SNP marker related to growth rate of penaeus vannamei and application thereof

Abstract

The application discloses a SNP marker related to growth speed of Penaeus vannamei and application thereof, a nucleotide sequence of the SNP marker is shown in SEQ ID NO:1, and polymorphism of the SNP marker is reflected at the 501th position of the SEQ ID NO.1 sequence, and the polymorphism site is G or T. The SNP marker related to growth speed of Penaeus vannamei and application thereof can be used for early selection of breeding materials of Penaeus vannamei according to actual breeding requirements, further can effectively improve breeding efficiency and accuracy, improve the genetic level of a Penaeus vannamei breeding population, and thus can accurately and efficiently breed a fine variety of Penaeus vannamei.

Description

TECHNICAL FIELD

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

[0002] Penaeus vannamei (Boone, 1931) is an important aquatic animal. Litopenaeus vannamei In the past 30 years, artificial breeding techniques for Penaeus vannamei have been very mature, the scale of Penaeus vannamei culture has gradually expanded, and the rapid development of the Penaeus vannamei industry has important significance for promoting fishermen's income, meeting the demand of the public for aquatic products, and optimizing the structure of the aquatic breeding industry. However, germplasm degradation and lack of excellent varieties are the main bottlenecks for the sustainable and healthy development of the Penaeus vannamei industry. Therefore, cultivating new excellent varieties of Penaeus vannamei has become the key to the development of the Penaeus vannamei industry in China.

[0003] Traditional fish selection methods completely rely on phenotypes, and there are insurmountable obstacles such as long cycle and low efficiency. Molecular breeding, i.e. molecular marker assisted selection breeding, refers to the use of DNA molecular markers to select breeding materials and comprehensively improve important economic traits of selected species. It is a breeding method that combines traditional genetic breeding with modern molecular biology. Molecular breeding opens up a new way for fish breeding, and with the development of modern biotechnology, the role of molecular markers in fish breeding will become increasingly prominent. In the breeding of Penaeus vannamei, people hope to achieve early selection and improve breeding accuracy by selecting DNA markers closely related to growth traits and closely linked to quantitative traits, so as to achieve greater genetic progress.

[0004] However, at present, growth trait related molecular markers that can be effectively used for Penaeus vannamei breeding still need to be explored. The present application provides a SNP marker related to the growth rate of Penaeus vannamei. By detecting the SNP polymorphism of Penaeus vannamei, the growth rate of Penaeus vannamei can be effectively determined, thereby facilitating early screening of Penaeus vannamei, having the advantages of saving time, low cost and high accuracy. SUMMARY

[0005] To solve the above technical problems, one object of the present application is to provide a SNP marker related to the growth traits of Penaeus vannamei, which can be effectively used for the selection of Penaeus vannamei.

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

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

[0008] The inventors found that the body weight of Penaeus vannamei with homozygous TT genotype at the site is significantly lower than that of Penaeus vannamei with heterozygous GT genotype at the site. Further, according to the embodiments of the present application, by detecting the above-mentioned SNP of Penaeus vannamei, the growth rate thereof can be effectively determined, specifically, as described above, the body weight of Penaeus vannamei with homozygous TT genotype at the SNP site is significantly lower than that of Penaeus vannamei with heterozygous GT genotype at the SNP site, for example, when the genotype of the SNP site is TT, it can be determined that the to-be-tested Penaeus vannamei belongs to an individual with fast growth rate. Therefore, the inventors determined that the SNP marker of the present application is closely related to the growth rate of Penaeus vannamei, and can be effectively used for molecular marker assisted breeding of Penaeus vannamei. Further, the growth rate of breeding materials of Penaeus vannamei can be selected according to the actual breeding needs for early selection, which can further effectively improve the efficiency and accuracy of breeding, improve the genetic level of the breeding population of Penaeus vannamei, so that the excellent variety of Penaeus vannamei can be accurately and efficiently selected. In addition, according to some embodiments of the present application, the SNP marker of the present application is used for molecular marker assisted breeding of Penaeus vannamei, which has the advantages of early screening, time saving, low cost and high accuracy.

[0009] According to another aspect of the present application, a method for determining the growth rate of Penaeus vannamei is provided, the method comprising the following steps:

[0010] 1) extracting the genomic DNA of the to-be-tested Penaeus vannamei;

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

[0012] 3) sequencing analysis of the PCR amplification product, detecting the genotype at the 501bp of the amplification product, determining the genotype of the Penaeus vannamei to be tested, and the growth rate of the individual with TT genotype is significantly lower than that of the individual with GT genotype.

