 12000r for 10 minutes. Take the upper aqueous phase and extract it again with an equal volume of the above phenol-chloroform until there is no white precipitate between the aqueous phase and the organic phase;

[0039] (4) Extract once more with chloroform, remove the supernatant, add 2 times the volume of pre-cooled anhydrous ethanol to precipitate DNA, mix by inversion, let stand at 4°C for 30 min, centrifuge at 12000r for 10 min, wash the precipitate with 70% ethanol, centrifuge to dry the precipitate, and dissolve it in 50 μL sterile water. Store at 4°C for future use or at -20°C for long-term storage.

[0040] 1.3 Construction of a simplified genome sequencing (Restriction site Associated DNA sequencing, RAD-seq) library and sequencing to obtain SNP markers related to weight in white shrimp

[0041] Based on the MGI-Seq2000 high-throughput sequencing platform, the RAD simplified genome sequencing method was used to sequence the DNA samples of 300 individuals, generating a total of about 600Gb of data, with an average sequencing data volume of 2Gb for each individual. At the same time, the 300 individuals were also phenotyped for growth traits such as weight. The data were processed and screened using PLINK software, and then GWAS analysis was performed using EMMAX software based on a mixed linear model. A SNP site significantly associated with weight was found from 5,416 SNPs. The SNP site is located at the 501bp site of the sequence shown in SEQ ID NO: 1. In the SEQ ID NO.1 sequence, K is used to represent the site, and the base at this site is G or T. The weight of the white shrimp with a homozygous TT genotype at this site is significantly higher than that of the white shrimp with a heterozygous GT genotype at this site.

[0042] Example 2 Sequencing verification and application of SNP markers associated with growth rate of white shrimp

[0043] 2.1 Extraction of genomic DNA from the tail limbs of Penaeus vannamei

[0044] The whiteleg shrimp to be tested came from the whiteleg shrimp population in Example 1. 300 individuals were randomly selected, and genomic DNA was extracted according to the DNA extraction method described in Example 1.

[0045] 2.2 Amplification of nucleotide fragments containing SNP sites

[0046] The genomic DNA of each white shrimp to be tested obtained by the above extraction was used as a template, and the forward primer F: 5'-TTTCTGATCCTGTTTCTCTAGAT-3' (SEQ ID NO: 2) and the reverse primer R: 5'-CTATTGTGAGGCCAACGTGTGT-3' (SEQ ID NO: 3) were used to amplify the nucleotide fragment where the SNP to be tested was located. Among them, the PCR reaction system was calculated as 25 μL: 50-100 ng / μL template DNA 1 μL, 10 pmol / μL primers F and R 1 μL each, 10 mmol / L dNTP mix 2.0 μL, 5 U / μL TaqDNA polymerase 0.125 μL, 10×PCR reaction buffer 2.5 μL, and the balance was double distilled water; the PCR reaction conditions were: 94°C for 5 minutes; 94°C for 30 seconds, 53°C for 30 seconds, 72°C for 30 seconds, 30 cycles; 72°C for 5 minutes.

[0047] 2.3 Sequencing to identify SNP genotypes

[0048] Each PCR amplification product obtained in the above steps was sequenced in one direction on an ABI3730 sequencer to identify the genotype at 501 bp (i.e., the SNP marker of the present invention) in the sequence of SEQ ID NO: 1. The genotypes of the SNP site of the 300 tested white shrimp individuals and their weights are shown in Table 1 below.

[0049] Table 1 Genotypes of the SNP locus and their body weights of 300 individuals

[0050]

[0051]

[0052]

[0053]

[0054]

[0055] 2.4 Association analysis between SNP genotype and growth rate

[0056] Based on the results in Table 1, the SAS10.0 software Mixed program was used to perform linear simulation analysis on the association between the genotype of the SNP locus and the growth rate. The individual weight was used to represent the phenotypic value during the analysis, and the model used was as follows:

[0057] Y ijk =μ+G i +a j +e ijk .

[0058] Among them, Y ijk is the individual weight value, μ is the mean weight of the group, G i is the genotype effect vector, a j is a micro-effect multi-gene vector, e ijk is the random residual effect vector.

[0059] The results of the association analysis between the genotype of the SNP locus and growth rate are shown in Table 2 below.

[0060] Table 2 Genotype frequency of SNP loci and association analysis with body weight

[0061]

[0062] As shown in Table 2, the mean body weight of TT homozygous individuals is greater than that of GT heterozygous individuals.

[0063] The results of association analysis shown in Table 2 show that the difference between the mean weight of individuals with TT genotype and the mean weight of individuals with GT genotype is extremely significant (P<0.01). Furthermore, it is proved that the 501st base G or T from the 5' end of the nucleotide sequence shown in SEQ ID NO: 1 is significantly correlated with the growth rate of white shrimp, and is a SNP marker related to the growth rate of white shrimp. The growth rate of individuals with TT genotype marked by this SNP is significantly higher than that of individuals with GT genotype.

[0064] In summary, the above embodiments are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A SNP marker related to the growth rate of Penaeus vannamei, characterized in that: The nucleotide sequence of the SNP marker is shown in SEQ ID NO:

1. The polymorphism of the SNP marker is reflected in the 501st position of the SEQ ID NO. 1 sequence, and the polymorphic site is G or T.

2. A method for determining the growth rate of Penaeus vannamei, characterized in that: The method comprises the following steps: 1) Extracting genomic DNA of Penaeus vannamei to be tested; 2) using the genomic DNA of the tested white shrimp as a template, and performing a PCR amplification reaction using primers with nucleotide sequences as shown in SEQ ID NO: 2-3; 3) The PCR amplification product was sequenced and analyzed to detect the genotype at the 501 bp position of the amplification product to determine the genotype of the tested white shrimp. The growth rate of the TT genotype individual was significantly higher than that of the GT genotype individual.

3. The method for determining the growth rate of Penaeus vannamei according to claim 2, characterized in that: The reaction system of the PCR amplification reaction used in step 2) is calculated as 25 μL, including: 1 μL of 50-100 ng / μL of the tested white shrimp DNA, 1 μL of each of the upstream and downstream primers shown in SEQ ID NO: 2-3 at 10 pmol / μL, 2.0 μL of 10 mmol / L dNTP mix, 0.125 μL of 5 U / μL TaqDNA polymerase, 2.5 μL of 10×PCR reaction buffer, and the balance is double distilled water.

4. The method for determining the growth rate of Penaeus vannamei according to claim 2, characterized in that: The reaction program of the PCR amplification reaction used in step 2) is: 94°C, 5 minutes; 94°C, 30 seconds; 53°C, 30 seconds; 72°C, 30 seconds; 30 cycles in total; incubation at 72°C for 5 minutes.

5. A primer pair for detecting SNP markers related to the growth rate of Penaeus vannamei, characterized in that: The nucleotide sequences of the primer pairs are shown in SEQ ID NOs: 2-3.

6. A kit for detecting SNP markers related to growth rate of Penaeus vannamei, characterized in that: The kit comprises the primer pair as shown in claim 5.

7. Application of a SNP marker in detecting growth traits of Penaeus vannamei, characterized in that: Use of the SNP marker associated with the growth rate of Penaeus vannamei as claimed in claim 1 in detecting the growth traits of Penaeus vannamei.

8. The use of the SNP marker according to claim 7 in detecting growth traits of Penaeus vannamei, characterized in that: The growth rate of individuals with TT genotype marked by the SNP is significantly higher than that of individuals with GT genotype.

9. A product containing the SNP marker associated with the growth rate of Penaeus vannamei as claimed in claim 1, characterized in that: The product is any one of the following: 1) Products for detecting SNP polymorphism or genotype associated with growth rate of Penaeus vannamei; 2) Identify or assist in identifying products for the growth cycle of Penaeus vannamei; 3) Products used for breeding of Penaeus vannamei.

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