[0013] Specifically, the reaction system of the PCR amplification reaction used in step 2) is 25 μL, including: 50-100 ng / μL of the DNA of the Penaeus vannamei to be tested 1 μL, 10 pmol / μL of each of the upper and lower primers shown in SEQ ID NO: 2-3 1 μL, 10 mmol / L of dNTP mix 2.0 μL, 5 U / μL of Taq DNA polymerase 0.125 μL, 10×PCR reaction buffer 2.5 μL, and the rest is double distilled water.

[0014] Specifically, the reaction procedure of the PCR amplification reaction used in step 2) is: 94℃, 5 minutes; 94℃, 30 seconds; 53℃, 30 seconds; 72℃, 30 seconds; a total of 30 cycles; 72℃, 5 minutes.

[0015] Preferably, the method for sequencing the PCR amplification product of the present application 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 application, at least one of the first generation gene sequencing, the second generation high-throughput gene sequencing or the third generation high-throughput gene sequencing can be used to sequence the PCR amplification product. In this way, the sequencing results can be obtained in a high-throughput, rapid, efficient and accurate manner.

[0016] Another aspect of the present application also provides a primer pair for detecting the SNP marker related to the growth rate of Penaeus vannamei, and the nucleotide sequence of the primer pair is shown in SEQ ID NO: 2-3. The primer pair of the present application can effectively amplify the fragment of the above-mentioned SNP marker related to the growth rate of the Penaeus vannamei to be tested by PCR, and then the detection of the SNP marker can be effectively realized by sequencing, the genotype of the SNP marker site of the Penaeus vannamei to be tested can be determined, and the growth rate of the Penaeus vannamei to be tested can be effectively determined. Specifically, the growth rate of the Penaeus vannamei with homozygous TT genotype at the SNP marker site is significantly lower than that of the Penaeus vannamei with heterozygous GT genotype at the SNP marker site, for example, when the genotype of the SNP site is GT, it can be determined that the Penaeus vannamei to be tested belongs to the individual with fast growth rate. Therefore, the primer pair for detecting the SNP marker of the present application described above can be effectively used for the molecular marker assisted breeding of Penaeus vannamei, and then it can assist in early breeding of excellent varieties of Penaeus vannamei in a short time, low cost and high accuracy.

[0017] Still another aspect of the present application provides a kit for detecting the SNP marker related to the growth rate of Penaeus vannamei, which comprises the primer pair as shown in claim 5. By using the primer pair comprised in the kit of the present application, the polymorphism of the above-mentioned SNP marker related to the growth rate of the Penaeus vannamei to be tested can be effectively detected, the genotype of the SNP marker site of the Penaeus vannamei to be tested can be determined, and the growth rate of the Penaeus vannamei to be tested can be effectively determined. Specifically, the growth rate of the Penaeus vannamei with the genotype of TT at the SNP marker site is significantly lower than that of the Penaeus vannamei with the genotype of GT at the SNP marker site, for example, when the genotype of the SNP site is GT, it can be determined that the Penaeus vannamei to be tested belongs to the individual with fast growth rate. Therefore, the kit for detecting the aforementioned SNP marker of the present application can be effectively used for the molecular marker assisted breeding of the Penaeus vannamei, and can further assist in early breeding of the Penaeus vannamei of excellent variety with short time, low cost and high accuracy.

[0018] Still another aspect of the present application provides the application of the SNP marker in detecting the growth trait of Penaeus vannamei, for example, the application of the aforementioned SNP marker related to the growth rate of Penaeus vannamei in detecting the growth trait of Penaeus vannamei.

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

[0020] The present application further provides a product containing the aforementioned SNP marker related to the growth rate of Penaeus vannamei, which is any one of the following products:

[0021] 1) a product for detecting the polymorphism or genotype of the SNP related to the growth rate of Penaeus vannamei;

[0022] 2) a product for identifying or assisting in identifying the growth cycle of Penaeus vannamei;

[0023] 3) a product for breeding Penaeus vannamei.

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

[0025] 1. The present application provides a SNP marker based on the species of Penaeus vannamei for the first time, which can be effectively used for the molecular marker assisted breeding of Penaeus vannamei in cooperation with the upstream and downstream primers, and the growth rate of Penaeus vannamei can be effectively determined by detecting the polymorphism of the SNP, thereby facilitating the early screening of Penaeus vannamei;

[0026] 2. The application provides a SNP marker related to the growth rate of Penaeus vannamei, which can select the growth rate according to the actual breeding needs, thereby early selecting the breeding materials of Penaeus vannamei, further effectively improving the efficiency and accuracy of breeding, improving the genetic level of the breeding population of Penaeus vannamei, so as to accurately and efficiently breed excellent varieties of Penaeus vannamei.

[0027] 3. The SNP marker provided by the application is not limited by the age and gender of Penaeus vannamei, and can be used for early breeding of Penaeus vannamei, which can significantly promote the breeding process of Penaeus vannamei. DETAILED DESCRIPTION

[0028] The technical solutions of the application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the application. The materials, instruments and reagents used in the following embodiments, unless otherwise specified, can be obtained from commercial channels. The technical means used in the embodiments, unless otherwise specified, are conventional means known to those skilled in the art.

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

[0030] The application will be described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and should not be construed as limiting the application. If the specific techniques or conditions are not specified in the embodiments, they are in accordance with the conventional experimental conditions, such as the molecular cloning experimental manual (Green MR & Sambrook J, Molecular cloning: a laboratory manual (Fourth Edition), 2012), or the conditions recommended by the manufacturer's instructions. If the reagents or instruments used are not specified by the manufacturer, they are conventional products that can be obtained from commercial channels.

[0031] Example 1: Obtaining of a SNP marker related to the growth rate of Penaeus vannamei

[0032] 1.1 Obtaining of a Penaeus vannamei population

[0033] The population used is Penaeus vannamei hatched on March 10, 2020, from a certain Penaeus vannamei breeding farm in Hainan. On April 12, 2020, 20000 shrimp fry (total length 0.8 cm) were transferred to an effective water body of 10 m 3Indoor cement pool cultivation. On August 8, 2020, 300 individuals were randomly selected from the cement pool, and tail fins 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 Penaeus vannamei genomic DNA extraction

[0035] In this experiment, the genomic DNA in the fin of Penaeus vannamei was extracted by the conventional phenol-chloroform method, and the specific steps were as follows:

[0036] (1) Take 0.3-0.5 g of tail fin in a 1.5 mL Eppendorf tube, cut it, and dry it on a clean bench for 20 min;

[0037] (2) After the ethanol evaporates, wash with TE buffer (10 mmol / mL Tris, 1 mmol / mL EDTA, SDS 5%, pH=8.0) for 1-2 times, then add 600 μL of DNA extraction solution (0.001 mol / L Tris-Cl, 0.1 mol / L EDTA, SDS 5%, pH=8.0) and 3 μL of proteinase k (200 mg / ml), 55 ℃ water bath digestion for 3 h, shake the centrifuge tube every 10 min for the first 30 min, and digest until the liquid in the tube is clear;

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

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

[0040] 1.3 Construction of a simplified genomic sequencing (Restriction site Associated DNA sequencing, RAD-seq) library and sequencing to obtain Penaeus vannamei weight-related SNP markers

[0041] Based on MGI-Seq2000 high-throughput sequencing platform, the DNA samples of 300 individuals were sequenced by using RAD simplified genome sequencing method, and about 600 Gb of data was generated, with an average of 2 Gb of sequencing data per individual. Meanwhile, the 300 individuals were also identified for growth traits such as body weight. The data were processed and screened by using PLINK software, and then GWAS analysis was performed by using EMMAX software based on mixed linear model, and one SNP site significantly related to body weight was found from 5,416 SNPs. The SNP site is located at the 501 bp site of the sequence shown in SEQ ID NO: 1, and the site is represented by K in the SEQ ID NO. 1 sequence, and the base at this site is G or T. The body weight of the Penaeus vannamei with homozygous TT genotype at this site is significantly lower than that of the Penaeus vannamei with heterozygous GT genotype at this site.

[0042] Example 2 Sequencing verification and application of SNP marker related to growth rate of Penaeus vannamei

[0043] 2.1 Extraction of genomic DNA in the tail of the Penaeus vannamei to be tested

[0044] The Penaeus vannamei to be tested were randomly selected from the Penaeus vannamei population in Example 1, and the 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 Penaeus vannamei to be tested obtained by the foregoing 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 fragments containing the SNP sites. The PCR reaction system was as follows: 50-100 ng / μL template DNA 1 μL, 10 pmol / μL primers F and R each 1 μL, 10 mmol / L dNTP mix 2.0 μL, 5U / μL Taq DNA polymerase 0.125 μL, 10×PCR reaction buffer 2.5 μL, and the rest was double distilled water; the PCR reaction conditions were as follows: 94℃ for 5 minutes; 94℃ for 30 seconds, 53℃ for 30 seconds, 72℃ for 30 seconds, 30 cycles; 72℃ for 5 minutes.

[0047] 2.3 Sequencing to identify SNP site genotypes

[0048] The PCR amplification products obtained in the above steps were subjected to single-direction sequencing on an ABI3730 sequencer to identify the genotype at 501 bp in the sequence of SEQ ID NO: 1 (i.e. the SNP marker of the application). The genotypes of the SNP site of 300 Penaeus vannamei individuals to be tested and their body weights are shown in Table 1 below.

[0049] Table 1: Genotype of the SNP site of 300 individuals and their body weights

[0050]

[0051] 2.4 Association analysis of SNP site genotype and growth rate

[0052] Based on the results in Table 1, the SAS 10.0 software Mixed program was used to perform linear simulation analysis of the association between the genotype of the SNP site and the growth rate, wherein the individual body weight was used as the phenotypic value in the analysis, and the following model was used:

[0053] Y ijk = μ + G i + a j + e ijk .

[0054] wherein Y ijk is the individual body weight value, μ is the population body weight mean, G i is the genotype effect vector, a j is the micro-effect polygene vector, and e ijk is the random residual effect vector.

[0055] The results of the association analysis of the genotype of the SNP site and the growth rate are shown in Table 2 below.

[0056] Table 2: Genotype frequency of the SNP site and association analysis with body weight

[0057]

[0058] As shown in Table 2, the mean body weight of the TT homozygous individuals is smaller than that of the GT heterozygous individuals.

[0059] The correlation analysis result shown in Table 2 shows that the difference between the mean of the body weight of the individual with the TT genotype and the mean of the body weight of the individual with the GT genotype reaches a significant level (P<0.01). Further, it is proved that the nucleotide sequence shown in SEQ ID NO: 1 is significantly related to the growth rate of the P. vannamei from the 501st base G or T at the 5' end, and is a SNP marker related to the growth rate of the P. vannamei, and the growth rate of the individual with the TT genotype of the SNP marker is significantly lower than that of the individual with the GT genotype.

[0060] To sum up, the above-mentioned embodiments are only the preferred embodiments of the present application, and are not used to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for determining the growth rate of Penaeus vannamei, characterized by, The method comprises the following steps: 1) extracting the genomic DNA of the Penaeus vannamei to be tested; 2) using the genomic DNA of the Penaeus vannamei to be tested as a template, performing a PCR amplification reaction by using primers with nucleotide sequences as shown in SEQ ID NO: 2-3; 3) performing sequencing analysis on the PCR amplification product, detecting the genotype at the 501st position of the sequence shown in SEQ ID NO: 1, determining the genotype of the Penaeus vannamei to be tested, and the growth speed of an individual with a TT genotype is significantly lower than that of an individual with a GT genotype.

2. The method of determining the growth rate of P. vannamei according to claim 1, characterized in that, The reaction system of the PCR amplification reaction used in step 2) is 25 μL, including: 1 μL of 50-100 ng / μL of the DNA of the Penaeus vannamei to be tested, 1 μL of 10 pmol / μL of the upper and lower primers shown in SEQ ID NO: 2-3, 2.0 μL of 10 mmol / L of dNTP mix, 0.125 μL of 5 U / μL of Taq DNA polymerase, 2.5 μL of 10×PCR reaction buffer, and the rest is double distilled water.

3. The method of determining the growth rate of P. vannamei according to claim 1, wherein, The reaction program of the PCR amplification reaction used in step 2) is: 94℃ for 5 minutes; 94℃ for 30 seconds; 53℃ for 30 seconds; 72℃ for 30 seconds; a total of 30 cycles; 72℃ for 5 minutes.

4. The use of a primer for detecting a SNP marker in detecting the growth rate of Penaeus vannamei, characterized in that, The nucleotide sequence of the SNP marker is shown in SEQ ID NO: 1, and the polymorphism of the SNP marker is reflected at the 501st position of the SEQ ID NO. 1 sequence, and the polymorphic site is G or T; The growth speed of an individual with a TT genotype of the SNP marker is significantly lower than that of an individual with a GT genotype.

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